A novel kindred with inherited STAT2 deficiency and severe viral illness

Abstract

Autosomal recessive signal transducer and activator of transcription 2 (STAT2) deficiency was first reported in 2 siblings. One developed disseminated vaccine-strain measles following routine immunization with measles-mumps-rubella but recovered. The younger sibling died in infancy from a probable viral infection.1Hambleton S. Goodbourn S. Young D.F. Dickinson P. Mohamad S.M. Valappil M. et al.STAT2 deficiency and susceptibility to viral illness in humans.Proc Natl Acad Sci U S A. 2013; 110: 3053-3058Crossref PubMed Scopus (42) Google Scholar Pedigree analysis showed incomplete clinical penetrance for the severe viral infection phenotype in this kindred, giving rise to questions regarding the completeness of the STAT2 defect. Two other siblings with autosomal recessive STAT2 deficiency were also reported to suffer from febrile illness following measles-mumps-rubella immunization; one went on to develop opsoclonus-myoclonus syndrome and suffered from severe neurological impairment but was alive at reporting at 2.5 years.2Shahni R. Cale C.M. Anderson G. Osellame L.D. Hambleton S. Jacques T.S. et al.Signal transducer and activator of transcription 2 deficiency is a novel disorder of mitochondrial fission.Brain. 2015; 138: 2834-2846Crossref PubMed Scopus (12) Google Scholar His sibling was alive and well as reported at age 2 years, 3 months. STAT2 deficiency was complete and was associated with impaired type I interferon signaling. The 6 reported patients with autosomal recessive STAT1 deficiency suffer from a broader phenotype, including mycobacterial disease due to the additional disruption of type II interferon signaling via STAT1-STAT1 complexes (gamma-activated factor [GAF]).3Dupuis S. Jouanguy E. Al-Hajjar S. Fieschi C. Al-Mohsen I.Z. Al-Jumaah S. et al.Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency.Nat Genet. 2003; 33: 388-391Crossref PubMed Scopus (445) Google Scholar, 4Vairo D. Tassone L. Tabellini G. Tamassia N. Gasperini S. Bazzoni F. et al.Severe impairment of IFN-γ and IFN-α responses in cells of a patient with a novel STAT1 splicing mutation.Blood. 2011; 118: 1806-1817Crossref PubMed Scopus (73) Google Scholar Here we describe 2 siblings with compound heterozygous STAT2 mutations who suffered from severe viral illnesses since infancy. One succumbed at the age of 7 years from an infection with an unidentified agent, whereas the other is well at age 11 years. Detailed clinical history of the 2 described patients can be found in this article's Online Repository and in Table E1, Table E2, Table E3 at www.jacionline.org. Whole-exome sequencing was performed on patient 2 (P2), the parents, and the younger healthy sibling. After filtering out common polymorphisms, we found that P2 was compound heterozygous for c.C1528T and c.G1576A variants in STAT2 (Fig 1, A and B). PCR analysis showed the specific absence of STAT2 cDNA transcripts in primary fibroblasts from P2 (see Fig E1 in this article's Online Repository at www.jacionline.org). STAT1 and STAT2 expression and phosphorylation were examined in primary fibroblasts of P2, the parents, the younger healthy brother, and an unrelated control. Immunoblotting for STAT2 and Y-690-phosphorylated STAT2 after stimulation with IFN-α demonstrated absence of expression of STAT2 protein in P2 fibroblasts, as neither full length (Fig 1, C) nor truncated protein (data not shown) was detectable. By contrast, STAT1 expression and phosphorylation after stimulation with IFN-γ was normal (Fig 1, C). These results indicate that the c.C1528T variant (p.R510X) drives nonsense-mediated decay. The c.G1576A variant, by contrast, encodes a variant (p.G526A) at a splice junction. Transfection of a prespliced c.G1576A variant into a STAT2 knockout fibrosarcoma cell line (U6A) resulted in normal STAT2 expression and normal