Association Study of IL2/IL21 and Fcgriia: Significant Association with the IL2/IL21 Region in Scandinavian Coeliac Disease Families

ORIGINAL ARTICLE Association study of IL2/IL21 and FcgRIIa: significant association with the IL2/IL21 region in Scandinavian coeliac disease families

S Adamovic1, SS Amundsen2, BA Lie3, AH Gudjo´nsdo´ttir4, H Ascher4,5,JEk6, DA van Heel7, S Nilsson8, LM Sollid2,3 and A˚ Torinsson Naluai1 1Department of Medical and Clinical Genetics, Institute of Biomedicine, Sahlgrenska Academy, Gothenburg University, Go¨teborg, Sweden; 2Centre for Immune Regulation, Institute of Immunology, University of Oslo, Oslo, Norway; 3Institute of Immunology, Rikshospitalet University Hospital, Oslo, Norway; 4Department of Paediatrics, The Queen Silvia Children’s Hospital, Sahlgrenska University Hospital/O¨ stra, Gothenburg, Sweden; 5The Nordic School of Public Health, Go¨teborg, Sweden; 6Department of Paediatrics, Buskerud Central Hospital, Drammen, Norway; 7Centre for Gastroenterology, Institute of Cell and Molecular Science, Queen Mary University of London, UK and 8Department of Mathematical Sciences, Chalmers University of Technology, Go¨teborg, Sweden

The first genome-wide association study performed in a UK coeliac disease (CD) case-control cohort revealed association with a linkage disequilibrium block containing the KIAA1109/Tenr/IL2/IL21 genes. Also recently, an association with a non- synonymous polymorphism in FcgRIIa (CD32a) was reported in CD with an unusually strong P-value. We aimed to replicate the reported associations with the single nucleotide polymorphisms rs13119723 A4G and rs6822844 G4T in the KIAA1109/Tenr/ IL2/IL21 region and rs1801274 G4A in the FcgRIIa gene in a family sample consisting of 325 Swedish/Norwegian families using the robust transmission disequilibrium test. The family sample used in this study included 100 families with two or more children affected by CD and 225 families with one affected child. We could confirm significant association between the polymorphisms rs13119723 A4G and rs6822844 G4T located in the KIAA1109/Tenr/IL2/IL21 region and CD (P-value 0.001 and 0.002, respectively). However, we found no association with the FcgRIIa rs1801274 G4A polymorphism (P-value ¼ 0.3). In conclusion, our results support the KIAA1109/Tenr/IL2/IL21 region as a true CD susceptibility region. Genes and Immunity (2008) 9, 364–367; doi:10.1038/gene.2008.27; published online 17 April 2008

