A novel peripherin gene (PRPH) mutation identified in one sporadic amyotrophic lateral sclerosis patient

Neurobiology of Aging 32 (2011) 552.e1–552.e6 www.elsevier.com/locate/neuaging

A novel peripherin gene (PRPH) mutation identified in one sporadic amyotrophic lateral sclerosis patient Lucia Corradoa,*1, Yari Carlomagnoa,1, Luca Falascoa, Simona Mellonea, Michela Godia, Emanuela Covab, Cristina Ceredab, Lucia Testac, Letizia Mazzinic, Sandra D’Alfonsoa a b

Department Medical Sciences, Interdisciplinary Research Center of Autoimmune Diseases (IRCAD), University of Eastern Piedmont, Novara, Italy Laboratory of Experimental Neurobiology, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), Neurological Institute “C. Mondino”, Pavia, Italy c Department of Neurology, “A. Avogadro” University, Maggiore della Carita Hospital, Novara, Italy Received 9 September 2009; received in revised form 7 January 2010; accepted 16 February 2010

Abstract Motor neurons in amyotrophic lateral sclerosis (ALS) are characterized by the presence of inclusion bodies composed of intermediate filament (IF) proteins. Peripherin protein is as components of these inclusions and rare mutations in peripherin gene (PRPH) were identified in sporadic ALS cases. The aim of this study was to further define the spectrum of PRPH mutations in a cohort of 122 Italian ALS patients. We screened the coding sequence, the exon/intron boundaries, and the 5=–3= un-translated regions (UTRs) in 122 ALS patients. Eighteen sequence variations were detected. Seven variants were not identified in a panel of at least 245 matched controls, including 2 missense variations, namely p.R133P and p.D141Y, each identified in one heterozygous patient. p.R133P was newly identified whereas p.D141Y was previously described in one homozygous sporadic ALS patient. These 2 variants were predicted to have a deleterious effect on protein structure or function. This work contributes to determine the role of PRPH gene variants in ALS. Further studies are necessary to define the mechanisms through which the mutant peripherin could cause ALS phenotype. © 2011 Elsevier Inc. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Mutation; Peripherin; PRPH gene; Sporadic

1. Introduction Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease that affects motor neurons of the brain stem and spinal cord, resulting in paralysis and death within 2–5 years. Although most cases of ALS are sporadic (SALS), some families demonstrate a clinically indistinguishable form of ALS with clear Mendelian inheritance and high penetrance (FALS). Pathogenic mutations in the SOD1 gene have been found in approximately 20% of all FALS pedigrees (Rosen et al., 1993), while variants in the TARDBP gene account for additional ⬃ 5% of cases

* Corresponding author at: University of Eastern Piedmont, Department Medical Sciences, Via Solaroli, 17, 28100 Novara, Italy. Tel: 03 ⫹39 0321 660606. E-mail address: [email protected] (L. Corrado). 1 These authors contributed equally to this work. 0197-4580/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2010.02.011

(Lagier-Tourenne and Cleveland, 2009). Moreover, at least 9 disease loci and pathogenic mutations in 5 other genes (ALS2, SETX, VAPB, DNCT1, and ANG) have been described in isolated families (Beleza-Meireles and AlChalabi, 2009; Pasinelli and Brown, 2006). Recently, mutations in the FUS gene, located on chromosome 16, have been identified in ⬃ 5% of FALS patients that tested negative for SOD1 and TARDBP mutations (Kwiatkowski et al., 2009; Vance et al., 2009). A hallmark of motor neurons in ALS is the presence of inclusion bodies composed of intermediate filament (IF) proteins in the pericaryon and axon of motor neurons. Neurofilament and peripherin proteins were identified as components of these inclusions. Peripherin (PRPH) is associated with ubiquitinated inclusions, specifically round inclusions and Lewy body-like inclusions, hyaline conglomerate inclusions, as well as with axonal spheroids, which occur in proximal axons of diseased motor neurons (Corbo and Hays, 1992; He and Hays,

