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Abstracts

THEME 5 GENETICS

Pages 110-118 | Published online: 18 Oct 2010

P95 H63D POLYMORPHISM IN THE HEMOCHROMATOSIS GENE IS ASSOCIATED WITH SPORADIC AMYOTROPHIC LATERAL SCLEROSIS IN CHINA

XU Y, HE X, YAO X

West China Hospital, Sichuan University, Chengdu, Sichuan Province, China

E-mail address for correspondence: [email protected]

Keywords: HFE gene, single nucleotide polymorphism

Background and purpose: H63D(His63Asp) polymorphism in HFE gene has been reported as a risk factor for amyotrophic lateral sclerosis (ALS) in Europe and America, whereas no data has been obtained for Asia. Herein, we investigated the frequency of this polymorphism in a Chinese population.

Methods: A total of 195 individuals with sporadic ALS (sALS) from three centers in China and 405 unrelated healthy controls were recruited. The HFE H63D polymorphism was determined by restriction fragment length polymorphism (RFLP) analysis.

Results: Sporadic ALS was significantly related to H63D polymorphism in heterozygous carriers (odds ratio 3.10, CI: 1.49 to 6.47, P=0.002).

Conclusions: The HFE H63D polymorphism may contribute to the development of sporadic ALS in China.

P96 TWO NOVEL ANGIOGENIN (ANG) GENE MUTATIONS AND THEIR PATHOGENESIS

AKIMOTO C1, MORITA M1, SAKASHITA E2, ENDO H2, NAKANO I1

1Neurology, Shimotsuke, Tochigi, Japan, 2Biochemistry, Shimotsuke, Tochigi, Japan

E-mail address for correspondence: [email protected]

Keywords: angiogenin, V105A, P (−4) Q

Background: Mutations of ANG have been found in ALS, and recent studies revealed angiogenin protects motoneurons from hypoxic injury.

Objectives: We screened the ANG gene in sporadic ALS cases, and examined synthesis of mutated angiogenin protein in HeLa cells.

Methods: Screening of the ANG gene: We scanned the coding regions of the ANG gene by high resolution melting (HRM) analysis and sequenced the samples indicated to include mutations. Synthesis of ANG protein: We constructed mammalian expression plasmid vectors with C terminal FLAG tags including wild and mutated type ANG genes. The resultant plasmid DNAs were transfected into HeLa cells, and then angiogenin protein synthesis was examined by Western blotting and immunocytochemistry.

Results: We identified two novel mutations (V105A and P (−4) Q) in 184 sporadic ALS patients. One was present in the mature protein region, and the other in the signal peptide sequence. Based on the results of expression vector, we verified the mature size angiogenin protein was synthesized with the P (−4) Q mutated ANG gene (mutated type), but the early secretion was reduced compared with the wild type. Both the mutated and wild type angiogenin proteins were localized to the endoplasmic reticulum (ER) and Golgi apparatus, but transportation of the mutated type from the ER to the Golgi apparatus was slightly obstructed. We speculate that it is one of the reasons for the delayed secretion.

Discussion and conclusion: The prevalence of the ANG mutation in sporadic ALS was 1.08% in our Japanese group, which was almost the same (0.3∼1.0%) as in European countries. In the case of an angiogenin synthesis state like the hypoxic one, the early secretion of the P (−4) Q protein is reduced with the consequent increase in neuronal damage. Judging from our experiment, the mutation in the signal peptide sequence in the ANG gene could be pathogenic.

References:

P97 IDENTIFICATION AND CHARACTERIZATION OF A NOVEL SOD1 SPLICE SITE MUTATION ASSOCIATED WITH FAMILIAL ALS

BIRVE A1, NEUWIRTH C3, WEBER M3, MARKLUND S2, NILSSON A-C1, JONSSON A2, ANDERSSON R1, ANDERSEN P1

1Department of Pharmacology and Clinical Neuroscience, Umea, Sweden, 2Department of Medical Biosciences, Umea, Sweden, 3Neuromuscular Diseases Unit, St. Gallen, Switzerland

E-mail address for correspondence: [email protected]

Keywords: SOD1, intronic mutation, splice variant

Background: More than 145 mutations have been found in the gene CuZn-Superoxide dismutase (SOD1) in patients with amyotrophic lateral sclerosis (ALS). The vast majority are single nucleotide substitutions in the coding region causing missense mutations. Most mutations result in a protein with reduced dismutation. Four intronic mutations have been associated with ALS previously, all in intron 4. In this study we report the identification and characterization of a novel intronic mutation in intron 3 found in a Swiss patient with familial ALS.

Objectives: The purpose of this study was to identify the suspected SOD1 mutation in a 42-year-old FALS patient with significantly low SOD1 enzymatic activity in erythrocytes. The patient was DNA-sequence analysed in SOD1 but there was no sequence aberration found in the coding regions. This called for a further investigation of the SOD1 gene.

Methods: The SOD1 gene of the FALS patient was analysed with DNA sequencing and Reverse Transcription-PCR. Interned based splicing algorithm tools were used to find potential splice site mutations. SOD1 activity was measured and Western immunoblotting was used to examine the presence and characteristics of SOD1 proteins.

Results: Sequence analysis revealed that the patient was heterozygous for a thymine to guanine mutation 7 bp upstream of exon 4 (c.240–7T>G). Splicing analysis tools revealed a potential novel splice site that would add 6 bp to the mRNA. This mRNA would insert Ser and Ile between Glu78 and Arg79 in the SOD1 protein (SOD1 E78_R79insSI). Both the predicted mutant transcript and the mutant protein were found to be highly expressed in fibroblasts from the patient and the SOD activity was approximately normal in these cells.

Discussion: The two mRNAs, wild type and mutant, as well their respective proteins appeared to be expressed in equal amounts. These findings show that the usage of the alternative splice site must be near 100%. Comparison of SOD1 protein and activity analyses in fibroblasts suggests that, despite the insertion of two novel amino acids into the metal ion binding loop IV, the mutant SOD1 has roughly normal enzymatic activity, close to native structure and is relatively stable. However, since the enzymic activity in erythrocytes was ∼50% of controls it seems the mutant protein shows the reduced stability typical of ALS-linked mutant SOD1s.

