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Objective: Angiogenin (ANG) is a pro-angiogenic and neurotrophic factor with an important role in stress-induced injury, by promoting neovascularization and neuronal survival. Identification of loss-of-function mutations and evidence of beneficial effect of ANG administration in transgenic SOD1G93A mice have linked ANG to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), stimulating interest in considering circulating ANG levels as an ALS disease biomarker although robust evidence is still lacking. Aim of our study was to assess differences of ANG levels in the cerebrospinal fluid (CSF) of a large cohort of patients with ALS and frontotemporal dementia (FTD) compared to controls and to explore correlations between ANG content and disease-related clinical variables. Methods: ANG levels were measured in CSF samples using a commercially available ELISA kit in 88 patients affected with ALS and/or FTD and 46 unrelated individuals (control group). Results: ANG levels didn't differ significantly between cases and controls. Patients with FTD or ALS-FTD showed significantly increased CSF concentration of ANG compared to ALS patients without dementia and controls in a multivariate regression model (p < 0.001). No correlations were found in ALS/FTD patients between ANG levels and clinical parameters, including age, presence of C9orf72 repeat expansion, body mass index (BMI). Conclusions: our findings highlight a role of ANG as CSF biomarker useful to identify ALS patients with concurrent FTD and suggest that it should be further explored as potential biomarker for FTD. [ABSTRACT FROM AUTHOR]

Background Optineurin (OPTN), a causative gene of hereditary primary open-angle glaucoma, has been recently associated with amyotrophic lateral sclerosis (ALS) with mainly autosomal recessive, but also dominant, traits. To further define the contribution of OPTN gene in ALS, we performed a mutational screening in a large cohort of Italian patients. Methods A group of 274 ALS patients, including 161 familial (FALS) and 113 sporadic (SALS) cases, were screened for OPTN mutations by direct sequencing of its coding sequence. All patients fulfilled the El Escorial criteria for probable or definite ALS and were negative for mutations in SOD1, ANG, TARDBP and FUS→TLS genes. Results The genetic analysis revealed six novel variants in both FALS and SALS patients, all occurring in an heterozygous state. We identified three missense (c.844A→C p.T282P, c.941A→T p.Q314L, c.1670A→C p.K557T), one nonsense (c.67G→T p. G23X) and two intronic mutations (c.552&plus;1delG, c.1401 &plus;4A→G). The intronic c.552&plus;1delG variant determined a splicing defect as demonstrated by mRNA analysis. All mutations were absent in 280 Italian controls and over 6800 worldwide glaucoma patients and controls screened so far. The clinical phenotype of OPTN-mutated patients was heterogeneous for both age of onset and disease duration but characterised by lower-limb onset and prevalence of upper motor neuron Conclusion In this cohort, OPTN mutations were present both in FALS (2/161), accounting for 1.2% cases, and in SALS patients (4/113), thereby extending the spectrum of OPTN mutations associated with ALS. The study further supports the possible pathological role of optineurin protein in motor neuron disease. [ABSTRACT FROM AUTHOR]

Repeat expansions in the C9orf72 gene are the most common genetic cause of (ALS) and frontotemporal dementia (FTD). Like other genetic forms of neurodegeneration, pinpointing the precise mechanism(s) by which this mutation leads to neuronal death remains elusive, and this lack of knowledge hampers the development of therapy for C9orf72-related disease. We used an agnostic approach based on genomic data (n = 41,273 ALS and healthy samples, and n = 1,516 C9orf72 carriers) to overcome these bottlenecks. Our drug-repurposing screen, based on gene- and expression-pattern matching and information about the genetic variants influencing onset age among C9orf72 carriers, identified acamprosate, a γ-aminobutyric acid analog, as a potentially repurposable treatment for patients carrying C9orf72 repeat expansions. We validated its neuroprotective effect in cell models and showed comparable efficacy to riluzole, the current standard of care. Our work highlights the potential value of genomics in repurposing drugs in situations where the underlying pathomechanisms are inherently complex. VIDEO ABSTRACT., Competing Interests: Declaration of interests B.J.T. holds patents on clinical testing and therapeutic intervention for the hexanucleotide repeat expansion of C9orf72., (Published by Elsevier Inc.)

