Muddassar M. Mirza

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.

Evidence for a NOD2-independent susceptibility locus for inflammatory bowel disease on chromosome 16p

Heritable predisposition to inflammatory bowel disease (IBD) has been demonstrated by epidemiological and genetic analysis. Linkage of IBD to broad regions of chromosome 16 has been established by analysis of multiple populations. NOD2, located on proximal 16q, was recently identified as an IBD gene. As the linkage regions on chromosome 16 are large, we have investigated the possibility that NOD2 is not the only IBD gene located on this chromosome. A high-density experiment using 39 microsatellite markers was performed to identify additional regions of association, and to indicate areas of interest for further investigation. A triple-peaked configuration of the linkage curve with peak logarithm of odds (lod) scores of 2.7, 3.2, and 3.1 was observed on proximal 16p, proximal 16q, and central 16q, respectively. The cohort was stratified by coding individuals carrying the NOD2 single nucleotide polymorphism (SNP)8 and SNP13 “unknown.” Significance at the central peak, corresponding to the genomic location of NOD2, then decreased from 3.2 to 1.2. The maximal lod scores on the proximal p-arm (lod = 2.1) and central q-arm (lod = 2.6) changed only moderately. An exploratory association analysis (TRANSMIT) yielded a strong lead at D16S3068 (P = 0.00028). The region around this marker was further investigated by using anonymous SNPs. An associated haplotype containing three SNPs was identified (peak significance P = 0.00027, IBD phenotype). On stratification based on NOD2 genotype, this significance increased to P = 0.0001. These results confirm the importance of NOD2 and provide evidence for a second IBD gene located on chromosome 16p.

Genetic evidence for interaction of the 5q31 cytokine locus and the CARD15 gene in Crohn disease.

A common haplotype spanning 250 kb in the cytokine gene cluster on chromosome 5q31 has recently been reported to be strongly associated with Crohn disease (CD) in Canadian families. We have replicated this finding by both the transmission-disequilibrium test (TDT) (P=.016) and in a case-control association study (P=.008) in a large European cohort of patients with CD, although the increase in disease risk was small (odds ratio 1.49 for homozygotes, 95% CI 1.11-2.0). No association was detected in families or individuals with ulcerative colitis (UC). Stratification of offspring with CD in the TDT sample by mutation status in the CD susceptibility gene CARD15 showed that the association with the 5q31 risk haplotype was present only in offspring with at least one of the known CARD15 disease susceptibility alleles (P=.044). The 5q31 risk haplotype frequency was 53.1% in unrelated individuals with CD who had one or two CARD15 mutations versus 43.7% in control subjects (P=.0001) but was not significantly elevated in individuals with CD who had no CARD15 mutations (45.4%, P=.41). Kaplan-Meier survival analysis of age at disease onset showed a significantly earlier onset in homozygotes for the 5q31 risk haplotype (P=.0019). These findings suggest that genetic variants at the 5q31 (IBD5) locus may hasten the onset of Crohn disease and cooperate with CARD15 in disease causation.

Novel isoforms of the CARD8 ( TUCAN ) gene evade a nonsense mutation

CARD8 (TUCAN) is implicated in the regulation of apoptosis and inflammation, and is a positional and functional candidate gene for inflammatory bowel disease (IBD). Recent investigations have reported conflicting results of association between a CARD8 nonsynonymous SNP, rs2043211, and IBD. SNP rs2043211 results in an A>T transversion in the CARD8 template strand, which introduces a stop codon polymorphism (Cys10Stop), and genotyping of the Cys10Stop variant revealed that 9% of the control population was homozygous for the ‘Stop’ allele. The effect of the Stop allele on mRNA and protein expression of the two known isoforms of this gene was investigated. IBD patients homozygous for the Stop allele showed somewhat reduced expression of CARD8 mRNA, but, contrary to expectation, expressed a 48 kDa protein isoform. A search of the EST database and reverse transcription-PCR analysis revealed a novel coding exon and three novel CARD8 mRNA isoforms that are conserved in primates. The isoforms of CARD8 differ in their N-termini, resulting in diverse predicted molecular weights (47, 48, 51, 54 and 60 kDa) and multiple outcomes for the variant including Cys10Stop, Cys34Stop, Phe52Ile and Phe102Ile; one isoform may arise through transcription and translation initiated downstream of rs2043211 to yield a novel protein isoform of ∼47 kDa. The multiple isoforms and differing consequences for a predicted stop codon polymorphism underline the importance of detailed analysis of the effects of proposed functional variants on gene expression.

