Deciphering the co-occurrence of germline variants in tumour suppressor genes and viral DNA detection in breast cancer: A case-control study from Pakistan

Abstract Breast cancer is the most common female malignancy globally. Being a multifactorial disease, the study investigated germline variants in tumour suppressor genes (TSGs) and viral DNA detection in breast cancer and their associations. In this case-control study, 640 women (320 breast cancer patients and 320 healthy individuals) were screened for germline variants in TSGs ( BRCA1 , BRCA2 , TP53 and PTEN ) using Sanger sequencing. Furthermore, PCR-based detection of HPV, EBV and HCMV was performed. Associations were determined using multivariable logistic regression and false discovery rate correction was performed using the Benjamini-Hochberg procedure. Age > 50 years (aOR = 2.42, 95% CI: 1.73–3.40; p < 0.001) and a positive family history of breast cancer (aOR = 4.84, 95% CI: 2.29–11.48; p < 0.001) were associated with increased odds of breast cancer. Sanger sequencing identified 18 germline variants. Five variants were classified as pathogenic/likely pathogenic (P/LP) according to ACMG/AMP guidelines. EBV was the most prevalent virus, detected in 38.8% of patients, followed by HCMV (30.6%) and HPV (5.3%). Permutation-based enrichment analysis identified significant enrichment of the BRCA1 -EBV-HCMV co-occurrence pattern (2.87-fold enrichment, q = 0.0049). A family history of breast cancer was significantly associated with P/LP variant carrier status for BRCA1 (aOR = 8.06, 95% CI: 1.66–39.23; q = 0.047). After age adjustment and FDR correction, only BRCA2 P/LP variants were significantly associated with HPV positivity (aOR = 20.69, 95% CI: 2.95-148.51; q = 0.049). HER2-positive tumours were less frequent among EBV-positive patients than EBV-negative patients ( p = 0.0175). Exploratory pathway enrichment analysis identified significant enrichment of the KEGG breast cancer pathway (FDR = 5.89 × 10⁻⁸) among the analysed virus-host interacting genes. The findings demonstrate the co-occurrence and statistical associations between TSG variants and viral detection in breast cancer, warranting further functional studies to clarify their biological significance.