Friday, October 21, 2011


Nature | Letter

Mutations of optineurin in amyotrophic lateral sclerosis

Nature
465,
223–226
(13 May 2010)
doi:10.1038/nature08971
Received
Accepted
Published online
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 cord1. Most cases of ALS are sporadic, but about 10% are familial. Genes known to cause classic familial ALS (FALS) are superoxide dismutase 1 (SOD1)2, ANG encoding angiogenin3, TARDP encoding transactive response (TAR) DNA-binding protein TDP-43 (ref. 4) and fused in sarcoma/translated in liposarcoma (FUS, also known as TLS)5, 6. 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)7, 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-κB), 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-κB 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.

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We analysed six Japanese individuals from consanguineous marriages who had ALS; two of them were siblings, the others were from independent families. We used homozygosity mapping, which has been shown to identify a locus of a disease-causing gene from as few as three individuals8. We performed a genome-wide scan of single nucleotide polymorphisms (SNPs) by using the GeneChip Human Mapping 500K Array Set (Affymetrix), and selected for the run of homozygous SNPs (RHSs) more than 3 centimorgans in length. Under this condition, the RHSs are able to retrieve more than 98% of the entire length of the autozygous segments created as a result of a first-cousin or second-cousin marriage (Supplementary Information)8. We extracted RHSs of six individuals (Supplementary Fig. 1a). A region (hg18: 12,644,480–15,110,539) in chromosome 10, which was an overlap among four subjects, was chosen as the primary candidate region (Supplementary Fig. 1b). Assuming that subjects ii, iii, v and vi had the same disease gene, the chance that the overlap had the disease gene was Pii+iii+v+vi = 0.935 (Supplementary Information). We listed up to 17 candidate genes in the region and sequenced their exons (Supplementary Fig. 1c). We detected a deletion of exon 5 in the OPTN (also known as FIP-2 (ref. 9)) gene in two siblings (Fig. 1a, family 1, subjects 1 and 2). PCR with a forward primer of exon 4 and a reverse primer of intron 5 revealed a 2.5-kilobase (kb) band in the control, V-3 and IV-1, and a 0.7-kb band in IV-1, subject 1 and subject 2 (Fig. 1b). Direct sequence analysis of the short band showed the joining of the 5′ part of AluJb in intron 4 and the 3′ part of AluSx in intron 5 with 12-base-pair (bp) microhomology (Fig. 1c). Thus, the deletion resulted from Alu-mediated recombination. We also found a homozygous nonsense c.1502C>T mutation (Q398X, exon 12) in the gene in one individual with ALS (Fig. 1d, e, family 2, subject 3). For the other three subjects, we found neither mutations nor copy number changes in the OPTN gene, although we did not completely exclude the possibility of mutations in introns or intergenic regions in the gene. We extended our analysis of OPTN to ten additional individuals from consanguineous marriages who had ALS, 76 individuals with familial ALS and 597 individuals with sporadic ALS (SALS). We found the Q398X mutation in a sporadic individual (subject 4, family 3; Fig. 1d). Subjects 3 and 4, who were not related according to their family history, shared their haplotype for a 0.9-megabase (Mb) region (hg18: chr10: 12,973,261–13,879,735) containing the OPTN gene (Supplementary Table 1). We investigated a total of 170 copies of chromosome 10 from 85 Japanese subjects genotyped for the HapMap3 project, and found that the incidental length of haplotype sharing around OPTN gene was at most 320 kb. Given that a haplotype sharing of 0.9 Mb rarely occurs by chance, the mutation is likely to have been derived from a single ancestor (Supplementary Fig. 1d). Subjects 1 and 2 shared their haplotype for an 8.3-Mb region (hg18: chr10: 6,815,934–14,842,351), which contained the OPTN gene and was different from that in subjects 3 and 4 (Supplementary Table 1).
Figure 1: Exon 5 deletion, nonsense and missense mutations of the OPTN gene.
Exon 5 deletion, nonsense and missense mutations of the OPTN gene.
