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Autosomal recessive (AR) STAT1 deficiency is a severe inborn error of immunity disrupting cellular responses to type I, II, and III IFNs, and IL-27, and conferring a predisposition to both viral and mycobacterial infections. We report the genetic, immunological, and clinical features of an international cohort of 32 patients from 20 kindreds: 24 patients with complete deficiency, and 8 patients with partial deficiency. Twenty-four patients suffered from mycobacterial disease (bacillus Calmette–Guérin = 13, environmental mycobacteria = 10, or both in 1 patient). Fifty-four severe viral episodes occurred in sixteen patients, mainly caused by Herpesviridae viruses. Attenuated live measles, mumps, and rubella and/or varicella zoster virus vaccines triggered severe reactions in the five patients with complete deficiency who were vaccinated. Seven patients developed features of hemophagocytic syndrome. Twenty-one patients died, and death was almost twice as likely in patients with complete STAT1 deficiency than in those with partial STAT1 deficiency. All but one of the eight survivors with AR complete deficiency underwent hematopoietic stem cell transplantation. Overall survival after hematopoietic stem cell transplantation was 64%. A diagnosis of AR STAT1 deficiency should be considered in children with mycobacterial and/or viral infectious diseases. It is important to distinguish between complete and partial forms of AR STAT1 deficiency, as their clinical outcome and management differ significantly.

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The transcription factor STAT1 governs cellular responses to type I, II, and III IFNs and IL-27. Autosomal recessive (AR) STAT1 deficiency (Online Mendelian Inheritance in Man accession number 613796) is an inborn error of immunity (IEI) responsible for both viral and intramacrophagic bacterial diseases, including mycobacterial diseases in particular. AR complete STAT1 deficiency due to biallelic STAT1 null mutations was first described in 2003 (1). Its clinical infectious phenotype is probably as severe as that of SCID (16). It may be considered a severe innate immunodeficiency, but with the caveat that the defect is not restricted to innate leukocytes, instead also affecting adaptive leukocytes and even cell types other than leukocytes, as the receptors for type I and III IFNs are ubiquitously and broadly expressed, respectively, and control a major arm of nonleukocytic cell–intrinsic immunity. Loss-of-function (LOF) STAT1 alleles result in a complete absence of STAT1 expression and function, in terms of the responses to type I, II, and III IFNs and IL-27 (1, 46). Seven unrelated patients with AR complete STAT1 deficiency have been described to date. All suffered from disseminated mycobacterial infection and/or life-threatening viral infections within the first few months of life. The prognosis of this condition is clearly poor, as four of these patients died before the age of 2 y (1, 4, 7). Hematopoietic stem cell transplantation (HSCT) was performed in five patients, including the three survivors, all of whom were still well, up to 9 y after HSCT, at their last reported follow-up visit (37). Some patients display AR partial STAT1 deficiency because they carry one (n = 1) or two (n = 4) hypomorphic mutant alleles (810). STAT1-dependent cellular responses to type I, II, and III IFNs and IL-27, which are mediated by both IFN-γ activated factor (GAF) and IFN-stimulated gene factor 3 (ISGF3), were impaired but not abolished in these five patients (810). The genetic, immunological, and clinical features of patients with AR STAT1 deficiency, whether complete or partial, remain largely unknown. We report, in this study, the molecular, cellular, and clinical features of 32 patients with AR STAT1 deficiency.

The study was conducted in accordance with the Helsinki Declaration, with informed consent obtained from the parents of patients, who were followed up in their home country in accordance with local regulations. All experiments were performed at the French INSERM, with the approval of the appropriate institutional review board. A detailed questionnaire was completed by the physicians caring for the patients, including demographic data, clinical features, and biological and microbiological results, and the data were sent to T.L.V. and J.B. Patients were included if they received a genetic diagnosis of AR STAT1 deficiency or if they displayed consistent clinical manifestations in childhood (bacillus Calmette–Guérin [BCG] infection or life-threatening viral infection) and their sibling(s) received a genetic diagnosis. The following laboratory data were collected: hemogram; serum Ig (IgM, IgG, IgA, and IgE) levels; T, B, and NK cell counts; T cell proliferation assays; neutrophil respiratory burst (dihydrorhodamine and/or NBT assays); viral serological tests; and biological parameters for hemophagocytic lymphohistiocytosis (HLH) (fibrinogen, triglycerides, ferritin and soluble CD25 levels, and NK cell degranulation). Disseminated BCG infection (BCG-osis) was diagnosed according to the European Society for Immunodeficiencies diagnostic criteria (https://esid.org/content/download/12914/369208/file/BCG.doc). Environmental mycobacteria (EM) infection was diagnosed according to the American Thoracic Society/Infectious Diseases Society of America criteria (11). Patients were considered to have a probable EM infection if they had at least one sign or symptom suggestive of EM infection, with consistent radiological imaging results and a favorable clinical response to classic antimycobacterial treatment. The criteria for severe viral infection were a need for hospitalization and/or transfer to an intensive care unit. HLH was diagnosed if at least five of the eight diagnostic criteria were fulfilled, in accordance with the HLH-2004 trial (12).

