WO2009103992A1

WO2009103992A1 - Genetic variation associated with coeliac disease

Info

Publication number: WO2009103992A1
Application number: PCT/GB2009/000475
Authority: WO
Inventors: David Van Heel; Karen Hunt; Cisca Wijmenga; Owen Ross Mcmanus; Panagiotis Deloukas
Original assignee: Queen Mary & Westfield College; Universitair Medisch Centrum Groningen; The Provost Fellows And Scholars Of The College Of Queen Elizabeth Near Dublin; Genome Research Limited
Priority date: 2008-02-19
Filing date: 2009-02-19
Publication date: 2009-08-27
Also published as: GB0803004D0

Abstract

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 single nucleic polymorphisms (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.

Description

GENETIC VARIATION ASSOCIATED WITH COELIAC DISEASE

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.

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.

Several recent immunological advances have identified dominant epitopes, the role of tissue transglutaminase, and the crystal structures of DQ molecules binding wheat peptides as factors predisposing to coeliac disease. However, the primary factors, both environmental and genetic, predisposing to disease in the -30% of the population carrying susceptible HLA-DQ types are mostly unknown.

A single nucleotide polymorphism (SNP) 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. When a SNP occurs in a protein coding sequence, it may give rise to a change in amino acid and thus affect the polypeptide sequence encoded. Such SNPs are termed non-synonymous, whereas synonymous SNPs do not lead to a change in the encoded polypeptide sequence.

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.

There thus exists a need in the art to identify methods for accurately identifying individuals at 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.

According to a first aspect of the invention there is thus provided 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 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.

In humans, 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).

The International System for Human Cytogenetic Nomenclature (ISCN) is fixed by the Standing Committee on Human Cytogenetic Nomenclature. The basic terminology for banded chromosomes was decided at a meeting in Paris in 1971, and is therefore often referred to as the Paris 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 (SNP) 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.

The 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 present in the sequences of SEQ ID NOs: 1 to 13.

Within these 13 SNPs, the following alleles are indicative of coeliac disease (i.e. more common in coeliac disease than in control subjects):

T in SEQ ID NO: 1 (rs2816316) G in SEQ ID NO: 2 (rsl3015714)

A in SEQ ID NO: 3 (rs917997)

T in SEQ ID NO: 4 (rs6441961)

G in SEQ ID NO: 5 (rs 17810546)

C in SEQ ID NO: 6 (rs9811792) C in SEQ ID NO: 7 (rs9851967)

T in SEQ ID NO: 8 (rs 13076312)

T in SEQ ID NO: 9 (rsl464510)

T in SEQ ID NO: 10 (rs 1559810)

A in SEQ ID NO: 11 (rs 1738074) T in SEQ ID NO: 12 (rs3184504)

G in SEQ ID NO: 13 (rs653178) Accordingly, the risk of coeliac disease is increased in individuals possessing one of more of the above alleles. In other words, 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 human chromosomal regions now identified by the inventors as being associated with coeliac disease are in addition to the region 4q27 that was previously identified by the inventors (van Heel et al, supra). Accordingly, in some embodiments of the invention, 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). Accordingly, in these embodiments of the invention, 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.

Within these 8 SNPs, the following alleles are indicative of coeliac disease (i.e. more common in coeliac disease than in control subjects): T in SEQ ID NO: 14 (rsl 1938795) A in SEQ ID NO: 15 (rsl3151961) A in SEQ ID NO: 16 (rsl3119723)

T in SEQ ID NO: 17 (rsl 1734090) A in SEQ ID NO: 18 (rs7684187) G in SEQ ID NO: 19 (rsl 2642902) G in SEQ ID NO: 20 (rs6822844) C in SEQ ID NO: 21 (rs6840978) Accordingly, the risk of coeliac disease is increased in individuals possessing one or more of the above alleles. In other words, 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 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.

The 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.

Accordingly, in one embodiment of the invention, 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.

As set out above, 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.

In this embodiment, 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. In this embodiment of the invention, 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.

Typically, in this embodiment of the invention, 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.

The present invention can also be used in combination with previously reported HLA typing to diagnose coeliac disease. 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).

In the methods of the present invention, 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.

Typically, 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. In this embodiment, 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. For example, 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.

