DNA analysis of benign adult familial myoclonic epilepsy reveals associations between the pathogenic TTTCA repeat insertion in SAMD12 and the nonpathogenic TTTTA repeat expansion in TNRC6A.

Benign adult familial myoclonic epilepsy (BAFME) is an autosomal dominant disease characterized by adult-onset tremulous hand movement, myoclonus, and infrequent epileptic seizures. Recently, intronic expansion of unstable TTTCA/TTTTA pentanucleotide repeats in SAMD12, TNRC6A, or RAPGEF2 was identified as pathological mutations in Japanese BAFME pedigrees. To confirm these mutations, we performed a genetic analysis on 12 Japanese BAFME pedigrees. A total of 143 participants, including 43 familial patients, 5 suspected patients, 3 sporadic nonfamilial patients, 22 unaffected familial members, and 70 unrelated controls, were screened for expanded abnormal pentanucleotide repeats in SAMD12, TNRC6A, RAPGEF2, YEAT2, MARCH6, and STARD7. DNA samples were analyzed using Southern blotting, long-range polymerase chain reaction (PCR), repeat-primed PCR, and long-range PCR followed by Southern blotting. Of the 51 individuals with clinically diagnosed or suspected BAFME, 49 carried a SAMD12 allele with an expanded TTTCA/TTTTA pentanucleotide repeat. Genetic and clinical anticipation was observed. As in previous reports, the one patient with homozygous mutant alleles showed more severe symptoms than the heterozygous carriers. In addition, screening for expanded pentanucleotide repeats in TNRC6A revealed that the frequency of expanded TTTTA repeat alleles in the BAFME group was significantly higher than in the control group. All patients who were clinically diagnosed with BAFME, including those in the original family reported by Yasuda, carried abnormally expanded TTTCA/TTTTA repeat alleles of SAMD12. Patients with BAFME also frequently carried a TTTTA repeat expansion in TNRC6A, suggesting that there may be unknown factors in the ancestry of patients with BAFME that make pentanucleotide repeats unstable.

Mutations of optineurin in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) has its onset in middle age and is a progressive disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1), ANG encoding angiogenin, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS). However, these genetic defects occur in only about 20-30% of cases of FALS, and most genes causing FALS are unknown. Here we show that there are mutations in the gene encoding optineurin (OPTN), earlier reported to be a causative gene of primary open-angle glaucoma (POAG), in patients with ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Analysis of cell transfection showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B (NF-kappaB), and the E478G mutation revealed a cytoplasmic distribution different from that of the wild type or a POAG mutation. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic inclusions. Furthermore, TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also noticeably immunolabelled by anti-OPTN antibodies. Our findings strongly suggest that OPTN is involved in the pathogenesis of ALS. They also indicate that NF-kappaB inhibitors could be used to treat ALS and that transgenic mice bearing various mutations of OPTN will be relevant in developing new drugs for this disorder.

Clinicopathologic study on an ALS family with a heterozygous E478G optineurin mutation.

We investigated a family manifesting amyotrophic lateral sclerosis (ALS) with a heterozygous E478G mutation in the optineurin (OPTN) gene. Clinically, slow deterioration of motor function, mood and personality changes, temporal lobe atrophy on neuroimaging, and bizarre finger deformity were noted. Neuropathologically, TAR DNA-binding protein 43 (TDP-43)-positive neuronal intracytoplasmic inclusions were observed in the spinal and medullary motor neurons. In these cells, the immunoreactivity of nuclear TDP-43 was reduced. Consecutive sections revealed that the inclusions were also reactive with anti-ubiquitin and anti-p62 antibodies, but noticeably negative for OPTN. In addition, TDP-43/p62-positive glial cytoplasmic inclusions (GCIs) were scattered throughout the spinal cord and the medullary motor nuclei. Furthermore, Golgi fragmentation was identified in 70% of the anterior horn cells (AHCs). The presence of AHCs with preserved nuclear TDP-43 and a fragmented Golgi apparatus, which are unrecognizable in sporadic ALS, indicates that patients with the E4787G OPTN mutation would manifest Golgi fragmentation before loss of nuclear TDP-43. In the neocortex, GCIs were sparsely scattered among the primary motor and temporal cortices, but no neuronal TDP-43-positive inclusions were detected. In the amygdala and the ambient gyrus, argyrophilic grains and ballooned neurons were seen. The thorough neuropathologic investigations performed in this work demonstrated that OPTN-positive inclusion bodies, if any, were not prominent. We postulate that optineurinopathy is closely linked with TDP-proteinopathy and speculate that this heterozygous E478G mutation would cause ALS by acting through a dominant-negative mechanism.

