Retarded DSB repair kinetics suggestive of augmented radiation sensitivity and genetic instability in Wiskott-Aldrich syndrome patients

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Pathak, Rajesh Kumar Chaurasia, B.K. Sapra, Pallavi Gaikwad, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5719467/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Wiskott-Aldrich Syndrome (WAS) is a rare X-linked recessive disorder characterized by microthrombocytopenia, eczema, combined immunodeficiency, and an increased risk of malignancies. Previous research has highlighted genomic instability in WAS patients; however, the specific dynamics of double-strand break (DSB) repair in these individuals are not fully understood. Phosphorylation of γH2AX serves as an early indicator of DSBs, while 53BP1 binds to damaged chromatin in a γH2AX-dependent manner, stabilizing DNA ends and facilitating repair and hence serves as a marker for assessing DNA repair kinetics. In this study, we investigated the kinetics of DSBs in WAS patients and their carrier mothers using markers such as γH2AX and 53BP1. To evaluate radiation sensitivity, lymphocytes from WAS patients were exposed to 2 Gy of gamma radiation, and repair kinetics were recorded over 24 hours. Immunofluorescence staining for γH2AX and 53BP1 was performed on both irradiated and non-irradiated lymphocytes from patients, carrier mothers, and healthy controls at various time points. Notably, WAS patients exhibited a significant increase in baseline levels of γH2AX and 53BP1 (16 to 24 times higher) compared to healthy controls. Post-radiation decay model revealed a significantly prolonged DSB repair rate in WAS patients, with a repair half-life 1.5 to 1.9 times longer than that of controls. Additionally, at 24 hours, WAS patients showed 37–39% and 33–43% excess γH2AX and 53BP1 foci per cell, respectively, indicating increased genomic instability and radiosensitivity. This study provides the first evidence of delayed repair kinetics in WAS patients, which may impact clinical decisions regarding radiation or genotoxic exposure during diagnosis and treatment. Wiskott-Aldrich syndrome (WAS) DSB repair kinetics DSB repair half-time genetic instability radiosensitivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Wiskott-Aldrich syndrome (WAS), first identified in 1937 by Alfred Wiskott and Robert Aldrich reported that this disease manifests as microthrombocytopenia, eczema and a primary immunodeficiency disorder with various clinical presentations, including susceptibility to autoimmunity and malignancies ( 1 , 2 ). This X-linked rare disorder primarily affects males, although rare cases of female carriers with variable expressivity have been documented ( 3 ). The incidence is approximately 4 in 1 million live male births in the U.S., with occasional cases occurring in females ( 4 ). WAS is caused by mutations in the gene encoding the WAS protein (WASp), which is located on the X chromosome (Xp11.22-p11.23) ( 5 ). WASp, which is primarily expressed in hematopoietic cells ( 6 ) regulates actin cytoskeleton dynamics, cell motility, and immune synapse formation, resulting in both quantitative and qualitative deficiencies in T and B cells ( 7 – 8 ). The expression of WASp depends on type and location of mutation in WAS gene ( 9 ). More than 440 genetic mutations have been linked to WAS, with missense mutations being the most common, followed by splice mutations, deletions, and nonsense mutations ( 10 ). Children with WAS have a greater risk of developing malignancies, with reported incidences of up to 13%, most commonly EBV-related B-cell lymphomas or leukemia, which typically appear at approximate age of 9.5 y. ( 11 ). DNA double-strand breaks (DSBs) caused by ionizing radiation or chemotherapy represent some of the most severe types of DNA damage ( 12 ). If left unrepaired, DSBs can lead to genome instability or cell death, highlighting the critical necessity of prompt and accurate DSB repair ( 13 ). Two essential DSB repair pathways have evolved in human cells: nonhomologous end-joining (NHEJ) and homologous recombination (HR) ( 14 ). In mammalian cells, NHEJ serves as the primary DNA repair pathway and is active throughout the cell cycle. It predominantly handles DSB repair outside of the S and G2 phases and manages approximately 80% of DSB repair within the S and G2 phases ( 15 , 16 ). Recent research has shown the emerging roles of WASH (Wiskott-Aldrich syndrome protein and SCAR homologue) and its intricate components within the nucleus ( 17 – 20 ). A study conducted in Drosophila cells revealed the essential role of nuclear F-actin, facilitated by the Arp2/3 complex, in relocating heterochromatin breaks to the nuclear periphery for subsequent repair. Interestingly, the depletion of WASH led to a defect in this relocation process, suggesting its potential involvement in DSB repair ( 21 ). Moreover, Wang et al. (2022) revealed an interaction between WASH and Ku proteins, indicating a regulatory role for WASH in DSB repair pathways in 3T3 mouse fibroblasts ( 22 ). The timeframe for repairing DSBs is critical for maintaining genomic integrity ( 23 ). In healthy human cells, the standard DSB repair duration typically spans from 2 to 4 hours under optimal conditions ( 24 ). However, in instances of impaired DSB repair systems, this timeframe can be significantly prolonged or altered. Previous studies have reported the presence of unrepaired DSBs and genomic instability in cells with WAS mutations ( 25 ). However, the precise kinetics of DSB repair and the half-life of DSB repair processes remain unclear or elusive. The speed and accuracy of DSB repair are vital determinants of cell survival and the prevention of genomic instability and malignancy ( 26 ). Furthermore, they play a crucial role in assessing the sensitivity of WAS patients to genotoxic agents such as radiation, chemotherapeutics, etc. ( 27 ). WAS patients (30–50%) often require Bone Marrow Transplant (BMT) due to immunodeficiency and thrombocytopenia ( 28 ). Conditioning regimens, such as whole-body irradiation, precede BMT to prepare recipients. In WAS, the type and location of the mutation significantly influence disease severity, which in turn affects the degree of radiation sensitivity. This variability in radiation sensitivity is observed among patients with different mutations, reflecting the diverse impact that these genetic changes have on the condition ( 29 ). Studies on DSB repair efficiency and radiation sensitivity in patients with diverse mutations in the WAS gene are currently limited or unavailable. However, leveraging this information could enhance personalized treatment strategies, potentially including dose adjustments based on hypersensitivity assessments, to improve radiation therapy outcomes in this population. This study investigated the kinetics of DSB repair (utilizing γH2AX and 53BP1 and their colocalization) in the lymphocytes of four WAS patients with different mutations in the WAS gene. The DSB repair half-lives in lymphocytes were estimated, and the presence of spontaneous unrepaired DSBs was measured. However, despite having a heterozygous WAS mutation, carrier mothers exhibited normal repair kinetics. These insights are crucial for evaluating genomic instability and radiation sensitivity and tailoring personalized BMT strategies for the effective management of WAS patients. Materials and methods Study design: We assessed DSB repair kinetics, the repair half-life, and radiation sensitivity in four WAS patients and compared them with carrier mothers and a healthy control. Chemicals: 4′,6-Diamidino-2-phenylindole (DAPI) with antifade (ProLong™ Diamond Antifade Mountant with DAPI) was procured from Invitrogen, USA. Tween 20, poly L-lysine, and Histopaque-1077 were obtained from Sigma‒Aldrich, USA. Paraformaldehyde,1,1,3,3-tetraethoxypropane, Triton X-100 and Fetal calf serum (FCS) were procured from Gibco Life Technologies, USA. Mouse anti-phospho-histone γH2AX (ser-139) human monoclonal IgG was procured from Millipore, USA. FITC-labelled rabbit anti-mouse IgG was procured from Invitrogen (USA). Rabbit anti-phospho-53BP1 human monoclonal IgG and Texas Red-labelled goat anti-rabbit IgG were procured from Cell Signalling Technology (CST) (Massachusetts, USA) and Invitrogen (USA), respectively. Ethical approval and sample collection : Institutional ethics committee (IEC) of ICMR- National Institute of Immunohaematology, Mumbai, India has approved this study. Inform consent was obtained to collect 3 ml peripheral blood samples from patients and family members in heparin vacutainers. Preparation and irradiation of peripheral blood mononuclear cells (PBMCs): Peripheral blood mononuclear cells (PBMCs) were isolated from patients, carrier mothers and healthy controls using heparin vacutainers via density gradient centrifugation ( 30 ). After centrifugation, the milky buffy coat containing PBMCs was collected and washed (twice) with RPMI. PBMCs containing pellet was resuspended in fresh complete RPMI media. Lymphocytes were then exposed to 2 Gy of gamma irradiation using a Blood Irradiator (Dose rate: 0.54 Gy/min) ( 31 ). After irradiation, PBMCs were washed and allowed to recover for 24 hours in fresh complete RPMI media. Kinetics of DSB repair foci, γH2AX and 53BP1: The kinetics of DSB repair foci (γH2AX and 53BP1) and their colocalization were investigated for up to 24 hours following irradiation in lymphocytes obtained from the enrolled subjects (WAS patients and carrier mothers). After irradiation, the PBMCs were incubated under optimal conditions (37°C, 95% relative humidity, and 5% CO 2 ) for up to 24 hours. PBMCs were sampled at various time points (0, 0.083, 0.17, 0.25, 0.5, 1, 2, 4, 8, 16, and 24 hours) post irradiation to quantify the presence of DSB repair foci. Immunofluorescence-staining of γH2AX and 53BP1-DSB-repair proteins: Immunostaining for γH2AX and 53BP1 was performed following our own optimized protocol ( 32 , 33 ). After post-irradiation incubation, PBMCs were fixed in 4% paraformaldehyde at 4°C for 30 minutes, washed with PBS, and deposited on poly-L-lysine-coated coverslips to adhere for 1 hour. The cells were permeabilized with 0.5% Triton X-100, washed, and blocked with 5% FCS in PBS for 1 hour. Primary antibodies against γH2AX and 53BP1 were added (dilution-1:200), followed by incubation with Alexa Fluor 488 and Texas Red secondary antibodies (dilution-1:400). Cells were mounted using DAPI with antifade solution for imaging. Detection and quantification of γH2AX and 53BP1 foci using confocal microscopy: The mounted slides were imaged using a Leica SP8 confocal fluorescence microscope with Leica Application Suite-X (LAS-X). γH2AX foci were visualized with a green filter, 53BP1 foci with a red filter, and nuclei with a blue filter. Images were captured and overlaid to show colocalization of γH2AX and 53BP1 in the nuclei. Approximately 300 lymphocytes per individual were examined at each of the 11 incubation time points. Statistical analysis: The experiments were performed in three sets, with data presented as mean ± SD. Statistical analysis used Student’s t-test to assess differences among incubation time points and between subjects, with a significance threshold of p < 0.05 ( 34 ). Trends were modelled using the least squares method. Results Case history and pathophysiological conditions