In one embodiment of the present invention, 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. In this embodiment, 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. Accordingly, 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.

Typically, 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. In this embodiment, 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.

For example, 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, SEQ ID NO: 13 and SEQ ID NO: 20 are used in combination.

The above-mentioned combinations of SNPs can also be used in combination with previously reported HLA typing to diagnose coeliac disease. 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).

In the methods of the invention, 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. For example, 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. In some embodiments of the invention, however, 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.

In some embodiments of the invention, it will be necessary to compare the sample taken from the subject to a control sample. The control sample is typically a nucleic acid sample taken from a subject who is known not to be suffering from coeliac disease. However, it 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.

To determine the presence or absence of one or more SNPs in one or more of the above-mentioned human chromosomal regions, and typically to determine whether one or more of the above-mentioned SNPs is present in 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. For example, 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.

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.

In one embodiment, the nucleic acid is analysed by sequencing of the isolated nucleic acid. Again, sequencing of the isolated nucleic acid can be carried out by any suitable method known in the art.

In the methods of the present invention, one or more SNPs is detected. SNPs can be detected by a variety of methods known in the art. For example, 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. In one embodiment, the invention is used to diagnose individuals with one or more medical symptoms of coeliac disease, such as diarrhoea and/or fatigue. In other embodiments, 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. Typically, the presence of one or more SNPs in one or more of said human chromosomal regions is indicative of coeliac disease.

Typically, the one or more SNPs are selected from the group consisting of the SNPs present in SEQ ID NOs: 1 to 13. In this embodiment of the invention, the presence of one or more of the following alleles is indicative of coeliac disease:

T in SEQ ID NO: 1 (rs2816316)

G in SEQ ID NO: 2 (rsl3015714)

A in SEQ ID NO: 3 (rs917997)

T in SEQ ID NO: 4 (rs6441961) G in SEQ ID NO : 5 (rs 17810546)

C in SEQ ID NO: 6 (rs9811792)

C in SEQ ID NO: 7 (rs9851967)

T in SEQ ID NO: 8 (rsl3076312)

T in SEQ ID NO: 9 (rsl464510) T in SEQ ID NO: 10 (rsl 559810)

A in SEQ ID NO: 11 (rsl 738074)

T in SEQ ID NO: 12 (rs3184504) G in SEQ ID NO: 13 (rs653178)

In this embodiment of the invention, 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.

In some embodiments of the invention, 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. In this embodiment of the invention, the presence of one or more SNPs in this chromosomal region is indicative of coeliac disease.

Typically, in this embodiment of the invention, the presence of one or more of the following alleles is indicative of coeliac disease:

T in SEQ ID NO: 14 (rsl 1938795) A in SEQ ID NO: 15 (rsl 3151961)

A in SEQ ID NO: 16 (rsl3119723)

T in SEQ ID NO: 17 (rsl 1734090)

A in SEQ ID NO: 18 (rs7684187)

G in SEQ ID NO: 19 (rsl2642902) G in SEQ ID NO: 20 (rs6822844)

C in SEQ ID NO: 21 (rs6840978)

In this embodiment, 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.

In a second aspect, 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.

In a third aspect, 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.

In a fourth aspect, 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.

Preferred features for the second, third and fourth aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as limiting on the invention. Reference is made to a number of Figures, in which:

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 IL18RΛP 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, CCRl, CCR3 (Illumina probe

1980750), SCHIPl, IL2, IL21, TAGAP (two probes) and Tenr were not detected above background in intestinal tissue and are not shown. 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. Representative (n>3) semi-quantitative RT-PCR experiments for RGS-I, CCR5, CCR9 and β-actin on small intestinal intraepithelial lymphocytes (IELs); TCRγδ⁺CD4-CD8- [γδ DN]; TCRγδ⁺CD4- CD8 αα ⁺ [γδ CD8αα]; TCRαβ⁺CD4OD8 αα ⁺ [αβ CD8αα]; TCRαβ⁺CD4- CD8αβ⁺ [αβ CD8αβ]; TCR γδ⁺ cells [γδ] from thymus and spleen, CD4⁺CD8αβ⁺ [DP], TCRαβ⁺CD4⁺CD8αβ- [SP CD4] and TCRαβ⁺CD4-

