THEME 7 GENETICS

P155 GENETIC COUNSELLING IN ALS: FACTS AND UNCERTAINTIES

Chiò A¹

Mora G²

Sabatelli M³

Battistini S⁴

Corbo M¹

Caponnetto C⁵

Mandich P⁵

Penco S⁶

Conforti F⁷

Zollino M³

Mandrioli J⁸

Restagno G⁹

Surbone A¹⁰

^aUniversity of Turin, Turin, Italy

^bS. Maugeri Foundation, IRCCS, Milan, Italy

^cCatholic University, Rome, Italy

^dUniversity Siena, Siena, Italy

^eUnversity of Genoa, Genoa, Italy

^fNiguarda Hospital, Milan, Italy

^gInstitute of Neurological Sciences, National Research Council, Cosenza, Italy

^hUniversity of Modena, Modena, Italy

ⁱAzienda Ospedaliera Citta’ della Salute e della Scienza, Torino, Italy

^jDepartment of Medicine, New York University, New York, USA

Email address for correspondence: [email protected]

Keywords: genetic counselling

Background: The clinical approach to patients with amyotrophic lateral sclerosis (ALS) has been largely modified by the identification of several novel genes, detection of gene mutations in apparently sporadic patients, and discovery of the strict genetic and clinical relation of ALS with frontotemporal dementia (FTD). Consequently, clinicians are increasingly called to provide genetic testing and counselling both for ALS patients and their relatives.

Methods: A review of the existing literature on the genetics of ALS, and of other neurological late-onset, life-threatening disorders, and cancer will be presented and critically analysed.

Results: Following the Consensus meeting in Corteranzo, Italy, of a group of neurologists, geneticists, psychologists and ethicist with specific interest in genetics of ALS, detailed clinical suggestions were drafted and are now in press. We will present and discuss the consensus recommendations to enable neurologists and ALS specialists to provide optimal multidisciplinary clinical and genetic counselling to patients and families.

Genetic testing should be offered to ALS patients with a first- or second-degree relative with ALS, FTD or both, and it should be discussed with, but not offered to, all other ALS patients, with special emphasis on its major uncertainties in apparently sporadic ALS patients. The presence of co-morbid FTD in ALS patients should be assessed in order to establish their decision-making capacity, especially when signing informed consent for future genetic studies. Presently, genetic testing should not be proposed to asymptomatic at risk subjects, unless they specifically request it and/or are enrolled in research programs. We discourage parents from requiring ALS genetic analysis for their children and recommend that they be explained that genetic testing removes the child's ability to make an informed decision about testing when they reach adulthood and it carries major potential psychosocial implications.

Genetic counselling in ALS should take into account (1) uncertainties about the pathogenicity and penetrance of some genetic mutations; (2) possible presence of mutations of different genes in the same individual; (3) poor genotypic and phenotypic correlation in most ALS genes; and (4) phenotypic pleiotropy of some genes. Though psychological, social, ethical and legal implications of genetic testing are still relatively unexplored in ALS, a wealth of empiric data is available for other diseases such as cancer. We therefore also recommend in ALS a multidisciplinary counselling addressing all relevant medical, psychological and social issues, including sharing tests results to family members and risk for genetic discrimination. The ramifications of genetic testing on biological relatives should be made clear before any subject is tested. The meaning and value of the ‘Right Not to Know’ of both patients and family members should be familiar to all clinicians and discussed with all patients choosing to undergo genetic testing, and during the decision-making process regarding disclosure of their genetic testing results.

P156 WHOLE BLOOD GENE EXPRESSION PROFILES DISCRIMINATE ALS PATIENTS FROM HEALTHY CONTROLS

van Rheenen W

Saris C

Diekstra F

Groen E

Medic J

Schellevis R

Sodaar P

van Es M

Blauw H

van Vught P

Veldink JH

van den Berg LH

Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Centre Utrecht, Utrecht, The Netherlands

Email address for correspondence: [email protected]

Keywords: biomarker, gene-expression, micro-array

Introduction: Amyotrophic lateral sclerosis is often diagnosed per exclusionem. Although median survival after disease onset is 3 years, there is a considerable inter-individual variance. Biomarkers that provide diagnostic or prognostic information, and that can be used to monitor disease progression in clinical trials are highly needed in this disease (1). In the field of oncology, gene-expression profiling of tumor tissues has proven to yield valuable information for tumor classification, treatment options and prognosis (2).

Methods: We used whole blood gene expression profiling in order to try to identify a gene expression profile that is characteristic for ALS. Included were 397 ALS patients and 645 control subjects divided into a training set, test set and an independent validation set to ensure robustness of results. Whole blood messenger RNA was hybridized to Illumina HumantHT-12 v3 and v4 BeadChips. Data were corrected applying surrogate variable analysis. The nearest shrunken centroid algorithm was used to train, test and validate our model.

Results: We have identified a set of 106 genes that discriminated ALS patients from control subjects with high accuracy correctly classifying 89% of the samples in our test set and 80% in the validation set. Gene ontology analysis of gene functions revealed that the 106 genes were mainly involved in RNA binding, an important process in motor neuron disease biology. Survival could not be predicted reliably.

Discussion: Clinical applicability will be further studied comparing ALS patients with patients suffering from a condition that mimics ALS. Furthermore longitudinal studies will be able to observe changes in expression of these genes to monitor disease progression.

Conclusion: We here show that whole blood gene expression profiles can be used to reliably discriminate ALS patients from controls.

References

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P157 CHANGES IN MICRO-RNAS’ EXPRESSION DISCLOSE NOVEL LINKS BETWEEN ALS AND INFLAMMATION

Parisi C¹

Arisi I²

D’Ambrosi N¹

Storti AE²

Brandi R²

Ricci C³

Battistini S³

D’Onofrio M²

Volonté C¹

^kCellular Biology and Neurobiology Institute, CNR/FSL, Rome, Italy

^lEuropean Brain Research Institute EBRI “Rita Levi-Montalcini”, Rome, Italy

^mDepartment of Medical, Surgical and Neurological Sciences, University of Siena, Siena, Italy

Email address for correspondence: [email protected]

Keywords: microglia, microRNAs, P2X7 receptor

Background: Micro-RNAs regulate gene expression at the post-transcriptional level and are emerging as key modulators of the immune system whose dysfunction contributes to the progression of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) (1). ALS is a non-cell-autonomous disease targeting both motor neurons and neighbouring glia, with microgliosis substantially contributing to neurodegeneration. While purinergic P2X7 receptor has a recognized role in neuroinflammation and ALS pathogenesis (2), its mechanistic signalling is only partially known. Despite several studies, which have identified a prominent and complex role of miRNAs as key modulators of signal propagation, there are only a few works on miRNAs regulation of purinergic systems and ALS (3).

Objectives: The aim of this work was to provide a map of those miRNAs that are differentially expressed in ALS, and to identify their implication in the pathogenesis. We examined primary microglia cultures from SOD1-G93A mice and human serum. We compared microRNAs transcriptional profiling in resting conditions and after activation of P2X7 receptor.

Results: We identified a strong upregulation of immune- enriched microRNAs transcriptome in ALS resting microglia and after P2X7 receptor stimulation, and recognized miR-22, miR-155, miR-125b and miR-146b as important microglia activators. In particular, by luciferase assays and lentiviral based microRNAs overexpression, we proved that miR-365 and miR-125b, suppressing the IL-6/STAT3 pathway, determine an increase of TNFalpha transcription. Furthermore, since TNFalpha upregulates miR-125b, we might recognize the induction of miR-365 and miR-125b as the gateway of a vicious cycle culminating in abnormal TNFalpha release. Finally, by analyzing circulating miRNAs levels in serum of sporadic ALS patients, we found a significant decrease of miR-195, miR-134 and miR-150, known to be involved in immune system functions and P2X7 receptor regulation.

Discussion and conclusion: These results strengthen the impact of microRNAs in modulating genes linked to inflammation and ALS. Moreover, they identify some specific micro-RNAs that might directly contribute to the outcome of ALS and might act as diagnostic markers or novel therapeutics for the disease.

Acknowledgements:

We acknowledge Italian Ministry for Education, University and Research in the framework of the Flagship Project NanoMAX, FIRB RBAP10L8TY, and Fondazione Roma for financial support.

References

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P158 HOMOZYGOSITY ANALYSIS IN AMYOTROPHIC LATERAL SCLEROSIS

Mok K¹

Laaksovirta H^2,3

Tienari P^2,3

Peuralinna T³

Myllykangas L⁴

Chiò A⁵

Traynor BJ³

Nalls M³

Shoai M¹

Gurunlian N¹

Shatunov A⁶

Restagno G⁷

Mora G⁸

Leigh P⁹

Shaw CE⁶

Morrison K¹⁰

Shaw PJ¹¹

Al-Chalabi A⁶

Hardy J¹

Orrell R¹

ⁿReta Lila Weston Research Laboratories, Department of Molecular Neuroscience, and Department of Clinical Neuroscience, UCL Institute of Neurology, London, UK

^oHelsinki University Central Hospital, Department of Neurology, Molecular Neurology Research Program, Biomedicum, University of Helsinki, Helsinki, Finland

^pMolecular Genetics Section and Neuromuscular Diseases Research Group, Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, USA

^qDepartment of Pathology, Haartman Institute, University of Helsinki and HUSLAB, and Folkhalsan Institute of Genetics, Helsinki, Finland

^rDepartment of Neuroscience, University of Turin and Azienda Ospedaliera Universitaria San Giovanni Battista, Turin, Italy

^sMedical Research Council Centre for Neurodegeneration Research, King's College London, Institute of Psychiatry, London, UK

^tMolecular Genetics Laboratory, Azienda Ospedaliera OIRM-Sant’Anna, Turin, Italy

^uFondazione Salvatore Mangeri, IRCCS Scientific Institute of Milan, Milan, Italy

^vBrighton and Sussex Medical School, Trafford Centre for Biomedical Research, University of Sussex, Falmer, UK

^wSchool of Clinical and Experimental Medicine, University of Birmingham and Queen Elizabeth Hospital, University Hospitals Birmingham NHS Foundation Trust, Birmingham, UK

^xDepartment of Neuroscience, The Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield, UK

Email address for correspondence: [email protected]

Keywords: genetics, homozygosity, recessive variant

Background: Amyotrophic lateral sclerosis (ALS) can be familial or sporadic, with dominant and recessive inheritance recognized in familial cases. Nevertheless, sporadic ALS may be caused by rare homozygous recessive mutations with no family history.

Objectives: To determine any excess of homozygosity in ALS cases and potential regions that may carry recessive disease-associated variants.

Methods: We studied a multinational pooled analysis of GWAS data in five ALS cohorts. PLINK v1.07 was used to determine the homozygous segments and any potential recessive variant leading to the disease. Association analysis was performed with PLINK algorithm with case control permutation.

Results: Two thousand and seventeen ALS cases and 6918 controls were studied in the pooled analysis. There were more regions of homozygosity segments per case (p = 1 × 10 ⁻⁵), a greater proportion of cases harboured homozygosity (p = 2 × 10 ⁻⁵), a longer average length of segment (p = 1 × 10 ⁻⁵), a longer total genome coverage (p = 1 × 10 ⁻⁵`), and a higher rate of these segments overlapped with RefSeq gene regions (p = 1 × 10 ⁻⁵), in ALS patients than controls. Positive associations were found in the chromosome 21 SOD1 region, and also chromosome 1 2.9–4.8Mb.

