Tests for genetic interactions in type 1 diabetes: linkage and stratification analyses of 4,422 affected sib-pairs

Abstract

Objective: Interactions between genetic and environmental factors lead to immune dysregulation causing type 1 diabetes and other autoimmune disorders. Recently, many common genetic variants have been associated with type 1 diabetes risk, but each has modest individual effects. Familial clustering of type 1 diabetes has not been explained fully and could arise from many factors, including undetected genetic variation and gene interactions.

Research design and methods: To address this issue, the Type 1 Diabetes Genetics Consortium recruited 3,892 families, including 4,422 affected sib-pairs. After genotyping 6,090 markers, linkage analyses of these families were performed, using a novel method and taking into account factors such as genotype at known susceptibility loci.

Results: Evidence for linkage was robust at the HLA and INS loci, with logarithm of odds (LOD) scores of 398.6 and 5.5, respectively. There was suggestive support for five other loci. Stratification by other risk factors (including HLA and age at diagnosis) identified one convincing region on chromosome 6q14 showing linkage in male subjects (corrected LOD = 4.49; replication P = 0.0002), a locus on chromosome 19q in HLA identical siblings (replication P = 0.006), and four other suggestive loci.

Conclusions: This is the largest linkage study reported for any disease. Our data indicate there are no major type 1 diabetes subtypes definable by linkage analyses; susceptibility is caused by actions of HLA and an apparently random selection from a large number of modest-effect loci; and apart from HLA and INS, there is no important susceptibility factor discoverable by linkage methods.

FIG. 1.

Genome-wide linkage analyses of the final T1DGC cohort. Nonparametric linkage calculations of 6,090 SNPs segregating in 1,487 new affected sib-pair families were performed using Merlin (12). Kong and Cox LOD scores (14) are shown for each chromosome. The peak LOD on chromosome 6 was over 130, but the scale is truncated to show linkage scores for other loci.

FIG. 2.

Evidence of linkage from the entire T1DGC ASP family collection. A: Pedigree files were merged from the latest set of T1DGC families and those reported earlier (4,5) (which are shown in red). LOD scores for the entire dataset were calculated as described in Fig. 1 and shown in blue.The scale is truncated at 4, so it does not indicate the height of the peaks on chromosome 6 (~400) (see B) and 11 (~5). B: Linkage of chromosome 6 markers in the T1DGC family collection. LOD scores were calculated for the complete T1DGC family collection (solid green line) and compared with the cohort 3 families (dashed purple line). In addition, LOD scores were calculated correcting for linkage to HLA: the ELOD score based on decay of linkage from HLA is shown as a dashed blue line and ELOD + 3.6 as a dotted red line. The increase in observed LOD above ELOD in the region ~90–100 cM is consistent with additional type 1 diabetes susceptibility gene(s) mapping to this region.

FIG. 3.

Genome-wide linkage analysis of siblings sharing 2 (IBD) or 1/0 (non-IBD) HLA haplotypes IBD. Pedigree files of T1DGC sets 1 and 2 were constructed using SibShipper (www.sysgen.org.au) from ASPs that shared 2 HLA haplotypes IBD and from the remaining set of siblings that shared one or zero alleles IBD (for convenience, the latter set is referred to as non-IBD). LOD scores were calculated for each set as described in Fig. 1. The scores for the IBD ASPs are shown in red and non-IBD ASPs are shown in blue.

FIG. 4.

Linkage analysis of HLA IBD and HLA non-IBD sib-pairs for markers on chromosome 19 of ASPs from T1DGC sets 1 and 2. A: LOD scores on chromosome 19 are shown for the total dataset (black), HLA IBD siblings (red), and HLA non-IBD siblings (blue). Stratification according to HLA sharing indicates two different linkage peaks on this chromosome. B: ASPs who were HLA IBD (red boxes) or non-IBD (blue boxes) were compared for differences on chromosomes 19p (position <40 cM), 19q (40–80 cM), and 19qtel (>80 cM) via residuals from differenced autoregressive models as described in research design and methods. Relative variation in residuals across the chromosome differed between subgroups (P < 10⁻¹⁵), suggesting differing linkage profiles. P values for differences between HLA IBD and non-IBD ASPs for the three regions of chromosome 19 are 19p, P = 0.00002; 19q, P = 1.3 × 10⁻¹²; and 19tel, P = 0.06. Boxes represent the mid-50% of values; horizontal lines indicate means.

FIG. 5.

Chromosome 6 analyses, with correction for HLA linkage. ASP families from T1DGC sets 1 and 2 stratified on the basis of sex (upper panels: left, female ASPs; right, male ASPs) or INS genotype (lower panel: left, INS homozygous for the A genotype; right, at least one copy of the protective T allele). Observed LOD scores are shown in solid green lines. ELOD scores (dashed blue lines) are shown correcting for HLA linkage. The ELOD + 3.6 score (dotted red lines) indicates the threshold for linkage of a type 1 diabetes risk locus independent of HLA.