WO2007058986A2 - Methodes destinees au diagnostic du trouble depressif majeur et a l'identification d'agents therapeutiques efficaces pour le traitement du trouble depressif majeur - Google Patents

Methodes destinees au diagnostic du trouble depressif majeur et a l'identification d'agents therapeutiques efficaces pour le traitement du trouble depressif majeur Download PDF

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WO2007058986A2
WO2007058986A2 PCT/US2006/043886 US2006043886W WO2007058986A2 WO 2007058986 A2 WO2007058986 A2 WO 2007058986A2 US 2006043886 W US2006043886 W US 2006043886W WO 2007058986 A2 WO2007058986 A2 WO 2007058986A2
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promoter
crebl
cell
gene
dna sequence
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WO2007058986A3 (fr
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George Zubenko
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University Of Pittsburgh Of The Commonwealth System Of Higher Education
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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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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

  • MDD Major depressive disorder
  • the lifetime prevalence of MDD is between 5% and 10%, with women twice as likely to be affected as men (American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders, 4 th ed., American Psychiatric Association, Washington, D. C. (1994)), Robins et al., Psychiatric Disorders in America: The Epidemiologic Catchment Area Study, The Free Press, New York (1991), and U.S. Dept. of Health and Human Services, Mental Health: a Report of the Surgeon General - Executive Summary, U.S. Dept. of Health and Human Services Administration, Center for Mental Health Services, National Institutes of Health, National Institutes of Mental Health, Rockville, MD (1999)).
  • Suicide has been reported to occur in 10-15% of MDD patients who were previously hospitalized for depression: a death rate that is three orders of magnitude greater than that reported for the entire American population (Angst et al., J. Clin. Psychiatry, ⁇ 50(Suppl. 2): 57-62 (1999) and U.S. Public Health Service, The Surgeon General's Call to Action to Prevent Suicide, Washington, D. C. (1999)).
  • an even greater absolute increase in age- specific mortality from natural causes has been reported for patients who suffer from MDD and their family members.
  • the CREBl gene encodes a 43 kDa cAMP-responsive element-binding protein (CREB) consisting of 341 amino acids, which is a member of the basic leucine zipper family of transcription factors (see, e.g., Mayr et al., Nat. Rev. MoI. Cell. Biol, 2: 599-609 (2001)).
  • CREB cAMP-responsive element-binding protein
  • Phosphorylated CREB molecules induce transcription of genes whose promoters include a cAMP-responsive element (CRE).
  • CRE cAMP-responsive element
  • several growth factor and stress signals stimulate CREB-mediated transcription by promoting the phosphorylation of CREB (see, e.g., Mayr et al., supra).
  • the CREBl gene consists of a 5' untranslated region (i.e., a promoter) and nine exons. Exons 2-9 encode a 43 kDa cAMP-responsive element-binding protein (CREB) consisting of 341 amino acids (see, e.g., Mayr et al., supra). Phosphorylated CREB molecules induce transcription of genes whose promoters include a cAMP-responsive element (CRE).
  • CRE cAMP-responsive element
  • 341 amino acid CREB protein which is known in the art as isoform B
  • additional isoforms are synthesized by alternative mRNA splicing that are tissue-specific and differentially expressed during development (see, e.g., Hoeffler et al., Molecular Endocrinol, 4: 920-930 (1990), and Waeber et al., MoI. Endocrinol, 5: 1418- 1430 (1991)).
  • CREBl is ubiquitously expressed in human tissues and its target genes encode biosynthetic enzymes and receptors for neurotransmitters, neuropeptides, growth factors, transcription factors, and proteins that regulate the cell cycle and intercellular signaling and transport.
  • alterations in CREBl gene expression likely affect multiple organ systems in addition to the brain.
  • NCBI National Center for Biotechnology Information
  • NM_134442 accession numbers NM_134442
  • NM_004379 accession numbers NM_004379
  • human CREBl amino acid sequences have been published by the NCBI with accession numbers NP_604391 (isoform B) and NP_004370 (isoform A).
  • the promoter region of the human CREBl gene also has been characterized (see Meyer et al., Endocrinology, 132: 770-780 (2003)).
  • the CREBl promoter contains 65% guanine and cytosine residues and lacks the TATA and CAAT box elements typically found in most eukaryotic genes.
  • the promoter also contains three SP-I binding sites, four NF- ⁇ B binding sites; it also contains three cAMP-responsive enhancer (CRE) sequences, suggesting that the expression of the CREBl gene is positively autoregulated in trans.
  • CRE cAMP-responsive enhancer
  • the sequence of the CREBl promoter is disclosed in Meyer et al., supra, with a minor update provided by the National Center for Biotechnology Information (see National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institutes of Health. Entrez Human Map Viewer, Build 30, June 24, 2002. Chromosome 2 Map), and is set forth in Figure 1.
  • the CREBl promoter includes most of the untranslated exon 1 (nucleotides 1-131) and extends 1080 nucleotides from the major transcriptional start site in the 5' direction (- 1080).
  • nucleotide positions of a eukaryotic gene the transcription start site is denoted "+1.” Nucleotides upstream of the transcription start site are denoted with a “minus” (-) sign, while nucleotides downstream of the transcription start site are denoted with a “plus” (+) sign.
  • the -1080 to +131 nucleotide sequence is identical to the -1264 to -51 nucleotide sequence of the promoter region described in Meyer et al., supra, which was originally numbered relative to the invariant translational start site of the cloned cDNA sequence.
  • CREB also has been implicated in neuronal plasticity, cognition, and long-term memory (see, e.g., Weeber et al., Neuron, 33: 845-848 (2002)).
  • Other studies demonstrate that CREB synergistically interacts with nuclear estrogen receptors (see, e.g., Lazennec et al., J. Steroid Biochem. MoI. Biol, 77: 193-203 (2001), McEwen et al., J. Appl. Physiol, 91: 2785-2801 (2001), and Tremblay et al., J Steroid Biochem. MoI. Biol, 77: 19-27 (2001)), suggesting a mechanism by which CREB facilitates sex-specific patterns of gene expression that manifest themselves in the sex specificity of the susceptibility locus for MDD identified by the linkage studies discussed above.
  • the invention provides a method for determining the risk of a human developing major depressive disorder (MDD) or a related disorder, which method comprises (a) obtaining a sample from a human, and (b) screening the sample for the presence of at least one DNA sequence variation in the promoter of a CREBl gene, wherein the presence of a DNA sequence variation in the promoter of a CREBl gene indicates a risk of developing major depressive disorder or related disorders as compared to a human that comprises a wild- type CREBl promoter.
  • MDD major depressive disorder
  • the invention also provides a method of diagnosing major depressive disorder (MDD) or related disorders in a human, which method comprises (a) obtaining a sample from a human, and (b) screening the sample for the presence of at least one DNA sequence variation in the promoter of a CREBl gene, wherein the presence of at least one DNA sequence variation in the promoter of the CREBl gene is indicative of MDD or a related disorder in the human.
  • MDD major depressive disorder
  • the invention further provides a method of identifying a candidate drug for treating major depressive disorder (MDD) or related disorders.
  • the method comprises (a) preparing a nucleic acid construct comprising the promoter of a CREBl gene operably linked to a reporter gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, (b) introducing the nucleic acid construct into an expression system, (c) contacting the expression system with at least one test compound, and (d) assaying for restoration of at least a portion of the function of the promoter, whereupon restoration of at least a portion of the function of the promoter indicates the test compound is a candidate drug for treatment of major depressive disorder or a related disorder.
  • the cell can be transfected with the nucleic acid construct, in which embodiment, the method comprises (a) preparing a nucleic acid construct comprising the promoter of a CREBl gene operably linked to a reporter gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, (b) introducing the nucleic acid construct into an isolated cell, (c) contacting the cell with at least one test compound, and (d) assaying for restoration of at least a portion of the function of the promoter, whereupon restoration of at least a portion of the function of the promoter indicates the test compound is a candidate drug for treatment of major depressive disorder or related disorders.
