NZ624231B2 - Assays and methods for selecting a treatment regimen for a subject with depression - Google Patents

Assays and methods for selecting a treatment regimen for a subject with depression Download PDF

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NZ624231B2
NZ624231B2 NZ624231A NZ62423112A NZ624231B2 NZ 624231 B2 NZ624231 B2 NZ 624231B2 NZ 624231 A NZ624231 A NZ 624231A NZ 62423112 A NZ62423112 A NZ 62423112A NZ 624231 B2 NZ624231 B2 NZ 624231B2
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seq
allele
snp
nucleic acid
acid sequence
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NZ624231A
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NZ624231A (en
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Maurizio Fava
Jr Harold O Koch
David Kronlage
George Papakostas
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Alfasigma Spa
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Priority claimed from PCT/US2012/065084 external-priority patent/WO2013074676A2/en
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Publication of NZ624231B2 publication Critical patent/NZ624231B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Abstract

assay for selecting a treatment regimen for a human subject with depression, comprising (a) subjecting a test sample previously obtained from a human subject, who is diagnosed as having depression or having a risk for depression, to at least one genotyping assay adapted to determine the genotypes of at least two loci. The at least two loci are: i. position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), where the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR) or ii.position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), where the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR). The assay furhter includes b) detecting from the genotypes of said at least two loci the presence of a single nucleotide polymorphism (SNP) selected from the following: i. a SNP677 at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP2756 at position 2756 of SEQ ID NO.2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; and iii. a combination of at least one SNP677 T allele and at least one SNP2756 G allele; and if at least one of T allele at position SNP677 or at least one G allele at position SNP2756 or both at least one T allele at position SNP677 and at least one G allele at position SNP2756 is detected, then selecting a treatment regimen comprising an effective amount of a folate-containing compound. of at least two loci. The at least two loci are: i. position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), where the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR) or ii.position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), where the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR). The assay furhter includes b) detecting from the genotypes of said at least two loci the presence of a single nucleotide polymorphism (SNP) selected from the following: i. a SNP677 at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP2756 at position 2756 of SEQ ID NO.2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; and iii. a combination of at least one SNP677 T allele and at least one SNP2756 G allele; and if at least one of T allele at position SNP677 or at least one G allele at position SNP2756 or both at least one T allele at position SNP677 and at least one G allele at position SNP2756 is detected, then selecting a treatment regimen comprising an effective amount of a folate-containing compound.

Description

ASSAYS AND METHODS FOR SELECTING A TREATMENT REGIMEN FOR A SUBJECT WITH DEPRESSION CROSS REFERENCE TO RELATED APPLICATIONS This Application claims the benefit under 35 U.S.C §119(e) of U.S. Provisional Application No. 61/559,541 filed November 14, 2011, the content of which is orated herein by reference in its ty.
TECHNICAL FIELD OF THE DISCLOSURE Embodiments of the inventions generally relate to assays, methods and systems for selecting a ent regimen for a subject with depression, e.g., major depressive disorder. The ions further relate to methods for treating a subject with depression, e.g., major depressive BACKGROUND Recent estimates te that more than 19 million Americans over the age of 18 years ence a depressive illness each year. It has been generally believed that there is some form of association between folate-deficiency states and depression [1, 2 and 3], which in turn helps to explain prior observations on the myriad neuropsychiatric presentations of megaloblastic anemia [4].
Recently, the relevance of folate in other medical conditions, in particular neural tube defects [5] and cardiovascular disease, [6] and potential antidepressant efficacy of agents marketed as dietary supplements or "nutraceuticals," [7 and 8] such as S-adenosyl-methionine , hypericum perforatum (St. John’s wort), and 3-fatty acids, has been increasingly recognized. The field has gradually moved toward researching the impact of folate deficiency, replacement, and supplementation on the course and management of depressive disorders, in particular major depressive disorder, [9] and putative roles of folate in central–nervous-system function. [10, 11 and 12]. While significant advances in the treatment of sion have been made in the past , as many as 29% to 46% of patients with depression taking an anti-depressant are still partially or totally resistant to the treatment. Those who suffer from treatment-resistant depression have almost no alternatives. As not every ent regimen is effective for each individual, there is a strong need to identify markers that can facilitate ion of an appropriate treatment regimen for a subject with depression. [0003a] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. [0003b] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, ising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.
SUMMARY A significant portion of patients with depression, such as major depressive disorders, show only partial or no response to conventional antidepressant drugs, e.g., selective serotonin ke inhibitors. Thus, there is a strong need to develop effective antidepressant therapies and/or to stratify patients with sion such that they can receive appropriate antidepressant therapies.
Aspects of various embodiments described herein stem from the ery of single nucleotide rphisms (SNPs), peripheral biomarkers and/or clinical features that are associated with an efficacy response to the use of a folate-containing nd for treatment of depression, e. g., major depressive disorders, as a monotherapy or as an adjunct to an antidepressant drug. In some embodiments, the inventors have also shown that these markers or conditions described herein can also be used to select a more effective treatment for subjects with treatment-resistant sion (TRD), e. g., resistant to at least one selective serotonin reuptake inhibitor (SSRI).
Particularly, one or a combination of biomarkers that can be indicative of a patient (e. g., with major depressive ers and/or TRD) suitable for a treatment regimen comprising a -containing compound include, but are not limited to, at least one or more SNPs identified by rs numbers as follows: rs1801133 t in methylenetetrahydrofolate reductase (MTHFR); rs2274976 present in MTHFR; rs1805087 present in methionine synthase (MTR); 394 present in methionine synthase reductase (MTRR); 737 present in calcium channel, voltage-dependent, L- type, alpha 1C subunit (CACNAlC); rs1883729 present in DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); rs7163862 t in GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); rs12659 present in reduced folate carrier protein ; rs202676 present in folate hydrolase (prostate-specific membrane antigen) (FOLHl); 29l present in reduced folate carrier protein (RCFl); rs1051266 t in reduced folate carrier protein 1 (RCFl); 267 present in GTP cyclohydrolase l (GCHl); rs7639752 t in choline-phosphate cytidylytransferase A (PCYTlA); rs6275present in dopamine receptor D2 (DRDZ); rs1079596 present in DRDZ; rs11240594 present in DRDZ; rs4633 present in catechol-O-methyltransferase (COMT); rs4680 present in COMT; rs250682 present in dopamine active transporter (DAT, or SLC6A3); rs2277820 present in formiminotransferase cyclodeaminase (FTCD); 225 present in methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFDl); and any combinations thereof; and/or expression of at least one of s-adenosyl methionine (SAM), s-adenosyl homocysteine (SAH), 4-hydroxynonenal (4-HNE), high sensitivity c-reactive protein (hsCRP), and any combinations thereof. Additionally or alternatively, determination of whether a human subject is obese or not (e. g., by measurement of a BMI value) can also be used as a biomarker to select an appropriate treatment regimen (e.g., comprising a folate-containing compound or not) for a patient with depression or a risk for depression. Any individual or combinations of such biomarkers disclosed herein can be used to identify patients, who are diagnosed as having depression or having a risk for depression, for receiving a treatment regimen sing a folate-containing compound. In some embodiments, a -containing compound can be used in the e of an anti-depressant drug for treatment of depression (e.g., major depressive disorders) in subjects selected for carrying at least one or more biomarkers described herein. In alternative embodiments, a folate-containing compound can be used alone or in combination (e.g., as an adjunct) with an anti-depressant drug for treatment of depression (e. g., major sive disorders) in subjects selected for carrying at least one or more biomarkers described herein. In one embodiment, the epressant drug can include a selective serotonin ke inhibitor (SSRI). Examples of the SSRI include, but are not limited to, tine, citalopram, paroxetine, lopram, sertraline, and any ations thereof.
Accordingly, provided herein generally relate to assays, methods, systems, and kits for selecting a treatment regimen for a t with depression or a risk for depression, treating a subject with depression or a risk for depression, and/or improving the effectiveness of a treatment regimen recommended for or administered to a subject with depression or a risk for depression.
Provided herein also relate to folate-comprising compositions for use in treatment of sion in a subject (e.g., a human subject) selected to carry at least one (e.g., at least two or more) or any combinations of the biomarkers or ions described herein. [0006a] In a first aspect of the invention there is provided an assay for ing a treatment regimen for a human subject with depression, comprising: (a) subjecting a test sample previously obtained from a human t, who is diagnosed as having depression or having a risk for depression, to at least one genotyping assay adapted to determine the genotypes of at least two loci, wherein said at least two loci are: i. position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each ndently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii.position 2756 of SEQ ID NO. 2 or on 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and (b) detecting from the genotypes of said at least two loci the presence of a single nucleotide polymorphism (SNP) selected from the following: i. a SNP677 at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP2756 at position 2756 of SEQ ID NO.2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; and iii. a combination of at least one SNP677 T allele and at least one SNP2756 G allele; and if at least one of T allele at position SNP677 or at least one G allele at position SNP2756 or both at least one T allele at position SNP677 and at least one G allele at position SNP2756 is detected, then selecting a treatment regimen comprising an effective amount of a folate-containing compound. [0006b] In a second aspect of the invention there is provided an assay for selecting a treatment n for a human subject having depression and currently taking an antidepressant, comprising: (a) subjecting a test sample of the human subject, who is diagnosed as having depression or having a risk for depression, to at least one analysis to determine ters of at least two biomarkers from: i. genotype of a SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii.genotype of a SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iii. genotype of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); iv. genotype of a SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each ndently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); v. genotype of a SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, n the SEQ ID NO. 11 is a portion of a genomic c acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C)); vi. genotype of a SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, wherein the SEQ ID NO. 12 is a n of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B)); vii. genotype of a SNP locus at rs7163862 (position 27 of SEQ ID NO. 13, n the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 ck regulatory protein (GCHFR)) ; viii. genotype of a SNP locus at rs12659 ion 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic c acid sequence of reduced folate r protein (RCF2)); ix. genotype of a SNP locus at rs202676 ion 27 of SEQ ID NO. 15, wherein the SEQ ID NO. 15 is a n of a c nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1)); x. genotype of a SNP locus at 291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)); xi.genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17, n the SEQ ID NO. 17 is a portion of a genomic c acid sequence of reduced folate carrier protein (RCF1)); xii. genotype of a SNP locus at rs8007267 (position 27 of SEQ ID NO. 18, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid ce of GTP cyclohydrolase 1 (GCH1)); xiii. genotype of a SNP locus at rs7639752 (position 27 of SEQ ID NO. 19, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A A)); xiv. genotype of a SNP locus at rs6275 (position 27 of SEQ ID NO. 20, wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2)); xv. genotype of a SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a c nucleic acid sequence of dopamine or D2 (DRD2); xvi. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvii. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23, wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT)); xviii. genotype of a SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a n of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. genotype of a SNP locus at rs250682 (position 27 of SEQ ID NO. 25, wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 (SLC6A3)); xx. genotype of a SNP locus at rs2277820 (position 27 of SEQ ID NO. 26, n the SEQ ID NO. 26 is a portion of a c nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD)); and xxi. genotype of a SNP locus at rs2236225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a portion of a genomic c acid sequence of methylenetetrahydrofolate ogenase (NADP+ dependent) 1 (MTHFD1)); xxii. level of expression of SAM and SAH; xxiii. level of expression of 4-HNE; xxiv. level of expression of hsCRP; and any combinations thereof; (b) detecting from the ters of said at least two biomarkers, the presence of at least one ion selected from the following: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele; iii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; iv. a SNP at position 66 of SEQ ID NO. 3 or on 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele; v. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele; vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” ; vii. a SNP at rs7163862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; viii. a SNP at rs12659 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele; ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; x. a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” ; xi. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; xii. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one e “T” allele; xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19 ) comprising at least one alanine “A” allele; xiv. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele; xv. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele; xvi. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) sing at least one cytosine “C” allele; xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xix. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele; xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one e “T” allele; xxi. a SNP at 225 (position 1958 of SEQ ID NO. 27) comprising at least one alanine “A” allele; xxii. obesity (e.g., defined by a BMI value of at least 30 kg/m2 or r); xxiii. an expression level ratio of SAM to SAH smaller than a pre-determined reference ratio; xxiv. an expression level of 4-HNE greater than a termined reference value; xxv. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample; and any ations thereof, and and if at least one of said conditions is detected, then ing a treatment regimen comprising an effective amount of a folate-containing compound. [0006c] In a third aspect of the invention there is provided use of a folate-comprising composition in combination with an anti-depressant in the preparation of a medicament for use in the treatment of depression in a human subject who carries at least the following single nucleotide polymorphisms: a. a SNP677 at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by 133) comprising at least one thymine “T” allele; and b. a SNP2756 at on 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele. [0006d] In a fourth aspect of the invention there is provided use of a folate-comprising composition in combination with an anti-depressant in the preparation of a medicament for use in the treatment of depression in a human subject who carries at least two of the following conditions or any combination f: a. at least one thymine "T" allele at SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a c nucleic acid ce of methylenetetrahydrofolate ase (MTHFR); b. at least one alanine "A" allele at SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or on 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); c. at least one guanine "G" allele at SNP locus at position 2756 of SEQ ID NO.2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); d. at least one guanine "G" allele at SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a c nucleic acid sequence of methionine synthase reductase (MTRR); e. at least one e "A" allele at SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, e-dependent, L type, alpha 1C subunit (CACNA1C)); f. at least one alanine "A" allele at SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta B)); g. at least one thymine "T" allele at SNP locus at rs7163862 (position 27 of SEQ ID NO. 13, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 ck regulatory protein (GCHFR)); h. at least one thymine "T" allele at SNP locus at rs12659 (position 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2)); i. at least one guanine "G" allele at SNP locus at rs202676 (position 27 of SEQ ID NO. , wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate ase (prostate-specific membrane n) 1 (FOLH1)); j. at least one alanine "A" allele at SNP locus at rs2297291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)); k. at least one alanine "A" allele at SNP locus at rs1051266 (position 27 of SEQ ID NO. 17), wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1); l. at least one thymine "T" allele at SNP locus at rs8007267 (position 27 of SEQ ID NO. 18, n the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1)); m. at least one alanine "A" allele at SNP locus at rs7639752 ion 27 of SEQ ID NO. 19, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of e-phosphate cytidylyltransferase A (PCYT1A)); n. at least one thymine "T" allele at SNP locus at rs6275 (position 27 of SEQ ID NO. , wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2)); o. at least one thymine "T" allele at SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), n the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); p. at least one e "A" allele at SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a n of a c nucleic acid sequence of dopamine receptor D2 (DRD2); q. at least one cytosine "C" allele at SNP locus at rs4633 (position 27 of SEQ ID NO. 23, n the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT)); r. at least one guanine "G" allele at SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); s. at least one cytosine "C" allele at SNP locus at rs250682 (position 27 of SEQ ID NO. , wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitter transporter, dopamine), member 3 (SLC6A3)); t. at least one thymine "T" allele at SNP locus at rs2277820 (position 27 of SEQ ID NO. 26, wherein the SEQ ID NO. 26 is a n of a genomic c acid sequence of inotransferase cyclodeaminase (FTCD)); and u. at least one alanine "A" allele at SNP locus at rs2236225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a n of a c nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFD1)); v. obesity (e.g., defined by a BMI value of at least 30 kg/m2 or greater); w. an expression level ratio of SAM to SAH smaller than a pre-determined reference ratio; x. an expression level of 4-HNE greater than a pre-determined reference value; and y. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample.
In a further aspect, provided herein is an assay for selecting a treatment regimen for a human subject having depression or having a risk for depression. The assay comprises: (a) subjecting a test sample of a human subject, who is diagnosed as having depression or having a risk for depression, to at least one analysis to determine parameters of at least two biomarkers from the following: (i) genotype of a SNP locus at on 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); (ii) genotype of a SNP locus at position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8 (identified by rs2274976), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid ce of MTHFR; (iii) genotype of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a n of a genomic nucleic acid sequence of methionine synthase (MTR); (iv) genotype of a SNP locus at on 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); (v) genotype of a SNP locus at position 27 of SEQ ID NO. 11 ified by rs1006737), wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C); (vi) pe of a SNP locus at position 27 of SEQ ID NO. 12 (identified by rs1883729), wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA ine-5)-methyltransferase 3 beta (DNMT3B); (Vii) pe of a SNP locus at position 27 of SEQ ID NO. 13 (identified by rs7163862), wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP ydrolase 1 feedback tory protein (GCHFR); (viii) genotype of a SNP locus at position 27 of SEQ ID NO. 14 (identified by rs12659), wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein ; (ix) genotype of a SNP locus at position 27 of SEQ ID NO. 15 (identified by rs202676), wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); (x) genotype of a SNP locus at position 27 of SEQ ID NO. 16 (identified by rs2297291), wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of d folate carrier protein ; (xi) genotype of a SNP locus at position 27 of SEQ ID NO. 17 (identified by rs1051266), wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); (xii) genotype of a SNP locus at position 27 of SEQ ID NO. 18 (identified by rs8007267), n the SEQ ID NO. 18 is a portion of a genomic c acid sequence of GTP cyclohydrolase 1 (GCH1); (xiii) genotype of a SNP locus at position 27 of SEQ ID NO. 19 (identified by rs7639752), wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline- phosphate cytidylyltransferase A (PCYTlA); (xiv) genotype of a SNP locus at position 27 of SEQ ID NO. 20 (identified by rs6275), wherein the SEQ ID NO. 20 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); (xv) genotype of a SNP locus at position 27 of SEQ ID NO. 21 (identified by rs1079596), wherein the SEQ ID NO. 21 is a portion of a c nucleic acid sequence of dopamine or D2 (DRD2); (xvi) genotype of a SNP locus at position 27 of SEQ ID NO. 22 (identified by rs11240594), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); (xvii) genotype of a SNP locus at position 27 of SEQ ID NO. 23 ified by ), wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol- O-methyltransferase (COMT); (xviii) genotype of a SNP locus at position 27 of SEQ ID NO. 24 ified by rs4680), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol- O-methyltransferase (COMT); 2012/065084 (xix) genotype of a SNP locus at position 27 of SEQ ID NO. 25 (identified by rs250682), n the SEQ ID NO. 25 is a portion of a genomic c acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3); (xx) genotype of a SNP locus at position 27 of SEQ ID NO. 26 (identified by rs2277820), wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); (xxi) genotype of a SNP locus at position 27 of SEQ ID NO. 27 (identified by rs2236225), wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l 1)); (xxii) level of sion of SAM and SAH; (xxiii) level of expression of 4-HNE; (xxiv) level of expression of hsCRP; and any combinations thereof; and (b) detecting, ally with a non-human e, from the determined ters of at least two biomarkers, the ce of at least one condition selected from the following: (A) a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine "T" allele; (B) a SNP at position 1793 of SEQ ID NO. 1 or on 27 of SEQ ID NO. 8 (identified by rs2274976) comprising at least one alanine "A" allele; (C) a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087) comprising at least one guanine "G" allele; (D) a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394) comprising at least one guanine "G" allele; (E) a SNP at position 27 of SEQ ID NO. 11(identified by rs1006737) comprising at least one alanine "A" allele; (F) a SNP at position 27 of SEQ ID NO. 12 (identified by rs1883729) comprising at least one alanine "A" allele; (G) a SNP at position 27 of SEQ ID NO. 13(identified by rs7l63862) comprising at least one thymine "T" allele; (H) a SNP at position 27 of SEQ ID NO. 14 (identified by rsl2659) comprising at least one thymine "T" allele; (I) a SNP at position 27 of SEQ ID NO. ntified by rs202676) comprising at least one guanine "G" allele; (J) a SNP at position 27 of SEQ ID NO. 16 (identified by rs229729l) comprising at least one e "A" allele; (K) a SNP at position 27 of SEQ ID NO. 17 (identified by at rs1051266) comprising at least one alanine "A" allele; (L) a SNP at position 27 of SEQ ID NO. 18 (identified by rs8007267) comprising at least one thymine "T" allele; (M) a SNP at position 27 of SEQ ID NO. 19 (identified by 752) comprising at least one alanine "A" allele; (N) a SNP at position 27 of SEQ ID NO. 20 (identified by rs6275) comprising at least one thymine "T" allele; (0) a SNP at position 27 of SEQ ID NO. 21 (identified by 596) comprising at least one thymine "T" allele; (P) a SNP at position 27 of SEQ ID NO. 22 (identified by rsl 1240594) comprising at least one alanine "A" allele; (Q) a SNP at position 27 of SEQ ID NO. 23 (identified by rs4633) comprising at least one ne "C" allele; (R) a SNP at position 27 of SEQ ID NO. 24 ified by rs4680) comprising at least one guanine "G" allele; (S) a SNP at position 27 of SEQ ID NO. 25 ified by rs250682) comprising at least one cytosine "C" allele; (T) a SNP at position 27 of SEQ ID NO. 26 (identified by rs2277820) comprising at least one thymine "T" allele; (U) a SNP at position 1958 of SEQ ID NO. 27 (identified by rs2236225) comprising at least one alanine "A" allele; (V) an expression level ratio of SAM to SAH smaller than a termined reference ratio; (W) an expression level of 4-HNE greater than a pre-determined nce value; (X) an expression of hsCRP greater than about 2.3 mg per liter as measured in a plasma sample; and any combinations thereof.
Based on the analysis results from step (b), if at least one of the conditions described above is detected, the assay can further se selecting for the human t and optionally stering to the human subject a treatment regimen comprising an effective amount of a folatecontaining compound.
In some embodiments of this aspect and all other aspects described herein, any of the SNPs described herein can comprise one or two folate-respsonive alleles. By way of example only, a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 ified by rs1801133) can comprise one thymine "T" allele, or two thymine “T” alleles.
Any combinations of at least two biomarkers (i) to (xxiv) described herein can be determined in a test sample for the assays bed herein. Exemplary combinations of at least two biomarkers described herein can comprise genotypes of at least two SNP loci; or genotype of at least one SNP loci and expression level of at least one indicated biomarker (e. g., 4-HNE); or expression level of at least two indicated kers (e. g., SAM, SAH). Depending upon selected combinations of at least two biomarkers described herein, the test sample can be subjected to one or more analyses, e.g., including, but not limited to, genotyping assays, expression assays (e. g., protein and/or transcript levels), or any combinations thereof. ingly, in some embodiments, the assay can comprise subjecting a test sample from a human subject, who is sed as having depression or having a risk for depression to at least one genotyping assay adapted to ine genotypes of at least two loci, wherein said at least two loci are: (i) position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) and (ii) position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087). In such embodiment, ion of at least one SNP at either position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) sing at least one thymine “T” allele or position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9) comprising at least one guanine “G” allele, or detection of both aforementioned SNPs indicates selection and optional administration of a treatment regimen comprising an effective amount of a folate-containing compound to the human subject.
In some embodiments, the genotyping assay can comprise the step of amplifying the test sample with a set of primers flanking any one of the SNPs described . In some embodiments, at least two (e. g., at least three, at least four, at least five or more) sets of primers amplifying at least two (e. g., at least three, at least four, at least five or more) of the SNPs can be used in a lex amplification assay.
In some embodiments, the test sample can be subjected to determine parameters of at least three, at least four, at least five or more biomarkers (i) to (xxiv) bed herein. For example, in some embodiments, the assay can se: (a) subjecting a test sample of a subject to one or more than one analyses (e. g., genotyping and/or expression analyses) to determine the presence or absence of at least one of the following conditions: i. an expression ratio of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) smaller than a pre-determined reference ratio; ii. expression of 4-hydroxynonenal (4-HNE) greater than a pre-determined reference value; iii. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO: 1 comprising at least one thymine “T” allele, wherein the SEQ ID NO: 1 is a portion of a genomic nucleic acid sequence of enetetrahydrofolate reductase (MTHFR); iv. a SNP at position 2756 of SEQ ID NO: 2 comprising at least one guanine “G” allele, wherein the SEQ ID NO: 2 is a n of a genomic c acid sequence of methionine synthase (MTR); and v. a SNP at position 66 of SEQ ID NO: 3 comprising at least one guanine “G” allele, wherein the SEQ ID NO: 3 is a portion of a c nucleic acid sequence of methionine synthase reductase (MTRR); and if at least one of these conditions is determined to be present, the assay can r comprise selecting for the human subject and optionally administering to the human subject a treatment regimen comprising an effective amount of a folate-containing compound.
In some embodiments of this aspect and all other aspects described herein, when the expression ratio of SAM to SAH is smaller than the pre-determined nce ratio, e.g., smaller than 3.0, or smaller than about 2.8 as ed in a plasma sample, the subject can be recommended for and/or optionally administered with a treatment regimen comprising a folate-containing compound. In one embodiment, the expression ratio of SAM to SAH being smaller than 2.71, as ed in a plasma sample, is indicative of a subject recommended for and/or optionally administered with a treatment regimen comprising a folate-containing compound. In some embodiments, if the expression ratio of SAM to SAH is at least or greater than 2.71 as measured in a plasma sample, the subject is not recommended for nor stered with a treatment n comprising a folate-containing compound. Depending on the test sample , e.g., a blood sample vs. a buccal sample, the pre- determined reference ratio for a blood plasma sample can be different from that for, e.g., a buccal sample.
In some embodiments of this aspect and all other aspects described herein, when the expression of 4-HNE is greater than the pre-determined reference value, e.g., greater than about 3 mg per liter, or greater than about 3.2 mg per liter of plasma or higher as ed in a plasma sample, the subject can be recommended for and/or optionally administered with a treatment regimen sing a -containing compound. In one embodiment, if the expression of 4-HNE is at least 3.28 mg per liter of plasma as measured in a plasma sample or higher, a subject can be recommended for and/or ally stered with a ent regimen comprising a folate-containing compound. In some ments, if the expression of 4-HNE is less than 3.28 mg per liter of plasma as measured in a plasma sample or lower, the subject is not recommended for nor administered with a treatment regimen comprising a folate-containing compound. Depending on the test sample source, e.g., a blood sample vs. a buccal sample, the pre-determined reference value for a plasma sample can be different from that for, e.g., a buccal sample.
In some embodiments of this aspect and all other aspects described herein, the assay can further comprise determining if the human subject is obese or not. If the human subject is determined to be obese, then selecting for the human subject and ally administering to the human subject a treatment regimen comprising an effective amount of a folate-containing compound.
Methods of determining y in a human subject are known in the art and can include, but are not limited to, body mass index (BMI) measurement, measurement of abdominal fat (e.g., by waist circumference or waist-hip ratio), measurement of body fat, skinfold thickess, underwater weighing (densitometry), air-displacement plethysmography, computerized tomography (CT) and magnetic resonance imaging (MRI), and dual energy X-ray absorptiometry (DEXA), and any combinations thereof. For e, in one embodiment, the assay can further comprise measuring body mass index (BMI) of the human subject to determine if the human subject is obese or not, and if the BMI value of at least 30 kg/m2 or greater is measured in the subject, then selecting for the human subject and optionally stering to the human subject a treatment regimen sing an effective amount of a folate-containing compound. In some embodiments, if the human subject has a BMI value of less than 30 kg/mz, it may not be ble to recommend or administer to the human t a treatment n comprising a folate-containing compound.
In some embodiments, the assay can further comprise determining the presence or absence of a SNP at position 1298 of the SEQ ID NO. 1 comprising at least one cytosine “C” allele, wherein the presence of the SNP at position 1298 of the SEQ ID NO: 1 comprising at least one cytosine “C” is indicative of the subject recommended for and optionally administered with a treatment regimen comprising a folate-containing compound.
In some embodiments, the assay can further comprise determining expression of high-sensitivity c-reactive protein (hsCRP), wherein the hsCRP expression greater than about 2.3 mg per liter of plasma, as measured in a plasma , is indicative of the subject recommended for and optionally administered with a treatment regimen comprising a folate-containing compound. In some embodiments, if the expression of hsCRP is lower than 2.3 mg per liter of , as measured in a plasma sample, then the subject is not ended for nor administered with a treatment regimen comprising a folate-containing compound. Depending on the test sample source, e.g., a blood sample vs. a buccal sample, the hsCRP expression a plasma sample can be different from that in, e.g., a buccal .
While detection of the presence of at least one condition (A)-(X) described herein in a test sample of a human subject with depression is generally sufficient to indicate the human t be amenable to a treatment regimen comprising an effective amount of a folate-containing compound, in some embodiments, it can be desirable to detect the presence of at least two conditions, three ions, four conditions or more ponding to the selected kers in order to select for or administer to the human subject a treatment regimen comprising an effective amount of a folate- containing compound. In some ments, if none of these conditions (A) —(X) described herein occurs in the human subject, the subject is not recommended for a treatment regimen comprising a folate-containing compound.
Accordingly, in some embodiments of this aspect and all other aspects described herein, the test sample can be analyzed to determine at least one or at least two, at least three, at least four, at least five or six of the conditions (A)-(X) provided herein. For example, in some embodiments, the test sample can be analyzed to determine if the subject has at least the SNPs located at the positions 677 and 2756 of the MTHFR and MTR loci, tively. In some embodiments, the test sample can be analyzed to determine if the subject is obese (e.g., whether the subject has at least the BMI value of at least 30 kg/mz), and at least one or both of the SNPs located at the positions 2756 and 66 of the MTR and MTRR loci, respectively. In some embodiments, the test sample can be analyzed to determine if the subject is obese (e. g., whether the subject has at least the BMI value of at least 30 kg/mz), and at least one or both of the SNPs located at the positions 677 and 2756 of the MTHFR and MTR loci, tively. In other embodiments, the test sample can be analyzed to determine if the subject has at least the BMI value of at least 30 kg/m2 and the SNP located at the position 2756 of the MTR locus. In some embodiments, the test sample can be analyzed to determine if the subject has at least the SAM/SAH ratio smaller than the termined reference ratio and the SNP located at the position 2756 of the MTR locus. In some embodiments, the test sample can be analyzed to determine if the subject has at least the 4-HNE expression r than the pre-determined standard and the SNPs located at the positions 2756 and 66 of the MTR and MTRR loci, respectively.
It is envisioned that any combinations of all the conditions (A)-(X) can be analyzed in the assay as described herein.
In some embodiments, if at least one or at least two, including at least three or more, of the conditions ) provided herein are determined to be present in the test sample, a treatment regimen comprising a folate-containing compound is recommended or selected and optionally administered to the human subject.
In some embodiments, if the human subject satisfies at least one, including at least two, at least three or more, of the conditions (A)-(X) described herein (and an tor of a human subject being obese, e.g., a BMI value of at least 30 kg/m2 or greater), the t can be administered or prescribed with a folate-containing compound.
Some embodiments of the assays described herein can be included as part of a treatment strategy, e. g., to select an appropriate treatment regimen for a human subject diagnosed as having or having a risk for depression. Accordingly, another aspect provided herein relates to methods of ng a human subject diagnosed as having, or having a risk, for depression. In some embodiments, a method for treating a human t with depression can comprise ming one or more embodiments of the assay provided herein. In some ments, if the subject satisfies at least one of the conditions described in the assay provided herein, the treatment method can further comprise administering or prescribing the subject with a treatment regimen comprising an effective amount of a folate-containing compound.
Accordingly, in one embodiment, the method can comprise determining in a test sample of a human subject parameters of at least two or more (including at least three, at least four, at least five or more) biomarkers (i) to (xxiv) described herein; and administering to the human subject a ent n comprising an effective amount of a folate-containing compound, if the presence of at least one or more (including at least two, at least three, at least four, at least five or more), or any combinations of the conditions ) bed herein is ed in the test sample.
In particular embodiments, the method can comprise determining in a test sample of a human subject genotypes of at least two loci at position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) and at position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9); and administering to the human subject a treatment regimen sing an effective amount of a folatecontaining compound if either at least one or both of the following conditions is/are detected: (i) at least one thymine “T” allele present at position 677 of SEQ ID NO. 1 (or on 27 of SEQ ID NO. 7); and (ii) at least one guanine “G” allele present at position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9). In these embodiments, the method can further se determining the presence or absence of any of the conditions ) described herein.
In some embodiments, a method for treating a human subject with depression can comprise administering a composition comprising an ive amount of a folate-containing compound to the human subject, who is sed as having, or having a risk for, depression, and is further determined to carry at least one or more (including at least two, at least three, at least four or more), or any combinations of the conditions (A)-(X) described herein.
In certain embodiments, the method can se administering a composition comprising an effective amount of a folate-containing compound to the human subject, who is diagnosed as having, or having a risk for, depression, and is further determined to carry at least one of the following SNPs or a combination thereof: (i) a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine "T" allele, and (ii) a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 sing at least one guanine "G" allele. In these embodiments, the method can further comprise determining the presence or absence of any of the conditions (A)-(X) described herein.
In some embodiments, the subject administered with a treatment regimen comprising a folate-containing compound can be further determined to be obese (e. g., with a BMI value of at least about 30 kg/m2 or higher).
In some embodiments of this aspect and all other aspects described herein, a folate- containing nd can be administered in an amount effective to reduce at least one symptom (e.g., but not limited to, low mood, insomnia, ion, anxiety and/or weight loss) ated with depression, e.g., major depressive disorders. In some embodiments, the effective amount of a folate- containing nd can provide at least about 0.1 to about 1 mg/kg body weight per day administration to the human subject. In some embodiments, the effective amount of a folate- containing compound can provide at least about 7.5 mg/day to about 50 mg/day administration to the human subject. In one embodiment, the effective amount of a folate-containing compound can provide at least about 15 mg/day of folate administration to the human subject.
The effective amount of the folate-containing nd can be administered to a ed human subject as a single daily dose, or alternatively, in more than one divided doses per day via any suitable administration route, e. g., oral administration.
The effective amount of folate administered to a ed human subject for ent of depression as described herein is significantly higher than the typical amount taken as a dietary supplement (between 50-600 ). In some embodiments, the effective amount of folate administered to a selected human subject is at least about 2-fold, at least about 5-fold, at least about -fold, at least about d, at least about 50-fold, at least about lOO-fold, at least about ld, at least about 500-fold, at least about lOOO-fold or more than the typical amount taken as a dietary supplement. Accordingly, in some embodiments, the -containing compound is desirable to be formulated in slow-release or sustained release composition. For example, in one embodiment, the composition comprising a folate-containing compound can be formulated to release the effective amount of the folate-containing compound over a period between 3-6 hours or 4-5 hours post- administration.
Any art-recognized folate-containing compound can be selected and/or optionally administered to a human subject selected to carry at least one or more conditions (A)-(X) described . In some embodiments, the folate-containing compound can comprise a L-methylfolate compound. In one embodiment, the folate-containing nd can se - methyltetrahydrofolate or a derivative thereof.
In some embodiments of this aspect and all other aspects described , the treatment regimen can further comprise selecting and optionally administering an antidepressant drug.
In some embodiments, the anti-depressant drug can e a selective serotonin reuptake inhibitor, including, but not limited to, fluoxetine, citalopram, paroxetine, escitalopram, sertraline, and any combinations thereof.
In these embodiments, an antidepressant drug can be administered in the same composition as the folate-containing compound. In alternative ments, the antidepressant drug and the folate-containing compound can be administered in separate compositions at the same time (e.g., concurrently) or sequentially (e. g., one after the other), or in any temporal administration regimen, Where the nt effect of the -containing compound increases the efficacy of the pressant drug as compared to the efficacy Without the folate-containing compound. In some embodiments, the anti-depressant drug and/or the folate-containing nd can be administered in a single dose or in divided doses. The number of dosages administered over a period of time (e.g., per day) for the antidepressant drug and the -containing compound can be the same or different. The antidepressant drug and the folate-containing compound can be administered via the same or different routes.
In some embodiments of this aspect and all other aspects described herein, the adjuvant effect of the folate-containing compound administered in combination with an antidepressant drug can be additive.
In some ments of this aspect and all other aspects bed herein, the adjuvant effect of the folate-containing compound administered in combination with an antidepressant drug can be synergistic.
A further aspect provided herein is a method of determining and/or improving the effectiveness of an anti-depressant drug administered to a human subject, e.g., by determining if the human subject is amenable to folate or a derivative thereof as an nt, e. g., using the assay described herein. In some ments of this aspect, the method can r comprise stering or prescribing the subject with a compound containing an ive amount of folate as an nt to the epressant drug, if the subject satisfies at least one of the conditions (A)-(X) provided herein.
An exemplary effective amount of folate is about 7.5 mg/day to about 50 my day, or in one embodiment, the effective amount is at least about 15 mg/day. In one embodiment, the epressant drug is a ive serotonin reuptake inhibitor, for example, without limitations, fluoxetine, citalopram, paroxetine, escitalopram, sertraline, and any combinations thereof.
Yet another aspect provided herein is a method of improving the effectiveness or efficacy of an anti-depressant drug taken by a subject, e.g., by identifying the subject with at least one of the conditions as determined in the assay described herein. Accordingly, in some embodiments of this aspect, the method can further comprise administering or prescribing the t with a compound containing an effective amount of folate as an nt to the anti-depressant drug, if the subject satisfies at least one of the conditions determined in the assay provided herein. An ary effective amount of folate is at least about 15 mg/day. In one embodiment, the anti-depressant drug is a selective serotonin reuptake inhibitor, for example, without limitations, fluoxetine, citalopram, paroxetine, escitalopram, sertraline, and any ations thereof.
Computer systems for use in any aspects of the assays and/or methods described herein are also provided. For e, one embodiment provided herein is a computer system for ing data from at least one test sample obtained from at least one subject. The system comprises: (a) a determination module configured to receive at least one test sample and perform at least one analysis on at least one test sample to determine parameters of at least two biomarkers (i) to (xxiv) described herein; (b) a storage device configured to store output data from the determination module; (c) a computing module, e.g., a non-human machine, comprising specifically-programmed instructions to determine from the output data the presence of at least one condition (A) to (X) described ; and (d) a display module for displaying a content based in part on the data output from the computing module, wherein the content comprises a signal indicative of the presence of at least one condition (A) to (X) described herein, and optionally the absence of any one of the conditions (A) to (X) described herein, or a signal indicative of the absence of all of the conditions (A) to (X) described herein.
In some embodiments, the determination module can be configured to perform at least one ping analysis on at least one test sample to determine the genotypes of at least two loci comprising position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) and position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9). In these embodiments, the computing module can be configured to determine the presence of at least one SNP located at position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) comprising at least one thymine “T” allele, and/or at position 2756 of SEQ ID NO. 2 (or on 27 of SEQ ID NO. 9) comprising at least one guanine “G” allele.
In another embodiment, the determination module can be configured to perform at least one analysis on at least one test sample to determine the presence or absence of at least one of the following conditions: i. an expression ratio of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) smaller than a pre-determined reference ratio; ii. expression of 4-hydroxynonenal (4-HNE) greater than a pre-determined reference value; iii. a single tide polymorphism (SNP) at position 677 of SEQ ID NO. 1 (or at position 27 of SEQ ID NO. 7) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate ase (MTHFR); iv. a SNP at position 2756 of SEQ ID NO. 2 (or at position 27 of SEQ ID NO. 9) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine se (MTR); and V. a SNP at position 66 of SEQ ID NO. 3 (or at position 27 of SEQ ID NO. 10) comprising at least one e “G” allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a c nucleic acid sequence of methionine synthase reductase (MTRR).
In these embodiments, the determination module can be further configured to ine the ce or absence of at least one other condition (A)-(X) described herein. For example, in some embodiments, the determination module can be further ured to determine expression of high-sensitivity c-reactive protein (hsCRP). In some ments, the determination module can be further configured to determine the ce or absence of a SNP at position 1298 of the SEQ ID NO. 1 comprising at least one cytosine “C” allele.
In some embodiments, the ination module can be configured to analyze at least one test sample to determine the presence or absence of at least two of the ions provided above.
In some embodiments, the determination module can further comprise a comparison module adapted to compare the data output from the determination module with reference data stored on the storage device.
In some embodiments, the storage device can be further configured to store physical information of at least one subject, for example, comprising indicators of whether a test subject is obese, e. g., BMI of at least one subject).
In some embodiments, the t yed on the y module can further comprises the BMI value or a signal indicative of whether the BMI value is at least 30 kg/m2 or not.
In some embodiments, the content displayed on the display module can further comprise a signal indicative of the subject recommended to receive a treatment regimen comprising a folate-containing compound, or a signal tive of the subject recommended to receive an alternative treatment regimen without a folate-containing compound.
A tangible and non-transitory (e.g., not transitory forms of signal transmission) computer readable medium having computer readable instructions recorded thereon to define re modules for implementing a method on a computer is also provided herein. In one embodiment, the computer readable storage medium comprises: (a) instructions for comparing the data stored on a storage device with reference data to provide a ison result, wherein the comparison identifies the presence or absence of at least one condition (A)-(X) bed herein; and (b) instructions for displaying a content based in part on the data output from the determination module, wherein the content comprises a signal indicative of the presence of at least one of the conditions ) described herein, and optionally the absence of any one of these ions (A)-(X) described herein.
In other embodiments, the content can comprise a signal tive of the absence of all of the ions (A)-(X) described herein.
In some embodiments, the instructions can be specifically programmed to perform a comparison to identify the presence of at least one SNP located at position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) comprising at least one thymine “T” allele, and/or at position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9) comprising at least one guanine “G” allele.
In other embodiments, the instructions can be specifically mmed to perform a comparison to identify one of the following conditions: i. an expression ratio of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) smaller than a pre-determined reference ratio; ii. expression of 4-hydroxynonenal (4-HNE) greater than a pre-determined reference value; iii. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO: 1 comprising at least one thymine “T” allele, wherein the SEQ ID NO: 1 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iv. a SNP at position 2756 of SEQ ID NO: 2 comprising at least one e “G” allele, wherein the SEQ ID NO: 2 is a portion of a genomic c acid sequence of methionine synthase (MTR); and v. a SNP at position 66 of SEQ ID NO: 3 comprising at least one guanine “G” allele, wherein the SEQ ID NO: 3 is a n of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); In these embodiments, the computer le medium can further comprise instructions to identify the presence or e of at least one other condition (A)-(X) bed herein. For example, in one embodiment, the computer readable medium can r comprise ctions to identify the presence or absence of a SNP at position 1298 of the SEQ ID NO: 1 comprising at least one cytosine “C” . In some embodiments, the computer readable medium can further comprise instructions to compare expression of high-sensitivity c-reactive protein (hsCRP) with the reference data.
In some embodiments, the computer readable medium can further comprise instructions to determine or calculate if the subject is obese (e.g., whether the subject has BMI of at least 30 kg/m2 or not), based on input data of the subject’s physical features (e.g., weight and ).
In some embodiments, the er readable medium can further comprise ctions to display a ement to determine whether a subject is obese (e.g., a BMI value) or a signal indicative of whether the subject is obese (e.g., whether the BMI value is at least 30 kg/m2 or not).
In some embodiments, the computer readable medium can further comprise instructions to display a signal tive of the subject recommended to receive a treatment regimen sing a folate-containing compound, or a signal indicative of the subject recommended to receive an alternative treatment n without a folate-containing compound.
Based on the fication of SNPs and/or peripheral markers associated with a response to the use of a folate-containing compound, one aspect described herein also provides for the design and preparation of detection reagents needed to identify the SNPs and/or peripheral markers disclosed herein in a test sample of a subject. For example, the detection reagents can be designed and prepared to identify SNPs in MTHFR locus and MTR locus and optionally MTRR locus involved in assays and methods described herein, and/or e expression levels of SAM, SAH and 4-HNE in a test sample. Examples of detection ts that can be used to identify the sed SNPs in a test sample can include a primer and a probe, wherein the probe can selectively hybridize the SNP- containing nucleic acid molecules, as compared to a nucleic acid molecule which does not contain the SNP at the same tide position. Examples of detection regents that can be used to measure expression levels of serum or plasma proteins (e. g., SAM, SAH and/or 4-HNE) in a test sample can include antibodies against such proteins, or a primer and a probe, wherein the probe specifically hybridizes to a nucleic acid le corresponding to such proteins.
In one embodiment, a kit can comprise an oligonucleotide array affixed with a plurality of oligonucleotide probes that ogate, e. g., no more than 30 SNPs, wherein the SNPs comprise at least two or any combinations of the conditions ) described herein (e.g., but not limited to, a combination of conditions (A) and (C)); and an optional container containing a detectable label (e.g., comprising a cent molecule) to be conjugated to a nucleotide le derived from a test sample of a human subject; and at least one reagent. Examples of a reagent that can be included in the kit can include, without limitations, a restriction enzyme, a universal adaptor to be conjugated to a nucleotide molecule, a primer complementary to the universal adaptor, a wash agent, and any ations thereof.
In an alternative embodiment, a kit can comprise a plurality of oligonucleotide primers that bind to at least one allele of no more than 30 SNPs, wherein each subset of 2012/065084 oligonucleotide primers that bind to a specific allele of a SNP is labeled with a distinct reporter, and wherein said SNPs se at least two or any combinations of the SNP ions (A)-(U) described herein (e. g., but not d to a combination of conditions (A) and (C)); and at least one reagent, e.g., but not limited to, free nucleotide bases, a polymerase, or both.
In some embodiments, the kit can further comprise at least one reagent to determine sion of at least one biomarker described herein (e.g., SAM, SAH, 4-HNE and hsCRP). For example, in one embodiment, the kit can further comprise a solid substrate support affixed with at least one protein-based binding moiety (e. g., an antibody) that ically binds to one or more of the biomarkers described herein. Exemplary solid substrate support can include, but not limited to, a microtiter plate for ELISA, a dipstick, a magnetic bead, or any combinations thereof. In another embodiment, the kit can further comprise at least one primer designed to probe one or more biomarkers described herein.
The assays, methods, systems and/or kits described herein can be performed and/or used by a third-party service provider. For e, a third-party service er can provide and charge for a service offered to determine the presence or absence of at least one ion (A)-(X) in a test sample of a human subject, e.g., to facilitate selection of a treatment regimen for a human subject with depression. Accordingly, methods for selecting a treatment regimen for a human subject are also ed herein. For example, the method comprises (a) obtaining a test sample from a human subject diagnosed as having, or having a risk, for sion; (b) subjecting the test sample to at least one analysis to determine parameters of at least two biomarkers (i) —(xxiv) described herein (e. g., but not limited to, a combination of biomarkers (i) and (iii)); (c) determining, from the parameters of the selected biomarkers, the presence of at least one condition (A)-(X) (e.g., but not limited to, either one or both of conditions (A) and (C)); and (d) providing a result output setting forth Whether at least one of the conditions (A)-(X) is detected in the test sample. If at least one condition is present, the method can further comprise selecting and ally administering a treatment regimen comprising an ive amount of a folate-containing compound to the human subject.
In some ments, the step (b) of the method can further comprise ally packing and shipping the test sample to a test facility, e. g., a third-party CLIA-certified service provider.
In some ments, the step (d) of the method is performed by a non-human machine.
The test sample for use in the assays, methods, s or kits described herein can be derived from a biological sample of the subject, e.g., a blood sample or plasma or serum sample from the subject. In some embodiments, the test sample can comprise a urine sample. In some embodiments, the test sample can comprise a buccal sample. In some embodiments, the test sample can comprise a saliva sample.
If the test sample is a nucleic acid , the test sample can be subjected to at least one analysis selected from the group consisting of allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, sequencing, 5’ nuclease ion, molecular beacon assay, DNA chip analysis, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism, polymerase chain reaction (PCR), real-time quantitative PCR, and any combinations thereof.
If the test sample is a protein sample, the test sample can be subjected to at least one analysis selected from the group consisting of n blot, enzyme linked ance assay, mass spectrometry, immunoassay, flow cytometry, immunohistochemical analysis, and any combinations thereof.
A still further aspect provided herein relates to uses of a folate-comprising composition in the ent of depression in a human subject who carries at least one of the conditions ) described herein (e.g., but not limited to, either one or both ions (A) and (C)). r aspect provided herein relates to a folate-comprising composition in combination with an anti-depressant for use in the treatment of depression in a human subject who carries at least one of the conditions (A)-(X) bed herein (e. g., but not limited to, either one or both conditions (A) and (C)). In some embodiments of these aspects described herein, the folate-comprising composition can se at least about 5 mg of folate (e. g., about 7.5 mg to about 50 mg of ). In some embodiments, the folate-comprising composition can be formulated for a pre-determined release profile (e.g., a sustained release). In some embodiments, the human subject is an adult.
Embodiments of s aspects described herein can be employed for use in a human subject diagnosed as having, or having a risk for, any form of depression. In one embodiment, various aspects described herein can be employed for use in a human subject diagnosed as having, or having a risk for, a major depressive disorder. In some embodiments, the human subject can be further determined to be resistant to at least one anti-depressant monotherapy. In some embodiments, the human subject is an adult.
In some embodiments, various aspects described herein can be employed for use in a human subject who is currently taking an antidepressant. In these embodiments, the human subject who is determined to satisfy at least one (including, e.g., at least two, at least three or more) of the conditions ) can be selected and/or administered with a ent regimen comprising a folate- containing compound. In some embodiments, the treatment regimen does not include an antidepressant. In some embodiments, the treatment regimen can include the same antidepressant that the human subject is currently taking or a ent antidepressant.
BRIEF DESCRIPTION OF THE DRAWINGS Figs. lA-lB show schematic diagrams of exemplary study designs of analyzing the efficacy of a -containing nd, e. g., as an adjunct to a selective serotonin reuptake inhibitor (SSRI) for treating a t with depression or identifying biomarkers or conditions indicative of a subject with sion recommended for a treatment regimen comprising a folate-containing compound and an SSRI. Fig. 1A shows an exemplary study design, in which 7.5 mg/day L- methylfolate (e. g., 6(S)methyltetrahydrofolate, abbreviated as 6(S)MTHF as bed herein) is stered as an adjunct to an SSRI. Fig. 1B shows an exemplary study design, in which 15 mg/day L-methylfolate (e.g., 6(S)MTHF) is administered as an adjunct to an SSRI.
Fig. 2 shows a tic diagram of an exemplary double-blind, placebo-controlled study of 6(S)MTHF among SSRI-resistant outpatients with major depressive disorder (MDD) using sequential parallel comparison. As an example, 10% t rate is assumed. The t response and non-response rates provided in this figure are only meant to indicate the relative efficacy effect, but do not mean to be construed as or limited by the absolute values of the ted percentages.
Figs. 3A-3E show s of treating MDD patients with a folate-containing nd, e.g., as an adjunct to an SSRI in Trial 2. Fig.3A shows the response rates of MDD patients treated with either a folate-containing compound or a placebo, in conjunction with an SSRI, after 30- day treatment. A responder is a MDD patient with at least 50% reduction in 7 after treatment. Fig.3B shows the mean change in scores of various neuropsychological tests (e. g., HDAM- 17, QIDS-SR, and CGI-S) for MDD patients treated with either a folate-containing compound or a placebo, in conjunction with an SSRI, after 30-day treatment. Fig. 3C shows percents of MDD patients in remission, as measured by HDAM-l7, after treated with either a folate-containing compound or a placebo, in conjunction with an SSRI, after 30-day treatment. Fig. 3D shows percents of MDD patients in remission, as measured by QIDS-SR, after treated with either a folate-containing compound or a placebo, in conjunction with an SSRI, after 30-day treatment. Figs. 3C—3D show that remission rates after 30 days of adjuvant 15 mg/day L-methylfolate and an SSRI, compared to placebo and the SSRI, was not significant in depressed patients who inadequately respond to SSRI monotherapy. Fig. 3E shows percent of MDD patients discontinued from Trial 2, indicating no significant difference in discontinuation rates due to overall adverse events.
Figs. 4A-4B show effects of a single genetic polymorphism (SNP) in the MTHFR gene (MTHFR C677T) on the efficacy of the treatment comprising a folate-containing compound (e. g., 6(S)MTHF) and ally an SSRI in a patient with depression. Fig. 4A shows cy results as measured by 8 (28-item Hamilton Depression Rating Scale). Fig. 4B shows efficacy results as measured by CGI-S (Clinical Global Impression-Severity).
Figs. 5A-5B show s of obesity (i.e., BMI is at least 30 kg/m2 or above) on the efficacy of the treatment comprising a -containing compound (e. g., 6(S)MTHF) and optionally an SSRI in a patient with sion. Fig. 5A shows efficacy results as measured by HAMD-28 (28-item Hamilton Depression Rating Scale) with a chi-square of 3.94 for the treatment WO 74676 effect. Fig. 4B shows efficacy s as measured by CGI-S (Clinical Global Impression-Severity) with a chi-square of 10.03 for the treatment .
Fig. 6 is a block diagram showing an exemplary system for use in the s described here, e.g., for selecting a treatment n for a subject with depression.
Fig. 7 is an exemplary set of instructions on a computer readable storage medium for use with the systems described .
Figs. 8A-8B show mean changes in HAMD-28 in MDD patients ng at least one rare variant on the indicated gene, as opposed to fully normal on the respective gene, when they were treated with a folate-containing compound, e.g., as an t to an SSRI. Fig. 8A is a bar graph showing the mean change in HAMD-28 with respect to various SNP biomarkers as indicated. Fig. 8B is a table showing the results as shown in Fig. 8A and the corresponding loci in chromosomes. The term “prevalence” as used in Fig. 8B refers to the total tage of MDD patients who carry the SNP as indicated in this particular study.
