Publications by authors named "Masafumi Matsuo"

266 Publications

Usefulness of functional splicing analysis to confirm precise disease pathogenesis in Diamond-Blackfan anemia caused by intronic variants in .

Pediatr Hematol Oncol 2021 Feb 24:1-16. Epub 2021 Feb 24.

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.

Diamond-Blackfan anemia (DBA) is mainly caused by pathogenic variants in ribosomal proteins and 22 responsible genes have been identified to date. The most common causative gene of DBA is [NM_001022.4]. Nearly 180 variants have been reported, including three deep intronic variants outside the splicing consensus sequence (c.72-92A > G, c.356 + 18G > C, and c.411 + 6G > C). We also identified one case with a c.412-3C > G intronic variant. Without conducting transcript analysis, the pathogenicity of these variants is unknown. However, it is difficult to assess transcripts because of their fragility. In such cases, in functional splicing assays can be used to assess pathogenicity. Here, we report functional splicing analysis results of four deep intronic variants identified in our case and in previously reported cases. One splicing consensus variant (c.411 + 1G > A) was also examined as a positive control. Aberrant splicing with a 2-bp insertion between exons 5 and 6 was identified in the patient samples and minigene assay results also identified exon 6 skipping in our case. The exon 6 skipping transcript was confirmed by further evaluation using quantitative RT-PCR. Additionally, minigene assay analysis of three reported deep intronic variants revealed that none of them showed aberrant splicing and that these variants were not considered to be pathogenic. In conclusion, the minigene assay is a useful method for functional splicing analysis of inherited disease.
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http://dx.doi.org/10.1080/08880018.2021.1887984DOI Listing
February 2021

Urinary titin as a biomarker in Fukuyama congenital muscular dystrophy.

Neuromuscul Disord 2021 03 13;31(3):194-197. Epub 2021 Jan 13.

Department of Pediatrics, School of Medicine, Tokyo Women's Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162-8666, Japan. Electronic address:

Fukuyama congenital muscular dystrophy (FCMD) is the second most prevalent childhood-onset muscular dystrophy in Japan. It is an autosomal recessive disorder caused by the fukutin mutation (FKTN), characterized by muscle wasting and brain abnormalities. So far, serum creatine kinase (CK) is recognized as the only biomarker for FCMD. Recently, an ELISA assay to quantify the N-terminal fragment of titin in urine was developed. Urinary titin concentration is elevated in patients with Duchenne muscular dystrophy (DMD) compared to normal controls. Levels vary according to age with excellent sensitivity and specificity for detecting DMD, and they can be used as a diagnostic and disease progression marker. In this study, we measured the urinary titin concentration of 18 patients with FCMD. It was remarkably higher than normal controls and correlated with CK. Especially in homozygotes, the score for gross motor function measure, which is a quantitative motor scale for FCMD, was correlated with urinary titin concentration. Elevated urinary titin concentrations were thought to be reflective of a common pathophysiology with DMD. Urinary titin concentrations can assist with making the diagnosis of FCMD and to estimate the patient's motor function at that point.
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http://dx.doi.org/10.1016/j.nmd.2021.01.005DOI Listing
March 2021

Urinary Titin N-Fragment as a Biomarker of Muscle Atrophy, Intensive Care Unit-Acquired Weakness, and Possible Application for Post-Intensive Care Syndrome.

J Clin Med 2021 Feb 6;10(4). Epub 2021 Feb 6.

Emergency and Critical Care Medicine, Tokushima University Hospital, 2-50-1 Kuramoto, Tokushima 770-8503, Japan.

Titin is a giant protein that functions as a molecular spring in sarcomeres. Titin interconnects the contraction of actin-containing thin filaments and myosin-containing thick filaments. Titin breaks down to form urinary titin N-fragments, which are measurable in urine. Urinary titin N-fragment was originally reported to be a useful biomarker in the diagnosis of muscle dystrophy. Recently, the urinary titin N-fragment has been increasingly gaining attention as a novel biomarker of muscle atrophy and intensive care unit-acquired weakness in critically ill patients, in whom titin loss is a possible pathophysiology. Furthermore, several studies have reported that the urinary titin N-fragment also reflected muscle atrophy and weakness in patients with chronic illnesses. It may be used to predict the risk of post-intensive care syndrome or to monitor patients' condition after hospital discharge for better nutritional and rehabilitation management. We provide several tips on the use of this promising biomarker in post-intensive care syndrome.
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http://dx.doi.org/10.3390/jcm10040614DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915692PMC
February 2021

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the Gene.

Int J Mol Sci 2020 Nov 30;21(23). Epub 2020 Nov 30.

Research Center for Locomotion Biology, Kobe Gakuin University, Kobe 651-2180, Japan.

