Publications by authors named "Hisahide Nishio"

145 Publications

Phenotypes of SMA patients retaining SMN1 with intragenic mutation.

Brain Dev 2021 Apr 20. Epub 2021 Apr 20.

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

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by homozygous deletion or intragenic mutation of the SMN1 gene. It is well-known that high copy number of its homologous gene, SMN2, modifies the phenotype of SMN1-deleted patients. However, in the patients with intragenic SMN1 mutation, the relationship between phenotype and SMN2 copy number remains unclear.

Methods: We have analyzed a total of 515 Japanese patients with SMA-like symptoms (delayed developmental milestones, respiratory failures, muscle weakness etc.) from 1996 to 2019. SMN1 and SMN2 copy numbers were determined by quantitative polymerase chain reaction (PCR) method and/or multiplex ligation-dependent probe amplification (MLPA) method. Intragenic SMN1 mutations were identified through DNA and RNA analysis of the fresh blood samples.

Results: A total of 241 patients were diagnosed as having SMA. The majority of SMA patients showed complete loss of SMN1 (n = 228, 95%), but some patients retained SMN1 and carried an intragenic mutation in the retaining SMN1 (n = 13, 5%). Ten different mutations were identified in these 13 patients, consisting of missense, nonsense, frameshift and splicing defect-causing mutations. The ten mutations were c.275G > C (p.Trp92Ser), c.819_820insT (p.Thr274Tyrfs*32), c.830A > G (p.Tyr277Cys), c.5C > T (p.Ala2Val), c.826 T > C (p.Tyr276His), c.79C > T (p.Gln27*), c.188C > A (p.Ser63*), c.422 T > C (p.Leu141Pro), c.835-2A > G (exon 7 skipping) and c.835-3C > A (exon 7 skipping). It should be noted here that some patients with milder phenotype carried only a single SMN2 copy (n = 3), while other patients with severe phenotype carried 3 SMN2 copies (n = 4).

Conclusion: Intragenic mutations in SMN1 may contribute more significantly to clinical severity than SMN2 copy numbers.
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http://dx.doi.org/10.1016/j.braindev.2021.03.006DOI Listing
April 2021

Dried Blood Spot Screening System for Spinal Muscular Atrophy with Allele-Specific Polymerase Chain Reaction and Melting Peak Analysis.

Genet Test Mol Biomarkers 2021 Apr;25(4):293-301

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

Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance caused by homozygous deletions. Although SMA has been considered as incurable, newly developed drugs improve life prognoses and motor functions of patients. To maximize the efficacy of the drugs, SMA patients should be treated before symptoms become apparent. Thus, newborn screening for SMA is strongly recommended. In this study, we aim to establish a new simple screening system based on DNA melting peak analysis. A total of 124 dried blood spot (DBS) on FTA ELUTE cards (51 -deleted patients with SMA, 20 carriers, and 53 controls) were punched and subjected to direct amplification of and (reference gene). Melting peak analyses were performed to detect deletions from DBS samples. A combination of allele-specific polymerase chain reaction (PCR) and melting peak analyses clearly distinguished the DBS samples with and without . Compared with the results of fresh blood samples, our new system yielded 100% sensitivity and specificity. The advantages of our system include (1) biosafe collection, transfer, and storage for DBS samples, (2) obviating the need for DNA extraction from DBS preventing contamination, (3) preclusion of fluorescent probes leading to low PCR cost, and (4) fast and high-throughput screening for deletions. We demonstrate that our system would be applicable to a real-world newborn screening program for SMA, because our new technology is efficient for use in routine clinical laboratories that do not have highly advanced PCR instruments.
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http://dx.doi.org/10.1089/gtmb.2020.0312DOI Listing
April 2021

Assessment of Spinal Muscular Atrophy Carrier Status by Determining Copy Number Using Dried Blood Spots.

Int J Neonatal Screen 2020 May 29;6(2). Epub 2020 May 29.

