Spatiotemporal Gradient of Cortical Neuron Death Contributes to Microcephaly in Knock-In Mouse Model of Ligase 4 Syndrome.

Cells of the developing central nervous system are particularly susceptible to formation of double-stranded DNA breaks (DSBs) arising from physiological and/or environmental insults. Therefore, efficient repair of DSBs is especially vital for maintaining cellular health and proper functioning in the developing brain. Here, increased expression of DSB initiating and nonhomologous end joining repair machinery in newborn neurons in the developing brains of both mouse and human are demonstrated. In parallel, the first characterization is provided of the brain phenotype in the Lig4R278H/R278H (Lig4R/R) mouse model of DNA Ligase 4 (LIG4) syndrome, in which a hypomorphic Lig4 mutation, originally identified in patients, impedes nonhomologous end joining. It is shown that Lig4R/R mice develop nonprogressive microcephaly, resulting primarily from apoptotic death of newborn neurons that is both spatially and temporally specific during peak cortical neurogenesis. This apoptosis leads to a reduction in neurons throughout the postnatal cerebral cortex, but with a more prominent impact on those of the lower cortical layers. Together, these findings begin to uncover the pathogenesis of microcephaly in LIG4 syndrome and open avenues to more focused investigations on the critical roles of DSB formation and repair in vulnerable neuronal populations of the brain.

Inherited DOCK2 Deficiency in Patients with Early-Onset Invasive Infections.

Background Combined immunodeficiencies are marked by inborn errors of T-cell immunity in which the T cells that are present are quantitatively or functionally deficient. Impaired humoral immunity is also common. Patients have severe infections, autoimmunity, or both. The specific molecular, cellular, and clinical features of many types of combined immunodeficiencies remain unknown. Methods We performed genetic and cellular immunologic studies involving five unrelated children with early-onset invasive bacterial and viral infections, lymphopenia, and defective T-cell, B-cell, and natural killer (NK)-cell responses. Two patients died early in childhood; after allogeneic hematopoietic stem-cell transplantation, the other three had normalization of T-cell function and clinical improvement. Results We identified biallelic mutations in the dedicator of cytokinesis 2 gene (DOCK2) in these five patients. RAC1 activation was impaired in the T cells. Chemokine-induced migration and actin polymerization were defective in the T cells, B cells, and NK cells. NK-cell degranulation was also affected. Interferon-α and interferon-λ production by peripheral-blood mononuclear cells was diminished after viral infection. Moreover, in DOCK2-deficient fibroblasts, viral replication was increased and virus-induced cell death was enhanced; these conditions were normalized by treatment with interferon alfa-2b or after expression of wild-type DOCK2. Conclusions Autosomal recessive DOCK2 deficiency is a new mendelian disorder with pleiotropic defects of hematopoietic and nonhematopoietic immunity. Children with clinical features of combined immunodeficiencies, especially with early-onset, invasive infections, may have this condition. (Supported by the National Institutes of Health and others.).

N-WASP is required for B-cell-mediated autoimmunity in Wiskott-Aldrich syndrome.

Mutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott-Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling by modulating B-cell receptor (BCR) clustering and internalization. We have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-cell-dependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS.

Effect of repeated cycles of steam sterilization on the integrity of cannulated surgical screws.

The process of repeated handling and steam sterilization of bone screws and allows debris to deposit on the surface and inside cannulated screws. Individually packaged screws could alleviate these potential problems. This research intended to evaluate the effect that multiple cycles of sterilization may have, if any, on the mechanical integrity of cannulated screws. The results indicate that the mechanical integrity of the screws tested was not compromised.

EXTL3 mutations cause skeletal dysplasia, immune deficiency, and developmental delay.

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.

Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes.

Mutations in the Recombination Activating Gene 1 (RAG1) can cause a wide variety of clinical and immunological phenotypes in humans, ranging from absence of T and B lymphocytes to occurrence of autoimmune manifestations associated with expansion of oligoclonal T cells and production of autoantibodies. Although the mechanisms underlying this phenotypic heterogeneity remain poorly understood, some genotype-phenotype correlations can be made. Currently, mouse models of Rag deficiency are restricted to RAG1-/- mice and to knock-in models carrying severe missense mutations. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system is a novel and powerful gene-editing strategy that permits targeted introduction of DNA double strand breaks with high efficiency through simultaneous delivery of the Cas9 endonuclease and a guide RNA (gRNA). Here, we report on CRISPR-based, single-step generation and characterization of mutant mouse models in which gene editing was attempted around residue 838 of RAG1, a region whose functional role had not been studied previously.

Broad-spectrum antibodies against self-antigens and cytokines in RAG deficiency.

Patients with mutations of the recombination-activating genes (RAG) present with diverse clinical phenotypes, including severe combined immune deficiency (SCID), autoimmunity, and inflammation. However, the incidence and extent of immune dysregulation in RAG-dependent immunodeficiency have not been studied in detail. Here, we have demonstrated that patients with hypomorphic RAG mutations, especially those with delayed-onset combined immune deficiency and granulomatous/autoimmune manifestations (CID-G/AI), produce a broad spectrum of autoantibodies. Neutralizing anti-IFN-α or anti-IFN-ω antibodies were present at detectable levels in patients with CID-G/AI who had a history of severe viral infections. As this autoantibody profile is not observed in a wide range of other primary immunodeficiencies, we hypothesized that recurrent or chronic viral infections may precipitate or aggravate immune dysregulation in RAG-deficient hosts. We repeatedly challenged Rag1S723C/S723C mice, which serve as a model of leaky SCID, with agonists of the virus-recognizing receptors TLR3/MDA5, TLR7/-8, and TLR9 and found that this treatment elicits autoantibody production. Altogether, our data demonstrate that immune dysregulation is an integral aspect of RAG-associated immunodeficiency and indicate that environmental triggers may modulate the phenotypic expression of autoimmune manifestations.