WO2009103992A1 - Variation génétique associée à la maladie coeliaque - Google Patents

Variation génétique associée à la maladie coeliaque Download PDF

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WO2009103992A1
WO2009103992A1 PCT/GB2009/000475 GB2009000475W WO2009103992A1 WO 2009103992 A1 WO2009103992 A1 WO 2009103992A1 GB 2009000475 W GB2009000475 W GB 2009000475W WO 2009103992 A1 WO2009103992 A1 WO 2009103992A1
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seq
snps
coeliac disease
coeliac
sample
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PCT/GB2009/000475
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English (en)
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David Van Heel
Karen Hunt
Cisca Wijmenga
Owen Ross Mcmanus
Panagiotis Deloukas
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Queen Mary & Westfield College
Universitair Medisch Centrum Groningen
The Provost Fellows And Scholars Of The College Of Queen Elizabeth Near Dublin
Genome Research Limited
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Publication of WO2009103992A1 publication Critical patent/WO2009103992A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the diagnosis of coeliac disease, and in particular to human chromosomal regions and specific single nucleotide polymorphisms (SNPs) within those regions which are associated with coeliac disease. These human chromosomal regions and SNPs can thus be used to predict the occurrence of coeliac disease in a patient.
  • SNPs single nucleotide polymorphisms
  • Coeliac disease (alternative spelling: celiac disease) is a common heritable condition with a prevalence of approximately 1% in Western populations. In coeliac disease,
  • HLA-DQ2 or -DQ8 restricted T cell responses occur to dietary proteins (glutens) in cereals such as wheat, rye and barley. This leads to small intestinal inflammation and intestinal villous atrophy, with consequent clinical features such as chronic diarrhoea and fatigue. Sufferers have serum antibodies to gluten and show delayed hypersensitivity to gluten.
  • a single nucleotide polymorphism is a DNA sequence variation which occurs when a single nucleotide of the genome differs between members of a species or between different chromosomes in an individual.
  • SNP single nucleotide polymorphism
  • the present inventors have previously carried out a genome-wide association study and identified a number of SNPs in the chromosomal region harbouring the IL2 and IL21 genes which are associated with susceptibility to coeliac disease in humans (van Heel et al, Nature Genetics 39, 827-829, 2007). This indicates that genetic variation in this region of chromosome 4q27 predisposes to coeliac disease. However, it is expected that the IL2-IL21 locus explains less than 1% of the increased familial risk for coeliac disease.
  • the present inventors have now identified seven further human chromosomal regions, and a number of SNPs within these regions, which correlate with increased risk for coeliac disease.
  • a method of diagnosing coeliac disease comprising analysing a sample of nucleic acid from a human subject to determine the presence or absence of one or more SNPs in one or more human chromosomal regions selected from the group consisting of Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24.
  • the present invention meets a hitherto unmet need for a method of accurately identifying individuals at risk for coeliac disease.
  • the invention advantageously provides a number of SNPs which are associated with coeliac disease and can thus be used individually or in combination to determine whether an individual is at risk for or suffering from coeliac disease.
  • the diploid chromosome number is 46, with 23 chromosomes being inherited from each parent via the sperm and the egg. Homologous chromosomes form pairs with one chromosome from each parent. Human cells thus contain 23 pairs of chromosomes. Of these, the autosomes are numbered from 1 to 22, with the other pair being the sex chromosomes, either XX (female) or XY (male).
  • ISCN International System for Human Cytogenetic Nomenclature
  • Each human chromosome has a short arm and a long arm. The short arm is designated p and the long arm is designated q.
  • Each arm of the chromosome is divided into regions labelled pi, p2, p3 etc., and ql, q2, q3, etc., counting outwards from the centromere. Regions are delimited by specific landmarks, which are consistent and distinct morphological features, such as the ends of the chromosome arms, the centromere and certain bands. Regions are further divided into bands.
  • the seven further human chromosomal regions that the present inventors have now identified as being associated with coeliac disease are Iq31, 2ql l-2ql2, 3p21, 3q25- 3q26, 3q28, 6q25 and 12q24.
  • a single nucleotide polymorphism is a DNA sequence variation which occurs when a single nucleotide of the genome differs between members of a species or between different chromosomes in an individual. Typically, each SNP has only two different alleles. In other words, for each SNP there is typically only two different nucleotide variants.
  • SNPs are identified herein using by their GenBank accession number, as available in the National Center for Biotechnology Information (NCBI) database.
  • the GenBank accession number "rs " indicates the position of a SNP within the human genome and the sequence surrounding the SNP, and may be readily identified by a person skilled in the art using the NCBI database.
  • the specific sequences corresponding to the rs number of the SNP within the NCBI database may change over time, but this will be indicated on the NCBI database and will be readily identified by a person skilled in the art.
  • sequences including the SNPs for use in the present invention are also identified herein by their sequence identifiers, SEQ ID NOs: 1 to 21. These sequences are shown in Figure 7.
  • the nucleotide sequences of SEQ ID NOs: 1 to 21 are polymorphic sequences.
  • a polymorphic sequence is a polynucleotide sequence including a polymorphic site representing a SNP.
  • the sequences set out in SEQ ID NOs: 1 to 21 include both alleles of the SNP.
  • the SNP alleles are shown in square brackets in the sequences of SEQ ID NOs: 1 to 21 shown in Figure 7.
  • the SNPs within the seven new human chromosomal regions that the present inventors have identified as being associated with coeliac disease are rs2816316 (SEQ ID NO: 1), rsl3015714 (SEQ ID NO: 2), rs917997 (SEQ ID NO: 3), rs6441961 (SEQ ID NO: 4), rs 17810546 (SEQ ID NO: 5), rs9811792 (SEQ ID NO: 6), rs9851967 (SEQ ID NO: 7), rsl3076312 (SEQ ID NO: 8), rsl464510 (SEQ ID NO: 9), rsl559810 (SEQ ID NO: 10), rsl738074 (SEQ ID NO: 11), rs3184504 (SEQ ID NO: 12) and rs653178 (SEQ ID NO: 13). Accordingly, in the methods of the invention, the one or more SNPs are typically selected from the group consisting of the SNPs
  • the risk of coeliac disease is increased in individuals possessing one of more of the above alleles.
  • the risk of coeliac disease is increased in individuals who are homozygous for one or more of the above alleles or who are heterozygous.
  • the methods of the invention further comprise analysing a sample of nucleic acid from the human subject to determine the presence or absence of one or more SNPs in the human chromosomal region 4q27.
  • the SNPs within the 4q27 region that were identified as being associated with coeliac disease are rsl 1938795 (SEQ ID NO: 14), rsl3151961 (SEQ ID NO: 15), rsl3119723 (SEQ ID NO: 16), rsl 1734090 (SEQ ID NO: 17), rs7684187 (SEQ ID NO: 18), rsl2642902 (SEQ ID NO: 19), rs6822844 (SEQ ID NO: 20) and rs6840978 (SEQ ID NO: 21).
  • the one or more SNPs are typically selected from the group consisting of the SNPs present in the sequences of SEQ ID NOs: 14 to 21.
  • the region 4q27 and the SNPs rs 11938795 (SEQ ID NO: 14), rsl3151961 (SEQ ID NO: 15), rsl3119723 (SEQ ID NO: 16), rsl 1734090 (SEQ ID NO: 17), rs7684187 (SEQ ID NO: 18), rsl2642902 (SEQ ID NO: 19), rs6822844 (SEQ ID NO: 20) and rs6840978 (SEQ ID NO: 21) can therefore be used in the methods of the invention in addition to the seven new human chromosomal regions and 13 new SNPs which have been identified by the inventors.
  • SNPs for use in the present invention and their cytogenetic locations are set out in the following Table:
  • the present invention provides a method of diagnosing coeliac disease, said method comprising analysing a sample of nucleic acid from a human subject to determine the presence or absence of one or more SNPs in one or more human chromosomal regions selected from the group consisting of Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24.