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 FMv2v8, a modified clone of FMv2 carrying an insertion of exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.
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http://dx.doi.org/10.3390/ijms21239136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729581PMC
November 2020

Dystrophin Dp71ab is monoclonally expressed in human satellite cells and enhances proliferation of myoblast cells.

Sci Rep 2020 10 13;10(1):17123. Epub 2020 Oct 13.

Research Center for Locomotion Biology, Kobe Gakuin University, 518 Arise, Ikawadani, Nishi, Kobe, 651-2180, Japan.

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, transfection of the Dp71 expression plasmid encoding all 18 exons did not enhance myoblast proliferation. These findings indicated that Dp71ab, but not Dp71, is a molecular enhancer of myoblast proliferation and that transfection with Dp71ab may generate a high yield of stem cells for DMD treatment.
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http://dx.doi.org/10.1038/s41598-020-74157-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553993PMC
October 2020

Cellular senescence-mediated exacerbation of Duchenne muscular dystrophy.

Sci Rep 2020 10 12;10(1):16385. Epub 2020 Oct 12.

Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan.

Duchenne muscular dystrophy (DMD) is a progressive disease characterised by chronic muscle degeneration and inflammation. Our previously established DMD model rats (DMD rats) have a more severe disease phenotype than the broadly used mouse model. We aimed to investigate the role of senescence in DMD using DMD rats and patients. Senescence was induced in satellite cells and mesenchymal progenitor cells, owing to the increased expression of CDKN2A, p16- and p19-encoding gene. Genetic ablation of p16 in DMD rats dramatically restored body weight and muscle strength. Histological analysis showed a reduction of fibrotic and adipose tissues invading skeletal muscle, with increased muscle regeneration. Senolytic drug ABT263 prevented loss of body weight and muscle strength, and increased muscle regeneration in rats even at 8 months-the late stage of DMD. Moreover, senescence markers were highly expressed in the skeletal muscle of DMD patients. In situ hybridization of CDKN2A confirmed the expression of it in satellite cells and mesenchymal progenitor cells in patients with DMD. Collectively, these data provide new insights into the integral role of senescence in DMD progression.
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http://dx.doi.org/10.1038/s41598-020-73315-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550355PMC
October 2020

Pathological evaluation of rats carrying in-frame mutations in the dystrophin gene: a new model of Becker muscular dystrophy.

Dis Model Mech 2020 09 28;13(9). Epub 2020 Sep 28.

Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657, Japan.

Dystrophin, encoded by the gene on the X chromosome, stabilizes the sarcolemma by linking the actin cytoskeleton with the dystrophin-glycoprotein complex (DGC). In-frame mutations in cause a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), characterized by the reduced expression of truncated dystrophin. So far, no animal model with in-frame mutations in has been established. As a result, the effect of in-frame mutations on the dystrophin expression profile and disease progression of BMD remains unclear. In this study, we established a novel rat model carrying in-frame gene mutations (IF rats) and evaluated the pathology. We found that IF rats exhibited reduced expression of truncated dystrophin in a proteasome-independent manner. This abnormal dystrophin expression caused dystrophic changes in muscle tissues but did not lead to functional deficiency. We also found that the expression of additional dystrophin named dpX, which forms the DGC in the sarcolemma, was associated with the appearance of truncated dystrophin. In conclusion, the outcomes of this study contribute to the further understanding of BMD pathology and help elucidate the efficiency of dystrophin recovery treatments in Duchenne muscular dystrophy, a more severe form of X-linked muscular dystrophy.
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http://dx.doi.org/10.1242/dmm.044701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541341PMC
September 2020

The ACTN3 577XX Null Genotype Is Associated with Low Left Ventricular Dilation-Free Survival Rate in Patients with Duchenne Muscular Dystrophy.

J Card Fail 2020 Oct 10;26(10):841-848. Epub 2020 Aug 10.

Department of Pediatrics, Kobe University of Graduate School of Medicine, Kobe 650-0017, Japan.

Background: Duchenne muscular dystrophy (DMD) is a fatal progressive muscle-wasting disease caused by mutations in the DMD gene. Dilated cardiomyopathy is the leading cause of death in DMD; therefore, further understanding of this complication is essential to reduce morbidity and mortality.

Methods: A common null variant (R577X) in the ACTN3 gene, which encodes α-actinin-3, has been studied in association with muscle function in healthy individuals; however it has not yet been examined in relationship to the cardiac phenotype in DMD. In this study, we determined the ACTN3 genotype in 163 patients with DMD and examined the correlation between ACTN3 genotypes and echocardiographic findings in 77 of the 163 patients.

Results: The genotypes 577RR(RR), 577RX(RX) and 577XX(XX) were identified in 13 (17%), 44 (57%) and 20 (26%) of 77 patients, respectively. We estimated cardiac involvement-free survival rate analyses using Kaplan-Meier curves. Remarkably, the left ventricular dilation (> 55 mm)-free survival rate was significantly lower in patients with the XX null genotype (P < 0.01). The XX null genotype showed a higher risk for LV dilation (hazard ratio 9.04).