Figs. 9A-9C are result tables g effects of the presence or absence of an indicated condition, in MDD patients on the degree of depression, when the patients were treated with a treatment regimen comprising a folate-containing compound. The degree of depression was measured by Social Functioning Questionnaire (SFQ), Visual Analogue Scale (VAS), and Cognitive and Physical Function Questionnaire (CPFQ). Fig. 9A shows analyses on all samples. Fig. 9B shows analyses in biomarker subgroups. Fig. 9C shows analyses in genetic subgroups.
Figs. 10A-10E are result tables showing effects of the presence or absence of an indicated ion, in MDD patients on the degree of sion, when the patients were treated with a folate-containing compound (treatment group) or without a folate-containing compound (placebo group). The degree of depression was measured by Maier or HAMD-7 le of HAMD. Fig. 10A shows results of Maier SPCD. Fig. 10B shows Maier means. Fig. 10C shows HAMD-7 SPCD.
Fig. 10D shows HAMD-7 means. Fig. 10E shows a summary of the biomarker and genetic analyses on Maier scale.
ED DESCRIPTION OF THE INVENTION Recent estimates indicate that more than 19 million Americans over the age of 18 years experience a depressive illness each year. While significant advances in the treatment of depression have been made in the past , as many as 29% to 46% of patients with depression taking an anti-depressant are still partially or totally resistant to the treatment. Those who suffer from treatment-resistant depression have almost no alternatives. As not every treatment regimen is effective for each individual, there is a strong need to identify markers that can facilitate selection of an appropriate treatment regimen for a human t with depression In accordance with aspects of various embodiments described herein, at least 21 single nucleotide polymorphisms (SNPs) and 4 peripheral biomarker ters have been discovered for predicting the effectiveness or efficacy of a treatment regimen comprising a folate-containing compound. That is, these SNPs and serum/plasma biomarker parameters can be used to fy subjects with depression that would t from or d to a treatment regimen comprising a folate-containing compound, as compared to a treatment without a folate-containing compound. In particular, these SNPs and serum/plasma biomarker parameters can be used to identify a subject with treatment-resistant depression (e.g., a subject resistant to at least one selective serotonin reuptake inhibitor (SSRI)) who would benefit from or respond to a treatment regimen comprising a folate- containing compound, as compared to a treatment without the folate-containing compound. The folate-containing compound can be stered in the absence of an anti-depressant drug, or it can be provided as an adjuvant to an antidepressant drug. In some ments, the adjuvant effect of the folate-containing compound administered in combination with an antidepressant drug can additive. In other embodiments, the adjuvant effect of the folate-containing nd administered in combination with an antidepressant drug can be synergistic.
Specifically, the SNPs that can predict efficacy of administering to a human subject a folate-containing compound (e.g., alone or in a combination therapy to increase the efficacy of an antidepressant) for treatment of depression include at least one or a combination of SNPs identified by rs numbers as s: rs1801133 present in methylenetetrahydrofolate reductase (MTHFR); 976 present in MTHFR; rs1805087 present in methionine synthase (MTR); rs1801394 present in methionine se reductase (MTRR); rs1006737 present in calcium channel, voltage-dependent, L-type, alpha 1C subunit (CACNAIC); rs1883729 present in DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); rs7163862 present in GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); rsl2659 present in reduced folate carrier protein (RCF2); rs202676 present in folate ase (prostate-specific membrane antigen) (FOLHl); rs229729l present in reduced folate carrier protein (RCFl); rs1051266 present in reduced folate carrier protein 1 (RCFl); rs8007267 present in GTP cyclohydrolase l (GCHl); rs7639752 present in choline-phosphate cytidylytransferase A (PCYTlA); rs6275present in dopamine receptor D2 (DRD2); rs1079596 t in DRD2; rsl 1240594 present in DRD2; rs4633 present in catechol-O-methyltransferase ; rs4680 present in COMT; rs250682 present in dopamine active orter (DAT, or SLC6A3); rs2277820 present in inotransferase cyclodeaminase (FTCD); 225 present in methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFDl); and any combinations thereof.
Further, peripheral biomarker ters that can predict cy of administering to a human subject a folate-containing compound (e.g., alone or in a combination therapy to increase the efficacy of an antidepressant) for treatment of depression include ve expression levels between s- adenosyl nine (SAM) and s-adenosyl homocysteine (SAH), expression of 4-hydroxynonenal (4-HNE), expression of high-sensitivity c-reactive protein (hsCRP), and any combinations thereof.
Additionally, obesity has also been discovered to be predicative of effectiveness of a treatment regimen comprising a folate-containing compound (e.g., as a erapy or a ation therapy with an antidepressant). These genetic polymorphisms, eral biomarkers and clinical features 2012/065084 have been assessed on a human cohort, who has major depressive disorder and has shown resistance to anti-depressant monotherapies, e.g., has treatment-resistant depression (TRD), in ular, selective serotonin reuptake inhibitor (SSRI)-resistant sion.
Accordingly, some embodiments described herein are generally related to assays, methods, systems or kits for selecting a ent n for a subject with depression or identifying a subject with depression amenable for, or responsive to a treatment comprising a folate-containing compound. In some embodiments, the treatment can comprise a combination of a folate-containing compound and an antidepressant drug. In one embodiment, the assays, methods, systems and kits are directed to determining in a test sample from a human subject, e.g., a human subject sed as having, or having a risk for sion (e.g., but not limited to, major sive er) the presence or absence of at least one of single tide polymorphisms (SNPs) and/or peripheral biomarker parameters to t the response of a subject to a treatment comprising a folate- containing compound. If at least one of the conditions described herein is determined to be present in the test sample from the human subject, a treatment regimen comprising a folate-containing compound can be selected and optionally administered to the human subject. In some embodiments, the treatment regimen can further comprise an anti-depressant drug (e.g., an SSRI) to be administered, separately or concurrently, with a folate-containing compound.
Assaysfor selecting a treatment regimenfor a human subject with depression Accordingly, provided herein generally relate to assays, methods, systems, and kits for selecting a treatment n for a subject with depression or a risk for depression; for treating a t with depression or a risk for depression, and/or for improving the effectiveness of a treatment regimen recommended for and/or administered to a subject with sion or a risk for depression.
Provided herein also relate to folate-comprising compositions for use in treatment of depression in a subject (e.g., a human subject) selected to carry at least one (e.g., at least two or more) or any combinations of the biomarkers or conditions described herein.
One aspect described herein provides an assay for selecting a treatment regimen for a subject with depression, by identifying in a test sample from the subject pes of at least one of the SNPs and/or expression of at least one peripheral biomarker as described herein in order to determine the responsiveness of the human subject to a treatment regimen comprising a folate- containing compound. The assay comprises: (a) subjecting a test sample of a human subject, who is diagnosed as having depression or having a risk for depression, to at least one analysis to determine ters of at least two (including, e.g., at least three, at least four, at least five or more) of the biomarkers (i) to (xxiv): (i) genotype of a SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each 2012/065084 independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); (ii) genotype of a SNP locus at position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8 (identified by rs2274976), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of MTHFR; (iii) genotype of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by 087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); (iV) pe of a SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each ndently a portion of a c nucleic acid sequence of methionine synthase reductase (MTRR); (V) genotype of a SNP locus at on 27 of SEQ ID NO. 11 (identified by rs1006737), wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC); (Vi) genotype of a SNP locus at position 27 of SEQ ID NO. 12 (identified by rs1883729), wherein the SEQ ID NO. 12 is a portion of a genomic c acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); (Vii) genotype of a SNP locus at position 27 of SEQ ID NO. 13 (identified by rs7163862), wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); (Viii) genotype of a SNP locus at on 27 of SEQ ID NO. 14 (identified by rs12659), wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein ; (ix) genotype of a SNP locus at position 27 of SEQ ID NO. 15 (identified by rs202676), wherein the SEQ ID NO. 15 is a portion of a c nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); (x) genotype of a SNP locus at position 27 of SEQ ID NO. 16 ified by 291), wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); (xi) genotype of a SNP locus at position 27 of SEQ ID NO. 17 ified by rs1051266), wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); (xii) genotype of a SNP locus at position 27 of SEQ ID NO. 18 (identified by rs8007267), wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP ydrolase 1 (GCH1); (xiii) genotype of a SNP locus at position 27 of SEQ ID NO. 19 (identified by rs7639752), wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid ce of choline- phosphate cytidylyltransferase A (PCYT1A); (xiv) pe of a SNP locus at position 27 of SEQ ID NO. 20 (identified by rs6275), wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); (xv) genotype of a SNP locus at position 27 of SEQ ID NO. 21 (identified by rs1079596), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); (xvi) genotype of a SNP locus at position 27 of SEQ ID NO. 22 (identified by 0594), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); (xvii) genotype of a SNP locus at position 27 of SEQ ID NO. 23 (identified by rs4633), wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of ol- O-methyltransferase (COMT); (xviii) genotype of a SNP locus at position 27 of SEQ ID NO. 24 (identified by rs4680), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol- yltransferase (COMT); (xix) genotype of a SNP locus at position 27 of SEQ ID NO. 25 ified by rs250682), n the SEQ ID NO. 25 is a portion of a c nucleic acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3); (xx) genotype of a SNP locus at position 27 of SEQ ID NO. 26 (identified by rs2277820), wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); (xxi) genotype of a SNP locus at position 27 of SEQ ID NO. 27 (identified by rs2236225), n the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of enetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFD1)); (xxii) level of expression of SAM and SAH; (xxiii) level of expression of 4-HNE; (xxiv) level of expression of hsCRP; and any combinations f; and (b) detecting, optionally with a non-human machine, from the determined ters of at least two biomarkers, the presence of at least one condition (including, e.g., at least two conditions, at least three conditions, at least four conditions or more) ed from the ing conditions (A)-(X): (A) a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) sing at least one thymine "T" allele; (B) a SNP at position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8 (identified by rs2274976) comprising at least one alanine "A" allele; (C) a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087) comprising at least one guanine "G" allele; (D) a SNP at on 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394) comprising at least one e "G" allele; (E) a SNP at position 27 of SEQ ID NO. 11 (identified by rs1006737) comprising at least one alanine "A" allele; (F) a SNP at position 27 of SEQ ID NO. 12 ified by 729) comprising at least one alanine "A" allele; (G) a SNP at position 27 of SEQ ID NO. 13 (identified by rs7163862) comprising at least one thymine "T" allele; (H) a SNP at position 27 of SEQ ID NO. 14 (identified by rs12659) comprising at least one thymine "T" allele; (I) a SNP at position 27 of SEQ ID NO. 15 (identified by rs202676) comprising at least one guanine "G" allele; (J) a SNP at position 27 of SEQ ID NO. 16 (identified by rs2297291) comprising at least one alanine "A" ; (K) a SNP at position 27 of SEQ ID NO. 17 (identified by at 266) comprising at least one alanine "A" allele; (L) a SNP at position 27 of SEQ ID NO. 18 (identified by rs8007267) comprising at least one thymine "T" allele; (M) a SNP at position 27 of SEQ ID NO. 19 (identified by rs7639752) comprising at least one e "A" allele; (N) a SNP at position 27 of SEQ ID NO. 20 (identified by rs6275) comprising at least one thymine "T" allele; (0) a SNP at position 27 of SEQ ID NO. 21 (identified by rs1079596) comprising at least one thymine "T" allele; (P) a SNP at position 27 of SEQ ID NO. 22 (identified by rs11240594) sing at least one alanine "A" allele; (Q) a SNP at position 27 of SEQ ID NO. 23 (identified by ) comprising at least one cytosine "C" allele; (R) a SNP at position 27 of SEQ ID NO. 24 (identified by ) comprising at least one guanine "G" allele; (S) a SNP at position 27 of SEQ ID NO. 25 (identified by rs250682) comprising at least one cytosine "C" allele; (T) a SNP at position 27 of SEQ ID NO. 26 (identified by rs2277820) comprising at least one thymine "T" allele; (U) a SNP at position 1958 of SEQ ID NO. 27 (identified by rs2236225) comprising at least one alanine "A" allele; (V) an expression level ratio of SAM to SAH smaller than a pre-determined reference ratio; (W) an expression level of 4-HNE greater than a pre-determined reference value; (X) an expression of hsCRP greater than about 2.3 mg per liter as measured in a plasma sample; and any combinations f.
In some embodiments of this aspect and all other aspects described herein, any of the SNPs described herein can comprise one or two folate-respsonive alleles. By way of example only, a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) can comprise one e "T" allele, or two thymine “T” alleles. Without wishing to be bound by theory, a human subject determined to carry two folate-responsive alleles in a SNP locus described herein can show greater se to a treatment regimen comprising a folate-containing compound than a human subject with one folate-responsive allele in the same SNP locus. ing on the design of primers and probes, the SNP conditions (A)-(U) can be also represented by alleles complementary to the ponding folate-responsive alleles described herein. For example, instead of detecting the presence of a SNP at on 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine "T" allele, one of skill in the art can readily design primers and/or probes for the complementary sequence of SEQ ID NO. 7 to probe for a SNP at the same location comprising at least one “A” allele instead.
Accordingly, in some embodiments of this aspect and all other aspects described herein, the ce of at least one condition (including, e. g., at least two conditions, at least three conditions, at least four conditions or more) selected from the ions (A)-(U) can be indicated by detecting the presence of the complementary allele of the folate-responsive allele as shown above and also in Table 42 below.
Based on the analysis results from step (b), if at least one of the conditions (A)-(X) described above is detected, the assay can further comprise selecting for the human subject and ally administering to the human subject a treatment regimen comprising an effective amount of a folate-containing compound.
Any combinations of at least two kers (i) to (xxiv) described herein can be determined in a test sample for the assays described herein. Exemplary combinations of at least two biomarkers described herein can comprise genotypes of at least two SNP loci or more (e.g., including at least three SNP loci, at least four SNP loci, at least five SNP loci or more); or genotype of at least one SNP loci or more (e.g., including at least two SNP loci or more) and sion level of at least one peripheral biomarker or more (e. g., 4-HNE, SAM, SAH); or sion level of at least two peripheral biomarkers (e. g., 4-HNE, SAM, SAH). ing upon selected combinations of at least two biomarkers described , the test sample can be subjected to one or more es, e. g., including, but not limited to, genotyping assays, sion assays (e. g., protein and/or transcript levels), or any combinations thereof.
Accordingly, in some embodiments, the assay can comprise ting a test sample from a human subject, who is diagnosed as having depression or having a risk for depression to at least one genotyping assay adapted to determine genotypes of at least two loci, wherein said at least two loci are: (i) position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by 133) and (ii) position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087). In such embodiment, detection of at least one SNP at either position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) sing at least one thymine “T” allele or position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9) comprising at least one guanine “G” allele, or detection of both aforementioned SNPs indicates selection and optional administration of a treatment regimen comprising an effective amount of a folate-containing compound to the human subject.
In some embodiments, the test sample can be subjected to at least one genotyping assay adapted to determine genotypes of at least three loci, at least four loci, at least five loci, at least six loci, at least seven loci, at least eight loci, at least nine loci, at least ten loci or more. The onal loci to be interrogated can be selected from any combinations of the biomarkers (i)-(xxi).
In some embodiments, the ping assay can comprise the step of amplifying the test sample with a set of primers flanking any one of the SNPs described herein. In some embodiments, at least two (e. g., at least three, at least four, at least five or more) sets of primers amplifying at least two (e. g., at least three, at least four, at least five or more) of the SNPs can be used in a multiplex amplification assay.
In some embodiments, the test sample can be subjected to ine parameters of at least three, at least four, at least five or more biomarkers (i) to (xxiv) described herein. For example, in some embodiments, the assay can comprise: (a) subjecting a test sample of a subject to one or more than one analyses (e. g., genotyping and/or expression analyses) to determine the presence or e of at least one of the following conditions: i. an expression ratio of osyl methionine (SAM) to s-adenosyl homocysteine (SAH) smaller than a termined reference ratio; ii. expression of 4-hydroxynonenal ) greater than a pre-determined reference value; iii. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO: 1 (or at position 27 of SEQ ID NO. 7) comprising at least one thymine “T” allele, wherein the SEQ ID NO: 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase ); iv. a SNP at position 2756 of SEQ ID NO: 2 (or at position 27 of SEQ ID NO. 9) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and v. a SNP at position 66 of SEQ ID NO. 3 (or at position 27 of SEQ ID NO. 10) comprising at least one guanine “G” , wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a n of a genomic nucleic acid sequence of methionine synthase reductase (MTRR).
In these ments, detection of the presence of at least one of these conditions is indicative of the subject recommended for a ent regimen comprising a folate-containing compound. In some embodiments, if none of the conditions described herein is present, the subject is not recommended for a treatment regimen comprising a -containing compound.
In some ments of this aspect and all other aspects described herein, when the expression ratio of SAM to SAH is smaller than the pre-determined reference ratio, e.g., r than 3.0, or smaller than about 2.8 as measured in a plasma sample, the subject can be recommended for and optionally administered with a treatment regimen comprising a folate-containing compound. In one embodiment, the expression ratio of SAM to SAH being smaller than 2.71 (as measured in a plasma sample) is indicative of the subject recommended for and ally administered with a treatment regimen comprising a folate-containing compound. In some embodiments, if the expression ratio of SAM to SAH is at least or greater than 2.71 as measured in a plasma , the subject is not recommended for nor stered with a treatment regimen comprising a folate-containing compound. ing on the test sample source, e.g., a blood sample vs. a buccal sample, the pre- determined reference ratio for a blood plasma sample can be different from that for, e.g., a buccal sample.
In some embodiments of any aspects described herein, when the expression of 4-HNE is greater than the pre-determined reference value, e.g., greater than about 3 mg/L, or greater than about 3.2 mg per liter (as ed in a plasma sample), the subject can be recommended for and optionally administered with a treatment regimen comprising a -containing compound. In one embodiment, the expression of 4-HNE of at least 3.28 mg per liter or higher (as measured in a plasma sample) is indicative of a subject recommended for and optionally administered with a treatment regimen comprising a folate-containing compound. In some embodiments, if the expression of 4-HNE is less than 3.28 mg per liter of plasma as measured in a plasma sample or lower, the subject is not recommended for nor administered with a treatment regimen comprising a folate-containing compound. Depending on the test sample source, e.g., a blood sample vs. a buccal sample, the pre- determined reference value for a plasma sample can be different from that for, e.g., a buccal sample.
In some embodiments of this aspect and all other aspects bed herein, the assay can further comprise ining if the human subject is obese or not. If the human t is determined to be obese, then the human subject is selected for and optionally administered with a treatment regimen comprising an effective amount of a folate-containing compound. Methods of determining y in a human subject are known in the art and can include, but are not limited to, body mass index (BMI) measurement, measurement of abdominal fat (e.g., by waist circumference or waist-hip ratio), measurement of body fat, ld thickess, underwater weighing (densitometry), air- displacement plethysmography, computerized tomography (CT) and magnetic resonance imaging (MRI), and dual energy X-ray absorptiometry (DEXA), and any combinations thereof. y can be defined clinically in different ways. For example, obsesity can be defined by a body mass index (BMI) value of at least about 30 kg/m2 or higher. In another embodiment, ty can be d by excessive abdominal fat (e.g., indicated by wasit ference and/or waist-hip ratio). For example, excess abdominal fat can be clinically defined as a waist circumference > 40 inches (>102 cm) in men and >35 inches (>88 cm) in women.
Alternatively, abdominal obesity can be defined as a waist—hip ratio above 0.95 for males and above 0.80 for females. In some embodiments, obesity can be defined by body fat percentage, e. g., obesity is defined as a body fat tage of at least about 32% or more in women and at least about 25% or more in men.
In one embodiment, measurement of BMI can be used to determine whether a human t is obese. In such embodiment, the assay can further comprise ing body mass index (BMI) of the human subject to determine if the human subject is obese or not, and if the BMI value of at least 30 kg/m2 or greater is measured in the subject, then ing for the human subject and optionally administering to the human subject a treatment regimen comprising an effective amount of a folate-containing compound. In some embodiments, if the human subject has a BMI value of less than 30 kg/mz, it may not be ble to recommend or administer to the human subject a treatment regimen comprising a folate-containing compound.
In some embodiments, the assay can further comprise determining the presence or absence of a SNP at position 1298 of the SEQ ID NO: 1 comprising at least one cytosine “C” , wherein the presence of the SNP at position 1298 of the SEQ ID NO: 1 comprising at least one cytosine “C” is indicative of the subject recommended for and/or administered with a treatment regimen comprising a folate-containing compound.
In some ments, the assay can further comprise determining expression of ensitivity c-reactive protein (hsCRP), wherein the hsCRP expression greater than about 2.3 mg per liter (as measured in a plasma sample) is indicative of the subject recommended for a treatment regimen comprising an epressant drug and a folate-containing compound. In some embodiments, if the expression of hsCRP is lower than 2.3 mg per liter of plasma, as ed in a plasma sample, then the subject is not ended for nor administered with a treatment regimen comprising a folate-containing compound. Depending on the test sample source, e. g., a blood sample vs. a buccal sample, the hsCRP expression a plasma sample can be different from that in, e.g., a buccal .
In some embodiments of the assay described herein, a test sample can be analyzed to determine at least one or at least two, at least three, at least four, at least five, at least six of the conditions provided herein. For example, in some embodiments, the test sample can be analyzed to determine if the subject has at least the SNPs located at the positions 677 and 2756 of the MTHFR and MTR loci, respectively. In some embodiments, the test sample can be analyzed to determine if the subject is obese (e. g., whether the subject has at least the BMI value of at least 30 kg/mz), and the SNPs located at the positions 2756 and 66 of the MTR and MTRR loci, respectively. In other embodiments, the test sample can be analyzed to ine if the subject is obese (e.g., whether the subject has at least the BMI value of at least 30 kg/mz) and the SNP located at the position 2756 of the MTR locus. In some embodiments, the test sample can be analyzed to determine if the subject has at least the SAM/SAH ratio smaller than the pre-determined reference ratio and the SNP located at the position 2756 of the MTR locus. In some embodiments, the test sample can be analyzed to determine if the subject has at least the 4-HNE sion greater than the pre-determined reference value and the SNPs located at the positions 2756 and 66 of the MTR and MTRR loci, tively. As sed previously, any combinations of one or more of the conditions (A)-(X) described herein can be assayed at the same time or at different times.
] In some embodiments, if at least one or at least two, including at least three or more, of the conditions provided herein are determined to be present in the test sample of a human subject, a treatment regimen comprising a folate-containing compound is selected and optionally administered to the human t.
In some embodiments, if the human subject ies at least one, including at least two, at least three or more, of the conditions (A)-(X) described herein (and obesity, e.g., defined by a BMI value of at least 30 kg/m2 or greater), the subject can be administered or prescribed with a treatment regimen comprising a folate-containing nd.
In some embodiments, the treatment regimen can further se an anti-depressant drug (e.g., an SSRI) to be administered in combination (e. g. rently or tely) with a folatecontaining compound.
In some embodiments, the folate-containing compound can comprise a L- methylfolate nd. In one embodiment, the folate-containing compound can comprise - methyltetrahydrofolate or a derivative f.
The assays, methods, systems and/or kits described herein can be performed and/or used by a third-party service provider. For example, a third-party service provider can provide and charge for a service offered to determine the presence or absence of at least one condition (A)-(X) in a test sample of a human subject, e.g., to facilitate selection of a treatment regimen for a human subject with depression. Accordingly, methods for selecting a treatment regimen for a human subject are also provided herein. For example, the method comprises (a) obtaining a test sample from a human subject diagnosed as having, or having a risk, for sion; (b) subjecting the test sample to at least one analysis to ine parameters of at least two biomarkers (i) —(xxiv) described herein (e.g., but not limited to, a combination of biomarkers (i) and (iii)); (c) determining, from the parameters of the selected biomarkers, the presence of at least one condition (A)-(X) (e.g., but not limited to, either one or both of conditions (A) and (C)); and (d) providing a result output setting forth whether at least one of the conditions (A)-(X) is detected in the test sample. If at least one ion is present, the method can further comprise selecting and optionally administering a treatment n comprising an effective amount of a folate-containing compound to the human t.
In some embodiments, the step (b) of the method can further comprise optionally packing and shipping the test sample to a test facility, e.g., a third-party CLIA-certified service provider.
In some embodiments, the step (d) of the method is performed by a non-human machine.
Folate-Responsive Biomarkers (i)-(xxiv) and associated conditions (A)-(X) tive of a treatment regimen comprising afolate-containing compound Table 4lA-41B below indicates folate-responsive biomarkers (i)-(Xxiv) and associated conditions (A)-(X), the presence of at least one of which in a test sample of a human subject diagnosed as , or having a risk for, depression, indicates a treatment regimen comprising a folate-containing nd be selected for and optionally administered to the human subject.
Next page shows Table 41A: Folate-responsive biomarkers (i)-(xxi) used in assays, methods, systems and kits described herein, and corresponding folate-responsive conditions (A)-(U).
The ces of human origin shown in Table 41A ding complementary sequences thereof) can provide a basis for designing primers and probes for interrogation of the SNP biomarkers bed herein.
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F0:RN<I(Q§QH$KUE . _ .: . . . . . , a. , , l :4 :z. N v u: w mu 1: x u m aw) an: 11‘; w. mm. mm: XJX xx x.“ xxx:_ xx“.a , . 4.. _ um am [99.“ bcwnsrksr «2(2an. _ _ . 3' I .\ I 4 .4 x x < x 4 .1 x :- .~, 3: .x .\ r. z 3 4 z. x a. g, 1. ‘4 \ \ \‘ x y u x x x t. 7' x x /_ 'z' \ 3 .1 '\‘ .x , 3 iii < >: r: :r > z. 4 l 7 x :- x x -: :x’ a \- < :- :r x > x -. :6 iv 3 r. .\ I. I. x x .< x 4 A .\ :c r, x .\ .\ 4 4 3 .4 I, x < v I. \' 7. \ V x y w: x \‘ x z 7 u: x x. 7' x .\ z ’x .\ l .\ 'ri x z; x z 4 I. 1‘, x x I :v: x .x 3‘: x. x V I z x /_ :v' x 7, vi: r \- x -c ( < x x x a : ;v :v a x .V :- q ..- .V x u 2- - vit: ,4 x < > ,\ 3 .\ .x 7x, ,\ x x a x n 4 4 > x .4 .\ A a .\, 2E x z x z z y 1 x x 7 x x x x *x x \‘ 7 7 x 7. :v x '1’ ) 3‘ K 3 f if < X X 'K i .1 '1 F 2 X )1 :- i K' F X E )- \' .\:i x x \- x x r L» : :- x x u x u x- ‘- 3‘ x x l a: x .. >‘ xii .\ r, ,\ r: a .t. .-'. x .4 ;< 4 .\ 3a a ;< .\ .\ I. I. ,x ,1 I. x 3. xiii 7. \' z w < x x x ‘\‘ x < c 7 z N z r »: x 2 x x I .\’ xiv < x c r m :< :~ : :- :1 3‘ x x x a s e r > .x ‘ x x K > xv X r. X a a :1. .a. x 4 :4 A x .\’ x ;< x x A z x l x :v. \ 7. mi 7 >4 y \‘ \‘ \‘ \- >‘ c x »: .4 *1 -, y < \’ 2 x w l \’ xvii V 7' \‘ :1 :1 y .4 v; v 2 :a x \- \’ x '1 \‘ :a 7 \- 7 ’x x 7' map > -: > -< -< .4 .x‘ K x x < t: .w 3‘ x :v < -; , ac a: > a x24 x x a x x .\‘ .V .V .x x ,\ x .1. 7. x x I. 7. .V .r. x x r x x: x- y < :x :x v I x 7 2 > >: x < x 2 \- \~ > x , x \ >1 nxi )- .4 x -: >1 .4 x x < x 4 «a n s- : x .\ .k 4 -; 4 L: ) q sxii .c x : m x .x , r :- :1 a x x -; x z w 4 r > x a, .x viii \’ 2 x z x 2 I. m: t x a y x x 1-1 \’ V a a \ 1 x v :23»; 7 a: > x x \’ x x \' x ‘: x : :-' 2 x 7 > ‘1 >4 ’\‘ v. < 3m (8m) \- y < :r >' a a r: 7 z > x \ < x ;~ \- \~ > >‘ \ y x Embodiments of the various aspects described herein relate to determination of appropriate parameters (e.g., genotypes or sion level) of at least two of the biomarkers (i) to (xxiv) in a test sample of a human subject. In some embodiments, a physical biomarker (xxv) of obesity tor (e.g., BMI), as shown in Table 42, can also be measured, wherein obesity (e.g., defined by a BMI value of at least about 30 kg/m2 or greater) indicates a treatment regimen comprising a -containing compound be recommended for and/or administered to the human subject. As shown in Table 42, any one of the folate-responsive biomarkers (selected from biomarker (i) to (xxiv)) can be detected in ation with at least one other folate-responsive biomarker as indicated by a “x” symbol in the table, including, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least , at least twelve, at least thirteen, at least en, at least fifteen, at least n, at least een, at least eighteen, at least nineteen, at least twenty, at least twenty-one, at least twenty-two, at least twenty-three, at least twenty-four other folate responsive biomarkers. By way of example only, considering column 1 of Table 42, a folate responsive biomarker (i) (corresponding to a SNP at position 27 of SEQ ID NO. 7 or at position 677 of SEQ ID NO.1) can be detected with one or any combinations of other folate- 2012/065084 responsive biomarkers Xxv). For example, both folate-responsive biomarkers (i) and (iii) can be selected for detection in the assays, methods, systems and kits described herein. In another embodiment, a combination of three folate responsive biomarkers (i), (iii), and (xvii) can be selected for detection in the assays, methods, systems and kits described herein. In another embodiment, a ation of three folate responsive biomarkers (i), (iii), and (XXV) can be ed for detection in the , methods, systems and kits described herein.
] Methylenetetrahydrofolate reductase ). Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that in humans is encoded by the MTHFR gene. SEQ ID NO: 1 corresponds to a portion of the genomic nucleic acid ce of human wild-type or normal MTHFR gene obtained from NCBI database (NCBI Reference Sequence: NM_005957.4), wherein the tide at position 677 and 1298 of SEQ ID NO: 1 are normal (e. g., wild-type) “C” allele and “A” allele, respectively. Methylenetetrahydrofolate reductase catalyzes the conversion of 5,10- methylenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine. Genetic variation in this gene has been previously shown to influence susceptibility to occlusive vascular disease, neural tube defects, colon cancer, acute leukemia, Alzheimer’s or vascular dementia, and mutations in this gene are associated with methylenetetrahydrofolate reductase deficiency.
The mutation of the MTHFR tide at position 677 of SEQ ID NO: 1 from “C” allele to “T” allele (C677T) results in a change of amino acid residue from alanine to valine at position 222 of the corresponding amino acid sequence (SEQ ID NO: 4). Such amino acid substitution encodes a thermolabile enzyme with reduced activity. People with the thermolabile form of such enzyme generally have increased levels of homocysteine in their blood. Accordingly, in some embodiments, ion of an increase in levels of steine in a blood sample of a subject with patient can be an indicative of a SNP at position 677 (e.g., C677T) of the MTHFR gene (or SEQ ID NO: 1). In some embodiments, detection of valine at position 222 (e. g., A222V) of the corresponding amino acid sequence (SEQ ID NO: 4), e.g., by mass spectrometry, can indicate a SNP at position 677 (e. g., C677T) of the MTHFR gene (or SEQ ID NO: 1).
At nucleotide 1298 of the MTHFR, there are generally two ilities: “A” or “C”.
MTHFR 1298A (leading to a Glu at amino acid residue 429) is the most common while 1298C (leading to an Ala substitution at amino acid 429) is less common. In some embodiments, detection of alanine at position 429 ) of the corresponding amino acid sequence (SEQ ID NO: 4) can indicate a SNP at position 1298 of the MTHFR gene (or SEQ ID NO: l).Without wishing to be bound by theory, previous studies on human recombinant MTHFR have ed that the protein encoded by 1298C cannot be distinguished from 1298A in terms of activity, thermolability, FAD release, or the protective effect of 5-methyl-THF. (See, e.g.,Yamada K. et al. (2001). Proc. Natl. Acad. Sci. USA. 98 (26): 14853—8). It is believed that the C mutation (e.g., A1298C) does not appear to affect the MTHFR protein or result in thermolabile MTHFR, or affect steine levels.
Methods for detecting the SNPs of the MTHFR gene, e. g., C677T, A1298C, or G1793A are well known in the art, for examples, including the methods and primers used in US. Pat.
No.: US 6,833,243, which is incorporated herein by reference.
Methionine se (MTR). Methionine synthase also known as MS, MeSe, MetH is an enzyme that in humans is encoded by the MTR gene (5-methyltetrahydrofolate-homocysteine methyltransferase). SEQ ID NO: 2 corresponds to a portion of the genomic nucleic acid sequence of human wild-type or normal MTR gene obtained from NCBI database (NCBI Reference Sequence: NM_000254.2), wherein the nucleotide at position 2756 of SEQ ID NO: 2 is normal (e. g., ype) “A” allele. This enzyme is responsible for the regeneration of methionine from homocysteine.
Methionine synthase forms part of the osylmethionine (SAM) biosynthesis and regeneration cycle. A polymorphism in the MTR gene, an A-to-G transition at position 2756 (e.g., A2756G) of SEQ ID NO: 2 causes an amino acid substitution from aspartic acid to glycine at codon 919 (D919G) of the corresponding amino acid sequence (SEQ ID NO: 5). Accordingly, in some embodiments, detection of glycine at position 919 (e.g., D919G) of the corresponding amino acid sequence (SEQ ID NO: 5), e.g., by mass spectrometry, can te a SNP at position 2756 (e.g., A2756G) of the MTR gene (or SEQ ID NO: 2).
Methionine synthase reductase (MTRR). Methionine synthase reductase, also known as MSR, is an enzyme that in humans is encoded by the MTRR gene. SEQ ID NO: 3 ponds to a portion of the genomic nucleic acid ce of human wild-type or normal MTRR gene obtained from NCBI database (NCBI Reference Sequence: NM_002454.2), wherein the tide at position 66 of SEQ ID NO: 3 is normal (e. g., wild-type) “A” allele. Methionine is an essential amino acid required for protein synthesis and one-carbon metabolism. Its synthesis is catalyzed by the enzyme methionine synthase. Methionine se eventually becomes inactive due to the ion of its cob(I)alamin cofactor. Methionine synthase reductase regenerates a functional methionine se via reductive methylation, and is a member of the ferredoxin-NADP(+) reductase (FNR) family of electron transferases. MTRR rphism, an adenine-to-guanine mutation at position 66 (e.g., A66G) of SEQ ID NO: 3 converts an isoleucine to a methionine amino acid (I22M) at on 22 of the corresponding amino acid sequence (SEQ ID NO: 6). Accordingly, in some embodiments, ion of nine at position 22 (e. g., I22M) of the corresponding amino acid sequence (SEQ ID NO: 6), e. g., by mass spectrometry, can indicate a SNP at on 66 (e.g., A66G) of the MTRR gene (or SEQ ID NO: 3).
Catecholmethyltransferase (COMT). ol-O-methyltransferase is an enzyme responsible for the breakdown of dopamine and norepinephrine, e. g., in the prefrontal cortex.
Met/Met are more rapid metabolizers than Val/Val subjects in which are associated with cognitive dysfunction and disease pathology. In some embodiments, a hypometholated state has led to an pression of COMT and greater executive dysfunction. COMT polymorphism (identified by rs4680: SEQ ID NO. 24), an adenine-to-guanine mutation at position 27 of SEQ ID NO. 24 converts a valine (Val) to a methionine (Met) amino acid (Va1158Met) at the corresponding position of the amino acid sequence. Accordingly, in some embodiments, detection of methionine at position 158 (e. g., V22M) of the corresponding amino acid sequence (SEQ ID NO. 28), e.g., by mass ometry, can indicate a SNP at position 27 of SEQ ID NO. 24.
Reducedfolate carrier 1 &2 (RCFI and RCFZ). Reduced folate r 1 and 2 (at 1) are receptors that transport S-MTHF across various membranes including the choroid plexus and blood brain barrier.
Dopamine receptor D2 (DRD2). Taq1B and H313H are dopamine receptor polymorphisms that effect dopamine transmission, receptor density, and antipsychotic response.
DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B). DNA (cytosine-5)- transferase 3 beta is s gene encoding a DNA methyltransferase which is believed to function in de novo methylation, rather than maintenance methylation. e-phosphate Cytidylytransferase A A). Choline-phosphate Cytidylytransferase A (PCYTlA) is an enzyme that aids in the transformation of phosphatidylcholine to choline.
GTP cyclohydrolase I (GCH1). GTP ydrolase I is part of the folate and biopterin biosynthesis pathways. It is responsible for the ysis of guanosine triphosphate (GTP) to form 7,8-dihydroneopterin phosphate. GTPCH is encoded by the gene GCH1 and is the rate- ng enzyme in tetrahydrobiopterin (THB, BH4) thesis. GCH1 is an essential or in monamine synthesis and NO production.
Folate hydrolase (prostate-specific membrane n) (FOLH1). FOLH1 is also known as glutamate carboxypeptidase II, which is an enzyme that in humans is encoded by the FOLH1 (folate hydrolase 1) gene. GCPII is a class 11 membrane glycoprotein. It catalyzes the hydrolysis of N-acetylaspartylglutamate (NAAG) to ate and N-acetylaspartate (NAA).
Dopmaine active transporter (DA T). The dopamine transporter (also dopamine active transporter, DAT, SLC6A3) is a membrane-spanning protein that pumps the neurotransmitter dopamine out of the synapse back into cytosol, from which other transporters sequester DA and NE into vesicles for later storage and release.
GTP cyclohydrolase 1 feedback regulatory protein (GCHFR). GTP cyclohydrolase 1 feedback regulatory protein is an enzyme that in humans is encoded by the GCHFR gene. GTP cyclohydrolase 1 feedback regulatory protein binds to and mediates tetrahydrobiopterin inhibition of GTP cyclohydrolase 1 which aids in the production of de novo BH4 production.
Calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAIC). Gene CACNA1C encodes an alpha-1 t of a voltage-dependent calcium channel. Calcium channels mediate the influx of calcium ions into the cell upon membrane polarization.
Formiminotransferase cyclodeaminase (FTCD). Formiminotransferase cyclodeaminase is an enzyme that catalyzes the sion of inoglutamate and tetrahydrofolate into formiminotetrahydrofolate and ate.
Methylenetetrahydrofolate dehydrogenase (NADP+dependent) 1 (MTHFD 1).
Methylenetetrahydrofolate dehydrogenase (NADP+dependent) l is a tri-allelic gene that encodes a protein that ses three distinct enzymatic activities, methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formate—tetrahydrofolate ligase. Each of these activities catalyzes one of three sequential reactions in the interconversion of l-carbon derivatives of tetrahydrofolate, which are ates for methionine, ylate, and de novo purine syntheses. A common single nucleotide polymorphism (SNP) at nucleotide 1958 of the MTHFDl gene (or at position 27 of SEQ ID 27) causes a “G” to “A” transition, which results in an arginine to glutamate substitution at amino acid position 653 in the synthetase domain of the enzyme ( See, e.g., Hol et al., (1998) “Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, tetrahydrofolate synthetase) in patients with neural tube defects.” Clin Genet 53: 119-25).
S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH). S-adenosyl methionine, commonly known as SAM, or SAM-e, or AdoMet, is a natural compound found in all living cells. It is one of the most used enzymatic substrates in biochemical reactions, second only to the universal energy storage and transfer molecule, adenosyl triphosphate (ATP).
S-Adenosyl methionine is a common cosubstrate involved in methyl group ers.
It is made from adenosine triphosphate (ATP) and nine by methionine adenosyltransferase.
Transmethylation, transsulfuration, and ropylation are the metabolic ys that use SAM.
SAH is formed by the demethylation of S-adenosyl-L-methionine (SAM). Further s about SAM and SAH, including immunoassays for determining SAM, SAH and/or ratios thereof are described in US. Pat. App. No.: US 2009/0263879, which is incorporated herein by reference. 4-hydroxynonenal (4-HNE). 4-Hydroxynonenal, or 4-hydroxynonenal or 4-HNE or HNE, 02), is an 0., B-unsaturated hydroxyalkenal which is produced by lipid peroxidation in cells. 4-HNE is the primary 0., B-unsaturated hydroxyalkenal formed in this s. It is found throughout animal tissue, and in higher quantities during oxidative stress due to the increase in the lipid peroxidation chain reaction, due to the increase in stress . 4-HNE has been believed to play a key role in cell signal transduction, in a variety of pathways from cell cycle events to cellular adhesion. 4-HNE is also considered as possible causal agents of numerous diseases, such as chronic inflammation, neurodegenerative diseases, adult respiratory distress syndrome, atherogenesis, diabetes and different types of cancer.
Protein residues known to react with 4HNE via 1,4-addition are Cys, His, and Lys.
Thus, in some embodiments, sion levels of 4-HNE can be determined by measuring expression levels of 4-HNE s, e.g., 4-HNE-His. Commercial ELISA kits for measuring 4-HNE adducts, e.g., OxiSelectTM HNE-His Adduct ELISA Kit are available, e.g., from CellBioLabs.
Test sample and collection and preparation thereof Collections of test samples for at least one analysis performed in the assays and/or methods described herein are well known to those skilled in the art. In some embodiments, a test sample subjected to analysis performed in the assays and/or methods described herein are derived from a biological sample of a subject. The term “biological sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., cell lysate, a homogenate of a tissue sample from a subject or a fluid sample from a subject. The term “biological sample” also includes untreated or pre- d (or pre-processed) biological samples. In some embodiments, the biological sample can be a biological fluid, including, but not limited to, blood (including whole blood, plasma, cord blood and serum), lactation products (e.g., milk), amniotic fluids, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, perspiration, mucus, liquefied feces, synovial fluid, lymphatic fluid, tears, tracheal aspirate, and fractions thereof. In other embodiments, the biological sample can include cell lysate and fractions f. For e, cells (such as red blood cells, ets, white blood cells and any cells circulating in the ical fluid described herein) can be ted and lysed to obtain a cell lysate. In some embodiments, a test sample or a biological sample is a blood sample. In some embodiments, a test sample or a biological sample is a plasma sample. In some embodiments, a test sample or a biological sample is a saliva sample. In some embodiments, a test sample or a biological sample is a buccal sample. In some embodiments, a test sample or a biological sample is a urine sample.
A “biological sample” can contain cells from subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine, that can be used to e plasma/serum biomarker expression levels or determine SNPs. In some embodiments, the sample is from a resection, biopsy, or core needle biopsy. In addition, fine needle aspirate samples can be used. Samples can be either in-embedded or frozen tissue.
The sample can be ed by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e. g. isolated by r person). In addition, the biological sample can be freshly collected or a previously collected sample.
] In some embodiments, the test sample or the biological sample can be a frozen biological sample, e.g., a frozen tissue or fluid sample such as urine, blood, serum or plasma. The frozen sample can be thawed before employing methods, assays and systems described . After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein.
In some embodiments, a test sample or a biological sample can be a c acid product amplified after polymerase chain reaction (PCR). The nucleic acid product can include DNA, RNA and mRNA and can be isolated from a particular ical sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. s of isolating and analyzing nucleic acid variants as bed above are well known to one skilled in the art and can be found, for example in the Molecular Cloning: A Laboratory Manual, 3rd Ed., Sambrook and , Cold Spring Harbor Laboratory Press, 2001.
In some embodiments, the test sample or the biological sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the , including biomolecules (e. g., c acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. In addition, or alternatively, chemical and/or biological reagents can be employed to release nucleic acid or n from the sample.
The skilled artisan is well aware of methods and processes appropriate for pre- sing of test or biological samples, e. g., blood, required for determination of SNPs or expression levels of serum/plasma biomarkers as bed herein.
In some embodiments, the test sample or biological sample is a blood sample, e.g., whole blood, plasma, and serum. In some embodiments, the test sample or biological sample is a whole blood sample. In some embodiments, the test sample or biological sample is a serum sample. In some embodiments, the test sample or biological sample is a plasma sample. In some ments, the blood sample can be allowed to dry at room temperature from about 1 hour to overnight, or in the refrigerator (low humidity) for up to several months before subjected to analysis, e. g., SNP analysis.
See, for example, Ulvik A. and Ueland RM. (2001) Clinical Chemistry 47: 2050, for methods of SNP genotyping in unprocessed whole blood and serum by real-time PCR.
To collect a blood , by way of e only, the t’s blood can be drawn by trained l personnel directly into anti-coagulants such as citrate, EDTA PGE, and theophylline. The whole blood can be separated into the plasma portion, the cells, and platelets portion by refrigerated centrifugation at 3500 g for 2 minutes. After centrifugation, the supernatant is the plasma and the pellet is RBC. Since platelets have a tendency to adhere to glass, it is preferred that the collection tube be siliconized. Another method of isolating red blood cells (RBCs) is described in Best, CA et al., 2003, J. Lipid Research, 44:612-620.
Alternatively, serum can be collected from the whole blood. By way of example, about 15 mL of whole blood can be drawn for about 6 mL of serum. The blood can be collected in a hard plastic or glass tube; blood will not clot in soft plastic. The whole blood is allowed to stand at room temperature for 30 minutes to 2 hours until a clot has formed. Then, clot can be carefully separated from the sides of the container using a glass rod or wooden applicator stick and the rest of the sample can be left overnight at 4°C. After which, the sample can be centrifuged, and the serum can be transferred into a clean tube. The serum can be clarified by centrifugation at 1000 g for 10 minutes at 4°C. The serum can be stored at —80°C before analysis. In such embodiments, carotenoids may not be stable for long s of time. Detailed described of ing serum using collection tubes can be found in U. S. Patent No. 3,837,376 and is incorporated by reference. Blood collection tubes can also be sed from BD Diagnostic s, Greiner Bio-One, and Kendall Company.
The whole blood can be first separated into platelet-rich plasma and cells (white and red blood cells). Platelet rich plasma (PRP) can be isolated from the blood centrifugation of citrated whole blood at 200 g for 20 minutes. The et rich plasma is then transferred to a fresh polyethylene tube. This PRP is then centrifuged at 800 g to pellet the platelets and the supernatant (platelet poor plasma [PPP]) can be saved for analysis, e. g., by ELISA, at a later stage. Platelets can be then gently re-suspended in a buffer such as Tyrodes buffer containing lU/ml PGE2 and ed by centrifugation again. The wash can be repeated twice in this manner before removing the membrane fraction of platelets by centrifugation with Triton X, and lysing the pellet of et for platelet-derived PF4 analyses. Platelets can be lysed using 50 mM Tris HCL, 100-120 mM NaCl, 5 mM EDTA, 1% Igepal and Protease Inhibitor Tablet (complete TM mixture, Boehringer Manheim, Indianopolis, IN).
] In one embodiment, platelets are separated from whole blood and the SNPs or hsCRP transcripts can be ined rom. When whole blood is centrifuged as described herein to separate the blood cells from the plasma, a pellet is formed at the end of the centrifugation, with the plasma above it. Centrifugation separates out the blood components (RBC, WBC, and platelets) by their various densities. The RBCs are denser and will be the first to move to the bottom of the collection/centrifugation tube, followed by the smaller white blood cells, and finally the platelets. The plasma fraction is the least dense and is found on top of the pellet. The “buffy coat” which contains the majority of platelets will be sandwiched between the plasma and above the RBCs. Centrifugation of whole blood (with anti-coagulant, PGE and theophylline) can produce an ed platelet rich "buffy coat" that lies just above the buoy. The buffy coat contains the concentrated ets and white blood cells.
In another embodiment, ets can be separated from blood according to methods described in United States Patent No. 4,656,035 using lectin to agglutinate the ets in whole blood. Alternatively, the methods and apparatus described in U. S. Patent No. 7,223,346 can be used involving a platelet collection device comprising a centrifugal eparator container with a cavity having a longitudinal inner surface in order to collect the “buffy coat” enriched with platelets after centrifugation. As another alternative, the methods and apparatus as described in WO/2001/066l72 can be used. Each of these references is incorporated by nce herein.
In another embodiment, platelets can be isolated by the two methods described in A.
L. Copley and R. B. Houlihan, Blood, 1947, 2: 170-181, which is incorporated by reference herein.
Both methods are based on the principle that the platelet layer can be obtained by repeated fractional centrifugation.
In some ments, apparatus and related s are used to obtain the sample, for example, machines described in U.S. Pat. No. 4,120,448, 5,879,280 and 7,241,281, which are incorporated herein by reference.
Methods for collecting different types of a test sample are known in the art and can be employed to prepare a test sample for the assays and methods described herein.
SNPs. Polymorphisms and Alleles The genomes of all sms undergo spontaneous mutation in the course of their uing evolution, generating variant forms of progenitor genetic sequences (Gusella, Ann. Rev.
Biochem. 55, 831-854 ). The tence of multiple forms of a genetic sequence gives rise to genetic polymorphisms, including SNPs.
Approximately 90% of all polymorphisms in the human genome are SNPs. SNPs are single base positions in DNA at which different alleles, or alternative nucleotides, exist in a tion. The SNP position (interchangeably ed to herein as SNP, SNP site, SNP allele or SNP locus) is y preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations). An individual can be homozygous or heterozygous for an allele at each SNP position. A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP is an amino acid coding sequence.
A SNP can arise from a substitution of one nucleotide for another at the polymorphic site. Substitutions can be transitions or transversions. A transition is the replacement of one purine nucleotide by another purine tide, or one dine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine, or vice versa. A SNP can also be a single base insertion or deletion variant referred to as an "in/del" (Weber et al., "Human diallelic insertion/deletion polymorphisms", Am J Hum Genet r 2002;71(4):854-62).
A synonymous codon change, or silent mutation/SNP (the terms "SNP" and "mutation" are used herein interchangeably), is one that does not result in a change of amino acid due to the degeneracy of the genetic code. A substitution that changes a codon coding for one amino acid to a codon coding for a different amino acid (i.e., a non-synonymous codon change) is referred to as a missense mutation. A nonsense mutation results in a type of non-synonymous codon change in which a stop codon is formed, thereby g to premature termination of a polypeptide chain and a truncated protein. A read-through mutation is r type of non-synonymous codon change that causes the destruction of a stop codon, thereby ing in an extended polypeptide product. While SNPs can be bi-, tri-, or tetra-allelic, the vast majority of the SNPs are bi-allelic, and are thus often referred to as "bi-allelic markers", or "di-allelic markers".
] A major database of human SNPs is maintained at NCBI as dbSNP, and it contains data for unique human SNPs consisting of 1.78 x 108 submitted SNP (identified by an “ss” number) and 5.2 x 107 reference SNP ified by an “rs” number), as of Build History 135: human_9606 based on NCBI human genome build 37.3. The rs numbers are unique, do not change and allow analysis of the particularly identified SNP in any genetic sample. Throughout the specification, the SNPs described herein can also be identified by an “rs” . For example, the SNP at position 677 of SEQ ID NO: 1 can be identified by rs 1801133; the SNP at position 1298 of SEQ ID NO: 1 can be identified by rs 1801131; the SNP at position 2756 of SEQ ID NO: 2 can be identified by rs 1805087; The SNP at position 66 of SEQ ID NO: 2 can be identified by rs 1801394. With the “rs” numbers known for each SNP, one of skill in the art will be able to determine the position of a specific SNP within a respective chromosome.
While a SNP could conceivably have three or four alleles, nearly all SNPs have only two alleles. Analysis of the SNPs identified herein generally relies on the two alleles that are listed in connection with each SNP. For example, the SNPs at the MTHFR locus described herein are each indicated to have two alleles, “C” or “T” at the position 677 of SEQ ID NO. 1, and “A” or “C” at the position 1298 of SEQ ID NO. 1, wherein SEQ ID NO. 1 is a n of a genomic nucleic acid sequence of MTHFR. The presence of at least one allele “T” at position 677 of SEQ ID NO. 1 and/or at least one allele “C” at position 1298 of SEQ ID NO. 1 indicates that a subject with depression is recommended for a treatment n comprising a folate-containing compound. The SNP at the MTR locus described herein is indicated to have two alleles, “A” or “G”. The presence of at least one allele “G” at position 2756 of SEQ ID NO. 2, wherein SEQ ID NO: 2 is a portion of a genomic nucleic acid ce of methionine synthase (MTR), indicates the subject ended for a treatment regimen comprising a folate-containing nd. The SNP at the MTRR locus described herein is indicated to have two s, “A” or “G”. The presence of at least one allele “G” at position 66 of SEQ ID NO. 3, n SEQ ID NO. 3 is a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR), indicates the subject recommended for a treatment regimen comprising a folate-containing nd.
Those skilled in the art will readily recognize that nucleic acid molecules can be double-stranded molecules and that reference to a ular site on one strand refers, as well, to the corresponding site on a complementary strand. In defining a SNP position, SNP allele, or nucleotide sequence, reference to an adenine “A”, a thymine “T” ne “U”), a cytosine “C”, or a guanine “G” at a particular site on one strand of a nucleic acid molecule also defines the thymine “T” (uridine “U”), adenine “A”, e “G”, or cytosine “C” (respectively) at the corresponding site on a complementary strand of the nucleic acid molecule. Thus, reference can be made to either strand in order to refer to a particular SNP position, SNP allele, or nucleotide sequence. Probes and primers can be designed to ize to either strand and SNP genotyping methods disclosed herein can generally target either strand. ingly, the claims are intended to cover analysis of the te strand as well.
