Analysis of the ImmunoChip single nucleotide polymorphism (SNP) array in 2816 individuals, comprising the most common subtypes (oligoarticular and RF negative polyarticular) of juvenile idiopathic arthritis (JIA) and 13056 controls strengthens the evidence for association to three known JIA-risk loci (HLA, PTPN22 and PTPN2) and has identified fourteen risk loci reaching genome-wide significance (p < 5 × 10-8) for the first time. Eleven additional novel regions showed suggestive evidence for association with JIA (p < 1 × 10-6). Dense-mapping of loci along with bioinformatic analysis has refined the association to one gene for eight regions, highlighting crucial pathways, including the IL-2 pathway, in JIA disease pathogenesis. The entire ImmunoChip loci, HLA region and the top 27 loci (p < 1 × 10-6) explain an estimated 18%, 13% and 6% risk of JIA, respectively. Analysis of the ImmunoChip dataset, the largest cohort of JIA cases investigated to date, provides new insight in understanding the genetic basis for this childhood autoimmune disease.
Antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener’s granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis.
A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria.
We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti–proteinase 3 ANCA was associated with HLA-DP and the genes encoding α1-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P = 6.2×10−89, P = 5.6×10−12, and P = 2.6×10−7, respectively). Anti–myeloperoxidase ANCA was associated with HLA-DQ (P = 2.1×10−8).
This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA–associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA–associated vasculitis and myeloperoxidase ANCA–associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.)
VKORC1 and CYP2C9 are important contributors to warfarin dose variability, but explain less variability for individuals of African descent than for those of European or Asian descent. We aimed to identify additional variants contributing to warfarin dose requirements in African Americans.
We did a genome-wide association study of discovery and replication cohorts. Samples from African-American adults (aged ≥18 years) who were taking a stable maintenance dose of warfarin were obtained at International Warfarin Pharmacogenetics Consortium (IWPC) sites and the University of Alabama at Birmingham (Birmingham, AL, USA). Patients enrolled at IWPC sites but who were not used for discovery made up the independent replication cohort. All participants were genotyped. We did a stepwise conditional analysis, conditioning first for VKORC1 −1639G→A, followed by the composite genotype of CYP2C9*2 and CYP2C9*3. We prespecified a genome-wide significance threshold of p<5×10−8 in the discovery cohort and p<0·0038 in the replication cohort.
The discovery cohort contained 533 participants and the replication cohort 432 participants. After the prespecified conditioning in the discovery cohort, we identified an association between a novel single nucleotide polymorphism in the CYP2C cluster on chromosome 10 (rs12777823) and warfarin dose requirement that reached genome-wide significance (p=1·51×10−8). This association was confirmed in the replication cohort (p=5·04×10−5); analysis of the two cohorts together produced a p value of 4·5×10−12. Individuals heterozygous for the rs12777823 A allele need a dose reduction of 6·92 mg/week and those homozygous 9·34 mg/week. Regression analysis showed that the inclusion of rs12777823 significantly improves warfarin dose variability explained by the IWPC dosing algorithm (21% relative improvement).
A novel CYP2C single nucleotide polymorphism exerts a clinically relevant effect on warfarin dose in African Americans, independent of CYP2C9*2 and CYP2C9*3. Incorporation of this variant into pharmacogenetic dosing algorithms could improve warfarin dose prediction in this population.
National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.
Ankylosing spondylitis is a common, highly heritable inflammatory arthritis affecting primarily the spine and pelvis. In addition to HLA-B*27 alleles, 12 loci have previously been identified that are associated with ankylosing spondylitis in populations of European ancestry, and 2 associated loci have been identified in Asians. In this study, we used the Illumina Immunochip microarray to perform a case-control association study involving 10,619 individuals with ankylosing spondylitis (cases) and 15,145 controls. We identified 13 new risk loci and 12 additional ankylosing spondylitis–associated haplotypes at 11 loci. Two ankylosing spondylitis–associated regions have now been identified encoding four aminopeptidases that are involved in peptide processing before major histocompatibility complex (MHC) class I presentation. Protective variants at two of these loci are associated both with reduced aminopeptidase function and with MHC class I cell surface expression.
