From whole-exome sequencing to functional insight: Characterization of rare germline variants in lung cancer

Whole-exome sequencing (WES) has greatly expanded the identification of rare germline variants in hereditary cancer predisposition. However, the interpretation of variants of uncertain significance (VUS), particularly in novel candidate genes, remains a major challenge in lung cancer genetics.

To functionally characterize selected germline variants identified by WES in patients with familial or suspected hereditary lung cancer and provide evidence for their prioritization.

Three splicing variants in HGS, CDK12, and CEACAM5 genes were investigated by RT-PCR and Sanger sequencing to assess their effect on splicing. Candidate missense variants were analyzed through protein-protein interaction network analysis, gene expression profiling, and structure-based computational modeling to evaluate their potential impact on protein stability and molecular interactions.

RNA analysis confirmed that the CDK12 variant produced the previously reported in-frame one amino acid deletion, whereas the HGS variant activated a cryptic splice acceptor site, resulting in an in-frame deletion affecting a region containing a known ubiquitination site. No aberrant splicing was detected for CEACAM5. Network analyses highlighted convergence of three candidate genes (ERBB3, MAP2K1 and RASA2) on receptor tyrosine kinase signaling and the RAS/MAPK pathway. Structural analyses predicted local perturbations affecting membrane interactions, residue interaction networks, or protein stability for variants in the three previous genes and in USP4, supporting their functional relevance.

Integrating RNA-based functional assays with network and structural analyses provides a practical framework for interpreting and prioritizing rare germline VUS identified by WES, supporting the identification of candidate lung cancer susceptibility genes and improving genetic risk assessment.