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Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55×10−151). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.

Author Summary

Recent genome-wide association studies (GWAS) have identified genetic variants associated with lung diseases. The challenge now is to find the causal genes in GWAS–nominated chromosomal regions and to characterize the molecular function of disease-associated genetic variants. In this paper, we describe an international effort to systematically capture the genetic architecture of gene expression regulation in human lung. By studying lung specimens from 1,111 individuals of European ancestry, we found a large number of genetic variants affecting gene expression in the lung, or lung expression quantitative trait loci (eQTL). These lung eQTLs will serve as an important resource to aid in the understanding of the molecular underpinnings of lung biology and its disruption in disease. To demonstrate the utility of this lung eQTL dataset, we integrated our data with previous genetic studies on asthma. Through integrative techniques, we identified causal variants and genes in GWAS–nominated loci and found key molecular drivers for asthma. We feel that sharing our lung eQTLs dataset with the scientific community will leverage the impact of previous large-scale GWAS on lung diseases and function by providing much needed functional information to understand the molecular changes introduced by the susceptibility genetic variants.

The spontaneously hypertensive rat (SHR) is a widely used rodent model of hypertension and metabolic syndrome. Previously we identified thousands of cis-regulated expression quantitative trait loci (eQTLs) across multiple tissues using a panel of rat recombinant inbred (RI) strains derived from Brown Norway and SHR progenitors. These cis-eQTLs represent potential susceptibility loci underlying physiological and pathophysiological traits manifested in SHR. We have prioritized 60 cis-eQTLs and confirmed differential expression between the parental strains by quantitative PCR in 43 (72%) of the eQTL transcripts. Quantitative trait transcript (QTT) analysis in the RI strains showed highly significant correlation between cis-eQTL transcript abundance and clinically relevant traits such as systolic blood pressure and blood glucose, with the physical location of a subset of the cis-eQTLs colocalizing with “physiological” QTLs (pQTLs) for these same traits. These colocalizing correlated cis-eQTLs (c3-eQTLs) are highly attractive as primary susceptibility loci for the colocalizing pQTLs. Furthermore, sequence analysis of the c3-eQTL genes identified single nucleotide polymorphisms (SNPs) that are predicted to affect transcription factor binding affinity, splicing and protein function. These SNPs, which potentially alter transcript abundance and stability, represent strong candidate factors underlying not just eQTL expression phenotypes, but also the correlated metabolic and physiological traits. In conclusion, by integration of genomic sequence, eQTL and QTT datasets we have identified several genes that are strong positional candidates for pathophysiological traits observed in the SHR strain. These findings provide a basis for the functional testing and ultimate elucidation of the molecular basis of these metabolic and cardiovascular phenotypes.

Expression quantitative trait loci (eQTL) analysis can provide insights into the genetic regulation of gene expression at a genomic level and this information is proving extremely useful in many different areas of research. As a consequence of the role of the liver in drug metabolism and disposition, the study of eQTLs in primary human liver tissue could provide a foundation for pharmacogenomics. Thus far, four genome-wide eQTL studies have been performed using human livers. Many liver eQTLs have been found to be reproducible and a proportion of these may be specific to the liver. Already these data have been used to interpret and inform clinic genome-wide association studies, providing potential mechanistic evidence for clinical associations and identifying genes which may impact clinical phenotypes. However, the utility of liver eQTL data has not yet been fully explored or realized in pharmacogenomics. As further liver eQTL research is undertaken, the genetic regulation of gene expression will become much better characterized and this knowledge will create a rational basis for the prospective pharmacogenomic study of many drugs.

Genome-wide association studies have identified many genetic variants associated with complex traits. However, at only a minority of loci have the molecular mechanisms mediating these associations been characterized. In parallel, whilst cis-regulatory patterns of gene expression have been extensively explored, the identification of trans-regulatory effects in humans has attracted less attention. We demonstrate that the Type 2 diabetes and HDL-cholesterol associated cis-acting eQTL of the maternally-expressed transcription factor KLF14 acts as a master trans-regulator of adipose gene expression. Expression levels of genes regulated by this trans-eQTL are highly-correlated with concurrently-measured metabolic traits, and a subset of the trans-genes harbor variants directly-associated with metabolic phenotypes. This trans-eQTL network provides a mechanistic understanding of the effect of the KLF14 locus on metabolic disease risk, providing a potential model for other complex traits.