To identify susceptibility loci for visceral leishmaniasis we undertook genome-wide association studies in two populations; 989 cases and 1089 controls from India, and 357 cases in 308 Brazilian families (1970 individuals). The HLA-DRB1-HLA-DQA1 locus was the only region to show strong evidence of association in both populations. Replication at this region was undertaken in a second Indian population comprising 941 cases and 990 controls, resulting in Pcombined=2.76×10−17 and OR(95%CI)=1.41(1.30-1.52) across the three cohorts at rs9271858. A conditional analysis provided evidence for multiple associations within the HLA-DRB1-HLA-DQA1 region, and a model in which risk differed between three groups of haplotypes better explained the signal and was significant in the Indian discovery and replication cohorts. In conclusion the HLA-DRB1-HLA-DQA1 HLA class II region contributes to visceral leishmaniasis susceptibility in India and Brazil, suggesting shared genetic risk factors for visceral leishmaniasis that cross the epidemiological divides of geography and parasite species.
Background Human ageing is a complex, multifactorial process and early developmental factors affect health outcomes in old age.
Methods Metabolomic profiling on fasting blood was carried out in 6055 individuals from the UK. Stepwise regression was performed to identify a panel of independent metabolites which could be used as a surrogate for age. We also investigated the association with birthweight overall and within identical discordant twins and with genome-wide methylation levels.
Results We identified a panel of 22 metabolites which combined are strongly correlated with age (R2 = 59%) and with age-related clinical traits independently of age. One particular metabolite, C-glycosyl tryptophan (C-glyTrp), correlated strongly with age (beta = 0.03, SE = 0.001, P = 7.0 × 10−157) and lung function (FEV1 beta = −0.04, SE = 0.008, P = 1.8 × 10−8 adjusted for age and confounders) and was replicated in an independent population (n = 887). C-glyTrp was also associated with bone mineral density (beta = −0.01, SE = 0.002, P = 1.9 × 10−6) and birthweight (beta = −0.06, SE = 0.01, P = 2.5 × 10−9). The difference in C-glyTrp levels explained 9.4% of the variance in the difference in birthweight between monozygotic twins. An epigenome-wide association study in 172 individuals identified three CpG-sites, associated with levels of C-glyTrp (P < 2 × 10−6). We replicated one CpG site in the promoter of the WDR85 gene in an independent sample of 350 individuals (beta = −0.20, SE = 0.04, P = 2.9 × 10−8). WDR85 is a regulator of translation elongation factor 2, essential for protein synthesis in eukaryotes.
Conclusions Our data illustrate how metabolomic profiling linked with epigenetic studies can identify some key molecular mechanisms potentially determined in early development that produce long-term physiological changes influencing human health and ageing.
Ageing; metabolomics; epigenetics; twin studies; developmental origins of health and disease; birthweight
To gain further insight into the genetic architecture of psoriasis, we conducted a meta-analysis of three genome-wide association studies (GWAS) and two independent datasets genotyped on the Immunochip, involving 10,588 cases and 22,806 controls in total. We identified 15 new disease susceptibility regions, increasing the number of psoriasis-associated loci to 36 for Caucasians. Conditional analyses identified five independent signals within previously known loci. The newly identified shared disease regions encompassed a number of genes whose products regulate T-cell function (e.g. RUNX3, TAGAP and STAT3). The new psoriasis-specific regions were notable for candidate genes whose products are involved in innate host defense, encoding proteins with roles in interferon-mediated antiviral responses (DDX58), macrophage activation (ZC3H12C), and NF-κB signaling (CARD14 and CARM1). These results portend a better understanding of shared and distinctive genetic determinants of immune-mediated inflammatory disorders and emphasize the importance of the skin in innate and acquired host defense.
