突变基因SCN5A-V1279I与LQTS的相关性分析  

Correlation analysis between SCN5A-V1279I and LQTS

作　　者：杜进良 王跃兵[3] 刘凯 龙武 位斯杰 习严梅 瞿鹏飞 马琳[3] 李玉华 唐雪[3] 雷普平 Du Jinliang;Wang Yuebing;Liu Kai;Long Wu;Wei Sijie;Xi Yanmei;Qu Pengfei;Ma Lin;Li Yuhua;Tang Xue;Lei Puping(l.School of Forensic Medicine,Kunming Medical University,Kunming 650500,China;School of Forensic Medicine and Laboratory Medicine,Jining Medical University,Jining 272067,China;Yunnan Institute for Endemic Diseases Control and Prevention,Dali 671000,China)

机构地区：[1]昆明医科大学法医学院,云南昆明650500 [2]济宁医学院法医学与医学检验学院,山东济宁272067 [3]云南省地方病防治所,云南大理671000

出　　处：《中国法医学杂志》2023年第4期406-411,共6页Chinese Journal of Forensic Medicine

基　　金：昆医联合专项-重点项目(202101AY070001-008)。

摘　　要：目的 对SCN5A-V1279I突变基因进行生物信息学分析和蛋白质功能研究,探究该突变基因与LQTS的相关性。方法 对SCN5A-V1279I的氨基酸序列进行同源比对分析,判断该突变位点在生物进化中的保守性;评估该突变的致病性并预测其蛋白质三级结构。构建野生型(WT)及突变型(V1279I)体外表达载体,细胞免疫荧光实验观察外源蛋白的表达情况;利用全细胞膜片钳技术分析研究该突变对Nav1.5通道电生理功能的影响。结果 同源比对结果表明SCN5A-V1279I突变位点具有生物进化保守性;致病性预测结果表明Mutation Taster和PolyPhen-2预测V1279I为致病性突变,而Mutation Assessor预测V1279I突变具有中度功能影响。Swiss-model和PyMOL-2.6.0两种软件的预测结果一致表明,缬氨酸突变为异亮氨酸后可导致Nav1.5通道蛋白DⅢ结构域的S3段第1279号位点的氨基酸侧链发生延长折叠改变,而S3段所在的α螺旋整体跨膜结构没有发生扭结和蛋白质错误折叠。免疫荧光实验结果显示突变蛋白可顺利表达并整合到细胞膜上且荧光强度与WT组相比没有差异;全细胞膜片钳实验结果表明,V1279I突变组的Nav1.5通道电生理功能特性(I-V曲线、激活/失活曲线)与WT组相比没有明显差异。结论 基于目前对SCN5A-V1279I功能研究的实验结果,尚不能认定V1279I突变足以导致QT间期延长的发生。Objective Bioinformatics analysis and protein function study will be conducted on the SCNVAV1279I gene to explore the correlation between the mutant gene and LQTS.Methods The amino acid sequence homology of SCN5A-V1279 was analyzed to determine the conservatism of the mutation site in biological evolution.The pathogenicity of the mutation was evaluated and its protein tertiary structure was predicted.Wild type(WT)and mutant type(V12791)expression vectors were constructed in vitro,and the expression of exogenous proteins were observed by immunofluorescence assay.The effect of the mutation on the electrophysiological function of Nav1.5 channel was analyzed by whole-cell patch-clamp recording experiment.Results Homology alignment analysis indicated that the SCNVA-V12791 mutation site was evolutionally-conserved.The analysis of Mutation Taster and PolyPhen-2 showed that V1279I was pathogenic mutation,while being predicted by Mutation Assessor,V1279I mutation had medium functional impact.Swissmodel and PyMOL-2.6.0 predicted that the mutation of Valine to Isoleucine resulted in the amino acid side chain at the 1279th position in the S3 segment in DII domain of Nav1.5 channel protein was changed by extension and folding,while theα-helix transmembrane structure where S3 segment was located did not have kinks and protein misfolding.The results of immunofluorescence experiment showed that the mutant protein could be smoothly expressed and integrated into the cell membrane,and the fluorescence intensity was not different from that of WT group.The results of whole-cell patch-clamp recording showed that the electrophysiological functional properties(I-V curve and activation/inactivation curve)of the V1279I mutant were not significantly different from WT.Conclusion Based on the results of current functional studies on SCNVA-V1279I,it is not clear that V1279I mutation is suffcient to cause QT interval prolongation.

关 键 词：LQTS SCN5A V1279I 全细胞膜片钳技术 钠电流 

分 类 号：D919.1[医药卫生—法医学]