Masaaki Kato

FALS with FUS mutation in Japan, with early onset, rapid progress and basophilic inclusion

Mutations in the fused in sarcoma (FUS, also known as translated in liposarcoma) gene have been recently discovered to be associated with familial amyotrophic lateral sclerosis (FALS) in African, European and American populations. In a Japanese family with FALS, we found the R521C FUS mutation, which has been reported to be found in various ethnic backgrounds. The family history revealed 23 patients with FALS among 46 family members, suggesting a 100% penetrance rate. They developed muscle weakness at an average age of 35.3 years, followed by dysarthria, dysphagia, spasticity and muscle atrophy. The average age of death was 37.2 years. Neuropathological examination of the index case revealed remarkable atrophy of the brainstem tegmentum characterized by cytoplasmic basophilic inclusion bodies in the neurons of the brainstem. We screened 40 FALS families in Japan and found 4 mutations (S513P, K510E, R514S, H517P) in exon 14 and 15 of FUS. Even in Asian races, FALS with FUS mutations may have the common characteristics of early onset, rapid progress and high penetrance rate, although in patients with the S513P mutation it was late-onset. Degeneration in multiple systems and cytoplasmic basophilic inclusion bodies were found in the autopsied cases.

An In Vitro Model for Lewy Body-Like Hyaline Inclusion/Astrocytic Hyaline Inclusion: Induction by ER Stress with an ALS-Linked SOD1 Mutation

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) containing mutant Cu/Zn superoxide dismutase 1 (SOD1) are morphological hallmarks of familial amyotrophic lateral sclerosis (FALS) associated with mutant SOD1. However, the mechanisms by which mutant SOD1 contributes to formation of LBHI/Ast-HI in FALS remain poorly defined. Here, we report induction of LBHI/Ast-HI-like hyaline inclusions (LHIs) in vitro by ER stress in neuroblastoma cells. These LHI closely resemble LBHI/Ast-HI in patients with SOD1-linked FALS. LHI and LBHI/Ast-HI share the following features: 1) eosinophilic staining with a pale core, 2) SOD1, ubiquitin and ER resident protein (KDEL) positivity and 3) the presence of approximately 15–25 nm granule-coated fibrils, which are morphological hallmark of mutant SOD1-linked FALS. Moreover, in spinal cord neurons of L84V SOD1 transgenic mice at presymptomatic stage, we observed aberrant aggregation of ER and numerous free ribosomes associated with abnormal inclusion-like structures, presumably early stage neuronal LBHI. We conclude that the LBHI/Ast-HI seen in human patients with mutant SOD1-linked FALS may arise from ER dysfunction.

Exome sequencing identifies a novel TTN mutation in a family with hereditary myopathy with early respiratory failure

Myofibrillar myopathy (MFM) is a group of chronic muscular disorders that show the focal dissolution of myofibrils and accumulation of degradation products. The major genetic basis of MFMs is unknown. In 1993, our group reported a Japanese family with dominantly inherited cytoplasmic body myopathy, which is now included in MFM, characterized by late-onset chronic progressive distal muscle weakness and early respiratory failure. In this study, we performed linkage analysis and exome sequencing on these patients and identified a novel c.90263G>T mutation in the TTN gene (NM_001256850). During the course of our study, another groups reported three mutations in TTN in patients with hereditary myopathy with early respiratory failure (HMERF, MIM #603689), which is characterized by overlapping pathologic findings with MFMs. Our patients were clinically compatible with HMERF. The mutation identified in this study and the three mutations in patients with HMERF were located on the A-band domain of titin, suggesting a strong relationship between mutations in the A-band domain of titin and HMERF. Mutation screening of TTN has been rarely carried out because of its huge size, consisting of 363 exons. It is possible that focused analysis of TTN may detect more mutations in patients with MFMs, especially in those with early respiratory failure.

