Animal model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset polyalanine disorder characterized clinically by progressive ptosis, dysphagia, and limb weakness and pathological hallmarked by unique intranuclear inclusions in the muscles. It is caused by heterozygous expansion of a 10-alanine stretch to 12-17 alanine residues in the N-terminus of the poly(A)-binding protein, nuclear 1 (PABPN1). Although PABPN1 is a major component of the inclusions in OPMD, the associated pathogenic mechanism is undetermined. No animal models of OPMD have been discovered in nature; therefore, we generated transgenic mice expressing human PABPN1 (hPABPN1) using a chicken beta-actin (CAG) promoter. While transgenic mice lines expressing normal hPABPN1 did not show myopathic changes, lines expressing high levels of expanded hPABPN1 with a 13-alanine stretch showed myopathy phenotype with aging. The latter mice disclosed intranuclear inclusions consisting of aggregated mutant hPABPN1 and scattered rimmed vacuoles restricted in the muscles. In particular, the nuclear inclusions closely resembled those of OPMD muscles on electron microscopy, and myopathic changes were more prominent in the eyelid and pharyngeal muscles. The results demonstrated that we had established the first transgenic OPMD model mouse. Recently, two other transgenic mice expressing mutated hPABPN1 with a 17-alanine stretch have been generated; however, the transgenic mouse using its natural promoter did not show myopathy phenotype, and the other using the human skeletal actin (HSA1) promoter disclosed quite different intranuclear inclusions from those of human OPMD muscles. Our transgenic OPMD model mouse appears to have more dramatic alterations in myofiber viability, but is useful for elucidating of molecular mechanisms and establishing therapeutic trials.

A Japanese family with FEOM1-linked congenital fibrosis of the extraocular muscles type 1 associated with spinal canal stenosis and refinement of the FEOM1 critical region.

We identified a Japanese family with congenital fibrosis of the extraocular muscles, including 24 affected individuals through five generations. To determine their form of congenital fibrosis of the extraocular muscles, we performed clinical and linkage studies. DNA typing for linkage to the FEOM1 (12p11.2-q12) and FEOM3 (16qter) loci was performed on genomic DNA, using fluorescent microsatellite polymorphic markers. All affected individuals shared the common manifestations of congenital fibrosis of the extraocular muscles type 1 including congenital ptosis, infraducted globe position in primary gaze, and upward gaze palsy in both eyes. Unexpectedly, we found apparent spinal canal stenosis in the cervical spine in all affected family members who were examined. Genetic analysis revealed linkage to the FEOM1 locus with a maximum lod score of 4.42 at theta of zero. One affected family member harbored a recombination event between D12S345 and D12S1692, narrowing the FEOM1 locus from the published 3-cM region flanked by D12S1584 and D12S1668 to a 2.1-cM region flanked by D12S345 and D12S1668. Thus, we have established that this family segregates congenital fibrosis of the extraocular muscles type 1 as an autosomal dominant trait and that their disorder both maps to and refines the FEOM1 locus. This is the first clinical and genetic report of such a family in the Japanese population and the first report of spinal involvement in congenital fibrosis of the extraocular muscles.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and CADASIL-like disorders in Japan.

We reviewed 12 patients from 11 Japanese families diagnosed as having CADASIL from 1998 to 2001. The age of onset of focal neurologic deficits ranged from 38 to 71 years (mean: 50.4 +/- 9.8 years). Japanese CADASIL patients rarely had migraine and frequently presented with symptoms of dementia at diagnosis. Notch3 mutations were concentrated in exons 3, 4, and 5. Cysteine was replaced by another amino acid or vice versa in the majority of Japanese CADASIL patients. However, in 2 families, the mutations were not related to cysteine. In the prospective study, 2030 patients with stroke were hospitalized in 6 hospitals with stroke units in the Kumamoto district from 1999 to 2001. Among them, 14 patients fulfilled the criteria of being less than 60 years of age, showing lacunar strokes and/or TIA, presence of a family history, and no risk factors of stroke. One of these 14 patients was diagnosed as having CADASIL by DNA analysis. However, if hyperlipidemia was excluded from the list, 16 patients fulfilled the criteria and 2 patients were diagnosed as having CADASIL by DNA analysis. It was suspected that the incidence of CADASIL is not so rare in Japan. There were some families with CADASIL-like features, but without Notch3 mutations or GOM, suggesting the need for genetic analysis in the future.

