EP1240354A2 - Einzelnukleotidpolymorphismen in genen - Google Patents

Einzelnukleotidpolymorphismen in genen

Info

Publication number
EP1240354A2
EP1240354A2 EP00959964A EP00959964A EP1240354A2 EP 1240354 A2 EP1240354 A2 EP 1240354A2 EP 00959964 A EP00959964 A EP 00959964A EP 00959964 A EP00959964 A EP 00959964A EP 1240354 A2 EP1240354 A2 EP 1240354A2
Authority
EP
European Patent Office
Prior art keywords
individual
vascular disease
nucleotide
nucleic acid
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00959964A
Other languages
English (en)
French (fr)
Inventor
Eric S. Lander
Michele Gargill
James S. Ireland
Stacey Bolk
George Q. Daley
Jeanette J. Mccarthy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Whitehead Institute for Biomedical Research
Millennium Pharmaceuticals Inc
Original Assignee
Whitehead Institute for Biomedical Research
Millennium Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Whitehead Institute for Biomedical Research, Millennium Pharmaceuticals Inc filed Critical Whitehead Institute for Biomedical Research
Publication of EP1240354A2 publication Critical patent/EP1240354A2/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the variant form may confer an evolutionary advantage or disadvantage relative to a progenitor form, or may be neutral.
  • a variant form confers a lethal disadvantage and is not transmitted to subsequent generations of the organism.
  • a variant form confers an evolutionary advantage to the species and is eventually incorporated into the DNA of many or most members of the species and effectively becomes the progenitor form.
  • a restriction fragment length polymo ⁇ hism is a variation in DNA sequence that alters the length of a restriction fragment (Botstein et al., Am. J. Hum. Genet. 32, 314-331 (1980)).
  • the restriction fragment length polymo ⁇ hism may create or delete a restriction site, thus changing the length of the restriction fragment.
  • RFLPs have been widely used in human and animal genetic analyses (see WO 90/13668; W090/11369; Donis-Keller, Cell 51, 319-337 (1987); Lander et al., Genetics 121, 85-99 (1989)). When a heritable trait can be linked to a particular RFLP, the presence of the RFLP in an individual can be used to predict the likelihood that the animal will also exhibit the trait.
  • VNTR variable number tandem repeat
  • polymo ⁇ hisms take the form of single nucleotide variations between individuals of the same species. Such polymo ⁇ hisms are far more frequent than RFLPs, STRs and VNTRs. Some single nucleotide polymo ⁇ hisms (SNP) occur in protein-coding nucleic acid sequences (coding sequence SNP (cSNP)), in which case, one of the polymo ⁇ hic forms may give rise to the expression of a defective or otherwise variant protein and, potentially, a genetic disease.
  • SNP single nucleotide polymo ⁇ hisms
  • cSNP protein-coding nucleic acid sequences
  • genes in which polymo ⁇ hisms within coding sequences give rise to genetic disease include ⁇ -globin (sickle cell anemia), apoE4 (Alzheimer's Disease), Factor V Leiden (thrombosis), and CFTR (cystic fibrosis).
  • cS Ps can alter the codon sequence of the gene and therefore specify an alternative amino acid. Such changes are called “missense” when another amino acid is substituted, and "nonsense" when the alternative codon specifies a stop signal in protein translation. When the cSNP does not alter the amino acid specified the cSNP is called "silent".
  • Single nucleotide polymo ⁇ hisms occur in noncoding regions. Some of these polymo ⁇ hisms may also result in defective protein expression (e.g., as a result of defective splicing). Other single nucleotide polymo ⁇ hisms have no phenotypic effects. Single nucleotide polymo ⁇ hisms can be used in the same manner as
  • Single nucleotide polymo ⁇ hisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymo ⁇ hism.
  • the greater frequency and uniformity of single nucleotide polymo ⁇ hisms means that there is a greater probability that such a polymo ⁇ hism will be found in close proximity to a genetic locus of interest than would be the case for other polymo ⁇ hisms.
  • the different forms of characterized single nucleotide polymo ⁇ hisms are often easier to distinguish than other types of polymo ⁇ hism (e.g., by use of assays employing allele-specific hybridization probes or primers).
  • the invention relates to a gene which comprises a single nucleotide polymo ⁇ hism at a specific location.
  • the invention relates to the variant allele of a gene having a single nucleotide polymo ⁇ hism, which variant allele differs from a reference allele by one nucleotide at the site(s) identified in the Table and Fig. 3.
  • Complements of these nucleic acid sequences are also included.
  • the nucleic acid molecules can be DNA or RNA, and can be double- or single-stranded. Nucleic acid molecules can be, for example, 5-10, 5-15, 10-20, 5-25, 10-30, 10-50 or 10-100 bases long.
  • the invention further provides allele-specific oligonucleotides that hybridize to the reference or variant allele of a gene comprising a single nucleotide polymo ⁇ hism or to the complement thereof. These oligonucleotides can be probes or primers.
  • the invention further provides a method of analyzing a nucleic acid from an individual.
  • the method determines which base is present at any one of the polymo ⁇ hic sites shown in the Table and/or Fig. 3.
  • a set of bases occupying a set of the polymo ⁇ hic sites shown in the Table and /or Fig. 3 is determined.
  • This type of analysis can be performed on a number of individuals, who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymo ⁇ hic site or sites in the individuals tested.
  • the invention further relates to a method of predicting the presence, absence, likelihood of the presence or absence, or severity of a particular phenotype or disorder associated with a particular genotype.
  • the method comprises obtaining a nucleic acid sample from an individual and determining the identity of one or more bases (nucleotides) at polymo ⁇ hic sites of genes described herein, wherein the presence of a particular base is correlated with a specified phenotype or disorder, thereby predicting the presence, absence, likelihood of the presence or absence, or severity of the phenotype or disorder in the individual.
  • thrombospondins are a family of extracellular matrix (ECM) glycoproteins that modulate many cell behaviors including adhesion, migration, and proliferation.
  • ECM extracellular matrix
  • Thrombospondins also known as thrombin sensitive proteins or TSPs
  • TSPs thrombin sensitive proteins
  • SNPs in these genes which are associated with premature coronary artery disease (CAD)(or coronary heart disease) and myocardial infarction (MI) have been identified and represent a potentially vital marker of upstream biology influencing the complex process of atherosclerotic plaque generation and vulnerability.
  • CAD premature coronary artery disease
  • MI myocardial infarction
  • the invention relates to the TSP gene SNPs identified as described herein, both singly and in combination, as well as to the use of these SNPs, and others in TSP genes, particularly those nearby in linkage disequilibrium with these SNPs, for diagnosis, prediction of clinical course and treatment response for vascular disease, development of new treatments for vascular disease based upon comparison of the variant and normal versions of the gene or gene product, and development of cell-culture based and animal models for research and treatment of vascular disease.
  • the invention further relates to novel compounds and pharmaceutical compositions for use in the diagnosis and treatment of such disorders.
  • the vascular disease is CAD or MI.
  • the invention relates to isolated nucleic acid molecules comprising all or a portion of the variant allele of TSP-1 (e.g., as exemplified by SEQ ID NO: 1), and to isolated nucleic acid molecules comprising all or a portion of the variant allele of TSP-4 (e.g., as exemplified by SEQ ID NO: 3).
  • Preferred portions are at least 10 contiguous nucleotides and comprise the polymo ⁇ hic site, e.g., a portion of SEQ ID NO: 1 which is at least 10 contiguous nucleotides and comprises the "G" at position 2210, or a portion of SEQ ID NO: 3 which is at least 10 contiguous nucleotides and comprises the "C" at position 1186.
  • the invention further relates to isolated gene products, e.g., polypeptides or proteins, which are encoded by a nucleic acid molecule comprising all or a portion of the variant allele of TSP-1 or TSP-4 (e.g., SEQ ID NO: 1 or SEQ ID NO: 3, respectively).
  • the invention also relates to nucleic acid molecules which hybridize to and/or share identity with the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site.
  • the invention further relates to isolated proteins or polypeptides comprising all or a portion of the variant amino acid sequence of TSP-1 (e.g., as exemplified by SEQ ID NO: 2), and to isolated proteins or polypeptides comprising all or a portion of the variant amino acid sequence of TSP-4 (e.g., as exemplified by SEQ ID NO: 4).
  • Preferred polypeptides are at least 10 contiguous amino acids and comprise the polymo ⁇ hic amino acid, e.g., a portion of SEQ ID NO: 2 which is at least 10 contiguous amino acids and comprises the serine at residue 700, or a portion of SEQ ID NO: 4 which is at least 10 contiguous amino acids and comprises the proline at residue 387.
  • the invention further relates to isolated nucleic acid molecules encoding such proteins and polypeptides, as well as to antibodies which bind, e.g., specifically, to such proteins and polypeptides.
  • the invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with the presence of one or more of (a) a G at nucleotide position 2210 of SEQ ID NO: 1 ; or (b) a C at nucleotide position 1186 of SEQ ID NO: 3 in an individual.