For the opposite-strand analysis, the SNPs at the MTHFR locus is allele “A” at position 677 or allele “G” at position 1298 of the complementary sequence of SEQ ID NO. 1, wherein SEQ ID NO. 1 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); while the SNP at the MTR locus is allele “C” at position 2756 of the mentary sequence of SEQ ID NO. 2, wherein SEQ ID NO. 2 is a portion of a c c acid sequence of methionine synthase (MTR); and the SNP at the MTRR locus is allele “C” at on 66 of the complementary sequence of SEQ ID NO. 3, wherein SEQ ID NO. 3 is a portion of a c c acid sequence of methionine synthase reductase (MTRR).
Identification method of SNPs can be of either a positive-type (inclusion of an ) or a negative-type (exclusion of an allele). Positive-type methods determine the identity of a nucleotide ned in a rphic site, whereas negative-type methods determine the identity of a nucleotide not present in a polymorphic site. Thus, a wild-type site can be identified either as wild- type or not mutant. For example, at a biallelic polymorphic site where the wild-type allele contains thymine and the mutant allele contains cytosine, a site can be positively determined to be either thymine or cytosine or negatively determined to be not thymine (and thus cytosine) or not cytosine (and thus thymine).
Methodsfor detecting the SNPS disclosed herein According to one aspect described herein, a method for determining whether a t is homozygous for a polymorphism, heterozygous for a polymorphism, or lacking the polymorphism altogether (i.e. homozygous wildtype) is encompassed. As an exemplary embodiment only, a method to detect the C>T variance at position 677 of SEQ ID NO: 1, a method for determining the allele, heterozygous for the C— and T-alleles, or homozygous for the C—allele or the T-allele at the SNP loci are provided. Substantially any method of detecting any allele of the SNPs described herein, such as restriction enzyme digestion, allele-specific probe hybridization, allele-specific primer extension, allele specific amplification, sequencing, 5’ nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, and single-stranded conformational rphism, can be used.
] In one embodiment, an allelic discrimination method for fying the pes of SNPs of a human described herein can be used. Such a method may involve the use of distinct oligonucleotide probes, for example one complementary to a sequence having a major allele and another complementary to a sequence having a minor allele. The allelic discrimination method also involves use of at least one, and preferably a pair of amplification primers for amplifying a nce region of the MTHFR, MTR or MTRR locus of a subject. The reference region includes at least a portion of the human MTHFR, MTR or MTRR locus.
] For full-length genes and entire protein-coding sequences, a SNP flanking sequence can be, for example, up to about 10 Kb, 9 Kb, 8 Kb, 7 Kb, 6Kb, 5 Kb, 4 Kb, 3 Kb, 2 Kb, 1 Kb on either side of the SNP. Furthermore, in such instances, the isolated nucleic acid molecule comprises exonic sequences (including protein-coding and/or non-coding exonic sequences), but may also include intronic sequences. Thus, any protein coding sequence may be either contiguous or separated by introns. The ant point is that the nucleic acid is isolated from remote and rtant flanking sequences and is of appropriate length such that it can be subjected to the specific manipulations or uses described herein such as recombinant protein expression, preparation of probes and primers for assaying the SNP position.
The probe is ably a DNA oligonucleotide haVing a length in the range from about 20 to about 40 nucleotide residues, preferably from about 20 to about 30 nucleotide residues, and more preferably haVing a length of about 25 nucleotide residues. In one embodiment, the probe is rendered incapable of extension by a talyzing enzyme such as Taq polymerase, for example by haVing a fluorescent probe attached at one or both ends thereof. Although non-labeled ucleotide probes can be used in the kits and methods described herein, the probes are preferably detectably labeled. Exemplary labels include radionuclides, light-absorbing chemical moieties (e. g. dyes), fluorescent moieties, and the like. Preferably, the label is a fluorescent moiety, such as 6- yfluorescein (FAM), 6-carboxy-4,7,2',7'-tetrachlorofluoroscein (TET), rhodamine, JOE (2,7- oxy-4,5-dichlorocarboxyfluorescein), HEX (hexachlorocarboxyfluorescein), or VIC.
In some embodiments, the probe can comprise both a cent label and a fluorescence-quenching moiety such as 6-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA), or 4- (4'-dimethlyaminophenylazo)benzoic acid (DABCYL). When the fluorescent label and the fluorescence-quenching moiety are ed to the same oligonucleotide and separated by no more than about 40 nucleotide residues, and preferably by no more than about 30 nucleotide es, the fluorescent intensity of the fluorescent label is diminished. When one or both of the fluorescent label and the fluorescence-quenching moiety are separated from the oligonucleotide, the intensity of the fluorescent label is no longer diminished. Preferably, the probe for use in the assays, methods, systems and kits described herein can have a fluorescent label ed at or near (i.e. within about 10 nucleotide residues of) one end of the probe and a fluorescence-quenching moiety attached at or near the other end. Degradation of the probe by a PCR-catalyzing enzyme releases at least one of the fluorescent label and the fluorescence-quenching moiety from the probe, y discontinuing fluorescence ing and increasing the detectable intensity of the fluorescent . Thus, cleavage of the probe (which, as discussed above, is correlated with complete complementarity of the probe with the target portion) can be detected as an increase in fluorescence of the assay e.
If detectably different labels are used, more than one labeled probe can be used. For e, the assay mixture can contain a first probe which is completely complementary to the target portion of the polymorphism of the MTHFR, MTR, or MTRR gene and to which a first label is attached, and a second probe which is completely mentary to the target portion of the wildtype or major allele. In some embodiments, by way of example only, the assay mixture can contain a first probe which is completely complementary to the target portion of the polymorphism of the MTHFR gene and to which a first label is attached, and a second probe which is completely complementary to the target portion of another gene, e.g., MTR or MTRR. When two probes are used, the probes are detectably different from each other, , for example, detectably ent size, absorbance, excitation, or emission spectra, radiative on properties, or the like. For example, a first probe can be completely complementary to the target portion of the polymorphism and have FAM and TAMRA attached at or near opposite ends thereof. The first probe can be used in the methods, , systems and kits bed herein together with a second probe which is completely complementary to the target n of the wildtype allele and has TET and TAMRA attached at or near te ends thereof. Fluorescent enhancement of FAM (i.e. effected by cessation of fluorescence quenching upon degradation of the first probe by Taq polymerase) can be detected at one wavelength (e.g. 518 nanometers), and fluorescent enhancement of TET (i.e. effected by cessation of fluorescence quenching upon ation of the second probe by Taq polymerase) can be detected at a ent wavelength (e. g. 582 nanometers).
Any approach that detects mutations or polymorphisms in a gene can be used to detect the presence or absence of SNP kers described herein, including but not limited to single-strand conformational polymorphism (SSCP) analysis (Orita et al. (1989) Proc. Natl. Acad. Sci.
USA 86:2766-2770), heteroduplex analysis (Prior et al. (1995) Hum. Mutat. 5:263-268), oligonucleotide ligation (Nickerson et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-8927) and hybridization assays (Conner et al. (1983) Proc. Natl. Acad. Sci. USA 80:278-282). Traditional Taq polymerase PCR-based strategies, such as PCR-RFLP, allele-specific amplification (ASA) (Ruano and Kidd (1989) Nucleic Acids Res. 17:8392), single-molecule dilution (SMD) (Ruano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6296-6300), and coupled amplification and cing (CAS) (Ruano and Kidd (1991) Nucleic Acids Res. 19:6877—68 82), are easily performed and highly sensitive methods to determine ypes (Michalatos-Beloin et al. (1996) Nucleic Acids Res. 24:4841-4843; Barnes (1994) Proc. Natl. Acad. Sci. USA 91:5695-5699; Ruano and Kidd (1991) Nucleic Acids Res. 19:6877-6882).
Restriction Fragment Length Polymorphism Analysis In some embodiments, restriction enzymes can be utilized to identify ces or a polymorphic site using “restriction fragment length polymorphism” (RFLP) is (Lentes et al. , Nucleic Acids Res. 16:2359 ; and CK. McQuitty et al., Hum. Genet. 93:225 (1994)). In RFLP, at least one target polynucleotide is digested with at least one ction enzyme and the resulting restriction fragments are separated based on mobility in a gel. Typically, smaller fragments migrate faster than larger fragments. Consequently, a target polynucleotide that contains a particular restriction enzyme recognition site will be digested into two or more smaller fragments, which will migrate faster than a larger fragment lacking the ction enzyme site. Knowledge of the nucleotide sequence of the target polynucleotide, the nature of the polymorphic site, and knowledge of restriction enzyme recognition sequences guide the design of such assays. In another embodiment, restriction site analysis of ular nucleotide sequence to fy a nucleotide at a polymorphic site is determined by the presence or e of a restriction enzyme site. A large number of restriction enzymes are known in the art and, taken er, they are capable of recognizing at least one allele of many polymorphisms. However, such single nucleotide polymorphisms (SNPs) rarely result in changes in a restriction endonuclease site. Thus, SNPs are rarely detectable by restriction fragment length analysis.
Ligation Based Assays (e.g., Oligonucleotide Ligation Assay) A number of approaches use DNA ligase, an enzyme that can join two nt ucleotides hybridized to a DNA template. In Oligonucleotide Ligaton Assay (OLA) the sequence surrounding the mutation site is first amplified and one strand serves as a template for three ligation probes, two of these are ASO (allele-specific oligonucleotides) and a third common probe.
Numerous approaches cane be used for the detection of the ligated products, for example the ASOs with differentially labeled with fluorescent of hapten labels and ligated products ed by fluorogenic of colorimetric enzyme-linked immunosorbent assays (Tobe et al, Nucleic Acid Res, l996;24;3728-32). For electrophorosis-based systems, use of a morbidity modifier taqgs or variation in probe length coupled with fluorescence detection enables the multiplex genotyping of l single nucleotide substitutions in a single tube (Baron et al, 1997; Clinical Chem., 43;l984-6). When used on arrays, ASOs can be spotted at specific locations or addresses on a chip, PCR amplified DNA can then be added and ligation to d oligonucleotides at specific addresses on the array measured (Zhong et al, Proc Natl Acad Sci 2003; 100;11559-64).
Single-Base Extension Single base-extension or minisequencing involves annealing an oligonucleotide primer to the single strand of a PCR product and the addition of a single dideoxynucleotide by thermal DNA polymerase. The oligonucleotide is designed to be one base short of the mutation site. The dideoxynucleotide orated is complementary to the base at the mutation site. Approaches can use different fluorescent tags or haptens for each of the four different dideoxynucleotides (Pastinen et al, Clin Chem 1996, 42;l39l-7). The dideoxynucleotide differ in molecular weight and this is the basis for single-base extension methods utilizing mass-spectrometry, and genotyping based on the mass of the ed oligonucleotide primer, can be used, for example matrix-assisted laser adsorption/ tion time-of flight mass spectrometry or MALDI-TOF (Li et al, Electrophorosis, 1999,20;1258- 65), which is quantitative and can be used to calculate the relative allele abundance making the ch suitable for other applications such as gene dosage studies (for example for estimation of allele frequencies on pooled DNA samples).
Minisequencing or equencing by TOF can be performed by means known by persons skilled in the art. In a variation of the MALDI-TOF technique, some embodiments can use the om’s Mass Array Technology (www.sequenom.com) (Sauser et al, Nucleic Acid Res, 2000, 28;E13 and Sauser et al, Nucleic Acid Res 2000, 28: E100). and also the GOOD Assay (Sauer S et al, Nucleic Acid Res, 2000; 28, E13 and Sauer et al, Nucleic Acid Res, 2000;28:E100).
In some embodiments, variations of MALDI-TOF can be performed for analysis of variances in the genes associated with SNPs described herein. For example, MALDI and ospray ioinization (ESI) (Sauer S. Clin Chem Acta, 2006; 363; 93-105) can also be used in various aspects described herein.
Hybridization Based Genotyping (e.g., Allele-Specific Amplification (ASA)) Allele-specific Amplification is also known as amplification refectory mutation system (ARMS) uses allele specific oligonucleotides (ASO) PCR primers and is an well established and known PCR based method for genotyping (Newton et al, J Med Genet, 1991 ;28;248-51).
Typically, one of the two oligonucleotide primers used for the PCR binds to the mutation site, and amplification only takes place if the tide of the mutation is present, with a mismatch being refractory to amplification. The resulting PCR Products can be analyzed by any means known to persons skilled in the art. In a ion of the approach, termed mutagenically separated PCR (MS- PCR) the two ARMS primer of different lengths, one specific for the normal gene and one for the mutation are used, to yield PCR procures of different lengths for the normal and mutant alleles (Rust et al, Nucl Acids Res, 1993; 21 ;3623-9). Subsequent gel ophoresis, for example will show at least one of the two allelic products, with normal, mutant or both (heterozygote) genes. A further ion of this forms the basis of the Masscode SystemTM (www.bioserve.com) which uses small molecular weight tags covalently attached through a photo-cleavable linker to the ARMS primers, with each ARMS primers labeled with a tag of differing weight (Kokoris et al, 2000, 5;329-40). A catalogue of numerous tags allows simultaneous amplification/genotyping (multiplexing) of 24 different targets in a single PCR reaction. For any one mutation, genotyping is based on comparison of the relative abundance of the two relevant mass tags by mass spectrometry.
Normal or mutant alleles can be ped by ing the binding of allele- specific oligonucleotides (ASO) hybridization probes. In such embodiments, two ASO probes, one complementary to the normal allele and the other to the mutant allele are hybridized to PCR-amplified DNA ng the mutation site. In some embodiments, the amplified ts can be immobilized on a solid surface and hybridization to radiolabelled oligonucleotides such as known as a ‘dot-blot’ assay. In alternative embodiments, the binding of the PCR ts containing a fiable label (e.g., biotin or fluorescent labels) to a solid phase allele-specific ucleotide can be measured.
Alternatively, for a reverse hybridization assay, or “reverse ot” the binding of PCR ts ning a quantifiable label (for example but not limited to biotin or fluorescent labels) to a solid phase allele-specific oligonucleotide can be measured. In some embodiments, the use of microarrays comprising hundreds of ASO immobilized onto a solid support surfaces to form an array of ASO can also be used for large scale genotyping of multiple single polymorphisms simultaneously, for example Affymetrix GENECHIP® Mapping 10K Array, which can easily be performed by persons skilled in the art. nous assays Homogenous assays, also called “closed tube” arrays, genomic DNA and all the ts required for the ication and genotyping are added simultaneously. Genotyping can be achieved without any post-amplification processing. In some embodiments, one such nous assay is the 5’flurogenic nuclease assay, also known as the TAQMAN® Assay (Livak et al, Genet Anal, 1999; 14: 143-9) and in alternative embodiments Melting curve analyses of FRET probes are used. Such methods are carried out using “real-time” thermocyclers, and e two dual-labeled ASO hybridization probes complementary to normal and mutant alleles, where the two probes have different reported labels but a common quencher dye. In such ments, the changes in fluorescence characteristics of the probes upon binding to PCR products of target genes during ication enables “real-time” monitoring of PCR amplification and differences in affinity of the fluorogenic probes for the PCR products of normal and mutant genes s differentiation of genotypes. The approach uses two dual-labeled ASO hybridization probes complementary to the mutant and normal s. The two probes have different fluorescent reported dyes but a common quencher dye. When intact, the probes do not fluoresces due to the proximity of the reporter and quencher dyes. During ing phase of PCR, two probes compete for hybridization to their target sequences, downstream of the primer sites and are subsequently cleaved by 5’ nuclease activity of Thermophilis aquaticus (Taq) polymerase as the primer is extended, resulting in the separation of the er dyes from the quencher. Genotyping is determined by measurement of the fluorescent intensity of the two reporter dyes after PCR amplification. Thus, when intact the probes do not fluoresce due to the proximity of the quencher dyes, whereas during the annealing phase of the PCR the probes e for hybridization of the target sequences and the separation of one of the probes from the er which can be detected.
Melting-carve ofFRET hybridization Melting-curve analysis of FRET hybridization is another approach that can be used to detect the presence or absence of SNP biomarkers described herein. Briefly, the reaction includes two oligonucleotide probes which when in close proximity forms a fluorescent complex, where one probe often termed the “mutant ” probe is designed to specifically hybridize across the mutation site and the other probe (often referred to as the “anchor probe”) hybridizes to an nt site. scent light is emitted by the “donor” excites the “acceptor” fluorophore creasing a unique fluorogenic complex, which only forms when the probes bind to adjacent sites on the amplified DNA.
The “sensor” probe is complementary to either the normal or the mutant allele. Once PCR is complete, heating of the sample through the melting temperatures of the probe yields a fluorescent temperature curve which differs for the mutant and normal allele.
A variation of the FRET hybridization method is the LCGREENTM method, which obviates the requirement for fluorescent d probes ther. LCGREENTM is a sensitive highly fluorogenic double-stranded DNA (dsDNA) binding dye that is used to detect the dissociation of unlabelled probes (Liew et al, Clin Chem, 2004; 50;l 156-64 and Zhou et al, Clin Chem, 2005; 5l;l76l 2). The method uses unlabeled allele-specific oligonucleotides probes that are perfectly complementary either to the mutant or normal allele, and the mismatch of the ASO/template double strand DNA complex results in a lower melting temperature and an earlier reduction in fluorescent signal form the dsDNA binding dye with increasing temperature.
The OLA can also be performed by the use of FRET probes (Chen et al, Genome Res, l998;8: 549-56). In such an embodiment, the PCR/ligation mix contains PCR primers, a thermostable DNA polymerase t 5’ exonuclease activity (to t the cleavage of ligation probes during the ligation phase), a thermostable DNA ligase as well as the oligonucleotides for the ligation reaction. The ligation of the ASO each have a different acceptor fluorophore and the third ligation oligonucleotide which binds adj acently to the ASO has a donor fluorophore. The three ligation oligonucleotides are designed to have a lower melting temperature than the annealing temperature for the PCR primers in order to prevent their interference in PCR amplification. Following PCR, the temperature is lowered to allow ligation to proceed. Ligation results in FRET between donor and acceptor dyes, and alleles can be discerned by comparing the fluorescence emission of the two dyes.
Molecular Beacon Assays Further, variations of the homogenous PCR- and hybridization based techniques to detect polymorphisms can also be used to detect the presence or absence of SNP biomarkers described . For example, the use of Molecular Beacons (Tyagi et al, Nat Biotech 1998; 16; 49-53) and SCORPION® Probes (Thelwell et al, Nucleic Acid Res 2000;28;3752-6l). Molecular Beacons are sed of oligonucleotides that have fluorescent reporter and dyes at their 5’ and 3’ ends, with the central n of the ucleotide hybridizing across the target sequence, but the 5’ and 3’ flanking regions are complementary to each other. When not hybridized to their target ce, the ’ and 3’ flanking regions hybridize to form a stem-loop structure, and there is little cence e of the ity of the reported and the quencher dyes. However, upon hybridization to their target sequence, the dyes are separated and there is a large increase in the fluorescence. Mismatched probe-target hybrids dissociate at substantially lower atures than exactly matched complementary hybrids. There are a number of ions of the “molecular Beacon” approach. In some ments, such a variation includes use of SCORPION® Probes which are similar but incorporate a PCR primer sequence as part of the probe ell et al, Nucleic Acid Res 2000;28;3752 61). In another variation, x’ format gives a better fluorescent signal (Solinas et al, Nucleic Acid Res, 2001, 29; E96).
In another embodiment, rphisms can be detected by genotyping using a homogenous or real-time analysis on whole blood samples, without the need for DNA extraction or real-time PCR. Such a method is compatible with FRET and TAQMAN® (Castley et al, Clin Chem, 2005;51; 0) enabling extremely rapid ing for the particular polymorphism of interest.
Fluorescent Polarization (FP) In FP, the degree to which the emitted light remains polarized in a particular plane is proportional to the speed at which the molecules rotate and tumble in on. Under nt pressure, temperature and viscosity, FP is ly related to the molecular weight of a fluorescent species. Therefore, when a small fluorescent molecule is incorporated into a larger molecule, there is an increase in PP. PP can be used in for genotyping of polymorphisms of interest (Chen et al, Genome Res, 1999; 9: 492-8 and Latif et al, Genome Res, 2001; 11;436-40). FP can be utilized in 5’ nuclease assay (as described above), where the oligonucleotide probe is digested to a lower molecule weight species, for example is amenable to is by FP, but with the added benefit of not requiring a quencher. For example, Perkin-Elmers AcycloPrimeTM-FP SNP Detection Kit can be used as a PP minisequencing method. Following PCR amplification, unicoportated primers and nucleotides are degraded enzymatically, the enzymes heat inactivated and a minisequencing reaction using DNA polymerase and fluorescent-labeled dideoxynucleotides performed. FP is then measured, typically in a 96- to 386-well plate format on a FP-plate reader.
Pyrosequencing In some embodiments, the primer extension reaction and analysis is performed using PYROSEQUENCINGTM (Uppsala, Sweden) which essentially is sequencing by synthesis. A cing primer, designed directly next to the nucleic acid differing between the disease-causing mutation and the normal allele or the different SNP s is first hybridized to a single stranded, PCR amplified DNA template from the individual, and incubated with the enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase, and the substrates, adenosine 5/ phosphosulfate (APS) and rin. One of four deoxynucleotide triphosphates (dNTP), for example, corresponding to the nucleotide t in the mutation or polymorphism, is then added to the reaction. DNA polymerase catalyzes the incorporation of the dNTP into the standard DNA strand. Each incorporation event is accompanied by release of osphate (PPi) in a quantity lar to the amount of incorporated tide. Consequently, ATP ylase converts PPi to ATP in the presence of adenosine 5/ phosphosulfate. This ATP drives the rase-mediated conversion of luciferin to oxyluciferin that generates visible light in amounts that are proportional to the amount of ATP. The light produced in the luciferase-catalyzed reaction is detected by a charge coupled device (CCD) camera and seen as a peak in a PYROGRAMTM. Each light signal is proportional to the number of nucleotides incorporated and allows a clear ination of the presence or absence of, for e, the mutation or polymorphism. Thereafter, apyrase, a nucleotide degrading enzyme, continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added which corresponds to the dNTP present in for example the selected SNP.
Addition of dNTPs is performed one at a time. Deoxyadenosine alfa-thio triphosphate (dATPS) is used as a substitute for the natural deoxyadenosine triphosphate (dATP) since it is efficiently used by the DNA polymerase, but not recognized by the luciferase. For detailed information about reaction conditions for the PYROSEQUENCING, see, e.g. U.S. Patent No. 6,210,891, which is incorporated herein by reference.
INVADER® Assay Alternatively, an INVADER® assay can be used (Third Wave Technologies, Inc (Madison, WI)). This assay is generally based upon a structure-specific nuclease activity of a variety of enzymes, which are used to cleave a -dependent cleavage structure, y indicating the presence of ic nucleic acid ces or specific variations thereof in a sample (see, e.g. U.S.
Patent No. 6,458,535). For example, an INVADER® operating system (OS), provides a method for detecting and quantifying DNA and RNA. The INVADER® OS is based on a "perfect match" enzyme-substrate reaction. The INVADER® OS uses proprietary CLEAVASE® enzymes (Third Wave Technologies, Inc (Madison, WI)), which recognize and cut only the ic structure formed during the INVADER® process which structure differs between the different alleles selected for detection, i.e. the e-causing allele and the normal allele as well as between the different selected SNPs. Unlike the PCR-based methods, the INVADER® OS relies on linear amplification of the signal generated by the INVADER® process, rather than on exponential ication of the target.
In the INVADER® process, two short DNA probes hybridize to the target to form a structure ized by the CLEAVASE® enzyme. The enzyme then cuts one of the probes to release a short DNA "flap." Each released flap binds to a fluorescently-labeled probe and forms another cleavage structure. When the CLEAVASE® enzyme cuts the labeled probe, the probe emits a able fluorescence signal.
Mutations or polymorphisms can also be detected using allele-specific hybridization followed by a MALDI-TOF-MS detection of the different ization products. In the preferred embodiment, the detection of the enhanced or amplified nucleic acids representing the ent alleles is med using matrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF) mass spectrometric (MS) analysis described in the Examples below. This method differentiates the alleles based on their different mass and can be applied to analyze the products from the various above- described -extension methods or the INVADER® process.
Gel Migration-Based Methods (e. g., Single Stranded Conformation Polymorphism) In other embodiments, alterations in electrophoretic mobility are used to identify the particular allelic variant. For example, single strand mation polymorphism (SSCP) can be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sol USA ; Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to the sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. Alterations in the mobility of the resultant products are thus tive of a base change. Suitable controls and knowledge of the "normal" migration patterns of the ype alleles can be used to identify polymorphic variants. The DNA fragments can be d or ed with labeled probes. The sensitivity of the assay can be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in ce. In another preferred embodiment, the subject method utilizes heteroduplex analysis to separate double ed heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).
In yet another embodiment, the identity of the allelic variant is obtained by analyzing the movement of a nucleic acid comprising the rphic region in polyacrylamide gels containing a gradient of denaturant, which is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be ed to insure that it does not completely denature, for, example by adding a GC clamp of approximately 40 bp of high-melting GC rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) s Chem 265: 1275).
Other Assays Other methods for genetic ing can be used to detect the presence or absence of any of the SNP biomarkers described herein, for example, to detect mutations in c DNA, cDNA and/or RNA s. Methods commonly used, or newly developed or methods yet unknown are encompassed for use in detection of the presence or e of any of the SNP biomarkers bed herein. Examples of newly discovered methods e for e, but are not limited to; SNP mapping (Davis et al, Methods Mol y, 2006; 351;75-92); Nanogen Nano Chip, (keen-Kim et al, 2006; Expert Rev Mol Diagnostic, 6;287-294); Rolling circle amplification (RCA) combined with circularable oligonucleotide probes (c-probes) for the detection of nucleic acids (Zhang et al, 2006: 363;61-70), luminex XMAP system for detecting multiple SNPs in a single reaction vessel (Dunbar SA, Clin Chim Acta, 2006; 363;71-82; Dunbar et al, Methods Mol Med, 2005;114:147-1471) and enzymatic mutation detection methods (Yeung et al, Biotechniques, 2005; 38;749-75 8).
WO 74676 In one embodiment, one method of screening for point mutations is based on RNase cleavage of base pair ches in RNA/DNA or RNA/RNA heteroduplexes. As used herein, the term "mismatch" is defined as a region of one or more unpaired or mispaired nucleotides in a double- stranded RNA/RNA, RNA/DNA or DNA/DNA le. This definition thus includes mismatches due to insertion/deletion ons, as well as single or multiple base point mutations.
In such embodiments, protection from cleavage agents (such as a nuclease, hydroxylamine or osmium tetroxide and with dine) can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes (see, e.g., Myers et al. (1985) Science 230:1242). In general, the technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing a control nucleic acid, which is optionally labeled, e. g., RNA or DNA, comprising a tide sequence of the allelic variant of the gene of interest with a sample nucleic acid, e. g., RNA or DNA, obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves -stranded regions of the duplex such as duplexes formed based on ir mismatches between the control and sample strands. For instance, A duplexes can be treated with RNase and A hybrids treated with S1 nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with ylamine or osmium tetroxide and with piperidine in order to digest mismatched regions.
After digestion of the mismatched s, the resulting material is then ted by size on denaturing polyacrylamide gels to determine r the control and sample nucleic acids have an identical nucleotide sequence or in which nucleotides they are different. See, for example, U. S.
Patent No. 6,455,249, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzy. 217:286-295. In another embodiment, the control or sample nucleic acid is labeled for detection.
US. Patent 4,946,773 describes an RNaseA ch ge assay that involves ing single-stranded DNA or RNA test s to an RNA probe, and subsequent treatment of the nucleic acid duplexes with RNaseA. For the detection of mismatches, the single-stranded products of the RNaseA treatment, electrophoretically ted according to size, are compared to similarly treated control es. Samples containing smaller fragments (cleavage products) not seen in the control duplex are scored as positive. The use of RNaseI for mismatch detection is also described in literature from Promega Biotech. Promega markets a kit containing RNaseI that is reported to cleave three out of four known mismatches.
In one embodiment, a long-range PCR (LR-PCR) is used to detect mutations or polymorphisms. LR-PCR products are genotyped for mutations or polymorphisms using any genotyping methods known to one skilled in the art, and haplotypes inferred using mathematical approaches (e.g., Clark's algorithm (Clark (1990) Mol. Biol. Evol. 7:111-122).
For example, methods including complementary DNA (cDNA) arrays (Shalon et al., Genome Research 6(7):639-45, 1996; Bernard et al., Nucleic Acids Research 24(8):l435-42, 1996), solid-phase mini-sequencing technique (U.S. Patent No. 6,013,431, Suomalainen et al. M01.
Biotechnol. Jun; 15(2):123-31, 2000), ion-pair high-performance liquid chromatography (Doris et al.
J. togr. A can 8; 806(1):47-60, 1998), and 5' nuclease assay or real-time RT-PCR (Holland et al. Proc Natl Acad Sci USA 88: 7276—7280, 1991), or primer extension methods described in the U.S.
Patent No. 6,355,433, can be used.
Another method to detect mutations or polymorphisms is by using cence tagged dNTP/ddNTPs. In addition to use of the fluorescent label in the solid phase mini-sequencing method, a standard nucleic acid sequencing gel can be used to detect the fluorescent label incorporated into the PCR amplification product. A sequencing primer is designed to anneal next to the base differentiating the e-causing and normal allele or the selected SNP alleles. A primer extension reaction is performed using chain terminating dideoxyribonucleoside triphosphates (ddNTPs) labeled with a fluorescent dye, one label attached to the ddNTP to be added to the standard nucleic acid and another to the ddNTP to be added to the target c acid.
Others have described using the MutS protein or other DNA-repair enzymes for detection of single-base ches. Alternative methods for detection of on, insertion or substitution mutations that can be used in the practice of various aspects described herein are disclosed in U.S. Patents 5,849,483, 5,851,770, 5,866,337, 525 and 870, each of which is orated herein by reference.
In another embodiment, multiplex PCR procedures using allele-specific primers can be used to simultaneously y multiple regions of a target nucleic acid (PCT Application W089/10414), enabling amplification only if a ular allele is present in a sample. Other embodiments using alternative primer-guided nucleotide incorporation procedures for assaying polymorphic sites in DNA can be used, and have been described (Komher, J. S. et al., Nucl. Acids.
Res. 17:7779-7784 (1989); v, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M.N. et al., Proc. Nad. Acad. Sci. ) 88:1143-1147 (1991); Bajaj et al. (U.S. Patent No. 5,846,710); Prezant, T.R. et al., Hum Mutat. 1: 159-164 (1992); Ugozzoli, L. et al., GATA 112 47 (1992); Nyr6n, P. et al., Anal. Biochem. 208:171-175 ( 1993)).
Other known nucleic acid ication procedures include transcription- based amplification systems (Malek, L.T. et al., U.S. Patent 238; Davey, C. et al., European Patent Application 329,822; Schuster et al.) U.S. Patent 5,169, 766; , H.I. et al., PCT cation W089/06700; Kwoh, D. et al., Proc. Natl. Acad Sci. (U.S.A) 86:1173 Zl989); Gingeras, T.R. et al., PCT Application W08 8/10315)), or isothermal amplification methods (Walker, G.T. et al., Proc. Natl. 4cad Sci. (U.S.A) 89:392-396 (1992)) can also be used.
Another method to determine genetic variation is using “gene chips”. The use of microarrays comprising a multiplicity of sequences is becoming increasingly common in the art.
Accordingly, a microarray having at least one oligonucleotide probe, as described above, appended thereon, can be used for SNP genotyping to interrogate the presence or absence of at least one SNP described herein and/or additional alleles ated with responsiveness to a folate-containing compound.
Probes can be affixed to surfaces for use as " gene chips." Such gene chips can be used to detect genetic variations by a number of techniques known to one of skill in the art. In one technique, oligonucleotides are arrayed on a gene chip for determining the DNA sequence of a by the sequencing by hybridization approach, such as that outlined in US. Patent Nos. 6,025,136 and 6,018,041. The probes can also be used for fluorescent ion of a genetic sequence. Such techniques have been described, for e, in US. Patent Nos. 5,968,740 and 5,858,659. A probe also can be affixed to an electrode surface for the electrochemical detection of nucleic acid sequences such as described by Kayyem et al. US. Patent No. 5,952,172 and by , SC. et al. (1999) Nucleic Acids Res. 27:4830-4837.
Examples of identifying polymorphisms and applying that information in a way that yields useful information regarding patients can be found, for example, in US. Patent No. 6,472,157; US. Patent Application Publications 20020016293, 20030099960, 20040203034; WO 0180896, all of which are hereby incorporated by reference.
Determination of expression levels ofserqulasma biomarkers (e. g., SAM, SAH, 4-HNE, hsCRP) At least one of serum/plasma biomarkers as described herein (e.g., SAM, SAH, 4- HNE, and/or hsCRP) can be measured ing to methods to one skilled in the art. In some embodiments, sion levels of serum/plasma biomarkers (e.g., SAM, SAH, 4-HNE and/or hsCRP) can be determined by measuring protein levels. In some embodiments, sion levels of serum/plasma biomarkers (e.g., hsCRP) can be determined by measuring mRNA levels.
Determining sion level by measuring protein: By way of example only, the levels of serum/plasma kers (e.g., SAM, SAH, 4-HNE and/or hsCRP) can be measured by contacting a test sample with an antibody-based binding moiety that specifically binds to at least one of the plasma kers described herein, or to a fragment thereof. Formation of the antibody- protein complex is then detected by a variety of methods known in the art.
The term "antibody-based binding " or “antibody” can include immunoglobulin molecules and immunologically active determinants of immunoglobulin molecules, e.g., molecules that contain an antigen binding site which specifically binds (immunoreacts with) to the serum/plasma ns (e.g., SAM, SAH, 4-HNE and/or hsCRP). The term ody-based binding moiety” is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof which are also specifically reactive with the serum/plasma proteins (e.g., SAM, SAH, 4-HNE and/or hsCRP). Antibodies can be fragmented using conventional techniques. Thus, the term es segments of proteolytically-cleaved or recombinantly-prepared ns of an antibody molecule that are e of selectively reacting with a certain protein. Non- WO 74676 limiting examples of such lytic and/or recombinant fragments include Fab, F(ab’)2, Fab’ , Fv, dAbs and single chain antibodies (scFv) containing a VL and VH domain joined by a peptide linker.
The scFv’s can be covalently or non-covalently linked to form antibodies having two or more binding sites. Thus, "antibody-base binding moiety" includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies. The term "antibody-base binding moiety" is further intended to include humanized dies, bispecific antibodies, and chimeric molecules having at least one n binding determinant derived from an antibody molecule. In some embodiments, the dy-based g moiety can be detectably labeled.
"Labeled antibody", as used herein, includes antibodies that are labeled by a detectable means and include, but are not limited to, antibodies that are enzymatically, radioactively, fluorescently, and chemiluminescently labeled. Antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS. The detection and quantification of the serum/plasma proteins (e. g., SAM, SAH, 4-HNE and/or hsCRP) in test samples correlate to the intensity of the signal emitted from the detectably d antibody.
In some embodiments, the dy-based binding moiety can be detectably labeled by linking the antibody to an . The enzyme, in turn, when exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibodies against the serum/plasma proteins (e. g., SAM, SAH, 4-HNE and/or hsCRP) can include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V- steroid isomerase, yeast l dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, e oxidase, alactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate ogenase, glucoamylase and acetylcholinesterase.
Detection can also be accomplished using any of a y of other immunoassays.
For e, by radioactively labeling an antibody, it is possible to detect the antibody through the use of radioimmune assays. The radioactive isotope can be detected by such means as the use of a gamma r or a scintillation counter or by autoradiography. Isotopes which are particularly useful for the purpose of detection are 3H, 131I, 35S, 14C, and 125I.
It is also possible to label an antibody with a cent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. Examples of the most commonly used fluorescent labeling compounds include, but not d to, CYE dyes, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
An antibody can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
An antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of chemiluminescent labeling compounds can include, but not limited to, luminol, luciferin, inol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
Without limitations, levels of the serum/plasma ns (e.g., SAM, SAH, 4-HNE and/or hsCRP) can be detected by immunoassays, such as enzyme linked immunoabsorbant assay ), radioimmunoassay (RIA), Immunoradiometric assay (IRMA), Western blotting, immunocytochemistry or immunohistochemistry, each of which are described in more detail below.
In some embodiments, immunoassays such as ELISA or RIA can be used for determining expression levels of the serum/plasma proteins (e.g., SAM, SAH, 4-HNE and/or hsCRP). Antibody arrays or protein chips can also be employed, see for e US. Patent Application Nos: 2003/0013208A1; 2002/0155493A1; 017515 and US. Patent Nos: 6,329,209; 6,365,418, which are herein incorporated by reference. Commercially ble antibodies and/or immunoassays (such as ELISA) for detecting the serum/plasma proteins (e.g., SAM, SAH, 4-HNE and/or hsCRP), e.g., from Cell BioLabs, Abcam, Novus Biologicals, and Thermo Scientific Pierce Antibodies, can be used in the assays and/or methods described herein.
Immunoassays: The most common enzyme immunoassay is the "Enzyme-Linked Immunosorbent Assay (ELISA)." ELISA is a technique for ing and measuring the concentration of an antigen using a labeled (e. g. enzyme ) form of the antibody. There are different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in ds in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al., "Methods and Immunology", W. A.
Benjamin, Inc., 1964; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem., 22:895-904.
In a "sandwich ELISA", an antibody (e.g. anti-enzyme) is linked to a solid phase (i.e. a microtiter plate) and exposed to a biological sample containing antigen (e. g. enzyme). The solid phase is then washed to remove unbound antigen. A labeled antibody (e.g. enzyme linked) is then bound to the bound-antigen (if t) forming an antibody-antigen-antibody sandwich. Examples of enzymes that can be linked to the antibody are alkaline phosphatase, adish peroxidase, luciferase, urease, and B-galactosidase. The enzyme linked antibody reacts with a substrate to generate a colored on product that can be measured.
In a "competitive ELISA", antibody is incubated with a sample ning antigen (i.e. enzyme). The antigen-antibody mixture is then contacted with a solid phase (e. g. a microtiter plate) that is coated with n (i.e., enzyme). The more antigen present in the sample, the less free antibody that will be available to bind to the solid phase. A labeled (e.g., enzyme linked) secondary 2012/065084 antibody is then added to the solid phase to determine the amount of primary antibody bound to the solid phase.
In an "immunohistochemistry assay" a test sample is tested for specific proteins by ng the test sample to antibodies that are specific for the n that is being assayed. The antibodies are then ized by any of a number of methods to determine the presence and amount of the protein present. Examples of methods used to visualize antibodies are, for example, through enzymes linked to the antibodies (e.g., luciferase, alkaline phosphatase, horseradish peroxidase, or beta-galactosidase), or chemical methods (e.g., DAB/Substrate chromagen). The sample is then analysed microscopically, for example, by light microscopy of a sample stained with a stain that is detected in the visible um, using any of a y of such staining methods and reagents known to those skilled in the art.
] Alternatively, "Radioimmunoassays" can be employed. A radioimmunoassay is a technique for detecting and measuring the concentration of an antigen using a labeled (e.g.. radioactively or fluorescently d) form of the antigen. Examples of radioactive labels for antigens include 3H, 14C, and 125I. The concentration of antigen enzyme in a test sample or a ical sample can be measured by having the antigen in the biological sample compete with the labeled (e. g. radioactively) antigen for binding to an antibody to the antigen. To ensure competitive binding between the labeled antigen and the unlabeled antigen, the labeled antigen is present in a concentration ient to saturate the binding sites of the antibody. The higher the concentration of antigen in the sample, the lower the concentration of labeled antigen that will bind to the antibody.
In a radioimmunoassay, to determine the concentration of labeled antigen bound to antibody, the antigen-antibody complex must be ted from the free antigen. One method for separating the antigen-antibody complex from the free antigen is by precipitating the antigen-antibody complex with an anti-isotype antiserum. Another method for separating the antigen-antibody complex from the free antigen is by performing a "solid-phase radioimmunoassay" where the antibody is linked (e.g., covalently) to Sepharose beads, polystyrene wells, polyvinylchloride wells, or microtiter wells.
By comparing the concentration of labeled n bound to antibody to a standard curve based on samples having a known concentration of antigen, the tration of antigen in the biological sample can be determined.
An "Immunoradiometric assay" (IRMA) is an immunoassay in which the antibody reagent is radioactively labeled. An IRMA requires the production of a multivalent antigen conjugate, by techniques such as conjugation to a protein e. g., rabbit serum n (RSA). The multivalent n conjugate must have at least 2 antigen residues per molecule and the antigen residues must be of sufficient ce apart to allow binding by at least two dies to the antigen. For e, in an IRMA the multivalent antigen conjugate can be attached to a solid surface such as a plastic sphere.
Unlabeled "sample" antigen and antibody to antigen which is radioactively d are added to a test tube containing the multivalent antigen ate coated sphere. The antigen in the sample competes with the multivalent antigen conjugate for antigen antibody binding sites. After an appropriate incubation period, the unbound reactants are removed by washing and the amount of radioactivity on the solid phase is determined. The amount of bound radioactive antibody is inversely proportional to the concentration of antigen in the sample.
In some embodiments, Western ng (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)) can be used to measure sion levels of the serum/plasma ns (e.g., SAM, SAH, 4- HNE and/or hsCRP, n a ly treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Detectably labeled anti- enzyme antibodies can then be used to assess enzyme levels, where the intensity of the signal from the detectable label ponds to the amount of enzyme present. Levels can be quantified, for example by densitometry.
In addition to assays, the expression level of at least one of the plasma biomarkers can be determined by mass spectrometry such as MALDI/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry ), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, r magnetic resonance spectrometry, or tandem mass spectrometry (e. g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos: 20030199001, 20030134304, 20030077616, which are herein incorporated by reference. Mass spectrometry methods are well known in the art and have been used to quantify and/or identify molecules (see, e. g., Li et al. (2000) Tibtech 18:151-160; Rowley et al. (2000) s 20: 383-397; and Kuster and Mann (1998) Curr. Opin. Structural Biol. 8: 393-400).
In certain embodiments, a gas phase ion spectrophotometer is used. In other embodiments, laser-desorption/ionization mass spectrometry is used to analyze the sample. Modern laser desorption/ionization mass spectrometry ("LDI-MS") can be practiced in two main variations: matrix assisted laser desorption/ionization ("MALDI") mass spectrometry and surface-enhanced laser desorption/ionization ("SELDI"). In MALDI, the analyte is mixed with a solution containing a matrix, and a drop of the liquid is placed on the surface of a substrate. The matrix solution then co- crystallizes with the ical molecules. The substrate is inserted into the mass spectrometer. Laser energy is directed to the substrate surface where it desorbs and ionizes the biological les without significantly fragmenting them. See, e. g., U.S. Pat. No. 5,118,937 (Hillenkamp et al.), and U.S. Pat. No. 5,045,694 (Beavis & Chait).
In SELDI, the substrate surface is modified so that it is an active participant in the desorption process. In one variant, the surface is tized with adsorbent and/or e reagents that selectively bind the protein of interest. In another variant, the surface is derivatized with energy absorbing molecules that are not ed when struck with the laser. In another variant, the surface is derivatized with les that bind the protein of interest and that contain a photolytic bond that is broken upon application of the laser. In each of these methods, the derivatizing agent generally is localized to a ic location on the substrate surface where the sample is applied. See, e.g., US.
Pat. No. 060 and WO 98/59361. The two methods can be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix-containing liquid to the captured analyte to provide the energy absorbing material.
For additional information regarding mass spectrometers, see, e.g., Principles of Instrumental Analysis, 3rd edition., Skoog, rs College Publishing, Philadelphia, 1985; and Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th ed. Vol. 15 (John Wiley & Sons, New York 1995), pp. 1071-1094. Software programs such as the Biomarker Wizard program (Ciphergen Biosystems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra, e.g., comparing the signal strength of peak values from a of a test subject sample and a control sample (e.g., a normal healthy person). The mass spectrometers and their techniques are well known to those of skill in the art.
Determining expression level of a gene or protein by measuring mRNA: Real time PCR is an amplification technique that can be used to ine expression levels of mRNA corresponding to a protein of interest (e.g., hsCRP). (See, e.g., Gibson et al., Genome Research 6:995- 1001, 1996; Heid et al., Genome Research 6:986-994, 1996). Real-time PCR tes the level of PCR product accumulation during amplification. This technique permits quantitative evaluation of mRNA levels in multiple samples. For mRNA levels, mRNA can be extracted from a biological sample, e.g. a blood sample (such as white blood cells and/or platelets) and cDNA is prepared using standard techniques. ime PCR can be performed, for example, using a Perkin Elmer/Applied Biosystems (Foster City, Calif.) 7700 Prism instrument. Matching primers and fluorescent probes can be ed for genes of interest using, for example, the primer express program provided by Perkin Elmer/Applied Biosystems (Foster City, Calif.). Optimal concentrations of primers and probes can be initially determined by those of ordinary skill in the art, and control (for example, beta-actin) primers and probes can be obtained commercially from, for example, Perkin Elmer/Applied Biosystems (Foster City, Calif.). To quantitate the amount of the specific c acid of st in a , a standard curve is generated using a control. Standard curves can be generated using the Ct values determined in the real-time PCR, which are related to the initial concentration of the nucleic acid of interest used in the assay. Standard dilutions g from 101-106 copies of the gene of interest are generally sufficient. In on, a standard curve is generated for the control sequence. This permits standardization of initial content of the c acid of interest in a test sample to the amount of control for comparison purposes.
Methods of ime tative PCR using TaqMan probes are well known in the art. Detailed protocols for real-time tative PCR are provided, for example, for RNA in: Gibson et al., 1996, A novel method for real time quantitative RT-PCR. Genome Res., 10995-1001; and for DNA in: Heid et al., 1996, Real time quantitative PCR. Genome Res., 10:986-994. 2012/065084 The TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end. When the PCR product is ied in subsequent , the 5' nuclease activity of the polymerase, for example, AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, Perkin- Elmer).
In another embodiment, detection of RNA transcripts can be achieved by Northern blotting, wherein a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Labeled (e. g., radiolabeled) cDNA or RNA is then hybridized to the preparation, washed and analyzed by methods such as autoradiography.
Detection of RNA transcripts can further be accomplished using known amplification methods. For example, mRNA can be reverse-transcribed into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as bed in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA ed by symmetric gap lipase chain reaction (RT- AGLCR) as described by R. L. Marshall, et al., PCR s and Applications 4: 80-84 . One suitable method for detecting enzyme mRNA transcripts is bed in reference Pabic et. al.
Hepatology, 37(5): 1056-1066, 2003, which is herein incorporated by reference.
In situ ization visualization can also be employed, n a radioactively labeled antisense RNA probe is hybridized with target biomarkers in a test sample, washed, d with RNase and exposed to a sensitive emulsion for autoradiography. The samples can be stained with haematoxylin to demonstrate the histological composition of the , and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin can also be used.
Alternatively, mRNA expression can be detected on a DNA array, chip or a microarray. Oligonucleotides corresponding to enzyme are immobilized on a chip which is then hybridized with labeled nucleic acids of a test sample obtained from a patient. Positive hybridization signal is obtained with the sample ning ker transcripts. Methods of preparing DNA arrays and their use are well known in the art. (See, for example U.S. Patent Nos: 6,618,6796; 6,379,897; 377; 6,451,536; 548,257; U.S. 20030157485 and Schena et al. 1995 Science 20:467- 470; Gerhold et al. 1999 Trends in Biochem. Sci. 24, 168-173; and Lennon et al. 2000 Drug discovery Today 5: 59-65, which are herein incorporated by reference). Serial Analysis of Gene Expression (SAGE) can also be med (See for e U.S. Patent Application 20030215858).
Methodsfor treating a human subject with depression ] In accordance with various aspects bed herein, if a subject with depression is detected to have the presence of at least one of the conditions (A)-(X) determined in the assay described herein, the subject is recommended for and/or administered with a treatment regimen comprising a folate-containing compound. In such embodiments, the subject can be further administered or prescribed with a treatment n comprising an anti-depressant drug and a folate- ning compound. Accordingly, ed herein are also methods for treating a subject with depression. In some embodiments, the method comprises performing any embodiments of the assay for selecting a treatment regimen for a subject with depression described herein. In some embodiments, the method can further comprise ibing and/or administering a treatment regimen comprising a folate-containing compound to the selected human subject.
In some embodiments, a method for treating a human subject with depression can comprise administering a composition comprising an effective amount of a folate-containing compound to the human subject, who is diagnosed as having, or having a risk for, depression, and is further determined to carry at least one or more ding at least two, at least three, at least four or more), or any combinations of the conditions (A)-(X) described .
In certain embodiments, the method can comprise administering a composition comprising an effective amount of a folate-containing compound to the human subject, who is diagnosed as having, or having a risk for, sion, and is further determined to carry at least one of the following SNPs or a combination thereof: (i) a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine "T" allele, and (ii) a SNP at position 2756 of SEQ ID NO. 2 or on 27 of SEQ ID NO. 9 comprising at least one guanine "G" allele. In these embodiments, the method can further comprise determining the presence or absence of any of the conditions (A)-(X) bed .
In some embodiments, the subject administered with a ent regimen comprising a folate-containing compound can be further ined to be obese (e.g., with a BMI value of at least about 30 kg/m2 or higher).
The terms “treatment” and “treating” as used herein, with respect to treatment of a disease, means preventing the progression of the e, or altering the course of the disorder (for example, but are not limited to, slowing the progression of the er), or reversing a symptom of the disorder or reducing one or more symptoms and/or one or more mical markers in a t, preventing one or more symptoms from worsening or progressing, promoting recovery or improving prognosis. For example, in the case of treating depression, therapeutic treatment refers to alleviation of at least one symptom associated with depression. Measurable lessening includes any statistically significant decline in a measurable marker or symptom, such as measuring markers for depression in the blood as described later or assessing the degree of depression, e.g., using the criteria listed in DSM-IV or the efficacy measures as described in the Examples, e.g., HAMD-l7, 8, CGI, Maier or HAMD-7, after treatment. In one embodiment, at least one symptom of depression is 2012/065084 alleviated by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In another embodiment, at least one symptom is alleviated by more than 50%, e. g., at least about 60%, or at least about 70%. In one embodiment, at least one symptom is alleviated by at least about 80%, at least about 90% or greater, as compared to a control (e.g., a subject having the same or similar degree of sion as the treated subject is administered without a folate-containing compound, or a subject who has met none of the conditions described herein is administered with treatment regimen comprising a folate-containing nd). In some embodiments, at least one neuropsychological test is improved (e. g., HAMD-17 rating is decreased) by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%. In another ment, at least one neuropsychological test is improved (e.g., HAMD-17 rating is decreased) by more than 50%, e.g., at least about 60%, or at least about 70%. In one embodiment, at least one neuropsychological test is improved (e.g., HAMD-17 rating is decreased) by at least about 80%, at least about 90% or greater, as compared to a control (e.g., a subject having the same or similar degree of depression as the treated subject is administered without a -containing compound, or a subject who has met none of the conditions described herein is administered with treatment n comprising a folate-containing compound). In some embodiments, at least one symptom of depression can be alleviated by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% or higher within a period of at least about 10 days, including, e.g., at least about 20 days, at least about 30 days, at least about 40 days, or longer. In some embodiments, at least one neuropsychological test is improved (e. g., HAMD-17 rating is decreased) by at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% or higher within a period of at least about 10 days, including, e.g., at least about 20 days, at least about 30 days, at least about 40 days, or longer.
In some embodiments of this aspect and all other aspects described herein, a folate- containing nd can be administered in an amount effective to reduce at least one m (e.g., but not limited to, low mood, anhedonia, low energy, insomnia, agitation, anxiety and/or weight loss) associated with depression, e. g., major depressive disorders. In some embodiments, the effective amount of a folate-containing nd can provide at least about 0.1 to about 1 mg/kg body weight per day administration to the human subject. In some embodiments, the effective amount of a folate- containing compound can provide at least about 7.5 mg/day to about 50 mg/day administration to the human t. In one embodiment, the effective amount of a folate-containing compound can provide at least about 15 mg/day of folate administration to the human t.
] The effective amount of the folate-containing compound can be administered to a selected human subject as a single daily dose, or alternatively, in more than one d doses per day via any suitable administration route, e. g., oral administration. 2012/065084 In some embodiments of this aspect and all other aspects described herein, the treatment regimen can r comprise selecting and optionally administering an antidepressant drug.
In some embodiments, the anti-depressant drug can include a selective serotonin reuptake inhibitor, including, but not limited to, fluoxetine, citalopram, paroxetine, escitalopram, sertraline, and any ations f.
The subject with depression being treated with the methods described herein can be a t currently taking an antidepressant. Accordingly, the methods of treating a human subject with depression described herein can also be used to select a human subject to be treated with the ation of a folate-containing nd and an antidepressant to improve the effectiveness of an anti-depressant drug tly taken by a subject accordingly. Accordingly, if the human t currently taking an antidepressant is determined to have at least one (including, e.g., at least two, at least three or more) of the conditions (A)-(X) bed herein, the subject can be further administered or prescribed with a folate-containing compound as an adjuvant to the anti-depressant he/she is currently taking.
