WO2008070074A2 - Marqueurs génétiques de la schizophrénie - Google Patents

Marqueurs génétiques de la schizophrénie Download PDF

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WO2008070074A2
WO2008070074A2 PCT/US2007/024830 US2007024830W WO2008070074A2 WO 2008070074 A2 WO2008070074 A2 WO 2008070074A2 US 2007024830 W US2007024830 W US 2007024830W WO 2008070074 A2 WO2008070074 A2 WO 2008070074A2
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individual
schizophrenia
marker
allele
csf3
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PCT/US2007/024830
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WO2008070074A3 (fr
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Maria Athanasiou
Carol Reed
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Pgxhealth Llc
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    • 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
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to genetic markers predictive of disease. More specifically, this invention relates to certain variants of the genes encoding interleukin 3 receptor alpha (IL3RA), granulocyte colony stimulating factor (CSF3), 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A), placental bone morphogenetic protein (PLAB), albumin (ALB), the tyrosine kinase oncogene MET, and granulocyte colony stimulating factor 2 receptor (CSF2RA), each of which are associated with schizophrenia.
  • IL3RA interleukin 3 receptor alpha
  • CSF3 granulocyte colony stimulating factor
  • HTR2A 5-hydroxytryptamine receptor 2A
  • PLAB placental bone morphogenetic protein
  • ARB albumin
  • tyrosine kinase oncogene MET granulocyte colony stimulating factor 2 receptor
  • CSF2RA granulocyte colony stimulating factor 2 receptor
  • Schizophrenia is a devastating disease afflicting 1% of the population across varied geographical regions of origin. Characterized by delusional beliefs, auditory hallucinations, disorganized thought patterns and apathy, schizophrenia is a chronic disorder that begins in early adulthood. The social and financial implications of schizophrenia are high. Patients withdraw socially, have difficulty sustaining relationships and are often unable to work. The cost of this loss of productivity combined with the burden of medical care is estimated to be $32.5 billion per year in the United States (Rice, J. Clin. Psychiatry 62: Suppl. 4-6 Discussion 28-30 (1999)). Because of these costs, there are needed methods for accurately predicting and diagnosing, and effectively treating, this disease.
  • the IL3RA gene (also known as CDl 23) is located on the pseudoautosomal region of the X and Y chromosomes (Xp22.3; YpI 1.3).
  • the Interleukin 3 receptor alpha chain (I13r alpha) encoded by IL3RA is a membrane, single-pass protein that belongs to the type I cytokine receptor family. It is expressed on early hemopoietic progenitor cells, on certain committed myeloid progenitors, eosinophils and basophils.
  • interleukin3 113
  • 113 stimulates immature myelomonocytic cells in the hemopoietic system and causes differentiation of granulocytes, macrophages and eosinophils. 113 does not appear to be important for maintaining steady states population, but plays a key role in accelerating proliferation of these cells in response to inflammation and infection.
  • Interleukin 3 receptors are heterodimeric complexes formed by association of the I13r alpha subunit and a common beta subunit which is shared by the Interleukin 3, Interleukin 5 and Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF) receptors.
  • the I13r alpha chain is the major ligand-binding subunit and on its own does not appear to transduce any of the biological activities of the 113 receptor. It is the beta subunit that converts the ligand-bound alpha subunit into a high-affinity state and is important for almost all the signaling activity.
  • the 113 receptors Upon binding of 113 to its alpha chain, the 113 receptors generate signals that activate the JAK/STAT, the ras/MAP kinase pathway and the PI-3 Kinase pathway.
  • the beta subunit of 113 receptors is known to mediate this activation by activating the kinase JAK2 and presumably other less important kinases that remain to be identified.
  • CSF3 Granulocyte colony stimulating factor
  • CSF3 is encoded by a single gene located on chromosome 17 (17ql 1-22).
  • CSF3 is a secreted monomelic cytokine of the Interleukin 6 superfamily and is produced mainly by cells of monocyte/macrophage origin.
  • the CSF3 gene has four introns. Differential splicing leads to two different peptides that differ by the presence or absence of three amino acids. Expression studies show that both proteins have authentic CSF3 activity.
  • CSF3 Primary effects of CSF3 include stimulation of proliferation and differentiation of normal hemopoietic stem cells, acceleration of neutrophil reconstruction following radiation-or chemotherapy-induced myelosuppression, activation of effector functions in mature neutrophils and mobilization of bone marrow hemopoietic stem cells in the peripheral blood.
  • CSF3 is thought to interact with the immune system by altering T cell reactivity and modifying antigen presenting cells function.
  • CSF3 plays a key role in hematopoeisis by controlling the production, differentiation and function of granulocytic progenitors regulated by the transcription factors/oncogenes SPIl, MYB and others and, inducing the PRTN3 gene inducing the JUNB and/or TOP in the proliferative response of human myeloidleukemia, putatively involved in severe congenital neutropenia.
  • HTR2A The 5-Hydroxytryptamine (serotonin) receptor 2A (HTR2A) is encoded by a single gene located on chromosome 13 (13ql4-q21).
  • HTR2A is one of the several different receptors for serotonin, a biogenic hormon that functions as a neurotransmitter, a hormone and a mitogen.
  • HTR2A is a multi-pass membrane protein located at the postsynaptic thickening region of axo-dendritic synapses. This receptor mediates its action by association with a G protein that activates a phosphatidylinositol-calcium second messenger system. Epidemiological studies have shown that there is a strong genetic factor in predisposition to developing schizophrenia and mutations in the HTR2A gene have shown positive linkage to the disease.
  • the PLAB gene encodes the 308-amino acid placental bone morphogenetic protein.
  • the gene consists of two exons, and is located on chromosome 19 (19pl3.2- p 13.1).
  • the protein is a secreted member of the transforming growth factor beta (TGF- beta) superfamily highly expressed in the placenta, and regulated by steroid hormones.
  • TGF- beta transforming growth factor beta
  • the primary role of the bone morphogenetic proteins (BMPs) is in tissue differentiation and maintenance. BMPs are synthesized as precursor molecules that are processed at a dibasic cleavage site to release C-terminal domains containing a characteristic motif of 7 conserved cysteines in the mature protein.
  • PLAB is most similar to BMP-8 in the family of bone morphogenetic protein family.
  • PLAB inhibits the proliferation of primitive hematopoietic progenitors, and its high expression in the placenta suggests that PLAB may regulate embroyonic development.
  • PLAB and other TGF -beta family bone morphogenetic proteins are downstream in the pathway of p53 activation, which is known to have growth-suppressive and proapoptotic effects.
  • the ALB gene located on chromosome 4 (4ql l-ql3), encodes albumin, a soluble, monomelic protein of molecular weight 65,000.
  • the human ALB gene consists of 15 exons.
