EP1276899A2 - Polymorphismes associes aux maladies enteriques inflammatoires - Google Patents

Polymorphismes associes aux maladies enteriques inflammatoires

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Publication number
EP1276899A2
EP1276899A2 EP00986325A EP00986325A EP1276899A2 EP 1276899 A2 EP1276899 A2 EP 1276899A2 EP 00986325 A EP00986325 A EP 00986325A EP 00986325 A EP00986325 A EP 00986325A EP 1276899 A2 EP1276899 A2 EP 1276899A2
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EP
European Patent Office
Prior art keywords
allele
nucleic acid
polymoφhic
polymoφhisms
gene
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EP00986325A
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German (de)
English (en)
Inventor
Mark Daly
Thomas J. Hudson
Eric S. Lander
John Rioux
Kathy Siminovitch
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Ellipsis Biotherapeutics Corp
Whitehead Institute for Biomedical Research
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Ellipsis Biotherapeutics Corp
Whitehead Institute for Biomedical Research
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Publication of EP1276899A2 publication Critical patent/EP1276899A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • 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
    • 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
    • C12Q2600/172—Haplotypes

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 polymorphism 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 polymorphism 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)).
  • 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
  • polymorphisms take the form of single nucleotide variations between individuals of the same species. Such polymorphisms are far more frequent than RFLPs, STRs and VNTRs. Some single nucleotide polymorphisms (SNP) occur in protein-coding nucleic acid sequences (coding sequence SNP (cSNP)), in which case, one of the polymorphic forms may give rise to the expression of a defective or otherwise variant protein and, potentially, a genetic disease.
  • SNP single nucleotide polymorphisms
  • genes in which polymorphisms 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).
  • cSNPs 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".
  • Other single nucleotide polymorphisms occur in noncoding regions. Some of these polymorphisms 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 polymorphisms can be used in the same manner as RFLPs and VNTRs, but offer several advantages. Single nucleotide polymorphisms occur with greater frequency and are spaced more uniformly throughout the genome than other forms of polymorphism. 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).
  • LD linkage disequilibrium
  • the alleles 193, 156, 373, 140, 222, and 307 at markers GAhl8a, IRFlp, CAhl5a, CAhl7a, D5S1984, CSF2plO, respectively, define a haplotype conferring susceptibility to Crohn's disease (CD).
  • CD Crohn's disease
  • SNPs single nucleotide polymo ⁇ hisms
  • Table 3 shows the results of the SNP discovery analyses. 139 triads were genotyped for a total of 241 SNPs thus far, where at least 50 trios were fully genotyped. Using a C 2 value of 13 (corresponding to a p-value of 0.05) as threshold, 12 SNPs were found to have a significant level of association with CD and extended over a region of 250 kb, from IRF1 to prolyl4 hydroxylase. These were markers IGR2055a_l , IGR2060a_l , IGR2063b_l , IGR2069a_2,
  • this haplotype is defined by the alleles G, C, G, T, A, A, G, T, G, G, C, T at markers IGR2055a_l, IGR2060a_l, IGR2063b_l, IGR2069a_2, IGR2078a_l, IGR2096a_l, IGR2198a_l, IGR2230a_l, IGR2277a_l, IGR3081a_l, IGR3096a_l, PROLYLex3_l, respectively.
  • the frequency of this haplotype is estimated to be approximately 37% in the general population.
  • this haplotype is transmitted from heterozygous parents to CD patients at a ratio of 2.5:1.
  • the invention relates to a isolated gene or nucleic acid molecule 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 Table 3.
  • Complements of these nucleic acid segments are also included.
  • the segments can be DNA or RNA, and can be double- or single-stranded. Segments 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 a gene comprising a single nucleotide polymo ⁇ hism or to the complement of the gene. 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 Table 3. Optionally, a set of bases occupying a set of the polymo ⁇ hic sites shown in Table 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 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.
  • the phenotype is inflammatory bowel disease or Crohn's disease.
  • the Figure shows multipoint nonparametric linkage results for the IBD genome scan.
  • Multipoint LOD scores were calculated using the MAPMAKER/SIBS functions implemented in GENHUNTER 2.0.
  • the thick black line indicates the LOD score along the length of each chromosome, and the tick marks indicate the position of the microsatellite markers.
  • CD Crohn's disease
  • UC ulcerative colitis
  • CD is characterized by discontinuous, transmural inflammation affecting any part of the gastrointestinal tract and is manifested by abdominal pain, chronic diarrhea, weight loss, bowel obstructions and fistulae.
  • UC occurs as a continuous, mucosal inflammation affecting only the large intestine with primary symptoms including diarrhea, rectal bleeding and abdominal pain.
  • the search for susceptibility genes for these two diseases has resulted in the identification of two potential susceptibility loci.
