P171 ISOLATION AND IDENTIFICATION OF RNA TARGETS OF TDP-43 IN MOUSE BRAIN
NARAYANAN R, MANGELSDORF M, WALLACE R
Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia
Email address for correspondence: [email protected]
Keywords: TDP-43, genomics, gene regulation
Background and objectives: TARDBP gene mutations have been reported in both familial and sporadic ALS patients (1). TDP-43, a 43-KDa protein that binds to (TG)n repeats in DNA and corresponding (UG)n repeats in RNA, is encoded by the TARDBP gene. TDP-43 shuttles between the nucleus and cytoplasm and transports RNA (2). ALS patients without TARDBP mutations also display aggregation of TDP-43 suggesting that TDP-43 is important in the pathogenesis of ALS. The main objective of this work is to identify RNA targets of TDP-43 and to determine how these targets are misregulated in the presence of TARDBP mutations. This will help elucidate the underlying molecular mechanisms including the specific roles of TDP-43 in human disease and also help in developing better diagnostic and therapeutic opportunities. RNA targets of TDP-43 are candidate genes for searching for mutations in ALS patients where the cause remains unknown.
Methods and results: Combining both RNA-immunoprecipitation and microarray techniques (RIP-Chip), we identified more than 1000 potential RNA targets that bind to TDP-43 in mouse brain. A motif based sequence search using MEME suite revealed that the top TDP-43 targets identified by microarray contained (TG)n repeats corresponding to (UG) repeats in RNA. Reverse transcription polymerase chain reaction (RT-PCR) was then used to validate some of the binding targets identified using RIP-Chip. The RNA targets identified included the RNA binding protein gene, Celf4, the calcium/calmodulin dependent protein kinaseII alpha, Camk2a, Septin 3 and the glial high affinity glutamate transporter, Slc1a3. Interestingly, our experiments on brain tissue from mice suggest a possible role for TDP-43 in regulating genes involved in synaptic transmission such as synapsin II (Syn2) and synaptophysin (Syp). Immunohistochemistry results show that TDP-43 is localized in the neuromuscular junction of phrenic-diaphragm motor neuron in mouse.
Discussion and conclusions: It has been suggested that RNA transport for site-specific translation in neurons is probably essential for synaptic transmission (3). Our RIP-Chip results from mouse brain suggest that TDP-43 plays a role in the regulation/transport of mRNAs involved in synaptic plasticity and that any dysfunction of TDP-43 might result in disruption of synaptic transmission.
References
- Sreedharan J, Blair IP, Tripathi V, et al. Science 2008;319:1668–72.
- Strong MJ, Volkening K, Hammond R, et al. Mol Cell Neurosci. 2007:35(2):320–7.
- Kao Der-I, Aldridge GM, Weiler IJ, et al. PNAS 2010: 107(35):15601–6.
P172 ALTERED TDP-43 EXPRESSION IN LYMPHOCYTES FROM DEFINITE AND PROBABLE ALS PATIENTS WITHOUT TARDBP MUTATIONS
MOUGEOT J-L, PRICE A, BROOKS BR
Carolinas Medical Center, Charlotte, NC, USA
Email address for correspondence: [email protected]
Keywords: TDP-43, expression, PBMCs
Background: Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive lethal motor neuron disease. TDP-43 is a ubiquitous multifunctional protein encoded by TARDBP involved in RNA processing. TDP-43, normally expressed in nuclei, has been shown to accumulate within pathological ubiquitinated cytoplasmic inclusions in brain and spinal cord of sporadic ALS patients and non-SOD1 familial ALS patients. In addition, previous Western blot analyses have shown that mutations in TARDBP produce TDP-43 aggregates in lymphocytes of ALS patients.
Objectives: Our objective was to determine the expression of TDP-43 in peripheral blood mononuclear cells (PBMCs) (i.e. lymphocytes and monocytes) in definite and probable ALS patients compared to healthy controls by immunocytochemistry.
