Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity  Pavel Lobachevsky, Lisa Woodbine, Kuang-Chih Hsiao, Sharon Choo, Chris Fraser, Paul Gray, Jai Smith, Nickala Best, Laura Munforte, Elena Korneeva, Roger F. Martin, Penny A. Jeggo, Olga A. Martin  The Journal of Molecular Diagnostics  Volume 17, Issue 5, Pages 560-575 (September 2015) DOI: 10.1016/j.jmoldx.2015.05.004 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Analysis of post–3-Gy irradiation (IR) γ histone 2A isoform X (H2AX) foci kinetics in six reference cultures without [C1 (normal control)] and with known deficiencies in nonhomologous end-joining repair pathway [C2 to C6 (repair-deficient mutants)]. A: C2, DCLRE1C null. B: C3, leaky DCLRE1C heterozygous. C: C4, PRKDC heterozygous. D: C5, leaky LIG4. E: C6, XLF. Circles represent the principal mean foci numbers; error bars show the SEMs of the means before IR exposure, positioned at 0 hour and after IR exposure, at 0.5 (0.25 for C5), 2, 6, 24, 48, and 72 hours. Lines demonstrate the results of regression analysis of the repair kinetics using the two-component model according to Equation 2. Each panel shows data from C1 and one of the repair-deficient mutants. The Journal of Molecular Diagnostics 2015 17, 560-575DOI: (10.1016/j.jmoldx.2015.05.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Comparative analysis of post–3-Gy irradiation (IR) γ histone 2A isoform X (H2AX) foci kinetics in two reference cultures, C1 (normal control) and C2 (DCLRE1C null), and the five cell strains from patients with unknown radiosensitive status (P1 to P5). A–E: The repair kinetics of each patient's cell strain is shown in a separate panel, alongside with kinetics for the normal (C1) and DCLRE1C-deficient (C2) reference cells for comparison. Circles represent the principal mean foci numbers; error bars show the SEMs of the means before IR exposure, at 0 hour, and after IR exposure, at 0.5, 2, 6, 24, 48, and 72 hours. Lines demonstrate the results of regression analysis of the repair kinetics using the two-component model according to Equation 2. F: Kinetics of each cell culture (C1, C2, and P1 to P5). G: Representative laser confocal microscopy images of fibroblasts from normal (C1) and DCLRE1C-deficient (C2) donors and from the patient (P2) immunostained with anti–γ-H2AX antibody (green channel). Nuclei are counterstained with propidium iodide (red). An increased number of γ-H2AX foci is evident at 6 and 24 hours after IR in C2 and P2 cells compared with C1 cells. The Journal of Molecular Diagnostics 2015 17, 560-575DOI: (10.1016/j.jmoldx.2015.05.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 The radiosensitive (RS) map. A: The thick line represents Q values obtained from the nonlinear regression analysis (Equation 1) of the γ histone 2A isoform X (H2AX) foci kinetics of normal control cell culture C1 (Figure 1) using a range of fixed s values, with closed circles representing the best-fit s and Q values; the thin lines indicate SEMs of these Q values. The area between these lines can be defined as a normal zone. Open circles represent best-fit s and Q values from nonlinear regression analysis of the γ-H2AX foci kinetics of normal control cells (C1 and 48BR) obtained in seven independent experiments. The dashed line represents the boundary of the normal zone calculated as a set of best-fit Q values obtained from the regression of the kinetics data from the same seven independent experiments using a range of fixed s values, based on the kinetics data from the same seven independent experiments. B: The individual data points are best-fit s and Q values of the independent nonlinear regression analysis of the γ-H2AX foci kinetics of all of the investigated reference and patient cell cultures (C1 to C6, P1 to P5). Lines represent the normal zone corresponding to the dashed line in A. Data points above the normal zone indicate increased cellular RS status. C: The individual data points are best-fit s and Q values of the nonlinear regression analysis of the foci kinetics reported in seven RS–severe combined immunodeficiency or RS–combined immunodeficiency patients (DCLRE1C deficient: DB333,41 CJ176,41 AA5547; LIG4 deficient: 495GOS,48 411BR,49 230349; XLF deficient: F07/40248). Lines represent the normal zone. The Journal of Molecular Diagnostics 2015 17, 560-575DOI: (10.1016/j.jmoldx.2015.05.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: Radiation dose–response curves for cell survival of four reference cell cultures (C1, normal control; C2, C3, and C5, cells with known deficiencies). B–F: The five SCID patient cell cultures with unknown radiosensitivity (RS) status. Lines are generated by nonlinear regression using the linear-quadratic model. Sensitization factors (SFs) and significance versus normal control cells (C1) are as follows: C2, SF = 4.24 ± 0.92, P < 0.0003; C3, SF = 1.64 ± 0.20, P < 0.009; C5, SF = 6.26 ± 1.09, P = 0.0001; P1, SF = 1.50 ± 0.30, P = 0.074; P2, SF = 2.68 ± 0.79, P < 0.006; P3, SF = 1.18 ± 0.09, P = 0.082; P4, SF = 1.01 ± 0.10, P = 0.92; and P5, SF = 1.19 ± 0.53, P = 0.698. The Journal of Molecular Diagnostics 2015 17, 560-575DOI: (10.1016/j.jmoldx.2015.05.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Identification of mutational changes in cDNA in P2 and P5. A: Mutational changes in LIG4 (P2). Dye-terminator sequence figures illustrating the c.73C>T = p.R25X (in the presented figure, C was automatically selected) and c.845A>T = p.H282L mutational change in P2 and respective wild-type sequence from C1. B: Locations of the identified mutational changes in LIG4 in relation to important domains. C: Mutational changes in DCLRE1C (P5). Dye-terminator sequence figures illustrating the Δexon 11 mutational change in P5 and respective wild-type sequence from C1. The Journal of Molecular Diagnostics 2015 17, 560-575DOI: (10.1016/j.jmoldx.2015.05.004) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions