Yasuhiro Takeshima

Contributions of Japanese patients to development of antisense therapy for DMD

INTRODUCTIONnDuchenne muscular dystrophy (DMD) is a fatal progressive muscle wasting disease considered untreatable since its first description in 1868. In 1987, the dystrophin gene responsible for DMD was cloned. This paved the way for the development of therapies. Antisense oligonucleotide (AO)-mediated exon skipping therapy is now reaching the stage of marketing authorization. On the 20th anniversary of the proposal of AO-mediated exon skipping therapy for DMD, this review explores the contributions of Japanese patients.nnnRESULTSnIn 1990, a Japanese DMD patient was reported as having a small deletion within dystrophin exon 19 and complicating exon 19 skipping in the absence of any mutation at the consensus splice sites. This led to identification of a splicing enhancer sequence within exon 19. Remarkably, AOs against this sequence were shown to induce exon skipping. This encouraged us to propose AO-mediated exon skipping therapy for DMD in 1995. The therapys effectiveness was verified in a Japanese patient with a nonsense dystrophin mutation manifesting as Becker muscular dystrophy. The patient showed skipping of the nonsense mutation-encoding exon. Finally, a DMD patient carrying a deletion of exon 20 volunteered to undergo intravenous AO infusion, enabling us to obtain proof of concept. The findings from these three patients greatly facilitated studies on exon skipping therapy. As a result, more than 300 reports on AO-mediated exon skipping therapy for DMD have been published, including at least two a month during the last few years.nnnCONCLUSIONnWe greatly appreciate the important contributions of Japanese patients to development of the exon skipping therapy for DMD.

Rituximab treatment for relapsed opsoclonus-myoclonus syndrome.

INTRODUCTIONnOpsoclonus-myoclonus syndrome (OMS) is a rare neurological disorder that is associated with paraneoplastic diseases. Because OMS can frequently relapse, patients may be inflicted with neurological problems for a long time. Recently, rituximab (RTX) was introduced as a drug to treat OMS. To assess RTX treatment, we studied a patient who experienced recurrence of OMS.nnnCASE REPORTnA 2-year-old Japanese boy, who had left adrenal neuroblastoma, suddenly showed OMS symptoms, including ataxia and opsoclonus. Surgical resection of the tumor and subsequent steroid therapy ameliorated his symptoms. When OMS relapsed during the time when prednisolone was reduced, he was treated with full-dose RTX therapy (375 mg/m2/week) for 4 consecutive weeks. However, 1year later, he presented again with OMS symptoms. This time, we only administered an additional single dose of RTX treatment (375 mg/m2), allowing remission of OMS symptoms. During 2 years after the additional RTX treatment, OMS symptoms did not appear, even when prednisolone was reduced. He had no adverse events associated with RTX during the whole treatment period.nnnCONCLUSIONSnAn additional single-dose RTX therapy might be effective for relapsed OMS patients who were previously treated with full-dose RTX therapy.

Resveratrol enhances splicing of insulin receptor exon 11 in myotonic dystrophy type 1 fibroblasts.

INTRODUCTIONnMyotonic dystrophy type 1 (DM1) is characterized by splicing abnormalities caused by CUG expansion of the DMPK gene transcript. Splicing of exon 11 of the insulin receptor (IR) gene is deregulated to suppress exon 11 inclusion into mRNA in DM1. Consequently, the exon 11-deleted IR isoform that is less sensitive to insulin is predominantly produced, leading to glucose intolerance in DM1. Upregulation of exon 11 retaining full-length IR mRNA is a potential way to recover insulin sensitivity in DM1.nnnMETHODSnWe examined candidate chemicals for their ability to enhance inclusion of exon 11 of the IR gene in cultured cells by reverse transcription-PCR amplification of a fragment extending from exons 10 to 12 of IR mRNA.nnnRESULTSnWe revealed that resveratrol (RES) enhanced the percentage of exon 11-containing IR mRNA among the total IR mRNA in HeLa cells. The RES-mediated enhancement of exon 11 inclusion was cell-specific and highest in fibroblasts. We tested RES on four fibroblast samples from three generations of one DM1 family. In each sample, RES treatment significantly upregulated the percentage of exon 11-containing IR mRNA to levels higher than that of the control, irrespective of the length of the samples CTG repeat expansion.nnnDISCUSSIONnA natural compound, RES, was shown for the first time to upregulate the full-length IR mRNA in fibroblasts from DM1 cases. Our results provide the justification of RES as a leading compound to improve glucose tolerance in DM1.