  • Albumin is synthesized by the liver as preproalbumin that is cleaved from the N-terminus prior to release from the endoplasmic reticulum to form prealbumin. In the Golgi vesicles, prealbumin is cleaved to the mature form of albumin that is then secreted from the cell. Albumin is the most abundant protein in plasma accounting for approximately 60% of blood serum protein.
  • albumin The primary function of albumin is to transport water, ions (Ca 2+ , Na + , K + ), fatty acids, and drugs through the bloodstream.
  • Albumin has a key role in the regulation of blood and extracellular fluid volume. There are changes in the level of albumin found in the anterior cingulated cortex of the brain of schizophrenia patients. Low serum albumin levels have been associated with psychiatric diseases including major depression and schizophrenia. Decreased serum albumin levels can also be explained by liver or kidney disease, as well as malnutrition or a low protein diet.
  • MET metal proto-oncogene
  • MSTlR macrophage stimulating 1 receptor
  • CSF2RA encodes the alpha subunit of the heterodimeric colony stimulating factor 2 (CSF2) receptor.
  • CSF2 is a cytokine that controls the production, differentiation and function of granulocytes and macrophages.
  • CSF2RA has widespread expression in the human adult, with highest levels present in the immune system, as well as the brain, kidney and lungs.
  • the CSF2RA gene is located on the pseudoautosomal region of the X and Y chromosomes (Xp22.32; YpI 1). Multiple variants, both membrane-bound and soluble, of this cytokine receptor have been identified.
  • the gene is comprised of 15 exons over a span of 41,927 nucleotides.
  • the invention provides a method for testing an individual for susceptibility for schizophrenia comprising detecting the presence or absence in the individual of a IL3RA, CSF3, HTR2A, PLAB, ALB, MET and/or CSF2RA marker, and generating a test report for the individual, wherein if the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker is present, then the test report indicates that the individual is susceptible to schizophrenia, and if the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker is not present, then the test report indicates that the individual is not susceptible schizophrenia.
  • the invention provides a method of testing an individual for the presence or absence of a genetic marker that is associated with schizophrenia comprising determining the copy number of a polymorphism in the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA genes that is associated with schizophrenia, using the determined copy number to. assign to the individual the presence of absence of the marker, and generating a test report which indicates whether the marker is present or absent in the individual.
  • the invention provides a method of predicting whether an individual is susceptible to schizphrenia comprising determining the presence or absence in the individual of a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker, and making a prediction based on the results, wherein if the IL3RA, CSF3, HTR2A, PLAB, ALB, MET and/or CSF2RA marker is present, then the prediction is that the individual is likely to likely to develop schizophrenia, and if the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker is absent, the prediction is that the individual is not likely to develop schizophrenia.
  • the invention provides a kit for detecting a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker comprising a set of one or more oligonucleotides designed for identifying each of the alleles at each polymorphic site in the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker.
  • SEQ ID NO:1 illustrates a reference sequence for the IL3RA gene.
  • SEQ ID NO:2 illustrates a reference sequence for the CSF3 gene.
  • SEQ ID NO: 3 illustrates a reference sequence for the HTR2A gene.
  • SEQ ID NO:4 illustrates a reference sequence for the PLAB gene.
  • SEQ ID NO:5 illustrates a reference sequence for the ALB gene.
  • SEQ ID NO:6 illustrates a reference sequence for the MET gene.
  • SEQ ID NO:7 illustrates a reference sequence for the CSF2RA.
  • Allele is a particular form of a gene or other genetic locus, distinguished from other forms by its particular nucleotide sequence, the term allele also includes one of the alternative polymorphisms (e.g., a SNP) found at a polymorphic site.
  • a SNP alternative polymorphisms found at a polymorphic site.
  • allele refers to the form of a locus that is present on a single chromosome in a somatic cell obtained from an individual; if the locus is on an autosomal chromosome, then the somatic cell in the individual will normally have two alleles for the locus.
  • the individual is homozygous for that locus, and if the two alleles have different sequences, then the individual is heterozygous for the locus. If the locus is on a sex chromosome, then somatic cells from female individuals normally have two alleles, which may have the same or different sequences, while somatic cells from male individuals normally only has one allele for the locus.
  • Disease refers to an interruption, cessation, or disorder of one or more body functions, structures, systems or organs.
  • Gene is a segment of DNA that contains the coding sequence for a protein, wherein the segment may include promoters, exons, introns, and other untranslated regions that control expression.
  • Marker in the context of the present invention is a specific copy number of a specific polymorphism that is associated with schizophrenia. Preferred markers are those shown in Tables A-I through A-7 (Appendix A), as well as genetic markers that are highly correlated with any marker in Table A-I through A-7 (Appendix A) and/or are replaced by the same copy number of a substitute polymorphism, each of which is referred to herein as an alternate genetic marker.
  • a substitute polymorphism comprises a sequence that is similar to that of any of the markers shown in Tables A-I through A-7 (Appendix A), but in which the allele at one or more of the specifically identified polymorphic sites in that marker has been substituted with the allele at a different polymorphic site, whose substituting allele is in high linkage disequilibrium (LD) with the allele at the specifically identified polymorphic site.
  • a linked polymorphism is any type of polymorphism, including a haplotype, which is in high LD with any one of the markers shown in Appendix A.
  • Two particular alleles at different loci on the same chromosome are said to be in LD if the presence of one of the alleles at one locus tends to predict the presence of the other allele at the other locus.
  • Alternate genetic markers which are further described below, may comprise types of variations other than SNPs, such as indels, RFLPs, repeats, etc.
  • Gene is an unphased 5' to 3' sequence of the two alleles, typically a nucleotide pair, found at a set of one or more polymorphic sites in a locus on a pair of homologous chromosomes in an individual.
  • Genotyping is a process for determining a genotype of an individual.
  • Haplotype pair refers to the two haplotypes found for a locus in a single individual.
  • Haplotyping refers to any process for determining one or more haplotypes in an individual, including the haplotype pair for a particular set of PS, and includes use of family pedigrees, molecular techniques and/or statistical inference.
  • isolated is typically used to reflect the purification status of a biological molecule such as RNA, DNA, oligonucleotide, or protein, and in such context means the molecule is substantially free of other biological molecules such as nucleic acids, proteins, lipids, carbohydrates, or other material such as cellular debris and growth media. Generally, the term “isolated” is not intended to refer to a complete absence of such material or to an absence of water, buffers, or salts, unless they are present in amounts that substantially interfere with the methods of the present invention.
  • Locus refers to a location on a chromosome or DNA molecule corresponding to a gene, a physical feature such as a polymorphic site, or a location associated with a phenotypic feature.
  • Normal as used herein in connection with the quantity in a subject of any clinical parameter (such as any type of blood cell or one of its hematopoietic precursors) means a specific number or numerical range of that parameter that is typically observed in healthy subjects of similar age, weight, and or gender, or that would be understood by a clinical to be normal. Conversely, “abnormal” refers to a specific number or numerical range for a clinical parameter that is lower or higher than a normal number or normal numerical range, or that would be understood by a clinical to be abnormal.