STAT2 phosphorylation after IFN-α stimulation (see Fig E2 in this article's Online Repository at www.jacionline.org), indicating a defect in splicing drove the null expression phenotype. We confirmed that the c.G1576A variant in STAT2 was responsible for aberrant splicing by introduction of exon 16 and its intron boundaries into an exon-trapping plasmid system (Fig E2, C). This is supported by the Human Splice Finder (Bioinformatics Team INSERM FO Desmet, D Hamroun, M Lalande, G Collod-Beroud, C Beroud; http://www.umd.be/HSF3) (score 81.98—new acceptor) and by Splice-site analyzer (48.01 dG 1.8) software (ibis.tau.ac.il/ssat/SpliceSiteFrame.htm). With undetectable protein expression of STAT2 in patient cells, function was compromised. IFN-α-induced IFN-stimulated gene factor 3 complex–mediated binding to IFN-α sequence response element was specifically abolished in P2, whereas IFN-γ and IFN-α induced gamma activating factor-mediated (gamma-activating sequences interaction were not affected; see Fig E3 in this article's Online Repository at www.jacionline.org). Moreover, interferon-inducible genes (MxA, ISG15, and OAS1) were not upregulated following IFN-α stimulation in the patient's primary fibroblasts (Fig 1, D; see Fig E4 in this article's Online Repository at www.jacionline.org). We next tested susceptibility to infection with Vesicular stomatitis virus in vitro by measuring cell survival in primary fibroblasts from P2, her healthy sibling, and parents. Pretreatment with exogenous IFN-α and IFN-γ protected the primary fibroblasts of healthy family members, whereas for P2, pretreatment with IFN-γ was protective but IFN-α had no effect (Fig 1, E). Finally, lentiviral transduction of wild-type STAT2 in primary fibroblasts of the patient restored STAT2 expression, IFN-α-induced STAT2 phosphorylation (Fig 1, F), and upregulation of interferon-inducible target genes (MxA, ISG15, and OAS1) (Fig 1, G-I). Together, these results demonstrate that c.C1528T/c.G1576A STAT2 mutation cripples STAT2 expression and function of the IFN-stimulated gene factor 3–dependent IFN-α pathway, within the limit of detection of these in vitro assays, whereas STAT1 function and the GAF-dependent IFN-γ pathway were intact. Febrile illness following measles-mumps-rubella vaccination has been documented in 6 of 6 immunized patients in the 3 unrelated kindreds (see Table E3 in this article's Online Repository), highlighting the importance of type I interferon signaling in the initial immune response against measles infections.5Buchanan R. Bonthius D.J. Measles virus and associated central nervous system sequelae.Semin Pediatr Neurol. 2012; 19: 107-114Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar Severe complications of natural measles infection include primary measles encephalitis (1-3 cases per 1,000 cases of measles infection) and subacute sclerosing panencephalitis (4-11 cases per 100,000). Whether these cases are driven by STAT2 deficiency remains to be elucidated. Hambleton et al1Hambleton S. Goodbourn S. Young D.F. Dickinson P. Mohamad S.M. Valappil M. et al.STAT2 deficiency and susceptibility to viral illness in humans.Proc Natl Acad Sci U S A. 2013; 110: 3053-3058Crossref PubMed Scopus (42) Google Scholar reported in the first described family that the majority of childhood viral illnesses were remarkably mild. Although some childhood viral illnesses such as respiratory syncytial virus bronchiolitis had a relatively uneventful course in P2, she did, however, suffer from severe recurrent varicella and enterovirus infections requiring multiple hospital admissions. She also suffered from severe course of primary EBV infection with delayed EBV suppression in peripheral blood and cerebrospinal fluid, in line with the important role of type I interferon signaling in the initial immune response against EBV.6Munz C. Dendritic cells during Epstein Barr virus infection.Front Microbiol. 