Khdeda E, Naumann-Bartsch N, Khdeda N, Cramer G, Hildebrand L, Schiller P, Wagner P, Fahrmeier F, Hüffmeier U, Corradini S, Distel L, Kuhlmann L (2026)
Publication Type: Journal article
Publication year: 2026
Book Volume: 14
Article Number: 92
Journal Issue: 3
Background/Objectives: DNA polymerase ε (Pol ε), encoded by POLE1, plays a pivotal role in high-fidelity DNA replication and in coordinating DNA repair. While pathogenic exonuclease-domain variants are well established in cancer, biallelic POLE1 variants remain largely unexplored in non-malignant human cells. Methods: Here, we analyzed primary fibroblasts derived from a skin biopsy of a compound-heterozygous patient carrying two POLE1 variants. Western blot analysis confirmed detectable Pol ε protein levels, indicating preserved protein expression despite the underlying variants. Results: Nevertheless, functional alterations were observed across multiple independent assays. Compared with healthy control fibroblasts, this patient-derived Pol ε fibroblast line exhibited reduced clonogenic survival following ionizing radiation. Surviving fractions were consistently lower across radiation doses from 2 to 4 Gy, with an approximately twofold reduction at 2 Gy and progressively greater differences at higher doses. The isoeffect dose corresponding to 10% survival was reduced relative to pooled control fibroblasts. In addition, chromosomal breakage was increased, supporting altered processing of radiation-induced DNA damage in this cellular model. Live-cell imaging and senescence assays revealed delayed proliferation and an increased proportion of senescent or senescence-like cells under baseline and genotoxic stress conditions, including enhanced senescence-associated β-galactosidase activity. Flow-cytometric analysis demonstrated S phase accumulation and G2/M arrest, consistent with replication stress and cell-cycle perturbation. Immunofluorescence staining revealed increased γH2AX foci, consistent with persistent DNA double strand breaks. RAD51 foci formation was not reduced; instead, increased RAD51 recruitment was observed under combined cisplatin and irradiation treatment, arguing against a primary defect in RAD51-mediated homologous recombination. POLE1-variant fibroblasts also showed impaired proliferative recovery, reduced wound closure, increased γH2AX accumulation following cisplatin exposure, suggesting heightened susceptibility to DNA crosslinking stress. Conclusions: Collectively, these findings provide the first functional characterization of a patient-derived POLE1-variant fibroblast cell line and indicate that altered Pol ε function may influence cellular responses to genotoxic stress. While based on primary fibroblasts from a single compound-heterozygous patient, validation in additional patient-derived or isogenic models will be required to determine the broader relevance of these findings.
APA:
Khdeda, E., Naumann-Bartsch, N., Khdeda, N., Cramer, G., Hildebrand, L., Schiller, P.,... Kuhlmann, L. (2026). Functional Characterization of POLE1 Variant Fibroblasts Reveals Replication Stress and Increased Sensitivity to Genotoxic Stress. Diseases, 14(3). https://doi.org/10.3390/diseases14030092
MLA:
Khdeda, Enas, et al. "Functional Characterization of POLE1 Variant Fibroblasts Reveals Replication Stress and Increased Sensitivity to Genotoxic Stress." Diseases 14.3 (2026).
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