Genetic diagnosis was performed by Sanger sequencing, targeted next-generation sequencing, or whole-exome sequencing (WES). Genomic DNA was extracted from PBMCs after Ficoll-Paque Plus gradient purification or from whole blood. Patients P1, P2, P5, P6, P7, P22, P23, and P25–P29 were previously reported and diagnosed by Sanger sequencing or WES, respectively (1, 3, 4, 610, 13). Patients P3, P4, P8, and P15 were diagnosed by Sanger sequencing. Patients P20, P21, P24, P31, and P32 were diagnosed by targeted next-generation sequencing with the primary immunodeficiencies panel, as described elsewhere for P31 (14). Patients P11, P14, P16, P17, P18, and P19 were diagnosed by WES. For P9, P10, P12, P13, and P30, no genomic DNA was available. All exons of STAT1 and their flanking intronic regions were amplified by PCR, purified by ultracentrifugation through Sephadex G-50 Superfine resin (Amersham Pharmacia Biotech) and sequenced with the Big Dye Terminator 3.1 Cycle Sequencing Kit on an ABI Prism 3700 apparatus (Applied Biosystems). DNA was sheared with an S2 Ultrasonicator (Covaris). Briefly, an adapter-ligated library was prepared with the TruSeq DNA Sample Prep Kit (Illumina). Exome capture was performed with the SureSelect Human All Exon 50 Mb kit (Agilent Technologies). The potentially damaging impact of variants was assessed with in silico algorithms: combined annotation-dependent depletion (CADD; http://cadd.gs.washington.edu/score) and mutation significance cutoff (MSC; http://pec630.rockefeller.edu:8080/MSC/. MSC scores were generated with a 99% confidence interval based on CADD v1.3 scores (15). The likelihood-based ESTIAGE method was used to estimate the age of the most recent common ancestor (MRCA) of the mutation from the observed shared haplotypes (16). Recombination rates and haplotype frequencies were provided by the HapMap Project. All mutations found in STAT1 were confirmed by Sanger sequencing on genomic DNA. Familial segregation was made when or if DNA from relatives was available.

Total protein was extracted from EBV–B cells and SV40 fibroblasts, with or without stimulation with IFN-γ (1 × 105 IU/ml; Imukin), IFN-α2b (1 × 105 IU/ml; IntronA), or IL-27 (100 ng/ml; R&D Systems) for 30 min in lysis buffer supplemented with protease inhibitors. Protein fractions were separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were blocked by incubation in TBS supplemented with 0.1% Tween 20 and 5% skimmed milk powder for 60 min at room temperature and incubated with Abs against Tyr701–p-STAT1 (612132; BD Biosciences), the N-terminal (aa 69–169; 610116; BD Biosciences), or C-terminal part (data not shown) of STAT1α (STAT1). For overexpression experiments, U3C cells transiently expressing wild-type (WT) or mutant STAT1 were used, and immunostaining was performed as previously described (17).

Briefly, EBV–B cells were stimulated for 30 min with IFN-γ (1 × 105 IU/ml; Imukin), IFN-α2b (1 × 105 IU/ml; IntronA), or IL-27 (100 ng/ml; R&D Systems). Nuclear extract (10 µg protein) was incubated with P32-labeled αdATP IFN-γ activation sequence (GAS) (from FCGR1 promoter) or IFN-sensitive response elements (ISRE) (from ISG15 promoter) probes, and the products were subjected to electrophoresis in a polyacrylamide gel. The unlabeled probe was incubated with the cells for 30 min at 4°C before addition of the radiolabeled probe.

We performed quantitative real-time PCR (RT-qPCR) on EBV–B cells and SV40 fibroblasts with Applied Biosystems TaqMan RT-PCR assays to determine the levels of mRNA for STAT1, CXCL10, CXCL9, IFIT1, ISG15, and MXI using the TaqMan probes delivered by Applied Biosystems for these genes in an ABI PRISM 7700 Sequence Detection System. Activation was performed by incubation with 1 × 105 IU/ml IFN-α2b, 1 × 105 IU/ml IFN-γ, 100 ng/ml IL-27, or 20 ng/ml IFN-λ for 2 h. Relative levels of expression for these genes were determined with the equation 2−ΔΔCt, and the results obtained were normalized relative to those for transcripts of the housekeeping gene encoding β-glucuronidase (GUSB).