2014; 5: 308PubMed Google Scholar Overall, STAT2 deficiency may display incomplete penetrance for several viral infections and even for complicated live measles vaccine as evidenced in Table E3. Similar diversity in presentation has been reported in other innate immune disorders such as TLR3 deficiency.7Zhang S.Y. Herman M. Ciancanelli M.J. Pérez de Diego R. Sancho-Shimizu V. Abel L. et al.TLR3 immunity to infection in mice and humans.Curr Opin Immunol. 2013; 25: 19-33Crossref PubMed Scopus (123) Google Scholar Impaired response to IFN-α and life-threatening viral diseases in STAT2-deficient patients are reminiscent of the phenotype of patients with complete autosomal recessive STAT1 deficiency, who additionally suffer from severe infections with weakly virulent Mycobacteria and other intracellular bacteria.3Dupuis S. Jouanguy E. Al-Hajjar S. Fieschi C. Al-Mohsen I.Z. Al-Jumaah S. et al.Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1 deficiency.Nat Genet. 2003; 33: 388-391Crossref PubMed Scopus (445) Google Scholar The STAT1-deficiency viral phenotype, however, seems more severe and broad than that of STAT2 deficiency, as out of the 6 identified patients, 4 died in early infancy due to herpes simplex virus-1, cytomegalovirus, or unknown viruses, and 2 received hematopoietic stem cell transplantation, from which 1 succumbed and the other was well 91 days after.4Vairo D. Tassone L. Tabellini G. Tamassia N. Gasperini S. Bazzoni F. et al.Severe impairment of IFN-γ and IFN-α responses in cells of a patient with a novel STAT1 splicing mutation.Blood. 2011; 118: 1806-1817Crossref PubMed Scopus (73) Google Scholar Similar, albeit less severe, presentation has been observed patients with autosomal recessive partial STAT1 deficiency.8Chapgier A. Kong X.F. Boisson-Dupuis S. Jouanguy E. Averbuch D. Feinberg J. et al.A partial form of recessive STAT1 deficiency in humans.J Clin Invest. 2009; 119: 1502-1514Crossref PubMed Scopus (146) Google Scholar Comprehensive analysis of STAT1-deficient cells strongly suggests that the corresponding patients' viral susceptibility is due to impaired IFN-α/β response, whereas their susceptibility to intracellular bacteria is due to defective IFN-γ signaling.9Boisson-Dupuis S. Kong X.F. Okada S. Cypowyj S. Puel A. Abel L. et al.Inborn errors of human STAT1: allelic heterogeneity governs the diversity of immunological and infectious phenotypes.Curr Opin Immunol. 2012; 24: 364-378Crossref PubMed Scopus (202) Google Scholar These effects are separated out in patients with autosomal dominant STAT1 deficiency, where there is no deficiency in IFN-stimulated gene factor 3–dependent signaling to IFN-α and no lethal viral disease. Here, mutant alleles were shown to be dominant for IFN-γ-GAF activity but recessive for IFN-α–IFN-α sequence response element activity,E1Chapgier A. Boisson-Dupuis S. Jouanguy E. Vogt G. Feinberg J. Prochnicka-Chalufour A. et al.Novel STAT1 alleles in otherwise healthy patients with mycobacterial disease.PLoS Genet. 2006; 2: e131Crossref PubMed Scopus (143) Google Scholar explaining the clinical divergence at the molecular level. The differential IFN-stimulated gene signature for type I and II interferons is also a likely culprit. Moreover, IFN-λ or type III interferon, which plays a crucial role at the mucosal surfaces of lung and intestine, has been shown to depend on the same signaling pathways as type I interferons do. Therefore the IFN-λ arm of the innate immune system is likely to be impaired in patients with STAT2 deficiency.E2Sommereyns C. Paul S. Staeheli P. Michiels T. IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo.PLoS Pathog. 2008; 4: e1000017Crossref PubMed Scopus (600) Google Scholar, E3Zhou Z. Hamming O.J. Ank N. Paludan S.R. Nielsen A.L. Hartmann R. Type III interferon (IFN) induces a type I IFN-like response in a restricted subset of cells through signaling pathways involving both the Jak-STAT pathway and the mitogen-activated protein kinases.J Virol. 