EXOME SEQUENCING APPROACH TO IDENTIFY CAUSATIVE GENES FOR AMYOTROPHIC LATERAL SCLEROSIS

Introduzione: La Sclerosi laterale amiotrofica (SLA) \ue8 una malattia neurodegenerativa progressiva e fatale caratterizzata dalla perdita selettiva dei motoneuroni nella corteccia cerebrale, nel tronco cerebrale e midollo spinale. La maggior parte dei casi \ue8 costituita da forme sporadiche, mentre solo il 5-10% dei casi \ue8 rappresentato da forme familiari, causate da geni con modalit\ue0 di trasmissione mendeliana, generalmente autosomica dominante. Sono stati identificati pi\uf9 di 20 geni causativi delle forme familiari, che hanno contribuito a comprendere meglio i meccanismi patogenetici coinvolti e sottolineano la grande eterogeneit\ue0 genetica della malattia. Nonostante i numerosi progressi raggiunti, in circa il 40% dei casi familiari la causa genetica non \ue8 stata ancora identificata, mentre la componente genetica delle forme sporadiche \ue8 in gran parte sconosciuta. L\u2019 applicazione delle tecniche di sequenziamento di nuova generazione ed in particolare il sequenziamento della porzione codificante del genoma o esoma rappresenta un approccio innovativo e promettente per gli studi genetici sulla SLA. Lo scopo del presente progetto di ricerca di Dottorato \ue8 stato quello di identificare nuovi geni associati alle forme familiari e sporadiche di SLA mediante sequenziamento dell\u2019esoma come metodo alternativo per superare i limiti delle tecniche genetiche tradizionali. Metodi: Sono state utilizzate tre diverse strategie per l\u2019identificazione di geni causativi: I) sequenziamento dell\u2019esoma in combinazione con analisi di linkage in due grandi famiglie SLA a trasmissione dominante; II) analisi per varianti rare degli esomi di 363 casi familiari singoli; III) sequenziamento dell\u2019esoma in 32 casi di SLA sporadica e dei loro genitori non affetti (approccio dei trios). Risultati: I) Attraverso l\u2019approccio combinato di sequenziamento dell\u2019esoma ed analisi di linkage, abbiamo identificato il gene PFN1 (profilina-1), codificante per una proteina implicata nella regolazione dell\u2019actina, come nuovo gene causativo di SLA. Mutazioni a carico del gene PFN1 sono state osservate nel 2,6% dei pazienti SLA familiari e gli studi funzionali condotti sui mutanti hanno dimostrato una maggiore tendenza all\u2019aggregazione, una riduzione del legame all\u2019actina ed un effetto inibitorio sulla crescita assonale. II) L\u2019analisi delle varianti rare tra casi e controlli, applicata su un totale di 12.495 geni, ha portato all\u2019identificazione di TUBA4A (codificante per lalfa-tubulina 4a) come gene candidato caratterizzato da un eccesso significativo di varianti rare potenzialmente dannose nei 363 casi familiari analizzati. L\u2019analisi funzionale ha dimostrato per i mutanti di TUBA4A una capacit\ue0 ridotta di dimerizzazione con la beta-tubulina in vitro ed un\u2019alterata incorporazione nei microtubuli in vivo. Inoltre, il mutante tronco TUBA4A p.W407X ha mostrato una maggiore tendenza all\u2019aggregazione. III) Infine, analizzando l\u2019esoma di 32 pazienti con SLA sporadica e dei loro genitori non affetti, abbiamo identificato 25 mutazioni de novo in 16 dei 32 trios analizzati, con un tasso di mutazioni de novo pari a 0,78. Non sono stati identificati geni con molteplici mutazioni de novo nei trios sequenziati, ma le analisi bioinformatiche hanno mostrato possibili connessioni tra i geni candidati e la classificazione funzionale ha rilevato che le mutazioni de novo sono principalmente a carico di geni codificanti per trasportatori o per proteine con attivit\ue0 regolatoria sulle GTPasi. Conclusioni: I risultati ottenuti hanno dimostrato che il sequenziamento dell\u2019esoma, applicato con specifiche strategie di studio e di analisi, \ue8 un approccio efficace per l\u2019identificazione di nuovi geni causativi nella SLA. La scoperta dei due geni PFN1 e TUBA4A, codificanti per proteine coinvolte nel processo di polimerizzazione dell\u2019actina e dei microtubuli, fornisce ulteriori evidenze a supporto del coinvolgimento del citoscheletro nella patogenesi della SLA.Introduction: Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder caused by the loss of motor neurons in the cerebral cortex, brainstem and spinal cord. ALS occurs prevalently as sporadic forms (SALS), but a small proportion of cases (5-10%) displays a positive family history (FALS), generally with an autosomal dominant pattern of inheritance. To date, more than 20 causative genes have been identified in FALS, providing fundamental insights into the pathogenic mechanisms and underlying the great genetic heterogeneity of the disease. Despite these numerous advances, the genetic basis of nearly 40% of FALS remains to be identified, while the genetic component of SALS is largely unknown. A powerful and innovative tool for genetic studies in ALS is represented by next-generation sequencing and in particular by the targeted sequencing of the coding part of the genome or exome. Aim of this research project was to identify novel genes associated to FALS and SALS by applying complementary approaches all based on exome sequencing, which overcomes the limitations of traditional genetic strategies. Methods: Three different disease gene identification strategies were applied: I) exome sequencing associated to linkage analysis in two large ALS dominant pedigrees; II) exome-wide rare variant burden analysis on 363 unrelated index FALS cases; III) exome sequencing of 32 SALS and their unaffected parents (trio-design). Results: I) By performing exome-sequencing in combination with linkage analysis, we identified PFN1 (profilin-1), encoding for a protein regulating actin dynamics, as a novel ALS-causative gene. Mutations in PFN1 were observed in ~2.6% of FALS and functional studies demonstrated aggregation propensity, reduction of actin binding ability and axonal outgrowth inhibition of mutant PFN1 proteins. II) As a result of the unbiased case-control rare variant analysis, applied on a total 12.495 genes, we identified TUBA4A (Tubulin, Alpha 4a) as candidate gene showing a statistically significant excess of rare damaging variants in 363 index FALS cases sequenced. Functional analysis revealed that ALS-related mutants were defective in forming alpha/beta tubulin dimers in vitro and in incorporating into microtubules in vivo. In addition, the truncated mutant TUBA4A p.W407X showed aggregation propensities. III) By sequencing the exomes of 32 SALS patients and their unaffected parents, we identified 25 de novo mutations (DNMs) in 16 of 32 trios, with an overall DNM rate of 0.78. Although we did not find recurrently mutated genes in our ALS trios, bionformatic analysis showed potential inter-connections between the candidate genes. Functional classification revealed that DNMs are enriched in genes encoding for proteins involved in transport and in GTPase regulatory activity. Conclusions: Our findings indicate that exome-sequencing, combined with different strategies for study design and data analysis, is an effective and successful approach for the identification of novel ALS causative genes. The identification of PFN1 and TUBA4A genes, encoding for proteins implicated, respectively, in actin polymerization and microtubule formation, further supports a major role of cytoskeletal defects in ALS pathogenesis

