Reassessment of the Type I Diabetes Association of the OAS1 Locus

ORIGINAL ARTICLE Reassessment of the type I diabetes association of the OAS1 locus

H-Q Qu1,2, C Polychronakos1,2 and the Type I Diabetes Genetics Consortium 1Department of Pediatrics, McGill University, Montreal, Que´bec, Canada; 2Department of Human Genetics, McGill University, Montreal, Que´bec, Canada

To reassess the type I diabetes (T1D) association of the OAS1 locus, the Type I Diabetes Genetics Consortium (T1DGC) genotyped 11 tag single-nucleotide polymorphisms spanning B41 kb from the 50 to 30 flanking region. For each sample obtained from over 2000 affected sib-pair families from nine cohorts, the genotyping was performed on both the Illumina Golden Gate and Sequenom iPlex platforms. The data suggest that there may be a weak association with T1D for two OAS1 polymorphisms, rs3741981 and rs10774671, in populations of European descent. The OAS1 locus is close to a recently identified T1D-associated linkage disequilibrium (LD) block in human chromosome 12q24. Extended LD in populations earlier examined may account for the prior observation of an association of T1D with OAS1 variants. This possibility needs to be addressed further by fine mapping of the T1D association represented in 12q24. Genes and Immunity (2009) 10, S69–S73; doi:10.1038/gene.2009.95

Keywords: autoimmune disease; OAS1; genetic susceptibility; linkage disequilibrium; single-nucleotide polymorphism; type I diabetes

Introduction org) SNPs within and around OAS1 with minor allele frequency 41% at r2X0.80 (Supplementary Figure 1). The 20,50-oligoadenylate synthetase genes (OAS1, OAS2, Both the splicing site SNP (rs10774671) and the non- OAS3) that are located in human chromosome 12q24 synonymous SNP (nsSNP rs3741981) were included. encode a family of enzymes pivotal in innate anti-viral defense.1–4 OAS1 has a major function in the total constitutive activity of OAS enzymes.5 The first reported Results association of type I diabetes (T1D) with the OAS1 locus was with the single-nucleotide polymorphism (SNP) All 11 SNPs selected for the OAS1 locus were genotyped rs10774671.6 This SNP occurred in a region that involved by both the Illumina GoldenGate and the Sequenom a splicing site. The minor (G) allele of rs10774671 iPlex platforms. Of the 11159 individuals from 2298 introduces a splicing site and is associated with families (5003 affected), a total of 1477 individuals had no increased OAS enzyme activity; further, this SNP was genotypes from either platform because of unavailability associated with T1D susceptibility.6 of samples at the time of genotyping. In addition, 322 Although methodological limitations of that study individuals had only the Illumina genotypes and 401 were later identified,7 in our independent study on individuals had only Sequenom genotypes. As this rate candidate polymorphisms, the highly correlated of genotype availability is similar to that of other (r2 ¼ 0.69) non-synonymous (Ser162Gly) SNP rs3741981 candidate genes in the T1DGC experiment, these missing of the OAS1 gene was significantly associated with T1D genotypes seem to be unrelated to assay quality. risk.8 In two large cohorts of European descent, however, The genotyping quality assessment of each SNP no statistically significant association of OAS1 SNPs with (Table 1) eliminated rs7135579 because of low call rate T1D was observed.7 and significant deviation of the rs7135579 genotype To reassess the T1D association of the OAS1 locus, the distribution from Hardy–Weinberg Equilibrium expecta- Type I Diabetes Genetics Consortium (T1DGC) geno- tion. Both of the earlier associated OAS1 candidate SNPs, typed 11 tag SNPs. The 11 SNPs span B41 kb from the rs3741981 and rs10774671, have genotype distribution in 50 to 30 flanking region of the OAS1 gene and capture Hardy–Weinberg Equilibrium and exhibit highly consis- 95.9% of the available HapMap (http://www.hapmap. tent results between the genotyping platforms. The results of association analysis between the OAS1 SNPs and T1D are shown in Table 2. The discrepancies in results between the two technologies are largely because Correspondence: Dr C Polychronakos, Departments of Pediatrics of incomplete overlap of the DNA sets tested (7% of the and Human Genetics, Pediatric Endocrinology McGill University Health Center (Children’s Hospital), 2300 Tupper, Montre´al, Que´bec samples were genotyped in only one of the two Canada H3H 1P3. platforms), rather than technical differences between E-mail: [email protected] platforms as shown by the high concordance rates in Reassessment of the T1D association of the OAS1 locus Hi-Q Qu et al S70 Table 1 The 11 SNPs in the OAS1 locus studied by the T1DGC

