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      Genome-wide association study identifies variants in the MHC class I, IL10, and IL23R/IL12RB2 regions associated with Behçet's disease

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      1 , 2 , 1 , 1 , 3 , 1 , 1 , 4 , 1 , 3 , 3 , 3 , 3 , 3 , 5 , 6 , 7 , 7 , 4 , 8 , 8 , 2 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 4 , 1 , 2 , 3
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          Abstract

          Behçet's disease (BD) is a genetically complex disease of unknown etiology, characterized by recurrent inflammatory attacks affecting orogenital mucosa, eyes, and skin. We performed a GWAS with 311,459 SNPs in 1215 BD patients and 1278 healthy controls from Turkey. We confirmed the known HLA-B51 association and identified a second, independent association within the MHC Class I region. We also found one SNP with genome-wide evidence for association within the gene encoding interleukin-10 (IL10). A meta-analysis including a total of 2430 cases and 2660 controls established associations with the IL10 variant (rs1518111, P=3.54×10-18, odds ratio 1.45 with 95% confidence interval 1.34-1.58) and with a variant located between the interleukin-23 receptor (IL23R) and interleukin 12 receptor β2 (IL12RB2) genes (rs924080, P=6.69×10-9, odds ratio 1.28 with 95% confidence interval 1.18-1.39). The disease-associated IL10 variant (rs1518111 A allele) was associated with diminished mRNA expression and low protein production, suggesting novel therapeutic targets for BD. BD is prevalent in Middle Eastern countries with the highest rate (4 in 1000 individuals) found in Turkey. A genetic contribution to BD is supported by the high sibling recurrence risk ratio (λs), estimated from 11.4 to 52.5 in the Turkish population1. HLA-B51 is the most strongly associated known genetic factor2, however it accounts for less than 20% of the genetic risk, even in familial cases3, indicating that genetic factors remain to be discovered. Candidate gene studies and several small GWASs4,5 have examined BD genetics, but the studies have been generally underpowered, making interpretation and replication of the results problematic. We therefore performed a GWAS of 311,459 autosomal SNPs in 1215 BD cases and 1278 healthy controls from Turkey (Fig. 1, Supplementary Fig. 1, and Supplementary Table 1). Only SNP genotype data that met strict quality control standards (see Online Methods) were included. A principal components method (Online Methods) was used to evaluate population stratification in the cases and the controls. After correction for 6 PCs, λGC, a measure of genomic inflation, was reduced from 1.06 to 1.05 (Supplementary Fig. 2a, b, c). Correcting for 6 PCs in the absence of the strongly associated MHC region SNPs reduced λGC from 1.05 to 1.04 (Supplementary Fig. 2d). Given the minimal degree of population stratification, uncorrected data are presented. P < 5.0 × 10-8 was considered genome-wide significance. The most significantly associated SNPs (P value < 10-44) were located on chromosome 6 in the MHC region. To evaluate the contribution of the HLA-B51 type to BD, we determined the HLA-B types of 1190 of the cases and 1257 of the controls. Occurrence of the HLA-B51 type (one or two copies) was found in 59.1% of cases and only 29.3% of controls (odds ratio = 3.49 [95% CI = 2.95 to 4.12], P = 5.47 × 10-50). Within the HLA-B region the most significantly associated SNPs were located from telomeric to the HLA-B coding region to centromeric to MICA (encoding MHC Class I chain related sequence A), which has been suggested to be the source of the BD-HLA-B51 association6. We found the HLA-B51 type was more strongly associated with disease than was any genotyped SNP (HLA-B51 allele frequency = 0.352 in cases and 0.159 in controls, P = 1.44 × 10-54, Fig. 2). Strong linkage disequilibrium (LD) was observed between HLA-B51 and all the SNPs located from HLA-B to more than 62 kb centromeric to the MICA gene, despite spanning several blocks of LD (Supplementary Fig. 3). This LD pattern is observed because the HLA-B51 variant is found almost exclusively on a single extended haplotype. This extended haplotype occurred at 0.321 frequency in cases and 0.144 frequency in controls. Interestingly, the identical SNP haplotype, but lacking HLA-B51, occurred in cases and controls equally at 0.04 frequency, suggesting that HLA-B51 is required for the disease association in the HLA-B region. We next performed a conditional logistic regression analysis of the 292 SNPs from the MHC region with allelic Chi-squared P < 0.0001, specifying HLA-B51 as a covariate. None of the HLA-B/MICA region SNPs remained significantly associated with BD in the conditional analysis (Fig. 2). After accounting for the effect of HLA-B51, three SNPs within the HLA-A gene region with disease-associations retained genome-wide significance. The