Dystrophin gene analysis in Duchenne/Becker dystrophy in a Malaysian population using multiplex polymerase chain reaction

Dystrophinopathy is the commonest form of muscular dystrophy and comprises clinically recognized forms, Duchenne dystrophy and Becker dystrophy. Mutations in the dystrophin gene which consist of large gene deletions (65%), duplications (5%) and point mutations (30%) are responsible for reducing the amount of functional dystrophin protein in skeletal muscle fi bres leading to fi bre destruction and disease. The aims of this study are to investigate the detection rate, types and distribution of large gene deletions in Malaysian dystrophinopathy patients using the multiplex polymerase chain reaction (MPCR). MPCR of 18 “hot-spot deletion” regions along the dystrophin gene was performed on DNA from 48 muscle biopsy-confi rmed cases of dystrophinopathy. A positive detection rate of 58% (28/48) was observed, where 84% (16/19) Indian, 35% (6/17) Chinese and 50% (6/12) Malay ethnic groups showed deletions in their dystrophin genes. The Malaysian Indians appear to have a higher prevalence for large gene deletions compared to the Chinese and Malays. Further analyses of 42 confi rmed positive cases (present 28 plus previous 14 cases) by MPCR showed the majority of deletions were in the mid-distal region of the dystrophin gene (81% in exons 45-60). The MPCR is a specifi c and sensitive method for confi rmation of gene deletions responsible for dystrophinopathy. Neurology Asia 2010; 15(1) : 19 – 25 Address correspondence to: Professor Wong Kum Thong, Dept of Pathology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia. Telephone: 603-79675762, Fax: 60379556845, email: [email protected] INTRODUCTION The commonest type of muscular dystrophy is dystrophinopathy that includes the clinically more severe form of Duchenne dystrophy (DD) and the less severe form, Becker dystrophy (BD). Mutations in the large dystrophin gene that consists of 79 exons, result in reduced or absent functional dystrophin protein in skeletal muscles, leading to varying clinical severity. About 65% of DD/BD cases are the result of large scale gene deletions, while about 5% are due to duplications, with the remaining 30% arising from point mutations. Sequencing the dystrophin gene is time consuming because of the complex organisation of the introns and exons that spans 2.4 million base pairs (Mbp). However, it is fortunate that many of the large gene deletions within the dystrophin gene can be detected in specifi c “hotspot areas” of the gene. These “hotspots” are clustered in two main regions at the 5’ proximal portion of the gene (exons 1, 3, 4, 5, 8, 13, 19) and within the mid-distal region (exons 42 45, 47, 48, 50 53, 60). DNA amplifi cation by the multiplex polymerase chain reaction (MPCR) can detect 98% of the large scale deletions in the dystrophin gene. The MPCR differs from the standard PCR in that more than two primer sets are used to amplify different regions of target DNA in a single reaction. The advantage of MPCR is its rapidity as multiple target sequences are amplifi ed simultaneously. MPCR requires only a very small amount of DNA that can be extracted from chorionic villi, blood and muscle samples. The main objective of this study is to determine the detection rate of large gene deletions in Malaysian DD/BD patients using an established in-house MPCR technique. In addition, the multiracial composition of the Malaysian population could possibly lend itself to studying racial differences in dystrophinopathy, if any. There are currently no published reports of molecular analysis of the dystrophin gene in the Malaysian population. Neurology Asia April 2010