Expression of Kruppel-like factor 5 gene in human brain and association of the gene with the susceptibility to schizophrenia.

Genome-wide gene expression analysis using DNA microarray technology is a potential tool to search for unexpected genes that have a susceptibility to schizophrenia. We carried out a microarray analysis in the postmortem prefrontal cortex and found that the expression of the KLF5 gene, whose locus is on 13q21, was down-regulated in schizophrenia patients. This result was confirmed by a Western blot analysis. In a genetic study, we found that a polymorphism of the KLF5 gene (-1593T>C) was associated with schizophrenia. We identified neurons in the prefrontal cortex of human brain as sites of KLF5 expression by in situ hybridization and immunohistochemistry. KLF5 was immunohistochemically localized in granular and pyramidal cells in the hippocampus, which are the principal source of glutamatergic neurotransmission. These findings suggest that the KLF5 gene is a novel schizophrenia-susceptibility gene, and that the expression of the gene is involved in the pathophysiology of schizophrenia via glutamatergic neurotransmission.

Multiple Proteinopathies in Familial ALS Cases With Optineurin Mutations.

Optineurin (OPTN) is a causative gene in familial amyotrophic lateral sclerosis (ALS) with transactivation response element DNA-binding protein of 43 kDa (TDP-43) protein pathology. Here, we report multiple proteinopathies in familial ALS cases with OPTN mutations. We examined the TDP-43, tau, and α-synuclein pathology of ALS cases with OPTN mutations including 2 previously reported cases (Cases 1 and 2) and 1 newly autopsied case (Case 3) that was clinically diagnosed as ALS and Parkinson disease with a heterozygous E478G OPTN mutation. Pathologic examination of Case 3 showed motor neuron degeneration and depigmentation of the substantia nigra. Neurofibrillary tangles (NFTs) were seen in the hippocampus, pontine tegmentum, and spinal cord. Accumulation of multiple proteins including phosphorylated TDP-43-positive neuronal cytoplasmic inclusions, phosphorylated tau (AT8)-positive NFTs, and α-synuclein-positive Lewy bodies were observed in the substantia nigra. The other 2 cases had a similar distribution of tau pathology, but lacked synuclein pathology. Consecutive sections of Case 3 revealed pTDP-43, AT8, and α-synuclein-positive inclusions in the same neuron and double immunofluorescence staining showed aggregation of different proteins (tau and α-synuclein, or tau and TDP-43) in the same neuron. Our results support the notion that OPTN mutations may lead to multiple proteins aggregation and neuronal degeneration.

The effect of tau genotype on clinical features in FTDP-17.

The clinical phenotype of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) varies. This variability is seen not only between kindreds with different mutations but also in families sharing the same mutation. Inheritance of tau haplotype (H1) and genotype (H1/H1) has been established as a risk factor for some neurodegenerative disorders with parkinsonism. We assessed the effect of tau polymorphism on the clinical features of FTDP-17 in 61 cases from 30 separately ascertained families with four different tau mutations, including P301L, +16, N279K, and P301S. There were no significant differences of age at symptomatic onset and disease duration between H1/H1 and H1/H2 genotypes. The comparison between tau genotype and type of initial clinical sign showed an association between the H1/H1 genotype and parkinsonian phenotype and between the H1/H2 genotype and frontotemporal dementia phenotype (OR=11.7; 95% confidence interval, 1.4-98.7; P=0.008). Our results suggest that tau genotype does not influence the disease course. However, it may predispose to a specific clinical sign in the early stage of FTDP-17.

