Yoko Fukuda

Mutations in COQ2 in familial and sporadic multiple-system atrophy the multiple-system atrophy research collaboration

BACKGROUND Multiple-system atrophy is an intractable neurodegenerative disease characterized by autonomic failure in addition to various combinations of parkinsonism, cerebellar ataxia, and pyramidal dysfunction. Although multiple-system atrophy is widely considered to be a nongenetic disorder, we previously identified multiplex families with this disease, which indicates the involvement of genetic components. METHODS In combination with linkage analysis, we performed whole-genome sequencing of a sample obtained from a member of a multiplex family in whom multiple-system atrophy had been diagnosed on autopsy. We also performed mutational analysis of samples from members of five other multiplex families and from a Japanese series (363 patients and two sets of controls, one of 520 persons and one of 2383 persons), a European series (223 patients and 315 controls), and a North American series (172 patients and 294 controls). On the basis of these analyses, we used a yeast complementation assay and measured enzyme activity of parahydroxybenzoate-polyprenyl transferase. This enzyme is encoded by the gene COQ2 and is essential for the biosynthesis of coenzyme Q10. Levels of coenzyme Q10 in lymphoblastoid cells and brain tissue were measured on high-performance liquid chromatography. RESULTS We identified a homozygous mutation (M78V-V343A/M78V-V343A) and compound heterozygous mutations (R337X/V343A) in COQ2 in two multiplex families. Furthermore, we found that a common variant (V343A) and multiple rare variants in COQ2, all of which are functionally impaired, are associated with sporadic multiple-system atrophy. The V343A variant was exclusively observed in the Japanese population. CONCLUSIONS Functionally impaired variants of COQ2 were associated with an increased risk of multiple-system atrophy in multiplex families and patients with sporadic disease, providing evidence of a role of impaired COQ2 activities in the pathogenesis of this disease. (Funded by the Japan Society for the Promotion of Science and others.).

Mutations for Gaucher Disease Confer High Susceptibility to Parkinson Disease

BACKGROUND Increased frequency of pathogenic variants in GBA, the causative gene for Gaucher disease, has been suggested to be associated with Parkinson disease (PD). OBJECTIVES To conduct comprehensive resequencing of GBA to identify all sequence variants and to investigate the association of these variants with PD. DESIGN Case-control study. SETTING Multicenter university-based study. PARTICIPANTS Five hundred thirty-four patients with PD, 34 families in which multiple patients with PD are present, and 544 control subjects. MAIN OUTCOME MEASURES Disease status and GBA variations. RESULTS Comprehensive resequencing of GBA in 534 patients with PD and 544 controls revealed 27 sequence variants: 11 pathogenic variants associated with Gaucher disease, 11 nonsynonymous variants not associated with Gaucher disease, and 5 synonymous variants. Fifty patients with PD (9.4%) had 1 of the 11 pathogenic variants in the heterozygous state, whereas only 2 controls (0.37%) had such variants (odds ratio, 28.0). Among the pathogenic variants, R120W and L444P/RecNciI were highly prevalent, and each showed a significant association with PD. Furthermore, other rare pathogenic variants were found in 13 patients with PD but not in the controls, further confirming the role of these rare variants in the susceptibility to PD. Patients with PD carrying pathogenic variants were significantly younger than those not carrying them. In addition, concordance of PD states and pathogenic variants was observed in 8 multiplex families with PD. CONCLUSION Heterozygous pathogenic variants in GBA confer a high risk for sporadic PD, even for familial clustering, and are associated with significantly earlier age at onset of disease.

A homozygous mutation of C12orf65 causes spastic paraplegia with optic atrophy and neuropathy (SPG55).

Background Autosomal recessive hereditary spastic paraplegias (AR-HSP) constitute a heterogeneous group of neurodegenerative diseases involving pyramidal tracts dysfunction. The genes responsible for many types of AR-HSPs remain unknown. We attempted to identify the gene responsible for AR-HSP with optic atrophy and neuropathy. Methods The present study involved two patients in a consanguineous Japanese family. Neurologic examination and DNA analysis were performed for both patients, and a skin biopsy for one. We performed genome-wide linkage analysis involving single nucleotide polymorphism arrays, copy-number variation analysis, and exome sequencing. To clarify the mitochondrial functional alteration resulting from the identified mutation, we performed immunoblot analysis, mitochondrial protein synthesis assaying, blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis, and respiratory enzyme activity assaying of cultured fibroblasts of the patient and a control. Results We identified a homozygous nonsense mutation (c.394C>T, p.R132X) in C12orf65 in the two patients in this family. This C12orf65 mutation was not found in 74 Japanese AR-HSP index patients without any mutations in previously known HSP genes. This mutation resulted in marked reduction of mitochondrial protein synthesis, followed by functional and structural defects in respiratory complexes I and IV. Conclusions This novel nonsense mutation in C12orf65 could cause AR-HSP with optic atrophy and neuropathy, resulting in a premature stop codon. The truncated C12orf65 protein must lead to a defect in mitochondrial protein synthesis and a reduction in the respiratory complex enzyme activity. Thus, dysfunction of mitochondrial translation could be one of the pathogenic mechanisms underlying HSPs.

