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“X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia” (XMEN) disease is an inborn error of glycosylation and immunity caused by loss of function mutations in the magnesium transporter 1 ( MAGT1 ) gene. It is a multisystem disease that strongly affects certain immune cells. MAGT1 is now confirmed as a non-catalytic subunit of the oligosaccharyltransferase complex and facilitates Asparagine (N)-linked glycosylation of specific substrates, making XMEN a congenital disorder of glycosylation manifesting as a combined immune deficiency. The clinical disease has variable expressivity and impaired glycosylation of key MAGT1-dependent glycoproteins in addition to Mg 2+ abnormalities can explain some of the immune manifestations. NKG2D, an activating receptor critical for cytotoxic function against EBV, is poorly glycosylated and invariably decreased on CD8 + T cells and natural killer (NK) cells from XMEN patients. It is the best biomarker of the disease. The characterization of EBV-naïve XMEN patients has clarified features of the genetic disease that were previously attributed to EBV infection. Extra-immune manifestations, including hepatic and neurological abnormalities have recently been reported. EBV-associated lymphomas remain the main cause of severe morbidity. Unfortunately, treatment options to address the underlying mechanism of disease remain limited and Mg 2+ supplementation has not proven successful. Here, we review the expanding clinical phenotype and recent advances in glycobiology that have increased our understanding of XMEN disease. We also propose updating XMEN to “ X -linked M AGT1 deficiency with increased susceptibility to E BV-infection and N -linked glycosylation defect” in light of these novel findings.

Magnesium transporter 1 (MAGT1) critically mediates magnesium homeostasis in eukaryotes and is highly-conserved across different evolutionary branches. In humans, loss-offunction mutations in the MAGT1 gene cause X-linked magnesium deficiency with Epstein-Barr virus (EBV) infection and neoplasia (XMEN), a disease that has a broad range of clinical and immunological consequences. We have previously shown that EBV susceptibility in XMEN is associated with defective expression of the antiviral natural-killer group 2 member D (NKG2D) protein and abnormal Mg 2؉ transport. New evidence suggests that MAGT1 is the human homolog of the yeast OST3/ OST6 proteins that form an integral part of the N-linked glycosylation complex, although the exact contributions of these perturbations in the glycosylation pathway to disease pathogenesis are still unknown. Using MS-based glycoproteomics, along with CRISPR/Cas9-KO cell lines, natural killer cell-killing assays, and RNA-Seq experiments, we now demonstrate that humans lacking functional MAGT1 have a selective deficiency in both immune and nonimmune glycoproteins, and we identified several critical glycosylation defects in important immune-response proteins and in the expression of genes involved in immunity, particularly CD28. We show that MAGT1 function is partly interchangeable with that of the paralog protein tumorsuppressor candidate 3 (TUSC3) but that each protein has a different tissue distribution in humans. We observed that MAGT1dependent glycosylation is sensitive to Mg 2؉ levels and that reduced Mg 2؉ impairs immune-cell function via the loss of specific glycoproteins. Our findings reveal that defects in protein glycosylation and gene expression underlie immune defects in an inherited disease due to MAGT1 deficiency.MAGT1 is an evolutionally conserved Mg 2ϩ -specific ion transport facilitator found in all animals and has been shown to participate in the multienzyme complex responsible for enzymatic coupling of N-glycans onto peptide substrates (1, 2). Null mutations in the MAGT1 gene lead to the rare primary immunodeficiency "X-linked immunodeficiency with Mg 2ϩ defect, Epstein-Barr virus (EBV) 7 infection and neoplasia" (XMEN) disease (3,4). Here, we explore these dual roles by examining cells from both healthy and MAGT1-deficient humans.Mg 2ϩ is the most abundant divalent cation in eukaryotic cells, with intracellular concentrations ranging from 15 to 20 mM depending on the cell type. Most Mg 2ϩ is tightly bound to cellular substituents, especially nucleic acids, nucleoside triphosphates, and enzymes. The unbound intracellular free Mg 2ϩ is estimated to be 0.4 -1.0 mM or ϳ1-5% of the total Mg 2ϩ concentration in the cell (5, 6), and because Mg 2ϩ is the biologically active form of Mg, these intracellular concentra-

Purpose Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients. Methods Two different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients. Results Two young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection. Conclusion Astute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.

Mg2+ is required at micromolar concentrations as a cofactor for ATP, enzymatic reactions, and other biological processes. We show that decreased extracellular Mg2+ reduced intracellular Mg2+ levels and impaired the Ca2+ flux, activation marker up-regulation, and proliferation after T cell receptor (TCR) stimulation. Reduced Mg2+ specifically impairs TCR signal transduction by IL-2–inducible T cell kinase (ITK) due to a requirement for a regulatory Mg2+ in the catalytic pocket of ITK. We also show that altered catalytic efficiency by millimolar changes in free basal Mg2+ is an unrecognized but conserved feature of other serine/threonine and tyrosine kinases, suggesting a Mg2+ regulatory paradigm of kinase function. Finally, a reduced serum Mg2+ concentration in mice causes an impaired CD8+ T cell response to influenza A virus infection, reduces T cell activation, and exacerbates morbidity. Thus, Mg2+ directly regulates the active site of specific kinases during T cell responses, and maintaining a high serum Mg2+ concentration is important for antiviral immunity in otherwise healthy animals.

Purpose of review To describe the role of the magnesium transporter 1 (MAGT1) in the pathogenesis of “X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection, and neoplasia” (XMEN) disease and its clinical implications. Recent findings The magnesium transporter protein MAGT1 participates in intracellular Mg2+ homeostasis and facilitates a transient Mg2+ influx induced by activation of the T cell receptor (TCR). Loss-of-function mutations in MAGT1 cause an immunodeficiency named XMEN syndrome characterized by CD4 lymphopenia, chronic EBV infection, and EBV-related lymphoproliferative disorders. Patients with XMEN disease have impaired T cell activation and decreased cytolytic function of natural killer (NK) and CD8+ T cells due to decreased expression of the natural killer stimulatory receptor natural-killer group 2, member D (NKG2D). Patients may have defective specific antibody responses secondary to T cell dysfunction, but B cells have not been shown to be directly affected by mutations in MAGT1. Summary XMEN disease has revealed a novel role for free intracellular magnesium in the immune system. Further understanding of the MAGT1 signaling pathway may lead to new diagnostic and therapeutic approaches.

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'X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus-infection and N-linked glycosylation defect' (XMEN) disease is a recently described primary immunodeficiency marked by defective T and Natural Killer (NK) cells. Potentially curative hematopoietic stem cell transplant is associated with high mortality rates. We sought to develop an ex vivo targeted gene therapy approach for XMEN patients using CRISPR/Cas9/adeno-associated vector (AAV) to insert a therapeutic MAGT1 gene at the constitutive locus under the regulation of the endogenous promoter. Clinical translation of CRISPR/Cas9/AAV-targeted gene editing (GE) is hampered by low engraftable GE hematopoietic stem/progenitor cells (HSPCs). Here, we optimized GE conditions by transient enhancement of homology-directed repair while suppressing AAV-associated DNA damage response to achieve highly efficient (>60%) genetic correction in engrafting XMEN HSPCs in transplanted mice. Restored MAGT1-glycosylation function in human NK and CD8+ T cells restored NKG2D expression and function in XMEN lymphocytes for potential treatment of infections, and corrected HSPCs for long-term gene therapy, thus offering two efficient therapeutic options for XMEN poised for clinical translation.