Masafumi Matsuo
Kobe Gakuin University, Physical Therapy, Faculty Member
Identification of mutations in G6PD gene is performed as an epidemiologic investigation of G6PD deficiency in many countries. In order to understand the hereditary background of G6PD deficiency in a population, screening of mutations is... more
Identification of mutations in G6PD gene is performed as an epidemiologic investigation of G6PD deficiency in many countries. In order to understand the hereditary background of G6PD deficiency in a population, screening of mutations is required not only in exonic regions but also for intron and promoter regions. One hundred male neonatal samples diagnosed as with G6PD deficiency by newborn screening in Singapore were used in this study. The multiplex PCR using the multiple tandem forward primers and common reverse primer (MPTP) method was carried out to detect the common 11 mutations in south-east Asia such as Gaohe 95A>G, Orissa 131C>G, Vanua-Lava 383T>C, Mahidol 487G>A, Mediterranean 563C>T, Coimbra 592C>T, Viangchan 871G>A, Chatham 1003G>A, Union 1360C>T, Canton 1376G>T and Kaiping 1388G>A. Samples whose mutations were unidentified by MPTP method were scanned at cording region, intron and promoter region by direct sequencing.Out of 100 samples, 9...
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Background Fabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening,... more
Background Fabry disease is an X-linked lysosomal storage disorder caused by insufficient α-galactosidase A (GLA) activity resulting from variants in the GLA gene, which leads to glycosphingolipid accumulation and life-threatening, multi-organ complications. Approximately 50 variants have been reported that cause splicing abnormalities in GLA. Most were found within canonical splice sites, which are highly conserved GT and AG splice acceptor and donor dinucleotides, whereas one-third were located outside canonical splice sites, making it difficult to interpret their pathogenicity. In this study, we aimed to investigate the genetic pathogenicity of variants located in non-canonical splice sites within the GLA gene. Methods 13 variants, including four deep intronic variants, were selected from the Human Gene Variant Database Professional. We performed an in vitro splicing assay to identify splicing abnormalities in the variants. Results All candidate non-canonical splice site variants...
Research Interests: Genetics, Biology, Alternative splicing, Gene, Alternative Splicing, and 3 moreExon Skipping, Exon, and Splice
Dystrophin Dp71 is an isoform produced from the Dp71 promoter in intron 62 of the DMD gene, mutations in which cause Duchenne muscular dystrophy. Dp71 is involved in various cellular processes and comprises more than 10 isoforms produced... more
Dystrophin Dp71 is an isoform produced from the Dp71 promoter in intron 62 of the DMD gene, mutations in which cause Duchenne muscular dystrophy. Dp71 is involved in various cellular processes and comprises more than 10 isoforms produced by alternative splicing. Dp71ab, in which both exons 71 and 78 are deleted, has a hydrophobic C-terminus that is hydrophilic in Dp71. Therefore, Dp71ab is believed to have different roles from Dp71. Previously, we reported that Dp71ab enhanced the proliferation of human myoblasts. Here, we further characterized Dp71ab, focusing on the activation of cell proliferation. Dp71ab increased the proliferation of immortalized human myoblasts in a dose-dependent manner. In contrast, Dp71 suppressed proliferation in a dose-dependent manner. Consistent with these opposite effects, eGFP-tagged Dp71ab and mCherry-tagged Dp71 showed different cellular distributions, with Dp71ab mostly in the nucleus. Notably, human Dp71ab enhanced the proliferation of rat and mou...
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Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown.... more
Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are allelic disorders caused by mutations in the DMD gene. The full mutation spectrum of the DMD gene in Indonesian patients is currently unknown. Mutation-specific therapies are currently being developed, such as exon skipping or stop codon read-through therapy. This study was conducted with the aim of identifying the mutation spectrum of the DMD gene in Indonesia to guide future development and application of feasible therapeutic strategies. Methods: This study is a cross sectional study that enrolled 43 male patients with a clinical suspicion of DMD or BMD. Multiplex ligation-dependent probe amplification (MLPA) reaction was performed to screen for the common mutations in the DMD gene. Results: Out of 43 subjects, deletions accounted for 69.77% (n=30) cases, while duplications were found in 11.63% (n=5) cases. One novel duplication spanning exons 2 to 62 was identified. Deletion mutations clust...
