Differential role of nonhomologous end joining factors in the generation, DNA damage response, and myeloid differentiation of human induced pluripotent stem cells.

Nonhomologous end-joining (NHEJ) is a key pathway for efficient repair of DNA double-strand breaks (DSBs) and V(D)J recombination. NHEJ defects in humans cause immunodeficiency and increased cellular sensitivity to ionizing irradiation (IR) and are variably associated with growth retardation, microcephaly, and neurodevelopmental delay. Repair of DNA DSBs is important for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). To compare the specific contribution of DNA ligase 4 (LIG4), Artemis, and DNA-protein kinase catalytic subunit (PKcs) in this process and to gain insights into phenotypic variability associated with these disorders, we reprogrammed patient-derived fibroblast cell lines with NHEJ defects. Deficiencies of LIG4 and of DNA-PK catalytic activity, but not Artemis deficiency, were associated with markedly reduced reprogramming efficiency, which could be partially rescued by genetic complementation. Moreover, we identified increased genomic instability in LIG4-deficient iPSCs. Cell cycle synchronization revealed a severe defect of DNA repair and a G0/G1 cell cycle arrest, particularly in LIG4- and DNA-PK catalytically deficient iPSCs. Impaired myeloid differentiation was observed in LIG4-, but not Artemis- or DNA-PK-mutated iPSCs. These results indicate a critical importance of the NHEJ pathway for somatic cell reprogramming, with a major role for LIG4 and DNA-PKcs and a minor, if any, for Artemis.

XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency.

BACKGROUND: Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair mechanism in human cells. The final rejoining step requires DNA ligase IV (LIG4) together with the partner proteins X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor. Patients with mutations in genes encoding LIG4, XRCC4-like factor, or the other NHEJ proteins DNA-dependent protein kinase catalytic subunit and Artemis are DSB repair defective and immunodeficient because of the requirement for NHEJ during V(D)J recombination. OBJECTIVE: We found a patient displaying microcephaly and progressive ataxia but a normal immune response. We sought to determine pathogenic mutations and to describe the molecular pathogenesis of the patient. METHODS: We performed next-generation exome sequencing. We evaluated the DSB repair activities and V(D)J recombination capacity of the patient's cells, as well as performing a standard blood immunologic characterization. RESULTS: We identified causal mutations in the XRCC4 gene. The patient's cells are radiosensitive and display the most severe DSB repair defect we have encountered using patient-derived cell lines. In marked contrast, a V(D)J recombination plasmid assay revealed that the patient's cells did not display the junction abnormalities that are characteristic of other NHEJ-defective cell lines. The mutant protein can interact efficiently with LIG4 and functions normally in in vitro assays and when transiently expressed in vivo. However, the mutation makes the protein unstable, and it undergoes proteasome-mediated degradation. CONCLUSION: Our findings reveal a novel separation of impact phenotype: there is a pronounced DSB repair defect and marked clinical neurological manifestation but no clinical immunodeficiency.

NUDCD3 deficiency disrupts V(D)J recombination to cause SCID and Omenn syndrome.

Inborn errors of T cell development present a pediatric emergency in which timely curative therapy is informed by molecular diagnosis. In 11 affected patients across four consanguineous kindreds, we detected homozygosity for a single deleterious missense variant in the gene NudC domain-containing 3 (NUDCD3). Two infants had severe combined immunodeficiency with the complete absence of T and B cells (T -B- SCID), whereas nine showed classical features of Omenn syndrome (OS). Restricted antigen receptor gene usage by residual T lymphocytes suggested impaired V(D)J recombination. Patient cells showed reduced expression of NUDCD3 protein and diminished ability to support RAG-mediated recombination in vitro, which was associated with pathologic sequestration of RAG1 in the nucleoli. Although impaired V(D)J recombination in a mouse model bearing the homologous variant led to milder immunologic abnormalities, NUDCD3 is absolutely required for healthy T and B cell development in humans.

The many faces of Artemis-deficient combined immunodeficiency - Two patients with DCLRE1C mutations and a systematic literature review of genotype-phenotype correlation.