Conclusions: This study revealed that the ACTN3 XX null genotype was associated with a lower left ventricular dilation-free survival rate in patients with DMD. These results suggest that the ACTN3 genotype should be determined at the time of diagnosis of DMD to improve patients' cardiac outcomes.
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http://dx.doi.org/10.1016/j.cardfail.2020.08.002DOI Listing
October 2020

Urinary Titin Is a Novel Biomarker for Muscle Atrophy in Nonsurgical Critically Ill Patients: A Two-Center, Prospective Observational Study.

Crit Care Med 2020 09;48(9):1327-1333

Department of Nutrition and Metabolism, Tokushima University Graduate School of Biomedical Sciences, Tokushima, Japan.

Objectives: Although skeletal muscle atrophy is common in critically ill patients, biomarkers associated with muscle atrophy have not been identified reliably. Titin is a spring-like protein found in muscles and has become a measurable biomarker for muscle breakdown. We hypothesized that urinary titin is useful for monitoring muscle atrophy in critically ill patients. Therefore, we investigated urinary titin level and its association with muscle atrophy in critically ill patients.

Design: Two-center, prospective observational study.

Setting: Mixed medical/surgical ICU in Japan.

Patients: Nonsurgical adult patients who were expected to remain in ICU for greater than 5 days.

Interventions: None.

Measurements And Main Results: Urine samples were collected on days 1, 2, 3, 5, and 7 of ICU admission. To assess muscle atrophy, rectus femoris cross-sectional area and diaphragm thickness were measured with ultrasound on days 1, 3, 5, and 7. Secondary outcomes included its relationship with ICU-acquired weakness, ICU Mobility Scale, and ICU mortality. Fifty-six patients and 232 urinary titin measurements were included. Urinary titin (normal range: 1-3 pmol/mg creatinine) was 27.9 (16.8-59.6), 47.6 (23.5-82.4), 46.6 (24.4-97.6), 38.4 (23.6-83.0), and 49.3 (27.4-92.6) pmol/mg creatinine on days 1, 2, 3, 5, and 7, respectively. Cumulative urinary titin level was significantly associated with rectus femoris muscle atrophy on days 3-7 (p ≤ 0.03), although urinary titin level was not associated with change in diaphragm thickness (p = 0.31-0.45). Furthermore, cumulative urinary titin level was associated with occurrence of ICU-acquired weakness (p = 0.01) and ICU mortality (p = 0.02) but not with ICU Mobility Scale (p = 0.18).

Conclusions: In nonsurgical critically ill patients, urinary titin level increased 10-30 times compared with the normal level. The increased urinary titin level was associated with lower limb muscle atrophy, occurrence of ICU-acquired weakness, and ICU mortality.
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http://dx.doi.org/10.1097/CCM.0000000000004486DOI Listing
September 2020

Onset mechanism of a female patient with Dent disease 2.

Clin Exp Nephrol 2020 Oct 14;24(10):946-954. Epub 2020 Jul 14.

Department of Pediatrics, Hokkaido University Graduate School of Medicine, North 15, West 7, Sapporo, Hokkaido, 060-8638, Japan.

Background: Approximately 15% of patients with Dent disease have pathogenic variants in the OCRL gene on Xq25-26, a condition that is referred to as Dent disease 2 (Dent-2). Dent-2 patients sometimes show mild extrarenal features of Lowe syndrome, such as mild mental retardation, suggesting that Dent-2 represents a mild form of Lowe syndrome. To date, eight female patients with Lowe syndrome have been reported, but no female Dent-2 patients have been reported.

Methods: In this study, we performed genetic testing of the first female Dent-2 patient to detect the presence of an OCRL variant. Aberrant splicing was demonstrated by in vivo, in vitro, and in silico assays, and skewed X-chromosome inactivation (XCI) in our patient and asymptomatic mothers of three Lowe patients with the heterozygous OCRL variant was evaluated by HUMARA assays using genomic DNA and RNA expression analysis.

Results: Our patient had an OCRL heterozygous intronic variant of c.1603-3G > C in intron 15 that led to a 169-bp insertion in exon 16, yielding the truncating mutation r.1602_1603ins (169) (p.Val535Glyfs*6) in exon 16. HUMARA assays of leukocytes obtained from this patient demonstrated incompletely skewed XCI (not extremely skewed). On the other hand, the asymptomatic mothers of 3 Lowe patients demonstrated random XCI. These results may lead to our patient's Dent-2 phenotype.