Expression quantitative trait loci (eQTL), or genetic variants associated with changes in gene expression, have the potential to assist in interpreting results of genome-wide association studies (GWAS). eQTLs also have varying degrees of tissue specificity. By correlating the statistical significance of eQTLs mapped in various tissue types to their odds ratios reported in a large GWAS by the Wellcome Trust Case Control Consortium (WTCCC), we discovered that there is a significant association between diseases studied genetically and their relevant tissues. This suggests that eQTL data sets can be used to determine tissues that play a role in the pathogenesis of a disease, thereby highlighting these tissue types for further post-GWAS functional studies.

Background

Identification of expression quantitative trait loci (eQTLs) is an emerging area in genomic study. The task requires an integrated analysis of genome-wide single nucleotide polymorphism (SNP) data and gene expression data, raising a new computational challenge due to the tremendous size of data.

Results

We develop a method to identify eQTLs. The method represents eQTLs as information flux between genetic variants and transcripts. We use information theory to simultaneously interrogate SNP and gene expression data, resulting in a Transcriptional Information Map (TIM) which captures the network of transcriptional information that links genetic variations, gene expression and regulatory mechanisms. These maps are able to identify both cis- and trans- regulating eQTLs. The application on a dataset of leukemia patients identifies eQTLs in the regions of the GART, PCP4, DSCAM, and RIPK4 genes that regulate ADAMTS1, a known leukemia correlate.

Conclusions

The information theory approach presented in this paper is able to infer the dependence networks between SNPs and transcripts, which in turn can identify cis- and trans-eQTLs. The application of our method to the leukemia study explains how genetic variants and gene expression are linked to leukemia.

Expression quantitative trait loci (eQTL) analysis is a powerful approach toward identifying genetic loci associated with quantitative changes in gene expression. We applied genome-wide association analysis to a data set of >300 000 single-nucleotide polymorphisms and >48 000 mRNA expression phenotypes obtained by Illumina microarray profiling of 149 human surgical liver samples obtained from Caucasian donors with detailed medical documentation. Of 1226 significant associations, only 200 were validated when comparing with a previously published similar study. Potential explanations for low replication rate include differences in microarray platforms, statistical modeling, covariates considered and origin and collection procedures of tissues. Focused analysis revealed a subset of 95 associations related to absorption, distribution, metabolism and excretion of drugs. Of these, 21 were true replications and 74 were newly discovered associations in enzymes, transporters, transcriptional regulators and other genes. This study extends our knowledge about the genetics of inter-individual variability of gene expression with particular emphasis on pharmacogenomics.

Variation in gene expression is heritable and has been mapped to the genome in humans and model organisms as expression quantitative trait loci (eQTLs). We applied integrated genome-wide expression profiling and linkage analysis to the regulation of gene expression in fat, kidney, adrenal, and heart tissues using the BXH/HXB panel of rat recombinant inbred strains. Here, we report the influence of heritability and allelic effect of the quantitative trait locus on detection of cis- and trans-acting eQTLs and discuss how these factors operate in a tissue-specific context. We identified several hundred major eQTLs in each tissue and found that cis-acting eQTLs are highly heritable and easier to detect than trans-eQTLs. The proportion of heritable expression traits was similar in all tissues; however, heritability alone was not a reliable predictor of whether an eQTL will be detected. We empirically show how the use of heritability as a filter reduces the ability to discover trans-eQTLs, particularly for eQTLs with small effects. Only 3% of cis- and trans-eQTLs exhibited large allelic effects, explaining more than 40% of the phenotypic variance, suggestive of a highly polygenic control of gene expression. Power calculations indicated that, across tissues, minor differences in genetic effects are expected to have a significant impact on detection of trans-eQTLs. Trans-eQTLs generally show smaller effects than cis-eQTLs and have a higher false discovery rate, particularly in more heterogeneous tissues, suggesting that small biological variability, likely relating to tissue composition, may influence detection of trans-eQTLs in this system. We delineate the effects of genetic architecture on variation in gene expression and show the sensitivity of this experimental design to tissue sampling variability in large-scale eQTL studies.

