Statistical analysis of kinetics, distribution and co-localisation of DNA repair foci in irradiated cells: Cell cycle effect and implications for prediction of radiosensitivity

•H2AX foci kinetics as a tool to assess individual radiosensitivity.•Identification of minor defects in an SCID patient.•Role of the proliferating status of cells.•Focus distribution analysis and average focus number in major sub-population.•H2AX/53bp1 co-localisation as an additional parameter.

Detection of γ-H2AX foci as a measure of DNA double strand break induction and repair provides the basis of a rapid approach to establish individual radiosensitivity. However, the assignment of criteria to define increased radiosensitivity is not straightforward. Experimental end points, analytical methods and proliferative status of the cells sampled for analysis are important. All these issues are addressed in the present study, which was prompted by a clinical request to assess the radiosensitivity status of an SCID paediatric patient being considered for bone marrow transplantation.We investigated the kinetics of repair of radiation-induced γ-H2AX foci in proliferating and confluent cultures of skin fibroblasts obtained from the patient, and from normal and radiosensitive (Artemis-deficient) controls. As well as the standard approach of averaging foci per cell over the entire population (“standard average”), we also examined foci per cell frequency distributions and calculated average foci per cell values in the major Poisson-distributed subpopulation (“principal average”). This approach allowed to avoid distortions such as that due to the S/G2 population in proliferating cells, with focus numbers approaching twice the normal, and to detect subpopulations of cells with defects in focus formation and repair.From the “standard average” analysis and co-localisation of γ-H2AX foci with 53BP1 we assigned the patient's repair status as close-to-normal. However, analysis of “principal average”, foci per cell frequency distributions and survival curves challenged this initial conclusion.These studies indicate new dimensions of the γ-H2AX assay that, with further elaboration and exemplification, have the potential to augment its power to predict radiosensitivity.