a, Family 1. The filled circle or square indicate the affected individuals; the arrows indicate the probands. b, Agarose gel electrophoretogram. Subject 1 (V-1) and subject 2 (V-2) showed lack of exon 5 PCR product and shortened product of exon 4 to intron 5. c, Chromatogram with OPTN deletion of exon 5 and schematic structure of deleted gene. d, Families 2 and 3. Dots indicate heterozygous carriers. e, Chromatograms from index subjects with OPTN mutation of c.1502 C>T. Homozygous mutation is in red, and the mutation is indicated by using the single-letter amino-acid code. f, Family 4. *DNA sample could not be obtained. Numerals show the age at death. g, Chromatograms from index subjects with the OPTN mutation of c.1743A>G. The heterozygous mutation is marked by the square.
In the screening of ALS families, we identified a heterozygous missense mutation (c1743A>G, E478G, exon14, Fig. 1g) of OPTN in four individuals with ALS in two families with ALS. Subjects 5 and 6 were sisters, and the pedigree suggests that the mutation had an autosomal dominant trait with incomplete penetrance (Fig. 1f, family 4). Subjects 7 and 8 (family 5) were brothers. Although these families are not related according to their family history, subjects 5–8 shared their haplotype for 2.3 Mb (hg18: chr10: 11,460,985–13,703,017, Supplementary Table 3), again suggesting that the mutation was derived from a single ancestor. Indeed, the Q398X nonsense and E478G missense mutations were not observed in 781 healthy Japanese volunteers as well as in over 6,800 (including 1,728 Japanese) individuals in the glaucoma studies, where the entire coding region of the gene was investigated (Supplementary Table 2). Collectively, the mutation was absent over a total of 5,000 Japanese chromosomes. The deletion mutation was also absent in 200 Japanese, and not reported in the over 6,800 glaucoma individuals. The co-segregation of three different mutations of OPTN with the ALS phenotype strongly suggests that some mutations of OPTN cause ALS.
The eight individuals with mutations of OPTN showed onset from 30 to 60 years of age. Most of them showed a relatively slow progression and long duration before respiratory failure, although the clinical phenotypes were not homogeneous (see Supplementary Information).
The Q398X mutation causes a premature stop during translation, truncating the 577 amino-acid OPTN protein to one of 397 amino acids in length. This truncation results in a deletion of the coiled coil 2 domain10, which is necessary for binding to ubiquitin11, huntingtin12 (htt), myosin VI13 and the ubiquitinated receptor-interacting protein14. In the gene with the deletion of exon 5, if there was a transcript, the transcript splicing from exon 4 to exon 6 would cause a frame shift and make a stop codon (TGA in the ninth to eleventh codons in exon 6), which would be expected to translate a peptide 58 amino acids in length. The missense mutation (E478G) was located between coiled coil 2 domain and the leucine zipper domain. This glutamic acid is highly conserved among OPTN proteins of a wide range of species (Supplementary Fig. 2a), and is situated within the DFxxER motif, an ubiquitin-binding domain shared among OPTN, NF-κB essential molecule (NEMO), and A20 binding and inhibitor of NF-κB proteins (ABIN) (Supplementary Fig. 2b). The mutations in the DFxxER motif in ABIN reduce the binding to ubiquitin, which render them unable to inhibit NF-κB activation11. We investigated the ability of various mutations of OPTN to inhibit NF-κB-mediated transcriptional activation by performing a luciferase assay using NSC-34 cells (a mouse neuroblastoma and spinal-cord hybrid cell line) transfected with wild-type or mutant OPTN. E50K OPTN, which causes POAG7, downregulated the NF-κB activity, as did the wild type. On the other hand, both Q398X and E478G had no ability to inhibit NF-κB activity (Tukey–Kramer, P < 0.05). These tendencies were retained after stimulation with tumour-necrosis factor (TNF)-α (Fig. 2A). We also examined the subcellular localization of overexpressed Flag-tagged wild-type OPTN (wild type) and its mutants in cells (Fig. 2B). Immunofluorescence staining was performed with their antibodies against