  • the one or more SNPs are selected from the group consisting of the SNPs present in the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
  • the seven new human chromosomal regions and 13 new SNPs within those chromosomal regions which have been identified by the inventors as being associated with coeliac disease can be used in the methods of the invention in combination with the previously reported genetic region 4q27 and the SNPs within it.
  • the method of the invention further comprises analysing a sample of nucleic acid from the human subject to determine the presence or absence of one or more SNPs in the human chromosomal region 4q27.
  • the presence or absence of one or more SNPs in one or more human chromosomal regions selected from the group consisting of Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24 is determined, in addition to determining the presence or absence of one or more SNPs in the human chromosomal region 4q27.
  • the human chromosomal region 4q27 comprises one or more SNPs selected from the group consisting of the SNPs present in the following sequences: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.
  • HLA typing can be done, for example, by serology or genetic methods well known in the art, or by genotyping an HLA marker, for example the marker rs2187668 (van Heel et al, supra).
  • one or more SNPs selected from the group consisting of the SNPs present in SEQ ID NOs: 1 to 13 are used. Typically, more than one of these SNPs is used. In this embodiment of the invention, any combination of the listed SNPs can be used. For example, a combination of any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or even all 13 of these SNPs is used in a method of the invention. Each additional SNP provides additional information on the risk of coeliac disease.
  • At least one SNP from each chromosomal region is used, i.e. at least one SNP from each of the chromosomal regions Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24 which have been identified by the inventors as being associated with coeliac disease.
  • the SNPs present in SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8 and/or SEQ ID NO: 9 and/or SEQ ID NO: 10, SEQ ID NO: 1 1, and SEQ ID NO: 12 and/or SEQ ID NO: 13 are typically used in combination.
  • the SNPs present in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 are used in combination.
  • one or more SNPs selected from the group consisting of the SNPs present in SEQ ID NOs: 14 to 21 is used in addition to one or more of the SNPs present in SEQ ID NOs: 1 to 13.
  • any one of the SNPs present in SEQ ID NOs: 14 to 21 or a combination of any 2, 3, 4, 5, 6, 7 or even all 8 of these SNPs is used in addition to one or more of the SNPs present in SEQ ID NOs: 1 to 13.
  • Any combination of the SNPs present in SEQ ID NOs: 14 to 21 can thus be used together with any combination of the SNPs present in SEQ ID NOs: 1 to 13.
  • any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even all 21 of the SNPs present in SEQ ID NOs: 1 to 21 can be used in a method of the invention.
  • Each additional SNP provides additional information on the risk of coeliac disease.
  • At least one SNP from each chromosomal region is used, i.e. at least one SNP from each of the chromosomal regions Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 4q27, 6q25 and 12q24.
  • the SNPs present in SEQ ID NO: 1, SEQ ID NO: 2 and/or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 6, SEQ ID NO: 7 and/or SEQ ID NO: 8 and/or SEQ ID NO: 9 and/or SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and/or SEQ ID NO: 13, SEQ ID NO: 14 and/or SEQ ID NO: 15 and/or SEQ ID NO: 16 and/or SEQ ID NO: 17 and/or SEQ ID NO: 18 and/or SEQ ID NO: 19 and/or SEQ ID NO: 20 and/or SEQ ID NO: 21 are typically used in combination.
  • SNPs present in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 20 are used in combination.
  • HLA typing can be done, for example, by serology or genetic methods well known in the art, or by genotyping an HLA marker, for example the marker rs2187668 (van Heel et al, supra).
  • a sample of nucleic acid from a human subject is analysed to determine the presence or absence of one or more SNPs in one or more of the above-mentioned human chromosomal regions, typically to determine whether one or more of the above-mentioned SNPs is present in the nucleic acid sample.
  • the sample of nucleic acid used in the invention is typically a sample of DNA or RNA.
  • DNA includes cDNA synthesized from mRNA.
  • the sample of nucleic acid can be derived from any biological sample which contains the subject's nucleic acid.
  • the biological sample can be a sample of whole blood, plasma, serum, urine, sputum or lymph and can be obtained by any suitable means.
  • the genetic regions identified by the inventors are within the human genome and therefore the subject is a human subject.