Conclusion: The investigation suggests that the intronic mutation found in the FALS patient is causing the disease, and highlights the importance of wide exon flanking sequencing and transcript analysis combined with erythrocyte SOD activity analysis in comprehensive search for SOD1 mutations in ALS cases.

P98 L67P: A NOVEL EXON 3 SOD1 MUTATION IN AN ITALIAN ALS PATIENT

DEL GRANDE A1, LUIGETTI M1, CONTE A1, MADIA F2, MANCUSO I3, LATTANTE S3, MARANGI G3, STIPA G4, TONALI PA1, ZOLLINO M3, SABATELLI M1

1Catholic University, Department of Neurosciences, Institute of Neurology, Rome, Italy, 2Complesso Integrato Columbus, Rome, Italy, 3Catholic University, Institute of Human Genetics, Rome, Italy, 4Azienda ospedaliera S. Maria, servizio di Neurofisiopatologia, Terni, Italy

E-mail address for correspondence: [email protected]

Keywords: SOD1 mutation

Background: Mutations in SOD1 gene are responsible for 20% of familial ALS and more than 130 mutations have been identified so far. The majority of them are located in exons 4 and 5, less than one third are in exons 1 and 2, while only eight mutations (5%) have been reported among the 24 codons of exon 3, generally disclosing low penetrance and predominant lower motor neuron involvement.

Objectives: To analyze clinical and electrophysiological features of a patient affected by sporadic ALS bearing a novel mutation in exon 3 of the Cu/Zn superoxide dismutase gene.

Methods: After obtaining written informed consent from the patient and her parents, all five exons and flanking intron regions of the SOD1 gene were analyzed using direct genome sequencing.

Results: We describe a novel L67P mutation located in exon 3 of the Cu/Zn superoxide dismutase gene. The propositus was a 38 year old woman affected by slowly progressive ALS with onset at the age of 36 years and disclosing pure lower motor neuron signs. Motor Evoked Potentials were within normal limit. At clinical examination she was able to walk with a cane and presented moderate wasting and weakness of all four limbs, more evident in the distal muscles and in lower limbs. There were no bulbar signs and respiratory function was normal. There were no other cases of ALS in her family but the mutation was found also in her healthy father, aged 76.

Discussion and conclusions: A total of 9 mutations involving exon 3 of SOD1 have been described, including the present one. Our data confirms that variable penetrance and predominant lower motor neuron involvement are common features in patients bearing mutations in exon 3 of SOD1.

P99 PHENOTYPIC PRESENTATION OF I113T SOD1 ALS

DONKERVOORT S, AJROUD-DRISS S, SIDDIQUE N, SIDDIQUE T

Northwestern University, Chicago, IL, United States

E-mail address for correspondence: [email protected]

Keywords: SOD1, I113T, FALS

Over 140 disease causing mutations have been reported in Cu/Zn superoxide dismutase (SOD1). The I113T mutation has been reported in both familial amyotrophic lateral sclerosis (FALS) and apparently sporadic amyotrophic lateral sclerosis (SALS). Genetic counseling for I113T SOD1 ALS is challenging due to its reported reduced penetrance, as well as variability in disease onset and progress. I113T is one of the most common SOD1 mutations; however, published literature is limited to individual case reports. We present clinical data on the largest cohort of I113T SOD1 patients to date, comprised of 28 ALS patients from 18 different kindreds, as well as a comprehensive literature review. Our data illustrates wide variability in age of onset (ranging from 23 to 77 years) and disease duration (5 to 145 months). Extensive inter- and intra-familial phenotypical variability was observed. None of the I113T SOD1 ALS patients had frontotemporal dementia and/or apparently sporadic disease, although reduced penetrance was observed in 3 families with obligate carriers of 66, 77 and, 91 years of age. Interestingly, because of the atypical slow presentation in some patients, 4 patients were initially diagnosed with neuropathy, 1 with multiple sclerosis and 1 with asthma. This analysis presents the phenotypic expressions of this common SOD1 mutation, which will aid in providing appropriate genetic counseling and testing for patients and families with FALS.

P100 TARDBP GENE MUTATIONS AMONG CHINESE PATIENTS WITH SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

HUANG R, FANG D-F, MA M-Y, GUO X-Y, ZHAO B, ZENG Y, ZHOU D, YANG Y, SHANG H-F

Department of Neurology, West China Hospital of Sichuan University, Chengdu, Sichuan, China

E-mail address for correspondence: [email protected]

Keywords: TARDBP gene, mutation

Background: Recently, several TARDBP mutations have been identified in sporadic amyotrophic lateral sclerosis (SALS) patients of different ethnicity. No TARDBP mutation analysis among Chinese SALS patients has been reported.

Objective: This study aims to analyze the clinical features and mutations in the TARDBP gene among Chinese patients with SALS.

Methods: The clinical characteristics of patients diagnosed with adult-onset SALS were analyzed. The frequencies of TARDBP mutations and the association between these mutations and the clinical features of ALS were analyzed.

Results: One hundred and sixty-five patients were studied. The mean age of onset was 50.8±12.0 years. The mean diagnostic delay was 18.8±17.1 months. A novel missense mutation (p.N378S) and a novel silent change (p.A321A) were detected in two male patients, respectively. A new variant of c.1098C>G in exon 6 and two reported variants, g.IVS1+85C>T in intron 1 and c.57A>G in exon 2, were found. The frequency of the ‘G’ variant of c.57A>G in exon 2 and the ‘G’ variant of c.1098C>G in exon 6 were significantly lower in the patient group than in the control (P<0.001 and P=0.024, respectively).

Conclusions: To the best of our knowledge, this is the first study on TARDBP mutation among Chinese patients with SALS. Our findings provide evidence that the frequency of TARDBP gene mutations is rare among Chinese SALS patients (0.61%). Several polymorphisms may influence susceptibility to ALS.

P101 PHENOTYPES IN SWISS PATIENTS WITH FAMILIAL AND SPORADIC ALS CARRYING TARDP MUTATIONS

WEBER M1, MORITA M2, NEUWIRTH C1, TIERBACH J3, PERREN F4, ANDERSEN P5

1Neuromuscular Diseases Unit, Kantonsspital, St. Gallen, Switzerland, 2Department of Neurology, Jichi Medical University, Yakushiji, Shimotsuke-shi, Tochigi, Japan, 3Blood Collection Centre, Kantonsspital, St. Gallen, Switzerland, 4Department of Neurology, University Hospital, Geneva, Switzerland, 5Department of Neurology, University Hospital, Umeå, Sweden

E-mail address for correspondence: [email protected]

Keywords: TARDP mutations, phenotypes, Switzerland

Background: Recently mutations in the TARDP gene which codes for the TAR DNA binding protein 43 (TDP-43) have been identified in familial and sporadic ALS patients. The frequency of TARDP mutations seems different in various European populations and phenotypic variability is high.

