dystrophin基因第45 ~54外显子缺失连接片段的克隆和测序(英文)  

Cloning and sequencing of junction fragment with exons 45-54 deletion of dystrophin gene

作　　者：钟敏[1] 潘速跃[1] 陆兵勋[1] 李伟[1] 

机构地区：[1]南方医科大学南方医院神经内科,广州510515

出　　处：《中华医学遗传学杂志》2006年第2期138-141,共4页Chinese Journal of Medical Genetics

基　　金：广东省自然科学基金(001032)~~

摘　　要：目的通过对dystrophin基因第45-54外显子缺失后连接片段的克隆和测序分析，探讨dystrophin基因缺失的发生机理。方法先以外显子PCR反应检测证实1例杜氏肌营养不良症（Duchenne muscular dystrophy，DMD）患者第45～54外显子缺失，然后在第44和第54内含子上用PCR步移方法寻找断裂位点，最后用靠近断裂位点处设计的引物，以PCR法直接扩增dystrophin基因的缺失连接片段并测序，测序结果和正常内含子序列作对比分析。结果对扩增连接片段的PCR产物测序获得2716bp有效序列。本例基因缺失片段长达402kb。5’端断裂点位于第44内含子长散在元件（long interspersed elements，LINE）L1序列内，邻近基质附着区（matrix attachment region，MAR），3’端断裂位点在第54内含子较可能形成MAR的一个次级区域内，附近有拓扑异构酶Ⅱ识别位点，断裂点两旁存在6bp的回文序列。连接片段通过4bp的微小同源序列AGAG连接断裂点两端。结论由L1重复序列、断裂点附近拓扑异构酶Ⅱ酶切位点、MARs以及微小同源序列的非同源末端连接修复等综合因素引起的非同源基因重组可能是导致此一大片段基因缺失的重要原因。Objective To study the mechanisms of dystrophin gene deletion, the junction fragment with exons 45-54 deletion were cloned and sequenced. Methods A Duehenne muscular dystrophy （DMD） patient with exons 45-54 deletion has been substantiated by PCR amplification of the exons. Then we used a PCR-based gonome-walking method for localizing the breakpoints in introns 44 and 54. At last, the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoints in introns 45 and 54. The sequencing result of the deletion-junction fragment was compared with the normal intronie sequences. Results A total of 2716 bp sequence containing the junction fragment was obtained. The 5＇ breakpoint was located in LINE/L1 element of intron 44 and close to a matrix attachment region （MAR）. The 3＇ breakpoint was located in the minor potential MAR with topoisomerase Ⅱ cleavage sites around. Beside the 3＇ breakpoint there was a 6 bp palindromic sequence. A 4 bp mierohomologous sequence （AGAG） was in the joint of the deletion-junction fragment. Conclusion The nonhomologous recombination caused by L1 repeated element, topoisomerase Ⅱ cleavage sites, MARs and the nonhomologous end joining of mierohomologous sequence may be the important factors in this huge gene fragment deletion.

关 键 词：肌营养不良症 连接片段 基因缺失 

分 类 号：R346[医药卫生—基础医学]