WO2002022881A1 - Polymorphisme du promoteur de l'endotheline-1 - Google Patents

Polymorphisme du promoteur de l'endotheline-1 Download PDF

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WO2002022881A1
WO2002022881A1 PCT/US2001/028834 US0128834W WO0222881A1 WO 2002022881 A1 WO2002022881 A1 WO 2002022881A1 US 0128834 W US0128834 W US 0128834W WO 0222881 A1 WO0222881 A1 WO 0222881A1
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hypertension
diabetes mellitus
dependent diabetes
disease
insulin dependent
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PCT/US2001/028834
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David W. Moskowitz
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Dzgenes, Llc
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Priority to AU2001294559A priority Critical patent/AU2001294559A1/en
Priority to US10/380,024 priority patent/US20040191774A1/en
Publication of WO2002022881A1 publication Critical patent/WO2002022881A1/fr

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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
    • 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
    • C07K14/575—Hormones
    • C07K14/57536—Endothelin, vasoactive intestinal contractor [VIC]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to detection of individuals at risk for pathological conditions based on the presence of single nucleotide polymorphisms (SNPs) at positions 2239 and
  • Polymorphisms can be created when DNA sequences are either inserted or deleted from the genome, for example, by viral insertion.
  • Another source of sequence variation can be caused by the presence of repeated sequences in the genome variously termed short tandem repeats (STR), variable number tandem repeats (VNTR), short sequence repeats (SSR) or microsatellites. These repeats can be dinucleotide, trinucleotide, tetranucleotide or pentanucleotide repeats.
  • STR short tandem repeats
  • VNTR variable number tandem repeats
  • SSR short sequence repeats
  • Polymorphism results from variation in the number of repeated sequences found at a particular locus.
  • SNPs single nucleotide polymorphism
  • SNPs account for approximately 90% of human DNA polymorphism (Collins et al., Genome Res., 8:1229-1231, 1998). SNPs are single base pair positions in genomic DNA at which different sequence alternatives (alleles) exist in a population. In addition, the least frequent allele must occur at a frequency of 1% or greater.
  • single nucleotide polymorphism or “SNP” includes all single base variants and so includes nucleotide insertions and deletions in addition to single nucleotide substitutions (e.g. A->G). Nucleotide substitutions are of two types. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine for a pyrimidine or vice versa.
  • the typical frequency at which SNPs are observed is about 1 per 1000 base pairs (Li and Sadler, Genetics, 129:513-523, 1991; Wang et al., Science, 280:1077-1082, 1998; Harding et aX., Am. J. Human Genet, 60:772-789, 1997; Taillon-Miller et al., Genome Res., 8:748-754, 1998).
  • the frequency of SNPs varies with the type and location of the change. In base substitutions, two-thirds of the substitutions involve the C ⁇ ->T (G ⁇ ->A) type. This variation in frequency is thought to be related to 5-methylcytosine deamination reactions that occur frequently, particularly at CpG dinucleotides.
  • SNPs occur at a much higher frequency in non-coding regions than they do in coding regions.
  • SNPs can be associated with disease conditions in humans or animals.
  • the association can be direct, as in the case of genetic diseases where the alteration in the genetic code caused by the SNP directly results in the disease condition. Examples of diseases in which single nucleotide polymorphisms result in disease conditions are sickle cell anemia and cystic fibrosis.
  • the association can also be indirect, where the SNP does not directly cause the disease but alters the physiological environment such that there is an increased likelihood that the patient will develop the disease.
  • SNPs can also be associated with disease conditions, but play no direct or indirect role in causing the disease.
  • the SNP is located close to the defective gene, usually within 5 centimorgans, such that there is a strong association between the presence of the SNP and the disease state. Because of the high frequency of SNPs within the genome, there is a greater probability that a SNP will be linked to a genetic locus of interest than other types of genetic markers.
  • SNPs Disease associated SNPs can occur in coding and non-coding regions of the genome. When located in a coding region, the presence of the SNP can result in the production of a protein that is non-functional or has decreased function. More frequently, SNPs occur in non-coding regions. If the SNP occurs in a regulatory region, it may affect expression of the protein. For example, the presence of a SNP in a promoter region, may cause decreased expression of a protein. If the protein is involved in protecting the body against development of a pathological condition, this decreased expression can make the individual more susceptible to the condition. Numerous methods exist for the detection of SNPs within a nucleotide sequence.
  • SNPs can be detected by restriction fragment length polymorphism (RFLP) (U.S. Patent Nos. 5,324,631; 5,645,995). RFLP analysis of the SNPs, however, is limited to cases where the SNP either creates or destroys a restriction enzyme cleavage site. SNPs can also be detected by direct sequencing of the nucleotide sequence of interest. Numerous assays based on hybridization have also been developed to detect SNPs. In addition, mismatch distinction by polymerases and ligases has also been used to detect SNPs.
  • RFLP restriction fragment length polymorphism
  • SNPs can provide a powerful tool for the detection of individuals whose genetic make-up alters their susceptibility to certain diseases. There are four primary reasons why SNPs are especially suited for the identification of genotypes which predispose an individual to develop a disease condition.
  • SNPs are by far the most prevalent type of polymorphism present in the genome and so are likely to be present in or near any locus of interest.
  • SNPs located in genes can be expected to directly affect protein structure or expression levels and so may serve not only as markers but as candidates for gene therapy treatments to cure or prevent a disease.
  • SNPs show greater genetic stability than repeated sequences and so are less likely to undergo changes which would complicate diagnosis.
  • the increasing efficiency of methods of detection of SNPs make them especially suitable for high throughput typing systems necessary to screen large populations.
  • SNPs single nucleotide polymorphisms
  • HTN end stage renal disease due to hypertension
  • NIDDM non-insulin dependent diabetes mellitus
  • ESRD due to NIDDM end stage renal disease due to non-insulin dependent diabetes mellitus
  • lung cancer breast cancer, prostate cancer, colon cancer
  • atherosclerotic peripheral vascular disease due to hypertension ASPND due to HT ⁇
  • cerebrovascular accident due to hypertension CNA due to HT ⁇
  • cataracts due to hypertension cataracts due to HT ⁇
  • cardiomyopathy with hypertension HT ⁇ CM
  • MI myocardial infarction due to hypertension
  • MI atherosclerotic peripheral vascular disease due to non-insulin dependent diabetes mellitus
  • CNA due to NIDDM cerebrovascular accident due to non-insulin dependent diabetes mellitus
  • Ischemic CM ischemic cardiomyopathy with non-insulin dependent diabetes mellitus
  • one aspect of the present invention provides a method for diagnosing a genetic predisposition for HTN, ESRD due to HTN, NIDDM, ESRD due to NTDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPND due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HT ⁇ , HT ⁇ CM, MI due to HT ⁇ , ASPND due to ⁇ IDDM, CNA due to ⁇ IDDM, Ischemic CM, Ischemic CM with ⁇ IDDM, MI due to ⁇ IDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to IDDM, or seizure disorder in a subject, comprising obtaining a sample containing at least one polymicleotide from the subject, and analyzing the polynucleotide to detect a genetic polymorphis
  • the polymorphism is located in the EDN-1 gene.