Objective: To further define the phenotypic spectrum of TARDP mutations and their frequency in a European population.

Methods: A total of 246 patients diagnosed with ALS (221 sporadic cases, 43 FALS cases in 25 families) were screened for TARDP mutations. Except for one patient who was followed at the University of Geneva all patients were followed at the Kantonsspital St.Gallen. FALS cases carrying SOD mutations were excluded.

Results: In 4 patients TARDP mutations were identified. Two female ALS patients, one apparently sporadic and one familial case, carried the Asn352Ser mutation. Both had limb onset and a slowly progressive course of the disease. In the sporadic case with a survival of 8 years, bulbar muscles were spared until death despite severe respiratory insufficiency. The other patient died 7 years after disease onset. In a 44-year old female patient a novel mutation (Gly376Asp) was identified. Family history suggested an autosomal dominant trait with complete penetrance. Survival was 20 months in her and only 6 to 18 months (mean 13 months) in relatives. A fourth male case carried the Ala90Val mutation. None of the patients had cognitive impairment. The frequency of TARDP mutations was 1% in SALS and 9% in FALS.

Conclusion: The frequency of TARDP mutations in the Swiss population is higher compared to other European populations. The novel Gly376Asp mutation is associated with rapid disease progression while the Asn352Ser mutation is associated with slow disease progression.

P102 HOMOZYGOUS A382T MUTATION OF TARDBP GENE IN AN ITALIAN PATIENT WITH AN ATYPICAL ALS PHENOTYPE

MORELLI C, TILOCA C, COLOMBRITA C, TICOZZI N, DORETTI A, MESSINA S, RATTI A, SILANI V

Department of Neurology – Università degli Studi di Milano – IRCCS Istituto Auxologico Italiano, Milan, Italy

E-mail address for correspondence: [email protected]

Keywords: TARDBP, TDP-43, genetics

Background: A 44-year-old woman of Southern Italian origin, affected by ALS with bulbar onset and without familial history of motor neuron disease, was admitted to our ALS Centre. The neurological examination revealed upper and lower motor neuron signs in the bulbar and cervical regions and only upper motor neuron signs in the lumbosacral region (probable ALS according to El Escorial criteria). Electromyography showed active denervation in the right first dorsal interosseous muscle and chronic denervation in the left one. Cerebral MRI revealed periventricular and subcortical demyelinating lesions, without pathologic enhancement after gadolinium administration. Cervical MRI was normal. Numerous oligoclonal bands restricted to the cerebrospinal fluid were detected by isoelectrofocusing. Search for serum anti-HIV1/2 and anti-Borrelia burgdorferi antibodies was negative. A thrombophilic condition was excluded. Thus neuroradiological and laboratory tests suggested the presence of an inflammatory demyelinating disease of the CNS, in addition to features characteristic of ALS.

Objectives: We investigated the potential genetic contribution to this atypical ALS case with a demyelinating disease of the CNS by performing a mutational screening of the ALS causative genes SOD1, ANG, TARDBP and FUS.

Methods: After informed consent, blood was withdrawn and DNA was extracted according to standard procedures. Molecular analyses of SOD1, ANG, TARDBP and FUS genes were conducted by direct DNA sequencing. Array CGH (Comparative Genomic Hybridization) analysis was performed on the Agilent platform.

Results: Mutations in SOD1, ANG and FUS genes were excluded in our patient. Molecular analysis of TARDBP gene led to the identification of the homozygous missense mutation A382T (1144G>A) in exon 6. Furthermore, array-CGH analysis provided no evidence for deletions of the TARDBP genomic region.

Discussion and conclusion: Mutations in TARDBP gene account for 5% of familial ALS and 2% of sporadic ALS cases. Among the TARDBP gene mutations, the A382T variant represents the most frequent one, but to our knowledge this is the first report of an ALS patient carrying this mutation in an homozygous state.

We speculate that a dose-dependent mechanism could explain this peculiar phenotype. Absence of wild-type TDP-43 with loss of its physiological nuclear function or a double dose of the mutant protein could be related to the development of ALS complicated by a demyelinating process. This hypothesis could also be supported by determination of TDP-43 levels in the CSF of our patient, compared with those of patients carrying heterozygous or no TARDBP mutations.

P103 MULTIPLE SYSTEM DEGENERATION WITH BASOPHILIC INCLUSIONS IN JAPANESE ALS PATIENTS WITH FUS MUTATION

TATEISHI T1, HOKONOHARA T1,2, KIKUCHI H1, MIURA S4, OHYAGI Y1, IWAKI T3, FUKUMAKI Y2, KIRA J-I1

1Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Fukuoka, Japan, 2Division of Human Molecular Genetics, Research Center for Genetic Information, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Fukuoka, Japan, 3Department of Neuropathology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Fukuoka, Japan, 4Division of Respirology, Neurology and Rheumatology, Department of Medicine, Kurume University School of Medicine, Kurume, Fukuoka, Japan

E-mail address for correspondence: [email protected]

Keywords: fused in sarcoma/translated in liposarcoma, basophilic inclusion, glial cytoplasmic inclusion

Background: Approximately, 5–10% of cases are familial forms, which are usually transmitted by an autosomal dominant trait. Recently, Kwiatkowski and Vance reported mutations in a gene encoding another DNA/ RNA-binding protein called fused in sarcoma (FUS) specific for familial ALS.

Objectives: To clarify the clinical and pathological findings in a large Japanese familial ALS pedigree with an autosomal dominant inheritance pattern, and carrying a FUS R521C mutation.

Methods: We carried out clinical, neuropathological and genetic studies on a large pedigree with familial ALS. In six successive generations of this family, 16 individuals of both sexes were affected by progressive muscle atrophy and weakness, indicating an autosomal dominant trait.

