Eri Kondo

Pial-glial barrier abnormalities in fetuses with Fukuyama congenital muscular dystrophy

This report concerns light and electron microscopic studies on the central nervous system of a 20-week and an 18-week fetus with Fukuyama congenital muscular dystrophy (FCMD). The diagnosis of FCMD was established by prenatal molecular genetic analysis. Cerebral lesions containing neurites, subpial granular cells and glias, accompanied by cortical dysplasia were found in both cases. Small irregular defects, readily detectable by periodic acid-methenamine-silver staining or by immunohistochemical staining for S-100 protein, were observed in the cerebral surface. More severe dysplasia was evident at the areas with the larger defects. Surface defects were also observed in the cerebellum and brain stem, with brain tissue extruding into the leptomeninges. The pyramidal tract was aberrant in the pons and medulla oblongata. The spinal cord appeared normal by light microscopy. Electron microscopic examination revealed an abnormal configuration of the basement membrane and glial cytoplasmic membrane of the brain and spinal cord surfaces, including areas with no detectable defects by light microscopy. These findings suggest that abnormalities of the pial-glial barrier, especially the basement membrane and/or basement membrane-related structures, are involved in the genesis of cortical dysplasia.

Recessive RYR1 mutations in a patient with severe congenital nemaline myopathy with ophthalomoplegia identified through massively parallel sequencing.

Nemaline myopathy (NM) is a group of congenital myopathies, characterized by the presence of distinct rod‐like inclusions “nemaline bodies” in the sarcoplasm of skeletal muscle fibers. To date, ACTA1, NEB, TPM3, TPM2, TNNT1, and CFL2 have been found to cause NM. We have identified recessive RYR1 mutations in a patient with severe congenital NM, through high‐throughput screening of congenital myopathy/muscular dystrophy‐related genes using massively parallel sequencing with target gene capture. The patient manifested fetal akinesia, neonatal severe hypotonia with muscle weakness, respiratory insufficiency, swallowing disturbance, and ophthalomoplegia. Skeletal muscle histology demonstrated nemaline bodies and small type 1 fibers, but without central cores or minicores. Congenital myopathies, a molecularly, histopathologically, and clinically heterogeneous group of disorders are considered to be a good candidate for massively parallel sequencing.

Early Ultrastructural Changes in the Central Nervous System in Fukuyama Congenital Muscular Dystrophy

Electron microscopy of the central nervous system surface structure is described in two fetuses with Fukuyama congenital muscular dystrophy (FCMD). In addition to relatively large surface defects, many minute defects less than several micrometers in size associated with protrusion of glial cytoplasm were observed in the cerebrum. These findings were considered to represent early changes prior to cortical dysplasia. The basement membrane adjacent to the defects showed amorphous, wavy, or whorled configurations, and gradually disappeared. The glial cytoplasmic membrane seemed to be relatively well preserved in some areas where the basement membrane disappeared. On the other hand, both the basement membrane and cytoplasmic membrane became indistinct irregularly in areas without defects, including the spinal cord; similar lesions were found in the skeletal muscle. These observations confirm previous observations concerning defects of the pial-glial barrier of the brain surface, and may suggest the involvement of abnormal basement membrane or related structures, or both, in the genesis of the brain lesions of FCMD.

Prenatal diagnosis of Fukuyama type congenital muscular dystrophy by polymorphism analysis.

Fukuyama type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder characterized by a combination of primary muscular dystrophy of early infantile onset and brain malformation (lissencephaly type II). The identification of the FCMD gene locus at 9q31 opened the theoretical possibility of prenatal diagnosis. The authors conducted prenatal diagnosis in two unrelated FCMD families by analysis using nine microsatellite CA-repeat polymorphic markers flanking the FCMD locus, and calculated phenotype probabilities in fetuses with a computer program, LINKAGE. The fetus in family 1 showed a 99% probability of being healthy either as a normal homozygote or a heterozygote carrier and was born without signs of FCMD. In family 2, the fetus was diagnosed to have FCMD with at least 86% probability. The parents of this family decided to terminate the pregnancy and an abortus showed brain malformations characteristic of an FCMD fetus.

Localization of laminin subunits in the central nervous system in Fukuyama congenital muscular dystrophy: an immunohistochemical investigation.

