Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy.

Autosomal dominant oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease with a world-wide distribution. It usually presents in the sixth decade with progressive swallowing difficulties (dysphagia), eyelid drooping (ptosis) and proximal limb weakness. Unique nuclear filament inclusions in skeletal muscle fibres are its pathological hallmark. We isolated the poly(A) binding protein 2 gene (PABP2) from a 217-kb candidate interval on chromosome 14q11 (B.B. et al., manuscript submitted). A (GCG)6 repeat encoding a polyalanine tract located at the N terminus of the protein was expanded to (GCG)8-13 in the 144 OPMD families screened. More severe phenotypes were observed in compound heterozygotes for the (GCG)9 mutation and a (GCG)7 allele that is found in 2% of the population, whereas homozygosity for the (GCG)7 allele leads to autosomal recessive OPMD. Thus the (GCG)7 allele is an example of a polymorphism which can act either as a modifier of a dominant phenotype or as a recessive mutation. Pathological expansions of the polyalanine tract may cause mutated PABP2 oligomers to accumulate as filament inclusions in nuclei.

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.

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.

Presenile-onset cerebral adrenoleukodystrophy presenting as Balint's syndrome and dementia.

We describe a patient with presenile-onset cerebral adrenoleukodystrophy presenting as Balint's syndrome and dementia. There were demyelinating MRI changes in the parieto-occipital white matter bilaterally, including the splenium of the corpus callosum. Therapeutic trials using 1-deamino-(8-D-arginine)-vasopressin, very long-chain fatty acid-free diet, and gamma-globulin were of no benefit.

Oculopharyngeal muscular dystrophy in two unrelated Japanese families.

The occurrence of oculopharyngeal muscular dystrophy (OPMD) in Orientals is uncertain. We identified two unrelated Japanese families, including 30 affected individuals (14 men, 16 women, mean age 58 years) of OPMD through four generations, with complete penetrance. Their major clinical manifestations were late-onset bilateral ptosis and dysphagia. Histologic studies of slightly affected muscles reveal mild myogenic changes, occasional rimmed vacuoles, and small angulated fibers. By contrast, the severely involved cricopharyngeal muscle showed marked loss of fibers and massive proliferation of connective tissue. Ultrastructural studies of four different biopsied muscles disclosed subsarcolemmal intranuclear tubulofilamentous inclusions, identical to those of non-Japanese OPMD patients.

Genetic localization of the familial adult myoclonic epilepsy (FAME) gene to chromosome 8q24.

OBJECTIVE: To identify the genetic locus for the familial adult myoclonic epilepsy (FAME) gene. BACKGROUND: Idiopathic generalized epilepsy (IGE) represents a collection of disorders in which affected individuals present with recurring seizures that have diffuse onset on EEG. These individuals have no known structural cerebral lesions or other identifiable etiology. IGE accounts for approximately 40% of all epilepsies. FAME is a type of IGE characterized by autosomal dominant inheritance, adult onset, varying degrees of myoclonus in the limbs, rare tonic-clonic seizures, and a benign course. METHODS: We investigated four previously reported Japanese kindreds and performed a genome-wide screen with genetic linkage analysis. RESULTS: Clinical characterization and sampling of 30 individuals in four families revealed that 21 had the FAME phenotype. We defined a 4.6-cM region on chromosome 8q24 (maximum lod score of 4.86 at theta = 0) that contains the FAME gene. CONCLUSIONS: The identification and characterization of the FAME gene allows us to better understand the molecular basis of FAME. Such knowledge may provide clues to understanding the molecular basis of the clinically similar, and more common, juvenile myoclonic epilepsies, and other generalized seizure disorders that have thus far eluded genetic approaches.

Autosomal recessive oculopharyngodistal myopathy in light of distal myopathy with rimmed vacuoles and oculopharyngeal muscular dystrophy.

