EQTL discovery and their association with severe equine asthma in European Warmblood horses

Victor C Mason,Molly E Mccue,Vinzenz Gerber,Tosso Leeb,Robert J Schaefer

doi:10.1186/s12864-018-4938-9

Abstract

BackgroundSevere equine asthma, also known as recurrent airway obstruction (RAO), is a debilitating, performance limiting, obstructive respiratory condition in horses that is phenotypically similar to human asthma. Past genome wide association studies (GWAS) have not discovered coding variants associated with RAO, leading to the hypothesis that causative variant(s) underlying the signals are likely non-coding, regulatory variant(s). Regions of the genome containing variants that influence the number of expressed RNA molecules are expression quantitative trait loci (eQTLs). Variation associated with RAO that also regulates a gene’s expression in a disease relevant tissue could help identify candidate genes that influence RAO if that gene’s expression is also associated with RAO disease status.ResultsWe searched for eQTLs by analyzing peripheral blood mononuclear cells (PBMCs) from two half-sib families and one unrelated cohort of 82 European Warmblood horses that were previously treated in vitro with: no stimulation (MCK), lipopolysaccharides (LPS), recombinant cyathostomin antigen (RCA), and hay-dust extract (HDE). We identified high confidence eQTLs that did not violate linear modeling assumptions and were not significant due to single outlier individuals. We identified a mean of 4347 high confidence eQTLs in four treatments of PBMCs, and discovered two trans regulatory hotspots regulating genes involved in related biological pathways. We corroborated previous RAO associated single nucleotide polymorphisms (SNPs), and increased the resolution of past GWAS by analyzing 1,056,195 SNPs in 361 individuals. We identified four RAO-associated SNPs that only regulate gene expression of dexamethasone-induced protein (DEXI), however we found no significant association between DEXI gene expression and presence of RAO.ConclusionsThousands of genetic variants regulate gene expression in PBMCs of European Warmblood horses in cis and trans. Most high confidence eSNPs are significantly enriched near the transcription start sites of their target genes. Two trans regulatory hotspots on chromosome 11 and 13 regulate many genes involved in transmembrane cell signaling and neurological development respectively when PBMCs are treated with HDE. None of the top fifteen RAO associated SNPs strongly influence disease status through gene expression regulation.

Highlights

Severe equine asthma, known as recurrent airway obstruction (RAO), is a debilitating, performance limiting, obstructive respiratory condition in horses that is phenotypically similar to human asthma
We used a filtered subset of imputed Single nucleotide polymorphism (SNP) (1,056,195) to increase resolution in genome wide association studies (GWAS) and Expression quantitative trait loci (eQTL) analyses when we searched for eQTL SNP (eSNP) that are RAO associated SNPs
Gene and individual filtration Prior to eQTL analysis, we removed genes with mean read counts below a read count threshold determined using the Kolmogorov-Smirnov (KS) test (Table 1) (Fig. 1, Additional file 1: Figure S1) [26, 27]. This reduced the number of NCBI annotated genes analyzed to a mean value of 12,736 genes across the four treatments of peripheral blood mononuclear cells (PBMCs), and RNA sequenced (RNAseq) counts were normalized and variance stabilized for each treatment (Additional file 1: Figure S2)

Summary

Introduction

Known as recurrent airway obstruction (RAO), is a debilitating, performance limiting, obstructive respiratory condition in horses that is phenotypically similar to human asthma. Past genome wide association studies (GWAS) have not discovered coding variants associated with RAO, leading to the hypothesis that causative variant(s) underlying the signals are likely non-coding, regulatory variant(s). Regions of the genome containing variants that influence the number of expressed RNA molecules are expression quantitative trait loci (eQTLs). Differential expression and genome wide association (GWAS) studies that included samples used in this study, have identified differentially expressed genes and genomic regions suggestively associated (p ≤ 1e-5) with RAO [9, 11,12,13]. No RAO associated coding variants have been found that explain the GWAS signals, so it was hypothesized that the causative variant(s) underlying these signals were likely non-coding regulatory variant(s) [12]. Regulatory function can be inferred for genetic variants associated with gene expression levels

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Discussion

Conclusion