Background: The two genome-wide association studies published by us and by the Wellcome Trust Case–Control Consortium (WTCCC) revealed a number of novel loci, but neither had the statistical power to elucidate all of the genetic components of type 1 diabetes risk, a task for which larger effective sample sizes are needed.
Methods: We analysed data from two sources: (1) The previously published second stage of our study, with a total sample size of the two stages consisting of 1046 Canadian case–parent trios and 538 multiplex families with 929 affected offspring from the Type 1 Diabetes Genetics Consortium (T1DGC); (2) the Rapid Response 2 (RR2) project of the T1DGC, which genotyped 4417 individuals from 1062 non-overlapping families, including 2059 affected individuals (mostly sibling pairs) for the 1536 markers with the highest statistical significance for type 1 diabetes in the WTCCC results.
Results: One locus, mapping to a linkage disequilibrium (LD) block at chr15q14, reached statistical significance by combining results from two markers (rs17574546 and rs7171171) in perfect LD with each other (r2 = 1). We obtained a joint p value of 1.3×10−6, which exceeds by an order of magnitude the conservative threshold of 3.26×10−5 obtained by correcting for the 1536 single nucleotide polymorphisms (SNPs) tested in our study. Meta-analysis with the original WTCCC genome-wide data produced a p value of 5.83×10−9.
Conclusions: A novel type 1 diabetes locus was discovered. It involves RASGRP1, a gene known to play a crucial role in thymocyte differentiation and T cell receptor (TCR) signalling by activating the Ras signalling pathway.
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Type 1 diabetes is due to autoimmune destruction of the pancreatic beta cells, a process of multifactorial aetiology with a strong genetic component. Genome-wide association studies (GWAS) conducted by us1 and the Wellcome Trust Case Control Consortium (WTCCC)2 3 identified a number of loci which, when added to those previously known from the candidate gene approach, still leave a considerable part of the relative sibling risk (λS) for type 1 diabetes unexplained. This is consistent with the experience in other complex traits, which also involve a multiplicity of weak genetic effects for the detection of which the sample sizes of current GWAS studies have relatively low power. Multistage approaches and meta-analyses that increase effective sample size are therefore called for.
In a recent, three stage follow-up of our original GWAS, we reported the discovery of two additional novel loci, involving the UBASH3A and BACH2 genes,4 a finding independently reported in two other studies.5 6 In this report we are presenting new evidence suggesting another locus for type 1 diabetes risk that maps to the RASGRP1 gene, based on evidence obtained by combining our data with the Rapid Response 2 (RR2) project of the Type 1 Diabetes Genetics Consortium (T1DGC, http://www.t1dgc.org).7
The T1DGC RR2 study genotyped 4417 individuals from 1062 type 1 diabetes families, including 2059 affected siblings and both their parents for the 1536 markers with the highest statistical significance for type 1 diabetes in the WTCCC results. Genotyping was performed at the Sanger Institute on the Illumina Golden Gate platform. Most subjects were of European ancestry, with a median age at onset of 10 years (lower and upper quartiles at 6 years and 15.5 years).
In our study, we genotyped 1046 type 1 diabetes case–parent trios, collected in paediatric diabetes clinics in Montreal, Toronto, Ottawa and Winnipeg. The median age at onset is 8.4 years with lower and upper quartiles at 5.0 years and 11.8 years. Ethnic backgrounds were of mixed European descent, with the largest single subset (40%) being French Canadian. The Research Ethics Board of the Montreal Children’s Hospital and other participating centres approved the study, and written informed consent was obtained from all subjects. In addition, we genotyped 549 families with at least one child with type 1 diabetes and both parents (946 total affected). The median age at onset is 8 years with quartiles at 4 years and 13 years. The samples were collected in Europe, North America and Australia and most subjects were of European ancestry. Genotyping data from 11 overlapping families that were also included in the RR2 study were removed for analysis. As we previously described,4 we used the Illumina Golden Gate array to genotype 982 markers with p<0.05 in both the transmission disequilibrium test (TDT) and case–control phase of our original GWAS. In addition, 15 single nucleotide polymorphisms (SNPs) with p<0.1 in each of our two GWAS cohorts and p<0.01 in WTCCC were genotyped using mass spectrometry on the Sequenom iPlex platform.