  • the methods of the present invention are typically carried out on a sample of nucleic acid that has previously been taken from a human subject, and thus the taking of the nucleic acid sample does not typically form part of the methods of the invention.
  • the method also comprises taking the nucleic acid sample from the subject, for example by a cheek swab, by taking a urine sample or by taking a blood sample.
  • control sample is typically a nucleic acid sample taken from a subject who is known not to be suffering from coeliac disease.
  • the control sample may not be necessary to compare the sample to a control sample, since the SNP, and in particular the disease-associated allele, can be detected on its own, for example by sequencing of the nucleic acid sample.
  • the nucleic acid will typically be isolated from the subject.
  • Nucleic acid isolation can be carried out using any appropriate method known in the art.
  • DNA can be directly purified from tissues or cells or a specific region can be amplified using, for example, the polymerase chain reaction (PCR) and isolated.
  • PCR polymerase chain reaction
  • the isolated nucleic acid is then analysed to determine the presence or absence of one or more SNPs in one or more of the above-mentioned human chromosomal regions.
  • the nucleic acid is analysed by sequencing of the isolated nucleic acid.
  • sequencing of the isolated nucleic acid can be carried out by any suitable method known in the art.
  • one or more SNPs is detected.
  • SNPs can be detected by a variety of methods known in the art.
  • the nucleic acid can be sequenced to determine whether a disease-causing allele is present.
  • Hybridization- based methods can also be used to identify SNPs.
  • the nucleotides of the polymorphic site can be identified by hybridizing the DNA with a probe containing the sequence of the SNP site or a complementary probe thereof, and examining the degree of hybridization.
  • Enzyme-based methods such as restriction fragment length polymorphism, allele-specific polymerase chain reaction (PCR) and primer extension (for example the Infinium assay used in the Examples herein) can also be used.
  • the present invention provides a method for the diagnosis of coeliac disease.
  • the invention is used to diagnose individuals with one or more medical symptoms of coeliac disease, such as diarrhoea and/or fatigue.
  • the invention is used to diagnose individuals who do not currently have medical symptoms of coeliac disease, but are related to a known coeliac disease sufferer, or are concerned about the risk of coeliac disease for some other reason.
  • These embodiments of the invention allow preventative measures to be taken to avoid the subject suffering from symptoms of coeliac disease, for example by modification of the subject's diet.
  • the invention can also be used to diagnose coeliac disease in cases where diagnosis has previously been unclear.
  • the invention provides methods of diagnosing coeliac disease.
  • the methods of the invention can be used to diagnose coeliac disease in a human subject if it is found that there are one or more SNPs in one or more human chromosomal regions selected from the group consisting of Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24 in a sample of nucleic acid from that subject.
  • the presence of one or more SNPs in one or more of said human chromosomal regions is indicative of coeliac disease.
  • the one or more SNPs are selected from the group consisting of the SNPs present in SEQ ID NOs: 1 to 13.
  • the presence of one or more of the following alleles is indicative of coeliac disease:
  • At least one SNP from each chromosomal region is typically used, and in this embodiment the presence of the following alleles is indicative of coeliac disease: T in SEQ ID NO: 1, G in SEQ ID NO: 2 and/or A in SEQ ID NO: 3, T in SEQ ID NO: 4, G in SEQ ID NO: 5 and/or C in SEQ ID NO: 6, C in SEQ ID NO: 7 and/or T in SEQ ID NO: 8 and/or T in SEQ ID NO: 9 and/or T in SEQ ID NO: 10, A in SEQ ID NO: 11, and T in SEQ ID NO: 12 and/or G in SEQ ID NO: 13.
  • the method further comprises analysing a sample of nucleic acid from the human subject to determine the presence or absence of one or more SNPs in the human chromosomal region 4q27.
  • the presence of one or more SNPs in this chromosomal region is indicative of coeliac disease.
  • the presence of one or more of the following alleles is indicative of coeliac disease:
  • At least one of the above alleles is detected in combination with at least one of the above-mentioned alleles of SEQ ID NOs: 1 to 13.