Western blotting analysis of the beta-hexosaminidase alpha- and beta-subunits in cultured fibroblasts from cases of various forms of GM2 gangliosidosis.

OBJECTIVES: The GM2 gangliosidoses are a group of genetic disorders caused by the accumulation of ganglioside GM2 in neuronal cells. We examined the alpha- and beta-subunits of beta-hexosaminidases by a non-radioisotopes detecting system to evaluate whether it was a useful method for understanding of the pathophysiologies of GM2 gangliosidoses. MATERIALS AND METHODS: We investigated the alpha- and beta-subunits of beta-hexosaminidases in cultured fibroblasts from cases of various forms of GM2 gangliosidosis by means of Western blotting and a chemiluminescence detection system. RESULTS: In a patient with infantile Tay-Sachs disease [HEXA genotype, Int5-SA(g-1-->t)/Int5-SA(g-1-->t)], the mature alpha-subunit was undetectable. In a patient with infantile Sandhoff disease (HEXB genotype, C534Y/C534Y), the mature beta-subunit was deficient. However, a small amount of the mature beta-subunit was detected in a patient with adult Sandhoff disease (HEXB genotype, R505Q(+I207V)/R505Q(+I207V)), which may have resulted in the residual enzyme activity and mild clinical course. Normal amounts of alpha- and beta-subunits were detected in a patient with GM2 activator deficiency. CONCLUSION: This method is easy and sensitive for detecting target proteins, and is useful for clarification of the pathophysiologies of GM2 gangliosidoses.

The identification of eight novel glucocerebrosidase (GBA) mutations in patients with Gaucher disease.

Mutations in the gene encoding for the lysosomal enzyme glucocerebrosidase (GBA) result in Gaucher disease. In this study, seven novel missense mutations in the glucocerebrosidase gene (A136E, H162P, K198E, Y205C, F251L, Q350X and I402F) and a splice site mutation (IVS10+2T-->A) were identified by direct sequencing of three amplified segments of the glucocerebrosidase gene. Five of the novel mutations were found in patients with neuronopathic forms of Gaucher disease, two of which, K198E and F251L, appear to be associated with type 2 Gaucher disease.

Homozygosity and linkage disequilibrium mapping of autosomal recessive distal myopathy (Nonaka distal myopathy).

Autosomal recessive distal myopathy or Nonaka distal myopathy (NM) is characterized by its unique distribution of muscular weakness and wasting. The patients present with spared quadriceps muscles even in a late stage of the disease. The hamstring and tibialis anterior muscles are affected severely in early adulthood. We have localized the NM gene to the region between markers D9S319 and D9S276 on chromosome 9 by linkage analysis. To further refine the localization of the NM gene, we conducted homozygosity and linkage disequilibrium analysis for 14 patients from 11 NM families using 18 polymorphic markers. All of the patients from consanguineous NM families were found to be homozygous for six markers located within the region between markers D9S2178 and D9S1859. We also provided evidence for significant allelic associations between the NM region and five marker loci. Examination of the haplotype analysis identified a predominant ancestral haplotype comprising the associated alleles 199-160-154-109 (marker order: D9S2179-D9S2180-D9S2181-D9S1804), present in 60% of NM chromosomes and in 0% of parent chromosomes. On the basis of the data obtained in this study, the majority of NM chromosomes were derived from a single ancestral founder, and the NM gene is probably located within the 1.5-Mb region between markers D9S2178 and D9S1791.

Efficient repetitive gene delivery to skeletal muscle using recombinant adenovirus vector containing the Coxsackievirus and adenovirus receptor cDNA.