During the last decades, our knowledge about the genetic architecture of sporadic amyotrophic lateral sclerosis (sALS) has significantly increased. However, besides the recognized genetic risk factors, also the environment is supposed to have a role in disease pathogenesis. Epigenetic modifications reflect the results of the interaction between environmental factors and genes and may play a role in the development and progression of ALS. A recent epigenome-wide association study (EWAS) in blood identified differentially methylated positions mapping to 42 genes involved in cholesterol biosynthesis and immune-related pathways. Here we performed a genome-wide DNA methylation analysis in the blood of an Italian cohort of 61 sALS patients and 61 healthy controls. Initially, a conventional genome-wide association analysis was performed, and results were subsequently integrated with the findings from the previous EWAS using a meta-analytical approach. To delve deeper into the significant outcomes, over-representation analysis (ORA) was employed. Moreover, the epigenetic signature obtained from the meta-analysis was examined to determine potential associations with chemical compounds, utilizing the Toxicogenomic Database. Expanding the scope of the epigenetic analysis, we explored both epigenetic drift and rare epivariations. Notably, we observed an elevated epigenetic drift in sALS patients compared to controls, both at a global and single gene level. Interestingly, epigenetic drift at a single gene level revealed an enrichment of genes related to the neurotrophin signaling pathway. Moreover, for the first time, we identified rare epivariations exclusively enriched in sALS cases associated with 153 genes, 88 of whom with a strong expression in cerebral areas. Overall, our study reinforces the evidence that epigenetics may contribute to the pathogenesis of ALS and that epigenetic drift may be a useful diagnostic marker. Moreover, this study suggests the potential role of epivariations in ALS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Brusati, Peverelli, Calzari, Tiloca, Casiraghi, Sorce, Invernizzi, Carbone, Cavagnola, Verde, Silani, Ticozzi, Ratti and Gentilini.)

Introduction: Amyotrophic lateral sclerosis (ALS) individuals carrying the hexanucleotide repeat expansion (HRE) in the C9orf72 gene (C9Pos) have been described as presenting distinct features compared to the general ALS population (C9Neg). We aim to identify the phenotypic traits more closely associated with the HRE and analyse the role of the repeat length as a modifier factor., Methods: We studied a cohort of 960 ALS patients (101 familial and 859 sporadic cases). Motor phenotype was determined using the MRC scale, the lower motor neuron score (LMNS) and the Penn upper motor neuron score (PUMNS). Neuropsychological profile was studied using the Italian version of the Edinburgh Cognitive and Behavioral ALS Screen (ECAS), the Frontal Behavioral Inventory (FBI), the Beck Depression Inventory-II (BDI-II) and the State-Trait Anxiety Inventory (STAI). A two-step PCR protocol and Southern blotting were performed to determine the presence and the size of C9orf72 HRE, respectively., Results: C9orf72 HRE was detected in 55/960 ALS patients. C9Pos patients showed a younger onset, higher odds of bulbar onset, increased burden of UMN signs, reduced survival and higher frequency of concurrent dementia. We found an inverse correlation between the HRE length and the performance at ECAS ALS-specific tasks (P = 0.031). Patients also showed higher burden of behavioural disinhibition (P = 1.6 × 10 -4 ), lower degrees of depression (P = 0.015) and anxiety (P = 0.008) compared to C9Neg cases., Conclusions: Our study provides an extensive characterization of motor, cognitive and behavioural features of C9orf72-related ALS, indicating that the C9orf72 HRE size may represent a modifier of the cognitive phenotype., (© 2022. The Author(s).)

Background: Aggregates of TAR DNA-binding protein of 43 kDa (TDP-43) represent the pathological hallmark of most amyotrophic lateral sclerosis (ALS) and of nearly 50% of frontotemporal dementia (FTD) cases but were also observed to occur as secondary neuropathology in the nervous tissue of patients with different neurodegenerative diseases, including Parkinson's disease (PD) and atypical parkinsonism. Mutations of TARDBP gene, mainly in exon 6 hotspot, have been reported to be causative of some forms of ALS and FTD, with clinical signs of parkinsonism observed in few mutation carriers., Methods: Direct DNA sequencing of TARDBP exon 6 was performed in a large Italian cohort of 735 patients affected by PD (354 familial and 381 sporadic) and 142 affected by atypical parkinsonism, including 39 corticobasal syndrome (CBS) and 103 progressive sopranuclear palsy (PSP). Sequencing data from 1710 healthy, ethnically matched controls were already available., Results: Four TARDBP missense variants (p.N267S, p. G294A, p.G295S, p.S393L) were identified in four patients with typical PD and in two individuals with atypical parkinsonism (1 CBS and 1 PSP). None of the detected mutations were found in healthy controls and only the variant p.N267S was previously described in association to idiopathic familial and sporadic PD and to CBS., Conclusion: In this study we provide further insight into the clinical phenotypic heterogeneity associated with TARDBP mutations, which expands beyond the classical ALS and FTD diseases to include also PD and atypical parkinsonism, although with a low mutational frequency, varying considerably in different Caucasian populations. In addition, our study extends the spectrum of TARDBP pathogenetic mutations found in familial and sporadic PD., Competing Interests: AR received research funding from AriSLA. VS received compensation for consulting services and/or speaking activities from AveXis, Cytokinetics, Italfarmaco, Liquidweb Srl, and Novartis Pharma AG. He receives or has received research support from the Italian Ministry of Health, AriSLA, and E-Rare Joint Transnational Call. He is on the Editorial Board of Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration, European Neurology, American Journal of Neurodegenerative Diseases, and Frontiers in Neurology. NT received compensation for consulting services and/or speaking activities from Amylyx Pharmaceuticals, Zambon Pharma AG, and Italfarmaco. He received research funding from the Italian Ministry of Health and AriSLA. He is Associate Editor for Frontiers in Aging Neuroscience. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tiloca, Goldwurm, Calcagno, Verde, Peverelli, Calini, Zecchinelli, Sangalli, Ratti, Pezzoli, Silani and Ticozzi.)