Possible reduced penetrance of expansion of 44 to 47 CAG/CAA repeats in the TATA-binding protein gene in spinocerebellar ataxia type 17.

BACKGROUND: Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant cerebellar ataxia caused by expansion of CAG/CAA trinucleotide repeats in the TATA-binding protein (TBP) gene. Because the number of triplets in patients with SCA17 in previous studies ranged from 43 to 63, the normal number of trinucleotide units has been considered to be 42 or less. However, some healthy subjects in SCA17 pedigrees carry alleles with the same number of expanded repeats as patients with SCA17. OBJECTIVE: To investigate the minimum number of CAG/CAA repeats in the TBP gene that causes SCA17. DESIGN: We amplified the region of the TBP gene containing the CAG/CAA repeat by means of polymerase chain reaction and performed fragment and sequence analyses. PATIENTS: The subjects included 734 patients with SCA (480 patients with sporadic SCA and 254 patients with familial SCA) without CAG repeat expansions at the SCA1, SCA2, Machado-Joseph disease, SCA6, SCA7, or dentatorubral-pallidolluysian atrophy loci, with 162 healthy subjects, 216 patients with Parkinson disease, and 195 with Alzheimer disease as control subjects. RESULTS: Eight patients with SCA possessed an allele with more than 43 CAG/CAA repeats. Among the non-SCA groups, alleles with 43 to 45 repeats were seen in 3 healthy subjects and 2 with Parkinson disease. In 1 SCA pedigree, a patient with possible SCA17 and her healthy sister had alleles with 45 repeats. A 34-year-old man carrying alleles with 47 and 44 repeats (47/44) had developed progressive cerebellar ataxia and myoclonus at 25 years of age, and he exhibited dementia and pyramidal signs. He was the only affected person in his pedigree, although his father and mother carried alleles with mildly expanded repeats (44/36 and 47/36, respectively). In another pedigree, 1 patient carried a 43-repeat allele, whereas another patient had 2 normal alleles, indicating that the 43-repeat allele may not be pathologic in this family. CONCLUSIONS: We estimate that 44 CAG/CAA repeats is the minimum number required to cause SCA17. However, the existence of unaffected subjects with mildly expanded triplets suggests that the TBP gene mutation may not penetrate fully. Homozygosity of alleles with mildly expanded triplet repeats in the TBP gene might contribute to the pathologic phenotype.

[A case of myeloradiculitis as a complication of visceral larva migrans due to Ascaris suum].

A 35-year-old man noticed a tingling sensation and subsequent slight weakness in the distal part of the left lower extremities, which extended to the left thigh over the following 8 months, when he developed a urinary disturbance. He was admitted to a local hospital and diagnosed as having myelitis, because of the presence of a gadolinium (Gd)-enhanced lesion in the lumbar spinal cord corresponding to the tenth thoracic vertebra (Th10) level. The symptoms disappeared along with some residual sensory disturbance following intravenous administration of 1 g of methylprednisolone (IVMP) for 3 days. However, 4 months later, the patient gradually developed gait disturbance, dysuria, impotence, and fecal incontinence, and was admitted to our hospital in May 2001. A neurological examination revealed the presence of myeloradiculopathy causing a slight weakness in the left hamstring muscles, with positive Babinski and Lasegue signs on the same side. In addition, deep tendon reflexes were absent in the 4 extremities and vibration sense was moderately decreased in the lower extremities, though the results of electrophysiological tests of motor and sensory nerves were normal. A magnetic resonance imaging (MRI) study showed a Gd-enhanced lesion in the spinal cord at the Th8-9 vertebrae level, which was 1 to 2 vertebrae above the initially detected lesion. A lumbar puncture yielded cerebrospinal fluid (CSF) containing mononuclear cells at 7/mm3 that were comprised of an increasd number of CD4+CD25+ activated helper T cells; however, no myelin basic protein or oligoclonal IgG band was present. Serological examinations were negative for the presence of collagen/vascular disorders as well as viral infection due to CMV, EBV, HSV, VZV, and HTLV-1, however, positive for specific antibodies against Ascaris suum in both serum and the CSF, confirming the diagnosis of chronic myeloradiculitis due to visceral larva migrans. The patient was first treated with a single course of a daily 600-mg dose of oral albendazole for 4 weeks, which was resulted in restoration of muscle weakness, as well as disappearance of the Lasègue sign. However, in contrast to the clinical improvement, the CSF sample obtained immediately after completing the treatment showed a worsening trend, as the CSF cell count had increased with a manifestation of marked eosinophilia and CD4+CD25+ cells were also increased. Thereafter, 3-day IVMP treatment was performed twice in cojunction with 3 courses of oral albendazole therapy for the subsequent 4 months, which resulted in normalization of all laboratory measurements concerning the CSF along with a decrement trend in serum and CSF antibody titers specific to Ascaris suum. Our results suggest that neurological involvement due to visceral larva migrans can be efficaciously treated with not only helminthic drugs but also intravenous corticosteroids.