Flag and the Golgi matrix marker GM130. Confocal images showed close apposition of granular signals of wild-type OPTN or E50K with GM130 (see g and i in Fig. 2B)15, 16. E50K often shapes large granular structures near the Golgi apparatus. E478G rarely showed granular signals (see b in Fig. 2B); however, when closely observed, some of the signals were still closely localized to GM130 (see h in Fig. 2B). Western blotting using a lysate of transformed lymphoblasts showed that the 74-kDa band, corresponding to OPTN, was absent in subjects 3 and 4, but was present in the non-diseased mother and brother of subject 3 (Supplementary Fig. 3a). Quantitative PCR with reverse transcription revealed that the products were diminished to 58.0% in the heterozygote (III-2) and to 13.8% in the homozygote (subject 4) compared with the control levels (Supplementary Fig. 3b). In addition, cycloheximide recovered the decrease in the OPTN messenger RNA (mRNA) with the mutation (Supplementary Fig. 3c). Thus mRNA with this mutation, which bears a premature termination, might be degraded through nonsense-mediated mRNA decay in lymphoblasts.
Figure 2: Influence of OPTN mutations.
Influence of OPTN mutations.
A, Luciferase assay to assess the ability of various OPTNs to inhibit activation of NF-κB. The wild type and E50K have a similar NF-κB activation-inhibiting effect, whereas mock, Q398X and E478G types lack this effect. Error bars, standard deviations of triplicate assays. B, Localization of OPTN. Flag is the white signals in a–c and red signals in g–i. GM130 is the white signals in d–f and green signals in g–i. The wild type shows many fluorescent granules closely localized with the Golgi apparatus. E478G OPTN shows a reduced number of granules, and rarely co-localized with the Golgi apparatus. E50K OPTN granules have become large and closely localized with the Golgi apparatus. Scale bar, 10 μm.
The spinal cord from subject 5 with the E478G mutation revealed loss of myelin from the corticospinal tract and of the anterior horn cells (AHCs, Fig. 3a and Supplementary Fig. 4a, b). OPTN immunohistochemistry demonstrated increased staining intensity of the cytoplasm of the remaining AHCs and the neurites in the anterior horn (Supplementary Fig. 4c). Higher magnification of the motor neurons revealed intracytoplasmic eosinophilic inclusions (Fig. 3b, d). Intriguingly, these inclusions were distinctly immunopositive for OPTN (Fig. 3c, e). On the other hand, the cytoplasm of AHCs from control individuals was faintly labelled with anti-OPTN antibodies (Supplementary Fig. 5a, c), similar to the spinal-cord AHCs of mice (Supplementary Fig. 6b) and in contrast to the highly labelled sensory neurons in the dorsal root ganglia of mice (Supplementary Fig. 6d). In patients with sporadic ALS, the staining intensity for OPTN apparently increased not only in the cytoplasm of the remaining AHCs but also in their neurites (Supplementary Fig. 5b, d). In addition, distinctive intracytoplasmic inclusions were also noticeably OPTN immunolabelled in cases of sporadic and familial ALS; eosinophilic round hyaline inclusions from patients with SALS were immunopositive for OPTN (Fig. 3f, g, i, j). Re-staining of the same sections for ubiquitin, a known constituent of many neurodegenerative inclusions, revealed that these inclusions were also positive and faithfully matched the distribution of OPTN immunoreactivity (Fig. 3h, k). The anti-OPTN antibodies also stained skein-like inclusions (Fig. 3l, n), which were again mirrored with the anti-ubiquitin antibodies (Fig. 3m) and with the anti-TDP-43 antibodies (Fig. 3o). The distinct OPTN immunoreactivity of ubiquitin- and TDP-43-positive intracytoplasmic inclusions was confirmed on serial sections from patients with SALS (Supplementary Fig. 7). Moreover, SOD1-immunopositive Lewy-body-like hyaline inclusions from cases with SOD1 FALS were also immunopositive for OPTN (Fig. 3p–r). We found that OPTN antibody labelled both SOD1- and TDP-43-positive inclusions. As the staining of SOD1 and TDP-43 is generally mutually exclusive, OPTN staining appears to be a more general marker for inclusions in various types of ALS; therefore, the OPTN molecule might also be involved in a broader pathogenesis of ALS.