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation., Objective: To identify the genetic variants associated with juvenile ALS., Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism., Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members., Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway., Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.

Introduction: Previous studies have associated single-nucleotide polymorphisms (SNPs) in the gene encoding the detoxifying enzyme paraoxonase 1 (PON1) to the risk of sporadic ALS. Here, we aimed to assess the role of the coding rs662 (Q192R) SNP as a modifier of ALS phenotype., Materials and Methods: We genotyped a cohort of 409 patients diagnosed with ALS at our Center between 2002 and 2009 (269 males and 140 females; mean age at onset, 58.3 ± 37.5 years)., Results: We found PON1 to be a disease modifier gene in ALS, with the minor allele G associated both with bulbar onset (30.9% vs. 24.6%, p = 0.013) and independently with reduced survival (OR = 1.38, p = 0.012) under a dominant model. No association was found with gender or age at onset., Discussion: As this SNP is known to modify the detoxifying activity of paraxonase 1 with respect to different substrates as well as other activities of the protein, we hypothesize that the identified association might reflect specific motor neuron vulnerability to certain exogenous toxic substances metabolized less efficiently by the 192R alloenzyme, or to detrimental endogenous pathophysiological processes such as oxidative stress. Further exploration of this possible metabolic susceptibility could deepen our knowledge of ALS pathomechanisms.

Mutations in TANK binding kinase 1 (TBK1) have been linked to amyotrophic lateral sclerosis. Some TBK1 variants are nonsense and are predicted to cause disease through haploinsufficiency; however, many other mutations are missense with unknown functional effects. We exome sequenced 699 familial amyotrophic lateral sclerosis patients and identified 16 TBK1 novel or extremely rare protein-changing variants. We characterized a subset of these: p.G217R, p.R357X, and p.C471Y. Here, we show that the p.R357X and p.G217R both abolish the ability of TBK1 to phosphorylate 2 of its kinase targets, IRF3 and optineurin, and to undergo phosphorylation. They both inhibit binding to optineurin and the p.G217R, within the TBK1 kinase domain, reduces homodimerization, essential for TBK1 activation and function. Finally, we show that the proportion of TBK1 that is active (phosphorylated) is reduced in 5 lymphoblastoid cell lines derived from patients harboring heterozygous missense or in-frame deletion TBK1 mutations. We conclude that missense mutations in functional domains of TBK1 impair the binding and phosphorylation of its normal targets, implicating a common loss of function mechanism, analogous to truncation mutations., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Pathological repeat expansion (RE) of the C9orf72 hexanucleotide sequence is associated to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia disease continuum, although other heterogeneous clinical phenotypes have been documented. The occurrence of multiple sclerosis (MS) in some C9orf72 carriers with a more severe ALS disease course has suggested a possible modifying role for MS. However, C9orf72 RE seems not to play a role in MS pathogenesis. In this study, we screened C9orf72 in 189 Italian patients with primary progressive MS (PPMS), a rare clinical form characterized by less inflammation over neurodegenerative features. We failed to detect C9orf72 RE, but a significant representation of intermediate alleles (≥ 20 units) was observed in our PPMS cohort (2.1%) compared to healthy controls (0%, p < 0.05). In the normal range, allele distribution showed a trimodal pattern (2,5,8-repeat units) in PPMS and healthy controls with no significant difference. Our findings further demonstrate that C9orf72 RE is not genetically associated to MS spectrum, but suggest that intermediate alleles may represent risk factors as already reported for Parkinson disease., (Copyright © 2018. Published by Elsevier B.V.)

