单链构象多态性技术在家族性高胆固醇血症患者低密度脂蛋白受体基因13外显子点突变筛查中的应用

目的 探讨单链构象多态性(SSCP)技术在家族性高胆固醇血症患者低密度脂蛋白受体(LDLR)基因13外显子点突变筛查上的应用价值.方法 以16例临床诊断为家族性高胆固醇血症(FH)患者为研究对象,提取外周血DNA,扩增LDLR基因第13号外显子片段,组合最优条件进行SSCP电泳并银染.对异常条带进行DNA序列测定确定其突变性质和位置.结果 优化SSCP电泳条件为:不含甘油8%凝胶,在10℃条件下电泳;含5%甘油的8%凝胶,在常温条件下电泳(交联度均为49:1).凝胶厚度均不超过0.4 mm,电泳电压为5 V/cm,在此条件下进行SSCP电泳均可以得到满意电泳图谱.对发现的异常条带,结合DNA测序证实有4例患者LDLR基因第13外显子分别发生A606T,D601N,Y601D,或G636V错义突变,同时均存在1959碱基C→T同义突变.另外4例患者13外显子仅存在1959碱基C→T同义突变.结论 通过优化各种条件进行的PCR-SSCP银染方法,是高胆固醇血症病LDLR基因13外显子点突变初步筛查的有效手段.

SSCP screening of mutation in exon 13 of low density lipoprotein receptor gene in Chinese familial hypercholesterolemia patients

Objective To investigate the application of polymerase chain reaction and single strand conformation polymorphism analysis(PCR-SSCP)to the screening of gene mutation of exon 13 of the LDLR gene in familial hypercholesterolemia(FH).Methods Peripheral blood DNA of 16 clinically diagnosed FH patients was extracted and the exon 13 coding region of the LDLR gene was amplified by PCR.PCR products were separated by optimized SSCP electrophoresis and visualized by silver staining.DNA fragments with abnormal mobility were sequenced to determine the nature and position of mutations.Results The SSCP electrophoresis conditions were optimized as 8%polyaerylamide(degree of cross linking 49:1)gel without glycerin at a electrophoresis temperature of 10℃ or 8%polyacrylamide gel with 5%glycerin at room temperature,gel thickness of＜0.4 mm,and a voltage of 5 V/cm.DNA fragments were well resolved with the conditions and sequencing of the abnormal bands resuhed in detections of missense mutations of A606T,D601N,Y601D and G636V together with a synonymous mutation of 1959C→T in 4 patients and a sole synonymous mutation of 1959C→T in other 4 patients.Conclusion PCR-SSCP is an effective method for the screening of exon13 mutations of LDLR gene in FH patients.