WO2003087309A2 - Polymorphismes de bdnf et association avec un trouble bipolaire - Google Patents

Polymorphismes de bdnf et association avec un trouble bipolaire Download PDF

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WO2003087309A2
WO2003087309A2 PCT/US2003/010527 US0310527W WO03087309A2 WO 2003087309 A2 WO2003087309 A2 WO 2003087309A2 US 0310527 W US0310527 W US 0310527W WO 03087309 A2 WO03087309 A2 WO 03087309A2
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nucleotide
individual
ofthe
nucleotide position
nucleic acid
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WO2003087309A3 (fr
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Pamela Sklar
Eric S. Lander
Melvin G. Mcinnis
J. Raymond Depaulo, Jr.
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Whitehead Institute For Biomedical Research
The General Hospital Corporation
Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • 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

  • the human genome is made of about 3 billion pairs of bases (adenine (A), cytosine (C), thymine (T) and guanine (G)). In one out of every 1,000 of these pairs, one individual has e.g., an A, where another individual has, e.g., a T.
  • SNPs single nucleotide polymorphisms
  • Haplotypes are ancestral segments of chromosomes that contain many single letter genetic variations inherited together as a set or a block, and they can be used to decipher the genetic differences that make some people more susceptible to disease than others. Due to the size ofthe human genome, identifying a haplotype would make finding disease genes a manageable task. Instead of searching through a giant haystack of millions of SNPs, one could search through bundles of 10,000 to 50,000 bases each. One goal of modern science is to identify specific haplotypes and determine which haplotype(s) is associated with a specific phenotype. Modern psychiatry typically subdivides mood disorders into bipolar disorders
  • Symptoms of mania include expansive, elevated or irritable mood, inflated self-esteem, grandiosity, decreased need for sleep, increased talkativeness, racing thoughts, distractibility, increased goal-directed activity, and excessive involvement in pleasurable activities with a high potential for painful consequences.
  • Depressive symptoms include depressed mood, diminished interest or pleasure in activities, insomnia or hypersomnia, psychomotor agitation or retardation, fatigue or loss of energy, feelings of worthlessness, excessive guilt, inability to concentrate or act decisively, and recurrent thoughts of death or suicide.
  • bipolar disorders include schizo affective disorder, recurrent unipolar depression and hypomania (bipolar II disorder).
  • Neuropsychiatric disorders such as schizophrenia, attention deficit disorders, schizoaffective disorders, bipolar disorders and unipolar disorders, differ from neurological disorders in that anatomical or biochemical pathologies are readily detectable for the latter but not the former.
  • drugs which have been used to treat individuals with neuropsychiatric disorders including lithium salts, valproic acid and carbamazepine, have not been predictably effective in treatment regimens across a variety of patients. Treatment regimens are further complicated by the fact that clinical diagnosis currently relies on clinical observation and subjective reports.
  • Identification ofthe anatomical or biochemical defects which result in neuropsychiatric disorders is needed in order to effectively distinguish between the disorders and to allow the design and administration of effective therapeutic agents and treatment regimens for these disorders. Identification of individual SNPs and/or haplotypes which are associated with neuropsychiatric disorders will aid in the diagnosis and treatment of these disorders.
  • the invention relates to the SNPs identified as described herem, both singly and in combination, as well as to the use of these SNPs, and other SNPs nearby in linkage disequilibrium with these SNPs, for diagnosis, prediction of clinical course and treatment response for neuropsychiatric disorders, development of new treatments for neuropsychiatric disorders based upon comparison ofthe variant and normal (reference) versions of the gene or gene product, and development of cell culture-based and animal models for research and treatment of neuropsychiatric disorders.
  • the invention further relates to novel compounds and pharmaceutical compositions for use in the diagnosis and treatment of such disorders.
  • the BDNF gene has the nucleotide sequence of SEQ ID NO: 1.
  • the invention relates to a method for predicting the likelihood that an individual will have a neuropsychiatric disorder, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at nucleotide position 11,757 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene, wherein the presence of a cytosine at position 11,757 indicates that the individual has a greater likelihood of having a neuropsychiatric disorder than an individual having a guanine at that position.
  • BDNF brain-derived neurotrophic factor
  • the neuropsychiatric disorder is bipolar disorder and/or the individual is an individual at risk for development of bipolar disorder.
  • the invention relates to a method for predicting the likelihood that an individual will have a neuropsychiatric disorder, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at nucleotide position 14,569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene, wherein the presence of a guanine at position 14,569 indicates that the individual has a greater likelihood of having a neuropsychiatric disorder than an individual having an adenine at that position.
  • BDNF brain-derived neurotrophic factor
  • the neuropsychiatric disorder is bipolar disorder and/or the individual is an individual at risk for development of bipolar disorder.
  • the invention relates to a method for predicting the likelihood that an individual will have reduced symptomology associated with a neuropsychiatric disorder, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at nucleotide position 11,757 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene, wherein the presence of a guanine at position 11,757 indicates that the individual has a greater likelihood of having reduced symptomology associated with a neuropsychiatric disorder than an individual having a cytosine at that position.
  • BDNF brain-derived neurotrophic factor
  • the neuropsychiatric disorder is bipolar disorder.
  • the invention relates to a method for predicting the likelihood that an individual will have reduced symptomology associated with a neuropsychiatric disorder, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at nucleotide position 14,569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene, wherein the presence of an adenine at position 14,569 indicates that the individual has a greater likelihood of having reduced symptomology associated with a neuropsychiatric disorder than an individual having a guanine at that position, h a particular embodiment, the neuropsychiatric disorder is bipolar disorder.
  • BDNF brain-derived neurotrophic factor
  • the invention relates to a method of diagnosing or aiding in the diagnosis of a neuropsychiatric disorder in an individual comprising obtaining a nucleic acid sample from the individual and determining the nucleotide present at two or more of nucleotide positions -633, 196, 11757 and 14569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene, wherein presence of one or more of an adenine at nucleotide position -633, a guanine at nucleotide position 196, a cytosine at nucleotide position 11757 or a guanine at nucleotide position 14569 is indicative of increased likelihood of a neuropsychiatric disorder in the individual as compared with an individual having one or more of a thymine at nucleotide position -633, an adenine at nucleotide position 196, a guanine at nucleotide position 11757 or an BDNF)
  • the neuropsychiatric disorder is bipolar disorder.