Keywords: IL2; IL21; 4q27; FcgRIIa; celiac; autoimmunity

Introduction cohort, of which rs13119723*G located in the hypothetical protein KIAA1109, was present on a single strongly Coeliac disease (CD) or gluten-sensitive enteropathy is a associated haplotype in both blocks. In addition to CD, chronic inflammatory condition with autoimmune fea- both type 1 diabetes and rheumatoid arthritis also tures, affecting the small intestine in genetically predis- demonstrated association with this region in another posed individuals.1 HLA genes and non-HLA genes, recent study.3 In this study, the rs6822844*T showed a together with gluten and most likely other environmen- significantly decreased frequency in cases compared to tal factors, are involved in disease development. Re- controls and was present on the strongest associated cently, the first genome-wide association analysis in CD haplotype in all the three diseases. rs6822844 is located in revealed association with the KIAA1109/Tenr/IL2/IL21 between interleukin 2 (IL2) and interleukin 21 (IL21). gene region at chromosome 4q27 and was further Both IL2 and IL21 are highly interesting candidate genes confirmed in two independent case-control cohorts.2 for CD susceptibility4,5 as both cytokines are T-cell The genome-wide association study presented several derived and stimulate the T-cell activation and prolifera- associated single nucleotide polymorphisms (SNPs) in tion in an autocrine manner. IL2 promotes proliferation the 4q27 region all strongly significant (Po10À10). These of T-cells, B-cells and NK-cells. Fina et al.6 have recently SNPs map to a approximately 480 kb linkage disequili- shown that IL21 expression is enhanced in duodenal brium (LD) block. This LD block was further subdivided samples from untreated CD patients. Furthermore, into two blocks by finer haplotype analysis, of the UK neutralization of IL21 prevented gliadin driven T- b and interferon-g production in intestinal biopsies from CD patients. Correspondence: Dr A˚ Torinsson Naluai, Department of Medical Another recently proposed susceptibility gene for CD, and Clinical Genetics, The Sahlgrenska Academy, Go¨teborg also allegedly a common risk factor for autoimmune University, Box 435, 405 30 Go¨teborg, Sweden. E-mail: [email protected] disease, is FcgRIIa (CD32a) located in chromosome region 7 Received 9 January 2008; revised 11 March 2008; accepted 14 March 1q23. FcgRIIa is a low affinity activating Fcg receptor 2008; published online 17 April 2008 that binds immune complexes. Ligation of this Fc IL2/IL21: susceptibility to coeliac disease S Adamovic et al 365 receptor leads to phagocytosis, dendritic cell maturation, Genotyping increased stimulation of T cells and enhanced secretion of The three candidate SNPs were genotyped using Taq- inflammatory cytokines.8 FcgRIIa has two common Man technology and genotypes were generated by the alleles which differ by a histidine (H) to arginine (R) SDS v 2.2. 1 software (Applied Biosystems, Foster City, substitution at position 131 defined by the SNP rs1801274 CA, USA). At least two negative and three positive G4A (also known as FcgRIIa*A519G). Compared to the control samples (one of each genotype) were included in R131 allotype, the H131 allotype (rs1801274*G) has each PCR run, and additionally, 60 randomly selected higher avidity for complexed human IgG2 and IgG3.9 samples were run in duplicate to score genotype error Interestingly, individuals homozygous for rs1801274*G rate (estimated on the basis of the percentage of genotype (H131) were associated with CD (odds ratio of 1.81, calls deviating in the duplicates). Few individuals were P ¼ 10À6), whereas weaker association were seen for type not genotyped for some of the markers due to missing 1 diabetes and rheumatoid arthritis.7 DNA. The unequivocal identification of IL2, IL21 and FcgRIIa Pedcheck was performed to reveal Mendelian incon- as susceptibility genes in CD will have great implications sistencies. for the understanding of the pathogenesis of this disease. The understanding of the molecular mechanisms of CD Statistical analysis has made substantial advances in recent years,10 and Power for the three SNPs rs13119723, rs6822844 and disease associated genes and gene variants can be readily rs1801274 was calculated using the TDT power calculator tested in relevant disease models involving gluten- for discrete traits located at http://pngu.mgh. harvard. reactive T-cell clones and disease specific anti-gluten edu/~purcell/gpc using a one-sided test and signifi- and anti-transglutaminase antibodies. Apart from the cance level of 0.05. Genotype relative risks were obtained implication for CD, this work may help in understanding from the original publications.2,7 how these genes are involved in other autoimmune Transmission disequilibrium test14 analysis for SNPs as diseases. There is, thus, an imminent need of replication well as haplotypes was performed employing the of new, promising genetic findings of CD. GENEHUNTER v2.1 program.15 A pure test for associa- To our knowledge, only case-control studies have been tion with disease was performed with TDT using only performed to investigate associations with the KIAA1109/ the probands of the multiplex families (325 trio families). Tenr/IL2/IL21 and the FcgRIIa gene in CD. In a case- LD analysis between rs13119723 and rs6822844 was control study design there is always a possibility of false performed using Haploview version 3.32 (www.broad. positive associations due to population stratification and mit.edu/mpg/haploview). poorly matched controls. The present study aims at investigating if the relationship between the SNPs rs13119723 A4G, rs6822844 G4T and rs1801274 G4A Results and CD can be detected in the Swedish/Norwegian population using the robust transmission disequilibrium Before our study, we estimated the power of our total test (TDT). family set (325 families). The power to confirm rs13119723, rs6822844 and rs1801274, given the same effect as seen in the original publications, was 77, 75 and Materials and methods 82%, respectively. The genotype success rate of rs13119723, rs6822844 and rs1801274 was 91, 96 and Subjects 97%, respectively, and no deviation was seen for the This study included 325 nuclear CD families, 100 families genotyping controls. The Pedcheck analysis revealed with at least two affected siblings (multiplex families) three inconsistencies for rs13119723 and one inconsis- and 225 trio-families (additional information about these tency for each of the markers rs6822844 and rs1801274. families is available elsewhere).11,12 All affected family These pedigrees were removed from the analysis of the members strictly fulfilled the ESPGAN (European individual markers. When missing genotypes and Society of Pediatric Gastroenterology and Nutrition) Mendelian errors were taken into account, the remaining diagnostic criteria.13 completely genotyped trios subjected to TDT analysis