of the subjects: In our study, we focused on four male paediatric patients (aged 3 months to 14 years) along with their carrier mothers. Clinical, immunological and molecular features of these patients were reported by Gaikwad et al 2024 ( 9 ). These were also summarised in Table 1 . Mothers who carried the mutated gene did not manifest symptoms of the disease. Tragically, W1 patient passed away at the age of 5 months due to severe respiratory tract infection and thrombocytopenia. Two patients W2 and W3 are currently suffering from characteristic WAS symptoms and are awaiting BMT. While W4 had undergone BMT at the age of 14 years, leading to a notable improvement in his quality of life. Table 1 Depiction of pathophysiological symptoms, age, mutation details, and current status of the four patients diagnosed with WAS. Subjects Patients codes Age Symptoms Mutation Details Current Status W1 P31 5 Months Born with second degree consanguineous marriage. At 4 months of age c/o cough, fever and increased respiratory activity, thrombocytopenia. Presented with eczema and bloody stools Frameshift c.1266_1267insG (p.L425Pfs*70) Expired W2 P40 3 Months Born with non-consanguineous marriage, presented with history of repeated episodes of loose stool with blood, recurrent sepsis, anaemia with thrombocytopenia. Cytomegalovirus (CMV) positive. Upper GI endoscopy and ileocolonoscopy shows gastritis and pancolitis. Bone marrow aspiration showed normal results. Frameshift c.763_764insG (p. Q255Rfs*5) Awaiting BMT W3 P39 1 Year Eczema, thrombocytopenia, Lower Respiratory Tract Infections (LRTI) with primary oxygen requirement. Missense c.134C > T (p. T45M) Awaiting BMT W4 P21 14 years H/o bleeding manifestation (skin and mucosal bleeding) and recurrent respiratory tract infection and H/o thrombocytopenia. Missense c.223G > C (p.V75L) Transplanted 3.2 Baseline DSB (genomic instability) assessment: Results showed significantly elevated frequencies of γH2AX and 53BP1 foci in the lymphocytes of WAS patients (γH2AX: 16–24 times and 53BP1: 17–25 times) compared to those in the healthy controls (Table 2 ; Fig. 2 ). The frequencies of foci observed in the mothers were found to be within the range of the controls. Figure 3 is a representative image depicting the foci and their colocalization in the lymphocytes of a healthy control volunteer, the WAS patient, and carrier mother. A good colocalization (71–99%) between γH2AX and 53BP1 foci was observed in all the subjects. Table 2 Baseline data on γH2AX and 53BP1 foci/cell and their colocalization in lymphocytes from four WAS patients, their carrier mothers and four healthy controls. Subjects Baseline levels of foci/cell gH2AX foci/cell 53BP1 foci/cell Colocalization foci/cell WAS1 (W1) 3.4 ± 0.45 3.23 ± 0.39 3.2 ± 0.37 WAS2 (W2) 3.1 ± 0.32 2.6 ± 0.31 2.4 ± 0.27 WAS3 (W3) 2.19 ± 0.37 2.16 ± 0.25 2.11 ± 0.23 WAS4 (W4) 2.99 ± 0.44 2.91 ± 0.45 2.56 ± 0.41 Avg of W1-W4 2.92 ± 0.52 2.73 ± 0.46 2.57 ± 0.46 Mother-W1 0.147 ± 0.112 0.121 ± 0.101 0.118 ± 0.087 Mother-W2 0.135 ± 0.102 0.123 ± 0.089 0.112 ± 0.065 Mother-W3 0.123 ± 0.081 0.121 ± 0.063 0.12 ± 0.062 Mother-W4 0.189 ± 0.131 0.159 ± 0.131 0.149 ± 0.072 Avg of MW1-MW4 0.149 ± 0.029 0.131 ± 0.019 0.125 ± 0.017 Control 1 (C1) 0.141 ± 0.037 0.134 ± 0.041 0.121 ± 0.037 Control 2 (C2) 0.139 ± 0.068 0.129 ± 0.049 0.119 ± 0.045 Control 3 (C3) 0.137 ± 0.029 0.131 ± 0.031 0.123 ± 0.032 Control 4 (C4) 0.138 ± 0.053 0.126 ± 0.043 0.114 ± 0.051 Avg of C1-C4 0.139 ± 0.002 0.13 ± 0.003 0.119 ± 0.004 3.3 DSB repair dynamics and radiation sensitivity in lymphocytes of WAS patients: We have assessed abundance of γH2AX and 53BP1 foci, as well as their colocalization post gamma exposure in WAS patients and their carrier mothers along with healthy controls. Within 15 minutes post-irradiation, foci were detectable, with a statistically significant increase (p < 0.05) compared to their respective baseline levels. The fitted curves represented in Figs. 4 A-D. While Fig. 5 illustrates foci and their colocalization in the lymphocytes of a healthy control volunteer, the WAS4 patient, and carrier mother. The fitting analysis revealed a pattern where the number of foci initially increased and then followed a saturating exponential function, which can be expressed as \(\:Y=A(1-{e}^{-kt})\) where A , and k are constants, with values as listed in Table 3 . Similar pattern was reported earlier in control samples ( 32 , 33 ). The units of Y and A , are foci/cell , while k is expressed in h −1 and t represents time in h . The constant k , representing the rate of foci formation, showed significant variation among the four WAS patients as they exhibited a lower rate of foci formation compared to control. Foci formation reached saturation after 1 h in all subjects with a plateau observed up to 2 hours postirradiation. Subsequently, the yield of foci began to decrease in both WAS and control lymphocytes. The saturation yield of the foci was slightly lower in WAS patients compared to control. The W4 patient exhibited the lowest yield of foci, 11.85% for γH2AX and 11.31% for 53BP1 of the control. Table 3 Fitting parameters for the build-up of foci (γH2AX, 53BP1, and their colocalization) in lymphocytes obtained from four WAS patients and carrier mothers, along with healthy controls. The lymphocytes were exposed to 2 Gy of 60 Co-γ-rays, and foci were quantified up to 1-hour post-irradiation. Initial foci build up followed a single exponential growth pattern, mathematically expressed as \(\:\varvec{Y}=\varvec{A}(1-{\varvec{e}}^{-\varvec{k}\varvec{t}})\) . Subjects Fitting parameters for foci development γH2AX foci 53BP1 foci Colocalization of foci A (foci/cell) Build up constant k (h − 1 ) A foci/cell Build up constant k (h − 1 ) A foci/cell Build up constant k (h − 1 ) Control 29.49 ± 4.45 1.76 ± 0.43 27.42 ± 4.56 1.72 ± 0.45 26.39 ± 5.30 1.62 ± 0.51 W1 27.17 ± 3.82 1.79 ± 0.47 25.82 ± 3.70 1.78 ± 0.47 25.19 ± 4.14 1.83 ± 0.54 W2 24.35 ± 1.03 2.45 ± 0.21 23.29 ± 0.97 2.40 ± 0.19 21.97 ± 1.43 2.29 ± 0.29 W3 23.99 ± 1.40 2.26 ± 0.26 22.30 ± 1.17 2.26 ± 0.21 20.02 ± 0.96 2.37 ± 0.21 W4 24.46 ± 1.32 2.01 ± 0.19 22.94 ± 1.34 1.99 ± 0.20 22.65 ± 1.97 1.78 ± 0.26 MW4 26.55 ± 1.05 2.11 ± 0.15 23.99 ± 1.39 2.11 ± 0.22 24.66 ± 2.92 1.74 ± 0.34 In a similar manner, the kinetics of foci decay were examined from 2 to 24 hours of irradiation, and the data were best fitted with a single exponential decay pattern given by: \(\:Y={Y}_{0}+{A}_{1}{e}^{-k1t}\) . This decay model does not differentiate between fast and slow decay components individually; it combines both into a single component defined by the constant k1 , consistent with earlier findings ( 32 , 33 ). Parameter Y 0 , A 1 , k 1 and t, represent residual foci, peak foci, foci decay constant and time in h . The fitting parameters are presented in Table 4 . Table 4 Fitting parameters for the decay of foci (γH2AX, 53BP1, and their colocalization) in lymphocytes from four WAS patients, carrier mother of patient W4, and a healthy control. Lymphocytes were exposed to 2 Gy of 60 Co-γ-rays, and foci were quantified up to 24 hours post-irradiation. Foci decay kinetics followed a single exponential decay pattern, expressed as \(\:\varvec{Y}={\varvec{Y}}_{0}+{\varvec{A}}_{1}{\varvec{e}}^{-\varvec{k}1\varvec{t}}\) . The half-lives (T 1/2 ) of DSB repair foci (γH2AX, 53BP1, and their colocalization) were estimated for the aforementioned subjects using first-order kinetics, with T 1/2 =0.693/k1. Subjects Fitting parameters for foci decay and estimation of DSB repair half-life (T 1/2 ) γH2AX foci 53BP1 foci Colocalization of foci Y 0 (foci/ cell) A 1 (foci/ cell) Decay constant k 1 (h − 1 ) DSB repair half-life T 1/2 Y 0 (foci/cell) A 1 (foci/ cell) Decay constant k1 (h − 1 ) DSB repair half-life T 1/2 Y 0 (foci/ cell) A 1 (foci/ cell) Decay constant k 1 (h − 1 ) DSB repair half-life T 1/2 Control 5.23 ± 0.27 23.35 ± 0.89 0.233 ± 0.015 2.98 ± 0.2 4.59 ± 0.41 21.42 ± 1.33 0.225 ± 0.022 3.08 ± 0.34 4.06 ± 0.53 21.31 ± 1.68 3.21 ± 0.47 0.216 ± 0.028 W1 11.78 ± 0.35 11.92 ± 0.39 0.126 ± 0.012 5.50 ± 0.59 12.05 ± 0.24 10.21 ± 0.28 0.136 ± 0.011 5.11 ± 0.46 11.49 ± 0.19 10.45 ± 0.31 4.73 ± 0.39 0.146 ± 0.011 W2 12.38 ± 0.04 11.26 ± 0.06 0.142 ± 0.002 4.89 ± 0.07 11.17 ± 0.39 11.09 ± 0.47 0.134 ± 0.015 5.16 ± 0.67 10.55 ± 0.42 10.22 ± 0.54 5.04 ± 0.79 0.138 ± 0.019 W3 11.64 ± 0.03 11.03 ± 0.03 0.134 ± 0.001 5.18 ± 0.05 10.10 ± 0.52 11.13 ± 0.50 0.118 ± 0.016 5.88 ± 0.92 9.03 ± 0.19 10.27 ± 0.19 5.77 ± 0.37 0.12 ± 0.007 W4 8.91 ± 0.25 14.18 ± 0.38 0.156 ± 0.011 4.43 ± 0.33 8.59 ± 0.40 13.33 ± 0.54 0.155 ± 0.019 4.48 ± 0.62 7.70 ± 0.46 12.86 ± 0.62 4.62 ± 0.71 0.15 ± 0.019 MW4 5.01 ± 0.25 23.89 ± 0.83 0.254 ± 0.016 2.73 ± 0.18 4.34 ± 0.40 20.39 ± 1.11 0.215 ± 0.022 3.23 ± 0.36 3.83 ± 0.25 20.54 ± 0.88 3.04 ± 0.25 0.228 ± 0.017 The average decay rate of foci for WAS patients was observed to be ~ 1.5 to ~ 1.9 times slower than for control, indicating a slower pace of DSB repair. A slight variation is observed in the decay rates of foci for WAS patients indicative of the dependence of the repair on the type of gene mutation. After 24 hours, the residual foci in WAS patients were ~ 2 times higher than the control, again indicating significantly elevated levels of unrepaired DSBs in WAS patients. The half-lives ( T 1/2 ) of DSB repair foci in both WAS and control lymphocytes were determined using first-order kinetics, calculated with the expression T 1/2 = 0.693/ k 1 ( 32 , 33 ). Among four WAS patients, a slight variation in the half-lives of the foci and their colocalization was observed. This substantial difference in repair efficiency of WAS patients raises significant concerns regarding genomic stability in WAS patients, potentially heightening their vulnerability to radiation exposure and increasing their risk of developing malignancies. 