Results: Both sexes (11 men and 5 women) were affected. Mean age at onset was 40.6±13.8 years, and mean duration from onset to respiratory failure was 11.7±7.3 months. Three out of six patients examined, showed preferential involvement of the proximal upper extremities with flailing arms and subsequent spread of motor weakness to the lower extremities. Motor paralysis progressed rapidly in these patients, culminating in respiratory failure within 1 year. The missense mutation c.1561 C>T; p.R521C was found in exon 15 of FUS in the four patients examined. Neuropathological study of one autopsied case with the FUS mutation revealed multiple system degeneration in addition to upper and lower motor neuron involvement: the globus pallidus, thalamus, substantia nigra, cerebellum, inferior olivary nucleus, solitary nucleus, intermediolateral horn, Clarke's column, Onuf's nucleus, central tegmental tract, medial lemniscus, medial longitudinal fasciculus, superior cerebellar peduncle, posterior column, and spinocerebellar tract were all degenerated. Argyrophilic and basophilic neuronal or glial cytoplasmic inclusions immunoreactive for FUS, GRP78/BiP, p62 and ubiquitin were detected in affected lesions. FUS-positive neuronal and glial inclusions were more frequently and widely observed than basophilic, ubiquitin-, and silver-positive inclusions.

Discussion and conclusions: The FUS R521C mutation in this Japanese family caused familial ALS with pathological features of multiple system degeneration and neuronal basophilic inclusion.

P104 NOVEL MISSENSE AND TRUNCATING MUTATIONS IN FUS/TLS IN FAMILIAL ALS

WAIBEL S1, NEUMANN M2, RABE M1, MEYER T3, LUDOLPH AC1

1Department of Neurology, University of Ulm, Ulm, Germany, 2Institute of Neuropathology, University Hospital of Zurich, Zurich, Switzerland, 3Department of Neurology, Humboldt University Berlin, Berlin, Germany

E-mail address for correspondence: [email protected]

Keywords: FUS/TLS, genetics

Background: Mutations in the FUS/TLS gene have been associated with familial amyotrophic lateral sclerosis (fALS).

Methods: We analyzed the presence and frequency of FUS/TLS mutations in a German ALS cohort, including 133 sALS and 58 fALS patients by sequence analysis of exons 13–15.

Results: We identified two novel heterozygous FUS/TLS mutations in four German ALS families including the missense mutation K510R and the truncating mutation R495X. The truncating mutation was associated with an aggressive disease whereas the K510R variant showed a mild phenotype with a life expectancy of more than 8 years which was seen in each patient, including monozygotic twins. No mutation was detected in 133 sALS patients.

Conclusions: Mutations in FUS/TLS account for 7% (4 of 58) of fALS in our German cohort. In comparison to the frequency of SOD1 mutations these FUS/TLS variants are comparatively rare in our patients.

P105 IDENTIFICATION OF A FUS/TLS GENE MUTATION IN A COHORT OF CATALAN FALS PATIENTS

GAMEZ J1,2, SYRIANI E1,3, MORALES M1,3

1ALS Unit, Neurology Department, Hospital Universitari Vall d'Hebron, Barcelona, Spain, 2Autonomous University of Barcelona, Barcelona, Spain, 3University of Barcelona, Barcelona, Spain

E-mail address for correspondence: [email protected]

Keywords: fused in sarcoma (FUS/TLS), p.R521C, TARDBP

Background: A new causal gene for ALS was discovered in early 2009, when mutations in the fused in sarcoma/translated in liposarcoma (FUS/TLS) gene were found to segregate with ALS in families linked to chromosome 16. Two articles described 15 FUS/TLS mutations in 26 unrelated ALS pedigrees. Subsequent studies in other populations identified FUS/TLS mutants in familial ALS, some sporadic ALS cases and in one patient with frontotemporal lobar degeneration (FTLD). The emergence of these results in such a short timeframe suggests that mutations in FUS/TLS represent the second most common genetic cause of FALS (the first being SOD1 mutations). FALS linked to the FUS/TLS gene is designated as ALS6 (MIM 608030). There appears to be an allelic heterogeneity in ALS6, as at least a hundred sequence variants have been described to date, and pathogenicity has been described in 38 of these. Clinical-genetic characterisation of ALS6 is therefore essential for providing information on the phenotype associated with a given mutation, the distributions of FUS/TLS mutations in different ethnic groups and clarification of the genotype-phenotype correlation in patients with FUS/TLS gene mutations. No FUS/TLS mutations have been reported in the Spanish population to date.

Objectives: To investigate the prevalence of FUS/TLS mutations in a Catalan cohort of patients with familial ALS, in which we carried out a mutational study of SOD1 in 2006 (1).

Materials and methods: Twenty-five of the families not carrying SOD1 mutations were screened for FUS/TLS mutations using direct sequence. Mutations in the TARDBP genes were excluded beforehand.

Results: One of the 25 FALS pedigrees (4%) carried a C-to-T transition at nucleotide position 1561 (c.1561C>T) leading to a p.R521C sequence change at protein level. The phenotype in this family was characterized by a relatively young main age at onset (36.8 years old), predominantly lower motor neuron signs and variable survival.

Conclusions: This is the first ALS6 mutation identified in Spain. The prevalence of FUS/TLS mutations is similar to that reported in other countries. Our findings suggest that FUS/TLS mutations are the second most common cause of FALS in our population. The fact that p.R521C is the most common mutation described in patients from different ethnic backgrounds means that future research should focus on a worldwide haplotype study of pedigrees carrying p.R521C in order to investigate the possibility of a common founder.

Acknowledgements: JG was supported by a Spanish Fondo de Investigaciones Sanitarias grant (IP 10/01070).

Reference:

P106 IDENTIFICATION OF NOVEL FUS MUTATIONS IN SPORADIC AND FAMILIAL ALS CASES

BELZIL V1, DAOUD H1, ST-ONGE J1, DESJARLAIS A1, BOUCHARD J-P2, DuprÉ N2, CAMU W3, DION P1, ROULEAU G1

1Centre for Excellence in Neuromics, University of Montreal, the Centre Hospitalier de l'Université de Montréal (CHUM) and Ste-Justine Hospital, Montreal, Quebec, Canada, 2Faculty of Medicine, Laval University, Centre Hospitalier Affilié Universitaire de Québec – Enfant-Jésus Hospital, Quebec City, Quebec, Canada, 3Unité de Neurologie Comportementale et Dégénérative, Institute of Biology, Montpellier, France

E-mail address for correspondence: [email protected]

Keywords: FUS gene, splicing mutation

Background: Amyotrophic lateral sclerosis is the most common of the motor neuron diseases and is characterized by the degeneration of upper and lower motor neurons localized in the motor cortex, brain stem and spinal cord. So far, many mutations have been identified in SOD1, TARDBP, and more recently in FUS, all of which contribute to a better understanding of the pathological mechanisms involved in ALS. Specifically, mutations identified in FUS have been mostly found in FALS cases, specifically in the carboxy terminal of the protein.