We investigated two Japanese siblings presenting with oculopharyngodistal myopathy, whose healthy parents were consanguineous. To clarify their disease characteristics, we compared them with four patients with distal myopathy with rimmed vacuoles linked to chromosome 9p1-q1, and 36 patients with oculopharyngeal muscular dystrophy linked to 14q11.2-q13. The first symptom in the patients with autosomal recessive oculopharyngodistal myopathy was weakness of the tibialis anterior muscle. Their biceps muscles showed initial and advanced myogenic changes, with rimmed vacuoles in 3% and 6% of the muscle fibers, respectively. In contrast, patients with distal myopathy with rimmed vacuoles revealed many rimmed vacuoles, on average in 20% of the fibers, and their oculopharyngeal muscles were spared. None of the patients with oculopharyngeal muscular dystrophy showed distal dominant weakness and the occurrence of rimmed vacuoles was rare. Ultrastructural studies in groups of autosomal recessive oculopharyngodistal myopathy and distal myopathy with rimmed vacuoles disclosed a collection of cytoplasmic filaments of 16-18 nm, but oculopharyngeal muscular dystrophy-specific intranuclear inclusions of 8.5 nm were not found. Thus, the phenotype of autosomal recessive oculopharyngodistal myopathy is distinct from distal myopathy with rimmed vacuoles and oculopharyngeal muscular dystrophy, but shares some ultrastructural characteristics with distal myopathy with rimmed vacuoles and hereditary inclusion body myopathy.

Oculopharyngeal muscular dystrophy in Japan.

Oculopharyngeal muscular dystrophy (OPMD) in the European population has been frequently diagnosed, but except for one black family, the occurrence in other ethnic groups is uncertain. We identified two unrelated OPMD Japanese families, including 34 affected individuals. Major clinical manifestations were bilateral ptosis and dysphagia starting after age 40. Histologic studies of limb muscles revealed mild myogenic changes, occasional rimmed vacuoles, and small angulated fibers. By contrast, cricopharyngeal muscle showed a marked loss of fibers and massive proliferation of connective tissue. Intranuclear tubulofilamentous inclusions (ITFI) of 8.5 nm outer diameter were observed in 2-5% of the nuclei in four different biopsied muscles. One patient with recurrent aspirations underwent successful cricopharyngeal myotomy. Aerodynamic examination was useful to evaluate velopharyngeal closure function. Our investigations revealed that OPMD is a geographically widespread disorder, and ITFI may be the specific morphologic hallmark.

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.

Novel chloride channel gene mutations in two unrelated Japanese families with Becker's autosomal recessive generalized myotonia.

We investigated the skeletal muscle voltage-gated chloride channel gene (CLCN1) in two unrelated Japanese patients with Becker's myotonia congenita. The non-myotonic parents of each patient were consanguineous. The proband of each family shares generalized myotonia, transient weakness after rest, and leg muscle hypertrophy. However, the disease severity related to the degree of myotonia differed, even in view of the response to long train nerve stimulation tests. CLCN1 gene analysis revealed a novel Ala659Val missense mutation identified to be homozygous in the more severe patient, while a novel Gln445Stop nonsense mutation was present in the other patient. Both mutations were absent in 90 Japanese normal controls. This is the first report of Japanese cases of Becker's myotonia congenita with CLCN1 gene mutations.

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.

Spontaneous T cell proliferation and release of soluble interleukin-2 receptors in patients with HTLV-I-associated myelopathy.

Patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy (HAM) show increased serum levels of soluble interleukin-2 receptors (sIL-2R), a marker of T cell activation. We found that peripheral blood lymphocytes from HAM patients proliferated spontaneously and released sIL-2R when cultured in vitro. Spontaneous proliferation was observed in T cell populations (both CD4+ cells and CD8+ cells), but not in B cell-rich populations or monocyte-rich populations. There was a significant increase of IL-2 activity in the culture supernatants of peripheral blood mononuclear cells (PBMC) after 2-3 days cultivation. On the other hand, sIL-2R concentrations in the supernatants were much higher after 5 days of cultivation. Such spontaneous T lymphocytic proliferation and release of sIL-2R were also found in non-HAM HTLV-I carriers, but not as intensely as in HAM patients. HTLV-I infection causes T cell activation to release IL-2 and sIL-2R; such T cell responses may play a role in the pathogenesis of HTLV-I-associated myelopathy.

Increased human T lymphotropic virus type-1 (HTLV-1) proviral DNA in peripheral blood mononuclear cells and bronchoalveolar lavage cells from Japanese patients with HTLV-1-associated myelopathy.