Conclusions: This is the first report of a female patient clinically and genetically diagnosed with Dent-2 caused by an OCRL heterozygous splicing site variant and skewed XCI. Skewed XCI may be one of the factors associated with phenotypic diversity in female patients with Lowe syndrome and Dent-2.
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http://dx.doi.org/10.1007/s10157-020-01926-4DOI Listing
October 2020

Common risk variants in NPHS1 and TNFSF15 are associated with childhood steroid-sensitive nephrotic syndrome.

Kidney Int 2020 11 14;98(5):1308-1322. Epub 2020 Jun 14.

Department of Pediatrics, Chonnam National University Children's Hospital, Gwangju, Korea.

To understand the genetics of steroid-sensitive nephrotic syndrome (SSNS), we conducted a genome-wide association study in 987 childhood SSNS patients and 3,206 healthy controls with Japanese ancestry. Beyond known associations in the HLA-DR/DQ region, common variants in NPHS1-KIRREL2 (rs56117924, P=4.94E-20, odds ratio (OR) =1.90) and TNFSF15 (rs6478109, P=2.54E-8, OR=0.72) regions achieved genome-wide significance and were replicated in Korean, South Asian and African populations. Trans-ethnic meta-analyses including Japanese, Korean, South Asian, African, European, Hispanic and Maghrebian populations confirmed the significant associations of variants in NPHS1-KIRREL2 (P=6.71E-28, OR=1.88) and TNFSF15 (P=5.40E-11, OR=1.33) loci. Analysis of the NPHS1 risk alleles with glomerular NPHS1 mRNA expression from the same person revealed allele specific expression with significantly lower expression of the transcript derived from the risk haplotype (Wilcox test p=9.3E-4). Because rare pathogenic variants in NPHS1 cause congenital nephrotic syndrome of the Finnish type (CNSF), the present study provides further evidence that variation along the allele frequency spectrum in the same gene can cause or contribute to both a rare monogenic disease (CNSF) and a more complex, polygenic disease (SSNS).
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http://dx.doi.org/10.1016/j.kint.2020.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8101291PMC
November 2020

Pathogenic evaluation of synonymous COL4A5 variants in X-linked Alport syndrome using a minigene assay.

Mol Genet Genomic Med 2020 08 16;8(8):e1342. Epub 2020 Jun 16.

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.

Background: X-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.

Methods: In 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=).

Results: The results from our hybrid minigene assay were sufficient to predict splicing abnormalities; c.876 A>T cause 17-bp del and 35-bp del, c.2358 A>G cause exon 29 skipping, c.3906 A>G cause exon 42 skipping, which are very likely to cause pathogenicity. Further, patients carrying c.2358 A>G exhibited a mild phenotype that may have been associated with the presence of both normal and abnormally spliced transcripts.

Conclusion: The minigene system was shown to be a sensitive assay and a useful tool for investigating the pathogenicity of synonymous variants.
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http://dx.doi.org/10.1002/mgg3.1342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434753PMC
August 2020

Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5.

Nat Commun 2020 06 2;11(1):2777. Epub 2020 Jun 2.

Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.

Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.
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http://dx.doi.org/10.1038/s41467-020-16605-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265383PMC
June 2020

Intronic Alternative Polyadenylation in the Middle of the Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients.

Int J Mol Sci 2020 May 18;21(10). Epub 2020 May 18.

Research Center for Locomotion Biology, Kobe Gakuin University, Nishi, Kobe 6512180, Japan.

The 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 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 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 exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.
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http://dx.doi.org/10.3390/ijms21103555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278912PMC
May 2020

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1.

Clin Exp Nephrol 2020 Jul 22;24(7):606-612. Epub 2020 Mar 22.

Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo, 650-0017, Japan.

Background: In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants.

Methods: We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106-17T>G, c.393+4A>G, c.517-8A>G, c.517-3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results.

Results: We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106-17T>G).

Conclusion: We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.
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http://dx.doi.org/10.1007/s10157-020-01876-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935734PMC
July 2020

Contribution of Rare Variants of the Gene to the Missing Heritability of Serum Urate Levels.

Genetics 2020 04 31;214(4):1079-1090. Epub 2020 Jan 31.

Tohoku Medical Megabank Organization, Tohoku University, Sendai, Miyagi 980-8573, Japan.

Gout is a common arthritis caused by monosodium urate crystals. The heritability of serum urate levels is estimated to be 30-70%; however, common genetic variants account for only 7.9% of the variance in serum urate levels. This discrepancy is an example of "missing heritability." The "missing heritability" suggests that variants associated with uric acid levels are yet to be found. By using genomic sequences of the ToMMo cohort, we identified rare variants of the gene that affect the urate transport activity of URAT1. URAT1 is a transporter protein encoded by the gene. We grouped the participants with variants affecting urate uptake by URAT1 and analyzed the variance of serum urate levels. The results showed that the heritability explained by the variants of men and women exceeds 10%, suggesting that rare variants underlie a substantial portion of the "missing heritability" of serum urate levels.
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http://dx.doi.org/10.1534/genetics.119.303006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153932PMC
April 2020

Spinal Muscular Atrophy: Advanced Version of Screening System with Real-Time mCOP-PCR and PCR-RFLP for SMN1 Deletion.

