Search Results (195)

The potency of urate, an abundant human plasma antioxidant, in preventing oxidative damage caused by the carbonate radical anion CO₃^•−, was studied using quantum chemical calculations. The influence of microhydration of CO₃^•−/CO₃²⁻ and urate⁻/urate^• couples on the thermodynamic and kinetics of the one-electron oxidation process was investigated. Depending on the degree of microhydration, the estimated rate constant for one-electron transfer is in the range of 2.0–7.3 × 10⁹ M⁻¹ s⁻¹, in good agreement with the experimental value of 1.3 × 10⁹ M⁻¹ s⁻¹. Modeling using vertical detachment energy and electron affinity, the driving forces of single electron transfer revealed urate(H₂O)₆⁻ and CO₃(H₂O)₉^•− clusters as the most likely existing species in water. Molecular docking revealed a favorable interaction of urate with the catalytic pocket of SOD1. Urate binds more strongly to the anionic active center of SOD1 than the reference inhibitor LSC-1, indicating its potency to prevent HCO₃⁻-supported CO₃^•− formation. In contrast, the known OGG1 inhibitor TH13264 shows substantially stronger binding than urate, indicating urate’s weaker affinity toward the DNA repair enzyme catalytic pocket. The molecular dynamics data indicate that urate binding does not destabilize either SOD1 or OGG1. In light of increasing evidence that the major source of oxidative stress could be CO₃^•−, rather than the commonly assumed hydroxyl radical HO^•, the obtained results indicate the inherent ability of plasma to combat oxidative stress induced by this selective, milder oxidant. Such an ability with respect to the non-selective, highly reactive HO^• does not exist in vivo. Full article

Background: Prostate cancer is a heterogeneous disease with variable clinical outcomes. If localized, the patient may be cured. However, prostate cancer is lethal if recurrence/progression to metastatic castrate resistant disease occurs. Thus, there is an unmet need to further understand the molecular underpinnings of this progression. Epidemiologic studies show that increased risk of developing and dying from prostate cancer has been associated with elevated serum IGF-1 levels, hyperinsulinemia and metabolic syndrome. Alterations in insulin pathway genes, such as PTEN, FOXO, and PIK3CA, are mutated in up to 32%, 15%, and 11% of localized prostate tumors, respectively. We aimed to further characterize expression of insulin pathway genes in localized prostate cancers in an effort to (1) provide insights into potential mechanisms of progression to metastatic disease and (2) try to further enrich for those prostate tumors that portend worse survival outcomes. Methods: Using the multi-institutional Oncology Research Information Exchange Network (ORIEN) database, gene expression data was analyzed from localized prostate cancer tumors. The raw counts were first normalized, and 176 genes related to the insulin receptor and its downstream pathways were then subset and used for clustering using the non-negative matrix factorization (NMF). The NMF cluster analysis was performed in an attempt to separate gene expression into two groups. Gene Set Enrichment Analysis (GSEA) was then performed between the two groups that had been separated by cluster analysis to determine homology between other GSEA sets. Kaplan–Meier curves were used to assess median overall survival. Cox analysis was performed to generate the adjusted KM curve. Mediation analysis was conducted to determine the relationship between cluster status, TN stage, and survival. Results: Cluster analysis revealed two distinct groups of insulin gene expression, cluster 1 (n = 96) and cluster 2 (n = 337). Compared with cluster 2, cluster 1 consisted of decreased expression of PTEN (p < 0.001) and PIK3R1 (p < 0.001), along with increases in the expression of AKT1 (p < 0.001), IRS1/2 (p < 0.001), FASN (p < 0.001), IGFBP2 (p < 0.001), and MTOR (p < 0.001). GSEA analysis revealed changes in lipid metabolism and WNT secretion pathways in cluster 1. Cluster 2 GSEA showed pathway changes related to DNA damage repair and testosterone. Patient characteristics between clusters differed significantly in the T and N stages of tumor but not in other ways. In unadjusted analysis, median overall survival was estimated at 117 months and 232 months for cluster 1 and cluster 2, respectively (p < 0.05). The proportion of patients who went on to develop metastases (p < 0.05) or need chemotherapy (p < 0.05) was increased in cluster 1 compared to cluster 2. Repeat survival analysis adjusted for confounders (T stage, N stage, age at diagnosis, pathologic grade) showed no difference in survival between clusters. Mediation analysis showed that the contribution of cluster status to survival was independent of T or N stage. Conclusions: A subset of localized prostate cancer patients demonstrated linked insulin pathway changes that are consistent with prior studies describing a pattern of insulin dysregulation. Though the group characterized by insulin dysregulation initially showed worse survival outcomes, this difference disappeared when controlling for confounders. Though baseline differences in tumor stage seemed to most readily explain the difference in survival between clusters, mediation analysis showed that the effect of cluster status on survival was independent of tumor stage. This suggests that other confounders, such as pathologic grade or baseline age, may explain the survival difference. Full article