  • Nucleotide pair is the set of two nucleotides (which may be the same or different) found at a polymorphic site on the two copies of a chromosome from an individual.
  • Oligonucleotide refers to a nucleic acid that is usually between 5 and 100 contiguous bases in length, and most frequently between 10-50, 10-40, 10-30, 10-25, 10- 20, 15-50, 15-40, 15-30, 15-25, 15-20, 20-50, 20-40, 20-30 or 20-25 contiguous bases in length.
  • the sequence of an oligonucleotide can be designed to specifically hybridize to any of the allelic forms of a locus; such oligonucleotides are referred to as allele-specific probes. If the locus is a PS comprising a SNP, the complementary allele for that SNP can occur at any position within an allele-specific probe.
  • oligonucleotides useful in practicing the invention specifically hybridize to a target region adjacent to a PS with their 3' terminus located one to less than or equal to about 10 nucleotides from the PS, preferably ⁇ about 5 nucleotides.
  • Such oligonucleotides hybridizing adjacent to a PS are useful in polymerase-mediated primer extension methods and are referred to herein as "primer-extension oligonucleotides.”
  • the 3 '-terminus of a primer-extension oligonucleotide is a deoxynucleotide complementary to the nucleotide located immediately adjacent to the PS.
  • Phhased sequence refers to the combination of nucleotides present on a single chromosome at a set of polymorphic sites, in contrast to an unphased sequence, which is typically used to refer to the sequence of nucleotide pairs found at the same set of PS in both chromosomes.
  • Polymorphic site refers to the position in a genetic locus or gene at which a SNP or other nonhaplotype polymorphism occurs.
  • a PS is usually preceded by and followed by highly conserved sequences in the population of interest and thus the location of a PS is typically made in reference to a consensus nucleic acid sequence of thirty to sixty nucleotides that bracket the PS, which in the case of a SNP polymorphism is sometimes referred to as a context sequence for the SNP.
  • the location of the PS may also be identified by its location in a consensus or reference sequence relative to the initiation codon (ATG) for protein translation.
  • ATG initiation codon
  • the location of a particular PS may not occur at precisely the same position in a reference or context sequence in each individual in a population of interest due to the presence of one or more insertions or deletions in that individual as compared to the consensus or reference sequence.
  • it is routine for the skilled artisan to design robust, specific and accurate assays for detecting the alternative alleles at a polymorphic site in any given individual when the skilled artisan is provided with the identity of the alternative alleles at the PS to be detected and one or both of a reference sequence or context sequence in which the PS occurs.
  • any PS described herein by reference to a particular position in a reference or context sequence is merely for convenience and that any specifically enumerated nucleotide position literally includes whatever nucleotide position the same PS is actually located at in the same locus in any individual being tested for the presence or absence of a genetic marker of the invention using any of the genotyping methods described herein or other genotyping methods well-known in the art.
  • Polymorphism refers to one of two or more genetically determined alternative sequences or alleles that occur for a gene or a genetic locus in a population.
  • polymorphism includes, but is not limited to (a) a sequence of as few as one nucleotide that occurs at a polymorphic site (as defined above), which is also referred to herein as a single nucleotide polymorphism (SNP) and (b) a sequence of nucleotides that occur on a single chromosome at a set of two or more polymorphic sites in the gene or genetic locus of interest, which is also referred to herein as a haplotype.
  • SNP single nucleotide polymorphism
  • the different alleles of a polymorphism typically occur in a population at different frequencies, with the allele occurring most frequently in a selected population sometimes referenced as the "major" or "wildtype” allele. Diploid organisms may be homozygous or heterozygous for the different alleles that exist.
  • a biallelic polymorphism has two alleles, and the minor allele may occur at any frequency greater than zero and less than 50% in a selected population, including frequencies of between 1% and 2%, 2% and 10%, 10% and 20%, 20% and 30%, etc.
  • a triallelic polymorphism has three alleles.
  • polymorphisms include restriction fragment length polymorphisms (RPLPs), variable number of tandem repeats (VNTRs), dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, insertion elements such as AIu, and deletions of one or more nucleotides.
  • RPLPs restriction fragment length polymorphisms
  • VNTRs variable number of tandem repeats
  • dinucleotide repeats trinucleotide repeats
  • tetranucleotide repeats simple sequence repeats
  • insertion elements such as AIu
  • Schizophrenia refers to a disorder of humans that lasts for six months or more, and includes at least one month of two or more symptoms selected from: delusions, hallucinations, disorganized speech, grossly disorganized behavior, and catatonic behavior.
  • a person with "treatment-refractory schizophrenia” is a person who has failed to respond to a first-line therapeutic, which is any approved typical or atypical antipsychotic other than clozapine, and a second-line therapeutic, which is any approved typical or atypical antipsychotic other than the first-line therapeutic and clozapine, and who therefore is being treated with clozapine.
  • genetic markers according to the present invention are associated with schizophrenia, and are referred to herein as IL3RA, CSF3, HTR2A, PLAB, ALB, MET, or CSF2RA markers.
  • Each IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and CSF2RA marker of the invention is a combination of a particular polymorphism associated with the adverse hematological response and a copy number of that polymorphism.
  • the polymorphism is one of the markers shown in Tables A-I through A-7 in Appendix A, each of which contains a sequence for a specific set of PS in the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, or CSF2RA genes.
  • the locations of these marker PSs in the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, or CSF2RA genes are at positions corresponding to those identified in SEQ ID NOS: 1-7 (see Tables B-I through B-7 in Appendix B for a summary of the PS location and the alternative nucleotide alleles that occur at each PS).
  • nucleic acid molecules containing a particular gene may be complementary double stranded molecules and thus reference to a particular site on the sense strand refers as well to the corresponding site on the complementary antisense strand.
  • the genetic markers of the invention are based on the discovery by the inventors of associations between particular copy numbers of certain polymorphisms in the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA genes and schizophrenia. Individuals having the copy number indicated for each of the polymorphisms shown in Tables A-I through A-7 in Appendix A were more likely to develop schizophrenia relative to individuals having other copy numbers of those polymorphisms. Moreover, as shown in Tables 1 through 7 below, the association between the presence of these genetic markers and susceptibility to schizophrenia is statistically significant.
  • alternate genetic markers comprising a linked polymorphism are readily identified by determining the degree of LD between a marker in Tables A-I through A-7 in Appendix A and a candidate linked polymorphism located elsewhere in the relevant gene or chromosome.
  • the candidate substituting allele or linked polymorphism may be a polymorphism that is currently known.
  • Other candidate substituting alleles and linked polymorphisms may be readily identified by the skilled artisan using any technique well-known in the art for discovering polymorphisms.
  • the degree of LD between a genetic marker in Tables A-I through A-7 in Appendix A and a candidate alternate polymorphism may be determined using any LD measurement known in the art.