Methods: Following the identification of several FUS mutations in FALS and a few in SALS, we wanted to evaluate to proportion of FUS mutations in the FALS and SALS populations. Therefore, the 15 exons of the FUS gene were sequenced in a cohort of 475 French and French-Canadian SALS patients, 475 matched controls, and 156 FALS of mixed origin.

Results: Two missenses, two deletions, one frameshift, one splice mutation and one nonsense mutation in a total of 8 FALS/SALS patients were identified. Among those, one deletion (G175del) was found in a FALS patient, like previously reported, and one missense was reported to be found in one control (G226S). The five other variants, P18S, G144_Y149del, R503fs, R514_Y526del and Q519X were new. The last three variants were located in the carboxy terminal of the protein where the previously reported variants were mostly clustered. One of those is a splicing mutation that is shared by all nine affected members of a large Spanish family. In addition, one new missense (R383C) and one mutation previously identified in a FALS patient (R216C) were found in two control participants. Synonymous variants were found in five SALS and four control individuals.

Conclusion: Our study identified new mutations in SALS patients as well as a new splicing mutation in an ALS family from Spain. This finding will help to better understand the contribution of FUS mutations in the ALS pathology.

P107 DE NOVO TRUNCATING FUS GENE MUTATION AS A CAUSE OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

DEJESUS-HERNANDEZ M1, KOCERHA J1, FINCH N1, CROOK R1, BAKER M1, DESARO P2, JOHNSTON A2, RUTHERFORD N1, WOJTAS A1, KENNELLY K2, WSZOLEK Z2, GRAFF-RADFORD N2, BOYLAN K2, RADEMAKERS R1

1Department of Neuroscience, 2Department of Neurology; Mayo Clinic, Jacksonville, Florida, United States

E-mail address for correspondence: [email protected]

Keywords: FUS, de novo, mutation

Background: Mutations in the gene encoding fused in sarcoma (FUS) were recently identified as a novel cause of amyotrophic lateral sclerosis (ALS), emphasizing the genetic heterogeneity of ALS and providing novel insights into its pathogenesis.

Objective: To determine the frequency and spectrum of mutations in FUS in a consecutive clinical cohort of ALS patients ascertained at Mayo Clinic Florida.

Methods: We sequenced the superoxide dismutase (SOD1), TAR DNA-binding protein 43 (TARDBP) and FUS genes in 99 sporadic and 17 familial ALS patients ascertained at Mayo Clinic Florida. In one family, segregation analysis was performed to determine the origin of the FUS mutation. FUS transcript analysis was performed in two patients carrying novel FUS mutations. Subcellular localization of recombinant FUS proteins was studied in N2A cells using immunocytochemistry and immunoblot analysis.

Results: We identified two novel mutations in FUS in two out of 99 (2.0%) sporadic ALS patients and established the de novo occurrence of one FUS mutation. In familial patients, we identified three (17.6%) SOD1 mutations, while FUS and TARDBP mutations were excluded. The de novo FUS mutation (g.10747A>G; IVS13-2A>G) affects the splice-acceptor site of FUS intron 13 and was shown to induce skipping of FUS exon 14 leading to the C-terminal truncation of FUS (p.G466VfsX14). Subcellular localization studies showed a dramatic increase in the cytoplasmic localization of FUS and a reduction of normal nuclear expression in cells transfected with truncated compared to wild-type FUS. We further identified a novel in-frame insertion/deletion mutation in FUS exon 12 (p.S402_P411delinsGGGG) which is predicted to expand a conserved poly-glycine motif.

Discussion: We extend the mutation spectrum in FUS leading to ALS and describe the first de novo mutation in FUS.

P108 PRIORITIZATION OF ALS CANDIDATE GENES THROUGH INTEGRATIVE PATHWAY ANALYSIS

KENNA K1, HARDIMAN O1,2, BRADLEY D1

1Trinity College Dublin, Dublin, Ireland, 2Beaumont Hospital, Dublin, Ireland

E-mail address for correspondence: [email protected]

Keywords: candidate, sequencing, pathway

Background: Mutations in 8 genes have been demonstrated to co-segregate with familial ALS. Such variants cause disease through their influence on a single or multiple biological processes. Variation in other genes capable of influencing the same biological processes may also cause disease. For example genetic variation in APP, PSEN1 or PSEN2, can cause familial forms of Alzheimer's disease.

Continual advances in DNA sequencing based technologies provide the opportunity for significantly larger scale interrogation of the genomes of ALS patients for disease relevant variation. Our work is centred on elucidating the genetic basis for ALS in the Irish population by screening candidate genes selected primarily by; 1) position of their encoded proteins within the human protein interaction network relative to proteins encoded by known ALS genes, and 2) similarity of their proteins to ALS proteins in terms of sequence and annotation.

Methods: Candidate Selection: Experimentally verified human protein interactions were retrieved from online database and supplemented with interactions manually curated from the literature to create a model of the human protein interactome. First order interaction partners of known ALS proteins and the second order interaction partners achieving the best prioritization scores were selected as candidates. Predicted paralogs of ALS proteins were also included with second order interactors for prioritization.

Prioritization technique: An in-house version of Google's PageRank algorithm was written and used to score candidates based on position within our model of the human protein interactome relative to that of known ALS proteins. The online tool ToppGene was used to score candidates based on similarity to ALS proteins in terms of numerous annotation features based on protein function, localization and expression. Candidates were also scored by sequence similarity to known ALS proteins using BLASTp. Scores were integrated to an overall score. A weighted profile of genes potentially relevant to ALS was constructed based on various lines of evidence such as occurrence within a known ALS linkage region etc and used to adjust final prioritization.

