Recent developments in molecular therapies for Duchenne muscular dystrophy (DMD) demand accurate genetic diagnosis, because therapies are mutation specific. The KUCG (Kobe University Clinical Genetics) database for DMD and Becker muscular dystrophy is a hospital-based database comprising 442 cases. Using a combination of complementary DNA (cDNA) and chromosome analysis in addition to conventional genomic DNA-based method, mutation detection was successfully accomplished in all cases, and the largest mutation database of Japanese dystrophinopathy was established. Among 442 cases, deletions and duplications encompassing one or more exons were identified in 270 (61%) and 38 (9%) cases, respectively. Nucleotide changes leading to nonsense mutations or disrupting a splice site were identified in 69 (16%) or 24 (5%) cases, respectively. Small deletion/insertion mutations were identified in 34 (8%) cases. Remarkably, two retrotransposon insertion events were also identified. Dystrophin cDNA analysis successfully revealed novel transcripts with a pseudoexon created by a single-nucleotide change deep within an intron in four cases. X-chromosome abnormalities were identified in two cases. The reading frame rule was upheld for 93% of deletion and 66% of duplication mutation cases. For the application of molecular therapies, induction of exon skipping was deemed the first priority for dystrophinopathy treatment. At one Japanese referral center, the hospital-based mutation database of the dystrophin gene was for the first time established with the highest levels of quality and patient's number.
Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by a loss of the dystrophin protein. Control of dystrophin mRNA splicing to convert severe DMD to a milder phenotype is attracting much attention. Here we report a dystrophinopathy patient who has a point mutation in exon 31 of the dystrophin gene. Although the mutation generates a stop codon, a small amount of internally deleted, but functional, dystrophin protein is produced in the patient cells. An analysis of the mRNA reveals that the mutation promotes exon skipping and restores the open reading frame of dystrophin. Presumably, the mutation disrupts an exonic splicing enhancer and creates an exonic splicing silencer. Therefore, we searched for small chemicals that enhance exon skipping, and found that TG003 promotes the skipping of exon 31 in the endogenous dystrophin gene in a dose-dependent manner and increases the production of the dystrophin protein in the patient's cells.
Mutations in fukutin-related protein (FKRP) gene cause a wide spectrum of disease phenotypes including the mild limb-girdle muscular dystrophy 2I (LGMD2I), the severe Walker-Warburg syndrome, and muscle-eye-brain disease. FKRP deficiency results in α-dystroglycan (α-DG) hypoglycosylation in the muscle and heart, which is a biochemical hallmark of dystroglycanopathies. To study gene replacement therapy, we generated and characterized a new mouse model of LGMD2I harboring the human mutation leucine 276 to isoleucine (L276I) in the mouse alleles. The homozygous knock-in mice (L276I(KI)) mimic the classic late onset phenotype of LGMD2I in both skeletal and cardiac muscles. Systemic delivery of human FKRP gene by AAV9 vector in the L276I(KI) mice, at either neonatal age or at the age of 9 months, rendered body wide FKRP expression and restored glycosylation of α-DG in both skeletal and cardiac muscles. FKRP gene therapy ameliorated dystrophic pathology and cardiomyopathy such as muscle degeneration, fibrosis, and myofiber membrane leakage, resulting in restoration of muscle and heart contractile functions. Thus, these results demonstrated that the treatment based on FKRP gene replacement was effective.
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