Kobe J Med Sci 2019 Jul 16;65(2):E49-E53. Epub 2019 Jul 16.

Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.

Background: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion using dried blood spot (DBS), we developed a new combined system with real-time "modified competitive oligonucleotide priming"-polymerase chain reaction (mCOP-PCR) and PCR restriction fragment length polymorphism (PCR-RFLP). Although our real-time mCOP-PCR method is secured enough to be gene-specific, its amplification efficiency is not as good because the reverse primers carry a nucleotide mismatched with the sequence of the pre-amplified product. The mismatch has consequently been generated in the process of introducing a restriction enzyme site in the pre-amplified products for PCR-RFLP.

Method: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification). Pre-amplified products were used as template in the real-time mCOP-PCR, and, on the other hand, were digested with DraI enzyme (PCR-RFLP). To improve the amplification efficiency of mCOP-PCR, one nucleotide change was introduced in the original reverse primers (SMN1-COP and SMN2-COP) to eliminate the mismatched nucleotide.

Results: The real-time mCOP-PCR with a new primer (SMN1-COP-DRA or SMN2-COP-DRA) more rapidly and specifically amplified SMN1 and SMN2, and clearly demonstrated SMN1 deletion in an SMA patient. With the new primers, the amplification efficiencies of real-time mCOP-PCR were improved and the Cq values of SMN1 (+) and SMN2 (+) samples were significantly lowered.

Conclusion: In the advanced version of our screening system for homozygous SMN1 deletion using DBS, the real-time mCOP-PCR with newly-designed reverse primers demonstrated the presence or absence of SMN1 and SMN2 within a shorter time, and the results were easily tested by PCR-RFLP. This rapid and accurate screening system will be useful for detection of newborn infants with SMA.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012194PMC
July 2019

Spinal Muscular Atrophy: New Screening System with Real-Time mCOP-PCR and PCR-RFLP for SMN1 Deletion.

Kobe J Med Sci 2019 Jul 16;65(2):E44-E48. Epub 2019 Jul 16.

Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.

Background: Spinal Muscular Atrophy (SMA) is a common autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. More than 95% of SMA patients show homozygous deletion for the survival motor neuron 1 (SMN1) gene. For the screening of SMN1 deletion, it is necessary to differentiate SMN1 from its highly homologous gene, SMN2. We developed a modified competitive oligonucleotide priming-PCR (mCOP-PCR) method using dried blood spot (DBS)-DNA, in which SMN1 and SMN2-specific PCR products are detected with gel-electrophoresis. Next, we added a targeted pre-amplification step prior to the mCOP-PCR step, to avoid unexpected, non-specific amplification. The pre-amplification step enabled us to combine mCOP-PCR and real-time PCR. In this study, we combined real-time mCOP-PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) to develop a new screening system for detection of SMN1 deletion.

Methods: DBS samples of the subjects were stored at room temperature for a period of less than one year. Each subject had already been genotyped by the first PCR-RFLP using fresh blood DNA. SMN1/SMN2 exon 7 was collectively amplified using conventional PCR (targeted pre-amplification), the products of which were then used as a template in the real-time PCR with mCOP-primer sets. To confirm the results, the pre-amplified products were subject to the second PCR-RFLP.

Results: The real-time mCOP-PCR separately amplified SMN1 and SMN2 exon7, and clearly demonstrated SMN1 deletion in an SMA patient. The results of the real-time mCOP-PCR using DBS-DNA were completely consistent with those of the first and second PCR-RFLP analysis.

Conclusion: In our new system for detection of SMN1 deletion, real-time mCOP-PCR rapidly proved the presence or absence of SMN1 and SMN2, and the results were easily tested by PCR-RFLP. This solid genotyping system will be useful for SMA screening.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012196PMC
July 2019

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

Hum Genet 2020 Feb 9;139(2):247-255. Epub 2020 Jan 9.

Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo, 187-8502, Japan.

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.
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http://dx.doi.org/10.1007/s00439-019-02107-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981323PMC
February 2020

Schwann cell-specific Dp116 is expressed in glioblastoma cells, revealing two novel gene splicing patterns.

Biochem Biophys Rep 2019 Dec 10;20:100703. Epub 2019 Nov 10.

Research Center for Locomotion Biology, Kobe Gakuin University, Nishi, Kobe, 651-2180, Japan.

Background: The gene is one of the largest human genes, being composed of 79 exons. Dystrophin Dp116 expressed from the promoter in intron 55 is a Schwann cell-specific isoform. The pathophysiological roles of Dp116 are largely unknown, because of its limited expression. This study assessed the expression of Dp116 in glioblastoma cells and evaluated the splicing patterns of the gene in these cells.