Results: 283 first and 4140 second order interaction partners of ALS proteins were identified; 321 predicted paralogs were recovered based on an e-value threshold of 10; The co- ordinates of the exons of 600 genes have been submitted to Agilent for RNA capture probe design to allow for subsequent sequencing.

Conclusion: Current technologies allow much higher throughput screening of candidate genes for disease relevant variation. Genes capable of influencing similar biological processes to known ALS genes are worthy candidates, and we have integrated multiple approaches to prioritize the most plausible of such candidates for sequencing in ALS patients.

P109 IMMUNOLOGIC INVESTIGATIONS IN AMYOTROPHIC LATERAL SCLEROSIS: CYTOKINE POLYMORPHISMS AND RELATED mRNA LEVELS IN LYMPHOCYTES OF SALS PATIENTS

GAGLIARDI S1, BOIOCCHI C2, COVA E1, MILANI P1, ALVISI E3, CERONI M1,3, CUCCIA M1, CEREDA C1

1Laboratory of Experimental Neurobiology, IRCCS, Neurological Institute “C. Mondino”, Pavia, Italy, 2Department of Genetics and Microbiology, University of Pavia, Pavia, Italy, 3Division of General Neurology IRCCS, Neurological Institute “C. Mondino”, Pavia, Italy

E-mail address for correspondence: [email protected]

Keywords: cytokines, polymorphisms

Background: The implication of the immune system in ALS is supported by data on levels of cytokines that have been found increased in serum and in cerebrospinal fluid of ALS patients (1). Interleukin 7, 9, 12, 17 and IL-1beta (IL-1ß) levels were found higher in CSF (2) and Antigenic Tumor Necrosis Factor-α (TNF-α) have been found increased in sera from ALS patients (3).

Objectives: This study analyzed 23 genetic polymorphisms of the 13 cytokines genes and we evaluated lymphocyte mRNA levels of the genes that showed statistically significant differences in alleles and genotype frequencies between ALS patients and controls.

Methods: Allelic, genotypic and haplotype frequencies of the polymorphisms in IL-1α, IL-1β, IL1-R, IL1-RA, IL-4Rα, IL-12, IFN-g, TGF-β, TNF-α, IL-2, IL-4, IL-6, IL-10 genes were assessed in 60 ALS patients and 140 healthy controls by RFLP. A Real-Time Sybr Green qPCR was used for TNF-α, IL-1β and TGF-1β expression analysis in 35 SALS patients and 35 controls. Normalization was optimized using YWHAZ as a housekeeping gene.

Results: Our data showed statistically significant differences in CC/CT genotype distribution of IL-1β (P= 0.0347) and CG/GG genotypes of TGF-1β (P=0.013). Moreover, there was a decrease in CT genotype frequencies in the IL-1β gene (P=0.0149) and CG genotype in the TGF-1β gene (P=0.0126) in ALS patients compared to controls. Our data showed a significant increase of AA genotype (P=0.019) for -238 TNF-a polymorphism in patients compared to controls. Our study shows that TNF-α and IL-ß1mRNA levels from SALS lymphocytes were expressed at higher level than in controls (P<0.05). TGFß1 mRNA quantity was higher in SALS samples than in control, although this did not reach statistical significance. Gene polymorphisms were not associated with clinical features and mRNA level.

Discussion: Our data suggest a common function of TNF-α and IL-1ß in ALS. Increased TNF-α and IL-1ß gene expression may be due to neuroprotective properties. TNF-α could be a response to ROS increase, documented in ALS disease. Hydrogen peroxide is well known as an activator of TNF-α gene expression through NF-kb (3). In vitro experiments have demonstrated that TNF-α down-regulates hSOD1 promoter via JNK/AP-1 signalling pathway. The increase of TNF-α demonstrated in this work, might be one of the causes of reduced levels in lymphocytes of SOD1 observed in patients with sporadic ALS (4). As TNF-α, IL-1ß has a neuroprotective role by inducing NGF production by enhancing ROS level. We plan to deeply investigate the immunogenetics of ALS disease in order to identify the cytokines’ mechanisms in the development of this complex multifactorial disease.

References:

P110 INVESTIGATING THE GENETIC BASIS OF ALS AND OTHER MOTOR NEURON DISEASES: ANALYSIS OF KNOWN GENES AND SEARCH FOR NEW LOCI

WILLIAMS K1,2, DREW A1,2, SOLSKI J1, DURNALL J1, THOENG A1,2, WARRAICH S1,2, THOMAS V1,2, CRAWFORD J1,3, NICHOLSON G1,4, BLAIR I1,2

1ANZAC Research Institute, Sydney, NSW, Australia, 2University of Sydney, Sydney, NSW, Australia, 3Macquarie University, Sydney, NSW, Australia, 4Concord Hospital, Sydney, NSW, Australia

E-mail address for correspondence: [email protected]

Keywords: gene, linkage, next-generation sequencing

Background: The motor neuron disorders are a group of neurodegenerative diseases that cause the selective progressive death of motor neurons. MND ranges from the rapidly progressive fatal form, amyotrophic lateral sclerosis (ALS), to slowly progressive forms including the lower motor neuron disorder, hereditary motor neuropathy (HMN). Genetic and clinical overlaps between fatal and non-fatal MNDs imply shared pathogenic pathways. Familial ALS (FALS) accounts for approximately 10% of ALS cases with the remainder being sporadic (SALS). MND is genetically heterogeneous. To date, known genes account for a small proportion of cases.

Objectives: We aim to investigate known ALS/MND genes and identify new ALS/MND genes in large cohorts of Australian ALS and non-fatal MND families.

Methods: Large cohorts of Australian ALS families (n=147) and families with non-fatal MND (n=24) have been recruited. We are using a combination of traditional genetic linkage approaches together with next-generation sequencing strategies to search for new ALS/MND genes among families that are negative for all known ALS and other MND genes. Known ALS/MND genes have been the subject of mutation analysis among these cohorts.