most strongly associated SNP, rs9260997, located 50 kb centromeric to the HLA-A gene, had a regressor P value = 5.49 × 10-9, suggesting a highly significant association independent of the HLA-B51 association. Coincidentally, an association of markers within the HLA-A region, independent of HLA-B51, was recently described in Japanese BD patients with attribution of the association to the HLA-A26 allele5. In the association data (Fig. 1) we identified near genome-wide significance for one non-MHC SNP, rs936551 (P = 5.29 ×10-8), located at the telomeric end of the short arm of chromosome 4, within the promoter region of CPLX1, which encodes complexin-1, a regulator of exocytosis during vesicle membrane fusion. Additionally, one SNP located within the first intron of the IL10 gene, rs3024490, was strongly suggestive for association with a P value = 2.22 × 10-7 (Fig. 1). We also identified an additional 66 SNPs from an additional 49 chromosomal regions with suggestive evidence for disease association, P < 0.0001 (Supplementary Table 2). To better evaluate genetic associations within the CPLX1 and IL10 gene regions, we genotyped the same case and control samples for additional SNPs from the regions predicted by HapMap CEU data to be in LD with the disease-associated SNP. We also fine-mapped the IL23R/IL12RB2 region, which was identified in an independent GWAS of Behçet's disease in the Japanese population7 and within which we found three SNPs with p < 0.0001 (Supplementary Table 2). In the CPLX1 region, the additional genotyped SNPs increased the estimated coverage (at r2 greater than 0.8) of HapMap Phase II SNPs with minor allele frequency greater than 0.05 from 57% to 93%. The most significantly associated SNPs were located in the promoter region of the gene, although none were more significantly associated than rs936551 (Supplementary Fig. 4). In the IL10 gene region, we genotyped 27 additional SNPs resulting in coverage of 100% of the HapMap Phase II SNPs with greater than 5% minor allele frequency in the CEU samples. Five SNPs were found strongly associated with BD, with one SNP, rs1518111, with genome-wide significance (P = 1.88 × 10-8, Fig. 3a). The disease-associated SNPs were located in the promoter region and the first, second, and third introns of the gene (Supplementary Fig. 5) and all were in strong LD with one another. In the IL23R/IL12RB2 region we genotyped 11 additional SNPs increasing coverage from 58% to 88% of the HapMap Phase II SNPs with greater than 5% minor allele frequency in the CEU samples. No SNPs were found with stronger association than rs924080 from the GWAS analysis (Fig. 3b). We identified a recombination hotspot located in the intergenic region between IL23R and IL12RB2 (Fig. 3b), which is also seen in the HapMap Caucasian and Asian populations (data not shown). The BD-associated variants were located on the IL23R side of the hotspot, suggesting the disease association is more likely to influence IL23R than IL12RB2. Variants within the IL23R gene have been associated with ankylosing spondylitis8, psoriasis9, and inflammatory bowel disease10, diseases with some phenotypic overlap with each other and with BD. Neither of two IL23R coding variants associated with these seronegative diseases was associated with BD (Fig. 3b). We genotyped the BD-associated IL10, IL23R/IL12RB2, and CPLX1 SNPs in five additional collections of BD cases and controls from Turkey, the Middle East, Europe, and Asia (see Table 1 and Online Methods). A surrogate marker (rs11248047) was used in the place of the CPLX1 disease-associated SNP (rs936551), which failed quality control in the replication assay (both markers shown in Supplementary Fig. 4). The results are shown in Table 1. A number of the UK Caucasian and Middle Eastern Arab samples were previously tested for association of two IL10 promoter region SNPs with BD. Although an association with disease was reported in the UK collection, the association had not replicated in the Middle Eastern Arab collection11. The BD-associated IL10 SNP identified in the Turkish population, rs1518111, reached statistical significance after correction for three tests (P < 0.017) in the Middle Eastern Arab and Greek collections and was of nominal significance (P < 0.05) in the UK Caucasian collection (Table 1). For the most part, the IL23R/IL12RB2 and CPLX1 SNPs showed the same trend as found in the Turkish samples; however the associations did not reach statistical significance in these smaller collections. Association of these markers was not observed in the small Turkish replication collection. Although this lack of association could be explained by small sample size, an alternative explanation could be that the markers are associated with severe disease, as we have noted that disease manifestations in the replication collection (drawn from a Dermatology clinic) were milder than in the