Mutations in the Cu/Zn superoxide dismutase (Sod1) gene have been reported to cause adult-onset autosomal dominant Amyotrophic Lateral Sclerosis (FALS). In sporadic cases (SALS) de novo mutations in the Sod1 gene have occasionally been observed. The recent finding of a mutation in the VAMP/synaptobrevin-associated membrane protein B (VAPB) gene as the cause of amyotrophic lateral sclerosis (ALS8), prompted us to investigate the entire coding region of this gene in SALS patients. One hundred twenty-five unrelated patients with adult-onset ALS and 150 healthy sex-age-matched subjects with the same genetic background were analyzed.
Genetic analysis for all exons of the VAPB gene by DHPLC revealed 5 variant profiles in 83 out of 125 SALS patients. Direct sequencing of these PCR products revealed 3 nucleotide substitutions. Two of these were found within intron 3 of the gene, harbouring 4 variant DHPLC profiles. The third nucleotide variation (Asp130Glu) was the only substitution present in the coding region of the VAPB gene, and it occurred within exon 4. It was found in three patients out of 125. The frequency of the detected exon variation in the VAPB gene was not significantly different between patients and controls. In conclusion, our study suggests that VAPB mutations are not a common cause of adult-onset SALS.
Amyotrophic Lateral Sclerosis (ALS) is a progressive lethal disorder of motor neurons of the spinal cord and brain. More than 90% of ALS patients are sporadic (SALS), not showing any familial trait. Approximately 10% of cases are familial (FALS), and within these lie several distinct forms of the disease. The most common form of FALS is autosomal dominant for which to date 2 genes have been identified: ALS1, with adult-onset caused by mutations in the gene encoding the cytosolic antioxidant enzyme Cu, Zn-superoxide dismutase (Sod1) [1, 2]; and ALS4, a rare juvenile-onset disorder, associated with mutations of the senataxin gene (SETX) . An autosomal recessive, juvenile-onset form (ALS2), has been associated with mutations of the Alsin gene located at 2q33 . To date over 100 different missense mutations in the Sod1 gene  and up to eight described mutations in the Alsin gene have been reported .
By contrast, sporadic ALS is believed to be a multi-factorial disease in which modifying genes and environmental agents affect its clinical manifestation, but few associated genes have so far been identified [7–13]. De novo mutations in the Sod1 gene have only occasionally been observed in sporadic cases of ALS suggesting that mutations in this gene are a rare cause of non familial forms of ALS [14, 15].
The finding by Nishimura et al. of a missense mutation in the VAMP/synaptobrevin-associated membrane protein B (VAPB) gene in autosomal dominant motoneuron diseases (ALS8), prompted us to investigate the entire coding region of this gene in 125 SALS patients.
Following informed consent, blood was taken from 125 unrelated Caucasian patients from Southern Italy, who fulfilled the El Escorial criteria  for ALS. Samples were also taken from 150 controls, matched for age, sex and geographic region. Clinical information is provided in Table 1. Lymphocyte DNA was extracted from blood samples using standard procedure. Sod1 mutation-negative patients were screened using primer flanking the intron-exon regions of the VAPB gene. The PCR products were analyzed by Denaturing High Performance Liquid Chromatography (DHPLC) on a WAVE Nucleic Acid Fragment Analysis System (Transgenomic Inc., Mountain View, CA). Representative samples with abnormal profile in DHPLC were sequenced by the ABI Prism BigDye Terminator cycle sequencing ready reaction kit (Perkin Elmer- Applied Biosystems Inc., Foster City, CA).
