Publications by authors named "Toshio Saito"

71 Publications

Infection control in the respiratory care of coronavirus disease-19 patients with neuromuscular diseases.

Neurol Clin Neurosci 2021 Mar 1;9(2):159-165. Epub 2021 Feb 1.

Department of Neurology National Hospital Organization Hakone Hospital Odawara Japan.

Close contact is unavoidable in the care of patients with neuromuscular diseases (NMD). In addition, respiratory physiotherapy and noninvasive ventilation generate massive amounts of aerosols. Caring for a patient suffering from coronavirus disease-19 raises concerns about the risk of infection not only to the caregiver and/or medical staff but also to other individuals in contact with these personnel. We reviewed the points to be noted in infection control when a patient with neuromuscular diseases receiving respiratory care is infected with COVID-19 and summarizes the recommendation. Infected patients must be isolated in a negative-pressure or actively ventilated room. Clear zoning separating clean and infected areas should be performed for pathogen containment. Caregivers should wear appropriate personal protective equipment and thoroughly clean their hands. Leak-prevention measures and the use of proper respiratory circuits and filters with virus-removal performance are crucial to reducing aerosols in noninvasive ventilation. Although respiratory physiotherapy is essential, treatment should be minimized in consideration of the infection state and sputum status, and alternative therapies such as postural drainage should be carefully considered. Infection control is distinctly obligate; however, it impairs the quality of life and activity of daily living significantly. We should implement it with enough ethical consideration, adequate explanation, and patient consent. We hope that this paper will contribute to appropriate COVID-19 infection control in patients with neuromuscular diseases requiring respiratory care.
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http://dx.doi.org/10.1111/ncn3.12482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013339PMC
March 2021

[Changing medical care for amyotrophic lateral sclerosis patients and cause of death - review of muscular dystrophy wards (1999-2013)].

Rinsho Shinkeigaku 2021 Mar 23;61(3):161-165. Epub 2021 Feb 23.

Department of Neurology, National Hospital Organization Higashisaitama National Hospital.

We analyzed the records of inpatients with amyotrophic lateral sclerosis (ALS) treated at 27 specialized institutions for muscular dystrophy in Japan from 1999 to 2013 registered in a database on October 1 of each year. The total number of ALS inpatients in 1999 was 29, then that showed rapid increases in 2006 and 2007, and reached 164 in 2013. Age regardless of year was predominantly greater than 50 years. In 1999, the respirator dependent rate was 68.9% and then increased to 92.7% in 2013, while the oral nutritional supply rate was 41.4% in 1999 and decreased to 10.4% in 2013. The number of deaths from 2000 to 2013 was 118. Cause of death was respiratory failure in 26 of 30 patients who maintained voluntary respiration at the time of death and in 5 of 6 with non-invasive ventilation. On the other hand, the main cause of death in patients with tracheostomy invasive ventilation was respiratory infection, which was noted in 26 of 82, while other causes varied. It is expected that the number of ALS patients admitted to specialized institutions with muscular dystrophy wards will continue to increase.
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http://dx.doi.org/10.5692/clinicalneurol.cn-001536DOI Listing
March 2021

Psychological Case Conference Following the Death of a Patient With Neuromuscular Disease: A Source of Emotional Support for Participating Medical Staff.

J Patient Exp 2020 Oct 4;7(5):713-716. Epub 2019 Dec 4.

Graduate School of Human Sciences, Osaka University, Osaka, Japan.

Healthcare professionals involved in the treatment and care of patients with intractable diseases, such as muscular dystrophy, increasingly encounter situations that can elicit emotional distress for them as well as the patients. Therefore, medical professionals also need support. This article describes a psychological case conference of multidisciplinary professionals involved in the treatment of a deceased patient with Duchenne muscular dystrophy. The conference aimed to support medical professionals in reflecting on and sharing their thoughts, feelings, and conflicts. Such a practice could support medical professionals in reflecting patients' thoughts and sharing their personal experiences with other staff members, which may alleviate emotional and personal conflicts. Reflecting on their interactions and dealings with patients serves this supportive function. Psychological case conferences for medical staff may serve as an opportunity for participants to feel emotionally supported and may perhaps help prevent burnout.
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http://dx.doi.org/10.1177/2374373519892413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705834PMC
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

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

[Inpatients with facioscapulohumeral muscular dystrophy in specialized institutions in Japan from 1999 to 2013-Clinical condition changes and causes of death].