  • the construct alternatively can be integrated into the cellular genome, in which embodiment the method comprises (a) contacting a cell with test compound, wherein the cell comprises a cellular genome comprising a nucleic acid sequence operably linked to a promoter of a CREBl gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, and (b) assaying for restoration of at least a portion of the function of the promoter, whereupon restoration of at least a portion of the function of the promoter indicates the test compound is a candidate drug for treatment of major depressive disorder or related disorders.
  • the method involves assaying for least partial restoration of expression of CREBl target genes or least partial restoration of CREBl -associated functions, whereupon at least partial restoration of expression of CREBl target genes or at least partial restoration of
  • CREB-I associated functions indicates the test compound is a candidate drug for treatment of major depressive disorder or related disorders.
  • the invention provides a transgenic animal comprising a nucleic acid sequence encoding a CREBl gene operatively linked to a CREBl promoter comprising a DNA sequence variation at position -656 and/or -115.
  • the invention also provides an isolated or purified nucleic acid sequence comprising a CREBl promoter operably linked to a reporter gene, wherein the CREBl promoter comprises a DNA sequence variation at position -656 and/or -115.
  • the invention further provides an isolated cell comprising a cellular genome comprising a nucleic acid sequence operably linked to a promoter of a CREBl gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter.
  • a cell line comprising a clonal population of cells, wherein each cell comprises a nucleic acid sequence operably linked to a promoter of a
  • CREBl gene stably integrated into the genome of the cell, and wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter.
  • Figure 1 is the DNA sequence of the promoter of the human CREBl gene (SEQ ID NO:1). The DNA sequence is the positive strand and is shown in 5' to 3' orientation.
  • Figure 2 is a graph depicting the effect of gonadal steroid hormones on the basal activity of the wild-type and G(-656)A variant CREBl promoters in C6 cells. "N” indicates no hormone, "E” indicates 17 ⁇ -Estradiol, "P” indicates progesterone, and "T” indicates testosterone.
  • the wild-type promoter is represented by solid bars, and the variant promoter is represented by hatched bars.
  • the p-value of the genotype effect is p ⁇ 0.001, while the p- value for the hormone effect is p ⁇ 0.001.
  • Figure 3 is a graph depicting the effect of gonadal steroid hormones on the basal activity of the wild-type and A(-l 15)G variant CREBl promoters in C6 cells.
  • N indicates no hormone
  • E indicates 17 ⁇ -Estradiol
  • P indicates progesterone
  • T indicates testosterone.
  • the wild-type promoter is represented by solid bars
  • the variant promoter is represented by hatched bars.
  • the p-value of the genotype effect is p ⁇ 0.001, while the p- value for the hormone effect is p ⁇ 0.001.
  • Figure 4 is a graph depicting the effect of gonadal steroid hormones on the basal activity of the wild-type and G(-656)A variant CREBl promoters in CATH.a cells.
  • N indicates no hormone
  • E indicates 17 ⁇ -Estradiol
  • P indicates progesterone
  • T indicates testosterone.
  • the wild-type promoter is represented by solid bars
  • the variant promoter is represented by hatched bars.
  • Figure 5 is a graph depicting the effect of gonadal steroid hormones on the basal activity of the wild-type and A(-l 15)G variant CREBl promoters in CATH.a cells.
  • N indicates no hormone
  • E indicates 17 ⁇ -Estradiol
  • P indicates progesterone
  • T indicates testosterone.
  • the wild-type promoter is represented by solid bars
  • the variant promoter is represented by hatched bars.
  • the p-value of the genotype effect is p ⁇ 0.001
  • the p-value for the hormone effect is p ⁇ 0.001.
  • Figure 6 is a graph depicting the effect of gonadal steroid hormones on the activity of the wild-type and G(-656)A variant CREBl promoter following stimulation of the cAMP pathway in C6 cells.
  • N indicates no hormone
  • E indicates 17 ⁇ -Estradiol
  • P indicates progesterone
  • T indicates testosterone.
  • the wild-type promoter is represented by solid bars
  • the variant promoter is represented by hatched bars.
  • Figure 7 is a graph depicting the effect of gonadal steroid hormones on the activity of the wild-type and G(-656)A variant CREBl promoters following stimulation of the cAMP pathway in CATH.a cells.
  • N indicates no hormone
  • E indicates 17 ⁇ -Estradiol
  • P indicates progesterone
  • T indicates testosterone.
  • the wild-type promoter is represented by solid bars
  • the variant promoter is represented by hatched bars.
  • the p-value of the genotype effect is p ⁇ 0.001, while the p-value for the hormone effect is p ⁇ 0.001.
  • Figure 8 is a diagram of the pNTK+Nhel+INS 1+INS2 vector.
  • Figure 9 is an electropherogram showing the DNA sequence at the region of the CREBl promoter that was modified in the pNTK+NheI+INSl+INS2 vector, and the corresponding wild-type sequence.
  • the invention provides a method for determining the risk of a human developing major depressive disorder (MDD) or a related disorder and a method of diagnosing MDD or related disorders in a human.
  • the method comprises (a) obtaining a sample from a human, and (b) screening the sample for the presence of at least one DNA sequence variation in the promoter of a CREBl gene.
  • the presence of a DNA sequence variation in the promoter of a CREBl gene indicates a risk of developing major depressive disorder or related disorders as compared to a human that comprises a wild-type CREBl promoter, or alternatively, the presence of MDD or a related disorder in the human.
  • the invention is not limited to determining the risk of, diagnosing, or treating MDD, but also can be used to diagnose and treat other related disorders (e.g., mood disorders).
  • mood disorders related to MDD include, but are not limited to, bipolar disorder, dysthymia, cyclothymia, and mood disorders caused by other medical conditions (e.g., vascular or degenerative brain diseases, hypothyroidism, childbirth and menopause, and cancer), specific medications (e.g., antihypertensives, oral contraceptives and other steroids), and disorders associated with alcohol and/or drug abuse.
  • the invention extends to disorders of organ systems other than the nervous system, including, but not limited to, the cardiovascular, gastrointestinal, pulmonary, endocrine, reproductive, or immune systems, and disorders which often co-occur with mood disorders (see, e.g., Zubenko et al., Amer. J. Med. Genetics Part B (Neuropsychiatr. Genet), 123BU-1S (2003), Zubenko et al., Molecular Psychiatry, 7: 460-467 (2002), and Zubenko et al., Am. J. Med. Genet. (Neuropsychiatr. Genet), 105: 690-699 (2001)).
  • the term "risk” is synonymous with the term “predisposition,” and refers to the likelihood of acquiring a specific disease over the lifetime of an individual.
  • factors include, for example, genetics (e.g., disease-predisposing mutations), environment (e.g., pollution), and behaviors (e.g., smoking and alcohol intake).
  • genetics e.g., disease-predisposing mutations
  • environment e.g., pollution
  • behaviors e.g., smoking and alcohol intake
  • the "sample” can be any suitable sample, but preferably is a sample obtained from a mammal, preferably a human.
  • the sample can be a solid sample, such as a tissue sample.
  • a solid tissue sample can be obtained from any suitable organ, including but not limited to, skin, heart, lung, brain, etc.
  • the sample can be a fluid, such as a sample of body fluid. For instance, a section of whole tissue can be homogenized to liquefy the components found in the tissue.
  • suitable fluid samples include, but are not limited to, blood, saliva, serum, plasma, lymph, interstitial fluid, urine, milk (in the case of lactating females), and cerebrospinal fluid.
