WO2002012567A1 - Polymorphisme diagnostique du promoteur vhl - Google Patents

Polymorphisme diagnostique du promoteur vhl Download PDF

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WO2002012567A1
WO2002012567A1 PCT/US2001/024985 US0124985W WO0212567A1 WO 2002012567 A1 WO2002012567 A1 WO 2002012567A1 US 0124985 W US0124985 W US 0124985W WO 0212567 A1 WO0212567 A1 WO 0212567A1
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hypertension
disease
dependent diabetes
diabetes mellitus
insulin dependent
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PCT/US2001/024985
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English (en)
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David W. Moskowitz
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Dzgenes, Llc
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Priority to AU2001284779A priority Critical patent/AU2001284779A1/en
Priority to US10/344,908 priority patent/US20040166491A1/en
Priority to CA002418094A priority patent/CA2418094A1/fr
Priority to EP01963861A priority patent/EP1307595A4/fr
Publication of WO2002012567A1 publication Critical patent/WO2002012567A1/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
    • 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 520 and
  • vHL von Hippel-Lindau syndrome tumor suppressor gene
  • 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 polymorphisms
  • 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 al., Am. J. Human Genet., 60:772-789, 1997; Taillon-Miller et al, Genome
  • 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. In regard to location, 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. In this case, 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.
  • 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.
  • 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 associated with the development of various diseases, including colon cancer, hypertension (HTN), atherosclerotic peripheral vascular disease due to hypertension
  • ischemic cardiomyopathy due to HTN
  • cerebrovascular accident due to hypertension CVA due to HTN
  • cataracts due to hypertension cataracts due to hypertension
  • cardiomyopathy with hypertension HTN CM
  • myocardial infarction due to hypertension MI due to HTN
  • end stage renal disease due to hypertension ESRD due to HTN
  • non-insulin dependent diabetes mellitus NIDDM
  • atherosclerotic peripheral vascular disease due to non-insulin dependent diabetes mellitus
  • CVA due to NIDDM cerebrovascular accident due to non-insulin dependent diabetes mellitus
  • ischemic cardiomyopathy ischemic cardiomyopathy
  • CM ischemic cardiomyopathy with non-insulin dependent diabetes mellitus
  • MI myocardial infarction due to non-insulin dependent diabetes mellitus
  • COPD chronic obstructive pulmonary disease
  • COPD cholecystectomy
  • DJD degenerative joint disease
  • ESRD end stage renal disease and frequent de-clots
  • ESRD due to FSGS focal segmental glomerular sclerosis
  • ESRD due to NIDDM end stage renal disease due to insulin dependent diabetes mellitus
  • IDDM end stage renal disease due to insulin dependent diabetes mellitus
  • seizure disorder a method for diagnosing a genetic predisposition for the development of these diseases in individuals
  • one aspect of the present invention provides a method for diagnosing a genetic predisposition for colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NIDDM, Ischemic CM, Ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ESRD due to IDDM, or seizure disorder in a subject, comprising obtaining a sample containing at least one polynucleotide from the subject, and analyzing the polynucleotide to detect the genetic polymorphism wherein the presence or absence of
  • NTDDM NTDDM
  • Ischemic CM Ischemic CM with NIDDM
  • MI due to NIDDM
  • afib without valvular disease alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots
  • ESRD due to FSGS ESRD due to NIDDM
  • ESRD due to IDDM or seizure disorder.
  • the polymorphism is located in the vHL 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 colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NIDDM, Ischemic CM, Ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ESRD due to IDDM, or seizure disorder.