Rinsho Shinkeigaku 2019 Nov 26;59(11):716-722. Epub 2019 Oct 26.

Department of Neurology, National Hospital Organization Higashisaitama Hospital.

We analyzed the registration data of inpatients with facioscapulohumeral muscular dystrophy (FSHD) receiving care at 27 specialized institutions for muscular dystrophy in Japan from 1999 to 2013 using data from October 1 of each year. The number of inpatients of each year ranged from 63 to 72 (67.1 ± 3.3) throughout the study period. Those aged over 50 years gradually increased during the study period, while the oldest inpatient was 82.8 years old. Most could not walk. The rate of respirator dependency increased from 21.0% in 1999 to 71.0% in 2013, while the rate of patients receiving oral nutrition was 98.4% in 1999 and then reduced to 75.4% in 2013. There were 36 death cases reported in the database, including 15 patients with respiratory failure and 4 with heart failure. Our findings indicate that FSHD patients in a severe condition are impacted by respiratory and nutritional problems and their prognosis for survival is related to respiratory failure.
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http://dx.doi.org/10.5692/clinicalneurol.cn-001229DOI Listing
November 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

[Cerebral embolism in Duchenne muscular dystrophy after respiratory tract infection - Report of two cases].

Rinsho Shinkeigaku 2018 Oct 29;58(10):642-645. Epub 2018 Sep 29.

Department of Neurology, NHO Toneyama National Hospital.

We report cerebral embolism in 2 patients with Duchenne muscular dystrophy (DMD) after respiratory tract infection. A 31-year-old man (Case 1) was admitted to the hospital because of an upper respiratory tract infection, then suddenly developed left-sided hemiparesis. Transthoracic echocardiography revealed an intracardiac thrombus in the left ventricle, and, under assumption of cardioembolic stroke, oral anticoagulation was initiated. Case 2 was a 36-year-old man who developed dysphasia after increasing sputum. Based on brain CT scan findings, we confirmed a diagnosis of cerebral infarction. There was no recurrence in either case. Both cases developed cerebral infarction due to embolism after mild upper respiratory tract infections. DMD patients have various risk factors for thrombus and embolus, while physicians should also be aware of possible cerebral infarction and other coagulation disorders irrespective of respiratory and cardiac therapy.
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http://dx.doi.org/10.5692/clinicalneurol.cn-001135DOI Listing
October 2018

Autism spectrum disorders are prevalent among patients with dystrophinopathies.

Neurol Sci 2018 Jul 28;39(7):1279-1282. Epub 2018 Mar 28.

Graduate School of Human Sciences, Osaka University, Suita, Osaka, Japan.

Recent studies have reported a higher prevalence of autism spectrum disorders among patients with dystrophinopathies. The aim of this study was to investigate the prevalence of autism spectrum disorder (ASD) among those with dystrophinopathies. The possible role of dystrophin isoforms in patients was also explored. Fifty-six patients recruited from Toneyama National Hospital were included in this study (mean age = 12.9 years, SD = 5.2 years). Autistic symptoms were evaluated using the Pervasive Developmental Disorders/Autism Spectrum Disorders Rating Scale (PARS), a clinician rating scale. Eleven patients (19.6%; 95% confidence interval 10.2-32.4) met the criteria for ASD based on their PARS scores. Patients were separated into two groups based on the cumulative loss of dystrophin isoforms predicted from the mutation location. The prevalence of ASD was examined between these groups. Infantile and current autistic symptoms did not differ between the groups, except on one subscale of the PARS. This study revealed that there was a high prevalence of ASD in patients with dystrophinopathies.
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http://dx.doi.org/10.1007/s10072-018-3341-2DOI Listing
July 2018

Intron-retained transcripts of the spinal muscular atrophy genes, SMN1 and SMN2.

