MLPA And Exome Sequencing-Based Molecular Diagnostic Methods For Duchenne/Becker Muscular Dystrophy

Duchenne/Becker muscular dystrophy（DMD/BMD） can be divided into twotypes,DMD and BMD. DMD is the most common and lethal X-linked neuromusculardiseases, which is characterized by progressive muscle weakness and pseudohypertrophyof gastrocnemius. It has an incidence of1in3500live male births worldwide. Generally,the diease begins when the boys are3⁵years old, and the patients die before he istwenty years of life. The incidence of BMD is about1/18000, and the clinicalmanifestations are similar to DMD, but much milder. The dystrophin gene comprises of atleast79exons, spans more than2.5Mb of genomic DNA, is located at Xp21.2,which is thelargest known gene, containing eight independent tissue-specific promoters.Approximately75%of mutational events are represented by deletions or duplications ofone or more exons in the dystrophin gene, and the remaining cases by subtle mutations,including point mutations, small indels, and complex small rearrangements. In most cases,subtle mutations can cause premature translational termination, which in turn results in theformation of truncated peptide, and the happening of the disease.In the first part of this study, multiple ligation dependent probe amplification （MLPA）was used in the molecular diagnosis for patients from18pedigrees with clinical suspicionof DMD. One or more exons deletion mutations were detected in6families（with2casesbeing de novo mutations）, and in1family exon repeat was found.The second part of this study was the use of MLPA in the prenatal moleculardiagnosis for two high-risky DMD pregnancies. In the DMD gene of one fetus deletionmutation was detected, which was the same as that of the proband, and no mutation wasfound in the other.The third part of this study established a molecular diagnostic system for DMD subtlemutation. Second-generation sequencing was used in mutation detection of DMD gene fora patient with typical clinical manifestations of DMD, but without exon deletions orduplications in the dystrophin gene. Through the exome sequencing a mutationX₃1950196_C/G was found in the DMD gene, which was confirmed by Sangersequencing. This mutation located in a key splicing donor site of DMD gene, and mightaffect the intron recognition and splicing. The bioinformatics tools were used to predict theeffects of this mutation, and found that the5’end donor site of the mutation disappeared, and might lead to a change in the pathway of alternative splicing.In order to know the pathogenic mechanism of the mutation, hybrid minigenesplicing assay （HMSA） was used to find the effect of the mutation on the alternativesplicing of dystrophin gene at the cell level. The related flank sequence spanning themutation was inserted into the plasmid of pcDNATM3.1/Hygro （+）, and the recombinantplasmid was used to transiently transfect HeLa cells. The total RNA was extracted, andalternative splicing product analysed by RT-PCR. Compared product with normal control,the mutation caused two kinds of the products of transcripts:the first which was the majorproduct could be explained by the intron retention, and the second could be explained bythe alternative donor site. This result coincided with the prediction of bioinformatics. Bothsplicing types resulted in premature termination codon, which could not generatedystrophin with normal function.