Figure 3: Identification of OPTN in distinctive intracytoplasmic inclusions of subjects with ALS.
Identification of OPTN in distinctive intracytoplasmic inclusions of subjects with ALS.
a–e, Neuropathology of the lumbar spinal cord from subject 5. Klüver-Barrera (a) show loss of myelin from the corticospinal tract (arrow) and loss of motor neurons from the anterior horn (arrowhead). The cytoplasm of the remaining motor neurons contains an amorphous eosinophilic region (b, arrow). H&E, haematoxylin and eosin. The same neuron was re-stained with the anti-OPTN antibody (c, arrow). The eosinophilic retention occasionally appears to form a hyaline inclusion (d, arrow), which is intensely immunolabelled with the anti-OPTN antibody (e, arrow). f–k, Round hyaline inclusions of subjects with SALS (f, i) are immunolabelled with anti-OPTN-C and anti-OPTN-I antibodies (g and j, respectively). The sections were re-stained with anti-ubiquitin (Ub) antibodies (h, k). l–o, Skein-like inclusions of patients with SALS are reactive with the anti-OPTN-I and anti-OPTN-C antibodies (l, n). Re-staining of l with the anti-ubiquitin antibody (m) and n with anti-TDP-43 antibody (o). p–r, Lewy-body-like hyaline inclusion of a patient with FALS, stained with haematoxylin and eosin (p), anti-OPTN-C antibody (q) and SOD1 antibody (r). Scale bars, 200 µm (a), 20 µm (b–p).
The mutations of the OPTN gene cause both recessive and dominant traits, and the mechanism causing the disease may be different between the two traits. The Q398X nonsense mutation and probably the exon 5 deletion mutation cause a decrease in OPTN expression resulting from nonsense-mediated mRNA decay of the transcript carrying the nonsense OPTN mutations. Therefore, the mutated OPTN protein by itself is unlikely to disturb cell function or to be included in the inclusion body in the motor neuron cells. The mechanism of recessive mutations causing ALS is expected to be simply loss of function, and the heterozygote for the Q398X mutation does not develop the ALS phenotype. On the other hand, the E478G missense mutation increased the immunoreactivity for OPTN in the cell body and the neurites. The increased amount and different distribution of the mutated protein would disturb neuronal functions, and may accelerate the inclusion body formation as well as the increase and the different distribution of OPTN immunoreactivity in sporadic ALS. Thus the heterozygote for the E478G mutation will develop the disease.
The different impact on NF-κB signalling and the different intracellular localization of ALS- and POAG-linked mutated protein may explain the phenotypic divergence between the two diseases. Subject 3 with homozygotic Q398X also showed POAG, whereas subject 4 with the same mutation, and subjects 1 and 2 with the exon 5 deletion, did not show it. The prevalence of POAG in the population older than 40 years is 3.9% in Japan17.Considering this information, the ALS and glaucoma in subject 3 may accidentally coexist.
OPTN competes with NEMO for binding to the ubiquitinated receptor-interacting protein and negatively regulates TNF-α-induced activation of NF-κB14, which mediates an upregulation of OPTN, creating a negative feedback loop18. ALS-related OPTN mutations lacked the inhibitory effect towards NEMO, and thus exaggerated NF-κB activation. In sporadic ALS, a previous report showed that NF-κB, which is classified as a ‘cell death inhibitor’, is upregulated in motor neurons19. The upregulated NF-κB may induce the overexpression of OPTN, and may also cause neuronal cell death20. Thus NF-κB is a major candidate target for treating this disease. Additionally OPTN plays an important role in the maintenance of the Golgi complex, in membrane trafficking, in exocytosis, through its interaction with myosin VI and Rab8 (ref. 13), and in post-Golgi trafficking to lysosomes dependent on the Rab8/OPTN/htt complex21 (Supplementary Fig. 8). Interestingly, FUS/TLS has been reported to interact with myosin VI22 as well as with myosin V23. Impairment of intracellular trafficking of the complex including OPTN and/or FUS/TLS may cause inclusions in this neurodegenerative disorder.

