Abstract

Introduction

Type 1 diabetes (T1D), multiple sclerosis (MS), autoimmune thyroid disease (AITD) and rheumatoid arthritis (RA) are organ specific autoimmune diseases mediated by self-reactive T cells and other cells of the adaptive and innate immune systems. T1D is characterized by inflammation of the pancreatic islets of Langerhans with destruction of insulin producing ß-cells¹, while in MS and RA, there is selective white matter or joint tissue destruction, respectively^{2; 3}. Sibling and twin studies indicate a major genetic component of the familial clustering of these common diseases^4-6 with the major susceptibility loci in the HLA region^7-10. Recent genome-wide association (GWA) studies have successfully identified many non-HLA single nucleotide polymorphisms (SNPs) associated with common disease susceptibility¹¹. In addition to the identification of associated SNPs, these investigations are providing insight into genes and mechanisms shared among autoimmune diseases. Examples include STAT4 in RA and systemic lupus erythematosus (SLE)¹², and IL2RA in T1D¹³, Graves' disease¹⁴ and MS^{15; 16}.

We recently reported a GWA study of nsSNPs in T1D that provided strong statistical evidence for association at chromosome 18q22¹⁷. In 6,021 T1D cases and 6,088 controls, Gly307Ser, located in CD226, showed a P = 2.82 × 10⁻⁸ (Odds Ratio (OR) for minor allele = 1.16, 95% confidence interval (c.i.) = 1.10-1.22) and in 2,997 parent-child trios, with a P = 0.0281 (Relative Risk, RR, = 1.08, 95% c.i. = 1.00-1.16). Combining the results obtained from the case-control and family studies yielded a P = 1.38 × 10⁻⁸.

CD226 (also known as DNAX accessory molecule 1, DNAM-1) is a 67 kDa type I membrane protein involved in the adhesion and co-stimulation of T cells¹⁸. It belongs to the immunoglobulin supergene family of receptors, containing two Ig-like domains in the extracellular region and is constitutively expressed on the majority of natural killer (NK) cells, CD4⁺ and CD8⁺ T cells, monocytes, platelets and a subset of B cells¹⁸. Further, in an experimental model of MS, experimental autoimmune encephalomyelitis (EAE), anti-CD226 mAb treatment delayed the onset and reduced the severity of EAE¹⁹. The Gly307Ser variant could alter expression or signalling of CD226 as it occurs in the molecule's cytoplasmic tail¹⁷. It was, therefore, of interest to explore whether Gly307Ser was the causal variant in the region and a shared risk locus for autoimmune disease.

Here we report an initial fine-mapping study of the 18q22 region in both T1D and MS by means of exonic resequencing and a tag SNP mapping approach based around Gly307Ser. We provide no evidence against the hypothesis that the nonsynonymous SNP (Gly307Ser) is the causal variant in the 18q22 region for MS and T1D. Moreover, we extended this analysis to include RA (and additional AITD samples), suggesting that Ser³⁰⁷ predisposes to a range of human autoimmune diseases.

Results

In order to increase SNP density and detect as-yet-unknown SNPs in the coding region or identify SNPs that may disrupt intron/exon splice sites present in CD226, we resequenced the exonic regions in the roughly 50 kb linkage disequilibrium (LD) block (exons 4, 5, 6, and 7) containing Gly307Ser and 3 kb of 3′ flanking sequence in 32 individuals chosen from the HapMap CEPH collection. This led to 7.7 kb of DNA being resequenced and the identification of 13 SNPs in three exons and the 3′ flanking sequence (exon 5 was not sequenced due to PCR failures, see material and methods). When compared with the publicly available SNPs in dbSNP build 128, two SNPs were found to be novel polymorphisms. They were located in exon 6: a nsSNP (Ala279Leu, ss102661466) and a synonymous SNP (Gln282Gln, ss102661465) with minor allele frequencies (MAFs) of 0.065 and 0.078, respectively. As these variants are functional candidates, they were genotyped in the T1D case-control collection. The single locus tests provided little evidence of an association with T1D disease susceptibility: Ala279Leu P = 2.08 × 10⁻³ (OR = 1.15; 95% c.i. 1.05-1.26) and Gln282Gln P = 0.0298 (OR = 0.90; 95% c.i. 0.82-0.99) (Supplementary Information Table 1). Nor did the forward logistic regression analysis adding either novel SNP to Gly307Ser provide evidence (minimum P = 0.0542) of an independent association with T1D susceptibility, while Gly307Ser added significantly to both SNPs (minimum P = 8.10 × 10⁻⁶) indicating that neither novel SNPs are independently associated with T1D susceptibility.

