Identification of candidate genes encoding an LDL-C QTL in baboons

Cardiovascular disease (CVD) is the leading cause of death in developed countries, and dyslipidemia is a major risk factor for CVD. We previously identified a cluster of quantitative trait loci (QTL) on baboon chromosome 11 for multiple, related quantitative traits for serum LDL-cholesterol (LDL-C). Here we report differentially regulated hepatic genes encoding an LDL-C QTL that influences LDL-C levels in baboons. We performed hepatic whole-genome expression profiling for LDL-C-discordant baboons fed a high-cholesterol, high-fat (HCHF) diet for seven weeks. We detected expression of 117 genes within the QTL 2-LOD support interval. Three genes were differentially expressed in low LDL-C responders and 8 in high LDL-C responders in response to a HCHF diet. Seven genes (ACVR1B, CALCOCO1, DGKA, ERBB3, KRT73, MYL6B, TENC1) showed discordant expression between low and high LDL-C responders. To prioritize candidate genes, we integrated miRNA and mRNA expression profiles using network tools and found that four candidates (ACVR1B, DGKA, ERBB3, TENC1) were miRNA targets and that the miRNAs were inversely expressed to the target genes. Candidate gene expression was validated using QRT-PCR and Western blotting. This study reveals candidate genes that influence variation in LDL-C in baboons and potential genetic mechanisms for further investigation. Cardiovascular disease (CVD) is the leading cause of death in developed countries, and dyslipidemia is a major risk factor for CVD. We previously identified a cluster of quantitative trait loci (QTL) on baboon chromosome 11 for multiple, related quantitative traits for serum LDL-cholesterol (LDL-C). Here we report differentially regulated hepatic genes encoding an LDL-C QTL that influences LDL-C levels in baboons. We performed hepatic whole-genome expression profiling for LDL-C-discordant baboons fed a high-cholesterol, high-fat (HCHF) diet for seven weeks. We detected expression of 117 genes within the QTL 2-LOD support interval. Three genes were differentially expressed in low LDL-C responders and 8 in high LDL-C responders in response to a HCHF diet. Seven genes (ACVR1B, CALCOCO1, DGKA, ERBB3, KRT73, MYL6B, TENC1) showed discordant expression between low and high LDL-C responders. To prioritize candidate genes, we integrated miRNA and mRNA expression profiles using network tools and found that four candidates (ACVR1B, DGKA, ERBB3, TENC1) were miRNA targets and that the miRNAs were inversely expressed to the target genes. Candidate gene expression was validated using QRT-PCR and Western blotting. This study reveals candidate genes that influence variation in LDL-C in baboons and potential genetic mechanisms for further investigation. ERRATUMJournal of Lipid ResearchVol. 54Issue 8PreviewThe authors of “Identification of candidate genes encoding an LDL-C QTL in baboons” (J. Lipid Res. 2013 54:(7) 1776–1785) have informed the Journal that an author, Sassan Hafizi, was inadvertently omitted from the first published version of their manuscript. The initial authorship list read as follows: Full-Text PDF Open Access Cardiovascular disease (CVD), the leading cause of death in developed countries (1Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s.Nature. 1993; 362: 801-809Crossref PubMed Scopus (9982) Google Scholar), is commonly due to the development of atherosclerosis. Atherogenesis is a complex, multifactorial process attributable to the interaction of genotype and environmental factors, including diet. CVD is characterized by accumulation of lipoproteins, inflammatory cells, and fibrous tissues in the walls of the arteries, resulting in development of lesions (2McGill Jr, H.C. McMahan C.A. Herderick E.E. Tracy R.E. Malcom G.T. Zieske A.W. Strong J.P. Effects of coronary heart disease risk factors on atherosclerosis of selected regions of the aorta and right coronary artery. PDAY Research Group. Pathobiological Determinants of Atherosclerosis in Youth.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 836-845Crossref PubMed Scopus (257) Google Scholar, 3Kushwaha R.S. McGill Jr, H.C. Diet, plasma lipoproteins and experimental atherosclerosis in baboons (Papio sp.).Hum. Reprod. Update. 1998; 4: 420-429Crossref PubMed Scopus (33) Google Scholar). Upon rupture of advanced arterial plaques, occlusion of narrow arteries by thrombosis may lead to heart attack or stroke (4Libby P. Atherosclerosis: disease biology affecting the coronary vasculature.Am. J. Cardiol. 