  • the invention can be used to determine a predisposition to or diagnose MDD or related disorders in men or women
  • the invention preferably is used to determine MDD risk or diagnose MDD in women in view of the higher prevalence of MDD in women having a DNA sequence variation in the CREBl promoter as compared to men having the same DNA sequence variation.
  • the invention comprises screening the sample for the presence of at least one DNA sequence variation in the promoter of a CREBl gene.
  • DNA sequence variation is meant any change in a wild-type DNA sequence.
  • DNA sequence variations include mutations and polymorphisms.
  • mutation refers to any detectable and heritable change in a DNA sequence that causes a change in genotype and which is transmitted to daughter cells and to succeeding generations. Mutations include point mutations (e.g., nucleotide substitutions), insertions, deletions, inversions, and the like.
  • polymorphism refers to the regular and simultaneous occurrence of two or more alleles of a gene, where the frequency of the rarer alleles is greater than can be explained by recurrent mutation alone. Polymorphisms frequently are used in the art as markers for gene mapping or genotyping.
  • RFLP restriction fragment length polymorphism
  • SSLP simple-sequence length polymorphism
  • SNP single-nucleotide polymorphism
  • RAPD randomly amplified polymorphic DNA
  • the sample can be screened for any suitable DNA sequence variation in the promoter of the CREBl gene.
  • the sample can be screened for any one or combination of suitable DNA sequence variations (e.g., polymorphisms), so long as the combination of DNA sequence variations is indicative of a mood disorder or an enhanced risk of acquiring a mood disorder (e.g., MDD) or related disorders.
  • suitable DNA sequence variations are described herein and include polymorphisms (e.g., RPLPs, SSLPs, minisatellites, and single-nucleotide polymorphisms (SNP)).
  • the DNA sequence variation is a SNP.
  • the DNA sequence variations can be heterozygous or homozygous, depending upon the mode of inheritance of the mood disorder.
  • the sample preferably is screened for one or more (e.g., 1, 2, 3, or more) DNA sequence variations in the promoter of the CREBl gene. More preferably, the sample is screened for at least two DNA sequence variations in the CREBl promoter. In a particularly preferred embodiment of the invention, the sample is screened for a DNA sequence variation at position -656 and/or position -115 of the CREBl promoter.
  • the DNA sequence variation at position -656 of the CREBl promoter preferably comprises a guanine to adenine (G to A) substitution (i.e., transition), which eliminates a consensus binding motif for AP-2 and potentially additional transcriptional regulators and a potential CpG methylation site.
  • the DNA sequence variation at position - 115 of the CREBl promoter preferably comprises an adenine to guanine (A to G) substitution (i.e., transition), which eliminates a consensus binding motif for AP-4 and potentially additional regulatory sequences.
  • G to A guanine to adenine
  • a to G adenine to G substitution
  • a sample obtained from a human can be genotyped for the DNA sequence variation in the CREBl promoter using any suitable method known in the art, some of which are described herein. Such methods include, for example, polymerase chain reaction (PCR), DNA sequencing, and RFLP analysis.
  • PCR polymerase chain reaction
  • DNA sequencing DNA sequencing
  • RFLP analysis RFLP analysis
  • the invention also provides a method of identifying a candidate drug for treating major depressive disorder (MDD) or related disorders.
  • the method comprises (a) preparing a nucleic acid construct comprising the promoter of a CREBl gene operably linked to a reporter gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, (b) introducing the nucleic acid construct into an expression system, (c) contacting the expression system with at least one test compound, and (d) assaying for at least partial restoration of the function of the promoter, whereupon at least partial restoration of the function of the promoter indicates the test compound is a drug that treats major depressive disorder or a related disorder.
  • treat or “treatment,” as used herein, are synonymous with the terms “therapy” and “therapeutic,” and refer to the amelioration of a mood disorder (or a related disorder) itself, and the protection, in whole or in part, against further mood disorders (or related disorders), in particular MDD.
  • a mood disorder or a related disorder
  • a therapeutic refers to the amelioration of a mood disorder (or a related disorder) itself, and the protection, in whole or in part, against further mood disorders (or related disorders), in particular MDD.
  • any degree of protection from, or amelioration of, a mood or related disorder is beneficial to a patient, and that a drug need not completely eliminate the disorder to be consider an effective therapeutic.
  • a first step in the method of identifying a drug that treats MDD or a related disorder is the preparation of a nucleic acid construct comprising the promoter of a CREBl gene operably linked to a reporter gene.
  • the invention also provides an isolated or purified nucleic acid sequence comprising a CREBl promoter operably linked to a reporter gene.
  • reporter gene is meant a gene that encodes a product whose expression can be easily detected.
  • the reporter gene desirably is present in the nucleic acid construct as part of an expression cassette, i.e., a particular nucleotide sequence that possesses functions which facilitate subcloning and recovery of a nucleic acid sequence (e.g., one or more restriction sites) or expression of a nucleic acid sequence (e.g., polyadenylation or splice sites).
  • the reporter gene can be any suitable reporter gene known in the art. Suitable reporter genes include, but are not limited to, genes encoding luciferase, green fluorescent protein (GFP), ⁇ - galactosidase, chloramphenicol acetyltransferase (CAT), and ⁇ -glucuronidase (Gus).
  • the CREBl promoter within the construct comprises a DNA sequence variation which dysregulates the function of the promoter.
  • “dysregulate” is meant to disrupt the normal control of a promoter.
  • a promoter is dysregulated if its function is inhibited or upregulated beyond normal levels.
  • the CREBl promoter can comprise any suitable DNA sequence variation described herein, but preferably comprises a SNP at position -656 and/or position -115 of the CREBl promoter (see Fig. 1).
  • the CREBl promoter comprises a guanine to adenine (G to A) substitution at position -656 and a guanine (A to G) substitution at position -115.
  • the CREBl promoter can comprise any DNA sequence variation or combination of DNA sequence variations that dysregulate the function of the promoter, preferably to the extent that the dysregulation enhances the susceptibility of an individual harboring the sequence variation to developing MDD or a related disorder.
  • the DNA sequence variation can be located in a cAMP-responsive enhancer (CRE) sequence, an SP-I binding site, and/or a NFKB binding site present in the human CREBl promoter.
  • CRE cAMP-responsive enhancer
  • the nucleic acid construct preferably is introduced into an expression system.
  • expression system is meant a system which allows for the expression (i.e., transcription and/or translation ) of the nucleic acid construct.
  • the expression system can be an in vitro expression system or an in vivo expression system. Suitable in vitro expression systems include, for example, in vitro cell transfection systems and cell-free expression systems. In vitro expression systems are further described in, for example Sambrook et al., supra, Mantovani, Methods MoI. Biol, 31: 289-98 (1994), and Lyford et al., J Biol. Chan., 274(36): 25675-81 (1999).
  • the expression system is an in vivo expression system, such as a cell (cellular expression system) or an organism (e.g., a mouse or a human).
  • the invention further provides a method of identifying a drug that treats major depressive disorder (MDD) or related disorders comprising, by (a) preparing a nucleic acid construct comprising the promoter of a CREBl gene operably linked to a reporter gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, (b) introducing the nucleic acid construct into an cell, (c) contacting the cell with at least one test compound, and (d) assaying for at least partial restoration of the function of the promoter, whereupon at least partial restoration of the function of the promoter indicates the test compound is a candidate drug for treating major depressive disorder or related disorders.
  • MDD major depressive disorder
  • the inventive method comprises introducing the nucleic acid construct into a cell.
  • the cell can be "isolated” if it is substantially separated, produced apart from, or purified away from other cells in its natural environment.
  • a cell can be isolated if it is within a population of other similarly isolated cells, such as within a tissue culture well, tube or plate. It is specifically contemplated that the inventive method can be performed on populations of isolated cells.
  • the cell can be any cell that is capable of accepting, retaining, and expressing the nucleic acid sequence comprising a CREBl promoter operably linked to a reporter gene.