For Ab profiling by phage immunoprecipitation sequencing (PhIP-Seq), plasma samples were obtained from eight patients (P5, P15, P18, P19, P25–P27, and P32). For comparison, and as additional controls, we also tested 10% liquid IVIG from pooled human plasma (Privigen CSL Behring) and human IgG-depleted serum (supplier no HPLASERGFA5ML; Molecular Innovations) (18). The total IgG levels in the plasma samples were determined with a Human IgG total ELISA Ready-SET-Go kit (Thermo Fisher Scientific). Diluted plasma samples containing ∼4 μg of total IgG were incubated at 4°C overnight with 2 × 1010 PFUs of a modified version of the original PhIP-Seq phage library. This modified T7 phage library was used to display a total of 115,753 peptides, each 56 aa long, including the same viral peptides as the original PhIP-Seq phage library and additional peptides derived from the protein sequences of various microbial B cell Ags and allergens made available from the Immune Epitope Database and Analysis Resource (http://www.iedb.org). Subsequent steps were performed as previously described (18).

Venous blood samples from healthy controls and patients were collected into heparin-containing collection tubes. These samples were diluted 1:2 in RPMI 1640 (Gibco) supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco). We then dispensed 1 ml of each diluted blood sample into each of five wells (1 ml/well) of a 48-well plate (Nunc). These samples were incubated for 48 h at 37°C, under an atmosphere containing 5% CO2/95% air, and under different sets of activation: with medium alone, with live BCG (Mycobacterium bovis–BCG, Pasteur substrain) at a multiplicity of infection of 20 BCG cells per leukocyte or with BCG plus recombinant human IL-12 (20 ng/ml; R&D Systems) or BCG plus IFN-γ (Imukin). The supernatants were then collected 48 h later and subjected to ELISA with the human IFN-γ (Sanquin), IL-12p40 (R&D Systems), and IL-12p70 (R&D Systems) ELISA kits, according with the manufacturer’s instructions (1820).

Total RNA was extracted from EBV–B cells or SV40 fibroblasts with the RNeasy Mini Kit (QIAGEN), according to the manufacturer’s instructions. Reverse transcription was performed directly on 2 μg of RNA, with random primers and the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The STAT1 cDNA was amplified by PCR with the AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA) and specific primers flanking exons 2–9, 9–19, and 19–25, amplifying the full-length STAT1A and STAT1B sequences (3′ to 5′ untranslated region). The primers and conditions of PCR are available upon request. Sequencing products were analyzed on an ABI Prism 3700 apparatus (Applied Biosystems). Sequence files and chromatograms were analyzed with Genalys 2.8 software (Centre National de Génotypage, Evry, France). The RT-PCR product containing the STAT1 cDNA amplified with specific primers flanking exons 2–9 was cloned with the TOPO TA cloning 2.1 kit (Invitrogen), according to the manufacturer’s protocol, and used to transform bacteria.

For overexpression experiments, U3C null fibrosarcoma cells transiently expressed WT or mutant STAT1 alleles. A luciferase reporter assay was performed to assess the STAT1 mutations as previously described. The plasmids containing WT and/or mutant STAT1 genes, together with IRF1 reporter plasmids harboring five tandem GAS elements derived from IRF1 (TTCCCCGAA) and the pRL-SV40 plasmid, were used to transfect U3C cells with Lipofectamine LTX Reagent (Thermo Fisher Scientific, Waltham, MA). At 24 h after transfection, the cells were treated with IFN-γ (2, 10, or 100 IU/ml) for 16 h and subjected to luciferase reporter assays, performed with the Dual-Glo Luciferase Assay System (Promega) in accordance with the manufacturer’s protocol. The experiments were performed in triplicate, and the data are expressed in relative luciferase units.

SV40 fibroblasts were cultured in DMEM (Life Technologies) supplemented with 10% FBS. EBV–B cells or SV40 fibroblasts were washed in PBS and dispensed into a 96-well plate for labeling. The Aqua Live/Dead Fixable Dead Cell Stain kit from Invitrogen was used to exclude dead cells. The live cells were then left unstimulated or were stimulated by incubation for 20 min with IFN-γ (1 × 105 IU/ml; Imukin), IFN-α2b (1 × 105 IU/ml; IntronA), or IL-27 (100 ng/ml; R&D Systems). Intracellular staining was performed by incubation with the Cytofix/Cytoperm Plus Fixation/Permeabilization kit (BD Biosciences), with anti-STAT1–PE Abs (BD Biosciences), anti-Y701–pSTAT1–Alexa 468 Abs (BD Biosciences), or an equivalent concentration of isotype-matched control mAb (Becton Dickinson) in 2% FBS in PBS, for 1 h at 4°C. Gating was set on singlets and living cells in EBV–B cells and SV40 fibroblasts. Compensation was performed on single-stained nonstimulated samples. At least 5,000 cells for each condition and each sample were acquired on a Gallios cytometer (Beckman Coulter), and the results were analyzed with FlowJo v10 (Tree Star).