Clinical information of the studied populations
Age at Onseta (years)
58.9 (41–88, SD 10.5)
59.9 (40–78, SD 12.2)
a Mean age of onset (with lowest/highest and Standard Deviation values in parentheses);
b SALS: Sporadic Amyotrophic Lateral Sclerosis
DHPLC analysis of the VAPB gene [GenBank accession no. AL035455] detected five variant profiles in 83 out of 125 SALS patients. Direct sequencing of PCR products revealed 3 nucleotide substitutions. Two nucleotide substitutions, g.134585C>T and g.134688A>G were found within intron 3 of the gene, harbouring 4 variant DHPLC profiles, because of their combination in homozygous or heterozygous states. These polymorphisms were already present in the SNP database as rs 2234487 and rs 2234488, respectively . The frequencies in our SALS patients and in control population were 65% vs 64% for the rs 2234487 and 42% vs 45% for the rs 2234488, respectively.
The third nucleotide substitution was the only substitution present in the coding region of the VAPB gene and it occurred within exon 4 at nucleotide g.138864T>G (Asp130Glu).
The Asp130Glu variation is located between MSP and Coiled-coil domains, and involves an aminoacid residue of aspartic acid not conserved among different species. We identified this variation in heterozygous state in three out of 125 SALS patients (2.0%) and in three of 150 (2.4%) sex-age-matched healthy subjects from the same geographical area, suggesting that this aminoacidic substitution is not causative of the disease.
The data presented herein suggest that VAPB mutations are not a common cause of adult-onset SALS in Italian population. Further, the novel nucleotide variation identified in exon 4 of VAPB gene represents a polymorphism, probably linked to a restrict geographic area.
Institute of Neurological Sciences, National Research Council
Second Division of Neurology, Second University of Naples
Department of Neurology and Psychiatry, University of Palermo
Department of Neurological and Psychiatric Sciences, University of Bari
Department of Neurosciences, Psychiatric and Anaesthesiological Sciences, University of Messina
Institute of Neurology, University Magna Graecia
Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX, Rahmani Z, Krizus A, McKenna-Yasec D, Cayabyab A, Gaston SM, Berger R, Tanzi RE, Halperin JJ, Herzfeldt B, Ven den Bergh R, Hung WY, Bird T, Deng G, Mulder DW, Smyth C, Laing NG, Soriano E, Pericak-Vance MA, Haines J, Rouleau GA, Gusella JS, Horvitz HR, Brown RH: Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993, 362: 59-62. 10.1038/362059a0.View ArticlePubMed
Cudkowicz ME, McKenna-Yasek D, Sapp PE, Chin W, Geller B, Hayden DL, Schoenfeld DA, Hosler BA, Horvitz HR, Brown RH: Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann Neurol. 1997, 41: 210-221. 10.1002/ana.410410212.View ArticlePubMed
Chen YZ, Bennett CL, Huynh HM, Blair IP, Puls I, Irobi J, Dierick I, Abel A, Kennerson ML, Rabin BA, Nicholson GA, Auer-Grumbach M, Wagner K, De Jonghe P, Griffin JW, Fischbeck KH, Timmerman V, Cornblath DR, Chance PF: Chance, DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am J Hum Genet. 2004, 74: 1128-1135. 10.1086/421054.PubMed CentralView ArticlePubMed
Yang Y, Hentati A, Deng HX, Dabbagh O, Sasaki T, Hirano M, Hung WY, Ouahchi K, Yan J, Azim AC, Cole N, Gascon G, Yagmour A, Ben-Hamida M, Pericak-Vance M, Hentati F, Siddique T: The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat Genet. 2001, 29: 160-165. 10.1038/ng1001-160.View ArticlePubMed
