Michael Ransom

Genome-wide meta-analyses identify three loci associated with primary biliary cirrhosis

A genome-wide association screen for primary biliary cirrhosis risk alleles was performed in an Italian cohort. The results from the Italian cohort replicated IL12A and IL12RB associations, and a combined meta-analysis using a Canadian dataset identified newly associated loci at SPIB (P = 7.9 × 10−11, odds ratio (OR) = 1.46), IRF5-TNPO3 (P = 2.8 × 10−10, OR = 1.63) and 17q12-21 (P = 1.7 × 10−10, OR = 1.38).

Analysis and Application of European Genetic Substructure Using 300 K SNP Information

European population genetic substructure was examined in a diverse set of >1,000 individuals of European descent, each genotyped with >300 K SNPs. Both STRUCTURE and principal component analyses (PCA) showed the largest division/principal component (PC) differentiated northern from southern European ancestry. A second PC further separated Italian, Spanish, and Greek individuals from those of Ashkenazi Jewish ancestry as well as distinguishing among northern European populations. In separate analyses of northern European participants other substructure relationships were discerned showing a west to east gradient. Application of this substructure information was critical in examining a real dataset in whole genome association (WGA) analyses for rheumatoid arthritis in European Americans to reduce false positive signals. In addition, two sets of European substructure ancestry informative markers (ESAIMs) were identified that provide substantial substructure information. The results provide further insight into European population genetic substructure and show that this information can be used for improving error rates in association testing of candidate genes and in replication studies of WGA scans.

Analysis of East Asia Genetic Substructure Using Genome-Wide SNP Arrays

Accounting for population genetic substructure is important in reducing type 1 errors in genetic studies of complex disease. As efforts to understand complex genetic disease are expanded to different continental populations the understanding of genetic substructure within these continents will be useful in design and execution of association tests. In this study, population differentiation (Fst) and Principal Components Analyses (PCA) are examined using >200 K genotypes from multiple populations of East Asian ancestry. The population groups included those from the Human Genome Diversity Panel [Cambodian, Yi, Daur, Mongolian, Lahu, Dai, Hezhen, Miaozu, Naxi, Oroqen, She, Tu, Tujia, Naxi, Xibo, and Yakut], HapMap [ Han Chinese (CHB) and Japanese (JPT)], and East Asian or East Asian American subjects of Vietnamese, Korean, Filipino and Chinese ancestry. Paired Fst (Wei and Cockerham) showed close relationships between CHB and several large East Asian population groups (CHB/Korean, 0.0019; CHB/JPT, 00651; CHB/Vietnamese, 0.0065) with larger separation with Filipino (CHB/Filipino, 0.014). Low levels of differentiation were also observed between Dai and Vietnamese (0.0045) and between Vietnamese and Cambodian (0.0062). Similarly, small Fsts were observed among different presumed Han Chinese populations originating in different regions of mainland of China and Taiwan (Fsts <0.0025 with CHB). For PCA, the first two PCs showed a pattern of relationships that closely followed the geographic distribution of the different East Asian populations. PCA showed substructure both between different East Asian groups and within the Han Chinese population. These studies have also identified a subset of East Asian substructure ancestry informative markers (EASTASAIMS) that may be useful for future complex genetic disease association studies in reducing type 1 errors and in identifying homogeneous groups that may increase the power of such studies.

Immunochip analyses identify a novel risk locus for primary biliary cirrhosis at 13q14, multiple independent associations at four established risk loci and epistasis between 1p31 and 7q32 risk variants

To further characterize the genetic basis of primary biliary cirrhosis (PBC), we genotyped 2426 PBC patients and 5731 unaffected controls from three independent cohorts using a single nucleotide polymorphism (SNP) array (Immunochip) enriched for autoimmune disease risk loci. Meta-analysis of the genotype data sets identified a novel disease-associated locus near the TNFSF11 gene at 13q14, provided evidence for association at six additional immune-related loci not previously implicated in PBC and confirmed associations at 19 of 22 established risk loci. Results of conditional analyses also provided evidence for multiple independent association signals at four risk loci, with haplotype analyses suggesting independent SNP effects at the 2q32 and 16p13 loci, but complex haplotype driven effects at the 3q25 and 6p21 loci. By imputing classical HLA alleles from this data set, four class II alleles independently contributing to the association signal from this region were identified. Imputation of genotypes at the non-HLA loci also provided additional associations, but none with stronger effects than the genotyped variants. An epistatic interaction between the IL12RB2 risk locus at 1p31and the IRF5 risk locus at 7q32 was also identified and suggests a complementary effect of these loci in predisposing to disease. These data expand the repertoire of genes with potential roles in PBC pathogenesis that need to be explored by follow-up biological studies.