Using the Immunochip custom single nucleotide polymorphism (SNP) array, designed for dense genotyping of 186 genome wide association study (GWAS) confirmed loci we analysed 11,475 rheumatoid arthritis cases of European ancestry and 15,870 controls for 129,464 markers. The data were combined in meta-analysis with GWAS data from additional independent cases (n=2,363) and controls (n=17,872). We identified fourteen novel loci; nine were associated with rheumatoid arthritis overall and 5 specifically in anti-citrillunated peptide antibody positive disease, bringing the number of confirmed European ancestry rheumatoid arthritis loci to 46. We refined the peak of association to a single gene for 19 loci, identified secondary independent effects at six loci and association to low frequency variants (minor allele frequency <0.05) at 4 loci. Bioinformatic analysis of the data generated strong hypotheses for the causal SNP at seven loci. This study illustrates the advantages of dense SNP mapping analysis to inform subsequent functional investigations.
Economic variables such as income, education, and occupation are known to affect mortality and morbidity, such as cardiovascular disease, and have also been shown to be partly heritable. However, very little is known about which genes influence economic variables, although these genes may have both a direct and an indirect effect on health. We report results from the first large-scale collaboration that studies the molecular genetic architecture of an economic variable–entrepreneurship–that was operationalized using self-employment, a widely-available proxy. Our results suggest that common SNPs when considered jointly explain about half of the narrow-sense heritability of self-employment estimated in twin data (σg2/σP2 = 25%, h2 = 55%). However, a meta-analysis of genome-wide association studies across sixteen studies comprising 50,627 participants did not identify genome-wide significant SNPs. 58 SNPs with p<10−5 were tested in a replication sample (n = 3,271), but none replicated. Furthermore, a gene-based test shows that none of the genes that were previously suggested in the literature to influence entrepreneurship reveal significant associations. Finally, SNP-based genetic scores that use results from the meta-analysis capture less than 0.2% of the variance in self-employment in an independent sample (p≥0.039). Our results are consistent with a highly polygenic molecular genetic architecture of self-employment, with many genetic variants of small effect. Although self-employment is a multi-faceted, heavily environmentally influenced, and biologically distal trait, our results are similar to those for other genetically complex and biologically more proximate outcomes, such as height, intelligence, personality, and several diseases.
Recent genome-wide association studies (GWAS) have identified novel loci associated with sudden cardiac death (SCD). Despite this progress, identified DNA variants account for a relatively small portion of overall SCD risk, suggesting that additional loci contributing to SCD susceptibility await discovery. The objective of this study was to identify novel DNA variation associated with SCD in the context of coronary artery disease (CAD).
Methods and Findings
Using the MetaboChip custom array we conducted a case-control association analysis of 119,117 SNPs in 948 SCD cases (with underlying CAD) from the Oregon Sudden Unexpected Death Study (Oregon-SUDS) and 3,050 controls with CAD from the Wellcome Trust Case-Control Consortium (WTCCC). Two newly identified loci were significantly associated with increased risk of SCD after correction for multiple comparisons at: rs6730157 in the RAB3GAP1 gene on chromosome 2 (P = 4.93×10−12, OR = 1.60) and rs2077316 in the ZNF365 gene on chromosome 10 (P = 3.64×10−8, OR = 2.41).
Our findings suggest that RAB3GAP1 and ZNF365 are relevant candidate genes for SCD and will contribute to the mechanistic understanding of SCD susceptibility.
Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in the elderly. It is characterized by changes in joint structure including degeneration of the articular cartilage and its etiology is multifactorial with a strong postulated genetic component. We performed a meta-analysis of four genome-wide association (GWA) studies of 2,371 knee OA cases and 35,909 controls in Caucasian populations. Replication of the top hits was attempted with data from additional ten replication datasets. With a cumulative sample size of 6,709 cases and 44,439 controls, we identified one genome-wide significant locus on chromosome 7q22 for knee OA (rs4730250, p-value=9.2×10−9), thereby confirming its role as a susceptibility locus for OA. The associated signal is located within a large (500kb) linkage disequilibrium (LD) block that contains six genes; PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, beta), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like), and BCAP29 (the B-cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.
