Germline Variant Analysis in Cell Cycle-Related Genes Using Whole-Exome Sequencing (WES) and their Role in Breast Cancer Prognosis

Background and Objectives Breast cancer (BC) remains the most prevalent malignancy among women worldwide. Despite advances in early diagnosis and treatment, tumor heterogeneity and its complex pathogenesis continue to present significant challenges, contributing to high cancerrelated mortality. While germline variants are known to influence BC susceptibility, recent studies suggest that variants in genes regulating key cellular processes, such as the cell cycle, may also affect tumor phenotype, disease progression, and response to therapy. However, the role of these variants remains incompletely understood. Thus, the aim of this study was to analyze germline variants in cell cycle-related genes using whole-exome sequencing (WES) and evaluate their prognostic value in a group of BC patients. Material and Method This study included 52 patients diagnosed with either luminal A or triple-negative breast cancer from the Hospital of Lithuanian University of Health Sciences Kaunas Clinics. All participants were selected based on predefined criteria, including confirmed histopathological diagnosis, comprehensive medical data (age at diagnosis, tumor size (T), grade (G), lymph node involvement (N), estrogen and progesterone receptor status, presence of metastases, disease progression, mortality, and clinical outcomes), and absence of significant comorbidities. Peripheral blood samples were collected for genomic DNA extraction, followed by WES. Germline variant analysis was performed using the Franklin by Genoox platform. This study focused on missense variants with a minor allele frequency (MAF) of 5–20% (according to the 1000 Genomes Project) in 55 genes involved in cell cycle regulation (e.g., CDKN1A, ATM, AURKA, and others). Statistical analysis, including association and survival analysis, was performed using IBM SPSS Statistics version 30.0.0.0. A P-value of less than 0.05 was considered statistically significant. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (Approval Nos. BE-2-10 and P1-BE2-10/2014). Results In this study, germline variants of ATM rs1801516, ATR rs2229032, and TGFB1 rs1800471 were identified using WES analysis. The results showed that the frequency of genotypes was as follows: GG – 61.5%, GA – 36.5%, and AA – 1.9% for ATM rs1801516; CC – 75.0%, CT – 21.2%, and TT – 3.8% for ATR rs2229032; and CC – 96.2%, CG – 3.8%, and GG – 0.0% for TGFB1 rs1800471. All variants were distributed according to the Hardy-Weinberg equilibrium (p>0.05). The association analysis showed that ATM rs1801516 was associated with death (p<0.047); however, logistic regression analysis showed that the association was nonsignificant in both univariate and multivariate models. Survival analysis revealed that the same variant was associated with PFS, MFS, and OS (p<0.001). It was established that patients presenting the AA genotype were more likely to have a longer PFS, MFS, and OS (HR = 32.038, HR = 29.586, HR=60.698, respectively; p<0.05). The results remained statistically significant in multivariate analysis. Neither the ATR rs2229032 nor TGFB1 rs1800471 demonstrated any significant results in association and survival analysis. Conclusions and Recommendations In conclusion, the ATM rs1801516 was found to be significant for mortality and clinical outcomes in breast cancer patients and may have potential as a prognostic biomarker. However, due to the limited sample size, further studies with larger cohort are required to confirm these findings.