Decoding the Transcriptional Complexity of the Human BRCA2 DNA Repair Gene Using Hybrid-seq

BRCA2 plays a pivotal role in DNA repair and tumor suppression, with its dysregulation linked to breast and gynecological cancers. Despite the importance of BRCA2, its transcriptional complexity remains poorly understood due to the gene's size and intricate alternative splicing patterns. This study aims to comprehensively characterize the BRCA2 transcriptional landscape in breast, ovarian, and cervical cancers using a hybrid sequencing approach. A novel hybrid-seq method combining long-read nanopore sequencing and short-read NGS was applied to analyze BRCA2 transcripts from cancerous cell lines. Expression patterns were evaluated using the transcript-per-million (TPM) normalization method, and open reading frames (ORFs) of the identified transcripts were in silico characterized. Sequencing analysis led to the identification of 50 novel splice variants (BRCA2 sv.7-sv.56), expanding the known transcript repertoire of BRCA2 gene. Notably, transcript variants sv.9, sv.15, and sv.49 exhibited significant expression in breast and ovarian cancers, while others, such as sv.29 and sv.40, were specific to individual cancer types. Five cryptic exons (N1-N5) were unveiled, contributing to 10 unique splice variants. In silico analysis revealed that 19 novel transcripts retained coding potential, with some encoding BRCA2 isoforms harboring key functional domains. The identification of novel BRCA2 transcripts underscores the complexity of its regulation in cancer. These findings provide insights into the gene's potential role in tumorigenesis and highlight candidates for targeted therapies and diagnostic biomarkers.