2007; 81: 7749-7758Crossref PubMed Scopus (371) Google Scholar Finally, we observed a good response to high doses of intravenous immunoglobulin (IVIG) in P2 during infectious episodes. This response may in part be due to the anti-inflammatory effect of high doses of IVIG; however, it is probable that passive immunization may play a predominant role in controlling the ongoing viral infections. Therefore it could be argued that IgG replacement therapy might be beneficial for STAT2-deficient patients during childhood, until their adaptive immune system has sufficiently developed, as shown in patients with defects in the Toll-like receptor pathways.E4Casanova J.L. Abel L. Quintana-Murci L. Human TLRs and IL-1Rs in host defense: natural insights from evolutionary, epidemiological, and clinical genetics.Annu Rev Immunol. 2011; 29: 447-491Crossref PubMed Scopus (269) Google Scholar The history of P1 indeed demonstrates that patients may still be at risk for overwhelming viral illness beyond early childhood. Based on our limited experience, we recommend monthly IVIG substitution to prevent infections and high dose IVIG treatment in the course of severe (viral) infectious episodes with signs of emerging coagulopathy and/or immune dysregulation. The protective effect of in vitro infection with Vesicular stomatitis virus in P2's fibroblast culture by preincubation with IFN-γ invites speculation on the prophylactic or therapeutic use of IFN-γ. However, the long-term use of IFN-γ is not without side effects, and at the time of severe infection, its use may enhance macrophage activation. Therefore at present, IVIG seems to be the safer alternative. Our findings also add to the growing consensus that any fatal or life-threatening viral infection should not be seen simply as a case of “bad luck,” but should instead trigger comprehensive genetic analysis even in the absence of identifiable immunological (blood) anomalies.E5Casanova J.L. Severe infectious diseases of childhood as monogenic inborn errors of immunity.Proc Natl Acad Sci U S A. 2015; 112: E7128-E7137Crossref PubMed Scopus (146) Google Scholar This will allow for genetic counseling of the family, individualized directed treatment, and a better understanding of the immune system. We would like to thank Greet Wuyts, Doreen Dillaerts, and Irina Thiry for their technical assistance. We thank François Vermeulen for guiding the clinical care for these patients and Barbara Bosch for helping editing the text. We are very much indebted to the patient and her parents. Patient 1 (P1) was the second child of unrelated parents of Belgian European descent. The first 3 years of his life were characterized by frequent viral infections, each time necessitating admission to pediatric infectious diseases ward. At the time of adenovirus, enterovirus, and respiratory syncytial virus infection, he presented with high fever, malaise, pneumonitis, hepatitis, and pancytopenia and required in-patient supportive therapy. At the age of 24 months, he developed high fever 1 week after administration of the live measles-mumps-rubella vaccine, with a morbilliform rash, tonsillitis, conjunctivitis, lymphadenopathies, hepatosplenomegaly, and arthritis. Blood analysis showed pancytopenia and elevated liver enzymes suggestive of hepatitis. He recovered with supportive care. Basic immunological screening was normal. Physical and mental development was normal. From the age of 4 years, he had a relatively infection-free period. At the age 7 years, he succumbed to an infection with an unidentified agent. Clinically he suffered from a mild febrile illness, but after 48 hours, his condition suddenly deteriorated and he died of multiple organ failure in the context of disseminated intravascular coagulation. All cultures remained