  • kits for the detection of a polymorphism comprising, at a minimum, at least one polynucleotide of at least 10 contiguous nucleotides of SEQ ID NO: 1, or their complements, wherein the polynucleotide contains at least one polymorphic site associated with colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NTDDM, Ischemic CM, Ischemic CM with NIDDM, MI due to NTDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to
  • NIDDM NIDDM
  • ESRD due to IDDM ESRD due to IDDM
  • seizure disorder ESRD due to IDDM
  • Yet another aspect of the invention provides a method for treating colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NIDDM, Ischemic CM, Ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ESRD due to IDDM, or seizure disorder comprising, obtaining a sample of biological material containing at least one polynucleotide from the subject; analyzing the polynucleotides to detect the presence of at least one polymorphism associated with colon cancer, HTN,
  • ASPVD due to HTN CVA due to HTN
  • cataracts due to HTN, HTN CM, MI due to HTN
  • ESRD due to HTN
  • NTDDM ASPVD due to NIDDM
  • CVA due to NIDDM Ischemic CM, Ischemic CM with NIDDM
  • MI due to NIDDM
  • afib without valvular disease alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots
  • ESRD due to FSGS ESRD due to NIDDM, ESRD due to IDDM, or seizure disorder
  • Still another aspect of the invention provides a method for the prophylactic treatment of a subject with a genetic predisposition to colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NIDDM, Ischemic CM, Ischemic CM with NTDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, 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 polymorphis
  • Figure 1 shows SEQ ID NO: 1, the nucleotide sequence of the vHL gene as contained in GenBank Accession Number AF010238.
  • SEQ ID NO: 1 the nucleotide sequence of the vHL gene as contained in GenBank Accession Number AF010238.
  • 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.
  • the TATA box 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 eukaryotic 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, we feel that the numbering system used in this patent application is the least troublesome.
  • the transcription start site and first exon both begin at position 643.
  • 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 715.
  • the first SNP, C638 — > T is located at position 638 of the GenBank Accession
  • the 20 nucleotides surrounding the SNP are as follows: 5'- G ACT CGG GAG [C/T] GCG CAC GCA G - 3' (nucleotides 628-648 of SEQ ID NO. 1).
  • the second SNP, C638 --> T is located at position 638 of the GenBank Accession Number AF 010238.
  • the 20 nucleotides surrounding the SNP are as follows: 5'- G ACT CGG GAG [C/T] GCG CAC GCA G - 3' (nucleotides 628-648 of SEQ ID NO. 1).
  • AFIB atrial fibrillation without valvular disease
  • ASPVD atherosclerotic peripheral vascular disease
  • COPD chronic obstructive pulmonary disease
  • DJD degenerative joint disease, also know as osteoarthritis
  • DOL dye-labeled oligonucleotide ligation assay
  • ESRD end-stage renal disease
  • MADGE microtiter array diagonal gel electrophoresis
  • MI myocardial infarction
  • NIDDM noninsulin-dependent diabetes mellitus
  • OLA oligonucleotide ligation assay
  • SNP single nucleotide polymorphism
  • Polynucleotide and oligonucleotide are used interchangeably and mean a linear polymer of at least 2 nucleotides joined together by phosphodiester bonds and may consist of either ribonucleotides or deoxyribonucleo tides.
  • 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
  • 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 AF 010238 for the vHL 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.,
  • 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 colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NTDDM, ischemic CM, ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ESRD due to IDDM, and seizure disorder.
  • SNPs single nucleotide polymorphisms
  • 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
  • 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.
  • 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. In addition, 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., mAlu 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 D A 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 D ⁇ A 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.
  • HAT heteroduplex analysis
  • D ⁇ A 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 Hydroli k gels where the presence of the heteroduplex is observed as an additional band.
  • 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
  • C cytosine
  • 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. Detection of Known Polymorphisms
  • 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., 8:769-776, 1998, and will only be briefly reviewed here.
  • array hybridization assay an example of which is the multiplexed allele-specific diagnostic assay (MASDA) (U.S. Patent No.
  • 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.
  • the ASO probes contain complementary sequences flanking the target specific species so that a hairpin structure is formed.
  • the loop of the hairpin is complimentary to the target sequence while each arm of the hairpin contains either donor or acceptor dyes.
  • the hairpin structure When not hybridized to a donor sequence, the hairpin structure brings the donor and acceptor dye close together thereby extinguishing the donor fluorescence. When hybridized to the specific target sequence, however, the donor and acceptor dyes are separated with an increase in fluorescence of up to 900 fold.
  • Molecular beacons can be used in conjunction with amplification of the target sequence by PCR and provide a 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 5' end and form a mismatch near the 3' end if the proper allele is not present. Using this system, zygosity is determined based on the size and number of PCR products produced.
