Novel genetic and molecular regulation of HDL metabolism

Coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide. Low levels of high-density lipoprotein cholesterol (HDL-C) constitute a major independent risk factor for CAD, influenced by a combination of genetic and environmental factors. In this thesis, we aimed to advance our understanding of the genetic regulation of HDL-C, through the identification and characterization of novel candidate genes, and to delineate the molecular mechanisms underlying HDL biogenesis, in the hopes of better elucidating the complexities of HDL metabolism. First, we examined whether variation at the PCSK5 gene locus influences HDL-C levels. Through familial segregation analyses and two-stage genetic association studies in unrelated subjects of French Canadian descent and low HDL-C Finnish families (ntotal=883), we reported a region-wide significance of PCSK5 SNPs with HDL-C, suggesting that genetic variability at the PCSK5 locus regulates HDL-C levels, possibly through the inactivation of endothelial lipase. Second, to search for rare-low frequency variants responsible for low HDL-C, we used whole exome sequencing in a multigenerational French Canadian family (n=75) with HDL-C<5th age-sex specific percentile. Through this approach, we identified a complex combination of two non-synonymous variants, in the ATP-binding cassette transporter (ABCA1) and the lipoprotein lipase genes, predicted to be damaging and causing low HDL-C. These results emphasize the need for exome sequencing of complex lipid traits in unexplained familial cases. Third, we sought to characterize a novel genetic determinant involved in HDL-C regulation. Recent studies from our group identified the WW domain-containing oxidoreductase (WWOX) gene locus to be associated with low serum HDL-C levels in 9,798 subjects. In this study, we examined the role of WWOX in lipoprotein and HDL metabolism using a combination of in vivo functional studies, by means of total Wwox knock-out and Wwox liver-specific mouse models, gene microarray and next generation resequencing analyses in HDL-deficient French Canadian families. We demonstrated that the effects of WWOX on lipoprotein metabolism involve multiple mechanisms, including cholesterol homeostasis, apoA-I and ABCA1-mediated pathways and fatty acid/triglyceride metabolism. Fourth, with novel insight into genetic regulations of HDL metabolism, we focused on elucidating the mechanistic basis of nascent HDL formation. Specifically, we investigated the lipidation of ApoA-I and its interaction with an ABCA1/phosphatidylcholine(PC)-rich microdomain, the high-capacity binding site (HCBS). Using sucrose gradient fractionation, we also demonstrated that the ABCA1/ HCBS partitions to nonraft domains, thus playing a pivotal role in the selective desorption of PC molecules by apoA-I, creating an optimal environment for nascent HDL formation and cholesterol release.In summary, this work has collectively advanced our understanding of the molecular and genetic basis of HDL metabolism. It has brought to light novel genes governing plasma HDL-C levels in humans, highlighting the need to use multiple integrative genetic approaches to identify causal common and rare variants conferring susceptibility to low HDL-C. These findings have also helped elucidate the mechanistic basis of the nascent HDL genesis pathway. Together, this thesis has combined genetic and biochemical strategies to provide a more comprehensive understanding of the complexities of the HDL metabolism and cellular cholesterol transport, which may lead to the characterization of novel therapeutic targets for CAD.

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