Methods: Full-length Dp116 cDNA was PCR amplified from U-251 glioblastoma cells. Dp116 protein was analyzed by Western blotting.

Results: Full-length Dp116 cDNA, extending from exon S1 to exon 79, was PCR amplified to avoid confusion with other DMD isoforms. The full-length Dp116 transcript was amplified as nearly 3 kb in size. Western blotting of U-251 cell lysates revealed a signal at a position corresponding to vector-expressed Dp116 protein, indicating that Dp116 is expressed in glioblastoma cells. Sequencing of the amplified product revealed five splice variants, all skipping exon 78. The most abundant transcript lacked only exon 78 (Dp116b), whereas the second most abundant transcript lacked both exons 71 and 78 (Dp116ab). A third transcript lacking exons 71-74 and 78 was also identified (Dp116bc). Two novel splicing patterns were also observed, one with a deletion of exons 68 and 69 (Dp116bΔ68-69) and the other with a 100 bp deletion in the 5' terminal end of exon 75 (75s), which was produced by the activation of a cryptic splice acceptor site (Dp116b75s). However, the splicing patterns in glioblastoma cells of exons in Dp116 and Dp71 showed no significant differences.

Conclusions: Dp116 is expressed in glioblastoma cells as five splicing variants, with Dp116b being the most abundant. Two novel splicing patterns of exons were observed.
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http://dx.doi.org/10.1016/j.bbrep.2019.100703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849142PMC
December 2019

Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay.

Sci Rep 2019 09 3;9(1):12696. Epub 2019 Sep 3.

Department of Pediatrics, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo, Kobe, Hyogo, 650-0017, Japan.

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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http://dx.doi.org/10.1038/s41598-019-48990-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722096PMC
September 2019

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.

Hum Genet 2019 Jul 5;138(7):771-785. Epub 2019 Jun 5.

Department of Pathology, School of Medicine, Nankai University, 94 Weijin Road, Nankai District, Tianjin, 300071, China.

Modulation of dystrophin pre-mRNA splicing is an attractive strategy to ameliorate the severe phenotype of Duchenne muscular dystrophy (DMD), although this requires a better understanding of the mechanism of splicing regulation. Aberrant splicing caused by gene mutations provides a good model to study splicing regulatory cis-elements and binding proteins. In this study, we identified skipping of in-frame exon 25 induced by a nonsense mutation (NM_004006.2:c.3340A > T;p.Lys1114*) in the DMD gene. Site-directed mutagenesis study in minigenes suggested that c.3340A > T converts an exonic splicing enhancer sequence (ESE) to a silencer element (ESS). Indeed, RNA pull-down and functional study provided evidence that c.3340A > T abolishes the binding of the splicing enhancer protein Tra2β and promotes interactions with the repressor proteins hnRNP A1, hnRNP A2, and hnRNP H. By carefully analyzing the sequence motif encompassing the mutation site, we concluded that the skipping of exon 25 was due to disruption of a Tra2β-dependent ESE and the creation of a new ESS associated with hnRNP A1 and hnRNP A2, which in turn increased the recruitment of hnRNP H to a nearby binding site. Finally, we demonstrated that c.3340A > T impairs the splicing of upstream intron 24 in a splicing minigene assay. In addition, we showed that the correct splicing of exon 25 is finely regulated by multiple splicing regulators that function in opposite directions by binding to closely located ESE and ESS. Our results clarify the detailed molecular mechanism of exon skipping induced by the nonsense mutation c.3340A > T and also provide information on exon 25 splicing.
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http://dx.doi.org/10.1007/s00439-019-02036-2DOI Listing
July 2019

Titin fragment in urine: A noninvasive biomarker of muscle degradation.

Adv Clin Chem 2019 5;90:1-23. Epub 2019 Mar 5.

Research Center for Locomotion Biology, Kobe Gakuin University, Kobe, Japan; Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan.

Titin/connectin, encoded by the TTN gene, is the largest protein in humans. It acts as a molecular spring in the sarcomere of striated muscles. Although titin is degraded in the skeletal muscles of patients with muscular dystrophies, studies of titin have been limited by its mammoth size. Mutations in the TTN gene have been detected not only in skeletal muscle diseases but in cardiac muscle diseases. TTN mutations result in a wide variety of phenotypes. Recent proteome analysis has found that titin fragments are excreted into the urine of patents with Duchenne muscular dystrophy (DMD). Enzyme-linked immunosorbent assays (ELISAs) have shown that urinary titin is a useful noninvasive biomarker for the diagnosis and screening of not only DMD, but also of neuromuscular diseases, for predicting the outcome of cardiomyopathy and for evaluating physical activities. The development of ELISA systems to measure urinary titin has opened a door to studying muscle degradation directly and noninvasively. This review provides current understanding of urinary titin and future prospects for measuring this protein.
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http://dx.doi.org/10.1016/bs.acc.2019.01.001DOI Listing
June 2019

Molecular assay for an intronic variant in NUP93 that causes steroid resistant nephrotic syndrome.

