Eric R. Gamazon

Trait-Associated SNPs Are More Likely to Be eQTLs: Annotation to Enhance Discovery from GWAS

Although genome-wide association studies (GWAS) of complex traits have yielded more reproducible associations than had been discovered using any other approach, the loci characterized to date do not account for much of the heritability to such traits and, in general, have not led to improved understanding of the biology underlying complex phenotypes. Using a web site we developed to serve results of expression quantitative trait locus (eQTL) studies in lymphoblastoid cell lines from HapMap samples (http://www.scandb.org), we show that single nucleotide polymorphisms (SNPs) associated with complex traits (from http://www.genome.gov/gwastudies/) are significantly more likely to be eQTLs than minor-allele-frequency–matched SNPs chosen from high-throughput GWAS platforms. These findings are robust across a range of thresholds for establishing eQTLs (p-values from 10−4–10−8), and a broad spectrum of human complex traits. Analyses of GWAS data from the Wellcome Trust studies confirm that annotating SNPs with a score reflecting the strength of the evidence that the SNP is an eQTL can improve the ability to discover true associations and clarify the nature of the mechanism driving the associations. Our results showing that trait-associated SNPs are more likely to be eQTLs and that application of this information can enhance discovery of trait-associated SNPs for complex phenotypes raise the possibility that we can utilize this information both to increase the heritability explained by identifiable genetic factors and to gain a better understanding of the biology underlying complex traits.

Obesity-associated variants within FTO form long-range functional connections with IRX3

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition.

SCAN: SNP and copy number annotation

MOTIVATION Genome-wide association studies (GWAS) generate relationships between hundreds of thousands of single nucleotide polymorphisms (SNPs) and complex phenotypes. The contribution of the traditionally overlooked copy number variations (CNVs) to complex traits is also being actively studied. To facilitate the interpretation of the data and the designing of follow-up experimental validations, we have developed a database that enables the sensible prioritization of these variants by combining several approaches, involving not only publicly available physical and functional annotations but also multilocus linkage disequilibrium (LD) annotations as well as annotations of expression quantitative trait loci (eQTLs). RESULTS For each SNP, the SCAN database provides: (i) summary information from eQTL mapping of HapMap SNPs to gene expression (evaluated by the Affymetrix exon array) in the full set of HapMap CEU (Caucasians from UT, USA) and YRI (Yoruba people from Ibadan, Nigeria) samples; (ii) LD information, in the case of a HapMap SNP, including what genes have variation in strong LD (pairwise or multilocus LD) with the variant and how well the SNP is covered by different high-throughput platforms; (iii) summary information available from public databases (e.g. physical and functional annotations); and (iv) summary information from other GWAS. For each gene, SCAN provides annotations on: (i) eQTLs for the gene (both local and distant SNPs) and (ii) the coverage of all variants in the HapMap at that gene on each high-throughput platform. For each genomic region, SCAN provides annotations on: (i) physical and functional annotations of all SNPs, genes and known CNVs within the region and (ii) all genes regulated by the eQTLs within the region. AVAILABILITY http://www.scandb.org. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study

Summary Background 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. Methods 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. Findings 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). Interpretation 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. Funding National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.

Enrichment of cis-regulatory gene expression SNPs and methylation quantitative trait loci among bipolar disorder susceptibility variants