Large expansions of a noncoding GGGGCC repeat in the C9orf72 gene are the main cause of amyotrophic lateral sclerosis (ALS). The GGGGCC repeat is contiguous with another GC-rich region. Recent studies reported a significantly higher frequency of insertions/deletions within the GC-rich region in patients carrying the GGGGCC expansion. A GTGGT motif comprised within the GC-rich region, which joins two 100% GC sequences, was frequently deleted, supporting the hypothesis that these deletions could make the region more prone to slippage and pathological expansion. To confirm this hypothesis, we sequenced the GC-rich region adjacent the GGGGCC repeat in ALS patients, 116 C9orf72 expansion carriers, 219 non-carriers, and 223 healthy controls, from Italian and Turkish cohorts. Deletions were significantly more frequent in C9orf72 expansion carriers (6%) compared to non-carrier ALS patients (0.46%, OR =14.00, 95% CI =1.71-306.59, p = 0.003), to controls (0%, OR =16.29, 95% CI =2.12-725.99, p = 4.86 × 10 -4 ) and to the whole cohort of non-carriers (0.2%, OR =28.51, 95% CI =3.47-618.91, p = 9.58 × 10 -5 ). Among expansion carriers, deletions with or without the GTGGT motif were equally distributed (4 vs. 3). The frequency of insertions was not statistically different between C9orf72 expansion carriers and any other group including the whole cohort of non-carriers (p = 0.439, Fisher's exact test). Our data confirmed the association between deletions within GC-rich region and the GGGGCC expansion in Italian and Turkish cases, although we did not confirm a role of the GTGGT element deletion. Further studies will be therefore necessary to assess the causal relationships between contiguous deletions of the GC-rich region and the GGGGCC expansion.

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mutations in leucine-rich repeat kinase 2 gene ( LRRK2 ) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

Adult-onset neurological disorders are caused and influenced by a multitude of different factors, including epigenetic modifications. Here, using an ELISA kit selected upon careful testing, we investigated global 5-methylcytosine (5-mC) levels in sporadic and familial amyotrophic lateral sclerosis (sALS and fALS), spinocerebellar ataxia types 1 and 2 (SCA1 and SCA2), Huntington's disease, Friedreich's ataxia, and myotonic dystrophy type 1. We report a significant elevation in global 5-mC levels of about 2-7% on average for sALS (p < 0.01 [F(1, 243) = 9.159, p = 0.0027]) and various forms of fALS along with SCA1 (p < 0.01 [F(1, 83) = 11.285], p = 0.0012) and SCA2 (p < 0.001 [F(1, 122) = 29.996, p = 0.0001]) when compared to age- and sex-matched healthy controls. C9orf72 expansion carrier ALS patients exhibit the highest global 5-mC levels along with C9orf72 promoter hypermethylation. We failed to measure global 5-hydroxymethylcytosine (5-hmC) levels in blood, probably due to the very low levels of 5-hmC and the limitations of the commercially available ELISA kits. Our results point towards a role for epigenetics modification in ALS, SCA1, and SCA2, and help conclude a dispute on the global 5-mC levels in sALS blood., (© 2018 S. Karger AG, Basel.)

The genetic basis combined with the sporadic occurrence of amyotrophic lateral sclerosis (ALS) suggests a role of de novo mutations in disease pathogenesis. Previous studies provided some evidence for this hypothesis; however, results were conflicting: no genes with recurrent occurring de novo mutations were identified and different pathways were postulated. In this study, we analyzed whole-exome data from 82 new patient-parents trios and combined it with the datasets of all previously published ALS trios (173 trios in total). The per patient de novo rate was not higher than expected based on the general population (P = 0.40). We showed that these mutations are not part of the previously postulated pathways, and gene-gene interaction analysis found no enrichment of interacting genes in this group (P = 0.57). Also, we were able to show that the de novo mutations in ALS patients are located in genes already prone for de novo mutations (P < 1 × 10 -15 ). Although the individual effect of rare de novo mutations in specific genes could not be assessed, our results indicate that, in contrast to previous hypothesis, de novo mutations in general do not impose a major burden on ALS risk., (© 2017 Wiley Periodicals, Inc.)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases ( P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11-positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis., (Copyright © 2017, American Association for the Advancement of Science.)