Discussion and conclusion: The association in chromosome 21 SOD1 region is partly contributed by over-representation of a group of SOD1 D90A mutation carriers in one of the cohorts. A follow-up of the genes in chromosome 1 using next generation sequencing is currently on going.

Acknowledgements

This work was supported in part by the Intramural Research Programs of the NIH, the National Institute on Aging (Z01-AG000949-02), and the National Institute of Neurological Disorders and Stroke. Extramural NIH grants R01AG031278 and R01AG038791 supported some family assessments. The research leading to these results has received funding from the European Community’s Health Seventh Framework Programme (FP7/2007–2013) under grant agreement no. 259867. We thank the Motor Neurone Disease Association of Great Britain for several grants relating to this work (RWO, AAC, PJS, HM), the ALS Association, The Angel Fund, the ALS Therapy Alliance, and the Wellcome Trust (PJS) for support. This work was also funded by the Reta Lila Weston Foundation, and by an MRC returning scientist (JH) and fellowship (SPB) award, by Microsoft Research Foundation, the ALS Association, Helsinki University Central Hospital, the Finnish Academy, Ministero della Salute, Progetti Finalizzati 2007, Fondazione Vialli e Mauro for ALS, and Federazione Italiana Giuoco Calcio. The authors thank the NIHR specialist Biomedical Research Centre for Mental Health at the South London and Maudsley NHS Foundation Trust (SLaM) and the Institute of Psychiatry, King’s College London, and the National Institute for Health Research-funded University College London/University College London Hospitals Biomedical Research Centre.

P159 USING WHOLE GENOME SEQUENCING (WGS) TO SOLVE THE SPG27 QUESTION

Noreau A^1,2

Meijer I²

Girard S^1,2

Dionne-Laporte A¹

Cossette P²

Dion P^1,3

Rouleau G¹

^yMontreal Neurological Institute, McGill University, Montreal, Canada

^zFaculty of medicine

^aaDepartment of pathology and cellular biology, Université de Montreal, Montreal, Canada

Email address for correspondence: [email protected]

Keywords: SPG27, hereditary spastic paraplegia (HSP), whole genome sequencing (WGS)

Background: Hereditary spastic paraplegia (HSP) is a motor neuron disease for which the key symptoms are lower limb spasticity and weakness because of progressive neurodegeneration events. The group of HSP is genetically and clinically heterogeneous with different modes of inheritance reported (dominant, recessive, and X-linked) and various levels of symptom complexity; pure and complicated forms have been described. To date, over 50 spastic paraplegia loci were identified and 30 causatives genes have been identified. A previous linkage analysis on an autosomal recessive pure HSP family identified a locus (SPG27) on the 10q22.1–10q24.1 region; a locus partially overlapping the SPG9 locus.

Objective: To identify the causative gene, we performed a Whole Genome Sequencing (WGS) approach from Illumina, using their standard method. Three affected individuals were selected amongst the seven affected siblings. TruSeq kit was used as library and sequencing was done on HiSeq reaching a minimum of 30X coverage. Data analysis was performed using CASAVA (consensus sequence from ELAND alignment, called SNPs, and small indels).

Results: From a total of 68,994 variants shared by the three affected individuals, we filtered the variants list to keep only exonic variants that were not found in Exome Variant Server and also not found in dbSNP. We hand up with 23 rare variants to validate by traditional Sanger method.

Discussion: Finally, it will be possible to identify the causative gene by showing a perfect cosegregation with the clinical status of the whole family members.

Acknowledgments:

Anne Noreau holds a Doctoral Award – Frederick Banting and Charles Best Graduate Scholarship by the Canadian Institutes of Health Research (CIHR).

References

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P160 ALS MOLECULAR GENETIC ANALYSIS: EXPERIENCE AT GENOA'S MEDICAL GENETICS UNIT

Origone P^1,4

Verdiani S²

Mantero V^2,5

Bandettini di Poggio M²

Caponnetto C^2,3

Mancardi GL²

Mandich P²

^abDepartment of Internal Medicine

^acDepartment of Neuroscience, Rehabilitation, Ophtalmology, Genetics and Maternal Child Health, University of Genova, Genova, Italy

^adUO Neurology

^aeUO Medical Genetics of IRCCS AOU S. Martino-IST, Genova, Italy

^afDepartment of Neurology, A. Manzoni Hospital, Lecco, Italy

Email address for correspondence: [email protected]

Keywords: genetic analysis, mutation, expansion

Background: Research on genetic factors in ALS is rapidly advancing and recently new genes are recognized to be related to the disease. However, despite recent advances, most of the genes are still unknown. Considerable work is ongoing to discover such genes and elucidate specific therapy for mutation carriers.

Objective: Aim of the study was to review genetic features of a cohort of sporadic and familial ALS patients who underwent routine diagnostic genetics tests from 2000 to date, at the Medical Genetics Unit of the IRCCS AOU S. Martino-IST, Genova. Furthermore the analysis of candidate genes was performed.

Methods: After adequate information and signed written consent, 200 patients underwent DNA analysis for ALS. ALS clinical diagnosis was based on the revised El Escorial criteria. Family history was investigated, when possible, and demographic data and clinical characteristics of index cases were pointed out.

Exons and splice junctions of SOD1, TARDBP (exon 6); FUS-TLS (exons 6 and 15); FIG4, VCP (exons: 2, 3, 5, 6, and 14); OPTN (exons: 3, 4, 9, 10, 12, and 14) genes were examined by direct nucleotide sequence analysis. Pathological expansions in ATXN2 (CAG)n and C9ORF72 (GGGGCC)n were also analysed by TP-PCR.

Results: Out of 200 patients included in the study, 19 (9.5%) carried a mutation in one of the genes considered. All mutations were detected in heterozygous condition. Mutations found were eight SOD1 (42%), five in FALS and three in SALS patients; three TARDBP (15.8%), one FALS and the other SALS; two FUS-TLS (10.5%) in FALS patients; Pathological expansions in C9ORF72 were found in six patients (31.6%) three FALS and three SALS; No mutations in VCP, FIG4, and OPTN genes were found in the analyzed exons; No expansions were found in the ATXN2 gene.

Discussion: In our ALS series we found that 9.5% of patients carry a genetic mutation. In our population SOD1 mutations and C9ORF72 expansion were the most represented. The complexity of the C9ORF72 expanded phenotype, which includes cases with ALS, ALS-FTD, and FTD, changes the current notion of familial ALS, requiring collection of family members with different and often complex phenotypes. Furthermore, the presence of cognitive impairment adds more issues related to informed consent for genetic studies.

Conclusions: The overall percentage of our mutated ALS cases (9.5%) is in agreement with that discussed in the literature. Geneticists are increasingly facing questions about newly identified genes and their role in the ALS pathogenesis, as well as penetrance of individual gene mutations, severity and modifier factors of phenotypes. Genetic counselling is a useful setting for managing such problems, according to shared new guidelines which have to be discussed and identified at a national and international level.

Acknowledgements

The financial support of Regione Liguria Grant to P.M. is gratefully acknowledged.

P161 IMPROVING THE KNOWLEDGE OF ALS GENETICS: NOVEL SOD1 VARIANTS AND RECESSIVE FUS MUTATION

Bertolin C¹

Querin G¹

D’Ascenzo C¹

Boaretto F²

Salvoro C²

Vazza G²

Cagnin A¹

Pegoraro E¹

Sorarù G¹

Mostacciuolo M²

^agDepartment of Neurosciences

^ahDepartment of Biology; University of Padova, Padova, Italy

Email address for correspondence: [email protected]

Background: Mutations in C9ORF72, SOD1, TARDBP and FUS genes are currently considered as the most common genetic cause of familial (FALS) and sporadic (SALS) forms of ALS.

Objective: The aim of the study was to estimate the mutation frequency of these genes in a large cohort of ALS patients referred to the ALS Clinic of the University, Padova. Moreover, considering the emerging clinical and genetic overlap between ALS and frontotemporal dementia (FTD), a second panel of FTD patients was also investigated for mutations on C9ORF72, FUS and TARDBP genes.

Methods: We investigated a total of 354 unrelated patients, 45 of them with reported family history (FALS) and 309 classified as sporadic/isolated cases (SALS). We performed Sanger sequencing of SOD1, TARDBP and FUS genes. In addition, a cohort of 73 FTD patients (25% with positive family history) was also evaluated for point mutations on FUS and TARDBP genes. The expanded GGGGCC hexanucleotide repeats in the C9ORF72 gene was genotyped in all ALS/FTD patients.

Results: We identified several pathogenic mutations in ALS patients: six in SOD1, two in TARDBP and two in FUS. All but two of these mutations were already reported by other groups. A new SOD1 mutation (p.Glu41Gly) was detected in a FALS patient. We also identified a new single base deletion in homozygous state (p.Gly496Glyfs*31) in the FUS gene: the mutation carrier reported no family history for ALS, suggesting a recessive inheritance of the disease. No point mutations were identified on FTD patient panel. The frequencies of expanded repeats in C9ORF72 were similar in ALS and FTD patients: 22% in the familiar cases and about 5% in the sporadic patients.

Discussion and conclusion: Our mutation screening confirms C9ORF72 as the main causative gene both in ALS and FTD patients. Moreover, this work highlights the importance of molecular testing of SOD1, TARDBP, and FUS in ALS patients identifying a new SOD1 mutation and pointing out the chance of an autosomal recessive pattern of FUS mutation inheritance. Finally, our results suggest that mutations in TARDBP and FUS genes are rare causes of FTD.

P162 CHARACTERIZING THE GENETIC HETEROGENEITY OF ALS THROUGH MASSIVELY PARALLEL TARGETED RESEQUENCING

Kenna K¹

McLaughlin R¹

Byrne S¹

Heverin M¹

Kenny E¹

Cormican P¹

Morris D¹

Donaghy C²

Bradley D¹

Hardiman O¹

^aiTrinity College, Dublin, Ireland

^ajRoyal Victoria Hospital, Belfast, Ireland

Email address for correspondence: [email protected]

Keywords: genetic epidemiology, heterogeneity, oligogenic

Background: ALS is a highly heterogeneous disorder. A formal understanding of disease heterogeneity may greatly advance a variety of clinical and research efforts.

Objectives: (1) To describe the genetic heterogeneity of ALS in Ireland by characterizing the variation across 33 previously reported disease genes; (2) to compare the observed frequencies of disease variants with those reported by studies of representative patient cohorts from other European populations; and (3) to search for correlations in the occurrence of putative disease variants which may be indicative of oligogenic aetiology.

Methods: Four hundred and forty-four representative Irish ALS cases (50 fALS and 394 sALS) and 311 age and geographically matched controls were analyzed by multiplexed targeted high-throughput sequencing. Known and potential ALS variants were subsequently identified using an assortment of bioinformatic analyses.

Results: 17.1% of patients were found to carry known or potential high penetrance ALS variants. The frequency of Mendelian disease gene variants was 12.8% (C9orf72 8.78%; SETX 2 48%; ALS2 1.58%; FUS 0.45%; TARDBP 0.45%; OPTN 0.23%; VCP 0.23%; ANG, SOD1, VAPB 0%), while the frequency of low penetrance/tentative ALS gene variants was 4.7%. 9.7% of patients (30% of fALS and 7.1% of sALS) carried previously described ALS variants (C9orf72 8.78%; FUS 0.45%; TARDBP 0.45%). Only 1.58% of patients were found to carry multiple putative disease variants, yet this included all identified carriers of one established ALS variant (n = 2/2 sALS, p < 0.01). Comparison of our results with those from studies of other representative European cohorts revealed significant differences in the spectrum of disease variation (p = 1.7 × 10^-4), most notably regarding the frequencies of the C9orf72 hexanucleotide repeat expansion (p = 3.95 × 10^-4), SOD1 variants (p = 3.8 × 10^-3) and TARDBP variants (p = 0.035).

