The discovery of new and effective treatments for human cardiovascular disease (CVD) requires the identification and validation of novel disease mechanisms. Recently, studies of genomic variation entered a new phase, in which unbiased genome-wide association studies (GWAS) can identify novel genetic loci associated with common diseases. We have recently described 95 loci associated with blood lipid levels LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), or triglycerides (TG), which are strongly associated with risk for CVD. Much work will be needed to convert the novel associations into functional insights and, ultimately, therapies to reduce the risk of CVD. A key step is to determine how these genetic loci affect phenotypes in human tissue types relevant to lipid metabolism, principally liver and adipose. We have performed expression quantitative trait locus (eQTL) analyses of genotype vs. gene expression in surgical liver and adipose tissue samples from patients;from this work, we found strong associations between a number of lipid-associated tag single nucleotide polymorphisms (SNPs) and either hepatic or adipose expression of nearby genes. These observations suggest that causal SNPs in linkage disequilibrium (LD) with the tag SNPs directly influence the expression of causal genes that are responsible for changes in blood lipid levels in humans. Identifying these causal SNPs and causal genes would lead to insights into the molecular mechanisms by which the DNA variants drive phenotypic changes in liver and adipose and, ultimately, affect the risk of disease. Our general strategy is to combine several innovations to identify casual SNPs. We will: (1) perform high- throughput screening of candidate SNPs in eQTL loci for alteration of reporter gene expression in the appropriate tissue type, using a novel massively parallel reporter assay (MPRA), to prioritize SNPs for further study;(2) use human genome editing with cutting-edge TAL effector nuclease (TALEN) technology to alter each high-priority SNP in human pluripotent stem cells (hPSCs), so as to generate isogenic cell lines that differ only at the SNP;(3) differentiate the isogenic hPSCs into the appropriate tissue type;and (4) measure nearby gene expression to confirm that the SNP is truly causal for the eQTL. We propose to implement this general strategy for 57 lipid-associated loci with eQTLs in human liver and adipose. Success in completing this project will not only provide fresh new insights into the biology of lipid metabolism, but will also establish a new methodological paradigm by which investigators can determine which DNA sequence variants identified in next-generation human genetic studies underlie the genetic basis of complex phenotypes.

Public Health Relevance

To develop new therapies to prevent cardiovascular disease, the leading cause of death worldwide, we have performed genetic studies to identify novel cholesterol genes. We have identified numerous DNA variants associated with cholesterol;our challenge is to determine which of the DNA variants affect gene function. We will use novel methodologies to (1) rapidly screen through thousands of DNA variants to determine which ones affect gene expression and (2) insert these DNA variants into stem cells to determine which genes are affected and thus are likely to be cholesterol genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM104464-02
Application #
8642199
Study Section
Special Emphasis Panel (ZGM1-GDB-7 (CP))
Program Officer
Krasnewich, Donna M
Project Start
2013-04-01
Project End
2017-03-31
Budget Start
2014-04-01
Budget End
2015-03-31
Support Year
2
Fiscal Year
2014
Total Cost
$307,219
Indirect Cost
$52,440
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
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Lv, Wenjian; Qiao, Lyon; Petrenko, Nataliya et al. (2018) Functional Annotation of TNNT2 Variants of Uncertain Significance With Genome-Edited Cardiomyocytes. Circulation 138:2852-2854
Musunuru, Kiran; Bernstein, Daniel; Cole, F Sessions et al. (2018) Functional Assays to Screen and Dissect Genomic Hits: Doubling Down on the National Investment in Genomic Research. Circ Genom Precis Med 11:e002178
Wang, Xiao; Raghavan, Avanthi; Peters, Derek T et al. (2018) Interrogation of the Atherosclerosis-Associated SORT1 (Sortilin 1) Locus With Primary Human Hepatocytes, Induced Pluripotent Stem Cell-Hepatocytes, and Locus-Humanized Mice. Arterioscler Thromb Vasc Biol 38:76-82
Musunuru, Kiran (2017) Genome Editing: The Recent History and Perspective in Cardiovascular Diseases. J Am Coll Cardiol 70:2808-2821
Chadwick, Alexandra C; Wang, Xiao; Musunuru, Kiran (2017) In Vivo Base Editing of PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) as a Therapeutic Alternative to Genome Editing. Arterioscler Thromb Vasc Biol 37:1741-1747
Pashos, Evanthia E; Park, YoSon; Wang, Xiao et al. (2017) Large, Diverse Population Cohorts of hiPSCs and Derived Hepatocyte-like Cells Reveal Functional Genetic Variation at Blood Lipid-Associated Loci. Cell Stem Cell 20:558-570.e10
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Giani, Felix C; Fiorini, Claudia; Wakabayashi, Aoi et al. (2016) Targeted Application of Human Genetic Variation Can Improve Red Blood Cell Production from Stem Cells. Cell Stem Cell 18:73-78
Beaudoin, Mélissa; Gupta, Rajat M; Won, Hong-Hee et al. (2015) Myocardial Infarction-Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary Arteries. Arterioscler Thromb Vasc Biol 35:1472-1479