  • the method comprises obtaining a nucleic acid sample from the individual and determining the nucleotide present at one or more of the indicated nucleotide positions, wherein presence of one or more of (a) a G at nucleotide position 2210 of SEQ ID NO: 1 ; or (b) a C at nucleotide position 1186 of SEQ ID NO: 3 is indicative of increased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the reference nucleotide at one or more of said positions.
  • the disorder is a vascular disease selected from the group consisting of atherosclerosis, coronary heart or artery disease, MI, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.
  • the vascular disease is selected from the group consisting of CAD and MI.
  • the invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with one or more of (a) a G at nucleotide position 2210 of SEQ ID NO: 1; or (b) a C at nucleotide position 1186 of SEQ ID NO: 3 in an individual.
  • the method comprises obtaining a nucleic acid sample from the individual and determining the nucleotide present at one or more of the indicated nucleotide positions, wherein presence of one or more of (a) an A at nucleotide position 2210 of SEQ ID NO: 1; or (b) a G at nucleotide position 1186 of SEQ ID NO: 3 is indicative of decreased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the variant nucleotide at said position.
  • the disorder is a vascular disease selected from the group consisting of atherosclerosis, coronary heart or artery disease, MI, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.
  • the vascular disease is selected from the group consisting of CAD and MI.
  • the invention relates to a method for predicting the likelihood that an individual will have a vascular disease (or aiding in the diagnosis of a vascular disease), comprising the steps of obtaining a DNA sample from an individual to be assessed and determining the nucleotide present at one or more of nucleotide positions 2210 of SEQ ID NO: 1 or 1186 of SEQ ID NO: 3.
  • the presence of the reference nucleotide at one or more of these positions indicates that the individual has a lower likelihood of having a vascular disease than an individual having the variant nucleotide at one or more of these positions, or a lower likelihood of having severe symptomology.
  • the individual is an individual at risk for development of a vascular disease.
  • the invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with the presence of one or more of (a) a serine at amino acid position 700 of SEQ ID NO: 2; or (b) a proline at amino acid position 387 of SEQ ID NO: 4 in an individual.
  • the method comprises obtaining a biological sample containing the TSP-1 and/or TSP-4 protein or relevant portion thereof from the individual and determining the amino acid present at one or more of the indicated amino acid positions, wherein presence of one or more of (a) a serine at amino acid position 700 of SEQ ID NO: 2; or (b) a proline at amino acid position 387 of SEQ ID NO: 4 is indicative of increased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the reference amino acid at one or more of said positions.
  • the invention further relates to a method of diagnosing or aiding in the diagnosis of a disorder associated with one or more of (a) a serine at amino acid position 700 of SEQ ID NO: 2; or (b) a proline at amino acid position 387 of SEQ ID NO: 4 in an individual.
  • the method comprises obtaining a biological sample containing the TSP-1 and/or TSP-4 protein or relevant portion thereof from the individual and determining the amino acid present at one or more of the indicated amino acid positions, wherein presence of one or more of (a) an asparagine at amino acid position 700 of SEQ ID NO: 2; or (b) an alanine at amino acid position 387 of SEQ ID NO: 4 is indicative of decreased likelihood of said disorder in the individual as compared with an appropriate control, e.g., an individual having the variant amino acid at one or more of said positions.
  • the invention relates to a method for predicting the likelihood that an individual will have a vascular disease (or aiding in the diagnosis of a vascular disease), comprising the steps of obtaining a biological sample comprising the TSP-1 and/or TSP-4 protein or relevant portion thereof from an individual to be assessed and determining the amino acid present at one or more of amino acid positions 700 of SEQ ID NO: 2 or 387 of SEQ ID NO: 4.
  • the presence of the reference amino acid at one or more of these positions indicates that the individual has a lower likelihood of having a vascular disease than an individual having the variant amino acid at one or more of these positions, or a lower likelihood of having severe symptomology.
  • the individual is an individual at risk for development of a vascular disease.
  • the invention in another embodiment, relates to pharmaceutical compositions comprising a reference TSP-1 and/or TSP-4 gene or gene product, or active portion thereof, for use in the treatment of vascular diseases.
  • the invention further relates to the use of agonists and antagonists of TSP-1 and TSP-4 activity for use in the treatment of vascular diseases.
  • the vascular disease is selected from the group consisting of atherosclerosis, coronary heart or artery disease, MI, stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.
  • the vascular disease is selected from the group consisting of CAD and MI.
  • Figs. 1A-1D show the reference nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO: 2) sequences for TSP-1.
  • Figs. 2A-2C show the reference nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequences for TSP-4.
  • Fig. 3 shows a table providing detailed information about the SNPs identified herein. Column one shows the internal polymo ⁇ hism identifier. Column two shows the accession number for the reference sequence in the TIGR database
  • the present invention relates to a gene which comprises a single nucleotide polymo ⁇ hism (SNP) at a specific location.
  • the gene which includes the SNP has at least two alleles, referred to herein as the reference allele and the variant allele.
  • the reference allele (prototypical or wild type allele) has been designated arbitrarily and typically corresponds to the nucleotide sequence of the gene which has been deposited with GenBank or TIGR under a given Accession number.
  • the variant allele differs from the reference allele by one nucleotide at the site(s) identified in the Table.
  • the present invention also relates to variant alleles of the described genes and to complements of the variant alleles.
  • the invention also relates to nucleic acid molecules which hybridize to and/or share identity with the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site.
  • the invention further relates to portions of the variant alleles and portions of complements of the variant alleles which comprise (encompass) the site of the SNP and are at least 5 nucleotides in length. Portions can be, for example, 5-10, 5-15, 10- 20, 5-25, 10-30, 10-50 or 10-100 bases long.
  • a portion of a variant allele which is 21 nucleotides in length includes the single nucleotide polymo ⁇ hism (the nucleotide which differs from the reference allele at that site) and twenty additional nucleotides which flank the site in the variant allele. These nucleotides can be on one or both sides of the polymo ⁇ hism.
  • Polymo ⁇ hisms which are the subject of this invention are defined in the Table with respect to the reference sequence deposited in GenBank or TIGR under the Accession number indicated.
  • the invention relates to a portion of a gene (e.g., AT3) having a nucleotide sequence as deposited in GenBank (e.g., UI 1270) comprising a single nucleotide polymo ⁇ hism at a specific position (e.g., nucleotide 11918).
  • the reference nucleotide for AT3 is shown in column 8, and the variant nucleotide is shown in column 9 of the Table.
  • the nucleotide sequences of the invention can be double- or single-stranded.
  • the invention further provides allele-specific oligonucleotides that hybridize to the reference or variant allele of a gene comprising a single nucleotide polymo ⁇ hism or to the complement thereof. These oligonucleotides can be probes or primers.
  • the invention further provides a method of analyzing a nucleic acid from an individual.
  • the method determines which base is present at any one of the polymo ⁇ hic sites shown in the Table and/or Fig. 3.
  • a set of bases occupying a set of the polymo ⁇ hic sites shown in the Table and/or Fig. 3 is determined.
  • This type of analysis can be performed on a number of individuals, who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype is then correlated with a base or set of bases present at the polymo ⁇ hic site or sites in the individuals tested.
  • the invention further relates to a method of predicting the presence, absence, likelihood of the presence or absence, or severity of a particular phenotype or disorder associated with a particular genotype.
  • the method comprises obtaining a nucleic acid sample from an individual and determining the identity of one or more bases (nucleotides) at polymo ⁇ hic sites of genes described herein, wherein the presence of a particular base is correlated with a specified phenotype or disorder, thereby predicting the presence, absence, likelihood of the presence or absence, or severity of the phenotype or disorder in the individual.
  • a nucleic acid molecule or oligonucleotide can be DNA or RNA, and single- or double-stranded. Nucleic acid molecules and oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means. Preferred nucleic acid molecules and oligonucleotides of the invention include segments of DNA, or their complements, which include any one of the polymo ⁇ hic sites shown in the Table. The segments can be between 5 and 250 bases, and, in specific embodiments, are between 5-10, 5-20, 10-20, 10-50, 20-50 or 10-100 bases. For example, the segment can be 21 bases. The polymo ⁇ hic site can occur within any position of the segment.
  • the segments can be from any of the allelic forms of DNA shown in the Table.
  • the terms “nucleotide”, “base” and “nucleic acid” are intended to be equivalent.
