Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3 and ARL3 mutations.

ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3 and compound heterozygous ARL3 mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3 and compound heterozygous ARL3 mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3 and ARL3 mutations led to a decrease in cilium formation. The ARL3 mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3 mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3 , ARL3 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, and TGF-β signaling. Similar changes were observed in the proteomic results in ARPE-19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3 group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3 fibroblasts. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.