Research ArticleINTERFERON SIGNALING

Severe type I interferonopathy and unrestrained interferon signaling due to a homozygous germline mutation in STAT2

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Science Immunology  13 Dec 2019:
Vol. 4, Issue 42, eaav7501
DOI: 10.1126/sciimmunol.aav7501
  • Fig. 1 Neurological and systemic disease associated with excessive IFN activity.

    (A) Neuroimaging demonstrating calcifications [brainstem/hypothalamus (proband II:3, top), cerebral white matter/basal ganglia/midbrain/optic tract (sibling II:4, top and middle)], hemorrhages [occipital/subdural/subarachnoid (proband II:3, middle)], and cerebral white matter and cerebellar signal abnormality with parenchymal volume loss (both, bottom), accompanied by focal cystic change and cerebellar atrophy (sibling II:4). (B) Whole blood RNA-seq ISG profiles: controls (n = 5); proband II:3 (n = 4); and patients with mutations in: TREX1 (n = 6), RNASEH2A (n = 3), RNASEH2B (n = 7), RNASEH2C (n = 5), SAMHD1 (n = 5), ADAR1 (n = 4), IFIH1 (n = 2), ACP5 (n = 3), TMEM173 (n = 3), and DNASE2 (n = 3). (C) IFN scores (RT-PCR) of patients, parents, and n = 29 healthy controls. ****P < 0.001, ANOVA with Dunnett’s posttest. (D) Renal histopathology in proband (×400 magnification) showing TMA with extensive double contouring of capillary walls (silver stain, top left); endothelial swelling, mesangiolysis, and red cell fragmentation (top right); arteriolar fibrinoid necrosis (bottom left); and myxoid intimal thickening of an interlobular artery (bottom right, all hematoxylin and eosin). (E) Transcriptional response to JAK inhibitor (JAKi) ruxolitinib in both patients (RT-PCR).

  • Fig. 2 A homozygous missense variant in STAT2 consistent with autosomal recessive inheritance.

    (A) Pedigree, (B) capillary sequencing verification, (C) protein map, and (D) immunoblot (fibroblasts) showing normal expression of STAT2 protein. DBD, DNA binding domain; LD, linker domain; SH2, Src homology 2 domain; TAD, trans-activation domain.

  • Fig. 3 Heightened sensitivity to IFNα in cells bearing STAT2R148W.

    Unless stated, all data are from patient II:3 and control fibroblasts. (A) ISG expression (immunoblot, IFNα for 24 hours) and (B) densitometry analysis (n = 3, t test). MX1, MX dynamin like GTPase 1; IFIT1, IFN-induced protein with tetratricopeptide repeats 1; RSAD2, radical S-adenosyl methionine domain containing 2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) IFNB mRNA (RT-PCR) ± external polyinosinic:polycytidylic acid (poly I:C) treatment (25 μg/ml for 4 hours; n = 3, t test). US, unstimulated. (D) Antiviral protection assay (mCherry-PIV5). Twofold dilutions from IFNα (16 IU/ml), IFNγ (160 IU/ml) n = 7 replicates, representative of n = 2 experiments (two-way ANOVA with Sidak’s posttest). (E) Cytopathicity assay (IFNα for 72 hours; n = 3, t test). (F) As in (A), ISG expression in STAT2−/− U6A cells reconstituted with STAT2WT or STAT2R148W (immunoblot, IFNα for 24 hours). (G) As in (B), n = 3 to 4, t test. Data are presented as means ± SEM of repeat experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., nonsignificant.

  • Fig. 4 Prolonged STAT2 activation but no change to dephosphorylation rate.

    All data are from patient II:3 and control fibroblasts. (A) pSTAT2 time course [immunoblot, IFNα (1000 IU/ml)] and (B) densitometry analysis (n = 5 experiments, two-way ANOVA with Sidak’s posttest). (C) Immunofluorescence analysis [IFNα (1000 IU/ml); scale bar, 100 μm; representative of n = 3 experiments] with (D) image analysis of STAT2 nuclear translocation (n = 100 cells per condition, ANOVA with Sidak’s posttest). A.U., arbitrary units. (E) RNA-seq analysis of IFN-regulated genes (n = 3 controls) with (F) validation by RT-PCR (n = 3, two-way ANOVA with Sidak’s posttest). CPM, read counts per million. (G) pSTAT2 decay (immunoblot). IFNα (1000 IU/ml; 30 min) followed by extensive washing and treatment with 500 nM staurosporine (STAU). Times relative to STAU treatment. (H) No significant differences by densitometry analysis (n = 3, t test). Data are presented as means ± SEM of repeat experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 Prolonged proximal IFNAR signaling in STAT2R148W homozygosity but not heterozygosity.

    Time course of IFNα stimulation (1000 IU/ml) in EBV B cells from patient II:3 [homozygous (hom)], parent I:2 [heterozygous (het)], and n = 3 controls. (A) Immunoblot and (B) densitometry analyses. (C) Representative histograms (flow cytometry) and (D) mean fluorescence intensity (MFI). Data are means ± SEM of three repeat experiments (*P < 0.05, **P < 0.01, t test).

  • Fig. 6 STAT2R148W fails to support desensitization through its impaired interaction with USP18.