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Key Points Mutations registered in the database for autosomal Alport syndrome do not include synonymous variants.Certain synonymous variants can affect pre-mRNA splicing, and transcript analysis should be carried out to evaluate... more
Key Points Mutations registered in the database for autosomal Alport syndrome do not include synonymous variants.Certain synonymous variants can affect pre-mRNA splicing, and transcript analysis should be carried out to evaluate synonymous variants.Our in vivo and in vitro splicing assays showed that two of the four synonymous variants cause exon skipping. Background Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in COL4A3 and COL4A4 have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified. Methods We conducted in silico splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed in vitro splicing assays using minigene constructs and mRNA analysis of patient samples to determine the path...
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BackgroundX‐linked Alport syndrome (XLAS) is a progressive, hereditary glomerular nephritis of variable severity caused by pathogenic COL4A5 variants. Currently, genetic testing is widely used for diagnosing XLAS; however, determining the... more
BackgroundX‐linked Alport syndrome (XLAS) is a progressive, hereditary glomerular nephritis of variable severity caused by pathogenic COL4A5 variants. Currently, genetic testing is widely used for diagnosing XLAS; however, determining the pathogenicity of variants detected by such analyses can be difficult. Intronic variants or synonymous variants may cause inherited diseases by inducing aberrant splicing. Transcript analysis is necessary to confirm the pathogenicity of such variants, but it is sometimes difficult to extract mRNA directly from patient specimens.MethodsIn this study, we conducted in vitro splicing analysis using a hybrid minigene assay and specimens from three XLAS patients with synonymous variants causing aberrant splicing, including previously reported pathogenic mutations in the same codon. The variants were c.876 A>T (p.Gly292=), c.2358 A>G (p.Pro786=), and c.3906 A>G (p.Gln1302=).ResultsThe results from our hybrid minigene assay were sufficient to predi...
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X-linked Alport syndrome (XLAS) is a congenital renal disease caused by mutations in COL4A5. In XLAS cases suspected of being caused by aberrant splicing, transcript analysis needs to be conducted to determine splicing patterns and assess... more
X-linked Alport syndrome (XLAS) is a congenital renal disease caused by mutations in COL4A5. In XLAS cases suspected of being caused by aberrant splicing, transcript analysis needs to be conducted to determine splicing patterns and assess the pathogenicity. However, such analysis is not always available. We conducted a functional splicing assay using a hybrid minigene for seven COL4A5 intronic mutations: one was identified by us and six were found in the Human Gene Mutation Database. The minigene assay revealed exon skipping in four variants, exon skipping and a 10-bp insertion in one variant, and no change in one variant, which appeared not to be pathogenic. For one variant, our assay did not work. The results of all three cases for which transcript data were available were consistent with our assay results. Our findings may help to increase the accuracy of genetic test results and clarify the mechanisms causing aberrant splicing.
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Glomerulopathy with fibronectin deposits (GFND; OMIM: 601894) is a very rare inherited kidney disease caused by pathogenic variants in the FN1 gene. Only 9 exonic pathogenic variants in FN1, 9 at the heparin-binding site, and 1 at the... more
Glomerulopathy with fibronectin deposits (GFND; OMIM: 601894) is a very rare inherited kidney disease caused by pathogenic variants in the FN1 gene. Only 9 exonic pathogenic variants in FN1, 9 at the heparin-binding site, and 1 at the integrin-binding site have been reported. No intronic variants in FN1 have been detected. We found a pathogenic intronic variant in intron 36 (c.5888-2A>G) located at the heparin-binding site. To determine whether this mutation influences splicing processes, we conducted RT-PCR analysis and an in vitro splicing assay using minigene construction. RT-PCR using RNA extracted from leukocytes of the proband failed because of the low expression of FN1 mRNA in leukocytes. We conducted in vitro functional splicing analysis using minigenes and found that c.5888-2A>G caused a 12 bp deletion at exon 37 by the activation of a novel splicing acceptor site within exon 37. We were able to detect the same abnormal transcript in mRNA extracted from the patient...