3.4 DSB repair dynamics and radiation sensitivity in carrier mother of WAS patients: The dynamics of DSB repair foci were also examined in the lymphocytes of carrier mothers (MW1-MW4). Data for the carrier mother of the WAS-4 patient is shown in Table 3 and Table 4 , while data for the carrier mothers of WAS-1 to WAS-3 is not included. The foci induction and decay kinetics closely paralleled the trends observed in the control, with approximately 20% residual foci remaining even after 24 hours, which was consistent with the control data (Table 3 ) ( 32 , 33 ). The estimated half-lives for the foci, in the lymphocytes of the mothers was comparable to that of the control (Table 4 ). These findings suggest that the carrier mothers of WAS patients exhibited wild-type efficiency in repairing DSBs and did not show heightened radiation sensitivity. Discussion Previous reports have indicated that the pathophysiological conditions and severity of WAS patients vary significantly depending on the type and location of the mutation in the WAS gene ( 29 ). This study investigated the effects of radiation exposure on genome instability in four WAS patients possessing different WAS gene mutation. In the present study first time we have evaluated DSBs repair kinetics in WAS patients and their carrier mothers using markers such as γH2AX and 53BP1. We have noted ~ 16 to ~ 24 times elevated baseline levels of DSB markers (γH2AX and 53BP1) in lymphocytes of WAS patients compared to carrier mothers and healthy controls. A similar result has been reported by Wang et al. in in vitro WAS knockout cells ( 17 , 35 , 36 ). The baseline occurrence of DSBs arises from various endogenous and exogenous factors, triggering the generation of reactive oxygen species (ROS) and consequent DSBs in cells ( 37 , 38 ). Cellular processes, such as cell division and cell differentiation, can also provoke the formation of DSBs ( 39 ). Wild-type cells have the ability to repair these spontaneously induced DSBs, preventing their accumulation. However, WAS-mutant cells exhibit impaired DSB repair, potentially leading to the accumulation of these DSBs over time ( 17 , 27 ). Typically, WAS-mothers demonstrate normal basal levels of expression of DSB repair foci, although in rare instances, higher spontaneous yields have also been reported ( 3 ). However, our results demonstrated that the repair yield (at 24 h) of DSBs, indicated by γH2AX and 53BP1 foci, in the mothers was consistent with the typical range observed in controls and other documented human populations (0 to 0.49 foci/cell in populations from Germany, France, Cuba, India, and elsewhere) ( 32 , 33 , 40 , 41 ). Recent studies have elucidated the critical role of WAS protein (WASP) in DNA repair mechanisms, particularly emphasizing DSB repair ( 6 , 10 , 11 , 17 , 27 ). WASP deficiency hampers the assembly of actin filaments at DNA damage sites, impeding the recruitment of repair factors and compromising the competence of DSB repair pathways, including nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways ( 27 , 42 , 43 ). However, the kinetics of DSB repairs, such as the repair half-life and the dynamics of repair foci over time, are not adequately understood or explored. To assess the radiation sensitivity and genomic stability, DSBs were created by irradiating WAS lymphocytes with 2 Gy of gamma radiation and foci (γH2AX and 53BP1) formation and decay kinetics was studied. This study demonstrated that WAS lymphocytes exhibit a similar trend in the DSB repair process as control lymphocytes, with an initial rapid repair phase followed by a slower phase. Though, the pace of DSB repair was significantly reduced in WAS-lymphocytes than that in the control. Results showed presence of significantly higher residual foci after 24 hours reflecting the persistence of excess DSBs in WAS patients, compared to healthy control volunteers. These findings clearly indicate the accumulation of unrepaired DSBs in radiation-challenged WAS lymphocytes, suggesting increased radiosensitivity and genomic instability. Different levels of radiosensitivity were observed among the four WAS patients (Table 4 ). W1 exhibited the highest level of radiosensitivity, with a ~ 1.9-fold greater DSB repair half-life (slowest pace of DSB repair) than that of the controls, as evidenced by the data on γH2AX foci. Conversely, patient W4 exhibited a relatively lower level of sensitivity, with a ~ 1.5-fold increase in the half-life of DSB repair compared with that of the control. This differential sensitivity was due to frameshift mutations, as observed in W1, significantly impair WAS function compared to the milder impact of missense mutations, such as those seen in W4. Repairing complex DSBs is time-intensive, often taking hours to days. In contrast, 2 Gy of low-LET radiation (60Co-γ-ray) induces simpler DSBs, usually repaired within 2–3 hours ( 44 ). This study showed that WAS patients exhibit incomplete repair of simple DSBs for up to 24 hours (Table 4 ). Radiation exposure can result into compromised immune function, increasing susceptibility to infections and other complications ( 27 ). Understanding the extent of radiation sensitivity in WAS patients can guide healthcare providers in optimizing infection prevention strategies and providing timely interventions to minimize adverse effects. Owing to the accumulation of DSBs and genomic instability, WAS patients face an increased risk of developing malignancies ( 36 ). Radiation therapy is a commonly used treatment modality for various malignancies and autoimmune conditions ( 45 ). However, WAS patients may be at increased risk of developing radiation-induced toxicity due to underlying immunodeficiency and potential defects in DNA repair mechanisms ( 46 , 47 ). Moreover, hematopoietic stem cell transplantation (HSCT) is a potential curative option for WAS patients ( 48 ). Radiation conditioning regimens are commonly administered before HSCT to suppress the recipient's immune system and enhance the engraftment of donor cells ( 49 ). However, the increased radiation sensitivity of WAS patients raises significant concerns regarding the safety and tolerability of these conditioning regimens. Conducting an assessment of radiation sensitivity in WAS patients before HSCT can assist in risk stratification and treatment planning, thus ensuring the best possible outcomes while minimizing potential complications. This study demonstrated impaired or reduced DSB repair efficiency and the accumulation of spontaneous and induced DSBs in WAS lymphocytes. However, assessing the fidelity of the DSB repair process itself is another crucial aspect. Errors in the repair process may lead to misrepair products, contributing to chromosomal abnormalities and genomic instability ( 50 ). Cytogenetic markers such as dicentrics and/or chromosomal rearrangements arise from misrepaired DSBs. Investigating these cytogenetic markers can provide further insights into the accuracy of the DSB repair process in WAS patients. Conclusion This study is the first to demonstrate DSB repair kinetics in WAS patients and their carrier mothers. We uncovered the intricate relationship between four different WAS gene mutations and their effects on DNA repair dynamics and radiation sensitivity. By analyzing DSB repair in the lymphocytes of both patients and carrier mothers, we gained vital insights into DSB repair efficiency and the accumulation of unrepaired DSBs, which contribute to genomic instability in WAS. Our findings highlight the complex dynamics of DSB repair in WAS, offering valuable insights for clinical management and the potential to improve patient outcomes. Declarations Supplementary Information: None Acknowledgments: We extend our gratitude to the patients and their families for their invaluable cooperation and participation in this study. We also acknowledge the indispensable technical support provided by Mr. Shrikant Jagtap and other laboratory members. Additionally, we sincerely thank the referring clinicians for generously sharing clinical information, which played a crucial role in the successful completion of this research. Author contributions: RSP, RKC, and KBS conducted the experiments and collected the data. RKC, BKS, and MM conceptualized the project, analyzed the data, and drafted the manuscript. PG and UB contributed to writing the clinical details of the patients. Anjana Goel, N.N. Bhat, and Arshad Khan reviewed and provided critical edits to the manuscript. Funding: This research was supported by the host institute, Bhabha Atomic Research Centre, Mumbai, India. No external funding was received. Data Availability: All relevant data are provided within the manuscript. Code Availability: Not applicable. Ethical Approval: This study was approved by the Institutional Ethics Committee (IEC) of the National Institute of Immunohaematology (ICMR), Mumbai. Consent to Participate: Written informed consent was obtained from the patients' guardians. Consent for Publication: The patients' guardians provided signed informed consent for the publication of the data. Conflict of Interest: The authors declare no competing interests. References Wiskott A. Familiarer, angeborener morbus werlhofli. Monatsschrift Kinderheikunde. 1937;68:212–6. Dupuis-Girod S, Medioni J, Haddad E, Quartier P, Cavazzana-Calvo M, Le Deist F, de Saint Basile G, Delaunay J, Schwarz K, Casanova JL, Blanche S. Autoimmunity in Wiskott-Aldrich syndrome: risk factors, clinical features, and outcome in a single-center cohort of 55 patients. Pediatrics. 2003;111(5):e622–7. Andreu N, Pujol-Moix N, Martinez-Lostao L, Oset M, Muñiz-Diaz E, Estivill X, Volpini V, Fillat C. Wiskott–Aldrich syndrome in a female with skewed X-chromosome inactivation. Blood Cells Molecules Dis. 2003;31(3):332–7. Malik MA, Masab M. 2019. Wiskott-Aldrich Syndrome. Kwan SP, Lehner T, Hagemann T, Lu B, Blaese M, Ochs H, Wedgwood R, Ott J, Craig IW, Rosen FS. 1991. Localization of the gene for the Wiskott-Aldrich syndrome between two flanking markers, TIMP and DXS255, on Xp11. 22–Xp11. 3. Genomics, 10(1), pp.29–33. Parolini O, Berardelli S, Riedl E, Bello-Fernandez C, Strobl H, Majdic O, Knapp W. Expression of Wiskott-Aldrich syndrome protein (WASP) gene during hematopoietic differentiation. Blood J Am Soc Hematol. 1997;90(1):70–5. Derry JM, Ochs HD, Francke U. Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell. 1994;78(4):635–44. Gallego MD, Santamarı́a M, Peña J, Molina IJ. Defective actin reorganization and polymerization of Wiskott-Aldrich T cells in response to CD3-mediated stimulation. Blood J Am Soc Hematol. 1997;90(8):3089–97. Gaikwad P, Bargir UA, Jodhawat N, Dalvi A, Shinde S, Tamhankar P, Setia P, Kambli P, Dhawale A, Temkar L, Vedpathak D. 2025. Mutational Landscape of Patients with Wiskott Aldrich Syndrome: Update from India. Journal of Clinical Immunology, 45(1), p.56. Jin Y, Mazza C, Christie JR, Giliani S, Fiorini M, Mella P, Gandellini F, Stewart DM, Zhu Q, Nelson DL, Notarangelo LD. Mutations of the Wiskott-Aldrich Syndrome Protein (WASP): hotspots, effect on transcription, and translation and phenotype/genotype correlation. Blood. 2004;104(13):4010–9. Imai K, Morio T, Zhu Y, Jin Y, Itoh S, Kajiwara M, Yata JI, Mizutani S, Ochs HD, Nonoyama S. Clinical course of patients with WASP gene mutations. Blood. 2004;103(2):456–64. Kavanagh JN, Redmond KM, Schettino G, Prise KM. DNA double strand break repair: a radiation perspective. Antioxid Redox Signal. 2013;18(18):2458–72. Mladenov E, Magin S, Soni A, Iliakis G. June. DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation. Seminars in cancer biology. Volume 37. Academic; 2016. pp. 51–64. Ceccaldi R, Rondinelli B, D’Andrea AD. Repair pathway choices and consequences at the double-strand break. Trends Cell Biol. 2016;26(1):52–64. Beucher A, Birraux J, Tchouandong L, Barton O, Shibata A, Conrad S, Goodarzi AA, Krempler A, Jeggo PA, Löbrich M. ATM and Artemis promote homologous recombination of radiation-induced DNA double‐strand breaks in G2. EMBO J. 2009;28(21):3413–27. Mao Z, Bozzella M, Seluanov A, Gorbunova V. DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle. 2008;7(18):2902–6. Wang T, Du XH, Hong Y, Hong X, Fan L, Zhou JW, Sun H, Ge J, Billadeau DD, Deng ZH. 2022. WASH interacts with Ku to regulate DNA double-stranded break repair. IScience, 25(1). Verboon JM, Rincon-Arano H, Werwie TR, Delrow JJ, Scalzo D, Nandakumar V, Groudine M, Parkhurst SM. Wash interacts with lamin and affects global nuclear organization. Curr Biol. 2015;25(6):804–10. Xia P, Wang S, Huang G, Zhu P, Li M, Ye B, Du Y, Fan Z. WASH is required for the differentiation commitment of hematopoietic stem cells in a c-Myc–dependent manner. J Exp Med. 2014;211(10):2119–34. Kuznetsov NV, Almuzzaini B, Kritikou JS, Baptista MA, Oliveira MM, Keszei M, Snapper SB, Percipalle P, Westerberg LS. Nuclear Wiskott–Aldrich syndrome protein co-regulates T cell factor 1-mediated transcription in T cells. Genome Med. 2017;9:1–18. Caridi CP, D’Agostino C, Ryu T, Zapotoczny G, Delabaere L, Li X, Khodaverdian VY, Amaral N, Lin E, Rau AR, Chiolo I. Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature. 2018;559(7712):54–60. Naseem A, Steinberg Z, Cavazza A. Genome editing for primary immunodeficiencies: A therapeutic perspective on Wiskott-Aldrich syndrome. Front Immunol. 2022;13:966084. Lukas J, Lukas C, Bartek J. More than just a focus: The chromatin response to DNA damage and its role in genome integrity maintenance. Nat Cell Biol. 2011;13(10):1161–9. Osipov AN, Grekhova A, Pustovalova M, Ozerov IV, Eremin P, Vorobyeva N, Lazareva N, Pulin A, Zhavoronkov A, Roumiantsev S, Klokov D. 2015. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation. Oncotarget, 6(29), p.26876. García-Muse T, Aguilera A. R loops: from physiological to pathological roles. Cell. 2019;179(3):604–18. Iliakis G, Mladenov E, Mladenova V. 2019. Necessities in the processing of DNA double strand breaks and their effects on genomic instability and cancer. Cancers, 11(11), p.1671. Wen KK, Han SS, Vyas YM. Wiskott-Aldrich syndrome protein senses irradiation-induced DNA damage to coordinate the cell-protective Golgi dispersal response in human T and B lymphocytes. J Allergy Clin Immunol. 2020;145(1):324–34. Ozsahin H, Cavazzana-Calvo M, Notarangelo LD, Schulz A, Thrasher AJ, Mazzolari E, Slatter MA, Le Deist F, Blanche S, Veys P, Fasth A. Long-term outcome following hematopoietic stem-cell transplantation in Wiskott-Aldrich syndrome: collaborative study of the European Society for Immunodeficiencies and European Group for Blood and Marrow Transplantation. Blood J Am Soc Hematol. 2008;111(1):439–45. Kolluri R, Shehabeldin A, Peacocke M, Lamhonwah AM, Teichert-Kuliszewska K, Weissman SM, Siminovitch KA. Identification of WASP mutations in patients with Wiskott-Aldrich syndrome and isolated thrombocytopenia reveals allelic heterogeneity at the WAS locus. Hum Mol Genet. 1995;4(7):1119–26. Chaurasia RK, Shirsath KB, Sapra BK. 2021. Protocol for one-step selective lysis of red blood cells and platelets with long-term preservation of white blood cells (human) at ambient temperature. STAR protocols, 2(4), p.100834. Vijayalakshmi J, Chaurasia RK, Srinivas KS, Vijayalakshmi K, Paul SF, Bhat NN, Sapra BK. 2023. Establishment of ex vivo calibration curve for X-ray induced dicentric + ring and micronuclei in human peripheral lymphocytes for biodosimetry during radiological emergencies, and validation with dose blinded samples. Heliyon, 9(6). Chaurasia RK, Shirsath KB, Desai UN, Bhat NN, Sapra BK. 2022. Establishment of in vitro calibration curve for 60Co-γ-rays induced Phospho-53BP1 foci, rapid biodosimetry and initial triage, and comparative evaluations with γH2AX and cytogenetic assays. Frontiers in Public Health, 10, p.845200. Chaurasia RK, Bhat NN, Gaur N, Shirsath KB, Desai UN, Sapra BK. Establishment and multiparametric-cytogenetic validation of 60Co-gamma-ray induced, phospho-gamma-H2AX calibration curve for rapid biodosimetry and triage management during radiological emergencies. Volume 866. Mutation Research/Genetic Toxicology and Environmental Mutagenesis; 2021. p. 503354. Benjamin DJ, Berger JO, Johannesson M, Nosek BA, Wagenmakers EJ, Berk R, Bollen KA, Brembs B, Brown L, Camerer C, Cesarini D. Redefine statistical significance. Nat Hum Behav. 2018;2(1):6–10. Sarkar K, Han SS, Wen KK, Ochs HD, Dupré L, Seidman MM, Vyas YM. R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome. J Allergy Clin Immunol. 2018;142(1):219–34. Han SS, Wen KK, Vyas YM. Deficiency of Wiskott–Aldrich syndrome protein has opposing effect on the pro-oncogenic pathway activation in nonmalignant versus malignant lymphocytes. Oncogene. 2021;40(2):345–54. Tubbs A, Nussenzweig A. Endogenous DNA damage as a source of genomic instability in cancer. Cell. 2017;168(4):644–56. Sharma V, Collins LB, Chen TH, Herr N, Takeda S, Sun W, Swenberg JA, Nakamura J. 2016. Oxidative stress at low levels can induce clustered DNA lesions leading to NHEJ mediated mutations. Oncotarget, 7(18), p.25377. Chu G. Double strand break repair. J Biol Chem. 1997;272(39):24097–100. Eberlein U, Peper M, Fernández M, Lassmann M, Scherthan H. Calibration of the γ-H2AX DNA double strand break focus assay for internal radiation exposure of blood lymphocytes. PLoS ONE. 2015;10(4):e0123174. Bucher M, Duchrow L, Endesfelder D, Roessler U, Gomolka M. Comparison of inexperienced operators and experts in γH2A. X and 53BP1 foci assay for high-throughput biodosimetry approaches in a mass casualty incident. Int J Radiat Biol. 2020;96(10):1263–73. Notarangelo LD, Miao CH, Ochs HD. Wiskott-aldrich syndrome. Curr Opin Hematol. 2008;15(1):30–6. Thrasher AJ, Kinnon C. The Wiskott–Aldrich syndrome. Clin Experimental Immunol. 2000;120(1):2–9. Nikitaki Z, Velalopoulou A, Zanni V, Tremi I, Havaki S, Kokkoris M, Gorgoulis VG, Koumenis C, Georgakilas AG. Key biological mechanisms involved in high-LET radiation therapies with a focus on DNA damage and repair. Expert Rev Mol Med. 2022;24:e15. Kumari S, Mukherjee S, Sinha D, Abdisalaam S, Krishnan S, Asaithamby A. 2020. Immunomodulatory effects of radiotherapy. International journal of molecular sciences, 21(21), p.8151. Massaad MJ, Ramesh N, Geha RS. Wiskott-Aldrich syndrome: a comprehensive review. Ann N Y Acad Sci. 2013;1285(1):26–43. Candotti F. Clinical manifestations and pathophysiological mechanisms of the Wiskott-Aldrich syndrome. J Clin Immunol. 2018;38(1):13–27. Albert MH, Slatter MA, Gennery AR, Güngör T, Bakunina K, Markovitch B, Hazelaar S, Sirait T, Courteille V, Aiuti A, Aleinikova OV. Hematopoietic stem cell transplantation for Wiskott-Aldrich syndrome: an EBMT Inborn Errors Working Party analysis. Blood J Am Soc Hematol. 2022;139(13):2066–79. Barrett AJ, Savani BN. Stem cell transplantation with reduced-intensity conditioning regimens: a review of ten years experience with new transplant concepts and new therapeutic agents. Leukemia. 2006;20(10):1661–72. Aguilera A, Gómez-González B. Genome instability: a mechanistic view of its causes and consequences. Nat Rev Genet. 2008;9(3):204–17. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5719467","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":407688928,"identity":"7adcca3e-cb08-443f-b6f6-ddd7970ba0f0","order_by":0,"name":"Ranjana S. Pathak","email":"","orcid":"","institution":"GLA University","correspondingAuthor":false,"prefix":"","firstName":"Ranjana","middleName":"S.","lastName":"Pathak","suffix":""},{"id":407688929,"identity":"5d168283-8420-4cf2-b9bd-6e08f1446943","order_by":1,"name":"Rajesh Kumar Chaurasia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie3OIQ+CQBTA8XdjgwJS2XD4FXAECuOz3I3tKPIdLp2Fmf0YNKXdRsVZjVDI2MQRxKImwObm/dK97f13D0CSfhMGxMBxX7MyM/G+SuCZkGz2Vf5W1HV3DOODdqpbuAeEaYYYTZYljjyjjJI8jT0L7ShhygKPJhZgaiMukkxQFVBaeKDo7nhiVnHXcRG752ZuYmEKBhfYvQy/wK1wZiRVZBs8Wuf7RrEIo446fdiGXDsernyTorbtA900y/EEQMfvN+EA6sT+QBMfQz+9L0mS9H8eMGo/YXw/4SQAAAAASUVORK5CYII=","orcid":"","institution":"Bhabha Atomic Research Centre","correspondingAuthor":true,"prefix":"","firstName":"Rajesh","middleName":"Kumar","lastName":"Chaurasia","suffix":""},{"id":407688930,"identity":"df2c8244-7ba4-4e70-83c9-232ed670b0d4","order_by":2,"name":"B.K. 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Bhat","email":"","orcid":"","institution":"Bhabha Atomic Research Centre","correspondingAuthor":false,"prefix":"","firstName":"N.N.","middleName":"","lastName":"Bhat","suffix":""},{"id":407688940,"identity":"790d5c7a-8820-48ce-ad6b-f974fd9c1874","order_by":9,"name":"Arshad Khan","email":"","orcid":"","institution":"Bhabha Atomic Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Arshad","middleName":"","lastName":"Khan","suffix":""}],"badges":[],"createdAt":"2024-12-27 06:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5719467/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5719467/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75192538,"identity":"fd921a60-4e32-4120-9c0a-cf9ff6b4642e","added_by":"auto","created_at":"2025-01-31 18:57:40","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":118998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIllustration of the baseline frequency of gH2AX and 53BP1 foci, along with their colocalization, in lymphocytes of WAS patients, their carrier mothers and controls.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5719467/v1/0377af3f681a907fdd1636e6.jpg"},{"id":75192537,"identity":"f4e76c3d-9836-4cc7-9f8f-2ae6f016fb0d","added_by":"auto","created_at":"2025-01-31 18:57:40","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21453,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRepresentative image illustrating the baseline frequency of γH2AX and 53BP1 foci, as well as their colocalization, in lymphocytes obtained from a healthy control volunteer, mother of WAS 4 (M-W4) patient and the WAS (W4) patient. The baseline frequencies of γH2AX and 53BP1 foci were significantly greater in WAS patients than in healthy control volunteer. Foci frequencies in the mothers were within the control range.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5719467/v1/e0a39b260464cad853038fd3.jpg"},{"id":75192544,"identity":"edb5363d-5d3f-4ec0-ac0a-93446415ec28","added_by":"auto","created_at":"2025-01-31 18:57:40","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":365384,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eKinetics of foci (γH2AX, 53BP1 and their colocalization) buildup and decay in the subjects. Panels (A), (B), (C), (D) and (E) represent data for patients W1, W2, W3, W4, and the mother of the W4 patient, respectively, in comparison to the control volunteer. Foci quantification was carried out from 0 to 24 hours postirradiation. Error bars represent the standard deviation (SD) with a sample size of n = 5 × 3 and the number of cells scored per sample was N = 100.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5719467/v1/c055bbc4b885aa2a0ea8c376.jpg"},{"id":75192548,"identity":"749b8e43-5bcc-4bea-ba93-1412774de0f9","added_by":"auto","created_at":"2025-01-31 18:57:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33122,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eRepresentative images of lymphocytes showing residual γH2AX and 53BP1 foci, along with their colocalization, after 24 hours of irradiation. The lymphocytes of the carrier mother and control displayed residual foci while the WAS-4 patient showed a significantly greater number of residual foci, indicating persistent unrepaired DSBs.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5719467/v1/b020db409a8dc7b96c569361.jpg"},{"id":79510080,"identity":"2685dc96-a5a3-4b2b-96ac-c0c391058d5e","added_by":"auto","created_at":"2025-03-30 05:39:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13405258,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5719467/v1/4ab2728b-a2d0-4ae1-9ec5-54de8b21ab90.