In other embodiments, if the subject currently taking an antidepressant is determined to have at least one (including, e.g., at least two, at least three or more) of the conditions (A)-(X) described herein, the subject can be administered to a ent regimen comprising a folate- containing compound without the antidepressant.
] In some embodiments, the methods described herein can be used to treat at least one or more core symptoms of depression (e.g., but not limited to low or depressed mood, anhedonia (e.g., loss of interest or pleasure in nearly all activities), low energy levels) in a subject who is determined to have at least one (including, e.g., at least two, at least three or more) of the conditions (A)-(X) described herein.
Treatment regimen comprising afolate-eontaining compound A -containing compound included in a treatment regimen can be administered can be administered together via a single dosage form or by separate administration. In certain ments, the folate-containing compound can be administered in a single dosage form. For example, the single dosage form can be administered as a single tablet, pill, capsule for oral stration or a solution for parenteral administration. Alternatively, the folate-containing compound can be administered as separate itions, e.g., as separate tablets or solutions. The length of time between administration of a sub-dose of a folate-containing compound can be adjusted to achieve the d therapeutic effect.
In some embodiments, a treatment regimen comprising a folate-containing compound further comprise at least one anti-depressant (e.g., l, 2, 3 or more antidepressants). A treatment regimen comprising a folate-containing compound and at least one epressant can be administered together via a single dosage form or by separate administration. In certain embodiments, the antidepressant and the -containing compound are administered together in a single dosage form. For e, the single dosage form can be administered as a single tablet, pill, e for oral administration or a solution for eral administration. Alternatively, the antidepressant and the folate-containing compound can be administered as separate compositions, e.g., as separate tablets or solutions. The antidepressant can be administered at the same time as the folate-containing compound, or the antidepressant can be administered intermittently with the folate-containing compound. The length of time between administration of the antidepressant and the folate-containing compound can be adjusted to achieve the desired therapeutic effect. In particular, the folate-containing compound can be administered at any frequency or administration protocol to e the efficacy of the antidepressant drug, as compared to efficacy of the antidepressant drug alone (e. g., in the absence of the folate-containing compound).
] In some embodiments, the folate-containing compound can be administered only a few minutes (e. g., l, 2, 5, 10, 30, or 60 min) before or after administration of the antidepressant.
Alternatively, the folate-containing compound can be administered several hours (e.g., 2, 4, 6, 10, 12, 24, or 36 hr) before or after administration of the antidepressant. Depending on the half-lives of the antidepressant and the folate-containing compound, in certain embodiments, it can be advantageous to administer more than one dosage of the folate-containing compound between strations of the antidepressant. For example, the folate-containing compound can be administered at 3 hours and then again at 6 hours following administration of the pressant. Alternatively, it can be advantageous to administer more than one dosage of the antidepressant between administrations of the folate- ning nd. Importantly, in some embodiments, the eutic effect of each antidepressant and -containing compound can overlap for at least a portion of the duration of each therapeutic agent so that the overall therapeutic effect of the combination therapy is attributable in part to the combined of the combination therapy. In some embodiments, the folate-containing compound and the pressant can be administered in a pulse administration. In other embodiments, they can be administered as a pulse-chase administration, e. g., where a folate- containing compound is administered for a brief period of time (pulse), followed by administration of the antidepressant for a longer period of time (e.g., chase).
In some ments where the antidepressant and the folate-containing compound are administered in separate compositions, the antidepressant and the folate-containing compound can be administrated by the same or different routes. For example, the antidepressant can be administered by intravenous injection while the folate-containing compound can be administered orally, or vice versa. Alternatively, for example, both the antidepressant and -containing nd can be administered together by intravenous injection or by oral administration.
In any embodiments of the s described herein, the adjuvant effect of the folate-containing compound administered in combination with an antidepressant can be additive. The term “additive" as used herein in the context of one agent has an additive effect on a second agent, refers to an increase in effectiveness of a first agent in the presence of a second agent as compared to the use of the first agent alone. Stated in another way, the second agent can function as an agent which enhances the physiological response of an organ or organism to the presence of a first agent. Thus, a second agent will increase the effectiveness of the first agent by increasing an individual’s response to the presence of the first agent.
] In any embodiments of the methods described herein, the nt effect of the folate-containing compound administered in combination with an antidepressant can be istic.
The term “synergy" or “synergistic" as used herein refers to the interaction of two or more agents so that their combined effect is greater than each of their individual effects at the same dose alone.
In some embodiments, the treatment regimen can further comprise life-style advice, including, e.g., prescribing an se regime, dietary , and/or administering another ceutical agent effective in ent of depression.
Folate orfolate-wntaining compounds Any art-recognized folate-containing compound can be selected and/or optionally administered to a human t selected to carry at least one or more conditions (A)-(X) described herein. The term “folate-containing compound” as used herein refers to a nd containing an effective amount of at least one folate for use in the methods described herein. Folate is a form of the soluble vitamin B9. The term “folate” as used herein encompasses the naturally-occurring form of folate, folic acid (also known as vitamin B9 or folacin) and metabolites or derivatives thereof such as methylfolate, tetrahydrofolate, and methyltetrahydrofolate. The term "folate" as used herein can also refer to both pteroic acid monoglutamate (folic acid) and reduced forms such as dihydrofolates and ydrofolates, e. g. yltetrahydrofolic acid, 5-methyltetrahydrofolic acid, 5,10- methylenetetrahydrofolic acid, 5,l0-methenyltetrahydrofolic acid, lO-formyltetrahydrofolic acid and tetrahydrofolic acid, polyglutamates thereof, optical isomers thereof (e. g., lly pure natural isomers thereof, and also mixtures of optical isomers such as racemic mixtures), derivatives thereof, pharmaceutically acceptable salts and esters thereof, glucosamine salts thereof, and galactosamine salts thereof.
As used herein, the term "pharmaceutically acceptable salts and esters” refers to pharmacologically acceptable and pharmaceutically acceptable salts and esters. Pharmacologically and pharmaceutically acceptable salts can include, but are not limited to, alkali metal or alkaline earth metal salts, e.g., sodium, potassium, magnesium or calcium salts. Pharmacologically and pharmaceutically acceptable esters can e, but are not limited to, C1-C4 alkyl, C5 cycloalkyl oder C6 cycloalkyl, phenyl, Cl-C4 alkylphenyl, benzyl or Cl-C4-alkylbenzyl esters. The esters can be ters or diesters. Diesters can be homogeneous or heterogeneous. In some embodiments, pharmacologically and pharmaceutically acceptable esters can be homogeneous diesters such as C1- C4 dialkylesters, for example dimethyl- or diethylesters.
In some embodiments, the folate-containing compound can include at least one (including at least two, at least three or more) alkaline metal or alkaline earth metal salt of folate, e. g., but not d to, a calcium salt of folate.
In some embodiments, the folate-containing compound can include at least one (including at least two, at least three or more) amine salt and/or galactosamine salt of folate (including, e. g., folic acid and d folate, e. g., but not limited to, tetrahydrofolate, and derivatives thereof). Examples of glucosamine-folate and/or galactosamine-folate and derivatives there of, e.g., disclosed in US. Pat. US 7,947,662, can be administered to a human subject in the methods or ed in the compositions bed herein. In one embodiment, QUATREFOLIC® (Gnosis S.p.A, Milan, IT) or N-[4-[[[(6S)amino-l,4,5,6,7,8-hexahydromethyloxo pteridinyl]methyl]amino]benzoyl]-L-glutamic acid, glucosamine salt can be administered to a human subject in the methods or included in the compositions described herein.
Folic acid, folate and their metabolites or reduced folates, e.g., methylfolate, are substances that are characterized as vitamins, essential nutrients available in small amounts in leafy vegetables and other foods. Folic acid is itself not biologically active, but it can be biologically converted to tetrahydrofolate and other tives after its conversion to dihydrofolic acid in the liver in the ce of appropriate enzymes.
In the form of a series of tetrahydrofolate (THF) compounds, folate derivatives are substrates in a number of single-carbon-transfer reactions and also are ed in the synthesis of dTMP (2'—deoxythymidine-5'-phosphate) from dUMP (2'—deoxyuridine-5'-phosphate).
] The pathway leading to the formation of methylfolate such as tetrahydrofolate (THF) begins when folic acid (F), as folate, is reduced to dihydrofolate (DHF), which is then reduced to tetrahydrofolate (THF). The enzyme dihydrofolate reductase catalyses the last step. Accordingly, in some embodiments, for a subject with depression who is deficient in the enzyme dihydrofolate reductase, methyl folate, also known as Me-THF, N5-Methyl-THF, MTHF, 5-MTHF, L-methylfolate, and Levomefolic acid, or a pharmaceutically acceptable salt thereof (e. g., sodium salt, potassium salt, ium salt, calcium salt, glucosamine salt, or galactosamine salt), is more desirable for use as a -containing compound. For example, methyl folate calcium salt is available by prescription in the United States as DEPLIN® (L-methylfolate calcium salt). Methyl folate calcium salt is also available outside of the United States as METAFOLIN®, BODYFOLIN®, and NUTRIFOLIN®.
Additional examples of folates or folate-containing nds that can be administered to a subject in the methods or ed in the compositions described herein can include, but not limited to, the ones described in the US. Pat. Nos.: US 185; US 6,921,754; and US 7,947,662; and US. Pat. App. No.: US 2008/0064702, the contents of which are incorporated herein by reference.
Biological effects of folate on the central nervous : The role of folate in central—nervous-system function has been previously discussed, including the essential role of folate in the one-carbon cycle that furnishes SAMe, the principal methyl donor for a broad range of reactions involving the sis of neuroactive substances, the formation of membrane phospholipids, and the metabolism of nucleic acids [10]. When administered in parenteral and certain oral forms, SAM has been reported in European studies to have antidepressant efficacy greater than placebo and comparable to that of tricyclic antidepressants [41]. Folate also appears to influence the rate of synthesis of tetrahydrobiopterin [21], a cofactor in the hydroxylation of phenylalanine and tryptophan, rate-limiting steps in the biosynthesis of dopamine, norepinephrine, and serotonin, neurotransmitters postulated to play a role in the pathogenesis of depression. In addition, MTHF has been shown to bind to aptic glutamate receptors [42], where it may potentially modulate the release of other neurotransmitters, including the monoamines. Elevated levels of steine, resulting from folate deficiency, can play a role in mediating some of its neuropsychiatric complications by both generating elevated levels of S-adenosyl-homocysteine, which broadly inhibits methylation ons [43], and also possibly ng direct excitoxic effects via activity at the N- methyl-aspartate glutamate receptors [44].
] Low cerebrospinal fluid levels of the serotonin metabolite 5-hydroxyindole acetic acid and the dopamine metabolite homovanillic acid have been detected, though not always [45], among folate-deficient patients with sy [46], other neuropsychiatric disorders [47], and congenital folate-deficiency states [48]. Whether folate supplementation actually enhances monoamine sis or release or furnishes additional SAMe has not yet been ished. Human studies examining the impact of supraphysiologic doses of folate on cerebrospinal-fluid metabolites are lacking. Indeed, a previous study in rats yielded the paradoxical finding that folate supplementation and folate deficiency d brain serotonin. [47], which can in turn indicate the ility of a similarly complex pattern in humans.
Folate and depression: Neuropsychiatric and depressive symptoms, including apathy, fatigue, insomnia, irritability, and impaired concentration, have been discussed in clinical descriptions of folate-deficiency states ated with orption,[13] nvulsant-treated sy, [14, 15] megaloblastic anemia, [16] and dietary folate restriction [1]. Previous s have reported that as many as one-third of patients among psychiatric cohorts, mainly from the United Kingdom, exhibited low or deficient serum folate values, with generally comparable findings in the few studies that have assessed red blood cell (RBC) folate as a more accurate reflection of tissue folate stores [12, 17, 18]. In the subset of the studies in which depressed patients were ed with psychiatric or non-psychiatric control subjects, depressed patients were reported to have serum folate [2,19], RBC folate [21], or serum methyltetrahydrofolate (MTHF) [22] levels that were lower than levels in all other groups except for patients with alcoholism who had a r prevalence of low folate [20]. Furthermore, low serum or RBC folate and serum MTHF were often associated with greater symptom severity among sed patients [22, 23, 24, 25]. Among studies that failed to demonstrate such relationship between low folate and depression severity, an inverse relationship between folate and the duration of the depressive episode [26] or the length of alization [27] was observed.
Folate levels and response to antidepressant treatment: The ors have previously assessed serum folate and response to the selective serotonin reuptake inhibitor antidepressant, fluoxetine, among 213 adults with major depression [28]. Among those depressed, but otherwise healthy, outpatients, the prevalence of actual folate deficiency (defined as <1.5 ng/mL) was low (2%), whereas borderline low values (1.5- 2.5 ng/mL) were more common (17%). Individuals with low or deficient serum folate ted a 35% rate of sponse to an adequate course of fluoxetine compared with a 20% rate of sponse among those with serum folate levels in the normal range. A similar result was reported among 22 depressed patients older than 60 years old treated with nortriptyline or sertraline for whom there was an inverse relationship between RBC folate and antidepressant response [29]. Consistent with this finding, in an earlier study of 101 sed inpatients receiving a variety of treatments, including electroconvulsive therapy, antidepressants, or tryptophan, outcome was significantly poorer for patients with low serum folate [24]. r, for a small number of patients undergoing electroconvulsive therapy, serum MTHF did not appear to ate with response [22]. Thus, accurate and sensitive predictors of refractoriness to antidepressant treatment have remained elusive [30].
Folate and MTHF supplementation/augmentation: Godfrey et al.[31] administered MTHF (15 mg), an oral form of folate actively transported across the brain barrier, to 24 patients with major depression and low or deficient folate (RBC folate <200 [Lg/L). In this h, randomized, double-blind trial, those 13 depressed patients who received methylfolate were rated globally as having superior symptom improvement and social adjustment at 3 and 6 months when ed with the 11 ts who were assigned to placebo. However, this report was inconclusive, based on the relatively small sample and need for replication, stematic concomitant treatments (e.g., antidepressants, lithium, or no medications), symptom improvement on some but not all measures, and similar, gh somewhat less dramatic, response to MTHF among comparably d patients with schizophrenia, indicating that the positive effects of MTHF may not have been specific to depression. The prior reports extended previous work on folate replacement, reporting improved long-term neuropsychiatric outcome among folate-deficient patients with psychiatric [32] and gastrointestinal [13] disorders and in some, although not all, studies on nvulsant-treated patients with epilepsy [14].
The actual clinical nce of these previous studies, however, is likely to be increasingly limited. Recent work has reported that the ence of folate deficiency or borderline low values is lower among contemporary psychiatric cohorts than originally believed [28] and also that Western estimates may not be comparable to other parts of the world including Asia [33].
Moreover, most of the studies on folate and depression are based on data gathered before implementation of folic-acid—fortification programs and the widespread public awareness about the 2012/065084 possible health benefits of folate. In the United States, the Food and Drug Administration mandate requiring folic-acid fortification of all enriched grain products by 1998 appears to have exerted a rapid and ntial impact on the prevalence of low and deficient folate in the community, nearly eradicating low serum-folate values (<3 ng/mL) among 350 middle-aged and older adults in the Framingham Offspring Study Cohort [34].
A comparable study is needed on the prevalence of low folate among carefully diagnosed depressed cohorts in this postfortification era, ularly because the extent to which dietary intake butes to low folate among depressed duals is not well established [2, 24, 25, 28, 35, 36]. heless, the prevalence of low folate among depressed sample subjects is believed to be reduced well below the estimates prior to implementation of the folic-acid fortification programs.
If so, the more compelling clinical focus for clinicians who treat sion shifts from folate replacement to folate supplementation and to the question of whether supraphysiologic doses of folate, as monotherapy or augmentation of conventional agents, may confer antidepressant benefit among depressed, normofolatemic patients.
Despite tremendous advances in the development of safe and effective antidepressants, as many as 30—40% of depressed patients continue to be symptomatic and functionally impaired despite an adequate course of antidepressant therapy [30]. Thus, the development of novel treatment strategies has considerable public-health significance.
Correlational analyses had reported that, among individuals with major mood disorders, higher folate values predicted better acute [25] and long-term outcome [37]. In the latter study, duals with unipolar-depression or bipolar-disorder folate levels were boosted with daily low-dose folate supplementation (200 ug) together with pharmacologic prophylaxis of affective symptoms. r demonstration of the ial antidepressant t of folate comes from a double-blind study of 96 non—folate-deficient patients with senile dementia and depressive symptoms in which significant improvement of sion was reported among patients ized to MTHF (50 mg), similar to that observed on the active antidepressant comparator, trazodone (100 mg/d) [38].
Among 16 elderly non-demented subjects with major depression, an open trial of MTHF (50 mg) also resulted in ntial improvement in depressive symptoms, even among the 14 subjects who had normal baseline serum folate [39].
] The inventors have also previously assessed the efficacy of methylfolate as an adjunctive treatment among adults with MDD and inadequate response to an SSRI [40]. -two adults (59% female; mean age 45.2 +/- 11.0 years) with DSM-IV MDD, partial or nonresponse to an SSRI after at least 4 weeks of ent, and a 17—item Hamilton Depression Rating Scale (HAM-D- 17) score greater than or equal to 12 were enrolled in this 8-week prospective open trial. Exclusion criteria included current use of anticonvulsants or psychotropics other than an SSRI, or B12 deficiency. Folinic acid was selected as an oral, synthetic, highly bioavailable form of folate that is metabolized to MTHF after absorption and, unlike MTHF, is available for prescription in the United States. Thus, folinic acid was added to SSRIs at 15-30 mg/day. Folate levels rose from 28 +/- 19 ng/mL to 301 +/- 203 ng/mL (p < 0.001). HAM-D-17 scores among the 16 completers decreased from 19.1 +/— 3.9 to 12.8 +/- 7.0 (p < 0.01). 31% of completers and 27% of the intent-to-treat (ITT) sample achieved response (with at least 50% reduction in 17 scores), and 19% of completers and 18% of the ITT sample achieved remission (HAM-D-17 no r than 7). In this report, folinic acid appeared to be only modestly effective as an adjunct in SSRI-refractory depressed individuals with normal folate levels, and surprisingly a significant portion of depressed individuals still did not respond to the folinic acid as an adjunct to an antidepressant. Thus, folate augmentation in treatment- resistant depression does not produce an effective response in all individuals [49], and in particular, as many as 29% to 46% of MDD patients show only partial or non-response to an te course of an antidepressant [50]. In particular, these reports did not identify in which patients the folate argumentation was effective or those ts where the antidepressant drug efficacy was not enhanced by folate. In fact, none of these reports identify biomarkers or a screen method to identify those subjects in where folate can e the efficacy of the pressant drug.
The assays and/or methods described herein can be used as a screen to identify and select for particular patients with depression, where a treatment regimen sing an antidepressant and a -containing compound will be beneficial to enhance the therapeutic effect of the antidepressant drug.
In accordance with the assays and/or methods described , subjects with sion who have been ined to have the presence of at least one of the ions described herein (e.g., SNPs and/or plasma/serum biomarkers described herein) can benefit from the eutic effect of an antidepressant administered in combination with an effective amount of a folate- containing compound. In some embodiments, the folate-containing compound can comprise L- methylfolate. In some ments, the folate-containing compound can comprise 6(S) methyltetrahydrofolate (also known as 6(S)MTHF) or DEPLIN®.
] The effective amount of folate for use in the treatment methods described herein can vary, depending upon the types and/or dosage of the antidepressant (if any), types of , severity of depression, physical conditions of a subject (e.g., ages, genders, weights). The term “effective amount” as used herein generally refers to an amount of folate or a folate-containing compound that, when administered to a selected subject, can reduce at least one symptom associated with depression as described later, e.g., by at least about 5%, at least about 10%, at least about 20%, at least about %, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the treatment in the absence of a folate-containing compound.
In some embodiments, the term “effective ” as used herein refers to an amount of folate or a folate-containing compound, when administered to a selected subject in combination with an antidepressant, can increase the effect (e.g., efficacy or therapeutic effect) of the antidepressant, e. g., by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the treatment with antidepressant alone. Stated another way, the term tive amount” as used herein refers to an amount of folate or a folate-containing compound, when administered to a ed subject in combination with an antidepressant, can reduce at least one symptom associated with depression as described later, e.g., by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, as compared to the treatment with antidepressant alone.
In some embodiments, the effective amount of folate in the treatment regimen as described herein can range from about 1 mg/day to about 70 mg/day, from about 1 mg/day to about 50 mg/day, from about 2.5 mg/day to about 40 mg/day, from about 5 mg/day to about 40 , from about 5 mg/day to about 30 mg/day or from about 7 mg/day to about 15 mg/day. In some embodiments, the effective amount of folate in the treatment regimen as described herein can range from about 7.5 mg/day to about 50 mg/day. In some embodiments, the effective amount of folate in the ent regimen as described herein can range from about 7.5 mg/day to about 40 mg/day. In some embodiments, the effective amount of folate in a ent regimen can be about 15 .
Antidepressants As used herein, unless otherwise noted, the term "antidepressant" or “anti-depressant” or “antidepressant drug” refers to any pharmaceutical agent which treats depression. In some embodiments, the anti-depressant drug administered to the subject in accordance with the methods described herein can be any conventional pharmaceutical agent which is commonly ted for treating depression. Examples of antidepressants or antidepressant drugs can include, but are not limited to, mine oxidase inhibitors such as phenelzine, tranylcypromine, and moclobemide; tricyclics such as imipramine, amitriptyline, desipramine, nortriptyline, doxepin, protriptyline, trimipramine, chlomipramine, and amoxapine; tetracyclics such as maprotiline; non-cyclics such as nsine; triazolopyridines such as trazodone; serotonin reuptake inhibitors such as fluoxetine, sertraline, paroxetine, citalopram, and fluvoxamine; serotonin receptor antagonists such as done; serotonin noradrenergic reuptake inhibitors such as venlafaxine, and milnacipran; noradrenergic and specific serotonergic agents such as mirtazapine; noradrenaline ke tors such as reboxetine. Additional antidepressants that can be used in the methods described herein can include, but are not limited to, bupropion; natural products such as Kava-Kava, and St. John's Wort; dietary supplements such as osylmethionine; neuropeptides such as thyrotropin-releasing hormone; compounds targeting neuropeptide receptors such as neurokinin receptor antagonists; and hormones such as triiodothyronine. 2012/065084 In some ments, the antidepressant or the antidepressant drug can be a serotonin reuptake inhibitor (SR1) or selective serotonin reuptake inhibitor (SSRI). Examples of SRIs and/or SSRIs include, without limitations, citalopram, escitalopram, fluoxetine, R-fluoxetine, sertraline, paroxetine, fluvoxamine, venlafaxine, duloxetine, dapoxetine, nefazodone, mine, mine N-oxide, desipramine, pirandamine, dazepinil, nefopam, befuraline, fezolamine, femoxetine, clomipramine, cianoimipramine, litoxetine, cericlamine, seproxetine, WY 27587, WY 27866, imeldine, ifoxetine, tiflucarbine, viqualine, milnacipran, bazinaprine, YM 922, S 33005, F 98214TA, OPC 14523, alaproclate, cyanodothepine, trimipramine, quinupramine, dothiepin, amoxapine, nitroxazepine, McN 5652, McN 5707, 01 77, Org 6582, Org 6997, Org 6906, amitriptyline, amitriptyline N-oxide, nortriptyline, CL 255. 663, pirlindole, indatraline, LY 113.821, LY 214.281, CGP 6085 A, RU 25.591, napamezole, diclofensine, trazodone, EMD 68.843, BMY 42.569, NS 2389, remine, nitroquipazine, ademethionine, sibutramine and clovoxamine. The SRIs can be used in the form of the base or a pharmaceutically acceptable acid addition salt thereof.
In some embodiments, other eutic compounds that can cause an elevation in the extracellular level of 5-HT in the synatic cleft, e.g., tianeptine, can be used as an antidepressant in the methods described herein.
In some embodiments, the pressant or the antidepressant drug can be a selective serotonin reuptake inhibitor (SSRI). The term “selective serotonin reuptake inhibitor (SSRI)” as used herein, refers to an inhibitor of the monoamine transporters, which has stronger inhibitory effect at the serotonin transporter than the dopamine and the noradrenaline transporters. Examples of selective serotonin reuptake inhibitors (SSRIs) can include, t limitations, fluoxetine, citalopram, tine, escitalopram, sertraline, and any combinations thereof. onal SRIs and/or SSRIs that can be administered to a t with depression in combination with a folate-containing compound can include, for example, the ones described in the US. Pat. App. No.: US 2005/0054688, and US 2008/0138411; and US. Pat. Nos.: 6,720,003; 6,787,560; 7,893,261; and 7148238.
One d in the art would be able to readily determine recommended dosage levels for known and/or marketed antidepressant drugs by consulting appropriate references such as drug package s, FDA guidelines, and the ian's Desk Reference. In some embodiments, the antidepressant drug dose can range from 0.1 mg/day to about 1000 mg/day, from about 0.5 mg/day to about 500 , from about 1 mg/day to about 400 mg/day, from about 5 mg/day to about 300 mg/day, or from about 10 mg/day to about 200 mg/day. One of skill in the art can readily adjust dosage for each different antidepressant drug, ing on a number of factors such as types and/or potency of antidepressants, severity of depression, physical condition of a t (e.g., ages, genders, and weights), administration routes, other medications taken by a subject, and any combinations thereof.
Pharmaceutical compositionsfor treatment of depression For in viva stration to subjects who have met at least one (e.g., at least two, at least three or more) of the conditions (A)-(X) determined in the assays described herein, provided herein are pharmaceutical itions comprising a eutically effective amount of a folate- containing compound, and a pharmaceutically acceptable carrier.
In various embodiments, the therapeutically effective amount of a folate-containing compound or folate, optionally administered with an antidepressant or a pharmaceutically salt thereof, is sufficient to se the degree of improvement in at least one neuropsychological test, e. g., as ed by HAMD-17, HAMD-28 or other efficacy measures as described in the Examples, by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, as ed to the degree of improvement obtained in the absence of the folate-containing compound (e. g., with or without the antidepressant monotherapy). In some embodiments, the therapeutically effective amount of a folate-containing compound or folate, optionally administered with an antidepressant or a pharmaceutically salt thereof, is sufficient to increase the degree of improvement in at least one neuropsychological test, e.g., as measured by HAMD-17, 8 or other cy es as described in the es, by at least about 1-fold, at least about 2-fold, at least about 3- fold, at least about 4-fold, at least about 5-fold or more, as compared to the degree of improvement ed in the absence of the folate-containing nd (e.g., with or without the antidepressant monotherapy).
In some embodiments, a dose of a folate-containing nd or folate for administration to a human can be in the range of about 0.01 to about 50 mg per am body weight of the recipient per day, in the range of about 0.05 to about 5 mg per kilogram body weight per day, or in the range of about 0.1 to about 1 mg per kilogram body weight per day. In certain embodiments, the desired dose can be presented as one single unit dosage form, e. g., containing about 0.5 mg to about 500 mg, about 5 mg to about 250 mg, about 10 mg to about 100 mg, or about 10 mg to about 50 mg. In some embodiments, one single unit dosage form can provide about 1 mg to about 70 mg folate, about 5 mg to about 60 mg folate, or from about 7 mg to about 50 mg folate. In some embodiments, one single unit dosage form can provide about 7.5 mg to about 50 mg folate. In other embodiments, the desired dose can be presented in two, three, four, five or more sub-doses administered at appropriate intervals throughout the day. These sub-doses can be administered in unit dosage forms, for example, containing about 0.1 mg to about 250 mg, about 1 mg to about 100 mg, about 2 mg to about 20 mg, or about 2 mg to about 10 mg.
In some embodiments, the pharmaceutical composition can further comprise at least one antidepressant. In general, a dose of an antidepressant or a pharmaceutically acceptable salt thereof suitable for administration to a human is in the range of about 0.01 to 50 mg per kilogram body weight of the recipient per day, or in the range of 0.1 to 5 mg per kilogram body weight per day..
In certain embodiments, the desired dose can be presented as one single unit dosage form, e.g., containing about 1 mg to about 500 mg, or about 5 mg to about 300 mg. In other embodiments, the desired dose can be presented in two, three, four, five or more sub-doses administered at appropriate intervals throughout the day. These sub-doses can be administered in unit dosage forms, for example, containing about 0.1 mg to about 100 mg or about 1 mg to about 50 mg.
As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, le for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, surate with a reasonable t/risk ratio.
As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, ent, manufacturing aid (e. g., lubricant, talc magnesium, calcium or Zinc te, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (i) sugars, such as lactose, glucose and sucrose; (ii) starches, such as corn starch and potato starch; (iii) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (iv) powdered tragacanth; (v) malt; (vi) gelatin; (vii) lubricating agents, such as magnesium te, sodium lauryl sulfate and talc; (viii) excipients, such as cocoa butter and suppository waxes; (ix) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and n oil; (x) glycols, such as propylene glycol; (xi) polyols, such as in, sorbitol, mannitol and polyethylene glycol (PEG); (xii) esters, such as ethyl oleate and ethyl laurate; (xiii) agar; (xiv) ing agents, such as magnesium hydroxide and aluminum hydroxide; (xv) alginic acid; (xvi) pyrogen-free water; (xvii) ic saline; (xviii) Ringer's solution; (xix) ethyl alcohol; (xx) pH buffered solutions; (xxi) polyesters, rbonates and/or polyanhydrides; (xxii) bulking agents, such as ptides and amino acids (xxiii) serum component, such as serum albumin, HDL and LDL; (xxiv) C2-C12 alchols, such as ethanol; and (xxv) other non-toxic compatible substances employed in pharmaceutical ations. Wetting agents, coloring agents, e agents, coating agents, ning agents, flavoring agents, ing agents, preservative and antioxidants can also be present in the formulation.
Pharmaceutically acceptable carriers can vary in a composition described herein, ing on the administration route and formulation. For example, the pharmaceutically able composition described herein can be delivered via injection. These routes for administration (delivery) include, but are not limited to, subcutaneous or parenteral including intravenous, intraarterial, intramuscular, intraperitoneal, yocardial, and infusion techniques. In one embodiment, the pharmaceutical acceptable composition is in a form that is suitable for injection. In another ment, the pharmaceutical composition is formulated for ry by a catheter.
When administering a ceutical composition parenterally, it can be generally formulated in a unit dosage injectable form (solution, suspension, emulsion). The pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions. The carrier can be a solvent or dispersing medium containing, for example, water, cell culture medium, buffers (e.g., phosphate buffered ), polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof. In some embodiments, the pharmaceutical carrier can be a buffered solution (e.g. PB S).
In some embodiments, the pharmaceutical composition can be formulated in an emulsion or a gel.
In some embodiments, the pharmaceutical compositions described herein can be formulated for oral administration or for inhalation. For oral administration, suitable dosage forms can include tablets, troches, cachets, caplets, and capsules, including hard and soft gelatin capsules.
In some embodiments, both an antidepressant and a folate-containing compound can be formulated in a single pharmaceutical composition. For example, both an antidepressant and a folate-containing compound can be formulated in a single tablet for oral administration.
In some ments where the antidepressant and folate-containing nd are ated in a single composition, they can be released from the composition at the same time or at different times. By way of example only, if the -containing compound is formulated in an outer layer of a composition (e.g., a tablet or drug-delivery particle) while the pressant is formulated in an inner layer of the composition, the folate-containing compound could be ed from the composition first with a faster rate, while the antidepressant could be released from the composition later with a slower rate. On the other hand, if the antidepressant and the folate-containing compound are mixed nously within the composition, both can be released simultaneously from the composition.
In other embodiments, an antidepressant and a folate-containing compound can be formulated in separate pharmaceutical compositions for the same or different routes of administration during a therapy . For example, an pressant can be formulated for inhalation administration while a folate-containing compound can be formulated for oral administration. In other embodiments, both the antidepressant and folate-containing nd can be formulated for oral stration, e.g., in separate tablets.
The effective amount of folate administered to a selected human subject for treatment of depression as described herein is icantly higher than the typical amount taken as a y supplement (between 50-600 ug/day). In some embodiments, the effective amount of folate administered to a selected human t is at least about 2-fold, at least about 5-fold, at least about -fold, at least about 25-fold, at least about 50-fold, at least about ld, at least about 250-fold, at least about 500-fold, at least about 1000-fold or more than the typical amount taken as a dietary supplement. Accordingly, in some embodiments, the folate-containing compound is desirable to be formulated in slow-release or sustained release composition.
Accordingly, in some embodiments, the pharmaceutical compositions comprising a folate-containing compound (with or without an anti-depressant) can be formulated for sustained release or ned ry. In some embodiments, the pharmaceutical compositions can be formulated in controlled-release drug-delivery systems, e. g., to provide sustained release of a - containing compound (and optionally an anti-depressant). As used herein, the term "sustained release” or “sustained delivery" refers to continual delivery of a therapeutic agent in vivo over a period of time following administration. For example, sustained release can occur over a period of at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours, at least about 16 hours, at least about 24 hours following administration. In some embodiments, sustained release can occur over a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days following stration. In some embodiments, the release of a folate-containing compound from a drug-delivery system can be steady state (zero-order kinetics) with at least about 30% (e. g., ing at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more) of the folate-containing compound (and optionally an anti-depressant) released between about 3-6 hours post administration, or between about 4-5 hours post administration. In one embodiment, the release of a folate-containing compound (and ally an anti-depressant) from a drug-delivery system can be steady state (zero-order kinetics) with substantially full release (e.g., ~100%) of the folate-containing compound ed between about 3-6 hours post administration, or between about 4-5 hours post administration. In some embodiments, the folate-containing compound can be released from a drug-delivery system at a rate that is slow enough not to overload the intestinal absorption capacity of a patient’s duodenum (first 1/3 of the small ines where ~90% of the tion occurs for a folate-containing compound, e.g., L-MTHF). In some embodiments, the folate-containing compound and an epressant (if any) can be released from a drug-delivery system rently or separately, with the same or a different release rate.
Any drug delivery system (e.g., but not limited to polymer-based) that es a sustained release of a folate-containing nd (and optionally an anti-depressant) over a pre- determined period of time can be used for administration of a folate-containing compound (and optionally an anti-depressant). In one embodiment, the drug-delivery system can be a caplet design large enough to be blocked by a pyloric valve between the stomach and the duodenum, thus allowing the caplet to slowly and lly dissolve over a desirable period of time, e. g., over a period of about 2-3 hours, during which the folate-containing compound is steadily released from the caplet. As the caplet dissolves to a size that can get through the pyloric valve at which time it completes its steady state release (e.g., an additional period of time, e.g., an additional 2 hours), the caplet can continue to travel into the jejunum (the second third of the small intestines) where tion is minimal.
In some ments, a drug delivery system can use a blend of hydrophilic and hobic polymers to l release of a folate-containing compound (and optionally an anti- depressant) via diffusion through, and erosion of, a polymer matrix.
In some ments, a drug delivery system can comprise a folate-containing compound (and ally an anti-depressant) encapsulated in polymer-based particles. These folatecontaining r-based particles can be filled into capsules or single-dose sachets for additional control of release.
] Controlled-release (e.g., sustained release) drug ry systems for different administration methods (e.g., oral administration, injection, implantation, inhalation) are known in the art and can be adopted to deliver a folate-containing compound (and optionally an antidepressant) for the treatment methods described herein. See, e.g., International Pat. App. Nos. WO 2012/ l l 1961 (oral formulation), (injectable formulation); US. Pat. App. Nos. US 2012/0258161 (implantable formulation), US 2001/0038854, US 2001/0033866; and US. Pat. No. 8268347 (inhalation formulation), the t of which are incorporated herein by reference, for various types of drug-delivery systems to deliver an active agent via various administration routes.
Additionally, various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, ing agents, and buffers, can be added. tion of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it may be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like.
The compositions can also contain auxiliary substances such as g or emulsifying agents, pH buffering , g or viscosity enhancing additives, preservatives, colors, binders, and the like, depending upon the route of administration and the preparation desired.
Standard texts, such as “REMINGTON‘S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. With respect to compositions described herein, r, any vehicle, diluent, or additive used should have to be biocompatible with the antidepressant or a pharmaceutically acceptable salt f and/or a -containing compound.
The pharmaceutical compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the composition described herein can be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other nic or organic solutes. In one embodiment, sodium chloride is used in buffers containing sodium ions.
] Viscosity of the compositions can be maintained at the selected level using a pharmaceutically acceptable thickening agent. In one embodiment, methylcellulose is used because it is y and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The preferred concentration of the thickener will depend upon the agent selected. The important point is to use an amount which will achieve the selected viscosity. Viscous itions are normally prepared from solutions by the addition of such thickening agents.
Typically, any additives (in addition to the pressant and/or folate-containing compound) can be present in an amount of 0.001 to 50 wt % solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to rams to grams, such as about 0.0001 to about 5 wt %, about 0.0001 to about 1 wt %, about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt %, about 0.01 to about 10 wt %, and about 0.05 to about 5 wt %. For any therapeutic ition to be administered to a subject with ssion, and for any particular method of stration, it is preferred to ine toxicity, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e. g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan.
Those skilled in the art will recognize that the components of the compositions should be selected to be biocompatible with respect to the active agent, e. g., the antidepressant and/or folate- containing compound. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily d by reference to standard texts or by simple experiments (not involving undue experimentation).
The compositions described herein can be prepared by mixing the ingredients following generally-accepted procedures. For example, the ingredients can be mixed in an appropriate pharmaceutically acceptable carrier and the mixture can be ed to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to l tonicity. lly the pH can vary from about 3 to about 7.5. In some embodiments, the pH of the composition can be about 6.5 to about 7.5. Compositions can be administered in dosages and by techniques well known to those skilled in the medical and veterinary arts taking into consideration such factors as the age, sex, weight, and condition of the particular t, and the composition form used for administration (e.g., liquid). Dosages for humans or other mammals can be determined without undue experimentation by a skilled n.
] A still further aspect provided herein relates to uses of a folate-comprising composition in the treatment of depression in a human subject who carries at least one of the conditions (A)-(X) described herein (e.g., but not limited to, either one or both conditions (A) and (C)). Another aspect provided herein relates to a folate-comprising composition in combination with an anti-depressant for use in the treatment of depression in a human subject who carries at least one of the ions (A)-(X) described herein (e. g., but not limited to, either one or both conditions (A) and (C)). In some embodiments of these s described herein, the folate-comprising composition can comprise at least about 5 mg of folate (e. g., about 7.5 mg to about 50 mg of folate). In some embodiments, the folate-comprising composition can be formulated for a termined release profile (e.g., a sustained release). In some embodiments, the human subject is an adult.
Selection of subjects with depression In some embodiments, ts amenable to assays, methods and compositions described herein can be subjects that have been diagnosed with or suspected of having or developing depression. Accordingly, in some ments, subjects that have been diagnosed or suspected of having or developing with depression are selected prior to subjecting them to the assays, methods and/or itions bed herein. In some embodiments, a subject is selected for a treatment regimen comprising a folate-containing nd is being treated for depression. In some embodiments, the subject is specifically administered with a folate-containing nd to enhance (or as an adjuvant) the effect of the antidepressant drug, and not for r . For example, a female with depression wishing to become pregnant, or who is pregnant, or is lactating may take prenatal supplements containing folic acid in combination with an antidepressant drug. In such instance, a human subject amenable to the assays, methods and/or compositions described herein is ically selected for depression before performing the assays and/or methods described herein and/or administering the itions described herein.
In some embodiments, the subject is specifically administered with a - containing nd for treatment of depression, and not for another reason. For example, a human t diagnosed as having, or have a risk for, depression may take an effective amount of a folate- containing compound (with or without an anti-depressant). In such instance, a human subject amenable to the assays, methods, and/or compositions described herein is specifically selected for depression before performing the assays and/or methods bed herein and/or administering the compositions bed herein.
The phrase “having a risk for depression” or "suspected of having or developing depression" or “suspected of having or developing major depressive disorder” refers to a subject that presents one or more symptoms indicative of a depression including major depressive disorder (e.g., ained insomnia, fatigue, irritability, etc.) or is being screened for depression including major depressive disorder (e. g., during a routine physical), for example, in accordance with the criteria listed in DSM-IV or ICD-lO as discussed below.
As used , the term "depression" generally refers to a mental state of depressed mood characterized by feelings of sadness, despair and discouragement. In some embodiments, depression is a al m, and can include, but not d to, major depressive disorder (including single episode and recurrent), ar depression, treatment-refractory depression, resistant depression, anxious depression and dysthymia (also referred to as dysthymic er).
Further, the term "depression" can encompass any major depressive disorder, dysthymic er, mood disorders due to medical conditions with depressive features, mood ers due to medical conditions with major depressive-like es, substance-induced mood ers with depressive features and depressive disorder not otherwise specific as defined by their diagnostic criteria, as listed in the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) or any later edition thereof, or the World Health Organization's International Statistical Classification of Diseases and Related Health Problems (ICD-lO). In one embodiment, depression is major depressive disorder.
The DSM-IV and ICD-lO provides a common language and standard criteria for the classification of mental disorders, and have been ly used by a suitably trained general practitioner, or by a psychiatrist or psychologist for diagnosis of depression including major depressive disorders. Symptoms of depression can include, but are not limited to, problems concentrating, remembering, and/or making decisions, changes in eating and/or sleeping , a loss of interest in ble activities, difficulty going to work or taking care of daily responsibilities, feelings of guilt and/or hopelessness, slowed thoughts and/or speech, and preoccupation with thoughts of death or suicide. One of skill in the art can determine the score or rating of depression based on DSM-IV or ICD-lO.
Other scales or criteria for classification of mental disorders known in the art, e. g., Maier or HAMD-7 scale, or social functioning questionnaire (SFQ), visual analogue scale (VAS), and/or cognitive and physical function questionnaire (CPFQ) can also be used to determine the degree of depression.
During diagnosis for depression, the practitioner can also assess the patient’s medical history, discuss the subject’s current ways of regulating their mood (healthy or otherwise) such as l and drug use, and/or perform a mental state examination, which is an assessment of the person's t mood and thought content, in particular the presence of themes of hopelessness or pessimism, self-harm or suicide, and an absence of positive thoughts or plans. Additionally, a practitioner can generally perform a medical examination to rule out other non-cognitive causes of depressive symptoms. For e, blood tests measuring TSH and thyroxine can be used to exclude hypothyroidism; basic electrolytes and serum calcium to rule out a metabolic disturbance; and a full blood count including ESR to rule out a systemic infection or chronic disease. Testosterone levels can also be evaluated to se hypogonadism, a cause of depression in men.
Any c or biomarker methods known in the art can also be used for diagnosis of depression. For example, US. Pat. App. No. US 273153 describes that the presence of TG7AT haplotype can be indicative of predisposition to major depressive disorder. Additional marker gene for depression such as ATP2A2, SCYAS, STIPl, , GRBlO, CASP6, TSSCl, RAB9, NFATC3, TPR, and any others listed in, for example, US. Pat. App. No. US 2005/0239110 can also be used for diagnosing depression.
] In some embodiments, subjects amenable to assays, methods and compositions bed herein are subjects that have been diagnosed with or suspected of having or developing major depressive disorder. A major depressive episode is characterized by the ce of a severely depressed mood that persists for at least two weeks. Episodes can be ed or recurrent and can be categorized by a skilled practitioner as mild (few symptoms in excess of minimum criteria), moderate, or severe (marked impact on social or occupational functioning).
In some embodiments, subjects amenable to assays, methods and compositions described herein are subjects that have been diagnosed with major depressive disorder (MDD) and are resistant to antidepressant monotherapy, i.e., a treatment for depression with a single antidepressant only. The phrase “resistant to pressant monotherapy” is used herein in nce to a subject with depression being resistant to at least one antidepressant in one or more classes. This includes subjects with depression that are resistant to at least two, at least three, at least four or more antidepressants in one or more classes. In some embodiments, subjects amenable to assays, methods and compositions described herein are subjects that have been diagnosed with major depressive disorder (MDD) and are resistant to at least one serotonin reuptake tors (SRI), including at least one, at least two, at least three, at least four or more SRIs. In some embodiments, subjects amenable to assays, methods and compositions described herein are subjects that have been diagnosed with major depressive disorder (MDD) and are resistant to at least one ive serotonin reuptake inhibitor (SSRI), including at least two, at least three, at least four or SSRIs.
In some embodiments, the t selected for the , methods and compositions described herein have been in remission from depression and is now diagnosed with a relapse or a predisposition to a relapse. In other embodiments, the t ed for the assays, methods and compositions bed herein have been diagnosed with depression and is currently taking at least an antidepressant.
As used herein, a “subject” can mean a human or an . Examples of subjects include primates (e.g., humans, and monkeys). Generally, the animal is a vertebrate such as a primate, rodent, domestic animal or game . Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. A patient or a subject includes any subset of the foregoing, e.g., all of the above, or includes one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient9, cs , dual”, and “subject” are used interchangeably herein. A subject can be male or female. In some embodiments, the subjects le to the assays, methods and/or compositions described herein can be female subjects.
In certain embodiments, the subject is a human subject. Human subjects can come from any ethnicity or race, e.g., including, but not limited to, Caucasian, African American, Asian and Hispanic, n Indian and Alaska Native, Native Hawaiian and other Pacific Islander. In some embodiments, the human subjects amenable to the assays, s and/or itions described herein can be Caucasian subjects. As used herein, the term "Caucasian" refers to a member of the white race consisting of individuals of European, north African, or southwest Asian ry. In some embodiments, the human subjects amenable to the assays, methods and/or compositions described herein can be African an subjects. In some embodiments, the human subjects le to the assays, methods and/or compositions described herein can be Asian subjects. In some embodiments, the human subjects amenable to the assays, methods and/or compositions described herein can be Hispanic subjects.
In some embodiments, the human subjects amenable to the assays, methods and compositions described herein can be of any age. In some ments, the human ts amenable to the assays, methods and/or compositions described can be at an age of at least 18 years old. In other embodiments, human ts below 18 years can also be subjected to the assays, methods and/or compositions described .
Systems and computer readable mediafor use in the assays and/0r methods described herein Embodiments of a further aspect also provide for systems (and computer readable media for causing er systems) to perform an assay for selecting a treatment regimen for a subject with depression based on at least sequence information of the SNP biomarkers (i)-(xxi) described herein and/or expression levels of the peripheral biomarkers (xxii)-(xxiv).
A er system for obtaining data from at least one test sample obtained from at least one subject is ed. The system comprises: (a) a determination module configured to receive at least one test sample and perform at least one is on at least one test sample to determine parameters of at least two biomarkers (i) to (xxiv) described herein; (b) a storage device configured to store output data from the determination module; (c) a computing module, e.g., a non- human machine, sing specifically-programmed instructions to determine from the output data the presence of at least one condition (A) to (X) described herein; and (d) a display module for displaying a content based in part on the data output from the computing module, wherein the t comprises a signal indicative of the presence of at least one condition (A) to (X) described herein, and optionally the absence of any one of the conditions (A) to (X) described herein, or a signal indicative of the absence of all of the conditions (A) to (X) described herein. 2012/065084 In some embodiments, the ination module can be configured to m at least one genotyping analysis on at least one test sample to determine the genotypes of at least two loci sing position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) and position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9). In these embodiments, the computing module can be configured to determine the presence of at least one SNP located at position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) comprising at least one thymine “T” allele, and/or at position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9) comprising at least one guanine “G” allele.
In another embodiment, the determination module can be configured to perform at least one analysis on at least one test sample to determine the presence or absence of at least one of the following conditions: i. an expression ratio of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) smaller than a pre-determined reference ratio; ii. expression of 4-hydroxynonenal (4-HNE) greater than a pre-determined reference value; iii. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO: 1 comprising at least one thymine “T” allele, n the SEQ ID NO: 1 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iv. a SNP at position 2756 of SEQ ID NO: 2 comprising at least one guanine “G” allele, wherein the SEQ ID NO: 2 is a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and V. a SNP at position 66 of SEQ ID NO: 3 comprising at least one guanine “G” allele, wherein the SEQ ID NO: 3 is a portion of a genomic nucleic acid sequence of methionine se reductase (MTRR).
] In these embodiments, the determination module can be r configured to determine the presence or absence of at least one other condition (A)-(X) described herein. For example, in some embodiments, the determination module can be further configured to determine sion of high-sensitivity c-reactive protein (hsCRP). In some embodiments, the determination module can be further configured to determine the presence or e of a SNP at position 1298 of the SEQ ID NO. 1 comprising at least one cytosine "C" allele.
In some embodiments, the determination module of the computer system can be ured to analyze at least one test sample to ine the presence or absence of at least two of the conditions (i) — (V) bed above and expression of hsCRP.
In some embodiments, the determination module of the computer system can further comprise a ison module adapted to compare the data output from the determination module with reference data stored on the storage device. In some embodiments, the reference data can include, but not limited to, major and/or rare ts of alleles corresponding to the SNPs described herein, a pre-determined reference value of 4-HNE (e.g., at least 3.2 mg/L or above as ed in a plasma sample), a termined reference value of hsCRP (e.g., greater than 2.3 mg/L as measured in a plasma sample), a pre-determined reference ratio of SAM/SAH (e. g., less than 2.8 as measured in a plasma sample), and any combinations thereof.
Embodiments of the computer system described herein also comprises a storage device configured to store data output from the determination module; and a display module for displaying a content based in part on the data output from the ination module, wherein the content comprises a signal indicative of the presence of at least one of the conditions (i)-(v) described above, or a signal indicative of the absence of at least one of these conditions. In some embodiments, the content displayed on the display module can further comprise a signal indicative of the subject recommended to receive a treatment regimen comprising a folate-containing compound, or a signal tive of the subject ended to receive an alternative treatment regimen.
] In some embodiments, the storage device of the computer system can be further configured to store physical information of at least one subject to be . Examples of the physical information can include obesity tor (e.g., BMI) and/or gender of at least one tested subject. In such ments, the content yed on the display module of the er system can further comprise the obsesity indicator (e.g., the BMI value) or a signal indicative of whether the human subject is obese or not (e. g., whether the BMI value is at least 30 kg/m2 or not).
A le and non-transitory (e.g., no tory forms of signal transmission) computer readable medium having computer readable instructions recorded thereon to define software modules for implementing a method on a computer is also provided herein. In one embodiment, the computer readable storage medium comprises: (a) instructions for comparing the data stored on a storage device with reference data to provide a comparison result, wherein the comparison identifies the presence or absence of at least one condition (A)-(X) described herein; and (b) instructions for displaying a content based in part on the data output from the determination module, wherein the content comprises a signal indicative of the presence of at least one of the conditions (A)-(X) described herein, and optionally the absence of any one of these conditions (A)-(X) bed herein.
In other embodiments, the content can se a signal tive of the absence of all of the conditions (A)-(X) bed herein.
In some embodiments, the instructions can be specifically programmed to perform a comparison to identify the presence of at least one SNP located at position 677 of SEQ ID NO. 1 (or position 27 of SEQ ID NO. 7) comprising at least one thymine “T” allele, and/or at position 2756 of SEQ ID NO. 2 (or position 27 of SEQ ID NO. 9) sing at least one guanine “G” allele.
In other embodiments, the instructions can be specifically mmed to perform a comparison to identify one of the following conditions: i. an expression ratio of s-adenosyl nine (SAM) to s-adenosyl steine (SAH) smaller than a pre-determined reference ratio; ii. expression of 4-hydroxynonenal (4 HNE) greater than a pre-determined reference value; iii. a single nucleotide polymorphism (SNP) at on 677 of SEQ ID NO: 1 comprising at least one thymine “T” , wherein the SEQ ID NO: 1 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iv. a SNP at position 2756 of SEQ ID NO: 2 comprising at least one guanine “G” allele, wherein the SEQ ID NO: 2 is a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and V. a SNP at position 66 of SEQ ID NO: 3 comprising at least one guanine “G” allele, wherein the SEQ ID NO: 3 is a portion of a genomic nucleic acid sequence of methionine se reductase (MTRR); In these embodiments, the computer readable medium can further comprise instructions to identify the presence or absence of at least one other condition (A)-(X) described herein. For example, in one embodiment, the computer readable medium can further se instructions to fy the presence or absence of a SNP at position 1298 of the SEQ ID NO: 1 comprising at least one ne “C” allele. In some embodiments, the er readable medium can further comprise instructions to compare expression of high-sensitivity c-reactive protein (hsCRP) with the reference data, e.g., pre-determined reference value of hsCRP expression level r than 2.3 mg/L as ed in a plasma sample.
Embodiments of the display module of the computer readable medium described herein also comprise instructions for ying a content based in part on the data output from the comparison module, wherein the content comprises a signal indicative of the presence of at least one of the conditions, or a signal indicative of the absence of at least one of the ions. In some embodiments, the display module can further comprises instructions to display a signal indicative of the subject recommended to receive a treatment regimen comprising a folate-containing compound, or a signal tive of the subject recommended to receive an alternative treatment regimen.
