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For a closed, connected, and oriented 3-manifold with a non-singular flow, we construct its virtual knot diagram via a “flow-spine” of the manifold. Then, we introduce a coloring invariant of 3-manifolds through their virtual knot diagrams, and classify some 3-manifolds by using the invariant.

Introduction/Aims Due to muscular weakness and cardiopulmonary dysfunction, patients with muscular dystrophy (MD) have an increased risk of serious complications from coronavirus disease 2019 (COVID-19). Although vaccination is recommended, COVID-19 vaccination safety and immunogenicity in these patients are unknown. We investigated reaction frequency, post-vaccine antibody titers after two mRNA COVID-19 vaccine doses, and clinical predictors of antibody response among patients with MD.We recruited 171 inpatients with MD receiving two BNT162b2 mRNA COVID-19 vaccine doses from seven hospitals. Blood samples were obtained from 53 inpatients before the first dose and 28-30 days after the second dose, and antibody titers were measured.Overall, 104 (60.8%) and 115 (67.6%) patients experienced side effects after the first and second doses, respectively. These were generally mild and self-limited. Multiple logistic regression analysis showed that a bedridden state was associated with reduced side effects (OR: 0.29, 95% CI: 0.12-0.71). The antibody titers of all participants changed from negative to positive after two vaccine doses. The geometric mean titer (GMT) of the inpatients was 239 (95% CI: 159.3-358.7). Older age (RR=0.97, 95% CI: 0.95-0.99) and bedridden state (RR=0.27, 95% CI: 0.14-0.51) were associated with a lower antibody titer. Patients with myotonic dystrophy type 1 (DM1) had a lower GMT than patients with other MDs (RR=0.42, 95% CI: 0.21-0.85).COVID-19 vaccination is safe and immunogenic in inpatients with MD. Patients with DM1 appear to have a poorer COVID-19 antibody response than those with other MDs. This article is protected by copyright. All rights reserved.

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.

Background Rare neuromuscular diseases such as spinal muscular atrophy, spinal bulbar muscular atrophy, muscular dystrophy, Charcot-Marie-Tooth disease, distal myopathy, sporadic inclusion body myositis, congenital myopathy, and amyotrophic lateral sclerosis lead to incurable amyotrophy and consequent loss of ambulation. Thus far, no therapeutic approaches have been successful in recovering the ambulatory ability. Thus, the aim of this trial was to evaluate the efficacy and safety of cybernic treatment with a wearable cyborg Hybrid Assistive Limb (HAL, Lower Limb Type) in improving the ambulatory function in those patients. Results We conducted an open-label, randomised, controlled crossover trial to test HAL at nine hospitals between March 6, 2013 and August 8, 2014. Eligible patients were older than 18 years and had a diagnosis of neuromuscular disease as specified above. They were unable to walk for 10 m independently and had neither respiratory failure nor rapid deterioration in gait. The primary endpoint was the distance passed during a two-minute walk test (2MWT). The secondary endpoints were walking speed, cadence, and step length during the 10-m walk test (10MWT), muscle strength by manual muscle testing (MMT), and a series of functional measures. Adverse events and failures/problems/errors with HAL were also evaluated. Thirty patients were randomly assigned to groups A or B, with each group of 15 receiving both treatments in a crossover design. The efficacy of a 40-min walking program performed nine times was compared between HAL plus a hoist and a hoist only. The final analysis included 13 and 11 patients in groups A and B, respectively. Cybernic treatment with HAL resulted in a 10.066% significantly improved distance in 2MWT (95% confidence interval, 0.667–19.464; p = 0.0369) compared with the hoist only treatment. Among the secondary endpoints, the total scores of MMT and cadence at 10MWT were the only ones that showed significant improvement. The only adverse effects were slight to mild myalgia, back pain, and contact skin troubles, which were easily remedied. Conclusions HAL is a new treatment device for walking exercise, proven to be more effective than the conventional method in patients with incurable neuromuscular diseases. Trial registration: JMACTR, JMA-IIA00156

