脂蛋白脂酶基因突变（C310R／E396V）导致高三酰甘油血症的家系研究

Molecular mechanism of familial hypertriglyceridemia caused by lipoprotein lipase gene mutation （ C310R/ E396V）

目的本课题拟在细胞水平上对我们新发现的两个脂蛋白酯酶（lipoprotein lipase, LPL）基因突变p.C310R（c.T928C）和p.E396V（c.A1187T）进行分子机制研究，有助于构建我国家族性高三酰甘油血症患者的LPL基因突变型与表型关系谱，为高危人群提供精准早期诊断，同时为基因靶向治疗药物开发提供依据。方法提取先证者全体家族成员的外周血DNA，利用全外显子测序技术确定LPL突变位点，然后通过一代测序进行验证。细胞实验构建携带该突变基因位点的慢病毒表达载体，转染COS-1细胞，通过ELISA和酶荧光法分别检测细胞上清和裂解液内LPL的浓度和活性。通过RT-PCR检测该突变对于基因转录水平影响。结果全外显子测序结果显示先证者的LPL基因6号外显子有一个新的c.T928C杂合子突变（p.C310R），而其侄女则携带两个复合杂合子突变，其中一个突变位点与先证者相同，另一个突变位点位于8号外显子c.A1187T的突变（p.E396V）。细胞实验结果表明这两种突变均可以造成分泌出胞外的LPL活性下降，浓度减低（P〈0.05）。进一步的研究发现，C310R突变可以影响LPL基因转录后修饰，而E396V突变则影响LPL在细胞内转运。结论这两个位点的突变均为致病性基因突变，其最终通过减少分泌的LPL浓度和活性，影响体内三酰甘油的正常代谢。

ObjectiveThe purpose of this study is to investigate the molecular mechanisms of p. C310R（c.T928C） and p. E396V（c.A1187T） lipoprotein lipase（LPL） gene mutations in vitro, which may help to construct the spectrum of LPL gene mutations and phenotype. It also can provide accurate early diagnosis for high-risk population of familial hypertriglyceridemia and provide the basis for the development of gene targeted therapy. MethodsGenomic DNA was extracted from proband′s family members′ peripheral blood cells and screened by whole-exome sequencing to verify candidate gene variations. PCR products were afterwards directly sequenced again to confirm corresponding LPL variants. At the cellular level, lentiviruses containing LPL mutations were constructed and then transfected into COS-1 cells. Functional significance of the mutants was corroborated by analyzing LPL activity and mass in the cell medium and lysates via ELISA and enzyme-fluorescent method. mRNA was assayed by RT-PCR to confirm the effect on gene transcription.ResultsDNA sequence analysis revealed that the proband was a heterozygote for a novel c. T928C mutation in exon 6 of LPL gene, while his nephew was a compound heterozygote for the c. T928C mutation in exon 6 and a novel c. A1187T mutation in exon 8. In vitro studies, these two mutations can cause decreased activity and mass of extracellular LPL（P〈0.05）. Moreover, further investigation indicated that LPL C310R mutation tremendously affected post-transcriptional modification of LPL gene, whereas LPL E396V mutation dampened intracellular LPL trafficking.ConclusionBoth the mutations are pathogenic by reducing the activity and mass of LPL in the plasma, which affected normal metabolism of triglycerides.