CD8αβ⁺ [SP CD8] from thymus, and TCRαβ⁺CD4-CD8αβ⁺ [αβ CD8] from spleen. cDNA was normalised to β -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 )

Examples

INTRODUCTION

To identify additional coeliac disease susceptibility genes, the inventors recently tested 310,605 SNPs in a genome wide association study of 778 coeliac cases and

1,422 population controls from the United Kingdom (UKGWAS), using the Illumina

HumanHap300 BeadChip (van Heel et ah, supra). The only SNP outside the HLA region demonstrating genome-wide significance was rsl3119723 on 4q27, located in a -500 kb block of linkage disequilibrium (LD) containing the IL2 and IL21 genes. Independent replication of SNPs from the IL2-IL21 region was established in both

Dutch and Irish collections of coeliac patients and controls. It is estimated, using the current markers, that the IL2-IL21 region explains less than 1% of the increased familial risk to coeliac disease. Since a greater number of significantly associated

SNPs in the UKGWAS was observed than would be expected by chance, the inventors proceeded to study > 1,000 of the most significant UKGWAS association results in a further 1,643 coeliac cases and 3,406 controls from three independent

European coeliac disease collections. This two-stage strategy, involving a joint analysis of all data, substantially reduces the genotyping requirements versus performing whole genome genotyping on all samples and has been shown to maintain sufficient statistical power. ^"*--"

METHODS

Subjects. Detailed characteristics of UKGWAS, IRISH and DUTCH samples are provided in Table 3 below, and the inventors' previously published study (van Heel et ah, supra).

All subjects were of white Northern European origin. UK2 subjects were recruited similarly to the previously described UKGWAS subjects (van Heel et al., supra) - cases were recruited from hospital clinics and controls from the British 1958 Birth Cohort, except for n=374 cases and n=176 unrelated controls recruited direct through Coeliac UK advertisement. For DNA procedures see Supplementary Methods below. It was noted that HLA-DQ2.5cis genotype frequencies (Table 3 below, inferred from rs2187668) were similarly high across the coeliac collections (carriage rate range 87.4% to 89.1%), and similarly low across the control populations (carriage rate range 25.5% to 33.8%), suggesting broadly comparable phenotypic ascertainment across the four collections. Informed consent was obtained from all subjects. Ethical approval was from Oxfordshire REC B or East London and the City REC 1 (UKGWAS, UK2), the Medical Ethical Committee of the University Medical Center Utrecht (DUTCH), and the Institutional Ethics Committee of St James's Hospital (IRISH).

Marker selection and genotyping. Single non-HLA SNPs with two-tailed allele count χ² test P<0.00275, or if nsSNPs P<0.01, were selected from the inventors' published coeliac disease genome wide association study (van Heel et al., supra) of 310,605 post-quality control markers (Hardy- Weinberg equilibrium P>0.0001 in controls). Genotyping data and clustering of SNP genotypes was managed in BeadStudio. Samples with <95% call rate over 1025 SNPs, SNPs with <95% call rate over remaining samples, SNPs with poor amplification or poor genotype cloud clustering were excluded. Quality control steps. Using the 1025 SNP dataset, pairwise comparisons of identity by-descent were made for all samples (UKGWAS, UK2, IRISH, DUTCH) using PLINK (Purcell et al, Am J Hum Genet 81, 559-75, 2007). A higher proportion of 1 st degree relatives in the all sample dataset (98 pairs) was detected than in the initial

UK coeliac GWAS (11 pairs), and therefore in the current analyses the lowest call rate sample from each pair of 1° relatives from the entire study dataset was excluded. Minor but insignificant changes are therefore present in the UKGWAS dataset results compared to the inventors' previous publication (van Heel et al., supra).

Potential ethnic outlier samples were excluded (n=3, outliers had previously been excluded from the UKGWAS dataset) using the nearest neighbour allele sharing method in PLINK (samples with Z scores <-3 with >1 of 5 nearest neighbours excluded). A filter for SNP selection was applied for follow up GoldenGate genotyping based on Hardy Weinberg (HWE) equilibrium P>0.0001 in Infinium genotyped controls from the UKGWAS. HWE P values for each of the coeliac disease associated SNPs and 5 HLA tag markers in each of the follow-up UK2, IRISH and Dutch collections are shown in Table 4. Because of the prior filter step, no follow-up SNPs were specifically excluded based on HWE analysis. All of the top association findings were in HWE in controls.

