Abstract
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the DMD gene. The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families. Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS). Pathogenic variants in DMD were successfully identified in all cases, and 11 of them were novel. The most common mutations were intragenic deletions (69%), with two hotspots located in the 5′ end (exons 2–19) and the central of the DMD gene (exons 45–55), while point mutations were observed in 22% patients. Further, c.1149+1G>A and c.1150-2A>G were confirmed by hybrid minigene splicing assay (HMSA). This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein. Therefore, the clinical use of MLPA, NGS, and HMSA is an effective strategy to identify variants. Importantly, eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis (PGD). Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.
抽象
目 的
针对 100 例无亲缘关系的假肥大型肌营养不良症 (DMD/BMD) 患者, 联合应用多种检测技术进行遗传学诊断, 并且建立个体化产前诊断及胚胎植入前遗传学诊断方案, 以最大限度地降低 DMD/BMD 患儿的出生。
创新点
通过 minigene 剪接实验分析 DMD: c.1149+1G<A 和 c.1150−2A>G 突变是否导致剪接异常, 并确定 剪接方式。
方 法
收集 100 例无亲缘关系 DMD/BMD 患者的临床资 料, 应用多重连接依赖式探针扩增技术 (MLPA)、 第二代测序 (NGS)、minigene 剪接实验 (HMSA) 进行遗传学诊断, 并通过单体型分析及性别鉴定 进行胚胎植入前遗传学诊断。
结 论
联合应用多种检测技术可以尽早地对患者进行遗 传学诊断, 为临床遗传咨询和产前诊断及胚胎植 入前遗传学诊断提供了科学依据。
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Acknowledgments
The authors thank the patients for their voluntary involvement in this study. We are grateful to Dr. Jiong GAO (BGI Genomics, Shenzhen, China) for analyzing the data and modifying the article.
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Project supported by the National Key Research and Development Program of China (No. 2016YFC1000703), the Medicine and Health Science and Technology Plan Projects in Zhejiang Province (No. 2014KYA246), and the National Natural Science Foundation of China (Nos. 81801441 and 81300532)
Contributors
Min-yue DONG set up the theme and examined the manuscript. Yan-mei YANG performed the experiments and wrote the manuscript. Kai YAN, Bei LIU, Li-ya WANG, and Ying-zhi HUANG performed the experiments. Min CHEN was responsible for editing the grammar of the manuscript. Ye-qing QIAN guided the modification process. Yi-xi SUN and Hong-ge LI were responsible for collecting the patients. All authors read and approved the final manuscript and, therefore, had full access to all the data in the study and take responsibility for the integrity and security of the data.
Compliance with ethics guidelines
Yan-mei YANG, Kai YAN, Bei LIU, Min CHEN, Li-ya WANG, Ying-zhi HUANG, Ye-qing QIAN, Yi-xi SUN, Hong-ge LI, and Min-yue DONG declare that they have no conflict of interest.
All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 (5). Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for whom identifying information is included in this article.
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Comprehensive genetic diagnosis of patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and pathogenicity analysis of splice site variants in the DMD gene
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Yang, Ym., Yan, K., Liu, B. et al. Comprehensive genetic diagnosis of patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and pathogenicity analysis of splice site variants in the DMD gene. J. Zhejiang Univ. Sci. B 20, 753–765 (2019). https://doi.org/10.1631/jzus.B1800541
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DOI: https://doi.org/10.1631/jzus.B1800541