2006; 98: 3Q-9QAbstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Thus, CVD has profound economic and social impact. Dyslipidemia is a major variable in the etiology of atherosclerosis; high serum levels of LDL-cholesterol (LDL-C) and low serum levels of HDL-cholesterol (HDL-C) are associated with high risk of developing atherosclerosis in humans and experimental animals (3Kushwaha R.S. McGill Jr, H.C. Diet, plasma lipoproteins and experimental atherosclerosis in baboons (Papio sp.).Hum. Reprod. Update. 1998; 4: 420-429Crossref PubMed Scopus (33) Google Scholar, 5Barter P.J. Rye K.A. Molecular mechanisms of reverse cholesterol transport.Curr. Opin. Lipidol. 1996; 7: 82-87Crossref PubMed Scopus (171) Google Scholar, 6McGill Jr, H.C. McMahan C.A. Kruski A.W. Kelley J.L. Mott G.E. Responses of serum lipoproteins to dietary cholesterol and type of fat in the baboon.Arteriosclerosis. 1981; 1: 337-344Crossref PubMed Google Scholar). Because LDL-C is positively correlated with the extent and severity of atherosclerotic lesions (2McGill Jr, H.C. McMahan C.A. Herderick E.E. Tracy R.E. Malcom G.T. Zieske A.W. Strong J.P. Effects of coronary heart disease risk factors on atherosclerosis of selected regions of the aorta and right coronary artery. PDAY Research Group. Pathobiological Determinants of Atherosclerosis in Youth.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 836-845Crossref PubMed Scopus (257) Google Scholar), genetic variation underlying differing individual LDL-C serum concentrations is an important determinant of atherosclerosis. Significant information is available on a few genes that influence dyslipidemia (7Brown M.S. Goldstein J.L. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis.Annu. Rev. Biochem. 1983; 52: 223-261Crossref PubMed Google Scholar, 8Rust L. Tobacco prevention advertising: lessons from the commercial world.Nicotine Tob. Res. 1999; 1: S81-S89Crossref PubMed Scopus (7) Google Scholar, 9Willer C.J. Sanna S. Jackson A.U. Scuteri A. Bonnycastle L.L. Clarke R. Heath S.C. Timpson N.J. Najjar S.S. Stringham H.M. et al.Newly identified loci that influence lipid concentrations and risk of coronary artery disease.Nat. Genet. 2008; 40: 161-169Crossref PubMed Scopus (1324) Google Scholar), and microRNAs (miRNA) regulate their expression (10Rayner K.J. Esau C.C. Hussain F.N. McDaniel A.L. Marshall S.M. van Gils J.M. Ray T.D. Sheedy F.J. Goedeke L. Liu X. et al.Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides.Nature. 2011; 478: 404-407Crossref PubMed Scopus (598) Google Scholar); however, these genes account for only a small percentage of LDL-C variation (11Konigsberg L.W. Blangero J. Kammerer C.M. Mott G.E. Mixed model segregation analysis of LDL-C concentration with genotype-covariate interaction.Genet. Epidemiol. 1991; 8: 69-80Crossref PubMed Scopus (32) Google Scholar). In addition, despite the enormous economic and social constraints caused by dyslipidemia, there is still lack of a comprehensive understanding of underlying molecular genetic mechanisms regulating variation in LDL-C serum concentrations that, consequently, impairs efforts to develop genetically informed, personalized treatment. A systems biology approach has the potential to uncover gene networks underlying dyslipidemia-related genetic variation and provide insights to candidate therapeutic targets. Previously we used the baboon, a well-characterized model for atherosclerosis (2McGill Jr, H.C. McMahan C.A. Herderick E.E. Tracy R.E. Malcom G.T. Zieske A.W. Strong J.P. Effects of coronary heart disease risk factors on atherosclerosis of selected regions of the aorta and right coronary artery. PDAY Research Group. Pathobiological Determinants of Atherosclerosis in Youth.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 836-845Crossref PubMed Scopus (257) Google Scholar, 12McGill Jr, H.C. McMahan C.A. Kruski A.W. Mott G.E. Relationship of lipoprotein cholesterol concentrations to experimental atherosclerosis in baboons.Arteriosclerosis. 1981; 1: 3-12Crossref PubMed Google Scholar), to identify a cluster of QTLs on chromosome (chr) 11 (homolog of human chr 12, localized at genomic region, 12q13.13-q14.1) that encodes variation in multiple quantitative traits related to serum LDL-C (13Rainwater D.L. Cox L.A. Rogers J. VandeBerg J.L. Mahaney M.C. Localization of multiple pleiotropic genes for lipoprotein metabolism in baboons.J. Lipid Res. 2009; 50: 1420-1428Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In a pedigreed baboon population (n = 2,044), we phenotyped individuals for LDL-C serum concentrations and genotyped the baboons for microsatellite markers (n = 287) localized in a baboon genome linkage map (14Cox L.A. Mahaney M.C. Vandeberg J.L. Rogers J. A second-generation genetic linkage map of the baboon (Papio hamadryas) genome.Genomics. 