  • the cell can be a primary cell.
  • primary cell is meant that the cell does not replicate indefinitely in culture.
  • suitable primary cells include, but are not limited to, human brain cells, human embryonic kidney (HEK) cells, human retinal cells, and human embryonic retinal (HER) cells.
  • the cell can be a transformed cell.
  • the cell is "transformed” in that the cell has the ability to replicate indefinitely in culture.
  • suitable transformed cells include renal carcinoma cells, SY5 Y cells, neuroblastoma cells, C6 glial cells, CATH. a cells, HeLa cells, CHO cells, KB cells, HEK-293 cells, SW- 13 cells, MCF7 cells, and Vero cells. Often, transformed cells are part of cell lines.
  • cell line refers to a clonal population of cultured cells that have the potential to propagate indefinitely.
  • a cell culture propagates indefinitely if it can be passaged in culture for 10 or more generations (e.g., 10, 12, or 15 generations), preferably 20 or more generations (e.g., 20, 30, or 40 generations), more preferably 50 or more generations (e.g., 50, 60, or 80 generations), and most preferably 100 or more generations (e.g., 100, 200, or 500 generations).
  • the nucleic acid construct can be stably integrated into the cellular genome.
  • the cell preferably is a eukaryotic cell. More preferably, the cell is a mammalian cell. Most preferably, the cell is a human cell. Suitable human cells are known in the art and are commercially available from sources such as the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • the nucleic acid construct can be introduced into the cells using any suitable method known in the art (see, e.g., Sambrook et al., supra), such as transient transfection methods.
  • the method comprises (a) contacting a cell with at least one test compound, wherein the cell comprises a cellular genome comprising a nucleic acid sequence operably linked to a promoter of a CREBl gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, and (b) assaying for at least partial restoration of the function of the promoter, whereupon at least partial restoration of the function of the promoter indicates the test compound is a candidate drug for treating major depressive disorder or related disorders.
  • step (b) can alternatively, or in addition, comprise assaying for at least partially restored expression of CREBl target genes or at least partial restoration of CREBl -associated functions, whereupon at least partial restoration of expression of CREBl target genes or at least partial restoration of CREB-I associated functions indicates the test compound is a candidate drug for treating major depressive disorder or related disorders.
  • the cell can be isolated from an animal, preferably a mammal, and most preferably a human.
  • the cell can comprise a naturally-occurring CREBl gene and a CREBl promoter that comprises a DNA sequence variation as described herein, in which instance, the cell is not modified to contain the variant CREBl promoter.
  • the cell can be obtained from a transgenic animal (discussed below), in which a CREBl promoter comprising a DNA sequence variation has been introduced via gene targeting, or "knock-in", technology.
  • the cell can be a member of a population of cells, typically a cell line, into which a nucleic acid construct comprising the promoter of a CREBl gene operably linked to a reporter gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, has been introduced (e.g., as described above) such that the construct has integrated into the genome of the cells (e.g., using a suitable vector system for chromosomal integration).
  • a nucleic acid construct comprising the promoter of a CREBl gene operably linked to a reporter gene, wherein the promoter comprises a DNA sequence variation which dysregulates the function of the promoter, has been introduced (e.g., as described above) such that the construct has integrated into the genome of the cells (e.g., using a suitable vector system for chromosomal integration).
  • the method further comprises exposing the expression system (e.g., the nucleic acid construct, other molecules within the system, or the cell) to one or more test compounds being screened for a potential therapeutic effect on major depressive disorder or related disorders.
  • the expression system e.g., a cell
  • test compound e.g., 2, 3, 4, 5, or more compounds.
  • the test compound can be any molecule or substance, and can be, but need not be, a compound that is suspected of having a therapeutic effect on MDD or a related disorder. Suitable compounds include, for example, macromolecules (e.g., proteins, nucleic acids, carbohydrates) and small molecules.
  • Such compounds can be identified for testing in accordance with the inventive method by any suitable method, including selecting a compound based on (1) a previously-established effect on another mood disorder, or (2) the involvement of the compound in a biological pathway known to play a role in the etiology of a mood disorder.
  • the inventive method can be employed to assay randomly-selected compounds or batteries of compounds.
  • the test compound can contact the expression system in vitro or in vivo using any method known in the art, such as those described herein. Whatever method is used, the test compound desirably contacts the expression system (e.g., a cell) under conditions which allow for the test compound to interact with appropriate molecules and restore all or a portion of CREBl promoter activity.
  • test compound need not interact directly with a CREBl promoter sequence to restore all or a portion of CREBl promoter activity.
  • the test compound can affect another cellular process or biological pathway that is involved in CREBl gene expression. Such conditions will depend upon the compound being tested, as well as the method used to contact the expression system with the test compound. For example, it is contemplated that some compounds will interact directly with molecules that participate in forming complexes with the CREBl promoter DNA (e.g., within an in vitro expression system or by entering the cytoplasm to act within a cellular expression system).
  • test compound can act to restore CREBl promoter activity, particularly within a cellular expression system, by acting upstream of molecules that participate in forming complexes with the CREBl promoter DNA, for example by precipitating or interfering with signal transduction cascades.
  • the test compound need not enter the cells to "contact" the system, as some compounds can act by contacting transmembrane proteins, for example.
  • the test compound is considered a candidate drug for treating MDD or a related disorder if the test compound is capable of restoring all or a portion of the function of the CREBl promoter.
  • expression of a reporter gene at levels appropriate for a wild-type CREBl promoter as a result of exposure to the test compound indicates that the test compound has at least partially overcome the defects associated with the CREBl promoter DNA sequence variation and has restored all or a portion of the normal activity of the CREBl promoter.
  • Compounds that restore all or a portion of the function of the promoter are considered candidate drugs for treating MDD or a related disorder in that they are deemed suitable for further development as a potential therapeutic agent against MDD or a related disorder.
  • the test compound may or may not actually be found to be a viable therapeutic agent.
  • the inventive method provides information concerning test compounds that will enable the ordinarily skilled artisan to distinguish candidate drugs for treating MDD or a related disorder from those that are not suitable candidates for further development. For example, in the event that a particular test compound does not restore the normal function of the CREBl promoter (or a portion thereof), such a compound likely will not be selected for further development as a potential therapeutic against MDD or a related disorder.
  • Restoration of CREBl promoter activity within the context of the inventive method can be detected by assaying for expression of a reporter gene. Detection of reporter gene expression can be performed using any appropriate reporter gene assay known in the art that relies on colorimetric, fluorescent, luminescent, radiometric methods, or the like.
  • rtPCR can be conducted to assay for CREBl mRNA, while immunohistochemical techniques can be employed to assay for CREBl protein.
  • a test compound can be considered a candidate drug for treating MDD or a related disorder if the compound is capable of at least partially restoring expression of CREBl target genes or restoring of CREB-I associated functions.
  • a CREBl target gene includes any gene whose expression is regulated directly or indirectly by the CREBl protein.
  • CREBl target genes include, but are not limited to genes encoding metabolic proteins (e.g., lactate dehydrogenase), transcription factors (e.g., c-Fos), neuropeptides (e.g., Inhibin A), cell cycle regulators (e.g., cyclin A), growth factors (e.g., insulin), immune system regulators (e.g., interleukin 2), reproduction proteins (e.g., spermatid nuclear transition protein), signaling proteins (MKP-I), transport proteins (e.g., cystic fibrosis transmembrane conductance regulator), and structural proteins (e.g., fibronectin).
  • metabolic proteins e.g., lactate dehydrogenase
  • transcription factors e.g., c-Fos
  • neuropeptides e.g., Inhibin A
  • cell cycle regulators e.g., cyclin A
  • growth factors e.g., insulin
  • immune system regulators e.
  • a CREB-I -associated function is a cellular activity or process in which CREB-I participates, either directly or indirectly.
