P158 COMPLETE MUTATIONAL SPECTRUM OF KNOWN ALS GENES IN A LARGE COHORT OF FAMILIAL ALS CASES
Müller K1
Marroquin N1
Volk AE1,5
Hübers A1
Meyer T3
Andersen PM1,4
Meitinger T2
Ludolph AC1
Strom TM2
Weishaupt JH1
aUlm University, Ulm, Germany
bHelmholtz Institute, Munich, Germany
cHumboldt University Berlin, Berlin, Germany
dUmea University, Umea, Sweden
eUniversity Medical Center Hamburg-Eppendorf, Hamburg, Germany
Email address for correspondence: [email protected]
Keywords: next generation sequencing, common ALS genes, familial ALS
Recent advances in ALS genetics have led to the discovery of approximately 25 different genes mutated in ALS with a mostly autosomal-dominant pattern of inheritance. However, the complete spectrum of mutations in all known ALS genes, and their contribution to ALS has never been determined in a homogenous large cohort of familial ALS cases.
We thus combined Sanger sequencing, fragment length analysis, repeat-primed PCR, Southern blotting and whole exome sequencing to obtain a comprehensive profile of ALS gene mutations in more than 150 German ALS families.
We report the relative contribution of each ALS gene to familial ALS and surprisingly show a higher proportion of FUS cases (4%) compared to TARDBP (2.5%) in Germany. Moreover, we identified several novel mutations, and demonstrate absence of mutations in some recently described ALS genes. 55% of German familial ALS cases did not carry a mutation in any of the known ALS genes. 81% of the genetically defined cases showed mutations in the four most common ALS genes C9ORF72, SOD1, FUS and TARDBP, while the individual relative contribution of the other ALS genes in our cohort was very low (19%).
However, in these rare ALS genes several seemingly unique mutations that have not been described in other families were observed. Our data support the assumption that, beyond the 4 most frequent genes, ALS is a genetically highly heterogeneous disease.
P159 A DUTCH FAMILY WITH AUTOSOMAL RECESSIVE MOTOR NEURON DISEASE CAUSED BY OPTINEURIN MUTATIONS
Beeldman E1
van Ruissen F2
Baas F2
de Visser M1
van der Kooi A1
fDepartment of Neurology
gDepartment of Genome Analysis, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
Email address for correspondence: [email protected]
Keywords: optineurin (OPTN), case report, familial ALS
Background: Pathogenic mutations in the optineurin gene (OPTN) are infrequently found in patients with motor neuron disease (MND). We report the first Dutch family with autosomal recessive MND caused by mutations in the OPTN gene.
Case reports: A 21-year old healthy woman (2.2) presented with a 4-month history of weakness of her left leg and a bilateral foot drop. Neurological examination showed fasciculations, mild to moderate weakness of the upper and lower extremities, respectively, with symmetrical tendon reflexes and indifferent plantar responses. Electromyography (EMG) showed denervation in the lower extremities with signs of reinnervation in the right leg. Her symptoms progressed rapidly and she received a tracheostomy early in the disease course. She died 15 years after symptom onset. Her sister, a 27-year old healthy woman (2.1) and 4 months pregnant, presented with a 4-month history of fasciculations and weakness of the left arm. Neurological examination revealed a Trendelenburg’s sign on the left and atrophy with severe weakness of the proximal muscles of the left arm and mild weakness of the right arm and leg. Tendon reflexes were reduced on the left with an indifferent plantar response. She fulfilled the El Escorial criteria in the cervical and lumbosacral regions. She died 12 months after symptom onset due to acute respiratory failure. Their brother (2.3) presented to our hospital at age 38, with a one-year history of fasciculations. Neurological examination showed fasciculations of his back and extremities with mild proximal weakness of both arms. Tendon reflexes were symmetrical with normal plantar responses. On EMG, he fulfilled the El Escorial criteria in the cervical, thoracic and lumbosacral regions. Eighteen months after symptom onset he received non-invasive ventilation and a feeding tube. After thirty months he was completely bed-bound and slightly dysarthric. He lives at a nursing home and spends the weekends at home with his wife and kids. Genetic testing of patients 2.1 and 2.3 showed two mutations in the OPTN gene, a c.658delG and c.493C> T mutation. The c.658delG mutation was found in their father and the c.493C> T mutation in their mother. Both parents are alive and healthy. Genetic analysis of patient 2.2 was not available.
Discussion and conclusion: To the best of our knowledge this is the first Dutch family with autosomal recessive MND based on two mutations in the OPTN gene. The c.493C> T mutation has been described in a Danish MND family with a possible autosomal dominant inheritance. However, both mutations result in a stop codon, which most likely leads to an optineurin deficiency in our patients. It is therefore unlikely that a heterozygous c.493C> T mutation is pathogenic. Based on the clinical presentation and the family history, an autosomal recessive inheritance is highly probable in our patients.
P160 SPECTRUM OF MUTATIONS IN ALS GENES ON THE ISLAND OF SARDINIA
Chiò A1
Floris G2
Marrosu F2
Marrosu MG2
Murru MR2
Pugliatti M3
Parish LD3
Calvo A1
Moglia C1
Traynor BJ5
Renton AE5
Brunetti M1
Ossola I1
Barberis M1
Restagno G3
Borghero G2
hUniversity of Turin, Turin, Italy
iUniversity of Cagliari, Cagliari, Italy
jAOU Città della Salute e della Scienza, Torino, Italy
kUniversity of Sassari, Sassari, Italy
lNIH, Bethesda, USA
Email address for correspondence: [email protected]
Keywords: gene, Sardinia, epidemiology
Background: Sardinia, the second largest Mediterranean island, represents a genetic isolate; its population displays decreased genetic and allelic heterogeneity. We have already reported that ALS patients of Sardinian ancestry have frequency higher than expected of the TARDBP p.A382T missense mutation.
Aims: We report the genetics of a larger series of ALS patients of Sardinian ancestry extending our analysis to include other ALS genes and their clinical correlates.
Methods: All ALS patients of Sardinian ancestry were eligible to be included in the study. Patients were identified through the ITALSGEN consortium in the period 2008 to 2013. SOD1, TARDBP, FUS, OPTN and ANG genes were PCR amplified, sequenced using the Big-Dye Terminator v3.1 sequencing kit (Applied Biosystems Inc.), and run on an ABIPrism 3130 genetic analyzer. A repeat-primed PCR assay was used to screen for the presence of the GGGGCC hexanucleotide expansion in the first intron of C9ORF72. A cut-off of ≥ 30 repeats combined with a typical sawtooth pattern was considered pathological.
Results: Out of a total of 375 ALS cases of Sardinian ancestry, 155 (41.3%) carried mutations in one or more genes. Of these: 75 patients (20.0%) carried a TARDBP heterozygous p.A382T missense mutation; 3 (0.8%) a TARDBP homozygous p.A382T mutation; 10 (2.7%) a TARDBP heterozygous p.G295S mutation; one a TARDBP homozygous p.G295S mutation; and one a double heterozygous mutation (p.G295S and p.A382T) of TARDBP gene. 51 patients (13.6%) carried a hexanucleotide repeat expansion of the C9ORF72 gene and 8 (2.1%) a double mutation of TARDBP (p.A382T) and of C9ORF72. Four patients (1.1%) had a missense mutation of the SOD1 gene (p.A95G and p.A4T) and two a p.T622A missense mutation of the MATR3 gene.