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2004 Migheli et al., 1993; Xiao et al., 2006). Peripherin has the same tripartite structure common to all types of IF proteins comprising a predominantly alpha-helical rod domain flanked by non-alpha helical N-terminal head and C-terminal tail domains. The rod domain is highly conserved between different IF proteins and is subdivided into 4 coil domains (1A, 1B, 2A, and 2B) that are joined together by 3 non-alpha helical linker sequences. The rod domain mediates the formation of coiled-coil dimers, the first step of IF assembly (Steinert and Roop, 1988). Although peripherin is usually expressed at low levels in spinal motor neurons, there is an up-regulation of peripherin expression in ALS (Robertson et al., 2003). Over-expressing peripherin mice show a massive age-related motor neuron degeneration that is preceded by the presence of neuronal cytoplasmic peripherin aggregates (Beaulieu et al., 1999). Moreover, in the motor neurons of mutant SOD1G37R transgenic mice there is an abnormal expression of a peripherin neurotoxic splice variant (Per 61). Per 61 was generated by the retention of intron 4 and its upregulated expression induced peripherin aggregate formation and motor neuronal death (Landon et al., 1989, 2000; Robertson et al., 2003). An aberrant splice transcript, retaining intron 3 and 4, was found also in humans. This variant, called Per 28, is up-regulated at both mRNA and protein level in ALS and may be associated with inclusion body formation (Xiao et al., 2008). Moreover, rare PRPH gene mutations were identified in 2 sporadic ALS cases among a total of 124 investigated patients (Gros-Louis et al., 2004; Leung et al., 2004). One is a heterozygous frameshift mutation predicting a truncated peripherin species of 85 amino acids encompassing the head domain, and the other one is a homozygous p.D141Y mutation within the first linker sequence of the alpha helical rod domain. Both mutations are associated with peripherin aggregates that were also neurofilament immunoreactive in the residual motor neurons. The aim of this study was to further define the spectrum of PRPH mutations in a cohort of Italian ALS patients. 2. Methods 2.1. Subjects The 122 Italian ALS patients included in this study were referred to the ALS center in Novara (Italy) and came from different Italian regions. ALS diagnosis was performed according to the El Escorial revised criteria (Brooks et al., 2000). ALS was classified as familial (FALS) when at least one other member was clinically affected in the same pedigree. On this basis, 115 patients (80 male and 35 female; mean age at onset: 51.2 years, range: 20 –79 years) were classified as sporadic ALS cases (SALS), while 7 unrelated patients (3 males and 4 females; mean age: 51.5 years, range: 32–72) were classified as FALS. All ALS patients included in the study had been previously screened for Cu/Zn superoxide dismutase 1 (SOD1,