  • the terms “nucleotide sequence”, “nucleic acid sequence”, “nucleic acid molecule” and “segment” are intended to be equivalent.
  • Hybridization probes are oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Such probes include peptide nucleic acids, as described in Nielsen et al., Science 254, 1497-1500 (1991). Probes can be any length suitable for specific hybridization to the target nucleic acid sequence. The most appropriate length of the probe may vary depending upon the hybridization method in which it is being used; for example, particular lengths may be more appropriate for use in micro fabricated arrays, while other lengths may be more suitable for use in classical hybridization methods. Such optimizations are known to the skilled artisan. Suitable probes and primers can range from about 5 nucleotides to about 30 nucleotides in length.
  • probes and primers can be 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28 or 30 nucleotides in length.
  • the probe or primer preferably overlaps at least one polymo ⁇ hic site occupied by any of the possible variant nucleotides.
  • the nucleotide sequence can correspond to the coding sequence of the allele or to the complement of the coding sequence of the allele.
  • primer refers to a single-stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • the appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
  • linkage describes the tendency of genes, alleles, loci or genetic markers to be inherited together as a result of their location on the same chromosome. It can be measured by percent recombination between the two genes, alleles, loci or genetic markers.
  • polymo ⁇ hism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymo ⁇ hic marker or site is the locus at which divergence occurs.
  • Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
  • a polymo ⁇ hic locus may be as small as one base pair.
  • Polymo ⁇ hic markers include restriction fragment length polymo ⁇ hisms, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats, simple sequence repeats, and insertion elements such as Alu.
  • the first identified allelic form is arbitrarily designated as the reference form and other allelic forms are designated as alternative or variant alleles.
  • the allelic form occurring most frequently in a selected population is sometimes referred to as the wildtype form. Diploid organisms may be homozygous or heterozygous for allelic forms.
  • a diallelic or biallelic polymo ⁇ hism has two forms.
  • a triallelic polymo ⁇ hism has three forms.
  • Work described herein pertains to the resequencing of large numbers of genes in a large number of individuals to identify polymo ⁇ hisms which can predispose individuals to disease.
  • polymo ⁇ hisms in genes which are expressed in liver may predispose individuals to disorders of the liver.
  • SNPs may alter the function of the encoded proteins.
  • the discovery of the SNP facilitates biochemical analysis of the variants and the development of assays to characterize the variants and to screen for pharmaceutical that would interact directly with one or another form of the protein.
  • SNPs include silent SNPs
  • a single nucleotide polymo ⁇ hism occurs at a polymo ⁇ hic site occupied by a single nucleotide, which is the site of variation between allelic sequences.
  • the site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of the populations).
  • a single nucleotide polymo ⁇ hism usually arises due to substitution of one nucleotide for another at the polymo ⁇ hic site.
  • 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 by a pyrimidine or vice versa.
  • Single nucleotide polymo ⁇ hisms can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.
  • the polymo ⁇ hic site is occupied by a base other than the reference base. For example, where the reference allele contains the base "T" at the polymo ⁇ hic site, the altered allele can contain a "C", "G" or "A" at the polymo ⁇ hic site.
  • the invention also relates to nucleic acid molecules which hybridize to the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site.
  • Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • stringent conditions for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • 5X SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30°C, or equivalent conditions are suitable for allele-specific probe hybridizations.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleotide sequence and the primer or probe used.
  • the invention also relates to nucleic acid molecules which share substantial sequence identity to the variant alleles identified herein (or their complements) and which also comprise the variant nucleotide at the SNP site.
  • nucleic acid molecules and fragments which have at least about 60%, preferably at least about 70, 80 or 85%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98% identity with nucleic acid molecules described herein.
  • the percent identity of two nucleotide or amino acid sequences can be determined by aligning the sequences for optimal comparison pu ⁇ oses (e.g., gaps can be introduced in the sequence of a first sequence).
  • the length of a sequence aligned for comparison pu ⁇ oses is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%, 80% or 90% of the length of the reference sequence.
  • the actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non- limiting example of such a mathematical algorithm is described in Karlin et al., Proc. Natl. Acad. Sci.
  • an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.
  • an isolated nucleic acid comprises at least about 50, 80 or 90 percent (on a molar basis) of all macromolecular species present.
  • Some of the novel polymo ⁇ hisms of the invention are shown in the Table. Columns one and two show designations for the indicated polymo ⁇ hism. Column three shows the Genbank or TIGR Accession number for the wild type (or reference) allele. Column four shows the location of the polymo ⁇ hic site in the nucleic acid sequence with reference to the Genbank or TIGR sequence shown in column three. Column five shows common names for the gene in which the polymo ⁇ hism is located. Column six shows the polymo ⁇ hism and a portion of the 3' and 5' flanking sequence of the gene. Column seven shows the type of mutation; N, non-sense, S, silent, M, missense.
  • Polymo ⁇ hisms are detected in a target nucleic acid from an individual being analyzed.
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • tissue sample must be obtained from an organ in which the target nucleic acid is expressed.
  • the target nucleic acid is a cytochrome P450
  • the liver is a suitable source.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the first type of analysis is carried out to identify polymo ⁇ hic sites not previously characterized (i.e., to identify new polymo ⁇ hisms). This analysis compares target sequences in different individuals to identify points of variation, i.e., polymo ⁇ hic sites.
  • de novo characterization is carried out to identify polymo ⁇ hic sites not previously characterized (i.e., to identify new polymo ⁇ hisms).
  • This analysis compares target sequences in different individuals to identify points of variation, i.e., polymo ⁇ hic sites.
  • groups of individuals representing the greatest ethnic diversity among humans and greatest breed and species variety in plants and animals patterns characteristic of the most common alleles/haplotypes of the locus can be identified, and the frequencies of such alleles/haplotypes in the population can be determined. Additional allelic frequencies can be determined for subpopulations characterized by criteria such as geography, race, or gender.
  • the de novo identification of polymo ⁇ hisms of the invention is described in the Examples section.
  • the second type of analysis determines which form(s) of a characterized (known) polymo ⁇ hism are present in individuals under test. There are a variety of suitable procedures, which are discussed in turn.
  • Allele-specific probes for analyzing polymo ⁇ hisms is described by e.g., Saiki et al, Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele-specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the co ⁇ esponding segment from another individual due to the presence of different polymo ⁇ hic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles.
  • Some probes are designed to hybridize to a segment of target DNA such that the polymo ⁇ hic site aligns with a central position (e.g., in a 15-mer at the 7 position; in a 16-mer, at either the 8 or 9 position) of the probe.
  • This design of probe achieves good discrimination in hybridization between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymo ⁇ hisms within the same target sequence.
  • the polymo ⁇ hisms can also be identified by hybridization to nucleic acid arrays, some examples of which are described in WO 95/11995.
  • nucleic acid arrays Some examples of which are described in WO 95/11995.
  • One form of such arrays is described in the Examples section in connection with de novo identification of polymo ⁇ hisms.
  • the same array or a different array can be used for analysis of characterized polymo ⁇ hisms.
  • WO 95/11995 also describes subarrays that are optimized for detection of a variant form of a precharacterized polymo ⁇ hism.
  • Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence.
  • the second group of probes is designed by the same principles as described in the Examples, except that the probes exhibit complementarity to the second reference sequence.
  • a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (e.g., two or more mutations within 9 to 21 bases).
  • An allele-specific primer hybridizes to a site on target DNA overlapping a polymo ⁇ hism and only primes amplification of an allelic form to which the primer exhibits perfect complementarity. See Gibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers, resulting in a detectable product which indicates the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymo ⁇ hic site and the other of which exhibits perfect complementarity to a distal site. The single-base mismatch prevents amplification and no detectable product is formed.