  • a CREB-I -associated function need not be directly regulated by CREB-I. That is, genes encoding proteins which participate in CREB-I -associated functions need not comprise CREB-I binding sites.
  • CREB-I associated functions include, but are not limited to, metabolism, transcription, responses to neuropeptides and neurotransmitters, regulation of cell cycle/cell survival/DNA repair, response to growth factors, immune regulation, regulation of reproduction and development, cell signaling and communication pathways, inter and intracellular transport, and building structural components of cells (see Mayr et al., supra).
  • a particular compound does not restore the expression of a CREBl target gene or a CREBl -associated function
  • such a compound would likely not be selected for further development as a potential therapeutic against MDD or a related disorder, and would not be considered a candidate drug for treating MDD or a related disorder.
  • the invention further provides a transgenic animal comprising a nucleic acid sequence encoding a CREBl gene operatively linked to a CREBl promoter comprising a DNA sequence variation at position -656 and/or -115.
  • a "transgene” refers to a nucleic acid sequence which has been manipulated in vitro and which can be subsequently introduced into the genome of the same or a different species in either the native or modified forms, such that it is stably and heritably maintained in that genome.
  • the nucleic acid sequence preferably encodes a protein of interest, and is operably linked to sequences that regulate its expression (e.g., a promoter).
  • transgene also refers to the nucleic acid sequence and the regulatory sequences to which it is operably linked.
  • the transgene can further comprise other nucleic acid sequences which encode, for example, reporter genes in order to monitor expression of the transgene.
  • An organism into which a transgene has been introduced is termed a "transgenic organism.”
  • the transgenic animal can be any suitable animal, and preferably is a non-human animal. Suitable non-human animals include, but are not limited to, mice, rats, primates, rabbits, dogs, cats, horses, fish, cattle, swine, and sheep. In a preferred embodiment, the transgenic animal is a mouse. A transgene can be introduced into the germline of an animal using a variety of methods.
  • the transgene can be directly injected into the male pronucleus of a fertilized egg (see, e.g., Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, Cold Spring Harbor Press (1994)), resulting in the random integration into one locus of a varying number of copies of the transgene, usually in a head to tail array (see, e.g., Costantini and Lacy, Nature, 294: 92 (1981)).
  • the injected eggs are then re-transferred into the uteri of pseudopregnant recipient mothers.
  • Some of the resulting offspring may have one or several copies of the transgene integrated into their genomes, usually in one integration site.
  • transgenes can be introduced into an animal by gene targeting in embryonic stem (ES) cells.
  • ES embryonic stem
  • a targeting construct comprising the transgene is prepared using methods known in the art (see, e.g., Sambrook et al., supra).
  • a preferred, but non-limiting, example of a targeting construct useful for generating CREBl knock-in mice is the pNTK+NheI+INSl+INS2 plasmid depicted in Fig. 8.
  • the targeting construct may be introduced into an appropriate host cell using any method known in the art, such as, for example, microinjection, retrovirus mediated gene transfer, electroporation, bacterial protoplast fusion with intact cells, transfection, polycations, e.g., polybrene, polyornithine, etc., or the like (see, e.g., U.S. Patent 4,873,191, Van der Putten et al., Proc. Natl. Acad. Sd. USA, 82: 6148-6152 (1985), Thompson et al., Cell, 56: 313-321 (1989), Lo, MoI Cell.
  • any method known in the art such as, for example, microinjection, retrovirus mediated gene transfer, electroporation, bacterial protoplast fusion with intact cells, transfection, polycations, e.g., polybrene, polyornithine, etc., or the like (see, e.g., U.S. Patent 4,873,191, Van der Putten
  • the targeting construct is introduced into host cells by electroporation.
  • electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the construct.
  • the pores created during electroporation permit the uptake of macromolecules such as DNA.
  • any cell type capable of homologous recombination may be used in the invention.
  • target cells include cells derived from any of the non-human animals discussed above, as well as other eukaryotic organisms such as filamentous fungi, and higher multicellular organisms such as plants.
  • the targeting construct is introduced into embryonic stem (ES) cells, which are typically obtained from pre-implantation embryos cultured in vitro (see, e.g., Evans et al., Nature, 292: 154-156 (1981), Bradley et al., Nature, 309: 255-258 (1984), Gossler et al., Proc. Natl. Acad. Sd.
  • the ES cells are cultured and prepared for introduction of the targeting construct using methods well known to those of ordinary skill in the art (see, e.g., Robertson, E. J. ed., Teratocardnomas and Embryonic Stem Cells, a Practical Approach, IRL Press, Washington D.C. (1987), Bradley et al., Current Topics in Devel. Biol, 20: 357-371 (1986), Hogan et al., In: Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y.
  • the ES cells that are contacted with the targeting construct are derived from an embryo or blastocyst of the same species as the developing embryo into which they are to be introduced.
  • ES cells typically are selected for their ability to integrate into the inner cell mass and contribute to the germ line of an animal when introduced into the animal in an embryo at the blastocyst stage of development. Thus, any ES cell line having this capability is suitable for use in the invention.
  • the cells in which successful gene targeting has occurred are selected. Insertion of the targeting construct into the targeted gene typically is detected by identifying cells for expression of a marker gene included in the targeting construct.
  • the cells transformed with the targeting construct are subjected to treatment with an appropriate agent that selects against cells not expressing the selectable marker. Only those cells expressing the selectable marker gene survive and/or grow under certain conditions. For example, cells that express a neomycin resistance gene are resistant to the compound G418, while cells that do not express the neomycin resistance gene marker are killed by G418.
  • the targeting construct also comprises a screening marker such as GFP
  • homologous recombination can be identified through screening cell colonies under a fluorescent light. Cells that have undergone homologous recombination will have deleted the GFP gene and will not fluoresce.
  • Selected cells are then injected into a blastocyst (or other stage of development suitable for the purposes of creating a viable animal, such as, for example, a morula) of an animal (e.g., a mouse) to form chimeras (see e.g., Bradley In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, pp. 113-152 (1987)).
  • selected ES cells can be allowed to aggregate with dissociated mouse embryo cells to form the aggregation chimera.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, where the embryo is brought to term.
  • Chimeric progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA.
  • Transgenic animals may be identified by testing to ensure the required genotypic change has been effected. This can be accomplished using any suitable method known in the art, such as, for example, detecting the presence of the transgene by PCR with specific primers, or by Southern blotting with a transgene-specific probe.
  • heterozygous and homozygous transgenic mice can be compared to normal, wild-type mice to determine whether introduction of the transgene causes phenotypic changes, especially pathological changes.
  • heterozygous and homozygous mice may be evaluated for phenotypic changes by physical examination, necropsy, histology, clinical chemistry, complete blood count, body weight, organ weights, and cytological evaluation of bone marrow. Methods for generating transgenic mice are further described in, e.g., Jackson et al., eds., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000), and U.S. Patent 6,943,277.
  • the inventive transgenic animal comprises a nucleic acid sequence (i.e., a "transgene") encoding a CREBl gene operatively linked to a CREBl promoter comprising a DNA sequence variation, preferably at position -656 and/or -115.
  • a transgene a nucleic acid sequence
  • Descriptions of the CREBl gene, the CREBl promoter, and DNA sequence variation set forth above in connection with other embodiments of the invention also are applicable to those same aspects of the aforesaid transgenic animal.
  • the transgene incorporated into the animal is not limited to a nucleic acid sequence comprising these specific CREBl promoter DNA sequence variations.
  • the transgenic animal can comprise a nucleic acid sequence encoding a CREBl gene and/or CREBl promoter comprising any suitable DNA sequence variation that is associated with the development of a mood disorder.
  • the invention also provides a cell isolated from the transgenic animal and a cell line derived from the transgenic animal, both of which can be used in the method of identifying a drug that treats MDD or a related disorder described herein.