  • LD patterns in genomic regions are readily determined empirically in appropriately chosen samples using various techniques known in the art for determining whether any two alleles (e.g., between SNPs at different PSs or between two haplotypes) are in linkage disequilibrium (GENETIC DATA ANALYSIS II, Weir, Sinauer Associates, Inc. Publishers, Sunderland, MA, 1996). The skilled artisan may readily select which method of determining LD will be best suited for a particular sample size and genomic region.
  • ⁇ 2 is the measure of how well an allele X at a first locus predicts the occurrence of an allele Y at a second locus on the same chromosome. The measure only reaches 1.0 when the prediction is perfect (e.g., X if and only if Y).
  • the locus of a substituting allele or a linked polymorphism is in a genomic region of about 100 kilobases spanning the relevant gene, and more preferably, the locus is in the relevant gene.
  • Other preferred alternate genetic markers are those in which the LD or correlation between the relevant alleles (e.g., between the substituting SNP and the substituted SNP, or between the linked
  • 0 0 polymorphism and the haplotype has a ⁇ or r (the square of correlation coefficient) value, as measured in a suitable reference population, of at least 0.75, more preferably at least 0.80, even more preferably at least 0.85 or at least 0.90, yet more preferably at least 0.95, and most preferably 1.0.
  • Preferred genetic markers of the invention comprise any of the markers in Tables A-I through A-7 in Appendix A.
  • the schizophrenia is treatment-refractory schizophrenia.
  • the presence in an individual of a genetic marker of the invention may be determined by any of a variety of methods well known in the art that permits the determination of whether the individual has the required copy number of the polymorphism comprising the marker. For example, if the required copy number is 1 or 2, then the method need only determine that the individual has at least one copy of the polymorphism. In preferred embodiments, the method provides a determination of the actual copy number.
  • nucleic acid samples may be prepared from virtually any biological sample.
  • convenient samples include whole blood, serum, semen, saliva, tears, fecal matter, urine, sweat, buccal matter, skin and hair.
  • Preferred samples contain only somatic cells, and such samples would typically be required when the locus is on an autosomal or X chromosome.
  • Nucleic acid samples may be prepared for analysis using any technique known to those skilled in the art.
  • such techniques result in the production of genomic DNA sufficiently pure for determining the genotype or haplotype pair for a desired set of polymorphic sites in the nucleic acid molecule.
  • Such techniques may be found, for example, in Sambrook, et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York) (2001), incorporated herein by reference.
  • the copy number of the haplotype in the nucleic acid sample may be determined by a direct haplotyping method or by an indirect haplotyping method, in which the haplotype pair for the set of polymorphic sites comprising the marker is inferred from the individual's haplotype genotype for that set of PSs.
  • the way the nucleic acid sample is prepared depends on whether a direct or indirect haplotyping method is used.
  • Direct haplotyping methods typically involve treating a genomic DNA sample isolated from a blood or cheek sample obtained from the individual in a manner that produces a hemizygous DNA sample that contains only one of the individual's two alleles for the locus which, as readily understood by the skilled artisan, may be the same allele or different alleles, and detecting the nucleotide present at each PS of interest.
  • the nucleic acid sample may be obtained using a variety of methods known in the art for preparing hemizygous DNA samples, which include: targeted in vivo cloning (TIVC) in yeast as described in WO 98/01573, United States Patent No. 5,866,404, and United States Patent No.
  • any individual clone of the locus in that individual will permit directly determining the haplotype for only one of the two alleles; thus, additional clones will need to be examined to directly determine the identity of the haplotype for the other allele.
  • the haplotype for the other allele may be inferred if the individual has a known genotype for the PSs comprising the marker or if the frequency of haplotypes or haplotype pairs for the locus in an appropriate reference population is available.
  • Direct haplotyping of both alleles may be performed by assaying two hemizygous DNA samples, one for each allele, that are placed in separate containers.
  • the two hemizygous samples may be assayed in the same container if the two samples are labeled with different tags, or if the assay results for each sample are otherwise separately distinguishable or identifiable.
  • the samples are labeled with first and second fluorescent dyes, and a PS in the locus is assayed using an oligonuclotide probe that is specific for one of the alleles-and labeled with a third fluorescent dye, then detecting a combination of the first and third dyes would identify the nucleotide present at the PS in the first sample while detecting a combination of the second and third dyes would identify the nucleotide present at the PS in the second sample.
  • Indirect haplotyping methods typically involve preparing a genomic DNA sample isolated from a blood or cheek sample obtained from the individual in a manner that permits accurately determining the individual's genotype for each PS in the locus. The genotype is then used to infer the identity of at least one of the individual's haplotypes for the locus, and preferably used to infer the identity of the individual's haplotype pair for the locus.
  • the presence of zero, one or two copies of a haplotype of interest can be determined by comparing the individual's genotype for the PS in the marker with a set of reference haplotype pairs for the same set of PS and assigning to the individual a reference haplotype pair that is most likely to exist in the individual.
  • the individual's copy number for the haplotype comprising the marker is how many copies of that haplotype is in the assigned reference haplotype pair.
  • the reference haplotype pairs are those that are known to exist in the general population or in a reference population, or that are theoretically possible based on the alternative alleles possible at each PS.
  • the reference population may be composed of randomly-selected individuals representing the major ethnogeographic groups of the world.
  • a preferred reference population is one having a similar ethnogeographic background as the individual being tested for the presence of the marker.
  • the size of the reference population is chosen based on how rare a haplotype is that one wants to be guaranteed to see.
  • a particularly preferred reference population includes one or more 3 -generation families to serve as a control for checking quality of haplotyping procedures. If the reference population comprises more than one ethnogeographic group, the frequency data for each group is examined to determine whether it is consistent with Hardy- Weinberg equilibrium. Hardy- Weinberg equilibrium (D.L.
  • Assignment of the haplotype pair may be performed by choosing a reference haplotype pair that is consistent with the individual's genotype.
  • the frequencies of the reference haplotype pairs may be used to determine which of these consistent haplotype pairs is most likely to be present in the individual. If a particular consistent haplotype pair is more frequent in the reference population than other consistent haplotype pairs, then the consistent haplotype pair with the highest frequency is the most likely to be present in the individual.
  • the individual is assigned a haplotype pair containing this known haplotype and a new haplotype derived by subtracting the known haplotype from the possible haplotype pair.
  • the individual is preferably haplotyped using a direct molecular haplotyping method such as, for example, is disclosed in U.S. Patent No. 5,866,404, SMD, or allele- specif ⁇ c long-range PCR (Michalotos-Beloin et al., supra).
  • Marker X Indirect determination of the copy number of haplotypes present in an individual from her genotype is illustrated here for a hypothetical Marker X, which is associated with schizophrenia.
  • Marker X consists of one or two copies of Haplotype GA, which contains two polymorphic sites, PS-A and PS-B, in Gene Y on an autosomal chromosome.
  • frequency information may be used to determine the most probable haplotype pair and therefore the most likely number of copies of the marker haplotype in the individual, as described above.