Pooled sequencing of 531 genes in inflammatory bowel disease identifies an associated rare variant in BTNL2 and implicates other immune related genes

The contribution of rare coding sequence variants to genetic susceptibility in complex disorders is an important but unresolved question. Most studies thus far have investigated a limited number of genes from regions which contain common disease associated variants. Here we investigate this in inflammatory bowel disease by sequencing the exons and proximal promoters of 531 genes selected from both genome-wide association studies and pathway analysis in pooled DNA panels from 474 cases of Crohn’s disease and 480 controls. 80 variants with evidence of association in the sequencing experiment or with potential functional significance were selected for follow up genotyping in 6,507 IBD cases and 3,064 population controls. The top 5 disease associated variants were genotyped in an extension panel of 3,662 IBD cases and 3,639 controls, and tested for association in a combined analysis of 10,147 IBD cases and 7,008 controls. A rare coding variant p.G454C in the BTNL2 gene within the major histocompatibility complex was significantly associated with increased risk for IBD (p = 9.65x10−10, OR = 2.3[95% CI = 1.75–3.04]), but was independent of the known common associated CD and UC variants at this locus. Rare (<1%) and low frequency (1–5%) variants in 3 additional genes showed suggestive association (p<0.005) with either an increased risk (ARIH2 c.338-6C>T) or decreased risk (IL12B p.V298F, and NICN p.H191R) of IBD. These results provide additional insights into the involvement of the inhibition of T cell activation in the development of both sub-phenotypes of inflammatory bowel disease. We suggest that although rare coding variants may make a modest overall contribution to complex disease susceptibility, they can inform our understanding of the molecular pathways that contribute to pathogenesis.

Diverse effects of the CARD15 and IBD5 loci on clinical phenotype in 630 patients with Crohn's disease

Objectives Genetic variants at the CARD15 and IBD5 loci are strongly associated with Crohns disease (CD), but evidence of the effect of these variants on the clinical expression of CD is conflicting and has often been hampered by small sample sizes. We studied 630 well-characterized patients to clarify the genotype/phenotype relationship in CD. Methods Patients and healthy controls were genotyped for three common mutations in CARD15 and a marker of the IBD5 risk haplotype. Allele frequencies were compared between phenotypic subgroups using χ2 or Fishers exact tests. Genotype/phenotype analysis was carried out using multinomial logistic regression modelling allowing for adjustment for correlated or confounding factors. Results The mean age at diagnosis was significantly lower in carriers of the CARD15 or IBD5 risk alleles. After correction for age and smoking, CARD15 mutations were strongly associated with both ileal disease (P=8.8×10−6) and stenotic disease (P=0.003), but the association with stenotic disease appeared to be due to a confounding effect with ileal disease. CARD15 mutations were also associated with the presence of granulomas (P=5.7×10−5), which remained significant after adjustment for age at diagnosis and disease location (P=0.0047). The IBD5 risk haplotype frequency was significantly elevated in cases with perianal disease (P=0.028) and axial spondyloarthropathy (P=0.012). Conclusion Genetic variants at the CARD15 and IBD5 loci have diverse effects on clinical expression in CD. CARD15 mutations are significantly correlated with the presence of granulomas.

Sequence variation, linkage disequilibrium and association with Crohn's disease on chromosome 5q31

Chromosome 5q31 contains a cluster of genes involved in immune response, including a 250 kb risk haplotype associated with Crohns disease (CD) susceptibility. Recently, two functional variants in SLC22A4 and SLC22A5 (L503F and G-207C), encoding the cation transporters OCTN1 and OCTN2, were proposed as causal variants for CD, but with conflicting genetic evidence regarding their contribution. We investigated this locus by resequencing the coding regions of 10 genes in 24 CD cases and deriving a linkage disequilibrium (LD) map of the 27 single nucleotide polymorphisms (SNPs) detected. Ten SNPs representative of the LD groups observed, were tested for CD association. L503F in SLC22A4 was the only nonsynonymous SNP significantly associated with CD (P=0.003), but was not associated with disease in the absence of other markers of the 250 kb risk haplotype. Two other SNPs, rs11242115 in IRF1 and rs17166050 in RAD50, lying outside the 250 kb risk haplotype, also showed CD association (P=0.019 and P=0.0080, respectively). The RAD50 gene contains a locus control region regulating expression of the Th2 cytokine genes at this locus. Other as yet undiscovered SNPs in this region may therefore modulate gene expression and contribute to the risk of CD, and perhaps of other inflammatory phenotypes.

Exome Sequencing of 75 Individuals from Multiply Affected Coeliac Families and Large Scale Resequencing Follow Up

Coeliac disease (CeD) is a highly heritable common autoimmune disease involving chronic small intestinal inflammation in response to dietary wheat. The human leukocyte antigen (HLA) region, and 40 newer regions identified by genome wide association studies (GWAS) and dense fine mapping, account for ∼40% of the disease heritability. We hypothesized that in pedigrees with multiple individuals with CeD rare [minor allele frequency (MAF) <0.5%] mutations of larger effect size (odds ratios of ∼ 2–5) might exist. We sequenced the exomes of 75 coeliac individuals of European ancestry from 55 multiply affected families. We selected interesting variants and genes for further follow up using a combination of: an assessment of shared variants between related subjects, a model-free linkage test, and gene burden tests for multiple, potentially causal, variants. We next performed highly multiplexed amplicon resequencing of all RefSeq exons from 24 candidate genes selected on the basis of the exome sequencing data in 2,248 unrelated coeliac cases and 2,230 controls. 1,335 variants with a 99.9% genotyping call rate were observed in 4,478 samples, of which 939 were present in coding regions of 24 genes (Ti/Tv 2.99). 91.7% of coding variants were rare (MAF <0.5%) and 60% were novel. Gene burden tests performed on rare functional variants identified no significant associations (p<1×10−3) in the resequenced candidate genes. Our strategy of sequencing multiply affected families with deep follow up of candidate genes has not identified any new CeD risk mutations.