negative and no causal pathogen could be identified—viral pathogens were not sought. His younger sister P2 also suffered from frequent and severe viral infections in her first years of life, including severe Varicella zoster virus infection (6 months), Influenza A virus infection (age 7 months), rotavirus (age 8 months), recurrent Coxsackievirus, and adenovirus infections. Infections were accompanied by moderate thrombocytopenia, lymphopenia, neutropenia, monocytopenia, elevated levels of serum transaminases, and elevated percentage of T cells expressing HLA-DR (up to 50% of CD4(+) T cells) as well as hypergammaglobulinemia. Between infectious bouts, immunological screening could not demonstrate abnormalities in adaptive or innate immunity, with normal numbers of leucocyte subsets, normal lymphocyte proliferation tests, restoration of normal HLA-DR expression levels, normal immunoglobulin levels, and vaccine responses and normal activation of Toll-like receptor pathways (Table E1). Physical and mental development was normal. At age 18 months, she developed disseminated measles following routine immunization, complicated by hepatitis and pneumonitis. At this time, she received high-dose intravenous immunoglobulin (2 g/kg body weight) due to her poor condition and the emergence of coagulopathy, after which she recovered and became afebrile within 24 hours. At age 2.5 years, P2 suffered from severe EBV infection with high fever, malaise, tonsillitis, lymphadenopathy, and splenomegaly that again responded well to high-dose intravenous immunoglobulin treatment. There were no signs of macrophage activation. Over the next 3 years, repeated measurements by PCR showed persistent EBV presence in both blood and cerebrospinal fluid despite the presence of anti-EBV IgG (Table E2). At age 5 years she was admitted with a protracted course of Coxsackie B meningitis. From age 5 years until 11 years, she suffered from cutaneous plantar warts (not responding to acetyl salicylic acid and cryotherapy) and Molluscum contagiosum. From the age of 5 years, the frequency and severity of viral infections decreased. However, she suffered from recurrent chest infection requiring chronic antibiotic treatment with azithromycine, inhalation steroids, and daily chest physiotherapy. Computed tomography scan of the chest did not show irreversible lung damage. At age 11 years, P2 still experiences several mild viral infections per year, mostly upper respiratory infections. She has been off all medication since the age of 10 years. The study was performed in accordance with the modified version of the Helsinki declaration. The study was approved by the Ethics Committee of University Hospitals Leuven. Written informed consent was obtained prior to DNA isolation from blood of all family members and from bone marrow of the deceased patient. We performed whole-exome sequencing on the surviving patient, the unaffected parents, and the younger healthy sibling. Genomic DNA samples for whole-exome sequencing were prepared from heparinized peripheral blood using the QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany). Exome sequence libraries were prepared using a SeqCap EZ Human Exome Library v3.0 kit (Roche NimbleGen, Madison, Wis). Paired-end sequencing was performed on the Illumina HiSeq2000 (Genomics Core Facility, University of Leuven, Belgium). BWA software was used to align the sequence reads to the Human Reference Genome Build hg19 (Heng Li, http://bio-bwa.sourceforge.net). GATK Unified Genotyper (Broad Institute, https://software.broadinstitute.org/gatks/bestpractices) was used to identify single nucleotide variants and insertions/deletions. ANNOVAR (http://annovar.openbioinformatics.org) was used for annotation. A DNA sample of the deceased patient P1 was obtained from a bone marrow sample using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, Mo). The regions of