  • the first, called IBDl is a CD-susceptibility locus that lies in the pericentromeric region of chromosome 16 (Hugo et al, Nature 379:821-822 (1996)).
  • the second (IBD 2) is located in a 41 cM region surrounding marker D12S83 and appears to be linked to both CD and UC (Satsangi et al, Genetics 14:199-202 (1996)). These putative loci, however, have only been replicated in some, but not all, subsequent studies (Cavanaugh et al., Proc Natl Acad Sci USA 62:29X298 (1998); Cho et al, The National Academy of Sciences 95:1502-1501 (1998); Curren et al, Gastroenterology 115:1-1 (1998); Duerr et al, The American Society of Human Genetics 63:95-100 (1998); Rioux et al, Am. J. Hum. Genet. 63:1086-1094 (1998); Yang et al, Gastroenterology 709:440-448 (1995)), supporting the belief that there exists substantial genetic heterogeneity.
  • IBDl and IBD2 only account for a fraction of the heritability of IBD, suggesting that additional loci contribute to disease susceptibility.
  • susceptibility loci in a Canadian IBD population was assessed by studying families with multiple affected siblings (McLeod et al, Dis Colon Rectum 40:553-551 (1997)).
  • a genome-wide screen was performed on 181 IBD-affected sibling pairs (ASP) and 5 IBD-affected relative pairs (RP) from 163 families.
  • ASP 181 IBD-affected sibling pairs
  • RP IBD-affected relative pairs
  • 122 were CD pairs
  • 25 were UC pairs
  • 34 were "mixed" pairs (one sibling with either CD or UC, the other with CD, UC or IC).
  • All ASP and available parents 140 families had both parents available, 17 had one parent available, and 1 was missing both parents
  • All RP were genotyped with 312 microsatellite markers covering the genome with approximately 12 cM distance between markers. Simulations of this dataset indicated that the genome-wide threshold for suggestive linkage (the score expected to occur one time at random in a genome scan) was at a LOD of 2.0.
  • chromosome 3 had a peak LOD of 2.4 between markers D3S1766 and D#S1285, chromosome 5 a peak LOD of 3.0 between GATA68A03 and D5S816, chromosome 6 a peak LOD of 2.3 between D6S 1019 and D6S1017, and chromosome 19 a peak LOD of 4.6 between GATA21G05 and D19S586.
  • this chromosome 19 locus exceeds the threshold for genome-wide significance of 3.6 (Lander & Kruglyak, Nature Genetics 77:241-247 (1995)), and represents a novel IBD susceptibility locus.
  • This novel locus maps to an extended region on 19pl3 (Figure) that contains many different genes of immunologic interest such as intercellular adhesion molecule 1 (ICAMl), complement component 3 (C3), the thromboxane A2 receptor (TBXA2), leukotriene B4 hydro xylase (LTB4H), and the janus tyrosine kinases TYK2 and JAK3.
  • ICMl intercellular adhesion molecule 1
  • C3 complement component 3
  • TXA2 receptor thromboxane A2 receptor
  • LTB4H leukotriene B4 hydro xylase
  • janus tyrosine kinases TYK2 and JAK3.
  • this work has identified two novel susceptibility loci: a locus on chromosome 5q31-33 that confers susceptibility to CD and a locus on chromosome 19pl3 that confers susceptibility to IBD. Furthermore, particular SNPs within these loci have been identified which may be associated with disease susceptibility.
  • 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 Table 3.
  • the present invention also relates to variant alleles of the described genes and to complements of the variant alleles.
  • 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 Table 3.
  • the reference sequence for many of the genes or gene fragments is provided in Table 5.
  • sequences which are not present in Table 5 the skilled artisan can readily determine the specific location of the polymo ⁇ hism given the 3' and 5' nucleotide sequence flanking the polymo ⁇ hic site provided in Table 3 and the chromosomal loci information provided herein.
  • the nucleotide sequences of the invention can be double- or single-stranded.
  • the invention further provides allele-specific oligonucleotides that hybridize to a gene comprising a single nucleotide polymo ⁇ hism or to the complement of the gene. 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 Table 3.
  • a set of bases occupying a set of the polymo ⁇ hic sites shown in Table 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.
  • An oligonucleotide can be DNA or RNA, and single- or double-stranded. Oligonucleotides can be naturally occurring or synthetic, but are typically prepared by synthetic means. Preferred oligonucleotides of the invention include segments of DNA, or their complements, which include any one of the polymo ⁇ hic sites shown in Table 3. 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 Table 3.
  • nucleotide As used herein, the terms “nucleotide”, “base” and “nucleic acid” are intended to be equivalent.
  • nucleotide sequence As used herein, 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 microfabricated 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.
  • 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
  • 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, particularly IBD.
  • 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 on or another form of the protein.
  • SNPs may also alter the regulation of the gene at the transcriptional or post-transcriptional level.
