高胆固醇血症低密度脂蛋白受体基因点突变的研究  

A Study on Point Mutations of Lipoprotein Receptor Gene in Patients with Hypercholestemia

作　　者：朱大明[1] 陈在嘉[1] 陈保生[2] 陆宗良[1] 寇文镕[1] 徐义枢[1] 吕新跃[2] 曾武威[2] 杨跃进[1] 陈纪林[1] 高润霖[1] 杜怀明[3] 马咸成[3] 

机构地区：[1]中国医学科学院中国协和医科大学心血管病研究所阜外心血管病医院冠心病研究室,北京市100037 [2]中国医学科学院中国协和医科大学基础医学研究所 [3]天津医科大学附属医院

出　　处：《中国循环杂志》2001年第3期180-182,共3页Chinese Circulation Journal

基　　金：国家自然科学基金资助项目(39670312)

摘　　要：目的：建立低密度脂蛋白－受体（ＬＤＬ－Ｒ）基因点突变监测方法，从基因水平对３０例原发性高胆固醇血症患者进行筛选、明确诊断。 方法：将聚合酶链式反应－单链构象多态性（ＰＣＲ－ＳＳＣＰ）与银染技术相结合，用１９对引物对ＬＤＬ－Ｒ基因的全部１８个外显子进行检测，３０例患者中，对筛查出的突变用ＰＣＲ产物直接测序或克隆测序的方法明确突变的性质和位置。 结果：确立的ＰＣＲ－ＳＳＣＰ条件稳定，银染方法灵敏度高、重复性好，且避免了同位素污染。３０例患者中，发现１例定位于外显子１４的杂合子点突变患者，ＰＣＲ产物直接测序证实第６７１位密码子发生错义突变：ＣＣＣ→ＣＧＣ，导致脯氨酸→精氨酸；１例纯合子家族性高胆固醇血症患者外显子７发生点突变，克隆测序证实密码子３０８位发生错义突变：ＴＡＣ→ＴＧＣ，导致半胱氨酸→酪氨酸；２例杂合子患者外显子４的３’部分ＳＳＣＰ出现相同异常带型。查阅文献，测序证实的突变皆是新的突变。 结论：建立的ＰＣＲ－ＳＳＣＰ方法可用于高胆固醇血症患者ＬＤＬＲ基因点突变的筛检。研究结果从基因水平证实中国家族性高胆固醇血症患者ＬＤＬ－Ｒ基因突变具有多样性的特点。Objective: To establish a method to detect point mutations of low density lipoprotein receptor (LDL-R) gene for the screening and confirming diagnosis of familial hypercholesterolemia. Methods: Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining method was established to detect point mutations of LDL-R gene in 30 patients with hypercholestolemia using. 19 pairs of primers covering 18 exons. Direct seqencing of PCR product or clone sequencing was used to define the point mutations. Results: The conditions for PCR-SSCP were stable. Silver staining was sensitive, repetitive, and free of isotope contamination. A heterozygous FH patient with mutation in exon 14 was sereened out. Direct sequencing showed C→G transition in coden 671, so proline was changed to arginin. A homozygous familial hypercholesterolemia(FH)had point mutation in coden 308. Two heterozygous FHs showed the same abnormal bands in PCR-SSCP analysis in 3'half of exon 4. Reviewing the litera- ture, we found the sequencing comfirmed mutation were novel. Conclusions: The PCR-SSCP method enables the sereening of point mutations of LDL-R gene in FH patients. The results of study demonstrate that the mutations of LDL-R gene in FH patients in China are diverse.

关 键 词：高胆固醇血症 聚合酶链式反应一单链构象多态性 基因突变 低密度脂蛋白 

分 类 号：R589.2[医药卫生—内分泌]