Journal of medical genetics · 2016
Targeted massively parallel sequencing and histological assessment of skeletal muscles for the molecular diagnosis of inherited muscle disorders.
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Nishikawa Atsuko, Mitsuhashi Satomi, Miyata Naomasa, Nishino Ichizo
Аннотация
BACKGROUND: Inherited skeletal muscle diseases are genetically heterogeneous diseases caused by mutations in more than 150 genes. This has made it challenging to establish a high-throughput screening method for identifying causative gene mutations in clinical practice.
AIM: In the present study, we developed a useful method for screening gene mutations associated with the pathogenesis of skeletal muscle diseases.
METHODS: We established four target gene panels, each covering all exonic and flanking regions of genes involved in the pathogenesis of the following muscle diseases: (1) muscular dystrophy (MD), (2) congenital myopathy/congenital myasthenic syndrome, (3) metabolic myopathy and (4) myopathy with protein aggregations/rimmed vacuoles. We assigned one panel to each patient based on the results of clinical and histological analyses of biopsied muscle samples and performed high-throughput sequencing by using Ion PGM next-generation sequencer. We also performed protein analysis to confirm defective proteins in patients with major muscular dystrophies. Further, we performed muscle-derived cDNA analysis to identify splice-site mutations.
RESULTS: We identified possible causative gene mutations in 33% of patients (62/188) included in this study. Our results showed that the MD panel was the most useful, with a diagnostic rate of 46.2%.
CONCLUSIONS: Thus, we developed a high-throughput sequencing technique for diagnosing inherited muscle diseases. The use of this technique along with histological and protein analyses may be useful and cost-effective for screening mutations in patients with inherited skeletal muscle diseases.
AIM: In the present study, we developed a useful method for screening gene mutations associated with the pathogenesis of skeletal muscle diseases.
METHODS: We established four target gene panels, each covering all exonic and flanking regions of genes involved in the pathogenesis of the following muscle diseases: (1) muscular dystrophy (MD), (2) congenital myopathy/congenital myasthenic syndrome, (3) metabolic myopathy and (4) myopathy with protein aggregations/rimmed vacuoles. We assigned one panel to each patient based on the results of clinical and histological analyses of biopsied muscle samples and performed high-throughput sequencing by using Ion PGM next-generation sequencer. We also performed protein analysis to confirm defective proteins in patients with major muscular dystrophies. Further, we performed muscle-derived cDNA analysis to identify splice-site mutations.
RESULTS: We identified possible causative gene mutations in 33% of patients (62/188) included in this study. Our results showed that the MD panel was the most useful, with a diagnostic rate of 46.2%.
CONCLUSIONS: Thus, we developed a high-throughput sequencing technique for diagnosing inherited muscle diseases. The use of this technique along with histological and protein analyses may be useful and cost-effective for screening mutations in patients with inherited skeletal muscle diseases.