Dominant Mutations in RP1L1 Are Responsible for Occult Macular Dystrophy

Occult macular dystrophy (OMD) is an inherited macular dystrophy characterized by progressive loss of macular function but normal ophthalmoscopic appearance. Typical OMD is characterized by a central cone dysfunction leading to a loss of vision despite normal ophthalmoscopic appearance, normal fluorescein angiography, and normal full-field electroretinogram (ERGs), but the amplitudes of the focal macular ERGs and multifocal ERGs are significantly reduced at the central retina. Linkage analysis of two OMD families was performed by the SNP High Throughput Linkage analysis system (SNP HiTLink), localizing the disease locus to chromosome 8p22-p23. Among the 128 genes in the linkage region, 22 genes were expressed in the retina, and four candidate genes were selected. No mutations were found in the first three candidate genes, methionine sulfoxide reductase A (MSRA), GATA binding 4 (GATA4), and pericentriolar material 1 (PCM1). However, amino acid substitution of p.Arg45Trp in retinitis pigmentosa 1-like 1 (RP1L1) was found in three OMD families and p.Trp960Arg in a remaining OMD family. These two mutations were detected in all affected individuals but in none of the 876 controls. Immunohistochemistry of RP1L1 in the retina section of cynomolgus monkey revealed expression in the rod and cone photoreceptor, supporting a role of RP1L1 in the photoreceptors that, when disrupted by mutation, leads to OMD. Identification of RP1L1 mutations as causative for OMD has potentially broader implications for understanding the differential cone photoreceptor functions in the fovea and the peripheral retina.

ERBB4 Mutations that Disrupt the Neuregulin-ErbB4 Pathway Cause Amyotrophic Lateral Sclerosis Type 19

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by the degeneration of motor neurons and typically results in death within 3-5 years from onset. Familial ALS (FALS) comprises 5%-10% of ALS cases, and the identification of genes associated with FALS is indispensable to elucidating the molecular pathogenesis. We identified a Japanese family affected by late-onset, autosomal-dominant ALS in which mutations in genes known to be associated with FALS were excluded. A whole- genome sequencing and parametric linkage analysis under the assumption of an autosomal-dominant mode of inheritance with incomplete penetrance revealed the mutation c.2780G>A (p. Arg927Gln) in ERBB4. An extensive mutational analysis revealed the same mutation in a Canadian individual with familial ALS and a de novo mutation, c.3823C>T (p. Arg1275Trp), in a Japanese simplex case. These amino acid substitutions involve amino acids highly conserved among species, are predicted as probably damaging, and are located within a tyrosine kinase domain (p. Arg927Gln) or a C-terminal domain (p. Arg1275Trp), both of which mediate essential functions of ErbB4 as a receptor tyrosine kinase. Functional analysis revealed that these mutations led to a reduced autophosphorylation of ErbB4 upon neuregulin-1 (NRG-1) stimulation. Clinical presentations of the individuals with mutations were characterized by the involvement of both upper and lower motor neurons, a lack of obvious cognitive dysfunction, and relatively slow progression. This study indicates that disruption of the neuregulin-ErbB4 pathway is involved in the pathogenesis of ALS and potentially paves the way for the development of innovative therapeutic strategies such using NRGs or their agonists to upregulate ErbB4 functions.

SNP HiTLink: a high-throughput linkage analysis system employing dense SNP data

BackgroundDuring this recent decade, microarray-based single nucleotide polymorphism (SNP) data are becoming more widely used as markers for linkage analysis in the identification of loci for disease-associated genes. Although microarray-based SNP analyses have markedly reduced genotyping time and cost compared with microsatellite-based analyses, applying these enormous data to linkage analysis programs is a time-consuming step, thus, necessitating a high-throughput platform.ResultsWe have developed SNP HiTLink (SNPHi gh T hroughput Link age analysis system). In this system, SNP chip data of the Affymetrix Mapping 100 k/500 k array set and Genome-Wide Human SNP array 5.0/6.0 can be directly imported and passed to parametric or model-free linkage analysis programs; MLINK, Superlink, Merlin and Allegro. Various marker-selecting functions are implemented to avoid the effect of typing-error data, markers in linkage equilibrium or to select informative data.ConclusionThe results using the 100 k SNP dataset were comparable or even superior to those obtained from analyses using microsatellite markers in terms of LOD scores obtained. General personal computers are sufficient to execute the process, as runtime for whole-genome analysis was less than a few hours. This system can be widely applied to linkage analysis using microarray-based SNP data and with which one can expect high-throughput and reliable linkage analysis.