We conducted a systematic study of top susceptibility variants from a genome-wide association (GWA) study of bipolar disorder to gain insight into the functional consequences of genetic variation influencing disease risk. We report here the results of experiments to explore the effects of these susceptibility variants on DNA methylation and mRNA expression in human cerebellum samples. Among the top susceptibility variants, we identified an enrichment of cis regulatory loci on mRNA expression (eQTLs), and a significant excess of quantitative trait loci for DNA CpG methylation, hereafter referred to as methylation quantitative trait loci (mQTLs). Bipolar disorder susceptibility variants that cis regulate both cerebellar expression and methylation of the same gene are a very small proportion of bipolar disorder susceptibility variants. This finding suggests that mQTLs and eQTLs provide orthogonal ways of functionally annotating genetic variation within the context of studies of pathophysiology in brain. No lymphocyte mQTL enrichment was found, suggesting that mQTL enrichment was specific to the cerebellum, in contrast to eQTLs. Separately, we found that using mQTL information to restrict the number of single-nucleotide polymorphisms studied enhances our ability to detect a significant association. With this restriction a priori informed by the observed functional enrichment, we identified a significant association (rs12618769, Pbonferroni<0.05) from two other GWA studies (TGen+GAIN; 2191 cases and 1434 controls) of bipolar disorder, which we replicated in an independent GWA study (WTCCC). Collectively, our findings highlight the importance of integrating functional annotation of genetic variants for gene expression and DNA methylation to advance the biological understanding of bipolar disorder.

Genome-wide association study of Tourette's syndrome

Tourettes syndrome (TS) is a developmental disorder that has one of the highest familial recurrence rates among neuropsychiatric diseases with complex inheritance. However, the identification of definitive TS susceptibility genes remains elusive. Here, we report the first genome-wide association study (GWAS) of TS in 1285 cases and 4964 ancestry-matched controls of European ancestry, including two European-derived population isolates, Ashkenazi Jews from North America and Israel and French Canadians from Quebec, Canada. In a primary meta-analysis of GWAS data from these European ancestry samples, no markers achieved a genome-wide threshold of significance (P<5 × 10−8); the top signal was found in rs7868992 on chromosome 9q32 within COL27A1 (P=1.85 × 10−6). A secondary analysis including an additional 211 cases and 285 controls from two closely related Latin American population isolates from the Central Valley of Costa Rica and Antioquia, Colombia also identified rs7868992 as the top signal (P=3.6 × 10−7 for the combined sample of 1496 cases and 5249 controls following imputation with 1000 Genomes data). This study lays the groundwork for the eventual identification of common TS susceptibility variants in larger cohorts and helps to provide a more complete understanding of the full genetic architecture of this disorder.

Chemotherapeutic drug susceptibility associated SNPs are enriched in expression quantitative trait loci

Pharmacogenomics has employed candidate gene studies and, more recently, genome-wide association studies (GWAS) in efforts to identify loci associated with drug response and/or toxicity. The advantage of GWAS is the simultaneous, unbiased testing of millions of SNPs; the challenge is that functional information is absent for the vast majority of loci that are implicated. In the present study, we systematically evaluated SNPs associated with chemotherapeutic agent–induced cytotoxicity for six different anticancer agents and evaluated whether these SNPs were disproportionately likely to be within a functional class such as coding (consisting of missense, nonsense, or frameshift polymorphisms), noncoding (such as 3′UTRs or splice sites), or expression quantitative trait loci (eQTLs; indicating that a SNP genotype is associated with the transcript abundance level of a gene). We found that the chemotherapeutic drug susceptibility–associated SNPs are more likely to be eQTLs, and, in fact, more likely to be associated with the transcriptional expression level of multiple genes (n ≥ 10) as potential master regulators, than a random set of SNPs in the genome, conditional on minor allele frequency. Furthermore, we observed that this enrichment compared with random expectation is not present for other traditionally important coding and noncoding SNP functional categories. This research therefore has significant implications as a general approach for the identification of genetic predictors of drug response and provides important insights into the likely function of SNPs identified in GWAS analysis of pharmacologic studies.

