Severe Early-Onset Combined Immunodeficiency due to Heterozygous Gain-of-Function Mutations in STAT1

Abstract

Purpose: Loss and gain-of-function (GOF) mutations in human signal transducer and activator of transcription 1 (STAT1) lead to distinct phenotypes. Although recurrent infections are common to both types of STAT1 mutations, GOF mutations are distinguished by chronic mucocutaneous candidiasis and autoimmunity. However, the clinical spectra of STAT1 GOF mutations continue to expand. We here describe two patients with STAT1 GOF mutations presenting early in life with combined immunodeficiency (CID).

Methods: Clinical data and laboratory findings including immunophenotyping, level of interferon (IFN)-γ/IL-17(+) T cells, interferon-induced STAT1 phosphorylation, and JAK inhibitor assays were evaluated. Sequencing of STAT1 gene was performed by Sanger sequencer.

Results: Patient 1 (P1) had persistent oral candidiasis and cytomegalovirus (CMV) infection since 2 months of age and later developed cavitary lung lesions due to Mycobacterium tuberculosis. Patient 2 (P2) presented with oral candidiasis and recurrent pneumonia at 4 months of age and subsequently developed CMV pneumonitis. Both patients suffered heterozygous missense mutations in STAT1, leading to deleterious amino acid substitutions in the DNA binding domain (P1: c.1154C > T; p.T385M; P2. c.971G > T; p.C324F). Circulating CD4(+) T cells of both patients exhibited increased interferon-γ and decreased IL-17 expression as compared to controls. They also exhibited increased IFN-β and -γ-induced STAT1 phosphorylation that was reversed upon treatment with the JAK kinase inhibitor ruxolitinib.

Conclusion: STAT1 GOF mutations may present early in life with CID, consistent with the clinical heterogeneity of the disease. JAK kinase inhibitors may potentially be useful in some patients as adjunct therapy pending definitive treatment with bone marrow transplantation.

Keywords: STAT1; autoimmunity; combined immunodeficiency; gain-of-function mutation; mucocutaneous candidiasis; ruxolitinib.

Conflict of interest statement

Authors declare no conflict of interest.

Figures

Figure 1. Identification of de novo autosomal dominant STAT1 mutations in two patients with early onset CID

(A) Patient 1 (right site) and patient 2 (left site) families. The half-filled symbol indicates the heterozygous carrier; open symbols refer to wild type individuals. Males and females are distinguished by squares and circles, respectively. (B) Chest computed tomography of STAT1 GOF patients. Cavitary lung lesion (3.5 cm) in left lower lobe of patient 1 (Gray arrow). Bilaterally lobe infiltration, interstitial involvement and glossy patch appearances of patient 2 (Gray arrows).

Figure 2. Gain of function mutations in STAT1 (T385M, C324F) lead to STAT1 hyperphosphorylation

(A) Total p-STAT1 expression in CD4+ T cells stimulated with IFN-β (20 ng/mL) by flow cytometry in patients (P1, P2) and healthy control. (B) STAT1 expression in CD4+ T cells in patient P1 and control. (C) The dose response curve of STAT1 phosphorylation induced with IFN-β in patients and control CD4+ T cells. (D) Dephosphorylation kinetics of p-STAT1 in response to deprivation of IFN-β in CD4+ T cells represented as absolute MFI (D) and normalized to maximum expression prior to deprivation (E). **** p<0.0001 by two way ANOVA.

Figure 3. Exacerbated TH1 and impaired TH17 responses in STAT1 GOF mutations

(A) Flow cytometric analyses of IL-17 and IFN-γ secretion by peripheral CD4+ cells from controls and our patients. (B) The frequencies of IFN-γ and IL-17 producing CD4+ cells in three healthy controls and two patients. * p<0.05 and ** p<0.01 by unpaired two-tailed Student'st-test.

Figure 4. In vitro inhibition of STAT1 phosphorylation by Janus kinase inhibitor Ruxolitinib

(A) Expression of p-STAT1 in CD4+ T cells from healthy control and patient pretreated for 4 hours with 100 nM concentration of Ruxolitinib (black line) or vehicle (DMSO) then stimulated with IFN-β (20 ng/mL). The plain grays correspond to unstimulated cells. p-STAT1 mean fluorescence intensity (MFI) expressed in CD4+ T cells of healthy control and patient P1 as percent of maximum vehicle, Ruxolitinib 10 nM or 100 nM concentrations shown in (B). **** p<0.0001 two way ANOVA with posttest analysis.