Discussion: Our results represent the most extensive account of the relative and cumulative importance of identified ALS loci to date. They reveal that ˜17% of Irish ALS patients may carry high penetrance variants within the investigated genes, with the C9orf72 repeat expansion constituting the most common cause of disease. They also indicate that genetic susceptibility to ALS varies significantly across populations and that certain variants may not cause disease in isolation.

Conclusion: Efficient and simultaneous analysis of disease-related loci can be achieved with targeted high-throughput sequencing. Such analyses may prove highly important for the purposes of managing disease heterogeneity in clinical research settings and for identifying cases of oligogenic-based disease.

P163 A FAMILIAL ALS CASE CARRYING A NOVEL P.G147C SOD1 HETEROZYGOUS MISSENSE MUTATION

Canosa A¹

Restagno G²

Brunetti M²

Ossola I²

Barberis M²

Galmozzi F¹

Bertuzzo D¹

Chiò A¹

Tanel R³

Calvo A¹

^akUniversity of Torino, ‘Rita Levi Montalcini’ Neuroscience Department, ALS Center, Torino, Italy

^alA.O. Città della Salute e della Scienza, Laboratory of Molecular Genetics, Torino, Italy

^amU.O. Neurologia, Osp. Santa Chiara, A.P.S.S., Trento, Italy

Email address for correspondence: [email protected]

Keywords: SOD1, mutation, G147C

Background: Approximately 20% of FALS cases carry a mutation in the SOD1 gene. Some mutations at codon 147 have already been reported. We describe a familial case with a novel G147C missense mutation.

Case report: The index case (male), developed progressive weakness and wasting of the left hand at the age of 52, followed by motor impairment of the other hand. One year after onset the neurological exam showed mild spastic paraparesis with bilateral impairment of foot extension, weakness and wasting of both hands and brisk reflexes at lower limbs. The plantar response was absent bilaterally. The tongue was slightly hypotrophic, with evident fasciculations. The patient referred to diffuse cramps and fasciculations. Neither dysphagia and dysphonia, nor dyspnea was present. The MRI of brain and cervical spine were normal. The needle EMG showed signs of chronic and active denervation at upper and lower limbs and at the bulbar level. The FVC was 106%. Collecting the family history we found that two of the patient's brothers died from ALS respectively at the age of 46 and 50, with a spinal onset and a rapid disease course, and two siblings of 49 and 43 were alive and healthy. The patient's father died at the age of 76 from chronic kidney failure without any neurological impairment, while the mother was 83 years old and healthy. The genetic analysis on the index case revealed a point, G147C missense mutations of SOD1. The DNA of the other family members was not available. A diagnosis of clinically definite familial ALS with genetic confirmation was made. The patient is still alive, 14 months after the disease onset.

Discussion: To our knowledge, three other missense mutations of codon 147 of SOD1 have already been found. Andersen and colleagues first reported a p.G147R mutation in an ALS case of Icelandic origin. No further information was available. A p.G147D mutation was described in a French patient with spinal onset at the age of 73, 9-month disease course and unknown family history. Another group reported the same mutation in some Chinese familial cases, associated with a fast disease course. Finally, a p.G147S mutation was described by an Italian group in an apparently sporadic case with bulbar onset at the age of 56 and death from respiratory failure after 8 months.

The codon 147 encodes an aminoacidic residue which is highly conserved across species. This change was predicted to alter the normal function of SOD1 protein by molecular modelling studies. Most of the cases carrying mutations of the 147 codon show a rapidly progressive course, but we need further data to establish a possible genotype–phenotype correlation. This is the first report of an ALS case carrying a p.G147C heterozygous missense mutation of SOD1 gene.

P164 FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS WITH CYS111TYR MUTATION IN CU/ZN SUPEROXIDE DISMUTASE SHOWING WIDESPREAD LEWY BODY-LIKE HYALINE INCLUSIONS

Suzuki M

Oketa Y

Mikami H

Watanabe T

Ono S

Teikyo University Chiba Medical Center, Ichihara, Chiba, Japan

Email address for correspondence: [email protected]

Keywords: Cys111Tyr mutation, SOD-1, lewy body-like hyaline inclusions

Background: It is known that familial amyotrophic lateral sclerosis (FALS) with abnormalities of the SOD1 gene is neuropathologically characterized by the degeneration of middle root zone of the posterior column and the presence of Lewy body-like hyaline inclusions (LBHIs) in the lower motor neurons, in addition to the involvement of the upper and lower motor neurons. We report here an autopsy case of FALS with Cys111Tyr missense mutation in exon 4 of the SOD1 gene in which no pathological data have been available.

Case report: The patient’s mother died of ALS at age 66. Her first symptom was weakness of the leg muscles. Our patient (male), developed insidious muscle weakness in the right leg that progressively worsened thereafter. His gait gradually became disturbed from age 38. At age 39, he also experienced weakness in the arms and the left leg. At age 41, he presented dysarthria and dysphasia and needed tube-feeding. He became bedridden from the age of 41 years, but could communicate with facial, jaw and eye movements. At age 42, he developed difficulty in respiration. About 5 years after onset of the disease, at the age of 43 years, the patient died of respiratory failure.

Results: The main neuropathological findings of the spinal cord were a significant reduction in the number of anterior horn cells and pyramidal tract degeneration as well as degeneration of Clarke’s nuclei, spinocerebellar tract and middle root zone of the posterior column. In the medulla oblongata, the hypoglossal nuclei showed mild loss and shrinkage of nerve cells. In the pons, there was neuronal shrinkage and disappearance with gliosis in the trigeminal motor and facial nuclei. As seen in hematoxylin and eosin (HE) preparations, the LBHIs were eosinophilic or slightly paler inclusions and sometimes found eosinophilic cores with peripheral halos. LBHIs were seen in the anterior horn cells, Onufrowicz nucleus, Clarke’s nucleus, intermediolateral column, and posterior gray horn of the spinal cord. In addition, LBHIs were observed in the periaqueductal gray matter, nucleus raphe dorsalis, locus ceruleus, trigeminal motor nucleus, vestibular nucleus, dorsal vagal nucleus, hypoglossal nucleus, and reticular formation of the brain stem.

Discussion and conclusion: It is of particular interest that in our patient a lot of neuronal LBHIs were observed not only in the lower neurons but also in the nonmotor neurons. This would suggest the possibility that the neurons of the nonmotor system were impaired by the same morbid process that affected the motor neurons. These findings raise the question of whether the involvement of multiple systems in addition to motor neuron system can be considered to represent essential FALS lesions.

P165 CO-OCCURRENCE OF MULTIPLE SCLEROSIS (MS) AND AMYOTROPHIC LATERAL SCLEROSIS (ALS) IN A PATIENT CARRYING A P.D109Y MISSENSE MUTATION OF SOD1 GENE

Bertuzzo D¹

Ilardi A¹

Manera U¹

Moglia C¹

Calvo A¹

Cammarosano S¹

Valentini C²

Brunetti M³

Restagno G³

Chiò A¹

^anALS Center, ‘Rita Levi Montalcini’ Department of Neuroscience, University of Torino, Torino, Italy

^aoNeuoradiology Unit

^apLaboratory of Molecular Genetics, AO Città della Salute e della Scienza di Torino, Torino, Italy

Email address for correspondence: [email protected]

Keywords: ALS, SOD1, multiple sclerosis

Background: Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by selective loss of both upper and lower motor neuron with a fatal course. The majority of patients have a sporadic form of the disease (sALS), while about 10% of cases have a positive family history (fALS) for ALS or frontotemporal dementia. The second commonest ALS-causing gene is SOD1. Multiple sclerosis (MS) is a non-Mendelian, immune-mediated, inflammatory disease of the central nervous system, characterized by demyelination and axonal degeneration, leading to progressive neurological impairment. Although some reports have described the co-occurrence of ALS and MS, this association is still unexplained. We describe a 60-year-old female with definite ALS, carrying a p.D109Y missense mutation of SOD1, with biochemical and radiological signs consistent with a demyelinating disease.

Case report: A 60-year-old female referred to our clinic complaining weakness at right lower limb; she first became aware of mild, progressive impairment in walking 5 years prior to admission. She reported a 45-year-old male first-degree cousin diagnosed as having MS. Neurological examination revealed pyramidal right trait alteration; she had also weakness in her right hand with hypotrophy of the first dorsal interosseus muscle. No cranial nerve involvement was observed; no cerebellar or extrapiramidal signs were present. Her deep and superficial sensation was normal. She denied urinary symptoms.

The patient underwent full laboratory tests, which were unremarkable. Needle-EMG showed active denervation in right-hand muscles; somatosensory evoked potential study was abnormal at left lower limb, absent at the right side. Motor evoked potential study showed prolonged conduction time on the right side in the cortex-L5 tract. A genetic evaluation revealed a p.D109Y missense mutation of SOD1; she was diagnosed as having ALS. MRI scans of the brain and cervical spine showed multiple T2-high signal periventricular lesions of the white matter and an inflammatory lesion at C1–C2 posterior cords with no abnormal enhancement. Because of this finding, she underwent a lumbar puncture, which showed oligoclonal IgG bands and elevated IgG index. Therefore, the patient satisfied McDonald's diagnostic criteria for primary progressive multiple sclerosis. She was treated with a bolus of high-dose steroids with no clinical benefit.

Discussion: According to the best of our knowledge, this is the first case of SOD1-ALS with co-morbid features of MS. The relationship in the pathogenetic mechanism between ALS and MS has already been described but is poorly understood. Hemminki et al (2009) found that ALS patients’ offspring had an increased risk of MS and postulated that the shared familiar risk of MS with ALS suggest shared genetic basis. The possible association between these two disorders requires further investigation.

P166 A NEW MISSENSE MUTATION IN EXON 4 OF SOD1 GENE IN A PATIENT WITH SPORADIC ALS

Ricci C

Battistini S

Benigni M

Casali S

Giannini F

University of Siena, Siena, Italy

Email address for correspondence: [email protected]

Keywords: SOD1, mutation, bioinformatics analyses

Background: Superoxide dismutase 1 (SOD1) gene is the second most common mutated gene in amyotrophic lateral sclerosis (ALS), accounting approximately for 20% of familial ALS cases and 3% of sporadic cases (1). To date, more than 160 missense mutations of SOD1 have been reported. Here, we describe a novel missense mutation in exon 4 of the SOD1 gene in a patient without family history of ALS.

Objective: To describe a novel SOD1 mutation and to evaluate its impact on protein structure.

Method: After obtaining written consensus, DNA was extracted from peripheral blood and the SOD1 gene was analysed by PCR and sequencing. The effect of the novel SOD1 missense mutation on SOD1 protein was analyzed using PolyPhen, SIFT and PANTHER software. Modeling of the SOD1 variant was performed using the crystal structure of the normal SOD1 protein as reference.

Case report: The patient is a 78-year-old woman who at age 70 began to complain weakness of the left leg. During the subsequent years, the weakness spread to her right leg, leading the patient to be wheelchair confined. Physical and neurophysiological examination confirmed the involvement of only the lower motor neuron in lumbar-sacral district and no respiratory impairment, configuring a very slowly progressive flail-legs phenotype.