Department of Community Medicine and Social Healthcare Science, Division of Epidemiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

Spinal muscular atrophy (SMA) is a common neuromuscular disease with autosomal recessive inheritance. The disease gene, , is homozygously deleted in 95% of SMA patients. Although SMA has been an incurable disease, treatment in infancy with newly developed drugs has dramatically improved the disease severity. Thus, there is a strong rationale for newborn and carrier screening for SMA, although implementing SMA carrier screening in the general population is controversial. We previously developed a simple, accurate newborn SMA screening system to detect homozygous deletions using dried blood spots (DBS) on filter paper. Here, we modified our previous system to detect the heterozygous deletions of , which indicates SMA carrier status. The system involves a calibrator-normalized relative quantification method using quantitative nested PCR technology. Our system clearly separated the DBS samples with one copy (carrier status with a heterozygous deletion of ) from the DBS samples with two copies (non-carrier status with no deletion of ). We also analyzed DBS samples from SMA families, confirmed SMA in the affected children, and determined the carrier status of their parents based on the copy number. In conclusion, our system will provide essential information for risk assessment and genetic counseling, at least for SMA families.
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http://dx.doi.org/10.3390/ijns6020043DOI Listing
May 2020

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

Assessment of Spinal Muscular Atrophy Carrier Status by Determining Copy Number Using Dried Blood Spots.

Int J Neonatal Screen 2020 Jun 29;6(2):43. Epub 2020 May 29.

Department of Community Medicine and Social Healthcare Science, Division of Epidemiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan; (Y.O.S.W.); (E.T.E.N.); (M.S.).

Spinal muscular atrophy (SMA) is a common neuromuscular disease with autosomal recessive inheritance. The disease gene, , is homozygously deleted in 95% of SMA patients. Although SMA has been an incurable disease, treatment in infancy with newly developed drugs has dramatically improved the disease severity. Thus, there is a strong rationale for newborn and carrier screening for SMA, although implementing SMA carrier screening in the general population is controversial. We previously developed a simple, accurate newborn SMA screening system to detect homozygous deletions using dried blood spots (DBS) on filter paper. Here, we modified our previous system to detect the heterozygous deletions of , which indicates SMA carrier status. The system involves a calibrator-normalized relative quantification method using quantitative nested PCR technology. Our system clearly separated the DBS samples with one copy (carrier status with a heterozygous deletion of ) from the DBS samples with two copies (non-carrier status with no deletion of ). We also analyzed DBS samples from SMA families, confirmed SMA in the affected children, and determined the carrier status of their parents based on the copy number. In conclusion, our system will provide essential information for risk assessment and genetic counseling, at least for SMA families.
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http://dx.doi.org/10.3390/ijns6020043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423012PMC
June 2020

A Novel System for Spinal Muscular Atrophy Screening in Newborns: Japanese Pilot Study.

Int J Neonatal Screen 2019 Dec 12;5(4):41. Epub 2019 Nov 12.

Department of Community Medicine and Social Healthcare Science, Division of Epidemiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan; (M.S.); (E.T.E.N.); (Y.O.S.W.); (I.T.).

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by gene deletion/mutation. The drug nusinersen modifies mRNA splicing, increasing the production of the full-length SMN protein. Recent studies have demonstrated the beneficial effects of nusinersen in patients with SMA, particularly when treated in early infancy. Because nusinersen treatment can alter disease trajectory, there is a strong rationale for newborn screening. In the current study, we validated the accuracy of a new system for detecting deletion (Japanese patent application No. 2017-196967, PCT/JP2018/37732) using dried blood spots (DBS) from 50 patients with genetically confirmed SMA and 50 controls. Our system consists of two steps: (1) targeted pre-amplification of genes by direct polymerase chain reaction (PCR) and (2) detection of deletion by real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) using the pre-amplified products. Compared with PCR analysis results of freshly collected blood samples, our system exhibited a sensitivity of 1.00 (95% confidence interval [CI] 0.96-1.00) and a specificity of 1.00 (95% CI 0.96-1.00). We also conducted a prospective SMA screening study using DBS from 4157 Japanese newborns. All DBS tested negative, and there were no screening failures. Our results indicate that the new system can be reliably used in SMA newborn screening.
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http://dx.doi.org/10.3390/ijns5040041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510215PMC
December 2019

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

Clinical phenotypes of spinal muscular atrophy patients with hybrid SMN gene.