OMIM* 147450), angiogenin (ANG OMIM* 105850) TAR-DNA binding protein (TARDBP OMIM* 605078) and FUS (FUS/TLS OMIM* 137070) gene mutations (Corrado et al., 2007; Corrado et al., 2009, Corrado et al., 2010). Control DNA was obtained from 365 unrelated age- and regionally-matched Italian subjects (University and Hospital staff, blood donors) without reported history of neurological disorders. An additional 96 ALS patient samples (83 SALS and 13 FALS), recruited at Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Neurological Institute “C Mondino” (Pavia, Italy), were screened for exon 1, 5= flanking and 5= UTR of PRPH gene. The study has been approved by the local hospital Ethic Committees. A blood sample of the patients was drawn after informed consent to genetic analysis for research purposes. 2.2. Bioinformatic analysis The effect of missense, synonymous, and intronic variants on splicing was analyzed with Spliceview (http://zeus2.itb.cnr.it/ ⬃webgene/wwwspliceview_ex.html) and NNSplice (http://www. fruitfly.org/seq_tools/splice.html) programs. The effect of the detected PRPH missense mutations on protein structure or function was analyzed with 3 prediction programs: PolyPhen (http://genetics.bwh.harvard.edu/pph/), SNAP (http://cubic.bioc.columbia.edu/services/SNAP/submit. html), and SIFT (http://sift.jcvi.org/). Presence of ESE (exonic splice enhancer) was checked using ESE Finder (http://rulai.cshl.edu/tools/ESE), and RESCUE ESE (http://genes.mit.edu/burgelab/rescue-ese). The identification of putative transcription binding sites was performed using Promo 3.0 (http://alggen.lsi.upc.es/cgi-bin/ promo_v3/promo/promoinit.cgi?dirDB⫽TF_8.3). Codon usage analysis was performed using Codon Usage Database (www.kazusa.or.jp/codon). 2.3. Genetic analysis Genomic DNA from patients and controls was extracted from peripheral blood using standard methods. Nine primer pairs were designed from genomic DNA to amplify by polymerase chain reaction (PCR) the 9 coding exons and the intron/exon boundaries (including at least 50 base pairs of adjacent intronic sequences) of PRPH gene. Exons 1, 3, 6, 7, 3= flanking and 3= UTR were sequenced directly for all patients. The remaining exons (exons 2, 4, 5, 8, 9 and 5= UTR) were first screened by heteroduplex analysis (denaturing highperformance liquid chromatography; Wave Transgenomic, Glasgow, UK), as previously described (Mellai et al., 2003). To obtain a sensitivity and specificity near to 100% the analysis was performed at RTm and RTm ⫹ 2 °C (Jones et al., 1999). Exons with heteroduplex denaturing high-performance liquid chromatography (DHPLC) profiles were sequenced using the Big-Dye Terminator v3.1 sequencing kit (Applied Biosystems, Foster City, CA). All variants were confirmed

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Table 1 PRPH sequence variations identified in ALS patients and/or healthy controls Nucleotide change

Position

AA change

Patients n

c.-99 delT c.-24 G>C (rs74089010) c.26 G⬎A (rs57451017) c.63 C⬎T (rs58403142) c.398 G>C c.421G>T (rs58599399) c.547 T>C c.606⫹52 T⬎G c.702⫹ 27 T⬎C (rs2070760)

5= flanking 5=UTR Exon 1 Exon 1 Exon 1 Exon 1 Exon 2 Intron 2 Intron 3

— — p.R9Q p.F21F p.R133P p. D141Y p.L183L — —

c.706-13 C>T c.829 G⬎A (rs62636520) c.870ⴙ12 G>A c.1083 C⬎G (rs62636517) c.1107 A⬎G (rs73112143)

Intron 3 Exon 4 Intron 4 Exon 6 Exon 6

— p.A277T — p.L361L p.K369K

c.1217⫹27 c.1217ⴙ34 c.1217⫹43 c.1413⫹82

Intron 6 Intron 6 Intron 6 3’UTR

— — —

C⬎A G>A C⬎A C⬎A (rs7137584)

a

MAF Freq.

3/218 1/218 10/218 8/218 1/218 1/218 1/122 0/122 89/122 TT 30/122 TC 3/122 CC 1/122 2/122 1/122 1/122 87/122 AA 31/122 AG 4/122 GG 0/122 1/122 4/122 1/122

0.013 0.004 0.040 0.036 0.004 0.004 0.008 0 0.730 0.246 0.024 0.008 0.016 0.008 0.008 0.713 0.254 0.032 0.008 0.032

Controls n

0.006 0.002 0.022 0.018 0.002 0.002 0.004 0 0.150

0.004 0.008 0.008 0.004 0.160

0 0.004 0.020 0.004

a

2/365 0/365 11/365 12/365 0/260 0/260 0/260 1/260 164/260 TT 88/260 TC 8/260 CC 0/260 14/260 0/260 8/245 151/245 AA 85/245 AG 9/245 GG 2/243 0/245 22/245 nd

