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Background Two inflammatory disorders, type 1 diabetes and celiac disease, cosegregate in populations, suggesting a common genetic origin. Since both diseases are associated with the HLA class II genes on chromosome 6p21, we tested whether non-HLA loci are shared.
Methods We evaluated the association between type 1 diabetes and eight loci related to the risk of celiac disease by genotyping and statistical analyses of DNA samples from 8064 patients with type 1 diabetes, 9339 control subjects, and 2828 families providing 3064 parent–child trios (consisting of an affected child and both biologic parents). We also investigated 18 loci associated with type 1 diabetes in 2560 patients with celiac disease and 9339 control subjects.
Results Three celiac disease loci — RGS1 on chromosome 1q31, IL18RAP on chromosome 2q12, and TAGAP on chromosome 6q25 — were associated with type 1 diabetes (P<1.00x10–4). The 32-bp insertion–deletion variant on chromosome 3p21 was newly identified as a type 1 diabetes locus (P=1.81x10–8) and was also associated with celiac disease, along with PTPN2 on chromosome 18p11 and CTLA4 on chromosome 2q33, bringing the total number of loci with evidence of a shared association to seven, including SH2B3 on chromosome 12q24. The effects of the IL18RAP and TAGAP alleles confer protection in type 1 diabetes and susceptibility in celiac disease. Loci with distinct effects in the two diseases included INS on chromosome 11p15, IL2RA on chromosome 10p15, and PTPN22 on chromosome 1p13 in type 1 diabetes and IL12A on 3q25 and LPP on 3q28 in celiac disease.
Conclusions A genetic susceptibility to both type 1 diabetes and celiac disease shares common alleles. These data suggest that common biologic mechanisms, such as autoimmunity-related tissue damage and intolerance to dietary antigens, may be etiologic features of both diseases.
Celiac disease and anti–tissue transglutaminase antibodies occur more frequently in patients with type 1 diabetes than in the general population, depending on the age of the patient; at most, 10% of children and 2% of adults with type 1 diabetes have positive tests for such antibodies.11 An increasing incidence of celiac disease during recent decades has also been reported.8 It has been suggested that gluten consumption, along with gut permeability and inflammation, are factors in the development of type 1 diabetes.6,12 These results suggest that type 1 diabetes and celiac disease may share some causative genetic and environmental factors.
Genomewide association studies have recently identified eight chromosome regions outside the HLA region as being associated with celiac disease (P<5.00x10–7), findings that probably provide a representative view of the major genetic effects in the northern European population for this disorder.10 In patients with type 1 diabetes, 15 non-HLA regions have been established to date,1,13,14,15 and two other loci, IL7R on chromosome 5p13 and CD226 on chromosome 18q22, have been implicated in type 1 diabetes and multiple sclerosis.1,16,17 It has already been reported that the SH2B3 locus on chromosome 12q24 is shared between type 1 diabetes and celiac disease, with indications of such sharing in IL2–IL21 on chromosome 4q27 and CCR3 on chromosome 3p21.9,10 In addition, there is some evidence for association of the established type 1 diabetes loci, CTLA4 on chromosome 2q339,18 and PTPN22 on chromosome 1p13,19 in celiac disease. In this study, we evaluated the association between all these loci and type 1 diabetes and celiac disease, including the CCR5 32-bp insertion–deletion variant that we report here as a type 1 diabetes locus, in order to assess the genetic similarities and differences between these two inflammatory disorders. (See the Glossary and the Methods section in the Supplementary Appendix, available with the full text of this article at www.nejm.org.)