Background: Ultraviolet-C (UV-C) radiation is a high-energy physical mutagen capable of inducing DNA damage and oxidative stress, thereby generating genomic variability in photosynthetic organisms. However, its genome-wide effects in unicellular eukaryotic microalgae remain poorly characterized. This study developed a UV-C mutagenesis protocol in Chlamydomonas reinhardtii and evaluated its genomic and physiological impacts. Methods: Axenic cultures of Chlamydomonas reinhardtii (137c+) were exposed to UV-C (100–280 nm) for 12, 48, and 96 min. Viable colonies were analyzed by Random Amplification of Polymorphic DNA PCR (RAPD-PCR) to assess genetic variability, while chlorophyll content and the expression of stress-responsive genes were measured via spectrophotometry and Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), respectively. Results: UV-C treatment induced extensive genomic polymorphism with heterogeneous clustering patterns independent of exposure time, consistent with stochastic mutagenesis. Several mutants exhibited reduced chlorophyll content, indicating impaired photosynthetic efficiency. In contrast, one genotype (pop18) maintained wild-type chlorophyll levels despite marked genetic divergence, coupled with upregulation of antioxidant, DNA repair, and stress-response genes. Conclusions: Overall, UV-C irradiation represents an effective approach to generate non-directional genomic variability in Chlamydomonas reinhardtii, with evidence that random mutagenesis can drive functional reorganization of stress-response pathways, supporting its application in microalgal strain improvement. Full article

Cadmium exposure results in the impairment of pancreatic β-cells. The FTO protein, the product of the Fto gene, is a key regulator of diverse pathophysiological processes, including oxidative damage and cell death. However, it remains unclear whether Fto gene knockout affects cadmium-induced pancreatic β-cell damage, and the precise mechanisms involved are yet to be elucidated. Under conditions of cadmium exposure, Fto gene knockout was found to alleviate pancreatic β-cell damage significantly. Specifically, Fto gene knockout counteracted cadmium-induced cytotoxicity—manifested as reduced cell viability, increased apoptosis, and heightened lactate dehydrogenase (LDH) release—while simultaneously suppressing DNA damage and preserving cellular membrane integrity. On a molecular level, Fto gene knockout markedly mitigated cadmium-induced oxidative stress. This was achieved by curbing excessive reactive oxygen species (ROS) accumulation, lowering malondialdehyde (MDA) generation, and reducing 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, alongside restoring superoxide dismutase (SOD) activity. Furthermore, ER-Tracker Red staining revealed that cadmium treatment induced clustered aggregation of the endoplasmic reticulum (ER) and increased fluorescence intensity, suggesting the activation of endoplasmic reticulum stress (ERS). Conversely, Fto knockout ameliorated ER morphological abnormalities, thereby effectively antagonizing the excessive activation of ERS. In summary, our study elucidates the impact and underlying molecular mechanisms of the Fto gene in cadmium-induced toxicity in pancreatic β-cells from the perspectives of oxidative damage, ERS, and apoptosis. These findings identify the Fto gene as a potential molecular target for mitigating cadmium-induced toxicity in pancreatic β-cells, thereby providing a new theoretical basis for the prevention and treatment of cadmium-induced pancreatic β-cell injury. Full article

