Transcriptome-Wide Expression Quantitative Trait Loci Mapping For Atherosclerosis Susceptibility Reveals Surf4 As Novel Candidate Gene

Previous work in an F2 intercross between atherosclerosis-susceptible C57BL/6 and atherosclerosis-resistant BALB/cByJ (BALB) mice on the LDL-receptor deficient background revealed a quantitative trait locus (QTL) for atherosclerotic lesion size at rs33142586 on chromosome (Chr) 2 in female mice (n=183) (Burkhardt et al., Arterioscler Thromb Vasc Biol; 31:775-84, 2011). The aim of the present study was to identify genetic variants responsible for differences in atherosclerosis-susceptibility between the two strains. We hypothesized that the causal genes could be identified by transcriptome-wide expression analyses followed by functional validation. 25,697 transcripts were analyzed in aortas (n=165) and livers (n=176) of F2 mice using Illumina Ref8 expression arrays. Expression levels were correlated with single nucleotide polymorphism (n=31) on Chr2 by expression QTL (eQTL) mapping. Genes co-segregating with the atherosclerosis QTL were identified as potential candidate genes. Results were validated by quantitative real-time PCR (qRT-PCR) in livers (n=176) and bone marrow-derived macrophages (BMDM, n=184) of F2 mice. Expression patterns of candidate genes were examined in different tissues of F0 mice, followed by functional studies. eQTL mapping identified Surf4 as the only cis -regulated gene co-segregating with the Chr2 QTL in aortas (LOD array =17.6), livers (LOD array =11.5, LOD qRT-PCR =23.2) and BMDM (LOD qRT-PCR =15.7). Surf4 mRNA was significantly increased in F2 mice carrying the BALB allele at rs33142586 and BALB F0 mice. Increased Surf4 expression was associated with reduced atherosclerosis lesion size in F2 mice. Immunohistochemical staining revealed expression of Surf4 in vascular endothelial cells of the aorta. RNAi of Surf4 lead to increased staurosporine- and cycloheximide-induced apoptosis and decreased proliferation compared to control siRNA in RAW 264.7 cells, which are two key mechanisms of atherosclerosis. In conclusion, we identified Surf4 as a novel candidate gene of atherosclerosis susceptibility co-segregating a QTL for atherosclerosis on mouse Chr2. In current work, its functional role in atherogenesis is being further investigated in in-vitro experiments.