  • SNPs also enable the development of specific DNA, RNA, or protein-based diagnostics that detect the presence or absence of the polymo ⁇ hism in particular conditions.
  • 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.
  • Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • stringent conditions for example, at a salt concentration of no more than 1 M and a temperature of at least 25°C.
  • 5X SSPE 750 mM NaCl, 50 mM NaPhosphate, 5 mM
  • EDTA, pH 7.4 EDTA, pH 7.4
  • a temperature of 25-30°C, or equivalent conditions are suitable for allele-specific probe hybridizations.
  • Equivalent conditions can be determined by varying one or more 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.
  • 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.
  • 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.
  • Many of the methods described below 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.
  • LCR Iigase 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 polymo ⁇ hisms identified as described herein can be used as a platform for genotyping (i.e., determining the genotype of) individuals. This 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 corresponding 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).
  • the direct analysis of the sequence of polymo ⁇ hisms of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al, Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989); Zyskind et al, Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).
  • Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, (W.H. Freeman and Co, New York, 1992), Chapter 7.
  • Alleles of target sequences can be differentiated using single-strand conformation 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. 7.
  • 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 94:10156-61 (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.
  • 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.
  • 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.
  • p(ID) x 4 + (2xy) 2 + (2yz) 2 + (2xz) 2 + z 4 + y 4
  • the appropriate binomial expansion is used to calculate p(ID) and p(exc).
  • 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. If 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.
  • the polymo ⁇ hisms identified herein and shown in Table 3 are present in the chromosomal loci which have been identified as described herein as conferring susceptibility to IBD such as CD and UC.
  • 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. To perform such analysis, the presence or absence of a set of polymo ⁇ hisms (i.e.
  • a polymo ⁇ hic set is determined for a set of the individuals, some of whom exhibit a particular trait, and some of which exhibit lack of the trait.
  • 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. For example, it might be found that the presence of allele Al at polymo ⁇ hism A correlates with heart disease.
  • 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 patient can be motivated to begin simple life-style changes (e.g., diet, exercise) that can be accomplished at little cost to the patient but confer potential benefits in reducing the risk of conditions to which the patient may have increased susceptibility by virtue of variant alleles.
  • Identification of a polymo ⁇ hic set in a patient correlated with enhanced receptiveness to one of several treatment regimes for a disease indicates that this treatment regime should be followed.
  • correlations between characteristics and phenotype are useful for breeding for desired characteristics. For example, Beitz et al, US 5,292,639 discuss use of bovine mitochondrial polymo ⁇ hisms in a breeding program to improve milk production in cows.
  • each cow was assigned a value of 1 if variant or 0 if wildtype with respect to a prototypical mitochondrial DNA sequence at each of 17 locations considered.
  • Each production trait was analyzed individually with the following animal model:
  • Y ijkpn ⁇ + YS f + P j + X k + ⁇ , + ... ⁇ 17 + PE n + a n +e p
  • Y ij np is the milk, fat, fat percentage, SNF, SNF percentage, energy concentration, or lactation energy record
  • ⁇ is an overall mean
  • YS f 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 ⁇ 17 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,, 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.
  • the previous section concerns identifying correlations between phenotypic traits (e.g., IBD) and polymo ⁇ hisms that directly or indirectly contribute to those traits, such as those identified in Table 3.
  • 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 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, 0 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.
  • the invention further provides variant forms of nucleic acids and corresponding proteins.
  • the nucleic acids comprise one of the sequences described in Table 3, 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 Table 3, (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 Table 3. That position is occupied by the amino acid coded by the corresponding codon in any of the alternative forms shown in Table 3.
  • 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. See Hogan et al,
  • the present invention includes biologically active fragments of the polypeptides, or analogs thereof, including organic molecules which simulate the interactions of the peptides.
  • Bioly 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 Table 3.
  • 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.
  • LD linkage disequilibrium
  • the alleles 193, 156, 373, 140, 222, and 307 at markers GAM8a, IRFlp, CAM 5a, CAM 7a, D5S1984, CSF2plO, respectively, define a haplotype conferring susceptibility to Crohn's disease (CD).
  • CD Crohn's disease
  • SNPs single nucleotide polymo ⁇ hisms
  • markers IGR2055a_l, IGR2060a_l, IGR2063b_l , IGR2069a_2, IGR2078a_l, IGR2096a_l, IGR2198a_l, IGR2230a_l, IGR2277a_l, IGR3081a_l, IGR3096a_l, PROLYLex3_l see Table 4. Any of these best SNPs by themselves are in strong association with CD and fully explain the microsatellite LD observations.
  • this haplotype is defined by the alleles G, C, G, T, A, A, G, T, G, G, C, T at markers IGR2055a_l, IGR2060a_l, IGR2063b_l, IGR2069a_2,
  • telomere genotyping 296 triads were genotyped: 95 of these triads were derived from families used in the original identification of the IBD5 locus (only one triad per family), and 201 were from newly collected families.
