The advent of NGS has incredibly accelerated the step of gene discovery even in sporadic cases or very small families. In parallel, the development of web-based networks such as GeneMatcher (https://genematcher.org/statistics/), allows a researcher who identified a putative novel gene to share clinical and genetic data with the scientific community, to get easily in touch with other researchers around the world. My thesis project was based on the case report of two siblings, NG5393 and NG5394, affected by a not previously described neurodevelopmental syndrome characterized by optic nerve atrophy, spastic paraplegia, and epilepsy; at the MRI both siblings showed mild dilatation of the lateral ventricles, thin corpus callosum, and white matter changes. Given the negativity for L1CAM variants, a whole exome sequencing was performed, which identified a homozygous missense variant in FSD1L (c.409T>G, p.Leu137Val; M1). Sharing of clinical and genetic data through GeneMatcher led to the identification of one additional family, with similar phenotypes, carrying distinct pathogenic variants in the same gene (c.1251_1252insTAA, p.Thr418Ter; M2 and c.1228T>G, p.Phe410Val; M3). This family accounts for two Italian siblings, both deceased, of whom, only for one of them, 1655/19, the biological sample was available for study. FSD1L undergoes extensive alternative splicing, resulting in eight different isoforms, of whom isoform 206 is considered the canonical isoform. FSD1L presents three important domains (coiled-coil (COS), Fibronectin type III, and B30.2/SPRY) that are conserved and shared with its paralog FSD1, encoding for a centrosomal protein the FSD1 protein. Interestingly, the Fibronectin type III domain is also shared with L1CAM, a transmembrane molecule of adhesion whose alteration causes a neurodevelopmental syndrome including hydrocephalus and hypoplasia of the corpus callosum. To date, the function of the FSD1L gene is still unknown. To verify its correlation with the complex phenotype observed, induced Pluripotent Stem Cell (iPSC) previously reprogrammed from patient and control fibroblasts were differentiated into small molecule Neural Precursor Cells (smNPCs), a type of neural precursor that can be further differentiated into various subtypes of neurons. The characterization of smNPCs was accessed by Real-time PCR and Immunofluorescence analysis, ensuring the purity of the cell population obtained. The in vitro generation of smNPCs is a viable approach, to create a patient-specific cell model that is as suitable as possible for the study of the neurodevelopmental pathology under investigation. The experiments conducted in this thesis have helped develop an amount of data, that has laid the groundwork for conducting further functional studies aimed at reaching a complete understanding of the gene function and the causative pathogenetic mechanism of brain malformation.
L'avvento di NGS ha incredibilmente accelerato la fase di scoperta di geni anche in casi sporadici o in famiglie molto piccole. Parallelamente, lo sviluppo di reti basate sul web come GeneMatcher (https://genematcher.org/statistics/), consente a un ricercatore che ha identificato un presunto gene nuovo di condividere dati clinici e genetici con la comunità scientifica, per entrare facilmente in contatto con altri ricercatori in tutto il mondo. Il mio progetto di tesi era basato sul caso di due fratelli, NG5393 e NG5394, affetti da una sindrome del neurosviluppo non descritta in precedenza caratterizzata da atrofia del nervo ottico, paraplegia spastica ed epilessia; alla risonanza magnetica entrambi i fratelli hanno mostrato una lieve dilatazione dei ventricoli laterali, corpo calloso assottigliato, e alterazioni della sostanza bianca. Data la negatività per le varianti L1CAM, è stato eseguito un sequenziamento dell’intero esoma, che ha identificato una variante missenso omozigote in FSD1L (c.409T>G, p.Leu137Val; M1). La condivisione di dati clinici e genetici attraverso GeneMatcher ha portato all'identificazione di un ulteriore famiglia con fenotipi simili, portatrice di varianti patogene nello stesso gene (c.1251_1252insTAA, p.Thr418Ter; M2 e c.1228T>G, p.Phe4102Val; M3). In questa famiglia sono presenti due fratelli italiani, entrambi deceduti, di cui, solo per uno di loro, 1655/19, era disponibile il campione biologico per lo studio. FSD1L subisce un esteso splicing alternativo, dando origine a otto diverse isoforme, di cui l'isoforma 206 è considerata l'isoforma canonica. FSD1L presenta tre domini importanti (coiled coil (COS), Fibronectina di tipo III e B30.2/SPRY) che sono conservati