Defective V(D)J recombination and DNA double-strand break (DSB) repair severely impair the development of T-lymphocytes and B-lymphocytes. Most patients manifest a severe combined immunodeficiency during infancy. We report 2 siblings with combined immunodeficiency (CID) and immunodysregulation caused by compound heterozygous Artemis mutations, including an exon 1-3 deletion generating a null allele, and a missense change (p.T71P). Skin fibroblasts demonstrated normal DSB repair by gamma-H2AX analysis, supporting the predicted hypomorphic nature of the p.T71P allele. In addition to these two patients, 12 patients with Artemis-deficient CID were previously reported. All had significant morbidities including recurrent infections, autoimmunity, EBV-associated lymphoma, and carcinoma despite having hypomorphic mutants with residual Artemis expression, V(D)J recombination or DSB repair capacity. Nine patients underwent stem cell transplant and six survived, while four patients who did not receive transplant died. The progressive nature of immunodeficiency and genomic instability accounts for poor survival, and early HSCT should be considered.

DNA repair is limiting for haematopoietic stem cells during ageing.

Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of ageing. Here we address this question by taking advantage of the highly specific role of DNA ligase IV in the repair of DNA double-strand breaks by non-homologous end-joining, and by the discovery of a unique mouse strain with a hypomorphic Lig4(Y288C) mutation. The Lig4(Y288C) mouse, identified by means of a mutagenesis screening programme, is a mouse model for human LIG4 syndrome, showing immunodeficiency and growth retardation. Diminished DNA double-strand break repair in the Lig4(Y288C) strain causes a progressive loss of haematopoietic stem cells and bone marrow cellularity during ageing, and severely impairs stem cell function in tissue culture and transplantation. The sensitivity of haematopoietic stem cells to non-homologous end-joining deficiency is therefore a key determinant of their ability to maintain themselves against physiological stress over time and to withstand culture and transplantation.

Endogenously induced DNA double strand breaks arise in heterochromatic DNA regions and require ataxia telangiectasia mutated and Artemis for their repair.

Ataxia telangiectasia (ATM) mutated and Artemis, the proteins defective in ataxia telangiectasia and a class of Radiosensitive-Severe Combined Immunodeficiency (RS-SCID), respectively, function in the repair of DNA double strand breaks (DSBs), which arise in heterochromatic DNA (HC-DSBs) following exposure to ionizing radiation (IR). Here, we examine whether they have protective roles against oxidative damage induced and/or endogenously induced DSBs. We show that DSBs generated following acute exposure of G0/G1 cells to the oxidative damaging agent, tert-butyl hydroperoxide (TBH), are repaired with fast and slow components of similar magnitude to IR-induced DSBs and have a similar requirement for ATM and Artemis. Strikingly, DSBs accumulate in ATM(-/-) mouse embryo fibroblasts (MEFs) and in ATM or Artemis-defective human primary fibroblasts maintained for prolonged periods under confluence arrest. The accumulated DSBs localize to HC-DNA regions. Collectively, the results provide strong evidence that oxidatively induced DSBs arise in HC as well as euchromatic DNA and that Artemis and ATM function in their repair. Additionally, we show that Artemis functions downstream of ATM and is dispensable for HC-relaxation and for pKAP-1 foci formation. These findings are important for evaluating the impact of endogenously arising DNA DSBs in ATM and Artemis-deficient patients.

Biological network topology features predict gene dependencies in cancer cell-lines.

Motivation: Protein-protein interaction (PPI) networks have been shown to successfully predict essential proteins. However, such networks are derived generically from experiments on many thousands of different cells. Consequently, conventional PPI networks cannot capture the variation of genetic dependencies that exists across different cell types, let alone those that emerge as a result of the massive cell restructuring that occurs during carcinogenesis. Predicting cell-specific dependencies is of considerable therapeutic benefit, facilitating the use of drugs to inhibit those proteins on which the cancer cells have become specifically dependent. In order to go beyond the limitations of the generic PPI, we have attempted to personalise PPI networks to reflect cell-specific patterns of gene expression and mutation. By using 12 topological features of the resulting PPIs, together with matched gene dependency data from DepMap, we trained random-forest classifiers (DependANT) to predict novel gene dependencies. Results: We found that DependANT improves the power of the baseline generic PPI models in predicting common gene dependencies, by up to 10.8% and is more sensitive than the baseline generic model when predicting genes on which only a small number of cell types are dependent. Availability and implementation: Software available at https://bitbucket.org/bioinformatics_lab_sussex/dependant2. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.

XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex.

Magnetic Field Triggerable Macroporous PDMS Sponge Loaded with an Anticancer Drug, 5-Fluorouracil.