A distinct form of chronic kidney disease of unknown etiology (CKDu) has emerged in tropical regions of Sri Lanka, predominantly affecting individuals aged 30–60 years in the North Central Province. Unlike conventional chronic kidney disease (CKD), CKDu occurs independently of diabetes or hypertension and is characterized by tubulointerstitial damage, including tubular atrophy, interstitial inflammation, and fibrosis. Epidemiological studies showed familial clustering, suggesting an underlying genetic predisposition. This study aimed to identify genetic variants associated with CKDu in Sri Lankan populations using whole-exome sequencing (WES). Eighty-six individuals (47 CKDu patients and 39 controls) were recruited from endemic and non-endemic regions. Physiological, biochemical, and geographic parameters were recorded. DNA extracted from blood was subjected to WES to identify variants associated with CKDu. Results: A total of 171 unique variants across 121 genes were identified. Among the most prevalent genes were ATXN3, LFNG, PNLDC1, LINC02456, and HLA-DRB1. In the case–control comparison, only LFNG showed statistically significant enrichment in affected individuals, whereas signals in ATXN3, PNLDC1, and LINC02456 were not statistically significant, but have an association with renal dysfunction, and thus are included as hypothesis-generating variant observations. HLA-DRB1 variants showed trends toward a protective haplotype. LFNG showed the greatest prevalence in affected individuals (71.7%), followed by PNLDC1 (63%), ATXN3 (56%), FIP1L1 (41%), and HLA-DRB1 (32%). Conclusion: Findings suggest genetic variants in combination with environmental factors may contribute to CKDu susceptibility in the Sri Lankan population. We underscore the multi-factorial nature of CKDu and highlight the need for integrative genomic and environmental research to elucidate disease mechanisms and inform targeted prevention strategies. Full article

Background/Objectives: Exposure of cells to ionizing radiation induces isolated DNA lesions, including single-strand breaks, apurinic/apyrimidinic sites, and oxidized bases, as well as clustered damages of different complexity. The latter types of damage are difficult to repair, and the failure to process them accurately and efficiently is related to the induction of mutagenesis, genomic instability, cancer, and aging. Since various types of clustered lesions may occur simultaneously after radiation exposure, leading to a complex architecture of DNA damage, the study of the concomitant formation and the removal kinetics of clustered DNA damage is important to determine the mutagenic and, consequently, the carcinogenic potential of ionizing radiation. Methods: With the aim of capturing real-time coexisting lesion types and assessing the repair kinetics of clustered damages, the simultaneous determination of double-strand breaks, apurinic/apyrimidinic site clusters, and oxypurine clusters induced by γ-irradiation of Saccharomyces cerevisiae yeast cells was performed immediately after exposure and at time intervals during incubation in Liquid Holding Recovery conditions. Results: Ionizing radiation induced lethal and mutagenic events, leading to a dose-dependent linear increase in double-strand breaks, apurinic/apyrimidinic site clusters, and oxypurine clusters. The kinetic study showed that double-strand break frequencies declined during Liquid Holding Recovery, although a transient increase was detected at early time points. At 160 Gy, apurinic/apyrimidinic site clusters repair was evident, whereas at 400 Gy the frequency of damage increased before returning to the initial value at 24 h. In contrast, oxypurine clusters showed no net increase in repaired lesions over 24 h. Conclusions: The complex nature and topological characteristics of ionizing radiation-induced clustered DNA damage may influence lesion processing. Also, ionizing radiation may disrupt redox cellular homeostasis, leading to DNA damage and delayed effects. Full article

Background/Objectives: Proton therapy has emerged as an advanced radiotherapy modality due to its unique physical dose distribution and its distinct radiobiological properties. The finite range of protons in tissue enables highly conformal dose delivery with minimal exit dose, significantly reducing irradiation of surrounding normal tissues compared to photon-based radiotherapy. Beyond these physical advantages, proton beams exhibit a spatially varying linear energy transfer that increases toward the distal edge of the spread-out Bragg peak, leading to clustered and complex DNA damage that is more difficult for cancer cells to repair. Methods: This review integrates experimental, computational, and clinical evidence to examine how proton-induced DNA damage, relative biological effectiveness, oxygen effects, and non-targeted responses contribute to tumor control and normal tissue sparing. Results: Comparative analyses with photon intensity-modulated radiotherapy demonstrate consistent reductions in acute and late toxicities across multiple tumor sites, particularly in pediatric patients and in tumors located near critical organs. The review also discusses emerging technologies, including pencil beam scanning, image-guided and adaptive proton therapy, compact accelerator systems, and ultra-high dose rate FLASH proton therapy, which collectively aim to enhance treatment precision, biological effectiveness, and accessibility. Conclusions: Together, these developments support proton therapy as a rapidly evolving modality with significant potential to improve therapeutic outcomes in modern oncology. Full article

