Cerebellar and Brainstem Congenital Defects (CBCDs) are an heterogenous group of pathologies that arise from alteration of the posterior cranial fossa during embryonic development. Among CBCDs, Joubert Syndrome (JS) is one of the most common hereditary forms: it is a neurodevelopmental ciliopathy, with predominantly recessive inheritance, characterized by a peculiar cerebellar and brainstem malformation known as molar tooth sign (MTS). Thanks to Next Generation Sequencing (NGS) platforms, more than 40 JS causative genes have been identified, all encoding proteins of the primary cilium. Among these genes, the most frequently mutated are AHI1, CC2D2A, CEP290, CPLANE1, and TMEM67, accounting for half of JS cases. NGS technologies additionally aided in an increased identification of variants in disease-associated genes, however some of these variants are classified as "of uncertain significance" (VUS) according to American College of Medical Genetics (ACMG) criteria, leading to an inconclusive genetic diagnosis and complicating the understanding of syndrome pathogenesis. This thesis project concentrated on the evaluation of the pathogenic impact of missense variants found in JS patients, with the aim of assessing their phenotypic variability and obtain valuable insights to potentially reclassify VUS as pathogenic or benign. In detail, this thesis focused on CC2D2A gene variants, whose impact on the primary cilium phenotype was investigated thanks to a knockdown (KD) IMCD3 cell model obtained through lentiviral infection with an shRNA targeting CC2D2A. The aim was to establish a stably silenced cell model for transfection with mutagenized plasmids carrying variants of interest, in order to evaluate the variant impact on the primary cilium phenotype. Mutagenesis was performed on a large plasmid (over 10 kb) containing the canonical isoform of human CC2D2A transcript to produce mutagenized plasmids carrying one of the following missense variants: c.3509G>A (benign), c.3364C>T (likely pathogenic), c.3856T>C (VUS), c.4220A>G (VUS). Overall, the information obtained with this analysis can enhance our understanding of CC2D2A in JS pathology and will likely increase the detection rate of related genetic variants. Additionally, this data will enhance our capability to provide patients with precise genetic information and assess the risk of recurrence for carrier parents.
I difetti congeniti del cervelletto e del tronco encefalico (CBCDs) sono un gruppo eterogeneo di patologie causate da alterazioni della fossa cranica posteriore durante lo sviluppo embrionale. Tra i CBCDs, la Sindrome di Joubert (JS) è una delle forme ereditarie più comuni: una ciliopatia del neurosviluppo con ereditarietà prevalentemente recessiva, caratterizzata dal segno del dente molare (MTS), una malformazione del cervelletto e del tronco encefalico. Grazie a piattaforme di Next Generation Sequencing (NGS) oltre 40 geni causativi della JS sono stati identificati, tutti codificanti per proteine del cilio primario. I geni mutati più frequentemente sono AHI1, CC2D2A, CEP290, CPLANE1 e TMEM67, responsabili di circa la metà dei casi di JS. Le tecnologie NGS permettono inoltre l'identificazione di varianti in geni associati alla patologia; tuttavia, alcune di queste varianti sono classificate come "di significato incerto" (VUS) secondo i criteri dell'American College of Medical Genetics (ACMG), portando a diagnosi genetiche inconcludenti e complicando la comprensione della patogenesi della sindrome. Questo progetto di tesi si è concentrato sulla valutazione della patogenicità di varianti missenso riscontrate in pazienti affetti da JS, con l'obiettivo di valutarne la variabilità fenotipica e ottenere informazioni per potenzialmente riclassificare le VUS come patogeniche o benigne. In particolare, la tesi si è focalizzata su varianti del gene CC2D2A, il cui impatto sul fenotipo del cilio primario è stato valutato tramite un modello cellulare IMCD3 knockdown (KD) ottenuto tramite infezione lentivirale con shRNA mirato a silenziare CC2D2A. L’obiettivo era quello di creare un modello cellulare stabilmente silenziato in cui andare ad overesprimere plasmidi mutagenizzati contenenti varianti di interesse, al fine di valutare l'impatto delle varianti sul fenotipo del cilio primario. La mutagenesi è stata eseguita su un plasmide di grandi dimensioni (oltre 10 kb), contenente l’isoforma canonica del trascritto umano di CC2D2A, al fine di ottenere plasmidi contenenti una delle seguenti varianti missenso: c.3509G>A (benigna), c.3364C>T (probabilmente patogenica), c.3856T>C (VUS), c.4220A>G (VUS). Le informazioni ottenute da questa analisi possono migliorare la comprensione del ruolo di CC2D2A nella patologia della JS e aumentare il tasso di rilevamento delle varianti genetiche correlate. Inoltre, questi dati miglioreranno la nostra capacità di fornire ai pazienti informazioni genetiche precise e valutare il rischio di ricorrenza per genitori portatori.
Caratterizzazione funzionale di varianti missenso nel gene CC2D2A in pazienti affetti da Sindrome di Joubert: analisi del cilio primario tramite un modello knockdown in vitro
DAPELO, MIRIAM
2023/2024
Abstract
Cerebellar and Brainstem Congenital Defects (CBCDs) are an heterogenous group of pathologies that arise from alteration of the posterior cranial fossa during embryonic development. Among CBCDs, Joubert Syndrome (JS) is one of the most common hereditary forms: it is a neurodevelopmental ciliopathy, with predominantly recessive inheritance, characterized by a peculiar cerebellar and brainstem malformation known as molar tooth sign (MTS). Thanks to Next Generation Sequencing (NGS) platforms, more than 40 JS causative genes have been identified, all encoding proteins of the primary cilium. Among these genes, the most frequently mutated are AHI1, CC2D2A, CEP290, CPLANE1, and TMEM67, accounting for half of JS cases. NGS technologies additionally aided in an increased identification of variants in disease-associated genes, however some of these variants are classified as "of uncertain significance" (VUS) according to American College of Medical Genetics (ACMG) criteria, leading to an inconclusive genetic diagnosis and complicating the understanding of syndrome pathogenesis. This thesis project concentrated on the evaluation of the pathogenic impact of missense variants found in JS patients, with the aim of assessing their phenotypic variability and obtain valuable insights to potentially reclassify VUS as pathogenic or benign. In detail, this thesis focused on CC2D2A gene variants, whose impact on the primary cilium phenotype was investigated thanks to a knockdown (KD) IMCD3 cell model obtained through lentiviral infection with an shRNA targeting CC2D2A. The aim was to establish a stably silenced cell model for transfection with mutagenized plasmids carrying variants of interest, in order to evaluate the variant impact on the primary cilium phenotype. Mutagenesis was performed on a large plasmid (over 10 kb) containing the canonical isoform of human CC2D2A transcript to produce mutagenized plasmids carrying one of the following missense variants: c.3509G>A (benign), c.3364C>T (likely pathogenic), c.3856T>C (VUS), c.4220A>G (VUS). Overall, the information obtained with this analysis can enhance our understanding of CC2D2A in JS pathology and will likely increase the detection rate of related genetic variants. Additionally, this data will enhance our capability to provide patients with precise genetic information and assess the risk of recurrence for carrier parents.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28413