MAF Freq. 0.005 0 0.030 0.033 0 0 0 0.004 0.631 0.338 0.031 0 0.054 0 0.033 0.616 0.347 0.037 0.008 0 0.090

0.003 0 0.015 0.016 0 0 0 0.002 0.200

0 0.027 0 0.016 0.210

0.004 0 0.045

The two PCR fragments, containing exon 1, 5= UTR and 5= flanking sequences, were analyzed also in 96 additional patients and 115 controls, since in the samples analyzed for the remaining fragments we observed a slightly different frequency among patients and controls. The results of the combined dataset are displayed. Nucleotide numbering of PRPH variations reflects cDNA numbering with ⫹ 1 corresponding to the A of the ATG translation initiation codon in the GenBank reference sequence NM_006262.3. The initiation codon is codon 1. For the 1413⫹82 C⬎A variant (rs7137584) we did not genotype controls because the frequencies found in Italian ALS patients were substantially similar to those reported in dbSNP. Key: AA, Aminoacid; ALS, amyotrophic lateral sclerosis; dbSNP, database Single Nucleotide Polymophism; Freq., frequency; MAF, Minor Allele Frequency; nd, not determined; PCR, polymerase chain reaction; PRPH, peripherin; PRPH, peripherin gene; UTR, untranslated region. PRPH variants identified only in ALS cases are shown in bold. a Number of individuals carrying the variation (corresponding to heterozygous individuals, with the exception of c.705⫹ 27 T⬎C and c.1107 A ⬎ G for whom the 3 genotypes are indicated).

in at least 2 independent PCR and sequencing reactions. Variants detected in the patients were tested in a panel of matched controls. Nucleotide numbering of PRPH variations reflects cDNA numbering with ⫹ 1 corresponding to the A of the ATG translation initiation codon in the GenBank reference sequence NM_006262.3. The initiation codon is codon 1. 2.4. Statistical analysis The statistical significance of the difference of gene, genotype frequencies among ALS patients and controls was evaluated using the ␹2 test with Yates’s correction. When required by the small number of expected cases, the 2-tailed Fisher exact test was used. The ages at onset in ALS patients carrying PRPH mutations and in the whole ALS cohort were compared by the Mann-Whitney U test. 3. Results 3.1. Genetic analysis DNA samples from 122 unrelated ALS patients (7 FALS and 115 SALS cases) were screened for PRPH sequence variations. A total of 18 variants were detected (Table 1).

Eight of them occurred within the coding region (c.26 G⬎A p.R9Q, c.63 C⬎T p.F21F, c.398 G⬎C p.R133P, c.421G⬎T p.D141Y, c.547 T⬎C p.L183L, c.829 G⬎A p.A277T, c.1083 C⬎G p.L361L, c.1107 A⬎G p.K369K), 7 in the intronic regions, 2 in the 5= and 3= UTR and one in the 5= flanking region. To test whether any of these PRPH variants could be causative mutations or susceptibility factors for ALS, we compared their frequencies in ALS patients versus normal controls. 3.1.1. PRPH variants found only in ALS cases Seven nucleotide variants, detected each in one patient, were not identified in a panel of 245 matched controls. Among them, 5 were newly identified (p.R133P, p.L183L, c.706-13 C⬎T c.870⫹12 G⬎A, c.1217⫹34 G⬎A), while 2 variants were previously described (c.-24 G⬎C, p.D141Y) (Gros-Louis et al., 2004; Leung et al., 2004). Two out of 3 coding variants are predicted to result in an amino acid substitution (p.R133P, p.D141Y) at residues that are highly conserved between human, mouse, and rat peripherin proteins (data not shown). The third (p.L183L) is a synonymous variant. The remaining 4 variants are localized in 5= UTR (c.-24 G⬎C) and in intronic regions (c.706-13 C⬎T c.870⫹12 G⬎A, c.1217⫹34 G⬎A).