Statistical analysis was performed with GraphPad Prism, version 7.02 (GraphPad Software). Survival curves were plotted by the Kaplan–Meier method, and when necessary, curves were compared in log-rank tests. A p value <0.05 was considered statistically significant.

We studied a total of 20 unrelated index cases with mycobacterial and/or viral diseases originating from 13 countries, 10 previously described, and 10 new cases (Fig. 1A, Table I). Nine index cases were from the Middle East (Saudi Arabia, n = 6; Israel [of Arab origin], n = 1; Palestine, n = 1 [living in Germany]; Arab United Emirates, n = 1). The remaining index cases were from Pakistan (n = 3, living in Pakistan, Italy, and Spain, respectively) and from India (State of Maharashtra), Australia, Russia (Republic of Dagestan), Denmark, Japan, the United States, Turkey, and Guinea (living in Germany), with one patient from each from these countries (Table I). In total, 22 different mutations were identified in the 20 index cases and 10 new mutations. The 16 homozygous mutations were missense (n = 6), small frameshift deletions (n = 4), intronic essential splicing site (n = 2), nonsense (n = 2), a small frameshift insertion, and a small duplication, with compound heterozygous variants in three kindreds (Fig. 1A, 1B, Table I). CADD scores predicted all variants to be deleterious and were well above the MSC score for STAT1 (15) (Table I). The homozygous p.L407R and p.I648T variants affect residues conserved throughout evolution (Supplemental Fig. 1A). Familial segregation identified a total of 27 patients from 20 kindreds and was consistent with an AR trait (Fig. 1A, Supplemental Fig. 1). A founder effect in two kindreds from Saudi Arabia (recurrent c.1757_1758delAG variant) was found and estimated the MRCA to have occurred 37 generations ago (95% confidence interval [12–135]). Assuming a generation time of 25 y, the MRCA of the patients therefore lived 925 y ago (95% confidence interval [300–3375]). In addition to the 27 genetically diagnosed patients with AR STAT1 deficiency, five siblings of three index cases (from kindreds F, G, and S) were suspected to have AR STAT1 deficiency because they died during the first year of life from suspected BCG-osis or recurrent severe viral infections, and genetic analysis was therefore not possible for these individuals (Fig. 1A). Overall, these findings strongly suggest that 32 individuals suffered from AR STAT1 deficiency; 24 of these individuals were from 15 kindreds with complete deficiency, and eight were from five kindreds with partial deficiency.

FIGURE 1.

Autosomal recessive STAT1 deficiency. (A) Pedigrees of 32 patients from 20 kindreds with AR STAT1 deficiency. Each kindred is designated by a letter (A–T), each generation is designated by a Roman numeral (I–II), and each individual is designated by an Arabic numeral (1–7). The double lines connecting the parents indicate consanguinity. The probands are indicated by an arrow. Symbols in black indicate mycobacterial infection and/or severe viral infection. Mutated STAT1 alleles conferring AR complete deficiency are indicated in red, and those conferring AR partial deficiency are shown in blue. Individuals whose genetic status could not be evaluated are indicated by “E?” (B) Schematic representation of the coding region of the STAT1A gene containing 23 coding exons and encoding a 750-aa protein. The exons are numbered with Roman numerals (III–XXV). The protein domains are shown in yellow with N-terminal domain (N-ter), coiled-coil domain (CCD), DNA-binding domain (DBD), linker domain (LD), SH2 domain, tail segment domain (TS), and transcriptional activation domain (TA); mutations conferring AD partial deficiency are shown in green, AR partial deficiency in blue, and AR complete deficiency in red.

FIGURE 1.