Phenotypic heterogeneity within a new family with the MAPT p301s mutation.

Mutations in the gene encoding the microtubule-associated protein tau (MAPT) cause frontotemporal dementia and parkinsonism linked to chromosome 17. Clinical variability is seen not only among families with different mutations, but also among family members with the same mutation. We investigated a newly identified familial frontotemporal dementia and parkinsonism family. The disease was of early onset and was inherited as an autosomal dominant trait. Clinically, parkinsonism was the prominent and often early feature, and it preceded dementia. Three autopsied cases shared involvement predominantly in the frontal and temporal lobes and also in the subcortical nuclei, including substantia nigra, globus pallidus, and subthalamic nucleus, that microscopically consisted of neuronal loss, microvacuolation, and astrocytic fibrosis. Immunohistochemistry demonstrated neuropil threads, ballooned cells, and glial fibrillary tangles. Sequencing analysis of the MAPT gene showed an alteration in one allele, resulting in a P301S substitution. These findings suggest that the MAPT P301S mutation can cause pathologically subcortical-predominant, neuropil thread-rich, tau-containing lesions, which could result in consistent parkinsonism. Our study confirms the notion that the phenotype observed in affected individuals from P301S MAPT mutation families is heterogeneous and is broader than the phenotypes seen to date in affected family members carrying other MAPT mutations.

Contribution of the interleukin-1beta gene polymorphism in multiple system atrophy.

We studied genetic polymorphisms in the promoter region at position -511 of the interleukin (IL) -1beta gene (IL-1B-511) and at position -889 of the IL-1alpha gene (IL-1A-889), in 111 Japanese patients with multiple system atrophy (MSA) and 160 controls. The distribution of IL-1B-511 was significantly different between MSA patients and controls, because of the under-representation of patients with homozygotes for allele 2 (IL-1B-511*2), a high producer of IL-1beta. The frequency of IL-1A-889*2, a high secretor of IL-1alpha, was also decreased in MSA patients. Our findings suggest that abnormal cytokine expression may be implicated in the pathogenesis of MSA.

SCA8 repeat expansion: large CTA/CTG repeat alleles are more common in ataxic patients, including those with SCA6.

We analyzed the SCA8 CTA/CTG repeat in a large group of Japanese subjects. The frequency of large alleles (85-399 CTA/CTG repeats) was 1.9% in spinocerebellar ataxia (SCA), 0.4% in Parkinson disease, 0.3% in Alzheimer disease, and 0% in a healthy control group; the frequency was significantly higher in the group with SCA than in the control group. Homozygotes for large alleles were observed only in the group with SCA. In five patients with SCA from two families, a large SCA8 CTA/CTG repeat and a large SCA6 CAG repeat coexisted. Age at onset was correlated with SCA8 repeats rather than SCA6 repeats in these five patients. In one of these families, at least one patient showed only a large SCA8 CTA/CTG repeat allele, with no large SCA6 CAG repeat allele. We speculate that the presence of a large SCA8 CTA/CTG repeat allele influences the function of channels such as alpha(1A)-voltage-dependent calcium channel through changing or aberrant splicing, resulting in the development of cerebellar ataxia, especially in homozygous patients.