摘要
目的对临床诊断为Duchenne型肌营养不良（DMD）的患儿进行基因检测，分析抗肌萎缩蛋白基因（DMD基因）突变类型及变异大小，探讨DMD分子遗传学诊断策略。方法对临床诊断为DMD的28例患儿采用多重链接探针依赖扩增技术（MLPA）进行DMD基因检测，然后随机选择MLPA检测阳性的5例和阴性的1例采用DMD全基因捕获探针进一步行全基因组新一代测序（NGS），最后采用PCR和Sanger测序法对所得结果验证。结果采用MLPA技术对28例患儿进行检测，发现22例阳性结果，包括外显子大片段缺失突变16例，重复突变4例，同时合并缺失和重复突变2例；随机选择MLPA检测阳性的5例和阴性的1例采用NGS技术检测，发现3例外显子缺失突变（exon51del，chrX：31779857—31795289；exon53del，chrX：31685440-31747548；exork51-55del，chrX：31632504—31827732），及2例外显子重复突变（exon45dup，chrX：31970766—32023816；exon55dup，chrX：31540860—31649750），与MLPA结果一致，且精确定位了断裂点区域和片段大小，同时发现MLPA检测阴性的1例为点突变（exon54，chrX：31676176，C．7948delG，P．D2650IfsX12），该点突变经PCR扩增和Sanger测序验证，所得结果与NGS检测结果一致。结论DMD基因突变类型多样，包括外显子缺失和（或）重复、点突变等，应用MLPA技术能有效检出DMD基因外显子大片段缺失、重复以及杂合突变，应用NGS技术不仅能检出缺失和重复突变，并精确判断断裂点位置及大小，还能准确检出微小缺失和点突变，为临床DMD的早期诊断、预后判断及产前诊断提供遗传学帮助。
Objective To detect the mutation type and size variation of the dystrophy gene in Duchenne mus- cular dystrophy（DMD） patients, and discuss the strategy of molectilar genetic diagnosis for DMD. Methods Muhiplex ligation-dependent probe amplification（MLPA） and next-generation sequencing（NGS） were applied for genetic detec- tion in patients with clinical suspected DMD. Sanger sequencing was performed to confirm the results. Results Patho- genic mutations were found in 28 cases with DMD. Twenty-two of the 28 cases were found to be positive with MLPA a- nalysis, 16 cases with deletion mutations ,4 cases with duplication, and 2 cases with both deletion and duplication muta- tions. Then, NGS technology was performed to detect 5 cases with MLPA positive and 1 case with MLPA negative, which were chosen optionally, and 3 cases showed deletion （ exon51 del, chrX： 31779857-31795289; exon53 del, chrX： 31685440-31747548; exon51-55 del, chrX： 31632504-31827732） and 2 cases of duplication （ exon45 dup, chrX： 31970766-32023816;exon55 dup,chrX：31540860-31649750） ,which were consistent with MLPA analysis. Meanwhile, NGS could determine the location of break point and the size of DNA segments accurately and also detect one point mu- tation which was MLPA negative. In the end, Sanger sequencing was performed to confirm this point mutation. Conclu- sions The mutational spectrum of the DMD gene is complex, including deletion/duplication and point mutations, and MLPA is an efficient method for detecting paternal deletion and duplication of DMD ,while NGS can not only detect both deletion/duplication and point mutations, but also determine the location of break point and the size of gene segments accurately. Next-generation sequencing may be a powerful technology for early clinical diagnosis, prognostic judgment and prenatal diagnosis of DMD.
出处
《中华实用儿科临床杂志》
CAS
CSCD
北大核心
2013年第14期1099-1102,共4页
Chinese Journal of Applied Clinical Pediatrics
关键词
肌营养不良
多重链接探针依赖扩增技术
全基因组新一代测序
点突变
Muscular dystrophy
Multiplex ligation-dependent probe amplification
Next-generation sequencingtechnology
Point mutation