Abstract We have undertaken an immunohistochemical study of laminin subunits in the central nervous system (CNS) of fetuses and patients with Fukuyama congenital muscular dystrophy (FCMD) and of controls including five fetuses. Immunoreaction product deposits with antibodies to laminin α1, α2, β1 and γ1, and β-dystroglycan were detected on the surface and vessels of the CNS of controls. No staining with anti-α-sarcoglycan antibody was detected in the CNS. Neurons and glia did not react with any of the antibodies used. In utero expression of laminin subunits and β-dystroglycan seemed to be lower in the cerebrum than in the spinal cord. Moreover, immunostaining for laminin α2 and β1 tended to be weak on the fetal spinal cord surface. Expression of laminin subunits and dystrophin-associated proteins in the CNS may be modulated during development, as in the skeletal muscle. The distribution of immunoreaction product deposits was basically the same in FCMD and controls, although laminin α2 and β-dystroglycan expression appeared to be decreased in the CNS of the FCMD cases. Defects of the pial-glial barrier of the fetal brain surface have been considered the main cause of micropolygyria in FCMD, and these observations suggest that the co-localization and secondary loss of these proteins in association with the unknown product(s) of the FCMD gene might be involved in the CNS lesions of this disorder.

Fukuyama congenital muscular dystrophy: Cortical dysplasia of the cerebrum in a 20 week fetus

This report describes cerebral cortical dysplasia in a 20 week fetus, aborted as a result of the prenatal genetic analysis which provided evidence of Fukuyama congenital muscular dystrophy (FCMD). The histological appearance of the brain of this fetus was similar to that of previously described cases. However, cortical dysplasia was less severe in this 20 week fetus, and the interesting finding was the existence of a leptomeningeal lesion probably preceding dysplasia. In this case, abnormal findings were found mainly in the cerebral surface, and maturation of the brain seemed appropriate for the gestational age. Furthermore, periodic acid‐methenamine‐silver‐positive linear structure of the cerebral surface was disrupted irregularly. These findings suggest that defects in the pial‐glial barrier of the cerebral surface may play a cardinal role for the genesis of cortical dysplasia. It is suggested that the extracortical abnormal tissue would be detectable morphologically at least in the 12‐13th week because cells considered as subpial granular cells were contained in the lesion. This report on the brain of a fetus with FCMD provides information on relatively early central nervous system alterations in this disease, and may be of importance in clarifying their pathogenesis.

Target resequencing of neuromuscular disease-related genes using next-generation sequencing for patients with undiagnosed early-onset neuromuscular disorders.

Neuromuscular disorders are clinically and genetically heterogeneous diseases with broadly overlapping clinical features. Progress in molecular genetics has led to the identification of numerous causative genes for neuromuscular disorders, but Sanger sequencing-based diagnosis remains labor-intensive and expensive because the genes are large, the genotypes and phenotypes of neuromuscular disorders overlap and multiple genes related to a single phenotype exist. Recently, the advent of next-generation sequencing (NGS) has enabled efficient, concurrent examination of several related genes. Thus, we used NGS for target resequencing of neuromuscular disease-related genes from 42 patients in whom undiagnosed early-onset neuromuscular disorders. Causative genes were identified in 19/42 (45.2%) patients (six, congenital muscular dystrophy; two, Becker muscular dystrophy (BMD); three, limb-girdle muscular dystrophy; one, concurrent BMD and Fukuyama congenital muscular dystrophy; three, nemaline myopathy; one, centronuclear myopathy; one, congenital fiber-type disproportion; one, myosin storage myopathy; and one, congenital myasthenic syndrome). We detected variants of uncertain significance in two patients. In 6/19 patients who received a definitive diagnosis, the diagnosis did not require muscle biopsy. Thus, for patients with suspected neuromuscular disorders not identified using conventional genetic testing alone, NGS-based target resequencing has the potential to serve as a powerful tool that allows definitive diagnosis.

Corticosteroid therapy for duchenne muscular dystrophy: improvement of psychomotor function.