  • the present invention provides a method of testing for coeliac disease, said method comprising analysing a sample of nucleic acid from a human subject to determine the presence or absence of one or more SNPs in one or more human chromosomal regions selected from the group consisting of Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24.
  • the present invention provides a method of identifying one or more SNPs in a sample of nucleic acid, said method comprising analysing said sample of nucleic acid to determine the presence or absence of one or more SNPs in one or more human chromosomal regions selected from the group consisting of Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24.
  • the present invention provides a method of diagnosing coeliac disease, said method comprising analysing a sample of nucleic acid from a subject to determine the presence or absence of one or more SNPs in one or more human chromosomal regions selected from the group consisting of Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 and 12q24, wherein the presence of one or more SNPs in one or more of said human chromosomal regions is indicative of coeliac disease; thereby diagnosing coeliac disease in the subject.
  • Figure 1 shows quantile-quantile (Q-Q) plots for association results in follow-up samples.
  • Figure Ia is a Q-Q plot of association results (Cochran- Mantel-Haenszel test) for 1020 non-HLA SNPs in UK2, IRISH and DUTCH follow-up samples. Data points in light grey indicate SNPs shown in Table 2 with P overall ⁇ 5xlO -7 in all samples including UKGWAS. Straight line indicates expected results under null hypothesis.
  • Figure Ib is a Q-Q plot of residual association results (Cochran-Mantel-Haenszel test) for 992 non-HLA SNPs, excluding 28 SNPs mapping to the eight coeliac associated regions described in Table 2, in UK2, IRISH and DUTCH follow-up samples.
  • Figure 2 shows linkage disequilibrium structure and association results for eight non-HLA coeliac disease associated regions. Chromosomal positions based on NCBI build 36 coordinates, showing Ensembl (release 48) genes. Allele count P values are shown for SNPs analysed in UKGWAS samples (diamonds), and Cochran-Mantel-Haenszel association test P values (circles) for SNPs analysed in all UKGWAS, UK2, IRISH and DUTCH samples.
  • Figure 3 shows correlation of rs917997 genotype with whole blood cis IL18R ⁇ P mRNA expression. Expression levels (bars show group means) were determined in samples from gluten-free diet treated coeliac individuals.
  • Figure 4 shows cluster plots for most significantly associated SNPs.
  • R theta plots for the most significantly associated SNP from each of eight loci (Table 2) from Infinium (UKGWAS) and GoldenGate (UK2, Irish, Dutch) datasets.
  • Genotype call rates (all 7238 samples) were 99.88% for rs917997 and rsl464510, 99.97% for rsl738074 and rs653178, and complete (no missing data) for rs2816316, rs6441961, rs 17810546 and rs6822844.
  • Figure 5 shows expression of putative candidate genes in small intestinal tissue.
  • ILlRLl two probes
  • SLC9A4 4, CCRl
  • CCR3 Illumina probe
  • NC healthy normal controls
  • MO treated coeliac disease with (Marsh 0) normal intestinal histology
  • Mill untreated coeliac disease with villous atrophy (Marsh III).
  • Figure 6 shows expression profiling of T cell subsets in murine intestine, thymus and spleen.
  • cDNA was normalised to ⁇ -actin.
  • Negative control [-] is reaction in absence of cDNA.
  • Positive control [+] is reaction from mixed bulk IEL and splenic cDNA in excess.
  • Figure 7 shows the nucleotide sequences of the SNPs for use in the invention. The sequences are shown with reference to the NCBI accession number (rs )
  • Table 4 shows detailed association results. Table 4 indicates the alleles for the SNPs associated with coeliac disease.
  • rs2816316 SEQ ID NO: 1
  • rsl3015714 SEQ ID NO: 2
  • rs6441961 SEQ ID NO: 4
  • rs9811792 SEQ ID NO: 6
  • rs9851967 SEQ ID NO: 7
  • rsl3076312 SEQ ID NO: 8
  • rsl464510 SEQ ID NO: 9
  • rsl559810 SEQ ID NO: 10
  • rs3184504 SEQ ID NO: 12
  • rsl 1938795 SEQ ID NO: 14
  • rsl 1734090 SEQ ID NO: 17
  • rs6822844 SEQ ID NO: 20
  • rs6840978 SEQ ID NO: 21
  • SEQ ID NO: 1 SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 20 and SEQ ID NO: 21 are shown in this way in Table 4 as the complementary strand of the sequence shown in Figure 7. However, the alleles are in fact the same.