Result: We analysed 147 ALS families for mutations in SOD1, DCTN1, ANG, CHMP2B, VEGF, FIG4, TARDBP, FUS, and DAO. Haplotype analysis was also performed to identify any potential founder mutations. We determined that mutations in known ALS genes account for 20% of Australian ALS families, and comprises of SOD1 (14.3%), FUS (2.7%), ANG (2.0%), and TARDBP (1.4%) mutations. None of the identified mutations in SOD1 and ANG were present in a screen of 492 control chromosomes. Among families with identified mutations, variable phenotypic expressivity was observed with gene and mutation specific effects for disease onset and duration. To identify new loci for familial ALS and non-fatal MND, genome-wide linkage scans were performed in a subset of families that were negative for all known genes. An 8 cM genome-wide microsatellite scan was carried out by deCODE-Iceland using 166 individuals (affected, unaffected and obligate carriers). Subsequent analyses have yielded significant and suggestive linkage to several chromosomal regions. Families are undergoing fine genetic mapping using additional genetic markers to assist in refining loci and excluding false-positive regions. Target-region sequencing and exome capture-sequencing (NimbleGen capture-Solexa sequencing) have also been performed on selected families with data analysis underway.

Discussion and conclusions: The genetic defects are yet to be identified among 80% of ALS families (117/147 families) within our cohort. The chromosomal regions implicated from our genome-wide linkage scans do not overlap previously identified loci, implicating substantial genetic heterogeneity. Identification of novel ALS/MND genes will give insights into the biological basis of both familial and sporadic motor neuron degeneration, allow development of new disease models and provide new targets for therapeutic development.

P111 USING EXOME SEQUENCING TO IDENTIFY GENES CAUSATIVE FOR FAMILIAL ALS

BROWN, JR RH1, TICOZZI N2, GELLERA C3, LECLERC AL1, KEAGLE P1, MCKENNA-YASEK DM1, SAPP PC1, GLASS JD4, SILANI V1, LANDERS JE1

1Department of Neurology, University of Massachusetts Medical School, Worcester.MA, United States, 2Department of Neurology and Laboratory of Neuroscience, “Dino Ferrari” Center, Università degli Studi di Milano- IRCCS Istituto Auxologico Italiano, Milan, Italy, 3Units of Genetics of Neurodegenerative and Metabolic Diseases, Fondazione IRCCS Istituto Neurologico “Carlo Besta”, Milan, Italy, 4Department of Neurology, Emory University, Atlanta, GA, United States

E-mail address for correspondence: [email protected]

Keywords: next-generation sequencing, exome sequencing, gene identification

ALS is an adult-onset, rapidly progressive, and ultimately fatal neurodegenerative disease caused by the selective loss of motor neurons. To date, the underlying cause of familial ALS (FALS) has been identified only in ∼35% of all FALS cases. Approximately 20% of FALS cases are caused by mutations in SOD1, ∼5% in TARDBP and ∼5% in FUS; mutations in several other genes have been found in isolated families. Genetic studies have provided invaluable information towards the understanding of pathogenesis in both FALS and SALS. Undoubtedly, the discovery of novel FALS-associated genes will dramatically further our knowledge of the cellular pathways that lead to motor neuron degeneration.

To date, two strategies typically have been used to identify new genes in monogenic diseases: linkage analysis and candidate gene analysis. Linkage analysis requires large pedigrees composed of many affected individuals in different generations; this task is considerably difficult in ALS, a disease of adult life with a rapid disease course. On the other hand, candidate gene analysis, while feasible in cohorts of unrelated FALS cases, is flawed by a selection bias. Until now, it was not economically feasible to screen for rare variants at a genome-wide scale on large cohorts. However, the recent advances in automated short-read DNA sequencing offer new solutions to this problem. It is now possible to sequence only protein-coding regions of the genome (exomes) to reduce costs while enriching for discovery of a highly penetrant variant. Towards this end, we have embarked on an ongoing project to identify novel FALS variants using exome sequencing. In this presentation, the methodology of exome sequencing will be illustrated as well as the pitfalls of this approach. Recent results from ongoing studies in FALS families analyzed using exome capture and next-generation sequencing will be discussed.

P112 GENE SET ENRICHMENT ANALYSIS OF GENE EXPRESSION PROFILES OF LYMPHOCYTES FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS PREDICTS DEREGULATION OF THE UBIQUITIN/PROTEASOME PATHWAY

MOUGEOT J-L1,2, PRICE A1, ARMSTRONG E1, BROOKS BR1

1Carolinas Medical Center, Department of Neurology, Carolinas Neuromuscular/ALS-MDA Center, Charlotte, NC, United States, 2The University of North Carolina at Charlotte, Charlotte, NC, United States

E-mail address for correspondence: [email protected]

Keywords: microarray, GSEA, ubiquitination

Background: Recently, Saris et al. (2009) have found neurological disease-related molecular signatures in total blood from ALS patients compared to healthy controls using weighted gene co-expression network analysis. We applied gene set enrichment analysis (GSEA; BROAD Institute, Cambridge, MA) to a microarray dataset generated in our laboratory from purified lymphocytes from ALS patients and healthy controls.

Objectives: To analyze an existing (unpublished) microarray dataset produced in our laboratory using GSEA and to confirm predicted pathway alteration in peripheral blood mononuclear cells (PBMCs) at the protein level by Western blot analysis.

Methods: GSEA was applied on a microarray dataset obtained from lymphocytes isolated from definite sporadic ALS patients (ALS, n=11) and healthy controls (HC, n=11) using Agilent 4X44K Whole Human Genome Microarrays in a dual-mode reference design. The MIDAS/TM4 program was used for LocFit-LOWESS normalization and to generate two filtered datasets, DS3500 (10,147 probes) and DS7000 (7,199 probes), where both Cy3 and Cy5 integrated intensities were above one or two standard deviation(s) of their respective background. Log2 of ratio of classes and t-test were used as ranking metrics. Datasets were computed against generic human gene sets of the GSEA/MSigDB v2.0.5 database. GSEA analysis was also performed using keywords such as aging, mitochondrion, SOD1, proteasome and ubiquitin, to extract gene sets from MSigDB. Western blot analysis was used to determine total ubiquitination in PBMCs from additional ALS patients (n=10), HC (n=10) and patients with multiple sclerosis (MS, n=10).

