Saturation Genome Editing reveals the functional impact of RAD51D and XRCC2 variants

Area of Science:

• Genetics
• Genomic Medicine
• Cancer Genomics

Background:

• Germline variants in RAD51D and XRCC2 genes increase cancer risk and cause homologous recombination deficiency.
• Most RAD51D and XRCC2 variants are classified as variants of uncertain significance (VUS), limiting their clinical utility.

Purpose of the Study:

• To apply saturation genome editing (SGE) to functionally characterize RAD51D and XRCC2 variants.
• To assess the impact of these variants on cellular fitness and RNA expression.
• To improve the clinical utility of genetic testing for cancer risk.

Main Methods:

• Saturation genome editing (SGE) was used to assess the functional impact of thousands of RAD51D and XRCC2 variants.
• Cellular fitness assays were performed to measure the effect of variants on cell survival.
• RNA expression analysis was conducted to identify splice-altering variants.

Main Results:

• SGE accurately discriminated pathogenic from benign variants in RAD51D (AUC=0.994) and XRCC2 (AUC=1.000).
• RAD51D is highly sensitive to splice-altering variation, with 24% of loss-of-function missense variants acting through RNA-mediated mechanisms.
• XRCC2 showed lower sensitivity to splice-altering variation, with only 5% of variants acting through RNA-mediated mechanisms.

Conclusions:

• SGE provides robust, splice-resolved functional evidence for classifying RAD51D and XRCC2 variants.
• These findings enhance the clinical utility of genetic testing for individuals at risk of homologous recombination deficiency-related cancers.
• The study provides a valuable dataset for understanding the functional impact of genetic variation in DNA repair genes.