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The patients carrying the above PRPH sequence variants were negative for mutations in tested ALS genes (SOD1, ANG, TARDBP and FUS). In addition, 2 intronic variants (c.606⫹52 T⬎G; c.1217⫹ 27 C⬎A) were observed only in 2 different controls.

of frequency of codon usage from 12.9 for UUG to 39.6 for CUG codon. For c.63C⬎T and c.1107A⬎G the codon usage frequencies were not substantially different.

3.1.2. PRPH variants found both in ALS cases and controls Eleven PRPH variants were detected in ALS cases and also in healthy controls (Table 1). They are scattered along PRPH gene and include 2 missense, 3 synonymous, and 6 noncoding variants. For each of them we observed no significant differences of the allelic and genotype frequencies between patients and controls. For 2 sequence variants, c.705⫹ 27 T⬎C in intron 3 and c.1107 A⬎G in exon 6, we observed a not significant reduction of heterozygous genotypes in patients compared with controls (0.246 vs. 0.338, p value ⫽ 0.09 for c.705⫹ 27 T⬎C and 0.254 versus 0.347, p value ⫽ 0.09 for c.1107 A⬎G). The observed genotype frequencies are consistent with Hardy Weinberg Equilibrium (HWE). Interestingly, a similar trend was observed also in a previous study (Gros-Louis et al., 2004). The excess of homozygosity for 2 contiguous Single Nucleotide Polymorphisms (SNPs), might suggest the presence of a deletion spanning at least the region including these 2 variants. To detect the presence of a possible intragenic deletion of PRPH gene we performed a PCR amplification from genomic DNA of 83 patients, homozygous for both rs2070760 (intron 3) and c.1107 A⬎G, (exon 6) variants, using primers flanking the region of homozygous SNPs. No evidence of deletions was observed because the PCR fragments showed the expected size for all of tested patients. Three of the tested patients were heterozygous for variants localized between the PCR primers and the homozygous region. For the remaining 80 patients we cannot exclude the presence of a larger deletion involving PRPH gene.

PRPH variants were not associated with the tested ALS clinical features (age of onset and site of onset). In particular, patients carrying rare PRPH variants, not detected in the controls, showed a mean age of onset (56.6 years) not significantly different from that of the whole cohort (51.2 years, p ⬎ 0.05). Considering the 2 common PRPH variants (c.705⫹27T⬎C and c.1107 A⬎G), the mean age of onset was not significantly different among patients positive or negative for the minor allele (50.1 vs. 51.7 for c.705⫹27T⬎C, 50.5 vs. 51.5 for c.1107 A⬎G). Moreover, among patients carrying these PRPH variants the proportion of cases with a spinal versus a bulbar site of onset was not significantly different from the whole cohort.

3.2. In silico analysis The newly identified p.R133P as well as the already described p.D141Y mutations were predicted to have a deleterious effect on PRPH protein structure or function by three different prediction programs: PolyPhen (“probably damaging” Position-Specific Independent Count (PSIC) score 2.319 and “possibly damaging” PSIC 1.707, respectively), SNAP (“non-neutral” accuracy 82% and “non-neutral” accuracy 63%, respectively) and SIFT (“affect protein function” for both mutations). No evidence of alteration of splice sites or of exonic splice enhancer was observed for any of the intronic and synonymous variants. According to the PROMO 3.0 program, the nucleotide deletion c.-99delT determined the loss of DEAF-1 binding site and introduced a new putative binding site for Zic 2. The codon usage frequency analysis of the synonymous variants predicted for the c.547 T⬎C substitution a change