  • genotyping of one or more additional sites in Gene Y or nearby may be performed to resolve this ambiguity.
  • these one or more additional sites would need to have sufficient linkage with the alleles in at least one of the haplotypes in a possible haplotype pair to permit unambiguous assignment of that haplotype pair.
  • any of all of the steps in the indirect haplotyping method described above may be performed manually, by visual inspection and performing appropriate calculations, but are preferably performed by a computer-implemented algorithm that accesses data on the individual's genotype and reference haplotype pairs stored in computer readable format.
  • a computer-implemented algorithm that accesses data on the individual's genotype and reference haplotype pairs stored in computer readable format.
  • the haplotype pair in an individual may be predicted from the individual's genotype for that gene with the assistance of other reported haplotyping algorithms ⁇ e.g., Clark et al. 1990, MoI Bio Evol 7:111-22; Stephens, M. et al., (2001) Am J Hum Genet 68:978-989; WO 02/064617; Niu T. et al. (2002) Am J. Hum Genet 70:157-169; Zhang et al. (2003) BMC Bioinformatics 4(1):3) or through a commercial haplotyping service.
  • All direct and indirect haplotyping methods described herein typically involve determining the identity of at least one of the alleles at a PS in a nucleic acid sample obtained from the individual. To enhance the sensitivity and specificity of that determination, it is frequently desirable to amplify from the nucleic acid sample one or more target regions in the locus.
  • An amplified target region may span the locus of interest, such as an entire gene, or a region thereof containing one or more polymorphic sites. Separate target regions may be amplified for each PS in a marker.
  • PCR polymerase chain reaction
  • PCR may be carried out using materials and methods known to those of skill in the art (See generally PCR Technology: Principals and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N. Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al, Academic Press, San Diego, Calif, 1990); Matilla et al, Nucleic Acids Res. 19: 4967 (1991); Eckert et al., PCR Methods and Applications 1 : 17 (1991); PCi? (eds.
  • ligase chain reaction LCR
  • Genomics 4 560 (1989) and Landegren et al, Science 241 : 1077 (1988)
  • transcription amplification Kwoh et al, Proc. Natl. Acad. Sci. USA 86: 1173 (1989)
  • self-sustained sequence replication Guatelli et al, Proc. Nat. Acad. Sci. USA, 87: 1874 (1990)
  • isothermal methods (Walker et al, Proc. Natl. Acad. Sci. USA 89:392-6 (1992)); and nucleic acid-based sequence amplification (NASBA).
  • LCR ligase chain reaction
  • NASBA nucleic acid-based sequence amplification
  • the amplified target region is assayed to determine the identity of at least one of the alleles present at a PS in the region. If both alleles of a locus are represented in the amplified target, it will be readily appreciated by the skilled artisan that only one allele will be detected at a PS in individuals who are homozygous at that PS, while two different alleles will be detected if the individual is heterozygous for that PS.
  • the identity of the allele may be identified directly, known as positive-type identification, or by inference, referred to as negative-type identification.
  • a PS may be positively determined to be either guanine or cytosine for an individual homozygous at that site, or both guanine and cytosine, if the individual is heterozygous at that site.
  • the PS may be negatively determined to be not guanine (and thus cytosine/cytosine) or not cytosine (and thus guanine/guanine). Identifying the allele or pair of alleles at a PS may be accomplished using any technique known to those of skill in the art. Preferred techniques permit rapid, accurate assaying of multiple PS with a minimum of sample handling.
  • suitable techniques include, but are not limited to, direct DNA sequencing of the amplified target region, capillary electrophoresis, hybridization of allele-specific probes, single-strand conformation polymorphism analysis, denaturing gradient gel electrophoresis, temperature gradient electrophoresis, mismatch detection; nucleic acid arrays, primer specific extension, protein detection, and other techniques well known in the art. See, for example, Sambrook, et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, New York) (2001); Ausubel, et al, Current Protocols in Molecular Biology (John Wiley and Sons, New York) (1997); Orita et al, Proc. Nat. Acad. Sci.
  • the identity of allele(s) at a PS is determined using a polymerase-mediated primer extension method.
  • a polymerase-mediated primer extension method Several such methods have been described in the patent and scientific literature and include the "Genetic Bit Analysis” method (WO 92/15712) and the ligase/polymerase mediated genetic bit analysis (United States Patent No. 5,679,524. Related methods, are disclosed in WO 91/02087, WO 90/09455, WO 95/17676, and United States Patent Nos. 5,302,509 and 5,945,283. Extended primers containing the complement of the polymorphism may be detected by mass spectrometry as described in United States Patent No. 5,605,798.
  • Another primer extension method is allele-specific PCR (Ruano et al, 1989, supra; Rua ⁇ o et al., 1991, supra; WO 93/22456; Turki et al, J. Clin. Invest. 95:1635-41 (1995)).
  • multiple PSs may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in WO 89/10414.
  • Another primer extension method for identifying and analyzing polymorphisms employs single-base extension (SBE) of a fluorescently-labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label of the added base and the label of the primer.
  • SBE single-base extension
  • FRET fluorescence resonance energy transfer
  • the method uses a locus-specific oligonucleotide primer labeled on the 5' terminus with 5-carboxyfluorescein (FAM).
  • FAM 5-carboxyfluorescein
  • This labeled primer is designed so that the 3' end is immediately adjacent to the polymorphic site of interest.
  • the labeled primer is hybridized to the locus, and single base extension of the labeled primer is performed with fluorescently labeled dideoxyribonucleotides (ddNTPs) in dye- terminator sequencing fashion, except that no deoxyribonucleotides are present.
  • ddNTPs dideoxyribonucleotides
  • An increase in fluorescence of the added ddNTP in response to excitation at the wavelength of the labeled primer is used to infer the identity of the added nucleotide.
  • the accuracy and specificity of an assay designed to detect the identity of the allele(s) at any PS is typically validated by performing the assay on DNA samples in which the identity of the allele(s) at that PS is known.
  • a sample representing each possible allele is included in the validation process.
  • the validation samples will typically include a sample that is homozygous for the major allele at the PS, a sample that is homozygous for the minor allele at the PS, and a sample that is heterozygous at that PS.
  • test samples are typically also included as controls when performing the assay on a test sample (i.e., a sample in which the identity of the allele(s) at the PS is unknown).
  • the specificity of an assay may also be confirmed by comparing the assay result for a test sample with the result obtained for the same sample using a different type of assay, such as by determining the sequence of an amplified target region believed to contain the PS of interest and comparing the determined sequence to a context sequence based on the reference sequence in SEQ ID NOS: 1-7.
  • the length of the context sequence necessary to establish that the correct genomic position is being assayed will vary based on the uniqueness of the sequence in the target region (for example, there may be one or more highly homologous sequences located in other genomic regions).
  • the skilled artisan can readily determine an appropriate length for a context sequence for any PS using known techniques such as blasting the context sequence against publicly available sequence databases.