  • the joining by DNA ligases of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3' end. This sensitivity has been utilized in the oligonucleotide ligation assay (Landegren et al., Science, 241:1077-1080, 1988) and the ligase chain reaction (LCR; Barany, Proc. Natl. Acad. Sci. USA, 88:189-193, 1991).
  • OLA the sequence surrounding the SNP is first amplified by PCR, whereas in LCR, genomic DNA can be used as a template.
  • amplified DNA templates are analyzed for their ability to serve as templates for ligation reactions between labeled oligonucleotide probes (Samotiaki et al., Genomics, 20:238-242, 1994).
  • two allele-specific probes labeled with either of two lanthanide labels (europium or terbium) 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
  • thermostable ligase and a thermostable DNA polymerase without 5' nuclease activity. Because FRET occurs only when the donor and acceptor dyes are in close proximity, ligation is inferred by the change in fluorescence.
  • minisequencing In another method for the detection of SNPs termed 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.
  • 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.
  • 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.
  • 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
  • the sequence including the polymorphic site is amplified by PCR using one amplification primer which is biotinylated on its 5' end.
  • the biotinylated PCR products are captured in streptavidin-coated microtitration wells, the wells washed, and the captured PCR products denatured.
  • a sequencing primer is then added whose 3' end binds immediately prior to the polymorphic site, and the primer is elongated by a DNA polymerase with one single labeled dNTP complementary to the nucleotide at the polymorphic site. After the elongation reaction, the sequencing primer is released and the presence of the labeled nucleotide detected.
  • dye labeled dideoxynucleoside triphosphates ddNTPs
  • ddNTPs dye labeled dideoxynucleoside triphosphates
  • ddNTP incorporation of the ddNTP is determined using an automatic gel sequencer. Minisequencing has also been adapted for use with microarrays (Shumaker et al., Human Mut., 7:346-354, 1996). In this case, elongation (extension) 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.
  • extension (elongation) reaction is carried out using a polymerase, a labeled dNTP and noncompeting ddNTPs. Incorporation 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 colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NTDDM, CVA due to NIDDM, ischemic CM, ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ES
  • the genetic variant is located at one of the polymorphic sites contained in Table 13. In another embodiment, the genetic variant is one of the variants contained in Table 13 or the complement of any of the variants contained in Table 13. 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 ID NO: 1.
  • the sequence contains at least one polymorphic site associated with a disease, and in particular colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NIDDM, ischemic CM, ischemic CM with
  • the genetic variant is located at one of the polymorphic sites contained in Table 13. In another embodiment, the genetic variant is one of the variants contained in
  • 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 colon cancer HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NTDDM, ischemic CM, ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ESRD due to IDDM, or seizure disorder.
  • kits contain, at a minimum, at least one polynucleotide of at least 10 contiguous nucleotides of SEQ ID NO 1, or the complements of SEQ ID NO: 1.
  • the polynucleotide contains at least one polymorphic site, preferably, the polymorphic site is located at one of the sites contained in Table 13. Alternatively the 3' end of the polynucleotide is immediately 5' to a polymorphic site, preferably a polymorphic site located at one of the sites contained in Table 13.
  • the polymorphic site contains a genetic variant as denominated in Table 13, or the complement of any of the variants contained in Table 13.
  • 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.
  • kits 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 polymorphisms 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 colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NIDDM, ischemic CM, ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ESRD due to IDDM, or 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.
  • 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 colon cancer, HTN, ASPVD due to HTN, CVA due to HTN, cataracts due to HTN, HTN CM, MI due to HTN, ESRD due to HTN, NIDDM, ASPVD due to NIDDM, CVA due to NIDDM, ischemic CM, ischemic CM with NIDDM, MI due to NIDDM, afib without valvular disease, alcohol abuse, alcoholic cirrhosis, anxiety, asthma, COPD, cholecystectomy, DJD, ESRD and frequent de-clots, ESRD due to FSGS, ESRD due to NIDDM, ESRD due to IDDM, or seizure disorder due to the presence of one or more single nucleotide polymorphisms.