Table 4 shows detailed association results. Table 4 indicates the alleles for the SNPs associated with coeliac disease. For 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) and rs6840978 (SEQ ID NO: 21) the alleles shown in Table 4 do not correspond to the disease associated alleles specified in the description, claims and Figures herein. This is because the double stranded DNA sequence can be read in either strand direction. Therefore if a SNP is described as, for example, having A and C alleles, this identical to describing it as having T and G alleles. This is due to the complementarity of nucleic acid based pairing, in which the complement of A is T and vice versa and the complement of C is G and vice versa, which is well understood by a person skilled in the art. The alleles for the SNPs shown in 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.

Genotype statistical analysis. Cochran-Mantel-Haenszel allele count chi-squared association tests were performed using PLINK (Purcell et al, supra) with 4 clusters: UKGWAS (Infinium assay), UK2 (GoldenGate assay), IRISH and DUTCH collections. All P-values are two-tailed. Minimal evidence for bias was observed in the Cochran-Mantel-Haenszel test statistics due to population stratification or other factors (see λ_GC values <1.03 in RESULTS), and present uncorrected statistics. To test for differences in association across the datasets as manifested by heterogeneity of odds ratios at disease-associated regions, the Breslow-Day test was applied to SNPs in Table 2 (below). These SNPs, and HLA-DQ2.5cis (inferred by rs2187668 genotype), were also evaluated for possible epistatic (gene-gene) interaction in predisposing to coeliac disease. Breslow-Day and epistatic interaction tests were carried out as implemented in PLINK. Epistatic interactions were also tested for using the model of Howard et al (Am J Hum Genet 70, 230-6, 2002). For the PLINK analysis of interaction, genotypes at the SNPs are evaluated as allele "doses" (e.g. 0, 1, or 2) whereas for the model of Howard et al (supra) heterozygotes and homozygotes for the higher-risk allele are pooled together and compared against the lower-risk homozygote genotype. The PLINK interaction analysis and our analysis of the model of Howard et al (implemented in R statistical software, www.r-project.org) both tested for statistically significant departure from the model of log-additive odds ratios as evaluated by logistic regression.

The possible influence of stratification was also investigated by an alternative principle components approach in which each dataset was separately evaluated by EIGENSTRAT software (Price et al. Nat Genet 38, 904-9, 2006). After eliminating SNPs in the 9 regions showing association with coeliac disease, 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. For each case-control set, 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). This enabled us to achieve a corrected, study-wide Z-score and P value for each SNP that can be compared to the Cochran-Mantel-Haenszel P value and shows that all 9 regions remain significant at P <5xlO^-7 after correction by this alternative approach (Table 4). The corrected P values for SNPs not in the 9 coeliac-associated regions also generate a QQ-plot that shows no evidence of overdispersion and is almost identical to the QQ- plot (Figure Ib) of the Cochran-Mantel-Haenszel P values.

Calculation of familial clustering for each region is described in Supplementary Methods, below.

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.

Whole blood expression analysis. PAXgene blood 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.

Whole blood PAXgene expression data were obtained from 114 unique coeliac individuals who had been genotyped in the UKGWAS. To assess the quality of the

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

Pearson correlation all samples = 0.22) were correlated and removed n=2 outlier samples that had low correlation (Pearson correlation < 0.10). Analysis of transcripts mapping to the non-pseudoautosomal region of chromosome Y, allowed for determining sex, resulting in the removal of n=2 samples with mismatched gender. n=l further sample was removed where genotypes did not correlate as expected with cis expression level across the genome. Data from 109 individuals were analysed after applying these quality control criteria. ANOVA tests were performed to investigate possible cis effects of the strongest SNP associations on candidate gene expression from disease associated regions.

Intestinal biopsy expression analysis. Duodenal tissue biopsies were collected in RNAlater (Ambion), 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.

SUPPLEMENTARY METHODS

Subject DNA. 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. One marker (rs641941) failed in Irish but not Dutch WGA samples.