  • EIGENSTRAT was applied to the remaining SNPs (drawn from 310,605 SNPs for UKGWAS, or from 1025 SNPs for UK2, IRISH, DUTCH) to determine eigenvectors that correct for possible population stratification in cases and controls.
  • EIGENSTRAT calculates the Armitage Trend test chi-square statistic for each genotyped SNP and then applies a SNP-specific correction based on the eigenvectors that individually adjusts the value of Armitage chi-square. Following EIGENSTRAT correction, we also corrected with the residual genomic control factor for each collection and then combined the post-correction chi-squares across the 4 studies according to the meta-analysis method of Stouffer (Rosenthal, R. Meta-Analytic Procedures for Social Research, Sage Publications Inc., 1991).
  • P value threshold for genomewide statistical significance For statistical significance of disease association results in the combined genome scan (UKGWAS) and follow-up datasets (UK2, DUTCH, IRISH), the same genome- wide significance threshold (P ⁇ 5x10 -7 ) adopted by the WTCCC (Wellcome Trust Case Control Consortium, Nature 447, 661-78, 2007) was applied. It should be noted that this threshold is close to the conservative Bonferroni corrected significance threshold as suggested by Skol et al. (Nat Genet 38, 209-13, 2006) for a two-stage study such as this in which genome-scan and subsequent results from stage-2 followup samples are combined. Since 307,411 non-HLA SNPs were tested in this genome scan, the genomewide significance threshold based on Skol et al. criteria would be approximately 0.05/307,411 ⁇ 2 x 10 '7 .
  • RNA sample was collected from unselected Illumina Hap300 genotyped UKGWAS coeliac cases. All were selected on the basis of gluten free diet treatment for >6 months, to avoid possible bias in gene expression levels due to active inflammation. Notable features of this experimental design were: use of primary cells, analysis of expression from unstimulated leucocytes, and the possibility that the samples would be enriched for disease causal genetic variants compared to healthy controls. RNA was extracted using the
  • PAXgene protocol hybridised to Illumina HumanRef-8v2 expression arrays, scanned and processed in BeadStudio. Data were cubic spline normalised.
  • RNA for each sample 6,298 genes were assessed with two different probes present on the Illumina HumanRef8v2 chip. Intensities for all probes for each sample (Median
  • RNAlater RNA was extracted using TRIzol (Invitrogen) and glass beads, hybridised to HumanRef-8v2 arrays and analysed as for the PAXgene samples. Expression profiling of T cell subsets is described in Supplementary Methods, below.
  • DNA was extracted from whole blood, except for 1958 Cohort control samples which were lymphoblastoid cell line DNA, and 374 cases and 176 controls from the UK2 collection which were Oragene saliva DNA.
  • Whole genome amplified (WGA) blood DNA was used for 194 Irish cases and 18 Dutch cases. Genotype cluster theta values for WGA DNA were similar to blood DNA, for a small fraction of markers intensity (R) was lower.
  • R markers intensity
  • a control DNA sample was included on 96 well sample plates genotyped in both London (UK2, Irish collections) and the Netherlands (Dutch collections). Concordance between plates for this sample was 99.94% for 45 replicates of 1025 SNPs.
  • a further 9 control samples were genotyped once in both London and the Netherlands, concordance was 99.90% over 1025 SNPs.