  • the method works best when the mismatch is included in the 3'-most position of the oligonucleotide aligned with the polymo ⁇ hism because this position is most destabilizing to elongation from the primer (see, e.g., WO 93/22456).
  • Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.
  • Single-Strand Conformation Polymo ⁇ hism Analysis Alleles of target sequences can be differentiated using single-strand conformation polymo ⁇ hism analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al, Proc. Nat. Acad. Sci. 86, 2766-2770 (1989).
  • Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products.
  • Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence differences between alleles of target sequences.
  • An alternative method for identifying and analyzing polymo ⁇ hisms is based on single-base extension (SBE) of a fluorescently-labeled primer coupled with fluorescence resonance energy transfer (FRET) between the label of the added base and the label of the primer.
  • SBE single-base extension
  • FRET fluorescence resonance energy transfer
  • the method such as that described by Chen et al, (PNAS 94: 10756-61 (1997), inco ⁇ orated herein by reference) uses a locus- specific oligonucleotide primer labeled on the 5' terminus with 5-carboxyfluorescein (FAM). This labeled primer is designed so that the 3' end is immediately adjacent to the polymo ⁇ hic site of interest.
  • FAM 5-carboxyfluorescein
  • the labeled primer is hybridized to the locus, and single base extension of the labeled primer is performed with fluorescently labeled dideoxyribonucleotides (ddNTPs) in dye-terminator sequencing fashion, except that no deoxyribonucleotides are present.
  • ddNTPs dideoxyribonucleotides
  • An increase in fluorescence of the added ddNTP in response to excitation at the wavelength of the labeled primer is used to infer the identity of the added nucleotide.
  • this information can be used in a number of methods.
  • polymo ⁇ hisms of the invention are often used in conjunction with polymo ⁇ hisms in distal genes.
  • Preferred polymo ⁇ hisms for use in forensics are biallelic because the population frequencies of two polymo ⁇ hic forms can usually be determined with greater accuracy than those of multiple polymo ⁇ hic forms at multi-allelic loci.
  • the capacity to identify a distinguishing or unique set of forensic markers in an individual is useful for forensic analysis. For example, one can determine whether a blood sample from a suspect matches a blood or other tissue sample from a crime scene by determining whether the set of polymo ⁇ hic forms occupying selected polymo ⁇ hic sites is the same in the suspect and the sample. If the set of polymo ⁇ hic markers does not match between a suspect and a sample, it can be concluded (barring experimental error) that the suspect was not the source of the sample. If the set of markers does match, one can conclude that the DNA from the suspect is consistent with that found at the crime scene.
  • p(ID) is the probability that two random individuals have the same polymo ⁇ hic or allelic form at a given polymo ⁇ hic site. In biallelic loci, four genotypes are possible: AA, AB, BA, and BB.
  • the probability of identity p(ID) for a 3-allele system where the alleles have the frequencies in the population of x, y and z, respectively is equal to the sum of the squares of the genotype frequencies: p(ID) - x 4 + (2xy) 2 + (2yz) 2 + (2xz) 2 + z 4 + y 4
  • the object of paternity testing is usually to determine whether a male is the father of a child. In most cases, the mother of the child is known and thus, the mother's contribution to the child's genotype can be traced. Paternity testing investigates whether the part of the child's genotype not attributable to the mother is consistent with that of the putative father. Paternity testing can be performed by analyzing sets of polymo ⁇ hisms in the putative father and the child.
  • the set of polymo ⁇ hisms in the child attributable to the father does not match the set of polymo ⁇ hisms of the putative father, it can be concluded, barring experimental error, that the putative father is not the real father. If the set of polymo ⁇ hisms in the child attributable to the father does match the set of polymo ⁇ hisms of the putative father, a statistical calculation can be performed to determine the probability of coincidental match.
  • the cumulative probability of exclusion of a random male is very high. This probability can be taken into account in assessing the liability of a putative father whose polymo ⁇ hic marker set matches the child's polymo ⁇ hic marker set attributable to his/her father.
  • the polymo ⁇ hisms of the invention may contribute to the phenotype of an organism in different ways. Some polymo ⁇ hisms occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both beneficial and detrimental, depending on the circumstances. For example, a heterozygous sickle cell mutation confers resistance to malaria, but a homozygous sickle cell mutation is usually lethal. Other polymo ⁇ hisms occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single polymo ⁇ hism may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by polymo ⁇ hisms in different genes.
  • polymo ⁇ hisms predispose an individual to a distinct mutation that is causally related to a certain phenotype.
  • Phenotypic traits include diseases that have known but hitherto unmapped genetic components (e.g., agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-Aldrich syndrome, Fabry's disease, familial hypercholesterolemia, polycystic kidney disease, hereditary spherocytosis, von Willebrand's disease, tuberous sclerosis, hereditary hemorrhagic telangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome, osteogenesis imperfecta, and acute intermittent po ⁇ hyria).
  • diseases that have known but hitherto unmapped genetic components (e.g., agammaglobulimenia, diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy, Wiskott-
  • Phenotypic traits also include symptoms of, or susceptibility to, multifactorial diseases of which a component is or may be genetic, such as autoimmune diseases, inflammation, cancer, diseases of the nervous system, and infection by pathogenic microorganisms.
  • autoimmune diseases include rheumatoid arthritis, multiple sclerosis, diabetes (insulin-dependent and non-independent), systemic lupus erythematosus and Graves disease.
  • Some examples of cancers include cancers of the bladder, brain, breast, colon, esophagus, kidney, leukemia, liver, lung, oral cavity, ovary, pancreas, prostate, skin, stomach and uterus.
  • Phenotypic traits also include characteristics such as longevity, appearance (e.g., baldness, obesity), strength, speed, endurance, fertility, and susceptibility or receptivity to particular drugs or therapeutic treatments.
  • the correlation of one or more polymo ⁇ hisms with phenotypic traits can be facilitated by knowledge of the gene product of the wild type (reference) gene.
  • the genes in which cSNPs of the present invention have been identified are genes which have been previously sequenced and characterized in one of their allelic forms.
  • Correlation is performed for a population of individuals who have been tested for the presence or absence of a phenotypic trait of interest and for polymo ⁇ hic markers sets.
  • a set of polymo ⁇ hisms i.e. a polymo ⁇ hic set
  • the alleles of each polymo ⁇ hism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the trait of interest.
  • Correlation can be performed by standard statistical methods such as a K-squared test and statistically significant correlations between polymo ⁇ hic form(s) and phenotypic characteristics are noted.
  • allele Al at polymo ⁇ hism A correlates with heart disease.
  • allele Bl at polymo ⁇ hism B correlates with increased milk production of a farm animal.
  • Such correlations can be exploited in several ways.
  • detection of the polymo ⁇ hic form set in a human or animal patient may justify immediate administration of treatment, or at least the institution of regular monitoring of the patient.
  • Detection of a polymo ⁇ hic form correlated with serious disease in a couple contemplating a family may also be valuable to the couple in their reproductive decisions.
  • the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymo ⁇ hism from her husband to her offspring.
  • Y, jkpn ⁇ + YS, + P. + X k + ⁇ , + ... ⁇ 17 + PE n + a profession +e p
  • Y ljknp is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record
  • ⁇ is an overall mean
  • YS is the effect common to all cows calving in year-season
  • X k is the effect common to cows in either the high or average selection line
  • ⁇ , to ⁇ , 7 are the binomial regressions of production record on mtDNA D-loop sequence polymo ⁇ hisms
  • PE n is permanent environmental effect common to all records of cow n
  • a municipality is effect of animal n and is composed of the additive genetic contribution of sire and dam breeding values and a Mendelian sampling effect
  • e p is a random residual. It was found that eleven of seventeen polymo ⁇ hisms tested influenced at least one production trait. Bovines
  • D. Genetic Mapping of Phenotypic Traits The previous section concerns identifying correlations between phenotypic traits and polymo ⁇ hisms that directly or indirectly contribute to those traits.
  • the present section describes identification of a physical linkage between a genetic locus associated with a trait of interest and polymo ⁇ hic markers that are not associated with the trait, but are in physical proximity with the genetic locus responsible for the trait and co-segregate with it.
  • Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al, Proc. Natl. Acad. Sci.