  • examining the context sequence of about 30 to 60 bases on each side of the PS in known samples is typically sufficient to ensure that the assay design is specific for the PS of interest.
  • a validated assay may fail to provide an unambiguous result for a test sample. This is usually the result of the sample having DNA of insufficient purity or quantity, and an unambiguous result is usually obtained by repurifying or reisolating the DNA sample or by assaying the sample using a different type of assay.
  • the presence or absence of a marker of the invention may be detected by detecting, in a protein sample obtained from the individual, a polypeptide specified by the polymorphism comprising the marker.
  • the polypeptide may be detected using a monoclonal antibody specific for that polypeptide.
  • such determination may be made by consulting a data repository that contains sufficient information on the patient's genetic composition to determine whether the patient has the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker.
  • the data repository lists what IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker(s) are present and absent in the individual.
  • the data repository could include the individual's patient records, a medical data card, a file (e.g.
  • a medical data card is a portable storage device such as a magnetic data card, a smart card, which has an on-board processing unit and which is sold by vendors such as Siemens of Kunststoff Germany, or a flash-memory card.
  • the data repository is a file accessible by a computer; such files may be located on various media, including: a server, a client, a hard disk, a CD, a DVD, a personal digital assistant such as a Palm Pilot, a tape, a zip disk, the computer's internal ROM (read-only-memory) or the internet or worldwide web.
  • Other media for the storage of files accessible by a computer will be obvious to one skilled in the art.
  • IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA markers described herein support using these markers in a variety of methods and products, including, but not limited to, diagnostic methods and kits.
  • any of the methods or products claimed herein is not dependent on complete correlation between the presence of a genetic marker of the invention and the occurrence of schizophrenia, or upon whether a diagnostic method or kit is 100% accurate, or has an specific degree of accuracy, in determining the presence or absence of a genetic marker in every individual, or in predicting for every individual whether the individual is susceptible to schizophrenia.
  • the inventors herein intend that the terms "determine”, and “determining” and “predicting” should not be interpreted as requiring a definite or certain result; instead these terms should be construed as meaning that a claimed method or kit provides an accurate result for the majority of individuals, or that the result or prediction for any given individual is more likely to be correct than incorrect.
  • the accuracy of the result provided by a diagnostic method or kit of the invention is one that a skilled artisan or regulatory authority would consider suitable for the particular application in which the method or kit is used.
  • An individual to be tested in any of the methods described herein is a human subject in need of a diagnosis of schizophrenia or susceptibility to schizophrenia.
  • diagnostic methods and kits of the invention are useful in clinical diagnostic applications as well as in the methods of treatment described below.
  • diagnostic is not limited to clinical or medical uses, and that diagnostic methods and kits of the invention claimed herein are also useful in any research application in which it is desirable to test a subject for the presence or absence of any genetic marker described in Section II above.
  • the diagnostic methods and kits of the invention test for, or are designed to test for, respectively, the presence or absence of a set of IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA markers, which set may comprise a marker from Tables A- 1 through A-7 in Appendix A, or may comprise all IL3RA, CSF3, HTR2A, PLAB, ALB, MET and CSF2RA markers described herein.
  • any or all of the diagnostic methods claimed herein may be performed by a testing laboratory on an individual's biological sample provided directly by the individual or by any third party, such as the individual's physician, a relative of the individual, a person conducting a research study in which the individual is participating and the like.
  • the third party may have a commercial relationship with the testing laboratory, or may be totally independent thereof.
  • the testing laboratory is preferably a clinical laboratory who performs the diagnostic method in compliance with all applicable laws and regulations in the locality where the testing is performed as well as where the individual resides.
  • the testing laboratory does not know the identity of the individual whose sample it is testing; i.e., the sample received by the laboratory is anonymized in some manner before being sent to the laboratory.
  • the sample may be merely identified by a number or some other code (a "sample ID") and the results of the diagnostic method can be reported to the party ordering the test using the sample ID.
  • sample ID a number or some other code
  • the link between the identity of an individual and the individual's sample is known only to the individual or to the individual's physician. In other applications, such as research studies, the link may be broken prior to the testing laboratory sending a report of the results; thus, the results cannot be obtained by the individual or the individual's insurance company.
  • Kits of the invention which are useful for detecting the presence or absence of a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker in an individual, comprise a set of oligonucleotides designed for identifying each of the alleles at each PS in the marker.
  • the set of oligonucleotides is designed to identify the alleles at all polymorphic sites in all of the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA markers described herein.
  • the set of oligonucleotides is designed to identify both alleles at each PS in a set of IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA markers, with the marker set comprising a marker from Tables A-I through A-7 in Appendix A.
  • the oligonucleotides in the kit are either allele-specific probes or allele-specific primers.
  • the kit comprises primer- extension oligonucleotides.
  • the set of oligonucleotides is a combination of allele-specific probes, allele-specific primers, or primer-extension oligonucleotides.
  • the kit may comprise oligonucleotides designed for genotyping other PS, which may be in the IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA gene or at any other locus of interest in the human genome.
  • Oligonucleotides in kits of the invention must be capable of specifically hybridizing to a target region of a polynucleotide.
  • specific hybridization means the oligonucleotide forms an anti-parallel double-stranded structure with the target region under certain hybridizing conditions, while failing to form such a structure with non-target regions when incubated with the polynucleotide under the same hybridizing conditions.
  • the target region contains a PS in a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker, while in other embodiments, the target region is located one to 10 nucleotides from the PS.
  • each oligonucleotide in the kit will depend on the nature of the genomic region containing the PS as well as the type of assay to be performed with the oligonucleotide and is readily determined by the skilled artisan.
  • the polynucleotide to be used in the assay may constitute an amplification product, and thus the required specificity of the oligonucleotide is with respect to hybridization to the target region in the amplification product rather than in genomic DNA isolated from the individual.
  • the melting temperatures for the oligonucleotides in the kit will typically be within a narrow range, preferably less than about 5°C and more preferably less than about 2°C.
  • each oligonucleotide in the kit is a perfect complement of its target region.
  • An oligonucleotide is said to be a "perfect” or “complete” complement of another nucleic acid molecule if every nucleotide of one of the molecules is complementary to the nucleotide at the corresponding position of the other molecule.
  • perfectly complementary oligonucleotides are preferred for detecting polymorphisms, departures from complete complementarity are contemplated where such departures do not prevent the molecule from specifically hybridizing to the target region as defined above.
  • an oligonucleotide primer may have a non- complementary fragment at its 5' end, with the remainder of the primer being completely complementary to the target region.
  • non-complementary nucleotides may be interspersed into the probe or primer as long as the resulting probe or primer is still capable of specifically hybridizing to the target region.
  • each oligonucleotide in the kit specifically hybridizes to its target region under stringent hybridization conditions.
  • Stringent hybridization conditions are sequence-dependent and vary depending on the circumstances. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium.