Research Interests: Molecular Biology, Medicine, Medical Physiology, Splicing, Clinical Sciences, and 3 moreIntron, FN, and Exon
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Research Interests: Genetics, Human Genetics, Biology, Long Noncoding Rna, Intellectual Disability, and 13 moreMedicine, Brain, Humans, Fluorescence in situ hybridization, Male, Gene, Duchenne Muscular Dystrophy, Clinical Sciences, Dystrophin, XQ, Exon, Child preschool, and reverse transcriptase polymerase chain reaction
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Research Interests: Genetics, Molecular Biology, Molecular Evolution, Biology, Medicine, and 15 moreCase Study, Polymerase Chain Reaction, Promoter, Genetic Map, Gene, Biological evolution, Duchenne Muscular Dystrophy, Intron, Clinical Investigation, Exon, Nucleotides, DNA sequence, Nucleotide sequence, Sequence similarity, and Medical and Health Sciences
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Background Autosomal dominant pseudohypoaldosteronism type 1 (PHA1) is a rare inherited condition that is characterized by renal resistance to aldosterone as well as salt wasting, hyperkalemia, and metabolic acidosis. Renal PHA1 is caused... more
Background Autosomal dominant pseudohypoaldosteronism type 1 (PHA1) is a rare inherited condition that is characterized by renal resistance to aldosterone as well as salt wasting, hyperkalemia, and metabolic acidosis. Renal PHA1 is caused by mutations of the human mineralcorticoid receptor gene (MR), but it is a matter of debate whether MR mutations cause mineralcorticoid resistance via haploinsufficiency or dominant negative mechanism. It was previously reported that in a case with nonsense mutation the mutant mRNA was absent in lymphocytes because of nonsense mediated mRNA decay (NMD) and therefore postulated that haploinsufficiency alone can give rise to the PHA1 phenotype in patients with truncated mutations. Methods and Results We conducted genomic DNA analysis and mRNA analysis for familial PHA1 patients extracted from lymphocytes and urinary sediments and could detect one novel splice site mutation which leads to exon skipping and frame shift result in premature termination a...
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X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the gene. Genotype-phenotype correlation in male XLAS is relatively well established; relative to truncating mutations, nontruncating mutations... more
X-linked Alport syndrome (XLAS) is a progressive hereditary nephropathy caused by mutations in the gene. Genotype-phenotype correlation in male XLAS is relatively well established; relative to truncating mutations, nontruncating mutations exhibit milder phenotypes. However, transcript comparison between XLAS cases with splicing abnormalities that result in a premature stop codon and those with nontruncating splicing abnormalities has not been reported, mainly because transcript analysis is not routinely conducted in patients with XLAS. We examined transcript expression for all patients with suspected splicing abnormalities who were treated at one hospital between January of 2006 and July of 2017. Additionally, we recruited 46 males from 29 families with splicing abnormalities to examine genotype-phenotype correlation in patients with truncating (=21, from 14 families) and nontruncating (=25, from 15 families) mutations at the transcript level. We detected 41 XLAS families with abnor...
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Autosomal recessive distal renal tubular acidosis (dRTA) is a rare hereditary disease caused by pathogenic variants in the ATP6V0A4 gene or ATP6V1B1 gene, and characterized by hyperchloremic metabolic acidosis with normal anion gap,... more
Autosomal recessive distal renal tubular acidosis (dRTA) is a rare hereditary disease caused by pathogenic variants in the ATP6V0A4 gene or ATP6V1B1 gene, and characterized by hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia and nephrocalcinosis. Although several intronic nucleotide variants in these genes have been detected, all of them fell in the apparent splice consensus sequence. In general, transcriptional analysis is necessary to determine the effect on function of the novel intronic variants located out of splicing consensus sequences. In recent years, functional splicing analysis using minigene construction was used to assess the pathogenicity of novel intoronic variant in various field. We investigated a sporadic case of dRTA with a compound heterozygous mutation in the ATP6V0A4 gene, revealed by next generation sequencing. One variant was already reported as pathogenic; however, the other was a novel variant in intron 11...
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The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined... more
The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), comprises 79 exons that show multiple alternative splicing events. Intron retention, a type of alternative splicing, may control gene expression. We examined intron retention in dystrophin introns by reverse-transcription PCR from skeletal muscle, focusing on the nine shortest (all <1000 bp), because these are more likely to be retained. Only one, intron 40, was retained in mRNA; sequencing revealed insertion of a complete intron 40 (851 nt) between exons 40 and 41. The intron 40 retention product accounted for 1.2% of the total product but had a premature stop codon at the fifth intronic codon. Intron 40 retention was most strongly observed in the kidney (36.6%) and was not obtained from the fetal liver, lung, spleen or placenta. This indicated that intron retention is a tissue-specific event whose level varies among tissues. In two DMD patients, intron 40 retention was observed in one patient but not in...