Methods: Immunocytochemistry (ICC) experiments were performed using Shandon Cytospin™ centrifugation and fluorescence microscopy on Histopaque™ gradient-isolated PBMCs. The study group consisted of 9 definite ALS patients, 9 probable ALS patients, and 5 healthy control subjects. TDP-43 expression in lymphocytes and monocytes was determined by densitometry using ImageJ program. Confocal microscopy was used to determine cytoplasmic localization of TDP-43.
Results: Immunostaining of TDP-43 in monocytes was not significantly different between ALS patients and healthy controls. However, the relative expression of TDP-43 in lymphocytes versus monocytes (i.e. expression ratio) was increased in definite ALS patients compared to healthy controls (p < 0.05). In addition, the mean relative expression of TDP-43 in lymphocytes from probable ALS patients was intermediate compared to the mean relative expression of TDP-43 in lymphocytes from definite ALS patients and healthy controls. Cytoplasmic localization of TDP-43 in PBMCs was mostly detected in ALS patients with higher levels of TDP-43 expression and was not detected in PBMCs from healthy controls. No correlation was found between the expression of TDP-43 and the duration of the disease or the revised ALS functional rating scale (ALSFRS-R).
Discussion and conclusions: Increased relative expression of TDP-43 in lymphocytes of definite ALS patients compared to healthy controls is shown by immunohistochemistry for the first time in this study. The results support a role for TDP-43 in a systemic disease response without any association with the duration of the disease or the ALSFRS-R. TDP-43 expression alone is unlikely to represent a useful blood biomarker. However, the detection of a particular misfolded/unfolded form or abnormal translocation of TDP-43 in lymphocytes might still be indicative of disease-onset, severity or rate of progression.
P173 PHENOTYPIC HETEROGENEITY IN A LARGE CLUSTER OF SARDINIAN ALS CASES CARRYING A FOUNDER TARDBP A382T MISSENSE MUTATION
CHIO A1, BORGHERO G2, PUGLIATTI M3, TICCA A4, ORTU E5, CALVO A1, MOGLIA C1, BRUNETTI M6, OSSOLA I6, MARROSU MG2, MURRU MR2, FLORIS G2, CANNAS A2, PARISH LD3, COSSU P3, SOLINAS G9, SOTGIU A9, ABRAMZON Y7, JOHNSON J7, TRAYNOR BJ7,8
1Department of Neuroscience, University of Torino, Torino, Italy, 2Department of Neurology, Azienda Universitaria-Ospedaliera di Cagliari and University of Cagliari, Cagliari, Italy, 3Department of Neuroscience, University of Sassari, Sassari, Italy, 4Department of Neurology, Azienda Ospedaliera San Francesco, Nuoro, Italy, 5Department of Neurology, Ospedale di Ozieri, Ozieri, Italy, 6Laboratory of Molecular Genetics, Azienda Sanitaria Ospedaliera Ospedale Infantile Regina Margherita– Sant'Anna, Torino, Italy, 7Neuromuscular Diseases Research Group, Laboratory of Neurogenetics and Molecular Genetics Unit, National Institute on Aging, National Institutes of Health, Bethesda, MD, USA, 8Department of Neurology, Johns Hopkins Hospital, Baltimore, MD, USA, 9Department of Biomedical Science, University of Sassari, Sassari, USA
Email address for correspondence: [email protected]
Keywords: TARDBP, founder mutation, clinical heterogeneity
Background: Genetic studies of population isolates, such as Sardinia, are a powerful method to understand disease pathogenesis.
Objective: We undertook mutational screening of the SOD1, FUS and TARDBP genes in 197 patients of Sardinian ancestry diagnosed with ALS.
Methods: All patients underwent mutational analysis for SOD1, FUS and TARDBP. Specifically, all the coding exons and 50bp of the flanking intron-exon boundaries of SOD1, FUS and TARDBP were PCR amplified, sequenced using the Big-Dye Terminator v3.1 sequencing kit (Applied Biosystems Inc.), and run on an ABIPrism 3100Avant genetic analyzer.
