Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder (ND) characterized by heterogeneous clinical manifestations due to both genetic and phenotypic overlapping with other NDs, such as Frontotemporal dementia (FTD), Hereditary spastic paraplegia (HSP), Parkinson’s disease (PD) and other motor neuron diseases. NDs and particularly ALS can be considered as polygenic diseases, characterized either by oligogenic inheritance and genetic pleiotropy because a phenotypic trait is determined by more than one gene and even a single gene has multiple phenotypic manifestations. The aim of this work was to study the genetic architecture of sporadic ALS (sALS) patients to emphasize the importance of the genetic contribution in the disease onset. To do this, we took into consideration both pathogenic and genetic risk genes already associated to ALS. Additionally, we also analysed other genes involved in NDs and motor neuron diseases, principally FTD, HSP and PD, to investigate which is the relevance of comorbidity in the disease onset and to examine if variants in these genes may be helpful to clinicians in explaining atypical clinical presentations. An Italian cohort, of 80 sporadic ALS patients, was investigated through Whole Exome Sequencing. All the variants obtained were further investigated, analysing, for each sample, a panel of 277 genes related to ALS, FTD, HSP and PD. Among the 80 sALS cases analysed in this study, 97,5% presented at least one variant in the genes we took into consideration. Following the ACMG criteria, variants were prioritized according to their pathogenicity: eight (3,5%) mutations were found in ten patients and classified as pathogenic, while 12 (5,2%) variants, observed in eleven patients, were classified as likely pathogenic. 212 mutations (91,4%) were instead variants with uncertain significance (VUS). This cohort of ALS patients revealed a heterogeneous genetic architecture, with variants in several rarely mutated genes, especially in those genes which have been linked to other neurodegenerative disorders, underlying the genetic overlapping between neurodegenerative conditions. Interestingly, among the pathogenic and likely pathogenic variants, only six were identified in ALS causative genes, while the other fourteen mutations were found in genes associated to diverse HSP, PD and other NDs. Beyond NGS analysis for the detection of point mutations and small insertions and/or deletions, we analysed our cohort of sALS patients also for the hexanucleotide repeat expansion GGGGCC (G4C2)n within the C9ORF72 gene, which is the most common genetic cause of ALS so far. The 3,7% of the Italian cohort examined presented a pathogenic hexanucleotide repeat expansion as principal genetic alterations since none of these patients carried pathogenic or likely pathogenic variants in ALS causative genes. This experimental thesis has reaffirmed the heterogeneity of ALS-causative genes and risk factors and has highlighted the importance of genetic comorbidity studies. Additionally, this study has validated and emphasized the key role of genetics in the study of ALS, for the identification of pathogenic variants causative of the disease, but also all the genetic factors linked either to ALS and other NDs that predispose to and may explain not only the “classic” clinical phenotypes, but especially more complex phenotypes. For this reason, this experimental thesis fits into the context of works that evaluate how genetics and onset of NDs are strictly interlinked. Further analyses will be needed to confirm our results.
La sclerosi laterale amiotrofica (SLA) è una malattia neurodegenerativa progressiva caratterizzata dalla degenerazione dei motoneuroni, le cellule nervose cerebrali e del midollo spinale che permettono i movimenti della muscolatura volontaria. Negli ultimi anni la SLA ha iniziato ad esser considerata una malattia poligenica causata da molteplici varianti rare con effetti additivi o sinergici sulla presentazione della malattia, caratterizzata da eterogenee manifestazioni cliniche dovute sia alla sovrapposizione genetica sia fenotipica con altre malattie neurodegenerative, come FTD, HSP e PD. L'obiettivo di questo lavoro è quello di studiare l'architettura genetica di pazienti SLA sporadici, al fine di sottolineare l'importanza del contributo genetico nell'insorgenza della malattia. Per far questo, abbiamo preso in considerazione sia i geni causativi, sia i fattori di rischio già associati alla SLA; inoltre, abbiamo analizzato tutti i fattori genetici coinvolti in altre malattie neurodegenerative, principalmente FTD, HSP e PD, per studiare quale sia l'importanza della comorbidità nell'insorgenza della malattia e per esaminare se le varianti in questi geni possano essere utili ai medici al fine di chiarire atipiche presentazioni cliniche. Una coorte italiana di 80 pazienti SLA sporadici è stata reclutata dall' IRCCS "Fondazione Mondino" (Pavia, Italia). Per questo studio, è stata applicata la tecnologia WES, che consente l'analisi di tutte le regioni codificanti del genoma, con un output di oltre centomila varianti per paziente. Motivo per il quale, tutte le varianti ottenute sono state ulteriormente esaminate, analizzando per ogni campione un pannello di 277 geni, associati a SLA, FTD, HSP and PD e altri disturbi neuromuscolari. Tra gli 80 pazienti sporadici esaminati in questo studio, il 97,5% presenta almeno una variante nei geni presi in considerazione. Secondo i criteri ACMG, le varianti sono state prioritizzate a seconda della loro patogenicità: otto (3,5%) mutazioni sono state trovate in 10 pazienti e classificate come patogenetiche, invece 12 (5,2%) varianti, osservate in undici pazienti, sono state classificate come verosimilmente patogenetiche. 