Homozygous STAT2 gain-of-function mutation by loss of USP18 activity in a patient with type I interferonopathy

Abstract

Type I interferonopathies are monogenic disorders characterized by enhanced type I interferon (IFN-I) cytokine activity. Inherited USP18 and ISG15 deficiencies underlie type I interferonopathies by preventing the regulation of late responses to IFN-I. Specifically, USP18, being stabilized by ISG15, sterically hinders JAK1 from binding to the IFNAR2 subunit of the IFN-I receptor. We report an infant who died of autoinflammation due to a homozygous missense mutation (R148Q) in STAT2. The variant is a gain of function (GOF) for induction of the late, but not early, response to IFN-I. Surprisingly, the mutation does not enhance the intrinsic activity of the STAT2-containing transcriptional complex responsible for IFN-I-stimulated gene induction. Rather, the STAT2 R148Q variant is a GOF because it fails to appropriately traffic USP18 to IFNAR2, thereby preventing USP18 from negatively regulating responses to IFN-I. Homozygosity for STAT2 R148Q represents a novel molecular and clinical phenocopy of inherited USP18 deficiency, which, together with inherited ISG15 deficiency, defines a group of type I interferonopathies characterized by an impaired regulation of late cellular responses to IFN-I.

Graphical abstract

Figure 1.

Peripheral blood signature indicates autosomal recessive type I interferonopathy. (A) Clinical signs of immune pathology, clockwise from top: CT scan demonstrating calcifications of the frontal and parietal lobes, fistulizing adenitis of the axillary and inguinal nodes, and chest radiograph displaying bilateral opacities of the lungs. (B) mRNA expression of IFN-stimulated genes measured from whole-blood RNA isolated from healthy donors (HDs; n = 3), the patient’s mother, and the patient. Error bars represent one standard deviation. (C) Quantification of circulating IFN-α by digital ELISA (single-molecule array) in plasma from three healthy controls (HD) and the patient (P1). (D) t-Distributed stochastic neighbor embedding (tSNE) plots demonstrating the immunophenotype of PBMCs as determined by mass cytometry. (E) Quantification of immune cell populations of four healthy donors and the patient expressed as percent of total PBMCs, with age-matched healthy range in gray. (F) Histograms of CD169 expression, an IFN-stimulated gene, in classical monocytes and myeloid dendritic cells. (DC). Th, T helper cell. TEMRA, terminally differentiated effector memory cells re-expressing CD45RA.

Figure 2.

Homozygous mutation affecting the coiled-coil domain of STAT2 identified in a patient with lethal autoinflammation. (A) Family pedigree displaying affected individuals (shaded) and genotypes at position c.443 in STAT2. M indicates the STAT2 mutation and NA indicates individuals for whom the genotype was not available. (B) Crystal structure of murine STAT2 (blue) in complex with IRF9 (gray), demonstrating position of R148Q (red) in the coiled-coil domain, but outside of the STAT2/IRF9 interface. (C) Sanger sequencing of genomic DNA from the patient, his mother, and a healthy donor. (D) Schematic of STAT2 structure, indicating localization of the mutated residue and its conservation across mammals. CC, coiled-coil domain; DBD, DNA-binding domain; LD, linker domain; NTD, N-terminal domain; SH2, Src homology domains; TAD, transactivation domain.

Figure S1.

Genetic analysis of a patient with early-onset type I interferonopathy. (A) Schematic demonstrating variant analysis pipeline and results. (B) Prediction of functional impact of R148Q on STAT2 protein. CADD, combined annotation-dependent depletion. SIFT, sorting intolerant from tolerant.

Figure 3.

STAT2 R148Q leads to hyperinduction of ISGs, which can be rescued by WT STAT2. (A) STAT2 transcript levels in U6A cells (STAT2^−/−) reconstituted with an empty vector, WT STAT2 (WT), or R148Q STAT2 (RQ). Nonsignificant (n.s.) differences were determined by a P value > 0.05 from an unpaired two-tailed Student’s t test. (B) STAT2 protein levels by Western blot in HEK239T cells transfected with indicated plasmids. (C) Expression of ISGs in transduced U6A cells after 16 h of 100 IU/ml IFN-α stimulation. Statistical testing by Student’s t test. (D) Induction of ISGs in hTERT-immortalized fibroblasts. Statistical testing by Student’s t test. (E) Transfection of WT STAT2 (WT; teal), R148Q STAT2 (RQ; salmon), or both (purple) in U6A cells, followed by overnight stimulation with IFN-α. Statistical analysis performed by one-way ANOVA. All results are representative of at least three independent experiments each with biological triplicates. Error bars represent one standard deviation. P values for all statistical testing correspond to *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Figure S2.

