From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus

Journal name:
Nature
Volume:
466,
Pages:
714–719
Date published:
DOI:
doi:10.1038/nature09266
Received
Accepted

Abstract

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes.

At a glance

Figures

  1. Human chromosome 1p13 locus is preferentially associated with very small LDL and liver gene expression.
    Figure 1: Human chromosome 1p13 locus is preferentially associated with very small LDL and liver gene expression.

    a, Mean plasma lipid and lipoprotein particle levels in homozygotes for the minor haplotype of the 1p13 locus (minor allele of rs646776) versus homozygotes for the major haplotype (major allele of rs646776), normalized to the mean level in minor haplotype homozygotes, in the MDC-CC cohort (measured by ion mobility) and the PARC cohort (measured by gradient gel electrophoresis). LDL-L, large LDL; LDL-M, medium LDL; LDL-S, small LDL; LDL-VS, very small LDL. b, Relative gene positions in and around the 1p13 locus; * indicates position of rs646776. c, Mean expression of local genes in homozygotes for the major 1p13 haplotype (major allele of rs646776) versus heterozygotes versus homozygotes for the minor 1p13 haplotype (minor allele of rs646776), normalized to the mean level in major haplotype homozygotes, in samples of human liver, human subcutaneous adipose and human omental adipose. d, Mean expression of PSRC1, CELSR2, SORT1 and TCF7L2 (negative control) mRNA, standardized to B2M expression, and sortilin protein, standardized to α-tubulin, in samples of human liver from homozygotes for the major 1p13 haplotype (major allele of rs12740374) versus heterozygotes versus homozygotes for the minor 1p13 haplotype (minor allele of rs12740374) if available, normalized to the mean level in major haplotype homozygotes. P values derived from linear regression analyses or unpaired t-test. Error bars show s.e.m.

  2. rs12740374 is responsible for haplotype-specific difference in transcriptional activity.
    Figure 2: rs12740374 is responsible for haplotype-specific difference in transcriptional activity.

    a, Map of 1p13 SNPs genotyped in ~20,000 individuals of European descent relative to CELSR2 and PSRC1 genes. The six SNPs with strongest association with LDL-C (indicated with boxes), comprising a single haplotype, define the 6.1kb region between the stop codons of the two genes. b, Firefly luciferase expression from constructs transfected into Hep3B human hepatoma cells. Both the major (darker colours) and minor (lighter colours) haplotypes of the 6.1kb region were subcloned in forward and reverse orientations into a basal firefly luciferase construct with the SV40 promoter. Shown are ratios of firefly luciferase expression to Renilla luciferase expression (expressed from cotransfected plasmid), measured 48h after transfection, normalized to the mean ratio from the major haplotype, forward orientation construct. Error bars show s.e.m., n = 2. c, Both the major and minor haplotypes of a minimal 2.1kb region were subcloned into the basal construct. Single nucleotide alterations were introduced individually into the minor haplotype, changing minor alleles of SNPs into major alleles. Shown are ratios of firefly luciferase expression to Renilla luciferase expression normalized to the mean ratio from the major haplotype construct. Error bars show s.e.m., n = 4.

  3. rs12740374 alters a C/EBP transcription factor binding site.
    Figure 3: rs12740374 alters a C/EBP transcription factor binding site.

    a, The human DNA sequence surrounding rs12740374, major and minor alleles, and orthologous DNA sequence in mouse. The major allele of rs12740374 disrupts one of two core elements (position 2, 3 and 8, 9) in the predicted consensus binding site on which a C/EBP dimer binds21. b, Electrophoretic mobility shift assays (EMSA) with labelled probes matching the C/EBP consensus binding site18, the rs12740374 minor allele (T) sequence, and the rs12740374 major allele (G) sequence. Competition assays were performed with 100-fold excess of cold probe. Either of two C/EBPα antibodies was used to compete for binding and/or shift the protein–DNA complex. c, Relative firefly luciferase expression from constructs with haplotypes of 2.1kb region transfected into Hep3B cells. Single nucleotide alterations were introduced into constructs as indicated, altering rs12740374 and the three other core recognition nucleotides in the predicted C/EBP binding site. d, e, Relative firefly luciferase expression from constructs with haplotypes of 6.1kb region transfected into (d) Hep3B human hepatoma cells with or without concomitant transduction with A-C/EBP (dominant negative C/EBP) cDNA via lentivirus and (e) NIH 3T3 fibroblasts with or without concomitant transduction with C/EBPα cDNA via lentivirus. f, Relative SORT1 expression, determined as a ratio with B2M expression by qRT–PCR, in Hep3B cells (homozygous major (GG) at rs12740374) or SK-HEP-1 human hepatoma cells (heterozygous (GT) at rs12740374) with or without concomitant transduction with A-C/EBP cDNA via lentivirus. Error bars show s.e.m., n = 3 for each experiment.

  4. Overexpression or knockdown of Sort1 in mouse liver alters plasma lipids and lipoproteins.
    Figure 4: Overexpression or knockdown of Sort1 in mouse liver alters plasma lipids and lipoproteins.