A TARDBP p.A382T heterozygous missense mutation has been detected in eight of the 700 control samples (1.1%); the relative risk of developing ALS in a subject carrying this mutation was 66.2 (95% CI, 32.9-141.9).
Subjects carrying the C9ORF72 repeat expansion had a higher frequency of bulbar onset. The age at symptom onset differed between genetic subgroups; in particular, patients with co-occurrence of C9ORF72 and the TARDBP p.A382T missense mutation had ∼20 years lower age at onset. Frontotemporal dementia, identified in 51 patients (13.6%), was more frequent in patients with C9ORF72 mutations.
Discussion and conclusion: In Sardinian ALS patients genetic mutations accounted for 75% of FALS and 30% of apparently SALS, representing the largest proportion of ALS that is genetically explained in a population outside of Finland. The most common mutations were the p.A382T and p.G295S missense mutations in TARDBP and the pathogenic repeat expansion of C9ORF72. Several patients carried a double mutation, more commonly the combination of C9ORF72 and TARDBP p.A382T missense mutation, and a smaller number were homozygous for TARDBP missense mutations.
P161 TARDBP MUTATION MIMICS A DISTAL MOTOR NEUROPATHY IN A SARDINIAN PATIENT
Caldarazzo EI1
Carlesi C1
Lo Gerfo A1
Chico L1
Mancuso M1
Fogli A2
Simi P2
Siciliano G1
mDepartment of Clinical and Experimental Medicine, Neurological Clinic, University of Pisa, Italy
nDepartment of Medical Laboratories, Medical Genetics Laboratory, University Hospital of Pisa, Italy
Email address for correspondence: [email protected]
Keywords: TARDBP, p.A382T mutation, Sardinia
Background: TARDBP-related Amyotrophic Lateral Sclerosis (ALS) patients present an adult-onset, autosomal dominant clinically typical form of ALS. TARDBP mutations are also observed in both ALS-Fronto Temporal Dementia (FTD) and pure FTD cases. Since first TARDBP mutations were reported in familial ALS cases in 2008, over 40 mutations have been identified in several populations of different geographic origin.
Objectives: Here we report an atypical case of TARDBP-associated ALS patient, coming from Sardinia, an Italian island historically genetically segregated and distinct from other European populations.
Care report: A 50-yr old man came to our attention for a 10-year story of slowly progressive mild symmetrical limb distal hyposthenia and amyotrophy with cramps and fasciculation. No upper motor neuron sign or sensitive impairment was present. Electrophysiological examinations were consistent with second motor neurons damage. A psychiatric history of bipolar disorder was present without cognitive impairment. No family history of neuromuscular disorders.
Results: Genetic analysis revealed that the patient was carrying in heterozygosis the c.1144G-> A (p.A382T) pathogenic missense mutation of the TARDBP gene.
Discussion and conclusion: TARDBP p.A382T missense mutation accounts for approximately one-third of all ALS Sardinian cases. Despite a quite heterogeneous spectrum of resulting phenotypes, the flail arm variant of ALS occured with greater than expected frequency in these patients, although clinical presentation may also include forms of parkinsonism and FTD. To our knowledge, this is the first report of a distal motor neuropathies-like syndrome associated with this mutation.
References:
- Corrado L. et al. Hum Mutat. 2009 Apr; 30(4):688–94.
- Chiò A. et al. Arch Neurol. 2011 May; 68(5):594–8.
- Cannas A. et al. Neurogenetics. 2013 May; 14(2):161–6.
P162 ARHGEF28 GENE EXON 6/INTRON 6 JUNCTION MUTATIONS IN CHINESE AMYOTROPHIC LATERAL SCLEROSIS COHORT
Ma Y
Tang L
Chen L
Zhang B
Fan D
Peking University Third Hospital, Beijing, China
Email address for correspondence: [email protected]
Keywords: ARHGEF28 gene, RGNEF protein, ALS cohort
Objective: To analyze the intron 6, + 1 delG (GT> TT) mutation in Chinese patients with amyotrophic lateral sclerosis (ALS), investigate the incidence of this mutation, and identify the relationship between the genotype and phenotype in Chinese ALS patients.
Methods: We sequenced this mutation in 25 familial ALS (fALS) cases, 357 sporadic ALS (sALS) patients, and 442 healthy control subjects. We collected blood samples and screened for the intron 6, + 1 delG (GT> TT) mutation by extraction of genomic DNA, PCR, direct sequencing, and TA cloning.
Results: Two sALS patients exhibited the intron 6, + 1 delG (GT> TT) mutation of the ARHGEF28 gene. Thus, the incidence of the mutation was 0.52% (2 cases/382 cases) in all of the ALS patients and 0.56% (2 cases/357 cases) in the sALS subgroup. The clinical features of the mutation-positive patients were quite different from those reported in the literature. These characteristics differed in terms of sex, site of onset, cognitive function, and family history.
Conclusion: The intron 6, + 1 delG (GT> TT) mutation of the ARHGEF28 gene is present in the Chinese population; however, the resultant phenotype differs from those observed in other ethnic groups.
P163 FIVE NOVEL SQSTM1 MUTATIONS IN A CHINESE AMYOTROPHIC LATERAL SCLEROSIS COHORT
Yang Y
Tang L
Zhang N
Fan D
Peking University Third Hospital, Beijing, China
Email address for correspondence: [email protected]
Keywords: p62 protein, SQSTM1, novel mutation
Objective: The SQSTM1 gene encodes the p62 protein. Mutations in this gene have been previously reported in patients with familial and sporadic amyotrophic lateral sclerosis (ALS). The purpose of this study was to identify mutations in the SQSTM1 gene and to determine the survival time based on the progression rate of the ALSFRS-R score.
Methods: We sequenced the SQSTM1 gene in 471 Chinese patients with sporadic and familial ALS from 2011 to 2013. SQSTM1 gene mutations were screened using PCR and direct sequencing, and genotype-phenotype correlations and the progressive ALSFRS-R ratio were analyzed.
Results: Seven heterozygous missense mutations were detected in 471 ALS patients. We identified five novel missense mutations: c. 241 G> A p. E81K in the PB1 domain, c. 717 C> A p. N239K in the TRAF6 domain, c.889 G> A p. G297S and c. 1116 G> C p. E372D in the PEST2 domain, and c. 1162 C> T p. P388S in the UBA domain.
Conclusion: The SQSTM1 mutations present in Chinese patients suggest that this gene is involved in ALS cases worldwide. Most of the clinical phenotypes of this mutation varied greatly among different patients.
P164 EVIDENCE OF COMMON GENETIC VARIATION FOR ALS RISK IN CHINESE SAMPLES
Benyamin B1
He J2,1
Leo P1
Shah S1
Hemani G3
Cremin K1
Mangelsdorf M1
Wray N1
Xu H4
Bartlett PF1
Brown M1
Visscher P1
Fan D2
oThe University of Queensland, Brisbane, Australia
pPeking University Third Hospital, Beijing, China
qThe University of Bristol, Bristol, UK
rSecond Military Medical University, Shanghai, China
Email address for correspondence: [email protected]
Keywords: GWAS, methylation, gene
Background: Genetic factors are a major cause of ALS even in apparently sporadic cases. Currently, the known ALS genes explain a small proportion of sporadic cases. Except for age and sex, there are no specific biomarkers and environmental factors known that affect ALS. Thus, elucidating the genetic aetiology of ALS is a key to its treatment and cure.