Human T lymphotropic virus type-1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is endemic in tropical areas and in southwestern Japan, and has now been identified among risk groups in the United States and some European countries. Patients with HAM/TSP may also have T lymphocyte alveolitis related to the HTLV-1 infection. To quantify proportions of HTLV-1-infected cells, a fragment of HTLV-1 proviral DNA was amplified from peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage (BAL) cells obtained from patients with HAM/TSP, non-HAM/TSP HTLV-1 carriers with chronic pulmonary inflammation, and asymptomatic HTLV-1 carriers. The proportion of HTLV-1-infected cells in PBMC from patients with HAM/TSP was much higher (3-30%) than that from asymptomatic HTLV-1 carriers (mostly < 1%), based on findings with the quantitative polymerase chain reaction. In non-HAM/TSP carriers with chronic pulmonary inflammation, HTLV-1-infected cells in PBMC were also increased, but not as markedly as that seen in patients with HAM/TSP. Integration of HTLV-1 was also noted in BAL cells from patients with HAM/TSP or non-HAM/TSP carriers with chronic pulmonary inflammation. However, in patients with HAM/TSP, there was a marked increase in HTLV-1-infected cells in the lung (7.5-30% of BAL cells), as compared with findings in non-HAM/TSP carriers (< 5%). These results suggest that increased HTLV-1 proviral DNA loading may play an important role in the development of T lymphocyte alveolitis and myelopathy in patients with HAM/TSP.

An atypical case of adult Moyamoya disease with initial onset of brain stem ischemia.

We present an atypical case of adult moyamoya disease whose clinical onset consisted of ischemic symptoms of the brain stem. She initially presented with left hemisensory disturbance caused by a pontine lesion, followed by a myelopathy of the upper cervical spinal cord. Eight months later, she presented with left hemiplegia and disturbed consciousness. Magnetic resonance angiography showed significant narrowing of both horizontal portions of the middle cerebral arteries (M1). Conventional angiography revealed bilateral occlusion of the internal carotid arteries. Her anterior circulation was supplied from the vertebro-basilar system through Moyamoya vessels and leptomeningeal collaterals. The intracranial steal phenomenon was thought to be the reason for the preceding events in the brain stem and upper cervical spinal cord. In addition, transcranial color-coded duplex sonography (TCCS) showed identical findings to conventional angiography with antegrade flow in the proximal M1 and retrograde flow in the distal M1. Thus, TCCS was useful for diagnosing the M1 occlusion in this case of Moyamoya disease.

Decrease in Cu/Zn- and Mn-superoxide dismutase activities in brain and spinal cord of patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative disorder of motor neurons in the central nervous system (CNS). Mutation of the Cu/Zn-superoxide dismutase (SOD) gene on chromosome 21 has been found in some families with autosomal dominant familial ALS (FALS). We sought to determine whether there may be differences in the distribution and activity of SOD in the CNS of patients with sporadic ALS, and of control patients without neurological disorders. The frontal cortex, cerebellum, and spinal cord were obtained at autopsy on 5 patients with ALS and from 10 controls. Immunohistochemically, in the controls, the cytosols of the large pyramidal neurons of the cerebral cortex, anterior and posterior horn cells, and neurons of the nucleus thoracicus of spinal cord were stained homogeneously with anti-human Cu/Zn-SOD antibody, and in a granular manner with anti-human Mn-SOD antibody. Pia mater and epithelial cells of choroid plexus also stained well. Conversely, in the CNS of the ALS patients, most neurons were stained faintly, or not at all with both anti-Cu/Zn- and Mn-SOD antibodies, whereas the pia mater and the epithelial cells of choroid plexus stained intensely. There was no difference in total SOD activity in the entire CNS between ALS patients and controls, as determined by enzyme assay. Results suggest that, in cases of sporadic ALS, the activities of Cu/Zn- and Mn-SOD are decreased and superoxide produced within the neurons accumulates because of an insufficient elimination, leading to the development or acceleration of cell damage, ultimately producing neuronal degeneration and necrosis.

Cognitive event-related potentials and brain magnetic resonance imaging in HTLV-1 associated myelopathy (HAM).