European Population Genetic Substructure: Further Definition of Ancestry Informative Markers for Distinguishing among Diverse European Ethnic Groups

The definition of European population genetic substructure and its application to understanding complex phenotypes is becoming increasingly important. In the current study using over 4,000 subjects genotyped for 300,000 single-nucleotide polymorphisms (SNPs), we provide further insight into relationships among European population groups and identify sets of SNP ancestry informative markers (AIMs) for application in genetic studies. In general, the graphical description of these principal components analyses (PCA) of diverse European subjects showed a strong correspondence to the geographical relationships of specific countries or regions of origin. Clearer separation of different ethnic and regional populations was observed when northern and southern European groups were considered separately and the PCA results were influenced by the inclusion or exclusion of different self-identified population groups including Ashkenazi Jewish, Sardinian, and Orcadian ethnic groups. SNP AIM sets were identified that could distinguish the regional and ethnic population groups. Moreover, the studies demonstrated that most allele frequency differences between different European groups could be controlled effectively in analyses using these AIM sets. The European substructure AIMs should be widely applicable to ongoing studies to confirm and delineate specific disease susceptibility candidate regions without the necessity of performing additional genome-wide SNP studies in additional subject sets.

TYK2 protein-coding variants protect against rheumatoid arthritis and autoimmunity, with no evidence of major pleiotropic effects on non-autoimmune complex traits

Despite the success of genome-wide association studies (GWAS) in detecting a large number of loci for complex phenotypes such as rheumatoid arthritis (RA) susceptibility, the lack of information on the causal genes leaves important challenges to interpret GWAS results in the context of the disease biology. Here, we genetically fine-map the RA risk locus at 19p13 to define causal variants, and explore the pleiotropic effects of these same variants in other complex traits. First, we combined Immunochip dense genotyping (n = 23,092 case/control samples), Exomechip genotyping (n = 18,409 case/control samples) and targeted exon-sequencing (n = 2,236 case/controls samples) to demonstrate that three protein-coding variants in TYK2 (tyrosine kinase 2) independently protect against RA: P1104A (rs34536443, OR = 0.66, P = 2.3x10-21), A928V (rs35018800, OR = 0.53, P = 1.2x10-9), and I684S (rs12720356, OR = 0.86, P = 4.6x10-7). Second, we show that the same three TYK2 variants protect against systemic lupus erythematosus (SLE, Pomnibus = 6x10-18), and provide suggestive evidence that two of the TYK2 variants (P1104A and A928V) may also protect against inflammatory bowel disease (IBD; Pomnibus = 0.005). Finally, in a phenome-wide association study (PheWAS) assessing >500 phenotypes using electronic medical records (EMR) in >29,000 subjects, we found no convincing evidence for association of P1104A and A928V with complex phenotypes other than autoimmune diseases such as RA, SLE and IBD. Together, our results demonstrate the role of TYK2 in the pathogenesis of RA, SLE and IBD, and provide supporting evidence for TYK2 as a promising drug target for the treatment of autoimmune diseases.

Classical HLA-DRB1 and DPB1 alleles account for HLA associations with primary biliary cirrhosis

Susceptibility to primary biliary cirrhosis (PBC) is strongly associated with human leukocyte antigen (HLA)-region polymorphisms. To determine if associations can be explained by classical HLA determinants, we studied Italian, 676 cases and 1440 controls, genotyped with dense single-nucleotide polymorphisms (SNPs) for which classical HLA alleles and amino acids were imputed. Although previous genome-wide association studies and our results show stronger SNP associations near DQB1, we demonstrate that the HLA signals can be attributed to classical DRB1 and DPB1 genes. Strong support for the predominant role of DRB1 is provided by our conditional analyses. We also demonstrate an independent association of DPB1. Specific HLA-DRB1 genes (*08, *11 and *14) account for most of the DRB1 association signal. Consistent with previous studies, DRB1*08 (P=1.59 × 10−11) was the strongest predisposing allele, whereas DRB1*11 (P=1.42 × 10−10) was protective. Additionally, DRB1*14 and the DPB1 association (DPB1*03:01; P=9.18 × 10−7) were predisposing risk alleles. No signal was observed in the HLA class 1 or class 3 regions. These findings better define the association of PBC with HLA and specifically support the role of classical HLA-DRB1 and DPB1 genes and alleles in susceptibility to PBC.

Amerindian ancestry in Argentina is associated with increased risk for systemic lupus erythematosus.

Previous studies have demonstrated that in admixed populations, West African ancestry is associated with an increased prevalence of systemic lupus erythematosus (SLE). In the current study, the effect of Amerindian ancestry in SLE was examined in an admixed population in Argentina. The Argentine population is predominantly European with approximately 20% Amerindian admixture, and a very small (<2%) contribution from West Africa. The results indicate that Amerindian admixture in this population is associated with a substantial increase in SLE susceptibility risk (Odds Ratio=7.94, P=0.00006). This difference was not due to known demographic factors, including site of collection, age and gender. In addition, there were trends towards significance for Amerindian ancestry influencing renal disease, age of onset and anti-SSA antibodies. These studies suggest that populations with Amerindian admixture, like those with West African admixture, should be considered in future studies to identify additional allelic variants that predispose to SLE.