  • Linkage studies are typically performed on members of a family. Available members of the family are characterized for the presence or absence of a phenotypic trait and for a set of polymo ⁇ hic markers. The distribution of polymo ⁇ hic markers in an informative meiosis is then analyzed to determine which polymo ⁇ hic markers co-segregate with a phenotypic trait. See, e.g., Kerem et al, Science 245, 1073-1080 (1989); Monaco et al, Nature 316, 842 (1985); Yamoka et al, Neurology 40, 222- 226 (1990); Rossiter et al, FASEB Journal 5, 21-27 (1991).
  • Linkage is analyzed by calculation of LOD (log of the odds) values.
  • a lod value is the relative likelihood of obtaining observed segregation data for a marker and a genetic locus when the two are located at a recombination fraction ⁇ , versus the situation in which the two are not linked, and thus segregating independently (Thompson & Thompson, Genetics in Medicine (5th ed, W.B. Saunders Company, Philadelphia, 1991); Strachan, "Mapping the human genome” in The Human Genome (BIOS Scientific Publishers Ltd, Oxford), Chapter 4).
  • the likelihood at a given value of ⁇ is: probability of data if loci linked at ⁇ to probability of data if loci unlinked.
  • the computed likelihoods are usually expressed as the log 10 of this ratio (i.e., a lod score). For example, a lod score of 3 indicates 1000:1 odds against an apparent observed linkage being a coincidence.
  • the use of logarithms allows data collected from different families to be combined by simple addition. Computer programs are available for the calculation of lod scores for differing values of ⁇ (e.g., LIPED, MLINK (Lathrop, Proc. Nat. Acad. Sci. (USA) 81, 3443-3446 (1984)).
  • a recombination fraction may be determined from mathematical tables. See Smith et al, Mathematical tables for research workers in human genetics (Churchill, London, 1961); Smith, Ann. Hum. Genet. 32, 127-150 (1968). The value of ⁇ at which the lod score is the highest is considered to be the best estimate of the recombination fraction. Positive lod score values suggest that the two loci are linked, whereas negative values suggest that linkage is less likely (at that value of ⁇ ) than the possibility that the two loci are unlinked. By convention, a combined lod score of +3 or greater (equivalent to greater than 1000:1 odds in favor of linkage) is considered definitive evidence that two loci are linked.
  • Negative linkage data are useful in excluding a chromosome or a segment thereof from consideration. The search focuses on the remaining non- excluded chromosomal locations.
  • nucleic acids comprise one of the sequences described in the Table, column 5, in which the polymo ⁇ hic position is occupied by one of the alternative bases for that position.
  • Some nucleic acids encode full-length variant forms of proteins.
  • variant proteins have the prototypical amino acid sequences encoded by nucleic acid sequences shown in the Table, column 5, (read so as to be in- frame with the full-length coding sequence of which it is a component) except at an amino acid encoded by a codon including one of the polymo ⁇ hic positions shown in the Table. That position is occupied by the amino acid coded by the corresponding codon in any of the alternative forms shown in the Table.
  • Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter.
  • the promoter is a eukaryotic promoter for expression in a mammalian cell.
  • the transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host.
  • the selection of an appropriate promoter for example t ⁇ , lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected.
  • Commercially available expression vectors can be used.
  • Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.
  • the means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra.
  • a wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like.
  • gene product includes mRNA, peptide and protein products.
  • the protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, Methods in Enzymology Volume 104, Academic Press, New York (1984); Scopes, Protein Purification, Principles and Practice, 2nd Edition, Springer- Verlag, New York (1987); and Deutscher (ed), Guide to Protein Purification, Methods in Enzymology, Vol. 182 (1990). If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells.
  • the invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene and/or having one or both alleles of an endogenous variant gene inactivated.
  • Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote.
  • Inactivation of endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive selection marker. See Capecchi, Science 244, 1288-1292 (1989). The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals. Such animals provide useful drug screening systems.
  • the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides.
  • biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding.
  • Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
  • Antibodies that specifically bind to variant gene products but not to corresponding prototypical gene products are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.
  • kits comprising at least one allele-specific oligonucleotide as described herein.
  • the kits contain one or more pairs of allele-specific oligonucleotides hybridizing to different forms of a polymo ⁇ hism.
  • the allele-specific oligonucleotides are provided immobilized to a substrate.
  • the same substrate can comprise allele-specific oligonucleotide probes for detecting at least 10, 100 or all of the polymo ⁇ hisms shown in the Table.
  • kits include, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions.
  • the kit also contains instructions for carrying out the methods.
  • the thrombospondins are a family of extracellular matrix (ECM) glycoproteins that modulate many cell behaviors including adhesion, migration, and proliferation.
  • Thrombospondins also known as thrombin sensitive proteins or TSPs
  • TSPs are large molecular weight glycoproteins composed of three identical disulfide-linked polypeptide chains.
  • TSPs are stored in the alpha-granules of platelets and secreted by a variety of mesenchymal and epithelial cells (Majack et al, Cell Membrane 3:51-11 (1987)). Platelets secrete TSPs when activated in the blood by such physiological agonists such as thrombin. TSPs have lectin properties and a broad function in the regulation of fibrinolysis and as a component of the ECM, and are one of a group of ECM proteins which have adhesive properties.
  • TSPs bind to fibronectin and fibrinogen (Lahav et al, Eur J Biochem 145:151-6 (1984)), and these proteins are known to be involved in platelet adhesion to substratum and platelet aggregation (Leung, J Clin Invest 74: 1164-1112 (1986)). Recent work has implicated TSPs in response of cells to growth factors. Submitogenic doses of PDGF induce a rapid but transitory, increase in TSP synthesis and secretion by rat aortic smooth muscle cells (Majack et al, J Biol Chem 101: 1059-10 (1985)).
  • TSP mRNA levels rise rapidly in response to PDGF (Majack et al, JBiol Chem 262:8821-5 (1987)).
  • TSPs act synergistically with epidermal growth factor to increase DNA synthesis in smooth muscle cells (Majack et al, Proc Natl Acad Sci S3 :9050-4 (1986)), and monoclonal antibodies to TSPs inhibit smooth muscle cell proliferation (Majack et al, JBiol Chem 106:415-22 (1988)).
  • TSPs modulate local adhesions in endothelial cells
  • TSPs particularly TSP-1 primarily derived from platelet granules
  • TGFB-1 transforming growth factor beta-1
  • Thrombospondin (TSP) 4 and 1 emerged as important SNPs associated with premature CAD and MI.
  • CAD CAD
  • sequences for TSP-1 are shown in Figs. 1 A- ID.
  • Specific reference nucleotide (SEQ ID NO: 3) and amino acid (SEQ ID NO: 4) sequences for TSP-4 are shown in Figs. 2A-2C. It is understood that the invention is not limited by these exemplified reference sequences, as variants of these sequences which differ at locations other than the SNP sites identified herein can also be utilized. The skilled artisan can readily determine the SNP sites in these other reference sequences which correspond to the SNP sites identified herein by aligning the sequence of interest with the reference sequences specifically disclosed herein, and programs for performing such alignments are commercially available. For example, the ALIGN program in the GCG software package can be used, utilizing a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4, for example. Two SNPs have been specifically studied as described herein. The first
  • G334u4 is a change from A (reference nucleotide) to G (alternate or variant nucleotide) at nucleotide position 2210 of the nucleic acid sequence of TSP-1 (Figs. 1A-1D), resulting in a missense amino acid mutation from asparagine (reference) to serine (alternate) at amino acid 700.
  • the second SNP is a change from G (reference) to C (alternate) at nucleotide position 1186 of the nucleic acid sequence of TSP-4 (Figs. 2A-2C), resulting in a missense amino acid alteration from alanine (reference) to proline (alternate) at amino acid 387.
  • polymo ⁇ hism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymo ⁇ hic marker or site is the locus at which divergence occurs. Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
  • a polymo ⁇ hic locus may be as small as one base pair, in which case it is referred to as a single nucleotide polymo ⁇ hism (SNP).
  • the invention relates to a method for predicting the likelihood that an individual will have a vascular disease, or for aiding in the diagnosis of a vascular disease, or predicting the likelihood of having altered symptomology associated with a vascular disease, comprising the steps of obtaining a DNA sample from an individual to be assessed and determining the nucleotide present at one or more of nucleotide positions 2210 of the TSP-1 gene or 1186 of the TSP-4 gene. In a preferred embodiment, the nucleotides present at both of these nucleotide positions are determined.
  • the TSP-1 gene has the nucleotide sequence of SEQ ID NO: 1 and the TSP-4 gene has the nucleotide sequence of SEQ ID NO: 3.