Proton therapy offers superior dose localization, yet the biological effects of low-energy protons relevant to superficial tissues remain underexplored. We report the design and validation of a proton irradiation setup developed at the Tandem Accelerator of NCSR “Demokritos” for controlled radiobiological experiments. Monte Carlo simulations using Geant4 and Monte Carlo Damage Simulation (MCDS—Monte Carlo Damage Simulation) were used to determine proton energy spectra, linear energy transfer (LET), and predicted DNA damage yields. A single layer (15–20 μm in thickness) of human keratinocytes (HaCaT) was irradiated at doses from 0.65 to 3.65 Gy, and γ-H2AX foci were quantified as markers of tracks including one or more DNA double-strand breaks. The system achieved a uniform dose rate of 0.37 Gy/min, as calculated with Geant4, with a mean proton energy of 4.1 MeV (LET ≈ 8 keV/μm). A strong correlation (R² = 0.93) was observed between proton dose and γH2AX foci per nucleus (~10 foci/Gy), reflecting damage-inducing proton tracks rather than individual DNA double-strand breaks. At higher doses, an increased fraction of cells exhibited pan-nuclear γH2AX staining, characterized by a diffuse γH2AX signal throughout the nucleus and commonly associated with extensive or clustered DNA damage and global chromatin phosphorylation. These responses are consistent with the well-established dense ionization patterns produced by low-energy protons, as indicated by the LET spectrum and supported by MCDS-predicted clustered damage yields. While the γH2AX assay does not directly resolve simple versus complex DNA lesions, the agreement between Monte Carlo modeling and the observed cellular stress responses indicates that the irradiation platform reliably reproduces the expected biological signatures of low-energy proton exposure. Consequently, the developed system provides a robust experimental tool for systematic investigations of cellular radiosensitivity and radiotoxicity, with potential applications in skin dosimetry and radioprotection. Full article

Tankyrases (TNKS1 and TNKS2) are multifunctional enzymes of the poly(ADP-ribose) polymerase (PARP) family that regulate cellular homeostasis by catalyzing poly(ADP-ribosyl)ation and stabilizing protein–protein interactions through their ankyrin repeat clusters. By engaging with diverse sets of proteins, TNKSs act as central hubs that coordinate signaling and metabolic pathways. In this review, we discuss how TNKS –protein interactions underpin their roles across multiple biological pathways, including Wnt/β-catenin, YAP and SRC signaling, mTORC1 signaling, DNA damage repair (via PARP crosstalk and recruitment of repair factors), telomere maintenance, cell-cycle regulation, glucose metabolism, cytoskeleton rearrangement, autophagy, proteasomal degradation, and apoptosis. We highlight the structural basis of these interactions, emphasizing ankyrin repeat domain recognition motifs and the consequences of TNKS-mediated PARylation on protein stability and localization. By integrating findings from oncology, virology, and metabolism, we illustrate how TNKS functions as a nodal regulator linking genome stability, signaling fidelity, and metabolic control. The interplay between TNKS and these varied pathways is essential for the well-being of the organism, with its dysregulation having severe biological and clinical consequences, which are discussed here. Finally, we consider therapeutic implications of disrupting TNKS–protein interactions, with particular attention paid to selective small-molecule inhibitors and their translational potential in cancer, viral infections, and degenerative diseases. Full article

Background: Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterised by uncontrolled proliferation of lymphoid cells. Despite improved outcomes with modern chemotherapy, treatment resistance and adverse effects remain major clinical challenges. Curcumin, a natural compound from Curcuma longa, has shown anticancer potential in multiple malignancies, including leukemia. This systematic review aims to summarise preclinical and clinical evidence on the anti-leukemic effects and mechanisms of action of curcumin in ALL. Methods: A literature search was conducted in August 2025 across PubMed, Scopus, Ovid MEDLINE, and Web of Science according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines. Primary research involving in vitro, in vivo, and human studies examining curcumin’s anti-leukemic effects on ALL were included. Of the 2034 records screened, 26 articles met the inclusion and exclusion criteria. Results: Curcumin inhibited proliferation and induced cytotoxicity and apoptosis in ALL cells via reactive oxygen species generation, DNA damage, mitochondrial dysfunction, and caspase activation. It also inhibited the Janus kinase/signal transducer and activator of transcription (JAK/STAT) and phosphoinositol-3 kinase/protein kinase B (PI3K/AKT) signalling, downregulated breakpoint cluster region-Abelson (BCR-ABL), Wilms tumor 1 (WT1), and Multidrug resistance 1 (MDR1) mRNAs, and induced ceramide accumulation and autophagy. In vivo evidence was limited, and no human studies were identified. Conclusions: Curcumin exerts multi-targeted anti-leukemic effects in ALL. Clinical translation is constrained by its poor bioavailability and limited clinical data. Future research should focus on improving the bioavailability of curcumin via chemical or pharmaceutical modification, as well as conducting well-designed clinical trials. Full article

