A Double-Blind, Placebo-Controlled, Crossover Study of Magnesium Supplementation in Patients with XMEN Disease

Abstract

X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus (EBV) infection and N-linked glycosylation defect (XMEN) disease is an inborn error of immunity caused by loss-of-function mutations in the magnesium transporter 1 (MAGT1) gene. The original studies of XMEN patients focused on impaired magnesium regulation, leading to decreased EBV-cytotoxicity and the loss of surface expression of the activating receptor "natural killer group 2D" (NKG2D) on CD8⁺ T cells and NK cells. In vitro studies showed that supraphysiological supplementation of magnesium rescued these defects. Observational studies in 2 patients suggested oral magnesium supplementation could decrease EBV viremia. Hence, we performed a randomized, double-blind, placebo-controlled, crossover study in 2 parts. In part 1, patients received either oral magnesium L-threonate (MLT) or placebo for 12 weeks followed by 12 weeks of the other treatment. Part 2 began with 3 days of high-dose intravenous (IV) magnesium sulfate (MgSO₄) followed by open-label MLT for 24 weeks. One EBV-infected and 3 EBV-naïve patients completed part 1. One EBV-naïve patient was removed from part 2 of the study due to asymptomatic elevation of liver enzymes during IV MgSO₄. No change in EBV or NKG2D status was observed. In vitro magnesium supplementation experiments in cells from 14 XMEN patients failed to significantly rescue NKG2D expression and the clinical trial was stopped. Although small, this study indicates magnesium supplementation is unlikely to be an effective therapeutic option in XMEN disease.

Trial registration: ClinicalTrials.gov NCT02496676.

Keywords: Immunodeficiency; MAGT1; NKG2D; XMEN disease; congenital disorder of glycosylation; magnesium.

Fig. 1

Study design. A Diagram of the timeline for the XMEN clinical trial

Fig. 2

Screening, randomization, and follow-up. A Consort diagram of the XMEN clinical trial

Fig. 3

Primary Outcomes. A The percent and count of EBV⁺ B cells in XMEN01, as determined by EBV fluorescent in situ hybridization (FISH). B The EBV viral load by polymerase chain reaction (PCR) in XMEN01. Quantification of the flow cytometry mean fluorescence intensity (MFI) of NKG2D on CD8⁺ T cells (C) and NK cells (D) throughout part 1, the double-blinded, placebo-controlled crossover part of the clinical trial. NKG2D staining results from part 2, the open-label portion of the study is shown for CD8⁺ T cells (E) and NK cells (F). Measurements are shown during baseline (blue), on oral magnesium (green), on placebo (magenta), on IV magnesium (dark green), and post-study (gray). Healthy controls (HCs) are shown in black. In C–F, the dotted line shows the average MFI of the baseline for that part of the trial

Fig. 4

Limitations of supplementation. A Concentration of total blood, ionized blood, and urinary magnesium throughout the trial. Normal ranges for total and ionized blood magnesium are shown in gray. Measurements are shown during baseline (blue), on oral magnesium (green), on placebo (magenta), and on IV magnesium (dark green). Levels of alanine transaminase (ALT) (B) and aspartate transaminase (AST) (C) in the blood throughout the clinical trial. Normal ranges are shown in gray

Fig. 5

In vitro Mg²⁺ supplementation. Representative flow cytometric histogram of NKG2D staining on CD8⁺ T cells (A) and CD70 staining on pan T cells (B) from a healthy control (HC) and an XMEN patient. Cells were cultured in complete RPMI (cRPMI) supplemented with either 0 mM (black for HC and red for XMEN) or 5 mM Mg²⁺ (blue for HC and orange for XMEN). Isotype (NKG2D) or fluorescence minus one (CD70) is shown in gray. Quantification of 6 HCs and ≥ 14 XMEN is shown on the right. Error bars are mean ±S.D. Statistical analysis: Bayesian estimation supersedes the t-test (BEST) analysis. ***P(difference) ≥ 99.5%. ****P(difference) > 99.9%; n.s., not significant