Pseudoexons of the DMD Gene.
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ABSTRACT:
SUBMITTER: Keegan NP
PROVIDER: S-EPMC7175933 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
The DMD gene is the largest in the human genome, with a total intron content exceeding 2.2Mb. In the decades since DMD was discovered there have been numerous reported cases of pseudoexons (PEs) arising in the mature DMD transcripts of some individuals, either as the result of mutations or as low-frequency errors of the spliceosome. In this review, I collate from the literature 58 examples of DMD PEs and examine the diversity and commonalities of their features. In particular, I note the high fr ...[more]
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Project description:Pseudoexon (PE) inclusion has been implicated in various dystrophinopathies; however, its splicing characteristics have not been fully investigated. This study aims to analyze the splicing characteristics of dystrophin PEs and compare them with those of dystrophin canonical exons (CEs). Forty-two reported dystrophin PEs were divided into a splice site (ss) group and a splicing regulatory element (SRE) group. Five dystrophin PEs with characteristics of poison exons were identified and categorized as the possible poison exon group. The comparative analysis of each essential splicing signal among different groups of dystrophin PEs and dystrophin CEs revealed that the possible poison exon group had a stronger 3' ss compared to any other group. As for auxiliary SREs, different groups of dystrophin PEs were found to have a smaller density of diverse types of exonic splicing enhancers and a higher density of several types of exonic splicing silencers compared to dystrophin CEs. In addition, the possible poison exon group had a smaller density of 3' ss intronic splicing silencers compared to dystrophin CEs. To our knowledge, our findings indicate for the first time that poison exons might exist in DMD (the dystrophin gene) and present with different splicing characteristics than other dystrophin PEs and CEs.
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Project description:Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are two subtypes of muscular dystrophy diseases caused by pathogenic mutations in the DMD gene. Until now, more than 4,600 disease-causing mutations in DMD have been reported. However, only 33 mutations were deep intronic, cases with this type of mutations were limited. Methods: In this study, we used a combination of complementary DNA (cDNA) and target DNA sequencing analysis in addition to conventional whole-exome sequencing (WES). Results: Three novel hemizygous mutations IVS11 + 17811C > G (c.1331 + 17811C > G), IVS21 + 3252A > G (c.2803 + 3252A > G) and IVS40 + 362A > G (c.5739 + 362A > G) were identified in DMD patients, while a reported hemizygous mutation IVS62-285A > G (c.9225-285A > G) was found in the BMD patient. These DMD mutations lead to pseudoexon insertions, causing the generation of truncated and dysfunctional dystrophin. Conclusion: This study defines three novel and one reported intronic mutations, which can result in DMD/BMD. We also emphasize the need to combine WES and cDNA-based methods to detect the variant in the very large DMD gene in which the mutational spectrum is complex.
| S-EPMC8085517 | biostudies-literature
Project description:BackgroundDuchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder characterised by progressive irreversible muscle weakness, primarily of the skeletal and the cardiac muscles. DMD is characterised by mutations in the dystrophin gene, resulting in the absence or sparse quantities of dystrophin protein. A precise and timely molecular detection of DMD mutations encourages interventions such as carrier genetic counselling and in undertaking therapeutic measures for the DMD patients.ResultsIn this study, we developed a 2.1 Mb custom DMD gene panel that spans the entire DMD gene, including the exons and introns. The panel also includes the probes against 80 additional genes known to be mutated in other muscular dystrophies. This custom DMD gene panel was used to identify single nucleotide variants (SNVs) and large deletions with precise breakpoints in 77 samples that included 24 DMD patients and their matrilineage across four generations. We used this panel to evaluate the inheritance pattern of DMD mutations in maternal subjects representing 24 DMD patients.ConclusionHere we report our observations on the inheritance pattern of DMD gene mutations in matrilineage samples across four generations. Additionally, our data suggest that the DMD gene panel designed by us can be routinely used as a single genetic test to identify all DMD gene variants in DMD patients and the carrier mothers.