Autosomal recessive STAT1 deficiency. (A) Pedigrees of 32 patients from 20 kindreds with AR STAT1 deficiency. Each kindred is designated by a letter (A–T), each generation is designated by a Roman numeral (I–II), and each individual is designated by an Arabic numeral (1–7). The double lines connecting the parents indicate consanguinity. The probands are indicated by an arrow. Symbols in black indicate mycobacterial infection and/or severe viral infection. Mutated STAT1 alleles conferring AR complete deficiency are indicated in red, and those conferring AR partial deficiency are shown in blue. Individuals whose genetic status could not be evaluated are indicated by “E?” (B) Schematic representation of the coding region of the STAT1A gene containing 23 coding exons and encoding a 750-aa protein. The exons are numbered with Roman numerals (III–XXV). The protein domains are shown in yellow with N-terminal domain (N-ter), coiled-coil domain (CCD), DNA-binding domain (DBD), linker domain (LD), SH2 domain, tail segment domain (TS), and transcriptional activation domain (TA); mutations conferring AD partial deficiency are shown in green, AR partial deficiency in blue, and AR complete deficiency in red.

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Table I.

Genetic, demographic, and infectious phenotypes of the international cohort of patients with AR STAT1 deficiency

Patient [Kindred] (References)MutationCADD ScoreaOriginSexAge at Onset (mo)Follow- upMycobacterial Disease (BCG, EM)bViral InfectionsbOther Bacterial InfectionsbFungal Infectionb
P1 [A] (1c.1757_1758delAG/1757_1758delAG 35 Saudi Arabia Dead M. bovis-BCG HSV-1 — — 
P2 [B] (1p.L600P/L600P 34 Saudi Arabia Dead M. bovis-BCG NDf — — 
P3 [B] p.L600P/L600P 34 Saudi Arabia Dead M. bovis-BCG NDf — — 
P4 [B] p.L600P/L600P 34 Saudi Arabia Dead M. bovis-BCG — — — 
P5 [C] (4c.1928insA/1928insA 35 Pakistan Dead M. bovis-BCG Parainfluenza virus — — 
P6 [D] (5, 6p.Q124H/Q124H 28.5 Pakistan 10 Alive M. kansasii HSV-1, CMV, EBV, enterovirus — Aspergillus fumigatusd 
P7 [E] (3c.88delA/88delA 28.3 Australia Alive – HHV-6 — — 
P8[E] c.88delA/88delA 28.3 Australia e Alive – — — — 
P9 [F] Not genotyped — Saudi Arabia — Dead M. bovis-BCG — — — 
P10 [F] Not genotyped — Saudi Arabia — Dead – — — — 
P11 [F] c.1757_1758delAG/1757_1758delAG 35 Saudi Arabia Dead M. abscessus NDf Corynebacterium spp., α-haemolyticus streptococcus NDg 
P12 [G] Not genotyped — Russia Dead – — — — 
P13 [G] Not genotyped — Russia Dead – — — — 
P14 [G] c.541 + 1G>A/541 + 1G>A 29.4 Russia Dead M. bovis-BCG — B. circulans, P. aeruginosa, S. aureus C. parapsilosis 
P15 [G] c.541 + 1G>A/541 + 1G>A 29.4 Russia Alive – CMV — C. parapsilosis 
P16 [H] c.541 + 2dup/541 + 2dup 26.6 Turkey Dead M. bovis-BCG — — — 
P17 [I] c.769dup/769dup 35 India Dead M. bovis-BCG — — — 
P18 [I] c.769dup/769dup 35 India 2.5 Dead M. bovis-BCG — — — 
P19 [J] p.S62*/S62* 35 Saudi Arabia Alive M. scrofulaceum CMV Influenza B, rhinovirus, coronavirus 229E, bocavirus, norovirus, metapneumovirus K. pneumoniae C. tropicalis 
P20 [K] p.Q9*/E625* 36/42 USA Dead M. kansasii HHV-6, VZV,c RSV, rhinovirus, enterovirus, influenza A — C. parapsilosis, C. albicans, Cryptococcus neoformans 
P21 [L] c.693_696del/693_696del 35 United Arab Emirates 0.1 Dead Suspected EM VZV,c RSV — — 
P22 [M] (13c.542-8G>A/128 + 2T>G 2.3/25.3 Japan Alive M. bovis-BCG M. malmoense VZV,c RSV, metapneumovirus, influenza A, rotavirus Mycoplasma pneumoniae — 
P23 [N] (7c.1011_1012delAG/1011_1012delAG 35 Guinea Dead – VZV,c RSV, rhinovirus, enterovirus, parainfluenza virus Rothia dentocariosa, Streptococcus viridans, K. pneumoniae — 
P24 [O] p.E618*/E618* 47 Palestine Alive – CMV, HSV-1 S. aureus — 