Genotype concordance. Concordance between saliva DNA GoldenGate genotypes and blood DNA Infinium genotypes was 99.85% in n=4 subjects genotyped for 1025 SNPs on both platforms. 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.

Calculation of familial clustering due to the identified loci. For the most significant SNP in each locus in Table 2, genotype relative risk (GRR) 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

Genet 67, 1340-7, 2000). The calculated GRR and population frequency of the disease-associated allele were then used to calculate familial clustering in terms of the sibling relative risk (λ_s) contributed by the locus (McGinnis, supra). Familial clustering contributed by the 8 loci in Table 2 and by HLA were combined according to the multilocus multiplicative model of Risch (Am J Hum Genet 46, 222-8, 1990) and thus ∑log(λ_s,)/log(λ_s) is the proportion of the total disease clustering (λ_s) contributed by the identified loci (X₅₁). For this calculation, epidemiological estimates of 30 for λ_s and 3.3 for the λ_s, contributed by the HLA region (Bevan et al, J Med

Genet 36, 687-90, 1999) were used. Expression profiling of T cell subsets. 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:

CCR5-F 5'-GGTACTTGGCTATTGTCCATGCTG-S' (SEQ ID NO: 22)

CCR5-R 5'-ATGACAAGTAGAGGCAGGATCAGG-S' (SEQ ID NO: 23)

CCR7-F 5'-ATCATCCGTACCTTGCTCCAGGCAC-S ' (SEQ ID NO: 24)

CCR7-R 5'-TGTCAACCTGACTGGCCAGAATTGC-S ' (SEQ ID NO: 25)

RGS-I-F 5'-ACCTGAGATCGATGATCCCACATCT-S' (SEQ ID NO: 26) RGS-I-R 5'-CTGTCGATTCTCGAGTATGGAAGTC-S ' (SEQ ID NO: 27) β-actin-F 5'-TCCCTGTATGCCTCTGGTCGTACCAC-S ' (SEQ ID NO: 28) β-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. After exclusion of SNPs unlikely to be successful based on Illumina GoldenGate assay design criteria, and inspection of actual genotype clusters, 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 markers included 8 SNPs from the IL2-IL21 region that were reported to be associated to coeliac disease in the inventors' previous study (van Heel et al., supra), and 5 SNPs were additionally selected to tag coeliac disease associated HLA-DQ2/8 haplotypes (de Bakker et al, Nat Genet 38, 1166-72, 2006). Samples failing quality control criteria were excluded (see Methods, above), and 719 cases and 1,561 population controls from the UK (UK2 collection), 416 cases and 957 bloodbank controls from Ireland (IRISH), 508 cases and 888 bloodbank controls from the Netherlands (DUTCH) were analysed.

Observed association statistics for the UKGWAS SNPs in the follow-up collections markedly deviate from expected findings (Figure Ia). The inventors reported 21 non- HLA SNPs from 8 distinct chromosomal regions meeting a genome wide significance threshold in all 7,238 samples of /* overall < 5 x 10^-7 (Table 2 above). Results from the WTCCC (Wellcome Trust Case Control Consortium, supra) and other recent GWA studies have shown that the majority of markers at a P < 5 x 10^-7 "genome- wide" significance level will be true findings, although independent replication by other investigators is necessary for definitive validation. Only one of these eight regions, the IL2-IL21 region, has been previously reported in coeliac disease (van Heel et al., supra). Breslow-Day tests were non-significant for each of the eight regions implying consistent effect sizes and direction across the four collections, and accuracy of the reported Cochran-Mantel-Haenszel test odds ratios. The observation of generally weaker association evidence in the IRISH dataset (Table 4) is therefore likely to be a reflection of the smaller sample-size of this collection, rather than ethnic heterogeneity. No evidence was observed for gene-gene interactions (departure from log-additive effects) between these regions, nor between the HLA and these regions. Association statistics, linkage disequilibrium plots, and Ensembl genes for each of the eight regions are shown in Table 2 and Figure 2, and more detailed statistics are shown in Table 4. Coeliac disease HLA-DQ associations reflect the ability of antigen presenting cells to present toxic cereal epitopes to T cells. Remarkably, seven of the eight identified non-HLA regions also contain biologically plausible candidate genes involved in the immune response.

