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Summary
Despite the successes of human genome-wide association studies, the causal genes underlying most metabolic traits remain unclear. We used outbred heterogeneous stock (HS) rats, coupled with expression data and mediation analysis, to identify quantitative trait loci (QTLs) and candidate gene mediators for adiposity, glucose tolerance, serum lipids, and other metabolic traits. Physiological traits were measured in 1,519 male HS rats, with liver and adipose transcriptomes measured in >410 rats. Genotypes were imputed from low-coverage whole-genome sequencing. Linear mixed models were used to detect physiological and expression QTLs (pQTLs and eQTLs, respectively), using both single nucleotide polymorphism (SNP)– and haplotype-based models for pQTL mapping. Genes with cis-eQTLs that overlapped pQTLs were assessed as causal candidates through mediation analysis. We identified 14 SNP-based pQTLs and 19 haplotype-based pQTLs, of which 10 were in common. Using mediation, we identified the following genes as candidate mediators of pQTLs: Grk5 for fat pad weight and serum triglyceride pQTLs on Chr1, Krtcap3 for fat pad weight and serum triglyceride pQTLs on Chr6, Ilrun for a fat pad weight pQTL on Chr20, and Rfx6 for a whole pancreatic insulin content pQTL on Chr20. Furthermore, we verified Grk5 and Ktrcap3 using gene knockdown/out models, thereby shedding light on novel regulators of obesity.
About data processing
RNA from liver and adipose tissue was extracted from subsets of 430 and 415 rats, respectively, selected to maximize genetic diversity (e.g., no more than 1 or 2 rats per family), while encompassing the phenotypic spectrum of fat pad weights. Liver tissue RNA sequencing (RNA-seq) was run by the Genomics Core at the Medical College of Wisconsin, while adipose tissue RNA-seq was run by the Genomics Core at Wake Forest University School of Medicine.
For liver, poly-A libraries were prepared on the Illumina NeoPrep platform using the TruSeq Stranded mRNA Library Prep Kit for NeoPrep (catalog number NP-202–1001; Illumina). For adipose tissue, Ribo depletion libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero Gold Preparation kit (Illumina).
Libraries were run on an Illumina HiSeq 2500 to obtain 37-bp paired-end reads for liver and 75-bp single-end reads for adipose tissue. We used STAR (v2.6.1a) (19) to align reads to the reference Rn6.0, PICARD (v2.5.0) to remove PCR duplicates, and featureCount in R package Rsubread (20) to compute gene-level expression counts, which were later normalized by sequencing depth, gene length, and RNA composition using the DESeq2 R package (v1.24.0). We excluded very lowly expressed genes with average reads per sample less than one. The normalized expression of 18,358 genes from adipose and 16,796 genes from liver were then RINT-transformed for expression QTL (eQTL) mapping and further analyses.
Contributors
Author Contributions. W.V., R.M., and L.C.S.W. conceived experiments. K.H. and L.C.S.W. conducted animal experiments. K.H. and O.S. extracted DNA or RNA. M.T., A.C., and G.H. were responsible for RNA-seq. T.H.-L., W.L.C., and G.R.K. performed statistical analysis. T.H.-L., W.V., R.M., and L.C.S.W. were responsible for interpretation and presentation of analyzed results. S.K.D., B.M., N.K.S., and C.-C.C.K. conducted 3T3-L1 knockdown experiments. L.C.S.W. oversaw all experimental work. W.V. and R.M. oversaw statistical analysis. T.H.-L., W.V., R.M., and L.C.S.W. wrote the manuscript. All authors approved the final version of the manuscript. L.C.S.W. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Acknowledgment
The authors thank Apurva Chitre (Department of Psychiatry, University of California, San Diego) for assistance in creating Porcupine plot, Michael Scott (School of Biological Sciences, University of East Anglia) for helping with STITCH, and Leilei Cui (Genetics Institute, University College London) for helping with eQTL mapping.