In some embodiments, the comparison module of the computer readable medium can further comprise instructions to determine if a human t is obese (e.g., if BMI of at least one subject is at least 30 kg/m2 or not). In such embodiments, the display module can further comprise instructions to display the obesity tor (e. g., the BMI value) or a signal indicative of whether the subject is obese (e. g., whether the BMI value is at least 30 kg/m2 or not). ments of the s described herein have been described through functional s, which are defined by computer executable instructions ed on computer readable media and which cause a er to perform method steps when executed. The modules have been segregated by function for the sake of clarity. However, it should be understood that the modules need not correspond to discrete blocks of code and the described functions can be carried out by the execution of various code portions stored on various media and executed at various times.
Furthermore, it should be appreciated that the modules may perform other functions, thus the modules are not limited to having any particular functions or set of functions.
The er readable media can be any available tangible media that can be accessed by a computer. Computer readable media includes volatile and nonvolatile, removable and non-removable tangible media ented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer readable media includes, but is not limited to, RAM m access memory), ROM (read only memory), EPROM (eraseable programmable read only memory), EEPROM (electrically eraseable programmable read only memory), flash memory or other memory technology, CD-ROM (compact disc read only memory), DVDs al versatile disks) or other optical storage media, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage media, other types of volatile and non-volatile memory, and any other tangible medium which can be used to store the desired information and which can accessed by a computer ing and any suitable combination of the foregoing.
In some embodiments, the computer readable storage media 700 can e the “cloud” , in which a user can store data on a remote server, and later access the data or perform further analysis of the data from the remote server.
Computer-readable data embodied on one or more computer-readable media, or computer readable medium 700, may define instructions, for example, as part of one or more programs, that, as a result of being executed by a er, ct the computer to perform one or more of the functions described herein (e.g., in relation to system 600, or computer readable medium 700), and/or various embodiments, variations and combinations thereof. Such instructions may be written in any of a plurality of programming languages, for example, Java, J#, Visual Basic, C, C#, C++, Fortran, Pascal, Eiffel, Basic, COBOL assembly language, and the like, or any of a y of combinations thereof. The computer-readable media on which such instructions are embodied may reside on one or more of the components of either of system 600, or computer readable medium 700 described herein, may be distributed across one or more of such components, and may be in transition there between.
The computer-readable media can be transportable such that the instructions stored thereon can be loaded onto any computer resource to implement the assays and/or methods described herein. In addition, it should be appreciated that the instructions stored on the computer readable media, or computer-readable medium 200, described above, are not limited to instructions embodied as part of an application program running on a host computer. Rather, the ctions may be embodied as any type of computer code (e.g., software or microcode) that can be employed to m a computer to implement the assays and/or methods described herein. The computer able instructions may be written in a suitable er language or combination of several languages. Basic computational y s are known to those of ordinary skill in the art and are described in, for example, Setubal and is et al., Introduction to Computational Biology Methods (PWS Publishing Company, Boston, 1997); Salzberg, s, Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics : Application in Biological Science and Medicine (CRC Press, London, 2000) and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysis of Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001).
The functional modules of certain embodiments of the system described herein can include a determination module, a storage device, and a display module. In some embodiments, the system can further include a comparison module. The functional s can be executed on one, or multiple, computers, or by using one, or le, computer networks. The determination module 602 has computer executable instructions to e sequence information in computer readable form. As used herein, "sequence information" refers to any nucleotide and/or amino acid sequence, including but not limited to full-length nucleotide and/or amino acid ces, partial nucleotide and/or amino acid sequences, or mutated sequences. Moreover, information "related to" the sequence information includes detection of the presence or absence of a ce (e.g., detection of a mutation or deletion), determination of the concentration of a sequence in the sample (e.g., amino acid ce expression levels, or nucleotide (RNA or DNA) expression levels), and the like. The term “sequence information” is ed to include the presence or absence of post-translational modifications (e.g. orylation, glycosylation, summylation, farnesylation, and the like).
As an example, determination modules 602 for ining sequence ation may include known s for ted sequence analysis including but not limited to i FMBIO® and Hitachi FMBIO® II Fluorescent Scanners (available from Hitachi Genetic Systems, Alameda, California); Spectrumedix® SCE 9610 Fully Automated 96-Capillary Electrophoresis c Analysis Systems (available from uMedix LLC, State e, Pennsylvania); ABI PRISM® 377 DNA Sequencer, ABI® 373 DNA Sequencer, ABI PRISM® 310 Genetic Analyzer, ABI PRISM® 3100 Genetic Analyzer, and ABI PRISM® 3700 DNA Analyzer (available from Applied Biosystems, Foster City, California); Molecular Dynamics FluorImagerTM 575, SI Fluorescent Scanners, and Molecular Dynamics FluorImagerTM 595 Fluorescent Scanners (available from Amersham ences UK Limited, Little Chalfont, Buckinghamshire, England); GenomnyCTM DNA Sequencing System (available from Genomyx Corporation r City, California); and Pharmacia ALFTM DNA Sequencer and Pharmacia ALFexpressTM (available from Amersham Biosciences UK Limited, Little Chalfont, Buckinghamshire, England).
Alternative methods for determining sequence ation, i.e. determination modules 602, include systems for protein and DNA analysis. For example, mass spectrometry systems including Matrix Assisted Laser Desorption Ionization — Time of Flight (MALDI-TOF) systems and TOF-MS ProteinChip array profiling systems; systems for analyzing gene expression data (see, for example, published US. Patent Applicaion, Pub. No. US. 2003/0194711); systems for array based expression analysis: e.g., HT array systems and cartridge array systems such as GeneChip® AutoLoader, Complete GeneChip® Instrument System, ip® Fluidics Station 450, GeneChip® Hybridization Oven 645, GeneChip® QC Toolbox Software Kit , GeneChip® Scanner 3000 7G plus Targeted Genotyping System, GeneChip® Scanner 3000 7G Whole-Genome ation System, GeneTitanTM Instrument and GeneChip® Array Station (each available from Affymetrix, Santa Clara, California); automated ELISA systems (e.g., DSX® or DS2® able from Dynax, Chantilly, VA) or the Triturus® (available from Grifols USA, Los Angeles, California), The Mago® Plus (available from Diamedix Corporation, Miami, Florida) ; Densitometers (e. g. X-Rite Spectro Densitometer® (available from RP ImagingTM, Tucson, Arizona), The HYRYSTM 2 HIT densitometer (available from Sebia Electrophoresis, Norcross, Georgia); automated Fluorescence insitu hybridization s (see for e, United States Patent 6,136,540); 2D gel imaging s coupled with 2-D imaging re; microplate s; Fluorescence activated cell sorters (FACS) (e.g. Flow Cytometer FACSVantage SE, (available from Becton Dickinson, Franklin Lakes, New Jersey); and radio isotope analyzers (e.g. scintillation counters).
The sequence information of SNPs and/or expression level information of /serum biomarkers determined in the determination module can be read by the storage device 604. As used herein the “storage device” 604 is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the system described herein can include stand-alone computing apparatus, data telecommunications networks, including local area networks (LAN), wide area networks (WAN), Internet, et, and et, and local and distributed computer processing systems. Storage devices 604 also include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage media, magnetic tape, optical storage media such as CD-ROM, DVD, electronic storage media such as RAM, ROM, EPROM, EEPROM and the like, l hard disks and hybrids of these categories such as ic/optical storage media. The storage device 604 is adapted or configured for having recorded thereon ce information or expression level information. Such information may be provided in digital form that can be itted and read electronically, e. g., via the Internet, on diskette, via USB (universal serial bus) or via any other suitable mode of communication, e. g., the “cloud”.
As used herein, "expression level information" refers to any nucleotide and/or amino acid expression level information, including but not limited to ength nucleotide and/or amino acid sequences, partial tide and/or amino acid sequences, or mutated sequences. Moreover, information "related to" the expression level information includes detection of the ce or absence of a sequence (e. g., presence or e of an amino acid sequence, nucleotide sequence, or post translational modification), determination of the concentration of a sequence in the sample (e. g., amino acid sequence levels, or nucleotide (RNA or DNA) expression , or level of post translational modification), and the like. In some embodiments, the expression level information also includes arithmetic manipulation of sion levels of at least two or more biomarkers (e. g., expression ratio of SAM to SAH).
As used herein, "stored" refers to a process for encoding information on the storage device 604. Those skilled in the art can readily adopt any of the tly known methods for recording information on known media to te manufactures comprising the ce information or expression level information.
A variety of software programs and formats can be used to store the sequence information or expression level information on the e device. Any number of data sor structuring formats (e.g., text file or database) can be employed to obtain or create a medium having recorded thereon the sequence information or expression level information.
By providing sequence information and/or expression level information in computer- readable form, one can use the sequence information and/or expression level ation in readable form in the comparison module 606 to compare a specific ce or expression profile with the reference data within the storage device 604. For example, search programs can be used to identify fragments or regions of the sequences that match a particular sequence (reference data, e.g., sequence information of major or rare alleles corresponding to the SNPs described herein) or direct comparison of the determined expression level can be compared to the reference data expression level (e.g., median expression level information obtained from a population of subjects). The comparison made in computer-readable form provides a er readable comparison result which can be processed by a variety of means. t 608 based on the comparison result can be retrieved from the determination module 600 or the comparison module 606 to indicate the presence or e of at least one SNP and serum/plasma biomarkers described herein.
In one embodiment the reference data stored in the storage device 604 to be read by the determination module 600 or the ison module 606 is sequence information data obtained from a l biological sample of the same type as the biological sample to be tested. Alternatively, the reference data are a database, e.g., a part of the entire genome sequence of an organism, or a protein family of sequences, or an expression level profile (RNA, protein or e). In one embodiment, the reference data are sequence information and/or sion level profiles that are used to facilitate determining whether a subject with depression should be recommended for a treatment regimen comprising a folate-containing compound.
In one embodiment, the reference data are one or more reference polynucleotide, or polypeptide sequences. In some embodiments, the reference polynucleotide ces can be derived from nucleotide sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and a portion there of comprising the tide at the corresponding SNP location, and ments f. In some embodiments, the reference polypeptide sequences can be derived from amino acid sequences selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and a portion thereof comprising an amino acid residue at the corresponding mutation location.
In one embodiment, the reference data are electronically or digitally ed and annotated from databases including, but not limited to GenBank (NCBI) protein and DNA databases such as genome, ESTs, SNPS, Traces, Celara, Ventor Reads, Watson reads, HGTS, and the like; Swiss Institute of Bioinformatics ses, such as ENZYME, PROSITE, 2DPAGE, Swiss- Prot and TrEMBL databases; the Melanie software package or the ExPASy WWW server, and the like; the SWISS-MODEL, Swiss-Shop and other network-based computational tools; the Comprehensive Microbial Resource database (available from The Institute of Genomic Research).
The resulting information can be stored in a relational data base that may be employed to determine homologies between the nce data or genes or proteins within and among s.
The “comparison module” 606 can use a variety of available software programs and formats for the comparison operative to compare sequence ation determined in the determination module 602 to nce data. In one embodiment, the ison module 606 is configured to use pattern recognition ques to compare sequence ation from one or more entries to one or more reference data patterns. The comparison module 606 may be ured using existing commercially-available or freely-available software for comparing patterns, and may be optimized for particular data isons that are conducted. The ison module 606 provides computer readable information related to the sequence information that can include, for example, detection of the presence or absence of a sequence (e.g., detection of a mutation or on (protein or DNA), information regarding distinct alleles, detection of post-translational modification, or omission or repetition of sequences); determination of the concentration of a sequence in the sample (e. g., amino acid sequence/protein expression levels, or nucleotide (RNA or DNA) expression levels, or levels of post-translational modification), or determination of an expression profile.
In one embodiment, the comparison module 606 permits the prediction of protein sequences from polynucleotide sequences, s tion of open reading frames (ORF), or permits prediction of homologous sequence information in comparison to reference data, i.e., homologous protein domains, homologous DNA or RNA sequences, or homologous exons and/or introns.
In one embodiment, the comparison module 606 uses sequence information alignment programs such as BLAST (Basic Local Alignment Search Tool) or FAST (using the Smith- Waterman algorithm) may be employed individually or in combination. These algorithms determine the alignment between similar regions of ces and a percent identity between sequences. For example, alignment may be calculated by matching, bases-by-base or amino acid-by amino-acid.
The comparison module 606, or any other module of the system described herein, may include an operating system (e.g., UNIX) on which runs a relational database management system, a World Wide Web application, and a World Wide Web . World Wide Web application includes the executable code necessary for generation of se language statements (e.g., Structured Query Language (SQL) statements). Generally, the executables will e embedded SQL statements. In addition, the World Wide Web application may include a configuration file which contains pointers and addresses to the various software entities that comprise the server as well as the various external and internal databases which must be accessed to service user requests. The Configuration file also directs requests for server resources to the appropriate hardware--as may be ary should the server be distributed over two or more separate computers. In one embodiment, the World Wide Web server supports a TCP/IP protocol. Local networks such as this are mes referred to as "Intranets." An advantage of such Intranets is that they allow easy communication with public domain databases residing on the World Wide Web (e.g., the GenBank or Swiss Pro World Wide Web site). Thus, in a particular ment, users can directly access data (via Hypertext links for example) residing on Internet databases using a HTML interface provided by Web rs and Web s. In another embodiment, users can directly access data residing on the “cloud” provided by the cloud computing service providers.
In one embodiment, the comparison module 606 performs comparisons with mass- spectrometry spectra, for example comparisons of peptide fragment sequence information can be carried out using spectra processed in MATLAB with script called “chalign” (see for example WO2007/022248, herein incorporated by nce) and s” (Spectrum Square Associates, Ithaca, NY), or Ciphergen Peaks 2.1TM software. The processed spectra can then be aligned using alignment algorithms that align sample data to the control data using minimum entropy algorithm by taking baseline corrected data (see for example WIPO Publication WO2007/022248, herein incorporated by reference). The comparison result can be further processed by calculating ratios.
Protein expression profiles can be discerned.
In one embodiment, computational algorithms are used in the comparison module 606 such as expectation-maximization (EM), subtraction and PHASE are used in methods for statistical tion of haplotypes (see, e.g., Clark, A.G. Mol Biol Evol 7:111-22 (1990); Stephens, M., Smith, N.J. & ly, P. AmJ Hum Genet 68:978-89 ; Templeton, A.R., Sing, C.F., Kessling, A. & Humphries, Genetics 120:1145-54 (1988)).
] Various algorithms are available which are useful for comparing data and identifying the predictive gene signatures. For example, algorithms such as those fied in Xu et al., Physiol.
Genomics 11:11-20 (2002). There are numerous software available for detection of SNPs and polymorphisms that can be used in the comparison module, including, but not limited to: HaploSNPer, a web-based m for detecting SNPs and alleles in user-specified input sequences from both diploid and polyploid species (available on the world-wide web at bioinformatics.nl/tools/haplosnper/; see also Tang et al., BMC Genetics 9:23 (2008)); Polybayes, a tool for SNP discovery in redundant DNA ces , GT., et al., Nature Genetics 23(4):452-6 (1999); SSAHA-SNP, a polymorphism detection tool that uses the SSAHA alignment algorithm (available from Wellcome Trust Sanger Institute, Cambridge, United Kingdom, see also Ning 2., et al., Genome ch 11(10): 1725-9 ); red, A SNP discovery package built on phred, phrap, and consed tools (available on the world-wide web, see Nickerson, DA et al., Nucleic Acids ch 25(14):2745-51 (1997)); NovoSNP, a graphical Java-based program (PC/Mac/Linux) to identify SNPs and indels (available on the world-wide web, see Weckx, S. et al., Genome Research 436-442 ); SNPdetectorTM, for automated identification of SNPs and mutations in fluorescence-based resequencing reads (available from Affymetrix, Santa Clara, California), see also Zhang et al. PLoS Comput Biol (5):e53 (2005). SNPdetector runs on inux platform and is available publicly; Affymetrix (Santa Clara, California) has multiple data analysis software that can be used, for example Genotyping ConsoleTM Software, GeneChip® Sequence Analysis re (GSEQ), GeneChip® Targeted Genotyping Analysis Software (GTGS) and Expression ConsoleTM Software.
In one embodiment, the comparison module 606 compares gene expression profiles.
For example, detection of gene expression profiles can be determined using Affymetrix Microarray Suite software n 5.0 (MAS 5.0) (available from Affymetrix, Santa Clara, California) to analyze the relative abundance of a gene or genes on the basis of the intensity of the signal from probe sets, and the MAS 5.0 data files can be transferred into a database and analyzed with Microsoft Excel and GeneSpring 6.0 software (available from Agilent Technologies, Santa Clara, California). The detection algorithm of MAS 5.0 re can be used to obtain a comprehensive overview of how many transcripts are detected in given samples and allow a comparative analysis of two or more microarray data sets.
In one embodiment, the comparison module 606 compares protein sion profiles. Any available comparison software can be used, including but not limited to, the Ciphergen Express (CE) and Biomarker Patterns Software (BPS) package (available from Ciphergen Biosystems, Inc., Freemont, California). Comparative analysis can be done with protein chip system software (e.g., The Proteinchip Suite (available from Bio-Rad Laboratories, Hercules, California). thms for fying sion profiles can include the use of optimization algorithms such as the mean variance algorithm (e. g. IMP Genomics algorithm available from JMP re Cary, North Carolina).
In one ment, pattern ison software can be used to determine whether patterns of expression or mutations are indicative of the presence or the absence of the ions detected in a test sample of a subject.
The comparison module 606 provides computer readable comparison result that can be processed in computer readable form by predefined criteria, or ia defined by a user, to provide a content based in part on the comparison result that may be stored and output as requested by a user using a y module 610. The display module 610 enables display of a content 608 based in part on the comparison result for the user, wherein the content 608 is a signal tive of the presence of at least one of the conditions bed herein or a signal indicative of the e of at least one of the conditions described herein. Such signal, can be for example, a display of content 608 indicative of the presence or absence of at least one of the conditions on a computer monitor, a printed page of t 608 indicating the presence or absence of at least one of the ions from a printer, or a light or sound indicative of the presence or absence of at least one of the conditions.
In various embodiments of the er system described herein, the comparison module 606 can be integrated into the determination module 602.
The content 608 based on the ison result can also include an expression profile of one or more plasma/serum biomarkers described herein. In one embodiment, the content 608 based on the comparison includes a sequence of a particular gene or protein and a determination of the ce of one or more ons, or specific post-translational modification. In one embodiment, the content 608 based on the comparison result is merely a signal indicative of the presence or absence of at least one of the conditions bed herein. In some ments, the content 608 can be a signal indicative of an obesity indicator (e.g., a BMI value) or a signal indicative of whether the subject is obese (e. g., whether the BMI value is at least 30 kg/m2 or not). In some embodiments, the content 608 can be a signal indicative of the subject recommended to receive a treatment regimen comprising a folate-containing compound, or a signal indicative of the subject recommended to receive an alternative treatment regimen.
In one embodiment, the content 608 based on the comparison result is displayed a on a computer monitor. In one embodiment, the content 608 based on the comparison result is displayed through printable media. The display module 610 can be any suitable device configured to e from a computer and display er readable information to a user. Non-limiting examples include, for example, general-purpose computers such as those based on Intel PENTIUM-type processor, la PowerPC, Sun UltraSPARC, Hewlett-Packard PA-RISC processors, any of a variety of processors available from Advanced Micro Devices (AMD) of Sunnyvale, California, or any other type of processor, visual display devices such as flat panel displays, cathode ray tubes and the like, as well as computer rs of various types.
In one embodiment, a World Wide Web browser is used for providing a user interface for display of the content 608 based on the comparison result. It should be understood that other modules of the system described herein can be adapted to have a web browser interface. h the Web browser, a user may construct requests for retrieving data from the comparison module. Thus, the user will typically point and click to user interface elements such as buttons, pull down menus, scroll bars and the like conventionally employed in graphical user interfaces. The requests so formulated with the user's Web browser are transmitted to a Web application which formats them to produce a query that can be employed to extract the pertinent information related to the sequence information, e. g., display of an indication of the presence or absence of mutation or deletion (DNA or protein); display of sion levels of an amino acid sequence in); display of nucleotide (RNA or DNA) expression ; y of sion, SNP, or on profiles, or haplotypes, or display of information based thereon. In one embodiment, the sequence information of the reference sample data is also displayed.
In any ments, the comparison module can be executed by a computer ented software as discussed earlier. In such embodiments, a result from the comparison module can be displayed on an electronic y. The result can be yed by graphs, numbers, characters or words. In additional ments, the s from the comparison module can be transmitted from one location to at least one other location. For example, the comparison results can be transmitted via any electronic media, e.g., internet, fax, phone, a “cloud” system, and any combinations thereof. Using the “cloud” system, users can store and access personal files and data or perform r analysis on a remote server rather than physically carrying around a storage medium such as a DVD or thumb drive.
The system 600, and computer readable medium 700, is merely illustrative embodiments for performing assays for selecting a treatment regimen for a subject with depression, based on expression level information or sequence information, and is not intended to limit the scope of the inventions described herein. Variations of system 600, and computer readable medium 700, are possible and are intended to fall within the scope of the inventions described herein.
] The modules of the machine, or used in the computer readable medium, may assume numerous configurations. For example, function may be provided on a single machine or distributed over multiple machines.
Kits Based on the identification of SNPs and/or peripheral markers associated with a se to the use of a folate-containing compound, one aspect described herein also provides for the design and preparation of detection reagents needed to identify the SNPs and/or peripheral markers sed herein in a test sample of a subject. For example, the detection reagents can be ed and prepared to fy SNPs in MTHFR locus and MTR locus and optionally MTRR locus involved in assays and methods described , and/or measure expression levels of SAM, SAH and 4-HNE in a test sample. Examples of detection reagents that can be used to identify the disclosed SNPs in a test sample can include a primer and a probe, wherein the probe can selectively hybridize the SNP- containing nucleic acid molecules, as compared to a nucleic acid molecule which does not contain the SNP at the same nucleotide on. Examples of detection regents that can be used to measure expression levels of peripheral proteins (e. g., SAM, SAH and/or 4-HNE) in a test sample can include antibodies against such proteins, or a primer and a probe, wherein the probe specifically hybridizes to a nucleic acid molecule corresponding to such proteins.
Accordingly, ed herein include kits for selecting a treatment regimen for a subject with depression. In some embodiments, the kits can be used for monitoring the efficacy response of a subject treated with a combination y comprising a folate-containing compound, for example as shown in Example 5. The kits can include at least one reagent specific for detecting for the presence or absence of SNP s (i)-(xxi) described herein (e. g., MTHFR C677T, MTR A2756G, and/or MTRR A66G) and/or antibodies specific for detecting peripheral biomarkers (xxii) — (xxiv) (e.g., SAM, SAH, and 4-HNE), and instructions for determining that the subject is recommended for a treatment n comprising a folate-containing compound if the presence of at least one of the conditions bed herein is detected in a test sample (e.g., a blood sample, a saliva sample, or a buccal ) of the subject, for example, the procedures as shown in Example 5. The kit can optionally include a nucleic acid for detection of the gene of interest.
In one embodiment, a kit can comprise an oligonucleotide array affixed with a plurality of oligonucleotide probes that interrogate no more than 30 SNPs (including no more than 25 SNPs, no more than 20 SNPs, no more than 15 SNPs, no more than 10 SNPs, no more than 5 SNPs or less), wherein the SNPs comprise at least two or any combinations of the conditions (A)-(U) described herein (e.g., but not limited to, a combination of conditions (A) and (C)); and an optional container containing a detectable label (e.g., comprising a cent molecule) to be ated to a nucleotide molecule derived from a test sample of a human subject; and at least one reagent. es of a reagent that can be included in the kit can e, without limitations, a restriction enzyme, a universal adaptor to be conjugated to a nucleotide molecule, a primer complementary to the universal adaptor, a wash agent, and any combinations thereof.
In some embodiments, the plurality of oligonucleotide probes affixed to an oligonucleotide array can interrogate about 2-30 SNPs, e.g., about 3-25 SNPs, about 3-20 SNPs, about 3-10 SNPs, or about 3-5 SNPs, wherein the SNPs comprise at least two or any combinations of the conditions (A)-(U) described herein (e.g., but not limited to, a combination of conditions (A) and (C)).
In an alternative embodiment, a kit can comprise a plurality of oligonucleotide primers that bind to at least one allele of no more than 30 SNPs (including no more than 25 SNPs, no more than 20 SNPs, no more than 15 SNPs, no more than 10 SNPs, no more than 5 SNPs or less), wherein each subset of ucleotide s that bind to a ic allele of a SNP is labeled with a ct reporter, and wherein said SNPs comprise at least two or any combinations of the SNP conditions (A)-(U) described herein (e.g., but not limited to a combination of ions (A) and (C)); and at least one t, e.g., but not limited to, free tide bases, a polymerase, or both.
In some embodiments, the plurality of oligonucleotide primers can bind to at least one allele of about 2-30 SNPs, e.g., about 3-25 SNPs, about 3-20 SNPs, about 3-10 SNPs, or about 3-5 SNPs, wherein the SNPs comprise at least two or any combinations of the conditions (A)-(U) described herein (e.g., but not limited to, a combination of conditions (A) and (C)).
Additional reagents included in the kit can vary with the selection of a ping assay, e.g., but not limited to, allele-specific probe hybridization, allele-specific primer extension, allele-specific ication, sequencing, 5’ nuclease digestion, molecular beacon assay, DNA chip analysis, oligonucleotide ligation assay, size analysis, -stranded conformation polymorphism, polymerase chain reaction (PCR), real-time quantitative PCR, and any combinations thereof.
In some embodiments, the kit can further comprise at least one reagent to determine sion of at least one biomarker described herein (e.g., SAM, SAH, 4-HNE and . For example, in one embodiment, the kit can further comprise a solid substrate support affixed with at least one protein-based binding moiety (e. g., an antibody) that specifically binds to one or more of the biomarkers described herein. Exemplary solid substrate support can include, but not limited to, a microtiter plate for ELISA, a dipstick, a ic bead, or any combinations thereof. Different solid substrate supports can be selected based on various types of expression assays, e.g., but not d to, western blot, enzyme linked absorbance assay, mass spectrometry, immunoassay, flow cytometry, immunohistochemical analysis, and any combinations f.
In another embodiment, the kit can further comprise at least one primer designed to probe one or more biomarkers described herein.
Embodiments of the various aspects described herein can also be bed by any one of the following numbered paragraphs. 1. An assay for selecting a treatment regimen for a human subject with depression, comprising: (a) subjecting a test sample from a human subject, who is sed as haVing depression or haVing a risk for depression, to at least one genotyping assay adapted to determine the genotypes of at least two loci, wherein said at least two loci are: i. on 677 of SEQ ID NO. 1 or on 27 of SEQ ID NO. 7 (identified by 133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each ndently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and (b) ing from the genotypes of said at least two loci the presence of a single nucleotide polymorphism (SNP) selected from the following: i. a SNP677 at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” ; ii. a SNP2756 at position 2756 of SEQ ID NO.2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; and iii. a combination of at least one SNP677 T allele and at least one SNP2756 G allele; and if at least one of T allele at position SNP677 or at least one G allele at position SNP2756 or both at least one T allele at position SNP677 and at least one G allele at position SNP2756 is detected, then selecting, and optionally administering, a treatment regimen comprising an effective amount of a folate-containing compound. 2. The assay of paragraph 1, wherein if r SNP677 T allele nor SNP2756 G allele is detected then selecting a treatment regimen without a folate-containing compound. 3. An assay for selecting a ent regimen for a human subject haVing depression, sing: subjecting a test sample of the human subject, who is diagnosed as haVing depression or haVing a risk for depression, to at least one assay to detect the ce or absence of at least one of the following conditions: i. an expression ratio of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) smaller than a pre-determined nce ratio; ii. expression of 4-hydroxynonenal (4-HNE) greater than a pre- determined reference value; iii. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of enetetrahydrofolate reductase (MTHFR); iV. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” , wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and V. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. comprising at least one guanine “G” allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each ndently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); and if at least one of said ions is detected to be present then recommending that a treatment regimen comprising a folate-containing compound be selected; and if none of these conditions is determined to be present then recommending a ent regimen t a folate-containing compound. 2012/065084 4. The assay of any one of the preceding paragraphs, further comprising ining a parameter of at least one biomarker from the following: i. genotype of a SNP locus at position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8 ified by rs2274976), wherein SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR) ; ii. genotype of a SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), n the SEQ ID NO. 3 and SEQ ID NO. are each independently a portion of a genomic nucleic acid ce of nine synthase reductase (MTRR); iii. pe of a SNP locus at 737 (position 27 of SEQ ID NO. 11), wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC); iV. genotype of a SNP locus at rs1883729 (position 27 of SEQ ID NO. 12), wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta B); . genotype of a SNP locus at rs7163862 (position 27 of SEQ ID NO. 13), wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); Vi. genotype of a SNP locus at rs12659 (position 27 of SEQ ID NO. 14), wherein the SEQ ID NO. 14 is a n of a genomic nucleic acid sequence of reduced folate carrier protein (RCFZ); Vii. genotype of a SNP locus at rs202676 (position 27 of SEQ ID NO. 15), wherein the SEQ ID NO. 15 is a portion of a c nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); Viii. genotype of a SNP locus at rs2297291 (position 27 of SEQ ID NO. 16), wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF 1 ); ix. genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17 ), wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid ce of reduced folate carrier protein (RCF 1 ); 2012/065084 X. genotype of a SNP locus at rs8007267 (position 27 of SEQ ID NO. 18), wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); xi. genotype of a SNP locus at rs7639752 (position 27 of SEQ ID NO. 19), wherein the SEQ ID NO. 19 is a portion of a c nucleic acid sequence of choline- phosphate cytidylyltransferase A (PCYTlA); xii. genotype of a SNP locus at rs6275 (position 27 of SEQ ID NO. 20), wherein the SEQ ID NO. 20 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); xiii. genotype of a SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xiV. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); XV. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23), wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O- methyltransferase (COMT); xvi. genotype of a SNP locus at rs4680 ion 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O- methyltransferase (COMT); xvii. genotype of a SNP locus at rs250682 (position 27 of SEQ ID NO. 25, n the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 transmitted transported, dopamine), member 3 3); xviii. genotype of a SNP locus at rs2277820 (position 27 of SEQ ID NO. 26), wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase ; xix. genotype of a SNP locus at 225 (position 27 of SEQ ID NO. 27), wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFDl); XX. an obesity indicator (e.g., a BMI value); xxi. expressions of SAM and SAH; xxii. expression of 4-HNE; xxiii. expression of hsCRP; and any ations thereof.
. The assay of paragraph 4, r comprising determining, from the determined parameter of said at least one biomarker, the presence of at least one condition from the following: i. a SNP at position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8 comprising at least one alanine “A” allele; ii. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele; iii. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) sing at least one alanine “A” allele; iV. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele; V. a SNP at rs7163862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; Vi. a SNP at rs12659 ion 27 of SEQ ID NO. 14) sing at least one thymine “T” allele; Vii. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; Viii. a SNP at rs2297291 ion 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele; ix. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; x. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele; xi. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele; xii. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele; xiii. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele; xiV. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xv. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele; xvi. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xvii. a SNP at 82 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele; xviii. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele; and xix. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) sing at least one alanine “A” allele; xx. obesity (e.g., defined by a BMI value of 30 kg/m2 or greater); xxi. an expression ratio of SAM to SAH smaller than a pre-determined reference ratio; xxii. an expression of 4-HNE greater than a pre-determined reference value; xxiii. an expression of hsCRP greater than about 2.3 mg per liter as measured in a plasma sample; and any ations f; and if at least one of the condition is detected, then selecting, and optionally administering, a treatment n comprising an effective amount of a folate-containing compound. 6. An assay for selecting a treatment n for a human subject having depression, comprising: (a) subjecting a test sample of the human subject, who is sed as having depression or having a risk for depression, to at least one analysis to determine parameters of at least two biomarkers from: i. genotype of a SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid ce of methylenetetrahydrofolate reductase (MTHFR); ii. genotype of a SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic c acid sequence of methylenetetrahydrofolate reductase (MTHFR); iii. genotype of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of nine synthase (MTR); iV. genotype of a SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 ified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); V. genotype of a SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC)); Vi. pe of a SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B)); Vii. genotype of a SNP locus at rs7163862 (position 27 of SEQ ID NO. 13, wherein the SEQ ID NO. 13 is a portion of a c nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein )) ; Viii. genotype of a SNP locus at rs12659 (position 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier n (RCF2)); ix. genotype of a SNP locus at rs202676 ion 27 of SEQ ID NO. 15, wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1)); X. genotype of a SNP locus at 291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)) ; xi. genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17, wherein the SEQ ID NO. 17 is a n of a genomic c acid sequence of reduced folate carrier protein (RCF1)); xii. genotype of a SNP locus at rs8007267 (position 27 of SEQ ID NO. 18, n the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1)); xiii. genotype of a SNP locus at rs7639752 (position 27 of SEQ ID NO. 19, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A)); xiv. genotype of a SNP locus at rs6275 (position 27 of SEQ ID NO. 20, wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2)); xv. genotype of a SNP locus at 596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvi. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); xvii. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23, n the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT)); xviii. genotype of a SNP locus at rs4680 (position 27 of SEQ ID NO. 24), n the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. genotype of a SNP locus at rs250682 (position 27 of SEQ ID NO. 25, wherein the SEQ ID NO. 25 is a n of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3)); xx. genotype of a SNP locus at rs2277820 (position 27 of SEQ ID NO. 26, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase eaminase (FTCD)); and xxi. pe of a SNP locus at 225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid ce of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFD1)); xxii. an obesity indicator (e. g., a BMI value); xxiii. level of expression of SAM and SAH; xxiv. level of expression of 4-HNE; xxv. level of expression of hsCRP; and any combinations thereof; (b) detecting, optionally with a non-human machine, from the ters of said at least two biomarkers, the presence of at least one ion selected from the following: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele; iii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; iV. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele; . a SNP at 737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele; Vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele; Vii. a SNP at rs7163862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; Viii. a SNP at rs12659 (position 27 of SEQ ID NO. 14) comprising at least one e “T” allele; ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; . a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele; xi. a SNP at 266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; xii. a SNP at rs8007267 ion 27 of SEQ ID NO. 18) sing at least one thymine “T” allele; xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19 ) comprising at least one alanine “A” allele; xiV. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele; XV. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one e “T” allele; xvi a SNP at rsl 1240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xvii. a SNP at rs4633 ion 27 of SEQ ID NO. 23) comprising at least one ne “C” allele; xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xix. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele; XX. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele; xxi. a SNP at rs2236225 ion 1958 of SEQ ID NO. 27) comprising at least one alanine “A” allele; xxii. obesity (e.g., defined by a BMI value of at least 30 kg/m2 or greater); xxiii. an sion level ratio of SAM to SAH smaller than a termined reference ratio; xxiv. an expression level of 4-HNE greater than a pre-determined nce value; XXV. an expression of hsCRP greater than about 2.3 mg per liter of plasma as ed in a plasma ; and any combinations thereof, and and if at least one of said conditions is detected, then selecting and optionally administering a treatment n comprising an effective amount of a folate- containing compound. 7. The assay of any of the preceding paragraphs, wherein the pre-determined reference ratio is about 2.8 if measured in a plasma sample. 8. The assay of any of the preceding paragraphs, wherein the pre-determined reference ratio is about 3.0 if measured in a plasma sample. 9. The assay of any of the preceding paragraphs, wherein the predetermined reference value is about 3.0 mg per liter of plasma if measured in a plasma sample.
. The assay of any of the preceding paragraphs, wherein the predetermined reference value is about 3.2 mg per liter of plasma as measured in a plasma sample. 11. The assay of any of the preceding paragraphs, wherein the test sample is analyzed to determine at least two of the conditions. 12. The assay of any of the preceding paragraphs, wherein the test sample is analyzed to determine at least three of the ions. 13. The assay of any of the preceding paragraphs, wherein the test sample comprises a blood sample. 14. The assay of any of the preceding paragraphs, wherein the test sample comprises a urine sample.
. The assay of any of the preceding paragraphs, wherein the test sample comprises a buccal sample. 16. The assay of any of the preceding paragraphs, wherein the test sample comprises a saliva sample. 17. The assay of any of the preceding paragraphs, wherein the genotyping comprises the step of amplifying the test sample with a set of primers flanking any one of the SNPs. 18. The assay of paragraph 17, n at least two sets of s ying at least two of the SNPs are used in a multiplex ication assay. 19. The assay of any of the preceding paragraphs, wherein the test sample comprises a protein sample, and the test sample comprising a protein sample is subjected to at least one analysis selected from the group consisting of n blot, enzyme linked absorbance assay, mass spectrometry, immunoassay, flow cytometry, immunohistochemical analysis, and any combinations thereof.
. The assay of any of the preceding paragraphs, n the treatment regimen further comprises selecting and optionally administering an antidepressant drug. 21. The assay of paragraph 20, wherein the anti-depressant drug comprises a selective serotonin reuptake inhibitor. 22. The assay of any of the preceding paragraphs, n the depression is major depressive disorder. 23. A method for treating a human subject with depression, comprising administering a composition sing an effective amount of a folate-containing compound to a human t, who is diagnosed to have depression or have a risk for sion, and is further determined to carry at least one of the following single nucleotide polymorphisms (SNPs) or a combination thereof: i. a SNP at on 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); and ii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, n the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR). 24. A method for treating a human subject with depression, comprising a. determining the genotypes of at least two loci in a biological sample of a subject, who is diagnosed as having depression or having a risk for depression, wherein said at least two loci are: i. SNP677, wherein the SNP677 is position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), n the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); and ii. SNP2756, wherein the SNP2756 is position 2756 of SEQ ID N02 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each ndently a n of a genomic c acid sequence of methionine synthase (MTR); and b. administering a treatment regimen comprising a composition comprising an effective amount of a folate-containing compound to the subject if at least one of the ing conditions is detected: i. at least one thymine “T” allele at SNP677; ii. at least one e “G” allele at SNP2756; or iii. at least one thymine “T” allele at SNP677 and at least one guanine “G” allele at SNP2756.
. A method of ing the effectiveness of an anti-depressant drug administered to a human subject, comprising administering a composition comprising an effective amount of a folate- containing compound, in combination with the anti-depressant drug, to the human subject if the human subject is determined to carry any one of the ing single nucleotide polymorphisms (SNPs) or a combination thereof: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine "T" allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a n of a genomic nucleic acid sequence of enetetrahydrofolate reductase (MTHFR); and ii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine "G" allele, n the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR). 26. The method of any of the preceding paragraphs, wherein the subject is further determined to carry at least one of the following conditions or any combinations thereof: i. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele; ii. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one e "G" allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a n of a genomic c acid sequence of methionine se reductase (MTRR); iii. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC); iV. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one e “A” allele, n the SEQ ID NO. 12 is a portion of a genomic c acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); . a SNP at rs7l63862 (position 27 of SEQ ID NO. 13) sing at least one thymine “T” allele, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); Vi. a SNP at rsl2659 (position 27 of SEQ ID NO. 14) sing at least one thymine “T” allele, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2); Vii. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” , wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane n) 1 (FOLHl); Viii. a SNP at rs229729l (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 16 is a portion of a c nucleic acid sequence of reduced folate carrier protein (RCFl); 2012/065084 ix. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) sing at least one alanine “A” allele, wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); . a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid ce of GTP cyclohydrolase 1 (GCHl); xi. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele, n the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A); xii. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele), wherein the SEQ ID NO. 20 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); xiii. a SNP at 596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele ), wherein the SEQ ID NO. 21 is a n of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xiV. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” , ), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); XV. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele, wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase ; xvi. a SNP at rs4680 (position 27 of SEQ ID NO. 24) sing at least one guanine “G” allele, wherein the SEQ ID NO. 24 is a portion of a genomic c acid sequence of catechol-O-methyltransferase (COMT); xvii. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele, wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute r family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3);; xviii. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase eaminase (FTCD); and xix. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFDl); XX. obesity (e.g., defined by a BMI value of at least 30 kg/m2 or r); xxi. an expression ratio of SAM to SAH smaller than a pre-determined ratio; xxii. an expression of 4-HNE greater than a pre-determined standard; and xxiii. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample. 27. A method for treating a human subject with depression, sing stering a composition comprising an effective amount of a folate-containing compound to the human subject haVing been diagnosed with depression or with a risk for sion, and is further determined to carry at least one of the following conditions or any ations f: 1. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate ase (MTHFR); ii. a SNP at rs2274976 ion 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); iV. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); . a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 11 is a portion of a c nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC); 2012/065084 Vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); Vii. a SNP at rs7163862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 13 is a n of a genomic nucleic acid ce of GTP ydrolase 1 feedback regulatory protein (GCHFR); Viii. a SNP at rs12659 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2); ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one e “G” allele, wherein the SEQ ID NO. 15 is a portion of a c nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); . a SNP at 291 (position 27 of SEQ ID NO. 16) sing at least one alanine “A” allele, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); xi. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 17 is a n of a genomic c acid sequence of reduced folate carrier protein (RCFl); xii. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid ce of GTP cyclohydrolase 1 (GCH1); xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 19 is a portion of a genomic c acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A); xiV. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele), wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of ne receptor D2 (DRD2); XV. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele, ), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvi. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele, ), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele, wherein the SEQ ID NO. 23 is a portion of a genomic c acid ce of catechol-O-methyltransferase (COMT); xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” , wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. a SNPat rs250682 (position 27 of SEQ ID NO. 25) comprising at least one ne “C” allele, wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute r family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3);; xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); and xxi. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFDl); xxii. obesity (e.g., d by a BMI value of at least 30 kg/m2 or greater); xxiii. an expression ratio of SAM to SAH smaller than a pre-determined ratio; xxiv. an expression of 4-HNE greater than a pre-determined standard; and xxv. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample. 28. A method of treating depression in a human subject comprising detecting at least one of the following conditions in a biological sample from the human subject and if any one of them is present then stering to the human subject a treatment regimen comprising an effective amount of a folate-containing compound, wherein said at least one of the conditions is selected from the ing: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by 133) comprising at least one thymine “T” allele; ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele; iii. a SNP at position 2756 of SEQ ID N02 or on 27 of SEQ ID NO. 9 sing at least one guanine “G” allele; iV. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele; . a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele; Vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one e “A” allele; Vii. a SNP at rs7163862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; Viii. a SNP at rs12659 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele; ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; . a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele; xi. a SNP at 266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; xii. a SNP at rs8007267 ion 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele; xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19 ) sing at least one alanine “A” allele; xiV. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele; XV. a SNP at 596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele; xvi. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele; xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xix. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one ne “C” allele; xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele; xxi. a SNP at rs2236225 (position 1958 of SEQ ID NO. 27) comprising at least one alanine “A” allele; xxii. obesity (e. g., defined by a BMI value of at least 30 kg/m2 or greater); xxiii. an expression level ratio of SAM to SAH smaller than a pre-determined reference ratio; xxiv. an expression level of 4-HNE greater than a pre-determined reference value; xxv. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample; and any combinations thereof. 29. The method of any of the preceding paragraphs, wherein the subject is further ined to carry at least two of the conditions or more.
. The method of any of the preceding paragraphs, n the effective amount of the folate- containing compound ranges from about 0.1 to about 1 mg/kg body weight per day. 31. The method of any of the preceding paragraphs, wherein the effective amount of the folate- containing compound is about 7.5 mg/day to about 50 mg/day. 32. The method of any of the preceding paragraphs, wherein the effective amount of the folate- ning compound is about 15 mg/day. 33. The method of any of the preceding paragraphs, wherein the effective amount of the folate- containing compound is administered as a single daily dose. 34. The method of any of the preceding paragraphs, wherein the effective amount of the folate- containing compound is administered in more than one divided doses per day.