discovery collection used for the GWAS, which was obtained from a Rheumatology clinic. While analyzing these data we learned that another group had also performed a GWAS for Behçet's disease7. We exchanged data for cross validation purposes and found that the IL10 and the IL23R/IL12RB2 SNP associations were also strongly supported by the data obtained from the Japanese population, whereas the CPLX1 association was not observed. A meta-analysis of all the association data (including a total of 2430 BD cases and 2660 controls) provided strong evidence for associations of the IL10 and IL23R/IL12RB2 loci with BD (Table 1). Association of the CPLX1 variant failed to replicate in any of the additional collections and failed to reach genome-wide significant association in a meta-analysis of the data. Analysis of even larger numbers of replication samples will be required to establish whether the CPLX1 locus contributes to BD susceptibility. Several SNP haplotypes of the IL10 gene promoter have been reported to be associated with regulation of the gene's expression, but the reported allelic effects have been inconsistent and difficult to reconcile12-14. We therefore tested for a difference in expression of the gene from the disease-associated haplotype and other haplotypes by measuring allelic imbalance of the rs1518111 variant in pre-mRNA from monocytes isolated from 8 healthy individuals heterozygous for the disease-associated allele. In 8 donors, the pre-mRNA transcript with the rs1518111 A allele was found at reduced levels compared with the G allele. Expression from the A bearing chromosome was 35% of expression from the G bearing chromosome (Fig. 4a). To assess whether this expression difference was relevant in terms of cytokine production, we activated mononuclear cells from healthy Turkish donors with lipopolysaccharide (LPS) and found significantly lower amounts of IL-10 protein in supernatants from donors homozygous for the rs1518111 A allele compared with individuals with one or two G alleles (Fig. 4b). We also cultured monocytes from ethnically diverse healthy donors from the United States and stimulated them with two Toll-like receptor and nucleotide-binding oligomerization domain containing receptor ligands, the lipoprotein, Pam3Cys and muramyl dipeptide. Individuals homozygous for the BD-associated rs1518111 A allele produced significantly less IL-10 than individuals with one or two G alleles (Fig. 4c), whereas no statistically significant variation was found in TNFα production (data not shown). Taken together, these data suggest that a genetic predisposition for low IL-10 expression is a risk factor for BD. There are extensive data in mouse models linking reduction in IL-10 with inflammation15. Variants in the IL10 gene region have been shown to be associated with ulcerative colitis (UC)16, type I diabetes17, systemic lupus erythematosus18, and severe juvenile rheumatoid arthritis (JRA)12, and IL-10 receptor mutations were recently shown to cause early onset enterocolitis19. Interestingly, the SNP found in the UC, type 1diabetes, and lupus studies, rs3024505, was not associated with BD (Fig. 3a) and the BD-associated IL10 variants were not associated with these three diseases, suggesting a different IL-10-related disease mechanism in BD compared with these other diseases. On the other hand, the IL10 association with severe JRA is the same as for BD. Variants from several candidate genes have been examined for association with BD. Although no variants within these genes were found with genome-wide significant association in the Turkish GWAS, 6 of the 12 genes examined contained at least one SNP with nominal significance (P < 0.05, Supplementary Table 3). A much larger sample collection will be required to determine whether any of these genes harbor true BD susceptibility alleles. In summary, this GWAS provides new insights into the genetic factors that contribute to BD. It not only supports the association of HLA-B51 as the primary MHC association with BD, but also reveals another independent MHC Class I association telomeric to the HLA-B gene. Most importantly, this study demonstrates that common variations in the IL10 gene and in the vicinity of the IL23R/IL12RB2 genes predispose to BD. Functional analyses indicate that the disease-associated IL10 variants cause decreased expression of this anti-inflammatory cytokine, and thereby, possibly in concert with commensal microorganisms20, result in an inflammation-prone state, thus suggesting a mechanistic hypothesis and possible therapeutic targets. Data accession The genotype data for the 311,459 SNPs in 1215 Behçet's disease cases and 1278 healthy controls from Turkey have been deposited in the National Institutes of Health database of genes and phenotypes, dbGaP (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gap), accession number: phs000272.v1.p1 Supplementary Material 1

