Mechanisms underlying morphological and functional changes of cilia in fibroblasts derived from patients bearing ARL3^T31A and ARL3^T31A/C118F 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(T31A) and compound heterozygous ARL3(T31A/C118F) 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(T31A) and compound heterozygous ARL3(T31A/C118F) mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3(T31A) and ARL3(T31A/C118F) mutations led to a decrease in cilium formation. The ARL3(T31A/C118F) mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3(T31A) mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3(T31A), ARL3(T31A/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, and TGF-beta 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(T31A/C118F) group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3(T31A/C118F) 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.