Synopsis

The combined application of genome-wide expression profiling from microarray experiments with genetic linkage analysis enables the mapping of expression quantitative trait loci (eQTLs), which are primary control points for gene expression across the genome. This approach has been called “genetical genomics”, and recent technological and methodological advances have made its large-scale application feasible in humans and model organisms. Using this approach, the authors have carried out an extensive analysis of the genetic architecture underlying variation in gene expression using a panel of 30 rat recombinant inbred strains. The results are used to explore the relationship between heritability of gene expression, cis- and trans-acting genetic effects, tissue heterogeneity, and statistical cut-offs of significance, which are important factors for large-scale eQTL studies. By examining large eQTL data from four tissues, the authors provide a detailed picture of cis- and trans-eQTL features that may help understanding of the genetic regulation of transcription on a genomic scale. The results also show the sensitivity of this approach to discriminate between cis and trans regulation and the value of the rat system in studying large eQTL datasets from multiple tissues.

Aims/hypothesis

While genome-wide association studies (GWASs) have been successful in identifying novel variants associated with various diseases, it has been much more difficult to determine the biological mechanisms underlying these associations. Expression quantitative trait loci (eQTL) provide another dimension to these data by associating single nucleotide polymorphisms (SNPs) with gene expression. We hypothesised that integrating SNPs known to be associated with type 2 diabetes with eQTLs and coexpression networks would enable the discovery of novel candidate genes for type 2 diabetes.

Methods

We selected 32 SNPs associated with type 2 diabetes in two or more independent GWASs. We used previously described eQTLs mapped from genotype and gene expression data collected from 1,008 morbidly obese patients to find genes with expression associated with these SNPs. We linked these genes to coexpression modules, and ranked the other genes in these modules using an inverse sum score.

Results

We found 62 genes with expression associated with type 2 diabetes SNPs. We validated our method by linking highly ranked genes in the coexpression modules back to SNPs through a combined eQTL dataset. We showed that the eQTLs highlighted by this method are significantly enriched for association with type 2 diabetes in data from the Wellcome Trust Case Control Consortium (WTCCC, p = 0.026) and the Gene Environment Association Studies (GENEVA, p = 0.042), validating our approach. Many of the highly ranked genes are also involved in the regulation or metabolism of insulin, glucose or lipids.

Conclusions/interpretation

We have devised a novel method, involving the integration of datasets of different modalities, to discover novel candidate genes for type 2 diabetes.

Background

Complexity and noise in expression quantitative trait loci (eQTL) studies make it difficult to distinguish potential regulatory relationships among the many interactions. The predominant method of identifying eQTLs finds associations that are significant at a genome-wide level. The vast number of statistical tests carried out on these data make false negatives very likely. Corrections for multiple testing error render genome-wide eQTL techniques unable to detect modest regulatory effects.

We propose an alternative method to identify eQTLs that builds on traditional approaches. In contrast to genome-wide techniques, our method determines the significance of an association between an expression trait and a locus with respect to the set of all associations to the expression trait. The use of this specific information facilitates identification of expression traits that have an expression profile that is characterized by a single exceptional association to a locus.

Our approach identifies expression traits that have exceptional associations regardless of the genome-wide significance of those associations. This property facilitates the identification of possible false negatives for genome-wide significance. Further, our approach has the property of prioritizing expression traits that are affected by few strong associations. Expression traits identified by this method may warrant additional study because their expression level may be affected by targeting genes near a single locus.

Results

We demonstrate our method by identifying eQTL hotspots in Plasmodium falciparum (malaria) and Saccharomyces cerevisiae (yeast). We demonstrate the prioritization of traits with few strong genetic effects through Gene Ontology (GO) analysis of Yeast. Our results are strongly consistent with results gathered using genome-wide methods and identify additional hotspots and eQTLs.

Conclusions

New eQTLs and hotspots found with this method may represent regions of the genome or biological processes that are controlled through few relatively strong genetic interactions. These points of interest warrant experimental investigation.