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Project description:OBJECTIVE:Duchenne/Becker muscular dystrophy (DMD/BMD) is the most common genetic neuromuscular disease in children, resulting from a defect in the DMD gene located on Xp21.2. The new emerging treatment using exon skipping strategy is tailored to specific mutations, thus molecular diagnostics are particularly important. This study aimed to detect the DMD gene deletion in Indonesian DMD/BMD patients and analyze the potential amenability by exon skipping therapy. RESULTS:Thirty-four male patients were enrolled in this study, 23 of them (67.6%) underwent muscle biopsy and showed the absence or partially expressed dystrophin protein in immunohistochemistry staining. All patients had very high serum CK levels (10.529?±?9.97 IU/L). Multiplex PCR revealed the DMD gene deletions in 15 (44.1%) cases. Seventy-eight percent of deletions were clustered in the hot-spot region of exon 43 to 52. Furthermore, seven (20.5%) patients were potentially amenable to exon skipping treatment. Therefore, multiplex PCR is one feasible method to detect DMD gene deletion in Indonesian DMD/BMD patients that can further determine the potential amenability of exon skipping therapy. In addition, this study is the first report of DMD gene deletion analysis in Indonesia.
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Project description:Understanding pre-mRNA splicing is crucial to accurately diagnosing and treating genetic diseases. However, mutations that alter splicing can exert highly diverse effects. Of all the known types of splicing mutations, perhaps the rarest and most difficult to predict are those that activate pseudoexons, sometimes also called cryptic exons. Unlike other splicing mutations that either destroy or redirect existing splice events, pseudoexon mutations appear to create entirely new exons within introns. Since exon definition in vertebrates requires coordinated arrangements of numerous RNA motifs, one might expect that pseudoexons would only arise when rearrangements of intronic DNA create novel exons by chance. Surprisingly, although such mutations do occur, a far more common cause of pseudoexons is deep-intronic single nucleotide variants, raising the question of why these latent exon-like tracts near the mutation sites have not already been purged from the genome by the evolutionary advantage of more efficient splicing. Possible answers may lie in deep intronic splicing processes such as recursive splicing or poison exon splicing. Because these processes utilize intronic motifs that benignly engage with the spliceosome, the regions involved may be more susceptible to exonization than other intronic regions would be. We speculated that a comprehensive study of reported pseudoexons might detect alignments with known deep intronic splice sites and could also permit the characterisation of novel pseudoexon categories. In this report, we present and analyse a catalogue of over 400 published pseudoexon splice events. In addition to confirming prior observations of the most common pseudoexon mutation types, the size of this catalogue also enabled us to suggest new categories for some of the rarer types of pseudoexon mutation. By comparing our catalogue against published datasets of non-canonical splice events, we also found that 15.7% of pseudoexons exhibit some splicing activity at one or both of their splice sites in non-mutant cells. Importantly, this included seven examples of experimentally confirmed recursive splice sites, confirming for the first time a long-suspected link between these two splicing phenomena. These findings have the potential to improve the fidelity of genetic diagnostics and reveal new targets for splice-modulating therapies.
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Project description:We investigated the molecular mechanisms for in-frame skipping of DMD exon 39 caused by the nonsense c.5480T>A mutation in a patient with Becker muscular dystrophy. RNase-assisted pull down assay coupled with mass spectrometry revealed that the mutant RNA probe specifically recruits hnRNPA1, hnRNPA2/B1 and DAZAP1. Functional studies in a human myoblast cell line transfected with DMD minigenes confirmed the splicing inhibitory activity of hnRNPA1 and hnRNPA2/B1, and showed that DAZAP1, also known to activate splicing, acts negatively in the context of the mutated exon 39. Furthermore, we uncovered that recognition of endogenous DMD exon 39 in muscle cells is promoted by FUSE binding protein 1 (FUBP1), a multifunctional DNA- and RNA-binding protein whose role in splicing is largely unknown. By serial deletion and mutagenesis studies in minigenes, we delineated a functional intronic splicing enhancer (ISE) in intron 38. FUBP1 recruitment to the RNA sequence containing the ISE was established by RNA pull down and RNA EMSA, and further confirmed by RNA-ChIP on endogenous DMD pre-mRNA. This study provides new insights about the splicing regulation of DMD exon 39, highlighting the emerging role of FUBP1 in splicing and describing the first ISE for constitutive exon inclusion in the mature DMD transcript.
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Project description:The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.
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