Genetic variants that associate with DNA methylation at CpG sites (methylation quantitative trait loci, meQTLs) offer a potential biological mechanism of action for disease associated SNPs. We investigated whether meQTLs exist in abdominal subcutaneous adipose tissue (SAT) and if CpG methylation associates with metabolic syndrome (MetSyn) phenotypes. We profiled 27,718 genomic regions in abdominal SAT samples of 38 unrelated individuals using differential methylation hybridization (DMH) together with genotypes at 5,227,243 SNPs and expression of 17,209 mRNA transcripts. Validation and replication of significant meQTLs was pursued in an independent cohort of 181 female twins. We find that, at 5% false discovery rate, methylation levels of 149 DMH regions associate with at least one SNP in a ±500 kilobase cis-region in our primary study. We sought to validate 19 of these in the replication study and find that five of these significantly associate with the corresponding meQTL SNPs from the primary study. We find that none of the 149 meQTL top SNPs is a significant expression quantitative trait locus in our expression data, but we observed association between expression levels of two mRNA transcripts and cis-methylation status. Our results indicate that DNA CpG methylation in abdominal SAT is partly under genetic control. This study provides a starting point for future investigations of DNA methylation in adipose tissue.
Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin) deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip). Three loci located outside the Human Leukocyte Antigen (HLA) region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.
While there is now broad consensus that narcolepsy-hypocretin deficiency results from a highly specific autoimmune attack on hypocretin cells, little is understood regarding the initiation and progression of the underlying autoimmune process. We have taken advantage of a unique high-density genotyping platform (the ImmunoChip) designed to study variants in genes known to be important to autoimmune and inflammatory diseases. Our study of nearly 2000 narcolepsy cases compared to 10,000 controls underscored important roles for HLA DQB1*06:02 and the T cell receptor alpha genes and implicated two additional genes, Cathepsin H and TNFSF4/OX40L, in disease pathogenesis. These findings are particularly important, as these encoded proteins have key roles in antigen processing, presentation, and T cell response, and they suggest that specific interactions at the immunological synapse constitute the pathway to the disease. Further studies of these genes and encoded proteins may therefore reveal the mechanism leading to this highly selective and unique autoimmune disease.
Little is known about genetic contributors to higher than usual warfarin dose requirements, particularly for African Americans. This study tested the hypothesis that the γ-glutamyl carboxylase (GGCX) genotype contributes to warfarin dose requirements >7.5 mg/day in an African American population.
A total of 338 African Americans on a stable dose of warfarin were enrolled. The GGCX rs10654848 (CAA)n, rs12714145 (G>A), and rs699664 (p.R325Q); VKORC1 c.-1639G>A and rs61162043; and CYP2C9*2, *3, *5, *8, *11, and rs7089580 genotypes tested for their association with dose requirements >7.5 mg/day alone and in the context of other variables known to influence dose variability.
The GGCX rs10654848 (CAA) 16 or 17 repeat occurred at a frequency of 2.6% in African Americans and was overrepresented among patients requiring >7.5mg/day versus those who required lower doses (12% vs 3%, p=0.003; odds ratio 4.0, 95% CI, 1.5–10.5). The GGCX rs10654848 genotype remained associated with high dose requirements on regression analysis including age, body size, and VKORC1 genotype. On linear regression, the GGCX rs10654848 genotype explained 2% of the overall variability in warfarin dose in African Americans. An examination of the GGCX rs10654848 genotype in warfarin-treated Caucasians revealed a (CAA)16 repeat allele frequency of only 0.27% (p=0.008 compared to African Americans).
These data support the GGCX rs10654848 genotype as a predictor of higher than usual warfarin doses in African Americans, who have a 10-fold higher frequency of the (CAA)16/17 repeat compared to Caucasians.