  • genotype relative risk was calculated for the observed allelic odds ratio assuming a multiplicative mode of inheritance and population frequency of the disease-associated allele equal to the frequency observed in controls (Risch & Teng, Genome Res 8, 1273-88, 1998; McGinnis, Am J Hum
  • T cell populations from the small intestine, thymus and spleen of healthy adult C57B1/6 mice were stained with antibodies and FACS sorted on a MoFIo machine (Cytomation) as previously described (Shires et al, Immunity 15, 419-34, 2001). Total RNA preparation, reverse transcription, and semiquantitative PCR were also undertaken as previously described (Shires et al, supra). Primer sequences were:
  • RGS-I-F 5'-ACCTGAGATCGATGATCCCACATCT-S' (SEQ ID NO: 26)
  • RGS-I-R 5'-CTGTCGATTCTCGAGTATGGAAGTC-S ' (SEQ ID NO: 27)
  • ⁇ -actin-F 5'-TCCCTGTATGCCTCTGGTCGTACCAC-S ' (SEQ ID NO: 28)
  • ⁇ -actin-R 5'-CAGGATCTTCATGAGGTAGTCTGTCAG-S' SEQ ID NO: 29
  • RESULTS 1,164 non-HLA SNPs were initially selected from the UKGWAS for follow up, comprising 1,088 single SNPs with association results of P ⁇ 0.00275 and 76 additional non-synonymous SNPs (nsSNP) with association results between P>0.00275 and P ⁇ 0.01.
  • nsSNP non-synonymous SNPs
  • 1,025 SNPs were analysed in follow-up collections. These collections comprised coeliac cases and controls of Northern European origin and of similar phenotypes to the UKGWAS samples. This design represents an unbiased search of the genome for susceptibility variants.
  • the data was inspected for possible bias due to population differences between cases and controls, genotyping artefact, missing genotype data or other factors.
  • the overall genotype call rate across all samples and SNPs was high at 99.94% (details for each of the coeliac disease associated SNPs and 5 HLA tag markers in Table 5). Inspection of cluster plots for the most associated SNPs from each of the eight non-HLA coeliac associated regions ( Figure 4), consistent findings from two assay chemistries (Infinium and GoldenGate) in the UK populations, and multiple associated SNPs for some regions suggested that results were not due to laboratory generated false positive findings.
  • Independent replication, with the same allele and direction, of the inventors' previous report (van Heel et al., supra) is provided by the UK2 collection (Table 4, rs6822844 P UK2 0.0017).
  • This SNP is among a cluster of 8 associated SNPs that are in a block of strong linkage disequilibrium containing four genes (KIAAl 109-AD ADl -IL2-IL21). Both IL2 and IL21 are strong candidate genes because of their role in T cell activation.
  • RGS family genes attenuate the signalling activity of G-proteins by acting as GTPase activating proteins.
  • RGSl acts to regulate chemokine receptor signaling and is known to be involved in B-cell activation and proliferation.
  • RGS1 'A mice have enhanced B cell movement into and out of lymph nodes (Han et al, Immunity 22, 343-54 2005), and heightened dendritic cell migratory response to chemokines (Shi et al, J Immunol 172, 5175-84, 2004). RGSl was found to be expressed in human small intestinal biopsies (Figure 5).
  • the IL- 18 pathway is highly relevant as mature IL- 18 induces T cell interferon- ⁇ synthesis, a key cytokine involved in the mucosal inflammation of coeliac disease.
  • IL- 18 binds to targeted cells through a receptor comprising an ⁇ chain (IL18R ⁇ , IL18R1) and a ⁇ chain (IL18R ⁇ , AcPL, IL18RAP).
  • IL18R ⁇ , IL18R1 binds to targeted cells through a receptor comprising an ⁇ chain (IL18R ⁇ , IL18R1) and a ⁇ chain (IL18R ⁇ , AcPL, IL18RAP).
  • Mature IL- 18 is expressed in the intestinal mucosa of active, treated and latent coeliac patients but not in healthy controls (Salvati et al, Gut 50, 186-90, 2002).
  • IL18RAP is strongly expressed in unstimulated T cells and NK cells (GNF SymAtlas, Su et al, supra), and is expressed in small intestinal biopsies (Figure 5).
  • a large cis effect (ANOVA P 3.2 x 10 -5 ) of rs917997 genotypes was observed on the level of
  • ILl 8RAP mRNA expression in whole blood from treated coeliac patients (Figure 3), accounting for 16.1% of the population variance in ILl 8RAP expression.
