Abstract
Ovarian cancer, a complex and aggressive malignancy, remains a significant challenge in clinical oncology due to its heterogeneous nature and limited therapeutic options. In this study, across Pakistani ovarian cancer patients, we conducted a comprehensive analysis of mutations within the BRCA1 and BRCA2 genes to elucidate their potential implications in ovarian cancer susceptibility and progression. Employing Next-Generation Sequencing (NGS), we conducted a comprehensive mutational analysis of BRCA1/2 genes. Kaplan Meier analysis was used to analyze the effect of pathogenic mutations on the survival outcomes of ovarian cancer patients. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Immunohistochemistry (IHC) analyses were conducted to analyze the downstream effect of the pathogenic mutations. Targeted bisulfite sequencing (bisulfite-seq) analysis facilitated the investigation of epigenetic contributions to gene expression regulation. Enrichment analysis was conducted to uncover significant Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with BRCA1/2. Exploring DrugBank, we identified potential drugs capable of modulating BRCA1/2 expression regulation. NGS analysis identified three clinically significant pathogenic mutations within the BRCA1 gene and two within the BRCA2 gene, shedding light on their potential involvement in ovarian cancer susceptibility and progression. Kaplan Meier analysis unveiled poor overall survival (OS) associated with the identified pathogenic mutations, accentuating their prognostic value. Expression analysis using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and IHC demonstrated a significant up-regulation of BRCA1 and BRCA2 genes in ovarian cancer samples harboring pathogenic mutations. Bisulfite-seq revealed a significant hypomethylation within promoter regions of mutated BRCA1 and BRCA2 genes in ovarian cancer samples, compared to non-mutated cases with pathogenic mutations, indicating the role of epigenetics in expression dysregulation as well. By uncovering clinically significant pathogenic mutations in BRCA1/2 genes and establishing their link with up-regulated gene expression, this study significantly advances our understanding of ovarian cancer's underlying causes in the Pakistani population.