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. To date, more than 20 genes have been implicated in ALS, and of these, the 2 most frequently mutated are the superoxide dismutase 1 (SOD1) gene and the chromosome 9 open reading frame 72 (C9ORF72) gene. In this study, we aimed to investigate the contribution of these 2 Mendelian genes to the development of the disease in Hungarian ALS patients (n = 66). Direct sequencing of the SOD1 gene revealed a novel (p.Lys91ArgfsTer8) and 3 recurrent heterozygous mutations (p.Val14Met, p.Asp90Ala, and p.Leu144Phe) in 5 patients. The novel p.Lys91ArgfsTer8 mutation led to a frameshift causing the addition of 8 new amino acids, including a premature stop codon at position 99. The GGGGCC hexanucleotide repeat expansion of the C9ORF72 gene was present in 1 ALS patient. This study represents the first genetic analysis of 2 major ALS causative genes in a cohort of Hungarian ALS patients and contributes to the further understanding of the genetic and phenotypic diversity of ALS., (Copyright © 2017 Elsevier Inc. All rights reserved.)

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G 4 C 2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G 4 C 2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72 -associated ALS (c9ALS). Therapeutics that target G 4 C 2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G 4 C 2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G 4 C 2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G 4 C 2 RNA and downstream G 4 C 2 RNA-mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G 4 C 2 RNA-based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention., (Copyright © 2017, American Association for the Advancement of Science.)

This study presents the Italian validation of the recently developed Edinburgh Cognitive and Behavioural ALS Screen (ECAS), a short screen for cognitive/behavioural alterations in patients with amyotrophic lateral sclerosis (ALS). We evaluated the psychometric properties of the ECAS Italian version in terms of reliability and convergent validity for both cognitive and behavioural features. Furthermore, we investigated the relationship with affective and clinical variables, in addition to ECAS usability and patients' insight into cognitive/behaviour changes. Finally, correlations between genetic and cognitive/behavioural data were analysed. We recruited 107 patients with ALS. Normative data were collected on 248 healthy subjects. Participants were administered the ECAS and two standard cognitive screening tools (FAB, MoCA), two psychological questionnaires (BDI, STAI/Y) and an ad hoc usability questionnaire. The FBI was also carried out with caregivers. Results showed that the ECAS Italian version discriminated well between patients and controls. The most prevalent deficit occurred in executive functions and fluency. Correlations were observed between the ECAS and standard cognitive screening tools and between the ECAS carer interview and the FBI, supporting its full convergent validity. In conclusion, the ECAS Italian version provides clinicians with a rapid, feasible and sensitive tool, useful to identify different profiles of cognitive-behavioural impairment in ALS.

To identify genetic factors contributing to amyotrophic lateral sclerosis (ALS), we conducted whole-exome analyses of 1,022 index familial ALS (FALS) cases and 7,315 controls. In a new screening strategy, we performed gene-burden analyses trained with established ALS genes and identified a significant association between loss-of-function (LOF) NEK1 variants and FALS risk. Independently, autozygosity mapping for an isolated community in the Netherlands identified a NEK1 p.Arg261His variant as a candidate risk factor. Replication analyses of sporadic ALS (SALS) cases and independent control cohorts confirmed significant disease association for both p.Arg261His (10,589 samples analyzed) and NEK1 LOF variants (3,362 samples analyzed). In total, we observed NEK1 risk variants in nearly 3% of ALS cases. NEK1 has been linked to several cellular functions, including cilia formation, DNA-damage response, microtubule stability, neuronal morphology and axonal polarity. Our results provide new and important insights into ALS etiopathogenesis and genetic etiology.

To elucidate the genetic architecture of amyotrophic lateral sclerosis (ALS) and find associated loci, we assembled a custom imputation reference panel from whole-genome-sequenced patients with ALS and matched controls (n = 1,861). Through imputation and mixed-model association analysis in 12,577 cases and 23,475 controls, combined with 2,579 cases and 2,767 controls in an independent replication cohort, we fine-mapped a new risk locus on chromosome 21 and identified C21orf2 as a gene associated with ALS risk. In addition, we identified MOBP and SCFD1 as new associated risk loci. We established evidence of ALS being a complex genetic trait with a polygenic architecture. Furthermore, we estimated the SNP-based heritability at 8.5%, with a distinct and important role for low-frequency variants (frequency 1-10%). This study motivates the interrogation of larger samples with full genome coverage to identify rare causal variants that underpin ALS risk.