Brain Dev 2018 Sep 23;40(8):670-677. Epub 2018 Mar 23.

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

Background: The SMN genes, SMN1 and SMN2, are highly homologous genes which are related to the development or clinical severity of spinal muscular atrophy. Some alternative splicing patterns of the SMN genes have been well documented. In 2007, an SMN1 transcript with a full sequence of intron 3 was reported as the first intron-retained SMN transcript.

Methods: Intron-retained SMN transcripts in various cells and tissues were studied using reverse transcription (RT)-PCR. HeLa cells were used for subcellular localization of the transcripts and protein expression analysis with Western blotting.

Results: Two intron-retained SMN transcripts were detected, which contain full sequences of intron 2b or intron 3. These transcripts were produced from SMN1 and SMN2, and ubiquitously expressed in human cells and tissues. Western blotting analysis showed no proteins derived from the intron-retained transcripts. Fractionation analysis showed that these intron-retained transcripts were localized mainly in the nucleus. Contrary to our expectation, the intron-retained transcript levels decreased during the treatment of cycloheximide, an inhibitor of nonsense-mediated decay (NMD), suggesting that they were not targets of NMD.

Conclusion: Intron 2b-retained SMN transcript and intron3-retained SMN transcript were ubiquitously expressed in human cells and tissues. The intron-retained transcripts were mainly localized in the nucleus and decreased through non-NMD pathway.
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http://dx.doi.org/10.1016/j.braindev.2018.03.001DOI Listing
September 2018

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

Kobe J Med Sci 2017 Dec 18;63(3):E80-E83. Epub 2017 Dec 18.

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

Background: Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA.

Aim: To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA.

Methods: DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus.

Results: The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP.

Conclusion: Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first-tier diagnostic method of SMA.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826024PMC
December 2017

Gender Effects on the Clinical Phenotype in Japanese Patients with Spinal Muscular Atrophy.

Kobe J Med Sci 2017 Oct 16;63(2):E41-E44. Epub 2017 Oct 16.

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

Background: Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a mutation in SMN1. SMA is classified into three subtypes (types 1, 2, 3) based on achieved motor milestones. Although NAIP and SMN2 are widely accepted as SMA-modifying factors, gender-related modifying factors or gender effects on the clinical phenotype are still controversial.

Methods: A total of 122 Japanese patients with SMA, of which SMN1 was homozygously deleted, were analyzed from the perspective of the achieved motor milestone, NAIP status and SMN2 copy number.

Results: A predominance of male patients was observed in SMA type 3 (the walker group) without NAIP-deletion or with high SMN2 copy number (3 or 4 copies).

Conclusion: We suggest the presence of gender-related modifiers on disease severity in SMA patients. The modifiers may contribute only in the presence of NAIP and a high copy number of SMN2.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826018PMC
October 2017

New, Improved Version of the mCOP-PCR Screening System for Detection of Spinal Muscular Atrophy Gene (SMN1) Deletion.

Kobe J Med Sci 2017 Sep 7;63(2):E37-E40. Epub 2017 Sep 7.

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

Background: Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results.

Aim: To establish an improved version of the mCOP-PCR screening system without non-specific amplification.

Methods: DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels.

Results: No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system.

Conclusion: An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826017PMC
September 2017

Validation of The Individualized Neuromuscular Quality of Life in Japanese patients with myotonic dystrophy.

Muscle Nerve 2018 Jan 17. Epub 2018 Jan 17.

Department of Neurology, National Hospital Organization Toneyama National Hospital, Toyonaka, Japan.

Introduction: The Individualized Neuromuscular Quality of Life (INQoL) is used to measure the quality of life (QoL) of patients with neuromuscular disease. We conducted this study to translate and validate the Japanese version of the INQoL in patients with myotonic dystrophy.

Methods: Forward and backward translation, patient testing, and psychometric validation were performed. We used the 36-Item Short Form Health Survey (SF-36) and the modified Rankin scale for concurrent validation.

