Masafumi Matsuo

Germ-line mutation of KCNQ2, p.R213W, in a Japanese family with benign familial neonatal convulsion

Background: Benign familial neonatal convulsion (BFNC) is an autosomal‐dominantly inherited epilepsy of neonates. The KCNQ2 and KCNQ3 genes have been cloned as the responsible genes for BFNC. Detection of mutations in these genes is helpful for confirmation of BFNC or differential diagnosis of convulsive disorders in the neonatal period.

Epigallocatechin gallate inhibits sphere formation of neuroblastoma BE(2)-C cells

ObjectivesA growing number of epidemiological studies have demonstrated that the consumption of green tea inhibits the growth of a variety of cancers. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, has been shown to have an anti-cancer effect against many cancers. Most cancers are believed to be initiated from and maintained by a small population of tumor-initiating cells (TICs) that are responsible for chemotherapeutic resistance and tumor relapse. In neuroblastoma, an aggressive pediatric tumor that often relapses and has a poor prognosis, TICs were recently identified as spheres grown in a serum-free non-adherent culture used for neural crest stem cell growth. Although EGCG has been reported to induce growth arrest and apoptosis in neuroblastoma cells, its effect on neuroblastoma TICs remains to be defined.MethodsGene expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The effects of EGCG on cell proliferation, apoptosis, and sphere formation were determined by cell counting, propidium iodide staining, and sphere (>100xa0μm in diameter) counting, respectively.ResultsNeuroblastoma BE(2)-C cells showed increased expression of stem cell markers (nanog homeobox [NANOG] and octamer-binding transcription factor 4 [OCT4]), as well as decreased expression of neuronal differentiation markers (Cu2+-transporting ATPase alpha polypeptide [ATP7A] and dickkopf homolog 2 [DKK2]) in spheres grown in serum-free non-adherent culture, compared to parental cells grown in conventional culture. Although EGCG induced growth arrest and apoptosis in the parental cells in a dose-dependent manner, it was not effective against spheres. However, EGCG potently inhibited sphere formation in the BE(2)-C cells.ConclusionsThe present results suggest that EGCG may inhibit the development of TICs in BE(2)-C cells.

Involvement of aldehyde dehydrogenase 1A2 in the regulation of cancer stem cell properties in neuroblastoma

Despite the introduction of 13-cis-retinoic acid (13-cis-RA) into the current chemotherapy, more than half of high-risk neuroblastoma patients have experienced tumor relapses driven by chemoresistant cancer stem cells (CSCs) that can be isolated by their ability to grow as spheres. Although aldehyde dehydrogenase (ALDH) has been used to characterize CSCs in certain cancers, ALDH remains elusive in neuroblastoma. In the present study, we determined ALDH activity and expression of its 19 isoforms in spheres and parental cells of neuroblastoma. ALDH activity and several ALDH isoforms were consistently induced in spheres of different neuroblastoma cells. While ALDH1A2, ALDH1L1 and ALDH3B2 expression was consistently induced in spheres and associated with the sphere and colony formation, only ALDH1A2 expression was significantly correlated with the poor prognosis of neuroblastoma patients. ALDH1A2 expression was further associated with the growth and undifferentiation of neuroblastoma xenografts and the resistance of neuroblastoma cells to 13-cis-RA. These results suggest that ALDH1A2 is involved in the regulation of CSC properties in neuroblastoma.

Deletion analyses of SMN1 and NAIP genes in Malaysian spinal muscular atrophy patients

Background: The survival motor neuron 1 (SMN1) gene has been recognized to be responsible for spinal muscular atrophy (SMA) because it is homozygously deleted in more than 90% of SMA patients, irrespective of their clinical severity, whereas the neuronal apoptosis inhibitory protein (NAIP) gene is now considered to be a modifying factor of the severity of SMA. In Malaysia, it remains to be elucidated whether deletion of the SMN1 gene is also a main cause of SMA or whether deletion of the NAIP gene is found in the SMA patients.

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.