Type 1 diabetes association was tested by the Family Based Association Test (FBAT) software (http://www.biostat.harvard.edu/∼fbat/fbat.htm).8 Considering most of the T1DGC families have multiple siblings, the option of the empirical variance was used in the FBAT statistics to permit a robust but unbiased test of genetic association. As 1536 SNPs were tested in the RR2 study, we used a conserved significance threshold corrected for multiple comparisons at 3.26×10−5.
RESULTS AND DISCUSSION
Overlap in the markers selected in the two projects was determined either by identity of SNPs or, in cases of physical proximity (<1 Mb), by linkage disequilibrium (LD (r2 value >0.8). After excluding known type 1 diabetes loci, there was only one locus nominally significant (p<0.05) in both projects. It involves a locus evaluated in the RR2 cohort by SNP rs17574546 (p = 3.41×10−3) and in our set by rs7171171 (p = 8.40×10−5, table 1).
The genotype calling rate of rs17574546 in the RR2 samples is 99.8%, and for rs7171171 in our own samples is 99.9%. No Mendelian error was found in either. As these SNPs are in perfect LD (r2 = 1) we performed a direct combined analysis which showed p = 1.30×10−6. This exceeds by more than an order of magnitude the corrected significance level. The odds ratio (OR) (95% confidence interval (CI)) estimated on the combined family dataset is 1.22 (95% CI 1.12 to 1.33), while the OR in the WTCCC case–control set is 1.21 (95% CI 1.09 to 1.33) (p = 2.67×10−4). The meta-analysis of these two results gives an OR of 1.21 (95% CI 1.14 to 1.30) and p = 5.83×10−9, a significance level accepted for genome-wide studies. Based on these results, we can conclude that the RASGRP1 locus is associated with type 1 diabetes. It is interesting to note that rs17574546 and rs7171171 both have D’ = 0.902, and r2 = 0.553, with rs8035957, an SNP that narrowly failed to reach significance in our previous report.1
This novel type 1 diabetes association signal maps to an LD block at Chr15q14, ∼13 kb upstream of the transcription start site of the RASGRP1 gene (supplemental fig 1), and has no LD with any known type 1 diabetes locus. As type 1 diabetes is caused by the autoimmune destruction of pancreatic β cells, it is interesting that the RASGRP1 gene has an important immune function. RASGRP1 (NCBI GeneID: 10125) encodes calcium and DAG regulated RAS guanyl releasing protein 1 (RasGRP1).9 RasGRP1 plays crucial roles in thymocyte differentiation and T cell receptor (TCR) signalling by activating the Ras signalling pathway. RasGRP1-null mutant mice have approximately normal numbers of immature thymocytes, but a marked deficiency of mature, single positive (CD4+CD8− and CD4−CD8+) thymocytes.10 Transgenic expression of RasGRP1 induces the maturation of double negative thymocytes and enhances the production of CD4−CD8+ thymocytes.11 In addition, RasGRP1 has a dramatic effect on the development and function of CD4+CD25+ regulatory T cells (Treg). In the absence of RasGRP1, the development of CD4+CD25+ Treg in the thymus is severely impaired, whereas the peripheral expansion and function of CD4+CD25+ Treg are greatly increased.12 CD4+CD25+ Treg plays a critical role in maintaining immune homeostasis and inhibiting autoimmune reaction of type 1 diabetes and other autoimmune diseases.13 As the transfer of CD4+CD25+ CD4+CD25+ Treg cells can prevent type 1 diabetes in the recipient NOD mice,14 knowledge of the role of genes involved in the generation of this subset in type 1 diabetes may play an important role in the development of preventive interventions.
This research utilises resources provided by the Type 1 Diabetes Genetics Consortium, a collaborative clinical study sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institute of Allergy and Infectious Diseases (NIAID), National Human Genome Research Institute (NHGRI), National Institute of Child Health and Human Development (NICHD), and Juvenile Diabetes Research Foundation International (JDRF) and supported by U01 DK062418. We thank all the patients, their parents and the healthy control subjects for their participation in the study.
▸ An additional figure is published online only at http://jmg.bmj.com/content/vol46/issue8
Funding: This work was funded by the Children’s Hospital of Philadelphia, the Juvenile Diabetes Research Foundation International and Genome Canada. HQQ is supported by a fellowship from the Canadian Institutes of Health Research.
Competing interests: None.
Patient consent: Obtained.
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