Identification of germline AEN polymorphisms as risk factors in cervical cancer

Background: Inherited genetic variation contributes 27-36% of cervical cancer risk. While genome-wide association studies (GWAS) and candidate gene approaches have identified both risk and protective alleles, mostly associated with immune and DNA repair pathways, much remains unknown about the role of rare or low-frequency germline variants across the exome. Methods: We performed gene-based association testing on whole-exome sequencing from blood-derived DNA of 105 Norwegian cervical cancer patients and 263 cancer-free European female controls (1000 Genomes). High-quality and high-confidence variants were identified following GATK best practices for variant calling, and a gene-based sequence kernel association test (SKAT) was performed to identify significant gene-level associations. The presence and linkage of significant variants were further examined in a cohort of 9,286 Norwegian newborn genotypes. To assess the potential functional impact and the effects on cancer risk of the identified variants, vector constructs containing wild-type and variants introduced into C4I cervical cancer cells and cell proliferation rates were measured. Results: The Apoptosis Enhancing Nuclease ( AEN ), a TP53 -related gene, showed significant germline association with cervical cancer (SKAT-RC p = 3.69×10⁻⁹). The signal was driven by two low-frequency variants, rs61752779 and rs118097475, which were about 9-fold more common in cases than controls. Linkage analysis showed that these two variants were strongly correlated (r 2 = 0.996) in cervical cancer patients and similarly linked in the Norwegian newborns (r 2 = 0.948). Functional validation revealed that overexpression of the wildtype AEN construct in the C4I cervical cancer cell resulted in reduced cellular proliferation. However, overexpression of an AEN construct with both variants resulted in increased cell proliferation compared with the wildtype AEN construct. Conclusions: This study provides both statistical and experimental evidence supporting the role of AEN as a tumor suppressor gene and suggests that two highly linked germline variants may contribute to cervical cancer risk.