A novel mutation in PNPLA2 causes neutral lipid storage disease with myopathy and triglyceride deposit cardiomyovasculopathy: A case report and literature review

Mutations in PNPLA2 cause neutral lipid storage disease with myopathy (NLSDM) or triglyceride deposit cardiomyovasculopathy (TGCV). We report a 59-year-old patient with NLSDM/TGCV presenting marked asymmetric skeletal myopathy and cardiomyovasculopathy. Skeletal muscle and endomyocardial biopsies showed cytoplasmic vacuoles containing neutral lipid. Gene analysis revealed a novel homozygous mutation (c.576delC) in PNPLA2. We reviewed 37 genetically-proven NLSDM/TGCV cases; median age was 30 years; distribution of myopathy was proximal (69%) and distal predominant (16%); asymmetric myopathy (right>left) was reported in 41% of the patients. Frequently-affected muscles were posterior compartment of leg (75%), shoulder girdle to upper arm (50%), and paraspinal (33%). Skeletal muscle biopsies showed lipid accumulation in 100% and rimmed vacuoles in 22%. Frequent comorbidities were cardiomyopathy (44%), hyperlipidemia (23%), diabetes mellitus (24%), and pancreatitis (14%). PNPLA2 mutations concentrated in Exon 4-7 without apparent genotype-phenotype correlations. To know the characteristic features is essential for the early diagnosis of NLSDM/TGCV.

FUS/TLS-immunoreactive neuronal and glial cell inclusions increase with disease duration in familial amyotrophic lateral sclerosis with an R521C FUS/TLS mutation.

Abstract Basophilic inclusions (BIs) are pathological features of a subset offrontotemporal lobar degeneration disorders, including sporadic amyotrophic lateral sclerosis (ALS) and familial ALS (FALS). Mutations in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene have recently been identified as a cause of FALS. The FUS/TLS-immunoreactive inclusions are consistently found in cases of frontotemporal lobar degeneration with BIs; however, the association between ALS cases with BIs and FUS/TLS accumulation is not well understood. We used immunohistochemistry to analyze 3 autopsy cases of FALS with the FUS/TLS mutation and with BIs using anti-FUS/TLS antibodies. The disease durations were 1, 3, and 9 years. As the disease duration becomes longer, there were broader distributions of neuronal and glial FUS/TLS-immunoreactive inclusions. As early as 1 year after the onset, BIs, neuronal cytoplasmic inclusions and glial cytoplasmic inclusions were found in the substantia nigra in addition to the anterior horn of the spinal cord. Glial cytoplasmic inclusions are found earlier and in a wider distribution than neuronal cytoplasmic inclusions. The distribution of FUS/TLS-immunoreactive inclusions in FUS/TLS-mutated FALS with BIs was broader than that of BIs alone, suggesting that the pathogenetic mechanism may have originated from the FUS/TLS proteinopathy.

Marked reduction of the Cu/Zn superoxide dismutase polypeptide in a case of familial amyotrophic lateral sclerosis with the homozygous mutation.

We identified a missense mutation of the Cu/Zn superoxide dismutase (SOD) gene (Leu126Ser) in a Japanese family with ALS that included a patient with the homozygous mutation. The content of the Cu/Zn SOD polypeptide in erythrocytes was markedly reduced in the case with the homozygous mutation compared to those with the heterozygous mutation. We speculated that this reduction of the mutant Cu/Zn SOD molecule might be related to the severe clinical phenotype of the case.

GNE myopathy associated with congenital thrombocytopenia: A report of two siblings

GNE myopathy is an autosomal recessive muscular disorder caused by mutations in the gene encoding the key enzyme in sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE/MNK). Here, we report two siblings with myopathy with rimmed vacuoles and congenital thrombocytopenia who harbored two compound heterozygous GNE mutations, p.V603L and p.G739S. Thrombocytopenia, which is characterized by shortened platelet lifetime rather than ineffective thrombopoiesis, has been observed since infancy. We performed exome sequencing and array CGH to identify the underlying genetic etiology of thrombocytopenia. No pathogenic variants were detected among the known causative genes of recessively inherited thrombocytopenia; yet, candidate variants in two genes that followed an autosomal recessive mode of inheritance, including previously identified GNE mutations, were detected. Alternatively, it is possible that the decreased activity of GNE/MNK itself, which would lead to decreased sialic content in platelets, is associated with thrombocytopenia in these patients. Further investigations are required to clarify the association between GNE myopathy and the pathogenesis of thrombocytopenia.

Hoarseness due to bilateral vocal cord paralysis as an initial manifestation of familial amyotrophic lateral sclerosis.

