Matthew J. Simmonds

Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes

The Wellcome Trust Case Control Consortium (WTCCC) primary genome-wide association (GWA) scan on seven diseases, including the multifactorial autoimmune disease type 1 diabetes (T1D), shows associations at P < 5 × 10−7 between T1D and six chromosome regions: 12q24, 12q13, 16p13, 18p11, 12p13 and 4q27. Here, we attempted to validate these and six other top findings in 4,000 individuals with T1D, 5,000 controls and 2,997 family trios independent of the WTCCC study. We confirmed unequivocally the associations of 12q24, 12q13, 16p13 and 18p11 (Pfollow-up ≤ 1.35 × 10−9; Poverall ≤ 1.15 × 10−14), leaving eight regions with small effects or false-positive associations. We also obtained evidence for chromosome 18q22 (Poverall = 1.38 × 10−8) from a GWA study of nonsynonymous SNPs. Several regions, including 18q22 and 18p11, showed association with autoimmune thyroid disease. This study increases the number of T1D loci with compelling evidence from six to at least ten.

CD226 Gly307Ser association with multiple autoimmune diseases

Genome-wide association studies provide insight into multigenic diseases through the identification of susceptibility genes and etiological pathways. In addition, the identification of shared variants among autoimmune disorders provides insight into common disease pathways. We previously reported an association of a nonsynonymous single nucleotide polymorphism (SNP) rs763361/Gly307Ser in the immune response gene CD226 on chromosome 18q22 with type 1 diabetes (T1D) susceptibility. Here, we report efforts toward identifying the causal variant by exonic resequencing and tag SNP mapping of the 18q22 region in both T1D and multiple sclerosis (MS). In addition to the analysis of newly available samples in T1D (2088 cases and 3289 controls) and autoimmune thyroid disease (AITD) (821 cases and 1920 controls), resulting in strong support for the Ser307 association with T1D (P=3.46 × 10−9) and continued potential evidence for AITD (P=0.0345), we provide evidence for association of Gly307Ser with MS (P=4.20 × 10−4) and rheumatoid arthritis (RA) (P=0.017). The Ser307 allele of rs763361 in exon 7 of CD226 predisposes to T1D, MS, and possibly AITD and RA, and based on the tag SNP analysis, could be the causal variant.

Negligible impact of rare autoimmune-locus coding-region variants on missing heritability

Genome-wide association studies (GWAS) have identified common variants of modest-effect size at hundreds of loci for common autoimmune diseases; however, a substantial fraction of heritability remains unexplained, to which rare variants may contribute. To discover rare variants and test them for association with a phenotype, most studies re-sequence a small initial sample size and then genotype the discovered variants in a larger sample set. This approach fails to analyse a large fraction of the rare variants present in the entire sample set. Here we perform simultaneous amplicon-sequencing-based variant discovery and genotyping for coding exons of 25 GWAS risk genes in 41,911 UK residents of white European origin, comprising 24,892 subjects with six autoimmune disease phenotypes and 17,019 controls, and show that rare coding-region variants at known loci have a negligible role in common autoimmune disease susceptibility. These results do not support the rare-variant synthetic genome-wide-association hypothesis (in which unobserved rare causal variants lead to association detected at common tag variants). Many known autoimmune disease risk loci contain multiple, independently associated, common and low-frequency variants, and so genes at these loci are a priori stronger candidates for harbouring rare coding-region variants than other genes. Our data indicate that the missing heritability for common autoimmune diseases may not be attributable to the rare coding-region variant portion of the allelic spectrum, but perhaps, as others have proposed, may be a result of many common-variant loci of weak effect.

The HLA Region and Autoimmune Disease: Associations and Mechanisms of Action

The HLA region encodes several molecules that play key roles in the immune system. Strong association between the HLA region and autoimmune disease (AID) has been established for over fifty years. Association of components of the HLA class II encoded HLA-DRB1-DQA1-DQB1 haplotype has been detected with several AIDs, including rheumatoid arthritis, type 1 diabetes and Graves’ disease. Molecules encoded by this region play a key role in exogenous antigen presentation to CD4+ Th cells, indicating the importance of this pathway in AID initiation and progression. Although other components of the HLA class I and III regions have also been investigated for association with AID, apart from the association of HLA-B*27 with ankylosing spondylitis, it has been difficult to determine additional susceptibility loci independent of the strong linkage disequilibrium (LD) with the HLA class II genes. Recent advances in the statistical analysis of LD and the recruitment of large AID datasets have allowed investigation of the HLA class I and III regions to be re-visited. Association of the HLA class I region, independent of known HLA class II effects, has now been detected for several AIDs, including strong association of HLA-B with type 1 diabetes and HLA-C with multiple sclerosis and Graves’ disease. These results provide further evidence of a possible role for bacterial or viral infection and CD8+ T cells in AID onset. The advances being made in determining the primary associations within the HLA region and AIDs will not only increase our understanding of the mechanisms behind disease pathogenesis but may also aid in the development of novel therapeutic targets in the future.