BACKGROUND Of the numerous clinical trials for Duchenne muscular dystrophy, only the corticosteroid prednisolone has shown potential for temporal improvement in motor ability. In this study, the effects of prednisolone on intellectual ability are examined in 29 cases of Duchenne muscular dystrophy because little information has been reported. And also, motor functions and cardiac functions were evaluated. METHODS The treated group was administered prednisolone (0.75 mg/kg) orally on alternate days and the compared with the untreated control group. Gene mutations were investigated. The patients were examined for intelligence quotient adequate for age, brain natriuretic peptide, creatine kinase, and manual muscle testing before treatment and after the period 6 months to 2 years. RESULTS Intelligence quotient scores of the treated increased to 6.5 ± 11.9 (mean ± standard deviation) were compared with the controls 2.1 ± 4.9 (P = 0.009). Intelligence quotient scores of the patients with nonsense point mutations improved significantly (21.0 ± 7.9) more than those with deletion or duplication (1.9 ± 9.0; P = 0.015). Motor function, such as time to stand up, of those treated improved significantly and brain natriuretic peptide level was reduced to a normal level after treatment in 15 patients (73%). CONCLUSIONS Our results demonstrate the effectiveness of prednisolone in improving intellectual impairment as well as in preserving motor function and brain natriuretic peptide levels. We presume that prednisolone has a read-through effect on the stop codons in the central nervous systems of Duchenne muscular dystrophy because intelligence quotient of point mutation case was improved significantly.

Immunocytochemical dystrophin staining in a boy with severe Duchenne type muscular dystrophy and his younger sister with floppy infant syndrome

Immunocytochemical dystrophin staining in a boy with severe Duchenne type muscular dystrophy and his younger sister with floppy infant syndrome Eri Kondo, Kiyoko Ikeya, Kayoko Saito, Makiko Osawa, Sawako Sumita, Keiko Shishikura, Haruko Suzuki, Yoshito Hirayama, Yumi Arai and Yukio Fukuyama (Department of Pediatrics, Tokyo Women’s Medical College, Tokyo, Japan) Introduction: Immunocytochemical dystrophin staining in congenital muscular dystrophy (CMD) shows weak or no staining in some necrotic and regenerating fibers, despite that the majority of muscle fibers show normal positive staining. We carried out immunocytochemical dystrophin staining of muscles in a pair of siblings with different clinical phenotypes. In a boy, ‘severe’ Duchenne type muscular dystrophy (DMD) was clinically suspected, and brain malformations were found at autopsy. The boy’s younger sister was a floppy infant. Dystrophin staining of both their muscle membranes was positive. So we considered them to have CMD rather than DMD. However, dystrophin staining of the boy’s skeletal muscles was weak or C-terminal anti-dystrophin antibodies were absent. Case 1: A boy had hyperCKemia (1000-8000 mu/ml) at birth, and his motor development was delayed from infancy. He started to walk unaided at 2 years 1 month. His peak motor function, attained at about age 5, was to step up and down stairs. Progressive muscle weakness and joint contractures followed. He was wheelchair-bound at 7 and unable to shuffle at 13 years 8 months, though he could still sit unaided. Facial muscle involvement was apparent and pseudohypertrophy of the calves was marked. Mild brain atrophy was demonstrated on CT. At age 14, after an upper respiratory tract infection, he became dyspneic and a cardiac arrest ensued. Case 2: The younger sister of case 1 was first brought to our department at age 8 months. She could not sit unaided, tendon reflexes were absent and CK was 6000 mu/ml; a floppy infant. Her muscle weakness was more pronounced than that of her brother. Method: Skeletal muscle from case 1 was obtained at autopsy 5 h after death. Case 2 underwent muscle biopsy at 1 year 7 months. Immunocytochemistry was performed on frozen sections, using dystrophin staining panels. Western blot analysis was also performed. Results: Skeletal muscle membrane dystrophin was positive with N-terminal anti-dystrophin antibodies (POO, 2-5E2, PO41, but with rod domain and C-terminal antibodies (P23, 4-4C5, P34), many membranes showed weak or no staining. The dystrophin molecular weight was normal, 400 kD, as judged on Western blotting. In case 2, the muscle membrane dystrophin was stained positive with six anti-dystrophin antibodies. Discussion: The result of immunocytochemical dystrophin analysis differed between these CMD siblings, suggesting a postmortem change in the autopsied muscle, though these changes were not typical of other autopsied specimens. Dystrophin staining was weak or negative with C-terminal antibodies in the boy. The Cterminal domain of dystrophin generally binds with a muscle cell membrane through dystrophin associated glycoprotein (DAG). We plan to examine abnormalities of the other cytoskeletal proteins such as DAG.