Lethal Influenza in Two Related Adults with Inherited GATA2 Deficiency

Abstract

The pathogenesis of life-threatening influenza A virus (IAV) disease remains elusive, as infection is benign in most individuals. We studied two relatives who died from influenza. We Sanger sequenced GATA2 and evaluated the mutation by gene transfer, measured serum cytokine levels, and analyzed circulating T- and B-cells. Both patients (father and son, P1 and P2) died in 2011 of H1N1pdm IAV infection at the ages of 54 and 31 years, respectively. They had not suffered from severe or moderately severe infections in the last 17 (P1) and 15 years (P2). A daughter of P1 had died at 20 years from infectious complications. Low B-cell, NK- cell, and monocyte numbers and myelodysplastic syndrome led to sequence GATA2. Patients were heterozygous for a novel, hypomorphic, R396L mutation leading to haplo-insufficiency. B- and T-cell rearrangement in peripheral blood from P1 during the influenza episode showed expansion of one major clone. No T-cell receptor excision circles were detected in P1 and P3 since they were 35 and 18 years, respectively. Both patients presented an exuberant, interferon (IFN)-γ-mediated hypercytokinemia during H1N1pdm infection. No data about patients with viremia was available. Two previously reported adult GATA2-deficient patients died from severe H1N1 IAV infection; GATA2 deficiency may predispose to life-threatening influenza in adulthood. However, a role of other genetic variants involved in immune responses cannot be ruled out. Patients with GATA2 deficiency can reach young adulthood without severe infections, including influenza, despite long-lasting complete B-cell and natural killer (NK) cell deficiency, as well as profoundly diminished T-cell thymic output.

Keywords: GATA2; H1N1; Immunodeficiency; immunological memory; influenza A virus.

Fig. 1

Novel missense mutation in GATA2. a Pedigree of the family of the GATA2-deficient patients. Patients (P1, P2, and P3) and their relatives are indicated by a black square or circle. GATA2 genotypes at residue 396 (R396L, mutants; wt, wild-type; E?, unknown) are indicated. The index patient is indicated by an arrow. b Electropherograms showing a heterozygous G>T substitution at nucleotide 16913 (exon 7) of GATA2 in P1. c Alignment of the portion of the human GATA2 molecule containing residue 396 and the corresponding regions in other species. Residue 396 is indicated in gray and by a thick arrow. Other residues in this region, found to be mutated (T354M, N371K, R396Q, R396W, R398W, and R398Q) in previously reported patients with autosomal dominant GATA2 deficiency are indicated by a thin arrow

Fig. 2

Expression and transcriptional activity of the R396L allele in overexpression system. a Expression of GATA2 protein. HEK293T cells were transfected with an empty vector (EV), a construct encoding a GATA2 WT (wild type) DDK-tagged or mutated GATA2 variants (R396L, R396Q, R398W) for 48 h. Protein expression of GATA2 isoforms 1 (480 amino acids (aa)) and 2 (466 aa) was evaluated by western blotting with an anti-GATA2 antibody and an anti-DDK tag. GAPDH was included for normalization. NT corresponds to no transfected cells. The results shown are representative of three independent assays. b Functional characterization of the R396L allele. GATA2-mediated luciferase activity was measured in HEK293T cells. The GATA2-dependent transactivation potential was evaluated with GATA2 luciferase reporter vector. Cells were transiently transfected with (+) or without (−) promyelocytic leukemia protein (PML), empty vector (EV), and different GATA2-vectors (WT, R396L, R396Q, or R398W). The results shown are representative of three independent assays, each performed in triplicate

Fig. 3

Analysis of the impact of heterozygosity for the R396L allele in overexpression system. GATA2-mediated luciferase activity was measured in HEK293T cells. a Transient co-transfection of cells with promyelocytic leukemia protein (PML) (+) and different amounts of vectors (empty vector (EV), wild type (WT), R396L, EV/WT, EV/R396L, WT/R396L, EV/R396Q, WT/R396Q, EV/R398W, or WT/R398W). b Transient co-transfection of cells with PML and constant amount of the GATA2-WT vector with decreasing amounts of GATA2 mutant (R396L, R396Q, or R398W) vectors were used in the experiment. The results shown are representative of three independent assays, each performed in triplicate. Statistical analysis was performed using GraphPad Prism version 5.03 by means of an unpaired t test (Wilcoxon matched-pairs signed-rank test). Data were considered significant when p < 0.01

Fig. 4

Peripheral B lymphocytes and production of antibody against influenza A virus in patient 1 during H1N1pdm infection. a B-cell phenotype from P1, from one patient with severe acute respiratory failure due to H1N1pdm infection (S-H1N1) and from one healthy control (HC). Plasmacytoid B-cells are indicated by rectangles. Acquisition was stopped when 20,000 lymphocytes were gated based on CD45 expression and side scattering. b Measurement of neutralizing antibodies against H1N1pdm (thin line) and a seasonal IAV strain (dashed line) in P1. Influenza human antibody standard 09/194 to A/California/07/09-like viruses with a neutralization titer of 1/516 (The National Institute for Biological Standards and Control, UK) was used for titer normalization. Seroconversion is considered to occur when the neutralization titer increase is higher than fourfold. Microneutralization titers at day 4 were lower than 1/8 (the limit of detection of the analysis). c Spectratyping analysis of the B-cell receptor heavy chain (IGH) repertoire in P1 and in an adult healthy control (HC)

Fig. 5

Analysis of peripheral T lymphocytes. a Analysis of IFN-γ- and IL-17A-producing T-cells in response to polyclonal activation in P1, in patients suffering from severe acute respiratory failure due to H1N1pdm infection (S-H1N1pdm; N = 5) and in healthy controls (HC, N = 5). b Production of IFN-γ, IL-2, IL-4, and IL-17A by PBMC after polyclonal activation. The experiments were performed when the patient was attended at the intensive care unit during the severe H1N1pdm infection. c Spectratyping analysis of the T-cell receptor gamma gene (TCRG) repertoire. Analysis performed in DNA from whole blood from P1 and P3 at different ages and from one representative healthy control (HC)

Fig. 6

Serum cytokine and chemokine levels. Sera from P1 (filled square) and P2 (filled triangle) were obtained 6 and 4 days after admission to the intensive care unit, respectively. Samples from patients with severe acute respiratory failure due to H1N1pdm infection (S-H1N1pdm; N = 6) obtained 5.4 days (range 3–7) after hospital admission and seven healthy controls (HC) are also included. No differences were observed when serum levels of IL-1-β, IL-2, IL-4, IL-12p70, IL-17A, TNF-α, and CCL5 (RANTES) were compared