Results: We found a c.1144G > A (p.A382T) missense mutation of the TARDBP gene in a quarter of cases (n = 46, 23.4%). The mutation was identified in 17 (39.5%) out of 43 fALS and 29 (18.8%) out of 154 apparently sALS. Considering 15 healthy carriers of the mutation, we calculated that the penetrance of the p.A382T mutation in the Sardinian population was 79% (95% c.i. 56-93) at 80 years. Patients carrying the A382T missense mutation had a better prognosis than patients who did not carry the mutation (3-year survival rate, 80.7% vs. 63.9%, p = 0.03). The presence of the A382T mutation remained independently significant also in Cox multivariable analysis.
The mutated cases carried a 94-SNP (663Kb) risk haplotype across the TARDBP locus on chromosome 1p36.22, indicating they shared a common ancestor. Flail arm and pyramidal phenotypes were significantly more frequent in patients carrying the p.A382T mutation than in those not carrying this mutation. Mutated patients were more frequently affected by frontotemporal lobar dementia (p = 0.02). Three of these patients developed extrapyramidal symptoms several years after their initial presentation with motor weakness.
Conclusions: We found that the TARDBP p.A382T missense mutation accounts for approximately one fourth of all ALS cases in this island population. These ALS patients share a large risk haplotype across the TARDBP locus indicating that they have a common ancestor. The phenotype of the mutated patients is quite heterogeneous, indicating that, even in presence of a founder mutation, other factors, genetics or environmental in nature, play a role in determining the ALS phenotype. Sardinian patients carrying the TARDBP p.A382T missense mutation have a better prognosis than patients who do not carry this mutation. The identification of mutated cases with extrapyramidal features expand the clinical spectrum associated with mutations in the TARDBP gene to include basal ganglia dysfunction.
P174 SCREENING FOR TARDBP MUTATIONS IN CHINESE SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
LI X, LIN Y, PENG Y, ZUO Z, XIE M, LIU M, CUI L
Peking Union Medical College Hospital, Beijing, China
Email address for correspondence: [email protected]
Keywords: TARDBP gene, Chinese sporadic, high-resolution melting
Objective: Sporadic Amyotrophic Lateral Sclerosis (ALS) accounts for about 90% of patients. TDP-43 protein is coded by Transactive response DNA binding protein (TARDBP) gene and abnormal TDP-43 protein inclusion has been found in ALS patients. Many recent studies found TARDBP gene mutations in familiar and sporadic ALS. Our study is to screen TARDBP gene mutation in Chinese sporadic ALS patients.
Methods: We recruited 137 cases of sporadic ALS and extracted genomic DNA from blood samples. The coding region of TARDBP exon 6, was amplified by polymerase chain reaction (PCR). The PCR products were genotyped using high-resolution melting technology (HRM) and some of them were sequenced.
Results: We observed one heterozygous missense mutation (p.Gly348Val, c.G1043T) in one Chinese individual and one silent mutation (1098C > G) in two Chinese individuals with sporadic ALS. The mutation was not reported before. No mutation was found in 90 control Chinese individuals.
Conclusions: One heterozygous missense mutation and one silent mutation in TARDBP was found in 137 Chinese cases. Our data indicates that genetic variation in TARDBP (0.73%) may not be a common cause of sporadic ALS in Chinese population.
P175 NINE PEDIGREES WITH MUTATIONS IN FUS/TLS IN FAMILIAL ALS CONTAINING A NEW TRUNCATING MUTATION
WAIBEL S1, NEUMANN M2, RABE M1, MÜLLER G4, MEYER T3, LUDOLPH AC1
1Department of Neurology, University of Ulm, Germany, 2Institute of Neuropathology, Zurich, Switzerland, 3Department of Neurology, Charité University Hospital, Berlin, Germany, 4Department of Neurology, Gießen, Germany
Email address for correspondence: [email protected]
Keywords: FUS, genetics, familial
Background: Mutations in the FUS/TLS gene have been associated with familial amyotrophic lateral sclerosis (fALS).
Methods: We analyzed the presence and frequency of C-terminal FUS/TLS mutations in a German ALS cohort, including 184 fALS and 247 sALS patients by sequence analysis of exons 5, 6, 13 -15.
Results: We identified nine pedigrees containing seven heterozygous FUS/TLS mutations and two truncating mutation in German ALS families, including the R521H, K510R, R514G, R495X and G478X. The truncating mutation were both associated with an aggressive disease course whereas the R521H, K510R and R514G mutation showed a mild phenotype with disease duration ranging from 6 to 8 years.