    (A) Desensitization assay (immunoblot, fibroblasts) with (B) pSTAT densitometry analysis (pSTAT/tubulin, ratio to unprimed; n = 4, ANOVA with Sidak’s posttest). (C) Schematic of USP18 mechanism of action and proposed model of STAT2R148W pathomechanism. (D) Modeling of exposed WT (R148)/mutant (W148) residue, demonstrating charge-change (blue, positive; red, negative) and possible steric restriction. (E) Coimmunoprecipitation of USP18 by STAT2 in U6A cells expressing STAT2WT or STAT2R148W with (F) densitometry analysis (USP18/STAT2, ratio to WT; one-sample t test). Data are means ± SEM (**P < 0.01, ****P < 0.0001). IB, immunoblot.

  • Fig. 7 USP18 insensitivity in cells bearing STAT2R148W.

    All data are from patient II:3 and control fibroblasts. (A) STAT phosphorylation in USP18 and vector expressing fibroblasts (immunoblot) with (B) pSTAT densitometry analysis (pSTAT/tubulin, ratio to unprimed; n = 3, ANOVA with Sidak’s posttest). (C) Immunofluorescence analysis of STAT2 nuclear translocation [IFNα (1000 IU/ml 30 min); representative of n = 3 experiments] with (D) image analysis (n = 100 cells per condition, ANOVA with Sidak’s posttest). (E) Time course of STAT phosphorylation upon IFNα stimulation (1000 IU/ml for 0, 1, 6, and 24 hours) of cells transduced with USP18 shRNA or nontargeting (NT) shRNA with (F) densitometry analysis of pSTAT2 (n = 3, t test). Data are means ± SEM (**P < 0.01, ***P < 0.001, ****P < 0.0001).

  • Table 1 Clinical phenotype of affected individuals.

    HLH, hemophagocytic lymphohistiocytosis; EEG, electroencephalogram.

    Clinical featureProband (II:3)Sibling (II:4)
    Premature birthYesYes
    Neonatal thrombocytopeniaYesNo
    Apnea requiring respiratory supportYesYes
    SeizuresYesAbnormal EEG
    Developmental delayYesYes
    White matter changesYesYes
    Intracranial calcificationYesYes
    Intracerebral hemorrhageYesYes
    Cerebellar hypoplasiaNoYes
    Sterile feversYesNo
    HLH-like inflammationYesNo
    HyperferritinemiaYesBorderline
    HypertensionYesBorderline
    Acute kidney injuryYesNo
    ProteinuriaYesYes
    TMAYesPossible
    Elevated D-dimersYesYes
    High ISG scoreYesYes
    Age at death20 months3 months

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/42/eaav7501/DC1

    Materials and Methods

    Supplementary case summary

    Fig. S1. Ingenuity pathway analysis of whole blood RNA-seq data.

    Fig. S2. Longitudinal series of laboratory parameters.

    Fig. S3. Multiple sequence alignment of STAT2.

    Fig. S4. Factor H genotyping and mutant factor H purification strategy.

    Fig. S5. Functional analysis of factor H Tyr779Cys variant.

    Fig. S6. Immunoblot analysis of MX1 expression in PBMCs.

    Fig. S7. Transduction of STAT2-deficient primary fibroblasts.

    Fig. S8. Prolonged STAT2 phosphorylation in PBMCs.

    Fig. S9. STAT2 immunofluorescence image analysis.

    Fig. S10. STAT phosphorylation is not prolonged in patient cells in response to IFNγ or IL-6.

    Fig. S11. RT-PCR analysis of gene expression in whole blood.

    Fig. S12. STAT2R148W does not impair regulation of STAT1 signaling.

    Fig. S13. Phosflow gating strategy.

    Table S1. Laboratory parameters, patients II:3 and II:4.

    Table S2. Rare variants segregating with disease.

    Table S3. Digital ELISA detection of IFNα protein concentration.

    Table S4. Phenotypes of monogenic defects of USP18 expression and/or function.

    Table S5. RT-PCR primers and probes.

    Table S6. Antibodies.

    Data file S1. Raw data (Excel).

    References (5159)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Supplementary case summary
    • Fig. S1. Ingenuity pathway analysis of whole blood RNA-seq data.
    • Fig. S2. Longitudinal series of laboratory parameters.
    • Fig. S3. Multiple sequence alignment of STAT2.
    • Fig. S4. Factor H genotyping and mutant factor H purification strategy.
    • Fig. S5. Functional analysis of factor H Tyr779Cys variant.
    • Fig. S6. Immunoblot analysis of MX1 expression in PBMCs.
    • Fig. S7. Transduction of STAT2-deficient primary fibroblasts.
    • Fig. S8. Prolonged STAT2 phosphorylation in PBMCs.
    • Fig. S9. STAT2 immunofluorescence image analysis.
    • Fig. S10. STAT phosphorylation is not prolonged in patient cells in response to IFNγ or IL-6.
    • Fig. S11. RT-PCR analysis of gene expression in whole blood.
    • Fig. S12. STAT2R148W does not impair regulation of STAT1 signaling.
    • Fig. S13. Phosflow gating strategy.
    • Table S1. Laboratory parameters, patients II:3 and II:4.
    • Table S2. Rare variants segregating with disease.
    • Table S3. Digital ELISA detection of IFNα protein concentration.
    • Table S4. Phenotypes of monogenic defects of USP18 expression and/or function.
    • Table S5. RT-PCR primers and probes.
    • Table S6. Antibodies.
    • References (5159)

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    Other Supplementary Material for this manuscript includes the following:

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