DNA-protecting proteins (Dps) are crucial for safeguarding chromosomal DNA in starved cells during the stationary phase under stressful conditions. In previous research, the two Dps proteins in Deinococcus geothermalis, Dgeo_0257 (Dps3) and Dgeo_0281 (Dps1), were found to complement each other in protecting DNA from oxidative damage. This study investigates the physiological changes and transposition of insertion sequences (ISs) in a double-knockout (DK) mutant lacking both dps genes. Comparisons between the wild-type and mutant strains revealed significant phenotypic differences in viability under oxidative stress conditions induced by hydrogen peroxide and ferrous ions, particularly during the stationary phase. Notably, oxidative stress triggered the transposition of the IS families IS701 and IS5, with IS66 being transposed exclusively in the DK mutant into a gene encoding phytoene desaturase. Transcriptomic analysis using RNA-seq revealed substantial fold changes in gene expression across the genome. For example, the dgeo_1459–1460 gene cluster, which encodes a DUF421 domain-containing protein and a hypothetical protein, was highly upregulated under both oxidative and non-oxidative conditions. Interestingly, catalase, encoded by a single gene in D. geothermalis, was upregulated in the DK mutant during the stationary phase, with expression levels exceeding those observed in the single dps gene-deficient mutants. Conversely, a prominent downregulation of the Fur family regulator was detected. These findings highlight the growth phase-dependent physiological adaptation of the dps-DK mutant and reveal a novel IS transposition event of the ISBst12 group involving the IS66 family. Therefore, this study provides new observations into the influence of DNA-protective protein deficiency on oxidative stress responses and IS transposition in D. geothermalis, as well as the regulatory mechanisms of the catalase induction pathway, raising the need for further investigation into the role of OxyR. Full article

Background: In nuclear medicine, numerous cancer types are treated via internal irradiation with radiopharmaceuticals, including low-LET (linear energy transfer) beta-emitting radionuclides like Lu-177. In most cases, such treatments lead to low-dose exposure of organ systems with β-irradiation, which induces only few isolated DSBs (double-strand breaks) in the nuclei of hit cells, the most threatening DNA damage type. That damaging effect contrasts with the clustering of DNA damage and DSBs in nuclei traversed by high-LET particles (α particles, ions, etc.). Methods: After in-solution β-irradiation for 1 h with Lu-177 leading to an absorbed dose of about 100 mGy, we investigated the spatial nano-organization of chromatin at DSB damage sites, of repair proteins and of heterochromatin marks via single-molecule localization microscopy (SMLM) in PBMCs. For evaluation, mathematical approaches were used (Ripley distance frequency statistics, DBScan clustering, persistent homology and similarity measurements). Results: We analyzed, at the nanoscale, the distribution of the DNA damage response (DDR) proteins γH2AX, 53BP1, MRE11 and pATM in the chromatin regions surrounding a DSB. Furthermore, local changes in spatial H3K9me3 heterochromatin organization were analyzed relative to γH2AX distribution. SMLM measurements of the different fluorescent molecule tags revealed characteristic clustering of the DDR markers around one or two damage foci per PBMC cell nucleus. Ripley distance histograms suggested the concentration of MRE11 molecules inside γH2AX-clusters, while 53BP1 was present throughout the entire γH2AX clusters. Persistent homology comparisons for 53BP1, MRE11 and γH2AX by Jaccard index calculation revealed significant topological similarities for each of these markers. Since the heterochromatin organization of cell nuclei determines the identity of cell nuclei and correlates to genome activity, it also influences DNA repair. Therefore, the histone H3 tri methyl mark H3K9me3 was analyzed for its topology. In contrast to typical results obtained through photon irradiation, where γH2AX and H3K9me3 markers were well separated, the results obtained here also showed a close spatial proximity (“co-localization”) in many cases (minimum distance of markers = marker size), even with the strictest co-localization distance threshold (20 nm) for γH2AX and H3K9me3. The data support the results from the literature where only one DSB induced by low-dose low LET irradiation (<100 mGy) can remain without heterochromatin relaxation for subsequent repair. Full article