  • the presence of one or more of a G (the variant nucleotide) at position 2210 of SEQ ID NO: 1 or a C (the variant nucleotide) at position 1186 of SEQ ID NO: 1186 indicates that the individual has a greater likelihood of having a vascular disease, or a greater likelihood of having severe symptomology associated with a vascular disease, than if that individual had the reference nucleotide at one or more of these positions.
  • the presence of one or more of an A (the reference nucleotide) at position 2210 of SEQ ID NO: 1 or a G (the reference nucleotide) at position 1186 of SEQ ID NO: 3 indicates that the individual has a reduced likelihood of having a vascular disease or a likelihood of having reduced symptomology associated with a vascular disease than if that individual had the variant nucleotide at one or more of these positions.
  • the individual is an individual at risk for development of a vascular disease.
  • the individual exhibits clinical symptomology associated with a vascular disease.
  • the individual has been clinically diagnosed as having a vascular disease.
  • Vascular diseases include, but are not limited to, atherosclerosis, coronary heart disease, myocardial infarction (MI), stroke, peripheral vascular diseases, venous thromboembolism and pulmonary embolism.
  • the vascular disease is CAD or MI.
  • the genetic material to be assessed can be obtained from any nucleated cell from the individual.
  • genomic DNA virtually any biological sample (other than pure red blood cells) is suitable.
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, skin and hair.
  • tissue sample For assay of cDNA or mRNA, the tissue sample must be obtained from a tissue or organ in which the target nucleic acid is expressed.
  • Many of the methods described herein require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al, Academic Press, San Diego, CA, 1990); Mattila et al, Nucleic Acids Res. 19, 4967 (1991); Eckert et al, PCR Methods and
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • the latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1, respectively.
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • the nucleotide which occupies the polymo ⁇ hic site of interest can be identified by a variety of methods, such as Southern analysis of genomic DNA; direct mutation analysis by restriction enzyme digestion; Northern analysis of RNA; denaturing high pressure liquid chromatography (DHPLC); gene isolation and sequencing; hybridization of an allele-specific oligonucleotide with amplified gene products; single base extension (SBE).
  • determination of the allelic form of TSP is carried out using SBE-FRET methods as described herein, or using chip-based oligonucleotide arrays as described herein.
  • the invention also relates to a method for predicting the likelihood that an individual will have a vascular disease, or for aiding in the diagnosis of a vascular disease, or predicting the likelihood of having altered symptomology associated with a vascular disease, comprising the steps of obtaining a biological sample comprising TSP-1 and/or TSP-4 protein or relevant portion thereof from an individual to be assessed and determining the amino acid present at one or more of amino acid positions 700 of the TSP-1 gene product (e.g., as exemplified by SEQ ID NO: 2) or 387 of the TSP-4 gene product (e.g., as exemplified by SEQ ID NO: 4). In a preferred embodiment, the amino acids present at both of these amino acid positions are determined.
  • the term "relevant portion" of the TSP-1 and TSP-4 proteins is intended to encompass any portion of the protein which comprises the polymo ⁇ hic amino acid positions.
  • the presence of one or more of a serine (the variant amino acid) at position 700 of SEQ ID NO: 2, or a proline (the variant amino acid) at position 387 of SEQ ID NO: 4 indicates that the individual has a greater likelihood of having a vascular disease, or a greater likelihood of having severe symptomology associated with a vascular disease, than if that individual had the reference amino acid at one or more of these positions.
  • the presence of one or more of an asparagine (the reference amino acid) at position 700 of SEQ ID NO: 2, or an alanine (the reference amino acid) at position 387 of SEQ I D NO: 4 indicates that the individual has a reduced likelihood of having a vascular disease or a likelihood of having reduced symptomology associated with a vascular disease, than if that individual had the varaint amino acid at one or more of these positions.
  • the individual is an individual at risk for development of a vascular disease.
  • the individual exhibits clinical symptomology associated with a vascular disease.
  • the individual has been clinically diagnosed as having a vascular disease.
  • the biological sample contains protein molecules from the test subject.
  • In vitro techniques for detection of protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • in vivo techniques for detection of protein include introducing into a subject a labeled anti-protein antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • Polyclonal and/or monoclonal antibodies that specifically bind to variant gene products but not to corresponding reference gene products, and vice versa, are also provided.
  • Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof comprising the variant portion.
  • Monoclonal antibodies are screened as are described, for example, in Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988); Goding, Monoclonal antibodies, Principles and Practice (2d ed.) Academic Press, New York (1986). Monoclonal antibodies are tested for specific immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene product. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.
  • the polymo ⁇ hisms of the invention may be associated with vascular disease in different ways.
  • the polymo ⁇ hisms may exert phenotypic effects indirectly via influence on replication, transcription, and translation. Additionally, the described polymo ⁇ hisms may predispose an individual to a distinct mutation that is causally related to a certain phenotype, such as susceptibility or resistance to vascular disease and related disorders.
  • the discovery of the polymo ⁇ hisms and their correlation with CAD and MI facilitates biochemical analysis of the variant and reference forms and the development of assays to characterize the variant and reference forms and to screen for pharmaceutical agents that interact directly with one or another form of the protein.
  • these particular polymo ⁇ hisms may belong to a group of two or more polymo ⁇ hisms in the TSP gene(s) which contributes to the presence, absence or severity of vascular disease.
  • An assessment of other polymo ⁇ hisms within the TSP gene(s) can be undertaken, and the separate and combined effects of these polymo ⁇ hisms, as well as alternations in other, distinct genes, on the vascular disease phenotype can be assessed.
  • Correlation between a particular phenotype, e.g., the CAD or MI phenotype, and the presence or absence of a particular allele is performed for a population of individuals who have been tested for the presence or absence of the phenotype.
  • Correlation can be performed by standard statistical methods such as a Chi-squared test and statistically significant correlations between polymo ⁇ hic form(s) and phenotypic characteristics are noted. This correlation can be exploited in several ways. In the case of a strong correlation between a particular polymo ⁇ hic form, e.g., the variant allele for TSP-1 and/or TSP-4, and a disease for which treatment is available, detection of the polymo ⁇ hic form in an individual may justify immediate administration of treatment, or at least the institution of regular monitoring of the individual. Detection of a polymo ⁇ hic form correlated with a disorder in a couple contemplating a family may also be valuable to the couple in their reproductive decisions.
  • the female partner might elect to undergo in vitro fertilization to avoid the possibility of transmitting such a polymo ⁇ hism from her husband to her offspring.
  • immediate therapeutic intervention or monitoring may not be justified.
  • the individual can be motivated to begin simple life-style changes (e.g., diet modification, therapy or counseling) that can be accomplished at little cost to the individual but confer potential benefits in reducing the risk of conditions to which the individual may have increased susceptibility by virtue of the particular allele.
  • identification of a polymo ⁇ hic form correlated with enhanced receptiveness to one of several treatment regimes for a disorder indicates that this treatment regimen should be followed for the individual in question.
  • CAD CAD
  • MI genetic locus associated with a trait of interest
  • Such analysis is useful for mapping a genetic locus associated with a phenotypic trait to a chromosomal position, and thereby cloning gene(s) responsible for the trait. See Lander et al, Proc. Natl. Acad.
  • the invention in another embodiment, relates to pharmaceutical compositions comprising a reference TSP-1 and/or TSP-4 gene or gene product for use in the treatment of vascular disease, e.g., CAD and MI.
  • a reference TSP gene product is intended to mean gene products which are encoded by the reference allele of the TSP gene.
  • the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides.
  • Biologically active fragments include any portion of the full-length polypeptide which confers a biological function on the variant gene product, including ligand binding, and antibody binding.
  • Ligand binding includes binding by nucleic acids, proteins or polypeptides, small biologically active molecules, or large cellular structures.
  • the polypeptide or protein, or fragment thereof, of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition.
  • the particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions.
  • the optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and will depend on the ultimate pharmaceutical formulation desired.
  • Methods of introduction of exogenous peptides at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow release polymeric devices.