  • CREBl coding regions (1026 total nucleotides), splice junctions of all 9 exons, and the 5' regulatory regions, of all women affected or unaffected with MDD from an extended RE-MDD pedigree ("family A") were sequenced, with the goal of identifying sequence variants that were associated with the development of unipolar mood disorders.
  • family A extended RE-MDD pedigree
  • family A yielded a peak multipoint logarithm of the odds (LOD) score of 3.77 at the marker D2S2208 for female relative pairs affected by unipolar mood disorders, and had the potential to generate a significant LOD score between a variant CREBl allele that was co-segregating with mood disorders (or their absence).
  • LOD odds
  • This analysis revealed a single nucleotide polymorphism (SNP) at position -656 (denoted G(-656)A) of the CREBl promoter consisting of a guanine to adenine transition that eliminates a consensus binding motif for AP-2, a transcriptional activator, other potential regulatory sequences, and a potential CpG methylation site in the CREBl promoter.
  • SNP single nucleotide polymorphism
  • Rat glioma cell line C6 obtained from the ATCC (Manassas, VA) (Accession No. CCL-107), was grown in Ham's F12 medium (Kaighn's modification) supplemented with 2.5g/L sodium bicarbonate, 15% horse serum, and 2.5% fetal bovine serum at 37 0 C, 5% CO2, 100% humidity.
  • C6 cells were seeded in 60 mm cell culture dishes (Corning Inc., Corning, NY) at a density of 8 x 10 5 cells/dish using medium that lacked or contained physiological concentrations (100 nM) of a gonadal steroid hormone (i.e., 17 ⁇ -estradiol, progesterone, or testosterone, (Sigma, St. Louis, MO)).
  • a gonadal steroid hormone i.e., 17 ⁇ -estradiol, progesterone, or testosterone, (Sigma, St. Louis, MO)
  • the resulting cultures had reached a density of approximately 50% confluence when transfection was initiated.
  • Transfection was performed with the lipid-based transfection reagent FuGENE® 6 (Roche Applied Science, Indianapolis, IN), using methods that were optimized for C6 cells.
  • the cells were transfected with an equimolar mixture of a CAT reporter construct comprising (a) either the wild-type CREBl promoter, a G(-656)A polymorphic CREBl promoter, or a A(-l 15)G polymorphic CREBl promoter, and (b) a pSV- ⁇ - galactosidase control vector (Promega Corporation, Madison, WI) that constitutively expresses ⁇ -galactosidase, and was included to adjust for potential differences in transfection efficiency across experiments.
  • the CAT expression plasmids containing the wild-type or variant CREBl promoters were constructed, then confirmed by restriction mapping and automated DNA sequencing, as described herein. Sham transfections employing the native pCAT®3 -basic vector (Promega Corporation, Madison, WI) were performed to control for any background level of reporter or ⁇ -galactosidase activity.
  • the largest hormonal effect was reflected by a significant elevation of basal promoter activity in the presence of 17 ⁇ -estradiol compared to the no hormone condition (p ⁇ 0.001, post hoc Tukey HSD).
  • the greatest effect of genotype on promoter activity was observed in the presence of 17 ⁇ -estradiol.
  • both the hormonal environment and the A(-l 15)G variant had significant effects on CREBl promoter activity, and a significant hormone by genotype interaction was observed (two-way ANOVA; all three p values ⁇ 0.001).
  • the A to G transition at position -115 resulted in a significant reduction in basal CREBl promoter activity, with the greatest effect of genotype occurring in the presence of 17 ⁇ -estradiol.
  • Both the G(-656)A and A(-l 15)G promoter variants exert the greatest effects on basal CREBl promoter activity in C6 cells grown in the presence of 17 ⁇ -estradiol.
  • This example demonstrates the effect of gonadal steroid hormones on the basal activity of the wild-type and polymorphic CREBl promoters in a mouse neuronal cell line.
  • the CATH.a cell line was developed from a brain stem tumor of a transgenic mouse expressing the SV40 T antigen under the control of the tyrosine hydroxylase promoter, exhibits a neural, noradrenergic phenotype, and resembles Locus Ceruleus (LC) neurons in their signal transduction profile (see, e.g., Suri et al., J Neurosci., 13: 1280-1291 (1993), and Widnell et al., Proc. Natl. Acad.
  • LC Locus Ceruleus
  • This cell line was obtained from the ATCC (Accession No. CRL-11179) and grown in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 10 mM HEPES, 4.5 g/L glucose, 1 mM sodium pyruvate, 2 g/L sodium bicarbonate, 8% horse serum, and 4% fetal bovine serum, at 37°C, 5% CO 2 , 100% humidity.
  • CATH.a cells were seeded in 60 mm cell culture dishes (Corning Inc., Corning, NY) at a density of 1 x 10 6 cells/dish using medium that lacked or contained physiological concentrations (10OnM) of a gonadal steroid hormone (i.e., 17 ⁇ -estradiol, progesterone, or testosterone).
  • CATH.a cells were transfected using the transfection reagent LipofectamineTM 2000 (Invitrogen Corp., Carlsbad, CA) optimized for CATH.a cells.
  • the cells were transfected with an equimolar mixture of a CAT reporter construct comprising (a) either the wild-type CREBl promoter, a G(-656)A polymorphic CREBl promoter, or a A(-l 15)G polymorphic CREBl promoter, and (b) a pSV- ⁇ -galactosidase control vector (Promega Corporation, Madison, WI) that constitutively expresses ⁇ -galactosidase and was included to adjust for potential differences in transfection efficiency across experiments.
  • the CAT expression plasmids containing the wt or variant CREBl promoters were constructed, then confirmed by restriction mapping and automated DNA sequencing, as described herein. Sham transfections employing the native pCAT®3- Basic Vector (Promega Corp., Madison, WI) were performed to control for any background level of reporter or ⁇ -galactosidase activity.
  • Negligible CAT specific activity was found in cells that lacked the CREBl promoter-CAT reporter constructs.
  • the CREBl promoter-mediated expression of the CAT gene in each experimental condition was determined by normalizing CAT specific activity to ⁇ - galactosidase specific activity in each lysate, after subtraction of the specific activities observed for similarly-treated sham-transfected cells. Each experiment was performed six times and the results expressed as mean ⁇ standard deviation (SD).
  • SD standard deviation
  • the basal activity of the wild-type CREBl promoter was several -fold higher in CATH.a cells compared to C6 cells in the absence of gonadal steroid hormones and in all of the hormone conditions tested (compared to Example 2).
  • the largest hormonal effect was attributable to a significant elevation of basal promoter activity in the presence of testosterone (p ⁇ 0.001, post hoc Tukey HSD).
  • This example demonstrates the effects of gonadal steroid hormone and activation of the cAMP signaling pathway on wild-type and polymorphic CREBl promoter activity in rat glioma cells.
  • C6 cells were grown in the absence or presence of gonadal steroids and transfected with equimolar amounts of (a) either the wild-type or G(-656)A variant CREBl promoter-CAT reporter constructs, and (b) a pSV- ⁇ -galactosidase control vector (Promega Corporation, Madison, WI) that constitutively expresses ⁇ -galactosidase and was included to adjust for potential differences in transfection efficiency across experiments, as described in Example 2.
  • a pSV- ⁇ -galactosidase control vector Promega Corporation, Madison, WI
  • cAMP signaling pathway was induced by replacement of the transfection medium with the identical growth medium (+/- steroids) containing 10 ⁇ M forskolin and 0.25 mM 3-isobuyl-l-methylxanthine (IBMX) (Sigma, St. Louis, MO).
  • IBMX 3-isobuyl-l-methylxanthine
  • Forskolin increases intracellular cAMP levels by direct stimulation of adenylate cyclase
  • IBMX inhibits the breakdown of cAMP by inhibition of phosphodiesterase. This condition simulates the activation of G protein-coupled neurotransmitter/growth factor receptors on brain cells grown in the presence of different gonadal hormones.
