Shiro Ozasa

Genotype–phenotype correlations in alternating hemiplegia of childhood

Objective: Clinical severity of alternating hemiplegia of childhood (AHC) is extremely variable. To investigate genotype–phenotype correlations in AHC, we analyzed the clinical information and ATP1A3 mutations in patients with AHC. Methods: Thirty-five Japanese patients who were clinically diagnosed with AHC participated in this study. ATP1A3 mutations were analyzed using Sanger sequencing. Detailed clinical information was collected from family members of patients with AHC and clinicians responsible for their care. Results: Gene analysis revealed 33 patients with de novo heterozygous missense mutations of ATP1A3: Glu815Lys in 12 cases (36%), Asp801Asn in 10 cases (30%), and other missense mutations in 11 cases. Clinical information was compared among the Glu815Lys, Asp801Asn, and other mutation groups. Statistical analysis revealed significant differences in the history of neonatal onset, gross motor level, status epilepticus, and respiratory paralysis in the Glu815Lys group compared with the other groups. In addition, 8 patients who did not receive flunarizine had severe motor deteriorations. Conclusions: The Glu815Lys genotype appears to be associated with the most severe AHC phenotype. Although AHC is not generally seen as a progressive disorder, it should be considered a disorder that deteriorates abruptly or in a stepwise fashion, particularly in patients with the Glu815Lys mutation.

Novel mutation in splicing donor of dystrophin gene first exon in a patient with dilated cardiomyopathy but no clinical signs of skeletal myopathy

One cause of X-linked dilated cardiomyopathies is mutation of the dystrophin gene. We report the case of a young boy who suffered from dilated cardiomyopathy caused only by dystrophin-deficient cardiac muscle, but who did not present with any clinical signs of skeletal myopathy. Sequence analysis of the patients dystrophin gene revealed the presence of a novel single point mutation at the first exon—intron boundary, inactivating the 5′ splice site consensus sequence of the first intron. The lack of muscle weakness observed clinically can be explained by expression of the brain and Purkinje dystrophin isoforms in skeletal muscle.

Immobility reduces muscle fiber necrosis in dystrophin deficient muscular dystrophy.

Duchenne/Becker muscular dystrophy is a progressive muscle disease, which is caused by the abnormality of dystrophin. Spina bifida is characterized by paralysis of the feet, with most of the upper extremities not being affected. We report here on the first case of Becker muscular dystrophy coinciding with spina bifida. The muscle biopsy specimens of the patient showed dystrophic changes in upper extremities, but clearly less in lower extremities. The results show that the restriction of excessive exercise is important for dystrophin deficiency disease.

Exon-skipping events in candidates for clinical trials of morpholino

Background:  Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by abnormalities in the DMD gene. The majority of DMD patients have out‐of‐frame deletion(s), which disrupt the reading frame; while some cases of DMD are caused by duplication or nonsense mutation(s). Most patients with BMD have in‐frame deletion(s), which preserve the reading frame. The phenotype of BMD is generally milder than that of DMD. Antisense morpholino‐mediated exon skipping, which changes out‐of‐frame deletions to in‐frame deletions, is a promising therapeutic approach for DMD. It is necessary, however, to confirm the exon‐skipping event in cells of DMD patients before the clinical trial.

Estimation of muscle strength from actigraph data in Duchenne muscular dystrophy

The purpose of this study was to evaluate the utility of a wrist actigraph for estimating muscle strength in Duchenne muscular dystrophy patients.

A 2-bp deletion in exon 74 of the dystrophin gene does not clearly induce muscle weakness

Duchenne muscular dystrophy (DMD) is caused by mutation of the dystrophin gene. Cases of dystrophinopathy with a 2-bp deletion in the dystrophin gene commonly result in DMD. We report here a case of dystrophinopathy in a 9-years-old boy with a 2-bp deletion in exon 74 of the dystrophin gene; however, the boy had no clear clinical signs of muscle weakness. Immunohistochemical studies with N-terminal (DYS3) and rod-domain anti-dystrophin (DYS1) antibodies revealed that the dystrophin signals were weaker than in the control sample (non-dystrophinopathy) at the sarcolemma of myofibers, and the studies with C-terminus anti-dystrophin antibody (DYS2) were negative. Our patients mutation is located between the binding sites of alpha-syntrophin and alpha-dystrobrevin. These results suggest that this mutation does not clearly induce muscle weakness at least through the age of 9 years.