To improve adenovirus-mediated gene delivery to skeletal muscle, we have used a recombinant adenovirus vector encoding the human Coxsackievirus and adenovirus receptor (hCAR). Because CAR is expressed at a lower level in rodent myoblasts and muscle fibers than in other tissues, we expected that elevated expression of CAR in skeletal muscle would improve the efficacy of adenovirus-mediated gene transfer. Since the mouse myoblasts, C2C12 cells, showed low sensitivity to infection by recombinant adenovirus 5, we initially infected these cells at a high multiplicity of infection (MOI) of 250 with the recombinant adenovirus containing hCAR cDNA and LacZ gene. Subsequent infection by recombinant adenovirus containing the marker gene, green fluorescence protein, became efficient even at a low MOI of 25. Thus, elevated hCAR expression in mouse muscle fibers made a second virus inoculation at low doses possible. We also demonstrated that the elevated hCAR expression did not influence muscle membrane integrity. Our results suggest that co-expression of CAR and a therapeutic gene by adenovirus vector constitutes a novel strategy to advance gene therapy for hereditary muscle diseases.

A novel CLCN1 mutation: P480T in a Japanese family with Thomsen's myotonia congenita.

At least 50 disease-causing mutations in the skeletal muscle voltage-gated chloride channel gene (CLCN1), almost all of which originate from Caucasian families, have been identified. We investigated a Japanese family with Thomsen's myotonia congenita that included 16 affected individuals (8 men and 8 women) through five generations. Polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) screening of 11 members showed an aberrant conformer in exon 13 of CLCN1 complementary DNA (cDNA) in 8 affected and 1 unaffected members. By sequence analysis, we identified a C-to-A transition at nucleotide position 1438, resulting in a substitution of proline for threonine at amino acid position 480 (P480T), the same position of the original mutation (P480L) in Thomsen's disease. The P480T mutation was novel and absent in 100 normal controls. Seven of the 8 affected individuals were heterozygous; another, from affected parents, was homozygous. Clinically, myotonia in the homozygous patient was more severe than that in heterozygous patients, probably due to the gene dosage effect. On a long-train nerve-stimulation test at a rate of 3 Hz, M-wave responses in the homozygous patient showed marked decrement followed by recovery. In contrast, the heterozygous patients showed just a slight decrement or no changes, and none of 2 patients with myotonic muscular dystrophy or 2 normal controls revealed any decrement. Thus, the long-train nerve-stimulation test at a low stimulus frequency may be a useful tool to assess the disease-severity/genotype relationship in myotonia congenita.

Susac's syndrome: beneficial effects of corticosteroid therapy in a Japanese case.

Susac's syndrome is a rare disorder characterized by the triad of microangiopathy of the brain and retina with hearing loss. More than 50 affected individuals have been reported worldwide, all Caucasians. We herein identify the first Japanese patient with Susac's syndrome. A 36-year-old man developed recurrent subacute encephalopathy, bi- a lateral sensorineural hearing loss, and retinal arteriolar occlusions, caused by microangiopathy from a year previously. T2-weighted MRI showed multiple high-signal lesions ti predominantly in the periventricular white matter. During the exacerbated phase both high-dose intravenous methyl-prednisolone and oral prednisone therapy produced beneficial effects. He showed definite remission within 2 years from the disease onset.

High signal in the spinal cord on T2-weighted images in rapidly progressive tropical spastic paraparesis.

We report a 59-year-old woman with human T-cell lymphotrophic virus type-I (HTLV-I) associated myelopathy/tropical spastic paraparesis who showed high signal in the cervical and thoracic spinal cord on T2-weighted and contrast enhancement on T1-weighted images.

Mutations of the notch3 gene in non-caucasian patients with suspected CADASIL syndrome.

The Notch3 gene has been recently identified as a causative gene for cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). To investigate the genetic contribution of Notch mutations in familial cases with vascular leukoencephalopathy, we screened 13 patients from 11 unrelated families, which were selected on the basis of magnetic resonance imaging findings and positive family history. We identified three different missense mutations in 5 patients from 4 families. Two (Arg90Cys and Arg133Cys) are the same as previously reported in Caucasian patients, the other (Cys174Phe) is a novel mutation causing a loss of a cysteine in epidermal-growth-factor-like repeats of Notch3. These data indicate that the CADASIL Notch3 mutations were found in approximately 35% of familial cases with leukoencephalopathy, suggesting genetic heterogeneity of the disease.

Clinical investigation of the lesions responsible for sensory disturbance in Minamata disease.

