Mechanisms Underlying Morphological and Functional Changes of Cilia in Fibroblasts Derived from Patients Bearing ARL3T31A and ARL3T31A/C118F Mutations

ARL3 plays a crucial role in cilia development, while mutations in ARL3 are closely associated with ciliopathies. In a previous study, we reported distinct phenotypes of retinal dystrophy in patients with single ARL3T31A and compound ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated fibroblasts from patients carrying single ARL3T31A and compound ARL3T31A/C118F mutations, systematically evaluated their cilia morphology and function, and explored the underlying molecular mechanisms by RNA-sequencing. Our results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilia incidence. The compound ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the single ARL3T31A mutation did not induce significant changes in fibroblasts. Compared to ARL3T31A, ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM-receptor interaction, focal adhesion, axon guidance, and TGF-β signaling. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, which were further validated. Additionally, we observed that IQUB had a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. These findings suggest that the ARL3T31A/C118F mutation downregulates IQUB, disrupting TGF-β signaling cascades and leading to cilia elongation in ARL3T31A/C118F cells. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of single and compound mutations in genetics.