Objectives: We aim to discover novel genes affecting ALS using a genome-wide association study (GWAS) in a Chinese ALS case-control cohort.
Methods: We used genome-wide single nucleotide polymorphisms (SNPs) data from Illumina OmniZhongHua-8 V1 genotyping arrays from 1,324 cases and 3,115 controls. After quality control, we performed a number of analyses in a cleaned dataset of 1,243 cases and 2,854 controls. These include: (i) a genome-wide association analysis to identify SNPs associated with ALS using PLINK software; (ii) GREML analysis to estimate the proportion of the phenotypic variance in ALS liability due to common SNPs; (iii) gene-based analysis to identify genes associated with ALS. Genome-wide methylation data from the Illumina 450K array was also available in a subset of samples (501 cases and 198 controls).
Results: There were no genome-wide significant SNPs and genes associated with ALS. However, we estimated that 17% (SE: 0.05; p = 6 × 10-5) of the phenotypic variance in ALS liability was due to common SNPs. The top associated SNP was within GNAS (Guanine Nucleotide Binding Protein (G Protein), Alpha Stimulating Activity, rs4812037; p = 7 × 10-7). GNAS was also the most associated gene from gene-based study (p = 2 × 10-5). The analysis of methylation data showed that rs4812037 was differently methylated in 2 out 173 GNAS probes (cg10797197; P = 1 × 10-16 and cg17696847; p = 2 × 10-8).
Discussion and conclusion: The failure to identify a genome-wide (GW) significant variant is likely due to the limited power of this study to find variants with small effects. However, a significant proportion (17%) of ALS liability that can be explained by common SNPs indicated that with an increase in sample size, GW significant variants can be identified. To achieve that, we are currently performing a meta-analysis of our Chinese GWAS results with the largest European ALS GWAS of 6100 cases and 7100 controls (the results are pending). The significant associations between the top SNP and the two GNAS probes also showed that GNAS is an interesting candidate, and methylation data can provide biological support for the identified genetic variant-disease associations.
Acknowledgments: We acknowledge funding support from the Australian Research Council (ARC) and MND Research Institute of Australia. Ji He was funded by a grant from the National Natural Sciences Foundation of China (81030019).
P165 C9ORF72 HEXANUCLEOTIDE REPEAT EXPANSIONS ARE RARE AND HYPER-METHYLATED IN CHINESE SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
He J1,2
Tang L1
Benyamin B2
Shah S2
Hemani G2
Liu R1
Ye S1
Liu X1
Ma Y1
Zhang H1
Cremin K3
Leo P3
Wray N2
Visscher P2
Xu H4
Brown M3
Bartlett PF2
Mangelsdorf M2
Fan D1
sPeking University Third Hospital, Beijing, China
tQueensland Brain Institute, University of Queensland, Brisbane, Australia
uUniversity of Queensland Diamantina Institute, Brisbane, Australia
vShanghai Changzheng Hospital, Second Military Medical University, Shanghai, China
Email address for correspondence: [email protected]
Keywords: C9ORF72, haplotype, methylation
Background: A hexanucleotide repeat expansion (HRE) in the C9ORF72 gene has been identified as the most common mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients among Western populations.
Methods: We performed fragment-length and repeat-primed PCR to determine GGGGCC copy number and expansion within the C9ORF72 gene in 1,092 sporadic ALS (sALS) and 1,062 controls from China. We performed haplotype analysis of 23 SNPs within and surrounding the C9ORF72 gene. Lastly, CpG methylation was assessed in the region of C9ORF72 gene.
Results: The C9ORF72 HRE was found in three sALS patients (0.3%) but not in control subjects (p = 0.25, Fisher’s exact test). Two cases with the HRE did not harbor four risk alleles that have previously been determined to be strongly associated with ALS in Caucasian populations. Several risk alleles (including rs2814707 and rs384992) of the 20-SNP consensus risk founder haplotype in Caucasians demonstrated that two of the three cases shared a novel haplotype carrying the repeat expansion. Two of the three HRE carriers showed hyper-methylation of the CpG island upstream of the repeat that was not detected in other sALS patients (p < 10−8) or controls.
Discussion and conclusion: The low frequency (1.8%) of the 20-SNP consensus risk haplotype and the distinct allele distribution in Chinese sALS patients compared to Caucasian populations indicates that the C9ORF72 HRE is not from the same single founder haplotype involved in Caucasian populations. The extreme methylation pattern of the CpG island upstream of the repeat in two of the HRE carriers is consistent with the repeat expansions being causal ALS mutations.
Acknowledgements: National Natural Sciences Foundation of China (81030019), Australian Research Council Linkage Grant LPl10200926, National Health and Medical Research Council Senior Principal Research Fellowships.
P166 ANALYSIS OF C9ORF72 REPEAT EXPANSION IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS FROM SOUTHWEST OF CHINA
Shang H
Chen Y
Chen X
West China Hospital, Sichuan University, Chengdu, Sichuan, China
Email address for correspondence: [email protected]
Keywords: C9ORF72, repeat expansion, Southwest of China
An intronic GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene was identified as the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in Western populations. Using the repeat-primed polymerase chain reaction analysis, we screened for C9ORF72 in a cohort of sporadic ALS patients of Chinese origin (n = 640).
No pathogenic repeats (> 30 repeats) were detected in either the patients or control subjects, indicating that the pathogenic expansions of C9ORF72 might be a rare cause of ALS in Southwest of China.
To the best of our knowledge, this is the first and largest study to investigate the correlation between C9ORF72 and ALS patients from Southwest of China. Additionally, the results of this study suggest that it would seem pointless to screen for this pathogenic expansion in Chinese patients with this fatal neurodegenerative disease.
P167 A BLINDED COMPARATIVE STUDY ON THE RELIABILITY OF GENETIC TESTING FOR THE GGGGCC-REPEAT EXPANSION IN C9ORF72 PERFORMED IN 14 LABORATORIES
Akimoto C1
Volk AE2
Nordin A1
Andersen PM1
Kubisch C2
wDepartment of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden
xInstitute of Human Genetics, Ulm University, Ulm, Germany
Email address for correspondence: [email protected]
Keywords: genetic testing, C9ORF72, RP-PCR
Background: The GGGGCC-repeat expansion in C9ORF72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9ORF72 have relied on repeat-primed PCR (RP-PCR) for detection of an expansion, a cheap and fast method but it cannot provide size measurements for large expansions and may be inaccurate.
Objectives: To analyse the reliability of the RP-PCR technique for genotyping the GGGGCC-repeat expansion in C9ORF72.
Method: To investigate the inherent limitations of RP-PCR, we compared methods and results of 14 laboratories, which genotyped DNA from 78 ALS and FTD patients in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone. Southern blot was performed in three laboratories as a reference.
Results: The mean sensitivity of RP-PCR alone was 94.3% (71.7-100%) and the mean specificity was 97.3% (87.5-100%). Combining the results of RP-PCR and amplicon-length analysis, 100% sensitivity and specificity were found in only five laboratories (A-E, 35.7%), whereas sensitivity and specificity above 95% were found in seven laboratories (A-E, G and L, 50%). Using PCR-based techniques, only 5 of the 14 laboratories got results in full accordance with Southern blot analysis.
Conclusion: There was a high degree of false positive and false negative results. We recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. Southern blot should be the gold standard and obligatory in a clinical diagnostic setting.
