title: Multiple Hepatic Regulatory Variants at the GALNT2 GWAS Locus Associated with High-Density Lipoprotein Cholesterol.
abstract: Genome-wide association studies (GWASs) have identified more than 150 loci associated with blood lipid and cholesterol levels; however, the functional and molecular mechanisms for many associations are unknown. We examined the functional regulatory effects of candidate variants at the GALNT2 locus associated with high-density lipoprotein cholesterol (HDL-C). Fine-mapping and conditional analyses in the METSIM study identified a single locus harboring 25 noncoding variants (r(2) > 0.7 with the lead GWAS variants) strongly associated with total cholesterol in medium-sized HDL (e.g., rs17315646, p = 3.5 × 10(-12)). We used luciferase reporter assays in HepG2 cells to test all 25 variants for allelic differences in regulatory enhancer activity. rs2281721 showed allelic differences in transcriptional activity (75-fold [T] versus 27-fold [C] more than the empty-vector control), as did a separate 780-bp segment containing rs4846913, rs2144300, and rs6143660 (49-fold [AT(-) haplotype] versus 16-fold [CC(+) haplotype] more). Using electrophoretic mobility shift assays, we observed differential CEBPB binding to rs4846913, and we confirmed this binding in a native chromatin context by performing chromatin-immunoprecipitation (ChIP) assays in HepG2 and Huh-7 cell lines of differing genotypes. Additionally, sequence reads in HepG2 DNase-I-hypersensitivity and CEBPB ChIP-seq signals spanning rs4846913 showed significant allelic imbalance. Allelic-expression-imbalance assays performed with RNA from primary human hepatocyte samples and expression-quantitative-trait-locus (eQTL) data in human subcutaneous adipose tissue samples confirmed that alleles associated with increased HDL-C are associated with a modest increase in GALNT2 expression. Together, these data suggest that at least rs4846913 and rs2281721 play key roles in influencing GALNT2 expression at this HDL-C locus.
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Tamara S
authors.initials: TS
authors.lastname: Roman
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Amanda F
authors.initials: AF
authors.lastname: Marvelle
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Marie P
authors.initials: MP
authors.lastname: Fogarty
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Swarooparani
authors.initials: S
authors.lastname: Vadlamudi
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Arlene J
authors.initials: AJ
authors.lastname: Gonzalez
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Martin L
authors.initials: ML
authors.lastname: Buchkovich
authors.affiliation: Department of Biostatistics and Center for Statistical Genetics, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Jeroen R
authors.initials: JR
authors.lastname: Huyghe
authors.affiliation: Department of Biostatistics and Center for Statistical Genetics, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Fuchsberger
authors.affiliation: Department of Biostatistics and Center for Statistical Genetics, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Anne U
authors.initials: AU
authors.lastname: Jackson
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biomedical Engineering, Center for Public Health Genomics, University of Virginia, Charlottesville, VA 22908, USA.
authors.forename: Mete
authors.initials: M
authors.lastname: Civelek
authors.affiliation: Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Deparment of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Aldons J
authors.initials: AJ
authors.lastname: Lusis
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Gaulton
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Praveen
authors.initials: P
authors.lastname: Sethupathy
authors.affiliation: Computational Medicine, Institute of Health Sciences, University of Oulu, 90014 Oulu, Finland.
authors.forename: Antti J
authors.initials: AJ
authors.lastname: Kangas
authors.affiliation: Computational Medicine, Institute of Health Sciences, University of Oulu, 90014 Oulu, Finland; Nuclear Magnetic Resonance Metabolomics Laboratory, School of Pharmacy, University of Eastern Finland, 70211 Kuopio, Finland.
authors.forename: Pasi
authors.initials: P
authors.lastname: Soininen
authors.affiliation: Computational Medicine, Institute of Health Sciences, University of Oulu, 90014 Oulu, Finland; Nuclear Magnetic Resonance Metabolomics Laboratory, School of Pharmacy, University of Eastern Finland, 70211 Kuopio, Finland; Oulu University Hospital, 90220 Oulu, Finland; Computational Medicine, School of Social and Community Medicine and Medical Research Council Integrative Epidemiology Unit, University of Bristol, Bristol BS8 2BN, UK.
authors.forename: Mika
authors.initials: M
authors.lastname: Ala-Korpela
authors.affiliation: Department of Medicine, University of Eastern Finland and Kuopio University Hospital, 70210 Kuopio, Finland.
authors.forename: Johanna
authors.initials: J
authors.lastname: Kuusisto
authors.affiliation: National Human Genome Research Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Francis S
authors.initials: FS
authors.lastname: Collins
authors.affiliation: Department of Medicine, University of Eastern Finland and Kuopio University Hospital, 70210 Kuopio, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Laakso
authors.affiliation: Department of Biostatistics and Center for Statistical Genetics, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Boehnke
authors.affiliation: Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address: mohlke@med.unc.edu.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Mohlke
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism; D000483:Alleles; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics* / Q000378:metabolism; D008076:Cholesterol, HDL / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D002874:Chromosome Mapping; D024202:Electrophoretic Mobility Shift Assay; D005787:Gene Frequency; D017930:Genes, Reporter; D015894:Genome, Human; D055106:Genome-Wide Association Study; D006239:Haplotypes; D056945:Hep G2 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D061251:Primary Cell Culture; D011485:Protein Binding; D040641:Quantitative Trait Loci; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D022782:CCAAT-Enhancer-Binding Protein-beta; D008076:Cholesterol, HDL; D002843:Chromatin; D008156:Luciferases; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C545012:CEBPB protein, human