Results: Molecular analysis showed a heterozygous mutation g.1161G> A in the SOD1 gene. The mutation of GTG to ATG at codon 118 in exon 4 determined a substitution of methionine for valine in SOD1 protein (V118M). Bioinformatics analysis demonstrated the possible damaging effect of this substitution on protein structure/function. Modeling of mutant SOD1 showed a destabilization of secondary structure in the amino-acid sequence around the residue 118, involving also His120 and His46, localized in the SOD1 catalytic site.

Discussion and conclusion: We identified a novel mutation V118M in a sporadic ALS patient. The mutation, in exon 4, involves an amino-acid residue highly conserved in different species. The V118M substitution is localized in the b-7 strand of the b-barrel. Such b-barrel mutations usually result in local perturbations able to alter the protein structure by affecting the monomer stability, or the dimer interface, or both at the same time (2). In this case, the V118M mutation can perturb the secondary structure of the protein, destabilizing also the His120 and His46, which bind copper ion in the catalytic site. The metal binding alteration is known to diminish the metal coordination and lead to altered SOD1–SOD1 interactions (3). Thus, the V118M can result both in a failure of protein folding and/or genesis of toxic intracellular aggregates.

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P167 GENETIC BACKGROUNDS OF ADULT ONSET LOWER MOTOR NEURON SYNDROME

Mashiko T¹

Morita M¹

Tetsuka S¹

Akimoto C¹

Higuchi Y²

Hashiguchi A²

Takashima H²

Nakano I³

^aqJichi Medical University, Tochigi, Japan

^arKagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan

^asTokyo Metropolitan Neurological Hospital, Tokyo, Japan

Email address for correspondence: [email protected]

Keywords: lower motor neuron syndrome, adult-onset, hereditary motor neuropathy

Background: Lower motor neuron (LMN) syndrome is clinically characterized by signs of lower motor neuron dysfunction without signs of upper motor neuron (UMN). Whether LMN syndrome is a subtype of amyotrophic lateral sclerosis (ALS) or not is still a controversial matter today. Some LMN syndrome patients show relentless progression and poor prognosis with dyspnea or dysphagia similar to ALS, while the others show extremely slow clinical course different from ALS. In this retrospective study, we disclose the backgrounds of these slowly progressive LMN syndromes by means of both clinical and genetic methods.

Methods: We reviewed the medical records of 302 patients who had been diagnosed as having motor neuron disease at our institute over a period of 15 years from around 1997. Out of 302 patients, we selected patients who had been showing only LMN signs without UMN signs for more than 6years, focusing on their initial symptoms as unilateral weakness in the upper or lower extremity, bilateral weakness in the upper or lower extremities, and others. Primarily, we analyzed SMN, known as a causative gene for spinal muscular atrophy (SMA). This SMN analysis was performed for the purpose of checking the mutation of SMN for causing slowly progressive LMN syndrome characterized as proximal and symmetrical weakness in the lower extremities in adult cases. Subsequently, we analyzed the genes which cause hereditary motor neuropathy.

Results: One hundred and eight patients showed LMN signs without UMN signs at their first visit. However, UMN signs or acute exacerbation appeared to most of them in the follow-up, and finally 11 patients were diagnosed as having slowly progressive LMN syndromes. About their initial symptoms, 10 cases showed unilateral weakness in their upper or lower extremity, and only one case showed bilateral weakness in the lower extremities. Genetic analysis was carried out in nine cases upon their informed consent. None of them had SMN mutations, but five of them had the mutations in the genes including DCTN1, HSPB1, GARS and TRPV4, which are known to cause hereditary axonal motor neuropathy.

Conclusion: Our study showed that cases, clinically diagnosed as adult onset LMN syndromes, were different from SMA from both clinical and genetic aspects, and were composed of genetically heterogenic backgrounds including motor neuropathy.

P168 LOWER MOTOR NEURON DISEASE WITH PREDOMINANT RESPIRATORY FAILURE AND WITHOUT DEMENTIA CAUSED BY A NOVEL MAPT MUTATION IN AN ITALIAN KINDRED: CLINICAL, GENETIC AND PATHOLOGICAL CHARACTERIZATION

Di Fonzo A¹

Trezzi I¹

Milena C³

Ronchi D¹

Gallia F²

Salani SA¹

Bordoni A¹

Corti S¹

Bosari S³

Zuffardi O⁴

Bresolin N¹

Nobile-Orazio E²

Comi G P¹

^atDino Ferrari Centre, Neuroscience Section, Department of Pathophysiology and Transplantation, University of Milan, IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Milan, Milan, Italy

^auDepartment of Medical Biotechnology and Translational Medicine (BIOMETRA), 2nd Neurology, Humanitas Clinical and Research Center, IRCCS Humanitas Clinical Institute, University of Milan, Milan, Italy

^avDivision of Pathology, IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico, Milan, Milan, Italy

^awDepartment of Molecular Medicine, University of Pavia. Pavia, Italy and National Neurological Institute C. Mondino, University of Pavia, Pavia, Italy

Email address for correspondence: [email protected]

Keywords: amyotrophyc lateral sclerosis, MAPT mutations, tauopathy

Case report: We report the genetic and clinicopathological characterization of a large Italian kindred with an adult onset progressive respiratory failure and proximal weakness of the upper limbs, and evidence of lower motor neuron degeneration.

A genome wide linkage analysis showed association to chromosome 17q21. A novel Asp348Gly mutation cosegregating with the disease was identified in the MAPT gene using exome sequencing.

The mutation does not act modifying tau interactions with microtubules. Human neuroblastoma cell lines overexpressing mutated Asp348Gly Tau isoforms displayed a consistent reduction in neurites length and arborisation. Neuropathology of an affected subject showed motoneuron loss and atrophy of the spinal anterior horns with accumulation of phosphorylated tau within the surviving motor neurons. The R3–R4 tau staining showed pathology similar to those observed in familial MAPT cases. Our data broaden the phenotype of tauopathies to include lower motor neuron disease and implicate the defect in the tau degradation pathway in motor neuron degeneration.

P169 PHENOTYPIC VARIABILITY ASSOCIATED WITH THE R155C VCP GENE MUTATION

Battistini S

Ricci C

Benigni M

Casali S

Giannini F

University of Siena, Siena, Italy

Email address for correspondence: [email protected]

Keywords: VCP, mutation, distal lower motor neuron disease

Background: Mutations in the valosin-containing protein (VCP) gene, on chromosome 9p13.3-p12, were identified as the genetic cause of an unusual syndrome characterized by Inclusion body Myopathy with Paget disease and Frontotemporal Dementia (IBMPFD)(1). Mutations in the VCP gene have been recently reported as a cause of 1–2% of familial amyotrophic lateral sclerosis (ALS) cases (2). To date, 24 VCP mutations have been reported. Among these, 10 missense mutations have been identified in ALS cases, including five detected in both IBMPFD and ALS and five in only ALS cases. Here we report the R155C mutation in the VCP gene in an Italian patient with only lower motor neuron signs in a family with autosomal dominant inheritance.

Objectives: To further describe the diversity of phenotypes associated with mutations in the VCP gene.

Methods: All the 17 coding exons of VCP gene were analyzed using PCR and sequencing.

Case report: The patient is a 59-year-old woman followed for 11 years since presenting with progressive weakness of left hand beginning at age 29 and muscle cramps. Her symptom gradually progressed with prominent distal and symmetrical distribution weakness and muscle atrophy in upper and lower limbs. Currently she is unable to walk. No upper motor impairment neither bulbar nor cognitive involvement were detected. Her mother developed MND at 31 years of age with late onset bulbar signs and died due to respiratory failure at age 53. Anamnestic data revealed three likely affected relatives with evidence of an AD inheritance. No mutations were found in SOD1, C9ORF72, TDP-43 and FUS genes in the patient. Molecular analysis showed a heterozygous mutation c.463C> T in the VCP gene (codon 155 in exon 5) determining a substitution of arginine to cysteine in the VCP protein (R155C). No DNA was available from the other affected relatives.

Discussion and conclusion: We identified the R155C mutation in a patient belonging to a family with autosomal dominant transmission MND. The mutation is known to be pathogenetic. Arginine155 in exon 5 is the most common mutational hot spot and the R155C is the most frequent mutation found in IBMPFD pedigrees. To date, this mutation has been previously found in only one familial ALS in which detailed clinical information is not available (3). Phenotypic variability associated with VCP mutations has been reported between and within families with FTD, IBM (Inclusion Body Myopathy), PDB (Paget’s disease), ALS or a combination of these diseases. Our data extend the range of phenotype associated with VCP mutations to include patients with slowly progressive distal lower motor neuron disease.

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P170 SOMATIC VARIATION OF THE ATAXIN-2 CAG REPEAT IN MOTOR NEURON DISEASE

East S

Bäumer D

Tseu B

Clift A

Peppiatt J

Talbot K

Ansorge O

Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK

Email address for correspondence: [email protected]

Keywords: ataxin-2, expansion, mosaicism

Background: Expansion of the polyglutamine-encoding CAG tract in the ataxin-2 gene (ATXN2) to more than 33 repeats length causes spinocerebellar ataxia 2 (SCA2), and intermediate expansions of length 27–33 are overrepresented in motor neuron disease (MND) (1). Normal alleles (generally 22 or 23 repeats) and MND-associated alleles contain at least one CAA interruption (2), also coding for glutamine, whereas SCA2 tracts are pure CAG.

Objectives: Since repeat tract instability can result in somatic mosaicism, we tested the hypothesis that abnormal ATXN2 CAG stretches may be a more common feature of MND than has been inferred from blood DNA, and that repeat tract genotype may influence selective vulnerability of characteristic brain regions to degeneration.

Methods: DNA was extracted from frozen post-mortem spinal cord, frontal cortex and cerebellum of 15 cases of classical MND, 10 cases of MND-C9orf72 and 10 normal controls. The ATXN2 CAG repeat tract was PCR-amplified and cDNA products from both alleles were co-sequenced in both directions. Protein expression was studied in parallel using immunohistochemistry on fixed paraffin sections.

Results: Somatic mosaicism of the ATXN2 CAG repeat was detected in a minority of cases from all three groups. Variation generally involved repeats of 22 and 23 lengths with one or two CAA interrupts, none of which is likely to be involved in MND pathogenesis. However, in four MND cases where no mosaicism was detected, we did detect rare tract genotypes: two MND-C9orf72 cases had a pure CAG tract of 23 lengths while one case of classical MND displayed a 34-repeat (in the SCA2 range) with one CAA interrupt. A 37-repeat with one CAA interruption was also seen in a MND-frontotemporal dementia case. Preliminary analysis suggests a tendency towards excessive clumping of ataxin-2 protein in MND spinal cord.

Discussion and conclusion: Somatic variation involving intermediate length and expanded ATXN2 CAG repeats is unlikely to be a common feature of MND. Establishing the relevance to MND neuropathology of the 23-repeat pure CAG tracts and the full expansions requires further work.

Acknowledgements

This research was funded by the Oxford Biomedical Research Centre.

