Sundaram Medical Foundation, Chennai, India.
Indian J Med Res. 2010 Sep;132:303-11.
BACKGROUND & OBJECTIVES: Duchenne (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive disorders, caused by mutations in the dystrophin gene. Genetic diagnosis of the proband becomes crucial, and forms the base for carrier analysis, genetic counselling, prediction of natural history and prognosis, and eligibility for therapeutic strategies. Traditional multiplex PCR assay is the common method used in India to detect DMD gene deletions, mainly in the hot-spot region. Deletions of exons outside the usual 18 or 21 exons in the hot-spot, duplications and carrier analysis are often left without precise genetic diagnosis and require efficient dosage/quantitative analysis. In this study we evaluated the efficacy of using multiplex PCR (mPCR) of 30 exons followed by multiplex ligation-dependent probe amplification (MLPA), to study deletions and duplications in the DMD gene in patients clinically diagnosed as BMD/DMD.
Using an algorithm of mPCR and MLPA which was less invasive and cost-effective, we performed retrospective and prospective analysis on 150 male patients.
Multiplex PCR could pick up deletions in 103 of the 150 cases. MLPA was able to detect deletions and duplications including nine additional mutations. Further, the borders of the deletions and duplications were more accurately defined by this recent methodology, which enables one to determine the effect of the mutation on the reading frame. In all, including the single exon deletions, MLPA was efficient in accurately confirming mutations in 35 per cent of all cases. Ten novel mutations were identified in this study. Overall, this approach confirmed mutations in 75 per cent of the patients in our study.
INTERPRETATION & CONCLUSIONS: The systematic approach/algorithm used in this study offers the best possible economical mutation analysis in the Indian scenario.
杜兴氏肌营养不良症（DMD）和贝克肌营养不良症（BMD）是 X 连锁隐性疾病，由肌营养不良蛋白基因突变引起。对先证者进行基因诊断至关重要，这为携带者分析、遗传咨询、自然病史和预后预测以及治疗策略的资格提供了基础。传统的多重聚合酶链反应（PCR）检测是印度常用的检测 DMD 基因缺失的方法，主要集中在热点区域。热点区域以外的外显子缺失、重复和携带者分析通常无法进行精确的基因诊断，需要进行有效的剂量/定量分析。本研究评估了使用 30 个外显子的多重 PCR（mPCR）联合多重连接依赖性探针扩增（MLPA），对临床上诊断为 BMD/DMD 的患者的 DMD 基因缺失和重复进行研究的效果。
我们使用一种侵袭性较小且经济有效的 mPCR 和 MLPA 算法，对 150 名男性患者进行回顾性和前瞻性分析。
多重 PCR 可在 150 例病例中的 103 例中检测到缺失。MLPA 能够检测缺失和重复，包括 9 种额外的突变。此外，这种新方法更准确地定义了缺失和重复的边界，使人们能够确定突变对阅读框的影响。总的来说，包括单外显子缺失在内，MLPA 能够准确确认 35%的所有病例中的突变。本研究共发现 10 种新突变。总体而言，这种方法在我们的研究中确认了 75%的患者的突变。
本研究中使用的系统方法/算法为印度的情况提供了最佳的经济突变分析。