interest in exon 17 of STAT2 were sequenced using the primers 5′-GTTCTGCCCTGTGGGACAGATAG-3′ and 5′-CTCAATTGCCTGGGCTTCAGTTC-3′. Sanger sequencing was performed on an ABI 3730 XL Genetic Analyzer (Applied Biosystems, Foster City, Calif) at the LGC Genomics Facility in Berlin, Germany. Sequencing data were analyzed using CLC Main Workbench 6.9.1 (CLC Bio, Aarhus, Denmark). No DNA sample was available from the older healthy brother. Total RNA was extracted with Trizol reagent (Life Technologies, Carlsbad, Calif) from primary fibroblasts and/or SV40 fibroblasts left unstimulated or stimulated with 103 IU/mL IFN-α (Thermo Fisher Scientific, Waltham, Mass) or IFN-γ (R&D Systems, Abingdon, United Kingdom) for 6 hours. mRNA was then reverse transcribed directly with Superscript Vilo cDNA synthesis kit (Life Technologies). STAT1, STAT2, and STAT3 transcripts were evaluated by PCR. STAT2 qPCR (Taqman gene expression array) was performed for distinct cDNA fragments: ex8-9: HS01013120-g1;ex16-17: HS01013132-m1; ex19-20: HS01013123-m1. For the determination of MxA (Hs00895608), ISG15 (Hs00192713), and OAS1 (Hs00973635) transcript levels, Taqman gene expression assays (Applied Biosystems) was used. The results were normalized to values from the endogenous GUSB (Hs00939627; Applied Biosystems). COS-7 cells (ATTC) were transfected with pSPL3 plasmids containing STAT2 exon16 (wild-type or G1576A mutation) with a part of the introns at 5′ and 3′ end. RT-PCR amplified products were analyzed by agarose electrophoresis and visualized with GelRed staining (Biotium, Inc). Dermal primary fibroblasts were obtained from skin biopsies of the patient, family members, and an unrelated control and cultured in Dulbecco modified Eagle medium with 10% FBS and supplemental penicillin and streptomycin. Primary fibroblast cultures were stimulated with IFN-α2 (104 U/mL) or IFN-γ (103 U/mL) for 30 minutes or left unstimulated. Cells were lysed in radioimmunoprecipitation assay buffer after stimulation, and immune blotting was performed using anti-STAT1 p84/p91 (M-22, sc-592; Santa Cruz Biotechnology, Dallas, Tex), anti-STAT2 (22/Stat2, 610187; BD Biosciences, San Jose, Calif), anti-phosphoSTAT1 (Tyr701, sc-7988; Santa Cruz Biotechnology), anti-phosphoSTAT2 (Tyr690 AF2890; R&D Systems, Minneapolis, Minn), and anti-β actin (A5441; Sigma-Aldrich) antibodies. All Western blot images were captured and quantified with a ChemiDoc MP imager and Image Lab software (Bio-Rad Laboratories, Hercules, Calif) after adding Pierce ECL Western blotting substrate (Thermo Fisher Scientific). SV40 fibroblasts and fibrosarcoma cell cultures were stimulated with IFN-α (105 U/mL) or IFN-γ (104 U/mL) for 30 minutes or left unstimulated. Nuclear extracts, electrophoretic mobility shift assay, and supershift assays were performed as previously described.E1Chapgier A. Boisson-Dupuis S. Jouanguy E. Vogt G. Feinberg J. Prochnicka-Chalufour A. et al.Novel STAT1 alleles in otherwise healthy patients with mycobacterial disease.PLoS Genet. 2006; 2: e131Crossref PubMed Scopus (143) Google Scholar Primary fibroblasts were seeded in 96-well plates at 2 × 104 cells per well in Dulbecco modified Eagle medium with 2.5% FBS and supplemental penicillin and streptomycin, and they were stimulated with IFN-α (103 U/mL) or IFN-γ (103 U/mL) for 24 hours or left unstimulated. Cells were infected with Vesicular stomatitis virus (strain Indiana, MOI 0.005). Percentage of cell survival was evaluated by measuring the mitochondrial dehydrogenase activity (fluorometric cell cytotoxicity assay kit ab112119; Abcam, Cambridge, United Kingdom) 3 days after viral infection. The codon optimized coding sequence for human STAT2 isoform 1 (NP_005410.1. or NM_005419.3) was ordered in 2 parts as gBlocks, with or without the p.R510X mutation engineered in. Sequences were cloned into the HIV-based lentiviral vector transfer plasmid, pCHMWS_eGFP_T2A_Fluc-IRES-Puro