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P171 A RARE MOTOR NEURON DELETERIOUS MISSENSE MUTATION IN THE DPYSL3 (CRMP4) GENE IS ASSOCIATED WITH ALS

Blasco H^1,2

Bernard-Marissal N³

Vourc’h P^1,2

Guettard Y-O²

Sunyach C³

Augereau O²

Khederchah J²

Mouzat K⁴

Antar C^1,2

Gordon P H⁶

Veyrat-Durebex C^1,2

Besson G⁵

Andersen P M⁸

Salachas F⁶

Meininger V⁶

William C⁷

Pettmann B³

Andres C R¹

Corcia P^1,9

^axUNITE INSERM U930, Universite Francois Rabelais, Tours, France

^ayCHRU Bretonneau, Laboratoire de Biochimie et Biologie Moléculaire, Tours, France

^azUniversite de la Méditerranée, Marseille, France

^baCHU de Nime, Laboratoire de Biochimie, Nime, France

^bbCHU Grenoble, centre SLA, Grenoble, France

^bcAPHP, La pitié Salpétrière, Paris, France

^bdCHU Montpellier, Gui-de Chauliac, Montpellier, France

^beUmea University, department of neuroscience, Umea, Sweden

^bfCentre SLA, service de neurologie et neurophysiologie, Tours, France

Email address for correspondence: [email protected]

Keywords: ALS, CRMP4, risk factor

Background: The dihydropyrimidinase-like 3 (DPYSL3) or Collapsin Response Mediator Protein 4a (CRMP4a) expression is modified in neurodegeneration and is involved in several ALS-associated pathways including axonal transport, glutamate excitotoxicity and oxidative stress.

Objective: The objective of the study was to analyze CRMP4 as a risk factor for ALS.

Methods: We analyzed the CRMP4 gene in French ALS patients (n = 468) and matched-controls (n = 394).We subsequently examined a variant in a Swedish population (184 SALS and 186 controls), and evaluated its functional effects on axonal growth and survival in motor neuron cell culture.

Results: The rs147541241:A> G missense mutation occurred in higher frequency among French ALS patients (Odds ratio = 2.99) but the association was not confirmed in the Swedish population. In vitro expression of mutated CRMP4 in motor neurons reduced axonal growth and accelerated cell death compared to wild-type protein.

Discussion and conclusion: Thus, the association between the rs147541241 mutation and ALS was limited to the French population, highlighting the geographic particularities of genetic influences (risks and contributors). The identified variant appears to shorten motor neuron survival through a detrimental effect on axonal growth and CRMP4 could act as a key unifier in transduction pathways leading to neurodegeneration through effects on early axon development.

P172 MECHANISMS OF A NOVEL PHOSPHORYLATION SITE MUTATION IN PROFILIN 1

Ingre C²

Landers JE³

Rizik N¹

Volk A⁴

Akimoto C²

Birve A²

Keagle P³

Piotrowska K³

Press R²

Andersen PM²

Ludolph AC¹

Weishaupt JH¹

^bgUlm University, Ulm, Germany

^bhUmea University, Umea, Sweden

^biUniversity of Massachusetts Medical School, Worcester, USA

^bjDepartment of Human Genetics, Ulm, Germany

Email address for correspondence: [email protected]

Keywords: PFN1, microRNAs, cytoskeleton

Background: Profilin 1 is a central regulator of actin dynamics. Mutations in the gene profilin 1 (PFN1) have very recently been shown to be the cause of a subgroup of amyotrophic lateral sclerosis (ALS). Previously, we performed a large screen of US, Nordic and German familial as well as sporadic ALS and fronto-temporal dementia (FTLD) patients for PFN1 mutations in order to get further insight into the spectrum and pathogenic relevance of this gene for the complete ALS/FTLD continuum.

Methods: Four hundred and twelve familial and 260 sporadic ALS cases, as well as 16 ALS/FTLD cases from Germany, the Nordic Countries and the US were screened for PFN1 mutations. Phenotypes of patients carrying PFN1 mutations were studied, and cell biological consequences of a novel PFN1 mutation were studied.

Results: In a German ALS family we identified the novel heterozygous PFN1 mutation p.Thr109Met, which was absent in controls. This novel mutation abrogates a phosphorylation site in profilin 1. The ALS patients with mutations in PFN1 displayed spinal onset motor neuron disease without overt cognitive involvement. PFN1 mutations were absent in patients with motor neuron disease and dementia, and in patients with only FTLD. We furthermore studied the cell biological, epigenetic and biochemical consequences of our novel profilin 1 phosphorylation site mutation.

Conclusions: We provide further evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. Patients carrying PFN1 mutations reported so far represent the classical ALS end of the ALS-FTLD spectrum. The novel p.Thr109Met mutation provides additional proof-of-principle that mutant proteins involved in the regulation of cytoskeletal dynamics can cause motor neuron degeneration. Finally, we present data outlining the cell biological consequences and mechanisms of our PFN1 phosphorylation site mutation.

P173 SCREENING OF THE PFN1 GENE IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS AND IN FRONTOTEMPORAL DEMENTIA

Tiloca C^1,2

Ticozzi N^1,2

Pensato V³

Bagarotti A⁴

Del Bo R^1,5

Gagliardi S⁶

Lauria G³

Corti S^1,5

Galimberti D^1,5

Ceroni M^6,7

Siciliano G⁸

Cereda C⁶

Scarpini E^1,5

Sorarù G⁹

Comi GP^1,5

Corrado L⁴

Gellera C³

Ratti A^1,2

Landers JE¹⁰

Silani V^1,2

^bkUniversity of Milan, Milan, Italy

^blIRCCS Istituto Auxologico Italiano, Milan, Italy

^bmFondazione IRCCS Istituto Neurologico ‘Carlo Besta’, Milan, Italy

^bn’A. Avogadro’ University, Novara, Italy

^boIRCCS Fondazione ‘Ca Granda’ Ospedale Maggior Policlinico, Milan, Italy

^bpIRCCS Istituto Nazionale Neurologico ‘C. Mondino’, Pavia, Italy

^bqUniversity of Pavia, Pavia, Italy

^brUniversity of Pisa, Pisa, Italy

^bsUniversity of Padua, Padua, Italy

^btUniversity of Massachusetts Medical School, Worcester, MA, USA

Email address for correspondence: [email protected]

Keywords: PFN1, profilin1, genetics

Background: Mutations in the profilin 1 (PFN1) gene, encoding a protein regulating filamentous actin growth through its binding to monomeric G-actin, have been recently identified in familial amyotrophic lateral sclerosis (ALS). Functional studies performed on ALS-associated PFN1 mutants demonstrated aggregation propensity and alterations in growth cone and cytoskeletal dynamics (1).

Objectives: To determine the effective contribution of PFN1 mutations to sporadic ALS (SALS) in the Italian population.

Methods: We screened a large cohort of 1168 Italian SALS patients and also included 203 frontotemporal dementia (FTD) cases because of the great clinical and genetic overlap between these two neurodegenerative diseases. The three coding exons of PFN1 were analyzed by direct sequencing.

Results: We detected the previously described p.E117G variant in one SALS patient and the novel synonymous change p.G15G in another individual, but none in a panel of 1512 control subjects. Previous screening of PFN1 gene in ALS suggests that p.E117G likely represents a less pathogenic variant according to both frequency data in control subjects and cases, and functional experiments (1).

Discussions and conclusion: Our results suggest that PFN1 mutations in SALS and in FTD patients are rare, at least in the Italian population.

Acknowledgements

Research support was provided by AriSLA, the Italian Ministry of Health, Fondazione Cariplo, NIH/NINDS, and the “Amico Canobio” Association. We thank Dr Cecilia Carlesi for her contribution to clinical data.

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P174 PFN1 MUTATIONS ARE AN UNCOMMON CAUSE OF FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS

Calvo A¹

Restagno G²

Brunetti M²

Barberis M²

Ossola I²

Moglia C¹

Canosa C¹

Fuda G¹

Traynor BJ³

Johnson JO³

Drory V⁴

Rogaeva E⁵

Zinman L⁶

Sendtner M⁷

Drepper C⁷

Chiò A¹

^buRita Levi Montalcini Department of Neuroscience, University of Torino, Torino, Italy

^bvLaboratory of Molecular Genetics, AO Città della Salute e della Scienza, Torino, Italy

^bwNeuromuscular Diseases Research Unit, Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA

^bxDepartment of Neurology, Tel-Aviv Sourasky Medical Center, Tel-Aviv, Israel

^byDivision of Neurology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada

^bzDepartment of Medicine, Sunnybrook Health Sciences Centre, Toronto, Ontario, Canada

^caInstitute for Clinical Neurobiology, University of Würzburg, Würzburg, Germany

Email address for correspondence: [email protected]

Keywords: profilin, genetics, familial amyotrophic lateral sclerosis

Background: PROFILIN 1 (PFN1) mutations have been reported to underlie approximately 1% of familial amyotrophic lateral sclerosis (FALS). Recently, a number of study found that these mutations are very rare in FALS, SALS (sporadic amyotrophic lateral sclerosis) and FTD.

Objective: The aim of this study was to analyze for PFN1 mutations a large cohort of patients with FALS.

Methods and results: A complete spectrum of PFN1 mutations was assessed using exome sequence data generated for 209 FALS patients, 69 from Italy (ITALSGEN Consortium), 92 from USA, 13 from Israel, 27 from Germany, and 8 from Canada. The patients were diagnosed in accordance with the EI Escorial revised criteria. No mutations in PFN1 were detected.

Conclusions: Our results suggest that PFN1 mutations in FALS are not a common cause of disease.

References

• Wu CH et al. Mutations in the profilin 1 gene cause familial amyotrophic lateral sclerosis. Nature 2012 Aug 23; 488(7412): 499–503.
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P175 ANALYSIS OF HNRNP A1, A2/B1 AND A3 GENES IN ALS PATIENTS

Calini D¹

Del Bro R^2,3

Corrado L⁴

Gagliardi S⁵

Pensato V⁶

Verde F^1,2

Corti S^2,3

Mazzini L^4,7

Milani P⁵

Castellotti B⁶

Bertolin C⁸

Sorarù G⁸

Cereda C⁵

Comi GP^2,3

D’Alfonso S⁴

Gellera C⁶

Ticozzi N^1,2

Landers JE⁹

Ratti A^1,2

Silani V^1,2

^cbIRCCS Istituto Auxologico Italiano, Milano, Italy

^ccUniversità degli Studi di Milano, Milano, Italy

^cdIRCCS Foundation Ca’Granda Ospedale Maggiore Policlinico, Milano, Italy

^ce‘A. Avogadro’ University, Novara, Italy

^cfIRCCS National Neurological Institute ‘C. Mondino’, Pavia, Italy

^cgFondazione IRCCS Istituto Neurologico “Carlo Besta”, Milano, Italy

^chMaggiore della Carita` Hospital, Novara, Italy

^ciUniversity of Padua, Padova, Italy

^cjUniversity of Massachusetts Medical School, Worcester, MA 01605, USA

Email address for correspondence: [email protected]

Keywords: mutational analysis, hnRNPA, prion-like

Background: Mutations in the prion-like domain (PrLD) of hnRNP A1 and A2/B1 genes were recently identified in two families with inclusion body myopathy (IBM) associated with Paget’s disease of the bone (PDB), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (IBMPFD/ALS) as well as in one familial and one sporadic ALS patient (1). The biological evidence that hnRNP A1 and A2/B1 proteins are physiological TDP-43 binding partners further reinforces the importance of these genetic findings (2). The other family member hnRNP A3 was recently shown to be sequestered in p62-positive/TDP-43-negative pathological inclusions of C9orf72-mutated patients and, similarly to hnRNP A1 and A2/B1, hnRNPA3 protein contains a PrLD (3).

Objectives: Molecular analysis of hnRNP A1, A2/B1 and A3 genes in a cohort of 221 familial (FALS) and 622 sporadic (SALS) Italian patients.

Methods: PCR amplification and Sanger sequencing.