Results: The Japanese INQoL was administered to 90 adult patients. The coefficients for internal consistency and test-retest reliability were adequately high in most domains (Cronbach α 0.88-0.96 and intraclass coefficient 0.64-0.99). INQoL domains were moderately to strongly associated with relevant SF-36 subscales (Spearman's ρ -0.23 to -0.74). Symptom severity, disease duration, employment status, and use of a ventilator influenced overall QoL.

Discussion: The INQoL is a reliable and validated measure of QoL for Japanese patients with myotonic dystrophy. Muscle Nerve, 2018.
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http://dx.doi.org/10.1002/mus.26071DOI Listing
January 2018

A Pilot Study of Tranilast for Cardiomyopathy of Muscular Dystrophy.

Intern Med 2018 Feb 1;57(3):311-318. Epub 2017 Nov 1.

Department of Neurology, National Hospital Organization Toneyama National Hospital, Japan.

Objective Heart failure is currently the most serious complication of muscular dystrophy. The transient receptor potential cation channel, subfamily V, member 2 (TRPV2) is a stretch-sensitive Ca channel. In damaged myocytes or cardiomyocytes, TRPV2 translocates to the cytoplasmic membrane and enhances Ca influx, triggering cell damage. Evidence suggests that the inhibition of TRPV2 may be a new therapeutic target in heart failure. We found that tranilast, which is widely used as an anti-allergic drug, inhibits TRPV2. A pilot study was conducted to assess the safety and efficacy of tranilast in muscular dystrophy patients with cardiomyopathy. Methods After obtaining informed consent, two muscular dystrophy patients with advanced heart failure took tranilast (300 mg/day) for three months. Blood tests, echocardiography, electrocardiography (ECG), Holter ECG, analyses of the TRPV2 expression in peripheral mononuclear cells, and circulating micro ribonucleic acid profiling were performed to assess the safety and efficacy of tranilast. Results The brain natriuretic peptide levels decreased after treatment. The expression of TRPV2 on the cytoplasmic membrane of peripheral mononuclear cells was enhanced before treatment and was decreased after treatment. Some heart-related micro ribonucleic acids (miR-208a-5p, miR-223-3p) were elevated and then decreased after treatment. Some adverse events, including the potentiation of warfarin, the worsening of renal dysfunction, an increased heart rate and premature ventricular contractions, were observed. Conclusion Tranilast can inhibit TRPV2 and can be effective for treating heart failure, even in patients with muscular dystrophy. Although careful attention is needed, the inhibition of TRPV2 can be a new treatment target for cardiomyopathy. A multi-center trial is planned.
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http://dx.doi.org/10.2169/internalmedicine.8651-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827307PMC
February 2018

Spinal muscular atrophy carriers with two SMN1 copies.

Brain Dev 2017 Nov 1;39(10):851-860. Epub 2017 Jul 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. Over 95% of SMA patients have homozygous deletions of the SMA-causative gene, SMN1. Thus, SMA carriers are usually diagnosed based on SMN1 copy number, with one copy indicating SMA carrier status. However, two SMN1 copies do not always exclude carrier status. In this study, we identified SMA carriers with two SMN1 copies.

Subjects And Methods: From 33 families, 65 parents of genetically confirmed SMA patients were tested to determine SMA carrier status. Molecular genetic analyses, including multiplex ligation-dependent probe amplification (MLPA) assay, were performed using blood samples from family members.

Results: Of the 65 parents, three parents from three families had two SMN1 copies. Accordingly, the frequency of carriers with two SMN1 copies was 4.6%. Two of these families were further studied. Patient 1 was homozygous for SMN1 deletion. Patient 1's mother had two SMN1 copies on one chromosome, with deletion of SMN1 on the other chromosome ([2+0] genotype). Patient 1 inherited SMN1-deleted chromosomes from both parents. Patient 2 was compound heterozygous for two SMN1 mutations: whole-gene deletion and intragenic missense mutation, c.826T>C (p.Tyr276His). Patient 2's father had two SMN1 copies with the same intragenic mutation in one copy ([1+1] genotype, intragenic mutation). Patient 2 inherited the chromosome with an SMN1 mutation from the father and SMN1-deleted chromosome from the mother.