Methods

Ethical considerations

The study was approved by the institutional review boards of the participating institutions. All examinations were performed after having obtained informed consent from all subjects or their families.

Subjects

Neurologists performed the clinical diagnosis. The mean age at onset of subjects with ALS was 59.9 years (range 10–85 years, including 14 cases confirmed by autopsy). The possibility of mutation of SOD1 was excluded.

Screening for the mutation of OPTN

A list of PCR primer pairs used to amplify individual OPTN in the regulatory regions (~1,000 bases upstream from transcription start sites), non-coding exons, coding exons and the surrounding sequences (50–100 bases) of the exons or intron 4 and 5 is provided in Supplementary Table 4. Deletion of Exon 5 was checked by using exon 4 forward and intron 5 reverse primer pairs. Direct sequence of the joining part was performed by using intron 4-2 forward primer or intron 5-6 reverse primer. Screening for the c.1502C>T mutation was performed by analysing restriction-fragment length polymorphism or direct sequencing on 781 healthy control subjects (mean age 62.3 years; range 30–100 years). Exon 12 was amplified and then restricted with MseI, and thereafter the products were electrophoresed in 2% agarose gel. The wild type was digested into 204-, 106-, 14- and 12-bp fragments, and the mutant type (204 bp) into 169 + 35-bp fragments. The c.1743A>G mutation was determined by direct sequencing. In the Affymetrix Mapping 500K, there were 11 SNPs in the OPTN gene. However, there are no SNP markers between exon 2 and exon 12 of OPTN, and additional quantitative PCR analysis of all exons of the OPTN gene was performed.

Luciferase assay

We investigated the activity of NF-κB by using the luciferase assay. Four types of complementary DNA (cDNA) from OPTN were inserted into separate pDNR (Clontech). These were wild (IMAGE clone 3831267), Q398X (recessive), E478G (dominant) and E50K (which causes glaucoma) types. pDNR vector was used as mock. NSC-34 cells were co-transfected with NF-κB reporter ((Igk)3 conaluc plasmid) (a gift from S. Yamaoka) and pDNR-OPTN by using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured 5 h after either PBS or TNF-α (10 ng ml-1, R&D) stimulation by using a Dual-Luciferase Reporter Assay System (Promega). Consistent results were obtained by conducting three independent experiments.

Localization of OPTN

We investigated the localization of OPTN by using a 3×Flag tag. This was inserted into pcDNA3 (Invitrogen), and three types of OPTN cDNA (wild, E478G, E50K) were inserted after the 3×Flag tag. These plasmids were used to transfect NSC-34 cells with the aid of Lipofectamine 2000 (Invitrogen). GM130 (BD Transduction Laboratories) was used as a marker of the Golgi apparatus.