Conclusions: Mutations in FUS/TLS account for 8.7 % (16 of 184) of fALS in our German cohort.
P176 MUTATIONAL ANALYSIS OF THE FUS/TLS GENE IN A CATALAN ALS POPULATION
GAMEZ J1, SYRIANI E1,2, BADIA M1, MORALES M2
1Neurology Department, Hospital Universitari Vall d'Hebron, Institut de Recerca (VHIR) Autonomous University of Barcelona, Barcelona, Spain, 2Structural Synaptic Plasticity Laboratory, CIBIR, Logroño, Spain
Email address for correspondence: [email protected]
Keywords: FUS/TLS, epidemiology, MIM 608030
Background: Previous epidemiological studies suggest that mutations in the FUS/TLS gene are the second most common genetic cause of familial ALS (FALS). A predominantly autosomal dominance inheritance pattern was observed in these pedigrees harboring the FUS/TLS mutational variants, and in a few FALS patients with an autosomal recessive inheritance pattern. Sporadic ALS (SALS) cases have occasionally been described.
Genetic characterization of ALS6 families should provide information on the distribution of FUS/TLS mutations in different ethnic groups. The prevalence of FUS/TLS gene mutations in Catalonia, a Spanish region of 7,000,000 inhabitants, has not been studied.
Objective: To determine the prevalence of FUS/TLS gene mutations in a Catalan ALS population, and to analyze the genotype-phenotype relationship.
Materials and methods: 30 different FALS pedigrees and 124 sporadic ALS patients were screened for FUS/TLS gene mutations by direct sequence analysis using the methodology previously described.
Results: Clinical information about the disease's characteristics was available for 120 individuals in the FALS group (65 males and 55 females), with a male-female ratio of 1.18:1. The disease began with limb onset in 62.5% of cases, bulbar onset in 11.7%, and simultaneous bulbar and limb onset in 12.5%. No clear data concerning the exact site of onset was available in the remaining patients (13.3%). The mean age at onset was 49.3 years old (S.D. 12.0; median 50.0; range 21-81 years). A FUS/TLS mutant was identified in two of the 30 FALS pedigrees studied. The mutations found in this group were p.R521C and p.K510E. The phenotype in the p.R521C family was characterized by a young age at onset (38.2 years old), proximal limb girdle weakness, predominant lower motor neuron signs and dropped head. Survival time ranged from 10 to 36 months. Obligate asymptomatic carriers were detected. The phenotype in the p.K510E family was of an early onset (<40 years old), predominant lower motor neuron disease with survival of less than one year.
The mutational variant p.R522R was identified in a 74-year-old woman with bulbar onset in the sporadic ALS (SALS) group. The prevalence of FUS/TLS gene mutations in our FALS population was 6.6%, while in the SALS group it was 0.8%.
Conclusions: The prevalence of FUS/TLS mutations in FALS in Catalonia (6.6%) is similar to levels in other European populations, making ALS6 the second most common form of FALS in our population. In the SALS group, our results (0.8%) are similar to other epidemiological studies.
The high percentages of FUS/TLS mutations in other FALS populations and the presence of obligated asymptomatic carriers increases the probability that other genetic factors are involved in this disease's pathogenesis/epidemiology.
Acknowledgements: JG was supported by a Spanish FIS-FEDER grant (FIS 10/01070). MM received grants from Fundación Ramón Areces and Fundación Reina Sofía-CIEN.
P177 FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS WITH GLY93SER MUTATION IN CU/ZN SUPEROXIDE DISMUTASE: A CLINICAL AND NEUROPATHOLOGICAL STUDY
OKETA Y, ONO S
Teikyo University Chiba Medical Center, Ichihara, Chiba, Japan
Email address for correspondence: [email protected]
Keywords: Gly93Ser mutation, SOD-1, posterior column
Background: Familial amyotrophic lateral sclerosis (FALS) with abnormalities of the Cu/Zn superoxide dismutase (SOD-1) gene is neuropathologically characterized by the degeneration of middle root zones 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 Gly93Ser missence mutation in exon 4 of the SOD1 gene in which no clinical or detailed pathological data have been available.