  • Another aspect of the present invention provides an isolated nucleic acid sequence comprising at least 10 contiguous nucleotides from SEQ ID NO: 1, or their complements, wherein the sequence contains at least one polymorphic site associated with a disease and in particular HTN, ESRD due to HTN, NIDDM, ESRD due to NTDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPND due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HTN, HTN CM, MI due to HTN, ASPVD due to NTDDM, CNA due to ⁇ IDDM, Ischemic CM, Ischemic CM with ⁇ IDDM, MI due to ⁇ IDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to TDDM,
  • kits for the detection of a polymorphism comprising, at a minimum, at least one polynucleotide of at least 10 contiguous nucleotides of SEQ D NO: 1, or their complements, wherein the polynucleotide contains at least one polymorphic site associated with HTN, ESRD due to HTN, NTDDM, ESRD due to NTDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPND due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HT ⁇ , HT ⁇ CM, MI due to HT ⁇ , ASPND due to
  • ⁇ IDDM CNA due to ⁇ IDDM
  • Ischemic CM Ischemic CM with ⁇ TDDM
  • MI due to ⁇ TDDM
  • afib without valvular disease alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots
  • ESRD due to FSGS ESRD due to IDDM, or seizure disorder.
  • Yet another aspect of the invention provides a method for treating HT ⁇ , ESRD due to HT ⁇ , NTDDM, ESRD due to NIDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPND due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HT ⁇ , HT ⁇ CM, MI due to HT ⁇ , ASPND due to ⁇ TDDM, CNA due to ⁇ TDDM, Ischemic CM, Ischemic CM with ⁇ IDDM, MI due to ⁇ IDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to IDDM, or seizure disorder comprising, obtaining a sample of biological material containing at least one polynucleotide from the subject; analyzing the polynucleotide to detect the presence of at least one polymorphism associated with HT ⁇ ,
  • CM MI due to HT ⁇
  • ASPND due to ⁇ IDDM
  • CNA due to ⁇ IDDM
  • Ischemic CM Ischemic CM with ⁇ IDDM
  • MI due to ⁇ IDDM
  • afib without valvular disease alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots
  • ESRD due to FSGS ESRD due to TDDM, or seizure disorder
  • Still another aspect of the invention provides a method for the prophylactic treatment of a subject with a genetic predisposition to HT ⁇ , ESRD due to HT ⁇ , ⁇ TDDM, ESRD due to ⁇ IDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPVD due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HT ⁇ , HT ⁇ CM, MI due to HT ⁇ , ASPND due to NTDDM, CNA due to ⁇ TDDM, Ischemic CM, Ischemic CM with ⁇ TDDM, MI due to ⁇ IDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to TDDM, or seizure disorder comprising, obtaining a sample of biological material containing at least one polynucleotide from the subject; analyzing the polynucleo
  • Figure 1 shows SEQ X ⁇ D NO: 1, the nucleotide sequence of the EDN-1 promoter region as contained in GenBank Accession Number J05008.1. Position of the single nucleotide polymorphisms (SNPs) are here given according to the numbering scheme in GenBank Accession Number J05008.1. Thus, all nucleotides will be positively numbered, rather than bear negative numbers reflecting their position upstream from the RNA polymerase II binding site (a TATA box in about half of eukaryotic genes), the transcription initiation site (a variable number of nucleotides downstream of, i.e. 3' to, the
  • the translation start site or the first codon of the encoded protein (the "A” of the "ATG” codon for methionine, the first amino acid of every protein). Since not all genes are fully annotated, and not all promoter sequences contain elements far downstream such as the "ATG” encoding the first methionine in the translated protein, the numbering system used in this patent application is less troublesome.
  • the TATA box is located at position 3577.
  • the first exon begins at position 3608.
  • the position of the ATG codon for the first amino acid (methionine) of the protein is at position 3876.
  • the first SNP mentioned below is located at position 2239 of Accession Number J05008.1. According to the annotation of Accession Number J05008.1, the transcription start site is position 3608. Therefore, the T2239->G SNP would be -1369 relative to the transcription start position. Further, according to the annotation of Accession Number J05008.1, the position of the "A" of the ATG codon for the first amino acid (methionine) of the protein, i.e.- the translation start site, is at position 3876. The T2239- G SNP corresponds to -1637 with reference to the translation initiation site (the "A" of the first encoded "ATG").
  • the second SNP mentioned below is located at position 2657 according to the numbering scheme of GenBank Accession Number J05008.1. Again, according to the annotation of Accession Number J05008.1, the transcription start site is position 3608. Therefore, the A2657->C SNP would be -951 relative to the transcription start position. Further, according to the annotation of Accession Number J05008.1, the position of the "A" of the ATG codon for the first amino acid (methionine) of the protein, i.e.- the translation start site, is at position 3876. The A2657->C SNP corresponds to -
  • DJD degenerative joint disease, also know as osteoarthritis
  • DOL dye-labeled oligonucleotide ligation assay
  • ESRD end-stage renal disease
  • FSGS focal segmental glomerular sclerosis
  • HTN hypertension
  • MASDA multiplexed allele-specific diagnostic assay
  • MADGE microtiter array diagonal gel electrophoresis
  • MI myocardial infarction
  • NTDDM noninsulin-dependent diabetes mellitus
  • Sequence means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide.
  • Polymorphism refers to a set of genetic variants at a particular genetic locus among individuals in a population.
  • Promoter means a regulatory sequence of DNA that is involved in the binding of RNA polymerase to initiate transcription of a gene.
  • a “gene” is a segment of DNA involved in producing a peptide, polypeptide, or protein, including the coding region, non- coding regions preceding ("leader”) and following (“trailer”) coding region, as well as intervening non-coding sequences ("introns") between individual coding segments ("exons").
  • a promoter is herein considered as a part of the corresponding gene. Coding refers to the representation of amino acids, start and stop signals in a three base “triplet” code. Promoters are often upstream (“5' to”) the transcription initiation site of the gene.
  • Gene therapy means the introduction of a functional gene or genes from some source by any suitable method into a living cell to correct for a genetic defect.
  • Reference allele or “reference type” means the allele designated in the Gen Bank sequence listing for a given gene, in this case Gen Bank Accession Number J05008.1 for the endothelin-1 gene.
  • Genetic variant or “variant” means a specific genetic variant which is present at a particular genetic locus in at least one individual in a population and that differs from the reference type.
  • patient and “subject” are not limited to human beings, but are intended to include all vertebrate animals in addition to human beings.
  • the terms “genetic predisposition”, “genetic susceptibility” and “susceptibility” all refer to the likelihood that an individual subject will develop a particular disease, condition or disorder. For example, a subject with an increased susceptibility or predisposition will be more likely than average to develop a disease, while a subject with a decreased predisposition will be less likely than average to develop the disease.
  • a genetic variant is associated with an altered susceptibility or predisposition if the allele frequency of the genetic variant in a population or subpopulation with a disease, condition or disorder varies from its allele frequency in the population without the disease, condition or disorder (control population) or a control sequence (reference type) by at least 1%, preferably by at least 2%, more preferably by at least 4% and more preferably still by at least 8%.
  • an odds ratio of 1.5 was chosen as the threshold of significance based on the recommendation of Austin et al. in Epidemiol. Rev., 16:65-76, 1994. "[EJpidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios ⁇ 1.5)." Id. at 66.
  • isolated nucleic acid means a species of the invention that is the predominate species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods).
  • allele frequency means the frequency that a given allele appears in a population.
  • the present application provides single nucleotide polymorphisms (SNPs) in a gene associated with HTN, ESRD due to HTN, NTDDM, ESRD due to NTDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPND due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HT ⁇ , HT ⁇ CM, MI due to HT ⁇ , ASPND due to ⁇ IDDM, CNA due to ⁇ IDDM, Ischemic CM, Ischemic CM with ⁇ IDDM, MI due to ⁇ IDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to TDDM, or seizure disorder.