This study aims to prepare, optimize, and characterize magnetic-field-sensitive sugar-templated polydimethylsiloxane (PDMS) sponges for localized delivery of an anticancer drug, 5-fluorouracil (FLU). For this purpose, different concentrations of carbonyl iron (CI) and magnetite Fe3O4 nanopowders were embedded as magnetosensitive materials in PDMS resins for the fabrication of macroporous sponges via a sugar-template process. The process is environmentally friendly and simple. The fabricated interconnected macroporous magnetic particles loaded PDMS sponges possess flexible skeletons and good recyclability because of their recoverability after compression (deformation) without any breakdown. The prepared magnetic PDMS sponges were evaluated for their morphology (SEM and EDS), porosity (absorbency), elastic modulus, deformation under a magnetic field, thermostability, and in vitro cell studies. All physicochemical and magnetomechanical analysis confirmed that the optimized magnetic-field-sensitive PDMS sponge can provide an efficient method for delivering an on-demand dose of anticancer drug solutions at a specific location and timing with the aid of controlled magnetic fields.

3D Printed Calcium Phosphate Cement (CPC) Scaffolds for Anti-Cancer Drug Delivery.

One of the main applications of bone graft materials is filling the gap after the surgical removal of bone cancer or tumors. Insufficient healing commonly leads to non-union fracture which could lead to cancer resurgence or infection. Emerging 3D printing of on-demand bone graft biomaterials can deliver personalized solutions with minimized risk of relapse and recurrence of cancer after bone removal surgery. This research aims to explore 3D printed calcium phosphate cement (CPC) based scaffolds as novel anti-cancer drug delivery systems to treat bone cancer. For the study, various 3D printed CPC based scaffolds (diameter 5 mm) with interconnected pores were utilized. Various optimized polymeric solutions containing a model anticancer drug 5-fluorouracil (5-FU) was used to homogenously coat the CPC scaffolds. Both hydrophilic Soluplus (SOL) and polyethylene glycol (PEG) and a combination of both were used to develop stable coating solutions. The surface morphology of the coated scaffolds, observed via SEM, revealed deposition of the polymeric solution represented by a semi-smooth surface as opposed to the blank scaffolds that showed a smoother surface. An advanced surface analysis conducted via confocal microscopy showed a homogenous distribution of the drug throughout the coated scaffolds. Solid-state analysis studied by applying differential scanning calorimetry (DSC) and X-ray diffraction (XRD) revealed semi-crystalline nature of the drug whereas mechanical analysis conducted via texture analysis showed no evidence in the change of the mechanical properties of the scaffolds after polymeric solutions were applied. The FTIR analysis revealed no major intermolecular interactions between 5-FU and the polymers used for coatings except for F2 where a potential nominal interaction was evidenced corresponding to higher Soluplus content in the formulation. In vitro dissolution studies showed that almost 100% of the drug released within 2 h for all scaffolds. Moreover, in vitro cell culture using two different cell lines (Hek293T-human kidney immortalized cell line and HeLa-human bone osteosarcoma epithelial cell line) showed significant inhibition of cell growth as a function of decreased numbers of cells after 5 days. It can be claimed that the developed 5-FU coated 3D printed scaffolds can successfully be used as bone graft materials to potentially treat bone cancer or bone neoplasm and for personalized medical solutions in the form of scaffolds for regenerative medicine or tissue engineering applications.

Novel 3D printed device with integrated macroscale magnetic field triggerable anti-cancer drug delivery system.

With the growing demand for personalized medicine and medical devices, the impact of on-demand triggerable (e.g., via magnetic fields) drug delivery systems increased significantly in recent years. The three-dimensional (3D) printing technology has already been applied in the development of personalized dosage forms because of its high-precision and accurate manufacturing ability. In this study, a novel magnetically triggerable drug delivery device composed of a magnetic polydimethylsiloxane (PDMS) sponge cylinder and a 3D printed reservoir was designed, fabricated and characterized. This system can realize a switch between "on" and "off" state easily through the application of different magnetic fields and from different directions. Active and repeatable control of the localized drug release could be achieved by the utilization of magnetic fields to this device due to the shrinking extent of the macro-porous magnetic sponge inside. The switching "on" state of drug-releasing could be realized by the magnetic bar contacted with the side part of the device because the times at which 50%, 80% and 90% (w/w) of the drug were dissolved are observed to be 20, 55 and 140 min, respectively. In contrast, the switching "off" state of drug-releasing could be realized by the magnetic bar placed at the bottom of the device as only 10% (w/w) of the drug could be released within 12 h. An anti-cancer substance, 5-fluorouracil (FLU), was used as the model drug to illustrate the drug release behaviour of the device under different strengths of magnetic fields applied. In vitro cell culture studies also demonstrated that the stronger the magnetic field applied, the higher the drug release from the deformed PDMS sponge cylinder and thus more obvious inhibition effects on Trex cell growth. All results confirmed that the device can provide a safe, long-term, triggerable and reutilizable way for localized disease treatment such as cancer.

Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK.

DNA non-homologous end-joining (NHEJ) is a major mechanism for repairing DNA double-stranded (ds) breaks in mammalian cells. Here, we characterize the interaction between two key components of the NHEJ machinery, the Ku heterodimer and the DNA ligase IV/Xrcc4 complex. Our results demonstrate that Ku interacts with DNA ligase IV via its tandem BRCT domain and that this interaction is enhanced in the presence of Xrcc4 and dsDNA. Moreover, residues 644-748 of DNA ligase IV encompassing the first BRCT motif are necessary for binding. We show that Ku needs to be in its heterodimeric form to bind DNA ligase IV and that the C-terminal tail of Ku80, which mediates binding to DNA-PKcs, is dispensable for DNA ligase IV recognition. Although the interaction between Ku and DNA ligase IV/Xrcc4 occurs in the absence of DNA-PKcs, the presence of the catalytic subunit of DNA-PK kinase enhances complex formation. Previous studies have shown that DNA-PK kinase activity causes disassembly of DNA-PKcs from Ku at the DNA end. Here, we show that DNA-PK kinase activity also results in disassembly of the Ku/DNA ligase IV/Xrcc4 complex. Collectively, our findings provide novel information on the protein-protein interactions that regulate NHEJ in cells.

The rate of X-ray-induced DNA double-strand break repair in the embryonic mouse brain is unaffected by exposure to 50 Hz magnetic fields.

PURPOSE: Following in utero exposure to low dose radiation (10-200 mGy), we recently observed a linear induction of DNA double-strand breaks (DSB) and activation of apoptosis in the embryonic neuronal stem/progenitor cell compartment. No significant induction of DSB or apoptosis was observed following exposure to magnetic fields (MF). In the present study, we exploited this in vivo system to examine whether exposure to MF before and after exposure to 100 mGy X-rays impacts upon DSB repair rates. MATERIALS AND METHODS: 53BP1 foci were quantified following combined exposure to radiation and MF in the embryonic neuronal stem/progenitor cell compartment. Embryos were exposed in utero to 50 Hz MF at 300 µT for 3 h before and up to 9 h after exposure to 100 mGy X-rays. Controls included embryos exposed to MF or X-rays alone plus sham exposures. RESULTS: Exposure to MF before and after 100 mGy X-rays did not impact upon the rate of DSB repair in the embryonic neuronal stem cell compartment compared to repair rates following radiation exposure alone. CONCLUSIONS: We conclude that in this sensitive system MF do not exert any significant level of DNA damage and do not impede the repair of X-ray induced damage.

Evaluation of Severe Combined Immunodeficiency and Combined Immunodeficiency Pediatric Patients on the Basis of Cellular Radiosensitivity.

Pediatric patients with severe or nonsevere combined immunodeficiency have increased susceptibility to severe, life-threatening infections and, without hematopoietic stem cell transplantation, may fail to thrive. A subset of these patients have the radiosensitive (RS) phenotype, which may necessitate conditioning before hematopoietic stem cell transplantation, and this conditioning includes radiomimetic drugs, which may significantly affect treatment response. To provide statistical criteria for classifying cellular response to ionizing radiation as the measure of functional RS screening, we analyzed the repair capacity and survival of ex vivo irradiated primary skin fibroblasts from five dysmorphic and/or developmentally delayed pediatric patients with severe combined immunodeficiency and combined immunodeficiency. We developed a mathematical framework for the analysis of γ histone 2A isoform X foci kinetics to quantitate DNA-repair capacity, thus establishing crucial criteria for identifying RS. The results, presented in a diagram showing each patient as a point in a 2D RS map, were in agreement with findings from the assessment of cellular RS by clonogenic survival and from the genetic analysis of factors involved in the nonhomologous end-joining repair pathway. We provide recommendations for incorporating into clinical practice the functional assays and genetic analysis used for establishing RS status before conditioning. This knowledge would enable the selection of the most appropriate treatment regimen, reducing the risk for severe therapy-related adverse effects.