Brain Dev 2021 Feb 6;43(2):294-302. Epub 2020 Oct 6.

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

Background: Spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of SMN1 exons 7 and 8. However, exon 8 is retained in some cases, where SMN2 exon 7 recombines with SMN1 exon 8, forming a hybrid SMN gene. It remains unknown how the hybrid SMN gene contribute to the SMA phenotype.

Method: We analyzed 515 patients with clinical suspicion for SMA. SMN1 exons 7 and 8 deletion was detected by PCR followed by enzyme digestion. Hybrid SMN genes were further analyzed by nucleotide sequencing. SMN2 copy number was determined by real-time PCR.

Results: SMN1 exon 7 was deleted in 228 out of 515 patients, and SMN1 exon 8 was also deleted in 204 out of the 228 patients. The remaining 24 patients were judged to carry a hybrid SMN gene. In the patients with SMN1 exon 7 deletion, the frequency of the severe phenotype was significantly lower in the patients with hybrid SMN gene than in the patients without hybrid SMN gene. However, as for the distribution of SMN2 exon 7 copy number among the clinical phenotypes, there was no significant difference between both groups of SMA patients with or without hybrid SMN gene.

Conclusion: Hybrid SMN genes are not rare in Japanese SMA patients, and it appears to be associated with a less severe phenotype. The phenotype of patients with hybrid SMN gene was determined by the copy number of SMN2 exon 7, as similarly for the patients without hybrid SMN gene.
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http://dx.doi.org/10.1016/j.braindev.2020.09.005DOI Listing
February 2021

Phosphoethanolamine Elevation in Plasma of Spinal Muscular Atrophy Type 1 Patients.

Kobe J Med Sci 2020 04 1;66(1):E1-E11. Epub 2020 Apr 1.

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

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration or loss of lower motor neurons. The survival of motor neuron (SMN) 1 gene, which produces the SMN protein, has been identified as a responsible gene for the disease. SMN is ubiquitously expressed in any tissue and may play an important role on the metabolism in the human body. However, no appropriate biomarkers reflecting the alteration in the metabolism in SMA have been identified.

Methods: Low-molecular-weight metabolites were extracted from plasma of 20 human infants (9 SMA type 1 patients and 11 controls) and 9 infant mice (5 SMA-model mice, 4 control mice), and derivatized with N-methyl-N-trimethylsilyltrifluoroacetamide. Finally, the derivatized products were applied to Gas Chromatography/Mass Spectrometry apparatus. To confirm the metabolite abnormality in SMA type 1 patients, we performed SMN-silencing experiment using a hepatocyte-derived cell line (HepG2).

Results: We performed a comprehensive metabolomics analysis of plasma from the patients with SMA type 1 and controls, and found that phosphoethanolamine (PEA) was significantly higher in the patients than in the controls. HepG2 experiment also showed that SMN-silencing increased PEA levels. However, comprehensive metabolomics analysis of plasma from SMA-model mice and control mice showed different profile compared to human plasma; there was no increase of PEA even in the SMA-model mice plasma.

Conclusion: Our data suggested that PEA was one of the possible biomarkers of human SMA reflecting metabolic abnormalities due to the SMN protein deficiency.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447103PMC
April 2020

Determining the association between polymorphisms of the and genes with attention deficit hyperactivity disorder in children from Java Island.

Neurol Int 2020 Jul 10;12(1):8292. Epub 2020 Jul 10.