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Retarded DSB repair kinetics suggestive of augmented radiation sensitivity and genetic instability in Wiskott-Aldrich syndrome patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eWiskott-Aldrich syndrome (WAS), first identified in 1937 by Alfred Wiskott and Robert Aldrich reported that this disease manifests as microthrombocytopenia, eczema and a primary immunodeficiency disorder with various clinical presentations, including susceptibility to autoimmunity and malignancies (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). This X-linked rare disorder primarily affects males, although rare cases of female carriers with variable expressivity have been documented (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The incidence is approximately 4 in 1\u0026nbsp;million live male births in the U.S., with occasional cases occurring in females (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWAS is caused by mutations in the gene encoding the WAS protein (WASp), which is located on the X chromosome (Xp11.22-p11.23) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). WASp, which is primarily expressed in hematopoietic cells (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) regulates actin cytoskeleton dynamics, cell motility, and immune synapse formation, resulting in both quantitative and qualitative deficiencies in T and B cells (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The expression of WASp depends on type and location of mutation in \u003cem\u003eWAS\u003c/em\u003e gene (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). More than 440 genetic mutations have been linked to WAS, with missense mutations being the most common, followed by splice mutations, deletions, and nonsense mutations (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Children with WAS have a greater risk of developing malignancies, with reported incidences of up to 13%, most commonly EBV-related B-cell lymphomas or leukemia, which typically appear at approximate age of 9.5 y. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDNA double-strand breaks (DSBs) caused by ionizing radiation or chemotherapy represent some of the most severe types of DNA damage (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). If left unrepaired, DSBs can lead to genome instability or cell death, highlighting the critical necessity of prompt and accurate DSB repair (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Two essential DSB repair pathways have evolved in human cells: nonhomologous end-joining (NHEJ) and homologous recombination (HR) (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In mammalian cells, NHEJ serves as the primary DNA repair pathway and is active throughout the cell cycle. It predominantly handles DSB repair outside of the S and G2 phases and manages approximately 80% of DSB repair within the S and G2 phases (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent research has shown the emerging roles of WASH (Wiskott-Aldrich syndrome protein and SCAR homologue) and its intricate components within the nucleus (\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). A study conducted in \u003cem\u003eDrosophila\u003c/em\u003e cells revealed the essential role of nuclear F-actin, facilitated by the Arp2/3 complex, in relocating heterochromatin breaks to the nuclear periphery for subsequent repair. Interestingly, the depletion of WASH led to a defect in this relocation process, suggesting its potential involvement in DSB repair (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Moreover, Wang et al. (2022) revealed an interaction between WASH and Ku proteins, indicating a regulatory role for WASH in DSB repair pathways in 3T3 mouse fibroblasts (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe timeframe for repairing DSBs is critical for maintaining genomic integrity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). In healthy human cells, the standard DSB repair duration typically spans from 2 to 4 hours under optimal conditions (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). However, in instances of impaired DSB repair systems, this timeframe can be significantly prolonged or altered. Previous studies have reported the presence of unrepaired DSBs and genomic instability in cells with WAS mutations (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). However, the precise kinetics of DSB repair and the half-life of DSB repair processes remain unclear or elusive. The speed and accuracy of DSB repair are vital determinants of cell survival and the prevention of genomic instability and malignancy (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Furthermore, they play a crucial role in assessing the sensitivity of WAS patients to genotoxic agents such as radiation, chemotherapeutics, etc. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWAS patients (30\u0026ndash;50%) often require Bone Marrow Transplant (BMT) due to immunodeficiency and thrombocytopenia (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Conditioning regimens, such as whole-body irradiation, precede BMT to prepare recipients. In WAS, the type and location of the mutation significantly influence disease severity, which in turn affects the degree of radiation sensitivity. This variability in radiation sensitivity is observed among patients with different mutations, reflecting the diverse impact that these genetic changes have on the condition (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Studies on DSB repair efficiency and radiation sensitivity in patients with diverse mutations in the WAS gene are currently limited or unavailable. However, leveraging this information could enhance personalized treatment strategies, potentially including dose adjustments based on hypersensitivity assessments, to improve radiation therapy outcomes in this population.\u003c/p\u003e \u003cp\u003eThis study investigated the kinetics of DSB repair (utilizing γH2AX and 53BP1 and their colocalization) in the lymphocytes of four WAS patients with different mutations in the WAS gene. The DSB repair half-lives in lymphocytes were estimated, and the presence of spontaneous unrepaired DSBs was measured. However, despite having a heterozygous WAS mutation, carrier mothers exhibited normal repair kinetics. These insights are crucial for evaluating genomic instability and radiation sensitivity and tailoring personalized BMT strategies for the effective management of WAS patients.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design:\u003c/h2\u003e \u003cp\u003eWe assessed DSB repair kinetics, the repair half-life, and radiation sensitivity in four WAS patients and compared them with carrier mothers and a healthy control.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals:\u003c/h3\u003e\n\u003cp\u003e4\u0026prime;,6-Diamidino-2-phenylindole (DAPI) with antifade (ProLong\u0026trade; Diamond Antifade Mountant with DAPI) was procured from Invitrogen, USA. Tween 20, poly L-lysine, and Histopaque-1077 were obtained from Sigma‒Aldrich, USA. Paraformaldehyde,1,1,3,3-tetraethoxypropane, Triton X-100 and Fetal calf serum (FCS) were procured from Gibco Life Technologies, USA. Mouse anti-phospho-histone γH2AX (ser-139) human monoclonal IgG was procured from Millipore, USA. FITC-labelled rabbit anti-mouse IgG was procured from Invitrogen (USA). Rabbit anti-phospho-53BP1 human monoclonal IgG and Texas Red-labelled goat anti-rabbit IgG were procured from Cell Signalling Technology (CST) (Massachusetts, USA) and Invitrogen (USA), respectively.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical approval and sample collection\u003c/b\u003e:\u003c/p\u003e \u003c/p\u003e \u003cp\u003eInstitutional ethics committee (IEC) of ICMR- National Institute of Immunohaematology, Mumbai, India has approved this study. Inform consent was obtained to collect 3 ml peripheral blood samples from patients and family members in heparin vacutainers.\u003c/p\u003e\n\u003ch3\u003ePreparation and irradiation of peripheral blood mononuclear cells (PBMCs):\u003c/h3\u003e\n\u003cp\u003ePeripheral blood mononuclear cells (PBMCs) were isolated from patients, carrier mothers and healthy controls using heparin vacutainers via density gradient centrifugation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). After centrifugation, the milky buffy coat containing PBMCs was collected and washed (twice) with RPMI. PBMCs containing pellet was resuspended in fresh complete RPMI media. Lymphocytes were then exposed to 2 Gy of gamma irradiation using a Blood Irradiator (Dose rate: 0.54 Gy/min) (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). After irradiation, PBMCs were washed and allowed to recover for 24 hours in fresh complete RPMI media.\u003c/p\u003e\n\u003ch3\u003eKinetics of DSB repair foci, γH2AX and 53BP1:\u003c/h3\u003e\n\u003cp\u003eThe kinetics of DSB repair foci (γH2AX and 53BP1) and their colocalization were investigated for up to 24 hours following irradiation in lymphocytes obtained from the enrolled subjects (WAS patients and carrier mothers). After irradiation, the PBMCs were incubated under optimal conditions (37\u0026deg;C, 95% relative humidity, and 5% CO\u003csub\u003e2\u003c/sub\u003e) for up to 24 hours. PBMCs were sampled at various time points (0, 0.083, 0.17, 0.25, 0.5, 1, 2, 4, 8, 16, and 24 hours) post irradiation to quantify the presence of DSB repair foci.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence-staining of γH2AX and 53BP1-DSB-repair proteins:\u003c/h3\u003e\n\u003cp\u003eImmunostaining for γH2AX and 53BP1 was performed following our own optimized protocol (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). After post-irradiation incubation, PBMCs were fixed in 4% paraformaldehyde at 4\u0026deg;C for 30 minutes, washed with PBS, and deposited on poly-L-lysine-coated coverslips to adhere for 1 hour. The cells were permeabilized with 0.5% Triton X-100, washed, and blocked with 5% FCS in PBS for 1 hour. Primary antibodies against γH2AX and 53BP1 were added (dilution-1:200), followed by incubation with Alexa Fluor 488 and Texas Red secondary antibodies (dilution-1:400). Cells were mounted using DAPI with antifade solution for imaging.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetection and quantification of γH2AX and 53BP1 foci using confocal microscopy:\u003c/h2\u003e \u003cp\u003eThe mounted slides were imaged using a Leica SP8 confocal fluorescence microscope with Leica Application Suite-X (LAS-X). γH2AX foci were visualized with a green filter, 53BP1 foci with a red filter, and nuclei with a blue filter. Images were captured and overlaid to show colocalization of γH2AX and 53BP1 in the nuclei. Approximately 300 lymphocytes per individual were examined at each of the 11 incubation time points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eThe experiments were performed in three sets, with data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analysis used Student\u0026rsquo;s t-test to assess differences among incubation time points and between subjects, with a significance threshold of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Trends were modelled using the least squares method.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCase history and pathophysiological conditions of the subjects:\u003c/h2\u003e \u003cp\u003eIn our study, we focused on four male paediatric patients (aged 3 months to 14 years) along with their carrier mothers. Clinical, immunological and molecular features of these patients were reported by Gaikwad et al 2024 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). These were also summarised in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Mothers who carried the mutated gene did not manifest symptoms of the disease. Tragically, W1 patient passed away at the age of 5 months due to severe respiratory tract infection and thrombocytopenia. Two patients W2 and W3 are currently suffering from characteristic WAS symptoms and are awaiting BMT. While W4 had undergone BMT at the age of 14 years, leading to a notable improvement in his quality of life.