Using Regulatory and Epistatic Networks to Extend the Findings of a Genome Scan: Identifying the Gene Drivers of Pigmentation in Merino Sheep

Elsa García-Gámez,Sean M Mcwilliam,Juan José Arranz,Vicki Whan,Antonio Reverter,James Kijas,Sunil K Ahuja

doi:10.1371/journal.pone.0021158

Abstract

Extending genome wide association analysis by the inclusion of gene expression data may assist in the dissection of complex traits. We examined piebald, a pigmentation phenotype in both human and Merino sheep, by analysing multiple data types using a systems approach. First, a case control analysis of 49,034 ovine SNP was performed which confirmed a multigenic basis for the condition. We combined these results with gene expression data from five tissue types analysed with a skin-specific microarray. Promoter sequence analysis of differentially expressed genes allowed us to reverse-engineer a regulatory network. Likewise, by testing two-loci models derived from all pair-wise comparisons across piebald-associated SNP, we generated an epistatic network. At the intersection of both networks, we identified thirteen genes with insulin-like growth factor binding protein 7 (IGFBP7), platelet-derived growth factor alpha (PDGFRA) and the tetraspanin platelet activator CD9 at the kernel of the intersection. Further, we report a number of differentially expressed genes in regions containing highly associated SNP including ATRN, DOCK7, FGFR1OP, GLI3, SILV and TBX15. The application of network theory facilitated co-analysis of genetic variation with gene expression, recapitulated aspects of the known molecular biology of skin pigmentation and provided insights into the transcription regulation and epistatic interactions involved in piebald Merino sheep.

Highlights

The population history and genetic structure of domestic animals offer advantages for the identification of the genetic drivers associated with phenotypic change
The highest association (p = 8.4561027) was observed for SNP s49104 located in the region containing insulin-like growth factor binding protein 7 (IGFBP7) (OAR 6 Mb 78.9), the absence of a single and strong association peak confirmed a multigenic basis for ovine piebald (Figure S1)
We sought to interpret these genetic associations using gene expression obtained from five skin tissue types isolated from nonpigmented, piebald and recessive black individuals known to be under the control of Agouti (Figure 1A, 2A)

Summary

Introduction

The population history and genetic structure of domestic animals offer advantages for the identification of the genetic drivers associated with phenotypic change. Recent studies have identified complex traits such as skeletal morphology are under the control of a small number of genes of large effect [4]. To explore approaches which seek to incorporate GWAS into systems biology, we have merged SNP variation with analysis of differential gene expression to investigate the basis of a phenotypic trait in sheep. This was prompted by recent studies which exploit gene network theory and systems approaches to identify key genetic drivers [7,8,9,10]. Test matings indicate the condition is not consistent with a simple Mendelian mode of inheritance [12] and the location and extent of pigmentation in effected animals varies considerably, suggesting the coordinated action of multiple genes

Methods

Results

Conclusion