Cerebral malformation syndromes encompass a wide array of disorders characterized by abnormal brain development and accompanying clinical features. Among congenital brain malformations, abnormalities of the corpus callosum, a vital cerebral commissure beneath the cerebral cortex which plays a pivotal role in connecting the brain's two hemispheres, are among the commonest. Genes implicated in these malformations are associated with pathways involved in axon development, primary cilia formation, cell adhesion, proliferation, neuronal differentiation, and migration. Hydrocephalus is a pathological condition marked by the enlargement of ventricles and the accumulation of cerebrospinal fluid (CSF). This condition can arise from external factors or be congenital, often linked to neural tube defects (NTD) and malformative conditions that lead to the compression of cerebral aqueducts. Corpus callosum abnormalities and hydrocephalus often co-exist in a wide spectrum of syndromes, among which one of the most relevant is L1CAM syndrome . This is an X-linked congenital developmental disorder caused by mutations in the L1CAM gene (Xq28), which encodes the L1 cell adhesion molecule expressed primarily during nervous system development. In this context, the present thesis project focused on the characterization of a novel syndrome featuring corpus callosum anomalies and hydrocephalus. We first ascertained two siblings, NG5393 and NG5394, affected by a neurodevelopmental syndrome characterized by corpus callosum agenesis or hypoplasia, mild hydrocephalus, white matter abnormalities, optic nerve atrophy, spastic paraplegia, and epilepsy. These features were also observed in three fetuses from a French family and in two Italian siblings, both deceased. Since no mutations in L1CAM and related genes were detected with next-generation sequencing (NGS), a search for mutations in novel genes was made. Exome analysis and variant filtering led to the identification of candidate homozygous variants in the FSD1L gene, not previously associated with any disorder. In this study, we present the functional characterization of this newly identified gene, focusing of its expression profile and subcellular localization of its protein product. In-vitro analyses were therefore conducted on fibroblasts from patients and controls to assess the expression of the isoforms; for the localization of the FSD1L protein, we took advantage of the known homology of FSD1L with its paralog FSD1, involved in centrosome functions during cell division, as well as in primary cilium formation. Protocols for both transient and stable transfections were established in immortalized HTERT-RPE 1 and IMCD3 cell lines. Following overexpression, FSD1L was localized at the centrosome in transiently transfected HTERT-RPE 1 cells and showed cytoplasmatic localization, partially overlapping that of microtubules, in IMCD3 cells. While further analyses are required to extend these data, this stands as a promising starting point, laying the ground for further studies, such as attempting phenotype rescue in mutated fibroblasts by overexpressing wild type FSD1L. Further studies are underway to better define the functions of this gene and its actual involvement in this novel cerebral malformation syndrome.
Caratterizzazione funzionale di FSD1L, gene causativo di una nuova sindrome del neurosviluppo. Le malformazioni cerebrali congenite comprendono una vasta gamma di disturbi caratterizzati da uno sviluppo anomalo del cervello e da manifestazioni cliniche associate. Tra di esse, le anomalie del corpo calloso, una commessura cerebrale vitale situata sotto la corteccia cerebrale e che svolge un ruolo fondamentale nel collegamento tra i due emisferi cerebrali, sono tra le più comuni. I geni coinvolti in queste malformazioni sono associati a vie coinvolte nello sviluppo degli assoni, nella formazione dei cili primari, nell'adesione cellulare, nella proliferazione, nella differenziazione neuronale e nella migrazione. L'idrocefalo è una condizione patologica caratterizzata dall'ingrandimento dei ventricoli dovuto all'accumulo di liquido cerebrospinale (CSF). Questa condizione può derivare da fattori esterni o essere congenita, spesso associata a difetti del tubo neurale (NTD) e a condizioni malformative che portano alla compressione degli acquedotti cerebrali. Le anomalie del corpo calloso e l'idrocefalo spesso coesistono in un ampio spettro di sindromi diverse, tra cui la sindrome L1CAM, una malattia congenita legata al cromosoma X causata da mutazioni del gene L1CAM (Xq28), che codifica la molecola di adesione cellulare L1 espressa principalmente durante lo sviluppo del sistema nervoso. In questo contesto si inserisce il presente progetto di tesi, basato sullo studio di due fratelli, NG5393 e NG5394, affetti da una nuova sindrome di neurosviluppo caratterizzata da agenesia o ipoplasia del corpo calloso, lieve idrocefalo, alterazioni della sostanza bianca, atrofia del nervo ottico, paraplegia spastica ed epilessia. Un quadro malformativo simile è stato osservato anche in tre feti da una famiglia francese, e in due fratelli italiani, entrambi deceduti. Poiché non sono state rilevate mutazioni in L1CAM e in