In Japan, medical care for patients with muscular dystrophy has improved through multidisciplinary care provided by specialized institutions, resulting in a marked increase in life expectancy. Today, most patients with muscular dystrophy live in their own homes and receive medical care in various non-specialized institutions. Some muscular dystrophy patients have associated central nervous system disorders, which include neurodevelopmental syndromes. In addition, many patients are physically and mentally unstable during adolescence, when the transition from pediatric neurology to adult neurology occurs. Early opportunities to consult specialized institutions for rehabilitation or specific periods when pediatric and adult neurologists take care of patients together should be considered to facilitate this transition more easily.

The authors wish to make the following correction to this paper [...]

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.

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.

An improvement in efficacy treatment and development of the social support system has led to many patients with neurological disease being able to reach adulthood. Therefore health care for life from pediatrics to adulthood has become necessary. The Special Committee for Measures Against Transition from Pediatric to Adult Health Care of the Japanese Society of Neurology officially started to examine the current situation and issues of transition from pediatric to adult health care in July 2020. Pediatric neurologists and adult neurologists have an awareness of this issue of constructing a better transition from pediatric to adult health care. However, there are some tasks that need to be resolved in the medical system. We intend to improve the understanding of transition and assessment of medical service fees for transition in cooperation with the Japanese Society of Neurology and the Japanese Society of Child Neurology.

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Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disease characterized by defects of lower motor neurons. Approximately 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) gene deletion, while ~5% carry an intragenic SMN1 mutation. Here, we investigated the stability and oligomerization ability of mutated SMN1 proteins. Plasmids containing wild- and mutant-type SMN1 cDNA were constructed and transfected into HeLa cells. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar abundances of transcripts from the plasmids containing SMN cDNA, but Western blotting showed different expression levels of mutated SMN1 proteins, reflecting the degree of their instability. A mutated SMN1 protein with T274YfsX32 exhibited a much lower expression level than other mutated SMN1 proteins with E134K, Y276H, or Y277C. In immunoprecipitation analysis, the mutated SMN1 protein with T274YfsX32 did not bind to endogenous SMN1 protein in HeLa cells, suggesting that this mutation completely blocks the oligomerization with full-length SMN2 protein in the patient. The patient with T274YfsX32 showed a much more severe phenotype than the other patients with different mutations. In conclusion, the stability and oligomerization ability of mutated SMN1 protein may determine the protein stability and may be associated with the clinical severity of SMA caused by intragenic SMN1 mutation.

Spinal muscular atrophy (SMA) is a lower motor neuron disease, once considered incurable. The main symptoms are muscle weakness and muscular atrophy. More than 90% of cases of SMA are caused by homozygous deletion of survival motor neuron 1 (SMN1). Emerging treatments, such as splicing modulation of SMN2 and SMN gene replacement therapy, have improved the prognoses and motor functions of patients. However, confirmed diagnosis by SMN1 testing is often delayed, suggesting the presence of diagnosis-delayed or undiagnosed cases. To enable patients to access the right treatments, a screening system for SMA is essential. Even so, the current newborn screening system using dried blood spots is still invasive and cumbersome. Here, we developed a completely non-invasive screening system using dried saliva spots (DSS) as an alternative DNA source to detect SMN1 deletion. In this study, 60 DSS (40 SMA patients and 20 controls) were tested. The combination of modified competitive oligonucleotide priming-polymerase chain reaction and melting peak analysis clearly distinguished DSS samples with and without SMN1. In conclusion, these results suggest that our system with DSS is applicable to SMA patient detection in the real world.

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.

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.