3.3. Association with ALS clinical features

4. Discussion In this study we have detected 18 variants in PRPH gene in ALS cases. Five variants were newly identified and were absent in a panel of matched controls. Two missense variants are located within the linker region between coil 1A and coil 1B of the rod domain of peripherin. The p.R133P is newly identified and generates a nonconservative change. For this mutation we have not performed functional assays. Despite this, some remarks suggest that it might be involved in ALS pathogenesis. Firstly, it was not detected in 260 Italian controls as well as in over 290 controls reported worldwide (Gros-Louis et al., 2004; Leung et al., 2004). Secondly, this mutation affects evolutionary conserved residues. Thirdly, bioinformatics programs predicted that this variant had a deleterious effect on the protein structure/ function. Finally, the described mutation affects a residue of the rod domain where one previously described mutation (p.D141Y) in one ALS case, was reported (Leung et al., 2004). The male patient carrying p.R133P mutation is an apparently sporadic ALS case. He died at 72 years old for spinal onset ALS, which had been diagnosed 2 years before. DNA samples from other family members were not available for molecular analysis of the PRPH gene; therefore we were not able to define whether the mutation was inherited or de novo. Moreover, we identified the p.D141Y variant in one ALS patient and in none of the 260 tested controls. p.D141Y was previously described in homozygosity state in 1 sporadic ALS patient (Leung et al., 2004) and in heterozygosity state in 3 sporadic ALS patients, but also in 2 different control samples (Gros-Louis et al., 2004). Functional analysis performed by Leung et al. (2004) demonstrated that p.D141Y mutation did not abolish the ability of peripherin to assemble into filaments, but led to the formation of aggregates, also in heterozygous state. The in silico analysis indicates a

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negative effect on protein structure and functions, also for this mutation. Combining literature and our data, the variant was observed in heterozygosity in 4/342 patients and 2/550 controls. The excess of its frequency in ALS patients, although not significant, and the previously reported functional relevance also in heterozygous do not exclude that this could be a low penetrance variant involved in ALS susceptibility. Two intronic variants (c.706-13 C⬎T, c.870⫹12 G⬎A) localized in intron 3 and 4, nearly exon/intron boundaries, were detected only in ALS patients. We have no functional or in silico evidence of the involvement of these 2 variants in an aberrant splicing mechanism. Nevertheless these variants are interesting in light of recent findings that reported in mouse and in human different peripherin neurotoxic isoforms involved in ALS pathogenesis and generated through retention of intron 4 (Per 61) or intron 3 and 4 (Per 28) (Landon et al., 1989, 2000; Robertson et al., 2003; Xiao et al., 2008). The other variants detected exclusively in ALS patients are unlikely to have a functional impact on the protein, with the exception of the c.547 T⬎C variant, for whom a different codon usage frequency was predicted (from 12.9 to 39.6). Infrequent codons in mRNA appear to be slowly translated, whereas frequent codons are rapidly translated. Altered translation kinetics of a defined mRNA, due to synonymous codon substitutions, might drive the in vivo folding of the same polypeptide chain in a different conformation (Kimchi-Sarfaty et al., 2007; Komar, 2007). Accordingly, the usage of a frequent codon for the c.547 T⬎C variant might lead to an increase of expression and/or to a different folding of the protein which could be related to ALS pathogenesis. All these speculations derive from in silico analysis and thus require a confirmation with experimental data. If a functional role of p.R133P mutation will be confirmed, this is the third mutation identified in a total of 246 ALS patients worldwide (1.2%). This frequency is similar to that reported in SALS patients for the other genes mutated in an appreciable number of patients, such as SOD1, ANG, (Chiò et al., 2008; Fernández-Santiago et al., 2009; Gellera et al., 2008) and the more recently identified TARDBP (Lagier-Tourenne and Cleveland, 2010) and FUS (Corrado et al., 2010). Interestingly, all the PRPH mutations identified both in this and previous studies are localized in rod domain of the protein that is necessary for the IF assembly. The identified PRPH sequence variants may lead to the disease by a mechanism that involves the PRPH deposition process according to the general notion that variability at the gene that encodes the pathologically deposited species is a risk factor in neurological diseases involving protein deposition (Singleton and Hardy, 2004). In conclusion, this work contributes to determine the role of PRPH gene variants in ALS. Further studies on larger cohorts of ALS cases are necessary to estimate the involvement of PRPH mutations in