  • the nucleotide present at all four of nucleotide positions -633, 196, 11757 and 14569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene is determined.
  • the invention in another embodiment, relates to a method of diagnosing or aiding in the diagnosis of a neuropsychiatric disorder in an individual comprising obtaining a nucleic acid sample from the individual and determining the nucleotide present at two or more of nucleotide positions -633, 196, 11757 and 14569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene, wherein presence of one or more of a mymiiie at nucleotide position -633, an adenine at nucleotide position 196, a guanine at nucleotide position 11757 or an adenine at nucleotide position 14569 is indicative of decreased likelihood of a neuropsychiatric disorder in the individual as compared with an individual having one or more of an .
  • BDNF brain-derived neurotrophic factor
  • the neuropsychiatric disorder is bipolar disorder.
  • the nucleotide present at all four of nucleotide positions -633, 196, 11757 and 14569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene is determined.
  • the invention relates to a method for predicting the likelihood that an individual will have a neuropsychiatric disorder, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at two or more of nucleotide positions -633, 196, 11757 and 14569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene, wherein presence of one or more of an adenine at nucleotide position -633, a guanine at nucleotide position 196, a cytosine at nucleotide position 11757 or a guanine at nucleotide position 14569 is indicative of increased likelihood of a neuropsychiatric disorder in the individual as compared with an individual having one or more of a thymine at nucleotide position -633, an adenine at nucleotide position 196, a guanine at nucleotide position 117
  • the neuropsychiatric disorder is bipolar disorder.
  • the nucleotide present at all four of nucleotide positions -633, 196, 11757 and 14569 relative to the start codon ofthe brain-derived neurotrophic factor (BDNF) gene is determined, hi a particular embodiment, the neuropsychiatric disorder is bipolar disorder and/or the individual is an individual at risk for development of a neuropsychiatric disorder.
  • BDNF brain-derived neurotrophic factor
  • the invention in another embodiment, relates to a method for predicting the likelihood that an individual will have a neuropsychiatric disorder, or for aiding in the diagnosis of a neuropsychiatric disorder, or predicting the likelihood of having symptomology associated with a neuropsychiatric disorder, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at one or more ofthe nucleotide positions disclosed herem relative to the start codon ofthe BDNF gene.
  • the nucleotides present at one or more of these nucleotide positions are determ ed, in a particularly preferred embodiment the nucleotides present at two or more of these nucleotide positions are determined, in a more particularly preferred embodiment the nucleotides present at three or more of these nucleotide positions are determmed, in an even more particularly preferred embodiment the nucleotides present at four or more, five or more, six or more, seven or more, or eight of these nucleotide positions are determined.
  • the invention relates to a method for predicting the likelihood ofthe presence of one or more nucleotides comprising a haplotype associated with a neuropsychiatric disorder, comprising the steps of obtaining a nucleic acid sample from an individual to be assessed and determining the nucleotide present at one or more ofthe nucleotide positions disclosed herein relative to the start codon ofthe BDNF gene, hi a preferred embodiment, the nucleotides present at one or more of these nucleotide positions are determined, in a particularly preferred embodiment the nucleotides present at two or more of these nucleotide positions are determined, in a more particularly preferred embodiment the nucleotides present at three or more of these nucleotide positions are determined, in an even more particularly preferred embodiment the nucleotides present at four or more, five or more, six or more, seven or more, or eight of these nucleotide positions are determined.
  • the invention relates to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 2, wherein the nucleic acid sequence comprises a polymorphic site at nucleotide positions -1480, -633, 196, 3071, 9202, 11757, 12910 and 14569 relative to the start codon for the brain-derived neurotrophic factor (BDNF) gene and wherein the nucleotide at one or more ofthe polymorphic sites is different from a nucleotide at a corresponding position in a corresponding reference allele.
  • the nucleotide at one or more ofthe polymorphic sites is the variant nucleotide as shown in Figures 2A-2L.
  • the invention relates to a nucleic acid molecule comprising a portion ofthe nucleic acid sequence of SEQ ID NO: 2, which is at least ten nucleotides in length and which comprises at least one of the polymorphic sites selected from the group consisting of nucleotide positions -1480, -633, 196, 3071, 9202, 11757, 12910 and 14569.
  • the invention is related to an oligonucleotide microarray having immobilized thereon a plurality of probes, wherein at least one of said probes is an allele-specific oligonucleotide which hybridizes specifically to a nucleic acid molecule comprising at least 10 contiguous nucleotides ofthe nucleic acid sequence of SEQ ID NO: 2 and comprising at least one ofthe polymorphic sites at nucleotide positions -1480, -633, 196, 3071, 9202, 11757, 12910 and 14569 relative to the start codon.
  • an "allele-specific oligonucleotide” is one which hybridizes specifically to one polymorphic fonn of a nucleotide sequence and not to other fonns. Such allele-specific oligonucleotides can be used to distinguish between polymorphic forms.
  • Figures 1A through IL show the polynucleotide sequence of 29kb of genomic sequence ofthe region of chromosome 11 containing the BDNF gene (SEQ ID NO: 1).
  • Figures 2A through 2L show the location of SNPs that have been identified as described herein. 29 kb of genomic sequence in reverse complementarity relative to BDNF is shown (SEQ ID NO: 2). The box represents the start codon for the BDNF coding region. Positions of SNPs are indicated and their distance from the start codon for BDNF is assigned with regard to the 3' to 5' orientation of BDNF. The "n's" represent unknown bases and are indicated to aid in avoidance of primers which span these regions.
  • Figure 3 shows the results of genotyping of 8 BDNF SNPs in family-based samples.
  • Figure 4 shows estimated haplotype probabilities and chi-squared test of multimarker haplotypes using TRANSMIT.
