Fibroblast-based radiosensitivity assays as a clinically valuable tool for (severe) combined immunodeficiency
Elien Beyls1, Somara De Beul2, Victoria Bordon3
1Primary Immunodeficiency Research Lab (PIRL), Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium; Center for Primary Immunodeficiency, Jeffrey Modell Diagnosis and Research Center, Ghent University Hospital, Ghent, Belgium.
Abstract:
Genetic defects in one of the DNA double strand break (DSB) repair proteins lead to distinct human syndromes with severe clinical manifestations, including impaired neurological and immunological development, cancer proneness and sensitivity to ionizing radiation. Since diagnostic and therapeutic procedures frequently use DNA damaging agents, identification of radiosensitive individuals is imperative to optimize patient management. However, patients with a (severe) combined immunodeficiency (S)CID are often ineligible for lymphocyte-based radiosensitivity testing. Therefore, this study investigated the suitability of two fibroblast-based assays as alternative methods. DSB repair was evaluated following X-ray irradiation by an optimized cytokinesis-block micronucleus (MN) assay and the γH2AX focus test in fibroblasts from patients with a confirmed or suspected diagnosis of radiosensitive (S)CID. Using both assays, patients with a defect in Artemis were identified as radiosensitive while those with a RAG1/2 deficiency were not considered as radiosensitive. Although MN scoring was not feasible in irradiated fibroblasts deficient in XLF, LIG4 or NBS1, radiosensitivity could be readily demonstrated through impaired DNA DSB repair kinetics with the γH2AX focus assay in fibroblasts deficient in XLF or LIG4, but not in those deficient in NBS1. While both ATM defective fibroblasts clearly showed increased radiation-induced MN yields, one of the two fibroblast cell lines could not be identified as radiosensitive based on residual γH2AX focus levels. This study suggests that combining the fibroblast MN assay and γH2AX focus test can effectively exclude in vitro radiosensitivity in patients with a suspicion of radiosensitive (S)CID, particularly when lymphocyte-based radiosensitivity testing is not feasible.
Insights
Fibroblast assays can identify radiosensitive patients with (severe) combined immunodeficiency (SCID) when lymphocyte tests are unsuitable. Combining micronucleus and γH2AX focus assays effectively assesses DNA double strand break repair capacity.
Area of Science:
- Genetics
- Radiation Biology
- Immunology
Background:
- Genetic defects in DNA double strand break (DSB) repair proteins cause syndromes with neurological, immunological, and cancer issues.
- Identifying radiosensitive individuals is crucial for managing patients undergoing DNA-damaging treatments.
- Lymphocyte-based radiosensitivity testing is often not feasible for patients with (severe) combined immunodeficiency (SCID).
Purpose of the Study:
- To evaluate the suitability of two fibroblast-based assays for radiosensitivity testing in patients with suspected radiosensitive (S)CID.
- To assess DNA double strand break repair capacity in patient-derived fibroblasts after X-ray irradiation.
Main Methods:
- Optimized cytokinesis-block micronucleus (MN) assay.
- γH2AX focus assay.
- Evaluation of fibroblasts from patients with confirmed or suspected radiosensitive (S)CID.
Main Results:
- Artemis-deficient patients were identified as radiosensitive; RAG1/2-deficient patients were not.
- Micronucleus scoring was not feasible for XLF, LIG4, or NBS1 deficiencies.
- γH2AX focus assay detected radiosensitivity in XLF and LIG4 deficiencies, but not NBS1.
- ATM-deficient fibroblasts showed increased MN yields, but inconsistent γH2AX focus results.
Conclusions:
- Combining fibroblast micronucleus and γH2AX focus assays can effectively assess in vitro radiosensitivity.
- These assays provide a viable alternative for radiosensitivity testing when lymphocyte-based methods are not feasible for (S)CID patients.