To clarify the lesions responsible for sensory disturbance in Minamata disease (MD), we clinically investigated the characteristics of sensory disturbance. In all patients with the classical type MD, two-point discrimination was severely disturbed, but the involvement of superficial sensation was relatively mild. On short-latency somatosensory evoked potential study, the component corresponding to N20 was completely absent with normal N9, N11, and N13 components. Although 14 of 38 chronic MD patients demonstrated intact superficial sensation, 10 of these 14 showed mild to moderate disturbance in two-point discrimination. The two-point discrimination in chronic MD patients was significantly high irrespective of the disturbance of superficial sensation. These findings suggest that the sensory disturbance of MD patients may mainly be caused by a lesion in the sensory cortex rather than in the peripheral nerves. However, other foci could be also responsible for the sensory impairment, since 9 of 38 chronic MD patients showed intact two-point discrimination.

[A case of symptomatic cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)].

We here report a 42-year-old woman diagnosed cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) by age, family history, neurological findings, and neuro imagings. Magnetic resonance imaging well demonstrated the multiple lesions in the cerebrum including the brain stem, which was characteristic findings in CADASIL.

Nuclear accumulation of expanded PABP2 gene product in oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by (GCG) repeat expansions in exon 1 of the poly(A) binding protein 2 gene (PABP2). To elucidate the molecular mechanism underlying the disease, we raised an antiserum against a synthetic peptide fragment predicted from PABP2 cDNA. The peptide corresponded to amino acids 271-291 where a cluster of posttranslational arginine methylation occurs. We examined the subcellular localization of PABP2 in muscle specimens from five patients with OPMD, 14 patients with various neuromuscular disorders, and three normal controls. All Japanese patients with OPMD have been shown to have expanded (GCG)(8, 9, or 11) mutations in PABP2, as well as intranuclear tubulofilamentous inclusions (ITFI) of 8.5 nm. None of 50 separate Japanese control individuals were shown to have expanded (GCG) repeat in PABP2. Positive immunoreaction for polyclonal PABP2 was confined to the intranuclear aggregates of muscle fibers exclusively in patients with OPMD. Frequency of the nuclei positive for PABP2 (2%) was similar to that of ITFI detected by electron microscopy (2.5%). There was no apparent relationship between the frequency of PABP2-positive intranuclear aggregates and the severity of muscle fiber damage. In contrast, nuclear immunoreaction was not detected in any samples from normal controls or from other neuromuscular diseases. These results suggest the presence of molecular modification of the product of expanded (GCG) repeat in PABP2, since the synthetic antigen peptide may not recognize a highly dimethylated cluster of arginine residues of the native PABP2, but may recognize the mutated form. Nuclear accumulation of expanded PABP2 product implies a causative role for ITFI.

Arg133Cys mutation of Notch3 in two unrelated Japanese families with CADASIL.

OBJECTIVE: More than 80 unrelated, but all Caucasian, patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), originating from various communities around the world, have been molecularly identified. To clarify the occurrence of CADASIL in Orientals, we investigated Japanese families presenting as CADASIL. METHODS: We performed the PCR-SSCP and sequence analyses using genomic DNA, isolated from venous blood of participants under informed consent. PATIENTS: We identified two unrelated Japanese families with CADASIL, including 5 affected members through 2 generations. RESULTS: Each of the affected individuals developed recurrent strokes without risk factors resulting in progressive dementia, pseudobulbar palsy, and gait disturbances which started after the fifth decade of life. Although affected individuals had no vascular risk factors, they showed various degrees of narrowing of retinal arteries. Their MRI/CTs showed characteristics of the disease; bilateral small infarcts in the thalamus, basal ganglia, brain stem, and deep white matter in addition to the findings of leukoaraiosis. On SPECT imaging, there was severe hypoperfusion in the cortex as well as in the white matter. Ultrastructural studies revealed an abnormal deposition of granular osmiophilic materials (GOM) within the basal lamina of pericytes in muscular capillaries. On PCR-SSCP and sequence analyses, a heterozygous Arg133Cys mutation was present, in the affected individuals, in the exon 4 of Notch3 gene which is the hot spot region for CADASIL mutations in Caucasian families. None of the non-affected members nor the 50 Japanese normal controls revealed this mutation. CONCLUSION: Thus, our results confirm that CADASIL is a geographically widespread disorder caused by a Notch3 mutation.