  • Figure 5 is a schematic depicting the location of BDNF SNPs. Seventeen kb of genomic sequence are shown. The box represents the BDNF coding region. The arrow marks the amino terminus ofthe mature BDNF peptide. Positions of SNPs are indicated. Note that while a39 is not in the mature peptide, it is in a region highly conserved across species.
  • Figure 6 shows the association between the G allele (BDNF SNP a39) and bipolar disorder for all three samples.
  • Neurotrophic factors play a role in maintaining neurons and their differentiated phenotypes in the adult nervous system.
  • Nerve growth factor NGF
  • NGF Nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • the mature form of BDNF essentially corresponds to the C-terminal half of its precursor and comprises 119 ammo acids.
  • BDNF single nucleotide polymorphisms
  • SNPs single nucleotide polymorphisms
  • One ofthe haplotypes is transmitted significantly more often (overtransmitted) from heterozygous parents to children with bipolar disorder than would be expected by chance, while the other is transmitted less often (undertransmitted) than would be expected by chance.
  • polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymorphic marker or site is the locus at which divergence occurs.
  • a polymorphic locus may be as small as one base pair, in which case it is referred to as a single nucleotide polymorphism (SNP). Furthennore, two or more polymorphic markers can be inherited together to form a haplotype.
  • SNP single nucleotide polymorphism
  • a predictive marker is a polymorphic marker that is correlated with either an increased or reduced incidence of a phenotype (e.g., bipolar disorder).
  • an uncertain marker is a polymorphic marker which is not correlated with either an increased or reduced incidence of a phenotype.
  • One or more uncertain markers and one or more predictive markers, and various combinations thereof, can be inherited together to form a haplotype. The initial genotyping strategy used to genotype SNPs within BDNF is described in the Examples. In order to identify haplotypes with significant transmission disequilibrium to map and identify a true susceptibility allele, additional SNPs were obtained for genotyping.
  • SNPs were identified by this genotyping strategy using three test samples.
  • One test sample comprised a population of 136 trios (Hopkins sample), where the proband for 109 trios has BP1 or SAB disorder (80%) and 27 trios where the proband has bipolar II (BPII) (20%).
  • Another sample of 189 trios was obtained from the NIMH Genetics Initiative (NIMH sample), and a third sample of 145 trios (UK sample) was obtained from the UK collaboration between the University of Wales College of Medicine in Cambridge and the University of Birmingham in Biimingham. Twenty-three SNPs were chosen for genotyping based on allele frequency estimates from the resequencing.
  • Genotyping was successful for 10 (2 have a rare-minor allele frequency ⁇ 1%, 4 are monomorphic in the patient samples, and 9 failed in genotyping format).
  • the data for the 8 informative SNPs are presented in Figure 3, and their relative positions are indicated in Figure 2.
  • polymorphisms in the gene for BDNF, or polymorphisms in linkage disequilibrium with such polymorphisms form a haplotype that is correlated with incidence of neuropsychiatric disorders (e.g., bipolar disorder), hi particular, it has been discovered that one or more predictive SNPs are correlated with a reduced incidence of bipolar disorder in the sample population assessed as described herein.
  • neuropsychiatric disorders e.g., bipolar disorder
  • a predictive single nucleotide polymorphism from G to A at nucleotide position 196 (relative to the start codon ofthe BDNF gene as shown in Figure 2), or at a nucleotide position corresponding thereto, resulting in an amino acid change from valine to methiomne at amino acid position -63 (relative to the start ofthe mature protein), or at an amino acid position corresponding thereto, is correlated with a reduced incidence of bipolar disorder in the sample population assessed as described herein.
  • this polymorphism resides within the 132 amino acid precursor portion (the prepro portion) which is cleaved from the mature protein, it is in a region highly conserved across species.
  • BDNF genes and flanking nucleotide sequence can be found, for example, in primates, mice and/or dogs.
  • the corresponding positions can be identified, for example, by aligning the nucleotide or amino acid sequences of BDNF genes, as known to one of skill in the art, with SEQ ID NO: 2 and identifying the nucleotide position which is aligned with, e.g., nucleotide position 196 relative to the start codon ofthe BDNF gene.
  • a predictive single nucleotide polymorphism from T to A at nucleotide position -633 (relative to the start codon for BDNF; also referred to as a40) in Figure 2, or at a nucleotide position corresponding thereto, has been discovered.
  • Data has shown that there is a variation from random in the transmission ofthe reference (T) and variant (A) alleles from a parent who is heterozygous for the BDNF alleles to an offspring diagnosed with bipolar disorder.
  • a predictive single nucleotide polymorphism from C to G at nucleotide position 11757 (relative to the start codon for BDNF; also referred to as a20) in Figure 2, or at a nucleotide position corresponding thereto, has been discovered.
  • a predictive single nucleotide polymorphism from G to A at nucleotide position 14569 (relative to the start codon for BDNF; also referred to as al3) in Figure 2, or at a nucleotide position corresponding thereto has been discovered.
  • a SNP from G to A at nucleotide position 9202 (relative to the start codon for BDNF; also referred to as a22) in Figure 2 or at a nucleotide position corresponding thereto has been discovered
  • haplotype analysis performed as described herein reveals that 6 haplotypes, each with a probability of greater than 2%, account for 96.5% of the haplotype diversity (Figure 4).
  • Haplotype 6 is significantly overfransmitted to the bipolar probands, and haplotype 3 is undertransmitted in the Hopkins data set.
  • the invention relates to a method for predicting the likelihood that an individual will have a neuropsychiatric disorder, or for aiding in the diagnosis of a neuropsychiatric disorder, or predicting the likelihood of having reduced symptomology associated with a neuropsychiatric disorder, comprising the steps of obtaining a DNA sample from an individual to be assessed and determining the nucleotide present at one or more of nucleotide positions -633, 196, 11757 and 14569 relative to the start codon for the BDNF gene, h a preferred embodiment, the nucleotides present at one or more of these nucleotide positions are determined, in a particularly preferred embodiment the nucleotides present at two or more of these nucleotide positions are determined, in a more particularly preferred embodiment the nucleotides present at three or more of these nucleotide positions are determined, and in an even more particularly preferred embodiment the nucleotides present at four or more of these nucleotide positions are determined.