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

Attention deficit hyperactivity disorder (ADHD) is one of the most common neurobehavioural in the children. Genetic factor is known one of the factors which contributed in ADHD development. VNTR polymorphism in 3'UTR exon 15 of DAT1 gene and exon 3 of DRD4 gene are reported to be associated in ADHD. In this study we examine the association of ADHD with VNTR polymorphism of DAT1 and DRD4 gene in Indonesian children. Sixty-five ADHD children and 70 normal children (6-13 years of age), were included in the study, we matched by age and gender. ADHD was diagnosed by DSM-IV. We performed a casecontrol study to found the association between ADHD and VNTR polymorphism of DAT1 and DRD4 genes. The 10-repeat allele of DAT1 and 2-repeat allele of DRD4 were higher in Indonesian children. Although the frequency of these allele was higher, but it was similar both in ADHD and control groups. Neither DAT1 nor DRD4 gene showed showed significant difference in genotype distribution and frequency allele between both groups (p > 0.05) No association between ADHD and VNTR polymorphism of DAT1 and DRD4 genes found in Indonesian children. This data suggest that DAT1 and DRD4 do not contribute to etiology of ADHD in Indonesian children. Further studies are needed to clarify association between VNTR polymorphism of DAT1 and DRD4 genetic with ADHD of Indonesian children in larger sample size and family based study.
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http://dx.doi.org/10.4081/ni.2020.8292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378541PMC
July 2020

Survey of patients with spinal muscular atrophy on the island of Shikoku, Japan.

Brain Dev 2020 Sep 3;42(8):594-602. Epub 2020 Jun 3.

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

Background: Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder associated with spinal motor neuron loss and characterized by generalized muscle weakness. Only a few reports exist on SMA epidemiology in Japan. Additionally, nusinersen recently became available as a treatment for this condition. We estimated the prevalence of each type of SMA on Shikoku, Japan's fourth-largest major island.

Methods: We sent a questionnaire to all 131 hospitals in Shikoku that have pediatrics or neurology departments from March to September 2019, asking whether each hospital had SMA patients at that time. If so, we sent a second questionnaire to obtain more detailed information on the clinical data and treatment of each patient.

Results: A total of 117 hospitals (89.3%) responded to our first questionnaire, and 21 SMA patients were reported, 16 of whom had homozygous deletion of SMN1. Of the 21, nine had SMA type 1, five were type 2, five were type 3, one was type 4, and one was unidentified. The estimated prevalence for all instances of SMA and 5q-SMA was 0.56 and 0.43 per 100,000 people, respectively. Thirteen patients had received nusinersen therapy. Its outcomes varied from no obvious effects and being unable to sit to being able to sit independently.

Conclusion: Our data showed the prevalence of SMA types 2 and 3 was relatively low on Shikoku compared with previous reports from other countries, suggesting delayed diagnosis may affect the results. Remaining motor function may be one predicting factor. Greater awareness of SMA among clinicians and patients seems necessary for more accurate epidemiological studies.
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http://dx.doi.org/10.1016/j.braindev.2020.05.004DOI Listing
September 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

Newborn Screening for Spinal Muscular Atrophy: DNA Preparation from Dried Blood Spot and DNA Polymerase Selection in PCR.

Kobe J Med Sci 2019 Nov 14;65(3):E95-E99. Epub 2019 Nov 14.

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

Background: Polymerase chain reaction (PCR) analysis using DNA from dried blood spot (DBS) samples on filter paper is a critical technique for spinal muscular atrophy (SMA) newborn screening. However, DNA extraction from DBS is time-consuming, and elimination of PCR inhibitors from DBS is almost impossible.

Methods: Exon 7 of the two homologous SMA-related genes, survival motor neuron (SMN) 1 and SMN2, of five SMA patients and five controls were amplified by PCR with a punched-out circle of the DBS paper. Two types of DNA preparation methods were tested; DNA-extraction (extracted DNA was added in a PCR tube) and non-DNA-extraction (a punched-out DBS circle was placed in a PCR tube). As for the DNA polymerases, two different enzymes were compared; TaKaRa Ex Taq™ and KOD FX Neo™. To test the diagnostic quality of PCR products, RFLP (Restriction fragment length polymorphism) analysis with DraI digestion was performed, differentiating SMN1 and SMN2.