Methods
Study Subjects
In our study, patients with type 1 diabetes (www.childhood-diabetes.org.uk/grid.shtml) were under 16 years of age at the time of sample collection, with a mean age at diagnosis of 7.5 years (range, 0.5 to 16).1 A total of 9339 control samples were obtained from 6164 subjects in the British 1958 Birth Cohort (www.b58cgene.sgul.ac.uk) and from a collection of 3175 blood donors, established by the Wellcome Trust Case Consortium.13 The cohort of 2828 families (providing 3064 parent–child trios, consisting of one affected child and two biologic parents) included 468 multiplex families from the Diabetes UK Warren I repository, 331 multiplex families from the Human Biological Data Interchange in the United States, 881 multiplex and simplex families from Finland, 263 multiplex and simplex families from Northern Ireland, 124 simplex families from the Diabetes UK Warren III repository, 350 simplex families from Norway, and 411 simplex families from Romania (www-gene.cimr.cam.ac.uk/todd/dna-refs.shtml).1 The 2560 patients with celiac disease were recruited throughout England, Scotland, and Wales. DNA was extracted from peripheral-blood samples obtained from 1175 patients recruited from hospital outpatient clinics and from saliva samples obtained from 1385 patients recruited through an advertisement by Celiac UK.
The diagnosis of celiac disease was based on clinical symptoms, a current gluten-free diet, serologic analysis, a biopsy sample of the small intestine, and response to treatment. The mean age at diagnosis was 41.0 years (range, 3 months to 84 years); 75.1% of the patients were female. The Irish collection consisted of 416 patients with celiac disease and 957 control subjects, and the Dutch collection consisted of 507 patients with celiac disease and 888 control subjects.10 All patients with type 1 diabetes or celiac disease, control subjects, and parent–child trio families reported their race as white. The relevant research ethics committees approved the study, and written informed consent was obtained from all study subjects or their parents or guardians.
Genotyping
We genotyped single-nucleotide polymorphisms (SNPs) from 8 celiac disease loci — RGS1 on chromosome 1q31, IL18RAP on chromosome 2q12, CCR3 on chromosome 3p21, IL12A on chromosome 3q25, LPP on chromosome 3q28, IL2–IL21 on chromosome 4q27, TAGAP on chromosome 6q25, and SH2B3 on chromosome 12q24 — and from 15 type 1 diabetes loci — PTPN22 on chromosome 1p13, IFIH1 on chromosome 2q24, CTLA4 on chromosome 2q33, IL2–IL21 on chromosome 4q27, BACH2 on chromosome 6q15, IL2RA (CD25) on chromosome 10p15, PRKCQ on chromosome 10p15, INS on chromosome 11p15, ERBB3 on chromosome 12q13, SH2B3 on chromosome 12q24, CTSH on chromosome 15q24, CLEC16A on chromosome 16p13, PTPN2 on chromosome 18p11, UBASH3A on chromosome 21q22, and C1QTNF6 on chromosome 22q13 (for all gene names, see the Glossary in the Supplementary Appendix). We also genotyped SNPs from IL7R on chromosome 5p13, CD226 on chromosome 18q22, and the 32-bp insertion–deletion variant in CCR5 on chromosome 3p21.
Statistical Analysis
In our study, P<1.00x10–4 was determined to indicate statistical significance. This approach was conservative because established evidence needed to show that the two diseases being studied have a familial association (cosegregation), a clinical or epidemiologic association, or both and that they share some clinical and biologic phenotypes.20,21 We also required that the evidence for the locus association with the first disease be robust and convincing (i.e., P<5.00x10–7 in multiple populations) and that there be robust marker scoring and statistical analyses (see the Supplementary Appendix for details).
Results
Celiac Disease Loci in Type 1 Diabetes
We genotyped samples from 8064 patients with type 1 diabetes and 9339 control subjects and, where appropriate, samples from 2828 families. We chose the nine SNPs with the highest disease association from the eight non-HLA regions associated with celiac disease10 (Table 1, and Table 1 in the Supplementary Appendix). Three of these newly analyzed regions — RGS1 on chromosome 1q31, IL18RAP on chromosome 2q12, and TAGAP on chromosome 6q25 — showed strong evidence of association with type 1 diabetes (P<1.00x10–4) in case–control and family subjects. Therefore, along with the sharing in SH2B3 on chromosome 12q24 that was reported previously,10 four of these eight celiac disease loci are shared with those associated with type 1 diabetes (Figure 1A). The celiac disease–associated SNPs rs6441961 in CCR3 and rs6822844 in IL2–IL21 did not reach the threshold for significance for type 1 diabetes (P>1.00x10–4) (Table 1). The regions IL12A on chromosome 3q25 and LPP on chromosome 3q28 showed no evidence of association with type 1 diabetes (P>0.15) (Table 1 and Figure 1D).
