应用DHPLC技术检测非缺失型DMD致病基因的新突变  

Detection of new mutations in the dystrophin gene by denaturing high-performance liquid chromatography

作　　者：陈亚男[1] 周鑫[1] 金春莲[2] 徐岩[1] 林长坤[2] 曹丽华[1] 李宁[1] 张学[1] 罗阳[1] 

机构地区：[1]中国医科大学医学基因组学教研室 卫生部细胞生物学重点实验室,沈阳110001 [2]中国医科大学医学遗传学教研室

出　　处：《中华儿科杂志》2007年第6期413-416,共4页Chinese Journal of Pediatrics

基　　金："十五"国家科技攻关课题(2004BA720A04)

摘　　要：目的建立检测 Duchenne 型肌营养不良症(Duchenne muscular dystrophy,DMD)致病基因 dystrophin 突变的快速、敏感的微小突变筛查系统。方法选择经多重 PCR 检测未发现dystrophin 基因大尺度缺失的 DMD 患儿20例,年龄在2～10岁,以其基因组 DNA 为模板,通过 PCR反应,分别扩增 dystrophin 基因外显子及侧翼序列,采用变性高效液相色谱(DHPLC)技术进行突变筛查,对 DHPLC 峰形变异的外显子片段行 PCR 产物直接测序,确定突变的具体位点和类型。结果筛查了包括2个缺失热点区 dystrophin 基因的68个外显子和3′UTR 部分片段,分别在4例患儿中检测出该基因的4种致病突变:c.6808_6811delTTAA,c.4959_4960insA,c.8656C>T 和 c.8608C>T。前两例引起移码突变分别导致 p.Leu2270MeffsX9和 p.Ser1654LysfsX5,后两例为无义突变分别导致 p.R2886X 和 p.R2870X。其中 c.6808_6811delTTAA,c.4959_4960insA 和 c.8656C>T 为文献未曾报道的新突变。结论 DHPLC 可以作为有效地筛查 DMD 微小突变的检测方法。Objective Duchenne muscular dystrophy （DMD）is an X-linked recessive disease caused by dystrophin gene mutations; 55%-65% of these pathogenic mutations are large deletion and duplication mutations that can be detected by multiplexed polymerase chain reaction. However, finding the remaining micro-mutations （substitutions, deletions or insertions of one or several nueleotides ） cannot be achieved in this way. The aim of the present study was to detect mutations of the dystrophin gene in individuals with Ducherme muscular dystrophy（DMD） by denaturing high-performance liquid chromatography （DHPLC） and to establish a rapid and sensitive screening platform for micro-mutations leading to DMD. Methods Twenty patients negative for large deletions in the dystrophin gene by multiplex PCR were selected for further screening by DHPLC and 20 normal male without DMD family history as the control cohort. Dystrophin exons and their flanking sequences were individually amplified by genomie PCR and the amplieons showing abnormal DHPLC profile were directly sequenced to identify the position and the type of the mutations. Results After screening 68 exons covering the two deletion hotspots and 3＇UTR region, four pathogenic mutations, including c. 6808. 6811del TTAA, c. 4959_4960insA, c. 8656C 〉 T and c. 8608C 〉 T, were found in four DMD patients. Moreover, c. 6808_6811del TTAA, c. 4959_4960ins and c. 8656C 〉 T have not been reported previously. The first two frameshift mutations were predicted to produce premature stop eedons, p. Leu2270MeffsX9 and p. Ser1654LysfsX5, respectively. The remaining two were nonsense mutations, leading to p. R2886X and p. R2870X, respectively. Conclusion Three novel and one recurrent dystrophin mutations have been identified in Chinese DMD patients. This study has demonstrated that DHPLC is an effective screening method for micro-mutation associated with DMD.

关 键 词：肌营养不良 杜氏 色谱法 高效液相 突变 

分 类 号：R746[医药卫生—神经病学与精神病学]