P168 VARIATION IN SIZE OF THE C9ORF72 GGGGCC-REPEAT EXPANSION BETWEEN DIFFERENT TISSUES IN ALS AND FTD
Nordin A1
Akimoto C1
Forsberg K2
Graffmo K2
Alstermark H1
Brännström T2
Andersen P1
yDepartment of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden
zDepartment of Medical Biosciences, Umeå University, Umeå, Sweden
Email address for correspondence: [email protected]
Keywords: C9ORF72, repeat expansion, southern blot
Introduction: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders genetically linked to one another. The most common genetic cause for both conditions is a large GGGGCC-repeat expansion in C9ORF72 (Citation1–2). Repetitive sequences are often unstable which potentially can lead to changes in number of repeats between generations and during somatic cell division. Changes arising during somatic cell mitosis will result in populations of cells with varying expansion patterns, eg, one expansion size in neurons and another size in leucocytes. This could potentially give rise to problems regarding diagnosis, but might also explain some of the observed phenotypic differences among carriers of C9orf72-expansions.
Objectives: The aims of this study were 1) to determine whether the size of the GGGGCC-repeat expansion in C9ORF72 differs between different tissues in patients with ALS, FTD or ALS+ FTD and 2) to determine whether there are any correlations between repeat number and phenotype.
Methods: Using Southern blot we have analyzed a broad panel of tissues, consisting of autopsy material of both neuronal and non-neuronal origin with a focus on brain, spinal cord and muscle, from 17 individuals with ALS, PBP, FTD or ALS+ FTD, all with an expanded allele in blood. The number of repeats in each sample was determined and compared between different tissues within individuals. The expansion size pattern between individuals with different diagnoses was also compared.
Results: We see marked differences in expansion sizes between different tissues within patients, in some cases 1000–2000 repeats difference between tissues. No correlations between expansion size and disease phenotype were apparent. In line with previous findings (Citation3), we have found the expansion sizes in the cerebellum to be smaller than in other neuronal tissues and also observed patients which have a very small expansion size in blood, muscle and internal organs (approx. 60 repeats), but massive expansions in brain tissue.
Conclusions: There are differences in size of the C9ORF72 GGGGCC-repeat expansion between tissues of different origin. However, the expansion size pattern seems to be rather individual and not correlated to a specific disease phenotype.
References:
- DeJesus-Hernandez M. et al. Neuron 2011 Oct 20; 72(2):245–56.
- Renton AE. et al. Neuron 2011 Oct 20; 72(2):257–68.
- van Blitterswijk M. et al. Lancet Neurol. 2013 Oct; 12(10):978–88.
P169 SCREENING FOR C9ORF72 REPEAT EXPANSION IN AMYOTROPHIC LATERAL SCLEROSIS
Mosca L1
Tarlarini C1
Lunetta C2
Sansone V2
Penco S1
aaDepartment of Laboratory Medicine and Microbiology, Medical Genetics
abNEuroMuscular Omnicentre, Fondazione Serena Onlus, Niguarda Ca’ Granda Hospital, Milano, Italy
Email address for correspondence: [email protected]
Keywords: C9ORF72, genetics, molecular tests
Background: ALS is familial in 10–15% of cases (FALS), while for the majority of cases it is sporadic (SALS) (Citation1). To date more than 17 causative genes have been described; the most commonly mutated are: SOD1, FUS, TARDBP and C9ORF72. The C9ORF72 mutation is a polymorphic hexanucleotide (GGGGCC) repeat expansion located in intron 1 (Citation3); was and has been shown to be the most common genetic cause of FALS, FTD and ALS-FTD. The phenotype associated with the pathological expansion is extremely variable and, with few exceptions, the variant does not appear to be fully penetrant; this raises the possibility that the expansion may be a risk factor for disease and may not be capable of producing disease in isolation (Citation4).
Objectives: Determination of the prevalence of C9ORF72 repeat expansion in our cohort of 753 Italian ALS patients.
Methods: C9ORF72 expansion was analyzed by using the repeat primed PCR (RP-PCR) (Citation3) to detect repeat numbers of approximately maximum 60. The method is able to discriminate the repeat range detected in the normal population (0–20) from the higher mutated range (> 30). Genomic DNA extraction and PCR set-up have been performed on an automated Beckman Coulter Biomek NXP Workstation. Analysis was performed on an ABI 3730 DNA Analyzer (Applied Biosystems) and GeneMapper software (version 4.0, ABI). Positive subjects were defined in presence of a repeat number > 30 and/or the typical saw-tooth pattern with a 6-bp periodicity in RP-PCR.
Results: Our ALS cohort consisted in 51 FALS cases (6.8%) and 702 SALS patients (93.2%) (n = 753). FTD was present in 2 FALS (3.9%) and 16 SALS patients (2.3%). The C9ORF72 pathological expansion was identified in 38 ALS patients (5%). Interestingly, 2 of the C9ORF72 expansion carriers also presented a causative mutation in one of other ALS-associated genes (TARDBP and FUS, respectively).
Discussion and conclusion: C9ORF72 hexanucleotide repeat expansions have been found in 5% of all ALS cases and represent the commonest mutation in our population of Italian ancestry; these data are quite in agreement with previous reports (Citation5) (5% vs 6.7%) obtained in a prospectively ascertained, population-based epidemiologic series of cases identified through the Piemonte and Valle d’Aosta register for ALS (PARALS). Further efforts are needed to implement, in terms of intra- and inter-laboratory reproducibility, the method used for C9ORF72 analysis.
Acknowledgements: We thank SLAnciamoci Association and AISLA for supporting LM and part of the study.
References:
- Wijesekera LC, Leigh PN. Orphanet J Rare Dis. 2009; 4:3.
- Leblond CS. et al. Exp Neurol. 2014. pii: S0014–4886(14)00115-0.
- Renton AE. et al. Neuron 2011; 72:257–268.
- Cooper-Knock J, Shaw PJ, Kirby J. Acta Neuropathol. 2014; 127:333–345.
- Chiò A. et al. Neurology 2012; 79:1983–1989.
P170 IDENTIFICATION OF NEW GENETIC DETERMINANTS IN SPORADIC ALS
Couthouis J
Raphael A
Gitler A
Stanford University, Stanford, CA, USA
Email address for correspondence: [email protected]
Keywords: targeted sequencing, genetic determinants, yeast
Background: Most of the studies to identify causative genes in ALS have been carried out in familial ALS (fALS) and identified genes were then often confirmed to be mutated in sporadic ALS (sALS) cases as well (Citation1, Citation2). With very few large multi-generational ALS pedigrees available, new approaches are needed to expand the genetic landscape of ALS beyond fALS studies.
We previously performed a yeast screen of human RNA Recognition Motifs (RRMs) containing genes looking for candidates mimicking the behaviour of well-known ALS disease genes, TARDBP and FUS (Citation3, Citation4). We also previously used trios - families with an affected proband and two unaffected parents - to uncover de novo mutations that are only present in the affected proband (Citation5). These approaches allowed us to identify new ALS disease genes TAF15, EWSR1 and SS18L1/CREST and 62 other potential candidates.
Objectives: Here we wanted to take advantage of new high-throughput sequencing methods to study sporadic samples available to us to identify new ALS disease genes or variants.
Methods: We undertook a targeted sequencing approach of all exons in 169 known and candidate ALS disease genes in 242 sporadic ALS cases and 129 age-matched controls to try to identify novel variants linked to ALS.