Marker Physical position ILMN call rate ILMN HWE p SQNM call rate SQNM HWE p Concordance rate of ILMN vs SQNM

rs3741982 111791701 0.998 0.070 0.984 0.138 0.958 rs12177 111798145 0.999 0.514 0.982 0.443 0.957 rs2240193 111798381 0.999 0.179 0.985 0.393 0.993 rs2240191 111798451 0.997 0.743 0.970 0.006 0.989 rs12309946 111798975 0.998 0.869 0.992 0.857 1.000 rs4766662 111808419 0.984 0.457 0.981 0.214 0.978 rs3741981a 111811590 0.999 0.166 0.988 0.137 0.999 rs10774671a 111819913 1.000 0.188 0.994 0.162 0.999 rs7135579 111829602 0.930 5.58 Â 10À18 0.989 0.016 0.970 rs3803057 111831454 1.000 0.398 0.990 0.793 0.999 rs7967461 111832479 1.000 0.133 0.993 0.184 0.999

Abbreviations: HWE, Hardy–Weinberg equilibrium; ILMN, Illumina GoldenGate; SQNM, Sequenom iPlex; SNP, single-nucleotide polymorphism; T1D, type I diabetes; T1DGC, Type I Diabetes Genetics Consortium. aEarlier reported to be associated with T1D.6,8

Table 2 The T1D association test of the 11 OAS1 SNPs Table 3 The genotypic association test of rs3741981 and rs10774671 Marker Minor MAF ILMN Z ILMN P SQNM Z SQNM P allele Marker Genotype Frequency ILMN ILMN SQNM SQNM Z P Z P rs3741982 A 0.383 0.192 0.848 À0.510 0.610 rs12177 A 0.491 À0.294 0.768 0.699 0.484 rs3741981 A/A 0.327 0.599 0.549 0.441 0.659 rs2240193 T 0.066 À1.138 0.255 À1.734 0.083 A/G 0.481 À1.541 0.123 À1.858 0.063 rs2240191 A 0.170 À1.157 0.247 À1.134 0.257 G/G 0.192 1.507 0.132 2.133 0.033a rs12309946 A 0.040 0.508 0.611 0.408 0.683 rs4766662 A 0.222 À0.091 0.927 0.797 0.426 rs10774671 C/C 0.137 1.634 0.102 2.013 0.044a rs3741981 G 0.433 0.484 0.628 0.987 0.324 C/T 0.455 À1.087 0.277 À0.363 0.717 rs10774671a C 0.365 0.842 0.400 1.925 0.054 T/T 0.408 0.075 0.940 À1.051 0.293 rs7135579 A 0.103 À2.058b 0.040b À0.154 0.877 rs3803057 A 0.049 À0.871 0.384 À1.980 0.048 Abbreviations: ILMN, Illumina GoldenGate; SQNM, Sequenom rs7967461 G 0.370 0.859 0.390 1.784 0.074 iPlex. The genotypic type I diabetes (T1D) association was tested by the Abbreviations: ILMN, Illumina GoldenGate; MAF, minor allele family based association test (FBAT) (http://www.biostat.harvard. frequency; SQNM, Sequenom iPlex; SNP, single-nucleotide poly- edu/Bfbat/fbat.htm). morphism; T1D, type I diabetes. aPo0.05. aThe Type I Diabetes Genetics Consortium (T1DGC) genotyped the DNA antisense strand. The C allele of rs10774671 corresponds to the G allele in the sense strand, which produces a splicing site. T1D families and 4287 T1D cases vs 4735 controls from bPoor genotyping assay. the same population.7 Thus, the possible genotypic association still lacks statistical validation. The observed borderline significance may be due to the multiple Table 1. In these affected sib-pair families from the hypotheses implicitly tested. T1DGC, there was no evidence of a statistically significant allelic association of OAS1 SNPs with T1D risk. In our earlier study, the genotypic association with T1D was under a recessive model.8 In this model, only Discussion homozygosity for the minor allele of each of the three The above results suggest that association of the two OAS1 SNPs (rs3741981, rs10774671, and rs3177979) OAS1 SNPs, rs3741981 and rs10774671, with T1D in conferred an increased risk for T1D. The affected sib- populations of European descent is extremely weak, if pair family collection assembled by the T1DGC suggests present at all. This weak and inconsistent association the same result for OAS1 SNPs rs3741981 and rs10774671 may be due to long-range linkage disequilibrium (LD) (Table 3) in the Sequenom assay. In the Illumina assay, the with the recently replicated association with T1D in the genotypic association under the recessive model did not region of human chromosome 12q24, involving the reach statistical significance, although a trend in the intronic SNP rs1769673610 following the Wellcome Trust same direction as that observed with the Sequenom Case–Control Consortium study.9 This association was assay was seen. The same pattern of genotypic associa- also replicated in Stage 2 of our own genome-wide tion was also shown by the Wellcome Trust Case–Control association study (Hakonarson et al., unpublished data). Consortium study (P ¼ 0.020).9 The three reported OAS1 The T1D-associated SNP rs17696736 is located in a LD SNPs were not genotyped in the Wellcome Trust Case– block of over 1 Mb in length, which is only B300 kb from Control Consortium; however, SNP rs2660 has r2 ¼ 1 with the LD block containing OAS1. The LD between these rs10774671 (Supplementary Table 1). The genotypic two blocks is low in European-descent HapMap set association was not replicated, however, in the 1552 (Supplementary Figure 2).