To identify the genes and pathways that underlie cardiovascular and metabolic phenotypes we performed an integrated analysis of a mouse C57BL/6J x A/J F2 (B6AF2) cross by relating genome-wide gene expression data from adipose, kidney, and liver tissues to physiological endpoints measured in the population. We have identified a large number of trait QTLs including loci driving variation in cardiac function on chromosomes 2 and 6 and a hotspot for adiposity, energy metabolism, and glucose traits on chromosome 8. Integration of adipose gene expression data identified a core set of genes that drive the chromosome 8 adiposity QTL. This chromosome 8 trans eQTL signature contains genes associated with mitochondrial function and oxidative phosphorylation and maps to a subnetwork with conserved function in humans that was previously implicated in human obesity. In addition, human eSNPs corresponding to orthologous genes from the signature show enrichment for association to type II diabetes in the DIAGRAM cohort, supporting the idea that the chromosome 8 locus perturbs a molecular network that in humans senses variations in DNA and in turn affects metabolic disease risk. We functionally validate predictions from this approach by demonstrating metabolic phenotypes in knockout mice for three genes from the trans eQTL signature, Akr1b8, Emr1, and Rgs2. In addition we show that the transcriptional signatures for knockout of two of these genes, Akr1b8 and Rgs2, map to the F2 network modules associated with the chromosome 8 trans eQTL signature and that these modules are in turn very significantly correlated with adiposity in the F2 population. Overall this study demonstrates how integrating gene expression data with QTL analysis in a network-based framework can aid in the elucidation of the molecular drivers of disease that can be translated from mice to humans.

Expression quantitative trait loci (eQTLs) represent genetic control points of gene expression, and can be categorized as cis- and trans-acting, reflecting local and distant regulation of gene expression respectively. Although there is evidence of co-regulation within clusters of trans-eQTLs, the extent of co-expression patterns and their relationship with the genotypes at eQTLs are not fully understood. We have mapped thousands of cis- and trans-eQTLs in four tissues (fat, kidney, adrenal and left ventricle) in a large panel of rat recombinant inbred (RI) strains. Here we investigate the genome-wide correlation structure in expression levels of eQTL transcripts and underlying genotypes to elucidate the nature of co-regulation within cis- and trans-eQTL datasets. Across the four tissues, we consistently found statistically significant correlations of cis-regulated gene expression to be rare (<0.9% of all pairs tested). Most (>80%) of the observed significant correlations of cis-regulated gene expression are explained by correlation of the underlying genotypes. In comparison, co-expression of trans-regulated gene expression is more common, with significant correlation ranging from 2.9%–14.9% of all pairs of trans-eQTL transcripts. We observed a total of 81 trans-eQTL clusters (hot-spots), defined as consisting of ≥10 eQTLs linked to a common region, with very high levels of correlation between trans-regulated transcripts (77.2–90.2%). Moreover, functional analysis of large trans-eQTL clusters (≥30 eQTLs) revealed significant functional enrichment among genes comprising 80% of the large clusters. The results of this genome-wide co-expression study show the effects of the eQTL genotypes on the observed patterns of correlation, and suggest that functional relatedness between genes underlying trans-eQTLs is reflected in the degree of co-expression observed in trans-eQTL clusters. Our results demonstrate the power of an integrative, systematic approach to the analysis of a large gene expression dataset to uncover underlying structure, and inform future eQTL studies.

The recent success of genome-wide association studies (GWAS) is now followed by the challenge to determine how the reported susceptibility variants mediate complex traits and diseases. Expression quantitative trait loci (eQTLs) have been implicated in disease associations through overlaps between eQTLs and GWAS signals. However, the abundance of eQTLs and the strong correlation structure (LD) in the genome make it likely that some of these overlaps are coincidental and not driven by the same functional variants. In the present study, we propose an empirical methodology, which we call Regulatory Trait Concordance (RTC) that accounts for local LD structure and integrates eQTLs and GWAS results in order to reveal the subset of association signals that are due to cis eQTLs. We simulate genomic regions of various LD patterns with both a single or two causal variants and show that our score outperforms SNP correlation metrics, be they statistical (r2) or historical (D'). Following the observation of a significant abundance of regulatory signals among currently published GWAS loci, we apply our method with the goal to prioritize relevant genes for each of the respective complex traits. We detect several potential disease-causing regulatory effects, with a strong enrichment for immunity-related conditions, consistent with the nature of the cell line tested (LCLs). Furthermore, we present an extension of the method in trans, where interrogating the whole genome for downstream effects of the disease variant can be informative regarding its unknown primary biological effect. We conclude that integrating cellular phenotype associations with organismal complex traits will facilitate the biological interpretation of the genetic effects on these traits.

