Rfx6 identified as a candidate gene for whole pancreas insulin content in outbred heterogeneous stock rats

Abstract

Our laboratory has previously used outbred heterogeneous stock (HS) rats to fine-map metabolic and obesity traits. HS rats were created by interbreeding eight inbred founder strains and maintaining the colony to minimize inbreeding, thus enabling genetic fine-mapping to only a few Megabases. In the current work, we measured whole pancreas insulin (WPI) content in 1,003 adult male HS rats. The rats were genotyped using low coverage whole genome sequencing followed by imputation by STITCH, informed by high coverage sequencing of the HS founder strains. Physiological quantitative trait loci (pQTLs) were identified using both SNP-based and haplotype-based mapping methods (implemented in miQTL and R/qtl2, respectively), taking into account the observed genetic relationships among the HS rats. We found that WPI content strongly correlates with multiple metabolic traits, including visceral fat pad weight, fasting glucose and insulin levels, insulin in response to a glucose challenge and insulin sensitivity. We identified two pQTLs for WPI content, on rat chromosomes 10 and 20. In an effort to identify candidate genes underlying these loci, we incorporated gene expression measured through RNAseq from over 400 HS rats. Liver tissue was used because pancreatic tissue had been used to measure WPI content. RNAseq analysis was followed by identification of expression QTLs (eQTLs) and mediation analysis. The chromosome 20 locus covered 1.4 Mb and contained 25 genes, four of which mapped as cis-eQTLs. Rfx6 was identified as a full mediator of WPI content at this locus (e.g., when expression levels were included in the statistical model, the pQTL signal for whole pancreas insulin content nearly disappeared), and expression levels correlated with WPI content. In addition, both WPI and Rfx6 expression were driven by the WKY and ACI founder haplotypes, further implicating Rfx6 as a candidate gene within this locus. Rfx6 has previously been shown to be involved in beta cell development and insulin production and has been associated with mature onset diabetes of the young (MODY). The strong correlation between WPI content and multiple metabolic traits suggests that genetic variation in Rfx6 may contribute to metabolic dysfunction and/or diabetes susceptibility, which we will explore in future studies.