altri geni correlati tramite sequenziamento di nuova generazione (NGS), è stata effettuata la ricerca di mutazioni in nuovi geni. L'analisi dell'esoma e il conseguente filtraggio delle varianti hanno portato all'identificazione di varianti candidate in omozigosi nel gene FSD1L, fino ad allora non associato a patologia umana. In questo studio, presentiamo la caratterizzazione funzionale di questo gene focalizzandoci sul suo profilo di espressione e sulla localizzazione del suo prodotto proteico a livello cellulare. Sono state condotte analisi in vitro su fibroblasti di pazienti e controlli per valutare l'espressione delle isoforme; per la localizzazione di FSD1L, invece, le analisi si sono basate sull'omologia nota in letteratura con il suo paralogo FSD1, codificante per una proteina che regola alcune funzioni del centrosoma, durante la divisione cellulare, e la formazione del cilio primario. Sono stati stabiliti protocolli per trasfezioni transienti e stabili utilizzando linee cellulari immortalizzate HTERT-RPE 1 e IMCD3. A seguito dell'overespressione, è stata dimostrata la localizzazione della proteina FSD1L al centrosoma nelle cellule HTERT-RPE 1 trasfettate in modo transiente e la sua localizzazione citoplasmatica, con una parziale sovrapposizione con i microtubuli, nelle cellule IMCD3. Sebbene ulteriori analisi siano necessarie per confermare i dati, questo rappresenta un promettente punto di partenza. Inoltre, questo lavoro pone le basi per ulteriori esperimenti, come il rescue del fenotipo nei fibroblasti dei pazienti a seguito di overespressione di FSD1L wild type. Ulteriori studi sono attualmente in corso per definire meglio le funzioni di questo gene e il suo ruolo nello sviluppo del sistema nervoso.
Functional characterization of FSD1L, causative of a novel neurodevelopmental syndrome
CRIFFÒ, CARLOTTA
2022/2023
Abstract
Cerebral malformation syndromes encompass a wide array of disorders characterized by abnormal brain development and accompanying clinical features. Among congenital brain malformations, abnormalities of the corpus callosum, a vital cerebral commissure beneath the cerebral cortex which plays a pivotal role in connecting the brain's two hemispheres, are among the commonest. Genes implicated in these malformations are associated with pathways involved in axon development, primary cilia formation, cell adhesion, proliferation, neuronal differentiation, and migration. Hydrocephalus is a pathological condition marked by the enlargement of ventricles and the accumulation of cerebrospinal fluid (CSF). This condition can arise from external factors or be congenital, often linked to neural tube defects (NTD) and malformative conditions that lead to the compression of cerebral aqueducts. Corpus callosum abnormalities and hydrocephalus often co-exist in a wide spectrum of syndromes, among which one of the most relevant is L1CAM syndrome . This is an X-linked congenital developmental disorder caused by mutations in the L1CAM gene (Xq28), which encodes the L1 cell adhesion molecule expressed primarily during nervous system development. In this context, the present thesis project focused on the characterization of a novel syndrome featuring corpus callosum anomalies and hydrocephalus. We first ascertained two siblings, NG5393 and NG5394, affected by a neurodevelopmental syndrome characterized by corpus callosum agenesis or hypoplasia, mild hydrocephalus, white matter abnormalities, optic nerve atrophy, spastic paraplegia, and epilepsy. These features were also observed in three fetuses from a French family and in two Italian siblings, both deceased. Since no mutations in L1CAM and related genes were detected with next-generation sequencing (NGS), a search for mutations in novel genes was made. Exome analysis and variant filtering led to the identification of candidate homozygous variants in the FSD1L gene, not previously associated with any disorder. In this study, we present the functional characterization of this newly identified gene, focusing of its expression profile and subcellular localization of its protein product. In-vitro analyses were therefore conducted on fibroblasts from patients and controls to assess the expression of the isoforms; for the localization of the FSD1L protein, we took advantage of the known homology of FSD1L with its paralog FSD1, involved in centrosome functions during cell division, as well as in primary cilium formation. Protocols for both transient and stable transfections were established in immortalized HTERT-RPE 1 and IMCD3 cell lines. Following overexpression, FSD1L was localized at the centrosome in transiently transfected HTERT-RPE 1 cells and showed cytoplasmatic localization, partially overlapping that of microtubules, in IMCD3 cells. While further analyses are required to extend these data, this stands as a promising starting point, laying the ground for further studies, such as attempting phenotype rescue in mutated fibroblasts by overexpressing wild type FSD1L. Further studies are underway to better define the functions of this gene and its actual involvement in this novel cerebral malformation syndrome.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/20.500.14239/16406