African American; GGCX; warfarin
In order to assess whether gene expression variability could be influenced by several SNPs acting in cis, either through additive or more complex haplotype effects, a systematic genome-wide search for cis haplotype expression quantitative trait loci (eQTL) was conducted in a sample of 758 individuals, part of the Cardiogenics Transcriptomic Study, for which genome-wide monocyte expression and GWAS data were available. 19,805 RNA probes were assessed for cis haplotypic regulation through investigation of ∼2,1×109 haplotypic combinations. 2,650 probes demonstrated haplotypic p-values >104-fold smaller than the best single SNP p-value. Replication of significant haplotype effects were tested for 412 probes for which SNPs (or proxies) that defined the detected haplotypes were available in the Gutenberg Health Study composed of 1,374 individuals. At the Bonferroni correction level of 1.2×10−4 (∼0.05/412), 193 haplotypic signals replicated. 1000G imputation was then conducted, and 105 haplotypic signals still remained more informative than imputed SNPs. In-depth analysis of these 105 cis eQTL revealed that at 76 loci genetic associations were compatible with additive effects of several SNPs, while for the 29 remaining regions data could be compatible with a more complex haplotypic pattern. As 24 of the 105 cis eQTL have previously been reported to be disease-associated loci, this work highlights the need for conducting haplotype-based and 1000G imputed cis eQTL analysis before commencing functional studies at disease-associated loci.
In order to assess whether gene expression variability could be influenced by the presence of more than one cis-acting SNP, we have conducted a systematic genome-wide search for haplotypic cis eQTL effects in a sample of 758 individuals and replicated the findings in an independent sample of 1,374 subjects. In both studies, genome-wide monocytes expression and genotype data were available. We identified 105 genes whose monocyte expression was under the influence of multiple cis-acting SNPs. About 75% of the detected genetic effects were related to independent additive SNP effects and the last quarter due to more complex haplotype effects. Of note, 24 of the genes identified to be affected by multiple cis eSNPs have been previously reported to reside at disease-associated loci. This could suggest that such multiple locus-specific genetic effects could contribute to the susceptibility to human diseases.
A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers.
To identify previously unknown genetic loci associated with fasting glucose concentrations, we examined the leading association signals in ten genome-wide association scans involving a total of 36,610 individuals of European descent. Variants in the gene encoding melatonin receptor 1B (MTNR1B) were consistently associated with fasting glucose across all ten studies. The strongest signal was observed at rs10830963, where each G allele (frequency 0.30 in HapMap CEU) was associated with an increase of 0.07 (95% CI = 0.06-0.08) mmol/l in fasting glucose levels (P = 3.2 = × 10−50) and reduced beta-cell function as measured by homeostasis model assessment (HOMA-B, P = 1.1 × 10−15). The same allele was associated with an increased risk of type 2 diabetes (odds ratio = 1.09 (1.05-1.12), per G allele P = 3.3 × 10−7) in a meta-analysis of 13 case-control studies totaling 18,236 cases and 64,453 controls. Our analyses also confirm previous associations of fasting glucose with variants at the G6PC2 (rs560887, P = 1.1 × 10−57) and GCK (rs4607517, P = 1.0 × 10−25) loci.
DNA methylation is one of the most studied epigenetic marks in the human genome, with the result that the desire to map the human methylome has driven the development of several methods to map DNA methylation on a genomic scale. Our study presents the first comparison of two of these techniques - the targeted approach of the Infinium HumanMethylation450 BeadChip® with the immunoprecipitation and sequencing-based method, MeDIP-seq. Both methods were initially validated with respect to bisulfite sequencing as the gold standard and then assessed in terms of coverage, resolution and accuracy. The regions of the methylome that can be assayed by both methods and those that can only be assayed by one method were determined and the discovery of differentially methylated regions (DMRs) by both techniques was examined. Our results show that the Infinium HumanMethylation450 BeadChip® and MeDIP-seq show a good positive correlation (Spearman correlation of 0.68) on a genome-wide scale and can both be used successfully to determine differentially methylated loci in RefSeq genes, CpG islands, shores and shelves. MeDIP-seq however, allows a wider interrogation of methylated regions of the human genome, including thousands of non-RefSeq genes and repetitive elements, all of which may be of importance in disease. In our study MeDIP-seq allowed the detection of 15,709 differentially methylated regions, nearly twice as many as the array-based method (8070), which may result in a more comprehensive study of the methylome.