          Related collections

          Most cited references17

          • Record: found
          • Abstract: found
          • Article: not found

          A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes.

          We performed a multitiered, case-control association study of psoriasis in three independent sample sets of white North American individuals (1,446 cases and 1,432 controls) with 25,215 genecentric single-nucleotide polymorphisms (SNPs) and found a highly significant association with an IL12B 3'-untranslated-region SNP (rs3212227), confirming the results of a small Japanese study. This SNP was significant in all three sample sets (odds ratio [OR](common) 0.64, combined P [Pcomb]=7.85x10(-10)). A Monte Carlo simulation to address multiple testing suggests that this association is not a type I error. The coding regions of IL12B were resequenced in 96 individuals with psoriasis, and 30 additional IL12B-region SNPs were genotyped. Haplotypes were estimated, and genotype-conditioned analyses identified a second risk allele (rs6887695) located approximately 60 kb upstream of the IL12B coding region that exhibited association with psoriasis after adjustment for rs3212227. Together, these two SNPs mark a common IL12B risk haplotype (OR(common) 1.40, Pcomb=8.11x10(-9)) and a less frequent protective haplotype (OR(common) 0.58, Pcomb=5.65x10(-12)), which were statistically significant in all three studies. Since IL12B encodes the common IL-12p40 subunit of IL-12 and IL-23, we individually genotyped 17 SNPs in the genes encoding the other chains of these cytokines (IL12A and IL23A) and their receptors (IL12RB1, IL12RB2, and IL23R). Haplotype analyses identified two IL23R missense SNPs that together mark a common psoriasis-associated haplotype in all three studies (OR(common) 1.44, Pcomb=3.13x10(-6)). Individuals homozygous for both the IL12B and the IL23R predisposing haplotypes have an increased risk of disease (OR(common) 1.66, Pcomb=1.33x10(-8)). These data, and the previous observation that administration of an antibody specific for the IL-12p40 subunit to patients with psoriasis is highly efficacious, suggest that these genes play a fundamental role in psoriasis pathogenesis.
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            • Record: found
            • Abstract: found
            • Article: not found

            Sequence variants in IL10, ARPC2 and multiple other loci contribute to ulcerative colitis susceptibility.

            Inflammatory bowel disease (IBD) typically manifests as either ulcerative colitis (UC) or Crohn's disease (CD). Systematic identification of susceptibility genes for IBD has thus far focused mainly on CD, and little is known about the genetic architecture of UC. Here we report a genome-wide association study with 440,794 SNPs genotyped in 1,167 individuals with UC and 777 healthy controls. Twenty of the most significantly associated SNPs were tested for replication in three independent European case-control panels comprising a total of 1,855 individuals with UC and 3,091 controls. Among the four consistently replicated markers, SNP rs3024505 immediately flanking the IL10 (interleukin 10) gene on chromosome 1q32.1 showed the most significant association in the combined verification samples (P = 1.35 x 10(-12); OR = 1.46 (1.31-1.62)). The other markers were located in ARPC2 and in the HLA-BTNL2 region. Association between rs3024505 and CD (1,848 cases, 1,804 controls) was weak (P = 0.013; OR = 1.17 (1.01-1.34)). IL10 is an immunosuppressive cytokine that has long been proposed to influence IBD pathophysiology. Our findings strongly suggest that defective IL10 function is central to the pathogenesis of the UC subtype of IBD.
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              • Record: found
              • Abstract: found
              • Article: not found

              An investigation of polymorphism in the interleukin-10 gene promoter.

