一个家族性高胆固醇血症家系的低密度脂蛋白受体基因突变检测与功能分析  

Mutation screening and functional analysis of low density lipoprotein receptor in a familial hypercholesterolemia family

作　　者：程小欢[1] 郑芳[1] 周新[1] 熊陈岭[1] 丁军发[1] 陈永梅[1] 

机构地区：[1]武汉大学中南医院基因诊断中心,430071

出　　处：《中华医学遗传学杂志》2008年第1期55-58,共4页Chinese Journal of Medical Genetics

基　　金：2005年新世纪人才基金（NCET-05-0621）;湖北省杰出青年基金（2006ABB009）

摘　　要：目的检测一个家族性高胆固醇血症（familial hypercholesterolemia, FH）患者低密度脂蛋白受体（low density lipoprotein receptor, LDLR） 的基因突变，并对淋巴细胞表面LDLR功能进行检测。方法以一家系中4例临床诊断为FH患者的基因组DNA为模板，首先检测载脂蛋白B-100 （apolipoorotein B-100,ApoB100）基因R3500Q突变，以排除家族性ApoB100缺陷症（familial defective apolipoprotein B- 100, FDB） .然后用降落PCR方法，扩增LDLR基因的启动子和外显子片段，用单链构象多态性方法进行分析，对可疑外显子的PCR产物进行T／A克隆，测序分析。同时，用人淋巴细胞与荧光探针DiI标记的低密度脂蛋白发生结合反应，再通过流式细胞仪检测，显示具有功能性LDLR的淋巴细胞比例。结果该家系患者LDLR基因第10外显子均出现杂合突变，第462位密码子由TGG突变为TAG，导致在462位提前出现终止密码子（W462X）。具有功能性LDLR的淋巴细胞占总淋巴细胞比例患者为63．7％，健康对照者为77．3％，患者淋巴细胞LDLR活性约为健康对照者的82．4％，出现一定程度的降低。结论提示LDLR基因W462X突变可能是导致本FH家系患者患病的一个主要原因。Objective To screen the mutations of the low density lipoprotein receptor （LDLR） gene in a familial hypereholesterolemia （FH） family, and analyze the LDL-uptaking function of LDLR on lymphocytes of patients, Methods Genomie DNA was extracted from four affected members in a Chinese FH family. The presence of apoB100 gene R3500Q mutation which results in familial defective apolipoprotein B100 （FDB） was excluded first. Fragments of the LDLR gene were amplified by touch-down polymerase chain reaction （Touch-down PCR） and analyzed by single-strand eonformational polymorphism （SSCP）. The suspect fragments of the LDLR gene were cloned and sequenced. Furthermore, the lymphocytes bounded with fluorescent-labeled LDL （DiI-LDL） were measured by fluorescence flow cytometry. Results A nonsense mutation was identified in exon 10 of LDLR gene. This mutation gave rise to a premature stop codon （W462X）, resulting in the absence of most of the LDLR domains. It was detected in all the affected members of the FH family. The ratios of functional LDLR in lymphocytes from patients and normal controls were 63.7% and 77.3% respectively. As a result, the activity of the functional LDLR in patients was just 82.4% of that in the normal controls. Conclusion It is possible that the W462X mutation of LDLR gene is the main cause for the disease in this family.

关 键 词：家族性高胆固醇血症 低密度脂蛋白受体 流式细胞仪 

分 类 号：R589.2[医药卫生—内分泌] R394[医药卫生—内科学]