家族性高胆固醇血症纯合子家系低密度脂蛋白受体功能检测及基因突变分析

Analysis of low density lipoprotein receptor function and gene mutation in familial hypercholesterolemic patients

目的 分析家族性高胆固醇血症 (familialhypercholesterolemia ,FH)患者低密度脂蛋白受体(lowdensitylipoproteinreceptor ,LDLR)的功能改变及基因突变。 方法 分离FH患者外周血淋巴细胞 ,用流式细胞仪观察淋巴细胞结合和摄取荧光标记的低密度脂蛋白的情况。抽提FH患者外周血基因组DNA为模板 ,进行聚合酶链反应 单链构象多态性分析 ( polymerasechainreaction singlestrandconformationpolymor phism ,PCR SSCP)及DNA序列分析。 结果 对一家两例临床诊断为FH纯合子的患儿及其父母的外周血淋巴细胞LDLR功能进行了分析 ,发现均表现为低密度脂蛋白 (LDL)摄取和结合障碍。进一步从基因水平进行了研究 ,发现LDLR基因突变是位于第 6外显子编码第 2 97位氨基酸的碱基发生缺失 ,导致移码突变并使得终止密码子TGA在第 3 69位提前出现 ,从而不能表达正常的LDLR ,体内胆固醇的代谢发生障碍。结论 对 1例家族性高胆固醇血症纯合子家系应用流式细胞仪方法初步发现LDLR功能缺陷 ,进一步结合PCR SSCP方法证实其LDLR存在新的突变类型。

Objective: To investigate low density lipoprotein receptor (LDLR) function and gene mutation in Chinese patients with familial hypercholesterolemia (FH). Methods: Lymphocytes were isolated from 10 ml anticoagulated peripheral blood of the patients, then a flow-cytometric method (FCM) with 1, 1′-dioctadecyl-3, 3, 3′ 3-tetramethylindocarbocyanine perchlorate labelled low density lipoproetin (DiI-LDL) was used to identify the function of LDLR on the surface of lymphocytes. Genomic DNA was isolated from whole blood of FH patients and analyzed by PCR-single strand conformation polymorphism (SSCP) and nucleotide sequencing methods. Results: Defects of binding and uptaking of LDLR were identified by FCM in 2 FH patients in one family, and their parents were examined in the present study. Then they were analyzed genetically. The detected mutation was a deletion of A, which caused a frame shift in codon 297 of exon 6 and introduced a beforehand stop codon in codon 369. Conclusion: A novel mutation of LDL receptor gene was detected by the combination of FCM and PCR-SSCP methods.