P25 [P] (8p.P696S/P696S 15.64 Israel 24 Alive M. szulgai HSV-1, CMV, adenovirus, molluscum contagiosum Salmonella spp. — 
P26 (P] (8p.P696S/P696S 15.64 Israel Alive Suspected EM HSV-1, VZV, CMV Salmonella spp., E. coli, K. pneumoniae — 
P27 [Q] (10p.K201N/K201N 22.7 Saudi Arabia 72 Alive M. avium VZV — C. parapsilosis 
P28 [Q] (10p.K201N/K201N 22.7 Saudi Arabia Dead M. bovis-BCG — — — 
P29 [R] (9p.A46T/K211R 33/17.68 Denmark 174 Alive M. avium — — — 
P30 [S] Not genotyped — Saudi Arabia Dead M. bovis-BCG — — — 
P31 [S] p.L407R/L407R 28.8 Saudi Arabia Dead M. bovis-BCG Rhinovirus — C. parapsilosis, C. albicans 
P32 [T] p.I648T/I648T 31 Pakistan Alive M. avium CMV — — 
Patient [Kindred] (References)MutationCADD ScoreaOriginSexAge at Onset (mo)Follow- upMycobacterial Disease (BCG, EM)bViral InfectionsbOther Bacterial InfectionsbFungal Infectionb
P1 [A] (1c.1757_1758delAG/1757_1758delAG 35 Saudi Arabia Dead M. bovis-BCG HSV-1 — — 
P2 [B] (1p.L600P/L600P 34 Saudi Arabia Dead M. bovis-BCG NDf — — 
P3 [B] p.L600P/L600P 34 Saudi Arabia Dead M. bovis-BCG NDf — — 
P4 [B] p.L600P/L600P 34 Saudi Arabia Dead M. bovis-BCG — — — 
P5 [C] (4c.1928insA/1928insA 35 Pakistan Dead M. bovis-BCG Parainfluenza virus — — 
P6 [D] (5, 6p.Q124H/Q124H 28.5 Pakistan 10 Alive M. kansasii HSV-1, CMV, EBV, enterovirus — Aspergillus fumigatusd 
P7 [E] (3c.88delA/88delA 28.3 Australia Alive – HHV-6 — — 
P8[E] c.88delA/88delA 28.3 Australia e Alive – — — — 
P9 [F] Not genotyped — Saudi Arabia — Dead M. bovis-BCG — — — 
P10 [F] Not genotyped — Saudi Arabia — Dead – — — — 
P11 [F] c.1757_1758delAG/1757_1758delAG 35 Saudi Arabia Dead M. abscessus NDf Corynebacterium spp., α-haemolyticus streptococcus NDg 
P12 [G] Not genotyped — Russia Dead – — — — 
P13 [G] Not genotyped — Russia Dead – — — — 
P14 [G] c.541 + 1G>A/541 + 1G>A 29.4 Russia Dead M. bovis-BCG — B. circulans, P. aeruginosa, S. aureus C. parapsilosis 
P15 [G] c.541 + 1G>A/541 + 1G>A 29.4 Russia Alive – CMV — C. parapsilosis 
P16 [H] c.541 + 2dup/541 + 2dup 26.6 Turkey Dead M. bovis-BCG — — — 
P17 [I] c.769dup/769dup 35 India Dead M. bovis-BCG — — — 
P18 [I] c.769dup/769dup 35 India 2.5 Dead M. bovis-BCG — — — 
P19 [J] p.S62*/S62* 35 Saudi Arabia Alive M. scrofulaceum CMV Influenza B, rhinovirus, coronavirus 229E, bocavirus, norovirus, metapneumovirus K. pneumoniae C. tropicalis 
P20 [K] p.Q9*/E625* 36/42 USA Dead M. kansasii HHV-6, VZV,c RSV, rhinovirus, enterovirus, influenza A — C. parapsilosis, C. albicans, Cryptococcus neoformans 
P21 [L] c.693_696del/693_696del 35 United Arab Emirates 0.1 Dead Suspected EM VZV,c RSV — — 
P22 [M] (13c.542-8G>A/128 + 2T>G 2.3/25.3 Japan Alive M. bovis-BCG M. malmoense VZV,c RSV, metapneumovirus, influenza A, rotavirus Mycoplasma pneumoniae — 
P23 [N] (7c.1011_1012delAG/1011_1012delAG 35 Guinea Dead – VZV,c RSV, rhinovirus, enterovirus, parainfluenza virus Rothia dentocariosa, Streptococcus viridans, K. pneumoniae — 
P24 [O] p.E618*/E618* 47 Palestine Alive – CMV, HSV-1 S. aureus — 
P25 [P] (8p.P696S/P696S 15.64 Israel 24 Alive M. szulgai HSV-1, CMV, adenovirus, molluscum contagiosum Salmonella spp. — 
P26 (P] (8p.P696S/P696S 15.64 Israel Alive Suspected EM HSV-1, VZV, CMV Salmonella spp., E. coli, K. pneumoniae — 
P27 [Q] (10p.K201N/K201N 22.7 Saudi Arabia 72 Alive M. avium VZV — C. parapsilosis 
P28 [Q] (10p.K201N/K201N 22.7 Saudi Arabia Dead M. bovis-BCG — — — 
P29 [R] (9p.A46T/K211R 33/17.68 Denmark 174 Alive M. avium — — — 
P30 [S] Not genotyped — Saudi Arabia Dead M. bovis-BCG — — — 
P31 [S] p.L407R/L407R 28.8 Saudi Arabia Dead M. bovis-BCG Rhinovirus — C. parapsilosis, C. albicans 
P32 [T] p.I648T/I648T 31 Pakistan Alive M. avium CMV — — 
a

