Statins are widely used to reduce cardiovascular disease risk by lowering LDL cholesterol (C), but the magnitude of this effect varies widely among individuals. The overall objective of this proposal is to provide evidence in support of a novel pharmacogenetic mechanism contributing to this variation that involves alternative splicing of the mRNA for HMG-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis that is the target of statin inhibition. We have recently shown using lymphocyte cell lines derived from subjects in the Cholesterol and Pharmacogenetics (CAP) study, that increased magnitude of simvastatin-induced expression of a spliced HMGCR transcript lacking exon 13 in vitro is significantly correlated with smaller reductions in plasma total and LDL-C in response to simvastatin treatment of the same individuals in vivo. Moreover, we have used selective siRNA knockdown of the normal HMGCR transcript (containing exon 13) in cultured cells to provide preliminary evidence that the spliced variant encodes an HMGCR isoform that is relatively resistant to statin inhibition. Finally, we have found that the extent of exon 13 skipping is associated with a common HMGCR single nucleotide polymorphism (SNP) in intron 13 that we have also found to be associated with the magnitude of plasma LDL-C response to simvastatin. These observations lead to the hypotheses that: 1) the HMGCR mRNA without exon 13 encodes a statin resistant enzyme isoform and;2) the level of expression of this isoform modulates inhibition of cellular cholesterol synthesis in response to statin treatment. Overall, confirmation of these hypotheses would lead to the first demonstration that genetically-influenced variation in alternative splicing can contribute to inter-individual differences in statin response. To test these hypotheses, we will: 1) determine the effects of exon 13 deletion on HMGCR catalytic activity and sensitivity to statin inhibition using purified recombinant HMGCR catalytic domains with and without exon 13 both independently (1A) and as part of a heterogenous tetramer comprised of both HMGCR isoforms (1B);and 2) assess statin effects on HMGCR catalytic activity, cholesterol biosynthesis, and cellular cholesterol content in both cells over-expressing varying amounts of the HMGCR exon 13 splice variant (2A) and immortalized lymphocyte lines that express high versus low levels of the exon 13 splice variant in response to statin treatment (2B). Since HMGCR alternative splicing is genetically regulated, elucidating the basis for the relationship of HMGCR alternative splicing to statin response, would have a major impact on the field of pharmacogenetics by establishing a new role for post-transcriptional genetic regulation in modulating drug efficacy. Moreover since nearly 1/3 of statin-treated patients do not meet lipid-lowering goals, the identification of a statin-resistant HMGCR isoform directly related to its expression has potential utility in the emerging field of personalized medicine as a means of helping to predict an individual's response to statin treatment, with the potential of optimizing use of statin drugs in cardiovascular disease prevention. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to test a new genetically-influenced mechanism that can contribute to inter- individual variation in drug response. Specifically, we seek to test whether alternative splicing of HMG-CoA reductase, the target of statin inhibition, produces a statin-resistant isoform whose expression contributes to inter-individual differences in the magnitude of LDL cholesterol reduction observed in statin-treated patients. The results of this research could yield improvement in the ability to identify individuals most likely to achieve cardiovascular benefit from statin treatment, and new pharmacologic approaches for increasing statin efficacy.