Conclusion: SMA carriers with two SMN1 copies may be rare, but its possibility should be taken into consideration in carrier testing and counseling for SMA families or population-based carrier screening.
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http://dx.doi.org/10.1016/j.braindev.2017.06.002DOI Listing
November 2017

Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA.

Brain Dev 2017 Oct 15;39(9):774-782. Epub 2017 May 15.

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

Background: Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients.

Methods: A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes by PCR restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7.

Results: Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively.

Conclusion: We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening.
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http://dx.doi.org/10.1016/j.braindev.2017.04.015DOI Listing
October 2017

SMA mutations in SMN Tudor and C-terminal domains destabilize the protein.

Brain Dev 2017 Aug 31;39(7):606-612. Epub 2017 Mar 31.

Analytical Laboratory, Kobe Pharmaceutical University, Kobe, Japan.

Background And Purpose: Most spinal muscular atrophy (SMA) patients are homozygous for survival of motor neuron 1 gene (SMN1) deletion. However, some SMA patients carry an intragenic SMN1 mutation. Such patients provide a clue to understanding the function of the SMN protein and the role of each domain of the protein. We previously identified mutations in the Tudor domain and C-terminal region of the SMN protein in three Japanese SMA patients. To clarify the effect of these mutations on protein stability, we conducted expression assays of SMN with mutated domains.

Patients And Methods: Patients A and B carried a mutation in SMN1 exon 3, which encodes a Tudor domain, c.275G>C (p.Trp92Ser). Patient C carried a mutation in SMN1 exon 6, which encodes a YG-box; c.819_820insT (p.Thr274Tyrfs). We constructed plasmid expression vectors containing wild-type and mutant SMN1 cDNAs. After transfection of HeLa cells with the expression plasmids, RNA and protein were isolated and analyzed by reverse-transcription PCR and western blot analysis.

Results: The abundance of wild-type and mutant SMN1 transcripts in HeLa cells was almost the same. However, western blot analysis showed lower levels of mutant SMN proteins compared with wild-type SMN. In mutant SMN proteins, it is noteworthy that the level of the p.Thr274Tyrfs mutant was much reduced compared with that of the p.Trp92Ser mutant.

Conclusions: SMN mutations may affect the stability and levels of the protein.
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http://dx.doi.org/10.1016/j.braindev.2017.03.002DOI Listing
August 2017

Study of Duchenne muscular dystrophy long-term survivors aged 40 years and older living in specialized institutions in Japan.

Neuromuscul Disord 2017 Feb 25;27(2):107-114. Epub 2016 Nov 25.

Department of Neurology, National Hospital Organization Toneyama National Hospital, Toyonaka, Osaka, Japan.

The national muscular dystrophy wards database of Japan lists 118 long-term Duchenne muscular dystrophy (DMD) patients who were at least 40 years old as of October 1, 2013. To elucidate the clinical features of DMD patients aged 40 years and older, we obtained gene analysis and muscle biopsy findings, as well as medical condition information. Ninety-four of the registered patients consented to participate, of whom 55 meeting genetic or biochemical criteria confirming DMD were analyzed. The mean age at the time of the study was 43.6 ± 3.0 years, while at the time of independent ambulation loss it was 10.6 ± 1.5 years and at mechanical ventilation introduction it was 24.1 ± 5.5 years. All were receiving continuous ventilation support, 27 with non-invasive positive pressure ventilation and 28 with tracheal intermittent positive pressure ventilation. Thirty-eight were receiving β-blockers or a renin-angiotensin system inhibitor, while 9 were free from those agents. Forty had maintained oral nutrition. The 55 analyzed patients had survived into their 40s by receiving multidisciplinary intervention. Our findings emphasize the need of future studies to investigate disease modifiers and the mechanism of long-term survival. In addition, establishment of a worldwide care standard with focus on quality of life for adult males with DMD is important.
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http://dx.doi.org/10.1016/j.nmd.2016.11.012DOI Listing
February 2017

Alternative splicing of a cryptic exon embedded in intron 6 of and .

Hum Genome Var 2016 1;3:16040. Epub 2016 Dec 1.