STAT2 R148Q heightens target gene induction after stimulation with type I, but not type II, IFN. (A) Stimulation of transduced U6A cells with IFN-I for 4 h. (B) Induction of target genes by IFN-II stimulation (5 ng/ml) for 16 h in transduced U6A cells. (C) Induction of RSAD2 mRNA by IFN-α stimulation (100 IU/ml) for 16 h in transduced U6A cells. (D) Induction of IFI27 mRNA by IFN-α stimulation (100 IU/ml) for 16 h in hTERT-immortalized cells. All experiments were performed on three occasions with biological triplicates. Error bars represent one standard deviation. P values for all statistical testing correspond to **, P ≤ 0.01. n.s., not significant.

Figure 4.

Intact proximal, but not late, signaling with STAT2 R148Q owing to defective USP18-mediated negative regulation. (A) Analysis of STAT1 and STAT2 phosphorylation in dermal fibroblasts following 15-min stimulation with indicated doses (IU/ml) of IFN-α. Separate healthy donors (HD1 and HD2) and two biological replicates of patient fibroblasts were tested. (B) Immunofluorescence staining of STAT2 translocation in fibroblasts after 30-min stimulation with 1,000 IU/ml IFN-α. Scale bar represents 25 μm. (C) Nuclear translocation quantified in right panel. Statistical testing performed by one-way ANOVA, with n.s. indicating P values > 0.05. (D) Dephosphorylation of STAT1 and STAT2 in hTERT fibroblasts pulsed with IFN-α for 15 min, washed, and allowed to rest. (E) Transfection of indicated plasmids into HEK293T cells and subsequent immunoprecipitation (IP) of USP18-V5 to measure interaction with STAT2. (F) Recruitment of USP18 to the receptor in U6A cells transduced with negative control, WT STAT2, or mutant STAT2 and transfected with IFNAR2-Flag and USP18-V5. Immunoprecipitation performed against IFNAR2-Flag. (G) Band densitometry of relative USP18 quantities coimmunoprecipitated with IFNAR2 in three independent experiments. Statistical testing by repeated-measures ANOVA. (H) Analysis of negative regulatory capability by stimulating cells which had previously been stimulated (restim) or not (naive) with a primary stimulus of IFN-α for 12 h, washed, and allowed to rest for 6 h (rest) or 36 h before a restimulation with indicated doses of IFN-α. (I) IFN stimulation in U6A cells carrying WT STAT2 or R148Q STAT2 transduced with USP18 or without (empty). Stimulations were performed with indicated doses of IFN-α for 15 min. (J) Schematic demonstrating USP18 action in cells carrying WT STAT2 versus R148Q STAT2. All results are representative of three independent experiments. Error bars represent one standard deviation. P values for all statistical testing correspond to *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Figure S3.

STAT2 R148Q exhibits normal ISGF3 activity but fails to facilitate appropriate negative regulation. (A) Analysis of STAT1 and STAT2 phosphorylation in transduced U6A cells following 15-min stimulation with indicated doses (IU/ml) of IFN-α. (B) Flow cytometry for STAT1 phosphorylation in U6As with WT (HC) or R148Q (P) STAT2, stimulated (+) or not (−) with IFN-α for 15 min. (C) Immunofluorescence staining of STAT2 translocation in fibroblasts after 30-min and 12-h stimulation with 1,000 IU/ml IFN-α. Scale bar represents 25 μm. (D) Analysis of negative regulatory capability by stimulating cells that had previously been stimulated (restim) or not (naive) with a primary stimulus of IFN-α for 12 h, washed, and allowed to rest for 36 h before a restimulation with indicated doses of IFN-α. (E) IFN-α stimulation of healthy donor (HD), patient (Pat), or USP18-deficient fibroblasts that had previously been stimulated with IFN-α for 12 h and allowed to rest for 36 h. All results are representative of two or three independent experiments.