  • the first polymorphism is a T to G transversion at position 2239 and the second polymorphism is an A to C substitution at position 2657, both of the
  • the presence of genetic variants in the above genes or their control regions, or in any other genes that may affect susceptibility to disease is determined by screening nucleic acid sequences from a population of individuals for such variants.
  • the population is preferably comprised of some individuals with the disease of interest, so that any genetic variants that are found can be correlated with disease.
  • the population is also preferably comprised of some individuals that have known risk for the disease.
  • the population should preferably be large enough to have a reasonable chance of finding individuals with the sought-after genetic variant. As the size of the population increases, the ability to find significant correlations between a particular genetic variant and susceptibility to disease also increases.
  • the nucleic acid sequence can be DNA or RNA.
  • genomic DNA can be conveniently obtained from whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal cells, skin or hair.
  • target nucleic acid must be obtained from cells or tissues that express the target sequence.
  • One preferred source and quantity of DNA is 10 to 30 ml of anticoagulated whole blood, since enough DNA can be extracted from leukocytes in such a sample to perform many repetitions of the analysis contemplated herein.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the first type involves detection of unknown SNPs by comparing nucleotide target sequences from individuals in order to detect sites of polymorphism. If the most common sequence of the target nucleotide sequence is not known, it can be determined by analyzing individual humans, animals or plants with the greatest diversity possible. Additionally the frequency of sequences found in subpopulations characterized by such factors as geography or gender can be determined.
  • the presence of genetic variants and in particular SNPs is determined by screening the DNA and/or RNA of a population of individuals for such variants. If it is desired to detect variants associated with a particular disease or pathology, the population is preferably comprised of some individuals with the disease or pathology, so that any genetic variants that are found can be correlated with the disease of interest. It is also preferable that the population be composed of individuals with known risk factors for the disease. The populations should preferably be large enough to have a reasonable chance to find correlations between a particular genetic variant and susceptibility to the disease of interest.
  • the allele frequency of the genetic variant in a population or subpopulation with the disease or pathology should vary from its allele frequency in the population without the disease or pathology (control population) or the control sequence (reference type) by at least 1%, preferably by at least 2%, more preferably by at least 4% and more preferably still by at least 8%.
  • Determination of unknown genetic variants, and in particular SNPs, within a particular nucleotide sequence among a population may be determined by any method known in the art, for example and without limitation, direct sequencing, restriction length fragment polymorphism (RFLP), single-strand conformational analysis (SSCA), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis (HET), chemical cleavage analysis (CCM) and ribonuclease cleavage.
  • RFLP restriction length fragment polymorphism
  • SSCA single-strand conformational analysis
  • DGGE denaturing gradient gel electrophoresis
  • HET heteroduplex analysis
  • CCM chemical cleavage analysis
  • ribonuclease cleavage ribonuclease cleavage.
  • Direct sequencing has the advantage of determining variation in any base pair of a particular sequence.
  • RFLP analysis (see, e.g. U.S. Patents No. 5,324,631 and 5,645,995) is useful for detecting the presence of genetic variants at a locus in a population when the variants differ in the size of a probed restriction fragment within the locus, such that the difference between the variants can be visualized by electrophoresis. Such differences will occur when a variant creates or eliminates a restriction site within the probed fragment.
  • RFLP analysis is also useful for detecting a large insertion or deletion within the probed fragment. Thus, RFLP analysis is useful for detecting, e.g., an Alu sequence insertion or deletion in a probed DNA segment.
  • SSCPs Single-strand conformational polymorphisms
  • Double strands are first heat-denatured.
  • the single strands are then subjected to polyacrylamide gel electrophoresis under non-denaturing conditions at constant temperature (i.e., low voltage and long run times) at two different temperatures, typically 4-10°C and 23°C (room temperature).
  • constant temperature i.e., low voltage and long run times
  • the secondary structure of short single strands degree of intrachain hairpin formation
  • the method is empirical, but highly reproducible, suggesting the existence of a very limited number of folding pathways for short DNA strands at the critical temperature. Polymorphisms appear as new banding patterns when the gel is stained.
  • Denaturing gradient gel electrophoresis can detect single base mutations based on differences in migration between homo- and heteroduplexes (Myers et al., Nature, 313 :495-498, 1985).
  • the DNA sample to be tested is hybridized to a labeled reference type probe.
  • the duplexes formed are then subjected to electrophoresis through a polyacrylamide gel that contains a gradient of DNA denaturant parallel to the direction of electrophoresis.
  • Heteroduplexes formed due to single base variations are detected on the basis of differences in migration between the heteroduplexes and the homoduplexes formed.
  • heteroduplex analysis In heteroduplex analysis (HET) (Keen et al., Trends Genet.l:5, 1991), genomic DNA is amplified by the polymerase chain reaction followed by an additional denaturing step which increases the chance of heteroduplex formation in heterozygous individuals. The PCR products are then separated on Hydrolink gels where the presence of the heteroduplex is observed as an additional band.
  • HAT heteroduplex analysis
  • Chemical cleavage analysis is based on the chemical reactivity of thymine (T) when mismatched with cytosine, guanine or thymine and the chemical reactivity of cytosine (C) when mismatched with thymine, adenine or cytosine (Cotton et al., Proc. Natl. Acad. Sci. USA, 85:4397-4401, 1988).
  • Duplex DNA formed by hybridization of a reference type probe with the DNA to be examined is treated with osmium tetroxide for T and C mismatches and hydroxylamine for C mismatches.
  • T and C mismatched bases that have reacted with the hydroxylamine or osmium tetroxide are then cleaved with piperidine. The cleavage products are then analyzed by gel electrophoresis.
  • Ribonuclease cleavage involves enzymatic cleavage of RNA at a single base mismatch in an RNA:DNA hybrid (Myers et al., Science 230:1242-1246, 1985).
  • a 32 P labeled RNA probe complementary to the reference type DNA is annealed to the test DNA and then treated with ribonuclease A. If a mismatch occurs, ribonuclease A will cleave the RNA probe and the location of the mismatch can then be determined by size analysis of the cleavage products following gel electrophoresis.
  • the second type of polymorphism detection involves determining which form of a known polymorphism is present in individuals for diagnostic or epidemiological purposes.
  • several methods have been developed to detect known SNPs. Many of these assays have been reviewed by Landegren et al, Genome Res., 8:169-116, 1998, and will only be briefly reviewed here.
  • One type of assay has been termed an array hybridization assay, an example of which is the multiplexed allele-specific diagnostic assay (MASDA) (U.S. Patent No. 5,834,181; Shuber et al., Hum. Molec. Genet., 6:337-347, 1997).
  • MASDA multiplexed allele-specific diagnostic assay
  • samples from multiplex PCR are immobilized on a solid support.
  • a single hybridization is conducted with a pool of labeled allele specific oligonucleotides (ASO). Any ASOs that hybridize to the samples are removed from the pool of ASOs.
  • the support is then washed to remove unhybridized ASOs remaining in the pool. Labeled ASOs remaining on the support are detected and eluted from the support. The eluted ASOs are then sequenced to determine the mutation present.
  • ASO allele specific oligonucleotides
  • the TaqMan assay uses allele specific (ASO) probes with a donor dye on one end and an acceptor dye on the other end, such that the dye pair interact via fluorescence resonance energy transfer (FRET).
  • a target sequence is amplified by PCR modified to include the addition of the labeled ASO probe. The PCR conditions are adjusted so that a single nucleotide difference will effect binding of the probe.
  • a perfectly complementary probe is cleaved during the PCR while a probe with a single mismatched base is not cleaved. Cleavage of the probe dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence.