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. The median distribution of test statistics was assessed using genomic control (Devlin & Roeder, Biometrics 55, 997-1004, 1999) (λoc = 1.0 indicates a null distribution with no inflation of test statistics). In the full UKGWAS genome wide association scan dataset (using 767 non- first degree related cases and 1422 controls geno typed for 307,411 non-HLA SNPs) there was minimal evidence for inflation of test statistics (λoc = 1.03). In the UK2 IRISH DUTCH follow-up samples, genotyped for 1020 non-HLA SNPs there was also little evidence for inflation (λoc = 1.02). When 28 SNPs mapping to the eight non-HLA coeliac associated regions were also excluded there was no evidence for test statistic bias (λoc = 1.00) in the follow-up samples, and the residual Q-Q plot showed little deviation from expected results (Figure Ib). Furthermore, when an alternative principal components approach was used, each of the eight non-HLA regions again met the genome wide significance threshold (P overall < 5 x 10^-7) when corrected by EIGENSTRAT analysis (Table 6). Cis gene expression in whole blood RNA samples (n^KW) was correlated with SNPs from the eight non-HLA coeliac associated regions, to test for possible functional effects of these markers. Ei ght putative candidate genes from the regions were expressed above background levels in these samples. 14 pairs of SNPs and expressed gene probes were analysed (Table 7). Tissue and cell type expression were analysed based on published literature and the GNF SymAtlas database (Su et al, Proc Natl Acad Sci USA 101, 6062-7, 2004) for putative candidate genes from each region. Gene expression in small intestinal tissue (where coeliac disease manifests) from healthy controls, and treated and untreated coeliac disease individuals was analysed. Each region is summarised below.

4q27

A previously identified SNP rs6822844 (van Heel et al., supra), located -24 kb 3' of

IL21, showed the strongest association with coeliac disease in the current study (P overall=2.82 x 10^-13). 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. A SNP allele from this region was also reported in a recent type 1 diabetes GWAS (conferring susceptibility), and Graves' disease (conferring reduced risk) (Todd et al, Nat Genet 39, 857-64 2007). A further Dutch study suggested association of rs6822844 with type 1 diabetes and rheumatoid arthritis, in the same direction as the coeliac disease data (Zhernakova et al, Am J Hum Genet 81, 1284-8, 2007). These findings suggest the 4q27 region might represent a more general autoimmune locus, although whether effects are due to one or multiple causal variants and the exact nature of these effects is currently unclear.

Iq31 The most significant SNP outside the HLA and IL2-IL21 regions is rs2816316 (P overall = 2.58 x 10^'11) located within a ~70kb LD block containing the gene RGSl (regulator of G-protein signaling 1). rs2816316 maps 8kb distal to the 5' end of RGSl. 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). Interestingly, the present inventors previously observed RGSl to be strongly expressed in murine intestinal intra-epithelial lymphocytes (Pennington et al, Nat Immunol 4, 991-8, 2003), and now confirm that T cell RGSl expression appears to be specific to the intestinal intra-epithelial lymphocyte compartment and is not found in conventional splenic or thymic αβ T cells (Figure 6). Intestinal intra-epithelial lymphocytes play a key role in epithelial cell death and the development of villous atrophy in coeliac disease (Hue et al, Immunity 21, 367- 77, 2004).

2qll - 2ql2

Two associated SNPs, rs917997 (P overall=8.49 x 10^-10) and rsl3015714 (both in LD, r²=0.95 in UKGWAS controls) map to a ~400kb linkage disequilibrium block. Interestingly, in the WTCCC GWAS (Wellcome Trust Case Control Consortium, supra), Crohn's disease shows modest association (proxy SNP for rs917997, P~10^-4, Table 8). This LD block contains four genes, two of which are receptors for the IL- 18 protein (ILl 8RAP and ILl 8Rl). The IL- 18 pathway is highly relevant as mature IL- 18 induces T cell interferon-γ synthesis, a key cytokine involved in the mucosal inflammation of coeliac disease. IL- 18 binds to targeted cells through a receptor comprising an α chain (IL18Rα, IL18R1) and a β chain (IL18Rβ, 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.

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) was observed in a single control and the other new variant (c.l384+70_1384+71insT) was found in two coeliac individuals.

