Application of a radiosensitivity flow assay in a patient with DNA ligase 4 deficiency

• Published in: Blood advances 2018-08, Vol.2 (15), p.1828-1832
• Main Authors: Buchbinder, David, Smith, Matthew J., Kawahara, Misako, Cowan, Morton J., Buzby, Jeffrey S., Abraham, Roshini S.
• Format: Article
• Language: English
• Subjects: Biomarkers; DNA Ligase ATP - deficiency; DNA Ligase ATP - immunology; Female; Flow Cytometry; Gamma Rays; Histones - immunology; Humans; Immunobiology and Immunotherapy; Killer Cells, Natural - immunology; Killer Cells, Natural - pathology; Phosphorylation - immunology; Radiation Tolerance - genetics; Radiation Tolerance - immunology; Severe Combined Immunodeficiency - diagnosis; Severe Combined Immunodeficiency - genetics; Severe Combined Immunodeficiency - immunology; Severe Combined Immunodeficiency - pathology; T-Lymphocytes - immunology; T-Lymphocytes - pathology
• ISSN: 2473-9529; 2473-9537
• DOI: 10.1182/bloodadvances.2018016113

DNA ligase 4 deficiency (LIG4-SCID) causes lymphopenia (T-B-NK+) and a radiosensitive SCID (RS-SCID) phenotype. We demonstrate, for the first time, flow cytometric-based kinetic analysis of phosphorylated H2AX (γH2AX) in lymphocyte subsets, especially NK cells, for the assessment of LIG4-SCID. Measurement of phosphorylated (p) ATM, SMC1, and H2AX (γH2AX) was performed by flow cytometry to assess DNA repair defects in a 3-year-old girl. Functional assessment (phosphorylation) was measured in T and NK cells (B cells were absent) before irradiation (background control) or after low-dose (2Gy) irradiation (1 and 24 hours). We observed maximal γH2AX at 1 hour postirradiation, with dephosphorylation at 24 hours postirradiation in healthy control patients. The patient showed normal frequencies (percentage) of T cells and NK cells for γH2AX, but increased levels of γH2AX compared with control patients at 1 hour postirradiation. At 24 hours postirradiation, there was a lack of dephosphorylation in a substantial proportion of lymphocytes (with differences observed between T and NK cells) compared with healthy control patients. Although there was dephosphorylation of γH2AX at 24 hours in patient lymphocytes compared with 1 hour, the amount remained elevated at 24 hours compared with in control patients. The data from pATM and pSMC1 were uninformative. Flow-based kinetic analysis of γH2AX is a useful marker for the diagnosis of LIG4-SCID. •DNA ligase 4 deficiency is a defect causing lymphopenia (T-B-NK+) and a radiosensitive severe combined immunodeficiency phenotype.•Flow cytometric analysis of phosphorylation/dephosphorylation states of histone H2AX allows for in-depth lineage-specific assessment. [Display omitted]