Immunofluorescence microscopy

Cells were grown on glass-bottomed glass dishes (Matsunami) coated with poly-L-lysine and laminin (Sigma Aldrich) and transfected by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol; 24–48 h after transfection, the cells were fixed, blocked with normal serum and incubated with primary antibody at 4 °C overnight. Confocal images were acquired with an Olympus FV300 by using a ×100 oil immersion lens with a sequential-acquisition setting at a resolution of 512 pixels × 512 pixels with threefold magnification. Each cellular picture was generated by combining multiple optical images (10–15 slices, z-spacing of 0.2 μm) spanning 2–3 μm along the z-axis. Subcellular localization of Flag-tagged optineurin was verified by at least three independent experiments. More than 100 cells were photographed for each optineurin construct. The following antibodies were used: mouse monoclonal anti-GM130 (BD Transduction Laboratories, 1:1,000) and affinity-purified rabbit polyclonal anti-Flag (Sigma, 1:1,000).

Western blotting

We investigated the expression of OPTN by western blotting. Cell lysates were prepared from Epstein-Barr-virus immortalized B lymphocytes from subject 3, her brother and mother, and subject 4 by using standard protocols. Polyclonal antibodies recognizing the carboxy (C)-terminal part of OPTN (Cayman Chemical) and anti-rabbit IgG-HRP antibody (R&D Systems) were used. For the internal control, we used glyceraldehyde-3-phosphate dehydrogenase polyclonal antibody (IMGENEX).

Quantitative PCR with reverse transcription

Quantitative PCR with reverse transcription was performed by using THUNDERBIRD SYBR qPCR Mix (TOYOBO) and ABI 7900HT Fast Real Time PCR system (Applied Biosystems). Epstein-Barr-virus immortalized B lymphocytes were treated with cycloheximide (Sigma, 100 μg ml-1) for 2 h before RNA extraction.

Immunohistochemistry of mouse nervous tissue

Several antibodies were tested for their use in detecting mouse OPTN in tissue sections (data not shown). Among them, rabbit polyclonal antibodies raised against various peptides of human/mouse OPTN origin gave consistent and reasonable results. One such antibody was OPTN-C raised against the C-terminal part of OPTN, which is identical between human and mouse (amino acids 575–591; Cayman Chemical). Immunohistochemistry was performed on adult DBA/2 mouse. Mice were transcardially fixed with 4% paraformaldehyde in PBS, post-fixed in the same fixative overnight, and then dehydrated in 30% sucrose in PBS overnight. Frozen sections were obtained by using a cryostat and mounted onto 3-triethoxysilylpropylamine (TESPA)-coated glass slides. After air-drying, the slides were washed in PBS and blocked for 2 h at room temperature in 5% BSA/0.3% Triton X-100 containing PBS. The sections were then incubated overnight at 4 °C with primary antibodies against OPTN diluted in 1% BSA/1% normal goat serum/0.3% Triton X-100/PBS. After several washes in PBS, Alexa-594-conjugated secondary antibody (Invitrogen) in PBS was applied. Pictures were taken with a camera attached to a fluorescence microscope (BIOREVO BZ-9000; Keyence).

Histochemistry

Post-mortem material from one of the OPTN mutant cases (subject 5) was available. Sections (6 μm) of formalin-fixed, paraffin-embedded spinal cord were examined with Klüver–Barrera and haematoxylin and eosin staining. Some sections stained with haematoxylin and eosin were photographed, decolourized and immunostained with OPTN-C (mouse monoclonal, 1: 50,000) or OPTN-I (rabbit polyclonal, Cayman Chemical, 1:400). In addition, lumbar spinal cord tissue was obtained from clinically and neuropathologically proven cases of SALS (seven cases) and familial ALS with the A4V SOD1 mutation (FALS, three cases). Six age-matched normal individuals served as controls. After confirmation of complete removal of the OPTN antibody, we immunostained the same sections with the anti-ubiquitin antibodies (mouse monoclonal, Santa Cruz Biotechnology, 1:400; rabbit polyclonal, Sigma, 1:600), anti-TDP-43 antibodies (mouse monoclonal, Abnova, 1:1,000; rabbit polyclonal, Proteintech Group, 1:4,000) or anti-SOD1 antibodies (mouse monoclonal, Lab Vision Corporation, 1:50; rabbit polyclonal, Stressgen Biotechnologies, 1:2,000).