Genome-wide association and meta-analysis in populations from Starr County, Texas, and Mexico City identify type 2 diabetes susceptibility loci and enrichment for expression quantitative trait loci in top signals

Aims/hypothesisWe conducted genome-wide association studies (GWASs) and expression quantitative trait loci (eQTL) analyses to identify and characterise risk loci for type 2 diabetes in Mexican-Americans from Starr County, TX, USA.MethodUsing 1.8 million directly interrogated and imputed genotypes in 837 unrelated type 2 diabetes cases and 436 normoglycaemic controls, we conducted Armitage trend tests. To improve power in this population with high disease rates, we also performed ordinal regression including an intermediate class with impaired fasting glucose and/or glucose tolerance. These analyses were followed by meta-analysis with a study of 967 type 2 diabetes cases and 343 normoglycaemic controls from Mexico City, Mexico.ResultThe top signals (unadjusted p value <1 × 10−5) included 49 single nucleotide polymorphisms (SNPs) in eight gene regions (PER3, PARD3B, EPHA4, TOMM7, PTPRD, HNT [also known as RREB1], LOC729993 and IL34) and six intergenic regions. Among these was a missense polymorphism (rs10462020; Gly639Val) in the clock gene PER3, a system recently implicated in diabetes. We also report a second signal (minimum p value 1.52 × 10−6) within PTPRD, independent of the previously implicated SNP, in a population of Han Chinese. Top meta-analysis signals included known regions HNF1A and KCNQ1. Annotation of top association signals in both studies revealed a marked excess of trans-acting eQTL in both adipose and muscle tissues.Conclusions/InterpretationIn the largest study of type 2 diabetes in Mexican populations to date, we identified modest associations of novel and previously reported SNPs. In addition, in our top signals we report significant excess of SNPs that predict transcript levels in muscle and adipose tissues.

The missing association: sequencing-based discovery of novel SNPs in VKORC1 and CYP2C9 that affect warfarin dose in African Americans.

It is well recognized that the genetic variants VKORC–‐1–39, CYP2C9*2, and CYP2C9*3 contribute to warfarin dose response. This has led to warfarin dosing algorithms that include these polymorphisms and explains between 47% and 56% of variability in dose in Caucasians. However, these polymorphisms explain significantly less of the variance in dose among African Americans. In order to identify novel variations that affect warfarin dose in African Americans, we used a targeted resequencing strategy that examined evolutionarily conserved sequences and regions of putative transcriptional binding. Through ethnicity‐specific warfarin dose model building in 330 African Americans, we identified two novel genetic associations with higher warfarin dose, namely, VKORC–‐8–91 (rs61162043, P = 0.0041) and 18786 in CYP2C9 (rs7089580, P = 0.035). These novel finds are independent of the previous associations with these genes. Our regression model, encompassing both genetic and clinical variables, explained 40% of the variability in warfarin dose in African‐American subjects, significantly more than any model thus far.

Population differences in microRNA expression and biological implications.

Population differences observed for complex traits may be attributed to the combined effect of socioeconomic, environmental, genetic and epigenetic factors. To better understand population differences in complex traits, genome-wide genetic and gene expression differences among ethnic populations have been studied. Here we set out to evaluate population differences in small non-coding RNAs through an evaluation of microRNA (miRNA) baseline expression in HapMap lymphoblastoid cell lines (LCLs) derived from 53 CEU (Utah residents with northern and western European ancestry) and 54 YRI (African from Ibadan, Nigeria). Using the Exiqon miRCURYTM LNA arrays, we found that 16% of all miRNAs evaluated in our study differ significantly between these 2 ethnic groups (pBonferroni corrected<0.05). Furthermore, we explored the potential biological function of these observed differentially expressed miRNAs by comprehensively examining their effect on the transcriptome and their relationship with cellular sensitivity drug phenotypes. After multiple testing adjustment (false discovery rate (FDR)<0.1), we found that 55% and 88% of the differentially expressed miRNAs were significantly and inversely correlated with an mRNA expression phenotype in the CEU and YRI samples, respectively. Interestingly, a substantial proportion (64%) of these miRNAs correlated with cellular sensitivity to chemotherapeutic agents (FDR<0.05). Lastly, upon performing a genome-wide association study between SNPs and miRNA expression, we identified a large number of SNPs exhibiting different allele frequencies that affect the expression of these differentially expressed miRNAs, suggesting the role of genetic variants in mediating the observed population differences.