  • genetic material such as
  • DNA, cDNA, RNA or mRNA is obtained from a patient and screened for the presence of one or more SNPs associated either directly or indirectly to a disease, condition, disorder or other pathological condition.
  • 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.
  • SNP single nucleotide polymorphism
  • 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.
  • 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 Td 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 hrnis et al., eds., PCR Protocols, Academic Press, 1990.
  • the sense primer was 5'- CCA AAC CTT AGA GGG GTG AA -3' (SEQ ID NO: 2).
  • the anti-sense primer was 5'- CTC CGC GAT CCA GAC CAC -3' (SEQ ID NO: 3).
  • the PCR product produced spanned positions 441 to 711 of the human vHL gene (SEQ ID NO: 1).
  • PCR amplification Approximately 25 ng of template leukocyte genomic DNA was used for each PCR amplification. Twenty-five microliters of an aqueous solution of genomic DNA (1 ng/ul) was dispensed to the wells of a 96-well plate, and dried down at 70C for 15 min. The DNA was rehydrated with 7 ⁇ l of ultra-pure but not autoclaved water (Milli-Q, Millipore Corp.). PCR conditions were as follows: 5 min at 94°C, followed by 45 cycles, where each cycle consisted of 94°C for 45 seconds to denature the double-stranded DNA, then 64°C for 45 seconds for specific annealing of primers to the single-stranded DNA, then 72°C for 45 seconds for extension. After the 45th cycle, the reaction mixture was held at 72°C for 10 min for a final extension reaction.
  • Post-PCR clean-up was performed as follows. PCR reactions were cleaned to remove unwanted primer and other impurities such as salts, enzymes, and unincorporated 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.
  • the 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.
  • the samples were washed twice using "PE Buffer.” Vacuum pressure was applied between each step to remove the buffer. Filtrate was similarly discarded after each wash. After the last PE Buffer wash, maximum vacuum pressure was applied to the membrane plate to generate maximum airflow through the membrane in order to evaporate residual ethanol left from the PE Buffer.
  • the clean PCR product was then eluted from the filter using "EB Buffer.”
  • the filtrate contained the cleaned PCR product and was collected. All buffers were supplied as part of the Qiaquick-96 PCR Purification Kit.
  • the vacuum manifold was also purchased from Qiagen for exclusive use with the Qiaquick-96 Purification Kit.
  • 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. After shaking on a plate shaker for 10 minutes, 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.
  • 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 unincorporated dye-labeled terminators that can obscure data at the beginning of the sequence.
  • a precipitation protocol was used. To each sequencing reaction in the 96-well plate, 20 ⁇ l of Milli-Q water and 60 ⁇ l of 100% isopropanol was added. The plate was
  • the plate was spun in a plate centrifuge (Jouan) at 3,000 x g for 30 minutes.
  • the supernatant was discarded by inverting the plate onto several paper tissues (Kimwipes) folded to the size of the plate.
  • the inverted plate, with Kimwipes in place, was placed into the centrifuge (Jouan) and spun at 700 x g for 1 minute. The Kimwipes were discarded and the samples were loaded onto a sequencing gel.
  • Sequencing run settings were as follows: run module 48E-1200, 8 hr collection time, 2400 V electrophoresis voltage, 50 mA electrophoresis current, 200 W electrophoresis power, CCD offset of 0, gel temperature of 51 °C, 40 mW laser power, and CCD gain of 2.
  • Pyrosequencing is a 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 condition supplied by the manufacturer. Primers were designed to anneal within 5 bases of the polymorphism, 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 polymorphisms of interest.
  • 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.
  • 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 polymorphic 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 then 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.
  • Haldane's correction was used if the denominator was zero (multiplying all cells by 2 and adding 1).
  • An odds ratio incorporating Haldane's correction is indicated by a superscript "H.” If the odds ratio (OR) was > 1.5, the 95% confidence interval (C.I.) 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.
  • Haldane's correction was employed. An example of that calculation follows:
  • the susceptibility allele (S) is indicated; the alternative allele at this locus is defined as the protective allele (P).