Table 1 TDT results for markers in the regions 4q27 (KIAA1109/Tenr/IL2/IL21) and 1q23 (FcgRIIa)

Polymorphism Allele/Haplotype T/NT (%T) P-value OR 95% CI

KIAA1109/Tenr/IL2/IL21 rs13119723 A 69/36 (66%) 0.001 1.65 1.13–2.41 rs6822844 G 84/49 (63%) 0.002 1.55 1.11–2.16 rs13119723–rs6822844 AG 69/33 (68%) 0.0002

FcgRIIa rs1801274 G 153/142 (52%) 0.3 1.03 0.85–1.25

Abbreviations: CI, 95% confidence interval; OR, odds ratio; TDT, transmission disequilibrium test; T/NT, number of transmitted versus non- transmitted alleles; %T, the percentage of transmission. One member from each family was included in the analyses, resulting in a pure association test. P-values shown are uncorrected, one-sided and calculated using the binomial distribution.

Genes and Immunity IL2/IL21: susceptibility to coeliac disease S Adamovic et al 366 were: rs1801274 (n ¼ 315), rs13119723 (n ¼ 249) and contains four genes; however, considering the involve- rs6822844 (n ¼ 279). ment of IL2 and IL21 in inflammatory mechanisms and When testing single markers using TDT, rs13119723*A their modulating role in the immune response of the and rs6822844*G were significantly over-transmitted to inflamed intestinal mucosa, they are very good candi- the affected children; that is, 66%; P-value ¼ 0.001 and dates in which we should begin the search for functional 63%; P-value ¼ 0.002, respectively (Table 1). variants directly influencing disease. Further work When combining the SNPs in a haplotype analysis, should define the role of these cytokines in the already two haplotypes consisting of rs13119723*A— existing pathogenic models of CD. rs6822844*G and rs13119723*G—rs6822844*T accounted for over 90 % of the observed haplotypes and the haplotype rs13119723*A—rs6822844*G was significantly over-transmitted to affected children (68%; P-value Acknowledgements ¼ 0.0002) (Table 1). As seen in other populations, there We thank all the families participating in the study as was strong LD between rs13119723 and rs6822844, well as Britt-Marie Ka¨ck and The Celiac Society in 2 ¼ r 0.90 in our Scandinavian population. Sweden for help with collecting families and blood g The TDT analysis of the Fc RIIa polymorphism samples. We also thank the Swegene Genomics and rs1801274 showed no association (Table 1). Bioinformatics Core Facility in Go¨teborg where the significant part of this study (genotyping and statistic analysis) have been done. The work was supported by Discussion grants from the Foundation for Strategic Research, the Swedish Medical Research Council (Project no. K98-13X- Several studies have pointed out region IL2/IL21 on 12568-01A and K2000-27X-12568-03A), the Swedish g chromosome 4q27 and Fc RIIa at 1q23 as susceptibility Research Council (Project no. 2003-5560), the Research candidates in a number of autoimmune disorders with Council of Norway, the Gothenburg Medical Society, the chronic inflammatory features.16–20 Two recent publica- 2 7 European Commission (QLRT-1999-00037), EXTRA tions, one by van Heel et al. and one by Alizadeh et al., funds from the Norwegian Foundation for Health and reported association between CD and KIAA1109/Tenr/IL2/ Rehabilitation, the Swedish Society of Medicine, Frimur- g IL21 and Fc RIIa, respectively. Both of these studies were are-Barnhusdirektionen, the Foundation of the National based on a case-control approach, and one purpose of Board of Health and Welfare, Sigurd and Elsa Goljes our