Hereditary breast cancer accounts for 5–10% of all cases, with pathogenic variants in BRCA1/2 and other susceptibility genes playing a crucial role. This study elucidates the prevalence and spectrum of germline variants in 13 cancer predisposition genes among high—risk hereditary breast cancer patients from Southern Italy. We employed next-generation sequencing (NGS) to analyze 254 individuals selected through genetic counseling. Pathogenic or likely pathogenic variants were identified in 13% (34/254) of patients, with 54% of these variants occurring in non-BRCA1/2 genes. Notably, we observed a recurrent BRCA1 c.4964_4982del founder mutation, underscoring the importance of population-specific genetic screening. The spectrum of variants extended beyond BRCA1/2 to include PALB2, ATM, TP53, CHEK2, and RAD51C, highlighting the genetic heterogeneity of breast cancer susceptibility. Variants of uncertain significance were detected in 20% of patients, emphasizing the ongoing challenge of variant interpretation in the era of multi-gene panel testing. These findings not only enhance our understanding of the genetic landscape of breast cancer in Southern Italy but also provide a foundation for developing more targeted, population-specific approaches to genetic testing and counseling, ultimately contributing to the advancement of precision medicine in oncology.
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