The MSC CADD score for the STAT1 gene is 7.7. P9, P10, P12, P13, and P30 were not genotyped (E? in Fig. (1A).

b

Pretransplant infections.

c

VZV vaccine.

d

Positive Ag in blood and lung.

e

Preemptive.

f

Respiratory failure requiring ICU and mechanical ventilation.

g

Positive β-d-glucan and ultrasound suggestive of hepatosplenic fungal infection.

F, female; M, male; ND, nondocumented; RSV, respiratory syncytial virus; —, unavailable.

STAT1 is ubiquitously expressed in hematopoietic and nonhematopoietic cells (21). We evaluated the impact of five mutations not previously reported in patients’ cells. No cells were available from P17 and P18 (c.769dup), P20 (p.Q9*/p.E625*), P21 (c.693_696del), and P31 (p.L407R). We therefore evaluated the impact of the biallelic c.541 + 1G > A (P14), c.541 + 2dup (P16), and p.I648T (P32) mutations on STAT1 mRNA synthesis by RT-PCR on EBV–B cells. Amplification of the full-length of STAT1A cDNA (exons 1–25), and the exon 2–9 region revealed the presence of two transcripts in P14 and one in P16, both of lower m.w. than the WT transcript, suggesting exon skipping (Supplemental Fig. 1B). We determined the proportions of the different alleles by performing TOPO TA cloning of the STAT1 cDNA containing the exon 2–9 region from the WT sequence and the c.541 + 1G > A and c.541 + 2dup mutant sequences. Sequencing of the products revealed that the c.541 + 1G > A mutation mostly led to the splicing out of both exons 7 and 8 (82%), resulting in a transcript referred to as Δ_exon7-8 but, in some cases, led to the leaky splicing out of exon 7 alone (18%) (Δ_exon7) and an absence of the WT transcript, corresponding to the two bands previously identified (Supplemental Fig. 1C). For c.541 + 2dup, most of the transcripts displayed abnormal splicing out of exons 7 and 8 (98%). The Δ_exon7–8 resulted from an in-frame deletion in the CCD domain (c.463_633del [p.C155_K211del]), whereas the Δ_exon7 resulted in a frameshift (c.463_541del), potentially resulting in the production of a truncated protein (p.C155Nfs*24) (Supplemental Fig. 1D). We assessed also STAT1 mRNA levels in EBV–B cells from P14, P16, and P32, three patients carrying biallelic STAT1 null mutations (c.1758_1759delAG, c.1928insA and p.L600P) and 10 healthy controls. The STAT1 mRNA was barely detectable in the cells of P14 and P16 at levels similar to those in patients with complete STAT1 deficiency (4.5–16.5% of the levels found in controls), consistent with probable mRNA nonsense-mediated decay (Supplemental Fig. 1E). EBV–B cells carrying the biallelic p.I648T STAT1 mutation from P32 produced normal amounts of STAT1 mRNA, as shown by comparison with controls (Supplemental Fig. 1E). STAT1 mRNA expression in SV40 fibroblasts from P32 carrying the p.S62* mutation was much lower than those in controls (n = 4) (12.5% of control levels) (Supplemental Fig. 1F). Thus, biallelic c.541 + 1G > A, c.541 + 2dup, and p.S62* STAT1 mutations disrupt mRNA splicing and abolish the full-length transcript in EBV–B or SV40 fibroblasts from patients, and the p.I648T mutation decreases STAT1 mRNA levels in EBV–B cells from patients.