  • the odds ratio (OR) for the SS and SP genotypes is 1, since it is the reference group, and is not presented separately.
  • odds ratios > 1.5 the 95% confidence interval (CI.) is also given, in parentheses.
  • 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. "[E]pidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios ⁇ 1.5)." Id. at 66.
  • 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-
  • the observed genotype frequencies were 15% A/ A, 35% A/G, and 50% G/G, in fair agreement with those predicted for Hardy- Weinberg equilibrium.
  • the observed genotype frequencies were 11% A A, 32% A G, and 58% G/G, in close agreement with those predicted for Hardy- Weinberg equihbrium.
  • the observed genotype frequencies were 82% A/A, 12% A/G, and 6% G/G, in fair agreement with those predicted for Hardy- Weinberg equilibrium.
  • the observed genotype frequencies were 55% A/A, 25% A/G, and 20% G/G, in distant agreement with those predicted for Hardy- Weinberg equilibrium.
  • the odds ratio for the G allele as a risk factor for disease was 1.5 (95% CI, 0.3-6.7).
  • the odds ratio for the homozygote (GG) was 1.6 (95% CI, 0.1-28.1).
  • the heterozygote (GA genotype) had the same odds ratio of 1.6 (95%> CL,
  • the odds ratio for the G allele as a risk factor for disease was 2.1 (95%) CI, 0.3-14.3) relative to black men with hypertension but no renal disease.
  • the odds ratio for the homozygote (GG) was 5.7
  • the odds ratio for the G allele as a risk factor for disease was 1.9 (95% CI, 0.5-6.9) relative to black women with hypertension but no renal disease.
  • the odds ratio for the homozygote (GG) was 2.3 (95% CI, 0.3-20.1), while the heterozygote (GA genotype) had an odds ratio close to 1 (0.8).
  • the odds ratio for the G allele as a risk factor for disease was 3.0 (95% CI, 0.7-13.1) relative to white men with hypertension but no renal disease.
  • the odds ratio for the homozygote (GG) was 4.5 (95% CI, 0.3-65.2), whereas the heterozygote (GA genotype) had an odds ratio of 2.3 (95% CI, 0.2-22.1).
  • the odds ratio for the A allele as a risk factor for disease was 2.9 (95% CI, 0.6-14.8) relative to white women with hypertension but no renal disease.
  • the odds ratio for the homozygote (AA) was 4.3 (95%. CI, 0.5- 38.4) H .
  • the heterozygote (AG genotype) had essentially the same odds ratio of 4.1 (95% CL, 0.4-41.7)".
  • the odds ratio for the G allele as a risk factor for disease was 12.3 (95% CI, 1.6-95.4) H .
  • the odds ratio for the homozygote (GG) was 4.3 (95%> CI, 0.5-39.4)", while the heterozygote (GA genotype) actually had an odds ratio indistinguishable from 1 [0.5 (95% CI, 0-8.6)"].
  • the odds ratio for the G allele as a risk factor for disease was 6.0 (95% CI, 1.5-24.3).
  • the odds ratio for the homozygote (GG) was 10.5
  • the odds ratio for the A allele as a risk factor for disease was 2.8 (95% CI, 0.3-28.5)" relative to black men with NTDDM but no renal disease.
  • the odds ratio for the homozygote (AA) was 1.0 H
  • the heterozygote (AG genotype) had a higher odds ratio of 3.0 (95% CL, 0.3-33)".
  • the odds ratio for the A allele as a risk factor for disease was 1.5 (95%> CI, 0.4-6.3) relative to black women with NIDDM but no renal disease.
  • the odds ratio for the homozygote (AA) was 2.0 (95% CI, 0.1-27.4), whereas the heterozygote (AG genotype) had an odds ratio of 1.0.
  • the odds ratio for the G allele as a risk factor for disease was 1.5 (95% CI, 0.4-6.3) relative to white women with NIDDM but no renal disease.
  • the odds ratio for the homozygote (GG) was 1.7 (95% CI, 0.1-18.9), while the heterozygote (GA genotype) had a similar odds ratio of 2.5 (95% CI, 0.2-32.2).