Bulbar palsy is unusual as an initial manifestation of amyotrophic lateral sclerosis (ALS), although common in the advanced stages. In terms of bulbar palsy as a presenting symptom, dysarthria and dysphagia are of common features. Hoarseness, however, is an initial symptom of ALS in only a small number of patients. We report a 43‐year‐old female with hoarseness due to bilateral vocal cord paralysis as the first manifestation of ALS. Gene analysis revealed a heterozygous missense mutation in the SOD1 gene, which resulted in an amino acid substitution of isoleucine 149 by threonine. Hoarseness can be the initial symptom of ALS. Therefore, in cases of bilateral vocal cord paralysis of unknown etiology, ALS should be taken into consideration.

Proteasome dysfunction induces muscle growth defects and protein aggregation.

ABSTRACT The ubiquitin–proteasome and autophagy–lysosome pathways are the two major routes of protein and organelle clearance. The role of the proteasome pathway in mammalian muscle has not been examined in vivo. In this study, we report that the muscle-specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in profound muscle growth defects and a decrease in force production in mice. Specifically, developing muscles in conditional Rpt3-knockout animals showed dysregulated proteasomal activity. The autophagy pathway was upregulated, but the process of autophagosome formation was impaired. A microscopic analysis revealed the accumulation of basophilic inclusions and disorganization of the sarcomeres in young adult mice. Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways. The deletion of a component of the proteasome complex contributed to myofiber degeneration and weakness in muscle disorders that are characterized by the accumulation of abnormal inclusions.

Lower motor neuron disease caused by a novel FUS/TLS gene frameshift mutation

A 26-year-old Japanese man (patient 1, Fig. 1a) suffered from distal weakness of the right leg. After a few months, he began to stumble frequently and gradually developed distal weakness of the right upper limb. He was then admitted to our hospital. On physical examination, he was alert and his mental status was normal. He had no cognitive abnormalities. He had distal dominant muscular atrophy and weakness in the right extremities. His ocular movements were normal, and he did not display any signs of dysarthria, dysphasia, facial weakness, or tongue abnormalities. Muscle tonus was normal, no fasciculation was seen in any of the extremities and the deep tendon reflex was decreased to absent. He had no urinary or bowel disturbance, and no sensory impairment nor cerebellar ataxia. A nerve conduction study revealed a decrease in F-wave frequency in the right extremities. Electromyography (EMG) demonstrated denervation and renervation potentials in all extremities but fasciculation potentials were absent in all extremities. Routine blood analysis, cerebrospinal fluid analysis and spine MRI revealed no abnormalities. Approximately 6 months after the onset of symptoms, muscle weakness and atrophy had spread to all extremities symmetrically. Muscle weakness and atrophy increased rapidly without upper motor neuron signs, and the patient died of respiratory failure about 1 year after onset. Autopsy was not performed. The mother of patient 1, who was 52 years old (patient 2, Fig. 1a), and the maternal aunt, who was 42 years old (patient 3, Fig. 1a), both experienced weakness and wasting of the right leg in the first decade, and came to walk with dragging of the right leg. Neurological examinations revealed them to be objectively normal except for weakness and wasting of the right leg and a dragging gait. However, EMG demonstrated evidence of mild renervation potentials in all extremities. After progressing for several years, their subjective symptoms remained unchanged until their current ages, which were 52 years old in patient 2 and 42 years old in patient 3, respectively. We routinely performed the mutation analysis of superoxide dismutase 1 (SOD 1) gene, TAR-DNA binding protein 43 (TARDBP) gene and fused in sarcoma/translocation in liposarcoma (FUS/TLS) gene in all patients suspected of familial motor neuron disease with informed consent. Genetic analysis of patient 1 was thus performed. DNA extraction and genotyping were performed using standard protocols as described elsewhere [1]. No mutation of the SOD 1 gene and TARDBP gene was confirmed. We also sequenced all exons of FUS/TLS gene. The allelespecific cloning of the FUS/TLS gene was performed after cloning of the genomic DNA of both alleles, and we identified a novel heterozygous c.1420_1421insGT missense mutation located in exon 14 encoding the RNA-recognition motif (Fig. 1b), and result in an amino acid exchange from Glycine to Valine at protein 472, which is followed by reading frameshift at this point (p.Gly472ValfsX57). Exon 14 of the FUS/TLS gene was sequenced in 100 Japanese healthy controls and no mutation like this case was detected. No expansion of the androgen receptor CAG repeat number and no elongation M. Hara M. Minami (&) S. Kamei Division of Neurology, Department of Medicine, Nihon University School of Medicine, 30-1 Oyaguchikamicho, Itabashi-ku, Tokyo 173-8610, Japan e-mail: mm@med.nihon-u.ac.jp