Results: No variants in hnRNP A1, A2/B1 and A3 genes were found in 113 FALS individuals with no mutations in other ALS causative genes but a novel intronic variant in hnRNPA2/B1 (c.659-14insT) in one patient. This intronic variant was not predicted to alter hnRNPA2B1 splicing by in silico analysis. The screening of the PrLD-encoding exons of the three hnRNP genes in an additional panel of 108 FALS with known mutations in ALS-associated genes and of 622 SALS cases failed to find any mutation.

Discussion and conclusions: Our results suggest that mutations in hnRNP A1, A2/B1 and A3 genes are rare, at least in ALS disease.

Acknowledgements

Financial support was provided by AriSLA and the Italian Ministry of Health.

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P176 HNRNPA1 AND HNRNPA2B1 MUTATIONS IN ALS AND OTHER PROTEINOPATHIES IN THE NETHERLANDS

Seelen M¹

Visser A¹

Van Swieten J²

Schelten P²

Veldink JH¹

Van Es M¹

Van den Berg LH¹

^ckRudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, The Netherlands

^clVU University Medical Center, Amsterdam, The Netherlands

Email address for correspondence: [email protected]

Keywords: DNA analysis, multisystem proteinopathy, prion-like domain

Background: Recently mutations in hnRNPA1 and hnRNPA2B1 have been identified as a cause of ALS/IBMPFD (autosomal dominant multisystem proteinopathy which results in ALS, FTD, Paget’s disease and/or inclusion body myositis (IBM) with TDP-43 pathology) (1). Interestingly, hnRNPA1 and hnRNPA2B1 code for RNA-binding proteins and both contain a prion-like domain (PrLD), which is a shared feature with other ALS genes (TDP-43, FUS, EWSR1 and TAF15).

Objective: To determine the frequency of mutations in hnRNPA1 and hnRNPA2B1 in a large cohort of ALS patients, FTD patients and IBM patients from The Netherlands.

Methods: DNA samples from a total of 150 familial, 1,000 sporadic ALS patients and 500 controls were obtained from an ongoing, population-based, epidemiologic study in The Netherlands. We collected samples from 30 IBM patients that were referred to the neuromuscular clinic at the UMC Utrecht. Samples were obtained from 70 familial and 100 sporadic FTD patients that were seen at the VUMC dementia clinic. Genetic sequencing of the hnRNPA1 and hnRNPA2B1PrLD regions was determined using Sanger sequencing.

Preliminary results: After screening 1080 sporadic ALS cases and 70 familial FTD cases, we did not identify any pathogenic mutations. One potentially interesting splice variant was detected in a single case of familial FTD (splicing (uc003sxr.4:exon9:c.695-5A> G) which could perhaps affect the PrLD, follow-up of familial ALS, sporadic FTD and IBM cases is underway.

Discussion: Mutations in hnRNPA1 and hnRNPA2B1 appear to be a rare cause of ALS and FTD.

Reference:

• Kim et al. Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS. Nature 2013;495:467–73.
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P177 THE MOLECULAR BASIS OF ALS IN TURKEY

Ozoguz A¹

Uyan O¹

Birdal G¹

Agim ZS¹

Omur O¹

Lahut S¹

Iskender C¹

Saygi C¹

Kartal E¹

Parman Y²

Tan E⁴

Koc F³

Kotan D⁷

Ertas M⁹

Bilguvar K⁵

Gunel M⁵

Keskin O⁸

Ozcelik H⁶

Basak AN¹

^cmBogazici University, Department of Molecular Biology and Genetics, Neurodegeneration Research Laboratory, Istanbul, Turkey

^cnIstanbul University, Istanbul Medical School, Neurology Department, Istanbul, Turkey

^coCukurova University, Medical School, Neurology Department, Adana, Turkey

^cpHacettepe University, Medical School, Neurology Department, Ankara, Turkey

^cqYale University School of Medicine, Department of Neurosurgery, New Haven, CT, USA

^crUniversity of Toronto, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Department of Laboratory Medicine and Pathobiology, Toronto, Ontario, Canada

^csSakarya University, Faculty of Medicine, Department of Neurology, Sakarya, Turkey

^ctKoc University, Center for Computational Biology and Bioinformatics, College of Engineering, Istanbul, Turkey

^cuLIV Hospital, Neurology Department, Istanbul, Turkey

Email address for correspondence: [email protected]

Keywords: Turkey, genetics, molecular basis

Background: Recently the frequency of ALS mutations has been extensively investigated in several populations, however, a systematic analysis has not been reported in Turkey so far.

Objectives: To carry out a comprehensive genetic study in Turkish ALS patients.

Methods: A total of 362 Turkish ALS patients, 77 fALS, belonging to 54 families, and 285 sALS cases were screened for mutations in ALS genes. Patients were genotyped for SOD1 and UBQLN2 gene mutations via conventional PCR; for C9orf72 RP-PCR and partial southern blot analyses were performed. A subset of patients was also subjected to exome sequencing. Haplotype analysis was performed on patients carrying the SOD1-D90A mutation.

Results: SOD1 (14.8%), C9orf72 (11.0%) and UBQLN2 (3.7%) gene mutations were found to account for approximately 29.5% of fALS in Turkey. While no SOD1 mutations were shown so far in sALS patients; C9orf72 (3.9%) and UBQLN2 (0.7%) explained 4.6% of sALS in the cohort under study. Exomic sequencing revealed FUS, OPTN, SPG11 and PLEKHG5 mutations in four families. SOD1-D90A, which is known to occur both in dominant and recessive pedigrees, was shown to behave as a recessive trait in all three Turkish families in this study. A novel haplotype other than the common Scandinavian was detected.

Discussion and conclusion: In the framework of this study, we report a systematic screening of Turkish ALS patients for disease-causing mutations. Our results indicate that SOD1, C9Orf72 and UBQLN2 mutations are important genetic causes of ALS in the Turkish population. The frequency of SOD1 is consistent with other Mediterranean countries. Although mutational frequencies may change as additional Turkish patients are screened, the comparison of the above numbers with other population-based studies reflects both the different genetic background and the more heterogeneous nature of the Turkish population.

Acknowledgement: We would like to thank our patients and their families for their cooperation. We gratefully acknowledge the generous supports of Suna & Inan Kirac Foundation and Bogazici University Research Funds.

References

• Al-Chalabi A et al. Recessive amyotrophic lateral sclerosis families with the D90A SOD1 mutation share a common founder: evidence for a linked protective factor. Hum Mol Genet 1998;7:2045–2050.
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P178 EXTENSIVE GENETIC ANALYSIS IN A TAIWANESE COHORT WITH AMYOTROPHIC LATERAL SCLEROSIS

Lee YC

Tsai CP

Soong BS

Departement of Neurology, Taipei Veterans General Hospital, Taipei, Taiwan, Department of Neurology, National Yang-Ming University, Taipei, Taiwan

Email address for correspondence: [email protected]

Keywords: SOD1, TARDBP, FUS, C9ORF72, OPTN, VCP, UBQLN2,

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by relentlessly progressive loss of motor neurons. Mutations in at least 15 genes have been described to cause familial ALS (FALS), which may account for 10–20% of ALS cases.

Objectives: The aim of this study was to determine the frequency and spectrum of mutations in different genes in a Taiwanese ALS cohort of Chinese origin.

Methods: Mutational analyses of the SOD1, TARDBP, FUS, OPTN, VCP, UBQLN2, and PFN1 genes were carried out by direct sequencing in 147 unrelated patients with ALS, including 29 with FALS and 118 with sporadic ALS (SALS), diagnosed with probable or definite ALS at the Neurology Service of Taipei Veterans General Hospital, Taiwan. The CAG repeat sizes in ATXN2 and the GGGGCC hexanucleotide repeat expansion in C9ORF72 of the patients were also investigated.

Results: Mutations have been identified in 33 of the 147 patients (22.4%), including 21 with FALS (72.4%; 21/29) and 12 with SALS (10.2%; 12/118). Among the 29 FALS patients, 8 were found to have SOD1 mutations (p.Thr137Arg in 2 patients, and p.Leu8Val, p.Gly10Ala, p.Asp83Asn, p.Gly85Arg, p.Leu106Phe, and p.Gly138Glu in 1), 6 had TARDBP mutations (p.Met337Val in 4, p.Gly348Val, and p.Asn378Asp in 1), 5 had the C9ORF72 repeat expansion, and 2 had FUS mutations (p.His517Asp and p.Arg521His). Among the 118 SALS cases, 4 were found to have SOD1 mutations (p.Gly16Ser, p.Gly37Arg, p.Cys111Try, and p.Thr137Arg), 2 had FUS mutations (p.H517Asp and p.Arg521His), 2 had intermediate-length CAG expansions (32 and 33 CAG repeats), 2 had the C9ORF72 repeat expansion, 1 had a TARDBP mutation (p.Ser375Gly), and 1 had an OPTN mutation (p.Leu494Trp). No patient was found to have VCP, UBQLN2, or PFN1 mutation. Four of the above-mentioned mutations are novel, including p.Gly10Ala and p.Asp83Asn in SOD1, p.Ser375Gly in TARDBP, and p.Leu494Trp in OPTN.

Conclusion: This study clearly demonstrates the distribution and frequency of mutations in a Taiwanese ALS cohort of Chinese origin, and expands the spectrum and supports their global presence of mutations in the causative genes of ALS.

P179 IDENTITY-BY-DESCENT IN AN IRISH ALS COHORT

McLaughlin R

Kenna K

Bradley D

Hardiman O

Trinity College, Dublin, Ireland

Email address for correspondence: [email protected]

Keywords: identity-by-descent, rare variant discovery, genome-wide SNP analysis

Background: When two individuals share a common ancestor, parts of their genome show identity-by-descent (IBD), meaning that these genomic regions are identical, with the same single nucleotide polymorphisms (SNPs) and rare variants commonly inherited in both individuals. These regions are larger and more frequent in the genome, the closer the relationship is between the two individuals.

Mapping IBD regions can serve multiple purposes in the study of complex disease, two of which are highlighted here. First, if a genomic region shows a greater degree of within-patient IBD rather than showing within-control IBD, this points towards candidate disease loci. Second, the sum total of all identified IBD regions in a pair of individuals permits inference of the degree of relationship between the pair.

Results: We have mapped IBD regions in a large Irish ALS case–control genome-wide SNP dataset using the haplotype phasing programs BEAGLE and GERMLINE, and have identified a number of genomic regions that show excessive within-case IBD, hinting at possible sites harboring multiple rare ALS-causing variants which would be discoverable by targeted resequencing. We have also noted that clusters of individuals previously classified as having sporadic ALS are actually interrelated well above the population background level, at around the level of third cousins. These individuals represent affected members of the same extended pedigree and would be good candidates for disease variant discovery by exome or genome sequencing.

Discussion and conclusion: The identification of hidden relationships among apparently sporadic ALS cases challenges the distinction between familial and sporadic forms of the disease, and supports the hypothesis that genetic etiology underpins many cases of the disease.

P180 MOLECULAR GENETIC ANALYSIS IN RUSSIAN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS

Lysogorskaia E

Abramycheva N

Rossokhin A

Zakharova M

Illarioshkin S

Research Center of Neurology Russian Academy of Medical Sciences, Moscow, Russia

Email address for correspondence: [email protected]

Keywords: mutation analysis, molecular modeling, SOD1 gene

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons, the development of paralyses, and death from respiratory and bulbar failure.

Objective: About 10% of ALS cases are of familial. Mutations in 18 gene loci could determine ALS and more than 25 proteins are involved in ALS pathogenesis.

Methods: Two hundred and eight ALS patients (99 females and 109 males) from the Russian population were examined, including 9 patients from 8 unrelated families with a familial form of ALS. Sequence analysis was performed for the detection of SOD1, TARDBP, and ANG mutations. The associations with polymorphisms in VEGF and APOE were analyzed using RT-PCR and restriction analysis. Molecular modeling analysis was used to demonstrate the pathogenetic role of SOD1 mutations.

Results: Coding SOD1 mutations were detected in 50% of familial cases and 3% of sporadic cases of the disease. Gly16Ala, His48Arg, Leu84Val, Ans86Ser, Asp90Ala, Ser105Leu, Glu133Gly, and Leu144Phe were detected in the coding region. All of them are present in ALSoD database and led to moderate or significant changes of the SOD1 protein energy. Mutation in His49Arg increased the protein energy, and reconstruction of the respective model revealed spatial destabilization of the molecule and abnormal interaction with the metal ion inside the active center. The other seven mutations led to decrease in the protein energy and increase in the spatial stability of SOD1, which could be accompanied by increased propensity of the ‘inert’ mutant molecule to misfolding and cellular aggregation.

For the first time, intronic mutations c.–46C> T, c.*249T> C (*NT_011512.11) and c.169 + 50delAACAGTA (in two unrelated patients) were detected in ALS patients and were not present in 385 controls. Coding ANG mutations were detected in 1.5% of sporadic ALS. No coding mutations were detected in 6 exons of TARDBP, but c.715–126delG deletion was associated with the risk of ALS (OR = 1.53: 95% CI, 1.05–2.24; р = 0,026). The significant difference in the genotype distribution of VEGF was observed between ALS cases and controls (χ² = 11.1; р = 0.004). The significant association of the -2578A/A VEGF genotype with ALS was observed (OR = 2.01; 95% CI, 1.12–3.65; p = 0.018). Males carrying -2578A/A had more increased risk of ALS (OR = 2.26; 95% CI 1.01–5.12; p = 0.046). Any associations with APOE polymorphism were not observed.

Conclusion: In the Russian population of ALS patients, the spectrum of SOD1and ANG mutations was present. High frequency of SOD1 mutations in familial ALS was detected. The absence of coding TARDBP mutation was observed. Some significant associations were detected. The results of in silico analysis of the SOD1 gene mutations confirm the placement of ALS within the class of the conformational diseases.

P181 CONTRIBUTION OF MAJOR AMYOTROPHIC LATERAL SCLEROSIS-RELATED GENES TO THE ETIOLOGY OF THE DISEASE IN CHINA

Zou Z

Liu M

Li X

Cui L

Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China

Email address for correspondence: [email protected]

Keywords: genetics; mutation; Chinese

Background: Mutations in SOD1, ANG, TARDBP, FUS, VCP, C9ORF72, and PFN1 genes have been identified in amyotrophic lateral sclerosis patients (1,2).

Objectives: To determine the mutations in major amyotrophic lateral sclerosis (ALS)-related genes in a large cohort of Chinese familial ALS (FALS) and sporadic ALS (SALS) patients and the genotype–phenotype associations.

Methods: Screening for mutations of SOD1, ANG, TARDBP, FUS, VCP, C9ORF72, and PFN1 genes was consecutively carried out in 20 index FALS patients, 324 SALS patients, and 245 healthy controls admitted to Peking Union Medical College Hospital. Mutation rates of SOD1, ANG, TARDBP, FUS, and C9orf72 genes in ALS patients in different continents were calculated, and genotype–phenotype associations were analyzed by reviewing all published studies screening for mutations in these genes in ALS patients.

Results: Overall, mutations were detected in 35.0% (95%CI, 14.1%–55.9%) and 4.0% (95%CI, 1.9%–6.1%) of FALS and SALS patients, respectively. SOD1 (25.0%) and FUS (10.0%) mutations account for all mutations in FALS patients, whereas FUS (1.9%) and SOD1 (0.9%) were the most frequently mutated genes, followed by TARDBP (0.9%), and ANG (0.3%) in SALS patients. No mutations were detected in C9orf72, VCP, and PFN1. Patients with p.H46R mutation in SOD1 gene always manifested with weakness in the legs, the lower motor neuron signs usually dominate the clinical presentation and the disease progresses very slowly, with a mean survival of more than 17 years. Patients with mutations of p.P525L, p.R495X and nonsense mutations in FUS gene are associated with an early onset, a rapid disease progression, and short lifespan.

Discussion: The profile of major ALS-related genes mutations in Chinese ALS patients appears to be different from the that reported in Caucasian, in which C9orf72 and SOD1 are the most common mutated gene in both FALS and SALS patients, whereas FUS mutation is relatively less in Caucasian. The results suggest that there is an ethnic difference in the genetic background of ALS.

Conclusions: Mutations in major ALS-related genes are present in approximately 35.0% and 4.0% of Chinese FALS and SALS patients, respectively. SOD1 and FUS are the most frequently mutated genes in FALS patients, while FUS, SOD1, and TARDBP are the most common mutated gene in SALS patients in China. Some characterized clinical phenotypes are associated with some specific gene mutations.

Acknowledgements

The authors thank the patients and their families, as well as the healthy controls, for their cooperation in this study.

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P182 FLJ10986 IS NOT ASSOCIATED WITH AMYOTROPHIC LATERAL SCLEROSIS IN A LARGE CHINESE COHORT

Cai B

Fan D

Peking University Third Hospital, Beijing, China

Email address for correspondence: [email protected]

Keywords: rs6700125, rs6690993, FLJ10986

The single nucleotide polymorphisms (SNPs) rs6700125 and rs6690993 in FLJ10986 (FGGY) were recently reported to be a susceptibility factor for sporadic amyotrophic lateral sclerosis (SALS) in Caucasian populations in genome-wide association studies (GWAS). However, no such association was observed in other independent GWAS or replication studies in a European cohort or a small sample size of Chinese patients. We performed a large case–control study in a cohort consisting of 963 SALS cases and 1039 controls to examine the association between the two reported SNPs in FGGY and ALS in Chinese patients. Our results did not indicate any association of these SNPs with SALS and suggest that these FLJ10986 SNPs do not modulate the risk of SALS in the Chinese population.

P183 ASYMMETRICAL LATE-ONSET DISTAL HEREDITARY MOTOR NEUROPATHY IN A CHINESE FAMILY ASSOCIATED WITH AN HSPB1 MUTATION

Zhang Y¹

Liu X¹

Sun A¹

Song S²

Fan D¹

^cvPeking University Third Hospital, Beijing, China

^cwGenetesting Center, Peking University Health Science Center, Beijing, China

Email address for correspondence: [email protected]

Keywords: dHMN, late-onset, HSPB1

Distal hereditary motor neuropathy (dHMN) is a clinically and genetically heterogeneous group of disorders characterized by progressive, pure motor axonal neuropathy. Mutations in the small heat-shock protein HSPB1 are responsible for one form of dHMN. Here, we report a Chinese family with strikingly asymmetrical late-onset dominant dHMN. Three patients spanning 2 generations (the patient, a 53-year-old man, his brother and his mother) developed asymmetrical distal limb weakness and muscular atrophy after the fourth decade. Gene testing excluded mutations in MFN2, GJB1 and HSPB8. Two affected family members had a heterozygous mis-sense mutation, cDNA 379C→T(127Arg→Trp), in the HSPB1 gene.

P184 POLYMORPHISM RISK FACTOR STUDY IN FGGY GENE IN CHINESE SPORADIC AMYOTROPHIC LATERAL SCLEROSIS PATIENTS

Li X

Lin Y

Liu M

Xie M

Zhang J

Cui L

Peking Union Medical College hospital, Beijing, China

Email address for correspondence: [email protected]

Keywords: single nucleotide polymorphism, FGGY gene, susceptibility

Objective: Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the brain and spinal cord. Sporadic ALS (SALS) accounts for the majority of patients. There were several single nucleotide polymorphisms (SNPs) reported be associated with ALS susceptibility in whole genome association analysis. We study polymorphism of rs6700125 in FGGY carbohydrate kinase domain containing FGGY or FLJ10986 gene in SALS patient of Chinese Han origin.

Methods: We extracted the genomic DNA from the leukocytes of whole blood samples in 143 SALS patients and 153 normal controls. We applied two methods to analyze the samples, the first processed the asymmetric PCR in the presence of an unlabeled probe that contain the rs6700125 locus, and then genotyped the product on the LightScanner, confirming DNA sequence. The second-part genotyping was performed using a Sequenom chip-based MALDI-TOF mass spectrometry platform.

Results: Expression of rs6700125 that was strongly associated with susceptibility to SALS in different populations of European and American ancestry was not strongly associated with ALS in Chinese patients (χ^{2 =} 0.94;OR = 1.12;95% CI, 0.84–1.6).

Conclusions: In Chinese people, rs6700125 in FGGY gene are not associated with ALS susceptibility.

P185 C9ORF72 REPEAT EXPANSION IN CHINESE FAMILIAL AND SPORADIC ALS PATIENTS

Tang L

Liu R

Cai B

Liu X

Ye S

Ma Y

Chui D

Zhang H

Fan D

Peking University Third Hospital, Beijing, China

Email address for correspondence: [email protected]

Keywords: Chinese population; C9ORF72; repeat expansions

Objective: To identify the existence and distribution of C9ORF72 hexanucleotide (GGGGCC) repeat expansions as well as of the mutation-related clinical phenotypes in the amyotrophic lateral sclerosis (ALS) population from mainland China.

Methods: DNA samples and clinical data of 920 sporadic ALS (SALS) cases and 61 familial ALS (FALS) patients were collected during 2007–2012. Fluorescent fragment-length analysis and repeat-primed PCR were applied to detect the pathological GGGGCC repeat expansions.

Results: The C9ORF72 mutation was found in 3 SALS patients (0.3%, 3/920) and 0 FALS patients (0, 0/61). All of them are male, spinal-onset at the age of 54–58years, later bulbar-involved. The disease durations were from 15 to 40 months. The average C9ORF72 GGGGCC repeat numbers in 917 sALS patients without the C9ORF72 mutation is 3.89 ± 2.57 (range, 2–14).

Conclusions: This study clearly illustrates the existence and importance of the C9ORF72 repeat expansions in ALS population from mainland China, although the incidence was much lower than that in Western populations.

Acknowledgements

We thank Dr Yi-Chung Lee, Dr Ching-Paio Tsai, and Dr Yi-Chun Lu for their assistance with the repeat-primed PCR. This study was supported by grants from the National Natural Sciences Foundation of China (81030019) and Doctoral Fund of Chinese Ministry of Education (20100001110084).

P186 IDENTIFICATION OF GGGGCC HEXAMER REPEAT IN ALS LYMPHOBLASTOID CELL LINES

Sardone V^1,2

Lee Y¹

Smith BN¹

Vance C¹

Wright J¹

Cereda C³

Nishimura A¹

Shaw CE¹

^cxDepartment of Clinical Neuroscience, Institute of Psychiatry, King’s College London, London, UK

^cyDepartment of Brain and Behavioral Science, University of Pavia, Pavia, Italy

^czLab of Experimental Neurobiology, “C.Mondino” National Institute of Neurology Foundation, IRCCS, Pavia, Italy

Email address for correspondence: [email protected]

Keywords: C9ORF72, lymphoblasts, DNA

Background: Hexanucleotide GGGGCC repeat expansion within the first intron of C9ORF72 gene has been identified as a genetic cause for a significant proportion of ALS patients. A large study involving ALS cohorts from European, American, and non-white populations around the world found that the frequency of the C9ORF72 repeat expansion differed according to the geographical region. In particular, a genetic study of five European cohorts reported the C9ORF72 repeat expansion is around 26% of familial and 6% of sporadic ALS cases (1).

