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LDL胆固醇（C）来降低心血管疾病风险，但这种作用的程度在个体之间差异很大。本提案的总体目标是提供证据支持导致这种变异的新型药物遗传学机制，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靶点。我们最近使用来自胆固醇和药物遗传学（CAP）研究受试者的淋巴细胞系显示，体外辛伐他汀诱导的剪接HMGCR转录物（缺失外显子13）表达的增加幅度与体内辛伐他汀治疗相同个体后血浆总胆固醇和LDL-C的较小降低显著相关。此外，我们使用选择性siRNA敲除培养细胞中正常的HMGCR转录物（含有外显子13），以提供初步证据，即剪接变体编码对他汀类药物抑制具有相对抗性的HMGCR同种型。最后，我们发现外显子13跳跃的程度与内含子13中常见的HMGCR单核苷酸多态性（SNP）相关，我们还发现该SNP与血浆LDL-C对辛伐他汀的反应幅度相关。这些观察结果导致以下假设：1）不含外显子13的HMGCR mRNA编码他汀类药物耐药酶亚型; 2）该亚型的表达水平调节了对他汀类药物治疗的细胞胆固醇合成抑制。总体而言，这些假设的证实将导致第一次证明，在选择性剪接的遗传影响的变化可以有助于他汀类药物反应的个体间差异。为了检验这些假设，我们将：1）使用具有和不具有外显子13的纯化的重组HMGCR催化结构域，确定外显子13缺失对HMGCR催化活性和对他汀抑制的敏感性的影响，所述催化结构域独立地（1A）和作为由两种HMGCR同种型组成的异源四聚体的一部分（1B）;和2）评估他汀类药物对HMGCR催化活性，胆固醇生物合成，和过表达不同量的HMGCR外显子13剪接变体的两种细胞中的细胞胆固醇含量（2A）以及响应他汀治疗表达高水平对低水平外显子13剪接变体的永生化淋巴细胞系（2B）。由于HMGCR选择性剪接是遗传调节的，阐明HMGCR选择性剪接与他汀类药物反应的关系的基础，将通过建立转录后遗传调节在调节药物疗效中的新作用对药物遗传学领域产生重大影响。此外，由于近1/3的他汀类药物治疗的患者不能达到降脂目标，因此鉴定与其表达直接相关的他汀类药物耐药HMGCR同种型在个性化医疗的新兴领域中具有潜在的实用性，作为帮助预测个体对他汀类药物治疗的反应的手段，具有优化他汀类药物在心血管疾病预防中的使用的潜力。公共卫生相关性：本提案的目的是测试一种新的遗传影响机制，该机制可能导致药物反应的个体间差异。具体来说，我们试图测试是否选择剪接HMG-CoA还原酶，抑制他汀类药物的目标，产生他汀类药物耐药亚型，其表达有助于个体间差异的LDL胆固醇降低的幅度观察他汀类药物治疗的患者。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，以及增加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.