Importance: Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disorder with a poor prognosis and a median survival of 3 years. However, a significant proportion of patients survive more than 10 years from symptom onset., Objective: To identify gene variants influencing survival in ALS., Design, Setting, and Participants: This genome-wide association study (GWAS) analyzed survival in data sets from several European countries and the United States that were collected by the Italian Consortium for the Genetics of ALS and the International Consortium on Amyotrophic Lateral Sclerosis Genetics. The study population included 4256 patients with ALS (3125 [73.4%] deceased) with genotype data extended to 7 174 392 variants by imputation analysis. Samples of DNA were collected from January 1, 1993, to December 31, 2009, and analyzed from March 1, 2014, to February 28, 2015., Main Outcomes and Measures: Cox proportional hazards regression under an additive model with adjustment for age at onset, sex, and the first 4 principal components of ancestry, followed by meta-analysis, were used to analyze data. Survival distributions for the most associated genetic variants were assessed by Kaplan-Meier analysis., Results: Among the 4256 patients included in the analysis (2589 male [60.8%] and 1667 female [39.2%]; mean [SD] age at onset, 59 [12] years), the following 2 novel loci were significantly associated with ALS survival: at 10q23 (rs139550538; P = 1.87 × 10-9) and in the CAMTA1 gene at 1p36 (rs2412208, P = 3.53 × 10-8). At locus 10q23, the adjusted hazard ratio for patients with the rs139550538 AA or AT genotype was 1.61 (95% CI, 1.38-1.89; P = 1.87 × 10-9), corresponding to an 8-month reduction in survival compared with TT carriers. For rs2412208 CAMTA1, the adjusted hazard ratio for patients with the GG or GT genotype was 1.17 (95% CI, 1.11-1.24; P = 3.53 × 10-8), corresponding to a 4-month reduction in survival compared with TT carriers., Conclusions and Relevance: This GWAS robustly identified 2 loci at genome-wide levels of significance that influence survival in patients with ALS. Because ALS is a rare disease and prevention is not feasible, treatment that modifies survival is the most realistic strategy. Therefore, identification of modifier genes that might influence ALS survival could improve the understanding of the biology of the disease and suggest biological targets for pharmaceutical intervention. In addition, genetic risk scores for survival could be used as an adjunct to clinical trials to account for the genetic contribution to survival.

Exome sequencing is an effective strategy for identifying human disease genes. However, this methodology is difficult in late-onset diseases where limited availability of DNA from informative family members prohibits comprehensive segregation analysis. To overcome this limitation, we performed an exome-wide rare variant burden analysis of 363 index cases with familial ALS (FALS). The results revealed an excess of patient variants within TUBA4A, the gene encoding the Tubulin, Alpha 4A protein. Analysis of a further 272 FALS cases and 5,510 internal controls confirmed the overrepresentation as statistically significant and replicable. Functional analyses revealed that TUBA4A mutants destabilize the microtubule network, diminishing its repolymerization capability. These results further emphasize the role of cytoskeletal defects in ALS and demonstrate the power of gene-based rare variant analyses in situations where causal genes cannot be identified through traditional segregation analysis.

Identification of mutations at familial loci for amyotrophic lateral sclerosis (ALS) has provided novel insights into the aetiology of this rapidly progressing fatal neurodegenerative disease. However, genome-wide association studies (GWAS) of the more common (∼90%) sporadic form have been less successful with the exception of the replicated locus at 9p21.2. To identify new loci associated with disease susceptibility, we have established the largest association study in ALS to date and undertaken a GWAS meta-analytical study combining 3959 newly genotyped Italian individuals (1982 cases and 1977 controls) collected by SLAGEN (Italian Consortium for the Genetics of ALS) together with samples from Netherlands, USA, UK, Sweden, Belgium, France, Ireland and Italy collected by ALSGEN (the International Consortium on Amyotrophic Lateral Sclerosis Genetics). We analysed a total of 13 225 individuals, 6100 cases and 7125 controls for almost 7 million single-nucleotide polymorphisms (SNPs). We identified a novel locus with genome-wide significance at 17q11.2 (rs34517613 with P = 1.11 × 10(-8); OR 0.82) that was validated when combined with genotype data from a replication cohort (P = 8.62 × 10(-9); OR 0.833) of 4656 individuals. Furthermore, we confirmed the previously reported association at 9p21.2 (rs3849943 with P = 7.69 × 10(-9); OR 1.16). Finally, we estimated the contribution of common variation to heritability of sporadic ALS as ∼12% using a linear mixed model accounting for all SNPs. Our results provide an insight into the genetic structure of sporadic ALS, confirming that common variation contributes to risk and that sufficiently powered studies can identify novel susceptibility loci.

