家族性高胆固醇血症(FH)是一种与早期动脉粥样硬化和心血管疾病风险增加相关的遗传性疾病。LDLR有害突变与FH相关,然而,一些错义变异体在FH致病性中的作用仍有待阐明.这项研究探索了LDLR错义变体对蛋白质结构的预测性影响,并研究了它们对CRISPR/Cas9构建体转染的HepG2细胞中LDLR表达的功能影响。选择FH(n=287)和非FH患者(n=45),和血脂谱是从医疗记录中获得的。使用外显子靶向基因测序策略鉴定LDLR变体,考虑到其在较不费力的过程中增加与FH相关的最可能变体的鉴定步骤中的准确性的成本效益。LDLR变体是根据Clinvar和计算机预测工具中发现的相互冲突的致病性结果进行选择的。以及基于受影响的LDLR结构域和考虑小等位基因频率(MAF)<0.05的较不常见的变体。使用分子建模研究来预测LDLR错义变体对蛋白质结构的影响。使用CRISPR/Cas9系统构建重组LDLR变体并用于转染HepG2细胞。通过流式细胞术评估LDLR的表达和活性,西方印迹,和共聚焦显微镜。变异体rs121908039的分子对接分析(c.551G>Ap.C184Y),rs879254797(c.1118G>Ap.G373D),rs28941776(c.1646G>Ap.G549D),rs750518671(c.2389G>Cp.V797L),rs5928(c.2441G>Ap.R814Q)和rs137853964(c.2479G>Ap.V827I)揭示了蛋白质稳定性和分子内相互作用的改变。使用流式细胞术和蛋白质印迹进行功能测定以评估LDLR表达,而LDLR活性通过流式细胞术和显微镜分析进行评估。结果表明,变种p.G373D,p.V797L和p.R814Q降低了LDLR的表达和活性,改变了脂质的定位和积累,在转染的HepG2细胞中。FH患者血清总胆固醇较高,LDLc和甘油三酯比非FH组,但特别是FH携带者的变体p.G549D和p.V797L也显示与非FH组相比更高水平的LDLc。总之,变异p.C184Y增加稳定性和与EGF结构域的相互作用,因此损害LDLR表达。变体p.G373D和p.G549D显示能量变化,临床表现和LDLR活性降低。变体p.V797L对能量变化几乎没有影响,但与高LDLc水平相关。p.R814Q变体潜在地破坏受体锚定并且具有增加的胆固醇和LDL水平。p.V827I变体没有显示出显著的相互作用并且可以破坏受体锚定。
Familial Hypercholesterolemia (FH) is a genetic disorder associated with premature atherosclerosis and increased risk of cardiovascular diseases. LDLR deleterious mutations are associated with FH, however the role of some missense variants in FH pathogenicity remains to be elucidated. This study explored the predictive impact of LDLR missense variants on protein structure and investigated their functional effects on LDLR expression in HepG2 cells transfected with CRISPR/Cas9 constructs. FH (n = 287) and non-FH patients (n = 45) were selected, and lipid profile was obtained from medical records. LDLR variants were identified using an exon-targeted gene sequencing strategy, considering its cost-effective to increase accuracy in the identification step of the most likely FH-related variants in a less laborious process. LDLR variants were selected based on conflicting pathogenicity results found in Clinvar, in silico prediction tools, affected LDLR domains, and less common variants considering minor allele frequency < 0.05. Molecular modeling studies were used to predict the effects of LDLR missense variants on protein structure. Recombinant LDLR variants were constructed using CRISPR/Cas9 system and were used to transfect HepG2 cells. Functional assays in transfected cells were performed to assess LDLR expression using flow cytometry and western blotting, and LDLR activity using flow cytometry and confocal microscopy. The variants rs121908039 (c.551G>A, p.C184Y), rs879254797 (c.1118G>A, p.G373D), rs28941776 (c.1646G>A, p.G549D), rs750518671 (c.2389G>C, p.V797L), rs5928 (c.2441G>A, p.R814Q) and rs137853964 (c.2479G>A, p.V827I) were selected for molecular docking analysis. The p.C184Y exhibited a favorable energy change for protein stability due to its interaction with EGF-A/EGF-B regions; p.G373D and p.G549D displayed intermediate energy changes; and p.R814Q and p.V827I showed smaller energy changes. The results of functional assays showed that p.G373D, p.V797L and p.R814Q reduced LDLR expression and activity (p < 0.05). Microscopic analysis of the p.V797L and p.G373D variants revealed altered lipid localization and accumulation in transfected HepG2 cells. Carriers of p.G549D, p.V797L and p.R814Q had higher LDL cholesterol levels than non-FH group, and (p < 0.05). p.G373D and p.G549D were associated with clinical manifestations of FH. In conclusion, the p.C184Y, p.G373D, p.G549D and p.R814Q variants alter protein stability and intramolecular interactions, while p.V797L has a minimal impact on protein stability, and p.V827I has no significant intramolecular interactions. p.G373D, p.V767L and p.R814Q are associated with impaired LDLR expression and activity.