Posterior column ataxia with retinitis pigmentosa in a Japanese family with a novel mutation in FLVCR1

Posterior column ataxia with retinitis pigmentosa (PCARP) is an autosomal recessive neurodegenerative disorder characterized by retinitis pigmentosa and sensory ataxia. Previous studies of PCARP in two families showed a linkage to 1q31–q32. However, detailed investigations on the clinical presentations as well as molecular genetics of PCARP have been limited. Here, we describe a Japanese consanguineous family with PCARP. Two affected siblings suffered from childhood-onset retinitis pigmentosa and slowly progressive sensory ataxia. They also showed mild mental retardation, which has not been described in patients with PCARP. Parametric linkage analysis using high-density single nucleotide polymorphism arrays supported a linkage to the same locus. Target capture and high-throughput sequencing technologies revealed a novel homozygous c.1477G>C (G493R) mutation in FLVCR1, which cosegregated with the disease. A recent study has identified three independent mutations in FLVCR1 in the original and other families. Our results further confirmed that PCARP is caused by mutations in FLVCR1.

Autosomal-recessive complicated spastic paraplegia with a novel lysosomal trafficking regulator gene mutation

Background Autosomal-recessive hereditary spastic paraplegias (AR-HSP) consist of a genetically diverse group of neurodegenerative diseases characterised by pyramidal tracts dysfunction. The causative genes for many types of AR-HSP remain elusive. We tried to identify the gene mutation for AR-HSP with cerebellar ataxia and neuropathy. Methods This study included two patients in a Japanese family with their parents who are first cousins. Neurological examination and gene analysis were conducted in the two patients and two normal family members. We undertook genome-wide linkage analysis employing single nucleotide polymorphism arrays using the two patients’ DNAs and exome sequencing using one patients sample. Results We detected a homozygous missense mutation (c.4189T>G, p.F1397V) in the lysosomal trafficking regulator (LYST) gene, which is described as the causative gene for Chédiak–Higashi syndrome (CHS). CHS is a rare autosomal-recessive syndrome characterised by hypopigmentation, severe immune deficiency, a bleeding tendency and progressive neurological dysfunction. This mutation was co-segregated with the disease in the family and was located at well-conserved amino acid. This LYST mutation was not found in 200 Japanese control DNAs. Microscopic observation of peripheral blood in the two patients disclosed large peroxidase-positive granules in both patients’ granulocytes, although they had no symptoms of immune deficiency or bleeding tendency. Conclusions We diagnosed these patients as having adult CHS presenting spastic paraplegia with cerebellar ataxia and neuropathy. The clinical spectrum of CHS is broader than previously recognised. Adult CHS must be considered in the differential diagnosis of AR-HSP.

Adult-onset leukoencephalopathies with vanishing white matter with novel missense mutations in EIF2B2, EIF2B3, and EIF2B5.

Leukoencephalopathy with vanishing white matter (VWM) is a type of leukoencephalopathy with autosomal recessive inheritance. Magnetic resonance imaging (MRI) reveals diffuse leukoencephalopathy with lesions having cerebrospinal fluid (CSF)-like signals. The clinical presentations include progressive cerebellar ataxia, spasticity, and mental decline. The course is chronic progressive with episodes of rapid deterioration following a minor head trauma. Mutations in the five gene-encoding subunits of the translation initiation factor eIF2B, EIF2B1-5, have been identified as the causative mutations for VWM. Although the age at onset of VWM is usually 2–6 years, patients with adult onset have been described. All adult-onset cases except one have been found to be associated with mutations in EIF2B5 [1]. We report cases of adult-onset VWM with novel missense mutations in EIF2B2, EIF2B3, and EIF2B5, which showed decreased eIF2B activities.

A mutation database for amyotrophic lateral sclerosis

An amyotrophic lateral sclerosis (ALS) mutation database has been constructed as a publicly accessible online resource for recording the nucleotide and amino acid variants identified in genes associated with ALS, along with corresponding clinical conditions. The database currently consists of more than 600 entries, including about 180 unique variants found in 25 disease‐causative or disease‐related genes. In addition to published data collected from literature, novel variants identified by microarray resequencing in our laboratory are incorporated into the database. Every reported gene has a respective page that provides information on its variation positions with various statistics, clinical characteristics, and primary references, as well as gene‐sequence and protein‐structure information that will assist in assessing variation significance. Users can access a homology search function to find variations in arbitrary sequences of interest and to check if they have already been described in the database. This database is expected to fulfill an essential need in terms of integrating comprehensive information on genetic and clinical data related to ALS, which will subsequently deepen our understanding of the possible mechanisms of the disease, as well as help with the clinical practice and treatment of ALS. The database is accessible at: https://reseq.lifesciencedb.jp/resequence/SearchDisease.do?targetId=1. Data submission is open to all researchers and is highly encouraged. Hum Mutat 31:1003–1010, 2010.