Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS), characterized by a neurodegenerative component that leads to a progressive dysfunction of the nervous system, as well as a neuroinflammatory phase that corresponds to inflammatory attacks to the myelin sheath in the CNS by immune cells. Mitochondrial damage has already been evidenced in patients affected by primary progressive multiple MS (PPMS). Therefore it is hypothesized that dysfunctional mitochondria could lead to a higher risk of disease progression in MS patients. This study investigates the presence of alterations in genes of the mitochondrial DNA (mtDNA) that potentially have an impact on mitochondrial functionality, favoring impairment of mitochondria and resulting in neurodegeneration. A cohort of 279 patients, 113 diagnosed with benign relapsing-remitting multiple sclerosis (RRMS) and 166 diagnosed with primary progressive multiple sclerosis (PPMS), was included in the study. The DNA from each patient was extracted and subsequently mtDNA was amplified performing two PCR reactions, obtaining two mitochondrial amplicons for each sample. Starting from the two obtained amplicons libraries were prepared. The mtDNA from each library was sequenced with Next Generation Sequencing (NGS) approach, and after variant calling pipeline genetic variations in the mtDNA were identified. We focused our attention on single-nucleotide variants enriched in PPMS or RRMS predicted to be pathogenic according to multiple annotation datasets. Findings support a genetic alteration of subunits of the mitochondrial electron transport chain, which is essential for cellular respiration and energy production. We can speculate that these alterations lead in a decreased energy production favoring neurodegenerative processes typical of the progressive phase of multiple sclerosis. Further functional analysis will be required in order to evaluate the impact of the identified variants on mitochondrial function, to strengthen the results and consequently increase our knowledge on the pathogenesis of disease progression in multiple sclerosis.
Valutazione del coinvolgimento del danno mitocondriale nella sclerosi multipla primariamente progressiva mediante sequenziamento del DNA mitocondriale. La sclerosi multipla (SM) è una malattia cronica infiammatoria autoimmune del sistema nervoso centrale, caratterizzata da una componente neurodegenerativa che porta ad una progressiva disfunzione del sistema nervoso, e da una fase neuroinfiammatoria che corrisponde agli attacchi infiammatori alla guaina mielinica nel sistema nervoso centrale da parte delle cellule immunitarie. Il danno mitocondriale è già stato evidenziato in pazienti affetti dalla sclerosi multipla primariamente progressiva (SMPP). È quindi ipotizzato che mitocondri danneggiati e non funzionali potrebbero portare ad un aumentato rischio di progresso della malattia nei pazienti con sclerosi multipla. In questo studio viene investigata la presenza di alterazioni nei geni del DNA mitocondriale (mtDNA) che hanno un potenziale impatto sulla funzionalità del mitocondrio, favorendo il danno mitocondriale e causando il processo neurodegenerativo. Una coorte di 279 pazienti, 113 diagnosticati con una forma benigna di sclerosi multipla recidivante-remittente (SMRR) e 166 con una diagnosi di sclerosi multipla primariamente progressiva, è stata inclusa nel presente studio. Il DNA di ogni paziente è stato estratto e successivamente il DNA mitocondriale è stato amplificato con due reazioni di PCR, ottenendo due ampliconi mitocondriali per ogni campione. Partendo dai due ampliconi ottenuti sono state preparate le librerie. Il DNA mitocondriale in ogni libreria è stato sequenziato mediante l’approccio di Next Generation Sequencing (NGS), e dopo la chiamata delle varianti è stato possibile identificare le varianti genetiche presenti nel DNA mitocondriale. Ci siamo focalizzati sulle varianti a singolo nucleotide arricchite nei pazienti PP e RR previste come patogenetiche secondo vari datasets di annotazione. I risultati supportano una alterazione genetica delle subunità della catena di trasporto degli elettroni nel mitocondrio, essenziale per il processo di respirazione cellulare e la produzione di energia. Possiamo ipotizzare che queste alterazioni portino ad una diminuzione nella produzione di energia favorendo i processi neurodegenerativi tipici della fase progressiva della sclerosi multipla. Ulteriori analisi funzionali sono necessarie per valutare l’impatto delle varianti identificate sulla funzionalità del mitocondrio, per rafforzare i risultati e di conseguenza aumentare la nostra conoscenza sulla patogenesi della progressione di malattia nella sclerosi multipla.
Assessment of the involvement of mitochondrial impairment in primary progressive multiple sclerosis through sequencing of mitochondrial DNA.
DOYLE, FRANCESCA AISLINN
2021/2022
Abstract
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS), characterized by a neurodegenerative component that leads to a progressive dysfunction of the nervous system, as well as a neuroinflammatory phase that corresponds to inflammatory attacks to the myelin sheath in the CNS by immune cells. Mitochondrial damage has already been evidenced in patients affected by primary progressive multiple MS (PPMS). Therefore it is hypothesized that dysfunctional mitochondria could lead to a higher risk of disease progression in MS patients. This study investigates the presence of alterations in genes of the mitochondrial DNA (mtDNA) that potentially have an impact on mitochondrial functionality, favoring impairment of mitochondria and resulting in neurodegeneration. A cohort of 279 patients, 113 diagnosed with benign relapsing-remitting multiple sclerosis (RRMS) and 166 diagnosed with primary progressive multiple sclerosis (PPMS), was included in the study. The DNA from each patient was extracted and subsequently mtDNA was amplified performing two PCR reactions, obtaining two mitochondrial amplicons for each sample. Starting from the two obtained amplicons libraries were prepared. The mtDNA from each library was sequenced with Next Generation Sequencing (NGS) approach, and after variant calling pipeline genetic variations in the mtDNA were identified. We focused our attention on single-nucleotide variants enriched in PPMS or RRMS predicted to be pathogenic according to multiple annotation datasets. Findings support a genetic alteration of subunits of the mitochondrial electron transport chain, which is essential for cellular respiration and energy production. We can speculate that these alterations lead in a decreased energy production favoring neurodegenerative processes typical of the progressive phase of multiple sclerosis. Further functional analysis will be required in order to evaluate the impact of the identified variants on mitochondrial function, to strengthen the results and consequently increase our knowledge on the pathogenesis of disease progression in multiple sclerosis.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.
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https://hdl.handle.net/20.500.14239/14984