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

Both survival of motor neuron () genes are associated with spinal muscular atrophy; mutations in cause the disease, and modulates its severity. It is established that different alternative splicing of exon 7 occurs for and , and a cryptic exon was recently found in intron 6 of both genes. Here, we characterize this cryptic exon and clarify its alternative splicing pattern in control and spinal muscular atrophy cells.
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http://dx.doi.org/10.1038/hgv.2016.40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131094PMC
December 2016

The experiences of patients with Duchenne muscular dystrophy in facing and learning about their clinical conditions.

Int J Qual Stud Health Well-being 2016 5;11:32045. Epub 2016 Oct 5.

Graduate School of Human Sciences, Osaka University, Suita, Osaka, Japan.

Patients experience extreme difficulty when facing an intractable genetic disease. Herein, we examine the experiences of patients with Duchenne muscular dystrophy in facing and learning about their disease. A total of seven patients with Duchenne muscular dystrophy (age range: 20-48) participated. We conducted in-depth interviews with them about how they learned of their disease and how their feelings regarding the disease changed over time. Transcribed data were analysed using thematic analysis. The following themes emerged from this analysis: "experiences before receiving the diagnosis," "experiences when they learned of their condition and progression of the disease," "supports," and "desired explanations." Anxiety and worry were most pronounced when they had to transition to using wheelchairs or respirators due to disease progression; indeed, such transitions affect the patients psychological adjustment. In such times, support from significant others in their lives helped patients adjust.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5054085PMC
http://dx.doi.org/10.3402/qhw.v11.32045DOI Listing
April 2017

Renal dysfunction can be a common complication in patients with myotonic dystrophy 1.

J Neurol Sci 2016 Sep 15;368:266-71. Epub 2016 Jul 15.

Department of Neurology, National Hospital Organization Toneyama National Hospital, Toneyama 5-1-1, Toyonaka, Osaka 560-8552, Japan. Electronic address:

Although renal failure can be a life-threatening complication even in neuromuscular disorders (NMDs), renal dysfunction is easily overlooked because muscle atrophy decreases the serum creatinine level. Renal function was retrospectively assessed using cystatin C (CysC) in various NMDs to clarify the differences among diseases. As is in the general population, age was correlated to CysC, and female patients showed lower CysC levels. Although elevated CysC was frequent in myotonic dystrophy 1 (DM1: MIM 160900) and motor neuron disorders, an inter-disease comparison by sex adjusted for age showed that only DM1 had a higher CysC compared to other diseases. Multivariate linear regression with the stepwise method also suggested that the number of CTG repeats had an impact on CysC levels. In two autopsy DM1 cases, nephrosclerotic changes were observed even though they were in their forties. These facts suggested a disease-specific pathomechanism for renal dysfunction in DM1. Although further study is required, renal function should be carefully monitored in patients with DM1.
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http://dx.doi.org/10.1016/j.jns.2016.07.036DOI Listing
September 2016

Telomeric Region of the Spinal Muscular Atrophy Locus Is Susceptible to Structural Variations.

Pediatr Neurol 2016 05 30;58:83-9. Epub 2016 Jan 30.

Department of Community Medicine and Social Health Care, Kobe University Graduate School of Medicine, Kobe, Japan; Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe, Japan. Electronic address:

Background: Most patients with spinal muscular atrophy lack the survival motor neuron 1 gene (SMN1) in the telomeric region of the spinal muscular atrophy locus on chromosome 5q13. On the other hand, the copy number of SMN2, a centromeric homolog of SMN1, is increased in many of these patients. This study aimed to clarify the mechanism underlying these structural variations.

Methods: We determined the copy numbers of telomeric and centromeric genes in the spinal muscular atrophy locus of 86 patients and 22 control subjects using multiplex ligation-dependent probe amplification analysis. Then, we chose 74 patients lacking SMN1 exons 7 and 8, and compared their dataset with that of 22 control subjects retaining SMN1 exons 7 and 8.

Results: The SMN2 copy number was shown to vary widely and to correlate with the disease severity of the patients. Interestingly, telomeric NAIP and telomeric GTF2H2 showed similar tendencies. We also noted positive correlations among the copy number of SMN2 and the telomeric genes of the spinal muscular atrophy locus. However, the copy numbers of centromeric NAIP and centromeric GTF2H2 were stable among the patients, with both approximating a value of two.