cl.3, in frame with the peptide2A sequence. HIV-based viral vectors were produced by triple transient transfection of 293T producer cells. A Vesicular Stomatitis Virus Glycoprotein G envelope encoding plasmid, a packaging plasmid together with the respective transfer plasmids were transfected using polyethylenimine (Polysciences, Amsterdam, The Netherlands). After collecting the supernatant, the medium was filtered using a 0.45-μm filter (Corning Inc, Seneffe, Belgium) and concentrated using a Vivaspin 50,000 MW column (Vivascience, Bornem, Belgium). The vector containing concentrate was aliquoted and stored at −80°C. HIV-based vectors were used to transduce primary fibroblasts, in a serial dilution series, and subjected to puromycin selection (0.5 μg/mL). Vector dilutions that resulted in <30% eGFP (ehanced green fluorescent protein) positive cells (equivalent to 1 integrated copy/cell) were subjected to puromycin from day 5 onward and selected until all cells were green fluorescent protein positive. STAT2 expression was corroborated by Western blot analysis.Fig E2STAT2 knockout fibrosarcoma (U6A) cells were transfected with pMCSV empty vector or pMCSV vector containing wild-type STAT2, c.G1576A, c.C1528T, or cotransfection of both STAT2 variants. Expression of STAT2 (A) and STAT1 (B), and phosphorylated STAT2 or STAT1 following stimulation with or without IFN-α or IFN-γ respectively (30 minutes, 103 IU/mL) in whole cell extracts. β-ACTIN expression was used as loading control. (C) Exon trapping analysis: agarose gel electrophoresis of RT-PCR after transfection of COS-7 cells with pSPL3 plasmids containing wild-type (WT) or G1576A mutant (MT) STAT2 exon 16 and intron boundaries in triple. MW, Molecular weight marker; NT, not transfected.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Electrophoretic mobility shift assay (A and B) and electrophoretic mobility shift assay supershift (C and D) of nuclear extracts of SV40 fibroblasts and fibrosarcoma cell lines. The cells were left unstimulated or were stimulated with the indicated doses of IFN-α or IFN-γ. The radiolabeled interferon senstive response element probe (A and C) or gamma-activating sequences (B and D) probe were used, respectively. C, Healthy control; F, father; M, mother; S, sibling; STAT1 def., STAT1-deficient SV40 fibroblast cell line; STAT2−/−, STAT2 knockout fibrosarcoma cell line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Relative expression of ISG15 (A) and OAS1 (B) in primary fibroblasts and STAT1-deficient cell SV40 fibroblast cell lines after stimulation with IFN-α or IFN-γ. C, Healthy control; F, father; M, mother; S, sibling; STAT1 def., STAT1-deficient SV40 fibroblast cell line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Lymphocyte subsets and immunoglobulin measurements in P2Leucocyte subset or immunoglobulin2 y5 y10 yNeutrophils, per μL6,400 (1,500-8,500)2,600 (1,500-8,500)4,700 (1,800-8,000)Lymphocytes, per μL4,338 (2,700-11,900)2,880 (1,700-6,900)2,490 (1,500-6,500)T cells, per μL2,239 (1,400-8,000)1,668 (900-4,500)1,444 (700-4,200)CD4+ T cells, per μL1,384 (900-5,500)895 (500-2,400)822 (500-2,400)CD8+ T cells, per μL738 (400-2,300)637 (300-1,600)511 (300-1,600)NK cells, per μL152 (100-1,400)681 (100-1,000)586 (100-1,000)B cells, per μL1,048 (600-3,100)506 (200-2,100)647 (200-2,000)Switched memory B cells, %NA13.413.5IgG (g/L)16.2 (3.02-9.85)11.7 (4.78-11.29)11.8 (5.30-13.06)IgA (g/L)0.78 (0.13-1.08)0.89 (0.35-1.90)1.06 (0.60-2.70)IgM (g/L)1.32 (0.26-1.60)0.54 (0.34-1.34)0.49 (0.43-1.73)IgE (kU/L)47 (<91)1,177 (<224)1,127 (<331)Measles AbProtectiveProtectiveNAPoliomyelitis AbProtectiveNANAHepatitis B AbProtectiveAbsentNANA, Not applicable; NK, natural killer cell. Open table in a new tab Table E2EBV measurement by PCR in P2Age (y)2.5344.55.510EBV, log copies/mL4.732.994.253.074.33 (blood)Undetectable2.71 (CSF)CSF, Cerebrospinal