  • An alternative to the TaqMan assay is the molecular beacons assay (U.S. Patent No. 5,925,517; Tyagi et al., Nature Biotech., 16:49-53, 1998). In the molecular beacons method for real time detection of the presence of target sequences or can be used after amplification.
  • High throughput screening for SNPs that affect restriction sites can be achieved by Microtiter Array Diagonal Gel Electrophoresis (MADGE) (Day and Humphries, Anal. Biochem., 222:389-395, 1994).
  • MADGE Microtiter Array Diagonal Gel Electrophoresis
  • restriction fragment digested PCR products are loaded onto stackable horizontal gels with the wells arrayed in a microtiter format.
  • electrophoresis the electric field is applied at an angle relative to the columns and rows of the wells allowing products from a large number of reactions to be resolved.
  • PCR amplification of specific alleles PASA
  • ASA allele-specific amplification
  • ARMS amplification refractory mutation system
  • an oligonucleotide primer is designed that perfectly matches one allele but mismatches the other allele at or near the 3' end. This results in the preferential amplification of one allele over the other.
  • bi-PASA In another method, termed bi-PASA, four primers are used; two outer primers that bind at different distances from the site of the SNP and two allele specific inner primers (Liu et al., Genome Res., 7:389-398, 1997). Each of the inner primers has a non-complementary
  • two allele-specific probes labeled with either of two lanthanide labels compete for ligation to a third biotin labeled phosphorylated oligonucleotide and the signals from the allele specific oligonucleotides are compared by time-resolved fluorescence.
  • the oligonucleotides are collected on an avidin-coated 96-pin capture manifold. The collected oligonucleotides are then transferred to microtiter wells in which the europium and terbium ions are released. The fluorescence from the europium ions is determined for each well, followed by measurement of the terbium fluorescence.
  • DOL dye-labeled oligonucleotide ligation
  • DOL combines PCR and the oligonucleotide ligation reaction in a two-stage thermal cycling sequence with fluorescence resonance energy transfer (FRET) detection.
  • FRET fluorescence resonance energy transfer
  • labeled ligation oligonucleotides are designed to have annealing temperatures lower than those of the amplification primers. After amplification, the temperature is lowered to a temperature where the ligation oligonucleotides can anneal and be ligated together.
  • This assay requires the use of a thermostable ligase and a thermostable DNA polymerase without 5' nuclease activity.
  • minisequencing the target-dependent addition by a polymerase of a specific nucleotide immediately downstream (3') to a single primer is used to determine which allele is present (US Patent No. 5,846,710).
  • a polymerase of a specific nucleotide immediately downstream (3') to a single primer is used to determine which allele is present.
  • SNPs can be analyzed in parallel by separating locus specific primers on the basis of size via electrophoresis and determining allele specific incorporation using labeled nucleotides.
  • ddNTPs dye labeled dideoxynucleoside triphosphates
  • elongation primers are attached to a solid support such as a glass slide.
  • Methods for construction of oligonucleotide arrays are well known to those of ordinary skill in the art and can be found, for example, in Nature Genetics, Suppl., Vol. 21, January, 1999.
  • PCR products are spotted on the array and allowed to anneal.
  • the extension (elongation) reaction is carried out using a polymerase, a labeled dNTP and noncompeting ddNTPs.
  • incorporación of the labeled dNTP is then detected by the appropriate means.
  • extension is accomplished with the use of the appropriate labeled ddNTP and unlabeled ddNTPs (Pastinen et al, Genome Res., 7:606-614, 1997).
  • Solid phase minisequencing has also been used to detect multiple polymorphic nucleotides from different templates in an undivided sample (Pastinen et al., Clin. Chem., 42: 1391-1397, 1996).
  • biotinylated PCR products are captured on the avidin-coated manifold support and rendered single stranded by alkaline treatment.
  • the manifold is then placed serially in four reaction mixtures containing extension primers of varying lengths, a DNA polymerase and a labeled ddNTP, and the extension reaction allowed to proceed.
  • the manifolds are inserted into the slots of a gel containing formamide which releases the extended primers from the template.
  • the extended primers are then identified by size and fluorescence on a sequencing instrument. Fluorescence resonance energy transfer (FRET) has been used in combination with minisequencing to detect SNPs (U.S. Patent No. 5,945,283; Chen et al., Proc. Natl. Acad. Sci. USA, 94:10756-10761, 1997).
  • FRET Fluorescence resonance energy transfer
  • the extension primers are labeled with a fluorescent dye, for example fluorescein.
  • the ddNTPs used in primer extension are labeled with an appropriate FRET dye. Incorporation of the ddNTPs is determined by changes in fluorescence intensities.
  • the present invention provides a method for diagnosing a genetic predisposition for a disease.
  • a biological sample is obtained from a subject.
  • the subject can be a human being or any vertebrate animal.
  • the biological sample must contain polynucleotides and preferably genomic DNA. Samples that do not contain genomic DNA, for example, pure samples of mammalian red blood cells, are not suitable for use in the method.
  • the form of the polynucleotide is not critically important such that the use of DNA, cDNA, RNA or mRNA is contemplated within the scope of the method.
  • the polynucleotide is then analyzed to detect the presence of a genetic variant where such variant is associated with an increased risk of developing a disease, condition or disorder, and in particular HTN, ESRD due to HTN, NTDDM, ESRD due to NIDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ASPVD due to NIDDM, CVA due to NIDDM, Ischemic CM, Ischemic CM with NTDDM, MI due to NTDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to IDDM, or seizure disorder.
  • the genetic variant is at one of the polymorphic sites contained in Table 17. In another embodiment, the genetic variant is one of the variants contained in Table 17 or the complement of any of the variants contained in Table 17. Any method capable of detecting a genetic variant, including any of the methods previously discussed, can be used. Suitable methods include, but are not limited to, those methods based on sequencing, mini sequencing, hybridization, restriction fragment analysis, oligonucleotide ligation, or allele specific PCR.
  • the present invention is also directed to an isolated nucleic acid sequence of at least 10 contiguous nucleotides from SEQ ID NO: 1, or the complements of SEQ JD NO: 1.
  • the sequence contains at least one polymorphic site associated with a disease, and in particular HTN, ESRD due to HTN, NTDDM, ESRD due to NTDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPND due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HT ⁇ , HT ⁇ CM, MI due to HT ⁇ , ASPND due to ⁇ TDDM, CNA due to ⁇ IDDM, Ischemic CM, Ischemic CM with ⁇ IDDM, MI due to
  • the genetic variant is at one of the polymorphic sites contained in Table 17.
  • the genetic variant is one of the variants contained in Table 17 or the complement of any of the variants contained in Table 17.
  • the polymorphic site which may or may not also include a genetic variant, is located at the 3' end of the polynucleotide.
  • the polynucleotide further contains a detectable marker. Suitable markers include, but are not limited to, radioactive labels, such as radionuclides, fluorophores or fluorochromes, peptides, enzymes, antigens, antibodies, vitamins or steroids.
  • kits for the detection of polymorphisms associated with diseases, conditions or disorders and in particular HT ⁇ , ESRD due to HT ⁇ , ⁇ JDDM, ESRD due to ⁇ IDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPND due to HT ⁇ , CNA due to HT ⁇ , cataracts due to HT ⁇ , HT ⁇ CM, MI due to HT ⁇ , ASPND due to ⁇ TDDM, CNA due to ⁇ IDDM, Ischemic CM, Ischemic CM with ⁇ IDDM, MI due to ⁇ IDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to TDDM, or seizure disorder.
  • kits contain, at a minimum, at least one polynucleotide of at least 10 contiguous nucleotides of SEQ JD NO 1, or the complements of SEQ XD NO: 1.