Methods: The patient's aunt died of ALS at age 67. Her first symptom was weakness of the leg muscles. Weakness of the hand muscle was noted 1 year after the onset of the disease. Our patient developed insidious muscle weakness in the legs, and her gait gradually became disturbed from age 23. At age 24, she also experienced weakness in the arms. At age 30 she exhibited hoarseness due to bilateral cord palsy. At the same age, she presented with dysphasia. She became bedridden from the age of 36 years. About 16 years after onset of the disease, at the age of 39 years, the patient died of respiratory failure. She did not show dementia, opthalmolegia, or autonomic disturbances. During the course of her illness, deep tendon reflexes were decreased without pathological reflexes.
Results: Histological examination disclosed conspicuous changes throughout the spinal cord. In the anterior horns from cervical to lumbar segments, there was marked loss of neurons with gliosis, and there was absence of neuronal inclusion bodies in the remaining cells. Neither Bunina bodies nor LBHIs were present in the neurons. In the medulla oblongata, the hypoglossal nuclei showed mild loss and shrinkage of nerve cells. Marked atrophy and myelin pallor were present in the superior cerebellar peduncles. The dentate nuclei of the cerebellum showed neuronal loss accompanied by gliosis and grumose degeneration. The precentral gyrus showed mild loss and shrinkage of Betz cells. There was a moderate neuronal loss with gliosis in the red nucleus. The corticospinal tracts in both anterior and lateral funiculi showed a slight degree of myelin pallor. There were circumscribed areas of myelin loss in the posterior column from the cervical through the lumbar segments. There was also a marked myelin loss in the spinocerebellar tract. Prominent neuronal loss was noted in the Clarke's nuclei.
Discussion: The clinical characteristics in this case was onset of symptoms in the legs, frequently seen in FALS, and slow progression: 16 years survival. Based on clinical, genetic and pathological findings with a review of the literature, we suggest that degeneration of the dentatorubral system and the absence of LBHIs in our case are pathological features in FALS with the Gly93Ser mutation.
P178 A CASE OF ALS4 WITH A NOVEL SENATAXIN GENE R2136C MUTATION WITH PARTIALLY IMPROVED WEAKNESS AND SENSORY DISTURBANCE OF LIMBS BY IMMUNOTHERAPY
TATEISHI T1, SAIGA T1, KAWAMURA N1, NAGARA Y1, HASHIGUCHI T2, TAKASHIMA H2, HONDA H3, OHYAGI Y1, KIRA J-I1
1Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan, 2Department of Neurology and Geriatrics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan, 3Department of Neurology and Geriatrics, Kagoshima University Graduate School of Medical and Dental Sciences, Fukuoka, Japan
Email address for correspondence: [email protected]
Keywords: senataxin, ALS4, immunotherapy
Background: Mutations in the senataxin gene (SETX) were reported in patients with two distinct sydromes, familial amyotrophic lateral sclerosis (ALS) 4 and ataxia oculomotor apraxia type 2. Only three senataxin mutations, T3I, L389S, T1118I and R2136H, have been reported in ALS4 patients.
Objectives: The present study aimed to clarify clinical and pathological features of familial ALS4 with partially improved weakness and sensory disturbance of limbs by immunotherapy.
Methods: We carried out clinical, neuropathological, and genetic studies on a Japanese patient with the new senataxin gene (R2136C) mutation.