J Hum Genet 2019 Jul 23;64(7):673-679. Epub 2019 Apr 23.

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.

Advances in molecular genetics have revealed that approximately 30% of cases with steroid-resistant nephrotic syndrome (SRNS) are caused by single-gene mutations. More than 50 genes are responsible for SRNS. One such gene is the nucleoporin, 93-KD (NUP93). Thus far, few studies have reported mutations of NUP93 in SRNS. Here, we describe an NUP93 biallelic mutation in a 9-year-old boy with focal segmental glomerular sclerosis (FSGS). Notably, one mutation comprised an intronic variant; we conducted in vivo and in vitro analysis to characterize this variant. We found two heterozygous mutations in NUP93: c.2137-18G>A in intron 19 and a novel nonsense mutation c.727A>T (p.Lys243*) in exon 8. We conducted RNA sequencing and in vitro splicing assays by using minigene construction, combined with protein expression analysis to determine the pathogenicity of the intronic variant. Both RNA sequencing and in vitro splicing assay showed exon 20-skipping by the intronic variant. In protein expression analysis, aberrant subcellular localization with small punctate vesicles in the cytoplasm was observed for the intronic variant. Taken together, we concluded that c.2137-18G>A was linked to pathogenicity due to aberrant splicing. NUP93 variants are quite rare; however, we have shown that even intronic variants in NUP93 can cause SRNS. This study provides a fundamental approach to validate the intronic variant, as well as new insights regarding the clinical spectrum of SRNS caused by rare gene variants.
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http://dx.doi.org/10.1038/s10038-019-0606-4DOI Listing
July 2019

Identification of the shortest splice variant of Dp71, together with five known variants, in glioblastoma cells.

Biochem Biophys Res Commun 2019 01 5;508(2):640-645. Epub 2018 Dec 5.

Research Center for Locomotion Biology, Kobe Gakuin University, Nishi, Kobe, 651-2180, Japan; KNC Department of Nucleic Acid Drug Discovery, Faculty of Rehabilitation, Kobe Gakuin University, Nishi, Kobe 651-2180, Japan. Electronic address:

Background: Dystrophin Dp71 mRNA is produced from the most distal alternative promoter of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 is characterized by a wide variety of splice variants. In addition to being associated with cognitive disturbance in patients with DMD, Dp71 may also play a role in tumorigenesis. This study analyzed Dp71 transcripts in glioblastoma, the most common and most lethal type of cerebral malignancy.

Methods: Dp71 mRNA in the U-251 glioblastoma cell line was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). The amplified products were subcloned and sequenced.

Results: RT-PCR amplification of the 5' end of the Dp71 transcript yielded a product of expected size, indicating transcription from the Dp71 promoter in glioblastoma. Amplification of full-length Dp71, from exon G1 to DMD exon 79, yielded a product of expected size, as well as a faint, smaller sized band. Sequencing of 17 clones revealed six different alternatively spliced variants, with only one clone being of full-length Dp71 containing all 18 exons. Ten clones lacked exon 78 (Dp71b), indicating that Dp71b was a major type of Dp71 in glioblastoma. In addition, three clones lacked both exons 71 and 78 (Dp71ab), one clone lacked exons 71, 73 and 78 (Dp71ab △73), one clone lacked exons 71-74 and 78 (Dp71bc), and one clone lacked exons 68-76 and 78 (Dp71b△68-76). This novel transcript was the shortest Dp71 variant, with a predicted stop codon in exon 77 and was predicted to produce a 24.8 kDa protein, consisting of 216 amino acids including 15 amino acids from exon 77. This novel product was classified as Dp71g because of its unique C-terminal amino acid sequence.

Conclusions: Six splice variants of Dp71 were identified in glioblastoma cells, with Dp71b being the most abundant. Deletion of exon 78 was an apparent default splicing pathway in glioblastoma, being observed in 16 of 17 clones. Glioblastoma cells contained the shortest Dp71 transcript (Dp71b△68-76) identified to date, with a unique C-terminal amino acid sequence. These findings suggest the need to assess the function of Dp71 variants in glioblastoma.
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http://dx.doi.org/10.1016/j.bbrc.2018.11.168DOI Listing
January 2019

Can urinary titin be used for predicting Duchenne muscular dystrophy?

Clin Chim Acta 2019 Mar 2;490:162. Epub 2018 Nov 2.

Research Center for Locomotion Biology, Kobe Gakuin University, Nishi, Kobe 651-2180, Japan.

