Pezzella, Nunziana (2025) The puzzle of OFD1: one gene, several disorders. [Tesi di dottorato]

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Abstract

Pleiotropy, the phenomenon whereby a single gene influences multiple traits, is a fundamental principle underlying biological complexity. The X-linked OFD1 gene exemplifies this principle: variants give rise to a spectrum of multisystemic ciliopathies, including Oro-Facio-Digital Syndrome type 1 (OFD1), Joubert Syndrome type 10 (JBS10), Primary Ciliary Dyskinesia (PCD), and organ-specific disorders such as Retinitis Pigmentosa type 23 (RP23). Despite OFD1’s established role in ciliogenesis, the molecular mechanisms linking specific variants to distinct phenotypes remain incompletely understood. In this PhD project, I combined clinical and genetic analyses of patient cohorts, including 324 OFD1 variants, with in vitro functional studies using patient-derived fibroblasts, nasal brush cells, and HK-2 and BCi cell models expressing representative variants for each ciliopathy. We are performing a comprehensive phenotypic analysis of both published and newly collected patient data (53 new cases), by bioinformatic and statistical tools to develop a predictive model capable of clarifying genotype– phenotype correlations and anticipating the effects of novel variants. Remarkably, we identified a distinct and specific distribution of variants in JBS10, suggesting a correlation between variant position and phenotypic outcome. We also analysed how these variants impact protein function depending on whether the affected amino acid is buried or exposed, as well as their location within functional domains. We employed advanced imaging techniques, including confocal and expansion microscopy to map OFD1 localization across cellular compartments, while multi- omics analyses, metabolic assays, and bioinformatics interrogated downstream molecular effects and genotype-phenotype relationships. Our findings reveal that OFD1 localizes not only to basal bodies and centriolar satellites but also to mitochondria. The RP23-associated variant induces a qualitative, likely dominant- negative defect: although protein expression remains unaltered, mutant cells display hyper-elongated cilia and mitochondrial abnormalities, including swollen organelles, membrane hyper-polarization, increased maximal respiratory capacity, and elevated ROS, indicative of compensatory metabolic stress. In contrast, OFD1 loss-of-function models (KO and PCD) display mitochondrial hypo-polarization, reduced OCR, and downregulation of electron transport chain pathways, highlighting a dichotomy between qualitative and quantitative defects. In the PCD 7 model, the OFD1 variant also compromises centriolar satellite dynamics, distal appendage (DAPs) assembly, and axonemal morphology, leading to apical tip swelling and impaired mucociliary clearance. Transcriptomic and proteomic profiling further reveal coordinated perturbations in centriole-associated proteins, mitochondrial regulators, and oxidative phosphorylation components, suggesting convergence of structural and metabolic dysfunction. Collectively, this work defines OFD1 as a multi-compartmental, pleiotropic regulatory hub coordinating structural, metabolic, and signalling processes across centrosomes, cilia and mitochondria. The distinct phenotypes observed in our mutants reflect different pathogenic modalities perturbing a shared cilia–mitochondria axis, providing a mechanistic framework for the clinical diversity of OFD1-related disorders and highlighting potential therapeutic strategies targeting mitochondrial homeostasis and ciliary integrity.

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