. The method of any of the preceding paragraphs, wherein the administration is oral. 36. The method of any of the preceding paragraphs, wherein the composition is formulated to release at least a n of the folate-containing compound over a period of at least about 3-6 hours, upon the administration of the composition, wherein the release is ally a steady-state release. 37. The method of any of the ing paragraphs, further comprising selecting and ally administering an epressant drug in ation with the -containing compound. 38. The method of paragraph 37, wherein the anti-depressant drug ses a selective serotonin reuptake inhibitor. 39. The method of paragraph 38, wherein the selective serotonin reuptake tor is selected from the group consisting of fluoxetine, pram, paroxetine, lopram, sertraline, and any combinations thereof. 40. The method of any of the preceding paragraphs, wherein the subject who is diagnosed as having depression is resistant to at least one pressant monotherapy. 41. The method of any of the preceding paragraphs, wherein the depression is major depressive 42. The method of any of the ing paragraphs, wherein the test sample comprises a buccal sample. 43. The method of any of the preceding paragraphs, wherein the test sample comprises a saliva sample. 44. The method of any of the preceding paragraphs, wherein the test sample comprises a blood sample. 45. The method of any of the preceding paragraphs, wherein the test sample comprises a urine sample. 46. A computer system for obtaining data from at least one test sample obtained from at least one subject, the system comprising: (a) at least one determination module ured to receive said at least one test sample and perform at least one analysis on said at least one test sample to determine the presence or absence of at least one of the following conditions or any combinations thereof: i. an expression ratio of s-adenosyl methionine (SAM) to s-adenosyl homocysteine (SAH) smaller than a pre-determined reference ratio; ii. expression of 4-hydroxynonenal (4-HNE) r than a pre- determined reference value; iii. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” , wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each ndently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iv. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and V. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. sing at least one guanine “G” allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); (b) at least one storage device configured to store data output from said ination module; and (d) at least one display module for displaying a content based in part on the data output from said determination module, wherein the content comprises a signal indicative of the presence of at least one of these ions, and optionally the absence of any one of these ions, or a signal indicative of the e of all of these conditions. 47. The computer system of paragraph 46, wherein said ination module is configured to e said at least one test sample to ine the presence or e of at least two of the conditions. 48. The er system of paragraph 46 or 47, wherein said determination module further comprises a comparison module adapted to compare said data output from said determination module with reference data stored on said storage device. 49. A computer system for obtaining data from at least one test sample obtained from at least one subject, the system comprising: (a) a determination module configured to receive said at least one test sample and perform at least one genotyping analysis on said at least one test sample to determine the genotypes of at least two loci, wherein said at least two loci se: (i) position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); (ii) position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and (iii) optionally, position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are 2012/065084 each independently a portion of a genomic nucleic acid sequence of methionine se reductase (MTRR); (b) a storage deVice configured to store output data from said determination module; (c) a computing module comprising specifically-programmed instructions to determine from the output data the presence of at least one single nucleotide polyphormism (SNP) from the following: i. a SNP at position 677 of SEQ ID NO. 1 or on 27 of SEQ ID NO. 7 comprising at least one e “T” allele; ii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; and iii. optionally a SNP at the position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine “G” allele. (d) a y module for displaying a content based in part on the data output from said computing module, wherein the content comprises a signal indicative of the presence of at least one of these SNPs, and optionally the absence of any one of these SNPs, or a signal indicative of the absence of all of these SNPs. 50. The computer system of any one of the preceding paragraphs, wherein said determination module is further determine a parameter of at least one of the following biomarkers or any combinations thereof: i. pe of a SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each ndently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. genotype of a SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, wherein the SEQ ID NO. 11 is a n of a genomic nucleic acid sequence of calcium channel, e-dependent, L type, alpha 1C subunit (CACNAlC)); iii. genotype of a SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta B)); iV. genotype of a SNP locus at rs7l63862 (position 27 of SEQ ID NO. 13, wherein the SEQ ID NO. 13 is a portion of a genomic c acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR)) ; V. genotype of a SNP locus at rs12659 (position 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2)); Vi. genotype of a SNP locus at rs202676 (position 27 of SEQ ID NO. 20, wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific ne antigen) 1 (FOLH1)); Vii. genotype of a SNP locus at 291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)) ; Viii. genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17, wherein the SEQ ID NO. 17 is a portion of a genomic c acid sequence of reduced folate carrier protein (RCF1)); ix. genotype of a SNP locus at rs8007267 (position 27 of SEQ ID NO. 18, n the SEQ ID NO. 18 is a n of a genomic nucleic acid ce of GTP cyclohydrolase 1 (GCH1)); x. genotype of a SNP locus at rs7639752 (position 27 of SEQ ID NO. 19, n the SEQ ID NO. 19 is a portion of a genomic c acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A)); xi. genotype of a SNP locus at rs6275 (position 27 of SEQ ID NO. 20, wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine or D2 (DRD2)); xii. genotype of a SNP locus at 596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xiii. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xiV. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23, wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O- methyltransferase (COMT)); xv. genotype of a SNP locus at rs4680 (position 27 of SEQ ID NO. 24), n the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O- methyltransferase (COMT); WO 74676 xvi. genotype of a SNP locus at rs250682 (position 27 of SEQ ID NO. 25, wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3)); xvii. genotype of a SNP locus at rs2277820 (position 27 of SEQ ID NO. 26, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase eaminase ); and xviii. genotype of a SNP locus at rs2236225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of enetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFD1)); xix. an obesity indicator (e. g., a BMI value); xx. level of expression of SAM and SAH; xxi. level of expression of 4-HNE; xxii. level of expression of hsCRP. 51. The computer system of paragraph 50, wherein said computing module is further adapted to determine the presence of at least one of the following conditions or any combinations thereof: i. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one e “A” allele; ii. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele; iii. a SNP at rs1883729 ion 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele; iv. a SNP at rs7l63862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; v. a SNP at rsl2659 ion 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele; vi. a SNP at 76 (position 27 of SEQ ID NO. 15) comprising at least one e “G” allele; vii. a SNP at rs229729l (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele; viii. a SNP at rs105l266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; 2012/065084 ix. a SNP at 267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele; . a SNP at rs7639752 (position 27 of SEQ ID NO. 19 ) comprising at least one alanine “A” allele; xi. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele; xii. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele; xiii. a SNP at rsl 1240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xiv. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele; XV. a SNP at rs4680 ion 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xvi. a SNP at rs250682 ion 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele; xvii. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” ; xviii. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) sing at least one alanine “A” allele; xix. obesity (e.g., defined by a BMI value of at least 30 kg/m2 or greater); XX. an expression level ratio of SAM to SAH smaller than a pre-determined reference ratio; xxi. an expression level of 4-HNE greater than a pre-determined reference value; xxii. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample. 52. A computer system for obtaining data from at least one test sample obtained from at least one subject, the system comprising: (a) a determination module configured to receive said at least one test sample and perform at least one analysis on said at least one test sample to determine parameters of at least two biomarkers, wherein the ters of said at least two kers are selected from the following: . genotype of a single tide rphism (SNP) locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. genotype of a SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), n the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid ce of methylenetetrahydrofolate reductase ); iii. genotype of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic c acid sequence of methionine synthase (MTR); iV. genotype of a SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); . pe of a SNP locus at rs1006737 (position 27 of SEQ ID NO. 11), n the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC); Vi. genotype of a SNP locus at rs1883729 (position 27 of SEQ ID NO. 12), wherein the SEQ ID NO. 12 is a portion of a c nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); Vii. genotype of a SNP locus at rs7163862 (position 27 of SEQ ID NO. 13), wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory n (GCHFR); Viii. genotype of a SNP locus at rs12659 (position 27 of SEQ ID NO. 14), wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of d folate carrier protein (RCF2); ix. genotype of a SNP locus at rs202676 (position 27 of SEQ ID NO. 15), wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase ate-specific membrane antigen) 1 (FOLHl); . genotype of a SNP locus at rs2297291 (position 27 of SEQ ID NO. 16), wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); xi. genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17), wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate r protein (RCFl); xii. genotype of a SNP locus at rs8007267 (position 27 of SEQ ID NO. 18), wherein the SEQ ID NO. 18 is a n of a genomic nucleic acid sequence of GTP ydrolase 1 (GCHl); xiii. genotype of a SNP locus at rs7639752 (position 27 of SEQ ID NO. 19), wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate lyltransferase A (PCYTlA); xiV. genotype of a SNP locus at rs6275 (position 27 of SEQ ID NO. 20), wherein the SEQ ID NO. 20 is a portion of a genomic c acid sequence of dopamine or D2 (DRD2); XV. genotype of a SNP locus at 596 (position 27 of SEQ ID NO. 21), n the SEQ ID NO. 21 is a n of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvi. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a c nucleic acid sequence of dopamine receptor D2 (DRD2); xvii. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23), wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xviii. genotype of a SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of ol-O-methyltransferase (COMT); xix. genotype of a SNP locus at rs250682 (position 27 of SEQ ID NO. 25), wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3);; xx. genotype of a SNP locus at rs2277820 ion 27 of SEQ ID NO. 26), wherein the SEQ ID NO. 26 is a portion of a c nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); xxi. genotype of a SNP locus at rs2236225 (position 27 of SEQ ID NO. 27), wherein the SEQ ID NO. 27 is a portion of a genomic c acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFDl); xxii. sions of SAM and SAH; xxiii. expression of 4-HNE; xxiV. expression of hsCRP; and any combinations thereof. (b) a storage deVice configured to store output data from said determination ; (c) a computing module comprising specifically-programmed instructions to determine from the output data the presence of at least one condition from the following: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP at rs2274976 ion 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) sing at least one alanine “A” allele; iii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; iV. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele; V. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele; Vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) sing at least one alanine “A” allele; Vii. a SNP at rs7l63862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; Viii. a SNP at rsl2659 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele; ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; . a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele; xi. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; xii. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele; xiii. a SNP at 752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele; xiV. a SNP at rs6275 ion 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele; XV. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele; xvi. a SNP at 0594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele; xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xix. a SNP at rs250682 (position 27 of SEQ ID NO. 25) sing at least one cytosine “C” allele; XX. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele; and xxi. a SNP at rs2236225 ion 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele; xxii. an expression ratio of SAM to SAH smaller than a termined reference ratio; xxiii. an expression of 4-HNE r than a pre-determined reference value; xxiV. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample; and any combinations thereof. (d) a display module for displaying a content based in part on the data output from said computing module, wherein the content ses a signal indicative of the 2012/065084 presence of at least one of the conditions, and optionally the absence of any one of the conditions, or a signal indicative of the absence of all of the conditions. 53. The computer system of any of the preceding paragraphs, wherein the pre-determined reference ratio is about 3.0 as measured in a plasma sample. 54. The computer system of any of the preceding aphs, wherein the pre-determined reference ratio is about 2.8 as measured in a plasma sample. 55. The computer system of any of the preceding paragraphs, wherein the predetermined reference value is about 3.0 mg per liter as ed in a plasma sample. 56. The computer system of any of the preceding paragraphs, n the predetermined nce value is about 3.2 mg per liter as ed in a plasma sample. 57. The er system of any of the preceding paragraphs, wherein the determination module is configured to determine parameters of at least three kers or more. 58. The computer system of any of the preceding paragraphs, wherein the computing module is configured to determine the presence of at least two conditions or more. 59. The computer system of any of the preceding paragraphs, wherein said computing module further comprises a comparison module adapted to compare said output data from said determination module with reference data stored on said storage device. 60. The computer system of any of the preceding paragraphs, wherein the storage device is further configured to store physical information of said at least one subject. 61. The er system of paragraph 60, wherein the physical information comprises an obesity indicator (e.g., BMI) of said at least one subject. 62. The computer system of any of the preceding aphs, wherein the content displayed on said display module further comprises the obesity indicator (e.g., the BMI value) or a signal indicative of whether the subject is obese (e.g., whether the BMI value is at least 30 kg/m2 or not). 63. The computer system of any of the ing aphs, wherein the content displayed on said display module further comprises a signal indicative of the subject recommended to receive a treatment regimen sing a folate-containing compound, or a signal indicative of the subject recommended to receive an alternative treatment regimen without a folate-containing compound. 64. The computer system of any of the preceding paragraphs, wherein the depression is major depressive disorder. 65. A computer readable medium having computer le instructions recorded thereon to define software modules for implementing a method on a er, said computer readable storage medium comprising: (a) instructions for comparing the data stored on a storage device with reference data to provide a comparison result, n the comparison identifies the presence or absence of at least one of the following conditions or any ations thereof: i. an expression ratio of s-adenosyl nine (SAM) to s-adenosyl homocysteine (SAH) smaller than a pre-determined reference ratio; ii. expression of 4-hydroxynonenal (4-HNE) greater than a pre- determined reference value; iii. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele, n the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iv. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and v. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. comprising at least one guanine “G” allele, n the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); (b) ctions for displaying a content based in part on the data output from said determination module, wherein the t comprises a signal indicative of the presence of at least one of the conditions, and optionally the absence of any one of the conditions, or a signal indicative of the absence of all of the conditions. 66. A computer readable medium having computer readable instructions recorded thereon to define software s for implementing a method on a computer, said er readable storage medium comprising: (a) instructions for comparing the data stored on a storage device with reference data to provide a comparison result, wherein the ison identifies the presence or absence of at least one of the following conditions: i. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) sing at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of enetetrahydrofolate ase (MTHFR); ii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 sing at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and (b) instructions for ying a content based in part on the data output from said determination module, wherein the content ses a signal indicative of the presence of at least one of the conditions, and ally the absence of any one of the conditions, or a signal indicative of the e of all of the conditions. 67. The computer readable medium of any of the preceding paragraphs, further comprising instructions to identify the presence or absence of at least one of the following conditions or any combinations thereof: i. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 sing at least one guanine "G" allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); iii. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium l, voltage-dependent, L type, alpha 1C subunit (CACNAlC); iV. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); V. a SNP at 862 ion 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); Vi. a SNP at rsl2659 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” , wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid ce of d folate carrier protein (RCF2); vii. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid ce of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); viii. a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 16 is a n of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); ix. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); x. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); xi. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A); xii. a SNP at rs6275 ion 27 of SEQ ID NO. 20) comprising at least one thymine “T” , wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xiii. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one e “T” , wherein the SEQ ID NO. 21 is a portion of a c nucleic acid sequence of dopamine receptor D2 ; xiv. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xv. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele, wherein the SEQ ID NO. 23 is a portion of a genomic c acid ce of catechol-O-methyltransferase (COMT); xvi. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” , wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of ol-O-methyltransferase (COMT); xvii. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele, wherein the SEQ ID NO. 25 is a n of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3); xviii. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele, n the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); and xix. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) sing at least one alanine “A” allele, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFDl); xx. obesity (e. g., defined by a BMI value of at least 30 kg/m2 or r); xxi. an expression ratio of SAM to SAH r than a pre-determined ratio; xxii. an sion of 4-HNE greater than a pre-determined standard; and xxiii. an expression of hsCRP r than about 2.3 mg per liter of plasma as measured in a plasma . 68. A computer readable medium haVing computer readable instructions recorded thereon to define software modules for implementing a method on a computer, said computer readable storage medium comprising: (a) instructions for comparing the data stored on a storage deVice with reference data to provide a comparison result, wherein the comparison identifies the presence or absence of at least one of the following ions: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic c acid ce of methylenetetrahydrofolate reductase (MTHFR); iii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each ndently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); iV. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of nine synthase reductase (MTRR); V. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” , wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNA1C); Vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta B); Vii. a SNP at rs7163862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); Viii. a SNP at rs12659 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFZ); ix. a SNP at rs202676 ion 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase ate-specific ne antigen) 1 (FOLHl); x. a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 16 is a portion of a genomic c acid sequence of reduced folate carrier protein (RCFl); xi. a SNP at rs1051266 ion 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate r protein (RCFl); xii. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); xiii. a SNP at rs7639752 ion 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate lyltransferase A (PCYT1A); xiv. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 20 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); xv. a SNP at rs1079596 ion 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvi. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 22 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” , wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele, wherein the SEQ ID NO. 25 is a portion of a genomic c acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3); xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele, n the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase ; and xxi. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate ogenase (NADP+ dependent) 1 (MTHFD1); xxii. obesity (e. g., defined by a BMI value of at least 30 kg/m2 or greater); xxiii. an expression ratio of SAM to SAH r than a pre-determined ratio; xxiv. an expression of 4-HNE greater than a termined standard; xxv. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample; and any combinations thereof; and (b) instructions for displaying a t based in part on the data output from said determination module, wherein the content comprises a signal indicative of the 2012/065084 presence of at least one of the conditions, and optionally the absence of any one of the conditions, or a signal indicative of the absence of all of the conditions. 69. The computer readable medium of paragraph 68, r comprising instructions to display a signal indicative of the subject recommended to receive a treatment regimen comprising a folate- containing compound, or a signal indicative of the subject recommended to receive an alternative treatment regimen without a folate-containing compound. 70. The computer readable medium of any of the preceding aphs, wherein the sion is major depressive disorder. 71. A kit comprising: an oligonucleotide array affixed with a plurality of oligonucleotide probes that interrogate no more than 30 single nucleotide polymorphisms (SNPs), wherein said SNPs comprise at least two of the following SNPs or any combinations thereof: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate ase (MTHFR); ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each ndently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iii. a SNP at position 2756 of SEQ ID NO. 2 or on 27 of SEQ ID NO. 9 comprising at least one e “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a n of a genomic nucleic acid sequence of methionine synthase (MTR); iv. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid ce of methionine synthase reductase (MTRR); a SNP at 737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 11 is a n of a genomic nucleic acid sequence of calcium l, voltage-dependent, L type, alpha 1C subunit (CACNAlC); Vi. a SNP at rs1883729 ion 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid ce of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); Vii. a SNP at rs7163862 (position 27 of SEQ ID NO. 13) comprising at least one e “T” allele, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 ck tory protein (GCHFR); Viii. a SNP at 9 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein ; ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane n) 1 (FOLHl); a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid ce of reduced folate carrier protein (RCFl); xi. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); xii. a SNP at rs8007267 ion 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 ; xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 19 is a portion of a c nucleic acid sequence of e-phosphate cytidylyltransferase A (PCYT1A); xiV. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine or D2 (DRD2); XV. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvi. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele, wherein the SEQ ID NO. 23 is a portion of a genomic c acid sequence of catechol-O-methyltransferase (COMT); xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. a SNP at rs250682 (position 27 of SEQ ID NO. 25) sing at least one cytosine “C” , wherein the SEQ ID NO. 25 is a portion of a c nucleic acid sequence of solute r family 6 transmitted transported, ne), member 3 (SLC6A3); xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); and xxi. a SNP at rs2236225 ion 27 of SEQ ID NO. 27) comprising at least one alanine “A” , wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFDl); and an optional container containing a detectable label to be conjugated to a nucleotide molecule derived from a test sample of a subject; and at least one reagent. 72. A kit comprising: an oligonucleotide array affixed with a ity of oligonucleotide probes that interrogate no more than 5 single nucleotide rphisms (SNPs), said SNPs comprising: (i) a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); (ii) a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 ified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); an optional container containing a detectable label to be conjugated to a nucleotide molecule derived from a test sample of a subject; and at least one reagent. 73. The kit of paragraph 71 or 72, wherein the at least one reagent is selected from the group consisting of a restriction , a universal r to be conjugated to a nucleotide molecule, a primer complementary to the universal adaptor, a wash agent, and any combinations thereof. 74. The kit of any of paragraphs 71-73, wherein the detectable label comprises a fluorescent molecule. 75. A kit comprising: a plurality of ucleotide primers that bind to at least one allele of no more than 30 single nucleotide polymorphisms , wherein each subset of oligonucleotide s that bind to a ic allele of a SNP is labeled with a distinct reporter, and wherein said SNPs comprise at least two of the following SNPs or any combinations thereof: i. a single nucleotide rphism (SNP) at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 ified by rs1801133) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a n of a c nucleic acid sequence of methionine synthase (MTR); iV. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one e “A” allele, wherein the SEQ ID NO. 11 is a portion of a genomic c acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC); Vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” , wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); vii. a SNP at 862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 13 is a n of a genomic c acid sequence of GTP cyclohydrolase 1 ck regulatory protein (GCHFR); viii. a SNP at rs12659 (position 27 of SEQ ID NO. 14) sing at least one thymine “T” allele, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of d folate carrier protein (RCF2); ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); x. a SNP at rs2297291 (position 27 of SEQ ID NO. 16) sing at least one alanine “A” allele, wherein the SEQ ID NO. 16 is a n of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); xi. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein ; xii. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 18 is a portion of a c nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A); xiv. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one e “T” allele, wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of ne receptor D2 (DRD2); xv. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine or D2 (DRD2); xvi. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” , wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); XViii. a SNP at rs4680 ion 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one ne “C” allele, wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3); xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) sing at least one thymine “T” allele, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); and xxi. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l (MTHFDl); and at least one t. 76. A kit comprising: a plurality of oligonucleotide primers that bind to at least one allele of no more than 5 single nucleotide rphisms (SNPs), wherein each subset of ucleotide primers that bind to a specific allele of a SNP is d with a distinct reporter, and wherein said SNPs se the following SNPs: (i) a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid ce of methylenetetrahydrofolate reductase (MTHFR); (ii) a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each ndently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); and at least one reagent. 77. The kit of any of paragraphs 75-76, wherein said at least one reagent comprises free nucleotide bases, a rase, or both. 78. A kit for selecting a treatment regimen for a subject with depression, comprising at least one reagent for ining in a test sample of a human t diagnosed as haVing depression or haVing a risk for depression, the ce or e of the following SNPs : i. a single nucleotide polymorphism (SNP) at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” , wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); and ii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele, wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine se (MTR); and instructions for use in the assay of any of paragraphs 1-69, in the method of any of paragraphs 70-129, or in the system of any of paragraphs 130-171, or any combinations thereof. 79. The kit of paragraph 78, wherein said SNPs further comprises one or any combination of the ing: i. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 1 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele, wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); iii. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one e “A” allele, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid ce of calcium l, voltage-dependent, L type, alpha 1C subunit (CACNAlC); iV. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); V. a SNP at rs7l63862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 ck regulatory protein (GCHFR); Vi. a SNP at rs12659 (position 27 of SEQ ID NO. 14) comprising at least one e “T” allele, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2); Vii. a SNP at rs202676 ion 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 15 is a portion of a genomic c acid sequence of folate hydrolase (prostate-specific ne antigen) 1 (FOLHl); Viii. a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); ix. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” , wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid ce of reduced folate carrier protein (RCFl); x. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); xi. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one e “A” allele, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A); xii. a SNP at rs6275 (position 27 of SEQ ID NO. 20) sing at least one thymine “T” allele, wherein the SEQ ID NO. 20 is a n of a c nucleic acid ce of dopamine receptor D2 (DRD2); xiii. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele, wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xiV. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 22 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); xv. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” , wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xvi. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xvii. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” , wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitted orted, dopamine), member 3 (SLC6A3); xviii. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) sing at least one thymine “T” allele, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); and xix. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFDl). 80. The kit of any of the preceding paragraphs, further comprising a solid substrate support d with at least one protein-based binding moiety that specifically binds to a biomarker selected from the group consisting of SAM, SAH, 4-HNE and hsCRP. 81. The kit of paragraph 80, n the protein-based binding moiety comprises an antibody. 82. The kit of paragraph 80 or 81, wherein the solid substrate support is a microtiter plate for ELISA. 83. The kit of paragraph 80 or 81, wherein the solid substrate support is a dipstick. 84. The kit of paragraph 80 or 81, wherein the solid substrate support comprises a magnetic bead. 85. The kit of any of the preceding paragraphs, further comprising at least one primer designed to probe a biomarker selected from the group consisting of SAM, SAH, 4-HNE, and hsCRP. 86. The kit of any of the preceding paragraphs, wherein the depression is major depressive disorder. 87. A method for selecting a ent regimen for a human subject with depression, comprising: a. obtaining a test sample from the human subject sed as haVing depression; b. subjecting the test sample to at least one analysis to determine parameters of at least two biomarkers, wherein the parameters of said at least two kers are selected from the following: i. pe of a SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by 133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid ce of methylenetetrahydrofolate ase (MTHFR); ii. genotype of a SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iii. genotype of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); iV. genotype of a SNP locus at on 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); . genotype of a SNP locus at rs1006737 ion 27 of SEQ ID NO. 11), wherein the SEQ ID NO. 11 is a n of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC); Vi. pe of a SNP locus at rs1883729 (position 27 of SEQ ID NO. 12), wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); Vii. pe of a SNP locus at rs7163862 (position 27 of SEQ ID NO. 13), n the SEQ ID NO. 13 is a n of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein ); Viii. genotype of a SNP locus at rs12659 (position 27 of SEQ ID NO. 14), wherein the SEQ ID NO. 14 is a portion of a c nucleic acid sequence of reduced folate carrier protein (RCF2); ix. genotype of a SNP locus at rs202676 (position 27 of SEQ ID NO. 15), wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); . genotype of a SNP locus at rs2297291 (position 27 of SEQ ID NO. 16), n the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); xi. genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17, wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)); xii. genotype of a SNP locus at rs8007267 (position 27 of SEQ ID NO. 18), wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); xiii. genotype of a SNP locus at 752 (position 27 of SEQ ID NO. 19), wherein the SEQ ID NO. 19 is a portion of a c nucleic acid ce of choline-phosphate cytidylyltransferase A (PCYTlA); xiV. genotype of a SNP locus at rs6275 (position 27 of SEQ ID NO. 20), wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); XV. genotype of a SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvi. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvii. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23), wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xviii. genotype of a SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a c nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. genotype of a SNP locus at rs250682 (position 27 of SEQ ID NO. 250, wherein the SEQ ID NO. 25 is a n of a genomic nucleic acid ce of solute carrier family 6 (neurotransmitted transported, ne), member 3 (SLC6A3); XX. genotype of a SNP locus at rs2277820 (position 27 of SEQ ID NO. 26), wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of inotransferase cyclodeaminase (FTCD); xxi. genotype of a SNP locus at rs2236225 (position 27 of SEQ ID NO. 27), wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFDl); xxii. expressions of SAM and SAH; 2012/065084 xxiii. sion of 4-HNE; xxiV. expression of hsCRP; and any combinations thereof; and c. determining, from the parameters of said at least two biomarkers, the presence of at least one condition selected from the following: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele; iii. a SNP at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; iV. a SNP at on 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele; V. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele; Vi. a SNP at rs1883729 ion 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele; Vii. a SNP at rs7l63862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; Viii. a SNP at 9 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” allele; ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; x. a SNP at rs229729l (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” ; xi. a SNP at 266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; xii. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) sing at least one thymine “T” allele; xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele; xiV. a SNP at rs6275 (position 27 of SEQ ID NO. 20) comprising at least one thymine “T” allele; xv. a SNP at 596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele; xvi. a SNP at rsl 1240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele; xviii. a SNP at rs4680 ion 27 of SEQ ID NO. 24) sing at least one guanine “G” allele; xix. a SNP at 82 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele; xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele; and xxi. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele; xxii. an sion ratio of SAM to SAH smaller than a pre-determined reference ratio; xxiii. an expression of 4-HNE greater than a pre-determined reference value; xxiv. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample; and any combinations thereof; d. providing a result output setting forth whether at least one of said condition is detected from the test sample and if at least one condition is detected, then selecting and optionally administering a treatment n comprising an effective amount of a folate-containing compound to the human subject. 88. A method for selecting a ent regimen for a subject with depression, comprising: a. obtaining a test sample from the human subject diagnosed as having depression; b. subjecting the test sample to at least one analysis to determine parameters of at least two biomarkers, wherein the parameters of said at least two biomarkers comprise the following: i. pe of a SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a n of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. pe of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); c. determining, from the determined parameters of said at least two biomarkers, the presence of at least one condition of the following or a combination thereof : i. a SNP at position 677 of SEQ ID NO. 1 comprising at least one thymine “T” allele; ii. a SNP at position 2756 of SEQ ID NO. 2 sing at least one e “G” allele; d. providing a result output setting forth whether at least one of said condition is detected from the test sample and if at least one condition or both is detected, then selecting and optionally administering a ent regimen sing an effective amount of a folate-containing compound to the human t. 89. The method of aph 88, wherein the test sample is r subjected to determine a parameter of at least one of the following biomarkers or any combinations thereof: i. genotype of a SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); ii. genotype of a SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by 394), wherein the SEQ ID NO. 3 and SEQ ID NO. are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); iii. genotype of a SNP locus at rs1006737 (position 27 of SEQ ID NO. 11), wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C t (CACNAIC); iV. genotype of a SNP locus at rsl883729 (position 27 of SEQ ID NO. 12), wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B); V. genotype of a SNP locus at rs7l63862 (position 27 of SEQ ID NO. 13), n the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR); Vi. genotype of a SNP locus at rsl2659 (position 27 of SEQ ID NO. 14), wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid ce of reduced folate carrier protein (RCF2); 2012/065084 Vii. genotype of a SNP locus at rs202676 (position 27 of SEQ ID NO. 15), wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLHl); Viii. genotype of a SNP locus at rs2297291 (position 27 of SEQ ID NO. 16), wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF 1 ); ix. genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17, wherein the SEQ ID NO. 17 is a n of a c nucleic acid sequence of reduced folate carrier protein (RCF1)); . genotype of a SNP locus at rs8007267 (position 27 of SEQ ID NO. 18), wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); xi. genotype of a SNP locus at rs7639752 (position 27 of SEQ ID NO. 19), wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline- phosphate cytidylyltransferase A (PCYTlA); xii. genotype of a SNP locus at rs6275 ion 27 of SEQ ID NO. 20), wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid ce of dopamine receptor D2 (DRD2); xiii. genotype of a SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xiV. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), n the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 ; XV. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23), wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O- methyltransferase (COMT); xvi. genotype of a SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O- methyltransferase (COMT); xvii. pe of a SNP locus at rs250682 (position 27 of SEQ ID NO. 25), wherein the SEQ ID NO. 25 is a n of a c c acid ce of solute carrier family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3); xviii. genotype of a SNP locus at rs2277820 (position 27 of SEQ ID NO. 26), n the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD); xix. genotype of a SNP locus at rs2236225 (position 27 of SEQ ID NO. 27), wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) l l); xx. sions of SAM and SAH; xxi. sion of 4-HNE; and xxii. expression of hsCRP. 90. The method of paragraph 89 further comprising determining the presence of at least one of the following conditions or any combinations thereof: i. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele; ii. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" ; iii. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one e “A” allele; iV. a SNP at rsl883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele; V. a SNP at rs7l63862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; Vi. a SNP at rsl2659 (position 27 of SEQ ID NO. 14) comprising at least one e “T” allele; Vii. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; Viii. a SNP at rs229729l (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” ; ix. a SNP at rs1051266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; x. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele; xi. a SNP at 752 ion 27 of SEQ ID NO. 19) comprising at least one alanine “A” allele; xii. a SNP at rs6275 (position 27 of SEQ ID NO. 20) sing at least one thymine “T” allele; xiii. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” ; xiv. a SNP at rs11240594 (position 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xv. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele; xvi. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xvii. a SNP at rs250682 (position 27 of SEQ ID NO. 25) comprising at least one cytosine “C” allele; xviii. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele; and xix. a SNP at rs2236225 (position 27 of SEQ ID NO. 27) comprising at least one alanine “A” allele; xx. an expression ratio of SAM to SAH smaller than a pre-determined reference ratio; xxi. an expression of 4-HNE greater than a pre-determined reference value; and xxii. an expression of hsCRP r than about 2.3 mg per liter of plasma as measured in a plasma sample. 91. The method of any of the preceding paragraphs, wherein the predetermined nce value is about 3.0 mg per liter as measured in a plasma . 92. The method of any of the preceding paragraphs, wherein the predetermined reference value is about 3.2 mg per liter as measured in a plasma sample. 93. The method of any of the preceding aphs, wherein the step (b) further comprises optionally packing and shipping the test sample to a test facility. 94. The method of paragraph 93, wherein the test facility is a third-party CLIA-certified service provider. 95. The method of any of the preceding paragraphs, wherein the step (d) is med by a non- human machine. 96. The method of any of the preceding aphs, further comprising determining an obesity indicator (e.g., measuring a BMI value) of the subject. 97. The method of any of the preceding paragraphs, wherein at least three of the foregoing biomarker parameters are determined. 98. The method of any of the preceding paragraphs, wherein the depression is major depressive disorder. 99. A folate-comprising composition for use in the ent of depression in a human subject who carries at least one of the following single nucleotide polymorphisms or a combination thereof: a. at least one thymine “T” allele at SNP677, wherein the SNP677 is at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); and b. at least one e “G” allele at SNP2756, n the SNP2756 is at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine se (MTR). 100. A folate-comprising composition for use in the treatment of depression in a human t who carries at least one of the ing conditions or any combination thereof: a. at least one thymine "T" allele at SNP locus at position 677 of SEQ ID NO. 1 or on 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each ndently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase ); b. at least one alanine "A" allele at SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); c. at least one guanine "G" allele at SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine synthase (MTR); d. at least one guanine "G" allele at SNP locus at position 66 of SEQ ID NO. 3 or on 27 of SEQ ID NO. 10 (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a n of a genomic nucleic acid sequence of methionine synthase ase (MTRR); e. at least one alanine "A" allele at SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of m channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC)); f. at least one alanine "A" allele at SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, wherein the SEQ ID NO. 12 is a n of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B)); g. at least one thymine "T" allele at SNP locus at rs7163862 (position 27 of SEQ ID NO. 13, n the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR)) ; h. at least one thymine "T" allele at SNP locus at rs12659 (position 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2)); i. at least one guanine "G" allele at SNP locus at rs202676 (position 27 of SEQ ID NO. , n the SEQ ID NO. 15 is a portion of a c nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1)); j. at least one alanine "A" allele at SNP locus at rs2297291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid ce of reduced folate carrier protein (RCF1)) ; k. at least one alanine "A" allele at SNP locus at rs1051266 (position 27 of SEQ ID NO. 17), wherein the SEQ ID NO. 17 is a portion of a genomic c acid sequence of reduced folate carrier protein (RCFl); l. at least one thymine "T" allele at SNP locus at rs8007267 (position 27 of SEQ ID NO. 18, wherein the SEQ ID NO. 18 is a n of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1)); m. at least one alanine "A" allele at SNP locus at rs7639752 (position 27 of SEQ ID NO. 19, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A)); n. at least one e "T" allele at SNP locus at rs6275 (position 27 of SEQ ID NO. , wherein the SEQ ID NO. 20 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2)); 0. at least one thymine "T" allele at SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); p. at least one alanine "A" allele at SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); q. at least one cytosine "C" allele at SNP locus at rs4633 (position 27 of SEQ ID NO. 23, wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid ce of catechol-O-methyltransferase (COMT)); r. at least one e "G" allele at SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); s. at least one ne "C" allele at SNP locus at rs250682 (position 27 of SEQ ID NO. , wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute r family 6 (neurotransmitted transported, dopamine), member 3 (SLC6A3)); t. at least one thymine "T" allele at SNP locus at rs2277820 (position 27 of SEQ ID NO. 26, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD)); and u. at least one alanine "A" allele at SNP locus at rs2236225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFD1)); V. obesity (e. g., defined by BMI value of at least 30 kg/m2 or r); w. an sion level ratio of SAM to SAH smaller than a termined reference ratio; x. an expression level of 4-HNE greater than a pre-determined nce value; and y. an expression of hsCRP greater than about 2.3 mg per liter of plasma as ed in a plasma sample. 101. A folate-comprising composition in combination with an anti-depressant for use in the treatment of depression in a human t who carries at least one of the following single nucleotide polymorphisms or a combination thereof: a. a SNP677 at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one thymine “T” allele; and b. a SNP2756 at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele. 102. A folate-comprising ition in combination with an anti-depressant for use in the treatment of depression in a human subject who carries at least one of the following conditions or any ation thereof: a. at least one thymine "T" allele at SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a n of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); at least one alanine "A" allele at SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or on 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a n of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); at least one guanine "G" allele at SNP locus at position 2756 of SEQ ID N02 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a n of a genomic nucleic acid sequence of methionine synthase (MTR); at least one guanine "G" allele at SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), n the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); at least one alanine "A" allele at SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit (CACNAlC)); at least one alanine "A" allele at SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, n the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B)); at least one thymine "T" allele at SNP locus at rs7l63862 (position 27 of SEQ ID NO. 13, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR)) ; at least one e "T" allele at SNP locus at rsl2659 (position 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2)); i. at least one guanine "G" allele at SNP locus at rs202676 (position 27 of SEQ ID NO. , n the SEQ ID NO. 15 is a portion of a genomic nucleic acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1)); at least one alanine "A" allele at SNP locus at rs2297291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)) ; at least one alanine "A" allele at SNP locus at rs1051266 (position 27 of SEQ ID NO. 17), wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCFl); at least one thymine "T" allele at SNP locus at 267 (position 27 of SEQ ID NO. 18, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP ydrolase 1 (GCH1)); at least one alanine "A" allele at SNP locus at rs7639752 (position 27 of SEQ ID NO. 19, wherein the SEQ ID NO. 19 is a n of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A)); at least one thymine "T" allele at SNP locus at rs6275 (position 27 of SEQ ID NO. , wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of ne receptor D2 ); at least one thymine "T" allele at SNP locus at 596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); at least one alanine "A" allele at SNP locus at 0594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); at least one cytosine "C" allele at SNP locus at rs4633 (position 27 of SEQ ID NO. 23, wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT)); at least one guanine "G" allele at SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a n of a genomic nucleic acid ce of catechol-O-methyltransferase (COMT); at least one ne "C" allele at SNP locus at rs250682 (position 27 of SEQ ID NO. , wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 transmitted transported, dopamine), member 3 (SLC6A3)); t. at least one thymine "T" allele at SNP locus at rs2277820 (position 27 of SEQ ID NO. 26, wherein the SEQ ID NO. 26 is a n of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD)); and u. at least one alanine "A" allele at SNP locus at rs2236225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFD1)); V. obsesity (e. g., defined by BMI value of at least 30 kg/m2 or greater); W. an expression level ratio of SAM to SAH r than a pre-determined reference ratio; x. an expression level of 4-HNE greater than a pre-determined reference value; and y. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample. 103. The composition of any one of the ing paragraphs, n the folate-comprising composition comprises at least about 5mg of folate. 104. The composition of any one of the preceding paragraphs, wherein the folate-comprising composition comprises about 75-50 mg of folate. 105. The composition of any one of the preceding paragraphs, wherein the -comprising composition further comprises a pre-determined release profile. 106. The ition of paragraph 105, wherein the pre-determined release profile comprises a sustained release profile. 107. The composition of paragraph 106, wherein the ned release is a steady-state release. 108. The composition of paragraph 105, wherein the pre-determined release profile comprises a pulsatile release profile. 109. The ition of paragraph 105, wherein the pre-determined release profile comprises a chrono-controlled release profile. 110. The composition of any of paragraphs 105-109, wherein the -comprising composition is formulated to release at least 30% of the folate-containing nd over a period of at least about 3-6 hours, upon the administration of the ition. 111. The composition of any of the preceding paragraphs, wherein the depression is a major depressive disorder.
Some Selected Definitions For convenience, certain terms employed in the entire application (including the specification, examples, and appended claims) are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It should be understood that this invention is not limited to the particular ology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the e of describing particular ments only, and is not intended to limit the scope of the present invention, which is d solely by the claims.
Other than in the operating examples, or where otherwise ted, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used to described the present invention, in connection with percentages means 11%.
In one aspect, the t invention relates to the herein described compositions, methods, and respective component(s) thereof, as ial to the invention, yet open to the inclusion of unspecified elements, essential or not (“comprising”). In some ments, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the invention (“consisting essentially of”). This applies equally to steps within a described method as well as compositions and ents therein. In other embodiments, the inventions, compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the ent, composition or method (“consisting of”).
All patents, patent ations, and publications identified are sly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such ations that might be used in connection with the present ion. These publications are provided solely for their sure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or entation as to the contents of these documents is based on the information available to the applicants and does not tute any admission as to the correctness of the dates or contents of these documents.
The term “adjuvant” as used herein generally refers to any agent or entity which increases the effect of another agent or entity. In certain embodiments, the term “adjuvant” is used herein in reference to a -containing compound as an adjuvant to increase or enhance the effect (e.g., efficacy and/or therapeutic effect) of an antidepressant drug.
] As used herein, the term “polyglutamates” refers to folates that have at least two or more glutamate .
] The term “derivative" as used herein refers to a chemical substance related structurally to another, i.e., an "original" substance, which can be referred to as a "parent" nd.
A "derivative" can be made from the structurally-related parent compound in one or more steps. In some embodiments, the general physical and chemical properties of a derivative can be similar to or different from the parent nd.
As used here in the term “isomer" refers to compounds having the same molecular formula but differing in structure. Isomers which differ only in configuration and/or conformation are referred to as “stereoisomers." The term “isomer" is also used to refer to an enantiomer.
The term iomer" is used to describe one of a pair of molecular isomers which are mirror images of each other and non-superimposable. Other terms used to designate or refer to enantiomers include “stereoisomers" (because of the different arrangement or stereochemistry around the chiral center; although all enantiomers are stereoisomers, not all stereoisomers are omers) or “optical isomers" (because of the optical activity of pure enantiomers, which is the ability of different pure enantiomers to rotate plane polarized light in different directions). Enantiomers generally have identical physical ties, such as melting points and boiling points, and also have cal spectroscopic properties. Enantiomers can differ from each other with respect to their ction with plane polarized light and with t to biological activity.
The designations “R " and “ S " are used to denote the absolute configuration of the molecule about its chiral (s). The designations may appear as a prefix or as a suffix; they may or may not be separated from the isomer by a hyphen; they may or may not be hyphenated; and they may or may not be surrounded by parentheses. The designations or prefixes “(+) and (-)" are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) g that the compound is levorotatory (rotates to the left). A nd prefixed with (+) is dextrorotatory (rotates to the right).
The term “racemic mixture" refers to a mixture of the two omers of one compound, in any ratio. An ideal racemic mixture is one wherein there is a 50:50 mixture of both enantiomers of a compound such that the optical rotation of the (+) enantiomer cancels out the optical rotation of the (-) enantiomer.
The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., strand) of DNA, RNA or a derivative or analog f, comprising a nucleobase. A nucleobase includes, for e, a naturally occurring purine or pyrimidine base found in DNA (e.g. an adenine "A," a guanine "G" a thymine "T" or a cytosine "C") or RNA (e.g. an A, a G. an uracil "U" or a C). The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid." The term "oligonucleotide" refers to a le of between about 3 and about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule of greater than about 100 nucleobases in length.
The term "nucleic acid sequence" refers to a single or -stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5'—to the 3'—end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role. "Nucleic acid sequence" also refers to a consecutive list of abbreviations, s, characters or words, which represent nucleotides. In one embodiment, a nucleic acid can be a "probe" which is a vely short nucleic acid, usually less than 100 nucleotides in length.
The term "oligonucleotide," as used herein refers to s and probes bed herein, and is defined as a nucleic acid molecule comprised of at least two or more ribo- or ibonucleotides. The exact size of the oligonucleotide will depend on s factors and on the particular application and use of the ucleotide. The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and the method used. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe typically ns 15-25 or more tides, although it may n fewer nucleotides. The probes as disclosed herein are selected to be substantially complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non- complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target . Alternatively, non- complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarily with the sequence of the target nucleic acid to anneal therewith specifically.
In the context of some embodiments of various aspects described herein, the term " refers to a molecule which can detectably distinguish between target molecules differing in structure (e. g. nucleic acid or protein sequence). ion can be accomplished in a variety of different ways depending on the type of probe used and the type of target molecule. Thus, for example, detection may be based on discrimination on detection of specific binding. es of such specific binding include antibody binding and nucleic acid, antibody binding to protein, c acid binding to nucleic acid, or aptamer binding to protein or nucleic acid. Thus, for example, probes can include enzyme substrates, antibodies and dy fragments, and preferably nucleic acid hybridization probes.
The term "specifically hybridize" refers to the association between two singlestranded nucleic acid molecules of ient mentary sequence to permit such hybridization under pre-determined ions generally used in the art (sometimes the sequences are referred to as "substantially complementary"). In particular, the term ically hybridize also refers to hybridization of an oligonucleotide with a substantially complementary sequence as ed to non- mentary sequence.
The term “specifically” as used herein with reference to a probe which is used to specifically detect a sequence difference, refers to a probe that identifies a particular ce ence based on exclusive hybridization to the sequence difference under stringent ization conditions and/or on exclusive amplification or replication of the sequence difference.
In its broadest sense, the term "substantially” as used herein in respect to “substantially complementary", or when used herein with respect to a nucleotide sequence in relation to a reference or target nucleotide sequence, means a nucleotide sequence having a percentage of identity between the substantially complementary nucleotide sequence and the exact complementary sequence of the reference or target nucleotide sequence of at least 60%, at least 70%, at least 80% or 85%, at least 90%, at least 93%, at least 95% or 96%, at least 97% or 98%, at least 99% or 100% (the later being equivalent to the term "identical" in this t). For example, identity is assessed over a length of at least 10 nucleotides, or at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or up to 50 nucleotides of the entire length of the nucleic acid sequence to the reference sequence (if not specified otherwise below). Sequence comparisons can be carried out using default GAP analysis with the University of Wisconsin GCG, SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453; as defined above). A nucleotide ce "substantially complementary" to a reference nucleotide sequence izes to the reference nucleotide sequence under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions.
In its broadest sense, the term antially identical", when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference or target nucleotide sequence, wherein the percentage of identity n the substantially identical nucleotide ce and the reference or target nucleotide sequence is at least 60%, at least 70%, at least 80% or 85%, at least 90%, at least 93%, at least 95% or 96%, at least 97% or 98%, at least 99% or 100% (the later being equivalent to the term "identical" in this context). For example, ty is assessed over a length of 10-22 nucleotides, such as at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22 or up to 50 nucleotides of a nucleic acid sequence to the nce sequence (if not specified otherwise below).
Sequence comparisons are carried out using default GAP analysis with the University of Wisconsin GCG, SEQWEB application of GAP, based on the algorithm of Needleman and Wunsch (Needleman and Wunsch (1970) J Mol. Biol. 48: 443-453; as d . A nucleotide sequence "substantially identical" to a reference nucleotide sequence hybridizes to the exact complementary sequence of the reference nucleotide sequence (i.e. its corresponding strand in a double-stranded molecule) under low stringency conditions, preferably medium stringency conditions, most preferably high stringency conditions (as defined above). Homologues of a specific nucleotide sequence include nucleotide sequences that encode an amino acid sequence that is at least 24% identical, at least 35% identical, at least 50% identical, at least 65% cal to the reference amino acid sequence, as measured using the parameters described above, wherein the amino acid sequence d by the homolog has the same biological activity as the protein encoded by the specific nucleotide. The term “substantially non- identical” refers to a nucleotide ce that does not hybridize to the nucleic acid ce under stringent conditions. The term "substantially identical", when used herein with respect to a ptide, means a n corresponding to a reference polypeptide, wherein the polypeptide has ntially the same structure and function as the reference protein, e.g. where only changes in amino acids sequence not affecting the polypeptide function occur. When used for a polypeptide or an amino acid sequence, the percentage of identity between the substantially similar and the reference polypeptide or amino acid sequence is at least 24%, at least 30%, at least 45%, at least 60%, at least 75%, at least 90%, at least 95%, at least 99%, using default GAP analysis parameters as bed above. gues are amino acid ces that are at least 24% identical, more preferably at least % identical, yet more preferably at least 50% identical, yet more preferably at least 65% identical to the reference polypeptide or amino acid sequence, as measured using the parameters described above, wherein the amino acid sequence encoded by the homolog has the same biological activity as the reference polypeptide.
The term "primer" as used herein refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.
When presented with an riate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH, the primer may be extended at its 3' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield a primer ion product. The primer may vary in length depending on the ular conditions and requirement of the application. For example, in stic applications, the oligonucleotide primer is typically 15-25 or more tides in length.
The primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3' hydroxyl moiety of the primer in riate juxtaposition for use in the initiation of synthesis by a polymerase or similar . It is not required that the primer sequence represent an exact complement of the desired template. For example, a mplementary nucleotide ce may be attached to the 5' end of an otherwise complementary . Alternatively, non- complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient mentarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
The term "complementary" or “complement” as used herein refers to the broad concept of sequence complementarity n regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is anti-parallel to the first region if the residue is thymine or uracil.
Similarly, it is known that a cytosine residue of a first c acid strand is capable of base pairing with a residue of a second nucleic acid strand which is anti-parallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an anti-parallel fashion, at least one nucleotide e of the first region is capable of base pairing with a residue of the second region.
Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are ed in an anti-parallel fashion, such that at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% or at least 100% of the tide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all tide residues of the first portion are capable of base pairing with nucleotide residues in the second n.
The terms “variant”, “variance”, “mutation” or “polymorphism” are used interchangeably herein, and refer to a difference in nucleic acid sequence among members if a population of individuals. Polymorphisms can sometimes be referred to as e nucleotide polymorphism” or “SNP” when they vary at a single tide. In some embodiments, polymorphisms can be mous or nonsynonymous. Synonymous polymorphisms when present in the coding region or non-coding region typically do not result in an amino acid change, but can result in altered mRNA stability or altered alternative splice sites. Nonsynonymous polymorphism, when present in the coding , can result in the alteration of one or more codons resulting in an amino acid replacement in the amino acid chain. Such mutations and polymorphisms may be either heterozygous or homozygous within an dual. gous individuals have identical alleles at one or more corresponding loci on gous chromosomes, while heterozygous individuals have two different alleles at one or more corresponding loci on homologous chromosomes. A polymorphism is thus said to be ic," in that, due to the existence of the polymorphism, some members of a species carry a gene with one sequence (e. g., the normal or wild-type "allele"), whereas other members may have an altered sequence (e. g., the variant or, mutant "allele").
] The term "genotype" refers to the specific allelic composition of an entire cell or a certain gene, whereas the term "phenotype” refers to the detectable outward manifestations of a specific genotype.
The term "allele", as used herein, refers to one member of a pair of different forms of a gene. As used herein alleles refer to coding and to non-coding ces. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the t is said to be homozygous for the gene or allele. When a t has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or l nucleotides, and can include substitutions, deletions and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.
As used herein, the term “administer" refers to the ent of a composition into a subject by a method or route which results in at least l localization of the composition at a desired site such that desired effect is produced. Routes of administration suitable for the methods described herein can include both local and systemic administration. Generally, local administration results in a higher amount of an antidepressant (e.g., SSRI) and/or a folate-containing compound being delivered to a ic location (e.g., serotonin receptors in the central and/or eral nervous systems) as compared to the entire body of the subject, whereas, ic administration results in delivery of an pressant (e.g., SSRI) and/or a folate-containing compound to essentially the entire body of the subject. In some ments, the compositions described herein are stered to subjects with depression orally. In other embodiments, the compositions described herein can be administered to subjects with depression by injection.
EXAMPLES The es ted herein, in part, relate to the use of a folate-containing compound, e.g., 6(S)MTHF, alone or as an adjunct to an antidepressant drug for treating a patient with depression, e.g., major depressive disorder (MDD). The examples presented herein also relate to methods to identify genetic polymorphisms, peripheral biomarkers and clinical features involved in selecting patients with depression for receiving a folate-containing compound, e.g., as an adjunctive treatment to an antidepressant drug, e.g., a ive serotonin reuptake inhibitor (SSRI). Throughout this application, various publications are referenced. The sures of all of the publications and those nces cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The following examples are not intended to limit the scope of the paragraphs to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods which occur to the skilled artisan are intended to fall within the scope of the present invention.
Example I. A -blind, placebo controlled study of a folate-containing compound among SSRI— resistant outpatients with major depressive er (MDD) Exemplary Study Design Using the sequential parallel design [51] (see, for example, Figs. 1A-1B, and 2), the 60-day, double-blind treatment, either administration with a folate-containing nd (e. g., a 6(S)- ) or a placebo as an adjunct to an SSRI, can be divided into two phases of 30 days each, with assessments performed every 10 days. During the first phase of the double-blind treatment, eligible patients can be ized to 30 days of treatment with either 6(S)MTHF (15 mg/day) (n=19) or placebo , with a 2:33 ratio for random assignment to the treatment sequences drug/drug (referring to 6(S)MTHF), placebo/placebo, and placebo/drug. By way of example only, if there is a % ut rate during the first phase, 50 patients on placebo would te the first 30-day phase, 17 patients on 15 mg/day -MTHF would complete the first phase. Patients randomly assigned to the drug/drug sequence stay on the same 6(S)MTHF dose (15 mg/day) during the second phase, regardless of whether patients have responded during the first phase or have not. For those ly assigned to the placebo/placebo sequence, both responders and non-responders to placebo during the first phase (n=25) remain on placebo during the second phase. On the other hand, for those randomly assigned to the placebo/drug ce, both responders and non-responders to placebo during the first phase (n=25) go on to receive 15 mg/day 6(S)MTHF during the second phase. In some embodiments, some of the placebo-treated patients (e. g., about 27% of the placebo- treated patients) can respond during the first phase, and that a portion of the remaining placebo non- responders (e. g., about 9% of the placebo non-responders) during the first phase can go on to respond during the second phase, while a larger portion of 6(S)MTHF-treated patients (e.g., about 48% of the 6(S)-5 -MTHF-treated patients) can respond during the first phase, and that a portion of the placebo non-responders (e.g., about 35% of placebo non-responders) during the first phase can go on to respond to 15 mg/day 6(S)MTHF during the second phase. Data from the previous - blind, placebo-controlled study (Trial 1) can be pooled into the instant study (Trial 2) to provide a more te estimate of the placebo effect size, with a ed average from the two Phases 1 (one from Trial 1 and one from Trial 2) and two Phases 2 (one from Trial 1 and one from Trial 2). With 148 patients randomized to placebo during phase 1 or to stay on placebo during phase 2 following non-response to placebo in Trial 1; and with patients randomized to placebo during phase 1 (n=56) or to stay on placebo during phase 2 following non-response to placebo in Trial 2 (n=18) in Trial 2, and a weighted average response rate of approximately 19% across Trials 1 and 2 (total n=222); and patients randomized to 15 mg/day 6(S)MTHF during phase 1 (n=19) or to 15 mg/day 6(S) MTHF during phase 2 following non-response to placebo (n=18) and a weighted average response rate of approximately 41.5% (total n=37) in Trial 2, the statistical power to show a drug-placebo difference in response rates is greater than 0.8.
Subject Selection Inclusion Criteria: (1) Age 18-65; (2) Written informed consent; (3) Meet DSM-IV criteria (by Structured Clinical Interview for DSM-IV - /P) for MDD, t patients; (4) Quick Inventory of Depressive Symptomatology — Self-Rated (QIDS-SR) [52] score of at least 12 at both screen and baseline visits; (5) Patients treated with an SSRI at adequate doses, e. g., as shown in Table 1 (defined as 20mg/day or more of fluoxetine, citalopram, or paroxetine, 10 mg/day or more of escitalopram, and ay or more of sertraline) during the current episode for at least 8 weeks; and (6) During the baseline visit, patients must be on a stable dose of SSRI for the past 4 weeks.
In some embodiments, 40% of patients ed for Trial 2 study were on starting doses. In some embodiments, 90% of patients ed for Trial 2 study had not been maximized in therapeutic dose range.
Table I. Mean doses of SSRIS at baseline — Trial 2 _Dose (number of patients) mg (6) 40 mg (10) 60 mg (2) 35.5 mg mg (3) 40 mg (8) 60 mg (2) 38.5 mg mg (2) 30 mg (1) 40 mg (1) 27.5 mg mg (4) 20 mg (7) 30 mg (1) 17.5 mg 50 mg (4) 100 mg (9) 200 mg (1) 92.9 mg Exclusion Criteria: (1) Pregnant women or women of child bearing potential who are not using a medically accepted means of ception (to include oral contraceptive or implant, condom, diaphragm, spermicide, intrauterine device, tubal ligation, or partner with vasectomy); (2) Patients who no longer meet DSM-IV criteria for MDD during the baseline visit; (3) Patients who demonstrate a greater than 25% decrease in depressive symptoms as reflected by the QIDS-SR total score - screen to baseline; (4) Patients with serious suicide or homicide risk, as assessed by ting clinician; (5) Patients with unstable medical illness including cardiovascular, hepatic, renal, respiratory, endocrine, neurological, or logical disease; (6) Patients with the following DSM- IV diagnoses: nce use disorders active within the last six months, any bipolar disorder nt or past), any psychotic disorder (current or past); (7) ts with a history of a seizure disorder or clinical evidence of untreated hypothyroidism; (8) Patients requiring excluded medications (see Table 2 for details); (9) Patients with psychotic features in the current episode or a history of psychotic features, as assessed by SCID; (10) Patients with prior course of MTHF augmentation, or rance to MTHF at any dose; (1 1) Patients with any investigational psychotropic drug within the last 3 months; (12) Patients who have failed more than 2 adequate antidepressant trials during the current Major sive Episode. Some es of te dosage of an antidepressant trial include either greater than 150 mg of imipramine (or its tricyclic equivalent), greater than 60 mg of phenelzine (or its monoamine oxidase inhibitor equivalent), greater than 20 mg of fluoxetine (or its SSRI-equivalent), greater than 150mg of bupropion, greater than 300mg of trazodone (or nefazodone), or greater than 150 mg of venlafaxine. A trial of adequate duration was defined as one during which the patient was on any given antidepressant at an adequate dose for a minimum of 6 weeks; and (13) Patients with a history of antidepressant-induced nia.
] Human Subjects Involvement and Characteristics: A total of 75 individuals age 18- 65 with MDD are involved. Subjects must be medically stable as defined in the protocol bed herein. Patients excluded from the study include ts at acute risk for suicide, active substance abuse or dependence, with mild depression, inadequately treated with SSRIs or who have failed more than 2 , who have received MTHF for depression in the past, and those with psychosis or bipolar illness. Patients below the age of 18 or over the age of 65 are also excluded.
Drugs allowed or excluded as concomitant medications during the study Drugs that may be given to the patient include any iption or OTC medication such as aspirin, acetaminophen and cold ations not specifically excluded by the protocol.
Patients ing concomitant drug therapy with excluded drugs are discontinued from the study.
Table 2 shows a list of drugs allowed (Y) and not allowed (N) as concomitant medications. Some of the drug classes in Table 2 have a numeric value within parentheses, referring to additional notes shown below.
Table 2.‘ Concomitant drug therapy K VliSt'KrK‘K'iii‘ Kw Chm-n in: K31: ‘ IsiII'IHI-airteuté cf} ‘21? nix-KKK: Emma}mum K ‘I. I ‘ a} I Innmfhian HI: Iilcnmux 5in N (l) Benzodiazepine anxiolytics and non-benzodiazepine ve-hypnotics noted above are allowed only if subjects are on a stable regimen for at least 2 weeks prior to baseline at doses no greater than the ing or their equivalent: clonazepam 1.0 qd and zolpidem 10 mg qhs. (2) Patients must have been treated prior to study entry with adequate doses of SSRIs (e. g. minimum doses: fluoxetine/paroxetine/citalopram 20mg/day, escitalopram lOmg/day, sertraline 50 mg/day) for at least 8 weeks with only non-response. They must be on an SSRI at the time of study enrollment and they must have been at the current dose for at least 4 weeks at the ne Visit.
Patients must also agree to continue to take their SSRI medication at the same dose while being treated with 6(S)MTHF. If patients are on other tropic drugs as well, they must have been at the current dose of the psychotropic drug for at least 4 weeks, and they must also agree to continue to take their medication at the same dose while being treated with 6(S)MTHF. (3) Propranolol, metoprolol, acebutolol, ine, clonidine and aldomet are excluded. (4) Adequate thyroid replacement which has been stable for 6 months or more is able as is estrogen replacement for post-menopausal women or the use of oral contraceptives, the initiation of which does not coincide with the onset or exacerbation of depression. (5) Standard multivitamins with or without minerals are allowed (with no more than 400 mcg folate and 6 mcg B 12) if initiated at least 12 weeks prior to Baseline. Dietary supplements with putative CNS activity are excluded including SAMe, St. John's Wort, DHEA, Inositol, Ginko biloba and Omegafatty acids including DHA and Flax Seed Oil.
Subject Enrollment ] Seventy-five subjects enter double-blind treatment over 12 months (Trial 2, total ment Trials 1 and 2 is 225). This trial is conducted according to the FDA guidelines. Written informed consent is obtained from all patients before protocol-specified procedures are d out.
The subjects are drawn primarily from an outpatient sample of patients with MDD, diagnosed by the use of the Structured Clinical Interview for DSM-IV Axis I Disorders - Patient Edition (SCID-I/P).
At study entry, subjects must meet SCID ia for a depressive episode, and have a QIDS-SR score of at least 12 at both the screen and ne visits. In addition, their current major depressive e (MDE) must be considered resistant to SSRIs: during the current MDE, all patients must have received at least one prior trial of an SSRI at an adequate dose and duration, as defined by the MGH Antidepressant Treatment Response Questionnaire (MGH-ATR) [53]. The MGH-ATR defines an te trial of SSRIs as 10 mg or more of escitalopram, 20mg or more of fluoxetine, citalopram, or paroxetine, 50mg or more of sertraline for a minimum of 6 weeks. In addition, during the baseline visit, patients must be on a stable dose of SSRI for the past 4 weeks or more.
Study Procedures Once patients agree to participate in the study by signing the informed consent document, a full medical and psychiatric history is taken and a physical ation is performed.
Screen rating scales are performed. Screened and eligible ts are asked to return two weeks later for a baseline visit when they are randomized to double-blind treatment with placebo or 6(S)MTHF mg/day with the study design outlined above. The double-blind treatment lasts 60 days, during which patients are seen every 10 days (Visits 1 to 6 of Phase 1 — See Table 4 below). Subjects are ed randomization numbers in consecutive order. The randomization list is provided by a computer-generated random-number list and is ined by the research pharmacist. In addition, the presence of any side effect or adverse event is carefully documented with the SAFTEE-SI [54].
Reasons for premature tinuation, including rable side effects, are ed.
All concomitant medications taken during the study is recorded in the case report form, along with dosage information and start and stop dates. Patients requiring excluded drugs (see Table 2 for details) are discontinued from the study. Medication ment and clinical ratings are performed by the study clinicians.