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http://dx.doi.org/10.1016/j.cca.2018.10.045DOI Listing
March 2019

Receiver operating curve analyses of urinary titin of healthy 3-y-old children may be a noninvasive screening method for Duchenne muscular dystrophy.

Clin Chim Acta 2018 Nov 24;486:110-114. Epub 2018 Jul 24.

Research Center for Locomotion Biology, Kobe Gakuin University, Kobe, Japan; Department of Occupational Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan. Electronic address:

Background: Duchenne muscular dystrophy (DMD) is a progressive, fatal muscle wasting disease. Early detection of DMD by mass screening may enable the early treatment of these patients. We have reported that urinary titin concentration, an indicator of severe muscle wasting, is a diagnostic biomarker for DMD.

Methods: Urinary titin concentrations were measured in healthy 3-y-old children and, by comparison with concentrations in 4 DMD patients, and validated as a screening biomarker for DMD. Urine samples were obtained from 100 healthy Japanese children, 52 boys and 48 girls, and their urinary titin concentrations measured by ELISA.

Results: The mean ± SD urinary titin concentration was 1.5 ± 2.5 nmol/l, and the mean urinary titin concentration normalized to creatinine was 2.2 ± 4.1 pmol/mg creatinine, with no differences between boys and girls. Histograms and box-and-whisker plots showed that almost all titin and normalized titin concentrations were in narrow ranges, with one outlier in common. Receiver operating characteristic curve analysis showed that titin and normalized-titin concentrations from healthy 3-y-olds were completely separate from those of 3-y-old DMD patients.

Conclusions: These findings indicate that urinary titin may be an excellent non-invasive biomarker to screen for DMD.
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http://dx.doi.org/10.1016/j.cca.2018.07.041DOI Listing
November 2018

Detection of Splicing Abnormalities and Genotype-Phenotype Correlation in X-linked Alport Syndrome.

J Am Soc Nephrol 2018 08 29;29(8):2244-2254. Epub 2018 Jun 29.

Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan.

Background: 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.

Methods: 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.

Results: We detected 41 XLAS families with abnormal splicing patterns and described novel XLAS atypical splicing patterns (=14) other than exon skipping caused by point mutations in the splice consensus sequence. The median age for developing ESRD was 20 years (95% confidence interval, 14 to 23 years) among patients with truncating mutations and 29 years (95% confidence interval, 25 to 40 years) among patients with nontruncating mutations (=0.001).

Conclusions: We report unpredictable atypical splicing in the gene in male patients with XLAS and reveal that renal prognosis differs significantly for patients with truncating versus nontruncating splicing abnormalities. Our results suggest that splicing modulation should be explored as a therapy for XLAS with truncating mutations.
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http://dx.doi.org/10.1681/ASN.2018030228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6065097PMC
August 2018

Cardiac Dysfunction in Duchenne Muscular Dystrophy Is Less Frequent in Patients With Mutations in the Dystrophin Dp116 Coding Region Than in Other Regions.

Circ Genom Precis Med 2018 01;11(1):e001782

From the Department of Clinical Laboratory, Kobe University Hospital, Kobe, Japan (T.Y., S.K., I.S., T.I., N.H., J.S.); Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan (H.A., M. Matsumoto, M.N., K.I.); Department of Pathology, Medical School of Nankai University, Tianjin, China (Z.Z.); Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Kobe, Japan (Z.Z., M. Matsuo); and Department of Clinical Epidemiology, Hyogo College of Medicine, Nishinomiya, Japan (M.E.-S.).

Background: Duchenne muscular dystrophy (DMD), the most common inherited muscular disease in childhood, is caused by dystrophin deficiency because of mutations in the gene. Although DMD is characterized by fatal progressive muscle wasting, cardiomyopathy is the most important nonmuscle symptom threatening the life of patients with DMD. The relationship between cardiac involvement and dystrophin isoforms has not been analyzed.

Methods And Results: The results of 1109 echocardiograms obtained from 181 Japanese DMD patients with confirmed mutations in the gene were retrospectively analyzed. Patients showed an age-related decline in left ventricular ejection fraction. Patients were divided by patterns of dystrophin isoform deficiency into 5 groups. The cardiac dysfunction-free survival was significantly higher in the group with mutations in the Dp116 coding region than the others, whereas no significant differences in the other 3 groups. At age 25 years, the cardiac dysfunction-free rate was 0.6 in the Dp116 group, but only 0.1 in others. PCR amplification of Dp116 transcript in human cardiac muscle indicated promoter activation.

Conclusions: Left ventricular ejection fraction in DMD declined stepwise with age. Cardiac dysfunction was less frequent in Dp116-deficient than other patients with DMD. Dp116 transcript was identified in human cardiac muscle for the first time. These results indicate that Dp116 is associated with cardiac involvement in DMD.
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http://dx.doi.org/10.1161/CIRCGEN.117.001782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6319568PMC
January 2018