Results: In PCR using extracted DNA, sufficient amplification was achieved with TaKaRa Ex Taq™ and KOD FX Neo™, and there was no significant difference in amplification efficiency between them. In direct PCR with a punched-out DBS circle, sufficient amplification was achieved when KOD FX Neo™ polymerase was used, while there was no amplification with TaKaRa Ex Taq™. RFLP analysis of the direct PCR products with KOD FX Neo™ clearly separated SMN1 and SMN2 sequences and proved the presence of both of SMN1 and SMN2 in controls, and only SMN2 in SMA patients, suggesting that the direct PCR products with KOD FX Neo™ were of sufficient diagnostic quality for SMA testing.

Conclusion: Direct PCR with DNA polymerases like KOD FX NeoTM has potential to be widely used in SMA newborn screening in the near future as it obviates the DNA extraction process from DBS and can precisely amplify the target sequences in spite of the presence of PCR inhibitors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012323PMC
November 2019

Nested PCR Amplification Secures DNA Template Quality and Quantity in Real-time mCOP-PCR Screening for SMA.

Kobe J Med Sci 2019 Jul 16;65(2):E54-E58. 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 disorder caused by SMN1 gene deletion. SMA has been considered an incurable disease. However, a newly-developed antisense oligonucleotide drug, nusinersen, brings about a good outcome to SMA patients in the clinical trials. Now, a screening for SMA is required for early diagnosis and early treatment so as to give a better clinical outcome to the patients. We have invented a new technology, mCOP-PCR, for SMA screening using dried blood spot (DBS) on the filter paper. One of the problems encountered in SMA screening is poor quality and quantity of DNA extracted from DBS.

Methods: DNA was extracted from DBS of six individuals. Fresh blood DNA of each individual had already been genotyped using PCR/RFLP. The fragments including the sequence of SMN1/SMN2 exon 7 were pre-amplified with conventional PCR. To determine which pre-amplified product is a better template for the real-time mCOP-PCR, we did pre-amplification with a single PCR or pre-amplification with a nested PCR.

Results: The real-time mCOP-PCR using pre-amplified products with a single PCR brought about ambiguous results in some SMN1-carrying individuals. However, the results of real-time mCOP-PCR following pre-amplification with a nested PCR were completely matched with those of PCR-RFLP.

Conclusion: In our study on the real-time mCOP-PCR screening system for SMA, a nested PCR secured the DNA template quality and quantity, leading to unambiguous results of SMA screening.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012193PMC
July 2019

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

Intrathecal nusinersen treatment after ventriculo-peritoneal shunt placement: A case report focusing on the neurofilament light chain in cerebrospinal fluid.

Brain Dev 2020 Mar 28;42(3):311-314. Epub 2019 Dec 28.

Department of Pediatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan. Electronic address:

Background: In July 2018, a rare and serious adverse effect (AE), namely, communicating hydrocephalus unrelated to meningitis or bleeding, was reported in relation to five patients treated with nusinersen for spinal muscular atrophy (SMA). Some patients were managed using a ventriculo-peritoneal shunt (VPS) implant and continued to receive nusinersen treatment. However, there is limited information concerning the effectiveness and safety of nusinersen treatment for patients with a VPS.

Case Report: A female patient exhibited general hypotonia soon after birth and was diagnosed, using genetic analysis, with spinal muscular atrophy. She required permanent invasive ventilation from 2 months of age. She developed a progressive hydrocephalus and underwent placement of a VPS in infancy. Treatment with nusinersen was initiated when she was 7 years old. The neurofilament light-chain (NfL) concentration in the cerebrospinal fluid (CSF) decreased over time with nusinersen treatment. Twelve months have passed since the start of nusinersen treatment and no AEs have been observed.