Results: We sequenced all exons of 169 genes in 242 ALS patients and 129 age-matched controls from North America. We observed an overall enrichment in novel and rare variants in cases versus controls. Additionally, we identified new variants in known ALS disease genes, in genes associated with ALS, in genes identified through our previous RNA Recognition Motifs (Citation3,Citation4) and trios - families with an affected proband and two unaffected parents - studies (Citation5). While no single gene emerged as significantly enriched in our analysis, we did find several genes that trended towards more novel and rare variants in ALS patient samples versus controls.
Conclusion: We took advantage of affordable recently developed target sequencing methods to rapidly sequence a set of candidate genes in patients versus controls to ask if we could (Citation1) identify new mutations in known ALS disease genes, (Citation2) find further evidence that genes previously associated with ALS are indeed causative, (Citation3) find new mutations in candidates generated from our previously performed study.
References:
- Kabashi E. et al. Nat Genet. 2008; 40:572–574.
- van Es MA. et al. J Neurol Neurosurg Psychiatry. 2010; 81:562–566.
- Couthouis J. et al. Proc Natl Acad Sci U S A. 2011; 108:20881–20890.
- Couthouis J. et al. Hum Mol Genet. 2012; 21:2899–2911.
- Chesi A. et al. Nat Neurosci. 2013; 16:851–855.
P171 INVESTIGATING THE GENETIC BASIS OF AMYOTROPHIC LATERAL SCLEROSIS USING NEXT-GENERATION SEQUENCING TECHNIQUES
Fifita J1
Williams K1
Nicholson G1,2
Rowe D1
Blair I1
acAustralian School of Advanced Medicine, Macquarie University, Sydney, NSW, Australia
adMolecular Medicine Laboratory, Concord Hospital, Syndey, NSW, Australia
Email address for correspondence: [email protected]
Keywords: genetics, novel gene mutation, exome sequencing
Background: The majority of amyotrophic lateral sclerosis (ALS) cases occur sporadically, however 10% of cases show familial inheritance. ALS is genetically heterogeneous with mutations identified in over 15 genes. To date, known gene mutations account for about 60% of familial cases, and for about 5% of sporadic cases in Australian patient cohorts.
Objectives: We aim to identify novel gene mutations that cause familial ALS using exome sequencing, NGS bioinformatic pipelines and computer scripts, and high-throughput screening of control cohorts. Candidate sequence variants will be prioritised for functional studies using computational biology. Functional studies will use neuronal cell lines and zebrafish to examine the consequence of each candidate sequence variant.
Methods: A cohort of Australian ALS families (n = 201) has been recruited. An index patient from each family was screened for known ALS genes using both Sanger and exome sequencing. Three families negative for known ALS mutations were selected for gene discovery. Exome sequencing was used to search for novel ALS-linked sequence variants in these families. A custom bioinformatics pipeline was used to filter exome sequence variants and generate a list of candidate variants for each family. Variants were validated with Sanger sequencing. To prioritise variants, their functional effects will be predicted using programs such as MutationTaster, Polyphen, Pon-P2. Protein-protein interactions will be queried using DAPPLE and BioGRID.
Results: Analysis of known ALS genes has identified causative mutations in 62% of Australian familial ALS cases in our cohort (C9ORF72 39.3%, SOD1 15.4%, FUS 2.5%, TARDBP 2.0%, UBQLN2 1.0%, OPTN 0.5%, SS18L1 0.5%). Ten DNA samples from the three selected ALS families underwent exome capture and parallel sequencing. This identified approximately 250 000 variants in each of the three families. After initial filtering of the exome sequence data, 28, 37 and 24 candidate gene mutations remained in the three families. Validation, high-throughput genotyping of large numbers of Australian control samples and further filtering based on extended public SNP databases reduced candidate gene numbers to 13, 29 and 11(Citation5). Presently, data from protein predictions and gene function analysis is being collated to prioritise candidates for functional studies.
Discussion and conclusion: The genetic defects remain to be identified in around 38% of Australian familial ALS cases in our cohort. The identification of novel gene mutations and characterisation of their functional consequences will provide further insight into the genetic and pathological basis of motor neuron degeneration, providing new targets for diagnosis and therapeutic development. Furthermore, novel gene mutations will lead to the development of new in vitro and in vivo disease models, which may provide a platform for the full understanding of ALS pathogenesis.
P172 PROJECT MINE PHASE 1: IMPUTATION OF ALS GENOMES IN A GENOME WIDE ASSOCIATION STUDY
Consortium Mine
University Medical Center Utrecht, Utrecht, The Netherlands
Email address for correspondence: [email protected]
Keywords: GWAS, sequencing, imputation
Background: Genetic risk factors play an important role in ALS susceptibility. So far, genome wide association studies (GWAS) have identified 3 genetic risk factors (C9ORF72, UNC13A and a locus on 17q11.2). The C9ORF72 repeat expansion, being the most common cause of ALS, exhibits a clear founder effect and virtually all patients carrying the repeat expansion share a common haplotype at chromosome 9p21.2. Including 6,100 cases and 7,125 controls in the most recent GWAS meta-analysis, these studies are only moderately powered according to GWAS standards. To find new genetic risk loci in ALS we are conducting well powered GWAS and will improve imputation of ALS specific haplotypes by enriching our reference panel with high coverage whole genome sequencing data of 1,250 ALS patients.
Methods: Raw genotype data for all strata in previously published GWAS's in ALS were obtained. In addition 7,603 new patients and 3,811 new control subjects were genotyped on the Illumina OmniExpress array. Standard GWAS QC-measures for SNPs and individuals were performed per stratum and for all data combined. For the reference panel whole genome sequencing data at an average coverage of 40X was obtained for Dutch ALS patients and matched controls. After quality control this ALS-specific reference panel was phased using SHAPEIT2 software and was merged with publicly available reference panels (ie, 1000 Genomes, GoNL and UK10K). The GWAS strata will be imputed with the combined reference panel using IMPUTE2 and meta-analysed. To assess imputation accuracy masked whole genome sequencing data from Dutch familial ALS patients and trios were imputed using the different reference panels.
Results: In total 14,668 patients and 24,812 controls were included from 16 different nationalities. Final QC measures are currently assessed and the data will be prepared for imputation. When assessing the imputation accuracy, our ALS-specific reference panels is superior to 1000 Genomes and GoNL. Even for very rare alleles (< 0.5%) that are notoriously hard to impute concordance between imputed and sequenced genotypes of 74.6% is achieved using the ALS-genomes as reference panel compared to 69.6% and 47.1% for GoNL and 1000 Genomes respectively. When combining all reference panels the performance can only be improved by allowing a large number of haplotypes for imputation (78.7% when using 5000 haplotypes). Results on the association analysis and therefore possible new risk loci will be presented if available.
P173 AN UPDATE ON THE ALS ONLINE GENETICS DATABASE, ALSOD
Abel O1
Powell JF3
Andersen P2
Al-Chalabi A1
aeDepartment of Clinical Neuroscience Institute of Psychiatry, London, UK
afDepartment of Pharmacology and Clinical Neuroscience, Section for Neurology, Umeå University, Umea, Sweden
agDepartment of Neuroscience, King’s College London, London, UK
Email address for correspondence: [email protected]
Keywords: genetics, bioinformatics, ALSoD
Amyotrophic lateral sclerosis, also known as Lou Gehrig's disease, typically leads to death within 3–5 years of symptom onset. Understanding what causes ALS is an obstacle, but more research in this area, enhanced by advanced technology like high-throughput and next generation sequencing, is paving the way for better information and direction. The volume of data generated by genetics researchers has dramatically increased, largely because of increased opportunities for collaboration. ALSoD, a widely used online genetics database for collating, analysing and integrating ALS data, has been updated, with analytics tools able to portray the data graphically to users.