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Research Interests: Genetics, Human Genetics, Molecular Biology, Biology, Indonesia, and 15 moreMedicine, Human, Band, Humans, Polymerase Chain Reaction, Gene, Clinical Sciences, Molecular Characterization, Amino Acid Profile, Base Sequence, Erythrocyte Membrane, Exon, Nucleotides, Oligonucleotides, and Molecular Sequence Data
Research Interests: Genetics, Human Genetics, Biology, Medicine, Humans, and 15 moreAlternative splicing, Gene, Duchenne Muscular Dystrophy, Clinical Sciences, Introns, Intron, Alternative Splicing, Base Sequence, Exon Skipping, Dystrophin, Exon, Splice, Becker Muscular Dystrophy (BMD), Branch point, and Molecular Sequence Data
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Research Interests: Regeneration, Biology, Myogenesis, Immunohistochemistry, Medicine, and 15 moreBiopsy, In Situ Hybridization, Cell line, Humans, Mice, Muscular Dystrophy, Animals, Muscles, Duchenne Muscular Dystrophy, Molecular cloning, Amino Acid Sequence, Dystrophin, Molecular Sequence Data, reverse transcriptase polymerase chain reaction, and Medical and Health Sciences
Purpose: Mutations in the dystrophin (DMD) gene cause Duchenne or Becker muscular dystrophy (DMD/BMD).DMD contains a retina-specific promoter in intron 29. The short R-dystrophin transcript from this promoter has a retinaspecific exon 1... more
Purpose: Mutations in the dystrophin (DMD) gene cause Duchenne or Becker muscular dystrophy (DMD/BMD).DMD contains a retina-specific promoter in intron 29. The short R-dystrophin transcript from this promoter has a retinaspecific exon 1 (R1) joined to exon 30 of the DMD gene. It has been claimed that this is responsible for the ophthalmological
problems observed in DMD/BMD. This research characterizes the structure of the 5′-untranslated region (5′-UTR) of human R-dystrophin.
Methods: The 5′-UTR of the human R-dystrophin transcript was amplified from human retina and 20 other human tissue RNAs by reverse transcription polymerase chain reaction (RT–PCR). Amplified products were identified by sequencing. The translational activities of transcripts bearing differing 5′-UTRs were measured using a dual luciferase assay system.
Results: RT–PCR amplification of the R-dystrophin transcript from the retina using a conventional primer set revealed one product comprising exon R1 and exons 30 to 32 (R-dys α). In contrast, three amplified products were obtained when a forward primer at the far 5′-end of exon R1 was employed for RT–PCR. R-dys α, and a shorter form in which 98 bp was deleted from exon R1 (R-dys β), were the two major products. A minor, short form was also identified, in which 143 bp was deleted from exon R1 (R-dys γ). The two primary retinal products (R-dys α and β) encoded an identical open reading frame. The 98 bp deleted in R-dys β was identified as a cryptic intron that was evolutionarily acquired in higher mammals. The shorter R-dys β was expressed in several tissues with a wide range in expression level, while R-dys α was retina specific. The 5′-UTRs of R-dys α and β were examined for translational activity using a dual luciferase assay system. Unexpectedly, the 5′-UTR of R-dys β showed lower translational activity than that of R-dys α. This lower activity was presumed to be due to the removal of internal ribosome entry sites by activation of cryptic intron splicing.
Conclusions: An evolutionarily-acquired cryptic intron was identified in the 5′-UTR of the human R-dystrophin transcript. The two abundant R-dystrophin transcripts in the retina showed different translational activities in vitro owing to their differential splicing of the cryptic intron. This evolutionarily-acquired alternative splicing may act as a molecular switch that regulates translation of the R-dystrophin transcript.
problems observed in DMD/BMD. This research characterizes the structure of the 5′-untranslated region (5′-UTR) of human R-dystrophin.
Methods: The 5′-UTR of the human R-dystrophin transcript was amplified from human retina and 20 other human tissue RNAs by reverse transcription polymerase chain reaction (RT–PCR). Amplified products were identified by sequencing. The translational activities of transcripts bearing differing 5′-UTRs were measured using a dual luciferase assay system.
Results: RT–PCR amplification of the R-dystrophin transcript from the retina using a conventional primer set revealed one product comprising exon R1 and exons 30 to 32 (R-dys α). In contrast, three amplified products were obtained when a forward primer at the far 5′-end of exon R1 was employed for RT–PCR. R-dys α, and a shorter form in which 98 bp was deleted from exon R1 (R-dys β), were the two major products. A minor, short form was also identified, in which 143 bp was deleted from exon R1 (R-dys γ). The two primary retinal products (R-dys α and β) encoded an identical open reading frame. The 98 bp deleted in R-dys β was identified as a cryptic intron that was evolutionarily acquired in higher mammals. The shorter R-dys β was expressed in several tissues with a wide range in expression level, while R-dys α was retina specific. The 5′-UTRs of R-dys α and β were examined for translational activity using a dual luciferase assay system. Unexpectedly, the 5′-UTR of R-dys β showed lower translational activity than that of R-dys α. This lower activity was presumed to be due to the removal of internal ribosome entry sites by activation of cryptic intron splicing.