Further, we selected and tested a set of tag SNPs (see materials and methods) to investigate the association previously identified in the 18q22 region. Gly307Ser was still the most associated with T1D in 8,109 cases and 9,377 controls (P = 1.32 × 10⁻⁸; OR = 1.13, 95% c.i. 1.08-1.18) (Table 1). We conducted a forward logistic regression analysis testing the addition of each SNP to Gly307Ser and found that none added significantly. There was, therefore, no evidence for a known polymorphism (with a MAF > 0.05 and r² with Gly307Ser > 0.25) in the CD226 region that showed stronger association with T1D than Gly307Ser or had an independent effect on T1D susceptibility.

We then proceeded to test Gly307Ser in a cohort of MS samples consisting of 1,275 trios, 1,194 USA cases, 595 USA controls, 993 UK cases and 9,377 UK controls (UK controls are the same as in our T1D association study). The combined P-value was 4.20 × 10⁻⁴ (Table 2). In order to test the hypothesis that Gly307Ser was the causal variant in MS, we tested the same set of T1D tag SNPs in an extended set of 1,318 MS trios, 1,769 MS US cases, 2,508 US controls, and 1,003 MS UK cases and used the genotyping data already available for 9,377 UK controls. Consistent with our T1D study, we obtained no evidence against the hypothesis that CD226 Gly307Ser is the causal variant associated with MS in the 18q22 region (Table 3).

As Gly307Ser was found to be associated with both T1D and MS susceptibility, it was of interest to examine a collection of RA samples and an additional cohort of autoimmune thyroid disease cases. We tested Gly307Ser in 3,595 RA cases and 3,214 controls and obtained some evidence of association, at P = 0.017 (OR = 1.09; 95% c.i. 1.02-1.16) (Table 2). We obtained no evidence of heterogeneity of association between males and females (P = 0.90), nor between RF positive and RF negative cases (P = 0.86), nor between anti-CCP negative and anti-CCP positive cases (P = 0.45). This suggests Gly307Ser is associated with RA and not a sub-phenotype. Further, adding 821 AITD cases to our previously published data¹⁷ (N = 2,958, N = 5,431) we obtained potential evidence for association, at P = 0.0335 (OR = 1.08; 95% c.i. 1.00 - 1.15) (the controls are the same used in our T1D association study but matched geographically) (see Supplementary table 4 for Graves' disease and Hashimoto's disease results separately reported).

Discussion

GWA scans in common human autoimmune diseases have recently identified many loci associated with disease susceptibility. Understanding allelic heterogeneity and homogeneity among diseases provides insight into common gene function and pathways. Here, we examined the gene encoding CD226, a molecule expressed on the surface of haematopoietic cells that has independently been implicated in the pathogenesis of animal models of autoimmune diseases. Our resequencing efforts and tagging approach aimed at narrowing the association in the 18q22 region provided no evidence against the hypothesis that CD226 Gly307Ser is the causal variant in T1D and MS. In addition, the International Multiple Sclerosis Genetics Consortium (IMSGC) extended the evidence supporting an association of Gly307Ser with MS (see accompanying short report). In an additional sample of 3,610 MS cases, 324 controls and 1,036 trios, the IMSGC further validated Gly307Ser association with MS (P= 5.4 × 10⁻⁸) (Table XX in IMSGC paper, see editorial for complete analysis and overlap between studies). Taken together with our association study of the role of Gly307Ser in a collection of RA cases and additional data for Gly307Ser in AITD (P = 0.0345), we provide initial evidence for the CD226 gene to be shared among at least four common human autoimmune diseases.

We note, however, that until a more complete set of polymorphisms is identified and genotyped in a large collection of cases and control subjects, we cannot exclude another variant in LD with Gly307Ser being the causal variant. Future successful resequencing of exon 5 may provide as yet undiscovered variants that will need to be assessed for disease susceptibility. In addition, the CD226 region may harbour other, independent associations with susceptibility to disease that our tag mapping approach was not designed to identify, as has previously been shown for another T1D susceptibility locus containing the IL2RA gene²⁰, although the data indicate that this is not the case for common variants in the CD226 region.

CD226 is implicated in natural killer cell mediated cytoxicity as well as Th1 cell mediated immune response^{18; 19}. Phosphorylation of the cytoplasmic tail of CD226 assists in co-localization with LFA-1 and cell activation²¹. Our genetic association data now justify studies of the functional consequences of the Gly307Ser variant in adaptive and innate immune responses. We have previously hypothesized¹⁷ that the SNP could disrupt a splice site enhancer, or silencer, thereby altering RNA splicing, as has been demonstrated for other immune related genes (human CD45 and mouse Ctla4)²², resulting in either a putative CD226 isoform acting as a non-functional (non-signalling) protein, or with a novel function. Alternatively, this amino acid substitution could alter the signalling cascade by affecting the two known phosphorylation sites at positions 322 and 329^{21; 23}, which share a critical role in CD226 and the immune response.

Material and methods

Supplementary Material

Acknowledgments

Footnotes

Bibliography