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDepiction of pathophysiological symptoms, age, mutation details, and current status of the four patients diagnosed with WAS.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubjects\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePatients codes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSymptoms\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMutation Details\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCurrent Status\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5 Months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBorn with second degree consanguineous marriage. At 4 months of age c/o cough, fever and increased respiratory activity, thrombocytopenia. Presented with eczema and bloody stools\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrameshift\u003c/p\u003e \u003cp\u003ec.1266_1267insG\u003c/p\u003e \u003cp\u003e(p.L425Pfs*70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eExpired\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 Months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBorn with non-consanguineous marriage, presented with history of repeated episodes of loose stool with blood, recurrent sepsis, anaemia with thrombocytopenia. Cytomegalovirus (CMV) positive. Upper GI endoscopy and ileocolonoscopy shows gastritis and pancolitis. Bone marrow aspiration showed normal results.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFrameshift\u003c/p\u003e \u003cp\u003ec.763_764insG\u003c/p\u003e \u003cp\u003e(p. Q255Rfs*5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAwaiting\u003c/p\u003e \u003cp\u003eBMT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 Year\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEczema, thrombocytopenia, Lower Respiratory Tract Infections (LRTI) with primary oxygen requirement.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003cp\u003ec.134C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003cp\u003e(p. T45M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAwaiting\u003c/p\u003e \u003cp\u003eBMT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eW4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eH/o bleeding manifestation (skin and mucosal bleeding) and recurrent respiratory tract infection and H/o thrombocytopenia.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMissense\u003c/p\u003e \u003cp\u003ec.223G\u0026thinsp;\u0026gt;\u0026thinsp;C\u003c/p\u003e \u003cp\u003e(p.V75L)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTransplanted\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Baseline DSB (genomic instability) assessment:\u003c/h2\u003e \u003cp\u003eResults showed significantly elevated frequencies of γH2AX and 53BP1 foci in the lymphocytes of WAS patients (γH2AX: 16\u0026ndash;24 times and 53BP1: 17\u0026ndash;25 times) compared to those in the healthy controls (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The frequencies of foci observed in the mothers were found to be within the range of the controls. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e is a representative image depicting the foci and their colocalization in the lymphocytes of a healthy control volunteer, the WAS patient, and carrier mother. A good colocalization (71\u0026ndash;99%) between γH2AX and 53BP1 foci was observed in all the subjects.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline data on γH2AX and 53BP1 foci/cell and their colocalization in lymphocytes from four WAS patients, their carrier mothers and four healthy controls.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSubjects\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eBaseline levels of foci/cell\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003egH2AX foci/cell\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e53BP1 foci/cell\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eColocalization foci/cell\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWAS1 (W1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWAS2 (W2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWAS3 (W3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWAS4 (W4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAvg of W1-W4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMother-W1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.147\u0026thinsp;\u0026plusmn;\u0026thinsp;0.112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.121\u0026thinsp;\u0026plusmn;\u0026thinsp;0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.118\u0026thinsp;\u0026plusmn;\u0026thinsp;0.087\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMother-W2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.135\u0026thinsp;\u0026plusmn;\u0026thinsp;0.102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.123\u0026thinsp;\u0026plusmn;\u0026thinsp;0.089\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.112\u0026thinsp;\u0026plusmn;\u0026thinsp;0.065\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMother-W3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.123\u0026thinsp;\u0026plusmn;\u0026thinsp;0.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.121\u0026thinsp;\u0026plusmn;\u0026thinsp;0.063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.062\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMother-W4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.189\u0026thinsp;\u0026plusmn;\u0026thinsp;0.131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.159\u0026thinsp;\u0026plusmn;\u0026thinsp;0.131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.149\u0026thinsp;\u0026plusmn;\u0026thinsp;0.072\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAvg of MW1-MW4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.149\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.131\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.125\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl 1 (C1)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.141\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.134\u0026thinsp;\u0026plusmn;\u0026thinsp;0.041\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.121\u0026thinsp;\u0026plusmn;\u0026thinsp;0.037\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl 2 (C2)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.139\u0026thinsp;\u0026plusmn;\u0026thinsp;0.068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.129\u0026thinsp;\u0026plusmn;\u0026thinsp;0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.119\u0026thinsp;\u0026plusmn;\u0026thinsp;0.045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl 3 (C3)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.137\u0026thinsp;\u0026plusmn;\u0026thinsp;0.029\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.131\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.123\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl 4 (C4)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.138\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.126\u0026thinsp;\u0026plusmn;\u0026thinsp;0.043\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.114\u0026thinsp;\u0026plusmn;\u0026thinsp;0.051\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAvg of C1-C4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.139\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.119\u0026thinsp;\u0026plusmn;\u0026thinsp;0.004\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 DSB repair dynamics and radiation sensitivity in lymphocytes of WAS patients:\u003c/h2\u003e \u003cp\u003eWe have assessed abundance of γH2AX and 53BP1 foci, as well as their colocalization post gamma exposure in WAS patients and their carrier mothers along with healthy controls. Within 15 minutes post-irradiation, foci were detectable, with a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to their respective baseline levels. The fitted curves represented in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D. While Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates foci and their colocalization in the lymphocytes of a healthy control volunteer, the WAS4 patient, and carrier mother.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe fitting analysis revealed a pattern where the number of foci initially increased and then followed a saturating exponential function, which can be expressed as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Y=A(1-{e}^{-kt})\\)\u003c/span\u003e\u003c/span\u003e where \u003cem\u003eA\u003c/em\u003e, and \u003cem\u003ek\u003c/em\u003e are constants, with values as listed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Similar pattern was reported earlier in control samples (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The units of \u003cem\u003eY\u003c/em\u003e and \u003cem\u003eA\u003c/em\u003e, are \u003cem\u003efoci/cell\u003c/em\u003e, while \u003cem\u003ek\u003c/em\u003e is expressed in \u003cem\u003eh\u003c/em\u003e\u003csup\u003e\u0026minus;1\u003c/sup\u003e and \u003cem\u003et\u003c/em\u003e represents time in \u003cem\u003eh\u003c/em\u003e. The constant \u003cem\u003ek\u003c/em\u003e, representing the rate of foci formation, showed significant variation among the four WAS patients as they exhibited a lower rate of foci formation compared to control. Foci formation reached saturation after 1 h in all subjects with a plateau observed up to 2 hours postirradiation. Subsequently, the yield of foci began to decrease in both WAS and control lymphocytes. The saturation yield of the foci was slightly lower in WAS patients compared to control. The W4 patient exhibited the lowest yield of foci, 11.85% for γH2AX and 11.31% for 53BP1 of the control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFitting parameters for the build-up of foci (γH2AX, 53BP1, and their colocalization) in lymphocytes obtained from four WAS patients and carrier mothers, along with healthy controls. The lymphocytes were exposed to 2 Gy of \u003csup\u003e60\u003c/sup\u003eCo-γ-rays, and foci were quantified up to 1-hour post-irradiation. Initial foci build up followed a single exponential growth pattern, mathematically expressed as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{Y}=\\varvec{A}(1-{\\varvec{e}}^{-\\varvec{k}\\varvec{t}})\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSubjects\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eFitting parameters for foci development\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eγH2AX foci\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e53BP1 foci\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eColocalization of foci\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003e(foci/cell)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBuild up constant k (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003efoci/cell\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBuild up constant k (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003cp\u003efoci/cell\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eBuild up constant k (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e29.49\u0026thinsp;\u0026plusmn;\u0026thinsp;4.