Mutation analysis of dystrophin gene using multiplex ligation dependent probe amplification (MLPA)

Abstract

Duchenne muscular dystrophy (DMD), an allelic X-linked progressive muscle-wasting disease, and its allelic form Becker muscular dystrophy (BMD) are one of the most common single-gene disorders caused by mutations in the DMD gene (also dystrophin gene), the longest known human gene ranging 2.4 Mb, encoding a 427 kDa cytoskeletal protein called dystrophin. Due to the lack of reliable genetic diagnostic tool in Nepal the diagnosis of these genetic diseases are narrowed to phenotypic and clinical diagnoses. Besides, the lack of even the base line data of these genetic diseases has barred the progression of the further research of these genetic diseases in Nepal.This research has been carried out with the aim to introduce Multiplex ligation dependent probe amplification (MLPA) as one of the convenient molecular diagnostic tool in diagnosis of the genetic diseases as DMD/BMD.

In this research, DNA was extracted from the blood samples of DMD/BMD patients and from the normal male and female to be taken as the reference samples. DNA samples so extracted were then amplified in the thermocycler by using the MLPA assay. The PCR products of the test samples and the reference samples so obtained were run on the capillary electrophoresis (CE) and the data were analysed. Using an algorithm of MLPA, 26 total samples were assayed. The capillary electrophoresis run (ABI-310 genetic analyzer) demonstrated that it could pick up the deletions in 14 of the 21 test samples considered. Consequently, MLPA was efficient in accurately confirming mutations in about 67% of all cases. Most prevalent exonic deletion regions were found to be confined in the exon 7-14, the proximal zone and 45-53, the first half of C-terminal domain. The reading frame (in-frame or out-frame) were determined by using the “DMD exonic deletions/duplications reading frame checker 1.9” as recommended by MRCHolland, which need to be confirmed by sequencing.No novel mutations were identified in this study. Overall, this approach confirmed mutations in 67% of the patients in our study which is compatible with the recent studies in Chinese and Indian population. Among the 21 test samples used MLPA could not diagnose the mutation in some of the samples which were clinically diagnosed as DMD/BMD. This result aware us that in order to know the exact point of mutation and to know exactly which of the exon is deleted or duplicated further sequencing should be done. But still the efficiency of this MLPA assay makes it a rapid, robust, efficient and reliable genetic tool in the diagnosis of genetic disorders. The systematic approach/algorithm used in this study offers the best possible less invasive and effective mutation analysis in the context of Nepal.

Key words: Dystrophin gene, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), Multiplex Ligation Dependent Probe Amplification (MLPA), capillary electrophoresis (CE) 1