Regulation of key proteins by microRNAs (miRNAs) is an emergent field in biomedicine. Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is a relevant molecule for cardiovascular diseases, since it is the target of oral anticoagulant drugs and plays a role in soft tissue calcification. The objective of this study was to determine the influence of miRNAs on the expression of VKORC1. Potential miRNAs targeting VKORC1 mRNA were searched by using online algorithms. Validation studies were carried out in HepG2 cells by using miRNA precursors; direct miRNA interaction was investigated with reporter assays. In silico studies identified two putative conserved binding sites for miR-133a and miR-137 on VKORC1 mRNA. Ex vivo studies showed that only miR-133a was expressed in liver; transfection of miRNA precursors of miR-133a in HepG2 cells reduced VKORC1 mRNA expression in a dose-dependent manner, as assessed by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) as well as protein expression. Reporter assays in HEK293T cells showed that miR-133a interacts with the 3′UTR of VKORC1. Additionally, miR-133a levels correlated inversely with VKORC1 mRNA levels in 23 liver samples from healthy subjects. In conclusion, miR-133a appears to have a direct regulatory effect on expression of VKORC1 in humans; this regulation may have potential importance for anticoagulant therapy or aortic calcification.
The exon-junction complex (EJC) performs essential RNA processing tasks1-5. Here, we describe the first human disorder, Thrombocytopenia with Absent Radii6 (TAR), caused by deficiency in one of the four EJC subunits. A compound inheritance mechanism of a rare null allele and one of two low-frequency SNPs in the regulatory regions of RBM8A, encoding the Y14 subunit of EJC, causes TAR. We found that this mechanism explained 53 of 55 cases (P<5×10−228) with the rare congenital malformation syndrome. Fifty-one of those 53 carried a previously associated7 submicroscopic deletion of 1q21.1; two carried a truncation or frameshift null mutation in RBM8A. We show that the two regulatory SNPs result in reduction of RBM8A transcription in vitro and that Y14 expression is reduced in platelets from TAR cases. Our data implicate Y14 insufficiency, and presumably EJC defect, as the cause of TAR syndrome.
Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1–12, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64–133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene, ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.
We aimed to assess whether pri-miRNA SNPs (miSNPs) could influence monocyte gene expression, either through marginal association or by interacting with polymorphisms located in 3'UTR regions (3utrSNPs). We then conducted a genome-wide search for marginal miSNPs effects and pairwise miSNPs × 3utrSNPs interactions in a sample of 1,467 individuals for which genome-wide monocyte expression and genotype data were available. Statistical associations that survived multiple testing correction were tested for replication in an independent sample of 758 individuals with both monocyte gene expression and genotype data. In both studies, the hsa-mir-1279 rs1463335 was found to modulate in cis the expression of LYZ and in trans the expression of CNTN6, CTRC, COPZ2, KRT9, LRRFIP1, NOD1, PCDHA6, ST5 and TRAF3IP2 genes, supporting the role of hsa-mir-1279 as a regulator of several genes in monocytes. In addition, we identified two robust miSNPs × 3utrSNPs interactions, one involving HLA-DPB1 rs1042448 and hsa-mir-219-1 rs107822, the second the H1F0 rs1894644 and hsa-mir-659 rs5750504, modulating the expression of the associated genes.
As some of the aforementioned genes have previously been reported to reside at disease-associated loci, our findings provide novel arguments supporting the hypothesis that the genetic variability of miRNAs could also contribute to the susceptibility to human diseases.
