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

Abstract

DNA ligase 4 deficiency (LIG4-SCID) causes lymphopenia (T-B-NK1) and a radiosensitive SCID (RS-SCID) phenotype. We demonstrate, for the first time, flow cytometric-based kinetic analysis of phosphorylated H2AX (gH2AX) in lymphocyte subsets, especially NK cells, for the assessment of LIG4-SCID. Measurement of phosphorylated (p) ATM, SMC1, and H2AX (gH2AX) 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 gH2AX 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 gH2AX, but increased levels of gH2AX 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 gH2AX 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 gH2AX is a useful marker for the diagnosis of LIG4-SCID.