Method: The C9ORF72 repeat expansions are detected using repeat-primed PCR and/or Southern blotting techniques. The repeat PCR technique discriminates between the presence and absence of the expanded allele amplifying no more than 30 repeat copies; however, it does not allow the correct determination of the repeat number. On the other hand, Southern blot analysis allows the determination of the hexanucleotide repeat expansion size. The correlation between the GGGGCC repeat variation size in the C9ORF72 gene and the role of this transcript in different cell types is crucial for the understanding of the c9orf72-ALS pathology.

Results: Here, we report Southern blot analysis of B-lymphoblastoid cell lines derived from 22 ALS patients from the UK cohort. We have optimized a protocol that allows us the identification of the hexamer repeat size which ranges 3–23 kb. We have also observed that the majority of the lymphoblasts present different size of the hexamer repeat expansion indicating chromosome instability within each lymphoblastoid cell lines and underlying the genetic hetereogeneity of these cellular models.

Reference:

• Smith BN et al. Eur J Hum Genet 2013;21(1):102–8.
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P187 C9ORF72 REPEAT EXPANSIONS ARE SPECIFIC TO TDP-43 PROTEINOPATHIES

Ticozzi N^1,2

Tiloca C^1,2

Calini D¹

Gagliardi S³

Colombrita C²

Altieri A²

Cereda C³

Ratti A^1,2

Pezzoli G⁴

Borroni B⁵

Goldwurm S⁴

Padovani A⁵

Silani V^1,2

^daUniversity of Milan, Milan, Italy

^dbIRCCS Istituto Auxologico Italiano, Milan, Italy

^dcIRCCS Istituto Nazionale Neurologico ‘C. Mondino’, Pavia, Italy

^ddParkinson Institute, Istituti Clinici di Perfezionamento, Milan, Italy

^deUniversity of Brescia, Brescia, Italy

Email address for correspondence: [email protected]

Keywords: genetics, C9ORF72, FTLD

Background: Expansion of the hexanucleotide GGGGCC repeat (RE) in the C9ORF72 gene has been recently reported as the main genetic cause of familial and sporadic cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) (1,2). Although RE have been sporadically described also in other neurodegenerative diseases (3,4,5), a pathogenic role for C9ORF72 in these conditions has yet to be determined.

Objectives: To assess the frequency of C9ORF72 RE in patients with ALS-plus syndromes, amyloidopathies (Alzheimer's disease[AD]), tauopathies (progressive supranuclear palsy[PSP]and corticobasal degeneration[CBD]), and alpha-synucleinopathies (Parkinson's disease[PD]).

Methods: We screened a cohort comprised 9 patients with ALS-plus syndromes, 177 AD, 107 PSP, 103 CBD, and 190 PD cases. C9ORF72 RE detection was performed using a two-step protocol including a repeat-primed PCR as already described (6).

Results: We identified RE in 2 of 9 patients with ALS-plus syndromes (22.2%). The first patient belongs to an ALS-FTLD pedigree and was diagnosed with PSP superimposed to a lower motor neuron disease. The second patient had a predominantly upper motor neuron disease, and developed 2years after the onset of motor symptoms, features suggestive of a concurrent CBD. Conversely, screening for the C9ORF72 gene in PD, CBD, PSP, and AD cases was negative.

Discussions and conclusion: Our results indicate that C9ORF72 is probably not involved in the pathogenesis of neurodegenerative diseases other than that of ALS and FTLD. However, the high frequency of C9ORF72 RE in patients with ALS-plus syndromes suggests that, similarly to ALS-FTLD patients, individuals with motor neuron disease associated with extrapyramidal features should be screened for mutations in C9ORF72 gene, independently on their family history.

Acknowledgements

Research support was provided by AriSLA and the Italian Ministry of Health. The authors thank the Human Genetic Bank of Patients affected by PD and Parkinsonism (Telethon Genetic Biobank Network), and the Fondazione Grigioni per il Morbo di Parkinson.

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P188 SOMATIC HETEROGENEITY IN C9ORF72 EXPANSIONS AND THE EFFECT OF REPEAT LENGTH ON C9ORF72 TRANSCRIPTION

Cooper-Knock J¹

Higginbottom A¹

Connor-Robson N²

Bayati N¹

Bury J¹

Kirby J¹

Ninkina N²

Buchman V²

Shaw PJ¹

^dfSheffield Institute for Translational Neuroscience, Sheffield, UK

^dgSchool of Biosciences, Cardiff, UK

Email address for correspondence: [email protected]

Keywords: C9ORF72, transcriptome, repeat expansion length

Background: Toxic gain-of-function and haploinsufficiency are both proposed mechanisms of pathogenicity in autosomal dominant C9ORF72-related amyotrophic lateral sclerosis (ALS). Southern hybridization allows sizing of the GGGGCC expansion.

Objectives: To develop an optimized Southern hybridization-based protocol for the detection of the C9ORF72 expansion alongside independent quantification of size and variability in the number of repeats. Furthermore, identification of a patient with two expanded alleles of 50 ± 5 and > 2000 repeats allows investigation of the effect of repeat length on C9ORF72 mRNA expression, independent of compensation by a normal-length allele.

Methods: Our protocol achieves our objective by blotting an internal standard band which labels a non-expanded fragment within the C9ORF72 allele of each patient.

We have sized the C9ORF72 expansion in 40 ALS cases using DNA extracted from lymphoblastoid cell lines, blood, and central nervous system tissue (CNS). qRT-PCR was used to measure C9ORF72 mRNA expression in blood and lymphoblastoid cells and to determine its dependency on repeat number.

Results: We have demonstrated expansions of > 2000 repeats in the majority of ALS samples. Comparison between tissues of individual patients shows somatic heterogeneity with expansions in some CNS tissues of > 500 repeats longer than those in peripheral tissues.

Expression of C9ORF72 mRNA in blood of our patient with two expanded C9ORF72 alleles is equivalent to that of C9ORF72 mRNA in patients with a single expansion of > 2000 repeats, suggesting that the smaller allele is transcriptionally active. Supporting this conclusion, C9ORF72 mRNA levels in lymphoblastoid cells carrying an expansion of 50 ± 5 repeats were twice that in lines carrying more than 2000 repeats.

Discussion and conclusion: Somatic heterogeneity means evaluation of the pathogenicity will require sizing the expansion in the relevant tissue. However, performing both Southern hybridization and qRT-PCR in the blood cells of the same patients, we have shown that C9ORF72 expansions of 50 ± 5 repeats are transcriptionally active. If < 50 repeats are pathogenic, as has been proposed, then this is unlikely to be via haploinsufficiency.

Acknowledgements

This work was supported by Research Grants from the Welcome Trust (075615/Z/04/z) and RF State Programme (agreement no, 8829) to VLB, and EU Framework 7 (Euromotor No259867) to PJS and JK. JCK is supported by an MND Association/Medical Research Council Lady Edith Wolfson Fellowship award (MR/K003771/1). Samples used in this research were in part obtained from the UK National DNA Bank for MND Research, funded by the MND Association and the Wellcome Trust. We would like to thank people with MND and their families for their participation in this project.

P189 C9ORF72 REPEAT EXPANSION SIZE CORRELATES WITH AGE OF ONSET AND CAUSES AGGRESSIVE DISEASE PROGRESSION IN FALS, BUT IS RARE IN OTHER MOTOR NEURON DISEASES

Hübers A¹

Volk A²

Marroquin N²

Kubisch C²

Ludolph AC¹

Weishaupt JH¹

^dhDepartment of Neurology, University Hospital Ulm, Ulm, Germany

^diInstitute of Human Genetics, University Hospital Ulm, Ulm, Germany

Email address for correspondence: [email protected]

Keywords: C9ORF72, southern blot analysis, somatic instability

Background: An intronic GGGGCC-hexanucleotide repeat expansion in C9ORF72 was found to be the most frequently mutated ALS gene. However, most of the genetic studies on C9ORF72 so far solely rely on an indirect PCR-based methodology.

Objectives: In this study, we aimed to determine the frequencies and lengths of C9ORF72 repeat expansions (C9RE) in different motor neuron disease cohorts (ALS, adult SMA, HSP, and PLS).

Methods: We determined the frequency of C9RE using PCR and Southern blot analysis (SBA) in DNA of lymphoblastoid cell lines (LCLs) and EDTA-treated blood derived from ALS, adult SMA, PLS and HSP patients. Moreover, we studied the variability of C9RE comparing multiple independently generated LCLs from the same patient. C9RE were compared to clinical phenotypes.

Results: 24.6% of 175 familial ALS patients, but only 1 out of 30 PLS cases and none out of 22 adult SMA and 61 HSP cases, respectively, were found to have a SBA-confirmed C9RE. C9RE were highly unstable and had a strong tendency to be shorter in LCLs compared to those in EDTA blood. Stability of C9RE in LCLs seemed to differ inter-individually. We detected a significant correlation between C9RE length and age of onset of disease in ALS patients.

Conclusion: We provide the first comprehensive study on SBA-based determination of C9RE in different motor neuron diseases. C9RE are not a common cause of motor neuron diseases other than ALS. Instability of C9RE lengths questions the predictive value of blood C9RE for the relevant CNS cell types. Finally, longer C9RE in patients with a higher age of onset raise the hypothesis that C9RE might expand during lifetime.

P190 CAN A SIMPLE VISUAL MRI RATING ATROPHY SCALE DISTINGUISH CARRIERS OF THE C9ORF72 GENETIC MUTATION FROM NON-CARRIERS?

Devenney E^1,2

Tan R^1,2

Mioshi E^1,2

Kiernan MC^1,2

Hodges JR^1,2

Hornberger M^1,2

^djNeuroscience Research Australia, Sydney, New South Wales, Australia

^dkUniversity of New South Wales, Sydney, New South Wales, Australia

Email address for correspondence: [email protected]

Keywords: C9ORF72 mutation, visual rating scale, clinical tool

Background: Discovery of the C9ORF72 genetic mutation has confirmed the overlap between ALS and FTD with complex imaging analyses in this group revealing a distinct pattern of atrophy. Case selection for genetic testing of the C9ORF72 mutation can be difficult; family history alone is not a reliable predictor of mutation status as this mutation is also found in sporadic disease. Imaging analyses therefore show promise to differentiate between carriers and non-carriers; however, complex imaging techniques are not practical in the clinical setting.

Objective: To differentiate between mutation carriers and non-carriers of the C9ORF72 genetic mutation using a visual MRI rating scale which can be employed in a clinical setting.

Methods: MRI images of 46 participants (8 carriers, 16 non-carriers, and 22 controls) were rated in the following seven regions: anterior temporal lobe, orbitofrontal cortex, anterior cingulate, insula, basal ganglia, parietal lobe, and cerebellum. Areas of atrophy were rated on a five-point Likert scale by 2 raters blinded to the diagnosis.

Results: Mutation carriers had significantly more atrophy than controls in the parietal lobe (p < 0.05). In each of the other 6 regions, there was no significant difference in atrophy ratings between the groups. In contrast, non-carriers had significantly more atrophy in each region when compared to controls. On direct comparison between carriers and non-carriers, the latter group had significantly more atrophy than the former in each region except for the parietal region.

Conclusion: A simple visual rating scale is a useful clinical tool to aid in the selection of patients for C9ORF72 genetic testing. In addition, the parietal region has emerged as a potential marker of mutation status.

Acknowledgements

The authors are very grateful for the support from the Motor Neurone Disease Research Institute of Australia.