Cancer remains a leading cause of mortality worldwide, and new chemical leads are essential for developing potent anticancer therapies. Evidence suggests that Pseudomonas aeruginosa (Pa) may suppress tumorigenesis, although the underlying mechanisms remain largely unclear. This study characterized a novel small molecule quinolone, JH62 (E-2-(tridec-4-en-1-yl)-quinolin-4(1H)-one, C₂₂H₃₁NO), from Pa. JH62 exhibited broad-spectrum anticancer activity, inhibiting the proliferation of A549 lung cancer cells in a time- and dose-dependent manner with an IC₅₀ of 15 μM, while showed low cytotoxicity toward normal cells. In xenograft mice model, treatment with JH62 (10 mg/kg) reduced tumor weight and volume by 73% and 79%, respectively. Mechanistically, treatment with JH62 induced structural and functional disruption of mitochondria in cancer cells, triggered autophagic cell death, and did not cause DNA damage. Genetic analysis confirmed that JH62 biosynthesis depends on the pqsABCDE gene cluster and that JH62 positively regulates its own production. ADMET profiling further indicated promising drug-like properties for future development. These findings establish JH62 as a promising anticancer lead compound derived from microbial metabolism. Full article

The ferredoxin reductase (FDXR) gene is expressed as seven isoforms: 1–6 by alternative splicing and 7 by an alternative promoter according to the Entrez Gene Database. Previous studies showed that FDXR, primarily the mitochondrial isoform 1, plays a role in biosynthesis of sterols, heme, and iron–sulfur clusters. However, the biological functions of FDXR isoforms 3–7 have not been characterized. Here, we first examined the expression profile of various FDXR isoforms. We found that isoform 1 is the most abundant one, accounting for ~70% of total FDXR, whereas isoforms 4 and 7 account for ~10% and ~7%, respectively. We found that isoforms 1 and 4 are mainly localized in the mitochondria, whereas isoform 7, which lacks a mitochondria localization signal (MLS), is expressed in the cytosol. We also found that like the promoter 1 for isoforms 1-6, the P2 promoter for isoform 7 can be induced by DNA damage in a p53-dependent manner. To determine isoform-specific activity, we generated multiple MCF7 cell lines in which one or more FDXR isoforms are knocked out. While total FDXR-KO MCF7 cells are non-viable, cells deficient in isoforms 1–6, isoform 4, or isoform 7 remain viable but are defective in cell proliferation, DNA damage response, and repair. These data suggest that each FDXR isoform contributes to cell survival and that isoform 7 has extra-mitochondrial activity that may be sufficient for cell survival. Full article

Background: Germline alterations in DNA damage repair (DDR) genes represent a clinically important subset of prostate cancer (PCa), but real-world data from Middle Eastern and Turkish populations remain limited. We evaluated the prevalence and clinicopathologic associations of germline DDR variants in a single-center Turkish cohort. Methods: We retrospectively analyzed 122 men with histologically confirmed PCa who underwent germline multigene panel testing. Variants were classified according to ACMG/ClinVar criteria. Patients were grouped as pathogenic/likely pathogenic (P/LP), variants of uncertain significance (VUS), or variant-negative. Patients were grouped as variant-positive (P/LP or VUS/uncategorized) or clinically actionable variant–negative (benign/likely benign or no variant detected). Group comparisons used t-tests, chi-square or Fisher’s exact tests as appropriate. Results: The median age at diagnosis was 65.2 years (mean 64.6 ± 8.78). Overall, 37 patients (30.3%) carried at least one germline variant, including 12 (9.8%) with P/LP alterations and 24 (19.7%) with VUS; one patient (0.8%) harbored an uncategorized variant. The most frequently affected genes were CHEK2 (n = 8), BRCA1 (n = 6), BRCA2 (n = 6), ATM (n = 5), and APC (n = 4). Variant-positive status increased from 10.8% in ISUP 1–2 to 21.6% in ISUP 3 and 76.0% in ISUP 4–5, although this trend was not statistically significant (p = 0.391). Mean age at diagnosis and the prevalence of metastatic disease did not differ between variant-positive and clinically actionable variant–negative patients (64.2 vs. 65.7 years, p = 0.390; 66.7% vs. 64.6%, p = 0.842). Truncating DDR variants (RAD50, BRCA2, MSH3, NBN, CHEK2, ATM) occurred predominantly in ISUP 4–5 tumors. Conclusions: Germline DDR alterations—most notably in BRCA2, CHEK2, and ATM—were present in a substantial subset of Turkish men with PCa and showed a non-significant trend toward clustering in higher-grade disease. The high prevalence of VUS reflects limited genomic annotation in under-represented populations and underscores the need for longitudinal reinterpretation. These data support the clinical value of incorporating germline DDR testing into risk assessment and familial counseling, while larger cohorts integrating somatic profiling are needed to refine genotype–phenotype associations. Full article