Rheumatoid arthritis is an autoimmune disease with a complex etiology, leading to inflammation of synovial tissue and joint destruction. Through a genome-wide association study (GWAS) and two replication studies in the Japanese population (7,907 cases and 35,362 controls), we identified two gene loci associated with rheumatoid arthritis susceptibility (NFKBIE at 6p21.1, rs2233434, odds ratio (OR) = 1.20, P = 1.3×10−15; RTKN2 at 10q21.2, rs3125734, OR = 1.20, P = 4.6×10−9). In addition to two functional non-synonymous SNPs in NFKBIE, we identified candidate causal SNPs with regulatory potential in NFKBIE and RTKN2 gene regions by integrating in silico analysis using public genome databases and subsequent in vitro analysis. Both of these genes are known to regulate the NF-κB pathway, and the risk alleles of the genes were implicated in the enhancement of NF-κB activity in our analyses. These results suggest that the NF-κB pathway plays a role in pathogenesis and would be a rational target for treatment of rheumatoid arthritis.
Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting approximately 1% of the general adult population. More than 30 susceptibility loci for RA have been identified through genome-wide association studies (GWAS), but the disease-causal variants at most loci remain unknown. Here, we performed replication studies of the candidate loci of our previous GWAS using Japanese cohorts and identified variants in NFKBIE and RTKN2 gene loci that were associated with RA. To search for causal variants in both gene regions, we first examined non-synonymous (ns)SNPs that alter amino-acid sequences. As NFKBIE and RTKN2 are known to be involved in the NF-κB pathway, we evaluated the effects of nsSNPs on NF-κB activity. Next, we screened in silico variants that may regulate gene transcription using publicly available epigenetic databases and subsequently evaluated their regulatory potential using in vitro assays. As a result, we identified multiple candidate causal variants in NFKBIE (2 nsSNPs and 1 regulatory SNP) and RTKN2 (2 regulatory SNPs), indicating that our integrated in silico and in vitro approach is useful for the identification of causal variants in the post–GWAS era.
Genome-wide association studies have identified hundreds of loci for type 2 diabetes, coronary artery disease and myocardial infarction, as well as for related traits such as body mass index, glucose and insulin levels, lipid levels, and blood pressure. These studies also have pointed to thousands of loci with promising but not yet compelling association evidence. To establish association at additional loci and to characterize the genome-wide significant loci by fine-mapping, we designed the “Metabochip,” a custom genotyping array that assays nearly 200,000 SNP markers. Here, we describe the Metabochip and its component SNP sets, evaluate its performance in capturing variation across the allele-frequency spectrum, describe solutions to methodological challenges commonly encountered in its analysis, and evaluate its performance as a platform for genotype imputation. The metabochip achieves dramatic cost efficiencies compared to designing single-trait follow-up reagents, and provides the opportunity to compare results across a range of related traits. The metabochip and similar custom genotyping arrays offer a powerful and cost-effective approach to follow-up large-scale genotyping and sequencing studies and advance our understanding of the genetic basis of complex human diseases and traits.
Recent genetic studies have identified hundreds of regions of the human genome that contribute to risk for type 2 diabetes, coronary artery disease and myocardial infarction, and to related quantitative traits such as body mass index, glucose and insulin levels, blood lipid levels, and blood pressure. These results motivate two central questions: (1) can further genetic investigation identify additional associated regions?; and (2) can more detailed genetic investigation help us identify the causal variants (or variants more strongly correlated with the causal variants) in the regions identified so far? Addressing these questions requires assaying many genetic variants in DNA samples from thousands of individuals, which is expensive and timeconsuming when done a few SNPs at a time. To facilitate these investigations, we designed the “Metabochip,” a custom genotyping array that assays variation in nearly 200,000 sites in the human genome. Here we describe the Metabochip, evaluate its performance in assaying human genetic variation, and describe solutions to methodological challenges commonly encountered in its analysis.