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Functional analysis of variants in DMD exon/intron 10 predicted to affect splicing


Duchenne muscular dystrophy (DMD, MIM #310200) and Becker muscular dystrophy (BMD, MIM #300376) are X-linked recessive hereditary diseases caused by pathogenic variants in the DMD gene. Genetic testing of DMD identifies a certain number of variants of uncertain clinical significance (VUS) whose functional interpretations pose a challenge for gene-based diagnosis. To improve the accuracy of variant interpretation in public mutation repositories, we used computational tools to prioritize VUS and developed a cell-based minigene assay to confirm aberrant splicing. Using this procedure, we evaluated rare variants in exon and intron 10 of the DMD gene. We demonstrated that 16 variants, including both canonical and non-canonical splice sites, altered RNA splicing in variable patterns. Using the example of exon and intron 10 of the DMD gene, we demonstrated the utility of the in vitro minigene assay in the effective assessment of the spliceogenic effect for VUS identified in clinical practice and underlined the necessity of precise variant classification. This is the first systematic characterization of DMD splicing variants, besides, through our study, some undetermined variants are demonstrated to be pathogenic by altering RNA splicing of DMD.

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Fig. 1: Minigene construct.
Fig. 2: In vitro splicing results for potential splice-altering variants.


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We thank the GeneChem Co. Ltd. (Shanghai, China) for the construction of the vector and minigene plasmid.


This work was supported by the grant from National Natural Science Foundation of China (Grant No. 81700003), National Natural Science Foundation of China (Grant No. 81670003), the Fundamental Research Funds for the Central Universities (Grant No.2242018K40034), National Key Research and Development Program of China (Grant No. 2018YFF0215202) and Medical Science and Technology Program of Nanjing (Grant No. JQX20007).

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XZ, XC and LY conceived and designed the study, contributed to data analysis and interpretation, manuscript drafting and critical review for intellectual content and final approval of manuscript. XZ, JC and YM performed the experiment. SH, MC and LW co-designed experiments, contributed to critically revising for important intellectual content and discussed analyses, interpretation, and presentation. All authors contributed to data interpretation, revisions of the manuscript, and approved the final version of the manuscript.

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Correspondence to Xinxin Zhang or Long Yi.

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Zhang, X., Chen, X., Chen, J. et al. Functional analysis of variants in DMD exon/intron 10 predicted to affect splicing. J Hum Genet 67, 495–501 (2022).

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