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Type 1 Diabetes Loci in Celiac Disease
We analyzed the associations of the 18 loci that have been associated with type 1 diabetes in celiac disease (including the CCR5 insertion–deletion, rs333, and the SH2B3 locus on 12q24 that was previously recognized to be shared between the two diseases) by genotyping 19 SNPs and the rs333 variant in CCR5 in 2560 patients with celiac disease. We then compared the results with those from 9339 control subjects (Table 2 and Figure 1, and Table 3 in the Supplementary Appendix). The loci with the most significant associations were CTLA4 (rs3087243) with an odds ratio of 0.85 (P=1.26x10–6) and CCR5 (rs333) with an odds ratio of 0.79 (P=9.18x10–6). The findings in these two regions indicated that these are probably true effects, a conclusion supported by previous reports that these loci have been associated with both type 1 diabetes and celiac disease and other immune-mediated diseases.9,18,23,24,25,26,27,28
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We previously reported two independent associations with type 1 diabetes within the PTPN2 region marked by the SNPs rs1893217 and rs478582.1 Resequencing of the PTPN2 gene, genotyping, and analyses identified SNP rs45450798 in high linkage disequilibrium with rs1893217 (r2=0.97), so rs45450798 replaces rs1893217 as the most strongly associated SNP in the PTPN2 region. Logistic forward regression analysis revealed that rs45450798 explained the association at rs1893217 and, combined with rs478582, explained the association with type 1 diabetes of the PTPN2 chromosome region (Table 4 in the Supplementary Appendix). In the patients with celiac disease, the significance of the association of PTPN2 SNP rs45450798 (P=2.61x10–4) narrowly missed the threshold of P<1.00x10–4 (Table 2). Therefore, we analyzed the available but unpublished data for the two independent case–control sample sets from Ireland and the Netherlands, obtaining consistent support for the association of PTPN2 rs1893217 with celiac disease (P=0.045) (Table 6 in the Supplementary Appendix). Given the fact that PTPN2 has also been associated with another inflammatory disorder, Crohn's disease,29 it is highly likely that PTPN2 is also a celiac disease locus, bringing the total of non-HLA celiac disease loci from 8 to 11.
Six other regions showed nominal evidence of association with celiac disease (P<0.05). The most strongly associated SNP for the risk of type 1 diabetes in the IL2–IL21 region on chromosome 4q27, rs2069763 (a synonymous SNP in exon 1 of IL2), is weakly associated with celiac disease (P=0.02), which indicates that although this region is linked to both diseases, the genetic variants are different. The remaining regions are IL7R on chromosome 5p13 (P=0.007), BACH2 on chromosome 6q15 (P=0.003), PRKCQ on chromosome 10p15 (P=0.02), CD226 on chromosome 18q22 (P=0.01), and UBASH3A on chromosome 21q22 (P=0.009). Figure 1 illustrates the combined results of Table 1 and Table 2, with 15 loci showing some evidence for colocalization (assuming two susceptibility variants in the IL2–IL21 region on chromosome 4q27); of these, 7 have convincing evidence: RGS1 on chromosome 1q31, IL18RAP on chromosome 2q12, CTLA4 on chromosome 2q33, CCR5 on chromosome 3p21, TAGAP on chromosome 6q25, SH2B3 on chromosome 12q24, and PTPN2 on chromosome 18p11. At least five showed distinct differences — namely, a strong association in one disease and no or little evidence for association in the other disease (INS on chromosome 11p15, PTPN22 on chromosome 1p13, IL2RA on chromosome 10p15, LPP on chromosome 3q28, and IL12A on chromosome 3q25) (Figure 1C and 1D).