Mutations and other gene variants have been mapped to genomic coordinates, and the inclusion of dbSNP IDs has been implemented to facilitate the integration of data from numerous public sources. To increase the usability and functionality of ALSoD, population frequency of each variant found in the 1000 genome and EVS databases is displayed. To contribute to a better understanding of the pathogenesis of ALS, links to information on animal models are also available.
The database can now be viewed on mobile devices and for Android platforms, a mobile app is available. A more attractive genetic website can be built without extra expenses ie, changing the whole programming platform. A page to calculate the penetrance of the disease in a family is also included in the new version. Using the pdb ID, a user can interact with the molecular structures of mutations on the corresponding webpages.
P174 TWO ALS CASES CARRYING A NOVEL P.Q121G MISSENSE MUTATION IN EXON 5 OF SOD1 GENE
Tateishi T1,2
Hayashi S2
Iinuma K2
Murai H2
Kira J-I2
ahDepartment of Neurology, Iizuka Hospital, Iizuka, Japan
aiDepartment of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
Email address for correspondence: [email protected]
Keywords: SOD1, Q121G, mutation
Background: Genetically, 20% of FALS cases carry missense mutations in the SOD1 gene. 177 mutations have been reported to date.
Objectives: To describe two cases with a novel Q121G missense mutation in the SOD1 gene.
Methods: We carried out clinical and genetic studies on two Japanese patients with the SOD1 gene (Q121G) mutation.
Case reports: Patient 1 suffered from weakness of the right leg at age 69. Over the following year, the weakness spread to his left lower limb, leading to gait disturbance. He was admitted to our hospital at the age of 70. He was born the eighth of nine children, three of whom suffered from ALS in adult life. Neurological examination on admission revealed a proximal dominant moderate weakness in the bilateral lower limb muscles. Deep tendon reflexes were exaggerated in the upper limbs, and attenuated or lost in the lower limbs. The planter response was extensor on both sides. Vibration sense was moderately decreased in the lower extremities and urinary disturbance was noted. Muscle atrophy of tongue, dysphagia and dysarthria were not observed. EMG showed chronic neurogenic patterns, including giant motor unit potentials in the four limbs. Two years after onset, dysphagia appeared, and non-invasive ventilation was initiated because of decreased vital capacity. Five years after the onset, the respiratory disturbance became worse, resulting in death at 74 years of age.
Patient 2 noticed muscle weakness in his left foot at 66 years of age, which progressively deteriorated during the following two years. He developed urinary disturbance three years after the onset, and was admitted to our hospital. He has no family history of ALS. At admission, neurological examination demonstrated muscle weakness, fasciculation and atrophy of the bilateral lower limb, hyperreflexia in both upper limbs, and hyperreflexia in both lower limbs. The plantar response was flexor. Vibration sense was moderately decreased in the lower extremities and urinary disturbance was observed. Muscle atrophy of tongue, dysphagia and dysarthria were not observed. EMG showed acute neurogenic patterns in left upper limb and acute and chronic neurogenic patterns in left lower limb. The gait disturbance progressed, and the patient was unable to walk unaided four years after onset.
Molecular analysis showed the heterozygous (patient 1) and homozygous (patient 2) missense mutation c.362 A> G; p.Q121G was found in exon 5 of SOD1 gene.
Discussion and conclusion: To our knowledge, no other missense mutation of codon 121 of SOD1 has been found. These cases showed moderately decreased vibration sense in the lower extremities and urinary disturbance at an earlier stage of the disease. These symptoms may be a characteristic feature of this mutation, but we need further data to establish a genotype – phenotype correlation.
P175 DELETERIOUS VARIATIONS IN THE ESSENTIAL MRNA METABOLISM FACTOR, HGLE1, IN ALS PATIENTS
Kaneb HM1
Folkmann AW2
Belzil VV3
Jao L-E2
Leblond CS1
Girard SL3
Daoud H3
Noreau A3
Rochefort D1
Hince P1
Levert A1
Vidal S3
André-Guimont C3
Camu W4
Bouchard J-P5
Dupré N5
Rouleau GA1
Wente SR2
Dion PA1,3
ajMcGill University, Montreal, Quebec, Canada
akVanderbilt University School of Medicine, Nashville, Tennessee, USA
alUniversité de Montréal, Montreal, Quebec, Canada
amUnité de Neurologie Comportementale et Dégénérative, Institute of Biology, Montpellier, France
anLaval University, Centre Hopitalier Universitaire de Québec, Quebec City, Quebec, Canada
Email address for correspondence: [email protected]
Keywords: Gle1, RNA processing, genetics
Background: Causative mutations in the global RNA processing proteins TDP-43 and FUS (amongst others) as well as their aggregation in ALS patients have identified defects in RNA metabolism as an important feature of this disease. Lethal congenital contracture syndrome 1 (LCCS1) and lethal arthrogryposis with anterior horn cell disease (LAAHD) are autosomal recessive fetal motor neuron diseases that are caused by mutations in another global RNA-processing protein, hGle1(Citation1).
Objectives: To test the hypothesis that mutations in hGle1 are causative for ALS as well as for LCCS1/LAAHD
Methods: 173 unrelated familial ALS cases (FALS), 760 sporadic ALS (SALS) cases and 190 matched controls, of European descent, were screened for mutations in GLE1 via Sanger sequencing. Exons in which variants were identified were sequenced via the Sanger method in 285 further controls. All variants identified were then investigated in an independent whole exome sequencing dataset of 485 matched controls with no known neurodegenerative disorders.
Results: Although no excess of GLE1 variations was identified in ALS patients over controls we observed a striking absence of deleterious variations in controls and conversely identified two deleterious variations in ALS patients (one splice site (FALS) and one nonsense (SALS) variation). Functional analyses of these deleterious variations revealed them to be unable to rescue motor neuron pathology in zebrafish morphants lacking Gle1. Furthermore, in HeLa cells both mutations caused a depletion of hGle1 at the nuclear pore where it carries out an essential role in nuclear export of mRNA as well as reduced overall levels of hGle1 in the cytoplasm.
Discussion and conclusion: These results suggest a haploinsufficiency mechanism and highlight GLE1 as a strong candidate for a causative gene for ALS. This further supports the involvement of global defects in RNA metabolism in this disease.
Acknowledgements: This study was generously supported by the ALS division of the Muscular Dystrophy Association, the US ALS Association, the Canadian Institutes of Health Research, the National Institutes of Health and the March of Dimes. We would like to thank the patients involved as well as Annie Raymond, Pascale Thibodeau, Annie Levert, Anne Desjarlais and Pierre Provencher for technical support, sample collection and organization.
Reference
- Nousiainen HO. et al. Nat Genet. 2008; 40:155–7.