Evidence for malaria selection of a CR1 haplotype in Sardinia

Complement receptor 1 (CR1) levels have been associated with malarial susceptibility and/or severity of the disease in different population groups, and CR1 is a receptor for Plasmodium falciparum. In this study, multiple CR1 single-nucleotide polymorphisms (SNPs) showed strong evidence of population differentiation between Sardinian and other European ethnic groups. Cross population algorithms comparing haplotype structure and differences in haplotype and allele frequency distribution provided additional support for natural selection of CR1 in Sardinia. The predominant Sardinian CR1 haplotype included SNPs that are associated with decreased CR1 levels in Europeans and other population groups. Previous studies have shown that the SNPs within the dominant Sardinian haplotype have a significantly higher frequency in a malaria endemic compared with non-endemic regions in India. Together with the historical evidence of the prevalence of malaria in Sardinia, these data support the role of malaria leading to positive selection of this CR1 haplotype in Sardinia.

Independent Origin of c.57 C > T Mutation in MIR184 Associated with Inherited Corneal and Lens Abnormalities

MicroRNAs (miRNAs) are small non-coding RNAs that suppress post-transcriptional gene expression by base pairing with their target messenger RNAs (mRNAs) and inducing either translational repression or mRNA degradation of their targets 1. In contrast to the protein coding genes, germline sequence variants in mature miRNAs are extremely rare possibly due to the extreme conservation and importance of mature miRNAs as well really small size of these molecules (18–25 base pairs). Known variants were limited to the polymorphism in MIR125a 2 and the mutation in MIR96 causing autosomal dominant progressive hearing loss 3,4, until a recurring heterozygous mutation c.57 C>T in the seed region of MIR184 [MIM 613146] was repeatedly identified in the Northern Irish family with keratoconus and cataracts5, a family with EDICT (endothelial dystrophy, iris hypoplasia, congenital cataract, and stromal thinning) syndrome6, and most recently by our group in a five generation family with cataracts and varying corneal abnormalities including severe keratoconus and non-ectatic corneal thinning from Galicia, Spain7. The maternal line of the Galician family (Figure 1) has originated in the city of La Estrada in an autonomous community of Galicia in Northwest Spain. The paternal side of the family includes Eastern European (Russian/Ukranian Jewish, proband’s grandfather) and German (proband’s grandmother) roots. We performed targeted testing of the two rare polymorphisms located in close proximity to the c.57 C>T mutation previously described in Northern Irish family5 (Table 1, Supplementary Methods) in the founding member of the Galician family (Figure 1, individual 101). Neither of the two rare alleles identified in Northern Irish family was present in the tested individual. Then we performed genetic ancestry testing of this family by genotyping proband, his mother and maternal grandmother (all carrying c.57 C>T mutation) using HumanOmni 2.5 BeadChip and comparing SNP data with reference population genotypes using principal component analysis (PCA, Supplementary Methods, Supplementary Figure 1). PCA with subjects from different continents identified all members of the family as having European ancestry. Further investigation revealed proband’s mother and maternal grandmother having Iberian ancestry originating either in Spain or in Portugal; whereas the proband’s ancestry was found to be admixed between Iberian and Ashkenazi Jewish populations thus confirming their ancestry as identified by family history. Based on the absence of adjacent rare polymorphisms described in the Northern Irish family and by in-depth ancestral testing which conclusively showed lack of Irish genetic roots in this family we conclude that deleterious c.57 C>T MIR184 mutation arose independently in the Galician and Northern Irish families and thus represents a first observation of the recurrent germline mutation in the microRNA gene leading to genetic disease described up to date. We further hypothesized that genomic variants in the gene targets of MIR184 may act as nuclear modifier(s) leading to intra- and interfamily variability of age of onset and clinical symptoms. To identify SNPs which could potentially modify phenotype severity in the members of the Galician family we used MiRNASNP2.0 database to identify SNPs with the potential to influence structure and function of MIR184 8. We found that out of the total of five hundred and thirteen SNPs annotated in the database, eight SNPs, one located in MIR184 genomic region and seven in 3′ UTR regions of nuclear genes, were polymorphic in one or more of the three tested family members (Supplementary Table 1). The Proband (individual 301) affected with a more severe clinical presentation of the ocular symptoms was found to be a homozygote for the minor (potentially detrimental) allele for SNPs located in genes: IYD, C1orf158, and WSCD2. In addition, we performed analysis of predicted gene targets of MIR184 using MiRecords which combines results of eleven established miRNA target prediction programs9 and compared the results of such predictions between eight genes identified as candidate genes for keratoconus in recent studies 10–16 and seven genes known to be validated targets of MIR1845, 17–20 (Supplementary Table 2). We found that on average candidate genes were predicted to be MIR184 targets by 2 programs, whereas validated genes were predicted by an average of 2.4 programs (Mann-Whitney p-value = 0.4) implying that genes important for corneal disease such as keratoconus may be regulated by MIR184 and may contain phenotype modifying polymorphisms. Further analysis of genes expressed in the ocular tissues and potentially regulated by MIR184 will bring new understanding of microRNA regulation in health and disease and could suggest novel treatment options making therapeutic use of miRNA in the genetic eye diseases a real prospect. Figure 1 Pedigree structure of the Galician family. Genotyped individuals are identified by numbers. Table 1 Results of testing of rare variants identified in Northern Irish family in the Galician family.