HEK293 cells express dystrophin Dp71 with nucleus-specific localization of Dp71ab.

The dystrophin gene consists of 79 exons and encodes tissue-specific isoforms. Mutations in the dystrophin gene cause Duchenne muscular dystrophy, of which a substantial proportion of cases are complicated by non-progressive mental retardation. Abnormalities of Dp71, an isoform transcribed from a promoter in intron 62, are a suspected cause of mental retardation. However, the roles of Dp71 in human brain have not been fully elucidated. Here, we characterized dystrophin in human HEK293 cells with the neuronal lineage. Reverse transcription-PCR amplification of the full-length dystrophin transcript revealed the absence of fragments covering the 5′ part of the dystrophin cDNA. In contrast, fragments covering exons 64–79 were present. The Dp71 promoter-specific exon G1 was shown spliced to exon 63. We demonstrated that the Dp71 transcript comprised two subisoforms: one lacking exon 78 (Dp71b) and the other lacking both exons 71 and 78 (Dp71ab). Western blotting of cell lysates using an antibody against the dystrophin C-terminal region revealed two bands, corresponding to Dp71b and Dp71ab. Immunohistochemical examination with the dystrophin antibody revealed scattered punctate signals in the cytoplasm and the nucleus. Western blotting revealed one band corresponding to Dp71b in the cytoplasm and two bands corresponding to Dp71b and Dp71ab in the nucleus, with Dp71b being predominant. These results indicated that Dp71ab is a nucleus-specific subisoform. We concluded that Dp71, comprising Dp71b and Dp71ab, was expressed exclusively in HEK293 cells and that Dp71ab was specifically localized to the nucleus. Our findings suggest that Dp71ab in the nucleus contributes to the diverse functions of HEK293 cells.

Staurosporine allows dystrophin expression by skipping of nonsense-encoding exon

BACKGROUNDnAntisense oligonucleotides that induce exon skipping have been nominated as the most plausible treatment method for dystrophin expression in dystrophin-deficient Duchenne muscular dystrophy. Considering this therapeutic efficiency, small chemical compounds that can enable exon skipping have been highly awaited. In our previous report, a small chemical kinase inhibitor, TG003, was shown to enhance dystrophin expression by enhancing exon skipping.nnnPURPOSEnStaurosporine (STS), a small chemical broad kinase inhibitor, was examined for enhanced skipping of a nonsense-encoding dystrophin exon.nnnMETHODSnSTS was added to culture medium of HeLa cells transfected with minigenes expressing wild-type or mutated exon 31 with c.4303G>T (p.Glu1435X), and the resulting mRNAs were analyzed by RT-PCR amplification. Dystrophin mRNA and protein were analyzed in muscle cells treated with STS by RT-PCR and western blotting, respectively.nnnRESULTSnSTS did not alter splicing of the wild-type minigene. In the mutated minigene, STS increased the exon 31-skipped product. A combination of STS and TG003 did not significantly increase the exon 31-skipped product. STS enhanced skipping of exon 4 of the CDC-like kinase 1 gene, whereas TG003 suppressed it. Two STS analogs with selective kinase inhibitory activity did not enhance the mutated exon 31 skipping. When immortalized muscle cells with c.4303G>T in the dystrophin gene were treated with STS, skipping of the mutated exon 31 and dystrophin expression was enhanced.nnnCONCLUSIONSnSTS, a broad kinase inhibitor, was shown to enhance skipping of the mutated exon 31 and dystrophin expression, but selective kinase inhibitors did not.