Expansion of a GGGGCC repeat (RE) in the C9orf72 gene has been recently reported as the main genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Given the growing evidence of genetic and clinicopathologic overlap among ALS, FTD, and other neurodegenerative diseases, we investigated the occurrence of RE in a subset of 9 patients with ALS-plus syndromes, including Parkinson's disease (PD), progressive supranuclear palsy (PSP), corticobasal syndrome (CBS), and multiple system atrophy. We identified RE in 2 ALS-plus individuals (22.2%) displaying PSP and CBS features. On the basis of this finding, we extended our analysis to a cohort composed of 190 PD, 103 CBS, 107 PSP, and 177 Alzheimer's disease cases. We did not identify any RE in these patients, indicating that C9orf72 is in all probability not involved in the pathogenesis of these disorders. However, the high frequency of C9orf72 RE in patients with ALS-plus syndromes suggests that, similar to ALS-FTD patients, individuals with combined motor neuron and extrapyramidal features should be screened for RE, independent of their family history., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mutations in the profilin 1 (PFN1) gene, encoding a protein regulating filamentous actin growth through its binding to monomeric G-actin, have been recently identified in familial amyotrophic lateral sclerosis (ALS). Functional studies performed on ALS-associated PFN1 mutants demonstrated aggregation propensity, alterations in growth cone, and cytoskeletal dynamics. Previous screening of PFN1 gene in sporadic ALS (SALS) cases led to the identification of the p.E117G mutation, which is likely to represent a less pathogenic variant according to both frequency data in control subjects and cases, and functional experiments. To determine the effective contribution of PFN1 mutations in SALS, we analyzed a large cohort of 1168 Italian SALS patients and also included 203 frontotemporal dementia (FTD) cases because of the great overlap between these 2 neurodegenerative diseases. We detected the p.E117G variant in 1 SALS patient and the novel synonymous change p.G15G in another patient, but none in a panel of 1512 control subjects. Our results suggest that PFN1 mutations in sporadic ALS and in FTD are rare, at least in the Italian population., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objectives: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease mainly involving cortical and spinal motor neurones. Molecular studies have recently identified different mutations in the  ubiquilin-2 (UBQLN2) gene as causative of a familial form of X-linked ALS, 90% penetrant in women. The aim of our study was to analyse the UBQLN2 gene in a large cohort of patients with familial (FALS) and sporadic (SALS) amyotrophic lateral sclerosis, with or without frontotemporal dementia (FTD), and in patients with FTD., Methods: We analysed the UBQLN2 gene in 819 SALS cases, 226 FALS cases, 53 ALS-FTD patients, and 63 patients with a clinical record of FTD. Molecular analysis of the entire coding sequence was carried out in all FALS and ALS-FTD patients, while SALS and FTD patients were analysed specifically for the genomic region coding for the PXX repeat tract. Healthy controls were 845 anonymous blood donors and were screened for the PXX repeat region only., Results: We found five different variants in the UBQLN2 gene in five unrelated ALS patients. Three variants, including two novel ones, involved a proline residue in the PXX repeat region and were found in three FALS cases. The other two were novel variants, identified in one FALS and one SALS patient. None of these variants was present in controls, while one control carried a new heterozygous variant., Conclusions: Our data support the role of the UBQLN2 gene in the pathogenesis of FALS, being conversely a rare genetic cause in SALS even when complicated by FTD.

In amyotrophic lateral sclerosis (ALS) cerebrospinal fluid (CSF) analysis is usually performed to exclude inflammatory processes of the central nervous system. Although in a small subset of patients an intrathecal synthesis of IgG is detectable, usually there is no clear explanation for this evidence. This study investigates the occurrence of oligoclonal bands (OCBs) in the CSF of a large series of ALS patients, attempting a correlation with genotype data. CSF was collected from 259 ALS patients. CSF parameters were measured according to standard procedures, and detection of OCBs performed by isoelectric focusing. The patients were screened for mutations in SOD1, FUS, TARDBP, ANG, OPTN, and C9ORF72. We observed the presence of OCBs in the CSF of 9/259 ALS patients (3.5 %), and of disease-associated mutations in 12 cases. OCBs were significantly more frequent in mutation carriers compared to the remaining cohort (3/12 vs 6/247; p < 0.01). Among patients with OCBs, two patients had the TARDBP p.A382T mutation (one of which in homozygous state), and one the ANG p.P-4S variant. Both patients carrying the p.A382T mutation had an atypical phenotype, one of them manifesting signs suggestive of a cerebellar involvement, and the other presenting neuroradiological findings suggestive of an inflammatory disorder of the central nervous system. Our results suggest that ALS patients with OCBs may harbor mutations in disease-causing genes. We speculate that mutations in both TARDBP and ANG genes may disrupt the blood-brain barrier (BBB), promoting local immune responses and neuroinflammation. The role of mutant TARDBP and ANG genes on BBB integrity of ALS patients warrants further investigation.