Conclusion: Our findings suggested that the telomeric region of the spinal muscular atrophy locus appears to be susceptible to structural variation, whereas the centromeric region is stable. Moreover, according to our results, new SMN2 copies may be generated in the telomeric region of the spinal muscular atrophy locus, supporting the SMN1-to-SMN2 gene conversion theory.
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http://dx.doi.org/10.1016/j.pediatrneurol.2016.01.019DOI Listing
May 2016

A Rapid, Accurate and Simple Screening Method for Spinal Muscular Atrophy: High-Resolution Melting Analysis Using Dried Blood Spots on Filter Paper.

Clin Lab 2015 ;61(5-6):575-80

Background: Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutation of the survival of the motor neuron 1 (SMN1) gene. More than 95% of SMA patients carry a homozygous deletion of SMN1. SMA can be screened for by polymerase chain reaction and high-resolution melting analysis (PCR-HRMA) using DNA extracted from dried blood spots (DBSs) stored on filter paper. However, there are two major problems with this approach. One is the frequent poor quality/quantity of DNA extracted from DBSs on filter paper, and the other is the difficulty in designing primer sets or probes to separate allele-specific melting curves. In this study, we addressed these problems and established a rapid, accurate and simple screening system for SMA with PCR-HRMA using DNA extracted from DBSs on filter paper.

Methods: Seventy individuals were assayed in this study, 42 SMA patients and 28 controls, all of whom had been previously been screened for SMA by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) using DNA extracted from freshly collected blood. In this study, the DNA of each individual was extracted from dried blood that had been spotted onto cards and stored at room temperature (20 - 25 degrees C) for between 1 and 8 years. PCR amplification of 30 or 45 cycles was performed using 50 ng of DNA and was immediately followed by HRMA. SMN1 and SMN2 products were co-amplified using a previously designed primer set (R111 and 541C770) containing two single nucleotide differences.

Results: The absorbance ratio at 260/280 of DNA extracted from DBSs ranged from 1.49 to 2.1 (mean ± SD; 1.66 ± 0.12), suggesting high-purity DNA. Thirty cycles of PCR amplification were insufficient to amplify the target alleles; PCR with 45 cycles was, however, successful in 69 out of 70 samples. PCR-HRMA using the R111/541C770 primer set enabled separation of the normalized melting curves of the samples with no SMN1 from those with SMN1 and SMN2.

Conclusions: DBSs on filter paper can be a good source of DNA for the diagnosis of diseases and PCR-HRMA using DNA extracted from DBSs is an alternative method to detect the SMN1 deletion. These findings suggest that the SMA screening system using PCR-HRMA with DBSs on filter paper is practicable in a large population study over a long time period.
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http://dx.doi.org/10.7754/clin.lab.2014.141008DOI Listing
August 2015

[Two cases of hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P)].

Rinsho Shinkeigaku 2015 ;55(6):401-5

Department of Neurology, National Hospital Organization Toneyama National Hospital.

We, herein, report two independent cases with hereditary motor and sensory neuropathy with proximal dominant involvement (HMSN-P) inherited in an autosomal dominant fashion. Their common clinical features are slowly progressive proximal dominant muscular atrophy, fasciculations and mild to moderate distal sensory disturbance with areflexia. Nerve conduction study revealed an absence of sensory nerve action potentials, in contrast to almost normal compound muscle action potentials. Gene analysis in both patients elucidated heterozygous mutation (c.854C>T, p.Pro285Leu) in the TFG, which is an identical mutation, already described by Ishiura et al. Okinawa and Shiga are two foci of HMSN-P in Japan. Eventually, one patient is from Okinawa and the other is from a mountain village in Shiga prefecture. When we see a patient who has symptoms suggestive of motor neuron disease with sensory neuropathy, HMSN-P should be considered as a differential diagnosis despite the patient's actual resident place.
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http://dx.doi.org/10.5692/clinicalneurol.cn-000650DOI Listing
March 2016