Background and Aim: Spinal muscular atrophy (SMA) is a lower motor neuron disease with autosomal recessive inheritance caused by homozygous SMN1 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. Materials and Methods: A total of 124 dried blood spot (DBS) on FTA® ELUTE cards (51 SMN1-deleted patients with SMA, 20 carriers, and 53 controls) were punched and subjected to direct amplification of SMN1 and CFTR (reference gene). Melting peak analyses were performed to detect SMN1 deletions from DBS samples. Results: A combination of allele-specific polymerase chain reaction (PCR) and melting peak analyses clearly distinguished the DBS samples with and without SMN1. 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 SMN1 deletions. Conclusion: 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.

A Japanese woman first noticed dysarthria at the age of 23. She visited a hospital at the age of 32 and was diagnosed as having myotonic dystrophy clinically. She was diagnosed genetically as having myotonic dystrophy type 1 at 47 years old with 160-270 CTG repeats on the DMPK gene. At the age of 48, she needed non-invasive positive pressure ventilation because of hypoxia at night. Her gait function also deteriorated. She could not stand up from the supine position by herself. However, when she stood, she could walk without a cane for a short distance. She was admitted to our hospital to receive rehabilitation against progressive gait disturbance at the age of 53. She received gait training with hybrid assistive limb® (HAL®). We evaluated some parameters such as walking distance of 2-minute walk test (2MWT), gait speed /cadence/stride length of 10-meter walk test (10MWT), before and just after the course. The first course was performed in September 2017 and the second was done in May 2018 so the interval was about six months. After two courses of HAL® gait training, the distance on the 2-minute walk test increased from 111 m to 154 m, the average speed and the cadence of 10MWT improved from 2.01 m/s to 2.78 m/s and from 2.21 steps/s to 3.05 steps/s respectively. The score of the muscular disability quality of life (QOL) rating scale was also improved. The factors including "defecation," "breathing," and "ADL" suggest that the patient's physical abilities improved and she could move easily. Other factors such as "hope", "activity" and "human relationship" suggest that patient's mood improved after the HAL® training.It was suggested that HAL® gait training could improve QOL as well as gait function in patients with progressive neuromuscular disorder.

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.

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.

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.

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 Neo(™) 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.

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.

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.

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.

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.

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.

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.

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.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder that causes degeneration of anterior horn cells in the human spinal cord and subsequent loss of motor neurons. The severe form of SMA is among the genetic diseases with the highest infant mortality. Although SMA has been considered incurable, newly developed drugs—nusinersen and onasemnogene abeparvovec—improve the life prognoses and motor functions of affected infants. To maximize the efficacy of these drugs, treatments should be started at the pre-symptomatic stage of SMA. Thus, newborn screening for SMA is now strongly recommended. Herein, we provide some data based on our experience of SMA diagnosis by genetic testing in Japan. A total of 515 patients suspected of having SMA or another lower motor neuron disease were tested. Among these patients, 228 were diagnosed as having SMA with survival motor neuron 1 (SMN1) deletion. We analyzed the distribution of clinical subtypes and ages at genetic testing in the SMN1-deleted patients, and estimated the SMA incidence based on data from Osaka and Hyogo prefectures, Japan. Our data showed that confirmed diagnosis by genetic testing was notably delayed, and the estimated incidence was 1 in 30,000–40,000 live births, which seemed notably lower than in other countries. These findings suggest that many diagnosis-delayed or undiagnosed cases may be present in Japan. To prevent this, newborn screening programs for SMA (SMA-NBS) need to be implemented in all Japanese prefectures. In this article, we also introduce our pilot study for SMA-NBS in Osaka Prefecture.

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.

We show that two-bridge knots and alternating fibered knots admit no purely cosmetic surgeries, i.e., no pair of distinct Dehn surgeries on such a knot produce 3-manifolds that are homeomorphic as oriented manifolds. Our argument, based on a recent result by Hanselman, uses several invariants of knots or 3-manifolds; for knots, we study the signature and some finite type invariants, and for 3-manifolds, we deploy the $SL(2,\mathbb{C})$ Casson invariant., 13 pages, 3 figures; minor errors in Figures 2, 3 are corrected in V2