DESCRIPTION (provided by applicant): Despite the widespread use of cholesterol-lowering medications, principally the statin drugs, myocardial infarction remains the leading cause of death in the world. There is therefore a critical need for new medications for the prevention of myocardial infarction. We have used genome-wide association studies and exome sequencing studies in humans to identify a number of novel genes related to cholesterol metabolism. Recently, we applied exome sequencing to two healthy siblings in a family with an unusual lipid pattern that we have termed "familial combined hypolipidemia"-extremely low low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels. We discovered that the siblings were compound heterozygotes for two distinct nonsense mutations-S17X and E129X-in ANGPTL3 (encoding the angiopoietin-like 3 protein). ANGPTL3, a protein exclusively synthesized in liver and secreted into the bloodstream, has been reported to inhibit lipoprotein lipase (encoded by LPL) and endothelial lipase (encoded by LIPG), increasing plasma TG and HDL-C levels in rodents. Our finding of ANGPTL3 mutations highlights a role for the gene in LDL-C metabolism in humans, as well as implicating the gene as a potential therapeutic target for LDL-C reduction and prevention of MI. Having discovered a novel link between ANGPTL3 and LDL-C in humans, we now seek to define the mechanism by which the gene alters LDL-C in the blood. We found that carriers of ANGPTL3 nonsense mutations had decreased rates of very-low-density lipoprotein (VLDL) apolipoprotein B (apoB) production and increased fractional catabolic rates for LDL apoB. Thus, we hypothesize that ANGPTL3 acts directly in the human liver to regulate hepatocellular VLDL secretion and LDL clearance, thereby modulating LDL-C levels in the blood. To test this hypothesis, we seek to evaluate the effects of the S17X and E129X mutations on ANGPTL3 function in human-derived hepatocytes. We propose to do this in the most rigorous possible way by (1) using human genome editing with cutting-edge TAL effector nuclease (TALEN) technology to generate isogenic human pluripotent stem cell (hPSC) lines with or without the S17X or E129X mutations; (2) differentiating the hPSC lines into hepatocytes; and (3) assessing VLDL/LDL processing in the hepatocytes. We also propose to generate an ANGPTL3 reporter hepatocyte cell line with which to perform a small molecule screen for compounds that reduce ANGPTL3 expression and thereby reduce blood cholesterol levels. If successful, our approach of using genome-edited, hPSC-derived cells to study the effects of disease-associated mutations could be applied widely to a large variety of human genetic disorders.

PUBLIC HEALTH RELEVANCE: To develop new therapies to prevent heart attack, the leading cause of death worldwide, we have performed genetic studies to identify novel cholesterol genes. We discovered a family with extremely low cholesterol levels-a condition called familial combined hypolipidemia-to have two mutations in the ANGPTL3 gene using "exome" sequencing. Our goal is to understand how the ANGPTL3 mutations affect cholesterol levels and potentially apply that knowledge to develop a new type of cholesterol-lowering medication.