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e27.42\u0026thinsp;\u0026plusmn;\u0026thinsp;4.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e26.39\u0026thinsp;\u0026plusmn;\u0026thinsp;5.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e27.17\u0026thinsp;\u0026plusmn;\u0026thinsp;3.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e25.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e25.19\u0026thinsp;\u0026plusmn;\u0026thinsp;4.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e24.35\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e21.97\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e23.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e20.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e2.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e24.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e22.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e22.65\u0026thinsp;\u0026plusmn;\u0026thinsp;1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMW4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e26.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e23.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e24.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn a similar manner, the kinetics of foci decay were examined from 2 to 24 hours of irradiation, and the data were best fitted with a single exponential decay pattern given by: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Y={Y}_{0}+{A}_{1}{e}^{-k1t}\\)\u003c/span\u003e\u003c/span\u003e. This decay model does not differentiate between fast and slow decay components individually; it combines both into a single component defined by the constant \u003cem\u003ek1\u003c/em\u003e, consistent with earlier findings (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Parameter \u003cem\u003eY\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e, \u003cem\u003eA\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e, \u003cem\u003ek\u003c/em\u003e1 and t, represent residual foci, peak foci, foci decay constant and time in \u003cem\u003eh\u003c/em\u003e. The fitting parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFitting parameters for the decay of foci (γH2AX, 53BP1, and their colocalization) in lymphocytes from four WAS patients, carrier mother of patient W4, and a healthy control. Lymphocytes were exposed to 2 Gy of \u003csup\u003e60\u003c/sup\u003eCo-γ-rays, and foci were quantified up to 24 hours post-irradiation. Foci decay kinetics followed a single exponential decay pattern, expressed as \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varvec{Y}={\\varvec{Y}}_{0}+{\\varvec{A}}_{1}{\\varvec{e}}^{-\\varvec{k}1\\varvec{t}}\\)\u003c/span\u003e\u003c/span\u003e. The half-lives (T\u003csub\u003e1/2\u003c/sub\u003e) of DSB repair foci (γH2AX, 53BP1, and their colocalization) were estimated for the aforementioned subjects using first-order kinetics, with T\u003csub\u003e1/2\u003c/sub\u003e=0.693/k1.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eSubjects\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"12\" nameend=\"c13\" namest=\"c2\"\u003e \u003cp\u003eFitting parameters for foci decay and estimation of DSB repair half-life (T\u003csub\u003e1/2\u003c/sub\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eγH2AX foci\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003e53BP1 foci\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e \u003cp\u003eColocalization of foci\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003e\u003cem\u003e0\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(foci/ cell)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(foci/ cell)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDecay constant \u003cem\u003ek\u003c/em\u003e1 (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDSB repair half-life T\u003csub\u003e1/2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(foci/cell)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(foci/ cell)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eDecay constant \u003cem\u003ek1\u003c/em\u003e (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDSB repair half-life T\u003csub\u003e1/2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eY\u003c/em\u003e\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(foci/ cell)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(foci/ cell)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eDecay constant \u003cem\u003ek\u003c/em\u003e1 (h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eDSB repair half-life T\u003csub\u003e1/2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.233\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e21.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.225\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e3.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e4.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e21.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c13\"\u003e \u003cp\u003e0.216\u0026thinsp;\u0026plusmn;\u0026thinsp;0.028\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.126\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e12.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e10.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.136\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e5.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e11.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e10.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c13\"\u003e \u003cp\u003e0.146\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e12.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.142\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e11.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e11.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.134\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e5.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e10.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e10.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e5.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c13\"\u003e \u003cp\u003e0.138\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e11.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e11.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.134\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e10.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e11.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.118\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e9.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e10.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e5.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c13\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e8.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e14.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.156\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e4.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e8.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e13.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.155\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e4.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e7.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e12.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e4.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c13\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMW4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e23.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.254\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e2.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e4.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e20.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c8\"\u003e \u003cp\u003e0.215\u0026thinsp;\u0026plusmn;\u0026thinsp;0.022\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c9\"\u003e \u003cp\u003e3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c10\"\u003e \u003cp\u003e3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c11\"\u003e \u003cp\u003e20.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c12\"\u003e \u003cp\u003e3.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c13\"\u003e \u003cp\u003e0.228\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe average decay rate of foci for WAS patients was observed to be ~\u0026thinsp;1.5 to ~\u0026thinsp;1.9 times slower than for control, indicating a slower pace of DSB repair. A slight variation is observed in the decay rates of foci for WAS patients indicative of the dependence of the repair on the type of gene mutation. After 24 hours, the residual foci in WAS patients were ~\u0026thinsp;2 times higher than the control, again indicating significantly elevated levels of unrepaired DSBs in WAS patients.\u003c/p\u003e \u003cp\u003eThe half-lives (\u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e) of DSB repair foci in both WAS and control lymphocytes were determined using first-order kinetics, calculated with the expression T\u003csub\u003e1/2\u003c/sub\u003e = 0.693/\u003cem\u003ek\u003c/em\u003e1 (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Among four WAS patients, a slight variation in the half-lives of the foci and their colocalization was observed. This substantial difference in repair efficiency of WAS patients raises significant concerns regarding genomic stability in WAS patients, potentially heightening their vulnerability to radiation exposure and increasing their risk of developing malignancies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 DSB repair dynamics and radiation sensitivity in carrier mother of WAS patients:\u003c/h2\u003e \u003cp\u003eThe dynamics of DSB repair foci were also examined in the lymphocytes of carrier mothers (MW1-MW4). Data for the carrier mother of the WAS-4 patient is shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, while data for the carrier mothers of WAS-1 to WAS-3 is not included. The foci induction and decay kinetics closely paralleled the trends observed in the control, with approximately 20% residual foci remaining even after 24 hours, which was consistent with the control data (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). The estimated half-lives for the foci, in the lymphocytes of the mothers was comparable to that of the control (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These findings suggest that the carrier mothers of WAS patients exhibited wild-type efficiency in repairing DSBs and did not show heightened radiation sensitivity.