2006; 88: 274-281Crossref PubMed Scopus (50) Google Scholar). We performed genome scans and identified a QTL on chr 11 for LDL-C serum concentration. Using additional markers and LDL-C traits, we identified additional QTLs for multiple LDL-C related traits overlapping the LDL-C serum concentration QTL (Fig. 1). The identification of a chr 11 cluster of QTLs lead us to posit that a pleiotropic gene(s), discordantly expressed between low and high LDL-C responders, is responsible for variation in LDL-C serum concentration and that this gene(s) plays a role in lipid metabolism. The goal of this study was to identify candidate genes encoding variation in the chr 11 LDL-C serum concentration QTL. To augment detection of genetic variation influencing variation in LDL-C levels, we selected three pairs of half-sib baboons discordant for LDL-C serum concentrations and discordant for genotypes of markers within the QTL. That is, we selected related animals at extremes of a population of LDL-C measures in order to minimize overall genetic variation and maximize variation in the region of the genome encoding the QTL. The discordant baboons (low LDL-C, n = 3; high LDL-C, n = 3) were challenged with a high-cholesterol, high-fat (HCHF) diet for seven weeks. Biopsies were collected from liver, the primary organ for lipid metabolism, before and after the diet challenge. We performed whole-genome expression profiling to identify pathways and genes encoded within the QTL interval responsive to HCHF diet. Gene expression profiles that differed between baseline and HCHF diets and were discordant between low and high LDL-C baboons were considered candidates encoding LDL-C phenotypic variation. To further prioritize the candidate genes, we integrated expression profiles of genes discordant between low and high LDL-C baboons with miRNA expression profiles. Genes and miRNAs were integrated based on miRNA target sites located in the gene and inverse expression between the miRNA and targeted gene. Differential expression of prioritized candidate genes was validated by QRT-PCR and Western blot. We identified four candidate genes that influence variation in LDL-C four genes are in of and A In addition, the identified miRNA targets provide for identification of underlying baboon LDL-C variation in from pedigreed baboons were previously to identify LDL-C animals genotyped (14Cox L.A. Mahaney M.C. Vandeberg J.L. Rogers J. A second-generation genetic linkage map of the baboon (Papio hamadryas) genome.Genomics. 2006; 88: 274-281Crossref PubMed Scopus (50) Google Scholar, J. Mahaney M.C. S.M. S. S. L.A. A. et genetic linkage map of the baboon (Papio hamadryas) genome based on human microsatellite 2000; PubMed Scopus Google and are 11 to that have a of the of phenotypic and analysis of the pedigreed baboon three pairs of half-sib baboons with for LDL-C were for this differed by at for LDL-C serum In addition, of selected were discordant for at loci within the region of C.M. D.L. Cox L.A. J.L. Mahaney M.C. Rogers J. VandeBerg J.L. cholesterol response to dietary cholesterol is on baboon of human chromosome Thromb. Vasc. Biol. PubMed Scopus Google Scholar). this study we the detection of genes influencing LDL-C variation by genetic of serum baboons were with was from the artery and serum was by were at were at Research of the Research a by the for and of The and experimental Lipid and lipoprotein traits were as (13Rainwater D.L. Cox L.A. Rogers J. VandeBerg J.L. Mahaney M.C. Localization of multiple pleiotropic genes for lipoprotein metabolism in baboons.J. Lipid Res. 2009; 50: 1420-1428Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, D.L. Mahaney M.C. S.M. between measures of HDL and Thromb. Vasc. Biol. PubMed Scopus Google Scholar, D.L. Kammerer C.M. Mahaney M.C. Rogers J. Cox L.A. J.L. VandeBerg J.L. Localization of genes that in Full Text Full Text PDF PubMed Scopus Google Scholar). were on a commercial diet were fed a HCHF of from from diet for seven weeks. 