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Pseudoexon activating by a deep intronic variant and phenotype variation in a Chinese family with dystrophinopathy

Journal of Human Genetics volume 70, pages 483–488 (2025) Cite this article

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Abstract

Aberrant inclusion of pseudoexons (PE) in mature mRNA is a rare splicing defect contributing to Duchenne muscular dystrophy (DMD) pathogenesis. In this study, we described two affected males from a Chinese family who presented with progressive muscle weakness, elevated creatine kinase (CK) levels, and dystrophic changes on muscle pathology. Whole-genome sequencing followed by linkage-based filtering identified a shared deep intronic variant in intron 47 of DMD gene (c.6913-4037T>G), which activated a cryptic splice site and resulted in the inclusion of a 72 bp PE between exons 47 and 48. Patient induced pluripotent stem cells (iPSCs)-derived myotubes from the patient confirmed the presence of this PE, with a significant reduction in dystrophin expression compared to controls. Quantitative PCR revealed that aberrant transcripts comprised ~89% of total DMD transcripts in myotubes and ~97% in muscle, correlating with near-complete loss of dystrophin. Functional assays further showed impaired myotube fusion and altered calcium signaling. This study underscores the diagnostic complexity of intronic DMD variants and provides evidence supporting the pathogenicity of c.6913-4037T>G.

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Fig. 1: Pedigrees of the patients’ family and clinical phenotypes of the 17-year-old patient II-3.
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Fig. 2: Histopathology findings in patients II-3 and III-1.
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Fig. 3: Identification of the deep intronic variant in the DMD gene.
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Fig. 4: Dystrophin transcripts expression in differentiated iPSC-derived myotubes and muscle tissues from patient III-1.
The alternative text for this image may have been generated using AI.

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Acknowledgements

We extend our gratitude to all patients and volunteers for their participation in this study. The authors appreciate the support provided by Tonghai Dou, Aibei Xu, Xilu Wang, Dandan Dai, Tianfang Shi, JiMin Zhang, Xue Li from Amplicongene. We also thank Dr. Xihua Li, Wanhong He, Haijun Zhu, Sufen Zhang for their kind guidance on clinical database and molecular experiments.

Funding

Funding WZ and JX were supported by National Key R&D Program of China (2024YFC3406700, 2024YFC3406705), Clinical Research Project Supported by, Huashan Hospital, Fudan University (YN2023-013); National Natural Science Foundation of China (82171398, 82271437).

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Author notes

  1. These authors contributed equally: Xingyu Xia, Kexin Jiao.

Authors and Affiliations

  1. Department of Neurology, Huashan Hospital, Fudan University, Shanghai, China

    Xingyu Xia, Kexin Jiao, Nachuan Cheng, Ningning Wang, Bochen Zhu, Meining Diao, Jianying Xi, Chongbo Zhao & Wenhua Zhu

  2. Huashan Rare Disease Center, Shanghai Medical College, Huashan Hospital, Fudan University, Shanghai, China

    Xingyu Xia, Kexin Jiao, Nachuan Cheng, Mingshi Gao, Ningning Wang, Bochen Zhu, Meining Diao, Jianying Xi, Chongbo Zhao & Wenhua Zhu

  3. National Center for Neurological Disorders (NCND), Shanghai, China

    Xingyu Xia, Kexin Jiao, Nachuan Cheng, Mingshi Gao, Ningning Wang, Bochen Zhu, Meining Diao, Jianying Xi, Chongbo Zhao & Wenhua Zhu

  4. Department of Neurology, Children’s Hospital of Fudan University, Shanghai, China; National Children’s Medical Center, Shanghai, China

    Chaoping Hu

  5. Department of Pathology, Huashan Hospital, Fudan University, Shanghai, China

    Mingshi Gao

  6. Shanghai Key Laboratory of Maternal Fetal Medicine, Department of Fetal Medicine and Prenatal Diagnosis Center, Shanghai First Maternity and Infant Hospital, School of Medicine, Tongji University, Shanghai, China

    Shiyi Xiong

  7. Department of Neurology, Jing’an District Center Hospital of Shanghai, Shanghai, China

    Dongyue Yue

  8. Shanghai Amplicongene Co. Ltd, Shanghai, China

    Chengwen Chen

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  1. Xingyu Xia
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  2. Kexin Jiao
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  14. Wenhua Zhu
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Contributions

XYX and KXJ contributed equally to the draft of manuscript and data analysis. CPH, NCC, MSG, BCZ, MND, and DYY contributed to the acquisition and analysis of data. JYX, CBZ, and WHZ contributed to the design of the study and draft reviewing.

Corresponding author

Correspondence to Wenhua Zhu.

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The authors declared no competing interests.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Huashan Hospital, Fudan University and patient gave consent.

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Xia, X., Jiao, K., Hu, C. et al. Pseudoexon activating by a deep intronic variant and phenotype variation in a Chinese family with dystrophinopathy. J Hum Genet 70, 483–488 (2025). https://doi.org/10.1038/s10038-025-01361-x

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