212 varianti (91,4%) sono invece mutazioni dal significato incerto. Questa coorte di pazienti SLA ha rivelato un'eterogenea architettura genetica, con varianti in diversi geni raramente mutati, specialmente in quei geni che sono stati collegati ad altri disturbi neurodegenerativi, sottolineando la sovrapposizione genetica tra le condizioni neurodegenerative. Interessante notare che tra le varianti patogenetiche e verosimilmente patogenetiche, solamente sei sono state identificate in geni SLA causativi (TARDBP, SOD1, FIG4, OPTN, NEK1), quattordici mutazioni, invece, sono state descritte in geni associati ad altri disturbi neurodegenerativi, inclusi HSP e PD. In aggiunta all’analisi NGS per il rilevamento di mutazioni puntiformi e piccole inserzioni e/o delezioni, la nostra coorte di pazienti SLA sporadici è stata analizzata anche per l’individuazione dell'espansione esanucleotidica GGGGCC all’interno del gene C9ORF72. Il 3,7% della coorte italiana esaminata presenta un'espansione patogenetica della ripetizione esanucleotidica come principale alterazione, poiché nessuno di questi pazienti esibisce varianti patogenetiche o probabilmente patogenetiche in geni SLA causativi. Questa tesi sperimentale tende a riaffermare l’eterogeneità dei geni SLA causativi e dei fattori di rischio, sottolineando l’importanza degli studi sulla comorbidità genetica. Tra l’altro, questo studio ha validato ed enfatizzato il ruolo chiave della genetica nello studio della SLA, per l’identificazione di varianti patogenetiche causative della malattia, ma anche di tutti i fattori di rischio associati sia alla SLA sia ad altri disturbi neurodegenerativi, che predispongono e possono spiegare non solo i fenotipi classici, ma specialmente i fenotipi più complessi.
Sequenziamento dell'esoma per elucidare il contributo genetico nella diagnosi della Sclerosi Laterale Amiotrofica
CONTI, SILVIA
2019/2020
Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder (ND) characterized by heterogeneous clinical manifestations due to both genetic and phenotypic overlapping with other NDs, such as Frontotemporal dementia (FTD), Hereditary spastic paraplegia (HSP), Parkinson’s disease (PD) and other motor neuron diseases. NDs and particularly ALS can be considered as polygenic diseases, characterized either by oligogenic inheritance and genetic pleiotropy because a phenotypic trait is determined by more than one gene and even a single gene has multiple phenotypic manifestations. The aim of this work was to study the genetic architecture of sporadic ALS (sALS) patients to emphasize the importance of the genetic contribution in the disease onset. To do this, we took into consideration both pathogenic and genetic risk genes already associated to ALS. Additionally, we also analysed other genes involved in NDs and motor neuron diseases, principally FTD, HSP and PD, to investigate which is the relevance of comorbidity in the disease onset and to examine if variants in these genes may be helpful to clinicians in explaining atypical clinical presentations. An Italian cohort, of 80 sporadic ALS patients, was investigated through Whole Exome Sequencing. All the variants obtained were further investigated, analysing, for each sample, a panel of 277 genes related to ALS, FTD, HSP and PD. Among the 80 sALS cases analysed in this study, 97,5% presented at least one variant in the genes we took into consideration. Following the ACMG criteria, variants were prioritized according to their pathogenicity: eight (3,5%) mutations were found in ten patients and classified as pathogenic, while 12 (5,2%) variants, observed in eleven patients, were classified as likely pathogenic. 212 mutations (91,4%) were instead variants with uncertain significance (VUS). This cohort of ALS patients revealed a heterogeneous genetic architecture, with variants in several rarely mutated genes, especially in those genes which have been linked to other neurodegenerative disorders, underlying the genetic overlapping between neurodegenerative conditions. Interestingly, among the pathogenic and likely pathogenic variants, only six were identified in ALS causative genes, while the other fourteen mutations were found in genes associated to diverse HSP, PD and other NDs. Beyond NGS analysis for the detection of point mutations and small insertions and/or deletions, we analysed our cohort of sALS patients also for the hexanucleotide repeat expansion GGGGCC (G4C2)n within the C9ORF72 gene, which is the most common genetic cause of ALS so far. The 3,7% of the Italian cohort examined presented a pathogenic hexanucleotide repeat expansion as principal genetic alterations since none of these patients carried pathogenic or likely pathogenic variants in ALS causative genes. This experimental thesis has reaffirmed the heterogeneity of ALS-causative genes and risk factors and has highlighted the importance of genetic comorbidity studies. Additionally, this study has validated and emphasized the key role of genetics in the study of ALS, for the identification of pathogenic variants causative of the disease, but also all the genetic factors linked either to ALS and other NDs that predispose to and may explain not only the “classic” clinical phenotypes, but especially more complex phenotypes. For this reason, this experimental thesis fits into the context of works that evaluate how genetics and onset of NDs are strictly interlinked. Further analyses will be needed to confirm our results.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/11915