The discovery of expression quantitative trait loci (“eQTLs”) can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3′UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits. Many disease-associated genetic variants do not alter protein sequences and are difficult to precisely identify. Discovery of expression quantitative trait loci (eQTL), or correlations between genetic variants and gene expression levels, offers one means of addressing this challenge. However, eQTL studies in primary cells have several shortcomings. In particular, their reproducibility is largely unknown, the variables that generate unreliable associations are uncharacterized, and the resolution of their findings is constrained by linkage disequilibrium. We performed a three-way replication study of eQTLs in primary human livers. We demonstrated that ∼67% of cis-eQTL associations are replicated in an independent study and that known polymorphisms overlapping expression probes, SNP-to-gene distance, and unmeasured confounding variables all influence the replication rate. We fine-mapped 14 eQTLs and identified causative polymorphisms in the promoter or 3′UTR for 3 genes, suggesting that a considerable fraction of eQTLs are driven by proximal variants that are amenable to functional isolation. Finally, we found hundreds of overlaps between SNPs associated with complex traits and replicated eQTL SNPs. Our data provide both cautionary (i.e. non-reproducibility of many strong eQTLs) and optimistic (i.e. precise identification of functional non-coding variants) forecasts for future eQTL analyses and the complex traits that they influence.

Purpose: To assess the pharmacogenomic and pharmacokinetic determinants of skin rash and diarrhea, the two primary dose-limiting toxicities of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib.Patients and Methods: A prospective clinical study of 80 patients with non–small-cell lung cancer, head and neck cancer, and ovarian cancer was performed. Detailed pharmacokinetics and toxicity of erlotinib were assessed. Polymorphic loci in EGFR, ABCG2, CYP3A4, and CYP3A5 were genotyped, and their effects on pharmacokinetics and toxicities were evaluated.Results: A novel diplotype of two polymorphic loci in the ABCG2 promoter involving −15622C/T and 1143C/T was identified, with alleles conferring lower ABCG2 levels associated with higher erlotinib pharmacokinetic parameters, including area under the curve (P = .019) and maximum concentration (P = .006). Variability in skin rash was best explained by a multivariate logistic regression model incorporating the trough erlotinib plasma concentration (P = .034) and the EGFR intron 1 polymorphism (P = .044). Variability in diarrhea was associated with the two linked polymorphisms in the EGFR promoter (P < .01), but not with erlotinib concentration.Conclusion: Although exploratory in nature, this combined pharmacogenomic and pharmacokinetic model helps to define and differentiate the primary determinants of skin and gastrointestinal toxicity of erlotinib. The findings may be of use both in designing trials targeting a particular severity of rash and in considering dose and schedule modifications in patients experiencing dose-limiting toxicities of erlotinib or similarly targeted agents. Further studies of the relationship between germline polymorphisms in EGFR and the toxicity and efficacy of EGFR inhibitors are warranted.

Purpose: Long non-coding RNAs (lncRNAs) has been observed in various cancer types. Our bioinformatic analysis of existing database demonstrated overexpression of lncRNA THAP9-AS1 in pancreatic ductal adenocarcinoma (PDAC). We aimed to investigate the roles and mechanisms of THAP9-AS1 in PDAC. Experimental Design: The overexpression of THAP9-AS1 in samples of pancreatic cancer patients was characterized and was associated with clinical outcomes. The non-protein coding property of the THAP9-AS1 was verified. Various in vitro and in vivo experiments were performed to investigate the interaction between THAP9-AS1 and YAP signaling. Results: We demonstrated that lncRNA THAP9-AS1 is overexpressed in PDAC in multiple patient sample sets, which is significantly associated with poor outcome of PDAC patients. THAP9-AS1 promotes PDAC cells growth both in vitro and in vivo. THAP9-AS1 exerts its effects via enhancing YAP signaling. Ectopic YAP expression overcame the effects of THAP9-AS1 knockdown. Inversely, YAP knockdown diminished the effects of THAP9-AS1 overexpression. THAP9-AS1 acts as a competing endogenous RNA for miR-484, leading to YAP upregulation. Moreover, THAP9-AS1 binds to YAP protein and inhibits the phosphorylation mediated inactivation of YAP by LATS1. Reciprocally, YAP/TEAD1 complex promotes THAP9-AS1 transcription to form a feed-forward circuit. Importantly, THAP9-AS1 level positively correlates with YAP expression in PDAC tissues. YAP overexpression also predicts a poor outcome of PDAC patients. Conclusions: Our findings indicate that THAP9-AS1 plays an important role in PDAC growth via enhancing YAP signaling which in return also modulates THAP9-AS1 transcription. THAP9-AS1/YAP axis may serve as a potential biomarker and therapeutic target for PDAC treatment.

Nine different functional UGT1A enzymes are generated from a single UGT1A gene by alternative splicing, with each enzyme having a unique exon 1. SN-38, the active metabolite of the anticancer agent irinotecan, is metabolized by both UGT1A1 and UGT1A9. We aim to characterize the UGT1A9–UGT1A1 haplotypes in Asians and Caucasians and gain insights on their functional consequences. Asian and Caucasian individuals were genotyped for UGT1A1 and UGT1A9 variants. A higher frequency of the UGT1A9 −118T10 allele was observed in Asians compared to Caucasians, while the −275T>A and −2152C>T variants were relatively uncommon in Caucasians and not found in Asians. The strongest linkage disequilibrium (LD) was observed between the UGT1A1 −53 and −3156 and between the UGT1A9 −275 and −2152 loci. Lower LD was observed between the −118 UGT1A9 variant and the UGT1A1 variants. Fourteen UGT1A9–UGT1A1 haplotypes were found in Asians, seven of them found to be shared by both populations. Common UGT1A9–UGT1A1 diplotypes were defined, and a difference was observed across the SN-38 glucuronidation rates in Caucasian livers stratified by diplotypes. This study for the first time described common UGT1A9–UGT1A1 haplotypes, highlighting important ethnic differences between Asians and Caucasians. If the functional effect of these haplotypes can be confirmed, this haplotypic information would be applicable to the correct design of prospective clinical studies of irinotecan, as well as of other drugs primarily metabolized by both UGT1A1 and UGT1A9.