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To prioritize LDL-C QTL candidate genes, we used the in to the differentially expressed hepatic miRNAs in response to the HCHF diet with the expression profiles of the seven LDL-C QTL candidate genes to identify miRNA targets with expression profiles that are correlated with miRNA The LDL-C serum concentrations for the low and high LDL-C responders, before and at the of the diet are in and high LDL-C serum LDL-C for and HCHF LDL-C on LDL-C on HCHF LDL-C LDL-C are in Open in a are in low responders, we detected genes. these genes, were and were in response to the HCHF diet challenge. analysis pathways that were and in response to the HCHF diet challenge. pathways lipid metabolism, and pathways and and and high LDL-C responders, we detected genes. these genes, were and 117 were in response to the HCHF diet challenge. analysis that an of pathways (n = were or in response to the HCHF diet challenge. pathways factors, and metabolism. pathways and and metabolism of a of genes = were differentially expressed in the of high LDL-C responders in response to the HCHF diet with of low LDL-C responders = = We identified candidate genes encoding LDL-C concentration QTL in baboons. 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The in of genes in low LDL-C responders with high LDL-C responders that molecular mechanisms the HCHF response between the We that the candidate gene(s) encoding the LDL-C concentration QTL discordantly expressed between low and high LDL-C responders. the 2-LOD support interval of the LDL-C 117 of the genes were of were differentially expressed in response to HCHF diet in at of the LDL-C Seven of the genes were discordant between low and high LDL-C responders and were candidates encoding the LDL-C concentration QTL. factor and in lipoprotein metabolism L. and the implications for Thromb. Vasc. Biol. PubMed Scopus Google Scholar, R.E. Lipid the of high lipoprotein in lipid by cholesterol Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, L.L. J. R. J. responsive to dietary cholesterol hepatic and responsive primates Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, L.L. P. is a target for of coronary heart disease associated with Thromb. Vasc. Biol. PubMed Scopus Google Scholar, S. M.C. and in J. PubMed Scopus Google Scholar), were differentially expressed in response to the HCHF diet in we have identified candidate genes in lipid the identification of multiple related LDL-C trait QTLs overlapping the LDL-C concentration QTL that candidate genes underlying LDL-C concentration may pleiotropic and a role in networks related to lipid metabolism. We further that the candidate genes were regulated by miRNAs are that regulate gene of miRNAs has to in including and atherosclerosis A. A. J. et and of tissues in 2009; PubMed Scopus Google Scholar, S.C. of microRNAs in atherosclerosis and PubMed Scopus Google Scholar). Thus, we that candidate genes targeted by differentially expressed miRNAs in response to the diet in insights on mechanisms influencing dyslipidemia in baboons. we prioritized the LDL-C QTL candidate genes by with an miRNA target in the miRNAs response to HCHF diet in low and high LDL-C responders. we inverse expression profiles between gene expression and miRNA Using this we the of candidate genes encoding the LDL-C concentration QTL from to ERBB3, DGKA, and We validated candidate gene expression using we validated expression of candidate using Western and that expression is in with mRNA The baboon localized to on to the human or S. R. of with a with to Res. PubMed Scopus Google Scholar). and have to is associated with in human 2006; PubMed Scopus Google Scholar, S. is a of the and and J. PubMed Scopus Google Scholar). 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Using whole-genome expression QRT-PCR and Western for expression and of miRNA and mRNA expression we identified four candidate genes encoding an LDL-C concentration QTL on baboon chr 11 that may regulate lipid metabolism the and expression between low and high animals with inverse expression profiles of miRNAs these candidate genes their role in LDL-C metabolism. the four candidate genes identified in this are associated with lipid metabolism, these are genes in lipid that the chr 11 QTL for LDL-C concentration is encoded by multiple genes and that miRNAs regulate these genes. interaction are to the network the and the mechanisms by the HCHF diet regulate these miRNAs and we insights The of the candidate genes potential molecular genetic leading to dyslipidemia and atherosclerosis and that a systems biology approach for disease of LDL-C levels may of in the and including and and of and that leading to are candidate gene miRNA for the development of therapeutic are to due to of information on in the of

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