3p21

SNP rs6441961 (P overall = 3.14 x 10^-7) maps within a large cluster of chemokine receptor genes on 3p21, including CCRl, CCR2, CCRL2, CCR3, CCR5 and CCXCRl. 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. In the 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.

3q25 - 3q26 Two SNPs (rsl7810546, rs9811792) in a ~70kb linkage disequilibrium block show strong association to coeliac disease (rs 17810546 P overall = 1.07 x 10^-9). This region is immediately 5' of IL12A (interleukin- 12 A). Interestingly, the two associated SNPs (r²=0.l9 in UKGWAS controls) may represent independent association signals. SNP rs 17810546 also shows modest correlation with SNPs in both 1L12A and SCHIPl (schwannomin interacting protein 1). IL12A encodes the IL12p35 subunit that together with IL12p40 form IL12p70, i.e. the heterodimeric IL- 12 cytokine that has a broad range of biological activities on T and natural killer cells.

IL- 12 induces interferon-γ secreting ThI cells, one of the immunological hallmarks of coeliac disease.

3q28 Multiple correlated SNPs (rsl464510 P overall = 5.33 x 10^-9) within a -70 kb linkage disequilibrium block show association with coeliac disease. This block is either 5' of the RefSeq gene LPP, or intronic for other possible isoforms of LPP. The LPP gene shows very high expression in the small intestine (Figure 5), and may play a structural role at sites of cell adhesion in maintaining cell shape and motility. Relatively little is known about LPP, and how genetic variation in this region might predispose to coeliac disease is unclear.

6q25

SNP rsl738074 (P overall = 6.71 X 10^-8) on chromosome 6q25.3 maps to a -200 kb linkage disequilibrium block containing TAGAP (T-cell activation GTPase activating protein), itself within a larger region of weaker LD containing RSPH3 (radial spokehead-like 3). TAGAP is expressed in activated T cells (Mao et al, Genomics

83, 989-99, 2004), has three isoforms, and is a Rho GTPase-activating protein important for modulating cytoskeletal changes, although little is known about its role in immune function.

12q24

Association signals arise from two correlated SNPs rs653178 (P overall = 8 x 10- ) and rs3184504 (r²=0.99 in UKGWAS controls). These markers map in the vicinity of SH2B3 (also known as LNK) and ATXN 2, modest LD is seen over a broader region of -1Mb containing multiple other genes. Strong association with type 1 diabetes is reported in this region, with rs3184504 (same allele and direction as coeliac disease) entirely accounting for the association signal (Todd et al, supra). 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).

Tables 4 to 6 - Footnotes

All outputs are from PLINK, text has been edited in Excel.

SampleName GenotvpinqAssav

UKGWAS lnfinium

UK2 GoldenGate

IRISH GoldenGate

DUTCH GoldenGate

All samples genotyped for 1025 SNPs, including 5 HLA tag markers, only coeliac-associated

SNPs and HLA tag markers shown

Abbreviations for Table 4 - Detailed Association Results

A1 represents minor allele (whole sample) for specified collection

A2 represents minor allele (whole sample) for specified collection

F_A, F_U allele frequency in affecteds, unaffecteds

P ASSOC: two tailed P value from the 2x2 allele count chi-squared test

P CMH: two tailed P value from the 2x2xK Cochran-Mantel-Haenszel test

OR CMH: allelic odds ratio from the 2x2xK Cochran-Mantel-Haenszel test

Abbreviations for Table 5 - Detailed Call Rates

F_MISS fraction missing data (0.0=complete data, 1.0 = entirely missing data)

Claims

1. 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 single nucleic polymorphisms (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.

2. A method according to claim 1, wherein said 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.

3. A method according to claim 2, wherein said one or more SNPs comprises the SNPs present in the following sequences: 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, and SEQ ID NO: 12 and/or SEQ ID NO: 13.

4. A method according to any one of claims 1 to 3, wherein said method further comprises analysing a sample of nucleic acid from said human subject to determine the presence or absence of one or more SNPs in the human chromosomal region 4q27.

5. A method according to claim 4, wherein said one or more SNPs are 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.

6. A method according to claim 5, wherein said one or more SNPs comprise the SNPs present in the following sequences: 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.