Evaluation of Feasibility and Impact of Attacks against the 6top Protocol in 6TiSCH Networks

The 6TiSCH architecture has been gaining attraction as a promising solution to ensure reliability and security for communication in applications for the Industrial Internet of Things (IIoT). While many different aspects of the architecture have been investigated in literature, an in-depth analysis of the security features included in its design is still missing. In this paper, we assess the security vulnerabilities of the 6top protocol, a core component of the 6TiSCH architecture for enabling network nodes to negotiate communication resources. Our analysis highlights two possible attacks against the 6top protocol that can impair network performance and reliability in a significant manner. To prove the feasibility of the attacks in practice, we implemented both of them on the Contiki-NG Operating System and tested their effectiveness on a simple deployment with three Zolertia RE-Mote sensor nodes. Also, we carried out a set of simulations using Cooja in order to assess their impact on larger networks. Our results show that both attacks reduce reliability in the overall network and increase energy consumption of the network nodes

Profilin1 E117G is a moderate risk factor for amyotrophic lateral sclerosis

This is a post-peer-review, pre-copyedit version of an article published in Neurogenetics. The final authenticated version is available online at: J Neurol Neurosurg Psychiatry 2014;85:506–508. doi:10.1136/jnnp-2013-306761Background Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative disorders that share significant clinical, pathological and genetic overlap and are considered to represent different ends of a common disease spectrum. Mutations in Profilin1 have recently been described as a rare cause of familial ALS. The PFN1 E117G missense variant has been described in familial and sporadic cases, and also found in controls, casting doubt on its pathogenicity. Interpretation of such variants represents a significant clinical-genetics challenge. Objective and results Here, we combine a screen of a new cohort of 383 ALS patients with multiple-sequence datasets to refine estimates of the ALS and FTD risk associated with PFN1 E117G. Together, our cohorts add up to 5118 ALS and FTD cases and 13 089 controls. We estimate a frequency of E117G of 0.11% in controls and 0.25% in cases. Estimated odds after population stratification is 2.44 (95% CI 1.048 to ∞, Mantel-Haenszel test p=0.036). Conclusions Our results show an association between E117G and ALS, with a moderate effect size.PF is funded by MRC/MNDA Lady Edith Wolfson Fellowship. EMCF is funded by the UK Motor Neuron Disease Association. PF and EMCF are funded by the UK Medical Research Council and the Thierry Latran Foundation

Association of Variants in the SPTLC1 Gene with Juvenile Amyotrophic Lateral Sclerosis

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.

NEK1 variants confer susceptibility to amyotrophic lateral sclerosis

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

Exome-wide Rare Variant Analysis Identifies TUBA4A Mutations Associated with Familial ALS

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

Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis.

We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40-64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington's disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered

Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy

Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins

Genetic correlation between amyotrophic lateral sclerosis and schizophrenia

A. Palotie on työryhmän Schizophrenia Working Grp Psychiat jäsen.We have previously shown higher-than-expected rates of schizophrenia in relatives of patients with amyotrophic lateral sclerosis (ALS), suggesting an aetiological relationship between the diseases. Here, we investigate the genetic relationship between ALS and schizophrenia using genome-wide association study data from over 100,000 unique individuals. Using linkage disequilibrium score regression, we estimate the genetic correlation between ALS and schizophrenia to be 14.3% (7.05-21.6; P = 1 x 10(-4)) with schizophrenia polygenic risk scores explaining up to 0.12% of the variance in ALS (P = 8.4 x 10(-7)). A modest increase in comorbidity of ALS and schizophrenia is expected given these findings (odds ratio 1.08-1.26) but this would require very large studies to observe epidemiologically. We identify five potential novel ALS-associated loci using conditional false discovery rate analysis. It is likely that shared neurobiological mechanisms between these two disorders will engender novel hypotheses in future preclinical and clinical studies.Peer reviewe

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

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.Peer reviewe