  • the BDNF gene has the nucleotide sequence of SEQ ID NO: 1.
  • the presence of one or more of a thymine (the variant nucleotide) at the predictive marker position -633, an adenine (the variant nucleotide) at the predictive marker position 196, a guanine (the variant nucleotide) at the predictive marker position 11757, or an adenine (the variant nucleotide) at the predictive marker position 14569 indicates that the individual has a reduced likelihood of having a neuropsychiatric disorder or a likelihood of having reduced symptomology associated with a neuropsychiatric disorder than if that individual had the reference nucleotide at one or more of these positions.
  • the individual is an individual at risk for development of a neuropsychiatric disorder.
  • the individual exhibits clinical symptomology associated with a neuropsychiatric disorder
  • the individual has been clinically diagnosed as having a neuropsychiatric disorder.
  • the neuropsychiatric disorder is bipolar disorder.
  • the genetic material to be assessed can be obtained from any nucleated cell from the individual.
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable.
  • convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, skin and hair.
  • tissue sample must be obtained from an organ in which the target nucleic acid is expressed.
  • cells from the central nervous system such as cells ofthe hippocampus
  • heart, brain, lung and skeletal muscle are suitable sources for obtaining cDNA for the BDNF gene.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the nucleotides which occupy the polymorphic sites of interest can be identified by a variety methods, such as Southern analysis of genomic DNA; direct mutation analysis by restriction enzyme digestion; Northern analysis of RNA; denaturing high pressure liquid chromatography (DHPLC); gene isolation and sequencing; hybridization of an allele-specific oligonucleotide with amplified gene products; single base extension (SBE); or analysis ofthe BDNF protein.
  • Southern analysis of genomic DNA e.g., genomic DNA sequence of genomic DNA
  • direct mutation analysis by restriction enzyme digestion e.g., Southern analysis of genomic DNA
  • Northern analysis of RNA e.g., Southern analysis of RNA
  • denaturing high pressure liquid chromatography (DHPLC) e.g., gene isolation and sequencing
  • hybridization of an allele-specific oligonucleotide with amplified gene products e.g., single base extension (SBE); or analysis ofthe BDNF protein.
  • SBE single base extension
  • Allele-specific probes for analyzing polymorphisms is described by e.g., Saild et al, Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the con'esponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one ofthe alleles.
  • Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • stringent conditions for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • 5X SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM
  • EDTA, pH 7.4 EDTA, pH 7.4
  • a temperature of 25-30°C, or equivalent conditions are suitable for allele-specific probe hybridizations.
  • Equivalent conditions can be determined by varying one or more ofthe parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleotide sequence and the primer or probe used.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) ofthe probe. This design of probe achieves good discrimination in hybridization between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence. 2. Tiling Arrays
  • the polymorphisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in WO 95/11995.
  • WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymorphism.
  • Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant ofthe first reference sequence.
  • the second group of probes is designed by the same principles, except that the probes exhibit complementarity to the second reference sequence.
  • a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length ofthe probes (e.g., two or more mutations within 9 to 21 bases).
  • An allele-specific primer hybridizes to a site on target DNA overlapping a polymorphism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3 '-most position ofthe oligonucleotide aligned with the polymorphism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • the direct analysis ofthe sequence of polymorphisms ofthe present invention can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al, Recombinant DNA Laboratoi ⁇ Manual, (Acad. Press, 1988)).
  • Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.
  • Alleles of target sequences can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al, Proc. Nat. Acad. Sci. 86, 2766-2770 (1989).
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to fonn single stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.
  • An alternative method for identifying and analyzing polymorphisms is based on single-base extension (SBE) of a fluorescently-labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label ofthe added base and the label ofthe primer.
  • SBE single-base extension
  • FRET fluorescence resonance energy transfer
  • the method such as that described by Chen et al, (PNAS 94:10 '56-61 (1997), incorporated herein by reference) uses a locus- specific oligonucleotide primer labeled on the 5' tenninus with 5-carboxyfluorescein (FAM). This labeled primer is designed so that the 3' end is immediately ad acent to the polymorphic site of interest.
  • FAM 5-carboxyfluorescein
  • the labeled primer is hybridized to the locus, and single base extension ofthe labeled primer is performed with fluorescently labeled dideoxyribonucleotides (ddNTPs) in dye-tenninator sequencing fashion, except that no deoxyribonucleotides are present.
  • ddNTPs dideoxyribonucleotides
  • An increase in fluorescence ofthe added ddNTP in response to excitation at the wavelength of the labeled primer is used to infer the identity ofthe added nucleotide.
  • More than one phenotypic trait may be affected by the SNPs described herein.
  • other neuropsychiatric disorders which are believed to be alternate expressions of a bipolar disorder, including schizoaffective disorder, recurrent unipolar depression and hypomania (bipolar II disorder)
  • schizoaffective disorder including schizoaffective disorder, recurrent unipolar depression and hypomania (bipolar II disorder)
  • BDNF polymorphisms described herein may also be affected by the BDNF polymorphisms described herein.
  • the described polymorphisms may predispose an individual to a distinct mutation that is causally related to a certain phenotype, such as susceptibility or resistance to bipolar disorder.
  • the discovery of these polymorphisms and their correlation with bipolar disorder facilitates biochemical analysis ofthe variants and the development of assays to characterize the variants and to screen for pharmaceuticals that interact directly with one or another form ofthe protein.
  • a polymorphism may be one of a group of two or more polymorphisms in the BDNF gene, or in linkage disequilibrium with such polymorphisms, that form a haplotype which contributes to the presence, absence or severity ofthe neuropsychiatric disorder, e.g., bipolar disorder.
  • An assessment of other polymorphisms within the BDNF gene, or in linkage disequilibrium with such polymorphisms, can be undertaken, and the separate and combined effects of these polymorphisms on the neuropsychiatric disorder phenotype can be assessed.
