Chen Ya-nan, Zhou Xin, Jin Chun-lian, Xu Yan, Lin Chang-kun, Cao Li-hua, Li Ning, Zhang Xue, Luo Yang
The Research Center for Human Genomics and MOH Key Laboratory of Cell Biology, China Medical University, Shenyang 110001, China.
Zhonghua Er Ke Za Zhi. 2007 Jun;45(6):413-6.
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 nucleotides) cannot be achieved in this way. The aim of the present study was to detect mutations of the dystrophin gene in individuals with Duchenne 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.
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 genomic PCR and the amplicons showing abnormal DHPLC profile were directly sequenced to identify the position and the type of the mutations.
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 codons, p.Leu2270MetfsX9 and p.Ser1654LysfsX5, respectively. The remaining two were nonsense mutations, leading to p.R2886X and p.R2870X, respectively.
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.
杜氏肌营养不良症（DMD）是一种由抗肌萎缩蛋白基因突变引起的X连锁隐性疾病；其中55%-65%的致病突变是大片段缺失和重复突变，可通过多重聚合酶链反应检测到。然而，以这种方式无法找到其余的微小突变（一个或几个核苷酸的替换、缺失或插入）。本研究的目的是通过变性高效液相色谱法（DHPLC）检测杜氏肌营养不良症（DMD）患者抗肌萎缩蛋白基因的突变，并建立一个快速、灵敏的导致DMD的微小突变筛查平台。
选择20例经多重PCR检测抗肌萎缩蛋白基因大片段缺失为阴性的患者，通过DHPLC进行进一步筛查，并选取20例无DMD家族史的正常男性作为对照队列。通过基因组PCR分别扩增抗肌萎缩蛋白外显子及其侧翼序列，对显示异常DHPLC图谱的扩增子直接测序，以确定突变的位置和类型。
在筛查了覆盖两个缺失热点和3'UTR区域的68个外显子后，在4例DMD患者中发现了4个致病突变，包括c.6808_6811del TTAA、c.4959_4960insA、c.8656C>T和c.8608C>T。此外，c.6808_6811del TTAA、c.4959_4960ins和c.上的8656C>T此前尚未见报道。前两个移码突变预计分别产生提前终止密码子p.Leu2270MetfsX9和p.Ser1654LysfsX5。其余两个是无义突变，分别导致p.R2886X和p.R2870X。
在中国DMD患者中鉴定出3个新的和1个复发的抗肌萎缩蛋白突变。本研究表明，DHPLC是一种用于筛查与DMD相关微小突变的有效方法。