Discussion
Our findings and those reported previously1,10,14,15 provide convincing evidence that 21 non-HLA loci are associated with type 1 diabetes and 11 non-HLA loci are associated with celiac disease. Of these loci, we have identified three celiac disease loci as having an association with type 1 diabetes (RGS1, IL18RAP, and TAGAP) and two type 1 diabetes loci as having an association with celiac disease (CCR5 and PTPN2). Furthermore, our results provide confirmation of the importance of the CTLA4 region on chromosome 2q33. Seven of these chromosome regions are shared between the two diseases, suggesting that for an investigation of shared loci in two diseases that are known to cosegregate, the previous odds of 1000:1 against there being a true association at any tested candidate gene20,21 is too conservative (Supplementary Appendix).
Four alleles — RGS1 on chromosome 1q31, CTLA4 on chromosome 2q33, SH2B3 on chromosome 12q24, and PTPN2 on chromosome 18p11 — show the same direction of association in the two diseases, constituting evidence for shared causal variants. We know that this is not due to bias in ascertainment of the cases, nor is the use of a common set of control subjects a problem since we have consistent results from our family-based analyses (Supplementary Appendix).
The minor alleles of the SNPs rs917997 (IL18RAP on chromosome 2q12) and rs1738074 (TAGAP on chromosome 6q25) were negatively associated with type 1 diabetes, whereas these minor alleles were positively associated with celiac disease.10 These results may be interpreted in two ways: the causal variants in these two regions may have opposite biologic effects in type 1 diabetes and celiac disease, or there may be different causal variants for each disease in each region with the typed marker SNPs tagging these causal variants. For the regions of IL18RAP on chromosome 2q12 and TAGAP on chromosome 6q25, we have found no evidence for a second locus within these regions in genomewide association studies of type 1 diabetes13,15 (data not shown). Moreover, there is precedent for a causal variant having opposing effects in different diseases. For example, the minor allele of PTPN22 variant Arg620Trp on chromosome 1p13 predisposes a person to many immune-mediated diseases but is protective for Crohn's disease.30 Hence, we favor the possibility that the causal variants have opposite effects in patients with type 1 diabetes and celiac disease. In contrast, for chromosome 4q27, our data indicate that different causal variants are involved in type 1 diabetes and celiac disease, perhaps affecting different genes. The important immune-response genes, IL-231 and IL-21, are strong functional candidates. Before we can draw further conclusions, all the regions discussed here must be thoroughly resequenced from multiple persons to ascertain a complete catalogue of polymorphisms, followed by further genotyping to identify all the variants with the most association.
Nevertheless, the 32-bp insertion–deletion in CCR5 (rs333), which causes a loss of expression of the receptor,22 could well be the actual functional, causal variant involved. The disease associations of the two chemokine receptor genes, CCR3 and CCR5, suggest the central importance of lymphocyte trafficking in these organ-specific diseases. The development and anatomy of the small intestine and pancreas are close, and the gut immune system shares close connections with pancreatic lymph nodes, which have been linked to insulitis and destruction of beta cells.32 In patients with recent-onset type 1 diabetes, alterations in levels of two CCR5 ligands, CCL3 (MIP-1
) and CCL4 (MIP-1β), have been reported.33 In the NOD mouse model of type 1 diabetes, CCR5 and its ligand CCL4 have multiple reported roles in the development of disease.34
However, there are distinct differences in genetic susceptibility between patients with type 1 diabetes and those with celiac disease, including differences in PTPN22 on chromosome 1p13, in IL2RA on chromosome 10p15, and in INS on chromosome 11p15. Although there are shared predisposing alleles at the HLA-DQB1 gene for type 1 diabetes and celiac disease, there are distinguishing differences in the HLA-DQB1 genotype (Supplementary Appendix). One possibility is that there is a common genetic background with respect to autoimmunity and inflammation and that further combinations of more disease-specific variation at HLA and non-HLA genes, in interaction with epigenetic and environmental factors, determine the final clinical outcomes.