A Case Report of Congenital Fiber Type Disproportion with an Increased Level of Anti-ACh Receptor Antibodies.

Congenital fiber type disproportion (CFTD) is a form of congenital myopathy, which is defined by type 1 myofibers that are 12% smaller than type 2 myofibers, as well as a general predominance of type 1 myofibers. Conversely, myasthenia gravis (MG) is an acquired immune-mediated disease, in which the acetylcholine receptor (AChR) of the neuromuscular junction is blocked by antibodies. Thus, the anti-AChR antibody is nearly specific to MG. Herein, we report on a case of CFTD with increased anti-AChR antibody levels. A 23-month-old boy exhibited muscle hypotonia and weakness. Although he could walk by himself, he easily fell down and could not control his head for a long time. His blood test was positive for the anti-AChR antibody, while a muscle biopsy revealed characteristics of CFTD. We could not explain the relationship between MG and CFTD. However, we considered different diagnoses aside from MG, even when the patients blood is positive for the anti-AChR antibody.

Myoclonic Epilepsy With Ragged-Red Fibers Without Increased Lactate Levels

Myoclonic epilepsy associated with ragged-red fibers is one of the mitochondrial encephalomyopathies. Pathogenic mitochondrial DNA mutations have been identified in the mitochondrial transfer RNA (tRNA)(Lys) at positions 8344 and 8356. Characteristics of myoclonic epilepsy associated with ragged-red fibers include myoclonic epilepsy, generalized epilepsy, hearing loss, exercise intolerance, lactic acidosis, and ragged-red fibers. The elevated lactate level is one of the most important symptoms needed to make a diagnosis of mitochondrial encephalomyopathy. In the present case, however, myoclonic epilepsy was associated with ragged-red fibers but without increased lactate levels. Therefore, myoclonic epilepsy associated with ragged-red fibers should be suspected in a patient who has myoclonic epilepsy that is difficult to control with antiepileptic medications and who has other symptoms of mitochondrial disease, such as mental retardation, even if the patients lactate level is normal.

255. Embryonic Stem Cell-Mediated Regenerative Therapy for Duchenne Muscular Dystrophy

Introduction: Duchenne muscular dystrophy (DMD) is a severe muscle degenerative disorder caused by mutations in the dystrophin gene. Embryonic stem cell (ES cell) transplantation is one of the more promising therapies because ES cells are available in large quantities and can serve to systemically restore affected muscles of DMD patients. Although ES cells have shown to be capable of differentiating into various tissues and cell types, it has been difficult to induce differentiation of ES cells specifically into muscle fibers only. We are proposing a method to overcome this impediment by establishing genetically engineered ES cells which harbor a tetracycline-regulated expression vector encoding the myogenic transcriptional factor MyoD.

251. Dystrophin in Vascular Smooth Muscle Is Important for Duchenne Muscular Dystrophy Therapy

Duchenne muscular dystrophy (DMD) is an X-linked fatal disease caused by mutations of the gene encoding the cytoskeletal protein dystrophin, which is associated with a complex of proteins that spans the membrane and effectively links the cytoskeleton to the extracellular matrix. Despite the abundance of information about the molecular basis of this disease, there is currently no effective treatment for DMD because the mechanism by which dystrophin deficiency produces the clinical phenotype is poorly understood. Our previous data showed that dystrophin expression is normally detectable in smooth muscle cells (SMCs) as well as in skeletal muscle cells. Likewise, it is defective in SMCs of DMD patients and mdx mice, an animal model of DMD. Recent studies have revealed that focal multicellular myocytolytic lesions in muscular dystrophies may represent functional vascular abnormalities, regardless of whether or not they are primary. Although vascular dysfunction in DMD patients and mdx mice has been also reported, it is considered that the dystrophin deficiency of skeletal muscle and/or vascular endothelium, not vascular smooth muscle cells (VSMCs), is responsible for the vascular abnormalities.