Genet Med. 2025 Nov 13:101640. doi: 10.1016/j.gim.2025.101640. Online ahead of print.
ABSTRACT
PURPOSE: The RB1 gene encodes the retinoblastoma protein (pRB) playing a major role in cell cycle control, particularly by its interaction with E2F transcription factors. Familial forms of retinoblastoma are caused by germline pathogenic variants in the RB1 gene predisposing to retinoblastoma and other tumors. By analyzing the RB1 gene in retinoblastoma patients, missense variants often remain variants of uncertain significance (VUS).
METHODS: To classify RB1 VUS, we developed a functional assay evaluating their impact on the ability of pRB to inhibit the activity of the E2F1 promoter, with a luciferase reporter gene. A set of 14 pathogenic/likely pathogenic and benign/likely benign RB1 variants was used for validation.
RESULTS: We tested 16 VUS detected in retinoblastoma patients and found that nine VUS reduced the ability of pRB to inhibit E2F1 promoter. Among them, the (RB1) c. 2263T>G p.(Phe755Val) variant showed a reduced level of pRB on Western Blot, suggesting a defect in pRB stability. By applying the criterion PS3_moderate of the ACMG/AMP classification to this functional assay, five of the nine VUS with functional impact could be classified as likely pathogenic.
CONCLUSION: This functional assay can improve the molecular diagnosis of retinoblastoma predisposition by a better determination of pathogenic/likely pathogenic RB1 variants.
PMID:41241816 | DOI:10.1016/j.gim.2025.101640