  • Correlation between a particular phenotype, e.g., the bipolar phenotype, and the presence or absence of a particular allele is perfonned for a population of individuals who have been tested for the presence or absence ofthe phenotype. Correlation can be performed by standard statistical methods such as a Chi-squared test and statistically significant correlations between polymorphic form(s) and phenotypic characteristics are noted. For example, as described herein, it has been found that the presence ofthe BDNF variant allele having an A at polymorphic site 196, correlates negatively with bipolar disorder with a p-value of 0.0416.
  • This correlation can be exploited in several ways, h the case of a strong correlation, detection ofthe polymorphic form in an individual may justify immediate administration of treatment, or at least the institution of regular monitoring of the individual. Detection of a polymorphic form con-elated with a disorder in a couple contemplating a family may also be valuable to the couple in their reproductive decisions. For example, the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymorphism from her husband to her offspring, hi the case of a weaker, but still statistically significant correlation between a polymorphic form and a particular disorder, immediate therapeutic intervention or monitoring may not be justified.
  • the individual can be motivated to begin simple life-style changes (e.g., therapy or counseling) that can be accomplished at little cost to the individual but confer potential benefits in reducing the risk of conditions to which the individual may have increased susceptibility by virtue ofthe particular allele.
  • identification of a polymorphic form correlated with enhanced receptiveness to one of several treatment regimes for a disorder indicates that this treatment regime should be followed for the individual in question.
  • a physical linkage between a genetic locus associated with a trait of interest e.g., bipolar disorder
  • polymorphic markers that are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co-segregate with it.
  • Such analysis is useful for mapphig a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al, Proc. Natl. Acad. Sci. (USA) 83, 7353-7357 (1986); Lander et al, Proc. Natl. Acad. Sci.
  • Linkage studies are typically performed on members of a family. Available members ofthe family are characterized for the presence or absence of a phenotypic trait and for a set of polymorphic markers. The distribution of polymorphic markers in an informative meiosis is then analyzed to detennine which polymorphic markers co-segregate with a phenotypic trait. See, e.g., Kerem et al, Science 245, 1073-1080 (1989); Monaco et al, Nature 316, 842 (1985); Yamoka et al, Neurology 40, 222- 226 (1990); Rossiter et al, FASEB Journal 5, 21-27 (1991).
  • LOD log ofthe odds
  • a lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction ⁇ , versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome” in Ebe Human Genome (BIOS Scientific Publishers Ltd, Oxford), Chapter 4).
  • the likelihood at a given value of ⁇ is: probability of data if loci linked at ⁇ to probability of data if loci unlinked.
  • the computed likelihoods are usually expressed as the log 10 of this ratio (i.e., a lod score). For example, a lod score of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence.
  • the use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of ⁇ (e.g., LIPED, MLINK (Lathrop, Proc. Nat. Acad. Sci. (USA) 81, 3443-3446 (1984)).
  • a recombination fraction may be determined from mathematical tables. See Smith et al, Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith, Ann. Hum. Genet. 32, 127-150 (1968). The value of ⁇ at which the lod score is the highest is considered to be the best estimate ofthe recombination fraction. Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of ⁇ ) than the possibility that the two loci are unlinked. By convention, a combined lod score of +3 or greater (equivalent to greater than 1000:1 odds in favor of linkage) is considered definitive evidence that two loci are linked.
  • a variant gene product is intended to mean gene products which are encoded by the variant allele of a gene and includes, but is not limited to, a full-length, or substantially full length, variant gene product, or biologically active fragments ofthe gene product, or analogs thereof.
  • Variant gene products comprise at least one variant nucleotide at a polymorphic site disclosed herein.
  • Bioly active fragments include any portion ofthe encoded full-length polypeptide which confers a biological function on the variant gene product, including ligand binding and antibody binding.
  • Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
  • a variant gene product is also intended to mean gene products which have altered expression levels or expression patterns which are caused, for example, by the variant allele of a regulatory sequence(s).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • a variant polypeptide or protein, or fragment thereof, ofthe present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition.
  • a variant polypeptide or protein is one which is encoded by a variant gene product and preferable comprises a variant amino acid as described herein.
  • the particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the optimum concentration ofthe active ingredient(s) in the chosen medium can be detennined empirically, according to procedures well known to medicinal chemists, and will depend on the ultimate pha ⁇ naceutical formulation desired.
  • Methods of introduction of exogenous peptides at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents and treatment regimens.
  • Polyclonal and/or monoclonal antibodies that specifically bind to variant gene products (e.g., protein or polypeptide) but not to corresponding reference gene products are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof comprising the variant portion.
  • Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratoiy Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986).
  • Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product.
  • compositions comprising a nucleotide sequence encoding a variant gene product.
  • variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter.
  • the promoter is a eukaryotic promoter for expression in a mammalian cell.
  • the transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host.
  • trp for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters
  • trp trp
  • lac phage promoters
  • glycolytic enzyme promoters glycolytic enzyme promoters
  • tRNA promoters tRNA promoters
  • Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.
  • the means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra.
  • a wide variety of host cells can be employed for expression ofthe variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g. , mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like.
  • cells can be engineered to express a variant allele ofthe invention by gene therapy methods.
  • DNA encoding a variant gene product, or an active fragment or derivative thereof can be introduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal.
  • the cell population can be engineered to inducibly or constitutively express active variant gene product, hi a preferred embodiment, the vector is delivered to the bone marrow, for example as described in Corey et al (Science 244:1215-12%! (1989)).
  • the invention further relates to the use of compositions (i.e., agonists) which enliance or increase the level or activity of a variant gene product, or a functional portion thereof, or compositions which mimic the activity of a variant gene product, for use in the treatment of a phenotype.
  • compositions i.e., antagonists
  • the invention also relates to the use of compositions (i.e., antagonists) which reduce or decrease the level or activity of a varient gene product, or a functional portion thereof, for use in the treatment of a phenotype.
  • the invention also relates to constructs which comprise a vector into which a sequence ofthe invention has been inserted in a sense or antisense orientation.
  • a vector comprising a nucleotide sequence which is antisense to a variant allele may be used as an antagonist ofthe activity of a reference allele.
  • a vector comprising a nucleotide sequence of a variant allele may be used therapeutically to treat a phenotype.