P176 CHARACTERING NOVEL ALTERNATIVELY SPLICED ISOFORMS OF THE EPHA4 GENE
Mangelsdorf M1
Zhao J1
Narayanan RK1
Butler TJ1
Henderson RD2
Mccombe PA2
Bartlett PF1
aoQueensland Brain Institute, The University of Queensland, Queensland, Australia
apDepartment of Neurology, Royal Brisbane and Women’s Hospital, Queensland, Australia
Email address for correspondence: [email protected]
Keywords: EPHA4, modifier, TDP-43
Background: Alternative splicing is a common and essential phenomenon in human genes, and disruption of the process is associated with human genetic disease. There are currently > 20 genes in which mutations cause amyotrophic lateral sclerosis (ALS). A growing number of these play a role in RNA metabolism, including pre-mRNA splicing. We have identified EPHA4 as one gene whose pre-mRNA splicing is regulated by TDP-43.
Objectives: Given that EPHA4 has been implicated as a modifier of ALS and is regulated by TDP-43 we aimed to characterise alternative isoforms of EPHA4 and determine the role they play in ALS.
Methods and results: Analysis of the expressed sequence tags from the UniGene clusters of both mouse and human EPHA4 revealed the presence of possible novel isoforms. Reverse-transcriptase PCR validated the presence of two of the novel isoforms in both human and mouse brain and spinal cord. To determine if these isoforms encode protein we have performed Western blot hybridization using EPHA4 antibodies and have identified proteins consistent with the novel isoforms in mouse tissue. Expression of the novel isoforms in HEK293T cells has shown a similar localisation to the plasma membrane as the full length EPHA4 for one isoform, however the second shows a novel diffuse cytoplasmic localisation.
Discussion and conclusion: We have identified novel coding mRNA isoforms transcribed from the EPHA4 gene. We are now assessing the effect of the new isoforms on EPHA4 activation. Investigating the functional significance of these novel isoforms will be important to fully understand the contribution of EPHA4 to disease progression in ALS, and thus which may be a targets for disease therapy.
P177 SAITOHIN GENE RS62063857 VARIANT IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
Sonmez E1
Sazci A1
Ozel MD1
Idrisoglu HA2
aqUniversity of Kocaeli, Faculty of Medicine, Department of Medical Biology and Genetics, Kocaeli, Turkey
arUniversity of Istanbul, Istanbul Faculty of Medicine, Department of Neurology, Istanbul, Turkey
Email address for correspondence: [email protected]
Keywords: sALS, saitohin (STH), rs62063857
Background: Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease (MND), affecting either upper or lower motor neurons. Generally patients experience signs and symptoms of progressive muscle atrophy and weakness, problems with swallowing, leading to respiratory failure and death. Only 5–10% of patients survive more than 10 years and median survival is 3–5 years from onset (Citation1).
Objects: The aim of the study was to determine whether the saitohin (STH) gene rs62063857 variant is associated with sporadic amyotrophic lateral sclerosis (sALS).
Methods: We had 597 sporadic ALS patients and 423 controls to genotype. Genomic DNA was isolated from whole blood and a polymerase chain reaction and restriction fragment length polymorphism procedure was used to genotype the STH gene rs62063857 variant (Citation2). The amplified fragment was digested HifI restriction endonuclease and run on an 8% polyacrylimide gel followed by silver staining. SPSS statistical analysis was used in the calculation of genotype and allele frequencies and association.
Results: The satiohin gene rs62063857 variant was not associated with SALS (χ2 = 0.063, P = 0.969). The AA, AG and GG genotype frequencies in sALS were 61.5, 34.0 and 4.5% in cases and 60.8, 34.5 and 4.7% in controls respectively. The A and G allele frequencies were 78.48 and 21.52% in cases and 78.0 and 22.0% in the controls respectively. There was no association on gender (χ2 = 0.345, P = 0.842 in male and χ2 = 1.113, p = 0.573 in female).
Discussion and conclusion: Although ALS is a rare disease with a prevalence of 1–2 in 100,000 worldwide, it is a devastating disorder. In our earlier studies, we found that STH rs62063857 variant was associated with dementia (Citation3) and Parkinson's disease. In this particular study, we did not find any association between the STH gene rs62063857 variant and SALS. In this study, the hypothesis was that STH gene rs62063857 variant could be used as a biomarker to differentiate SALS from Dementia and Parkinson's Disease.
Acknowledgements: This study was supported by the Kocaeli University research fund to AS.
References:
- Leblond CS. et al. Exp Neurol. 2014; S0014-4886(14)00115-0.
- Conrad C. et al. Proc Natl Acad Sci U S A. 2002; 99(11):7751–7756.
- Sonmez E. et al. Neurobiol aging. 2014; 35: 718–719.
P178 CX3CR1 IS A MODIFYING GENE OF SURVIVAL AND PROGRESSION IN AMYOTROPHIC LATERAL SCLEROSIS
Salvado M1
Lopez-Lopez A2
Syriani E1
Morales M1
Rodríguez MJ2
Cazorla S1
Mahy N2
Vidal-Taboada JM2
Gamez J1
asALS Unit, Neuromuscular Disorders Clinic, VHIR. UAB, Barcelona, Spain
atBiochemistry and Molecular Biology Unit, Department of Physiological Sciences I, Faculty of Medicine - IDIBAPS, University of Barcelona, Barcelona, Spain
Email address for correspondence: [email protected]
Keywords: CX3CR1, influencing prognostic factors, modifying gene
Background: The reason why some ALS patients deteriorate much faster or survive much longer than others is unknown. An important challenge for ALS research is to determine how endogenous factors modify the disease to account for these different disease courses. The discovery of new biomarkers associated with different rates of progression and survival could provide new insights into the pathophysiological determinants of disease progression. Microglia activation and the crosstalk between immune cells appear to play a significant role in neuronal death. Microglial activation may modify disease progression and symptoms, and therefore disease outcome.
Objective: To investigate the association of functional variants of the human CX3CR1 gene (Fractalkine receptor) with the risk of ALS, the survival and the progression rate of the disease symptoms in a Spanish ALS cohort.
Methods: 187 ALS patients (142 sALS and 45 fALS) and 378 controls were recruited. We investigated CX3CR1 V249I (rs3732379) and T280M (rs3732378) genotypes and their haplotypes as predictors of survival, the progression rate of the symptoms (as measured by ALSFRS-R and FVC decline) and the risk of suffering ALS disease.
Results: The sALS patients with CX3CR1 249I/I or 249V/I genotypes presented a shorter survival time (42.2764.90) than patients with 249V/V genotype (67.6567.42; diff 225.49 months 95%CI (242.79, 28.18); p = 0.004; adj-p = 0.018). The survival time was shorter in sALS patients with spinal topography and CX3CR1 249I alleles (diff = 229.78 months; 95%CI (249.42, 210.14); p = 0.003). The same effects were also observed in the spinal sALS patients with 249I–280M haplotype (diff = 227.02 months; 95%CI (249.57, 24.48); p = 0.019). In the sALS group, the CX3CR1 249I variant was associated with a faster progression of the disease symptoms (OR = 2.58; 95%CI (1.32, 5.07); p = 0.006; adj-p = 0.027).
Discussion and conclusion: The progression rate of the disease symptoms and the survival time is affected in ALS patients with one or two copies of the CX3CR1 249I allele. The association evidenced herein is clinically relevant and indicates that CX3CR1 could be a disease-modifying gene in sALS.
Conclusion: The CX3CR1 is the most potent ALS survival genetic factor reported to date. These results reinforce the role of the immune system in ALS pathogenesis.