Author Summary

Genome-wide association studies have led to the identification of susceptibility loci for a variety of human complex traits. What is still largely missing, however, is the understanding of the biological context in which these candidate variants act and of how they determine each trait. Given the localization of many GWAS loci outside coding regions and the important role of regulatory variation in shaping phenotypic variance, gene expression has been proposed as a plausible informative intermediate phenotype. Here we show that for a subset of the currently published GWAS this is indeed the case, by observing a significant excess of regulatory variants among disease loci. We propose an empirical methodology (regulatory trait concordance—RTC) able to integrate expression and disease data in order to detect causal regulatory effects. We show that the RTC outperforms simple correlation metrics under various simulated linkage disequilibrium (LD) scenarios. Our method is able to recover previously suspected causal regulatory effects from the literature and, as expected given the nature of the tested tissue, an overrepresentation of immunity-related candidates is observed. As the number of available tissues will increase, this prioritization approach will become even more useful in understanding the implication of regulatory variants in disease etiology.

Genome-wide association studies (GWAS) and candidate gene studies in ulcerative colitis (UC) have identified 18 susceptibility loci. We conducted a meta-analysis of 6 UC GWAS, comprising 6,687 cases and 19,718 controls, and followed-up the top association signals in 9,628 cases and 12,917 controls. We identified 29 additional risk loci (P<5×10-8), increasing the number of UC associated loci to 47. After annotating associated regions using GRAIL, eQTL data and correlations with non-synonymous SNPs, we identified many candidate genes providing potentially important insights into disease pathogenesis, including IL1R2, IL8RA/B, IL7R, IL12B, DAP, PRDM1, JAK2, IRF5, GNA12 and LSP1. The total number of confirmed inflammatory bowel disease (IBD) risk loci is now 99, including a minimum of 28 shared association signals between Crohn’s disease (CD) and UC.

The integrated analysis of genotypic and expression data for association with complex traits could identify novel genetic pathways involved in complex traits. We profiled 19,573 expression probes in Epstein-Barr virus-transformed lymphoblastoid cell lines (LCLs) from 299 twins and correlated these with 44 quantitative traits (QTs). For 939 expressed probes correlating with more than one QT, we investigated the presence of eQTL associations in three datasets of 57 CEU HapMap founders and 86 unrelated twins. Genome-wide association analysis of these probes with 2.2 m SNPs revealed 131 potential eQTLs (1,989 eQTL SNPs) overlapping between the HapMap datasets, five of which were in cis (58 eQTL SNPs). We then tested 535 SNPs tagging the eQTL SNPs, for association with the relevant QT in 2,905 twins. We identified nine potential SNP-QT associations (P<0.01) but none significantly replicated in five large consortia of 1,097–16,129 subjects. We also failed to replicate previous reported eQTL associations with body mass index, plasma low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides levels derived from lymphocytes, adipose and liver tissue. Our results and additional power calculations suggest that proponents may have been overoptimistic in the power of LCLs in eQTL approaches to elucidate regulatory genetic effects on complex traits using the small datasets generated to date. Nevertheless, larger tissue-specific expression data sets relevant to specific traits are becoming available, and should enable the adoption of similar integrated analyses in the near future.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease characterized by loss of upper and lower motor neurons. ALS is considered to be a complex trait and genome-wide association studies (GWAS) have implicated a few susceptibility loci. However, many more causal loci remain to be discovered. Since it has been shown that genetic variants associated with complex traits are more likely to be eQTLs than frequency-matched variants from GWAS platforms, we conducted a two-stage genome-wide screening for eQTLs associated with ALS. In addition, we applied an eQTL analysis to finemap association loci. Expression profiles using peripheral blood of 323 sporadic ALS patients and 413 controls were mapped to genome-wide genotyping data. Subsequently, data from a two-stage GWAS (3,568 patients and 10,163 controls) were used to prioritize eQTLs identified in the first stage (162 ALS, 207 controls). These prioritized eQTLs were carried forward to the second sample with both gene-expression and genotyping data (161 ALS, 206 controls). Replicated eQTL SNPs were then tested for association in the second-stage GWAS data to find SNPs associated with disease, that survived correction for multiple testing. We thus identified twelve cis eQTLs with nominally significant associations in the second-stage GWAS data. Eight SNP-transcript pairs of highest significance (lowest p = 1.27×10−51) withstood multiple-testing correction in the second stage and modulated CYP27A1 gene expression. Additionally, we show that C9orf72 appears to be the only gene in the 9p21.2 locus that is regulated in cis, showing the potential of this approach in identifying causative genes in association loci in ALS. This study has identified candidate genes for sporadic ALS, most notably CYP27A1. Mutations in CYP27A1 are causal to cerebrotendinous xanthomatosis which can present as a clinical mimic of ALS with progressive upper motor neuron loss, making it a plausible susceptibility gene for ALS.

