Functional Characterization of Two Low-Density Lipoprotein Receptor Gene Mutations in Two Chinese Patients with Familial Hypercholesterolemia

BackgroundFamilial hypercholesterolemia (FH) is an autosomal dominant disease that primarily results from mutations in the low-density lipoprotein receptor (LDLR) gene. We investigated two unrelated Chinese FH patients using gene screening and functional analysis to reveal the pathogenicity and the mechanism by which these mutations cause FH.MethodsFirst, the LDLR gene was sequenced in these patients. Then, mutant receptors were transfected into human embryo kidney 293(HEK-293) cells, and a confocal laser-scanning microscope was used to observe the localization of mutant proteins. Further, the expression and the internalization activity were analyzed by flow cytometry. Finally, LDLR protein expression and stability was detected by western blot.ResultsTwo different LDLR class 2B mutations were detected in two patients. The C201F mutation is a known mutation. However, the G615V mutation is novel. Flow cytometry showed that the expression and internalization activity of the mutant LDLRs were reduced to 73.6% and 82.6% for G615V and 33.2% and 33.5% for C201F, respectively.ConclusionsThis study identified two LDLR mutations in Chinese patients with FH and analyzed the relationship between the genotype and phenotype of these patients. We found that these mutant LDLRs were defective in transport, which led to a reduction in cholesterol clearance. These results increase our understanding of the mutational spectrum of FH in the Chinese population.研究背景：家族性高胆固醇血症（Familial hypercholesterolemia, FH）是一种常染色体显性遗传病，其主要致病原因是低密度脂蛋白受体（low-density lipoprotein receptor, LDLR）基因发生突变。本研究通过基因筛查与功能分析，对2例无亲缘关系的中国FH患者展开研究，以阐明此类突变导致FH的致病性与分子机制。 研究方法：首先对2例患者的LDLR基因进行测序；随后将突变型LDLR受体转染至人胚肾293（HEK-293）细胞中，采用激光共聚焦显微镜观察突变蛋白的亚细胞定位；进一步通过流式细胞术分析突变受体的表达水平与内化活性；最后采用蛋白质印迹法（western blot）检测LDLR蛋白的表达量与稳定性。 研究结果：本研究在2例患者中检出2种不同的LDLR 2B类突变。其中C201F突变为已知突变，而G615V突变为新发突变。流式细胞术检测结果显示，G615V突变型LDLR的表达水平与内化活性分别降至正常的73.6%与82.6%，C201F突变型则分别降至33.2%与33.5%。 研究结论：本研究在中国FH患者中检出2种LDLR突变，并分析了这些患者的基因型与表型之间的关联。研究发现，此类突变型LDLR的转运功能存在缺陷，进而导致胆固醇清除能力下降。本研究结果有助于加深对中国人群FH突变谱的认知。