Results: The patient, a 41-year-old male, was born in normal delivery and had a tendency to fall in childhood. His feet showed pes cavus since childhood. He suffered from progressive weakness of lower limbs and gait disturbance from age 35. Within 1 year from onset, bilateral arm weakness developed and progressively worsened, and sensory and urinary disturbance also occurred. On first admission to our hospital at 37-year-old, he was diagnosed to have chronic inflammatory demyelinative polyneuropathy. Methylpredonisolone pulse therapy improved weakness of four limbs. The steroid pulse therapy was repeated at the time of exacerbations of weakness and sensory disturbance of limbs. Neurological examination at 41-year-old revealed a distal dominant severe weakness, atrophy and fasciculation in four limb muscles. Hyperreflexia was also noted in four limbs. The planter response was extensor type bilaterally. Sensory impairment of the lower limbs and urinary disturbance were noted. Neither tongue atrophy nor dysarthria was observed. A nerve conduction study revealed sensorimotor peripheral neuropathy; the conduction velocities of motor and sensory nerves and the amplitudes of compound muscle action potentials were severely reduced in all four limbs. Cervical MRI showed thickening and enhancement of nerve roots. Sural nerve biopsy showed a slight decrease in large myelinated fibers and endoneurial infiltration with lymphoid cells. Although the missense mutation R2136C was found in exon 17 of SETX in the patient, his genetically related parents and a sister did not have the mutation, suggesting the proband carried a de novo mutation.
Discussion and conclusions: This case showed distinct clinical features, as compared with previously reported cases, such a hypertrophy of nerve roots on MRI, inflammation of the biopsied sural nerve, and favorable response to immunotherapy. This case suggests that an immunological mechanism may be partly involved in SETX mutation.
P179 CLINICAL-GENETIC CHARACTERIZATION OF THE FIRST TWO SPANISH FAMILIES WITH SPASTIC PARAPLEGIA HARBORING A 6-EXON DELETION (EX10-16 DELETION) IN THE SPG4/SPAST GENE
GAMEZ J1, ÁLVAREZ V2, SÁNCHEZ-FERRERO E2,3, BEETZ C3, DÍAZ M2
1Neurology Department, Hospital Universitari Vall d’ Hebron, Institut de Recerca (VHIR) Autonomous University of Barcelona, Barcelona, Spain, 2Laboratorio de Genética Molecular. Hospital Universitario Central de Asturias, Oviedo, Spain, 3Institut für Klinische Chemie und Laboratoriumsdiagnostik, Universitätsklinikum, Jena, Germany
Email address for correspondence: [email protected]
Keywords: hereditary spastic paraplegia, SPG4, clinical-genetic relationships
Objectives: Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group in which at least 45 different loci have been identified. The inheritance pattern may be AD, AR, or linked to the X chromosome. The most frequent form of AD-HSP is that caused by mutations in the SPG4/SPAST gene. Its prevalence is 15-40%, depending on the population studied. Most mutations in this gene are “missense” and “non-sense” although “splice-site” mutations and small deletions/insertions have been described. However, large deletions are very unusual. Here we present the first two SPG4/SPAST Spanish families carrying a deletion of exons 10-16.
Patients and methods: We studied the phenotype, age of clinical onset, age of wheelchair dependence, neurophysiological examinations, and the Spastic Paraplegia Rating Scale (GeNeMove) scores in 10 members of two non-interrelated AD-HSP families. We carried out direct sequencing of the genes SPAST, ATL1, REEP1 and NIPA1 and the MLPA assay with the Salsa Kit P165 (MRC Holland) of the SPAST gene under standard conditions in 6 patients.
Results: The average age of clinical onset was 29.1 years old (range 6-50). All patients presented all the clinical features characteristic of pure forms, although five referred fecal incontinence as one of the initial symptoms, which was extremely debilitating in two. The average age of loss of gait autonomy and wheelchair confinement was 57.7 years old. The mean score on the GeNeMove scale was 22.3. The Barthel score was 68.6. In one of the families, we observed an intergenerational variability in the age of onset, suggesting a phenomenon of anticipation. TMS highlighted different degrees of conduction defects. In both families, we identified a deletion that covered exons 10-16 (c.1227-1729 + ?) of the SPAST gene that was confirmed using RNA.
Conclusions: This is the first description of a large SPG4/SPAST deletion in the Spanish population. The presence of fecal incontinence as one of the initial symptoms could be interpreted as a distinctive clinical finding for this mutation as it has not been described in the phenotype of other deletions described to date. Anticipation and almost complete penetrance were other characteristics associated with this deletion.
Acknowledgements: JG was supported by a Spanish Fondo de Investigaciones Sanitarias-FEDER grant (FIS 10/01070). VA was supported by grants from Spanish Fondo de Investigaciones Sanitarias PI08/0915 (European FEDER founds).