  • Cells were harvested at 0, 2, 4, 6, 12, 24, and 48 hours of activation, lysed, and assayed for CAT, ⁇ -galactosidase, and protein concentration as described in Example 2. Each experiment was performed six times and the results expressed as mean ⁇ SD.
  • This example demonstrates the effects of gonadal steroid hormone and activation of the cAMP signaling pathway on wild-type and polymorphic CREBl promoter activity in a mouse neuronal cell line.
  • CATH.a cells were grown in the absence or presence of gonadal steroids and transfected with equimolar amounts of (a) either the wild-type or G(-656)A variant CREBl promoter-CAT reporter constructs, and (b) a pSV- ⁇ -galactosidase control vector (Promega Corporation, Madison, WI) that constitutively expresses ⁇ -galactosidase and was included to adjust for potential differences in transfection efficiency across experiments, as described in Example 3.
  • a pSV- ⁇ -galactosidase control vector Promega Corporation, Madison, WI
  • cAMP signaling pathway was induced by replacement of the transfection medium with the identical growth medium (+/- steroids) containing 10 ⁇ M forskolin and 0.25 mM 3-isobuyl-l-methylxanthine (IBMX) (Sigma, St. Louis, MO).
  • IBMX 3-isobuyl-l-methylxanthine
  • Forskolin increases intracellular cAMP levels by direct stimulation of adenylate cyclase
  • IBMX inhibits the breakdown of cAMP by inhibition of phosphodiesterase. This condition simulates the activation of G protein-coupled neurotransmitter/growth factor receptors on brain cells grown in the presence of different gonadal hormones.
  • Cells were harvested at 0, 2, 4, 6, 12, 24, and 48 hours of activation, lysed, and assayed for CAT, ⁇ -galactosidase, and protein concentration as described in Example 2. Each experiment was performed six times and the results expressed as mean ⁇ SD.
  • This example demonstrates methods for identifying additional CREBl DNA sequence variations that confer susceptibility to mood disorders.
  • the strategy of sequencing the CREBl regions of two RE-MDD females from each family includes at least one carrier from 99% of families who were segregating CREBl risk alleles with phenocopy rates of 10%.
  • DNA sequencing is assessed by sequencing each region from both directions using each member of a PCR primer pair.
  • the potential introduction of DNA sequence changes during PCR amplification is reduced by the use of GeneAmp® High Fidelity Enzyme Mix (Applied Biosystems, Foster City, CA) an enzyme blend of AmpliTaq ® DNA Polymerase (Applied Biosystems, Foster City, CA) and a thermostable proofreading enzyme reported to increase the fidelity of PCR threefold over Taq DNA Polymerase.
  • GeneAmp® High Fidelity Enzyme Mix Applied Biosystems, Foster City, CA
  • AmpliTaq ® DNA Polymerase Applied Biosystems, Foster City, CA
  • thermostable proofreading enzyme reported to increase the fidelity of PCR threefold over Taq DNA Polymerase.
  • Each newly detected sequence variant is confirmed using an independently-prepared amplicon of the region that harbors the putative variant to exclude potential PCR or sequencing artifacts.
  • the female family members of probands who carry confirmed CREBl sequence polymorphisms are similarly genotyped by DNA sequencing to determine whether the identified polymorphism cosegregates with mood disorders among the women in those families.
  • the male members of families who meet these criteria are subsequently genotyped for the susceptibility alleles segregating in their families to support linkage analysis and to enable an assessment of the sex-specificity of the newly identified CREBl risk alleles.
  • RFLP-based assays are developed for each new risk allele based on the creation or elimination of a restriction site by the sequence variant, as described above for the G(-656)A and A(-115)G SNPs.
  • SNPs or deletions/insertions create or disrupt the recognition site of at least one currently available restriction endonuclease, as determined using Webcutter 2.0 (www. carolina.com/webcutter/carolina.asp; ⁇ Max Heiman (1997)), regardless of their location in regulatory regions, exons, or introns. More extensive sequence changes, if they are found, are often readily directly detectable as length polymorphisms.
  • the RFLP assays enable efficient screening of the remaining families in the collection for each risk allele. Positive results from the RFLP-based screening approach are evaluated using DNA sequencing to identify the specific sequence variation detected in each family.
  • DNA templates for sequencing CREBl regions have been produced by PCR amplification of genomic DNA (see Zubenko et al., MoI. Psychiatry, 8: 611-618 (2003)), employing oligonucleotide primers that were developed using the program Primer3 (see Rozen et al., PrimerS on the WWW for general users and for biologist programmers, In: Krawetz et al. (eds), Bioinformatics Methods and Protocols: Methods in Molecular Biology, Humana Press, Totawa, NJ, pp. 365-386 (2000)), and synthesized by Invitrogen Custom Primers (Invitrogen Corp., Carlsbad, CA).
  • This example demonstrates the construction of the pNTK+Nhel+INS 1+INS2 vector containing a mutagenized CREBl promoter. This plasmid is useful for generating CREBl knock-in mice.
  • Plasmid NTK contains origins of replication that enable propagation in bacteria, along with two selectable markers that confer resistance to ampicillin (Ap 1 ) or neomycin (neo). The neomycin gene also confers neomycin resistance in mammalian cells, while the TK-deficiency provides an opportunity for negative selection.
  • pNTK was transformed into competent bacteria. Colonies were chosen for DNA minipreps and screened by digestion with multiple enzymes (Sail, Nhel, Xhol, CIaI, BamHI), followed by resolution of the resulting restriction fragments by electrophoresis.
  • a reagent amount of pNTK was prepared and the structure of the prepared plasmid was again confirmed by digestion with multiple restriction enzymes (Sail, Nhel, Xhol, CIaI, BamHI).
  • An oligonucleotide adapter was designed and synthesized to convert the unique pNTK BamHI site to an Miel site.
  • pNTK was linearized by digestion with BamHI and purified by preparative gel electrophoresis.
  • the oligonucleotide adapter was ligated to the linearized pNTK plasmid. The products of this ligation were digested with Nhel and the modified plasmid was recovered by preparative gel electrophoresis.
  • the modified plasmid was recircularized by ligation and transformed into competent bacteria. Transformants were chosen and minipreps were performed. DNA preparations from transformants were screened by digestion with Nhel, and a reagent amount of the successfully modified pNTK was prepared.
  • Insert 1 Preparation of Insert 1 and 2. Plates were streaked with BAC Clone RP24-528i8, (BAC- PAC Resources, Oakland, CA) to obtain single colonies. Digestion of the BAC Clone with Alw44I produced DNA fragments that were purified by preparative gel electrophoresis. Two contiguous restriction fragments from mouse strain C57B1/6J were isolated from the BAC Clone. Insert 1 (6.3 kb) contained the CREBl promoter as well as Exon 1. Insert 2 (5.2 kb) consisted entirely of DNA from Intron 1. Oligonucleotide adapters were designed and synthesized to convert restriction sites at the ends of each fragment for cloning (i.e., Insert 1 becomes Sail, Insert 2 becomes Nhel).
  • a reagent quantity of pCR2.1-TOPO containing modified insert 2 was prepared.
  • the composition of the construct was confirmed by digestion with multiple restriction enzymes (Nhel, AfIII, CIaI, BamHI), followed by resolution of the DNA fragments by gel electrophoresis.
  • Insert 2 with Nhel ends was excised from this construct by Nhel digestion and recovered by preparative gel electrophoresis. Insert 2 with Nhel ends was then subcloned into modified pNTK, whose unique BamHI site had been converted to an Nhel site as described above [0094].
  • modified pNTK was digested with Nhel and treated with alkaline phosphatase to prevent recircularization of the plasmid.