Our results support further evaluation of the hypothesis that cereal and gluten consumption might be an environmental factor in type 1 diabetes, leading to the alteration of the function of the gut immune system and its relationship with the pancreatic immune system.6,12,32,35 Furthermore, insulin and its precursors are major targets of the T and B lymphocyte autoreactive response in type 1 diabetes. Thus, one might speculate that bovine insulin in infant foods could enhance anti-insulin responses,3 particularly if there are genetically determined defects in oral tolerance predisposing to type 1 diabetes. Conversely, genes that are classified as autoimmunity genes, because they are associated with type 1 diabetes, contribute to celiac disease.
Supported by Juvenile Diabetes Research Foundation International, the Wellcome Trust, the National Institute for Health Research's Cambridge Biomedical Research Centre, Celiac UK, the Celiac Disease Consortium (an innovative cluster approved by the Netherlands Genomics Initiative and partly funded by the Dutch Government), the European Union, the Netherlands Organization for Scientific Research, the Science Foundation Ireland, and the Irish Health Research Board.
Dr. McManus reports receiving grant support from Hitachi Europe; and Dr. van Heel, having an equity interest in and receiving consulting fees from NexPep. No other potential conflict of interest relevant to this article was reported.
We thank all the patients and control subjects for their participation in the study; the Avon Longitudinal Study of Parents and Children Laboratory in Bristol and the British 1958 Birth Cohort team, including S. Ring, R. Jones, M. Pembrey, W. McArdle, D. Strachan, and P. Burton for preparing and providing the control DNA samples; the Human Biological Data Interchange and Diabetes UK for data on multiplex families; the Norwegian Study Group for Childhood Diabetes for the collection of the Norwegian families, especially D. Undlien and K. Ronningen; D. Savage, C. Patterson, D. Carson, and P. Maxwell for the Northern Irish samples; J. Tuomilehto, L. Kinnunen, E. Tuomilehto-Wolf, V. Harjutsalo, and T. Valle of Genetics of Type 1 Diabetes in Finland for the Finnish family samples; C. Guja and C. Ionescu-Tirgoviste for the Romanian family samples; the Irish Blood Transfusion Service–Trinity College Dublin Biobank for DNA from Irish control subjects; the Barts and the London Genome Centre for genotyping support; J. Swift, R. Crimmins, P. Kumar, D.P. Jewell, L. Dinesen, S.P.L. Travis, K. Moriarty, P. Howdle, D.S. Sanders, G.K.T. Holmes, S. Sleet, and Celiac UK for the collection of British celiac samples; A. Monsuur, C.J. Mulder, M.L. Mearin, and W.H.M. Verbeek, for the recruitment of patients; G. Meijer and J. Meijer for their review of histologic analyses; K. Duran for DNA extraction; H. van Someren and F. Mulder for clinical database management; L. Franke, A. Zhernakova, M. Plateel, and the genotyping facilities at University Medical Center Groningen and University Medical Center Utrecht in the Netherlands for their technical assistance; F.M. Stevens, C. O'Morain, N.P. Kennedy, M. Abuzakouk, R. McLoughlin, K. Brophy, C. Feighery, J. McPartlin, D. Kelleher, A.W. Ryan, and G. Turner for sample collection and genotyping; and L. Wicker for comments on the manuscript.
Source Information
From the Juvenile Diabetes Research Foundation–Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom (D.J.S., V.P., N.M.W., J.D.C., K.D., J.H.M.Y., J.M.M.H., H.S., D.G.C., J.A.T.); the Department of Clinical Medicine, Institute of Molecular Medicine, Trinity College Dublin, Dublin (R.M.); the Genetics Department, University Medical Center and Groningen University, Groningen, the Netherlands (C.W.); and the Institute of Cell and Molecular Science, Barts and the London School of Medicine and Dentistry, London (G.A.H., P.C.D., K.A.H., D.A.H.).
This article (10.1056/NEJMoa0807917) was published at www.nejm.org on December 10, 2008.
Address reprint requests to Dr. Todd at the Juvenile Diabetes Research Foundation–Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0XY, United Kingdom, or at john.todd{at}cimr.cam.ac.uk.
References
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