  • HLF_01 A potential binding site for hepatic leukemia factor (HLF_01 as abbreviated by GENOMATIX; Hunger et al., Mol. Cell Biol, 14:5986-5996, 1994).
  • the consensus binding sequence for HLF_01 consists of the 10 nucleotides 5'- RTTACRYA4T-3' (SEQ ID NO: 4). This sequence occurs between nucleotides 512 and 521, inclusive, on the (+) strand, with a matrix score of 0.845 (where 1.000 represents a perfect match).
  • the A520 ⁇ >G SNP replaces the indicated A with a G.
  • HLF_01 binding sites occur on average 1.69 times per 1000 base pairs of random vertebrate genomic DNA.
  • VBPF PAR-type chicken vitellogenin promoter-b
  • the consensus binding sequence for VBPF consists of the ten nucleotides 5'-GTTACRTN_4N-3'. This sequence occurs between nucleotides 512 and 521, inclusive, on the (+) strand, with a matrix score of 0.884 (where 1.000 represents a perfect match).
  • the A520->G SNP replaces the indicated A with a G.
  • CEBP_C CCAAT/Enhancer Binding Protein
  • SEF1_C by GENOMATIX.
  • SEFl denotes a family of proteins which bind to a T cell-specific enhancer of the SL3-3 mouse leukemia virus, as well as to similar enhancers in cellular genes, including the T cell antigen receptor (Hallberg et al., Nucl Acids Res., 20(24):6495-6499, 1992; Thornell, J. Biol. Chem., 268(29):21946-21954, 1993).
  • the consensus binding sequence for SEFl consists of the 19 nucleotides complementary to 5'-
  • RACCAC GATATCCNTGTT-3' (SEQ ID NO: 6).
  • the complement is located on the (-) strand, across from nucleotides 514-532 on the (+) strand.
  • the match for this sequence has a score of 0.712, where 1.000 represents a perfect match.
  • the A520 ⁇ >G SNP replaces the indicated A with a G.
  • SEF1_C binding sites occur extremely rarely, less than 0.01 times per 1000 base pairs of random vertebrate genomic DNA.
  • 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.
  • Haldane's correction was used if the denominator was zero (multiplying all cells by 2 and adding 1).
  • An odds ratio incorporating Haldane's correction is indicated by a superscript "H.” 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. "[E]pidemiology in general and case-control studies in particular are not well suited for detecting weak associations (odds ratios ⁇ 1.5)." Id. at 66.
  • 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 equihbrium, as follows:
  • the observed genotype frequencies were 84% C/C, 16% C/T, and 0% T/T, in very close agreement with those predicted for Hardy- Weinberg equilibrium.
  • the observed genotype frequencies were 100% C/C, 0% C/T, and 0% T/T, in perfect agreement with those predicted for
  • the observed genotype frequencies were 100% C/C, 0% C/T, and 0% T/T, in perfect agreement with those predicted for
  • genotype frequencies were 82.0% C/C, 2.0% C/T, and
  • the odds ratio for the C allele as a risk factor for disease was 2.1 (95% CI, 0.2-25.3) relative to black men with hypertension but no renal disease.
  • the odds ratio for the homozygote (CC) was 3.1 H (95% CI, 0.3-28.3), while the heterozygote (CT genotype) had essentially the same odds ratio of 3.0 H (95% CI, 0.2-39.6).
  • the odds ratio for the T allele as a risk factor for disease was 3.2 (95% CI, 0.6-17.1).
  • the odds ratio for the homozygote (TT) was 2.1 H (95% CI, 0.2-24.0), whereas the heterozygote (TC genotype) had a much higher odds ratio of 22.5 (95%o CL, 1.5-335).
  • the odds ratio for the T allele as a risk factor for disease was 1.9 (95%> CI, 0.3-13.1).
  • the odds ratio for the homozygote (TT) was 3.0 H (95% CI, 0.2-52.1), whereas the heterozygote (TC genotype) had a smaller odds ratio of
  • the odds ratio for the T allele as a risk factor for disease was 9.4 H (95% CI, 0.9-94.6).