  • a significant allele dosage effect on expression ⁇ post-hoc regression testing for linear trend P ⁇ 0.0001) was also observed.
  • Individuals homozygous for the minor rs917997 A allele (which is more common in coeliac than control subjects) expressed the lowest levels of IL18RAP mRNA, heterozygotes intermediate and G allele homozygotes the highest levels.
  • ILl 8RAP The coding regions of ILl 8RAP were sequenced in 23 coeliac disease patients and 8 control individuals, and 19 variants were found, 17 of which were already in dbSNP. No variants (dbSNP or from resequencing) map to the region of the Illumina IL18RAP expression probe. None of the variants is predicted to have functional consequences.
  • One new variant c.1210+17 A>G
  • the other new variant c.l384+70_1384+71insT was found in two coeliac individuals.
  • rs6441961 lies 44kb 3' of the nearest gene (CCR3).
  • LD block definition is hampered by poor HapMap coverage of this region due to structural variation (Iafrate et al., Nat Genet 36, 949-51, 2004).
  • Chemokines and their receptors are critical for the recruitment of effector immune cells to the site of inflammation.
  • WTCCC GWAS Wellcome Trust Case Control Consortium, supra
  • type 1 diabetes shows modest association in the same direction with the same allele of SNP rs6441961 (P ⁇ 10 -5 , Table 8) suggesting a possible common mechanism between both immune-mediated diseases.
  • IL- 12 induces interferon- ⁇ secreting ThI cells, one of the immunological hallmarks of coeliac disease.
  • TAGAP is expressed in activated T cells (Mao et al, Genomics
  • Rho GTPase-activating protein important for modulating cytoskeletal changes, although little is known about its role in immune function.
  • SH2B3 (also known as LNK, lymphocyte adaptor protein) is strongly expressed in monocytes and dendritic cells, as well as to a lesser extent in resting B, T cells and NK cells (GNF SymAtlas, Su et al, supra). SH2B3 was found to be strongly expressed in the small intestine. Higher expression in inflamed coeliac biopsies may reflect leukocyte recruitment and activation ( Figure 5).
  • the rs3184504 marker is a non-synonymous SNP in exon 3 of SH2B3 leading to a R262W amino acid change in the pleckstrin homology domain. This domain may be important in plasma membrane targeting.
  • SH2B3 regulates T cell receptor, growth factor, and cytokine receptor-mediated signalling implicated in leukocyte and myeloid cell homeostasis (Fitau et al, J Biol Chem 281, 20148-59, 2006 and Li et al, J Immunol 164, 5199-206, 2000).
  • SH2B3 A mice have increased responses to multiple cytokines (Velazquez et al, J Exp Med 195, 1599-611, 2002).
  • A1 represents minor allele (whole sample) for specified collection
  • A2 represents minor allele (whole sample) for specified collection

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Abstract

L'invention porte sur un procédé de diagnostic de la maladie coeliaque, lequel procédé consiste à analyser un échantillon d'acide nucléique prélevé chez un sujet humain afin de déterminer la présence ou l'absence d'un ou plusieurs polymorphismes de nucléotide simple (PNS) dans une ou plusieurs régions chromosomiques humaines choisies dans le groupe composé de Iq31, 2ql l-2ql2, 3p21, 3q25-3q26, 3q28, 6q25 et 12q24.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010067381A1 (fr) * 2008-12-12 2010-06-17 Decode Genetics Ehf Variants génétiques en tant que marqueurs pour une utilisation dans le diagnostic, le pronostic et le traitement de l'éosinophilie, de l'asthme et de l'infarctus du myocarde
US20120046233A1 (en) * 2010-01-14 2012-02-23 Jason Robert Gotlib Mutations in the lnk gene in patients with myeloproliferative neoplasms and other hematolymphoid malignancies
US8945846B2 (en) * 2010-01-14 2015-02-03 The Board Of Trustees Of The Leland Stanford Junior University Mutations in the LNK gene in patients with myeloproliferative neoplasms and other hematolymphoid malignancies

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