Background

Analysis of expression quantitative trait loci (eQTL) aims to identify the genetic loci associated with the expression level of genes. Penalized regression with a proper penalty is suitable for the high-dimensional biological data. Its performance should be enhanced when we incorporate biological knowledge of gene expression network and linkage disequilibrium (LD) structure between loci in high-noise background.

Results

We propose a network-based group variable selection (NGVS) method for QTL detection. Our method simultaneously maps highly correlated expression traits sharing the same biological function to marker sets formed by LD. By grouping markers, complex joint activity of multiple SNPs can be considered and the dimensionality of eQTL problem is reduced dramatically. In order to demonstrate the power and flexibility of our method, we used it to analyze two simulations and a mouse obesity and diabetes dataset. We considered the gene co-expression network, grouped markers into marker sets and treated the additive and dominant effect of each locus as a group: as a consequence, we were able to replicate results previously obtained on the mouse linkage dataset. Furthermore, we observed several possible sex-dependent loci and interactions of multiple SNPs.

Conclusions

The proposed NGVS method is appropriate for problems with high-dimensional data and high-noise background. On eQTL problem it outperforms the classical Lasso method, which does not consider biological knowledge. Introduction of proper gene expression and loci correlation information makes detecting causal markers more accurate. With reasonable model settings, NGVS can lead to novel biological findings.

Genome-wide association studies (GWASs) have been used to identify genes that increase risk of psychiatric diseases. However, much of the variation in disease risk is still unexplained, suggesting that there are genes still to be discovered. Functional annotation of genetic variants may increase the power of GWASs to identify disease genes by providing prior information that can be used in Bayesian analysis or in reducing the number of tests. Genetic mapping of expression quantitative trait loci (eQTLs) is helping us to reveal novel functional effects of thousands of single nucleotide polymorphisms (SNPs). The published brain eQTL studies are reviewed here, and major methodological issues and their possible solutions are discussed. We emphasize the frequently-ignored problems of batch effects, covariates, and multiple testing, all of which can lead to false positives and false negatives. The future application of eQTL data to the GWAS analysis is also discussed.

Motivation: Analysis of expression quantitative trait loci (eQTL) significantly contributes to the determination of gene regulation programs. However, the discovery and analysis of associations of gene expression levels and their underlying sequence polymorphisms continue to pose many challenges. Methods are limited in their ability to illuminate the full structure of the eQTL data. Most rely on an exhaustive, genome scale search that considers all possible locus–gene pairs and tests the linkage between each locus and gene.

Result: To analyze eQTLs in a more comprehensive and efficient way, we developed the Graph based eQTL Decomposition method (GeD) that allows us to model genotype and expression data using an eQTL association graph. Through graph-based heuristics, GeD identifies dense subgraphs in the eQTL association graph. By identifying eQTL association cliques that expose the hidden structure of genotype and expression data, GeD effectively filters out most locus–gene pairs that are unlikely to have significant linkage. We apply GeD on eQTL data from Plasmodium falciparum, the human malaria parasite, and show that GeD reveals the structure of the relationship between all loci and all genes on a whole genome level. Furthermore, GeD allows us to uncover additional eQTLs with lower FDR, providing an important complement to traditional eQTL analysis methods.