  • the genetic variant is at one of the polymorphic sites contained in Table 17.
  • the 3' end of the polynucleotide is immediately 5' to a polymorphic site, preferably a polymorphic site selected from the sites in Table 17.
  • the genetic variant is one of the variants contained in Table 17 or the complement of any of the variants contained in Table 17.
  • the genetic variant is located at the 3' end of the polynucleotide.
  • the polynucleotide of the kit contains a detectable label.
  • Suitable labels include, but are not limited to, radioactive labels, such as radionuclides, fluorophores or fluorochromes, peptides, enzymes, antigens, antibodies, vitamins or steroids.
  • the kit may also contain additional materials for detection of the polymorphisms.
  • the kits may contain buffer solutions, enzymes, nucleotide triphosphates, and other reagents and materials necessary for the detection of genetic polymorphisms.
  • the kits may contain instructions for conducting analyses of samples for the presence of polymo ⁇ hisms and for interpreting the results obtained.
  • the present invention provides a method for designing a treatment regime for a patient having a disease, condition or disorder and in particular HTN, ESRD due to HTN, NIDDM, ESRD due to NIDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPVD due to HTN, CVA due to HTN, cataracts due to
  • HTN HTN CM
  • MI HTN
  • ASPVD due to NIDDM
  • CVA due to NTDDM
  • Ischemic CM Ischemic CM with NTDDM
  • MI due to NTDDM
  • afib without valvular disease alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de- clots
  • ESRD due to FSGS ESRD due to TDDM
  • seizure disorder caused either directly or indirectly by the presence of one or more single nucleotide polymorphisms.
  • genetic material from a patient for example, DNA, cDNA, RNA or mRNA is screened for the presence of one or more SNPs associated with the disease of interest.
  • a treatment regime is designed to counteract the effect of the SNP.
  • information gained from analyzing genetic material for the presence of polymorphisms can be used to design treatment regimes involving gene therapy. For example, detection of a polymorphism that either affects the expression of a gene or results in the production of a mutant protein can be used to design an artificial gene to aid in the production of normal, wild type protein or help restore normal gene expression.
  • the present invention is also useful in designing prophylactic treatment regimes for patients determined to have an increased susceptibility to a disease, condition or disorder, and in particular HTN, ESRD due to HTN, NIDDM, ESRD due to NTDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ASPVD due to NTDDM, CVA due to NTDDM, Ischemic CM, Ischemic CM with NTDDM, MI due to NTDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to TDDM, or seizure disorder due to the presence of one or more single nucleotide polymorphisms.
  • genetic material such as DNA, cDNA, RNA or mRNA
  • a treatment regime can be designed to decrease the risk of the patient developing the disease.
  • Such treatment can include, but is not limited to, surgery, the administration of pharmaceutical compounds or nutritional supplements, and behavioral changes such as improved diet, increased exercise, reduced alcohol intake, smoking cessation, etc.
  • SNPs are written as "reference sequence nucleotide” - "variant nucleotide.” Changes in nucleotide sequences are indicated in bold print.
  • Leukocytes were obtained from human whole blood collected with EDTA as an anticoagulant. Blood was obtained from a group of black men, black women, white men, and white women without any known disease.
  • Blood was also obtained from individuals with HTN, ESRD due to HTN, NTDDM, ESRD due to NTDDM, lung cancer, breast cancer, prostate cancer, colon cancer, ASPVD due to HTN, CNA due to HT ⁇ , cataracts due to HTN, HTN CM, MI due to HTN, ASPVD due to NIDDM, CVA due to NTDDM, Ischemic CM, Ischemic CM with NTDDM, MI due to NTDDM, afib without valvular disease, alcohol abuse, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to TDDM, or seizure disorder as indicated in the tables below.
  • Genomic DNA was purified from the collected leukocytes using standard protocols well known to those of ordinary skill in the art of molecular biology (Ausubel et al., Short Protocol in Molecular Biology, 3 rd ed., John Wiley and Sons, 1995; Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989; and Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, 1986). One hundred nanograms of purified genomic DNA were used in each PCR reaction.
  • Standard PCR reaction conditions were used. Methods for conducting PCR are well known in the art and can be found, for example, in U.S. Patent Nos 4,965,188, 4,800,159, 4,683,202, and 4,683,195; Ausbel et al., eds., Short Protocols in Molecular Biology, 3 rd ed., Wiley, 1995; and Innis et al., eds., PCR Protocols, Academic Press, 1990.
  • the first SNP T2239->G can be identified by PCR amplification of a specific region of the endothelin-1 promoter.
  • the sequence of the sense primer was 5' -CTC CAT CCC CAG AAA AAC TG-3', corresponding to nucleotides 2113 to 2132, inclusive.
  • the sequence of the anti-sense primer is 5'-AAG GAA GGT GGT GCT GAG AA-3 ' corresponding to nucleotides 2490 to 2509, inclusive. (SEQ JD NO: 3).
  • PCR product spanned positions 2113 to 2509, inclusive, of the EDN1 gene.
  • the second SNP A2657->C can be identified by PCR amplification of a specific region of the endothelin-1 promoter.
  • the sense primer was 5'- GGG GGA TTT CAA GGT TAG AT -3' (SEQ ID NO: 4).
  • the anti-sense primer was 5'- GAG AAG CCC CGA TAA GTT CTT T -3 ' (SEQ XD NO: 5).
  • the PCR product thus produced spanned positions 2390 to 2924 of the human EDN-1 gene (SEQ XD NO: 1).
  • the PCR reaction contained a total volume of 20 microliters ( ⁇ l), consisting of 10 ⁇ l of a premade PCR reaction mix (Sigma "JumpStart Ready Mix with RED Taq Polymerase”). Primers at 10 ⁇ M were diluted to a final concentration of 0.3 ⁇ M in the PCR reaction mix. Approximately 25 ng of template leukocyte genomic DNA was used for each PCR amplification. After an initial 5 minutes denaturation at 94°C, 35 cycles were performed consisting of 45 seconds of denaturation at 94°C, 45 seconds of hybridization at 62°C, 45 seconds of extension at 72°C, followed by a final extension step of 10 minutes at 72°C. Post-PCR clean-up was performed as follows.
  • PCR reactions were cleaned to remove unwanted primer and other impurities such as salts, enzymes, and uninco ⁇ orated nucleotides that could inhibit sequencing.
  • One of the following clean-up kits was used: Qiaquick-96 PCR Purification Kit (Qiagen) or Multiscreen-PCR Plates (Millipore, discussed below).
  • PCR samples were added to the 96-well Qiaquick silica-gel membrane plate and a chaotropic salt, supplied as "PB Buffer," was then added to each well.
  • PB Buffer caused the DNA to bind to the membrane.
  • the plate was put onto the Qiagen vacuum manifold and vacuum was applied to the plate in order to pull sample and PB Buffer through the membrane. The filtrate was discarded.
  • PCR samples were loaded into the wells of the Multiscreen-PCR Plate and the plate was then placed on a Millipore vacuum manifold. Vacuum pressure was applied for 10 minutes, and the filtrate was discarded. The plate was then removed from the vacuum manifold and 100 ⁇ l of Milli-Q water was added to each well to rehydrate the DNA samples. After shaking on a plate shaker for 5 minutes, the plate was replaced on the manifold and vacuum pressure was applied for 5 minutes. The filtrate was again discarded. The plate was removed and 60 ⁇ l Milli-Q water was added to each well to again rehydrate the DNA samples.
