摘要目的 从一个杜氏肌营养不良症家系DMD基因突变情况,分析该基因第19内含子受位剪切区域c.2381-3T＞C突变的性质.方法 提取该家系三代共5名成员的外周血基因组DNA,采用多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)排除携带者DMD基因大片段缺失与重复后,应用高通量测序技术检测DMD基因外显子及侧翼内含子剪切序列的突变,并用Sanger测序验证.针对发现的剪切位点突变构建mini-gene表达载体,评价突变对于DMD基因mRNA剪切的影响.结果 该家系第一代的祖母与第二代的两姐妹均携带DMD基因的无义突变c.8038C＞T(p.Arg2680Ter).第一代父亲为c.2381-3T＞C突变的半合子,第二代的两姐妹以及妹妹的女儿均携带c.2381-3T＞C杂合突变.在135名非DMD的成年男性中1人为c.2381-3T＞C突变的半合子.构建c.2381-3T＞C突变的mini-gene并分析表达产物未发现异常转录本.结论 DMD基因c.8038C＞T(p.Arg2680Ter)是该家系的致病突变.该家系非DMD男性以及对照群体中检出c.2381-3T＞C突变,以及该剪切突变的体外表达结果均提示这个已被人类基因突变数据库收录的DMD可疑致病突变属于罕见的单核苷多态,本身并不具备明显的致病效应.

abstractsObjective To clarify the nature of a DMD splice acceptor mutation c.2381-3T＞C.Methods Genomic DNA was extracted from 5 members of a family affected with DMD.For an obligatory carrier,after excluding gross deletion and duplication of the DMD gene with multiplex ligation-dependent probe amplification (MLPA) method,all coding and splice site sequences of the DMD gene were analyzed with Next Generation Sequencing followed by confirmation with targeted Sanger sequencing.Mutations of the carrier were detected in other 4 members.For the splice site mutation,mini-gene was constructed and expressed in vitro to detect the number of transcript and cDNA sequence.Results A known nonsense mutation (c.8038C＞T,p.Arg2680Ter) was identified in the carrier,her sister and the mother.The rest 4 members,except for the mother from the first generation,have all carried the c.2381-3T＞C mutation.The latter has been described as a splice site mutation to cause DMD.One of 135 male adults without DMD was also detected to have carried the c.2381-3T＞C mutation.No additional transcript was produced by the minigenes containing c.2381 3T＞C mutation.Conclusion The c.8038C＞T(p.Arg2680Ter)mutation of DMD gene probably underlies the disease in this family.The presence of the c.2381-3T＞ C mutation in a asymptomatic male and a non-DMD male control,together with the normal in vitro expression of the minigenecarrying the c.2381-3T＞C,strongly suggested that the c.2381-3T＞C mutation collected in the Human Gene Mutation Database is a rare SNP without significant pathogenicity.