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Abstract: We describe a gold nanoparticle-based technique for the detection of single-base mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene, a condition that can lead to neonatal jaundice and hemolytic anemia. The aim of this technique is to clearly distinguish different mutations frequently described within the Asian population from their wild-type counterparts and across different mutant variants. Gold nanoparticles of different sizes were synthesized, and each was conjugated with a single-strand DNA (ssDNA) sequence specific for a particular mutation in the G6PD gene. It was found that only mutant targets presented a characteristic band on the agarose gel, indicating the successful formation of dimeric nanostructures. No such dimer bands were observed for the wild-type targets. The difference in the relative dimer band levels allowed different mutant variants to be distinguished from one another. The technique was further validated using G6PD-deficient patient samples. This simple mutation detection method with direct result readout is amenable for rapid and mass screening of samples. [Copyright &y& Elsevier]

Mutations in the dysferlin ( DYSF) gene are associated with limb girdle muscular dystrophy type 2B and Miyoshi myopathy. In this study, we report the identification and characterization of a novel dysferlin transcript that we named DYSF_v1 (GenBank accession: DQ267935). This transcript differs from the currently known dysferlin transcript (GenBank accession: AF075575) in the sequence of the entire first exon which spans 232 bases. This unique first exon is derived from intron 1 of DYSF, and has an immediate upstream 5′ untranslated region containing CpG islands and sequences consistent with transcription factor binding sites. Exon 1 of DYSF_v1 shares 85% sequence homology and has similar genomic organization with the first exon of mouse dysferlin. Northern blot analysis showed that the DYSF_v1 transcript spans 7.5 kb and is expressed in human skeletal muscle, heart, placenta, brain, spleen, kidney, intestine, and lung tissues. DYSF_v1 retains phylogenic conservancy and shows similar expression pattern as the currently known human dysferlin. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Desmin myopathy was identified in a Chinese man with complete heart block and mild proximal and distal limb weakness. A novel heterozygous missense S13F mutation of the desmin gene was found to be associated with the myopathy. Family members carrying the mutation showed a similar or milder phenotype. The mutation is located at a protein kinase-C phosphorylation site within a highly conserved nonapeptide sequence in the head domain of the desmin protein. Expression of the mutant desmin cDNA in cell lines induced large desmin accumulations associated with preservation of a filamentous network. [Copyright &y& Elsevier]

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 Q10 levels, although decreased muscle coenzyme Q10 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. [Copyright &y& Elsevier]