  • the 60 ⁇ l of cleaned PCR product was transferred from the Multiscreen-PCR plate to another 96-well plate by pipetting.
  • the Millipore vacuum manifold was purchased from Millipore for exclusive use with the Multiscreen-PCR plates.
  • Cycle sequencing was performed on the clean PCR product using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin-Elmer). For a total volume of 20 ⁇ l, the following reagents were added to each well of a 96-well plate: 2.0 ⁇ l Terminator Ready Reaction mix, 3.0 ⁇ l 5X Sequencing Buffer (ABI), 5-10 ⁇ l template (30-90 ng double stranded DNA), 3.2 pM primer (primer used was the forward primer from the PCR reaction), and Milli-Q water to 20 ⁇ l total volume.
  • ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit Perkin-Elmer.
  • the following reagents were added to each well of a 96-well plate: 2.0 ⁇ l Terminator Ready Reaction mix, 3.0 ⁇ l 5X Sequencing Buffer (ABI), 5-10 ⁇ l template (30-90 ng double stranded DNA), 3.2 pM primer (primer used was the forward primer from the
  • the reaction plate was placed into a Hybaid thermal cycler block and programmed as follows: X 1 cycle: 1 degree/sec thermal ramp to 94°C, 94°C for 1 min; X 35 cycles: 1 degree/sec thermal ramp to 94°C, then 94°C for 10 sec, followed by 1 degree/sec thermal ramp to 50°C, then 50°C for 10 sec, followed by 1 degree/sec thermal ramp to 60°C, then 60°C for 4 minutes.
  • the cycle sequencing reaction product was cleaned up to remove the uninco ⁇ orated dye-labeled terminators that can obscure data at the beginning of the sequence. A precipitation protocol was used.
  • Pyrosequencing is another method of sequencing DNA by synthesis, where the addition of one of the four dNTPs that correctly matches the complementary base on the template strand is detected. Detection occurs via utilization of the pyrophosphate molecules liberated upon base addition to the elongating synthetic strand. The pyrophosphate molecules are used to make ATP, which in turn drives the emission of photons in a luciferin/luciferase reaction, and these photons are detected by the instrument. A Luc96 Pyrosequencer was used under default operating conditions supplied by the manufacturer. Primers were designed to anneal within 5 bases of the polymo ⁇ hism, to serve as sequencing primers.
  • Patient genomic DNA was subject to PCR using amplifying primers that amplify an approximately 200 base pair amplicon containing the polymo ⁇ hisms of interest.
  • One of the amplifying primers whose orientation is opposite to the sequencing primer, was biotinylated. This allowed selection of single stranded template for pyrosequencing, whose orientation is complementary to the sequencing primer.
  • Amplicons prepared from genomic DNA were isolated by binding to streptavidin-coated magnetic beads. After denaturation in NaOH, the biotinylated strands were separated from their complementary strands using magnetics. After washing the magnetic beads, the biotinylated template strands still bound to the beads were transferred into 96-well plates.
  • the sequencing primers were added, annealing was carried out at 95° for 2 minutes, and plates were placed in the Pyrosequencer.
  • the enzymes, substrates and dNTPs used for synthesis and pyrophosphate detection were added to the instrument immediately prior to sequencing.
  • the Luc96 software requires definition of a program of adding the four dNTPs that is specific for the location of the sequencing primer, the DNA composition flanking the SNP, and the two possible alleles at the polymo ⁇ hic locus. This order of adding the bases generates theoretical outcomes of light intensity patterns for each of the two possible homozygous states and the single heterozygous state.
  • the Luc96 software compares the actual outcome to the theoretical outcome and calls a genotype for each well. Each sample is also assigned one of three confidence scores: pass, uncertain, fail.
  • the results for each plate are output as a text file and processed in Excel using a Visual Basic program to generate a report of genotype and allele frequencies for the various disease and population cell groupings represented on the 96 well plate.
  • the susceptibility allele is indicated below, as well as the odds ratio (OR).
  • the allele which is present more often in the given disease category was chosen as the susceptibility allele.
  • the G allele was chosen as the susceptibility allele for black women with breast cancer because more of the individuals in that category had the G allele than had the T allele.
  • Haldane's correction multiplying all cells by 2 and adding 1 was used. If the odds ratio (OR) was > 1.5, the 95% confidence interval (C.I.) is also given.
  • the susceptibility allele (S) is indicated; the alternative allele at this locus is defined as the protective allele (P).
  • the odds ratio (OR) for each genotype (SS, SP; the odds ratio for the PP genotype is 1, since it is the reference group, and is not presented separately).
  • the 95% confidence interval (CI.) is also given, in parentheses. Where there was a "0" in a cell which produced a 0 in the denominator, Haldane's correction (multiplying all cells by 2 and adding 1) was used. As discussed above, an odds ratio of 1.5 is chosen as the threshold of significance based on the recommendation of Austin H et al. (Epidemiol. Rev. 16:65-76, 1994).
  • the observed genotype frequencies were 36% T/T, 45% T/G, and 18% C/C, in close agreement with those predicted for Hardy-Weinberg equilibrium.
  • the observed genotype frequencies were 36% T/T, 27% T/G, and 36% C/C, in rather distant agreement with those predicted for Hardy-Weinberg equilibrium.
  • the observed genotype frequencies were 89% T/T, 0% T/G, and 11% C/C, in rather distant agreement with those predicted for Hardy-Weinberg equilibrium.
  • the observed genotype frequencies were 75% T/T, 25% T/G, and 0% C/C, in close agreement with those predicted for Hardy-Weinberg equilibrium.
  • the odds ratio for the T allele as a risk factor for disease is 1.7 (95% CI, 0.5-5.7).
  • the odds ratio for the homozygote (TT) is 3.0 (95% CI,
  • the heterozygote (TG genotype) has an odds ratio of 2.0 (95% C.I., 0.1-29.8).
  • the odds ratio for the G allele as a risk factor for disease is 3.0 (95% CI, 0.3-33.5).
  • the odds ratio for the homozygote (GG) is 3.0 (95%
  • the heterozygote (GT genotype) has an odds ratio of 3.9 (95%> C.I., 0.3- 45.6). T hese data suggest that the G allele behaves as a dominant allele, with no additional effect of having two copies of the G allele (GG homozygote) as compared with having only one copy (GT heterozygote). For white men with lung cancer, the odds ratio for the G allele as a risk factor for disease is 3.7 (95% CI, 0.7-20.9). The odds ratio for the homozygote (GG) is only 1.5 (95%) CI, 0.3-9.1), whereas the heterozygote (GT genotype) has a remarkable odds ratio of 17.0 (95% C.I., 1.9-151).
  • the G allele behaves as a codominant allele.
  • the odds ratio for the G allele as a risk factor for disease is 3.0 (95% CI, 0.6-14.0).
  • the odds ratio for the homozygote (GG) is 5.2 (95% CI, 0.5-56.1), while the heterozygote (GT genotype) has an odds ratio of 2.2 (95% C.I., 0.6-7.6).
  • the odds ratio for the G allele as a risk factor for disease is 3.3 (95% CI, 0.6-18.3).
  • the odds ratio for the homozygote (GG) is actually less than 1, whereas the heterozygote (GT genotype) has a remarkable odds ratio of 23.2 (95% C.I., 2.7-201). These data suggest that the G allele behaves as a codominant allele.
  • the odds ratio for the T allele as a risk factor for disease is 1.6 (95% CI, 0.4-5.8).
  • the odds ratio for the homozygote (TT) is 1.5 (95% CI,
  • the odds ratio for the T allele as a risk factor for disease is 2.3 (95% CI, 0.7-8.3).