  • the pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents and treatment regimens.
  • compositions comprising a nucleotide sequence encoding reference or variant TSP-1 and/or TSP-4 gene products.
  • reference genes can be expressed in an expression vector in which a reference gene is operably linked to a native or other promoter.
  • the promoter is a eukaryotic promoter for expression in a mammalian cell.
  • the transcription regulation sequences typically include a heterologous promoter and optionally an enhancer which is recognized by the host.
  • the selection of an appropriate promoter for example t p, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected.
  • Commercially available expression vectors can be used.
  • Vectors can include host-recognized replication systems, amplifiable genes, selectable markers, host sequences useful for insertion into the host genome, and the like.
  • the means of introducing the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means include fusion, conjugation, transfection, transduction, electroporation or injection, as described in Sambrook, supra.
  • a wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene product to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post-translational modification, and the like.
  • cells can be engineered to express the reference allele of the invention by gene therapy methods.
  • DNA encoding the reference TSP gene product, or an active fragment or derivative thereof can be introduced into an expression vector, such as a viral vector, and the vector can be introduced into appropriate cells in an animal.
  • the cell population can be engineered to inducibly or constitutively express active reference TSP gene product.
  • the vector is delivered to the bone marrow, for example as described in Corey et al. (Science 244: 1215-1281 (1989)).
  • the invention further relates to the use of compositions (i.e., agonists) which enhance or increase the activity of the reference (or variant) TSP (e.g., TSP-1 or TSP-4) gene product, or a functional portion thereof, for use in the treatment of vascular disease.
  • compositions i.e., antagonists
  • the invention also relates to constructs which comprise a vector into which a sequence of the invention has been inserted in a sense or antisense orientation.
  • a vector comprising a nucleotide sequence which is antisense to the variant TSP-1 or TSP-4 allele may be used as an antagonist of the activity of the TSP-1 or TSP-4 variant allele.
  • a vector comprising a nucleotide sequence of the TSP-1 or TSP-4 reference allele may be used therapeutically to treat vascular diseases.
  • the term "vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) that serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • Preferred recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein .
  • the recombinant expression vectors of the invention can be designed for expression of a polypeptide of the invention in prokaryotic or eukaryotic cells, e.g., bacterial cells such as E. coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, supra.
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • a nucleic acid of the invention can be expressed in bacterial cells (e.g., E.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (supra), and other laboratory manuals.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a polypeptide of the invention.
  • the invention further provides methods for producing a polypeptide using the host cells of the invention.
  • the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a polypeptide of the invention has been introduced) in a suitable medium such that the polypeptide is produced.
  • the method further comprises isolating the polypeptide from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which a nucleic acid of the invention has been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous nucleotide sequences have been introduced into their genome or homologous recombinant animals in which endogenous nucleotide sequences have been altered.
  • Such animals are useful for studying the function and/or activity of the nucleotide sequence and polypeptide encoded by the sequence and for identifying and/or evaluating modulators of their activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • an "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing a nucleic acid of the invention into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the sequence can be introduced as a transgene into the genome of a non-human animal. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of a polypeptide in particular cells.
  • transgenic animals via embryo manipulation and microinjection, particularly animals such as mice
  • animals have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, U.S. Patent No. 4,873,191 and in Hogan, Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals.
  • a transgenic founder animal can be identified based upon the presence of the transgene in its genome and or expression of mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a transgene encoding the transgene can further be bred to other transgenic animals carrying other transgenes.
  • the invention also relates to the use of the variant and reference gene products to guide efforts to identify the causative mutation for vascular diseases or to identify or synthesize agents useful in the treatment of vascular diseases, e.g., CAD and MI.
  • Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al, Science, 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity in vitro, or in vitro activity.
  • Sites that are critical for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol, 224:899-904 (1992); de Vos et al. Science, 255:306-312 (1992)).
  • Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of proteins of the invention in clinical trials.
  • An exemplary method for detecting the presence or absence of proteins or nucleic acids of the invention in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the protein, or nucleic acid (e.g., mRNA, genomic DNA) that encodes the protein, such that the presence of the protein or nucleic acid is detected in the biological sample.
  • a preferred agent for detecting mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to mRNA or genomic DNA sequences described herein, preferably in an allele-specific manner.
  • the nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
  • oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • kits for detecting the presence of proteins or nucleic acid molecules of the invention in a biological sample can comprise a labeled compound or agent (e.g., nucleic acid probe) capable of detecting protein or mRNA in a biological sample; means for determining the amount of protein or mRNA in the sample; and means for comparing the amount of protein or mRNA in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect protein or nucleic acid.
  • the polymo ⁇ hisms shown in the Table were identified by resequencing of target sequences from individuals of diverse ethnic and geographic backgrounds by hybridization to probes immobilized to microfabricated arrays. The strategy and principles for design and use of such arrays are generally described in WO 95/11995.
  • a typical probe aoay used in this analysis has two groups of four sets of probes that respectively tile both strands of a reference sequence.
  • a first probe set comprises a plurality of probes exhibiting perfect complementarily with one of the reference sequences.
  • Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence. That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarily between the two.
  • For each probe in the first set there are three corresponding probes from three additional probe sets. Thus, there are four probes corresponding to each nucleotide in the reference sequence.
  • probes from the three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets.
  • probes were 25 nucleotides long. Arrays tiled for multiple different references sequences were included on the same substrate.
  • Genomic DNA was amplified in at least 50 individuals using 2.5 pmol each primer, 1.5 mM MgCl 2 , 100 ⁇ M dNTPs, 0.75 ⁇ M AmpliTaq GOLD polymerase, and 19 ng DNA in a 15 ⁇ l reaction.
  • Reactions were assembled using a PACKARD MultiPROBE robotic pipetting station and then put in MJ 96-well tetrad thermocyclers (96°C for 10 minutes, followed by 35 cycles of 96°C for 30 seconds, 59°C for 2 minutes, and 72°C for 2 minutes). A subset of the PCR assays for each individual were run on 3% NuSieve gels in 0.5X TBE to confirm that the reaction worked.
  • Low-density DNA chips (Affymetrix,CA) were hybridized following the manufacturer's instructions. Briefly, the hybridization cocktail consisted of 3M TMAC1, 10 mM Tris pH 7.8, 0.01% Triton X-100, 100 mg/ml herring sperm DNA (Gibco BRL), 200 pM control biotin-labeled oligo. The processed PCR products were denatured for 7 minutes at 100°C and then added to prewarmed (37°C) hybridization solution. The chips were hybridized overnight at 44°C.
  • Chips were washed in IX SSPET and 6X SSPET followed by staining with 2 ⁇ g/ml SARPE and 0.5 mg/ml acetylated BSA in 200 ⁇ l of 6X SSPET for 8 minutes at room temperature. Chips were scanned using a Molecular Dynamics scanner.
  • Chip image files were analyzed using Ulysses (Affymetrix, CA) which uses four algorithms to identify potential polymo ⁇ hisms.
  • Candidate polymo ⁇ hisms were visually inspected and assigned a confidence value: high confidence candidates displayed all three genotypes, while likely candidates showed only two genotypes (homozygous for reference sequence and heterozygous for reference and variant).
  • Some of the candidate polymo ⁇ hisms were confirmed by ABI sequencing. Identified polymo ⁇ hisms were compared to several databases to determine if they were novel. Results are shown in the Table.
  • Thrombospondin (TSP) 4 and 1 emerged as important SNPs associated with premature CAD and MI.
  • CAD CAD
  • COL7A1 collagen, type VII , alpha 1 (epidermolysis bullosa, dystrophic, dominant and

