两例成骨不全症患者候选剪接变异的致病性鉴定  

Identification of potential splicing variants in two Chinese patients with osteogenesis imperfecta

作　　者：米欢 李璐璐 曹一璇 杨涛[1] 翁习生 赵秀丽[1] Mi Huan;Li Lulu;Cao Yixuan;Yang Tao;Weng Xisheng;Zhao Xiuli(Department of Medical Genetics,Institute of Basic Medical Sciences,Chinese Academy of Medical Sciences&School of Basic Medicine Peking Union Medical College,Beijing 100005,China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础学院医学遗传学系,100005 [2]中国医学科学院基础医学研究所,北京协和医学院北京协和医院骨科100730

出　　处：《中华骨科杂志》2021年第9期576-583,共8页Chinese Journal of Orthopaedics

基　　金：国家重点研发计划(2016YFE0128400,2016YFC0905100);中国医学科学院医学与健康科技创新工程项目(2016-12M-3-003);国家自然科学基金(81871749)。

摘　　要：目的对2例中国汉族成骨不全症(osteogenesis imperfecta,OI)患者进行突变检测,对其候选剪接变异进行致病性鉴定。方法采用常规酚-氯仿法从2例中国汉族OI先证者的外周血中提取基因组DNA,通过全外显子组测序(WES)进行致病突变筛查;针对检出的可能引起RNA剪接异常的疑似致病变异,构建Minigene分析变异对于剪接的影响,进而确定其致病性。结果在先证者1中发现可能影响剪接的杂合变异:COL1A1:c.858+1858+5delGTAAG;先证者2同时存在COL1A2的两个变异,一个为可能引起异常剪接的杂合变异c.1405-7C>T,另一个为杂合错义变异c.2972G>T(p.G991V)。Minigene实验分析证实家系1的变异导致COL1A1基因第12外显子发生跳跃,家系2变异位点c.1405-7C>T对COL1A2的转录后剪接没有影响。先证者2的COL1A2基因的G991为一个高度保守的氨基酸,经生物信息学分析c.2972G>T(p.G991V)可能为真正的OI致病变异。结论先证者1的COL1A1剪接位点变异c.858+1858+5delGTAAG是导致OI的致病性变异;排除先证者2中COL1A2变异c.1405-7C>T的致病性,确定COL1A2基因变异c.2972G>T(p.G991V)为先证者2的致病原因。针对WES提示的剪接变异进行Minigene分析,不仅实现了突变致病性鉴定,丰富了OI的突变谱,而且为患者产前诊断和后续机制研究奠定了基础。Objective To identify pathogenicity of the potential splicing variants in two Chinese Han patients with osteogenesis imperfecta.Methods Genomic DNA was extracted using the conventional phenol-chloroform method;whole exome sequencing(WES)was used to analysis the disease-related variants in the two probands;Minigene assay was used to identify pathogenicity of the variants found in the patients'genome that possibly affect RNA splicing.Results Two potential splicing variants,c.858+1_858+5delGTAAG in intron 12 of COL1A1 and c.1405-7C>T in intron 24 of COL1A2,were found in proband 1 and proband 2,respectively.In addition,a missense mutation,c.2972G>T(p.G991V)in exon 45 of COL1A2,was detected in proband 2.Minigene assay revealed that the variant in proband 1 caused the skipping of exon 12,while the variant in proband 2 did not lead to aberrant splicing.G199 of the COL1A2 in proband 2 was a highly conserved amino acid site,and the results suggested that c.2972G>T(p.G991V)may be the real pathogenic variant by the means of bioinformatics analysis.Conclusion The variant c.858+1_858+5delGTAAG in COL1A1 was a causative variant that led to OI in proband 1,while the missense variant c.2972G>T(p.G991V)in COL1A2 was the cause of OI in proband 2,instead of the variant c.1405-7C>T.Minigene assay for potential splicing variants detected by WES could not only validate the pathogenicity of the candidate variants and enrich the mutation spectrum of OI,but also lay the foundation for patients'prenatal diagnosis and subsequent mechanism research.

关 键 词：骨发育不全 遗传学研究 剪接体 遗传变异 遗传关联研究 

分 类 号：R681.1[医药卫生—骨科学]