Genes and Immunity Reassessment of the T1D association of the OAS1 locus Hi-Q Qu et al S71 In the Canadian family dataset reporting T1D associa- ment (NICHD), and Juvenile Diabetes Research Founda- tion of the OAS1 variant,8 rs17696736 has D0 ¼ 0.085, tion International (JDRF) and supported by U01 r2 ¼ 0.005 with the splicing site SNP rs10774671, and DK062418. HQQ is supported by a fellowship from D0 ¼ 0.061, r2 ¼ 0.003 with the nsSNP rs3741981. How- the Canadian Institutes of Health Research. We thank ever, it is possible that the association signal in the Dr John Todd for the helpful comments. Genotyping was chromosome 12q24 region may originate in an unidenti- performed at the Broad Institute Center for Genotyping fied variant, with higher extended LD with the OAS1 and Analysis is supported by grant U54 RR020278 from SNPs. This may account for the T1D association in the the National Center for Research Resources. earlier reports.6,8 This issue needs to be addressed by fine mapping of the T1D association at 12q24.

Materials and methods References 1 Justesen J, Hartmann R, Kjeldgaard NO. Gene structure and Subjects 0 0 The T1DGC created a resource of 2295 affected sib-pair function of the 2 –5 -oligoadenylate synthetase family. Cell Mol Life Sci 57 families from nine cohorts for this experiment. SNPs in 2000; : 1593–1612. 2 Mashimo T, Lucas M, Simon-Chazottes D, Frenkiel MP, 21 candidate genes (including OAS1) were genotyped on Montagutelli X, Ceccaldi PE et al. A nonsense mutation in two platforms (Illumina GoldenGate and Sequenom the gene encoding 20–50-oligoadenylate synthetase/L1 isoform iPlex). Details of the sample, quality control, and other is associated with West Nile virus susceptibility in laboratory aspects of the data can be found in this volume (Brown mice. Proc Natl Acad Sci USA 2002; 99: 11311–11316. et al.11). The majority of the subjects were Caucasian 3 Perelygin AA, Scherbik SV, Zhulin IB, Stockman BM, Li Y, (81%) with 18% unknown ethnicity and 1% other (Asian, Brinton MA. Positional cloning of the murine flavivirus African American, Pacific Islander). resistance gene. Proc Natl Acad Sci USA 2002; 99: 9322–9327. 4 Lucas M, Mashimo T, Frenkiel MP, Simon-Chazottes D, Montagutelli X, Ceccaldi PE et al. Infection of mouse neurones Genotyping by West Nile virus is modulated by the interferon-inducible All 11 OAS1 SNPs were genotyped by both the Illumina 20–50 oligoadenylate synthetase 1b protein. Immunol Cell Biol GoldenGate and the Sequenom iPlex platforms. Of the 2003; 81: 230–236. 9682 individuals, 93% were genotyped in both platforms. 