fluid. Open table in a new tab Table E3Overview of mutations, clinical characteristics, viral illnesses, and outcome of reported STAT2-deficient patientsPatientMutationPhenotypeVirusTreatmentOutcome at latest follow-up reported1E7Hambleton S. Goodbourn S. Young D.F. Dickinson P. Mohamad S.M. Valappil M. et al.STAT2 deficiency and susceptibility to viral illness in humans.Proc Natl Acad Sci U S A. 2013; 110: 3053-3058Crossref PubMed Scopus (164) Google Scholarc.381+5G>C18 mo: disseminated vaccine strain measlesBlood and BAL vaccine strain measles (+)6 y, A&Wc.381+5G>CHSV gingivostomatitis, Influenza A pneumonia2E7Hambleton S. Goodbourn S. Young D.F. Dickinson P. Mohamad S.M. Valappil M. et al.STAT2 deficiency and susceptibility to viral illness in humans.Proc Natl Acad Sci U S A. 2013; 110: 3053-3058Crossref PubMed Scopus (164) Google Scholarc.381+5G>C10 wk: febrile illnessUnknownDeceased at 10 wkc.381+5G>C3E7Hambleton S. Goodbourn S. Young D.F. Dickinson P. Mohamad S.M. Valappil M. et al.STAT2 deficiency and susceptibility to viral illness in humans.Proc Natl Acad Sci U S A. 2013; 110: 3053-3058Crossref PubMed Scopus (164) Google Scholarc.381+5G>C—NA—Adult, A&Wc.381+5G>C4E7Hambleton S. Goodbourn S. Young D.F. Dickinson P. Mohamad S.M. Valappil M. et al.STAT2 deficiency and susceptibility to viral illness in humans.Proc Natl Acad Sci U S A. 2013; 110: 3053-3058Crossref PubMed Scopus (164) Google Scholarc.381+5G>C3 mo: bronchiolitis post-MMR FSND—6 y, A&Wc.381+5G>C5E7Hambleton S. Goodbourn S. Young D.F. Dickinson P. Mohamad S.M. Valappil M. et al.STAT2 deficiency and susceptibility to viral illness in humans.Proc Natl Acad Sci U S A. 2013; 110: 3053-3058Crossref PubMed Scopus (164) Google Scholarc.381+5G>CAdmitted for febrile viral illnessNo MMR—4 y, A&Wc.381+5G>C6E8Shahni R. Cale C.M. Anderson G. Osellame L.D. Hambleton S. Jacques T.S. et al.Signal transducer and activator of transcription 2 deficiency is a novel disorder of mitochondrial fission.Brain. 2015; 138: 2834-2846Crossref PubMed Scopus (54) Google Scholarc.1836C>A12 mo: post-MMR FSCSF viral PCR (–)IVIG2.5 y: spasticity, chorea, cortical visual impairmentc.1836C>A13 mo: opsoclonus-myoclonusAB2.5 y: fever, diarrhea, metabolic acidosis, meningoencephalitis, thrombocytopeniaAED7E8Shahni R. Cale C.M. Anderson G. Osellame L.D. Hambleton S. Jacques T.S. et al.Signal transducer and activator of transcription 2 deficiency is a novel disorder of mitochondrial fission.Brain. 2015; 138: 2834-2846Crossref PubMed Scopus (54) Google Scholarc.1836C>A13 mo: post-MMR FSCSF PCR mumps (+)IVIG27 mo?: A&Wc.1836C>A15 mo: fever, malaise, septic shock, metabolic acidosisCSF PCR HSV, VZV enterovirus (–)8 (P1)c.C1528T0-3 y: severe febrile episodesAdenovirus, enterovirus, RSVDeceased at 7 yc.G1576A24 mo: post-MMR FS7 y: febrile illness9 (P2)c.C1528T<2 y: recurrent severe viral infectionVZV, Coxsackie virus, HPV, enterovirus, Influenza A, adenovirus, rotavirus, Molluscum contagiosum—11 y: A&Wc.G1576A18 mo: post-MMR FSEBV2.5 y: fever, lymphoproliferationCoxsackie B5 y: meningitisAB, Antibiotics; AED, antiepileptic drugs; A&W, alive and well; BAL, broncho-alveolar lavage; CSF, cerebrospinal fluid; FS, febrile syndrome; HPV, human papilloma virus; HSV, herpes simplex virus; IVIG, intravenous immunoglobulin substitution; MMR, measles-mumps-rubella vaccine; NA, not applicable; ND, not done; RSV, respiratory syncytial virus; VZV, Varicella zoster virus. Open table in a new tab NA, Not applicable; NK, natural killer cell. CSF, Cerebrospinal fluid. AB, Antibiotics; AED, antiepileptic drugs; A&W, alive and well; BAL, broncho-alveolar lavage; CSF, cerebrospinal fluid; FS, febrile syndrome; HPV, human papilloma virus; HSV, herpes simplex virus; IVIG, intravenous immunoglobulin substitution; MMR, measles-mumps-rubella vaccine; NA, not applicable; ND, not done; RSV, respiratory syncytial virus; VZV, Varicella zoster virus.