  • the odds ratio for the homozygote (TT) was 3.3 H (95% CI, 0.2-56.6), whereas the heterozygote (TC genotype) had a three-fold higher odds ratio of 9.9 H (95% CL, 0.9-104).
  • the odds ratio for the C allele as a risk factor for disease was 13.4 H (95% CI, 1.5-123) relative to black men with NIDDM but no renal disease.
  • the odds ratio for the homozygote (CC) was 3.7 H (95% CI, 0.2-77.6), while the heterozygote (CT genotype) had an odds ratio of even less than 1 H .
  • the odds ratio for the T allele was 6.4 H (95% CI, 0.3 - 160.4).
  • the odds ratio for the homozygote (T/T) was 0.5 (95% CI, 0 - 7.9), while the odds ratio for the heterozygote (C/T) was 3.0 (95% CI, 0 - 473.1).
  • the odds ratio for the T allele was 2.7 (95% CI, 0.7 - 11.2), compared to African-Americans with MI due to NIDDM.
  • the odds ratio for the homozygote (T/T) was 0.4 (95% CI, 0 - 4.9), while the odds ratio for the heterozygote (C/T) was 1.5 (95% CI, 0.1 - 40.6).
  • vHL gene is significantly associated with ESRD due to IDDM in African- Americans, i.e. abnormal activity of the vHL gene predisposes African- Americans to ESRD due to IDDM.
  • the odds ratio for the C allele was 3.8 (95% CI, 0.9 - 15.2), compared to African-Americans with MI due to HTN.
  • the odds ratio for the homozygote (C/C) was 3.1 (95%> CI, 0.3- 38), while the odds ratio for the heterozygote (C/T) was 0.5 (95% CI, 0 -12.9).
  • WT1_B Wilm's Tumor transcription factor
  • the consensus binding sequence for WT1 B consists of the 13 nucleotides 5'-GNGTGGGSG;CGNS-3' (SEQ ID NO: 7). This sequence occurs on the (-) strand of the vHL promoter.
  • ->T SNP replaces the indicated G on the (-) strand with an A.
  • the complement of this sequence 5'-SNCG£SCCCACNC-3' (SEQ ID NO: 8), occurs on the (+) strand (nucleotides 634-646, inclusive).
  • the C638 ⁇ >T SNP replaces the indicated C with a T on the (+) strand.
  • the complement of this T is an A on the (-) strand.
  • the WT1_B binding sequence matches nucleotides 634-646 on the (-) strand with a matrix score of 0.907 (where 1.000 represents a perfect match). WT1_B binding sites occur on average 0.97 times per 1000 base pairs of random vertebrate genomic DNA. The effect of the C638 ⁇ >T SNP is predicted to be weakening of the WT1_B binding site, although it is unknown whether WT1_B acts as a transcriptional activator or repressor of the vHL gene.
  • the C638 ⁇ >T SNP disturbs a binding site for the Wilm's Tumor gene product, which itself is a transcription factor and tumor suppressor specific for a kidney tumor (nephroblastoma).
  • the vHL gene of course, is also a tumor suppressor involved rather specifically in renal cell cancer. This is the only WT1_B binding site in the vHL promoter (nucleotides 1-643 of Accession Number AF010238).
  • WT1_B binding site in the vHL promoter nucleotides 1-643 of Accession Number AF010238.
  • the unique WT1_B site effected by the C638->T SNP suggests that WTl acts as a transcriptional activator of the vHL gene.
  • the effect of the T allele is therefore predicted to be a decrease in transcription of the vHL gene.
  • the specificity of the interaction between WTl and vHL suggests that this effect of the T allele may be a potent one.