  • the term "vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • vectors Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors, expression vectors are capable of directing the expression of genes to which they are operably linked, hi general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors).
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retro viruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
  • Preferred recombinant expression vectors ofthe invention comprise a nucleic acid ofthe invention in a form suitable for expression ofthe nucleic acid in a host cell.
  • the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • the expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.
  • the recombinant expression vectors ofthe invention can be designed for expression of a polypeptide ofthe invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian, e.g., human, cells. Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a nucleic acid ofthe invention can be expressed in bacterial cells (e.g., E. coli), insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transfonning or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • a host cell ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide ofthe invention.
  • the invention further provides methods for producing a polypeptide using the host cells ofthe invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a polypeptide ofthe invention has been introduced) in a suitable medium such that the polypeptide is produced, hi another embodiment, the method further comprises isolating the polypeptide from the medium or the host cell.
  • the host cells ofthe invention can also be used to produce nonhuman transgenic animals.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid ofthe invention have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into their genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered.
  • Such animals are useful for studying the function and/or activity ofthe nucleotide sequence and for identifying and/or evaluating modulators of their activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes the exogenous nucleotide sequence.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome ofthe mature animal, thereby directing the expression of a gene product in one or more cell types or tissues ofthe transgenic animal.
  • an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.
  • a transgenic animal ofthe invention can be created by introducing a nucleic acid ofthe invention into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the sequence can be mtroduced as a fransgene into the genome of a non-human animal. If the exogenous nucleotide sequence encodes a protein, intronic sequences and polyadenylation signals can also be included in the fransgene to increase the efficiency of expression ofthe fransgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the fransgene to direct expression of a polypeptide in particular cells.
  • transgenic founder animal can be identified based upon the presence ofthe exogenous nucleotide sequence in its genome and/or expression of mRNA in tissues or cells ofthe animals. A transgenic founder animal can then be used to breed additional animals carrying the fransgene. Moreover, transgenic animals carrying a fransgene comprising the exogenous nucleotide sequence can further be bred to other transgenic animals carrying other transgenes.
  • the invention also relates to the use ofthe variant and reference SNPs to guide efforts to identify the causative mutation for a phenotype or to identify or synthesize agents useful in the treatment of a phenotype.
  • amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al, Science, 244:1081-1085 (1989)). The latter procedure introduces single alariine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro, or in vitro proliferative activity.
  • Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol, 224:899-904 (1992); de Vos et al. Science, 255:306-312 (1992)).
  • Another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the activity or function of SNPs ofthe invention in clinical trials.
  • An exemplary method for detecting the presence or absence of proteins or nucleic acids ofthe invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the protein, or nucleic acid (e.g., mRNA, genomic DNA), such that the presence ofthe protein or nucleic acid is detected in the biological sample.
  • a preferred agent for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein, preferably in an allele-specific manner.
  • the nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
  • suitable probes for use in the diagnostic assays ofthe invention are described herein.
  • Other agents for use in the diagnostic assays ofthe invention are antibodies described herein. These antibodies can be used to detect the presence or absence ofthe reference or variant gene product.
  • the invention also encompasses kits for detecting the presence of proteins or nucleic acid molecules ofthe invention in a biological sample.
  • the kit can comprise a labeled compound or agent (e.g., nucleic acid molecule, antibody, etc.) capable of detecting protein, DNA or mRNA in a biological sample; means for determining the amount of protein, DNA or mRNA in the sample; and means for comparing the amount of in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect protein or nucleic acid, hi a preferred embodiment the labeled compound or agent detects either the alternate or reference form ofthe protein, DNA or mRNA, but not both.
  • the invention further relates to an oligonucleotide micro array having immobilized thereon a plurality of oligonucleotide probes.
  • the microarray can contain one or more probes for the polymo ⁇ hic sites listed in Figure 2.
  • the preparation of such oligonucleotide microarrays is well known in the art.
  • the oligonucleotide probes hybridize only to one polymorphic form ofthe gene products disclosed herein.
  • labeled nucleic acid e.g., mRNA, genomic DNA
  • Genomic DNA and mRNA isolation from cells in culture is known in the art. (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, F.M., et al, Current Protocols in Molecular Biology, Greene Publishing Assoc.
  • the nucleic acid is then labeled by standard methods, (see above references).
  • the nucleic acid can be labeled with any appropriate molecule, for example fluorophores, biotin, radioactive nucleotide, and dye. h a preferred embodiment, the nucleic acid is fluorescently labeled.
  • the DNA microarray can be any high-density oligonucleotide microarray, for example, GeneChipTM HU 6800 (Affymetrix, Santa Clara, CA).
  • Hybridization of labeled mRNA to the DNA microarray is known to those of skill in the art (see Tamayo et al, 1999. Proc. Natl. Acad. Sci, USA, 96:2907-2912; Eisen et al, 1999. Methods Enzymol, 303:179-205). Quantitation of gene expression profiles from the hybridization of labeled mRNA/DNA microarray is performed by scanning the microarrays to measure the amount of hybridization at each position on the microarray with an Affymetrix scanner (Affymetrix, Santa Clara, CA ).
  • Ci and Mi are defined as relative steady-state mRNA levels, where i refers to the ith timepoint and n to the total number of timepoints ofthe entire timecourse.
  • ⁇ M and ⁇ M are defined as the mean and standard deviation ofthe control time course, respectively.
  • labeled RNA can be hybridized to a filter or other solid support containing target nucleic acids that comprise the polymorphic sites disclosed herein.
  • Hybridization and wash conditions should be stringent enough to ensure specific binding between labeled RNA and target sequences. Stringent hybridization and wash conditions are known in the art (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Ausubel, F.M., et al, Current Protocols in Molecular Biology, Greene Publishing Assoc. and
  • Quantitation of specific hybridization can be performed by any suitable method including scintillation counting and densitometry.