Conclusions: An evolutionarily-acquired cryptic intron was identified in the 5′-UTR of the human R-dystrophin transcript. The two abundant R-dystrophin transcripts in the retina showed different translational activities in vitro owing to their differential splicing of the cryptic intron. This evolutionarily-acquired alternative splicing may act as a molecular switch that regulates translation of the R-dystrophin transcript.
Research Interests: Genetics, Molecular Biology, Biology, Medicine, Molecular, and 15 moreHumans, Pubmed, Retina, Research article, Alternative splicing, Duchenne Muscular Dystrophy, Introns, Intron, Alternative Splicing, Base Sequence, Dystrophin, Exon, Protein Biosynthesis, Gene expression profiling, and Molecular Sequence Data
Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human... more
Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca(2+)) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca(2+) overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These resul...
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Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal... more
Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in ...
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Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of... more
Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested a...
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Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by deficiency in dystrophin, a protein product encoded by the DMD gene. Mitochondrial dysfunction is now attracting much attention as a central player in DMD... more
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by deficiency in dystrophin, a protein product encoded by the DMD gene. Mitochondrial dysfunction is now attracting much attention as a central player in DMD pathology. However, dystrophin has never been explored in human mitochondria. Here, we analyzed dystrophin in cDNAs and mitochondrial fractions of human cells. Mitochondrial fraction was obtained using a magnetic-associated cell sorting (MACS) technology. Dystrophin was analyzed by reverse transcription (RT)-PCR and western blotting using an antibody against the dystrophin C-terminal. In isolated mitochondrial fraction from HEK293 cells, dystrophin was revealed as a band corresponding to Dp71b and Dp71ab subisoforms. Additionally, in mitochondria from HeLa, SH-SY5Y, CCL-136 and HepG2 cells, signals for Dp71b and Dp71ab were revealed as well. Concomitantly, dystrophin mRNAs encoding Dp71b and Dp71ab were disclosed by RT-PCR in these cells. Primary culture...
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Dystrophin Dp71 is the smallest isoform of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 has also been shown to have roles in various cellular processes. Stem cell-based therapy may be effective in... more
Dystrophin Dp71 is the smallest isoform of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 has also been shown to have roles in various cellular processes. Stem cell-based therapy may be effective in treating DMD, but the inability to generate a sufficient number of stem cells remains a significant obstacle. Although Dp71 is comprised of many variants, Dp71 in satellite cells has not yet been studied. Here, the full-length Dp71 consisting of 18 exons from exons G1 to 79 was amplified by reverse transcription-PCR from total RNA of human satellite cells. The amplified product showed deletion of both exons 71 and 78 in all sequenced clones, indicating monoclonal expression of Dp71ab. Western blotting of the satellite cell lysate showed a band corresponding to over-expressed Dp71ab. Transfection of a plasmid expressing Dp71ab into human myoblasts significantly enhanced cell proliferation when compared to the cells transfected with the mock plasmid. However...
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The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody... more
The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exo...
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Dystrophin Dp71 is one of the isoforms produced by the gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was... more
Dystrophin Dp71 is one of the isoforms produced by the gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle. Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMD mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively. Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells...
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The Duchenne muscular dystrophy (DMD) gene is one of the largest genes in the human genome. The gene exhibits a complex arrangement of seven alternative promoters, which drive the expression of three full length and four shorter isoforms.... more
The Duchenne muscular dystrophy (DMD) gene is one of the largest genes in the human genome. The gene exhibits a complex arrangement of seven alternative promoters, which drive the expression of three full length and four shorter isoforms. Dp116, the second smallest product of the DMD gene, is a Schwann cell-specific isoform encoded by a transcript corresponding to DMD exons 56-79, starting from a promoter/exon S1 within intron 55. The physiological roles of Dp116 are poorly understood, because of its extensive homology with other isoforms and its expression in specific tissues. This review summarizes studies on Dp116, focusing on clinical findings and alternative activation of the upstream translation initiation codon that is predicted to produce Dp118.