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Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive disorders caused by mutations in the DMD gene. Emerging therapies targeting patients with specific mutations are now becoming a reality for many of these patients. Precise molecular diagnosis is essential to facilitate the identification of possible new treatments for patients in the local context. In this study, we screened 145 dystrophinopathic patients in Singapore and assessed their molecular status for eligibility to current emerging genetic therapies. Overall, 140 (96.5%) of all patients harbored pathogenic DMD mutations comprising 95 exonic deletions (65.5%), 14 exonic duplications (9.7%), and 31 pathogenic small mutations (21.4%). Nonsense and frameshift mutations constitute 83.9% of all the small mutations. We found 71% (103/145) of all Singaporean dystrophinopathy patients to be theoretically amenable for exon skipping, either through skipping of single (53.1%) or multiple exons (17.9%). This approach is applicable to 81.1% (77/95) of patients carrying deletions and 83.9% (26/31) of those with small mutations. Eteplirsen induced skipping of exon 51 is applicable to 12.4% of local patients. Nonsense read-through therapy was found to be applicable in another 12.4% of all patients. Mutation screening is crucial for providing insights into the underlying genetic signature of the disease in the local population and contributes toward existing information on DMD mutations in Asia and globally. This will guide future targeted drug development and clinical trial planning for this disease., (© 2019 Wiley Periodicals, Inc.)

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., (Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or intragenic mutation of SMN1. SMA is classified into several subtypes based on clinical severity. It has been reported that the copy number of SMN2, a highly homologous gene to SMN1, is associated with clinical severity among SMA patients with homozygous deletion of SMN1. The purpose of this study was to clarify the genotype-phenotype relationship among the patients without homozygous deletion of SMN1., Methods: We performed molecular genetic analyses of SMN1 and SMN2 in 112 Japanese patients diagnosed as having SMA based on the clinical findings. For the patients retaining SMN1, the PCR or RT-PCR products of SMN1 were sequenced to identify the mutation., Results: Out of the 112 patients, 106 patients were homozygous for deletion of SMN1, and six patients were compound heterozygous for deletion of one SMN1 allele and intragenic mutation in the retained SMN1 allele. Four intragenic mutations were identified in the six patients: p.Ala2Val, p.Trp92Ser, p.Thr274TyrfsX32 and p.Tyr277Cys. To the best of our knowledge, all mutations except p.Trp92Ser were novel mutations which had never been previously reported. According to our observation, clinical severity of the six patients was determined by the type and location of the mutation rather than SMN2 copy number., Conclusion: SMN2 copy number is not always associated with clinical severity of SMA patients, especially SMA patients retaining one SMN1 allele., (Copyright © 2013 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Nanoparticles are increasingly being used for applications in clinical diagnostics due to their unique physical and chemical properties. Gold nanoparticles, in particular, have unique optical properties allowing simplicity of detection methods. In this study, an assay based on dimeric assembly of gold nanoparticles was developed for discriminating single nucleotide mismatches. Only gel electrophoresis is needed for assay readout. No other sophisticated or expensive equipment is required. In addition, no false-positive was observed in the readout. We used this assay for genotyping mutations in the Duchenne muscular dystrophy (DMD) gene, the largest known in the human genome. Our results show that conjugating the gold nanoparticles with short DNA probes of 18 bases and 70 bases complimentary to target sequences allows specific discrimination between wild-type and mutant sequences for c.4150G > T (NM.004006.1) mutation in exon 30 of the DMD gene using a simple colorimetric detection. This method allows identification of both the patients as well as the carriers of the mutation who are at risk of transmitting the disease., (2010 Elsevier B.V. All rights reserved.)

The use of lipid-lowering statins has been associated with raised serum muscle enzymes and, occasionally, with rhabdomyolysis, especially in patients with pre-existing metabolic myopathies. The A3243G mutation is one of the most common mutations associated with mitochondrial disorders. A teenager harboring the A3243G mutation had the unusual association of hereditary glomerulopathy and recurrent episodes of raised creatine kinase levels with the use of lipid-lowering agents. Muscle biopsy showed both normal respiratory chain enzyme activities and normal coenzyme Q(10) levels, although decreased muscle coenzyme Q(10) concentration had been postulated to have a pathogenic role in statin-related myopathies. The close temporal relationship of statin administration and raised creatine kinase levels in this patient suggests caution in the use of statins in children and teenagers with mitochondrial myopathies.

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes. [ABSTRACT FROM AUTHOR]