  • SNP genotyping 139 triads were genotyped: 18 were derived from families used in the original identification of the IBD5 locus, and 121 were from the newly collected families.
  • Individuals affected by CD were identified by review of the clinical charts of all patients registered in the Mount Sinai Hospital Inflammatory Bowel Disease Centre patient database and from the Toronto Hospital for Sick Children IBD database. Written informed consent was obtained from all participants and ethics approval for this study was granted by the University of Toronto Ethics Committee.
  • Microsatellite Genotyping Genomic DNA was extracted from peripheral blood lymphocytes from probands and family members from 163 Caucasian pedigrees. The genome-wide scan, with an average inter-marker spacing of 12 cM, was carried out using a modified version of the Cooperative Human Linkage Centre (CHLC) Screening Set/version 6.0 that also included Genethon markers. These 312 loci were amplified using fluorescently-labeled primers (Research Genetics Inc., Huntsville AL) in separate polymerase chain reactions, and the products were then multiplexed into panels by pooling before electrophoresis on ABI 377 sequencers (PE Applied Biosystems, Foster City, CA).
  • CHLC Cooperative Human Linkage Centre
  • Fluorescent genotyping gels were analyzed in an automated system developed at the Whitehead Institute/MIT Center for Genome Research. Further details of the genotyping system have previously been described (Rioux et al, Gastroenterology 115:1062-1065 (1998)).
  • microsatellite LD mapping In the first phase of the microsatellite LD mapping, a total of 57 microsatellite markers were genotyped on 296 CD triads. Information regarding primer sequence, allele size range, and suggested amplification conditions for 55 of these genetic markers (all but IRFlpl and CSF2plO) can be obtained from the Genethon (http://www.genethon.fr/), Marshfield (http://research.marshfieldclinic.org/genetics/), or Genome Database (http://www.genethon.fr) World Wide Web sites. The markers IRFlpl, CSF2pl, and the 8 markers used in the 2nd stage of LD mapping, were designed during the course of this study. Genotypes for all of these markers were obtained as described above.
  • PCR assays were designed using Primer 3.0 to be approximately 700 bp in length, with 100 bp overlap with adjacent assays.
  • the -21 M13 forward and the -28 M13 reverse sequences were added to each of the forward and reverse PCR primers, respectively. These PCR primers were used to amplify 50 ng of genomic DNA from six CD patients, one unaffected family member, and one CEPH DNA as control.
  • PCR products were purified using the solid phase reversible immobilization (SPRI) method and then sequenced using the appropriate -21 Ml 3 or -28 Ml 3 DYEnamic Direct Cycle Sequencing kit (Amersham Pharmacia Biotech Ltd, Cleveland, OH). All sequencing reactions were run on ABI377 automated sequencers (PE Applied BioSystems, Foster City, CA); the gel files were processed using the BASS software, available on the Whitehead Institute/MIT Center for Genome Research FTP site. Sequences were base-called by the Phred program, and then the forward and reverse reads were assembled by the Phrap program. All traces were visually inspected by at least two observers.
  • SPRI solid phase reversible immobilization
  • SNP genotyping was performed using length-multiplexed single-base extension (LM-SBE) as previously described. Briefly, PCR primers were designed as close as possible to the SNPs identified in the current study, resulting in a product of a maximum length of 150 bp. Forward primers had T7 tails at their 5' ends and reverse primers had T3 tails at their 5' ends. These T7 and T3 tails were used for secondary amplification. Primer pairs were checked for homology to all amplicons and sorted into pools consisting of up to 50 primer pairs. Loci were subjected to two rounds of PCR amplification.
  • L-SBE length-multiplexed single-base extension
  • simulations were performed by generating artificial genotype data with the identical family structures. These simulations matched the datasets with respect to marker density, marker informativeness, the individuals genotyped, affected status, and the fraction of missing data.
  • Paffected is calculated from the frequency of the haplotype among the transmitted parental chromosomes and Pnormal is the frequency among untransmitted parental chromosomes.
  • Table 1 Summary of the first stage of LD mapping using microsatellite markers.
  • G Genethon
  • M Marshfield
  • U Utah
  • S designed by authors from genomic sequence.

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Abstract

La présente invention concerne des segments d'acide nucléique du génome humain, en particulier des segments d'acide nucléique issus d'un gène, comprenant des sites polymorphes. Cette invention concerne aussi des amorces et des sondes spécifiques aux allèles s'hybridant à des régions adjacentes à ces sites ou contenant ces sites . On utilise ces acides nucléiques, ces amorces et ces sondes dans des applications telles que les corrélations phénotypiques, la médecine légale, la recherche de paternité, la médecine et les analyses génétiques.
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