P180 AN AUTOSOMAL DOMINANT FAMILIAL MOTOR NEURON DISEASE WITH A NOVEL MUTATION OF A GENE ASSOCIATED WITH NF-KB SIGNALLING PATHWAY
KAJI R
Tokushima University, Tokushima, Japan
Email address for correspondence: [email protected]
Keywords: NF-kB, HMSNP, p65
We found a novel mutation of a gene in two families with motor neuron disease previously reported as HMSNP. The disease typically starts at 35-62 years of age in proximal muscles with prominent fasciculations, and progresses over 5-20 years symmmetrically affecting distal muscles as well. Later loss of vibratory sense becomes apprant at the feet on examination, but never presents sensory disabilities. Most patients die of respiratory failure or pneumonia. Autopsy findings included both upper and lower motor neuron invlovement where abnormal accumulation of the gene product was found. Immunohistochemical analysis of NF-kB p65 subunit showed its localization in the nucleus of neurons in the patient, whereas only cytoplasm was stained in normal spinal motor neurons. Mutant gene was found to affect NF-kB reporter expression in HEK293T cells on relative luciferease assay.
P181 GENE EXPRESSION ANALYSIS IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS AND MULTIFOCAL MOTOR NEUROPATHY
SHTILBANS A1, CHOI S-G1, FOWKES M1, KHITROV G1, SHAHBAZI M2, TING J1, ZHANG W1, SEALFON S1, LANGE D2
1Mount Sinai School of Medicine, New York, NY, USA, 2Hospital For Special Surgery, New York, NY, USA
Email address for correspondence: [email protected]
Keywords: mRNA, biomarker, microarray
Background: There are no reliable biologic markers for amyotrophic lateral sclerosis (ALS). Multifocal motor neuropathy (MMN) without conduction block can mimic ALS. Thus, a method to separate patients with ALS and MMN is needed.
Objectives: To confirm the previously identified gene expression pattern in muscles from patients with ALS and MMN compared to controls.
Methods: RNA extracted from skeletal muscles of 3-ALS, 3-MMN and 3-control patients were subjected to RT-PCR confirmation analysis based on our previously published genome-wide microarray gene expression data (1). Four additional ALS patients and four new controls were also used.
Results: Validation analysis of the most significant expression pattern differences confirmed our previous microarray data for leucine-rich repeat kinase-2 (LRRK-2), follistatin, collagen type XIX alpha-1, ceramide kinase-like, sestrin-3 which were overexpressed only in the ALS group. CXorf64 was increased in ALS and decreased in MMN compared to controls. Western blot analysis of LRRK-2 gene protein level from ALS muscles showed no obvious difference compared to controls.
Discussion and conclusions: The up-regulation of the above genes in the muscles of ALS patients relative to MMN and controls discovered previously by our microarray analysis is reproducible and statistically significant. Further studies are necessary to evaluate the identified genes in larger patient groups and different tissues.
Reference
- Shtilbans, et al. Ann Neurol., 2009.
P182 ATAXIN-2 INTERMEDIATE-LENGTH POLYGLUTAMINE: A POSSIBLE RISK FACTOR FOR CHINESE PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
HUANG R, CHEN Y, YANG Y, CHEN K, SONG W, PAN P, LI J, SHANG H
Department of Neurology, West China Hospital, SiChuan University, Chengdu, China
Email address for correspondence: [email protected]
Keywords: Ataxin-2 (ATXN2) gene, polyglutamine (polyQ) expansion
Background: Intermediate-length polyglutamine (polyQ) expansions in the Ataxin-2 (ATXN2) gene have recently been identified as a risk factor for sporadic amyotrophic lateral sclerosis (SALS). Our study aims to analyze (CAG)n expansions in the ATXN2 gene among Chinese patients with SALS.
Methods: All patients diagnosed with adult-onset sporadic ALS were consecutively followed up, and their clinical characteristics were collected. To measure the repeats length of ATXN2 polyQ, fluorescence-polymerase chain reaction products were analyzed on a 3100-Avant Genetic Analyzer Applied Biosystem using the ROC-500 size standard.