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  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un polymorphisme de nucléotide simple (SNP) associé aux états pathologiques et troubles suivants : cancer du colon, hypertension, maladie vasculaire périphérique athéroscléreuse due à l'hypertension, accident vasculaire cérébral dû à l'hypertension, cataractes dues à l'hypertension, cardiomyopathie hypertensive, infarctus du myocarde dû à l'hypertension, insuffisance rénale terminale due à l'hypertension, diabète non insulino-dépendant, maladie vasculaire périphérique athéroscléreuse due au diabète non insulino-dépendant, accident vasculaire cérébral dû au diabète non insulino-dépendant, cardiomyopathie ischémique, cardiomyopathie ischémique avec diabète non insulino-dépendant, infarctus du myocarde dû au diabète non insulino-dépendant, fibrillation auriculaire sans affection valvulaire, abus d'alcool, cirrhose alcoolique, anxiété, asthme, bronchopneumopathie chronique obstructive, cholécystectomie, pathologie articulaire dégénérative, insuffisance rénale terminale et décaillotages fréquents, insuffisance rénale terminale due à la sclérose glomérulaire segmentaire et focale, insuffisance rénale terminale due au diabète insulino-dépendant, insuffisance rénale terminale due au diabète non insulino-dépendant et trouble épileptique. L'invention concerne également des méthodes faisant appel au SNP pour déterminer la susceptibilité à ces maladies, des séquences nucléotidiques contenant le SNP, des kits permettant de déterminer la présence du SNP et des méthodes de traitement ou de prophylaxie reposant sur la présence du SNP.
PCT/US2001/024985 2000-08-09 2001-08-09 Polymorphisme diagnostique du promoteur vhl WO2002012567A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2001284779A AU2001284779A1 (en) 2000-08-09 2001-08-09 Vhl promoter diagnostic polymorphism
US10/344,908 US20040166491A1 (en) 2001-08-09 2001-08-09 Vhl promoter diagnostic polymorphism
CA002418094A CA2418094A1 (fr) 2000-08-09 2001-08-09 Polymorphisme diagnostique du promoteur vhl
EP01963861A EP1307595A4 (fr) 2000-08-09 2001-08-09 Polymorphisme diagnostique du promoteur vhl

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US22408400P 2000-08-09 2000-08-09
US60/224,084 2000-08-09

Publications (1)

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WO2002012567A1 true WO2002012567A1 (fr) 2002-02-14

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EP (1) EP1307595A4 (fr)
AU (1) AU2001284779A1 (fr)
CA (1) CA2418094A1 (fr)
WO (1) WO2002012567A1 (fr)

Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP2035439A1 (fr) * 2006-06-05 2009-03-18 Cancer Care Ontario Évaluation de risque pour cancer colorectal

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324631A (en) * 1987-11-13 1994-06-28 Timothy Helentjaris Method and device for improved restriction fragment length polymorphism analysis
US5645995A (en) * 1996-04-12 1997-07-08 Baylor College Of Medicine Methods for diagnosing an increased risk for breast or ovarian cancer
US6312890B1 (en) * 1993-05-14 2001-11-06 The United States Of America As Represented By The Department Of Health And Human Services Partial intron sequence of von hippel-lindau (VHL) disease gene and its use in diagnosis of disease

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6013436A (en) * 1994-07-08 2000-01-11 Visible Genetics, Inc. Compositions and methods for diagnosis of mutation in the von Hippel-Lindau tumor suppressor gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5324631A (en) * 1987-11-13 1994-06-28 Timothy Helentjaris Method and device for improved restriction fragment length polymorphism analysis
US6312890B1 (en) * 1993-05-14 2001-11-06 The United States Of America As Represented By The Department Of Health And Human Services Partial intron sequence of von hippel-lindau (VHL) disease gene and its use in diagnosis of disease
US5645995A (en) * 1996-04-12 1997-07-08 Baylor College Of Medicine Methods for diagnosing an increased risk for breast or ovarian cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1307595A4 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2035439A1 (fr) * 2006-06-05 2009-03-18 Cancer Care Ontario Évaluation de risque pour cancer colorectal
EP2035439A4 (fr) * 2006-06-05 2010-01-13 Cancer Care Ontario Évaluation de risque pour cancer colorectal
US8153369B2 (en) 2006-06-05 2012-04-10 Cancer Care Ontario Assessment of risk for colorectal cancer

Also Published As

Publication number Publication date
CA2418094A1 (fr) 2002-02-14
EP1307595A4 (fr) 2004-09-29
EP1307595A1 (fr) 2003-05-07
AU2001284779A1 (en) 2002-02-18

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