5 Bonnevie-Nielsen V, Field LL, Lu S, Zheng DJ, Li M, Martensen PM et al. Variation in antiviral 20,50-oligoadenylate 0 0 Statistics synthetase (2 5 AS) enzyme activity is controlled by a single- Data quality checks, using standard methods were nucleotide polymorphism at a splice-acceptor site in the OAS1 gene. Am J Hum Genet 2005; 76: 623–633. performed by the Coordinating Center of the T1DGC at 6 Field LL, Bonnevie-Nielsen V, Pociot F, Lu S, Nielsen TB, Wake Forest University. T1D association was tested by Beck-Nielsen H. OAS1 splice site polymorphism controlling the family based association test (http://www.biostat. antiviral enzyme activity influences susceptibility to type 1 harvard.edu/~fbat/fbat.htm).12 As the T1DGC families diabetes. Diabetes 2005; 54: 1588–1591. have multiple siblings (affected sib-pair families), the 7 Smyth DJ, Cooper JD, Lowe CE, Nutland S, Walker NM, option of the empirical variance was used in the FBAT Clayton DG et al. No evidence for association of OAS1 with statistics to permit a robust, but unbiased test of genetic type 1 diabetes in unaffected siblings or type 1 diabetic cases. association. Diabetes 2006; 55: 1525–1528. 8 Tessier MC, Qu HQ, Frechette R, Bacot F, Grabs R, Taback SP et al. Type 1 diabetes and the OAS gene cluster: association with splicing polymorphism or haplotype? J Med Genet 2006; Conflict of interest 43: 129–132. The authors declare no conflict of interest. 9 Wellcome Trust Case Control Consortium. Genome-wide association study of 14000 cases of seven common diseases and 3000 shared controls. Nature 2007; 447: 661–678. 10 Todd JA, Walker NM, Cooper JD, Smyth DJ, Downes K, Acknowledgements Plagnol V et al. Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes. Nat This research uses resources provided by the Type I Genet 2007; 39: 857–864. Diabetes Genetics Consortium, a collaborative clinical 11 Brown WM, Pierce JJ, Hilner JE, Perdue LH, Lohman K, Lu L et al. and the Type I Diabetes Genetics Consortium. study sponsored by the National Institute of Diabetes Overview of the Rapid Response data. Genes Immun 2009; and Digestive and Kidney Diseases (NIDDK), National 10(Suppl 1): S5–S15. Institute of Allergy and Infectious Diseases (NIAID), 12 Horvath S, Xu X, Laird NM. The family based association test National Human Genome Research Institute (NHGRI), method: strategies for studying general genotype—phenotype National Institute of Child Health and Human Develop- associations. Eur J Hum Genet 2001; 9: 301–306.

Genes and Immunity Reassessment of the T1D association of the OAS1 locus Hi-Q Qu et al S72 Supplementary Table 1 The genotypic association test of the WTCCC SNP rs2660

Group n A/A A/G G/G A/A + A/G G/G ×2 (P value)

2927 control 1177(40.2%) 1418(48.4%) 332(11.3%) 2595(88.7%) 332(11.3%) 5.4× (0.020)

1954

case 820(42%) 869(44.5%) 265(13.6%) 1689(86.4%) 265(13.6%)

The genotyping data were acquired from the Wellcome Trust Case-Control Consortium website

(http://www.wtccc.org.uk/info/summary_stats.shtml)1.

Reference

1. Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common

diseases and 3,000 shared controls. Nature 2007; 447: 661-678.

Genes and Immunity Reassessment of the T1D association of the OAS1 locus Hi-Q Qu et al S73

Genes and Immunity