              Interleukin-10 (IL-10) has been described as an anti-inflammatory cytokine and B-cell proliferation factor and has been implicated in autoimmunity, tumorigenesis and transplantation tolerance. We have identified three single base pair substitutions in the IL-10 gene promoter and have investigated whether this polymorphism correlates with IL-10 protein production in vitro.
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                Author and article information

                Journal
                9216904
                2419
                Nat Genet
                Nature genetics
                1061-4036
                1546-1718
                24 June 2010
                11 July 2010
                August 2010
                1 February 2011
                : 42
                : 8
                : 698-702
                Affiliations
                [1 ] Laboratory of Clinical Investigation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA
                [2 ] Istanbul Faculty of Medicine, Division of Rheumatology, Department of Internal Medicine, Istanbul University, Istanbul, Turkey
                [3 ] Department of Genetics, Institute for Experimental Medicine, Istanbul University, Istanbul, Turkey
                [4 ] Translational Immunology Section, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA
                [5 ] Istanbul Faculty of Medicine, Department of Ophthalmology, Istanbul University, Istanbul, Turkey
                [6 ] Istanbul Faculty of Medicine, Department of Neurology, Istanbul University, Istanbul, Turkey
                [7 ] University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA
                [8 ] Istanbul Faculty of Medicine, Department of Dermatology, Istanbul University, Istanbul, Turkey
                [9 ] School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, UK
                [10 ] Translational Breast Cancer Research Division Hematology/Oncology Northwestern University, Chicago, Illinois, USA
                [11 ] Department of Rheumatology, Athens Medical Center, Greece
                [12 ] Hadassah-Hebrew University Medical Center, Jerusalem, Israel
                [13 ] King's College, London, UK
                [14 ] Queen Mary's College, London, UK
                [15 ] University Hospital, Damascus, Syria
                [16 ] Centre for Integrated Genomic Medical Research, School of Immunity and Infection, University of Manchester, UK
                [17 ] Graduate School of Medical Science and Engineering, KAIST, Daejeon, Korea
                [18 ] Department of Dermatology, Yonsei University College of Medicine, Seoul, Korea
                [19 ] Lymphocyte Cell Biology Section, Molecular Immunology and Inflammation Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland, USA
                [20 ] Academic Unit of Ophthalmology, University of Birmingham, UK
                Author notes

                Author Contributions

                Study design: E.F.R., F.C., Y.K., M.J.O., B.D.K., D.U., W.C., C.I.A., J.O'S., M.Ga., D.L.K., A.G. Analysis: E.F.R., Y.K., M.J.O., B.Y., W.C., C.I.A., M.B.D., G.R.W., M.Ga., D.L.K., A.G., Sample procurement and data generation: E.F.R., F.C., Y.K., M.J.O., N.A., C.S., J.M.L., B.Y., B.D.K., A.C., O.A., Z.E., H.A., D.U., I.T.-T., G.A.-D., W.C., C.I.A., M.B.D., A.A.K., G.A., B.E., O.J.B., V.G.K., Ph.K., E.B.-C., M.S., F.F., M.Gh., W.E.R.O., Y.-H.C., D.B., J.O'S., G.R.W., M.Ga., D.L.K., A.G. Writing: E.F.R., Y.K., M.J.O., B.Y., C.I.A., J.O'S., M.Ga., D.L.K., A.G.

                Correspondence should be directed to E.F.R. ( remmerse@ 123456mail.nih.gov ).
                Article
                nihpa214962
                10.1038/ng.625
                2923807
                20622878
                a3d89aeb-8c0f-4dcf-a924-792ef3c75a6c

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                Funding
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: R01 ES009912-04A1 ||ES
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: P01 CA034936-19A29004 ||CA
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: P01 CA034936-14A19004 ||CA
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: ZIC AR041181-01 ||AR
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: Z99 HG999999 ||HG
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: Z01 AR041139-05 ||AR
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: Z01 AR041106-13 ||AR
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Funded by: National Cancer Institute : NCI
                Funded by: National Institute of Arthritis and Musculoskeletal and Skin Diseases : NIAMS
                Funded by: National Human Genome Research Institute : NHGRI
                Award ID: Z01 AR041083-19 ||AR
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