Contact: przytyck@ncbi.nlm.nih.gov

The identification of multiple signals at individual loci could explain additional phenotypic variance (‘missing heritability’) of common traits, and help identify causal genes. We examined gene expression levels as a model trait because of the large number of strong genetic effects acting in cis. Using expression profiles from 613 individuals, we performed genome-wide single nucleotide polymorphism (SNP) analyses to identify cis-expression quantitative trait loci (eQTLs), and conditional analysis to identify second signals. We examined patterns of association when accounting for multiple SNPs at a locus and when including additional SNPs from the 1000 Genomes Project. We identified 1298 cis-eQTLs at an approximate false discovery rate 0.01, of which 118 (9%) showed evidence of a second independent signal. For this subset of 118 traits, accounting for two signals resulted in an average 31% increase in phenotypic variance explained (Wilcoxon P< 0.0001). The association of SNPs with cis gene expression could increase, stay similar or decrease in significance when accounting for linkage disequilibrium with second signals at the same locus. Pairs of SNPs increasing in significance tended to have gene expression increasing alleles on opposite haplotypes, whereas pairs of SNPs decreasing in significance tended to have gene expression increasing alleles on the same haplotypes. Adding data from the 1000 Genomes Project showed that apparently independent signals could be potentially explained by a single association signal. Our results show that accounting for multiple variants at a locus will increase the variance explained in a substantial fraction of loci, but that allelic heterogeneity will be difficult to define without resequencing loci and functional work.

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10−9, odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4+ and CD19+ peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset.

Author Summary

Although recent genome-wide association study (GWAS) approaches have successfully contributed to disease gene discovery, many susceptibility loci are known to be still uncaptured due to strict significance threshold for multiple hypothesis testing. Therefore, prioritization of GWAS results by incorporating additional information is recommended. Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Considering that abnormalities in B cell activity play essential roles in SLE, prioritization based on an expression quantitative trait loci (eQTLs) study for B cells would be a promising approach. In this study, we report a GWAS and multi-stage replication studies for SLE examining 2,278 SLE cases and 31,948 controls in Japanese subjects. We integrated eQTL study into the results of the GWAS and identified AFF1 as a novel SLE susceptibility loci. We also confirmed cis-regulatory effect of the locus on the AFF1 transcript. Our study would be one of the initial successes for detecting novel genetic locus using the eQTL study, and it should contribute to our understanding of the genetic loci being uncaptured by standard GWAS approaches.

Several recent studies have reported that expression quantitative trait loci (eQTLs) may affect gene expression in a cell-dependent manner. In the current study, a genome-wide eQTL analysis was performed in whole blood samples collected from 76 Japanese subjects. RNA microarray analysis was performed for 3 independent sample groups that were genotyped in a genome-wide scan. The correlations between the genotypes of 534,404 autosomal single nucleotide polymorphisms (SNPs) and the expression levels of 30,465 probes were examined for each sample group. The SNP-probe pairs with combined correlation coefficients of all 3 sample groups corresponding to P<3.1×10−12 (i.e., Bonferroni-corrected P<0.05) were considered significant. SNP-probe pairs with a high likelihood of cross-hybridization and SNP-in-probe effects were excluded to avoid false positive results. We identified 102 cis-acting and 5 trans-acting eQTL regions. The cis-eQTL regions were widely distributed both upstream and downstream of the gene, as well as within the gene. The eQTL SNPs identified were examined for their influence on the expression levels in lymphoblastoid cell lines by using a public database. The results showed that genetic variants affecting expression levels in whole blood may have different effects on gene expression in lymphoblastoid cell lines. Further studies are required to clarify how SNPs function in affecting the expression levels in whole blood as well as in other tissues.

Recent genome-wide association studies (GWAS) have identified several gene variants associated with sporadic chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Many of these CLL/SLL susceptibility loci are located in non-coding or intergenic regions, posing a significant challenge to determine their potential functional relevance. Here, we review the literature of all CLL/SLL GWAS and validation studies, and apply eQTL analysis to identify putatively functional SNPs that affect gene expression that may be causal in the pathogenesis of CLL/SLL. We tested 12 independent risk loci for their potential to alter gene expression through cis-acting mechanisms, using publicly available gene expression profiles with matching genotype information. Sixteen SNPs were identified that are linked to differential expression of SP140, a putative tumor suppressor gene previously associated with CLL/SLL. Three additional SNPs were associated with differential expression of DACT3 and GNG8, which are involved in the WNT/β-catenin- and G protein-coupled receptor signaling pathways, respectively, that have been previously implicated in CLL/SLL pathogenesis. Using in silico functional prediction tools, we found that 14 of the 19 significant eQTL SNPs lie in multiple putative regulatory elements, several of which have prior implications in CLL/SLL or other hematological malignancies. Although experimental validation is needed, our study shows that the use of existing GWAS data in combination with eQTL analysis and in silico methods represents a useful starting point to screen for putatively causal SNPs that may be involved in the etiology of CLL/SLL.