Patients with Duchenne muscular dystrophy are significantly shorter than those with Becker muscular dystrophy, with the higher incidence of short stature in Dp71 mutated subgroup

Duchenne and Becker muscular dystrophy (DMD/BMD) are caused by mutations in the dystrophin gene and are characterized by severe and mild progressive muscle wasting, respectively. Short stature has been reported as a feature of DMD in the Western hemisphere, but not yet confirmed in Orientals. Height of young BMD has not been fully characterized. Here, height of ambulant and steroid naive Japanese 179 DMD and 42 BMD patients between 4 and 10 years of age was retrospectively examined using height standard deviation score (SDS). The mean height SDS of DMD was -1.08 SD that was significantly smaller than normal (pu2009<u20090.001), indicating short stature of Japanese DMD. Furthermore, the mean height SDS of BMD was -0.27 SD, suggesting shorter stature than normal. Remarkably, the mean height SDS of DMD was significantly smaller than that of BMD (pu2009<u20090.0001). In DMD higher incidence of short stature (height SDSu2009<u2009-2.5 SD) was observed in Dp71 subgroup having mutations in dystrophin exons 63-79 than others having mutations in exons 1-62 (27.8% vs. 7.5%, pu2009=u20090.017). These suggested that height is influenced by dystrophin in not only DMD but also BMD and that dystrophin Dp71 has a role in height regulation.

Cardiac involvement in Fukuyama muscular dystrophy is less severe than in Duchenne muscular dystrophy

BACKGROUNDnOne of the main complications in patients with muscular dystrophies is cardiac dysfunction. The literature on cardiac involvement in patients with Fukuyama congenital muscular dystrophy (FCMD) is limited.nnnAIMnTo compare cardiac involvement between patients with FCMD and Duchenne muscular dystrophy (DMD).nnnMETHODSnWe compared cardiac involvement between 30 patients with FCMD and 181 patients with DMD using echocardiography and serum biomarkers. All patients were receiving regular checkups at Kobe University Hospital. We used single regression analysis to compare echocardiographic parameters, age, and serum biomarkers.nnnRESULTSnAlmost all clinical and echocardiographic parameters were lower in patients with FCMD than DMD. The brain natriuretic peptide concentration in patients with FCMD showed no correlation with age or left ventricular ejection fraction (r=0.231, p=0.22 and r=0.058, p=0.76, respectively). A log-rank test revealed that the risk of left ventricular systolic dysfunction was lower in patients with FCMD than DMD (p=0.046, hazard ratio=0.348).nnnCONCLUSIONnThe clinical progression of cardiac dysfunction is significantly milder in patients with FCMD than DMD, while skeletal muscle involvement is significantly worse in patients with FCMD. These data suggest that the pathophysiological findings of FCMD can be explained by less severe cardiac dysfunction in FCMD than DMD.

Epidemiological survey and clinical investigation of pediatric IgA nephropathy

BackgroundSince school urinalysis screening was introduced in 1974, the number of cases requiring initiation of dialysis due to glomerulonephritis has been steadily decreasing and school urinalysis screening has been praised for contributing to the early detection and treatment of glomerulonephritis. However, the lack of nationwide epidemiological surveys is also a problem.MethodsWe conducted an epidemiological survey focusing on the frequency of occurrence of pediatric IgA nephropathy in Nishinomiya City. Subjects comprised 374,846 children who underwent school urinalysis screening from 2003 to 2012. Renal biopsy findings and clinical findings of these pediatric IgA nephropathy cases were retrospectively investigated.ResultsThere were 37 (mean 3.7/year) newly diagnosed cases of pediatric IgA nephropathy in Nishinomiya City. The IgA nephropathy onset rate per 100,000 children who underwent school urinalysis screening was 9.9xa0cases/year. Compared to the histologic low grade group, the histologic high grade group had significantly higher urinary P/C ratio (Pxa0<xa00.001). In the histologic high grade group, the number of cases of proteinuria remission 3xa0years after starting treatment was significantly higher in the group treated with steroids (Pxa0=xa00.045).ConclusionsOur study found that 9.9 cases of pediatric IgA nephropathy were diagnosed per 100,000 in the pediatric population, which is equivalent to or slightly more than past reports. IgA nephropathy, which poses a high histologic risk, presents with heavy proteinuria; but the proteinuria remission rate following steroid therapy is high 3xa0years after treatment, which suggests that administration of steroids results in an improved clinical outcome.