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ALS pathogenesis and to define the mechanisms through which the mutant peripherin could cause ALS phenotype. Disclosure statement None of the authors has potential conflicts of interest including any financial, personal or other relationships with other people or organizations. This study was approved by the local hospital Ethic Committees. A blood sample of the patients was drawn after informed consent to genetic analysis for research purposes. Acknowledgements We are grateful to all patients, their families, and the SLAGEN Consortium. S.D. was supported by Regione Piemonte (Ricerca Sanitaria Finalizzata Project-Grant, 2006/Grant, 2008, and Ricerca Sanitaria Applicata-CIPE Project), Eastern Piedmont University and the Italian Ministry of University and Research (PRIN Project). L.C. was partially supported by a fellowship from Soroptimist International Club Novara; L.C. is now supported by a fellowship from “Amico Canobio” Association. References Beaulieu, J.M., Nguyen, M.D., Julien, J.P., 1999. Late onset of motor neurons in mice overexpressing wild-type peripherin. J. Cell Biol. 147, 531–544. Beleza-Meireles, A., Al-Chalabi, A., 2009. Genetic studies of amyotrophic lateral sclerosis: controversies and perspectives. Amyotroph. Lateral Scler. 10, 1–14. Brooks, B.R., Miller, R.G., Swash, M., Munsat, T.L., 2000. 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 Mot. Neuron Disord. 1, 293–299. Chiò, A., Traynor, B.J., Lombardo, F., Fimognari, M., Calvo, A., Ghiglione, P., Mutani, R., Restagno, G., 2008. Prevalence of SOD1 mutations in the Italian ALS population. Neurology. 70, 533–537. Corbo, M., Hays, A.P., 1992. Peripherin and neurofilament protein coexist in spinal spheroids of motor neuron disease. J. Neuropathol. Exp. Neurol. 51, 531–537. Corrado, L., Battistini, S., Penco, S., Bergamaschi, L., Testa, L., Ricci, C., Giannini, F., Greco, G., Patrosso, M.C., Pileggi, S., Causarano, R., Mazzini, L., Momigliano-Richiardi, P., D’Alfonso, S., 2007. Variations in the coding and regulatory sequences of the angiogenin (ANG) gene are not associated to ALS (amyotrophic lateral sclerosis) in the Italian population. J. Neurol. Sci. 258, 123–127. Corrado, L., Ratti, A., Gellera, C., Buratti, E., Castellotti, B., Carlomagno, Y., Ticozzi, N., Mazzini, L., Testa, L., Taroni, F., Baralle, F.E., Silani, V., D’Alfonso, S., 2009. High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis. Hum. Mutat. 4, 688 – 694. Corrado, L., Del Bo, R., Castellotti, B., Ratti, A., Cereda, C., Penco, S., Sorarù, G., Carlomagno, Y., Ghezzi, S., Pensato, V., Colombrita, C., Gagliardi, S., Cozzi, L., Orsetti, V., Mancuso, M., Siciliano, G., Mazzini, L., Comi, G.P., Gellera, C., Ceroni, M., D’Alfonso, S., Silani, V., 2010. Mutations of FUS gene in sporadic amyotrophic lateral sclerosis. J. Med. Genet. 47,190 –194.

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Web References Spliceview (http://zeus2.itb.cnr.it/⬃webgene/wwwspliceview_ex.html) NNSplice (http://www.fruitfly.org/seq_tools/splice.html) PolyPhen (http://genetics.bwh.harvard.edu/pph/) SNAP (http://cubic.bioc. columbia.edu/services/SNAP/submit.html) SIFT (http://sift.jcvi.org/) ESE Finder (http://rulai.cshl.edu/tools/ESE) RESCUE ESE: (http://genes.mit.edu/burgelab/rescue-ese) Promo 3.0: (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi? dirDB⫽TF_8.3) Sage Database: (www.kazusa.or.jp/codon)