Regression Mapping of Association between the Human Leukocyte Antigen Region and Graves Disease

The human leukocyte antigen class II genes DRB1, DQB1, and DQA1 are associated with Graves disease (GD), but, because of strong linkage disequilibrium within this region, the primary etiological variant(s) remains unknown. In the present study, 871 patients with GD and 621 control subjects were genotyped at the DRB1, DQB1, and DQA1 loci. All three loci were associated with GD (P=1.45 x 10(-12), P=3.20 x 10(-5), and P=9.26 x 10(-12), respectively). Stepwise logistic-regression analysis showed that the association could be explained by either DRB1 or DQA1 but not by DQB1. To extend previous results, the amino acid sequence of the exon 2-encoded peptide-binding domain of DRB1 was predicted for each subject, and, by use of logistic regression, each position was analyzed for association with GD. Of 102 amino acids, 70 were uninformative; of the remaining 32 amino acids, 13 were associated with GD (P values ranged from 2.20 x 10(-4) to 1.2 x 10(-12)). The strongest association was at position beta 74. This analysis is consistent with the possibility that position beta 74 of exon 2 of the DRB1 molecule may have a specific and central role in autoantigen presentation by DRB1 to T lymphocytes. However, we cannot yet exclude a primary role for DQA1 or for other polymorphisms that affect DRB1 function or expression.

Alemtuzumab induction and sirolimus plus mycophenolate mofetil maintenance for CNI and steroid-free kidney transplant immunosuppression.

We performed a pilot study in which 22 kidney recipients (14 LD: 8 DCD) were given alemtuzumab induction (30 mg day 0 and 1), steroids (500 mg mp day 0 and 1, none thereafter), mycophenolate mofetil (MMF) maintenance (500 mg b.i.d) and sirolimus (concentration controlled 8–12 ng/mL). With a mean follow‐up of 15.9 months, patient survival is (21/22) 96% and graft survival (19/22) 87%. Acute rejections occurred in (8) 36.3% (two humoral). Of 19 surviving grafts, 18 (95%) remain steroid and 15 (79%) CNI‐free. At 1 year, mean creatinine was 1.43 mg/dL. Overall infection rates were low, but 2 patients developed severe acute respiratory distress syndrome (ARDS) at month 3 and 7, respectively, resulting in mortality in one and a graft loss in the other. No cancer or PTLD was observed. Leukopenia was common and MMF dose was reduced or eliminated in 6/22 (27%) patients. The reported higher than expected rate of acute rejection, leukopenia and possible pulmonary toxicity suggests excessive morbidity. Modifications such as an initial period of CNI use should be considered.

Unravelling the genetic complexity of autoimmune thyroid disease: HLA, CTLA-4 and beyond

The autoimmune thyroid diseases (AITDs) including Graves’ disease (GD) and autoimmune hypothyroidism (AIH) are the commonest of the autoimmune conditions affecting 2–5% of the western population. Twin studies have clearly demonstrated that AITDs are caused by a combination of both environmental and genetic factors. Association of the HLA class II region with AITD has been documented for over 20 years now, but the primary aetiological variant in this region remains unknown. More recently the CTLA‐4 gene region has been identified as the second locus conferring susceptibility to AITD. In contrast to HLA, a polymorphism of the CTLA‐4 gene, which encodes an important negative regulator of the immune system, has been identified as a candidate for a primary determinant for AITD. A large number of candidate gene and genome wide linkage studies have been involved in the search for the elusive ‘third’ locus. The thyroglobulin (Tg) gene in humans maps to chromosome 8q, which has been linked in family studies to AITD. A number of association studies in humans and the mouse model for AITD are beginning to implicate the Tg gene although convincing evidence for a primary causative role is still needed. The establishment of large DNA disease resources along with more detailed genetic maps and the development of faster, more effective, high throughput genotyping and sequencing methods, provides some sense of optimism that novel loci will be identified in the near future and the complex aetiology of AITD will be further unraveled.