For patients randomly assigned to the drug/drug sequence, the dose of 6(S)MTHF is 15 mg/day during both phases of the study. For patients randomly assigned to the placebo/drug sequence, the dose of 6(S)MTHF is 15 mg/day during the second phase of the study as well. All patients are asked to take one tablet of blinded study medication in the morning, in addition to their stable dose of g SSRI treatment. Each study medication tablet is either 15 mg of 6(S)MTHF or matching placebo. Therefore, for patients randomly assigned to the placebo/placebo ce, the tablets of study medication are placebo during both phases of the study. For ts randomly assigned to the drug/drug sequence, the tablets are 15 mg of 6(S)MTHF during both phases of the study. For patients randomly assigned to the placebo/drug sequence, the tablets are placebo during the first phase of the study, while the tablets are 15 mg of -MTHF during the second phase of the study.
Subjects unable to tolerate the study tions are withdrawn from the study. ts need to comply with the dosage regimen and to take all medications as instructed. All patients are instructed to return any excess medication at each Visit. A pill count is done to corroborate the study drug record. Protocol Violation is defined as less than 80% compliance by pill count Specimen collection procedure Blood samples should be taken from subjects after overnight fasting. The following specimens as shown in Table 3 are included for metabolic testing.
Table 3: Specimens and metabolic tests Specimen M etabntie Test m vnlume E’Ezmmu hiJEEIOCySmEH e, has-CREE, 3:13. We, .:=\Ej}i‘~v"E..»“=i. 2.5 mi mm, $2153 WMA Scrum it BEEP. {£1de 1 {BE WhoEe bEuuiE hemoEysztte Reel ecEE finite: i mE Who Ee h E0 :11d E‘sfii'EEilE-‘R and RES yping E mE Specimen Collection Instructions Plasma: About 5 ml of blood is withdrawn into a sodium n vacutainer tube.
Specimens should be centrifuged within one hour of collection at ~2000g for ~10 minutes. About 2ml of plasma are aliquoted into 2 plastic storage Vials (~1ml in each). The Vials are labeled and frozen at —80°C.
Serum: About 5 ml of blood is withdrawn into a vacutainer tube (plain red top), which contains no additive. The blood-containing tube is left to stand at room temperature for 20 about minutes and allowed to clot. The sample is then centrifuge at ~2000g for ~10 minutes.
Approximately 2ml of plasma is aliquoted into 2 plastic e Vials (~1ml in each). The Vials are labeled and frozen at —20°C or -80°C.
Whole blood Hemolysate: 100 [1L of fully suspended heparinized blood is accurately pipetted into a test tube with a pre-made solution, followed by vortexing and freezing at —20°C or - 80°C.
Whole Blood: About 5 mls of blood is awn into a EDTA vacutainer tube (lavender top). About 4 ml of whole blood is aliquoted into 2 plastic storage vials (~2 ml each). The vials are labeled and frozen at —20°C or -80°C.
Efficacy Measures ] Any neuropsychological tests can be used to measure efficacy response of a patient treated with a o or 6(S)MTHF in conjunction with an SSRI. The primary efficacy measure can e the change in l7—item on Rating Scale for Depression (HAM-D-l7) [55] score. In this study, a response is, for example, d as a 50% or greater reduction in HAM-D-l7 score from ne. A remission is, for example, defined as a HAM-D-l7 score less than 8 at endpoint.
Secondary measures of efficacy can include change in CGI-severity, with “clinical response defined” as CGI-S of l or 2 at endpoint. Patients with a CGI-I score greater than 5 at any post baseline visit or a 50 percent or greater worsening of depressive symptoms from baseline to that visit are discontinued from the study. Subjects are also discontinued from the study with any emergence of suicidality, homicidality, mania, or psychosis. Additional exemplary instruments that can be administered according to the study schedule include the following: (1) Structured Clinical Interview for DSM-IV: The SCID-I/P, administered by the clinician, proceeds by modules to diagnose the different Axis I disorders. Questions are asked exactly as written, and each is based on the individual criteria from DSM-IV. Answers are lly rated on a scale of 1-3 (1: doubtful, 2: probable, 3: te), and, based on the number of positive answers, a diagnosis is ined by a clinician. While the entire SCID-I/P is administered at screen, the mood module is administered at each follow up visit. (2) The MGH pressant Treatment History Questionnaire (MGH-ATR) [53]: The MGH-ATR es specific criteria for the adequate dose and adequate length of a trial for it to be considered a failure, thus allowing clinicians to systematically collect data aimed at assessing the degree of treatment-resistance of the current major depressive episode. (3) The 28-item Hamilton Depression Scale (HAM-D-28) [55]: This version allows scoring of the l7, 21-, 25-, and 28-item scales. This instrument is completed by the clinician by using a structured interview and defined anchor points, and aims to quantify the degree of depression over the past 7 days. The HAM-D is the most widely studied instrument for depression, and its ility and validity are high. (4) Clinical Global Impressions - ty and Improvement (CGI-S, CGI-I): These two instruments are scored 1-7 by the clinician based on ment of the t's clinical status. They measure, based on history and scores on other instruments: a) Depressive severity (CGI-S) and b) Clinical Improvement (CGI-I). Patient rated versions of both scales are also utilized (the PGI-S/I).
WO 74676 2012/065084 (5) QIDS-SR [52]: This is a brief (l6-item) self-report inventory of core depressive symptoms such as sleep, depressed mood, appetite, concentration, suicidal ideation, interest, energy, psychomotor retardation or agitation. (6) The Massachusetts General Hospital Cognitive and Physical Functioning Questionnaire: This is a brief (7-item) eport inventory to assess rates of significant cognitive symptoms, sleepiness, and fatigue. (7) The Massachusetts General Hospital Sexual Functioning Questionnaire [56]: This is a self-rating scale that es common symptoms of sexual dysfunction, such as reduced libido and orgasm ulties. (8) The Visual Analog Scale [57]: This is a brief (8-item) eport scale to measure painful symptoms of depression during treatment with the study medication (6(S)MTHF) or placebo.
Safety Measurements Once the patient has agreed to participate in the study by signing the informed consent document, vital signs (weight, and ng and supine pulse and blood pressure) are recorded at each visit and a physical exam is performed at screen and visit 6 (Day 60, or Phase I endpoint).
Consumptive habits (e. g., smoking, alcohol, and nated beverages) are recorded at baseline, Day , Day 60, Day 150, Day 240, Day 330, and Day 420 (or endpoint)(See Tables 4 & 5 below). A urine pregnancy test (for women of childbearing ial) is also administered at the screen visit and visit 3 (day 30). Pregnant women may not enroll in this study. onally, baseline blood samples are collected for the assessment of genetic polymorphism for (i) T677C allele for the methylenetetrahydrofolate reductase (MTHFR), (ii) A1298C allele for the MTHFR gene, (iii) A66G allele for the methionine synthase reductase gene, and (iv) A2746G allele for the methionine synthase gene.
Further, baseline, Day 30, Day 60, and Day 420 (or endpoint) blood samples are collected for the measurement of plasma folate, RBC folate, plasma homocysteine, vitamin B 12, MMA (methylmalonic acid), SAMe, asymmetrical dimethylarginine (ADMA), ialdehyde (MDA), 4-hydroxynonenal ), F2-Isoprostanes, and high-sensitivity C-Reactive Protein (hs- CRP). e side effects or events ] Documentation of the presence of any side effect or adverse event is completed at every visit using the SAFTEE-SI. Subjects can contact a clinician at any time between visits concerning adverse events or worsening of symptoms. Suicidal on is assessed at each visit.
Subjects who are felt by the study clinician to be at high risk for suicide are discontinued from the study and referred for hospitalization and further treatment if clinically indicated (See Tables 4 & 5 below).
Table 4. Schedule of ian/Subject Ratings & Plasma Tests — Double Blind Phase 1.,.._1 .3. a 1 1»3f0 “LWa» n 1.1f Emlym‘sm 1. "ham-‘- ‘1 .HE .11 .\ i. J1 1 31.3.11 . 1511111111 12 T,I.,1». NM :1. H.11 v. XXRXXKXKX\R Table 5. Schedule of Clinician/Subject Ratings & Plasma Tests — Follow Up Phase E's-lesi‘xitwettient' , ”.1 Elwin]. . £338- at“ a” IA: ./ {:3 HISLLIEH up{ 1%. .1. .1»; Groslairies \ Termination Acceptable reasons for early tinuation include the following: 1) request of patient, 2) on of physician, 3) serious adverse event, 4) protocol violation, 5) worsening of depression or al deterioration ing hospitalization. ary Data Management Clinical data at each visit are recorded using a standardized clinician assessment form and a set of patient rating scales. Edited and corrected data are added to a database that is ready to be used as input to statistical software (e. g., STATA) for data pment and analysis. All data are stored in locked file cabinets. No identifiers other than study ID’s are included in the data. Study staff and subjects may have the option to enter clinical data into the database directly, using DatStat IllumeTM, a platform for electronic data capture that streamlines data collection and management, and ensures data integrity, resulting in improved data quality. Subjects and/or ch staff enter survey responses into electronic assessment forms, and the ses are then transmitted securely via encrypted connection and stored in a secured database.
Exemplary Statistical es General considerations: Data are entered and error-checked at each clinical site involved in the study. Study staff and subjects may have the option to enter self-evaluations into the system directly using DatStat IllumeTM. The process of data entry is supervised by the DCRP staff. 2012/065084 Once the data set is entered and checked, analyses are conducted. Both a completer analysis of all patients finishing the trial and an intent-to-treat analysis examining all patients enrolled into the trial are used to define the severity of depression at endpoint. Examination of both study completers and all ts randomized can provide the broadest assessment of the s of treatment in trials of such kind. The data from Trial 2 can be pooled with those from Trial 1 with the sequential parallel comparison design, to provide an estimate of the placebo se based on a larger sample size.
According to the sequential parallel comparison design analytical plan, the effect of the active treatment is assessed using a e. Under the null hypothesis of no drug-placebo difference, the Z score has a mean of 0. Let pl, ql be the response rates to the first administration of drug and placebo respectively and let p2, q2 be the responses rates to the second ent. To analyze these data, a tic based on h = w(pl - ql) + (l - w)(p2 - q2) is used. The weight (W) and the randomization fraction (a) are chosen to maximize the power of the test, based on the ative hypothesis. The randomization fraction (a) can be involved in calculation of pl, ql, p2, and/or q2. For example, multiplying the total sample size by the randomization fraction (a) to obtain the denominator of the response rates, e. g., pl. The standard error for h requires a special formula because some of the same patients who are included in the estimation of p2, q2 are included in the estimation of pl, ql. The computation is facilitated by considering a table of outcomes, where in this case pl, p2, ql, q2 are the theoretical probabilities rather than the observed relative ncies. A more detailed description of the analyses is described in the Fava et al nce on the sequential el ison design [51].
Analysis ofStudy Attrition: A detailed analysis of the number and timing of study dropouts is calculated. ial differences between treatment groups in ts are examined with a Fisher's Exact test; if even weak trends (p < 0.05) indicative of differential dropout are detected, a more detailed al analysis is completed to illustrate the timing and magnitude of these differences. These t analyses can be used to better understand the outcomes depicted by the following analyses.
Analyses of the magnitude ofresponses: The ude of response between the two treatment groups can be measured by a decrease in baseline HAM-D-l7 scores. One-way analysis of covariance (adjusting for baseline HAM-D-l7 scores) can be used to assess differences in HAM-D-l7 scores at endpoint, pooling data from Trials 1 and 2 and from both phases by using the sequential parallel design.
Analyses ofpercentage onders and remitters: An exemplary statistical test for analysis of differences in proportion of responders in the treatment conditions is a Fisher's Exact test, pooling data from Trials 1 and 2 and from both phases by using the sequential parallel design.
Logistic regression is can also be carried out, with response or non-response and remission or non-remissions as the dependent variables, with the baseline HAM-D score, gender, and age, as the independent variables. Exploratory covariate analysis can be carried out to investigate any differences in remission rates seen with gender, age, and other variables.
] Analyses of the number ofadverse events: One-way analysis of variance can be used to assess differences in total number of SAFTEE-SI AEs between baseline and nt.
Analyses ofpredictors ofa greater 6(S)MTHF/placebo difierence in response rates: Folate levels are classified as either low (<=2.5 ng/ml) or normal; and homocysteine levels as either normal or elevated (>=13.2mmol/liter). The presence or absence of at least one T677C allele for the MTHFR gene are entered as a dichotomous le (present or absent). A 2X2 Analysis of Variance (ANOVA) can be used to test whether the presence of a low folate level predicts a r drug/placebo difference in response. Using folate as an example predicator, the presence or absence of a low serum-folate level along with treatment assignment can be entered in a 2x2, factorial ANOVA, using depression improvement scores (baseline minus endpoint HAM-D-17 scores) as the dependant variable. The effect of the predictor can be indicated by the interaction term, reflecting the potential differential effect of 6(S)MTHF on individuals with low vs. normal folate. Similar es (i.e. separate 2X2 ANOVA's) can be performed substituting the presence of low folate levels for the presence of other predictors as described herein. Similarly, the effect of other predictors can be indicated by the interaction term representing the potential modulating influence of those variables.
Example 2. Evaluation of the efiicacy 0f 6(S)MTHF as an augmentation strategy in MDD patients (Trial 1) Using the study design and sequential el design as described in Example 1, a 60-day, multi-center, double-blind, placebo-controlled study (Trial 1) on the efficacy of oral 6(S) MTHF augmentation of selective serotonin reuptake inhibitors (SSRIs) has been completed in 148 ts with major depressive disorder (MDD) resistant to treatment with SSRIs. The study involved the enrollment of a total of 148 patients with MDD over the course of 12 months across 10 medical centers or hospitals in the United States. Outpatients suffering from MDD were treated with either 7.5 mg/day of 6(S)MTHF or with placebo as an adjuvant to SSRIs for 60 days using the sequential parallel comparison design [51]. In accordance with the sequential parallel design [51], the 60-day, double-blind treatment was divided into two phases of 30 days each, with assessments performed every 10 days. As shown in Fig. 1A, during the first phase of double-blind treatment, 148 eligible patients were ized to 30 days of treatment with either 7.5 mg/day of 6(S)MTHF (“drug”) or placebo, with a 2:33 ratio for random assignment to the ent sequences drug/drug, placebo/placebo, and placebo/drug. Patients randomly ed to the drug/drug sequence had their -MTHF dose increased from 7.5 mg/day to 15 mg/day during the second phase, regardless of whether patients had responded during the first phase (n=12) or had not (n=20). For those ly assigned to the o/placebo sequence, both ders and non-responders to placebo during the first phase remained on o during the second phase. On the other hand, for those ly assigned to the placebo/drug sequence, both responders and non-responders to placebo during the first phase went on to receive 7.5 mg/day of 6(S)MTHF during the second phase.
The findings from Trial 1, as shown in Tables 6-7, te that the 7.5 mg/day 6(8)- -MTHF is not effective to act as an adjunct to the SSRIs. While 6(S)MTHF does not appear to cause any adverse side effect when administered with an SSRI, there was no significant difference in efficacy e (as measured by various parameters such as HDRS-l7, QIDS-SR and CGI-S) between MDD patients d with an SSRI in combination with either 7.5 mg/day 6(S)MTHF or placebo.
Table 6: Eflieaey results ofMDD patients having 7.5 mg/day 6(S)MTHF or placebo as an adjunct \\ \ \ “ \: \_-\\ 'm.\: \‘n\\fl\ \ ~~ '\\\\\\\\\\\\\ 3. * :43:- .w J Table 7. Adverse side- efieets of an SSRI administered with or without 6(S)MTHF ‘- ' - \- rm; - ‘ \u u:«\\\\ v. \ - - - ~ aaslrsnx-mzarxs 2‘ wt: 1 s °"‘\} a - Afiimei: mums f$ -_’.‘ ‘ ' «‘2 Mamba a '34- \\\ \x\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\~ However, an increase in dose of 6(S)MTHF from 7.5 mg/day to 15 mg/day in the ts assigned to the drug/drug sequence during the second phase demonstrated an significant increase in the se and remission rate, e. g., an increase by at least 2-fold, as compared to the group taking placebo with the SSRIs (24% vs 9%, p = 0.1: Such result is not included in Table 6).
Accordingly, a higher dose of oral 6(S)MTHF augmentation, 15 mg/day, was determined to be used in Trial 2.
Example 3. tion of the efiieaey of 6(S)MTHF as an tation strategy in MDD patients (Trial 2) Using the study design and sequential parallel design as described in Example 1, this Example 3 shows a 60-day, multi-center, double-blind, placebo-controlled pilot study (Trial 2) on the efficacy of a higher dose (15 mg qd) oral 6(S)MTHF augmentation of selective serotonin reuptake inhibitors (SSRIs) in 75 patients with major depressive disorder (MDD) resistant to ent with SSRIs. The design of Trial 2 (as shown in Fig. 1B) was identical to that of Trial 1, with the exception of the dosing of -MTHF, which was 15 mg/day throughout the trial for those patients assigned to the placebo-drug group and to the drug-drug group.
The critical clinical purpose of this study was to determine whether the use of a higher dose of oral 6(S)MTHF as an adjunct to SSRIs would be more effective than placebo as an adjunct to SSRIs in reducing depressive symptoms in outpatients with MDD with l or no response to SSRI treatment. An additional aim of the study was to demonstrate the safety and tolerability of 6(S)MTHF 15 mg/day augmentation. The study involves the enrollment of a total of 75 patients with MDD over the course of 12 months across 6 different medical centers or hospitals across the United . Outpatients ing from MDD were treated with either 6(S)MTHF 15 mg/day or with placebo for 60 days using the sequential parallel comparison design [51]. Fig. 2 shows the actual number of completers in each group by the end of the study.
Fig. 3A shows that there is a statistically significant difference in the percentage of responders (50% or greater reduction in 17 at endpoint) in the two treatment conditions (i.e., SSRI + 15 mg/day of 6(S)MTHF vs. SSRI + placebo) after 30 days. Using the sequential parallel comparison design [51], it was ined that the se rate is higher for the 6(S)MTHF group than the placebo group, with the response rate on placebo estimated from Trials 1 and 2.
Fig. 3B shows that there is a statistically significant difference between the two treatment conditions (i.e., SSRI + 15 mg/day of 6(S)MTHF vs. SSRI + placebo) after 30 days in the degree of improvement, as measured by the change in the 17-item Hamilton Depression Rating Scale (HAM-D-17) score from baseline to nt, QID-SR, or Clinical Global Impressions — Severity (CGI-S), using the sequential el ison design [51]. Using the sequential parallel comparison design [51], it was determined that there is a greater degree of reduction in the scores of HAM-D-17, QIDS-SR and CGI-S, tively, in the 6(S)MTHF group than in the placebo group, with the change on placebo estimated from Trials 1 and 2.
Figs. 3C-3D show that there is no significant difference in the percentage of remitters (HAM-D-17 score < 8 at end point or QIDS-SR score S 5 at end point) in the two treatment conditions (i.e., SSRI + 15 mg/day of 6(S)MTHF vs. SSRI + placebo) after 30 days.
The results of Figs. 3A-3D are summarized in Table 8 below.
Table 8. Eflicacy results ofMDD patients having 15 mg/day 6(S)MTHF or placebo as an adjunct ; 5:333.filth:-F1~F€=Sfrh¥ffi$$2f9 Table 9 shows that there are no significant differences in the number of e , as measured by the SAFTEE-SI between the two treatment groups (i.e., SSRI + 15 mg/day of 6(S)MTHF vs. SSRI + placebo).
Table 9. Adverse side- eflects of an SSRI administered with 15 mg/day of 6(S)MTHF or placebo “‘aaimi Mast 332523 3 {WWW I i7 85?“:3} mime. manna 5 {‘c .B‘Xfi . a: .. a: giam‘m {3.34 \\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\V “ r: :a E'.»S:S::?".i 'fafift'J‘EEE numb: "55': 15:3! receévexi simian:- w isnwtiws‘uikfie 35:23; a: some paint Mme. the 31:55:.
] As shown in Fig. 3E, there is no difference in discontinuation from the study due to overall adverse side-effects or exclusion events. The patient administered with 6(S)MTHF as an adjunct to his/her SSRI was removed from the trial due to mood elevation. The medical y of the patient indicated bipolar disorder which was not detected at the baseline visit.
Follow-up Study At the end of the double-blind study, both responders and non-responders who have completed the double-blind phase have the option of ing free, open-label adjunctive treatment with 15 mg/day of 6(S)MTHF for 12 months. Subjects who agree to receive open-label treatment with 6(S)MTHF for 12 months are assessed every three (3) months until the end of the follow-up phase. During each visit, patients are stered the 28, the CGI, the QIDS-SR, the SAFTEE-SI, the MGH-CPFQ, and the MGH-SFQ (See, e.g., Table 10 below). The dose of their concomitant SSRI can be adjusted during the 12 months of follow-up, as can the dose of 6(S) MTHF (e.g., up to 15 mg twice a day). Subjects are also allowed to change their antidepressant during the course of the follow-up, if deemed appropriate. For patients who refuse the 12 months of free -up care, a referral to a psychiatrist is offered.
Table 10 shows that remitters at the end of double-blind phase who received adjunctive treatment with 15 mg/day of 6(S)MTHF did not have a relapse during a 12-month maintenance phase.
Table 10. Follow-up s on remitters during the 12-month maintenance phase ers at end of double-blind Relapsers (HAM-D-17 > 15) during 12-month phase -17 < 8) entering nance phase, administered with 15 maintenance phase mg/day 6(S)MTHF as an adjuvant (1 subject had not relapsed at 9-month visit but did not return for 12-month visit) Example 4. Identification of biomarkersfor selecting patients with depressionfor a treatment comprising afolate-containing compound in combination with an SSRI A double-blind, placebo-controlled study of 6(S)MTHF among SSRI resistant outpatients with major depressive disorder (MDD) is performed, as described in Examples 1-3, to identify genetic polymorphisms, peripheral biomarkers and/or clinical features that are associated with a greater efficacy response when a patient is stered with a folate-containing compound (e. g., 6(S)MTHF) in addition to an antidepressant drug, e. g., an SSRI.
Figs. 4A-4B show the effect of the single genetic polymorphism (SNP) at the MTHFR gene (MTHFR C677T) on the cy of the treatment sing a folate-containing compound (e.g., 6(S)MTHF) and an SSRI in a patient with depression, as measured by HDRS-28 (28-item Hamilton Depression Rating Scale) and CGI-S (Clinical Global Impression-Severity), respectively. The findings of Figs. 4A-4B indicate that patients having at least one T allele (e. g., CT or TT) at the on 677 of the SEQ ID NO: 1, corresponding to a portion of the genomic sequence of the MTHFR gene, demonstrate a greater degree of improvement in the HDRS-28 or CGI-S test when they are treated with a folate-containing compound in combination with an SSRI, as compared to patients with no T allele ed at the position 677 of the SEQ ID NO: 1.
] Figs. SA-SB show the effect of obesity (i.e., BMI is at least 30 kg/m2 or above) on the efficacy of the treatment comprising a folate-containing compound (e. g., 6(S)MTHF) and an SSRI in a patient with depression, as measured by HAMD-28 (28-item Hamilton Depression Rating Scale) and CGI-S (Clinical Global Impression-Severity), respectively. The findings of Figs. SA-SB indicate that obese patients (i.e., with a BMI at least 30 kg/m2 or above) demonstrate a greater degree of improvement in the 8 or CGI-S test when they are treated with a folate-containing compound in combination with an SSRI, as compared to non-obese patients.
] Some exemplary predictors for greater efficacy response rates in ts receiving 6(S)MTHF in combination with their respective SSRIs in combination with SSRIs, can include low plasma and/or RBC folate levels, low plasma B12 and SAM levels, low ratios of SAM/SAH levels, an elevated plasma homocysteine level, presence of folate receptor autoantibodies (FRAs), trical dimethylarginine (ADMA), malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), high-sensitivity C-Reactive Protein (hs-CRP), prostanes (8-OH-Dg), brain d neutropic factor (BDNF) levels, and the following genetic polymorphisms: a) T677C allele for the methylenetetrahydrofolate reductase (MTHFR); b) A1298C allele for the MTHFR gene; c) A66G allele for the methionine synthase reductase gene; and d) A2746G allele for the nine synthase gene.
Various combinations of genetic and biomarkers were assessed for their effects on the efficacy of the treatment comprising a folate-containing compound (e. g., 6(S)MTHF) and an SSRI in ts with depression. Particularly, specific c and biomarkers that were assessed include the following: (a) BMI calculation of at least about 30 kg/mz; (b) an expression ratio of SAM/SAH smaller than 2.8 (e. g., 2.71) as measured in a plasma sample; (c) a level of 4-HNE no less than 3.28 ug/mL (as measured in a plasma sample); (d) presence of rare variants CT or TT at position 677 of SEQ ID NO: 1 ponding to a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase( MTHFR) (abbreviated as “MTHFR 677” below); (e) presence of rare ts AG or GG at position 2756 of SEQ ID NO: 2 corresponding to a portion of genomic nucleic acid sequence of methionine synthase (MTR) (abbreviated as “MTR 2756” below); and (f) presence of rare variants AG or GG at position 66 of SEQ ID NO: 3 corresponding to a n of genomic nucleic acid sequence of nine synthase reductase (MTRR) (abbreviated as “MTRR 66” below).
The s of how various combinations of the above conditions (a)-(f) affect the degree in improvement, as measured by 7 and HAMD-28 scores, when patients are treated with a folate-containing compound (e. g., 6(S)MTHF) and an SSRI, are shown in Tables 11-36 below. The efficacy effect is determined by measuring the mean change in the HAMD-17 score and HAMD-28 score by the end of Phase I and Phase II, as compared to the baseline.
Table 11. Effect of at least one rare variant on both MTHFR 677 and MTR 2756, as opposed to fully normal on both, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e. g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases MTHFR 677 CC and . 0.817 -0.571 0.866 0.722 (-2.694, 4.138) 0.679 MTR 2756 AA (29,13)* MTHFR 677 CT or TT . 0.006 -10.33 0.223 -18.71 (-24.33, -13.08) <0.001 and MTR 2756 AG or GG (8,4)* MTHFR 677 CC and -0.192 0 960 0.048 0.990 0.711 (-3.121, 4.543) MTR 2756 AA MTHFR 677 CT or TT -18.00 0.008 -13.00 0.167 -23.29 (-32.08, -14.50) <0.001 and MTR 2756 AG or * The numeric values within the parentheses correspond to the number of patients having the indicated condition Table 12. Effect of at least one rare variant on both MTR 2756 and MTRR 66 and baseline BMI of at least 30 kg/mz, as opposed to fully normal on both genes and ne BMI less than 30 kg/mz, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a -containing compound (e.g., 6(S)MTHF) and an SSRI. ””17 ------- MTR 2756 AA and All non-treated All non-treated MTRR 66 AA and BMI < kg/m2 (7,4) MTR 2756 AG or GG -13.667 0.029 -11.000 0.064 40.972 (—17.377, — and MTRR 66 AG or GG and BMI >= 30 kg/m2 (8,4) HAMD-28 MTR 2756 AA and MTRR 66 AA and BMI < kg/m2 MTR 2756 AG or GG -18.833 0.014 -12.000 0.115 -15.347 (-22.967,- and MTRR 66 AG or GG and BMI >= 30 kg/m2 Table 13. Effect of at least one rare variant on MTR 2756 and baseline BMI of at least 30 kg/mz, as opposed to fully normal on the gene and baseline BMI less than 30 kg/mz, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
Phase I Phase II Pooled phases 95% CI p- HAMD-17 MTR 2756 AA and BMI 3.196 6, 8.298) 0.220 < 30 kg/m2 (17,7) MTR 2756 AG or GG -11.714 (-15.859, -7.569) and BMI >= 30 kg/m2 (10,5) HAMD-28 MTR 2756 AA and BMI 3.352 (-2.692, 9.395) < 30 kg/m2 MTR 2756 AG or GG 3 (-19.453, -9.414) and BMI >= 30 kg/m2 Table 14. Effect of at least one rare variant on MTR 2756 and ne H smaller than a pre-determined reference ratio (e.g., the median ratio of a reference group), as d to fully normal on the gene and the baseline SAM/SAH no less than the pre-determined reference ratio, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e.g., -MTHF) and an SSRI.
Phase I Phase II Pooled phases Effect Effect p-Value Effect 95% CI e MTR 2756 AA and -1.556 0.510 (-3.764, 3.873) SAM/SAH >=median(24,12) MTR 2756 AG or GG and -9.800 0.007 (-12.163, 0.998) SAM/SAH < median (10,4) MTR 2756 AA and -1.278 0.662 9, 3.747) SAM/SAH >= median MTR 2756 AG or GG and 3 0.003 (-17.836, -5.530) SAM/SAH < median=2.71 (as measured in a plasma sample) ] Table 15. Effect of at least one rare variant on both MTR 2756 and MTRR 66 in combination with baseline SAM/SAH smaller than a pre-determined reference ratio (e.g., the median ratio of a reference group), as opposed to fully normal on both genes in combination with the baseline SAM/SAH no less than the pre-determined reference ratio, in MDD patients on the change in HAMD- 17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e. g., 6(S)- -MTHF) and an SSRI.
Phase II Pooled phases p-Value --—-— MTR 2756 AA and -0.400 0.962 -7.000 0.154 -3.98* (-8.03, 0.064) MTRR 66 AA and SAM/SAH >= median (63) MTR 2756 AG or GG -9.000 0.037 -6.500 0.084 -7.64* (-11.93, -3.34) and MTRR 66 AG or GG and SAM/SAH < median (83) HAMD-28 MTR 2756 AA and -1.000 0.916 -7.500 0.333 -3.74* (-8.60, 1.12) MTRR 66 AA and SAM/SAH >= median MTR 2756 AG or GG 1 0.019 -10.500 0.052 -11.62* (-16.14,-7.10) and MTRR 66 AG or GG and SAM/SAH < median=2.71 (as measured in a plasma Table 16. Effect of at least one rare variant on both MTR 2756 and MTRR 66 in ation with baseline HNE-His (or 4-HNE) no less than a predetermined level (e. g., a median level of a reference group), as opposed to fully normal on both genes in combination with the baseline HNE-His smaller than the predetermined level, in MDD patients on the change in HAMD-17 and HAMD-28 , when the patients are treated with a folate-containing compound (e. g., 6(S) MTHF) and an SSRI.
Phase I Phase II Pooled phases Effect p-value Effect p-value Effect 95% CI p-value HAMD-17 MTR 2756 AA and 3.000 0.750 -8.667 0.532 0.466 (-14.699, 15.630) 0.952 MTRR 66 AA and HNE- His < median (6,4) -7.200 0.131 -11.000 0.064 -7.942 (-14.328, -1.556) 0.015 MTR 2756 AG or GG and MTRR 66 AG or GG and HNE-His >=median (10,4) HAMD-28 MTR 2756 AA and 4.200 0.735 -9.000 0.500 -2.221 90, 17.549) 0.826 MTRR 66 AA and HNE- His < median=3.28 (as measured in a plasma sample) MTR 2756 AG or GG -9.800 0.072 -12.000 0.115 -10.241 (-17.365, -3.117) 0.005 and MTRR 66 AG or GG and HNE-His >= median Table 17. Effect of at least one rare variant on both MTHFR 677 and baseline BMI of at least about 30 kg/mz, as opposed to fully normal on the gene and the baseline BMI less than 30 kg/mz, in MDD patients on the change in HAMD-17 and HAMD-28 , when the patients are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases Effect e Effect p-value 95% CI p- value HAMD-17 MTHFR 677 CC and 4.500 0.360 -2.267 0.480 1.356 (4.388, 7.100) 0644 BMI < 30 kg/m2 (17,8) MTHFR 677 CT or TT -8.738 0.009 -7.333 0.129 -8.090 (-13.622, -2.558) and BMI >= 30 kg/m2 (13,6) HAMD-28 MTHFR 677 CC and 2.769 0.663 -3.000 0 415 -0.234 7, 6.750) BMI < 30 kg/m2 MTHFR 677 CT or TT -9.857 0.035 -8.667 0.077 -9.884 (-15.794, -3.974) and BMI >= 30 kg/m2 Table 18. Effect of at least one rare variant on both MTHFR 677 and baseline HNEHis (or 4-HNE) no less than a predetermined level (e.g., a median level of a reference group), as opposed to fully normal on the gene and the ne HNE-His smaller than the predetermined level, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are d with a folate-containing compound (e. g., 6(S)MTHF) and an SSRI.
Phase I Phase II Pooled phases Effect HAMD-17 MTHFR 677 CC and —1.471 0.695 -1.050 (-5.914, 3.814) HNE-His < median (20,1 1) MTHFR 677 CT or TT -7.143 0.050 0.215 -9.446 (-15.920, - and HNE-His >= median 2.972) (1 1,6) HAMD-28 MTHFR 677 CC and -2.821 0.559 -0.536 0.908 -1.340 (-6.933, 4.253) s < median=3.28 (as measured in a plasma sample) MTHFR 677 CT or TT -6.929 0.090 -7.500 0.223 -9.865 (-16.200, - and HNE-His >= median 3.529) Table 19. Effect of at least one rare variant on both MTR 2756 and baseline HNE-His (or 4-HNE) no less than a predetermined level (e.g., a median level of a nce group), as opposed to fully normal on the gene and the baseline HNE-His smaller than the ermined level, in MDD patients on the change in 7 and HAMD-28 scores, when the patients are treated with a -containing compound (e.g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases e Effect p-value 95% CI p-value MTR 2756 AA and HNE- 0.897 0.300 0.936 1.319 (-2.257, 4.895) His < median (26,13) MTR 2756 AG or GG 0.061 -8.417 0.048 -8.072 (-12.314, - and HNE-His >= median 3.831) (137) MTR 2756 AA and HNE- 0.835 -0 125 0.975 2.085 (-2.142, 6.312) His < median MTR 2756 AG or GG 0.026 -9.500 0.037 -9.508 (-13.855, - and HNE-His >= 5.161) median=3.28 (as measured in a plasma sample) Table 20. Effect of baseline H ratio smaller than a pre-determined reference ratio (e.g., a median ratio of a reference group) and baseline HNE-His (or 4-HNE) level no less than a predetermined level (e. g., a median level of a reference group), as opposed to the baseline SAM/SAH ratio no less than the pre-determined reference ratio and the baseline HNE-His level smaller than the predetermined level, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e. g., 6(S)MTHF) and an SSRI.
Effect p-Value Effect 95% CI e HAMD-17 SAM/SAH >= median -0.250 0.920 4.000 0.380 2.107 (-2.851, 7.064) and HNE-His < median (18,8) SAM/SAH < median -3.670 0.139 -9.000 0.031 -9.898 (-14.436, — and HNE-His >= median 5.361) (19,7) SAM/SAH >= median -0.750 0.822 4.500 0.388 2.413 (-3.079, 7.906) and HNE-His < median SAM/SAH < median -4.227 0.230 -12.000 0.004 -9.500 (-13.164, - and HNE-His >= median 5.830) Table 21. Effect of at least one rare variant on MTR 2756 and MTRR 66, as opposed to fully normal on both genes, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing nd (e. g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases Effect p-Value Effect p-Value 95% CI p-Value HAMD-17 MTR 2756 AA and 3.125 0.555 -8.000 0.451 (-10.45, 7.705) MTRR 66 AA (11,5) MTR 2756 AG or GG -6.900 0.091 -4.667 0.338 (-11.32, -2.638) and MTRR 66 AG or GG (15,7) HAMD-28 MTR 2756 AA and 5.000 0.454 -8.000 0 439 -3.032 (-13.50, 7.431) MTRR 66 AA MTR 2756 AG or GG -9.996 0.039 -6.583 0.263 -9.348 (-13.01, ) and MTRR 66 AG or GG Table 22. Effect of at least one rare variant on MTR 2756, as opposed to fully normal on the gene, in MDD patients on the change in HAMD-17 and 8 scores, when the patients are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases Effect p-Value 95% CI p-Value MTR 2756 AA (45,21) ---0 621 -0.427 0869 0590 (3418,2238) 0.683 MTR 2756 AG/GG 6 600 0.273 (-9.422, -1.932) (20,11) HAMD-28 MTR 2756 AA 0.409 0.859 -0.446 0869 (-2.974, 2.879) 0.975 MTR 2756 AG/GG -10.926 -5.300 0.160 -8.243 (-12.549, - 3.938) Table 23. Effect of baseline BMI of at least 30 kg/m2 and baseline SAM/SAH ratio smaller than a pre-determined reference ratio (e.g., a median ratio of a reference group), as opposed to the baseline BMI smaller than 30 kg/m2 and the baseline SAM/SAH ratio no less than the pre- determined reference ratio, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
Phase I Phase II Pooled phases Effect p-Value Effect p-Value 95% CI p-Value HAMD-17 BMI < 30 kg/m2 and 0.120 1.500 0.699 2.583 (-2.224, 7.389) SAM/SAH >= median (16,8) BMI >= 30 kg/m2 and -5.611 0.011 -9.200 0.005 (-10.404, — SAM/SAH < median 4.201) (21,9) BMI < 30 kg/m2 and 4.333 0.215 2.500 0.596 2.906 8, 8.970) SAM/SAH >= median BMI >= 30 kg/m2 and -6.306 0.049 0 0.005 -7.572 (-10.871, — SAM/SAH < 4.274) median=2.71 (as measured in a plasma sample) Table 24. Effect of at least one rare variant on MTRR 66 and baseline HNE-His (or 4- HNE) level no less than a predetermined level (e. g., a median level of a reference group), as opposed to fully normal on the gene and the baseline HNE-His level smaller than the predetermined level, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are d with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
Phase I Phase II Pooled phases Effect p-value Effect p-value Effect 95% CI p-value MTRR 66 AA and HNE- -1.167 0.844 -1.667 0.866 2.575 (-11.15, 16.30) His < median (8,5) MTRR 66 AG or GG and -5.958 0.018 -3.500 0.322 -4.850 (-9.033, -0.667) HNE-His >= median (24,11) MTRR 66 AA and HNE- -4.167 0.625 -2.000 0.835 -2.626 (-17.80, 12.55) His < median=3.28 (as measured in a plasma MTRR 66 AG or GG and -6.975 0.017 -4.071 0.335 -6.582 (-10.895, - HNE-His >= median 2.269) Table 25. Effect of at least one rare variant on MTRR 66 and baseline SAM/SAH smaller than a termined reference ratio (e.g., a median ratio determined from a nce group), as d to fully normal on the gene and the baseline SAM/SAH ratio no less than the pre- determined reference ratio, in MDD patients on the change in 7 and HAMD-28 scores, when the patients are treated with a folate-containing nd (e.g., 6(S)MTHF) and an SSRI.
Phase I Phase II Pooled phases Effect p-value Effect p-value 95% CI p-value MTRR 66 AA and -0.125 0.984 -3.000 0.554 -2.978 (-8.884, 2.928) SAM/SAH >= median (9,6) MTRR 66 AG or GG and -5.062 0.028 -6.190 0.042 -5.879 (-9.272, -2.487) SAM/SAH < median (24,10) MTRR 66 AA and 0.500 0.945 -3.333 0.523 -1.221 3, 4.581) SAM/SAH >= median MTRR 66 AG or GG and -6.498 0.031 -7.381 0.022 -6.579 (-10.257, - H < 2.901) median=2.71 (as measured in a plasma sample) Table 26. Effect of baseline BMI of at least about 30 kg/m2 and baseline s (or 4-HNE) level no less than a predetermined level (e. g., a median level determined from a nce group), as opposed to the baseline BMI smaller than 30 kg/m2 and the baseline HNE-His level smaller than the predetermined level, in MDD ts on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e. g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases Effect p-value Effect p-value Effect 95% CI p-value BMI < 30 kg/m2 and 0.307 3.667 0.382 4.738 (-0.935, 10.412) HNE-His < median (199) BMI >= 30 kg/m2 and -6.569 0.012 -4.233 0.242 -5.420 (-9.107, -1.732) HNE-His >= median (23,11) BMI < 30 kg/m2 and 4.606 0.335 1.833 0.708 4.253 (-2.634, 11.139) HNE-His < median BMI >= 30 kg/m2 and -6.510 0.055 -4.933 0.253 -5.996 (-9.831, ) HNE-His >= median Table 27. Effect of baseline BMI of at least about 30 kg/mz, as opposed to the baseline BMI smaller than 30 kg/mz, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI. The phrase “BMI>*< treat” as used in Table 27 refers to interaction of BMI with the treatment.
HAMD-17 BMI < 25 (14,7) 1.576 0.666 -1.400 0.761 -1.248 (-8.854, 6.359) =< BMI < 30 kg/m2 2.600 0.473 2.000 0.559 3.331 (-1.345, 8.001) (18,10) BMI >= 30 kg/m2 (40,21 ) 0.231 -4.600 (-7.219, -1.981) BMI*treat 0.619 -0.233 2, 0.036) BMI < 25 (14,7) 0.681 -1.018 (-8.587, 6.552) =< BMI < 30 kg/m2 1.000 2.054 (-3.884, 7.992) (18,10) BMI >= 30 kg/m2 (40,21) 0.262 -4.660 (-7.449, -1.871) BMI*treat 0.757 -0.270 (-0.581, 0.040) Table 28. Effect of ne SAM/SAH ratio smaller than a pre-determined reference ratio (e.g., a median ratio determined from a reference group), as opposed to the baseline SAM/SAH ratio no less than the pre-determined reference ratio, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a -containing compound (e. g., 6(S) MTHF) and an SSRI. The phrases “baseHAMD-17* treat” and “SAM/SAH*treat” as used in Table 28 refer to interaction of base 7 and SAM/SAH, respectively, with the treatment.
Phase II Pooled phases p-Value Effect p-Value Effect 95% CI p-Value baseHAMD-17*treat 0.165 0.722 -0.249 0.566 -0.025 (-0.553, 0.504) 0.927 SAM/SAH>=med -0.946 0.675 0.564 0.816 -0.202 (-3.111, 2.708) 0.892 SAM/SAH < med (-6.730, -0.514) (37,18) SAM/SAH*treat 0.495 0.755 1.483 0.298 0.882 (-0.913, 2.677) 0.335 H*treat 0.495 0.755 1.483 0.298 0.882 (-0.913, 2.677) 0.335 SAM/SAH >=med=2.71 -1.148 0.681 1.355 0.634 0.073 (-3.333, 3.480) 0.966 (as measured in a plasma WO 74676 —------- H< med -4.523 0.113 -7.325 0.014 -4.572 (-7.732, -1.413) 0.005 SAM/SAH*treat 0.972 0.636 1.806 0.271 1.112 (-0.927, 3.151) 0.285 Table 29. Effect of baseline HNE-his (or 4-HNE) no less than a predetermined level (e. g., a median level determined from a reference , as opposed to the baseline HNE-his smaller than the predetermined level, in MDD patients on the change in 7 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e. g., 6(S)MTHF) and an SSRI.
The phrase “HNE-his*treat” as used in Table 29 refers to interaction of HNE-his with the treatment.
Phase I Phase II Pooled phases Effect p-value Effect Effect 95% CI p-value HAMD-17 HNE-his >=med (37,20) 0.048 -4.030 0.086 -4.175 (-6.891, -1.459) HNE-his < med (36,19) . 0.532 0.631 0.824 0.260 (-2.900, 3.421) HNE-his >*‘treat . 0.754 2.027 0.188 .626 (- 1. 197, 2.449) HAMD-28 HNE-his >=med=3.28 (as (-7. 610, -1.499) measured in a plasma HNE-his < med . . . 0.961 -0.107 (-3.670, 3.457) HNE-his at . . . 0.184 0.685 (-1.440, 2.810) Table 30. Effect of at least one rare variant on MTHFR 677 and MTR 2756 in combination with baseline s (or 4-HNE) no less than a predetermined level (e. g., a median level determined from a reference group), as opposed to fully normal on both genes in combination with the baseline HNE-His smaller than the predetermined level, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a -containing compound (e. g., 6(S)MTHF) and an SSRI.
Effect p-value Effect p-value E—ffect95% CI value ””17 -----_- MTHFR 677 CC and (-5.972, 5.688) MTR 2756 AA and HNE-His < median (17,9) MTHFR 677 CT or TT and MTR 2756 AG or GG and HNE-His > median (4,2) MTHFR 677 CC and -2.333 .687 -0.053 MTR 2756 AA and HNE-His < median MTHFR 677 CT or TT -17.0 NA Not enough data and MTR 2756 AG or GG and HNE-His > median Table 31. Effect of at least one rare variant on MTHFR 677 and MTR 2756 in combination with baseline SAM/SAH ratio smaller than a pre-determined reference ratio (e.g., a median ratio determined from a nce , as opposed to fully normal on both genes in combination with the baseline SAM/SAH ratio no less than the pre-determined reference ratio, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing nd (e.g., 6(S)MTHF) and an SSRI.
Pooled phases Effect p-Value Effect e Effect 95% CI p- value MTHFR 677 CC and -6.429 0.284 4.000 0.346 -0.830 (-5.190, 3.530) 0.709 MTR 2756 AA and SAM/SAH >= median (15,8) MTHFR 677 CT or TT -12.667 0.070 Not enough data and MTR 2756 AG or GG and SAM/SAH < median (4,1) MTHFR 677 CC and -12.643 0.084 4.500 0.349 -4.472 (-10.619, 1.676) 0.154 MTR 2756 AA and -----—- MTHFR 677 CT or TT -18.000 0.070 Not enough data and MTR 2756 AG or GG and SAM/SAH < median ] Table 32. Effect of at least one rare variant on MTHFR 677 and MTR 2756 in combination with baseline BMI of at least about 30 kg/mz, as opposed to fully normal on both genes in combination with the baseline BMI smaller than 30 kg/mz, in MDD ts on the change in HAMD-l7 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e. g., -MTHF) and an SSRI.
Phase II Pooled phases HAMD-17 MTHFR 677 CC and MTR 2756 AA and BMI < 30 kg/m2 (10,5) MTHFR 677 CT or TT and MTR 2756 AG or GG and BMI >= 30 kg/m2 (5,2) HAMD-28 MTHFR 677 CC and . . All non-treated MTR 2756 AA and BMI < 30 kg/m2 MTHFR 677 CT or TT -23.500 0.021 -l7.0 NA Not enough data and MTR 2756 AG or GG and BMI >= 30 kg/m2 Table 33. Effect of at least one rare variant on MTRR 66 in combination with baseline BMI of at least about 30 kg/mz, as d to fully normal on the gene in combination with the baseline BMI smaller than 30 kg/mz, in MDD patients on the change in HAMD-l7 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
HAMD-17 MTRR 66 AA and BMI < 30 kg/m2 (10,7) MTRR 66 AG or GG and BMI >= 30 kg/m2 (29,16) MTRR 66 AA and BMI < 30 kg/m2 MTRR 66 AG or GG and BMI >= 30 kg/m2 Table 34. Effect of at least one rare variant on MTHFR 677 and baseline SAM/SAH ratio smaller than a pre-determined reference ratio (e. g., a median ratio determined from a reference group), as opposed to fully normal on the MTHFR 677 and the baseline SAM/SAH ratio no less than the pre-determined reference ratio, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the ts are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases p-Value Effect p-Value 95% CI p-Value MTHFR 677 CC and 0.827 2.333 0.443 1.075 (-3.250, 5.400) SAM/SAH >= median (21,12) MTHFR 677 CT or TT 0.059 2.333 0.751 -2.041 (-11.186, 7.104) and SAM/SAH < median (11,5) MTHFR 677 CC and 0.666 2.833 0.428 -0.764 0, 3.791) SAM/SAH >= median MTHFR 677 CT or TT 0.078 0.000 1.000 -3.533 (-11.408, 4.341) 0.379 and SAM/SAH < media“ - Table 35. Effect of at least one rare variant on both MTHFR 677 and MTRR 66, as opposed to fully normal on both, in MDD patients on the change in HAMD-17 and HAMD-28 scores, when the patients are treated with a folate-containing compound (e. g., 6(S)MTHF) and an SSRI.
Phase II Pooled phases Effect p-value Effect p-value Effect 95% CI p- value MTHFR 677 CC and 3.750 0.579 -12.25 0.116 -8.281 (-18.82, 2.262) 0.124 MTRR 66 AA (10,5) MTHFR 677 CT or TT -5.550 0.053 -5.250 0.069 -5.648 (-9.477, -1.818) 0.004 and MTRR 66 AG or GG (18,8) MTHFR 677 CC and 5.500 0 521 -13.00 0.047 -9.379 (-21.55, 2.789) 0.131 MTRR 66 AA MTHFR 677 CT or TT -5.275 0.141 -7.500 0.025 -6.264 (-10.36, -2.167) 0.003 and MTRR 66 AG or Table 36. l results combining all conditions from (a)-(f), when ts are treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
All Patients (-4.292, -0.236) All Patients -3.632 0.069 (-4.995,-0.482) 2012/065084 Table 37. y of s from Tables 11-19 and 36. ‘33 T'riai 2 Cs: mbinations of (2on Variable Pamsmgun Trial 2 Population Priority Combinations fl 1 2 3 4 5 6 T B 9 Variable L‘omba‘natians OVERALL D35 A+E+F {ME E+E E+E+F O+JE+F {3+5 {1er {3+5 N273 N24 «=4 :.— N28 M25 was N23 N: «=9 A EM5330 533% NA NA 553153 5 5‘3”: NA N:3 5N A ‘3 5“va NA NA E Lam-‘3 autism Ram; 53% NA. NA N33. NA 5333» .J “.2 “-5.: NA NA Nfi. N“.
C'- HESH NHNE 533%: NA NA NS}. NA N53. N31. 53 “ NA 5 14 '13; 53"}; D MTHFR 6?? C ’12??? 43%: NA 43% NA Nsb‘. NA Nix EN P4 4 :75; 41:3 NA .53: ('4. 8. 3e, 3. 8.
E MTR 2755 @5036 34% NA 534% 34% 343.“: 34% fin-337:: 34% NA NA. 34% F MTRR 66 MEGS ‘34‘3’3 NA NA ?:;3 NA NA Fifi: 73% NR Nr-K. NA COMB!NflTEON ‘35.. 100% TV}; 14% $333 3 '31-‘51 ‘I 3% it 3% 2m 25% 1 1’33 Fuuiefi Street 31cm iMMD-EE 12.3133 $3.298 235.34?" 44.433 41.683 -1 i .1523 43.343 -9.3=34 3.8535 3.5% wicked gm: \1: HAMBvEfi mm “£3,331 <1). (131: 8:31.36! 23.081 «mm 0.305 {1.9M 0.302 20.081 P‘uokm Eiiaci 352 * MAR-1&1 ? 3.263 48.719 -‘ifl..§?2 #13314 - 5.533 I? .540 3?.342 $.35“): $.44?) 43.6172 Fatima parasite iiiAMD-i? 6.029 30.001 0.051 «10.031 {3,096 20.001 5.611135 6.004 61.604 «0.801% Table 3 8. Summary of results from Tables 20-29.
“A Trial 2 Combinations of Code Variable anflafion Trial 2 Population Priority Combinaiians 1G 11 12 13 1.4 15 15 17’ 18 19 Variable: Combinations 8+3: £3}: E n+5 C+F EH? R“: A E C N231 N29 N231 M213 N215 M211 N212 M223 N213 N221 A 8ME230 52““.3 NR N33. NR ‘1‘ 5'33 NR N .553, 55% “J5% NA. NA. {3 Law 5:433:1an RAP-O 5 5‘: '23; NA NA 55 :33 NA 55 333 NA NA 5? ‘3 N91. (2 HEGH ANNE 53°23 5:33: NA NA NA 53% NA {33% NA Ms! 5W. 3‘} MTHFR '57? CTI'TT 49% NA Nzfi. NA NA NA NA NA NA NA. NA E MTR 2355 AGNES 34% NA 34% 343$} NA NA NA NA MR NA N3“ F MTRR 55 AEGIS T433 Nit. Fisk NA NA 3:73; Fifi NA NA M3. M53.
CflMBENATEON “1% 233% 253?. 31:32. 233?. 3fi'33 3-533. 2333 55% 5133 5153 gamed Efimi sue HAM 0328‘ 3.599 .3343 412-13 -~?.§?2 - 6.552 -6 .5133 .5_ 896 4.530 «4. 5.?2 «4.554 93mm p-w ism HAM{123 20.0631 (.EMIIEN 20.881 1 {1.003 film“! £1,062 3.9m 3.605 3 Passed iii-“set Size HEMEM? 3 26.9?6 255?? - ‘3362 4.85:} 25 RS: 25.42!) 24.696 23. S22 «M 7’5 PeuSed g-vaiue HAN!EMT 63.301 (L003 0.833 <0. cm: 8.323 {3.051 8.904 0.061 8,922 8.583 In addition to using HAMD- l 7 or HAMD-28 scales to evaluate the effects, other scales such as social functioning questionnaire (SFQ), visual analogue scale (VAS), cognitive and physical function questionnaire (CPFQ), Maier, and HAMD-7 subscales of HAMD were also used to analyze the effects and the results are shown in Figs. 9A-9C and Figs. lOA-lOE.