Establishment of a highly sensitive sandwich ELISA for the N-terminal fragment of titin in urine

Muscle damage and loss of muscle mass are triggered by immobilization, loss of appetite, dystrophies and chronic wasting diseases. In addition, physical exercise causes muscle damage. In damaged muscle, the N-terminal and C-terminal regions of titin, a giant sarcomere protein, are cleaved by calpain-3, and the resulting fragments are excreted into the urine via glomerular filtration. Therefore, we considered titin fragments as promising candidates for reliable and non-invasive biomarkers of muscle injury. Here, we established a sandwich ELISA that can measure the titin N-terminal fragment over a biologically relevant range of concentrations, including those in urine samples from older, non-ambulatory Duchenne muscular dystrophy patients and from healthy donors under everyday life conditions and after exercise. Our results indicate that the established ELISA could be a useful tool for the screening of muscular dystrophies and also for monitoring the progression of muscle disease, evaluating the efficacy of therapeutic approaches, and investigating exercise-related sarcomeric disruption and repair processes.

Minimal residual disease monitoring in neuroblastoma patients based on the expression of a set of real-time RT-PCR markers in tumor-initiating cells

Minimal residual disease (MRD) is derived from tumor-initiating cells (TICs) and is responsible for tumor relapse. Neuroblastoma is characterized by extreme tumor heterogeneity, and more than half of high-risk patients experience tumor relapse. To overcome tumor heterogeneity and achieve more sensitive detection of MRD, several sets of real-time RT-PCR markers have been reported for MRD monitoring in neuroblastoma patients from different centers. However, these markers vary across centers and are still being validated. In the present study, we validated the ability of 14xa0commonly used real-time RT-PCR markers to detect MRD based on their expression in neuroblastoma TICs, and we developed a novel MRD detection protocol, which scored the samples as MRD-positive when the expression of one of the 11xa0real-time RT-PCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B and TH) exceeded the normal range. By using this protocol, we prospectively monitored MRD in 73xa0bone marrowxa0(BM), 12xa0peripheral blood stem cell and 8xa0peripheral blood samples from 14 neuroblastoma patients treated at a single center. We scored 100, 56, 56 and 57% BM cytology-positive, elevated vanillylmandelic acid (VMA), elevated homovanillic acid (HVA) and elevated neuron-specific enolase (NSE) samples as MRD-positive, respectively. MRD was also positive in 48, 45, 46 and 43% of the BM cytology-negative and normal VMA, normal HVA and normal NSE samples, respectively. These results suggest that the present MRD detection protocol based on the expression of a set of 11xa0real-time RT-PCR markers in neuroblastoma TICs achieves sensitive MRD monitoring in neuroblastoma patients.

Rab15 alternative splicing is altered in spheres of neuroblastoma cells

Neuroblastoma is an aggressive pediatric tumor that accounts for 15% of cancer-related deaths in children. More than half of high-risk neuroblastoma patients develop tumor relapse that is lethal in most cases. A small population of tumor-initiating cells (TICs), recently identified from high-risk neuroblastoma patients as spheres, is believed to be responsible for tumor relapse. Rab family small G proteins are essential in controlling membrane traffic and their misregulation results in several cancers. Rab15 was originally isolated as a brain-specific Rab protein regulating the endocytic recycling pathway and was recently identified as a downstream target of the neural transcription factor Atoh1. Previously, we identified two alternatively spliced Rab15 isoforms in neuroblastoma cells and showed a significant correlation between Rab15 expression and neuronal differentiation. As aberrant alternative splicing is intimately associated with an increasing number of cancers, its use as a new diagnostic and/or prognostic biomarker has attracted considerable attention. In the present study, we explored cancer-associated changes of Rab15 alternative splicing in neuroblastoma TICs. We found that Rab15 alternative splicing generated two novel isoforms designated as Rab15(AN2) and Rab15(AN3) in addition to two known isoforms designated as Rab15(CN) and Rab15(AN1). Although both Rab15(AN2) and Rab15(AN3) contained premature termination codons, they were detected in not only neuroblastoma cells but also in normal human tissues. One isoform was predominantly expressed in the brain and testis, while the other isoform was more specifically expressed in the brain. In neuroblastoma, Rab15 isoform balance measured by the Rab15(CN)/Rab15(AN1+AN2+AN3) ratio was significantly decreased in spheres compared to parental cells. These results suggest that Rab15 alternative splicing may serve as a biomarker to discriminate TICs from non-TICs in neuroblastoma.

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.