Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype

Zhihong Wang; Yanhong Lin; Liping Qiu; Dezhu Zheng; Aizhen Yan; Jian Zeng; Fenghua Lan

doi:10.1515/cclm-2015-1042

Article

Hybrid minigene splicing assay verified the pathogenicity of a novel splice site variant in the dystrophin gene of a Chinese patient with typical Duchenne muscular dystrophy phenotype

Zhihong Wang , Yanhong Lin , Liping Qiu , Dezhu Zheng , Aizhen Yan , Jian Zeng and Fenghua Lan

Published/Copyright: March 17, 2016

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 54 Issue 9

Abstract

Background:

Duchenne muscular dystrophy (DMD) is typically caused by disrupting the reading frame of the dystrophin gene: approximately 70%–80% of mutational events are represented by deletions or duplications of one or more exons in the dystrophin gene, and the remaining cases by subtle mutations, including point mutations, small indels, small inversions, and complex small rearrangements. The dystrophin gene is the largest known gene with one of the highest known rates of new mutations.

Methods:

Deletions and duplications were detected in the DMD gene of the proband by using multiple ligation-dependent probe amplification (MLPA). Targeted next-generation sequencing (NGS) was used in the subtle mutation detection, followed by Sanger sequencing confirmation. The effect of the mutation on the splicing of the DMD gene was assessed by bioinformatics prediction and hybrid minigene splicing assay (HMSA).

Results:

Neither duplication nor deletion was found in the DMD gene of the proband. While a novel splice site mutation c.6762+1G>C was identified in the proband by NGS and Sanger sequencing, and his mother was heterozygous at the same site. Bioinformatics predicted that the 5′ donor splice site of intron 46 disappeared because of the mutation, which would lead to aberrant splicing and introduce premature stop codon. The HMSA results were in agreement with the prediction.

Conclusions:

The novel splice site mutation caused DMD in the proband by aberrant splicing. We suggested that combined applications of MLPA, NGS, HMSA and bioinformatics are comprehensive and effective methods for diagnosis and aberrant splicing study of DMD.

Keywords: bioinformatics; Duchenne muscular dystrophy; dystrophin gene; hybrid minigene splicing assay; next-generation sequencing

Corresponding author: Fenghua Lan, Research Center for Molecular Diagnosis of Genetic Diseases, Fuzhou General Hospital, 156 Xi’erhuanbei Road, Fuzhou City, Fujian Province 350025, P.R. China, Phone/Fax: +8659183721105, E-mail: fhlan@mail.fjmu.edu.cn

aZhihong Wang, Yanhong Lin and Liping Qiu contributed equally to this work.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2015-10-25

Accepted: 2016-2-15

Published Online: 2016-3-17

Published in Print: 2016-9-1

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Articles in the same Issue

https://doi.org/10.1515/cclm-2015-1042

Keywords for this article

bioinformatics; Duchenne muscular dystrophy; dystrophin gene; hybrid minigene splicing assay; next-generation sequencing