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A resolved discrepancy between multiplex PCR and multiplex ligation-dependent probe amplification by targeted next-generation sequencing discloses a novel partial exonic deletion in the Duchenne muscular dystrophy gene.

ABSTRACT: BACKGROUND:The genetic diagnosis of Duchenne muscular dystrophy (DMD) has been complicated by the large size of the gene and its heterogeneous mutational spectrum. Multiplex PCR and multiplex ligation-dependent probe amplification (MLPA) are two well-established mutation screening methods. Here, we applied targeted next-generation sequencing (NGS) to clarify discrepant results between multiplex PCR and MLPA in a Chinese patient with DMD. METHODS:MLPA was performed to confirm multiplex PCR results obtained previously. Targeted NGS was then used to analyze the full-length DMD gene including introns. RESULTS:Multiplex PCR had previously identified an apparent deletion of exon 43 in the patient with DMD, but current MLPA indicated that exon 43 was present. Targeted NGS to clarify the genetic diagnosis identified a novel mutation, c.6241_c.6290 + 1109del1159insAC, which caused partial deletion of exon 43. This mutation removed the annealing sequence of the exon 43 reverse primer in multiplex PCR but had no influence on the hybridization site of the MLPA probe. Therefore, the discrepancy between the two methods was caused by partial exonic deletion that escaped MLPA detection. CONCLUSION:Targeted NGS disclosed a novel partial exonic deletion in the DMD gene as the cause of discrepancy between multiplex PCR and MLPA. Targeted NGS could be used to provide a more accurate genetic diagnosis of DMD, particularly in cases of partial exonic deletions, which will be of benefit in patient management and the identification of disease carriers.

SUBMITTER: Liu C

PROVIDER: S-EPMC6816888 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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A resolved discrepancy between multiplex PCR and multiplex ligation-dependent probe amplification by targeted next-generation sequencing discloses a novel partial exonic deletion in the Duchenne muscular dystrophy gene.

Liu Chang C Deng Huiting H Yang Cheng C Li Xixi X Zhu Yanrong Y Chen Xiangfa X Li Hui H Li Shuo S Cui Hao H Zhang Xiaoyan X Tan Xiaoyue X Li Dong D Zhang Zhujun Z

Journal of clinical laboratory analysis 20180525 8

<h4>Background</h4>The genetic diagnosis of Duchenne muscular dystrophy (DMD) has been complicated by the large size of the gene and its heterogeneous mutational spectrum. Multiplex PCR and multiplex ligation-dependent probe amplification (MLPA) are two well-established mutation screening methods. Here, we applied targeted next-generation sequencing (NGS) to clarify discrepant results between multiplex PCR and MLPA in a Chinese patient with DMD.<h4>Methods</h4>MLPA was performed to confirm multi ...[more]

PMID: 29802662

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Project description:Dystrophinopathies cover a spectrum of rare progressive X-linked muscle diseases, arising from DMD mutations. They are among the most common pediatric muscular dystrophies, being Duchenne muscular dystrophy (DMD) the most severe form. Despite the fact that there is still no cure for these serious diseases, unprecedented advances are being made for the development of therapies for DMD. Some of which are already conditionally approved: exon skipping and premature stop codon read-through. The present work aimed to characterize the mutational spectrum of DMD in an Argentinian cohort, to identify candidates for available pharmacogenetic treatments and finally, to conduct a comparative analysis of the Latin American (LA) frequencies of mutations amenable for available DMD therapies. We studied 400 patients with clinical diagnosis of dystrophinopathy, implementing a diagnostic molecular algorithm including: MLPA/PCR/Sanger/Exome and bioinformatics. We also performed a meta-analysis of LA's metrics for DMD available therapies. The employed algorithm resulted effective for the achievement of differential diagnosis, reaching a detection rate of 97%. Because of this, corticosteroid treatment was correctly indicated and validated in 371 patients with genetic confirmation of dystrophinopathy. Also, 20 were eligible for exon skipping of exon 51, 21 for exon 53, 12 for exon 45 and another 70 for premature stop codon read-through therapy. We determined that 87.5% of DMD patients will restore the reading frame with the skipping of only one exon. Regarding nonsense variants, UGA turned out to be the most frequent premature stop codon observed (47%). According to the meta-analysis, only four LA countries (Argentina, Brazil, Colombia and Mexico) provide the complete molecular algorithm for dystrophinopathies. We observed different relations among the available targets for exon skipping in the analyzed populations, but a more even proportion of nonsense variants (∼40%). In conclusion, this manuscript describes the theragnosis carried out in Argentinian dystrophinopathy patients. The implemented molecular algorithm proved to be efficient for the achievement of differential diagnosis, which plays a crucial role in patient management, determination of the standard of care and genetic counseling. Finally, this work contributes with the international efforts to characterize the frequencies and variants in LA, pillars of drug development and theragnosis.

Dataset Information

A resolved discrepancy between multiplex PCR and multiplex ligation-dependent probe amplification by targeted next-generation sequencing discloses a novel partial exonic deletion in the Duchenne muscular dystrophy gene.

Publications

A resolved discrepancy between multiplex PCR and multiplex ligation-dependent probe amplification by targeted next-generation sequencing discloses a novel partial exonic deletion in the Duchenne muscular dystrophy gene.

Similar Datasets

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