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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Tuffery-Giraud S, Beroud C, Leturcq F, Yaou RB, Hamroun D, Michel-Calemard L, et al. Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase. Hum Mutat. 2009;30:934–45.
Bladen CL, Salgado D, Monges S, Foncuberta ME, Kekou K, Kosma K, et al. The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations. Hum Mutat. 2015;36:395–402.
Juan-Mateu J, Gonzalez-Quereda L, Rodriguez MJ, Baena M, Verdura E, Nascimento A, et al. DMD Mutations in 576 Dystrophinopathy Families: A Step Forward in Genotype-Phenotype Correlations. PLoS ONE. 2015;10:e0135189.
Aartsma-Rus A, Ginjaar IB, Bushby K. The importance of genetic diagnosis for Duchenne muscular dystrophy. J Med Genet. 2016;53:145–51.
Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Gappmaier E, Howard MT, et al. Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009;30:1657–66.
Fokkema IF, Taschner PE, Schaafsma GC, Celli J, Laros JF, den Dunnen JT. LOVD v.2.0: the next generation in gene variant databases. Hum Mutat. 2011;32:557–63.
Landrum MJ, Lee JM, Benson M, Brown G, Chao C, Chitipiralla S, et al. ClinVar: public archive of interpretations of clinically relevant variants. Nucleic Acids Res. 2016;44:D862–8.
Verhaart IEC, Aartsma-Rus A. Therapeutic developments for Duchenne muscular dystrophy. Nat Rev Neurol. 2019;15:373–86.
Jones HF, Bryen SJ, Waddell LB, Bournazos A, Davis M, Farrar MA, et al. Importance of muscle biopsy to establish pathogenicity of DMD missense and splice variants. Neuromuscul Disord. 2019;29:913–9.
Toksoy G, Durmus H, Aghayev A, Bagirova G, Sevinc Rustemoglu B, Basaran S, et al. Mutation spectrum of 260 dystrophinopathy patients from Turkey and important highlights for genetic counseling. Neuromuscul Disord. 2019;29:601–13.
Raj Joshi P, Sarangerel J, Munkhbayar R, Glaser D, Zierz S. Exon skipping in Duchenne Muscle dystrophy due to a silent p.Ser443= mutation in the DMD gene. J Clin Neurosci. 2020;76:229–32.
Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405–24.
Ito K, Patel PN, Gorham JM, McDonough B, DePalma SR, Adler EE, et al. Identification of pathogenic gene mutations in LMNA and MYBPC3 that alter RNA splicing. Proc Natl Acad Sci USA. 2017;114:7689–94.
Houdayer C, Dehainault C, Mattler C, Michaux D, Caux-Moncoutier V, Pages-Berhouet S, et al. Evaluation of in silico splice tools for decision-making in molecular diagnosis. Hum Mutat. 2008;29:975–82.
Soukarieh O, Gaildrat P, Hamieh M, Drouet A, Baert-Desurmont S, Frebourg T, et al. Exonic splicing mutations are more prevalent than currently estimated and can be predicted by using in silico tools. PLoS Genet. 2016;12:e1005756.
Gaildrat P, Killian A, Martins A, Tournier I, Frebourg T, Tosi M. Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants. Methods Mol Biol. 2010;653:249–57.
Fraile-Bethencourt E, Diez-Gomez B, Velasquez-Zapata V, Acedo A, Sanz DJ, Velasco EA. Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18. PLoS Genet. 2017;13:e1006691.
Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR. ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res. 2003;31:3568–71.
Yeo G, Burge CB. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol. 2004;11:377–94.
Vinci G, Lynch NJ, Duponchel C, Lebastard TM, Milon G, Stover C, et al. In vivo biosynthesis of endogenous and of human C1 inhibitor in transgenic mice: tissue distribution and colocalization of their expression. J Immunol. 2002;169:5948–54.
Duponchel C, Djenouhat K, Fremeaux-Bacchi V, Monnier N, Drouet C, Tosi M. Functional analysis of splicing mutations and of an exon 2 polymorphic variant of SERPING1/C1NH. Hum Mutat. 2006;27:295–6.
Zhang K, Yang X, Lin G, Han Y, Li J. Molecular genetic testing and diagnosis strategies for dystrophinopathies in the era of next generation sequencing. Clin Chim Acta. 2019;491:66–73.
Bai Y, Li S, Zong YN, Li XL, Zhao ZH, Kong XD. [Mutation screening of 433 families with Duchenne/Becker muscular dystrophy]. Zhonghua Yi Xue Za Zhi. 2016;96:1261–9.
Cartegni L, Chew SL, Krainer AR. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet. 2002;3:285–98.
Gurvich OL, Tuohy TM, Howard MT, Finkel RS, Medne L, Anderson CB, et al. DMD pseudoexon mutations: splicing efficiency, phenotype, and potential therapy. Ann Neurol. 2008;63:81–9.
Baralle D, Baralle M. Splicing in action: assessing disease causing sequence changes. J Med Genet. 2005;42:737–48.
Krawczak M, Thomas NS, Hundrieser B, Mort M, Wittig M, Hampe J, et al. Single base-pair substitutions in exon-intron junctions of human genes: nature, distribution, and consequences for mRNA splicing. Hum Mutat. 2007;28:150–8.
Deburgrave N, Daoud F, Llense S, Barbot JC, Recan D, Peccate C, et al. Protein- and mRNA-based phenotype-genotype correlations in DMD/BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene. Hum Mutat. 2007;28:183–95.
Flanigan KM, Dunn DM, von Niederhausern A, Soltanzadeh P, Howard MT, Sampson JB, et al. Nonsense mutation-associated Becker muscular dystrophy: interplay between exon definition and splicing regulatory elements within the DMD gene. Hum Mutat. 2011;32:299–308.
Witting N, Duno M, Vissing J. Becker muscular dystrophy with widespread muscle hypertrophy and a non-sense mutation of exon 2. Neuromuscul Disord. 2013;23:25–8.
Okubo M, Noguchi S, Hayashi S, Nakamura H, Komaki H, Matsuo M, et al. Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy. Hum Genet. 2020;139:247–55.
Winnard AV, Mendell JR, Prior TW, Florence J, Burghes AH. Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production. Am J Hum Genet. 1995;56:158–66.
Inacio A, Silva AL, Pinto J, Ji X, Morgado A, Almeida F, et al. Nonsense mutations in close proximity to the initiation codon fail to trigger full nonsense-mediated mRNA decay. J Biol Chem. 2004;279:32170–80.
Gurvich OL, Maiti B, Weiss RB, Aggarwal G, Howard MT, Flanigan KM. DMD exon 1 truncating point mutations: amelioration of phenotype by alternative translation initiation in exon 6. Hum Mutat. 2009;30:633–40.
Muntoni F, Torelli S, Ferlini A. Dystrophin and mutations: one gene, several proteins, multiple phenotypes. Lancet Neurol. 2003;2:731–40.
Zatz M, Pavanello RC, Lazar M, Yamamoto GL, Lourenco NC, Cerqueira A, et al. Milder course in Duchenne patients with nonsense mutations and no muscle dystrophin. Neuromuscul Disord. 2014;24:986–9.
Jin M, Li JJ, Xu GR, Wang N, Wang ZQ. Cryptic exon activation causes dystrophinopathy in two Chinese families. Eur J Hum Genet. 2020;28:947–55.
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).
The authors declare no competing interests.
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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). https://doi.org/10.1038/s10038-022-01035-y