    Adeno-associated virus 8 (AAV8) vectors either containing no gene, murine Sort1 cDNA or murine Psrc1 cDNA were administered via intraperitoneal injection; phosphate-buffered saline or siRNA duplex targeting firefly luciferase or mouse Sort1 and prepared in lipidoid formulation was administered weekly via tail vein injection at 2.0mgkg−1. Plasma samples were collected before injection and at various time points after injection, and were subjected: individually to analytical chemistry (Mira autoanalyser) to measure total cholesterol (a, e, g); as pooled samples to FPLC (d, f, h), from which fractions 10 to 26 were used to calculate LDL-C levels (a, e, g); individually to NMR to measure LDL particle concentrations (b). P values calculated with unpaired t-test, shown if P<0.05. Error bars show s.e.m. a–d, Apobec1−/−; APOB Tg mice (five mice per group). b, NMR measurements at 6weeks. c, Mice were injected intraperitoneally with Pluronic F-127 detergent to block VLDL triglyceride lipolysis and permit assessment of the rate of VLDL secretion. Plasma samples were collected at baseline, 1h, 2h and 4h after injection. VLDL particle concentrations were measured from pooled samples with NMR. e, f, Apobec1−/−; APOB Tg mice (five mice per group). g, h, Ldlr−/− mice (five mice per group).

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Author information

  1. These authors contributed equally to this work.

    • Kiran Musunuru,
    • Alanna Strong,
    • Sekar Kathiresan &
    • Daniel J. Rader

Affiliations

  1. Cardiovascular Research Center and Center for Human Genetic Research, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA

    • Kiran Musunuru,
    • Noemi E. Lee,
    • Tim Ahfeldt,
    • Nicolas Kuperwasser,
    • Vera M. Ruda,
    • James P. Pirruccello,
    • Kenechi G. Ejebe,
    • Chad A. Cowan &
    • Sekar Kathiresan
  2. Broad Institute, Cambridge, Massachusetts 02142, USA

    • Kiran Musunuru,
    • James P. Pirruccello,
    • Jennifer L. Hall,
    • Kenechi G. Ejebe,
    • Chad A. Cowan &
    • Sekar Kathiresan
  3. Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA

    • Kiran Musunuru
  4. Institute for Translational Medicine and Therapeutics, Institute for Diabetes, Obesity and Metabolism, and Cardiovascular Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Alanna Strong,
    • Katherine V. Sachs,
    • Xiaoyu Li,
    • Hui Li &
    • Daniel J. Rader
  5. Alnylam Pharmaceuticals, Inc., Cambridge, Massachusetts 02142, USA

    • Maria Frank-Kamenetsky,
    • Jamie Wong,
    • William Cantley,
    • Timothy Racie,
    • Victor Koteliansky &
    • Kevin Fitzgerald
  6. Department of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany

    • Tim Ahfeldt
  7. Laboratory of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Brian Muchmore &
    • Ludmila Prokunina-Olsson
  8. Program in Cardiovascular Translational Genomics, Lillehei Heart Institute, University of Minnesota, Minneapolis, Minnesota 55455, USA

    • Jennifer L. Hall
  9. Sage Bionetworks, Seattle, Washington 98109, USA

    • Eric E. Schadt
  10. Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada

    • Carlos R. Morales
  11. The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA

    • Sissel Lund-Katz &
    • Michael C. Phillips
  12. Department of Clinical Sciences, Skania University Hospital, Lund University, SE-20502 Malmö, Sweden

    • Marju Orho-Melander &
    • Olle Melander
  13. Children’s Hospital Oakland Research Institute, Oakland, California 94609, USA

    • Ronald M. Krauss

Contributions

K.M., A.S., M.F.-K., N.E.L., T.A., K.V.S., X.L., H.L., N.K., V.M.R., J.J.P., B.M., L.P.-O., J.L.H., E.E.S., C.R.M., S.L.-K., M.C.P., J.W., W.C., T.R., K.G.E., M.O.-M., O.M. and R.M.K. carried out experimental work and/or performed data analysis. V.K., K.F., C.A.C., S.K. and D.J.R. supervised the study. K.M., A.S., S.K. and D.J.R. conceived and designed the study. K.M. wrote the manuscript.

Competing financial interests

Competing interests: M.F.-K., J.W., W.C., T.R., V.K. and K.F. are employees of Alnylam Pharmaceuticals. R.M.K. has received research support from Quest Diagnostics related to work presented in this manuscript The other authors declare no competing financial interests.

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Comments

  1. Report this comment #13554

    kevin lu said:

    Wonderful work. Before we could develope any potential drugs targeting sortilin 1, we really have to sort out the relationship between the expression of sort1 in the liver and plasma VLDL/LDL cholesterol levels (negative association between hepatic sort1 expression and VLDL secretion in this nature paper). Based on the lastest paper from the cell metabolism (Kjolby M. etal), an opposite relationship (positive association between hepatic sort1 expression and VLDL secretion) was reported.

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