HMG-CoA reductase alternative splicing and LDL response to statin

描述（由申请人提供）：他汀类药物被广泛用于通过降低低密度脂蛋白胆固醇(C)来降低心血管疾病的风险，但这种效果的大小在个体之间差异很大。该提案的总体目标是为支持一种新的药物遗传机制提供证据，该机制涉及HMG-CoA还原酶（HMGCR） mRNA的选择性剪接，HMGCR是胆固醇生物合成中的限速酶，是他汀类药物抑制的目标。我们最近使用来自胆固醇和药物遗传学（CAP）研究对象的淋巴细胞系表明，辛伐他汀诱导的体外缺乏外显子13的剪接HMGCR转录物表达量的增加与体内相同个体的辛伐他汀治疗后血浆总含量和LDL-C的较小降低显著相关。此外，我们在培养细胞中使用选择性siRNA敲低正常HMGCR转录本（包含外显子13），以提供初步证据，证明剪接的变体编码的HMGCR亚型对他汀类药物抑制具有相对抗性。最后，我们发现外显子13跳跃的程度与13内含子中常见的HMGCR单核苷酸多态性（SNP）有关，我们还发现该多态性与血浆LDL-C对辛伐他汀的反应程度有关。这些观察结果导致以下假设：1)没有外显子13的HMGCR mRNA编码他汀类药物抗性酶亚型；2)这种异构体的表达水平调节了他汀类药物治疗对细胞胆固醇合成的抑制作用。总的来说，这些假设的证实将首次证明，受遗传影响的可变剪接变异可能导致他汀类药物反应的个体间差异。为了验证这些假设，我们将：1)确定外显子13缺失对HMGCR催化活性和他汀类药物抑制敏感性的影响，使用独立（1A）和由两种HMGCR亚型组成的异质四聚体（1B）的纯化重组HMGCR催化结构域（带和不带外显子13）；2)评估他汀类药物对HMGCR催化活性、胆固醇生物合成和细胞胆固醇含量的影响，这两种细胞过度表达不同数量的HMGCR外显子13剪接变体（2A），以及他汀类药物治疗后表达高水平和低水平外显子13剪接变体的永生化淋巴细胞系（2B）。由于HMGCR选择性剪接受基因调控，阐明HMGCR选择性剪接与他汀类药物反应关系的基础，将为转录后基因调控在调节药物疗效中的新作用奠定基础，从而对药物遗传学领域产生重大影响。此外，由于近1/3的他汀类药物治疗患者没有达到降脂目标，鉴定与其表达直接相关的他汀类药物耐药HMGCR异构体在新兴的个性化医疗领域具有潜在的实用性，可以帮助预测个体对他汀类药物治疗的反应，并有可能优化他汀类药物在心血管疾病预防中的应用。公共卫生相关性：本提案的目的是测试一种新的基因影响机制，这种机制可以促进药物反应的个体间差异。具体来说，我们试图测试HMG-CoA还原酶（他汀抑制的目标）的选择性剪接是否会产生他汀耐药异构体，其表达有助于他汀治疗患者观察到的LDL胆固醇降低幅度的个体差异。这项研究的结果可以提高识别最有可能从他汀类药物治疗中获得心血管益处的个体的能力，以及提高他汀类药物疗效的新药理学方法。

DESCRIPTION (provided by applicant): 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.