  • the odds ratio for the homozygote (TT) is 5.0 (95% CI, 0.4-64.4), whereas the heterozygote (TG genotype) has an odds ratio of 5.3 (95% CI, 0.4- 75.8).
  • the odds ratio for the G allele as a risk factor for disease is 3.0 (95% CI, 0.5-17.2).
  • the odds ratio for the homozygote (GG) is 1.9 (95% CI, 0.4-9.3), whereas the heterozygote (GT genotype) has an odds ratio of 5.7 (95% CI,
  • the odds ratio for the G allele as a risk factor for disease is 3.8 (95% CI, 0.8-17.2).
  • the odds ratio for the homozygote (GG) is 8.6 (95% CI, 0.9-83.8), whereas the heterozygote (GT genotype) has an odds ratio of only 1.7 (95%> CI, 0.5-6.2).
  • the odds ratio for the T allele as a risk factor for disease is 3.4 (95% CI, 0.6-19.2) as compared with white men with NIDDM but no renal disease.
  • the odds ratio for the homozygote (TT) is 5.7 (95%> CI, 0.6-54.1), while the heterozygote (TG genotype) has a similar odds ratio of 5.0 (95% CI, 0.4-59.7).
  • the odds ratio for the T allele as a risk factor for disease is a remarkable 18.3 (95% CI, 2.3-148) as compared with white women with NTDDM but no renal disease.
  • the odds ratio for the homozygote (TT) is 7.7 (95% CI, 0.8-75.3), while the heterozygote (TG genotype) has an odds ratio of only 0.7.
  • the odds ratio for the T allele was 2.1 (95% CI, 0.7 - 6.2), compared to Caucasians with MI due to NTDDM. Data were not sufficient to generate genotypic odds ratios of 1.5 or greater. These data further suggest that the EDN-1 gene is significantly associated with diabetic cardiomyopathy in Caucasians, i.e. abnormal activity of the EDN-1 gene predisposes Caucasians to diabetic cardiomyopathy.
  • the odds ratio for the G allele was 2.6 (95% CI, 0.8 - 9.1), compared to Caucasians with hypertension only.
  • the odds ratio for the homozygote (G/G) was 0.8 H (95% CI, 0 - 14.1), while the odds ratio for the heterozygote (T/G) was 2.3 H (95% CI, 0 - 137).
  • the odds ratio for the G allele was 1.7 (95% CI, 0.7 - 4.5).
  • the odds ratio for the homozygote (G/G) was 1.1 H (95% CI, 0.1 - 19.7), while the odds ratio for the heterozygote (T/G) was 2.7 H (95% CI, 0.1 - 75).
  • EDN-1 gene is significantly associated with ESRD due to FSGS in Caucasians, i.e. abnormal activity of the EDN-1 gene predisposes Caucasians to ESRD due to FSGS.
  • the odds ratio for the T allele was 1.6 (95% CI, 0.5 - 5.2).
  • the odds ratio for the homozygote (T/T) was 1.7 H (95% CI, 0.1 - 28.6), while the odds ratio for the heterozygote (T/G) was 1.3 H (95% CI, 0 - 38).
  • TCF-2 alpha The binding site of T-cell factor-2 alpha (TCF-2 alpha) is predicted to be disrupted by the T2239- G SNP (Quandt K et al., Nucleic Acids Res., 23:4878-4884, 1995).
  • TCF-2 alpha binds to a core sequence of five nucleotides, 5'-KjTKTC-3' (Waterman ML, et al. New Biology, 2(7):621-636, 1990).
  • a TCF-2 alpha binding site which occurs on average 3.91 times per 1000 base pairs of random genomic sequence in vertebrates, is predicted to occur at position 2236 to 2240 on the (-) strand of reference sequence J05008.1 (matrix score 1.000, with 1.000 being an identical match).
  • the T2239->G SNP replaces the indicated T with a G within the core binding sequence.
  • TCF-2 alpha is a transcriptional activator in lymphoid cells, although nothing is known of its activity in other cell types. Disruption of the TCF-2 alpha core binding site is expected to result in a decreased rate of transcription of the endothelin-1 gene.
  • the susceptibility allele is indicated below, as well as the odds ratio (OR). Where there was a "0" in a cell which produced a 0 in the denominator, Haldane's correction (multiplying all cells by 2 and adding 1) was used. If the odds ratio (OR) was > 1.5, the 95% confidence interval (CI.) is also given. An odds ratio of 1.5 was chosen as the threshold of significance based on the recommendation of Austin et al. in Epidemiol. Rev., 16:65-76, 1994. "[EJpidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios ⁇ 1.5)." Id. at 66. Odds ratios of greater than 1.5 are highlighted below. Table 13
  • Hypertension (HTN)
  • the susceptibility allele (S) is indicated; the alternative allele at this locus is defined as the protective allele (P).
  • the odds ratio for the PP genotype is 1, since it is the reference group, and is not presented separately.
  • CI. 95%> confidence interval
  • An odds ratio of 1.5 was chosen as the threshold of significance based on the recommendation of Austin et al., in Epidemiol. Rev., 16:65-76, 1994. "[Ejpidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios ⁇ 1.5)." Id. at 66.
  • Haldane's zero cell correction was used when the denominator contained a zero. Odds ratios of greater than 1.5 are highlighted below. Table 15
  • Hypertension (HTN)
  • Example 1 PCR and sequencing were conducted as described in Example 1.
  • the primers used were those in Example 1.
  • the control samples were in good agreement with Hardy- Weinberg equilibrium, as follows: For the Group I diseases, a frequency of 0.65 for the A allele ("p") and 0.35 for the
  • the observed genotype frequencies were 43 %> A/ A, 43% A/C, and 13%) C/C, in close agreement with those predicted for Hardy-Weinberg equilibrium.
  • the observed genotype frequencies were 55%> A/A, 40% A/C, and 5% C/C, in very close agreement with those predicted for Hardy-Weinberg equilibrium.
  • the observed genotype frequencies were 86% A/A, 10% A/C, and 5% C/C, in reasonably close agreement with those predicted for Hardy-Weinberg equilibrium.
  • the observed genotype frequencies were 79% A/A, 17% A/C, and 4% C/C, in reasonably close agreement with those predicted for Hardy-Weinberg equilibrium.
  • a frequency of 0.17 for the C allele ("p") and 0.83 for the A allele ("q") among Caucasian control individuals predicts genotype frequencies of 3.0% C/C, 28.0%> C/A, and
  • the odds ratio for the C allele as a risk factor was 3.9 (95% CI, 1.1-13).
  • the odds ratio for the homozygote (CC) was a remarkable 14.7 (95% CI, 1.2-185).
  • the heterozygote (CA genotype) had an odds ratio indistinguishable from 1 (odds ratio 0.5; 95%> CI. 0-5.3), suggesting that the C allele behaves as a recessive allele in this patient population.
  • the odds ratio for the C allele as a risk factor was 2.3 (95%> CI, 0.5-11).
  • the odds ratio for the homozygote (CC genotype) was 4.8 (95%o CI, 0.2-93).
  • the heterozygote (CA genotype) had an odds ratio indistinguishable from 1 (odds ratio 1.2; 95% CI. 0.1-14), suggesting that the C allele behaves as a recessive allele in this patient population.
  • the odds ratio for the C allele as a risk factor was 5.1 (95% CI, 1.3-20).
  • the C allele displayed a dosage effect, with the heterozygote (AC) having an odds ratio of 5.4 (95% CI, 0.7-42), and the homozygote (CC) an odds ratio of 7.2 (95% CI, 0.5-97).