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious reports have indicated that the pathophysiological conditions and severity of WAS patients vary significantly depending on the type and location of the mutation in the WAS gene (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). This study investigated the effects of radiation exposure on genome instability in four WAS patients possessing different WAS gene mutation. In the present study first time we have evaluated DSBs repair kinetics in WAS patients and their carrier mothers using markers such as γH2AX and 53BP1. We have noted\u0026thinsp;~\u0026thinsp;16 to ~\u0026thinsp;24 times elevated baseline levels of DSB markers (γH2AX and 53BP1) in lymphocytes of WAS patients compared to carrier mothers and healthy controls. A similar result has been reported by Wang et al. in \u003cem\u003ein vitro\u003c/em\u003e WAS knockout cells (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). The baseline occurrence of DSBs arises from various endogenous and exogenous factors, triggering the generation of reactive oxygen species (ROS) and consequent DSBs in cells (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Cellular processes, such as cell division and cell differentiation, can also provoke the formation of DSBs (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Wild-type cells have the ability to repair these spontaneously induced DSBs, preventing their accumulation. However, WAS-mutant cells exhibit impaired DSB repair, potentially leading to the accumulation of these DSBs over time (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTypically, WAS-mothers demonstrate normal basal levels of expression of DSB repair foci, although in rare instances, higher spontaneous yields have also been reported (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). However, our results demonstrated that the repair yield (at 24 h) of DSBs, indicated by γH2AX and 53BP1 foci, in the mothers was consistent with the typical range observed in controls and other documented human populations (0 to 0.49 foci/cell in populations from Germany, France, Cuba, India, and elsewhere) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRecent studies have elucidated the critical role of WAS protein (WASP) in DNA repair mechanisms, particularly emphasizing DSB repair (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). WASP deficiency hampers the assembly of actin filaments at DNA damage sites, impeding the recruitment of repair factors and compromising the competence of DSB repair pathways, including nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). However, the kinetics of DSB repairs, such as the repair half-life and the dynamics of repair foci over time, are not adequately understood or explored. To assess the radiation sensitivity and genomic stability, DSBs were created by irradiating WAS lymphocytes with 2 Gy of gamma radiation and foci (γH2AX and 53BP1) formation and decay kinetics was studied. This study demonstrated that WAS lymphocytes exhibit a similar trend in the DSB repair process as control lymphocytes, with an initial rapid repair phase followed by a slower phase. Though, the pace of DSB repair was significantly reduced in WAS-lymphocytes than that in the control. Results showed presence of significantly higher residual foci after 24 hours reflecting the persistence of excess DSBs in WAS patients, compared to healthy control volunteers. These findings clearly indicate the accumulation of unrepaired DSBs in radiation-challenged WAS lymphocytes, suggesting increased radiosensitivity and genomic instability.\u003c/p\u003e \u003cp\u003eDifferent levels of radiosensitivity were observed among the four WAS patients (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). W1 exhibited the highest level of radiosensitivity, with a\u0026thinsp;~\u0026thinsp;1.9-fold greater DSB repair half-life (slowest pace of DSB repair) than that of the controls, as evidenced by the data on γH2AX foci. Conversely, patient W4 exhibited a relatively lower level of sensitivity, with a\u0026thinsp;~\u0026thinsp;1.5-fold increase in the half-life of DSB repair compared with that of the control. This differential sensitivity was due to frameshift mutations, as observed in W1, significantly impair WAS function compared to the milder impact of missense mutations, such as those seen in W4.\u003c/p\u003e \u003cp\u003eRepairing complex DSBs is time-intensive, often taking hours to days. In contrast, 2 Gy of low-LET radiation (60Co-γ-ray) induces simpler DSBs, usually repaired within 2\u0026ndash;3 hours (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). This study showed that WAS patients exhibit incomplete repair of simple DSBs for up to 24 hours (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRadiation exposure can result into compromised immune function, increasing susceptibility to infections and other complications (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Understanding the extent of radiation sensitivity in WAS patients can guide healthcare providers in optimizing infection prevention strategies and providing timely interventions to minimize adverse effects.\u003c/p\u003e \u003cp\u003eOwing to the accumulation of DSBs and genomic instability, WAS patients face an increased risk of developing malignancies (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Radiation therapy is a commonly used treatment modality for various malignancies and autoimmune conditions (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). However, WAS patients may be at increased risk of developing radiation-induced toxicity due to underlying immunodeficiency and potential defects in DNA repair mechanisms (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Moreover, hematopoietic stem cell transplantation (HSCT) is a potential curative option for WAS patients (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Radiation conditioning regimens are commonly administered before HSCT to suppress the recipient's immune system and enhance the engraftment of donor cells (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). However, the increased radiation sensitivity of WAS patients raises significant concerns regarding the safety and tolerability of these conditioning regimens. Conducting an assessment of radiation sensitivity in WAS patients before HSCT can assist in risk stratification and treatment planning, thus ensuring the best possible outcomes while minimizing potential complications.\u003c/p\u003e \u003cp\u003eThis study demonstrated impaired or reduced DSB repair efficiency and the accumulation of spontaneous and induced DSBs in WAS lymphocytes. However, assessing the fidelity of the DSB repair process itself is another crucial aspect. Errors in the repair process may lead to misrepair products, contributing to chromosomal abnormalities and genomic instability (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Cytogenetic markers such as dicentrics and/or chromosomal rearrangements arise from misrepaired DSBs. Investigating these cytogenetic markers can provide further insights into the accuracy of the DSB repair process in WAS patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study is the first to demonstrate DSB repair kinetics in WAS patients and their carrier mothers. We uncovered the intricate relationship between four different WAS gene mutations and their effects on DNA repair dynamics and radiation sensitivity. By analyzing DSB repair in the lymphocytes of both patients and carrier mothers, we gained vital insights into DSB repair efficiency and the accumulation of unrepaired DSBs, which contribute to genomic instability in WAS. Our findings highlight the complex dynamics of DSB repair in WAS, offering valuable insights for clinical management and the potential to improve patient outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to the patients and their families for their invaluable cooperation and participation in this study. We also acknowledge the indispensable technical support provided by Mr. Shrikant Jagtap and other laboratory members. Additionally, we sincerely thank the referring clinicians for generously sharing clinical information, which played a crucial role in the successful completion of this research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRSP, RKC, and KBS conducted the experiments and collected the data. RKC, BKS, and MM conceptualized the project, analyzed the data, and drafted the manuscript. PG and UB contributed to writing the clinical details of the patients. Anjana Goel, N.N. Bhat, and Arshad Khan reviewed and provided critical edits to the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the host institute, Bhabha Atomic Research Centre, Mumbai, India. No external funding was received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are provided within the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode Availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Institutional Ethics Committee (IEC) of the National Institute of Immunohaematology (ICMR), Mumbai.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the patients\u0026apos; guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patients\u0026apos; guardians provided signed informed consent for the publication of the data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWiskott A. 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Nat Rev Genet. 2008;9(3):204\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Wiskott-Aldrich syndrome (WAS), DSB repair kinetics, DSB repair half-time, genetic instability, radiosensitivity","lastPublishedDoi":"10.21203/rs.3.rs-5719467/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5719467/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWiskott-Aldrich Syndrome (WAS) is a rare X-linked recessive disorder characterized by microthrombocytopenia, eczema, combined immunodeficiency, and an increased risk of malignancies. Previous research has highlighted genomic instability in WAS patients; however, the specific dynamics of double-strand break (DSB) repair in these individuals are not fully understood. Phosphorylation of γH2AX serves as an early indicator of DSBs, while 53BP1 binds to damaged chromatin in a γH2AX-dependent manner, stabilizing DNA ends and facilitating repair and hence serves as a marker for assessing DNA repair kinetics. In this study, we investigated the kinetics of DSBs in WAS patients and their carrier mothers using markers such as γH2AX and 53BP1. To evaluate radiation sensitivity, lymphocytes from WAS patients were exposed to 2 Gy of gamma radiation, and repair kinetics were recorded over 24 hours. Immunofluorescence staining for γH2AX and 53BP1 was performed on both irradiated and non-irradiated lymphocytes from patients, carrier mothers, and healthy controls at various time points. Notably, WAS patients exhibited a significant increase in baseline levels of γH2AX and 53BP1 (16 to 24 times higher) compared to healthy controls. Post-radiation decay model revealed a significantly prolonged DSB repair rate in WAS patients, with a repair half-life 1.5 to 1.9 times longer than that of controls. Additionally, at 24 hours, WAS patients showed 37\u0026ndash;39% and 33\u0026ndash;43% excess γH2AX and 53BP1 foci per cell, respectively, indicating increased genomic instability and radiosensitivity. This study provides the first evidence of delayed repair kinetics in WAS patients, which may impact clinical decisions regarding radiation or genotoxic exposure during diagnosis and treatment.\u003c/p\u003e","manuscriptTitle":"Retarded DSB repair kinetics suggestive of augmented radiation sensitivity and genetic instability in Wiskott-Aldrich syndrome patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 18:57:35","doi":"10.21203/rs.3.rs-5719467/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9aa7a735-f2a1-40a1-bd7b-e8d61d5f328a","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-30T05:23:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-31 18:57:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5719467","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5719467","identity":"rs-5719467","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}