  • Pedigrees were ascertained for a sample population of 136 trios (Hopkins samples) from inpatient and outpatient clinics in Maryland and Iowa. Most ofthe families have been previously described. The ascertainment criteria were: a treated bipolar I proband, at least 2 affected first-degree relatives and unilineal transmission. Probands had the following diagnoses: 106 BP1, 26 BP2 and 4 schizoaffective-manic (SA-M). All BP2 and SAM probands had a BP1 sibling and both siblings were used in the analyses.
  • SA-M schizoaffective-manic
  • SNPs in 76 candidate genes in the Hopkins sample of 136 parent-proband trios were genotyped.
  • the choice of SNPs for genotyping was governed by the following rationale. It was reasoned that missense SNPs, because they result in amino acid changes, are those that are most likely to affect function, and thus missense SNPs were focused on when available. If these SNPs are the causal mutations, then genotyping will directly identify an association with the underlying disease or phenotype. However, because blocks ofthe genome are consistently inherited together in a population (linkage disequilibrium), nearby linked SNPs can also reveal an association to the underlying causative SNP.
  • V66M brain-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • SNPs were identified from genomic DNA as previously described. 34 For those genes where genomic sequence was not available, the gene was amplified from RNA in an ethnically diverse panel of lymphoblastoid cell lines obtained from the Coriell cell repository. Approximately 50% of genes specific to the neuropsychiatry project could not be amplified from this source of RNA. To amplify those genes, brain RNA was obtained from the Stanley Foundation. Poly A+ RNA was purified using the Oligotex Direct mRNA kit (Qiagen, Valencia, CA).
  • Complementary DNA was transcribed from the polyA+ RNA using 200 ng RNA, random hexamers (750 ng) and SuperScript ⁇ RT (Life Technologies, Carlsbad, CA) in a reaction volume of 100 ⁇ l containing 5X Superscript buffer, 50 nmol dNTPs, and 1 ⁇ mol DTT. Each gene was divided into several primary transcripts of 1 kilobase. The primary transcripts were amplified in a volume of 10 ⁇ l. Each reaction contained 10X PCR bufferll, 30 nmol MgCl 2 , 2 nmol dNTPs, 1U AmpliTaq Gold (Perkin-Elmer, Boston, MA), and 2.5 mnol transcript specific primers.
  • PCR conditions were as follows: 96°C x 10 min followed by 35 cycles of 96 °C x 30 sec, 59 °C x 30 sec and 72 °C x 1 min.
  • Each primary transcript was subdivided into overlapping 500 base pair fragments that were separately amplified in a volume of 18 ⁇ l containing 10X PCR buffer II, 41.25 nmol MgCl 2 , 3 mnol dNTP's, 1.5U AmpliTaq Gold and 3.5 nmol fragment specific primers and amplified using the same PCR conditions.
  • PCR products were prepared for sequencing using solid-phase reversible immobilization (SPRI) using Bangs Estapor SuperParamagnetic Microspheres (Bangs Laboratories, Inc., IN and Seradyn Uniform Microparticles, MN) as described, (see DeAngelis M.M. et al, Solid- Phase Reversible Immobilization for the Isolation of PCR Products. Nucleic Acids Res 23: 4742-4743 (1995)). Sequencing was performed using BigDye Terminator Chemistry (Perkin-Elmer, Boston, MA) on a capillary ABI 3700.
  • SNP detection was as described previously (see Cargill M., et al, Characterization of Single- Nucleotide Polymorphisms in Coding Regions of Human Genes. Nat Genet 22: 231- 238 (1999)). Each fragment was sequenced in 32 individual D ⁇ A samples.
  • 29kb of contiguous genomic sequence was resequenced from genomic D ⁇ A as described, (see Cargikk M. et al, Characterization of Single- ⁇ ucleotide Polymorphisms in Coding Regions of Human Genes. Nat Genet 22: 231-238 (1999)). Sequence was obtained from the Human Genome Browser from human BAC AC068488.2_13. Six patients with BP1 and two control D ⁇ As obtained from the Coriell Cell Repository were resequenced. The bipolar patients used for resequencing were not included in the association analyses. Location of S ⁇ Ps are marked with respect to the ATG of BD ⁇ F.
  • Genotyping was performed by either single-base extension with fluorescent resonance energy transfer (SBE-FRET) (see Chen X. et al, Fluorescence Energy Transfer Detection as a Homogenous D ⁇ A Diagnostic Method. Pro c Natl Acad Sci USA 94: 10756-10761 (1997)), single-base extension with fluorescence polarization (SBE-FP) (see Chen X. and Kwok P.Y. Template-Directed Dye-Tenninator Incorporation (TDI) Assau: A Homogoneous D ⁇ A Diagnostic Method Based on Fluorescence Resonance Energy Transfer. Nucleic Acids Res 25 : 347-353 (1997)) using a modified protocol as described previously (Altshuler D. et al.
  • SBE-FRET fluorescent resonance energy transfer
  • SBE-FP single-base extension with fluorescence polarization
  • TDI Template-Directed Dye-Tenninator Incorporation
  • the PCR reaction contained AmpliTAQ Gold (4U, Perkin Ehner), dNTPs (0.5 mM), MgCl 2 (4 mM), genomic DNA (5 ng), specific locus primer mix (0.1 ⁇ M final concentration of each primer), biotinylated-T3 and biotinylated-T7 (0.125 ⁇ M) in the supplied buffer in a final volume of 20 ⁇ l using the following PCR conditions (95°C x 9 min, 37 cycles of 94°C x 30 sec, 55°C x 30 sec, and 72°C x 30 sec followed by a final extention of 72°C x 5min).
  • Primers used for genotyping can be found on the world- wide web at genome-wi.mit.edu. Genotyping of SNPs in BDNF was performed by mass spectrometry as follows. Primers were designed using SpectroDESIGNER software (Sequenom, CA) to have a Tm between 56 and 60 degrees with a mass range between 5000 and 8000 Da as described, (see Buetow K.H. et al, High-Throughput Development and Characterization of a Genomewide Collection of Gene-Based Single Nucleotide Polymo ⁇ hism Markers By Chip-Based Matrix- Assisted Laser Deso ⁇ tion/Ionization Time-of-Flight Mass Spectrometry.