Hand CK, Devon RS, Gros-Louis F, Rochefort D, Khoris J, Meininger V, Bouchard JP, Camu W, Hayden MR, Rouleau GA: Mutation screening of the ALS2 gene in sporadic and familial amyotrophic lateral sclerosis. Arch Neurol. 2003, 60: 1768-1771. 10.1001/archneur.60.12.1768.View ArticlePubMed
Figlewicz DA, Orrell RW: The genetics of motor neuron diseases. Amyotroph Lateral Scler Other Motor Neuron Disord. 2003, 4: 225-231. 10.1080/14660820310011287.View ArticlePubMed
Veldink JH, van den Berg LH, Cobben JM, Stulp RP, De Jong JM, Vogels OJ, Baas F, Wokke JH, Scheffer H: Homozygous deletion of the survival motor neuron 2 gene is a prognostic factor in sporadic ALS. Neurology. 2001, 56: 749-752.View ArticlePubMed
Rouleau GA, Clark AW, Rooke K, Pramatarova A, Krizus A, Suchowersky O, Julien JP, Figlewicz D: SOD1 mutations associated with accumulation of neurofilaments in amyotrophic lateral sclerosis. Ann Neurol. 1996, 39: 128-131. 10.1002/ana.410390119.View ArticlePubMed
Lambrechts D, Storkebaum E, Morimoto M, Del-Favero J, Desmet F, Marklund SL, Wyns S, Thijs V, Andersson J, van Marion I, Al-Chalabi A, Bornes S, Musson R, Hansen V, Beckman L, Adolfsson R, Pall HS, Prats H, Vermeire S, Rutgeerts P, Katayama S, Awata T, Leigh N, Lang-Lazdunski L, Dewerchin M, Shaw C, Moons L, Vlietinck R, Morrison KE, Robberecht W, Van Broeckhoven C, Collen D, Andersen PM, Carmeliet P: VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat Genet. 2003, 34: 383-394. 10.1038/ng1211.View ArticlePubMed
Kawahara Y, Ito K, Sun H, Aizawa H, Kanazawa I, Kwak S: RNA editing and the death of motor neurons: there is a glutamate-receptor defect in patients with amyotrophic lateral sclerosis. Nature. 2004, 427-801.
Drory VE, Birnbaum M, Korczyn AD, Chapman J: Association of APOE ε4 allele with survival in amyotrophic lateral sclerosis. J Neurol Sci. 2001, 190: 17-20. 10.1016/S0022-510X(01)00569-X.View ArticlePubMed
Kamel F, Umbach DM, Lehman TA, Park LP, Munsat TL, Shefner JM, Sandler DP, Hu H, Taylor JA: Amyotrophic lateral sclerosis, lead, and genetic susceptibility: polymorphisms in the delta-aminolevulinic acid dehydratase and vitamin D receptor genes. Environ Health Perspect . 2003, 111: 1335-1339.PubMed CentralView ArticlePubMed
Gellera C, Castellotti B, Riggio MC, Silani V, Morandi L, Testa D, Casali C, Taroni F, Di Donato S, Zeviani M, Mariotti C: Superoxide dismutase gene mutations in Italian patients with familial and sporadic amyotrophic lateral sclerosis: identification of three novel missense mutations. Neuromuscul Disord. 2001, 11: 404-410. 10.1016/S0960-8966(00)00215-7.View ArticlePubMed
Battistini S, Giannini F, Greco G, Bibbo G, Ferrera L, Marini V, Causarano R, Casula M, Lando G, Patrosso MC, Caponnetto C, Origone P, Marocchi A, Del Corona A, Siciliano G, Carrera P, Mascia V, Giagheddu M, Carcassi C, Orru S, Garre C: Penco SOD1 mutations in amyotrophic lateral sclerosis. Results from a multicenter Italian study. J Neurol. 2005, 252: 782-788. 10.1007/s00415-005-0742-y.View ArticlePubMed
Nishimura AL, Mitne-Neto M, Silva HC, Richieri-Costa A, Middleton S, Cascio D, Kok F, Oliveira JR, Gillingwater T, Webb J, Skehel P, Zatz M: A mutation in the vesicle-trafficking protein VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral sclerosis. Am J Hum Genet. 2004, 75: 822-831. 10.1086/425287.PubMed CentralView ArticlePubMed
Brooks BR, Miller RG, Swash M, Munsat TL: World Federation of Neurology Research Group on Motor Neuron Diseases. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord. 2000, 1: 293-299. 10.1080/146608200300079536.View ArticlePubMed
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