  • AC heterozygote
  • CC homozygote
  • the odds ratio for the reference A allele as a risk factor was 2.3 (95% CI, 0.6-9.3).
  • the odds ratio for the homozygote (AA genotype) was 3.7 H (95% CI, 0.4-34).
  • the heterozygote (AC genotype) had an odds ratio of 2.3 H (95%) CI, 0.2-22).
  • the A allele therefore behaves as a dominant allele, with an additive effect of increased allele dosage.
  • the effect of the A allele on disease is as expected for an additive model (3.7 ⁇ 2.3 + 2.3 -1).
  • the odds ratio for the C allele (the novel SNP) as a risk factor was 6.0 (95% CI, 1.5-25).
  • the C allele may be codominant with the A allele.
  • the odds ratio for the A allele as a risk factor was 3.5 (95% CI, 0.3-42.8).
  • the odds ratio for the AC heterozygous genotype was 4.0 (95%> CI, 0.3-55.5), and for the AA homozygous genotype was 1.9 H (95% CI, 0.1-34).
  • the A allele appears to be codominant with the C allele.
  • the odds ratio for the A allele was 2.0 H (95% CI, 0.2-18.8), relative to white women with hypertension but no renal disease.
  • the odds ratios for both the AC heterozygote and the AA homozygote were only 1.0 after the Haldane's correction, shedding no light on the mechanism of action of the A allele.
  • the odds ratio for the A allele at this locus was 2.3 (95% CI, 0.6-9.3).
  • the odds ratio for the heterozygote was 2.3 H (95% CI, 0.2-22), and for the AA homozygote was 3.7 H (95% CI, 0.4-34).
  • the odds ratio for the A allele at this locus was 2.6 (95% CI, 0.5-15.2).
  • the odds ratio for the heterozygote was 5.0 H (95% CI,
  • the odds ratio for the A allele at this locus was 7.5 (95% CI, 0.9-62.1).
  • the odds ratio for the heterozygote was indistinguishable from 1, and for the AA homozygote was 2.5 H (95% CI, 0.2-28).
  • the odds ratio for the C allele was 1.7 (95% CI, 0.7 - 3.9).
  • the odds ratio for the homozygote (C/C) was 1.6 (95% CI, 0.2 - 10.5), while the odds ratio for the heterozygote (CI A) was 2.1 (95% CI, 0.7 - 6.5).
  • C/C homozygote
  • CI A odds ratio for the heterozygote
  • the EDN-1 gene is significantly associated with alcohol abuse in Caucasians, i.e. abnormal activity of the EDN-1 gene predisposes Caucasians to alcohol abuse.
  • the odds ratio for the C allele was 1.6 (95% CI, 0.6 -
  • the odds ratio for the A allele was 1.8 (95%o CI, 0.7- 4.8) , compared to Caucasians with NTDDM only.
  • the odds ratio for the homozygote (A/ A) was 0.2 (95% CI, 0 - 1.9), while the odds ratio for the heterozygote (C/A) was 2.0 (95% CI, 0.4 -9.4).
  • COPD in African- Americans i.e. abnormal activity of the EDN-1 gene predisposes African- Americans to COPD .
  • EDN-1 gene is significantly associated with ESRD due to TDDM in African-Americans, i.e. abnormal activity of the EDN-1 gene predisposes African- Americans to ESRD due to IDDM .
  • EDN-1 gene is significantly associated with hypertensive cardiomyopathy in African- Americans, i.e. abnormal activity of the EDN-1 gene predisposes African- Americans to hypertensive cardiomyopathy .
  • CEBPB binds to a core sequence of four nucleotides, GMAA, in an overall sequence of 14 nucleotides (ref. Akira,
  • CEBPB_01 binding sites which occur on average 2.07 times per 1000 base pairs of random genomic sequence in vertebrates, are predicted to occur at positions 2647 to 2660 on the (+) strand of reference sequence J05008.1 (matrix score 0.952, with 1.0 being an identical match), as well as from position 2670 to 2657 on the (-) strand (matrix score 0.891 out of a possible 1.0). In neither case, however, does the C2657 ⁇ A SNP alter a nucleotide critical for binding.

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Abstract

L'invention concerne des polymorphismes de nucléotide simple (SNPs) associés à l'hypertension, à la néphropathie en phase terminale causée par l'hypertension, au diabète de type II, à la néphropathie en phase terminale causée par le diabète de type II, au cancer du poumon, au cancer du sein, au cancer de la prostate, au cancer du colon, à un acrosyndrome athéroscléreux causé par l'hypertension, à un accident vasculaire cérébral causé par l'hypertension, aux cataractes causées par l'hypertension, à la cardiomyopathie avec hypertension, à l'infarctus du myocarde causé par l'hypertension, à un acrosyndrome athéroscléreux causé par le diabète de type II, à un accident vasculaire cérébral causé par le diabète de type II, à la cardiomyopathie ischémique, à la cardiomyopathie ischémique avec diabète de type II, à l'infarctus du myocarde causé par le diabète de type II, à la fibrillation auriculaire sans valvulopathie, à l'abus d'alcool, à l'anxiété, à l'asthme, à la bronchopneumopathie chronique obstructive, à la cholécystectomie, au rhumatisme articulaire dégénératif, à la néphropathie en phase terminale et aux coagulopathies fréquentes, à la néphropathie en phase terminale causée par une sclérose glomérulaire segmentaire focale, à la néphropathie en phase terminale causée par le diabète de type II, au trouble épileptique. L'invention concerne également des méthodes permettant d'utiliser les SNP afin de déterminer une sensibilité à ces maladies; des séquences nucléotidiques contenant les SNP, des trousses permettant de déterminer la présence des SNP, et des méthodes de traitement ou prophylactiques fondées sur la présence des SNP.
PCT/US2001/028834 2000-09-11 2001-09-11 Polymorphisme du promoteur de l'endotheline-1 WO2002022881A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090602A2 (fr) * 2004-03-18 2005-09-29 Sucampo Ag Procédé servant à diagnostiquer ou à prédire la prédisposition vis-à-vis d'une neuropathie optique

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US5888819A (en) * 1991-03-05 1999-03-30 Molecular Tool, Inc. Method for determining nucleotide identity through primer extension

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US5888819A (en) * 1991-03-05 1999-03-30 Molecular Tool, Inc. Method for determining nucleotide identity through primer extension

Non-Patent Citations (2)

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Title
STEVENS ET AL.: "Genetic variability of the ET-1 and the ETA receptor genes in essential hypertension", JOURNAL OF CARDIOVASCULAR PHARMACOLOGY, vol. 23, no. SUPPL. 3, 1995, pages S9 - S12, XP002905176 *
TIRET ET AL.: "The Lys198Asn polymorphism in the endothelin-1 gene is associated with blood pressure in overweight people", HYPERTENSION, vol. 33, 1999, pages 1169 - 1174, XP002905177 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005090602A2 (fr) * 2004-03-18 2005-09-29 Sucampo Ag Procédé servant à diagnostiquer ou à prédire la prédisposition vis-à-vis d'une neuropathie optique
WO2005090602A3 (fr) * 2004-03-18 2006-04-13 Sucampo Ag Procédé servant à diagnostiquer ou à prédire la prédisposition vis-à-vis d'une neuropathie optique
JP2007529218A (ja) * 2004-03-18 2007-10-25 スキャンポ・アーゲー 視神経症に対する感受性を診断または予測するための方法
US8182990B2 (en) 2004-03-18 2012-05-22 Rusk Intellectual Reserve Ag Method for diagnosing or predicting susceptibility to optic neuropathy

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