  • Tm thermal melting point
  • stringent conditions include a salt concentration of at least about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 25° C for short oligonucleotide probes (e.g., 10 to 50 nucleotides).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
  • destabilizing agents such as formamide.
  • 5 x SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30° C are suitable for allele-specific probe hybridizations.
  • a preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4X sodium chloride/sodium citrate (SSC), at about 65-70°C (or alternatively hybridization in 4X SSC plus 50% formamide at about 42-50°C) followed by one or more washes in IX SSC, at about 65-70°C.
  • a preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in IX SSC, at about 65-70°C (or alternatively hybridization in IX SSC plus 50% formamide at about 42- 50°C) followed by one or more washes in 0.3X SSC, at about 65-70°C.
  • a preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4X SSC, at about 50-60 0 C (or alternatively hybridization in 6X SSC plus 50% formamide at about 40-45°C) followed by one or more washes in 2X SSC, at about 50-60°C. Ranges intermediate to the above-recited values, e.g., at 65-70°C or at 42-50°C are also intended to be encompassed by the present invention.
  • SSPE 0.15M NaCl, 1OmM NaH 2 PO 4 , and 1.25mM EDTA, pH 7.4
  • SSC 0.15M NaCl and 15mM sodium citrate
  • the hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-1O 0 C less than the melting temperature (T m ) of the hybrid, where T m is determined according to the following equations.
  • T m melting temperature
  • the oligonucleotides in kits of the invention may be comprised of any phosphorylation state of ribonucleotides, deoxyribonucleotides, and acyclic nucleotide derivatives, and other functionally equivalent derivatives.
  • the oligonucleotides may have a phosphate-free backbone, which may be comprised of linkages such as carboxymethyl, acetamidate, carbamate, polyamide (peptide nucleic acid (PNA)) and the like (Varma, in MOLECULAR BIOLOGY AND BIOTECHNOLOGY, A COMPREHENSIVE DESK REFERENCE, Meyers, ed., pp. 617-20, VCH Publishers, Inc., 1995).
  • the oligonucleotides may be prepared by chemical synthesis using any suitable methodology known in the art, or may be derived from a biological sample, for example, by restriction digestion.
  • the oligonucleotides may contain a detectable label, according to any technique known in the art, including use of radiolabels, fluorescent labels, enzymatic labels, proteins, haptens, antibodies, sequence tags and the like.
  • the oligonucleotides in the kit may be manufactured and marketed as analyte specific reagents (ASRs) or may be constitute components of an approved diagnostic device.
  • ASRs analyte specific reagents
  • the set of oligonucleotides in the kit have different labels to allow determining the identity of the alleles at two or more PSs simultaneously.
  • the oligonucleotides may also comprise an ordered array that is immobilized on a solid surface such as a microchip, bead, or glass slide ⁇ see, e.g., WO 98/20020 and WO 98/20019).
  • Kits comprising such immobilized oligonucleotides may be designed to perform a variety of polymorphism detection assays, including but not limited to probe hybridization and polymerase extension assays.
  • Kits of the invention may also contain other reagents such as hybridization buffer ⁇ e.g., where the oligonucleotides are to be used as allele-specific probes) or dideoxynucleotide triphosphates (ddNTPs; e.g. , where the alleles at the polymorphic sites are to be detected by primer extension).
  • Kits designed for use in polymerase-mediated genotyping assays may also contain a polymerase and a reaction buffer optimized for the polymerase-mediated assay to be performed.
  • Kits of the invention may also include reagents to detect when a specific hybridization has occurred or a specific polymerase- mediated extension has occurred.
  • Such detection reagents may include biotin- or fluorescent-tagged oligonucleotides or ddNTPs and/or an enzyme-labeled antibody and one or more substrates that generate a detectable signal when acted on by the enzyme. It will be understood by the skilled artisan that the set of oligonucleotides and reagents for performing the assay will be provided in separate receptacles placed in the kit container if appropriate to preserve biological or chemical activity and enable proper use in the assay.
  • each of the oligonucleotides and all other reagents in the kit have been quality tested for optimal performance in an assay designed to determine each of the alleles at the set of PSs comprising a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker.
  • the kit includes an instruction manual that describes the various ways the kit may be used to detect the presence or absence of a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker.
  • the set of oligonucleotides in the kit are allele- specific oligonucleotides.
  • ASO allele-specific oligonucleotide
  • allele-specificity will depend upon a variety of readily optimized stringency conditions, including salt and formamide concentrations, as well as temperatures for both the hybridization and washing steps.
  • an ASO will be perfectly complementary to one allele while containing a single mismatch for another allele.
  • the single mismatch is preferably within a central position of the oligonucleotide probe as it aligns with the polymorphic site in the target region ⁇ e.g., approximately the 7 th or 8 th position in a 15mer, the 8 th or 9 th position in a 16mer, and the 10 th or 11 th position in a 20mer).
  • the single mismatch in ASO primers is located at the 3' terminal nucleotide, or preferably at the 3' penultimate nucleotide.
  • ASO probes and primers hybridizing to either the coding or noncoding strand are contemplated by the invention.
  • the kit comprises a pair of allele-specific oligonucleotides for each PS to be assayed, with one member of the pair being specific for one allele and the other member member being specific for the other allele.
  • the oligonucleotides in the pair may have different lengths or have different detectable labels to allow the user of the kit to determine which allele-specific oligonucleotide has specifically hybridized to the target region, and thus determine which allele is present in the individual at the assayed PS.
  • the oligonucleotides in the kit are primer- extension oligonucleotides. Termination mixes for polymerase-mediated extension from any of these oligonucleotides are chosen to terminate extension of the oligonucleotide at the PS of interest, or one base thereafter, depending on the alternative nucleotides present at the PS.
  • any or all analytical and mathematical operations involved in performing the methods described herein or in using the kits and products described herein may be implemented by a computer.
  • the computer may execute a program that assigns the presence or absence of a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker to an individual based on genotype data inputted by an employee of a testing laboratory or by the treating physician.
  • the computer may output the diagnosis using the individual's genotype data for the polymorphic sites in a IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA marker, which may have been determined by the same or different computer program or input by the testing laboratory or the treating physician.
  • Data relating to the presence or absence of IL3RA, CSF3, HTR2A, PLAB, ALB, MET, and/or CSF2RA markers in an individual may be stored as part of a relational database (e.g., an instance of an Oracle database or a set of ASCII flat files) containing other clinical and/or genetic data for the individual.
  • a relational database e.g., an instance of an Oracle database or a set of ASCII flat files
  • These data may be stored on the computer's hard drive or may, for example, be stored on a CD-ROM or on one or more other storage devices accessible by the computer.
  • the data may be stored on one or more databases in communication with the computer via a network. Preferred embodiments of the invention are described in the following examples.
  • This example illustrates the inclusion and exclusion criteria in a case-control study to detect genetic markers associated with schizophrenia.