Donor ABCB1 Variant Associates with Increased Risk for Kidney Allograft Failure

The impact of variation within genes responsible for the disposition and metabolism of calcineurin inhibitors (CNIs) on clinical outcomes in kidney transplantation is not well understood. Furthermore, the potential influence of donor, rather than recipient, genotypes on clinical endpoints is unknown. Here, we investigated the associations between donor and recipient gene variants with outcome among 4471 white, CNI-treated kidney transplant recipients. We tested for 52 single-nucleotide polymorphisms (SNPs) across five genes: CYP3A4, CYP3A5, ABCB1 (MDR1; encoding P-glycoprotein), NR1I2 (encoding the pregnane X receptor), and PPIA (encoding cyclophilin). In a discovery cohort of 811 patients from Birmingham, United Kingdom, kidney donor CC genotype at C3435T (rs1045642) within ABCB1, a variant known to alter protein expression, was associated with an increased risk for long-term graft failure compared with non-CC genotype (hazard ratio [HR], 1.69; 95% confidence interval [CI], 1.20-2.40; P=0.003). No other donor or recipient SNPs were associated with graft survival or mortality. We validated this association in 675 donors from Belfast, United Kingdom (HR, 1.68; 95% CI, 1.21-2.32; P=0.002), and in 2985 donors from the Collaborative Transplant Study (HR, 1.84; 95% CI, 1.08-3.13; P=0.006). In conclusion, these data suggest that an ABCB1 variant known to alter protein expression represents an attractive candidate for future study and risk stratification in kidney transplantation.

Analysis of HLA class II genes in Hashimoto's thyroiditis reveals differences compared to Graves’ disease

Graves’ disease (GD) and Hashimotos thyroiditis (HT) represent the commonest forms of autoimmune thyroid disease (AITD) each presenting with distinct clinical features. Progress has been made in determining association of HLA class II DRB1, DQB1 and DQA1 loci with GD demonstrating a predisposing effect for DR3 (DRB1*03-DQB1*02-DQA1*05) and a protective effect for DR7 (DRB1*07-DQB1*02-DQA1*02). Small data sets have hindered progress in determining HLA class II associations with HT. The aim of this study was to investigate DRB1-DQB1-DQA1 in the largest UK Caucasian HT case control cohort to date comprising 640 HT patients and 621 controls. A strong association between HT and DR4 (DRB1*04-DQB1*03-DQA1*03) was detected (P=6.79 × 10−7, OR=1.98 (95% CI=1.51–2.59)); however, only borderline association of DR3 was found (P=0.050). Protective effects were also detected for DR13 (DRB1*13-DQB1*06-DQA1*01) (P=0.001, OR=0.61 (95% CI=0.45–0.83)) and DR7 (P=0.013, OR=0.70 (95% CI=0.53–0.93)). Analysis of our unique cohort of subjects with well characterized AITD has demonstrated clear differences in association within the HLA class II region between HT and GD. Although HT and GD share a number of common genetic markers this study supports the suggestion that differences in HLA class II genotype may, in part, contribute to the different immunopathological processes and clinical presentation of these related diseases.

GWAS in autoimmune thyroid disease: redefining our understanding of pathogenesis

The ability of the immune system to protect the body from attack by foreign antigens is essential for human survival. The immune system can, however, start to attack the bodys own organs. An autoimmune response against components of the thyroid gland affects 2–5% of the general population. Considerable familial clustering is also observed in autoimmune thyroid disease (AITD). Teasing out the genetic contribution to AITD over the past 40 years has helped unravel how immune disruption leads to disease onset. Breakthroughs in genome-wide association studies (GWAS) in the past decade have facilitated screening of a greater proportion of the genome, leading to the identification of a before unimaginable number of AITD susceptibility loci. This Review will focus on the new susceptibility loci identified by GWAS, what insights these loci provide about the pathogenesis of AITD and how genetic susceptibility loci shared between different autoimmune diseases could help explain disease co-clustering within individuals and families. This Review also discusses where future efforts should be focused to translate this step forward in our understanding of the genetic contribution to AITD into a better understanding of disease presentation and progression, and improved therapeutic options.