多重PCR检测DMD/BMD患者DMD基因外显子缺失的实验研究

Multiple PCR for detecting the deletion of DMD gene exon in patients with Duchenne/Becker muscular dystrophy

目的探索建立Duchenne/Becker肌营养不良(DMD/BMD)的稳定高效的基因诊断方法,为该疾病的临床鉴别诊断提供参考。方法收集2008年5月-2010年5月在第三军医大学新桥医院就诊的汉族男性DMD/BMD患者64例,年龄0.7～45(9.3±1.1)岁。抽取患者外周静脉血5ml,提取基因组DNA后,采用多重PCR(mPCR)扩增DMD/BMD患者DMD基因缺失热区内的18个外显子,确定DMD/BMD患者DMD基因外显子缺失类型。结果 64例DMD/BMD患者中,31例(48.44%)存在DMD基因缺失热区内的不同外显子缺失。其中,外显子50缺失频率最高,为35.48%(11/31);外显子47和43次之,分别为32.26%和25.81%(10/31和8/31),外显子45和49缺失率均为19.35%(6/31),外显子19和48缺失率均为16.13%(5/31)。缺失的外显子主要集中在DMD基因的中央缺失热区和5′端缺失热区。结论目前国内外采用的mPCR方法在DMD/BMD患者的临床诊断中发挥了重要作用。探索DMD基因其他缺失热点、重复突变热点或点突变热点,并以此为依据建立新的DMD/BMD基因诊断方法仍然是提高DMD/BMD基因诊断率的发展方向。

Objective To develop a consistent and effective gene diagnostic method for Duchenne/Becker muscular dystrophy （DMI）/BMD）, so as to provide a clinical reference for the differential diagnosis of DMD/BMD diseases. Methods Clinical data of sixtyfour male DMI）/BMI） patients, Han nationality, aged from 0. 7 to 45 （9. 34±1.13） years, were collected between May 2008 and May 2010 from the Departments of Neurology, Pediatrics, and Gynaecology and Obstetrics of affiliated hospitals of Third Military Medical University. Genomic DNA was extracted from 5ml of peripheral blood of each patient. Determination of exon deletion in DMD gene of DMD/BMI） patients was performed by amplifying 18 exon in hotspot deletion regions of DMD gene with multiple PCR （mPCR）, and then the deletion type of DMD gene exon was confirmed. Results Deletions of varied exons in hotspot deletion regions of DMD gene were found in 31 （48. 4%） out of 64 patients with DMD/BMD, among which exon 50 deletion ranked the top （35. 5%, 11/31）, followed by exon 47 and 43 deletion, accounting for 32. 3% （10/31） and 25.8% （8/31）, respectively. Exon 45 deletion was found in 6 out of the 31 patients （19. 3%）, while exon 49 deletion existed in same number of patients. Exon 19 deletion, as well as the 48 deletion, was respectively found in 5 out of the 31 patients （16. 1%）. In summary, the exon deletion happened mainly in the central and 5-end hotspot deletion region of DMD gene. Conclusions Multiple PCR, extensively used domestically and abroad, plays a vital role in assisting clinical diagnosis of DMD/BMD. It still is worthwhile to explore the other deletion hotpot, duplicated mutational hotpot and point mutation hotpot of DMD gene, and, based on them, new methods for DMD/BMD gene diagnosis can be established, thereby the diagnostic rate of DMD/BMI） disease can be elevated.