  • SpectroDESIGNER software Sequenom, CA
  • PCR amplification was performed as follows. Each reaction contained AmpliTAQ Gold (0.1U, Perldn Elmer), dNTPs (0.2 mM), MgCl 2 (1.5 mM), genomic DNA (5 ng), locus specific primers (0.2 ⁇ M final concentration of each primer), in the supplied buffer in a final volume of 6 ⁇ l using the following PCR conditions (92°C x 9 min, 46 cycles of 94°C x 20 sec, 56°C x 30 sec, and 72°C x 30 sec followed by a final extention of 72°C x 3 min).
  • dNTPs are removed by shrimp alkaline phosphatase (SAP) by adding 2 ⁇ l of SAP (0.3U) in Thennosequenase buffer and incubating at 37°C x 20 min, followed by inactivation at 85°C X 5 min.
  • SAP shrimp alkaline phosphatase
  • the homogeneous MassEXTEND reaction is performed by adding to the SAP -treated product 2 ⁇ l of a solution containing ddNTPs (0.50 ⁇ M each), dNTPs (0.50 ⁇ M each), MassEXTEND primers (0.6 nM), Thennosequenase buffer (Phannacia, Peapack, NJ), and Thennosequenase (0.063U/ ⁇ l).
  • the termination mix of ddNTPs and dNTPs is predicted by the SpectroDESIGNER software and is specific for each SNP genotyped.
  • the reaction is thermocycled under the following conditions: 94°C x 2min, 40 cycles of 94°C x 5 sec, 40°C x 5 sec, 72°C x 5 sec, then 72°C x 5 min.
  • SpectroCLEAN a proprietary ion-exchange resin, is added to remove salt.
  • the sample plate is rotated for 4 min at RT and then centrifuged for 1 min at 1400 ⁇ m.
  • SpectroCHIPs were analyzed in automated mode by a MassARRAY RT mass spectrometer (Bruker-Sequenom) (see Buetow K.H. et al. High-Throughput Development and Characterization of a Genomewide Collection of Gene-Based Single Nucleotide Polymo ⁇ hism Markers By Chip-Based Matrix- Assisted Laser Deso ⁇ tion/Ionization Time-of-Flight Mass Spectrometry.
  • SPECTROTYPER software Sequenom
  • DNA samples for which the SPECTROTYPER software could not define a genotype were subjected to clustering analysis by plotting the signal to noise ratio ofthe genotype-known and genotype-undetermined samples for each SNP. If the undetermined genotypes fell within clusters of known genotypes, genotypes were then obtained. The minimum acceptable signal to noise ratio was 5:1. Clusters were verified by two independent observers.
  • Genotyping data was assessed in the following manner. SNPs were used for TDT analysis only if they met the following criteria: 1) greater than 90% of attempted genotypes were successful, 2) parental alleles were in Hardy- Weinberg equilibrium, and 3) zero or one Mendelian inheritance eiror was detected. Several methods of genotyping were used for this study. For those SNPs genotyped by more ' than one method (23%), a consensus genotype was obtained and used for TDT analysis. Significance of single and two-marker haplotypes were analyzed using the Perml and Penn2 options in Genehunter2.0. Global analysis of multimarker haplotypes was performed using TRANSMIT v2.5.2 (see Clayton D. A
  • haplotype analysis was performed using TRANSMIT v2.5.2 and results are shown in Figure 4.
  • the program estimates the association from probabilities of a haplotype transmission to affected offspring even when there are uncertain marker haplotype assignments, hi the Hopkins dataset, 13 haplotypes were observed, of these only 6 were present with probabilities greater than 2% and these accounted for the vast majority ofthe haplotype diversity (96.5%).
  • haplotypes There are three common major haplotypes, 3, 5 and 6 with rarer haplotypes 2 and 4 differing by only a single marker from 3 and 5 respectively. Of these, haplotype 6 is significantly overtransmitted to the bipolar probands and haplotype 3 is undertransmitted.
  • haplotype 3 shows some excess transmission and haplotype 3 is undertransmitted.
  • BDNF has limited haplotype diversity in both samples which is nearly fully characterized by the SNPs genotyped.
  • a single undertransmitted haplotype is shared by these samples characterized by alleles of SNPs a39 and a20 that may mark a protective haplotype for bipolar disorder.
  • Probands from the NIMH sample, had the following diagnoses: 149 BPl, 1 BP2 and 5 SA-M. Diagnoses of BPl and SAM were established using DSMIH-R criteria and BP2 by the Research Diagnostic Criteria (RDC).
  • RDC Research Diagnostic Criteria

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Abstract

La présente invention concerne des procédés pour diagnostiquer et traiter des troubles neuropsychiatriques, notamment des troubles bipolaires, et des procédés pour identifier des composés utilisés pour diagnostiquer et traiter des troubles neuropsychiatriques. La présente invention concerne également de nouveaux composés et des compositions pharmaceutiques utilisés pour diagnostiquer et traiter des troubles neuropsychiatriques tels que des troubles bipolaires.
PCT/US2003/010527 2002-04-08 2003-04-07 Polymorphismes de bdnf et association avec un trouble bipolaire WO2003087309A2 (fr)

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AU2003223486A AU2003223486A1 (en) 2002-04-08 2003-04-07 Bdnf polymorphisms and association with bipolar disorder

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US37126002P 2002-04-08 2002-04-08
US60/371,260 2002-04-08

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474796A (en) * 1991-09-04 1995-12-12 Protogene Laboratories, Inc. Method and apparatus for conducting an array of chemical reactions on a support surface

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474796A (en) * 1991-09-04 1995-12-12 Protogene Laboratories, Inc. Method and apparatus for conducting an array of chemical reactions on a support surface

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VENTRIGLIA, M. ET AL.: 'Association between the BDNF 196 A/G polymorphism and sporadic Alzheimer's disease' MOLECULAR PSYCHIATRY vol. 7, 2002, pages 136 - 137, XP002977279 *

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WO2003087309A3 (fr) 2004-07-08
AU2003223486A1 (en) 2003-10-27
AU2003223486A8 (en) 2003-10-27

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