  • the linearized vector was separated from any undigested plasmid by preparative gel electrophoresis. Insert 2 with Nhel ends was ligated into modified pNTK at the introduced Nhel site and the product was transformed into competent bacteria. Transformants were selected and minipreps were performed. DNA preparations from transformants were screened by digestion with Nhel and Spel, followed by gel electrophoresis to detect the release of the 5.2 kb insert and to determine the orientation of Insert 2 in the plasmid. A reagent amount of pNTK containing Insert 2 subcloned into the unique Nhel site, and in the desired orientation, was prepared and again confirmed by restriction enzyme digestion with Nhel and Spel.
  • Cloning Insert 1 Sail adapters were ligated to the Alw44I ends of Insert lto generate a PCR-like overhang necessary for TOPO cloning. Modified Insert 1 was cloned into vector pCR-XL-TOPO (Invitrogen, Carlsbad, CA). Competent bacteria were transformed with the TOPO cloned construct. Transformants were selected and minipreps were performed. DNA preparations from transformants were screened by digestion with multiple restriction enzymes (Sail, Spel, BgIII, AfJII, BamHI, Pstl), followed by resolution of the resulting DNA fragments by gel electrophoresis. A reagent amount of the TOPO cloned construct was prepared and reconfirmed by digestion with restriction enzymes (Sail, Spel, BgIII, Afi ⁇ , BamHi, Pstl).
  • Transformants were selected and minipreps were performed. DNA preparations from transformants were screened by digestion with Sail and Spel, followed by resolution of the resulting DNA fragments by gel electrophoresis, to detect the release of the 6.3 kb Insert 1 and to determine the orientations of both Insert 1 and Insert 2 in the final construct.
  • the final construct is illustrated in Figure 8. Transcribed regions are shown by thick lines and the direction of transcription is shown by arrowheads. A reagent amount of this final construct was prepared, and the structure was confirmed by digestion with restriction enzymes Sail, Nhel, Spel, and Not!
  • the nucleotide sequence of the entire mouse CREBl promoter region ( Figure 8), including the human pathogenic sequence ( Figures 8 and 9), was confirmed by automated DNA sequencing.
  • the final construct will be digested with Notl and the linearized plasmid will be electroporated into mouse ES cells followed by isolation of neomycin resistant, TK-deficient ES clones that have undergone replacement of the wild-type CREBl promoter with the modified promoter by homologous recombination.

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Abstract

L'invention concerne une méthode permettant de déterminer le risque pour un humain de développer un trouble dépressif majeur ou un trouble associé et une méthode permettant de diagnostiquer un trouble dépressif majeur ou un trouble associé chez un humain. Ces deux méthodes consistent à analyser un prélèvement organique pour y détecter une variation au niveau de la séquence de l'ADN du promoteur d'un gène CREB1. L'invention concerne également des méthodes permettant d'identifier un médicament pouvant traiter le trouble dépressif majeur ou un trouble associé, un animal transgénique comprenant un gène CREB1 lié de manière fonctionnelle à une variante de promoteur CREB1, une séquence d'acide nucléique purifiée ou isolée comprenant une variante de promoteur CREB1 liée de manière fonctionnelle à un gène rapporteur, et une cellule isolée et une lignée cellulaire comprenant un gène lié de manière fonctionnelle à une variante de promoteur CREB1.
PCT/US2006/043886 2005-11-10 2006-11-10 Methodes destinees au diagnostic du trouble depressif majeur et a l'identification d'agents therapeutiques efficaces pour le traitement du trouble depressif majeur WO2007058986A2 (fr)

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US73609505P 2005-11-10 2005-11-10
US60/736,095 2005-11-10
US74915005P 2005-12-08 2005-12-08
US60/749,150 2005-12-08

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009114627A3 (fr) * 2008-03-12 2009-12-17 Ridge Diagnostics, Inc. Biomarqueurs d'inflammation pour la surveillance de troubles de dépression
US8158374B1 (en) 2006-09-05 2012-04-17 Ridge Diagnostics, Inc. Quantitative diagnostic methods using multiple parameters
US8440418B2 (en) 2008-11-18 2013-05-14 Ridge Diagnostics, Inc. Metabolic syndrome and HPA axis biomarkers for major depressive disorder

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050197312A1 (en) * 2004-03-03 2005-09-08 Kevin Fitzgerald Transcription factor RNA interference reagents and methods of use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050197312A1 (en) * 2004-03-03 2005-09-08 Kevin Fitzgerald Transcription factor RNA interference reagents and methods of use thereof

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BLENDY ET AL: "The Role of CREB in Depression and Antidepressant Treatment" BIOLOGICAL PSYCHIATRY, ELSEVIER SCIENCE, NEW YORK, NY, US, vol. 59, no. 12, 15 June 2006 (2006-06-15), pages 1144-1150, XP005512587 ISSN: 0006-3223 *
COVEN EMILY ET AL: "Cell type-specific regulation of CREB gene expression: Mutational analysis of CREB promoter activity" JOURNAL OF NEUROCHEMISTRY, vol. 71, no. 5, November 1998 (1998-11), pages 1865-1874, XP002435675 ISSN: 0022-3042 *
PAEZ-PEREDA ET AL: "New drug targets in the signaling pathways activated by antidepressants" PROGRESS IN NEURO-PSYCHOPHARMACOLOGY & BIOLOGICAL PSYCHIATRY, OXFORD, GB, vol. 29, no. 6, 8 June 2005 (2005-06-08), pages 1010-1016, XP005007795 ISSN: 0278-5846 *
SAKAI N, DUMAN RS: "Development and analysis of brain-specific and inducible CREB transgenic mice, using tetracycline regulated gene expression system." TWENTY-SECOND ANNUAL MEETING OF THE JAPAN NEUROSCIENCE SOCIETY. IN: NEUROSCIENCE RESEARCH, ELSEVIER, SHANNON, IR, vol. 35, 2 December 1999 (1999-12-02), page S134, XP002435676 ISSN: 0168-0102 *
SCHECHTER ET AL: "Innovative Approaches for the Development of Antidepressant Drugs: Current and Future Strategies" JOURNAL OF THE AMERICAN SOCIETY FOR EXPERIMENTAL NEUROTHERAPEUTICS, XX, XX, vol. 2, no. 4, October 2005 (2005-10), pages 590-611, XP005321004 ISSN: 1545-5343 *
ZUBENKO G S ET AL: "Genome-wide linkage survey for genetic loci that influence the development of depressive disorders in families with recurrent, early-onset, major depression." AMERICAN JOURNAL OF MEDICAL GENETICS. PART B, NEUROPSYCHIATRIC GENETICS : THE OFFICIAL PUBLICATION OF THE INTERNATIONAL SOCIETY OF PSYCHIATRIC GENETICS 15 NOV 2003, vol. 123, no. 1, 15 November 2003 (2003-11-15), pages 1-18, XP002435674 ISSN: 1552-4841 *
ZUBENKO G S ET AL: "Sequence variations in CREB1 cosegregate with depressive disorders in women." MOLECULAR PSYCHIATRY, vol. 8, no. 6, 2003, pages 611-618, XP002435673 ISSN: 1359-4184 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8158374B1 (en) 2006-09-05 2012-04-17 Ridge Diagnostics, Inc. Quantitative diagnostic methods using multiple parameters
US8450077B2 (en) 2006-09-05 2013-05-28 Ridge Diagnostics, Inc. Quantitative diagnostic methods using multiple parameters
WO2009114627A3 (fr) * 2008-03-12 2009-12-17 Ridge Diagnostics, Inc. Biomarqueurs d'inflammation pour la surveillance de troubles de dépression
US8440418B2 (en) 2008-11-18 2013-05-14 Ridge Diagnostics, Inc. Metabolic syndrome and HPA axis biomarkers for major depressive disorder

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