Auditory and visual cognitive event-related potentials (ERPs) were investigated in 14 patients with HTLV-1 associated myelopathy (HAM) and in 36 normal controls. In the HAM patients, the latencies of P300 and N200 by the auditory tone method were significantly delayed, and N100 by the auditory click method was significantly delayed in latency. No abnormal ERP components were observed with visual methods. While these auditory abnormal ERPs were present in the HAM patients, there was no evidence of visual abnormal ERPs. Abnormal lesions on the white matter were evident at magnetic resonance imaging (MRI) in 6 (75%) of 8 patients. There was no correlation between MRI lesions and the abnormalities of ERPs, but there was a significant correlation between bifrontal index on MRI and P300 amplitudes at Cz and Pz sites by auditory tone method. In one patient, atrophy of bilateral parietal lobes was seen on MRI and P300 latencies delayed using various methods. Therefore, the possibility that electrophysiological cognitive impairment in patients with HAM is related to brain atrophy rather than to white matter lesions requires attention.

PCR and immunoblot analyses of dystrophin in Becker muscular dystrophy.

The dystrophin gene was examined by PCR analysis in 30 Japanese patients with Becker muscular dystrophy (BMD). Fifteen PCR of these patients had exon deletion, generally, less than three exons. Muscle biopsies were also performed in 20 BMD patients (10 with sequence deletions, and 10 without detectable sequence deletions) in order to correlate PCR findings with immunoblot and immunostaining data. A patchy, heterogeneous membrane immunostaining pattern of reduced intensity was found, irrespective of the presence or absence of deletions. Immunoblotting studies demonstrated dystrophin of low molecular mass and quantity in BMD patients with deletion mutations, while a low quantity of dystrophin with an apparent wild type molecular mass was observed in nearly half the BMD patients without detectable deletions. However, these dystrophins were also found to have slightly abnormal molecular masses when the standard electrophoresis time was prolonged. This suggests that immunoblots and PCR data correlate well in patients with BMD. Additionally, it is suggested that immunoblot assays can detect abnormalities in dystrophin in the absence of detectable PCR deletions.

PCR and immunocytochemical analyses of dystrophin-positive fibers in Duchenne muscular dystrophy.

Immunocytochemical studies on serial sections of muscles from 19 patients with Duchenne muscular dystrophy (DMD) were done using seven kinds of antidystrophin antibodies that span dystrophin. Fifteen of the patients were screened for intragenic deletions by the multiplex polymerase chain reaction (PCR); 7 were also tested, using Southern blots. Dystrophin-positive fibers were detected in 10 of the 19 patients, occurring with a frequency of 0.06-75.7%, as clusters or as single fibers in certain muscle bundles. In 7 of the 10 patients, the fibers stained with all antibodies from the N- through the C-terminal region. However, in one patient, there were dystrophin-positive fibers that stained with an N-terminal antibody (DYS-3) and an antibody specific to the rod region (DYS-1), but not with C-terminal antibody (DYS-2). In 2 patients, there were two kinds of fibers: one that did not stain with DYS-1 or DYS-2 and another that stained with all the antibodies used. Single-fiber PCR analysis in 2 patients showed that the genotype of dystrophin-positive fibers differed from that of dystrophin-negative fibers. These results suggest that the majority of dystrophin-positive fibers are of the same clonal origin, but that some are derived from plural reverse mutations, each with a different translation-frame-repairing modality and somatic mosaicism.

Adult Sandhoff's disease: R505Q and I207V substitutions in the HEXB gene of the first Japanese case.

We describe a 31-year-old Japanese man with adult Sandhoff s disease presenting as spinocerebellar degeneration. There was a marked cerebellar atrophy on MRI, and proliferation of abundant PAS-positive foamy macrophages in the rectal mucosa. The activities of total beta-Hex, beta-Hex A, and beta-Hex B in leucocytes of the patient were 14%, 15%, and 6% of control values, respectively. However, oligosacchariduria or ultrastructural storage materials in liver tissue were nil. Direct sequencing of cDNA and genomic DNA, and restriction digestion revealed two different homozygous base substitutions in the HEXB gene: the G1514-->A substitution (R505Q) and the A619-->G substitution (1207V). The parents were consanguineous. His healthy mother, an enzymatic heterozygous carrier, was homozygous for 1207V, but heterozygous for R505Q mutation. Thus, the patient is probably homozygous for both base substitutions and a R505Q mutation may be linked to the phenotype of adult Sandhoff's disease.