Genome-wide gene expression profiling has been extensively used to generate biological hypotheses based on differential expression. Recently, many studies have used microarrays to measure gene expression levels across genetic mapping populations. These gene expression phenotypes have been used for genome-wide association analyses, an analysis referred to as expression QTL (eQTL) mapping. Here, eQTL analysis was performed in adipose tissue from 28 inbred strains of mice. We focused our analysis on “trans-eQTL bands”, defined as instances in which the expression patterns of many genes were all associated to a common genetic locus. Genes comprising trans-eQTL bands were screened for enrichments in functional gene sets representing known biological pathways, and genes located at associated trans-eQTL band loci were considered candidate transcriptional modulators. We demonstrate that these patterns were enriched for previously characterized relationships between known upstream transcriptional regulators and their downstream target genes. Moreover, we used this strategy to identify both novel regulators and novel members of known pathways. Finally, based on a putative regulatory relationship identified in our analysis, we identified and validated a previously uncharacterized role for cyclin H in the regulation of oxidative phosphorylation. We believe that the specific molecular hypotheses generated in this study will reveal many additional pathway members and regulators, and that the analysis approaches described herein will be broadly applicable to other eQTL data sets.

Author Summary

Genome-wide association (GWA) analyses seek to relate variation of phenotype to underlying (and presumably causative) variation in genotype. Recently, many GWA studies have identified candidate genes underlying disease phenotypes such as diabetes, heart disease, and cancer risk. Many groups have also performed GWA using variation in gene expression levels as the input phenotype. These expression QTL (eQTL) studies have provided important clues as to the genetic basis of gene expression regulation. Here, we perform an eQTL study in mouse adipose tissue. We then developed a systematic analysis method to relate these patterns of eQTL associations to biological pathways. Based on this approach, we identified putative roles for thousands of candidate upstream regulators and candidate pathway members in relation to specific biological pathways. Statistical analysis showed that these predictions were highly enriched for true genetic modulators of these pathways. Based on these predictions, we also experimentally validated a role for one particular gene, cyclin H, in the regulation of oxidative phosphorylation. These findings illustrate a new analysis method for relating eQTL studies to biological pathways and identify cyclin H as a novel key regulator of cellular energy metabolism.

Previous studies using SAGE (the Study of Addiction: Genetics and Environment) and COGA (the Collaborative Study on the Genetics of Alcoholism) genome-wide association study (GWAS) data sets reported several risk loci for alcohol dependence (AD), which have not yet been well replicated independently or confirmed by functional studies. We combined these two data sets, now publicly available, to increase the study power, in order to identify replicable, functional, and significant risk regions for AD. A total of 4116 subjects (1409 European-American (EA) cases with AD, 1518 EA controls, 681 African-American (AA) cases, and 508 AA controls) underwent association analysis. An additional 443 subjects underwent expression quantitative trait locus (eQTL) analysis. Genome-wide association analysis was performed in EAs to identify significant risk genes. All available markers in the genome-wide significant risk genes were tested in AAs for associations with AD, and in six HapMap populations and two European samples for associations with gene expression levels. We identified a unique genome-wide significant gene—KIAA0040—that was enriched with many replicable risk SNPs for AD, all of which had significant cis-acting regulatory effects. The distributions of −log(p) values for SNP-disease and SNP-expression associations for all markers in the TNN–KIAA0040 region were consistent across EAs, AAs, and five HapMap populations (0.369⩽r⩽0.824; 2.8 × 10−9⩽p⩽0.032). The most significant SNPs in these populations were in high LD, concentrating in KIAA0040. Finally, expression of KIAA0040 was significantly (1.2 × 10−11⩽p⩽1.5 × 10−6) associated with the expression of numerous genes in the neurotransmitter systems or metabolic pathways previously associated with AD. We concluded that KIAA0040 might harbor a causal variant for AD and thus might directly contribute to risk for this disorder. KIAA0040 might also contribute to the risk of AD via neurotransmitter systems or metabolic pathways that have previously been implicated in the pathophysiology of AD. Alternatively, KIAA0040 might regulate the risk via some interactions with flanking genes TNN and TNR. TNN is involved in neurite outgrowth and cell migration in hippocampal explants, and TNR is an extracellular matrix protein expressed primarily in the central nervous system.