  • the inclusion criteria for the cases were (1) an age of 18-75, (2) a diagnosis of schizophrenia, and (3) treatment with clozapine.
  • the inclusion criteria for the controls were (1) an age of 18-75 and (2) no diagnosis of schizophrenia.
  • the total numbers for the study were 85 cases and 40 controls.
  • Genomic DNA samples were isolated from blood samples obtained from each individual and amplified target regions containing the polymorphic sites in Tables B-I through B-7 in Appendix B were sequenced to determine the study subjects' genotypes at these polymorphic sites.
  • Tailed (Universal M 13 Forward and Reverse) PCR primers were designed using the seq ⁇ ences of SEQ ID NOS: 1-7, as appropriate.
  • Amplified PCR products were sequenced using Applied Biosystems' Big Dye® Terminator v 3.1 cycle sequencing kit according to manufacturer's instructions. The reaction products were then electrophoresed using an Applied Biosystems 3700 or 3730x1 DNA analyzer. Polymorphisms were identified using the Polyphred program, and confirmed by visual inspection.
  • Haplotypes were estimated from the unphased genotypes using a computer-implemented algorithm for assigning haplotypes to unrelated individuals in a population sample, essentially as described in WO 01/80156. In this method, haplotypes are assigned directly from individuals who are homozygous at all sites or heterozygous at no more than one of the variable sites. This list of haplotypes is then used to deconvolute the unphased genotypes in the remaining (multiply heterozygous) individuals. A quality control analysis was performed on the deduced haplotypes, which included analysis of the frequencies of the haplotypes and individual SNPs therein for compliance with principles of Hardy- Weinberg equilibrium.
  • This example illustrates analysis of the markers in Tables A-I through A-7 in Appendix A for association with disease status.
  • a proprietary algorithm was used as a tool for finding associations between markers and outcomes.
  • the clinical outcome was disease status.
  • a linear model was fitted on the genetic marker.

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Abstract

L'invention concerne des marqueurs du gène encodant le récepteur alpha de l'interleukine 3 (IL3RA), le facteur de stimulation des colonies de granulocytes (CSF3), le récepteur 2A de la 5-hydroxytryptamine (sérotonine) (HTR2A), la protéine morphogénétique osseuse placentaire (PLAB), l'albumine (ALB), et l'oncogène tyrosine kinase MET, et un récepteur de facteur 2 de stimulation des colonies de granulocytes (CSF2RA) et leur association avec la schizophrénie. Des compositions et procédés pour détecter et utiliser ces marqueurs sont également décrits.
PCT/US2007/024830 2006-12-04 2007-12-04 Marqueurs génétiques de la schizophrénie WO2008070074A2 (fr)

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Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8951939B2 (en) 2011-07-12 2015-02-10 Bio-Rad Laboratories, Inc. Digital assays with multiplexed detection of two or more targets in the same optical channel
US9089844B2 (en) 2010-11-01 2015-07-28 Bio-Rad Laboratories, Inc. System for forming emulsions
US9132394B2 (en) 2008-09-23 2015-09-15 Bio-Rad Laboratories, Inc. System for detection of spaced droplets
US9156010B2 (en) 2008-09-23 2015-10-13 Bio-Rad Laboratories, Inc. Droplet-based assay system
US9194861B2 (en) 2009-09-02 2015-11-24 Bio-Rad Laboratories, Inc. Method of mixing fluids by coalescence of multiple emulsions
US9222128B2 (en) 2011-03-18 2015-12-29 Bio-Rad Laboratories, Inc. Multiplexed digital assays with combinatorial use of signals
US9347059B2 (en) 2011-04-25 2016-05-24 Bio-Rad Laboratories, Inc. Methods and compositions for nucleic acid analysis
US9393560B2 (en) 2010-03-25 2016-07-19 Bio-Rad Laboratories, Inc. Droplet transport system for detection
US9399215B2 (en) 2012-04-13 2016-07-26 Bio-Rad Laboratories, Inc. Sample holder with a well having a wicking promoter
US9417190B2 (en) 2008-09-23 2016-08-16 Bio-Rad Laboratories, Inc. Calibrations and controls for droplet-based assays
US9492797B2 (en) 2008-09-23 2016-11-15 Bio-Rad Laboratories, Inc. System for detection of spaced droplets
US9500664B2 (en) 2010-03-25 2016-11-22 Bio-Rad Laboratories, Inc. Droplet generation for droplet-based assays
US9598725B2 (en) 2010-03-02 2017-03-21 Bio-Rad Laboratories, Inc. Emulsion chemistry for encapsulated droplets
US9764322B2 (en) 2008-09-23 2017-09-19 Bio-Rad Laboratories, Inc. System for generating droplets with pressure monitoring
US10512910B2 (en) 2008-09-23 2019-12-24 Bio-Rad Laboratories, Inc. Droplet-based analysis method
US11130128B2 (en) 2008-09-23 2021-09-28 Bio-Rad Laboratories, Inc. Detection method for a target nucleic acid

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8633015B2 (en) 2008-09-23 2014-01-21 Bio-Rad Laboratories, Inc. Flow-based thermocycling system with thermoelectric cooler
US8709762B2 (en) 2010-03-02 2014-04-29 Bio-Rad Laboratories, Inc. System for hot-start amplification via a multiple emulsion
CA2767113A1 (fr) 2010-03-25 2011-09-29 Bio-Rad Laboratories, Inc. Systeme de detection pour analyses a base de gouttelettes
EP2737089B1 (fr) 2011-07-29 2017-09-06 Bio-rad Laboratories, Inc. Caractérisation de banque par essai numérique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ERDMANN ET AL.: 'Systematic screening for mutations in the human serotonin-2A receptor gene: identification of two naturally occurring receptor variants and association analysis in schizophrenia' HUMAN GENETICS vol. 97, 1996, pages 614 - 619 *

Cited By (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US9089844B2 (en) 2010-11-01 2015-07-28 Bio-Rad Laboratories, Inc. System for forming emulsions
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US9885034B2 (en) 2011-04-25 2018-02-06 Bio-Rad Laboratories, Inc. Methods and compositions for nucleic acid analysis
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US10760073B2 (en) 2011-04-25 2020-09-01 Bio-Rad Laboratories, Inc. Methods and compositions for nucleic acid analysis
US10190115B2 (en) 2011-04-25 2019-01-29 Bio-Rad Laboratories, Inc. Methods and compositions for nucleic acid analysis
US11939573B2 (en) 2011-04-25 2024-03-26 Bio-Rad Laboratories, Inc. Methods and compositions for nucleic acid analysis
US8951939B2 (en) 2011-07-12 2015-02-10 Bio-Rad Laboratories, Inc. Digital assays with multiplexed detection of two or more targets in the same optical channel
US9399215B2 (en) 2012-04-13 2016-07-26 Bio-Rad Laboratories, Inc. Sample holder with a well having a wicking promoter

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