Results: Three hundred and forty-five patients with SALS were studied. The mean age of onset was 51.38 ± 12.45 years. ATXN2 polyQ with a repeat length greater than 27 was found to be weakly associated with ALS in our study. There was no significant difference in mean age of onset, gender, and onset site between the group of SALS patients with and without ATXN2 polyQ expansion greater than 27.
Conclusion: Our finding provides evidence that the ATXN2 polyQ expansion greater than 27 might be a risk factor for Chinese SALS patients.
P183 ASSOCIATION STUDY OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS IN CHINESE PATIENTS BY USE OF MALDI-TOF MASS SPECTROMETRY
LI X, LIN Y, XIE M, LIU M, CUI L
Peking Union Medical College Hospital, Chinese Academy of Medical Science, Beijing, China
Email address for correspondence: [email protected]
Keywords: case-control association analysis, single nucleotide polymorphism, MALDI-TOF genotyping
Objective: Sporadic amyotrophic lateral sclerosis (ALS) accounts for majority of patients. ALS is a kind of complex disorder. There were several SNPs reported to be associated with sporadic ALS in recently published genome-wide association studies (GWAS), but there are few data from Asian ALS populations and no report focuses on single nucleotide polymorphisms which may be associated with sporadic ALS of Chinese origin.
Methods: We have recruited 86 individuals with sporadic ALS and 94 matched controls for our study and extracted genomic DNA from blood samples. Genotypes were determined by a MALDI-TOF based approach followed by association analysis.
Results: Individual genotype data for 8 SNPs in Chinese population showed no significant association with sporadic ALS. Combining genotype data with published GWA, rs1942239 gained in strength of allelic association, and rs558889 deviated Hardy-Weinberg equilibrium at ALS case group which may be associated with susceptibility to sporadic ALS.
Conclusions: SNP rs1942239 and rs558889 may contribute to susceptibility of sporadic ALS in Chinese patients. The larger sample studies are warranted to confirm the association.
P184 FINDING THOSE ELUSIVE GENETIC VARIANTS IN SPORADIC ALS: ARE WE LOOKING AT THE RIGHT TISSUE?
PAMPHLETT R, MORAHAN J, LUQUIN N, YU B
University of Sydney, Sydney, NSW, Australia
Email address for correspondence: [email protected]
Keywords: somatic mutation, copy number variant, genome array
Background: The majority of analyses of blood DNA looking for a genetic basis for SALS have been unsuccessful. These include investigations of common genetic variants that could lead to disease susceptibility, or of rare variants that could be directly mutagenic. This lack of success may be because some SALS-causing mutations occur predominantly in brain tissue, and are either absent or are at presently undetectable levels in blood.
Objective: In an attempt to gauge the extent of genetic variability between blood and brain tissue in SALS, we looked for differences in genome-wide chromosomal copy number variants (CNVs) between these two tissues in SALS patients.
Methods: The 32 SALS patients were 22 male and 10 female Australians. Blood samples were taken during life, and patients also gave consent for post mortem removal of brain and spinal cord tissue. Genomic DNA was extracted from peripheral blood nucleated cells and from the cortex of the lateral frontal gyrus from frozen brain tissue. Genome-wide CNVs were compared between blood and brain from the SALS patients, as well as in 26 control brains (to exclude common brain-only polymorphisms) using Affymetrix 6.0 (1.8 million marker) arrays and Partek software.
Results: 410 CNVs were detected that were present in SALS brain but not blood DNA. These involved 94% of the SALS patients, with a median of 8 brain-CNVs per patient. Twenty-four of the brain-CNVs were rare, were not found in control brains, and overlapped with genes. Some of these genes are involved in processes suspected in SALS, e.g. apoptosis, glutamate metabolism, and RNA editing. Ten SALS brain-CNVs were found in more than one patient.
Discussion: Brain-predominant structural variants may be common and appear to be present in most patients with SALS. Many of these are likely to be brain-predominant private polymorphisms, and any mutagenicity of these variants remains to be tested.
Conclusion: Although with this number of subjects this needs to be regarded as a preliminary study further multi-tissue studies of this nature, with larger numbers of samples and using more sensitive methods of copy number detection, are warranted to look for brain-specific mutations that could underlie SALS.