Author information

Affiliations

  1. Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan

    • Hirofumi Maruyama,
    • Hiroyuki Morino,
    • Masaki Kamada &
    • Hideshi Kawakami

  2. Department of Neurology, Kansai Medical University, Moriguchi 570-8506, Japan

    • Hidefumi Ito,
    • Yoshimi Kinoshita &
    • Hirofumi Kusaka

  3. Department of Clinical Neuroscience, University of Tokushima Graduate School, Tokushima 770-8503, Japan

    • Yuishin Izumi,
    • Masaki Kamada,
    • Hiroyuki Nodera &
    • Ryuji Kaji

  4. Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, Saitama 350-1241, Japan

    • Hidemasa Kato

  5. Laboratory of Integrative Bioscience, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan

    • Yasuhito Watanabe &
    • Toru Takumi

  6. Faculty of Human Science, Hiroshima Bunkyo Women’s University, Hiroshima 731-0295, Japan

    • Hidenori Suzuki

  7. South Osaka Neurosurgical Hospital, Osakasayama 589-0011, Japan

    • Osamu Komure

  8. Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan

    • Shinya Matsuura

  9. Department of Neurology, Kobatake Hospital, Fukuyama 720-1142, Japan

    • Keitaro Kobatake

  10. Department of Neurology, Okayama University, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, Japan

    • Nobutoshi Morimoto &
    • Koji Abe

  11. Department of Neurology, Tohoku University School of Medicine, Sendai 980-8574, Japan

    • Naoki Suzuki &
    • Masashi Aoki

  12. Department of Neurology, Tokyo Metropolitan Neurological Hospital, Fuchu, Tokyo 183-0042, Japan

    • Akihiro Kawata &
    • Takeshi Hirai

  13. Department of Neurology, Haematology, Metabolism, Endocrinology and Diabetology, Yamagata University Faculty of Medicine, Yamagata 990-9585, Japan

    • Takeo Kato

  14. Department of Pathology, School of Medicine, Shiga University of Medical Science, Ohtsu 520-2192, Japan

    • Kazumasa Ogasawara

  15. Division of Neuropathology, Department of Pathology, Montefiore Medical Center, New York, New York 10467-2490, USA

    • Asao Hirano

  16. Department of Respiratory Medicine, Saitama Medical University, Saitama 350-0495, Japan

    • Koichi Hagiwara

  17. Present address: Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan.

    • Hidefumi Ito

Contributions

Author Contributions H. Kawakami designed and supervised the study. H.Mo. and K.H. extracted candidate genes. H.Ma. and M.K. performed sequencing analysis. H.Ma., H.Mo., Y.W., T.T., S.M., H. Kawakami and H.S. conducted molecular biological analysis. H.I., Y.K., H. Ku., H. Kato, K.O. and A.H. performed pathological analysis and provided pathological samples. Y.I., H.N., R.K., O.K., N.M., K.A., A.K., T.H, T.K., M.A., N.S. and K.K. collected clinical information and samples. H. Kawakami, H.Ma., H.I. and K.H. wrote the paper.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

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Editor's summary

Optineurin defects in ALS

About 10% of cases of the motor neuron disease amyotrophic lateral sclerosis (ALS) are familial, but the small number of mutations so far identified account for only around 20–30% of the those cases.…

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Materials

  1. ABI 7900HT Fast Real Time PCR

    TOYOBO

  2. BIOREVO BZ-9000

    Invitrogen

  3. Dual-Luciferase Reporter Assay System

    R&D

  4. FV300

    Olympus

  5. GeneChip Human Mapping 500K Array Set

    Affymetrix
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