study was to replicate the most associated SNPs from Memory Foundation, the Wilhelm and Martina Lundg- these publications in a family sample using TDT to avoid ren Research Foundation, Lundstro¨ms Research Founda- the possibility of a false positive finding due to tion and Ha¨ggquist Research Foundation. population stratification. Our result confirms the association with the KIAA1109/Tenr/IL2/IL21 region but not with the FcgRIIa polymorphism. As mentioned earlier, the IL2/IL21 region has been Conflict of interest implicated in several autoimmune diseases.17,18,20 More- over, many autoimmune disorders including type 1 The authors state no conflict of interest. diabetes are overrepresented in CD and vice versa.21 Therefore, one would expect that within a similar ethnical population, CD and associated autoimmune diseases should demonstrate associations with the same References alleles, and this seems to be the case in the KIAA1109/ 1 Sollid LM. Molecular basis of celiac disease. Annu Rev Tenr/IL2/IL21 region. In type 1 diabetes, fine mapping Immunol 2000; 18: 53–81. and resequencing of the Tenr/IL2/IL21 has been per- 2 van Heel DA, Franke L, Hunt KA, Gwilliam R, Zhernakova A, formed in an effort to localize the causal variant.22 No Inouye M et al. A genome-wide association study for celiac coding variants or obvious regulatory or splice variants disease identifies risk variants in the region harboring IL2 and were revealed, and the strong LD across the region IL21. Nat Genet 2007; 39: 827–829. restricted the fine-mapping to a 200-kb region. 3 Zhernakova A, Alizadeh BZ, Bevova M, van Leeuwen M, We were unable to confirm association with the Coenen MJH, Franke B et al. Novel association in chromosome FcgRIIa*A519G polymorphism in our Scandinavian CD 4q27 region to rheumatoid arthritis and confirmation to type 1 diabetes points to a general risk locus for autoimmune population. Several reasons could explain this lack of diseases. Am J Hum Genet 2007; 81: 1284–1288. reproducibility, the most likely being no or insignificant 4 Mehta DS, Wurster AL, Grusby MJ. Biology of IL-21 and the contribution of FcgRIIa to the genetic predisposition to IL-21 receptor. Immunol Rev 2004; 202: 84–95. CD in our population. As our study, and others, was 5 Wicker LS, Clark J, Fraser HI, Garner VE, Gonzalez-Munoz A, restricted to the investigation of the FcgRIIa*A519G Healy B et al. Type 1 diabetes genes and pathways shared by polymorphism only, variable LD between FcgRIIa*A519G humans and NOD mice. J Autoimmun 2005; 25 (Suppl): 29–33. and a yet unidentified causative variant in the studied 6 Fina D, Sarra M, Caruso R, Del Vecchio Blanco G, Pallone F, populations could possibly explain the discrepant Macdonald TT et al. Interleukin-21 contributes to the Mucosal results. It is also possible that the original finding T helper cell type 1 response in celiac disease. Gut 2007; 4: 288–291. was due to a false positive or an overestimated 7 Alizadeh BZ, Valdigem G, Coenen MJ, Zhernakova A, Franke effect, implying insufficient power to detect it in our B, Monsuur A et al. Association analysis of functional variants population. of the FcgRIIa and FcgRIIIa genes with type 1 diabetes, celiac We conclude that the chromosome region surrounding disease and rheumatoid arthritis. Hum Mol Genet 2007; 16: IL2/IL21 is a susceptibility region in CD. The region 2552–2559.

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