e condivisi con il suo paralogo FSD1, che codifica per una proteina centrosomiale, la proteina FSD1. È interessante notare che il dominio Fibronectina di tipo III è condiviso anche con L1CAM, una molecola transmembrana di adesione la cui alterazione provoca una sindrome del neurosviluppo che comprende idrocefalo e ipoplasia del corpo calloso. Ad oggi, la funzione del gene FSD1L è ancora sconosciuta. Per verificare la sua correlazione con il fenotipo complesso osservato, le cellule staminali pluripotenti indotte (iPSC) precedentemente riprogrammate dai fibroblasti di paziente e di controllo sono state differenziate in precursori neurali a piccole molecole (smNPCs), un tipo di precursore neurale che può essere ulteriormente differenziato in vari sottotipi di neuroni. La caratterizzazione di smNPCs è stata effettuata mediante Real-Time PCR e analisi di immunofluorescenza, garantendo la purezza della popolazione cellulare ottenuta. La generazione in vitro di smNPCs è un approccio valido per creare un modello cellulare paziente-specifico che sia il più possibile adatto allo studio della patologia del neurosviluppo in esame. Gli esperimenti condotti in questa tesi hanno contribuito a sviluppare una quantità di dati che ha posto le basi per la conduzione di ulteriori studi funzionali volti a raggiungere una comprensione completa della funzione genica e del meccanismo patogenetico causale della malformazione cerebrale.
Generazione e caratterizzazione di modelli di precursori neuronali peziente-specifico per studiare una nuova sindrome malformativa cerebrale causata da varianti recessive nel gene FSD1L
TONIOLO, MARTINA
2023/2024
Abstract
The advent of NGS has incredibly accelerated the step of gene discovery even in sporadic cases or very small families. In parallel, the development of web-based networks such as GeneMatcher (https://genematcher.org/statistics/), allows a researcher who identified a putative novel gene to share clinical and genetic data with the scientific community, to get easily in touch with other researchers around the world. My thesis project was based on the case report of two siblings, NG5393 and NG5394, affected by a not previously described neurodevelopmental syndrome characterized by optic nerve atrophy, spastic paraplegia, and epilepsy; at the MRI both siblings showed mild dilatation of the lateral ventricles, thin corpus callosum, and white matter changes. Given the negativity for L1CAM variants, a whole exome sequencing was performed, which identified a homozygous missense variant in FSD1L (c.409T>G, p.Leu137Val; M1). Sharing of clinical and genetic data through GeneMatcher led to the identification of one additional family, with similar phenotypes, carrying distinct pathogenic variants in the same gene (c.1251_1252insTAA, p.Thr418Ter; M2 and c.1228T>G, p.Phe410Val; M3). This family accounts for two Italian siblings, both deceased, of whom, only for one of them, 1655/19, the biological sample was available for study. FSD1L undergoes extensive alternative splicing, resulting in eight different isoforms, of whom isoform 206 is considered the canonical isoform. FSD1L presents three important domains (coiled-coil (COS), Fibronectin type III, and B30.2/SPRY) that are conserved and shared with its paralog FSD1, encoding for a centrosomal protein the FSD1 protein. Interestingly, the Fibronectin type III domain is also shared with L1CAM, a transmembrane molecule of adhesion whose alteration causes a neurodevelopmental syndrome including hydrocephalus and hypoplasia of the corpus callosum. To date, the function of the FSD1L gene is still unknown. To verify its correlation with the complex phenotype observed, induced Pluripotent Stem Cell (iPSC) previously reprogrammed from patient and control fibroblasts were differentiated into small molecule Neural Precursor Cells (smNPCs), a type of neural precursor that can be further differentiated into various subtypes of neurons. The characterization of smNPCs was accessed by Real-time PCR and Immunofluorescence analysis, ensuring the purity of the cell population obtained. The in vitro generation of smNPCs is a viable approach, to create a patient-specific cell model that is as suitable as possible for the study of the neurodevelopmental pathology under investigation. The experiments conducted in this thesis have helped develop an amount of data, that has laid the groundwork for conducting further functional studies aimed at reaching a complete understanding of the gene function and the causative pathogenetic mechanism of brain malformation.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28309