The clinical impact of deficiency in DNA non-homologous end-joining.

DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway in mammalian cells. Defects in NHEJ proteins confer marked radiosensitivity in cell lines and mice models, since radiation potently induces DSBs. The process of V(D)J recombination functions during the development of the immune response, and involves the introduction and rejoining of programmed DSBs to generate an array of diverse T and B cells. NHEJ rejoins these programmed DSBs. Consequently, NHEJ deficiency confers (severe) combined immunodeficiency - (S)CID - due to a failure to carry out V(D)J recombination efficiently. NHEJ also functions in class switch recombination, another step enhancing T and B cell diversity. Prompted by these findings, a search for radiosensitivity amongst (S)CID patients revealed a radiosensitive sub-class, defined as RS-SCID. Mutations in NHEJ genes, defining human syndromes deficient in DNA ligase IV (LIG4 Syndrome), XLF-Cernunnos, Artemis or DNA-PKcs, have been identified in such patients. Mutations in XRCC4 or Ku70,80 in patients have not been identified. RS-SCID patients frequently display additional characteristics including microcephaly, dysmorphic facial features and growth delay. Here, we overview the clinical spectrum of RS-SCID patients and discuss our current understanding of the underlying biology.

Reprint of "The clinical impact of deficiency in DNA non-homologous end-joining".

DNA non-homologous end-joining (NHEJ) is the major DNA double strand break (DSB) repair pathway in mammalian cells. Defects in NHEJ proteins confer marked radiosensitivity in cell lines and mice models, since radiation potently induces DSBs. The process of V(D)J recombination functions during the development of the immune response, and involves the introduction and rejoining of programmed DSBs to generate an array of diverse T and B cells. NHEJ rejoins these programmed DSBs. Consequently, NHEJ deficiency confers (severe) combined immunodeficiency - (S)CID - due to a failure to carry out V(D)J recombination efficiently. NHEJ also functions in class switch recombination, another step enhancing T and B cell diversity. Prompted by these findings, a search for radiosensitivity amongst (S)CID patients revealed a radiosensitive sub-class, defined as RS-SCID. Mutations in NHEJ genes, defining human syndromes deficient in DNA ligase IV (LIG4 Syndrome), XLF-Cernunnos, Artemis or DNA-PKcs, have been identified in such patients. Mutations in XRCC4 or Ku70,80 in patients have not been identified. RS-SCID patients frequently display additional characteristics including microcephaly, dysmorphic facial features and growth delay. Here, we overview the clinical spectrum of RS-SCID patients and discuss our current understanding of the underlying biology.

Increased apoptosis and DNA double-strand breaks in the embryonic mouse brain in response to very low-dose X-rays but not 50 Hz magnetic fields.

The use of X-rays for medical diagnosis is enhancing exposure to low radiation doses. Exposure to extremely low-frequency electromagnetic or magnetic fields is also increasing. Epidemiological studies show consistent associations of childhood leukaemia with exposure to magnetic fields but any causal relationship is unclear. A limitation in assessing the consequence of such exposure is the availability of sensitive assays. The embryonic neuronal stem and progenitor cell compartments are radiosensitive tissues. Using sensitive assays, we report a statistically significant increase in DNA double-strand break (DSB) formation and apoptosis in the embryonic neuronal stem cell compartment following in utero exposure to 10-200 mGy X-rays. Both endpoints show a linear response. We also show that DSB repair is delayed following exposure to doses below 50 mGy compared with 100 mGy. Thus, we demonstrate in vivo consequences of low-dose radiation. In contrast to these impacts, we did not observe any significant induction of DSBs or apoptosis following exposure to 50 Hz magnetic fields (100 or 300 µT). We conclude that any DSB induction by treatment with magnetic fields is lower than following exposure to 10 mGy X-rays. For comparison, certain procedures involving computed tomography scanning are equivalent to 1-5 mGy X-rays.

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

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

PRKDC mutations in a SCID patient with profound neurological abnormalities.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre- and postnatal neurogenesis.