Acknowledgements: This work was supported by a Marató de TV3 grant. JG was supported by Spanish Fondo de Investigaciones Sanitarias grants (PI10-01070-FEDER and FIS PI13-01272). JVT, NM and MJR were supported by grants SAF2008-01902 and IPT-010000-2010-35 from the Spanish Ministerio de Ciencia e Innovación (Micinn), and by 2009SGR1380 grant from the Government of Catalonia. MM and ES were supported by grant BFU2010-17537 from Micinn.
P179 INVOLVEMENT OF HOMEOBOX GENES IN C9ORF72-RELATED DISEASES
Van Blitterswijk M1
Wang X2
Asmann Y2
Baker M1
Brown P1
Josephs K3
Parisi J3
Knopman D3
Petersen R3
Boeve B3
Graff-Radford N4
Boylan K4
Rademakers R1
auDepartment of Neuroscience
avDepartment of Health Sciences Research
awDepartment of Neurology
axDepartment of Neurology, Mayo Clinic Jacksonville, FL, USA
Email address for correspondence: [email protected]
Keywords: C9ORF72, homeobox genes
Background: A GGGGCC-repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) is the most common genetic cause of frontotemporal dementia (FTD) and motor neuron disease (MND). Although previous studies have revealed reduced expression of C9ORF72 and the formation of RNA foci and dipeptide-repeat proteins, it is unclear how this eventually leads to neurodegeneration.
Objective: To elucidate the mechanisms underlying C9ORF72-related diseases we investigated the differential expression, alternative splicing and methylation profiles of individuals with C9ORF72 repeat expansions.
Methods: Thirty-two C9ORF72 expansion carriers were investigated (12 FTD, 10 FTD/MND and 10 MND) as well as 30 disease controls (10 FTD, 10 FTD/MND and 10 MND) and 20 controls without neurological disorders. The Whole-Genome DASL HT Assay was used to assess expression profiles, separately for the frontal cortex and cerebellum. Differentially expressed genes were identified using the lumi R package and analyzed with enrichment and network modules (MetaCore). To further investigate the frontal cortex we performed RNA-Seq analysis for 8 C9ORF72 expansion carriers (5 FTD and 3 FTD/MND) and 8 disease controls (6 FTD and 2 FTD/MND), including differential expression (DESeq2) and alternative splicing (multivariate analysis of transcript splicing (MATS)) analyses. We also studied genome-wide methylation profiles in the cerebellum of 15 C9ORF72 expansion carriers (5 FTD, 5 FTD/MND and 5 MND) in addition to 15 disease controls (5 FTD, 5 FTD/MND and 5 MND) and 15 controls; the Infinium Human Methylation450 BeadChip was used and data was analyzed with the lumi R package.
Results: In the cerebellum, we identified 40 differentially expressed genes that remained significant after FDR correction when comparing C9ORF72 expansion carriers to disease controls (eg, homeobox genes). Importantly, we observed enrichment for gene ontology (GO) processes involved in development (eg, organ morphogenesis and skeletal system development (FDR < 5E-09)). In the frontal cortex, only three genes were differentially expressed after correction for multiple testing, including homeobox genes and C9ORF72. All findings were comparable when focusing on disease subgroups, and when comparing cases to controls without neurological diseases; the upregulation of homeobox genes was validated with quantitative real-time PCR. To further investigate our findings, we also examined alternative splicing and methylation profiles, which provided additional evidence for the essential role of developmental processes (eg, 460 skipped exon events; enrichment for nervous system development and neuron projection morphogenesis (FDR < 5E-09)).
Discussion and conclusion: We are the first to describe involvement of homeobox genes in C9ORF72-related diseases, as supported by DASL arrays, RNA-Seq, and Methylation450 BeadChips. Based on our results, we speculate that homeobox genes might be a key player in the degeneration of neurons observed in C9ORF72 expansion carriers. Our findings, therefore, increase our understanding of C9ORF72-related diseases and reveal interesting targets for novel treatment strategies.
P180 A MITOCHONDRIAL ORIGIN FOR FRONTOTEMPORAL DEMENTIA AND AMYOTROPHIC LATERAL SCLEROSIS THROUGH CHCHD10 INVOLVEMENT
Bannwarth S1,2
Ait-El-Mkadem S1,2
Chaussenot A1,2
Genin EC.1
Lacas-Gervais S3
Fragaki K1,2
Berg-Alonso L1
Kageyama Y4
Serre V5
Moore D6
Verschueren A7
Rouzier C1,2
Le Ber I8,9
Lespinasse F1
N’Guyen K10
Brice AS8
Yu-Wai-Man P6,11
Sesaki H4
Pouget J7
Paquis-Flucklinger V1,2
ayIRCAN, UMR CNRS 7284/INSERM U1081/UNS, School of Medicine
azDepartment of Medical Genetics, National Centre for Mitochondrial Diseases
baJoint Center for Applied Electron Microscopy, Nice Sophia-Antipolis University, Nice, France
bbDepartment of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, USA
bcUMR7592 CNRS, Jacques Monod Institute, Paris Diderot University, Paris, France
bdWellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, International Centre for Life, Newcastle University, Newcastle, UK
beDepartment of Neurology, Timone Hospital, Marseille, France
bfSorbonne Université, UPMC Univ Paris 06, UM75, Inserm U1127, Cnrs UMR7225, Nice, France
bgNational Reference Centre on Rare Dementias, AP-HP, Groupe Hospitalier Pitié-Salpêtrière, Paris, France
bhDepartment of Medical Genetics, Timone Hospital, Marseille Teaching Hospital, Marseille, France
biNewcastle Eye Centre, Royal Victoria Infirmary, Newcastle upon Tyne, Newcastle, France
Email address for correspondence: [email protected]
Keywords: FTD-ALS, CHCHD10, mitochondrial DNA instability
Background: Mitochondrial DNA (mtDNA) instability disorders are responsible for a large clinical spectrum, among which amyotrophic lateral sclerosis-like symptoms and frontotemporal dementia are extremely rare.
Objectives: We report a large family with a late-onset phenotype including motor neuron disease, cognitive decline looking like frontotemporal dementia, cerebellar ataxia and myopathy.
Results: In all patients, muscle biopsy showed ragged-red and COX negative fibres with combined respiratory chain deficiency and abnormal assembly of complex V. The multiple mtDNA deletions found in skeletal muscle revealed a mtDNA instability disorder. Patient fibroblasts present with respiratory chain deficiency, mitochondrial ultrastructural alterations and fragmentation of the mitochondrial network. Interestingly, expression of matrix-targeted photoactivable GFP showed that mitochondrial fusion was not inhibited in patient fibroblasts. By whole-exome sequencing (WES), we identified a missense mutation (c.176C> T; p.Ser59Leu) in the CHCHD10 gene that encodes a coiled-coil helix protein, whose function is unknown. We show that CHCHD10 is a mitochondrial protein located in the intermembrane space and enriched at cristae junctions. Overexpression of CHCHD10 mutant allele in HeLa cells led to fragmentation of the mitochondrial network and ultrastructural major abnormalities including loss, disorganization and dilatation of cristae. The observation of a frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) phenotype in a mitochondrial disease led us to analyse CHCHD10 in a cohort of 21 families with pathologically proven FTD-ALS. We identified the same missense p.Ser59Leu mutation in one of these FTD-ALS families.
Discussion and conclusion: This work opens a novel field to explore the pathogenesis of FTD-ALS clinical spectrum by showing that mitochondrial disease may be at the origin of some of these phenotypes.
Reference:
- Bannwarth S. et al. Brain, in press.