Skipping of an exon with a nonsense mutation in the DMD gene is induced by the conversion of a splicing enhancer to a splicing silencer

Zhu, Yanrong; Deng, Huiting; Chen, Xiangfa; Li, Hui; Yang, Cheng; Li, Shuo; Pan, Xiaoying; Tian, Siqi; Feng, Shuxin; Tan, Xiaoyue; Matsuo, Masafumi; Zhang, Zhujun

doi:10.1007/s00439-019-02036-2

Cited by 14 publications

(17 citation statements)

References 58 publications

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“…In this context, current estimates indicate that at least 33% of disease-causing mutations alter pre-mRNA splicing [6], which is most probably an underestimation because: (i) NGS analysis (WES and gene panels) are mostly restrained to genetic variants located at the boundaries of canonical splice-site sequences; (ii) some synonymous substitutions, in fact, disrupt splicing regulatory elements such as ESEs/ISE (exonic/intronic splicing enhancers) and ESSs/ISSs (exonic/intronic splicing silencers) [7,8]; and (iii) genetic variants mapping at either non-canonical splice sites (NCSS) or hidden within large introns go mostly undetected.…”

Section: Introductionmentioning

confidence: 99%

Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants

Toulis

Cortés‐González

Castro-Miró

et al. 2020

Genes

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Aims: We aimed to validate the pathogenicity of genetic variants identified in inherited retinal dystrophy (IRD) patients, which were located in non-canonical splice sites (NCSS). Methods: After next generation sequencing (NGS) analysis (target gene panels or whole exome sequencing (WES)), NCSS variants were prioritized according to in silico predictions. In vivo and in vitro functional tests were used to validate their pathogenicity. Results: Four novel NCSS variants have been identified. They are located in intron 33 and 34 of ABCA4 (c.4774-9G>A and c.4849-8C>G, respectively), intron 2 of POC1B (c.101-3T>G) and intron 3 of RP2 (c.884-14G>A). Functional analysis detected different aberrant splicing events, including intron retention, exon skipping and intronic nucleotide addition, whose molecular effect was either the disruption or the elongation of the open reading frame of the corresponding gene. Conclusions: Our data increase the genetic diagnostic yield of IRD patients and expand the landscape of pathogenic variants, which will have an impact on the genotype–phenotype correlations and allow patients to opt for the emerging gene and cell therapies.

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Section: Introductionmentioning

confidence: 99%

Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants

Toulis

Cortés‐González

Castro-Miró

et al. 2020

Genes

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“…Previous reports have identified 11 nonsense mutations associated with exon skipping, in exon 25 (Fajkusova et al 2001;Santos et al 2014;Zhu et al 2019), in exon 27 (Shiga et al 1997), in exon 29 (Ginjaar et al 2000), in exon 31 (Disset et al 2006;Nishida et al 2011;Kevin et al 2011), in exon 37 (Hamed et al 2005), in exon 38 (Janssen et al 2005;Kevin et al 2011), and in exon 72 (Melis et al 1998). In this study, we identified 11 nonsense and three frameshift mutations causing exon skipping in exon 9, 25, 27, 31, 37, 38, 41, 72 and 74, indicating the exon skip event is not restricted to nonsense mutations, but also due to other mutations.…”

Section: Discussionmentioning

confidence: 99%

“…The skipping rates based on RT-PCR products from skeletal muscles might be overestimated, since unskipped products with nonsense/frameshift undergo degradation by nonsense-mediated RNA decay. Fourth, the minigene assay can be conducted in non-muscle cell line such as Hela cells (Nishida et al 2011;Zhu et al 2019), because this nonsense-mediated exon skipping was not related to skeletal muscle-specific alternative splicing, but it will probably be observed in other organs. By this method, in fact, exon skipping occurrence/non-occurrence was reproducibly observed in 89% of mutations.…”

Section: Discussionmentioning

confidence: 99%

“…The primer designs are shown in Supplementary Table S2. We constructed minigenes with wild-type fragment of each exon into H492-dys plasmid (Nishida et al 2011;Zhu et al 2019). Mutations were introduced by Quick Change sitedirected mutagenesis kit (Agilent Technologies, Palo Alto, CA, USA).…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…These exon skipping events by nonsense mutations are not specific to skeletal muscle, and they will be responsible for milder phenotype of not only skeletal muscle but also other organs in the patients. The minigene assay could be performed in HeLa cells as in previous reports (Nishida et al 2011;Zhu et al 2019). To understand these events and to simply evaluate the nonsense/frameshift mutations on the skipping rate of the corresponding exon, we analyzed exon skipping caused by the nonsense/frameshift mutations from a Japanese large cohort in artificial minigene experiments (Okubo et al 2016(Okubo et al , 2017.…”

Section: Introductionmentioning

confidence: 99%

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Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy

et al. 2020

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Duchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the DMD gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/ frameshift mutations in DMD and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in DMD in the Japanese Registry of Muscular Dystrophy. For each DMD mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in DMD gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in DMD and for interpreting the phenotypes of the BMD patients. Abbreviations BMD Becker muscular dystrophy DMD Duchenne muscular dystrophy ESE Exonic splicing enhancer ESS Exonic splicing silencer HSF Human Splicing Finder IMD Intermediate muscular dystrophy Remudy Registry of muscular dystrophy

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Functional characterization of ABCC8 variants of unknown significance based on bioinformatics predictions, splicing assays, and protein analyses: Benefits for the accurate diagnosis of congenital hyperinsulinism

et al. 2021

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ABCC8 encodes the SUR1 subunit of the β‐cell ATP‐sensitive potassium channel whose loss of function causes congenital hyperinsulinism (CHI). Molecular diagnosis is critical for optimal management of CHI patients. Unfortunately, assessing the impact of ABCC8 variants on RNA splicing remains very challenging as this gene is poorly expressed in leukocytes. Here, we performed bioinformatics analysis and cell‐based minigene assays to assess the impact on splicing of 13 ABCC8 variants identified in 20 CHI patients. Next, channel properties of SUR1 proteins expected to originate from minigene‐detected in‐frame splicing defects were analyzed after ectopic expression in COSm6 cells. Out of the analyzed variants, seven induced out‐of‐frame splicing defects and were therefore classified as recessive pathogenic, whereas two led to skipping of in‐frame exons. Channel functional analysis of the latter demonstrated their pathogenicity. Interestingly, the common rs757110 SNP increased exon skipping in our system suggesting that it may act as a disease modifier factor. Our strategy allowed determining the pathogenicity of all selected ABCC8 variants, and CHI‐inheritance pattern for 16 out of the 20 patients. This study highlights the value of combining RNA and protein functional approaches in variant interpretation and reveals the minigene splicing assay as a new tool for CHI molecular diagnostics.

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Skipping of an exon with a nonsense mutation in the DMD gene is induced by the conversion of a splicing enhancer to a splicing silencer

Cited by 14 publications

References 58 publications

Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants

Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants

Exon skipping induced by nonsense/frameshift mutations in DMD gene results in Becker muscular dystrophy

Functional characterization of ABCC8 variants of unknown significance based on bioinformatics predictions, splicing assays, and protein analyses: Benefits for the accurate diagnosis of congenital hyperinsulinism

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