AMBRA1 p.Gln30Arg Mutation, Identified in a Cowden Syndrome Family, Exhibits Hyperproliferative Potential in hTERT-RPE1 Cells.

Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the tumor suppressor. In the present study, we investigated the causative gene of CS in a family of (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of in the clinical manifestations of CS, we generated depletion and Q30R mutation in hTERT-RPE1 (humanTelomerase Reverse Transcriptase-immortalized Retinal Pigmented Epithelial cells) using the CRISPR-Cas9 gene editing system. We observed that both -depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a phenotype in -depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.