Additional SNP biomarkers were also evaluated for their effects on change in HAMD-28 in MDD ts with at least one rare variant as indicated below as opposed to fully 2012/065084 normal on the gene, when they were treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI. While Table 39 lists dual SNP biomarkers in an order of their decreasing effects in MDD patients (i.e., a SNP marker with a greater reduction in HAMD-28 is listed earlier in the table), it should not be construed as one SNP biomarker is more preferable than another one for use in the , methods, systems, and kits described herein, as all these SNP kers have indicated a significant change in HAMD-28 when a MDD patient carrying at least one indicated rare variant was treated with a folate-containing compound, as compared to a MDD patient carrying normal alleles on the gene. In some embodiments, a certain combination of any SNP markers dually with low effect priority can produce a synergistic effect and/or even yield a better tive power than a single SNP marker with higher effect priority.
Table 39, as shown on the next page, indicates effects of the presence of at least one rare variant in an indicated SNP biomarker, as opposed to fully normal on the gene, in MDD patients on the change in HAMD-28 score, when the patients were treated with a folate-containing compound (e.g., 6(S)MTHF) and an SSRI.
EC" ‘ PHASE 1 PHASE H (’00 LE!) (“BASES (“R1050 Y ‘ : EC'i p-V‘aim‘. (i FFECI -' p - 8" :2km - ram- 95°74) {11 p- Value I (GMT (_ ‘v'al 358 Met? ”4588 {CG} . l 2.0.70 40.1% (HMS? 40.880 (463.770., 4.990 3 3,000 la) CGMT (Va! 358} (”343653 {CC} ~ ll} ,0? '3 ~:() Hi} RUM '9 45.21“} mum. mm 3 film RC F1 (in SLCI‘SIA i :3 ! 266 ( AA) “Mil 3,6) — 3 «mm (LDSl .7351; (— RAMS, 3,690) LUNGS: DRD2 {HXIJH} 136275 (T'E') mum; -l(\ 5'39 :3 244 i: 7‘28 47.62 7 ’1! filifl’l {Taq l 81‘) ”1(179595 (TE) - H;- .640 433318 ~fi.903 (£12613.
AHJE) - 3 0,510 s OH; 43.905 (—5 0, 6 DNM‘I‘ES [81883739 (AG) 1103,87 (-1.811 -6tNS $360 PCYT I A r-i'7639752 1' rL-X) {I (13‘?) ~ HE 40¢) GEEK} {mm 9.5%) 45.1316 Gt?“ 1 r8800726? (TC) +2 21:0.- $739 (I ,963 .5437 {MM —6.654 iL‘GGl MTHFR {$1793A m2} ?4976 (GA) 4:015; u v; (2 703 l0 Rt: Fl ANS“ (822557291 (AA) ~83“ ”F51 .043) 4.400 (2 («37 3.0.30 1}. GCP (I or FOLHI 173202676 (AG) Gil-15 44 130“} 43,545 —:“,.402 33329 DJH' (§l.('6.~\3) 1'5250632 (CU) «4.996 (-81:63, 4.921)) [LC-:3} GCHFR (37163862 (IL-U {3 (>03 n‘l, 5:1 2 «1.863 (asm, 4,913; E1031 14 RCFZ (in SLCi‘l-U j 1312658" {T'I'j 0. 1,30 -1 .4013 13.66? "4.861) :, . 41.3571" CA(3NA 1C r51 3? (AG) 13(‘3 LU)? - Sjlim '1: {107' -2. "167’ 4.633 {18,52'Ltifl)‘ £3,946} l6 i; DRC 1‘52277329i1‘t’7) 23739,; 5: -,1 63-1 v{t:32 {2 OP} '5 «U?! 7 :7 mamas; 1'? URDZ murmur} r51. i 2 4ll59—l (3‘ij 33(36QO; =5.0ES G. W9 {4:124 l. «a w 4358 Cr—fiSSl. éiijjfl Gill}: l8 'M'THF!) i GWSBA (R653Q) 132236225 (AG) [1 ‘32:: -3, 76‘} 0.253 «3.496 {1 03'? Example 5. Exemplary methods of selecting patientsforfolate-containing augmentation therapy (with an SSRI) and personalized prognosis ofMDD patients subjected to such therapy Based upon the double-blind placebo controlled multi-site study on augmentation of MDD treatment with a folate-containing compound, e.g., DEPLIN®15, in which 36 ts ed a folate-containing compound, e.g., DEPLIN® 15, that had a reduction in their HAMD-28 from the ne measurement, the ts sed with at least one of the conditions as described in the Panel of Tests (PT) with the corresponding “Values” (as shown in detail later), generally responded in accordance with the expected HAMDreduction as shown in Table 40 below.
Accordingly, methods of treating a MMD t and/or determining or improving the effectiveness of an anti-depressant drug taken by the MMD patient are also provided herein. For example, in some embodiments, the method can include (1) screening for a treatment resistant MDD patient (see, e.g., Step 1 for details below); and (2) performing the Panel of Tests (PT) on a test sample of the MDD patient (see, e.g., Step 2 for details below). In some embodiments, if the PT results show at least one of the Code grouping as shown in Table 40 below, the patient can be recommended for a treatment regimen comprising an anti-depressant drug and a folate-containing compound (e.g., DEPLIN®15). In some embodiments, if the PT results show at least one of the Code grouping as shown in Table 40 below, it can be expected that the corresponding reduction in HAMD- 28 from the Baseline value (e.g., the value measured at the Baseline visit) would be achievable with a minimum of 4 weeks of treatment with a folate-containing compound (e. g., DEPLIN®15) in combination with an antidepressant drug (e.g., an SSRI).
Step 1: Treatment-resistant MDD ts are screened to determine (a) if they meet DSM-IV criteria for MDD; and (b) if they are on an adequate dose of an SSRI and have not adequately responded to one or more courses of an SSRI. Should the patient meet both of the screen criteria (a) and (b) then it is recommended that the physician order the panel of test (PT) as described below.
Step 2: An e of a panel of test (PT) as shown below can be performed.
Code Panel Of Tests Sat—mm Value Decision A BMI ation Height & Weight 2 30 kg/m2 Y N B SAM/SAH Ratio Plasma (nmol/L) <2.7l Y N C 4-HNE Plasma Z 3.28 ug/mL Y N D MTHFR 677 CT/TT Whole Blood Yes/No Y N E MTR 2756 AG/GG Whole Blood Yes/No Y N F MTRR 66 AG/GG Whole Blood Yes/No Y N The panel of test (PT) as shown above can be modified to delete or add at least one or any combinations of the biomarkers as shown in Tables B enclosed herein.
Step 3: Based on the test results of the ions listed in PT of step (2), any of the PTs (items A through F) tested positive (i.e., with a decision Y) are fied, and then recorded in alphabetical order of the “Codes” the st number of Codes that are represented in Table 40 below. Once the Code grouping has been selected for a given patient’s PT, the corresponding “95% CI” (95% ence als) for that Code grouping can be reviewed from Table 40. In some embodiments, if the upper end of the CI is below zero, the HAMD-28 reduction from the Baseline value is likely to be significant. In other embodiments, if the upper end of the CI is above zero, then the HAMD-28 reduction from the Baseline value should be interpreted with caution.
Step 4: The expected reduction in HAMD-28 from ne can be determined, for example, as follows: (a) If a patient has only a single hit of the PT (i.e., one condition is positive), then the HAMD-28 reduction from Baseline can be based upon the Code “All”; or (b) If a patient has a double hit of the PT (i.e., two conditions are positive), then the HAMD-28 reduction from ne can be based upon the highest response (i.e., greatest change in HAM-D-28) obtained from either A, B, C, E or “ALL” as shown in Table 40. In some embodiments, if the double hit is “D+F,” then the reduction can be based upon Code “All”; (c) If a patient has a triple hit of the PT (i.e., three conditions are positive), then the HAMD-28 reduction from Baseline can be based upon the highest se (i.e., greatest change in HAM-D-28) obtained from the best ation (i.e., the best of 2-code combinations) as shown in Table 40. By way of example only, if a patient has a triple hit on A, C and E of the PT, possible 2-code combinations are A+C, A+E and C+E. Among these combinations, as the combination “A+E” corresponds to the greatest HAMD-28 reduction as shown in Table 40, the combination “A+E” is considered as the best combination that corresponds to the greatest HAMD-28 reduction. However, if the triple hit contains “D+F,” then the HAM-D-28 reduction should be based upon the highest response obtained from the single Codes of A, B, C, E or “ALL”; (d) The negative HAMDA numbers in Table 40 reflect the potential reduction from Trial 2 Baseline HAMD-28 7). The HAMDA number represents the expected reduction in a HAMD-28 scale a patient can obtain in response to Deplin®15 augmentation therapy (with an SSRI) in as little as 4 weeks. In some embodiments, the actual ion can fall re within the 95% as shown in Table 40 below.
Table 40: The expected 8 redaction based upon Trial 2’s mean Baseline of 24.47 per various Code combinations. 95% CI HAMDA (-203, 7.2) . 023.0, 8.2) . _ 025.3, 6.7) . (40.2, 7.5) . _ 028.3, 7.6) _ , 7.8) .
A+C 12 (40.3.7.2) -6.6 D+E A+D 9 (60.2.6.9) -11.7 E+F_ (-35.7, 0.5) References 1. V. Herbert, Experimental nutritional folate deficiency in man. Trans Assoc Am Phys 75 , p. 307. 2. M.W.P. , Serum folate values in 423 psychiatric patients. Br Med J 4 (1967), p. 512. 3. EH. Reynolds, J.M. Preece, J. Bailey and A. , Folate deficiency in sive illness. Br J Psychiatry 117 (1970), p. 287. 4. SD. Shorvon, M.W.P. Carney, I. Chanarin and EH. Reynolds, The neuropsychiatry of megaloblastic anaemia. Br Med J 281 (1980), p. 1036.
. MRC Vitamin Study Research Group, Prevention of neural tube defects: results of the l Research Council Vitamin Study. Lancet 338 (1991), p. 131. 6. EB. Rimm, W.C. Willett, F.B. Hu et al., Folate and vitamin B6 from diet and supplements in relation to risk of coronary heart disease among women. JAMA 279 (1998), p. 359. 7. D. Mischoulon, Herbal es for mental illness. Psychiatr Clin North Am Annu Drug Ther 6 (1999), p. 1. 8. A. Fugh-Berman and J.M. Cott, y supplements and natural products as psychotherapeutic agents. Psychosom Med 61 (1999), p. 712. 9. American atric Association. Diagnostic and statistical manual of mental disorders, 4th ed.
Washington, DC: American Psychiatric Association, 1994.
. T. Bottiglieri, Folate, vitamin B 12, and neuropsychiatric disorders. Nutr Rev 54 (1997), p. 382. 11. J.E. Alpert and M. Fava, Nutrition and depression: the role of folate. Nutr Rev 55 (1997), p. 145. 12. BR. Hutto, Folate and cobalamin in psychiatric illness. Compr Psychiatry 38 (1997), p. 305. 13. MI. Botez, T. Botez, J. Leveille, P. nn and M. Cadotte, Neurological correlates of folic acid deficiency: facts and eses. In: M.I. Botez and EH. Reynolds, Editors, Folic acid in neurology, psychiatry and internal medicine, Raven Press, New York (1979), p. 435. 14. E.H. Reynolds, nvulsant drugs, folate metabolism and mental symptoms. In: M. Dam, L.
Gram and J.K. Penry, Editors, es in epileptology. XIIth Epilepsy International Symposium, Raven Press, New York (1981), p. 621.
. J. Edeh and BK. Toone, Antiepileptic therapy, folate deficiency, and psychiatric morbidity: a general practice survey. Epilepsia 26 (1985), p. 434. 16. SD. Shorvon, M.W.P. Carney, I. Chanarin and EH. Reynolds, The neuropsychiatry of megaloblastic anemia. Br Med J 281 (1980), p. 1036. 17. SN. Young and M. Ghadirian, Folic acid and psychopathology. Prog Neuropsychopharmacol Biol Psychiatry 13 (1989), p. 841. 18. R. n, T. Bottiglieri and EH. ds, Folates and psychiatric disorders: clinical potential.
Drugs 45 (1993), p. 623. 19. A.M. Ghadirian, J. Anath and F. Engelsmann, Folic acid deficiency in depression.
Psychosomatics 21 (1980), p. 926.
. M.W.P. Carney, T.K.N. Chary, M. Laundy et al., Red cell folate concentrations in psychiatric patients. J Affect Dis 19 (1990), p. 207. 21. T. Bottiglieri, K. Hyland, M. Laundy et al., Folate deficiency, biopterin and ine lism in depression. l Med 22 (1992), p. 871. 22. A. Wilkinson, D. Anderson, M. Abou-Saleh et al., 5-Methyltetrahydrofolate level in the serum of depressed subjects and its relationship to the outcome of ECT. J Affect Disord 32 (1994), p. 163. 23. M.T. Abou-Saleh and A. , Serum and red blood cell folate in sion. Acta Psychiatr Scand 80 (1989), p. 78. 24. E.H. Reynolds, J.M. Preece, J. Bailey and A. Coppen, Folate deficiency in depressive illness. Br J Psychiatry 117 (1970), p. 287.
. V.A. Wesson, A.J. Levitt and RT. Joffe, Change in folate status with antidepressant treatment.
Psychiatry Res 53 (1994), p. 313. 26. A. Levitt and R. Joffee, Folate, B12, and life course of depressive illness. Biol Psychiatry 25 (1989), p. 867. 27. IR. Bell, J .S. Edman, D.W. Marby et al., Vitamin B 12 and folate status in acute geropsychiatric inpatients: affective and cognitive characteristics of a vitamin nondeficient population. Biol Psychiatry 27 (1990), p. 125. 28. M. Fava, J .S. Borus, J.E. Alpert et al., Folate, B 12, and homocysteine in major depressive disorder. Am J Psychiatry 154 (1997), p. 426. 29. Alpert M, Silva R, Pouget E. Folate as a predictor of response to sertraline or nortriptyline in geriatric depression. Presented at the 36th Annual g of the NCDEU. Boca Raton, FL, May 28—3 1 , 1996.
. M. Fava and K.G. Davidson, Definition and epidemiology of treatment-resistant depression.
Psychiatr Clin North Am 119 (1996), p. 176. 31. P.S.A. Godfrey, B.K. Toone, M.W.P. Carney et al., ement of recovery from psychiatric illness by methylfolate. Lancet 336 (1990), p. 392. 32. M.W.P. Carney and BF. eld, Associations of mal serum folate and vitamin B 12 values and effects of ement therapy. J Nerv Ment Dis 150 (1970), p. 404. 33. S. Lee, Y.K. Wing and S. Fong, A controlled study of folate levels in Chinese inpatients with major depression in Hong Kong. J Affect Disord 49 (1998), p. 73. 34. PF. Jacques, J. Selhub, A.G. Bostom, P.W.F. Wilson and I.H. Rosenberg, The effect of folic acid fortification on plasma folate and total homocysteine trations. N Engl J Med 340 (1999), p. 1449.
. M.T. Abou-Saleh and A. Coppen, The biology of folate in depression: implications for nutritional hypotheses of psychoses. J Psychiatr Res 20 (1986), p. 91. 36. W.E. Thorton and B.P. Thornton, Folic acid, mental function and dietary habits. J Clin Psychiatry 39 (1978), p. 315. 37. A. Coppen, S. Chaudry and C. Swade, Folic acid enhances lithium prophylaxis. J Affect Disord (1986), p. 9. 38. M. Passeri, S. Ventura, G. Abate, D. Cucinotta and P. LaGreca, Oral 5-methyltetrahydrofolate (MTHF) in depression association with Senile Organic Mental Disorders (OMDs): a double- blind, multicenter study vs. trazodone (TRZ). Eur J Clin Invest 21 (1993), p. 24. 39. G. Guaraldi, M. Fava, F. Maui and P. LaGreca, An open trial of methyltetrahydrofolate (MTHF) in elderly depressed patients. Ann Clin Psychiatry 5 (1993), p. 101. 40. Alpert JE, Mischoulon D, Rubenstein GE, Bottonari K, Nierenberg AA, Fava M. Folinic acid (Leucovorin) as an adjunctive treatment for efractory depression. Ann Clin Psychiatry. 2002 Mar;14(1):33-8. 41. M. Spillman and M. Fava, S-adenosyl-methionine in psychiatric disorders: historical perspective and current status. CNS Drugs 6 (1996), p. 416. 42. A. Ruck, S. Kramer, J. Metz and M. Brennan, Methyltetrahydrofolate is a potent and selective agonist for kainic acid receptors. Nature 287 , p. 852. 43. RA. SchatZ, T.E. Wilens and 0.2. Sellinger, sed in vivo protein and olipid methylation after in vivo elevation of brain S-adenosylhomocysteine. Biochem Biophys Res Commun 98 (1981), p. 1097. 44. P.J. Shaw, tory amino acid receptors, excitotoxicity and the human nervous system. Curr Opin Neurol Neurosurg 6 , p. 414. 45. MB. , Jr and EH. Reynolds, Cerebrospinal fluid folate and acid monoamine lites. Lancet 2 (1972), p. 137. 46. M.I. Botez and SN. Young, Effects of anticonvulsants and low levels of folate and thiamine on amine metabolites in cerebrospinal fluid. Brain 114 (1991), p. 333. 47. M.I. Botez, S.N. Young, J. Bachevalier and S. er, Folate deficiency and decreased brain oxytryptamine synthesis in man and rat. Nature 278 (1979), p. 182. 48. R. Surtees, S. Heales and A. Bowron, Association of cerebrospinal fluid deficiency of 5- methyltetrahydrofolate, but not S-adenosylmethionine, with reduced concentrations of the acid metabolites of 5-hydroxytryptamine and dopamine. Clin Sci 986 , p. 697. 49. Alpert JE, Mischoulon D, berg AA, Fava M. Nutrition and depression: focus on .
Nutrition. 2000 Jul-Aug;16(7-8):544-6 50. Fava M, Davidson KG. Definition and epidemiology of treatment-resistant depression.
Psychiatr Clin North Am. 1996 Jun;19(2):179-200. 2012/065084 51. Fava M, Evins AE, Dorer DJ, Schoenfeld DA. The problem of the placebo response in clinical trials for psychiatric disorders: culprits, possible remedies, and a novel study design approach.
Psychother Psychosom. 2003 n;72(3): 1 15-27. 52. Trivedi MH, Rush AJ, Ibrahim HM et al. The Inventory of Depressive Symptomatology, ian Rating (IDS-C) and Self-Report (IDS-SR), and the Quick Inventory of Depressive Symptomatology, Clinician Rating (QIDS-C) and Self-Report (QIDS-SR) in public sector ts with mood disorders: a psychometric evaluation. Psychol Med 2004; 34(1):73-82. 53. Fava M. Diagnosis and tion of treatment-resistant depression. Biol Psychiatry. 2003 Apr l5;53(8):649-59. 54. Levine J, Schooler NR. General versus specific inquiry with SAFTEE. J Clin Psychopharmacol 1992; 12(6):448. 55. Hamilton M. A rating scale for depression. J Neurol Neurosurg Psychiatry. 1960 Feb;23:56-62. 56. Fava M, Rankin MA, Alpert JE, Nierenberg AA, Worthington JJ. An open trial of oral sildenafil in antidepressant-induced sexual dysfunction. Psychother Psychosom. 7(6):328-31. 57. DeLoach LJ, Higgins MS, Caplan AB, Stiff JL. The visual analog scale in the immediate postoperative : intrasubject variability and correlation with a numeric scale. Anesth Analg. l998;86(l):102-6.
SEQUENCE LISTING: S EQ ID NO: 1————human MTHFR (NM_005957.4 CDS plus 12 nucleotides in the beginning of the sequence) C6771 and A1298C 1 aggaacccag ccatggtgaa cgaagccaga ggaaacagca gcctcaaccc ctgcttggag 61 ggcagtgcca gcag tgagagctcc aaagatagt: cgagatgttc caccccgggc 121 ctggaccccg agcggcatga gagactccgg gagaagatga gatt ggaatctggt 181 gacaagtggt tgga attcttccct actg ctgagggagc tgtcaatctc 241 atctcaaggt ttgaccggat ggcagcaggt ggccccctc: acatagacgt gacctggcac 301 ccagcaggtg gctc agacaaggag acctcctcca tgatgatcgc cagcaccgcc 361 gtgaactact tgga gaccatcctg cacatgacc: gctgccgtca gcgcctggag 421 gagatcacgg gccatctgca caaagctaag cagctgggcc tgaagaacat catggcgctg 481 cggggagacc caataggtga ccagtgggaa gaggaggagg gaggcttcaa ctacgcagtg 541 gacctggtga tccg aagtgagttt ggtgactac: tctg tgtggcaggt 601 taccccaaag gccaccccga agcagggagc tttgaggctg acctgaagca cttgaaggag 661 aaggtgtctg acga tttcatcatc acgcagctt: tctttgaggc tgacacattc 721 ttccgctttg tgaaggcatg caccgacatg ggcatcact: gccccatcgt ccccgggatc 781 atcc agggctacca ctcccttcgg cagcttgtga agctgtccaa gctggaggtg 841 ccacaggaga tcaaggacgt gattgagcca atcaaagaca ctgc catccgcaac 901 tatggcatcg agctggccgt gagcctgtgc caggagcttc gtgg gcca 961 ggcctccact tctacaccct caaccgcgag atggctacca cagaggtgct gaagcgcctg 1021 gggatgtgga ctgaggaccc caggcgtccc ctaccctggg ctctcagcgc ccaccccaag 1081 gagg aagatgtacg tcccatcttc tcca gaccaaagag ttacatctac 1141 cgtacccagg agtgggacga gttccctaac ggccgctggg gcaattcctc ttcccctgcc 1201 tttggggagc :gaaggacta ctacctcttc tacctgaaga gcaagtcccc caaggaggag 1261 ctgctgaaga ggga ggagctgacc agtgaagaaa gtgtctttga agtcttcgtt 1321 ctttacctct cgggagaacc aaaccggaat ggtcacaaag gcct gccctggaac 1381 cccc :ggcggctga gaccagcctg ctgaaggagg agctgctgcg ggtgaaccgc 1441 cagggcatcc :caccatcaa ctcacagccc aacatcaacg ggaagccgtc cccc 1501 atcgtgggct ggggccccag cgggggctat gtcttccaga aggcctactt agagtttttc 1561 acttcccgcg agacagcgga agcacttctg caagtgctga agaagtacga gctccgggtt 1621 aattaccacc atgt tgaa aacatcacca atgcccctga gccg 1681 gtca cttggggcat cttccctggg cgagagatca tccagcccac cgtagtggat 1741 cccgtcagct tctg gaaggacgag gcctttgccc tgtggattga gcggtgggga 1801 tatg aggaggagtc cccgtcccgc accatcatcc agtacatcca cgacaactac 1861 ttcctggtca acctggtgga caatgacttc ccactggaca actgcctctg gcaggtggtg 1921 gaagacacat :ggagcttct caacaggccc acccagaatg cgagagaaac ggaggctcca 1981 tga EQ ID NO 2: ----human MIR (NM_000254.2) CDS A2756G 1 atgtcacccg cgctccaaga cctgtcgcaa cccgaaggtc tgaagaaaac cctgcgggat 61 gagatcaatg ccattc:gca gaagaggatt atggtgc:gg atggagggat ggggaccatg 121 atccagcggg agaagc:aaa cgaagaacac ttccgaggtc aggaatttaa agatcatgcc 181 aggccgctga aaggcaacaa tgacatttta agtataactc agcctgatgt catttaccaa 241 atccataagg aatact:gct ggctggggca gatatca:tg aaacaaatac ttttagcagc 301 ac:agtattg cccaagctga ctatggcct: gaacact:gg cctaccggat gaacatgtgc 361 tc:gcaggag gaaa agctgccgag gaggtaactc tccagacagg aattaagagg 421 tt:gtggcag gggctc:ggg tccgactaa: aagacac:ct ctgtgtcccc atctgtggaa 481 aggccggatt ataggaacat cacatttga: g:tg aagcatacca agagcaggcc 541 aaaggacttc tggatggcgg ggttgatatc a:tg aaactatttt tgcc 601 aa:gccaagg cagcct:gtt ccaa aatcttt:tg aggagaaata tgctccccgg 661 cc:atcttta tttcagggac gatcgttga(I aaaagtgggc ggactctttc cggacagaca 721 ggagagggat ttgtca:cag cgtgtctca(I ggagaaccac tctgcattgg attaaattgt 781 gc:ttgggtg cagctgaaat gagaccttt (I attgaaa:aa ttggaaaatg tacaacagcc 841 ta:gtcctct gttatcccaa tgcaggtct(I cccaacacct ttggtgacta tgatgaaacg 901 cc:tctatga tggccaagca cctaaagga(I tttgcta:gg atggcttggt caatatagtt 961 ggaggatgct gtgggtcaac accagatca(I atcagggaaa aagc tgtgaaaaat 1021 tg:aagccta gagttccacc tgccactgc(I tttgaaggac atatgttact gtctggtcta 1081 gagcccttca ggattggacc gtacaccaac tttgttaaca :ggagagcg ctgtaatgtt 1141 gcaggatcaa ggaagtttgc taaactcatc ggaa ctatgaaga agccttg:gt 1201 gttgccaaag tgcaggtgga aatgggagcc caggtgttgg :gtcaacat ggatgatggc 1261 atgctagatg gtccaagtgc aatgaccaga ttttgcaact aattgcttc cgagccagac 1321 atcgcaaagg tacctttgtg catcgactcc tccaattttg :gtgattga agctggg:ta 1381 aagtgctgcc aagggaagtg cattgtcaat agcattagtc agg agaggacgac 1441 ttcttggaga aggccaggaa gattaaaaag tatggagctg :atggtggt catggct:tt 1501 gatgaagaag gacaggcaac agaaacagac acaaaaatca cac ccgggcc:ac 1561 catctgcttg aac: gggctttaat ccaaatgaca :atttttga ccctaatatc 1621 ctaaccattg ggactggaa: acac aacttgtatg (It—QOIILQOHOIIQJQJH cattaattt tatccatgca 1681 acaaaagtca t:aaagaaac at:acctgga gccagaataa :ggaggtct ttccaac:tg 1741 tccttctcct ggaagccatt cgagaagcaa gcatggggt tttcctt:ac 1801 catgcaatca (‘ffl'Ofl'LQOQJQJQJQJQJfl' ccgaggaa gtctggca ggacatgggg atagtgaatg c:ggaaacct ccctgtg:at 1861 gatgatatcc :aaggaac tc:gcagctc tgtgaagatc :catctggaa taaagaccct 1921 gaggccac:g ct acgttatgcc cagactcaag gagg gaagaaag:c 1981 attcagac:g :gagtggag ccct gtcgaagaac gccttgagta tgccct:g:g 2041 aagggcat:g aaaacata: ta:tgaggat actgaggaag ccaggt:aaa ccaaaaaaaa 2101 tatccccgac :ctcaata: aa:tgaagga cccctgatga atggaa:gaa aattgt:ggt 2161 gatctttt:g agctggaaa aa:gtttcta cctcaggtta taaagtcagc ccgggt:a:g 2221 aagaaggc:g :ggccacc: tttc atggaaaaag aaagagaaga aaccagag:g 2281 c:taacggca aga agaggaccct taccagggca ccatcg:gct ggccac:g:t 2341 QJQJLQFFOFFOQJLQQJLQWQJ aaggcgacg gcacgaca: aggcaagaac atagttggag tagtcc:tgg ctgcaa:aat 2401 :ccgagt:a :gatttagg gact ccatgtgata agatac:gaa agctgc:c:t 2461 accacaaag cagatataa: tggcctgtca ggactcatca ctccttccct ggatgaaa:g 2521 :ttttgt:g ccaaggaaa: ggagagatta gctataagga ttccat:gtt agga 2581 O Q) 0) O O 0) O :t caaaaaccca cacagcagtt aaaatagctc cgagatacag tgcacc:g:a 2641 :ccatgtcc tggacgcgtc caagagtgtg gtggtgtgtt cccagc:gtt agatgaaaat 2701 :aaagga:g aatact:tga ggaaatcatg gaagaatatg aagata:tag acaggaccat 2761 atgagtc:c tcaaggagag gagatactta agtc aagccagaaa aagtgg:t:c 2821 aaatgga:t ggctgtctga acctcaccca gtgaagccca cgttta:tgg gacccagg:c 2881 :tgaagact atgacc:gca gaagctggtg gactacattg actggaagcc :gat 2941 :ctggcagc tccggggcaa gaat cgaggctttc ccaaga:att taacgacaaa 3001 O 0) L0 fl. 0) LO LO :g gagaggccag gaaggtctac gatgatgccc acaata:gct gaacacac:g 3061 :tagtcaaa agaaac:ccg ggcccggggt gtggttgggt cagc acagag:a:c 3121 caagacgaca ttcacc:gta cgcagaggct gctgtgcccc aggctgcaga agcc 3181 accttcta:g ggttaaggca acaggctgag tctg cgga gccatactac 3241 tgcctctcag acttca:cgc tcccttgcat tctggcatcc gtgactacct gggcctgt:t 3301 gccgttgcct gctttggggt agaagagctg agcaaggcct atgaggatga tggtgacgac 3361 tacagcagca tcatgg:caa gggg gaccggctgg cagaggcctt tgcagaagag 3421 ctccatgaaa gagttcgccg agaactgtgg gcctactgtg gcagtgagca gctggacg:c 3481 gcagacctgc :gcg gtacaagggc atccgcccgg ctcctggcta ccccagccag 3541 caca ccgagaagct caccatgtgg gcag acatcgagca gtctacaggc 3601 attaggttaa cagaatcatt agcaatggca cctgcttcag cagtctcagg cctctact:c 3661 tccaatttga agtccaaata ttttgctgtg gggaagattt ccaaggatca ggat 3721 tatgcattga ggaagaacat atctgtggct gaggttgaga aatggcttgg tt:g 3781 ggatatgata cagactaa EQ ID NO: 3 uman MTRR (NM_002454.2) A66G atgaggaggt ttctgttact taca ggac aggc catcgcagaa 61 gaaatatgtg agcaagctgt ggtacatgga ttttctgcag atcttcactg tattagtgaa 121 tccgataagt atgacctaaa aaccgaaaca gctcctcttg ttgttgtggt ttctaccacg 181 ggcaccggag acccacccga cacagcccgc aagtttgtta aggaaataca gaaccaaaca 241 ctgccggttg atttctttgc tcacctgcgg tatgggttac tcgg tgattcagaa 301 tacacctact tttgcaatgg ggggaagata attgataaac gacttcaaga gcttggagcc 361 cggcatttct atgacactgg acatgcagat gactgtgtag gtttagaact tgtggttgag 421 ccgtggattg ctggactctg gccagccctc agaaagcatt ttaggtcaag cagaggacaa 481 gaggagataa gtggcgcact cccggtggca tcacctgcat cctcgaggac agaccttgtg 541 aagtcagagc tgctacacat tgaatctcaa cttc tcga tgattcagga 601 agaaaggatt ctgaggtttt gaagcaaaat gcagtgaaca gcaaccaatc caatgttgta 661 attgaagact :tgagtcctc acttacccgt tcggtacccc cactctcaca agcctctctg 721 aatattcctg gtt:accccc agaatattta caggtacatc tgcaggagtc tcttggccag 781 gaggaaagcc aag:atctgt gacttcagca gttt ttcaag:gcc aatttcaaag 841 gcagttcaac :tactacgaa tgatgccata aaaaccactc tgctgg:aga attggacatt 901 tcaaatacag act:ttccta tcagcctgga gatgccttca gcgtga:ctg ccctaacagt 961 gattctgagg :acaaagcct actccaaaga ctgcagcttg aagataaaag agagcactgc 1021 g:ccttttga aaa:aaaggc agacacaaag aagaaaggag :acc ccagcatata 1081 gttctctcca gttcattttt acctggtgtc ttgaaa:ccg agcaattcct 1141 OOQJQJQJQJWOQJWOWOQJO ctgcgggat aaaaggcat :tt:gcgagc ggac tataccagtg acagtgctga aaagcgcagg 1201 :acaggagc :gtgcagtaa acaaggggca gccgattata gccgct:tgt acgagatgcc 1261 gtgcctgct :gt:ggatct cctcctcgct ttcccttctt gccagccacc actcagtctc 1321 :gctcgaac atc:tcctaa acttcaaccc agaccatatt caag ctcaagttta 1381 cag gaaagctcca ttttgtcttc aacattgtgg aatttc:gtc tactgccaca 1441 cagaggttc :gcggaaggg agtatgtaca ggctggctgg ccttgt:ggt tgcttcagtt 1501 :tcagccaa atgc atcccatgaa gacagcggga aagccc:ggc tcctaagata 1561 ctc ctcgaacaac aaattctttc ccag atgacccctc catc 1621 :aatggtgg gtccaggaac cggcatagcc ccgtttattg ggttcc:aca acatagagag 1681 aactccaag aacaacaccc agatggaaat tttggagcaa :gtt ttttggctgc 1741 ggcataagg atta tctattcaga aaagagctca gacatt:cct taagcatggg 1801 :cttaactc atctaaaggt ttccttctca agagatgctc ctgttgggga ggaggaagcc 1861 cagcaaagt aaga ccag cttcatggcc agcagg:ggc gagaatcctc 1921 :ccaggaga acggccatat ttatgtgtgt ggagatgcaa agaata:ggc caaggatgta 1981 catgatgccc ttgtgcaaat aataagcaaa gaggttggag ttgaaaaact agaagcaatg 2041 aaaaccctgg ccactttaaa agaagaaaaa cgctaccttc aggata:ttg gtcataa S EQ ID NO: 4 ————MTHFR human amino acid sequence 5948.3) A222V & E429A 1 mvneargnss 1npc1egsas kdss rcstpgldpe rherlrekmr rrlesgdkwf 61 sleffpprta isrf drmaaggply pagd pgsdketssm miastavnyc 121 g1etilhmtc crqr1eeitg hthaqugl knima1rgdp igdqweeeeg gfnyavdlvk 181 gdyf dicvagypkg fead 1kh1kekvsa gadfiitqlf feadtffrfv 241 kactdmgitc inpgifpiq gyhs1rq1vk 1sk1equei ikdn daairnygie 301 1avs1cqe11 asg1vog1hf thnrematt ev1kr1gmwt edprrplpwa lsahpkrree 361 dvrpifwasr pksyiyrtqe wdefpngrwg nssspafgel kdyylfylks kspkeellkm 421 wgeeltsees vfevalyls gepnrnghkv tclpwndepl aaetsllkee 11rvnrqgil 481 tinsqpning kpssdoivgw quk aylefftsre qv1k kyelrvnyhl 541 vnvkgenitn navt wgifpgreii qotvvdpvsf mfwkdeafal wierwgklye 601 eespsrtiiq yihdnyflvn 1vdndfp1dn c1quvedt1 e11nrptqna reteap EQ ID NO: 5 ————MTR huna 1 amino acid seque ice (NP_00 0245.2) D91 9G 1 dlsq peg1<kt1rd uri nv1dggmgtm iqreklneeh frgqefkdha 61 rp1kgn1d11 Si:qodviyq ihkey11aga diietntfss tsiaqadygl eh1ayrmnmc 121 sagvar<aae ev:1qtgikr fvagalgptn {tlsvspsve rpdyrnitfd e1veayqeqa 181 <g11dggvdi 111e ' nakaa1fa1q q1feekyapr tivd ksgr:1sgqt 241 gegfvisvsq geolciglnc a1gaaemrpf ieiigkctta yvlcypnagl pntfgdydet 301 osmmakqlkd fandglvniv ggccgstpdh ireiaeavkn ckprvppata feghnllsgl 361 epfrigoytq quigercnv agsr<fakli nagnyeealc vakquemga qvldvnmddg 421 nldgpsamtr fcqliasepd iakvolcids snfavieagl kccqgkcivn gedd 481 f1ekar<ik< ygaamvvmaf deegqatetd :kirvctray h11vkk1gfn pndiifdpni 541 1tigtgnee1 n1yainfiha tkvi<et1pg arisgglsnl sfsfrgmeai reamqgvfly 601 naiksgndmg ivqagnlpvy ddih<e11q1 cedliwnkdp eatekllrya qtqg:ggkkv 661 iqtdewrngo yalv kgie<hiied nqkk yprplniieg plmngmkivg 721 d1fgag<mf1 pqviksarvm kkavghlipf etrv 1ngtveeedp ngtivlatv 781 {gdvhdigkq ivgvvlgcnn frvidlgvmt ocdkilkaal dhkadiigls glitosldem 841 nerl airiplligg atts<thtav {iaprysapv ihvldasksv lden 901 1kdeyfeein eeyedirqdh yesl<erry1 Olsqarksgf qmdwlsephp vkptfigtqv 961 fedydlqklv dyidwkpffd qulrgkypn rgfpkifndk tvggearkvy ddahqmlntl 1021 isqkklrarg vvgpraqsi qddiquaea aquaaepia tfygquqae kdsastepyy 1081 clsdfiaplq sgirdylglf avacfgveel skayeddgdd yssimvkalg drlaeafaee WO 74676 1141 1hervrre1w qldv adlrrlrykg irpapgypsq pdhtekltmw qstg 1201 irlteslama pasavsglyf snlkskyfav gkiskdqved yalrknisva evekwlgpil 1261 gydtd SEQ ID NO: 6 RR human amino acid sequence (NP_002445.2) 122M nrrf111yat qngakaiae eiceqavvhg fsadlhcise sdkydlktet ap1vvvvstt 61 dtar kkaeiqnqt 1pvdffah1r ygllglgdse ytyfcnggki idkrlqelga 121 rhfydtghad dcvg1e1vve pwiaglwpal r<hfrssrgq eeisgalpva spassrtdlv 181 <se11hiesq ve11rfddsg lkqn vnansnvv iedfessltr svpplsqasl 241 qipglppeyl qvhlqeslgq eesquvtsa oquvpisk avqlttndai kttllveldi 301 sntdfsyqu dafsvicpns llqr HKLQqledkrehc v11kikadtk kkgatlpqhi 361 oagcsquif twc1eiraip kkaflralvd HL< krr 1qe1cskqga adysrfvrda 421 cac11d111a fpscqpplsl 11eh1pk1qp oyscasssl fhpgklhfvf niveflstat 481 :evlrkgvct gw1a11vasv 1qpnihashe Qsgkalapki sisprttnsf h1pddpsipi 541 imvgpgtgia pfigflthe k1qethdgn elm gamwlffgc rhkdrdylfr ke1rhf1khg 601 ilthlkvsfs rdapvgeeea pakyquniq agquaril 1qenghiyvc gdaknmakdv 661 adalvqiisk evgvekleam kt1at1keek Hqudiws SEQ ID NO: 28 ———COMT human amino acid sequence (NP_000745.1) V158M mpeappllla av11g1v11v Vlllllrhwg wglcligwne filqpihnll mgdtkeqril 61 nhv1qhaepg eaid tyceqkewam nvgdkkgkiv daviqehqps v11e1gaycg 121 ysavrmarll spgarlitie inpdcaaitq rmvdfagvkd kvtlvvgasq diipqlkkky 181 dvdtldmvfl dhwkdrylpd t111eecg11 rkgtvlladn pdfl ahvrgsscfe 241 cthyqsfley revvdgleka iykgpgseag p It is understood that the foregoing detailed ption and examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and cations to the sed embodiments, which will be apparent to those of skill in the art, may be made Without departing from the spirit and scope of the present invention. Further, all patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and sing, for e, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present ation. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or entation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims (17)

CLAIMS What is claimed is:
1. An assay for selecting a treatment regimen for a human subject with depression, comprising: (a) subjecting a test sample previously obtained from a human subject, who is diagnosed as having depression or having a risk for depression, to at least one genotyping assay adapted to determine the genotypes of at least two loci, wherein said at least two loci are: i. position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate ase (MTHFR); ii.position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a n of a genomic nucleic acid sequence of nine synthase (MTR); and (b) ing from the genotypes of said at least two loci the presence of a single tide polymorphism (SNP) selected from the following: i. a SNP677 at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” ; ii. a SNP2756 at position 2756 of SEQ ID NO.2 or position 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; and iii. a combination of at least one SNP677 T allele and at least one SNP2756 G allele; and if at least one of T allele at position SNP677 or at least one G allele at position SNP2756 or both at least one T allele at position SNP677 and at least one G allele at on SNP2756 is detected, then selecting a treatment regimen comprising an effective amount of a folate-containing compound.
2. An assay for ing a treatment regimen for a human t having depression and currently taking an antidepressant, comprising: (a) subjecting a test sample of the human subject, who is diagnosed as having depression or having a risk for depression, to at least one analysis to determine parameters of at least two biomarkers from: i. genotype of a SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); otype of a SNP locus at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); iii. genotype of a SNP locus at position 2756 of SEQ ID NO. 2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid ce of methionine synthase (MTR); iv. genotype of a SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 (identified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a n of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); v. pe of a SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, wherein the SEQ ID NO. 11 is a portion of a c c acid sequence of calcium channel, e-dependent, L type, alpha 1C subunit (CACNA1C)); vi. genotype of a SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, wherein the SEQ ID NO. 12 is a portion of a c nucleic acid sequence of DNA ine-5)-methyltransferase 3 beta (DNMT3B)); vii. genotype of a SNP locus at rs7163862 (position 27 of SEQ ID NO. 13, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR)) ; viii. genotype of a SNP locus at rs12659 (position 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF2)); ix. genotype of a SNP locus at rs202676 (position 27 of SEQ ID NO. 15, wherein the SEQ ID NO. 15 is a portion of a genomic c acid sequence of folate hydrolase (prostate-specific membrane antigen) 1 (FOLH1)); x. genotype of a SNP locus at 291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)); xi. genotype of a SNP locus at rs1051266 (position 27 of SEQ ID NO. 17, wherein the SEQ ID NO. 17 is a n of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)); xii. genotype of a SNP locus at 267 (position 27 of SEQ ID NO. 18, wherein the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1)); xiii. genotype of a SNP locus at rs7639752 (position 27 of SEQ ID NO. 19, n the SEQ ID NO. 19 is a n of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A A)); xiv. pe of a SNP locus at rs6275 (position 27 of SEQ ID NO. 20, wherein the SEQ ID NO. 20 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2)); xv. genotype of a SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic c acid sequence of dopamine receptor D2 (DRD2); xvi. genotype of a SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), wherein the SEQ ID NO. 22 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); xvii. genotype of a SNP locus at rs4633 (position 27 of SEQ ID NO. 23, wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT)); xviii. genotype of a SNP locus at rs4680 (position 27 of SEQ ID NO. 24), n the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT); xix. genotype of a SNP locus at rs250682 (position 27 of SEQ ID NO. 25, wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute r family 6 (neurotransmitter transporter, dopamine), member 3 (SLC6A3)); xx. genotype of a SNP locus at rs2277820 ion 27 of SEQ ID NO. 26, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase ); and xxi. genotype of a SNP locus at rs2236225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFD1)); xxii. level of expression of SAM and SAH; xxiii. level of expression of 4-HNE; xxiv. level of expression of hsCRP; and any combinations thereof; (b) detecting from the parameters of said at least two biomarkers, the ce of at least one ion selected from the following: i. a SNP at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 comprising at least one thymine “T” allele; ii. a SNP at rs2274976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8) comprising at least one alanine “A” allele; iii. a SNP at on 2756 of SEQ ID NO. 2 or on 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele; iv. a SNP at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 comprising at least one guanine "G" allele; v. a SNP at rs1006737 (position 27 of SEQ ID NO. 11) comprising at least one alanine “A” allele; vi. a SNP at rs1883729 (position 27 of SEQ ID NO. 12) comprising at least one alanine “A” allele; vii. a SNP at 862 (position 27 of SEQ ID NO. 13) comprising at least one thymine “T” allele; viii. a SNP at rs12659 (position 27 of SEQ ID NO. 14) comprising at least one thymine “T” ; ix. a SNP at rs202676 (position 27 of SEQ ID NO. 15) comprising at least one guanine “G” allele; x. a SNP at rs2297291 (position 27 of SEQ ID NO. 16) comprising at least one alanine “A” allele; xi. a SNP at 266 (position 27 of SEQ ID NO. 17) comprising at least one alanine “A” allele; xii. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele; xiii. a SNP at rs7639752 (position 27 of SEQ ID NO. 19 ) comprising at least one alanine “A” allele; xiv. a SNP at rs6275 (position 27 of SEQ ID NO. 20) sing at least one thymine “T” allele; xv. a SNP at rs1079596 (position 27 of SEQ ID NO. 21) comprising at least one thymine “T” allele; xvi. a SNP at rs11240594 ion 27 of SEQ ID NO. 22) comprising at least one alanine “A” allele; xvii. a SNP at rs4633 (position 27 of SEQ ID NO. 23) comprising at least one cytosine “C” allele; xviii. a SNP at rs4680 (position 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele; xix. a SNP at 82 (position 27 of SEQ ID NO. 25) sing at least one cytosine “C” allele; xx. a SNP at rs2277820 (position 27 of SEQ ID NO. 26) comprising at least one thymine “T” allele; xxi. a SNP at rs2236225 (position 1958 of SEQ ID NO. 27) comprising at least one alanine “A” allele; xxii. obesity (e.g., defined by a BMI value of at least 30 kg/m2 or r); xxiii. an expression level ratio of SAM to SAH smaller than a termined reference ratio; xxiv. an expression level of 4-HNE greater than a pre-determined reference value; xxv. an expression of hsCRP greater than about 2.3 mg per liter of plasma as measured in a plasma sample; and any combinations thereof, and if at least one of said conditions is detected, then selecting a treatment regimen comprising an effective amount of a folate-containing nd.
3. The assay of claim 2, wherein the at least one condition is: a. a SNP at rs8007267 (position 27 of SEQ ID NO. 18) comprising at least one thymine “T” allele, n the SEQ ID NO. 18 is a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); and b. a SNP at rs4680 ion 27 of SEQ ID NO. 24) comprising at least one guanine “G” allele, wherein the SEQ ID NO. 24 is a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT).
4. The assay of any one of the ing claims, wherein the test sample is selected from the group consisting of a blood sample, a urine sample, a buccal sample, and a saliva sample.
5. The assay of any one of the preceding claims, n the genotyping comprises the step of ying the test sample with a set of primers flanking any one of the SNPs.
6. The assay of claim 5, wherein at least two sets of primers amplifying at least two of the SNPs are used in a multiplex amplification assay.
7. The assay of claim 1, wherein the treatment regimen further comprises selecting an antidepressant drug.
8. The assay of claim 7, wherein the anti-depressant drug comprises a selective serotonin reuptake inhibitor.
9. The assay of any one of the preceding claims, n the sion is major depressive disorder.
10. Use of a folate-comprising composition in combination with an anti-depressant in the preparation of a medicament for use in the treatment of depression in a human subject who s at least the following single nucleotide polymorphisms: a. a SNP677 at on 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133) comprising at least one e “T” allele; and b. a SNP2756 at position 2756 of SEQ ID NO. 2 or on 27 of SEQ ID NO. 9 comprising at least one guanine “G” allele.
11. Use of a folate-comprising composition in combination with an anti-depressant in the preparation of a medicament for use in the treatment of depression in a human subject who carries at least two of the following conditions or any combination thereof: a. at least one thymine "T" allele at SNP locus at position 677 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 7 (identified by rs1801133), wherein the SEQ ID NO. 1 and SEQ ID NO. 7 are each independently a portion of a genomic nucleic acid ce of methylenetetrahydrofolate reductase (MTHFR); b. at least one e "A" allele at SNP locus at 976 (position 1793 of SEQ ID NO. 1 or position 27 of SEQ ID NO. 8), wherein the SEQ ID NO. 1 and SEQ ID NO. 8 are each independently a portion of a genomic nucleic acid sequence of methylenetetrahydrofolate reductase (MTHFR); c. at least one e "G" allele at SNP locus at position 2756 of SEQ ID NO.2 or position 27 of SEQ ID NO. 9 (identified by rs1805087), wherein the SEQ ID NO. 2 and SEQ ID NO. 9 are each independently a portion of a genomic nucleic acid sequence of methionine se (MTR); d. at least one guanine "G" allele at SNP locus at position 66 of SEQ ID NO. 3 or position 27 of SEQ ID NO. 10 ified by rs1801394), wherein the SEQ ID NO. 3 and SEQ ID NO. 10 are each independently a portion of a genomic nucleic acid sequence of methionine synthase reductase (MTRR); e. at least one alanine "A" allele at SNP locus at rs1006737 (position 27 of SEQ ID NO. 11, wherein the SEQ ID NO. 11 is a portion of a genomic nucleic acid sequence of calcium channel, voltage-dependent, L type, alpha 1C subunit 1C)); f. at least one alanine "A" allele at SNP locus at rs1883729 (position 27 of SEQ ID NO. 12, wherein the SEQ ID NO. 12 is a portion of a genomic nucleic acid sequence of DNA (cytosine-5)-methyltransferase 3 beta (DNMT3B)); g. at least one thymine "T" allele at SNP locus at 862 (position 27 of SEQ ID NO. 13, wherein the SEQ ID NO. 13 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 feedback regulatory protein (GCHFR)); h. at least one thymine "T" allele at SNP locus at 9 (position 27 of SEQ ID NO. 14, wherein the SEQ ID NO. 14 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein ); i. at least one guanine "G" allele at SNP locus at rs202676 (position 27 of SEQ ID NO. 15, wherein the SEQ ID NO. 15 is a portion of a genomic nucleic acid ce of folate ase (prostate-specific membrane antigen) 1 (FOLH1)); j. at least one alanine "A" allele at SNP locus at rs2297291 (position 27 of SEQ ID NO. 16, wherein the SEQ ID NO. 16 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1)); k. at least one alanine "A" allele at SNP locus at rs1051266 (position 27 of SEQ ID NO. 17), wherein the SEQ ID NO. 17 is a portion of a genomic nucleic acid sequence of reduced folate carrier protein (RCF1); l. at least one thymine "T" allele at SNP locus at rs8007267 (position 27 of SEQ ID NO. 18, n the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1)); m. at least one alanine "A" allele at SNP locus at rs7639752 (position 27 of SEQ ID NO. 19, wherein the SEQ ID NO. 19 is a portion of a genomic nucleic acid sequence of choline-phosphate cytidylyltransferase A (PCYT1A)); n. at least one thymine "T" allele at SNP locus at rs6275 (position 27 of SEQ ID NO. 20, wherein the SEQ ID NO. 20 is a n of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2)); o. at least one thymine "T" allele at SNP locus at rs1079596 (position 27 of SEQ ID NO. 21), wherein the SEQ ID NO. 21 is a portion of a genomic nucleic acid sequence of dopamine receptor D2 (DRD2); p. at least one alanine "A" allele at SNP locus at rs11240594 (position 27 of SEQ ID NO. 22), n the SEQ ID NO. 22 is a n of a genomic nucleic acid sequence of dopamine receptor D2 ; q. at least one cytosine "C" allele at SNP locus at rs4633 (position 27 of SEQ ID NO. 23, wherein the SEQ ID NO. 23 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT)); r. at least one guanine "G" allele at SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a c c acid sequence of catechol-O-methyltransferase (COMT); s. at least one ne "C" allele at SNP locus at rs250682 (position 27 of SEQ ID NO. 25, wherein the SEQ ID NO. 25 is a portion of a genomic nucleic acid sequence of solute carrier family 6 (neurotransmitter orter, ne), member 3 (SLC6A3)); t. at least one thymine "T" allele at SNP locus at rs2277820 (position 27 of SEQ ID NO. 26, wherein the SEQ ID NO. 26 is a portion of a genomic nucleic acid sequence of formiminotransferase cyclodeaminase (FTCD)); and u. at least one e "A" allele at SNP locus at rs2236225 (position 27 of SEQ ID NO. 27, wherein the SEQ ID NO. 27 is a portion of a genomic nucleic acid ce of methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 (MTHFD1)); v. obesity (e.g., defined by a BMI value of at least 30 kg/m2 or greater); w. an expression level ratio of SAM to SAH r than a pre-determined reference ratio; x. an expression level of 4-HNE greater than a pre-determined reference value; and y. an expression of hsCRP r than about 2.3 mg per liter of plasma as measured in a plasma .
12. The use of claim 11, n the at least two conditions are: a. at least one thymine “T” allele at SNP locus at rs8007267 (position 27 of SEQ ID NO. 18), n the SEQ ID NO. 18 is a portion of a genomic nucleic acid sequence of GTP cyclohydrolase 1 (GCH1); and b. at least one guanine “G” allele at SNP locus at rs4680 (position 27 of SEQ ID NO. 24), wherein the SEQ ID NO. 24 is a portion of a genomic nucleic acid sequence of catechol-O-methyltransferase (COMT).
13. The use of any one of claims 10 to 12, wherein the folate-comprising composition comprises at least about 5 mg of folate or about 7.5-50 mg of folate.
14. The use of any one of claims 10 to 13, wherein the depression is a major depressive disorder.
15. The use of any one of claims 10 to 14, wherein the folate-comprising composition includes an antidepressant.
16. An assay according to any one of claims 1 to 9, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
17. Use of a folate-comprising composition according to any one of claims 10 to 15, substantially as herein described with reference to any one or more of the es but excluding comparative examples.
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