Glycine plays a crucial role as a co-agonist of NMDA receptors in the neuronal circuit generating body movements in rat fetuses

Neuronal circuits generating fetal movements in mammals are localized in the brainstem and the spinal cord. It has been shown that glycine plays an important role through the strychnine-sensitive glycine receptors in these circuits. However, the role of glycine as the NMDA receptor co-agonist in fetal period is not fully understood. In this study, we examined the contribution of glycine to the perinatal rat spinal circuit generating forelimb movements utilizing isolated brainstem-cervical-spinal-cord preparations. In late embryonic-days-preparations, spontaneous motor bursts related to forelimb movements (forelimb-movement-related bursts; FMRBs) and respiration-related activity were observed. In neonatal preparations, spontaneous FMRBs were not observed but periodic motor bursts resembling the FMRBs could be induced after bath application of strychnine (strychnine-induced motor bursts; SIMBs). Both FMRBs and SIMBs were blocked by either the NMDA receptor antagonist APV or the antagonists of the glycine binding site of NMDA receptors [5,7-dichlorokynurenic acid (DCKA) or L-689560]. Furthermore, these motor bursts were facilitated by the glycine uptake blocker sarcosine. This effect of sarcosine was blocked by DCKA. The findings indicate that glycine plays a crucial role as a NMDA receptor co-agonist in generating spontaneous fetal motor activity before functioning as a classical inhibitory neurotransmitter in suppressing the fetal neuronal circuits.

Combining passive leg-lifting with transmural myocardial strain profile for enhanced predictive capability for subclinical left ventricular dysfunction in Duchenne muscular dystrophy

BACKGROUNDnWe previously reported that the transmural myocardial strain profile (TMSP) was an effective predictor for subclinical left ventricular (LV) dysfunction in patients with Duchenne muscular dystrophy (DMD) with preserved LV ejection fraction (LVEF), but its predictive power when used alone proved to be limited.nnnMETHODSnA total of 95 DMD patients with LVEF of 59±5% (all ≥55%) and age 11.3±3.0 years were analyzed retrospectively. Echocardiography was performed at baseline and 1-year follow-up, and all baseline measurements were repeated during a passive leg-lifting maneuver with legs elevated to approximately 45° from the horizontal position. TMSP of the posterior wall was evaluated from the mid-LV short-axis view. On the basis of our previous findings, TMSP with a notch was adopted as a predictor for evaluation of subclinical LV dysfunction in DMD patients whose LVEF remains preserved.nnnRESULTSnAt baseline, normal TMSP comprised 35 patients (37%), and the remaining 60 (63%) were classified as TMSP with a notch. Twenty-nine patients (48%) had developed LV wall motion abnormality at the 1-year follow-up, but this was observed only in the group of patients with TMSP with a notch at rest and also during passive leg-lifting. Furthermore, this group showed significantly more frequent development of LV wall motion abnormality at 1-year follow-up, with better sensitivity, specificity, and positive and negative predictive values for prediction of this abnormality than for other sub-groups.nnnCONCLUSIONSnMost DMD patients suffer from progressive skeletal muscle weakness, so that combining TMSP with passive leg-lifting may make TMSP even more effective as a simple and non-invasive predictor of LV subclinical dysfunction.