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP00959964A 1999-09-10 2000-09-07 Einzelnukleotidpolymorphismen in genen Withdrawn EP1240354A2 (de)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US15335799P 1999-09-10 1999-09-10
US153357P 1999-09-10
US22094700P 2000-07-26 2000-07-26
US220947P 2000-07-26
US22572400P 2000-08-16 2000-08-16
US225724P 2000-08-16
PCT/US2000/024503 WO2001018250A2 (en) 1999-09-10 2000-09-07 Single nucleotide polymorphisms in genes

Publications (1)

Publication Number Publication Date
EP1240354A2 true EP1240354A2 (de) 2002-09-18

Family

ID=27387432

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00959964A Withdrawn EP1240354A2 (de) 1999-09-10 2000-09-07 Einzelnukleotidpolymorphismen in genen

Country Status (3)

Country Link
EP (1) EP1240354A2 (de)
AU (1) AU7119400A (de)
WO (1) WO2001018250A2 (de)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0000995D0 (en) * 2000-01-18 2000-03-08 Zeneca Ltd Methods
US6774209B1 (en) 2000-04-03 2004-08-10 Dyax Corp. Binding peptides for carcinoembryonic antigen (CEA)
EP1403380A1 (de) * 2002-09-27 2004-03-31 Integragen Humanes fettleibigkeit Suszeptibilitätsgen und deren Verwendungen
EP1997913A1 (de) * 2002-05-15 2008-12-03 Integragen Menschliches Adipositas-Suszeptibilitäsgen und Verwendungen davon
JP4143756B2 (ja) * 2002-06-21 2008-09-03 財団法人名古屋産業科学研究所 心筋梗塞のリスク診断方法
CA2511012C (en) * 2002-12-20 2018-02-27 Applera Corporation Genetic polymorphisms associated with myocardial infarction, methods of detection and uses thereof
US20070059710A1 (en) * 2004-02-27 2007-03-15 Applera Corporation Genetic polymorphisms associated with stroke, methods of detection and uses thereof
WO2008018789A2 (en) * 2006-08-08 2008-02-14 Leiden University Medical Center Methods and means for diagnosing and treatment of osteoarthritis
EP2450444B1 (de) * 2006-11-30 2013-06-05 ARKRAY, Inc. Sonde zur Analyse eines Polymorphismus im beta2AR-Gen, Reagens, und Genanalyseverfahren für Adipositas
EP2322656A1 (de) * 2009-11-17 2011-05-18 Centre National de la Recherche Scientifique (C.N.R.S) Verfahren zur Diagnose von Hautkrankheiten
US20130137111A1 (en) * 2010-07-26 2013-05-30 Astellas Pharma Inc. Detection method of novel ret fusion

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5279966A (en) * 1992-04-02 1994-01-18 The Trustees Of Columbia University In The City Of New York Cloning, expression and uses of a novel secreted protein, F-spondin

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0118250A3 *

Also Published As

Publication number Publication date
WO2001018250A3 (en) 2002-07-25
AU7119400A (en) 2001-04-10
WO2001018250A2 (en) 2001-03-15

Similar Documents

Publication Publication Date Title
US5856104A (en) Polymorphisms in the glucose-6 phosphate dehydrogenase locus
EP0955382A2 (de) Polymorphismen in Verbindung mit Bluthochdruck
EP0941366A2 (de) Biallelische marker
US6869762B1 (en) Crohn's disease-related polymorphisms
US20060188875A1 (en) Human genomic polymorphisms
US20020037508A1 (en) Human single nucleotide polymorphisms
WO1998038846A2 (en) Genetic compositions and methods
WO2001066800A2 (en) Human single nucleotide polymorphisms
EP1240354A2 (de) Einzelnukleotidpolymorphismen in genen
EP0812922A2 (de) Polymorphismen in menschlicher mitochondriale Nukleinsäure
AU3363899A (en) Coding sequence polymorphisms in vascular pathology genes
US6833240B2 (en) Very low density lipoprotein receptor polymorphisms and uses therefor
WO2000058519A2 (en) Charaterization of single nucleotide polymorphisms in coding regions of human genes
WO1998058529A2 (en) Genetic compositions and methods
US20030039973A1 (en) Human single nucleotide polymorphisms
WO2001042511A2 (en) Ibd-related polymorphisms
EP1068354A2 (de) Biallelische marker
US20030175797A1 (en) Association of protein kinase C zeta polymorphisms with diabetes
EP1024200A2 (de) Genetische Zusammensetzungen und Methoden
WO2001038576A2 (en) Human single nucleotide polymorphisms
WO1999014228A1 (en) Genetic compositions and methods
WO2001034840A2 (en) Genetic compositions and methods
US20030232365A1 (en) BDNF polymorphisms and association with bipolar disorder
US7339049B1 (en) Polymorphisms in human mitochondrial DNA
US6913885B2 (en) Association of dopamine beta-hydroxylase polymorphisms with bipolar disorder

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020319

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

RIN1 Information on inventor provided before grant (corrected)

Inventor name: DALEY, GEORGE, Q.

Inventor name: LANDER, ERIC, S.

Inventor name: IRELAND, JAMES, S.

Inventor name: MCCARTHY, JEANETTE, J.

Inventor name: BOLK, STACEY

Inventor name: GARGILL, MICHELE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060404