Results: A total of 130 gene sets among 4995 were found significant (P<0.05). Chromosomal location gene set CHR5Q14 contained COX7C which polymorphisms may be associated with ALS. Four gene sets were significant in all comparisons ALS vs. HC: HSA04120_UBIQUITIN_MEDIATED_ PROTEOLYSIS, CANCER_NEOPLASTIC_META_UP, CALRES_RHESUS_DN and NOUZOVA_ CPG_H4_UP. Most enriched gene sets were associated with DNA repair, histone acetylation and ubiquitin-mediated proteosomal degradation. Mitochondria appeared marginally affected as lumen and matrix related gene sets were only significant using Log2 of ratio of classes as a metric. GSEA based on keywords (see Methods) also suggested alteration of ubiquitination/proteasome function. Western blot analysis of PBMCs from additional ALS and MS patients and HC subjects confirmed increased levels of ubiquitinated proteins in ALS as well as in MS patients.

Discussion and conclusions: GSEA was found to be a powerful method to reveal unifying biological themes regarding our ALS microarray dataset, as relevant biological differences are modest relative to the noise at the genome-wide scale. GSEA was predictive of changes in ubiquitination in PBMCs from ALS patients also occurring in MS patients. Thus, in the search for biomarkers that relate to ALS etiology, it is critical to include relevant disease controls in microarray studies.

P113 TOWARD DISCOVERY OF ALS-SPECIFIC GENE NETWORKS BY AUTOMATIC LITERATURE ANALYSIS

BUCCI EM1, POLI A1, NATALE M1, BONINO D1, MARCHIARO M1, GULLUSCI M1, MONTAGNOLI L1, CONSOLI P1, VINCI E1, ABRESCIA C2, BONGIOANNI P3,2

1Biodigitalvalley Srl, Pont Saint Martin (AO), Italy, 2NeuroCare onlus, Pisa (PI), Italy, 3Azienda Ospedaliero-Universitaria Pisana, Pisa (PI), Italy

E-mail address for correspondence: [email protected]

Keywords: bioinformatics, gene networks

Background: In former years, attention has been paid to the system biology of Amyotrophic Lateral Sclerosis (ALS), ie to the identification of relationships between different players in the cascade of molecular events regulating ALS. In particular, methods were developed to automatically parse the scientific literature, to identify co-occurring names of proteins/genes and to identify terms which qualify the relationships between them. However, since most of the available methods parse only scientific abstracts, the information obtained is often incomplete, due to the fact that only those proteins which are in the main scope of the paper are discussed, while often data on a number of other proteins are contained elsewhere in the relevant papers.

Objectives: To overcome these limitations, we focussed on the analysis of the figure captions contained in the scientific literature. We apply this method to obtain a robust biological network of genes involved in ALS.

Methods: The captions of a paper often contain an enriched amount of data on different proteins and on their interconnections, ie terms referring to proteins and simultaneously those referring to experimental methodologies. Analyzing captions allows identification of groups of proteins studied with a certain experimental technique, while at the same time characterizing the relationships among them. For example, proteins co-occurring in a caption describing a double-hybrid experiment are most likely binding partners, while proteins co-occurring in a caption describing a 2D-gel experiment are probably co-expressed in a given condition.

In our study, more than 2,000,000 papers were examined, using ad-hoc JAVA codes for parsing the pdf documents, identifying captions, and matching relevant terms. Identified genes were classified on the basis of their involvement in ALS only, in ALS and other neurodegenerative conditions, or in ALS and other non-neurodegenerative conditions.

Results: We found that: 1) by our method, some genes could be linked to ALS on the basis of publications preceding the demonstration of their direct involvement in ALS; 2) the obtained network matches quite well to the corresponding curated public-domain databases. Moreover, of relevance to ALS molecular biology, we found that: 3) new genes are identified, thus suggesting potential new targets for experimental studies; 4) some genes appear specifically connected to ALS, potentially differentiating this neurodegenerative condition from others.

Discussion and conclusions: Unravelling the intricacy of protein relationships defining the system biology of ALS involves building robust networks of protein biological interactions, including both genes already related to ALS and without recognized clinical relevance, but having biological meaning.

We found that supplementing usual methods of automatic literature analysis, with the analysis of the experimental data contained in captions, can produce better results, and leads to the identification of new potential targets for the molecular characterization of ALS.

P114 THE ALS ONLINE GENETICS DATABASE, ALSOD

ABEL O, POWELL J, ANDERSEN P, AL-CHALABI A

Department of Clinical Neuroscience, Institute of Psychiatry, King's College London, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: genome wide study, database, bioinformatics

Background: The ALS Online Genetics Database, ALSOD (http://alsod.iop.kcl.ac.uk/), is a central repository for genetic information on ALS. The initial aim of this database was to discover genotype: phenotype (G2P) correlations for SOD1 mutations. There has been considerable progress both technologically and scientifically since ALSOD was originally setup in 1999, presenting an opportunity for the collection and presentation of ALS genetic data in a single repository. The database is being transformed to meet these challenges.

Objectives: To provide a single, continuously updated central repository summarizing the current state of ALS genetics using open source standards and providing a service to the ALS research community, including automated meta analysis and integration of data from linkage and association studies.

Methods: The requirements of the ALS genetics research community were collected from the ALSOD feedback page. The database schema was restructured to allow for flexibility and expansion by changing table designs, rewriting queries and implementing appropriate stored procedures. Codes and scripts were written in programming languages like javascript, XML, C#, T-SQL and VB.NET integrated under the ASP.NET platform.

Results: The user registration process and data submission methods have been simplified and streamlined. ALSOD has been accessed over 141,000 times since January 2009 by users from 124 countries, with 11,604 accesses in the month of May alone. An overview of key published studies for approximately 43 ALS-related genes (http://alsod.iop.kcl.ac.uk/overview/index.aspx) is now included. Bioinformatics and analysis tools such as PLINK and Haploview have been integrated into the web-pages. Existing genome-wide association study data have been collected for meta-analysis and on-the-fly meta-analysis in which unpublished user-data is combined with existing studies confidentially and the result fed back in minutes. Links to gene variants, gene databases and relevant publications have been manually added to web-pages, but this process will be automated. Google AJAX search and Google Earth API have been combined to overlay geographical SOD1 mutations on the globe. This will be extended to other genes.

Discussion and conclusions: Advances in genetics and the fast pace of publications mean that a central resource summarizing all available data in an easily digestible form is essential. Integration with existing databases and analysis software means that ALSOD is a powerful resource for exploring existing genetic information for ALS. New tools are in development and these will be made live after initial beta-testing is passed. We welcome ideas for development or the addition of open source code from the user community. ALSOD is widely used by the ALS genetics research community. We aim to make it an indispensable tool for ALS research.

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