Conclusion: Nusinersen treatment may be effective and safe, even after placement of a VPS. NfL levels in the CSF could be valuable markers of disease activity/treatment response even in advanced stages of SMA.
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http://dx.doi.org/10.1016/j.braindev.2019.12.006DOI Listing
March 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

Establishment of the experimental procedure for prediction of conjugation capacity in mutant UGT1A1.

PLoS One 2019 15;14(11):e0225244. Epub 2019 Nov 15.

Department of Pharmacokinetics, Kyoto Pharmaceutical University, Kyoto, Japan.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is an enzyme that is found in the endoplasmic reticulum membrane and can reportedly have a large number of amino acid substitutions that result in the reduction of glucuronidation capacity. For example, adverse drug reactions when patients receive CPT-11 (irinotecan) such as in cancer chemotherapy are caused by amino acid substitutions in UGT1A1. We previously found that the extent of the docking when the hydroxyl residue of bilirubin was oriented toward UDP-glucuronic acid correlated with in vitro conjugation capacity. In this study, we analyzed the conformation of mutant UGT1A1s by means of structural optimization with water and lipid bilayers instead of the optimization in vacuo that we used in our previous study. We then derived a mathematical model that can predict the conjugation capacities of mutant UGT1A1s by using results of substrate docking in silico and results of in vitro analysis of glucuronidation of acetaminophen and 17β-estradiol by UGT1A1s. This experimental procedure showed that the in silico conjugation capacities of other mutant UGT1A1s with bilirubin or SN-38 were similar to reported in vitro conjugation capacities. Our results suggest that this experimental procedure described herein can correctly predict the conjugation capacities of mutant UGT1A1s and any substrate.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225244PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6857941PMC
March 2020

Nusinersen helps restore walking ability in childhood spinal muscular atrophy.

Pediatr Int 2019 Jul 9;61(7):728-729. Epub 2019 Jul 9.

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

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http://dx.doi.org/10.1111/ped.13867DOI 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

The Protective Effects of Levetiracetam on a Human iPSCs-Derived Spinal Muscular Atrophy Model.

Neurochem Res 2019 Jul 17;44(7):1773-1779. Epub 2019 May 17.

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu, 501-1196, Japan.

Spinal muscular atrophy (SMA) is an inherited disease characterized by progressive motor neuron death and subsequent muscle weakness and is caused by deletion or mutation of survival motor neuron (SMN) 1 gene. Protecting spinal motor neuron is an effective clinical strategy for SMA. The purpose of this study was to investigate the potential effect of an anti-epileptic drug levetiracetam on SMA. In the present study, we used differentiated spinal motor neurons (MNs) from SMA patient-derived induced pluripotent stem cells (SMA-iPSCs) to investigate the effect of levetiracetam. Levetiracetam promoted neurite elongation in SMA-iPSCs-MNs. TUNEL-positive spinal motor neurons were significantly reduced by levetiracetam in SMA-iPSCs-MNs. In addition, the expression level of cleaved-caspase 3 was decreased by levetiracetam in SMA-iPSCs-MNs. Furthermore, levetiracetam improved impaired mitochondrial function in SMA-iPSCs-MNs. On the other hand, levetiracetam did not affect the expression level of SMN protein in SMA-iPSCs-MNs. These findings indicate that levetiracetam has a neuroprotective effect for SMA.
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http://dx.doi.org/10.1007/s11064-019-02814-4DOI Listing
July 2019

Notch Signaling Mediates Astrocyte Abnormality in Spinal Muscular Atrophy Model Systems.

Sci Rep 2019 03 6;9(1):3701. Epub 2019 Mar 6.

Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of spinal motor neurons and muscle atrophy. The disease is mainly caused by low level of the survival motor neuron (SMN) protein, which is coded by two genes, namely SMN1 and SMN2, but leads to selective spinal motor neuron degeneration when SMN1 gene is deleted or mutated. Previous reports have shown that SMN-protein-deficient astrocytes are abnormally abundant in the spinal cords of SMA model mice. However, the mechanism of the SMN- deficient astrocyte abnormality remains unclear. The purpose of this study is to identify the cellular signaling pathways associated with the SMN-deficient astrocyte abnormality and propose a candidate therapy tool that modulates signaling. In the present study, we found that the astrocyte density was increased around the central canal of the spinal cord in a mouse SMA model and we identified the dysregulation of Notch signaling which is a known mechanism that regulates astrocyte differentiation and proliferation, in the spinal cord in both early and late stages of SMA pathogenesis. Moreover, pharmacological inhibition of Notch signaling improved the motor functional deficits in SMA model mice. These findings indicate that dysregulated Notch signaling may be an underlying cause of SMA pathology.
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http://dx.doi.org/10.1038/s41598-019-39788-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403369PMC
March 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

In silico and in vitro analyses of the pathological relevance of the R258H mutation of hepatocyte nuclear factor 4α identified in maturity-onset diabetes of the young type 1.

J Diabetes Investig 2019 May 10;10(3):680-684. Epub 2018 Dec 10.

Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.

Mutations of the hepatocyte nuclear factor 4α (HNF4α) gene give rise to maturity-onset diabetes of the young type 1. Although many such mutations have been identified in affected individuals, part of these mutations has been characterized with regard to their pathological relevance. We here identified a missense mutation (c.773G>A, p.R258H) of HNF4A in a mother and daughter with early-onset diabetes and impaired insulin secretion. In silico simulation and in vitro luciferase reporter analyses showed that the mutation impairs the stability of self-dimerization and the transactivation activity of HNF4α. Although arginine-258 does not appear to participate directly in dimerization, its mutation alters the electrostatic surface potential of the dimer interface. Our results thus suggest that this mutation impairs the function of HNF4α and thereby contributes to the pathogenesis of maturity-onset diabetes of the young type 1.
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http://dx.doi.org/10.1111/jdi.12960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497599PMC
May 2019

Incidence of infantile spinal muscular atrophy on Shikoku Island of Japan.

Brain Dev 2019 Jan 6;41(1):36-42. Epub 2018 Aug 6.

Department of Occupational Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Hyogo, Japan; Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Hyogo, Japan.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by homozygous mutations in the SMN1 gene. SMA has long been known to be the most common genetic cause of infant mortality. However, there have been no reports on the epidemiology of infantile SMA (types 1 and 2) based on genetic testing in Japan. In this study, we estimated the incidence of infantile SMA on Shikoku Island, which is a main island of Japan and consists of four prefectures: Ehime, Kagawa, Tokushima and Kochi.

Methods: A questionnaire was sent to 91 hospitals on Shikoku Island to investigate the number of SMA infants born from 2011 to 2015. A second questionnaire was then sent to confirm the diagnoses of SMA based on clinical and genetic features.

Results: Responses were received from all of the hospitals, and four patients were diagnosed with infantile SMA among 147,950 live births. We estimated the incidence of infantile SMA patients as 2.7 per 100,000 live births (95% confidence interval, 0.1-5.4). A comparison of the four prefectures indicated that the incidence of infantile SMA was significantly higher in Ehime Prefecture than in the other three prefectures; 5.6 per 100,000 live births (95% confidence interval, -0.7 to 11.9) in Ehime Prefecture and 1.1 per 100,000 live births (95% confidence interval, -1.0 to 3.1) in the other prefectures.

Conclusion: We estimated the incidence of infantile SMA in an isolated area of Japan. For more precise determination of the incidence of infantile SMA, further studies that include neonatal screening will be needed.
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http://dx.doi.org/10.1016/j.braindev.2018.07.016DOI Listing
January 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 Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System.

Int J Mol Sci 2018 May 23;19(6). Epub 2018 May 23.

Department of Physical Therapy, Faculty of Rehabilitation, Kobe Gakuin University, Nishi, Kobe 651-2180, Japan.

Background: 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.

Methods: 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.

Results: 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. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression.

Conclusions: Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.
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http://dx.doi.org/10.3390/ijms19061546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6032138PMC
May 2018