A hexanucleotide repeat expansion (RE) in C9ORF72 gene was recently reported as the main cause of amyotrophic lateral sclerosis (ALS) and cases with frontotemporal dementia. We screened C9ORF72 in a large cohort of 259 familial ALS, 1275 sporadic ALS, and 862 control individuals of Italian descent. We found RE in 23.9% familial ALS, 5.1% sporadic ALS, and 0.2% controls. Two cases carried the RE together with mutations in other ALS-associated genes. The phenotype of RE carriers was characterized by bulbar-onset, shorter survival, and association with cognitive and behavioral impairment. Extrapyramidal and cerebellar signs were also observed in few patients. Genotype data revealed that 95% of RE carriers shared a restricted 10-single nucleotide polymorphism haplotype within the previously reported 20-single nucleotide polymorphism risk haplotype, detectable in only 27% of nonexpanded ALS cases and in 28% of controls, suggesting a common founder with cohorts of North European ancestry. Although C9ORF72 RE segregates with disease, the identification of RE both in controls and in patients carrying additional pathogenic mutations suggests that penetrance and phenotypic expression of C9ORF72 RE may depend on additional genetic risk factors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.

Mutations in valosin-containing protein (VCP) gene, already known to be associated with the multisystemic disorder, inclusion body myopathy with Paget's disease and frontotemporal dementia (IBMPFD), have been recently found also in familial cases of amyotrophic lateral sclerosis (ALS). To further define the frequency of VCP mutations in ALS Italian population, we screened a cohort of 166 familial ALS and 14 ALS-frontotemporal dementia (FTD) individuals. We identified a previously reported synonymous mutation (c.2093A>C; p.Q568Q), 2 intronic variants (c.1749-14C>T; c.2085-3C>T), and 1 nucleotide change (c.2814G>T) in the 3' untranslated region (UTR). Bioinformatical analyses predicted no changes in splicing process or microRNA binding sites. Our results do not confirm a main contribution of VCP gene to familial ALS in the Italian population., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We have attempted to replicate a recently reported association of polymorphism rs10260404, in the Dipeptidyl-peptidase 6 gene (DPP6), with susceptibility to amyotrophic lateral sclerosis (ALS) in a large independent Italian cohort of 904 cases and 1036 controls. Minor allele frequency was 0.38 in cases and 0.39 in controls and no evidence of association with ALS was observed (P=0.638). Our negative results agree with those recently reported in additional Polish and Italian cohorts., (Copyright © 2009 Elsevier Inc. All rights reserved.)

Several RNA-processing genes have been implicated in the pathogenesis of Amyotrophic lateral sclerosis (ALS). In particular, causative mutations in the genes encoding for two DNA/RNA binding proteins, TAR DNA binding protein-43 (TDP-43) and fused in sarcoma/translocated in liposarcoma (FUS/TLS), were recently identified in ALS patients. These genetic findings and the presence of abnormal aggregates of these two RNA-binding proteins in ALS affected tissues suggest that molecular mechanisms regulating RNA metabolism are implicated in ALS pathogenesis through common pathways. In this review similarities and differences between TDP-43 and FUS/TLS proteins and their activities in physiological and pathological conditions will be discussed.

Amyotrophic lateral sclerosis (ALS) is a late onset, rapidly progressive and ultimately fatal neurodegenerative disease, caused by the loss of motor neurons in the brain and spinal cord. About 10% of all ALS cases are familial (FALS), and constitute a clinically and genetically heterogeneous entity. To date, FALS has been linked to mutations in 10 different genes and to four additional chromosomal loci. Research on FALS genetics, and in particular the discoveries of mutations in the SOD1, TARDBP, and FUS genes, has provided essential information toward the understanding of the pathogenesis of ALS in general. This review presents a tentative classification of all FALS-associated genes identified so far.

Dysfunction of the ubiquitin-proteasome system has recently been implicated in the pathogenesis of some untreatable myodegenerative diseases characterized by the formation of ubiquitinated inclusions in skeletal muscles. We have developed an in vitro model of proteasomal dysfunction by applying inhibitors of the proteasome to primary adult human skeletal muscle cultures. Our data show that proteasome inhibition causes both cytoplasmic accumulation of ubiquitinated inclusions and apoptotic death, the latter through accumulation of active caspase-3.