Six STAT1 mutants were previously studied and shown to be LOF (p.Q124H, c.1757_1758delAG, p.L600P, and c.1928insA) or hypomorphic (p.K201N and p.P696S) (1, 4, 6, 8, 10). We assessed the potential impact of reported but not fully experimentally tested (c.88delA, p.A46T, and c.1011_1012delAG) and new (p.Q9*, p.S62*, c.769dup, c.693_696del, p.L407R, p.E625*, and p.I648T) STAT1 mutant alleles in terms of STAT1 expression, phosphorylation, and GAS transcriptional activity in an overexpression system. We also tested the two misspliced cDNA transcripts (c.463_541del and c.463_633del) resulting from the c.541 + 1G > A and c.541 + 2dup splice mutations (Supplemental Fig. 2A–C). We first assessed the consequences of these transcripts in terms of protein levels and phosphorylation in STAT1-deficient U3C cells (a cell line deficient for STAT1) transiently transfected with a mock plasmid or empty vector, the WT, or mutant STAT1 alleles and the p.Y701C Mendelian susceptibility to mycobacterial disease (MSMD)–causing LOF STAT1 alleles (22) stimulated with IFN-γ (17) (Supplemental Fig. 2A). The c.463_633del, c.769dup, p.S62*, p.Q9*, p.E625*, and c.693_696del STAT1 mutants resulted in a complete abolition of STAT1 protein production and phosphorylation. The STAT1 p.L600P and c.463_541del mutants resulted in impaired protein production and the production of a truncated protein, respectively, together with an abolition of STAT1 phosphorylation. By contrast, the STAT1 p.A46T, p.L407R, and p.A648T mutants resulted in the production of normal amounts of protein, but phosphorylation after IFN-γ stimulation was impaired. We further investigated the IRF1 and GAS transcriptional activity of the mutants in a reporter assay (Supplemental Fig. 2B, 2C). Transient transfection of cells with c.88ddel, c.463_541del, c.463_633del, p.Q9*, p.E625*, c.693_696del, p.L600P, c.769dup, p.S62*, and c.1011_1012delAG STAT1 abolished the IFN-γ–induced GAS activation observed in cells transfected with the WT-STAT1 allele (Supplemental Fig. 2B). By contrast, p.A46T and p.I648T STAT1 resulted in a slight or severe impairment of GAS transcriptional activity after IFN-γ stimulation, respectively, but with residual activity. The p.L407R and p.I648T STAT1 mutants were strongly hypomorphic in transfected U3C cells in the IRF1 reporter assay (Supplemental Fig. 2C). The p.K211R mutant had no impact on STAT1 expression, phosphorylation, or GAS activity, but this mutant was predicted to lead to abnormal splicing in silico. Thus, the p.L407R, p.A46T, and p.I648T STAT1 mutants are hypomorphic, with residual phosphorylation and GAS or IRF1 transcriptional activity, whereas the c.88ddel, c.463_541del, c.463_633del, p.Q9*, p.E625*, c.693_696del, p.L600P, c.769dup, p.S62*, and c.1011_1012delAG STAT1 mutants are LOF, with a complete loss of phosphorylation and of STAT1-mediated transcriptional activity. Overall, based on these and previously reported data, 17 of these alleles are LOF, and 5 are hypomorphic.

We evaluated the effect of the STAT1 mutants in STAT-deficient U3C cells (Supplemental Figs. 2, 3). In addition, we evaluated the mutations at the protein level by performing Western blots or flow cytometry. A complete absence of STAT1 protein was observed in EBV–B cells from P14 and P16 and SV40 fibroblasts from P19, whereas P32 produced a mutant STAT1 protein, but in smaller amounts than the WT protein in controls (Fig. 2A–C). No STAT1 phosphorylation was detected after stimulation with IFN-γ, IFN-α2b, or IL-27 in EBV–B cells from P14 and P16 or in SV40 fibroblasts from P19. By contrast, STAT1 phosphorylation after stimulation with IFN-γ and IL-27 was impaired but not abolished in EBV–B cells from P32. In addition, STAT1 phosphorylation was impaired after exposure to a high dose of IFN-α2b in EBV–B cells from P32 (Supplemental Fig. 3B, 3C). The patient’s cells displayed impaired STAT1 phosphorylation in response to stimulation with IFN-γ relative to control and heterozygous cells (Supplemental Fig. 3B, 3C). In addition, heterozygous EBV–B cells from the parents of P2 or P14 (p.L600P/WT and c.541 + 1G > A/WT, respectively) displayed normal levels of STAT1 and of STAT1 phosphorylation after stimulation on Western blot, reflecting an absence of haploinsufficiency at the STAT1 locus (Supplemental Fig. 3D). We then assessed the DNA-binding activity of GAS and ISGF3 containing the WT and mutated STAT1 proteins by EMSA on EBV–B cells from P14, P16, and P32 after stimulation with IFN-γ, IL-27, or IFN-α2b. EMSA revealed that GAF DNA-binding activity after IFN-γ or IL-27 stimulation was abolished in P14’s and P16’s EBV–B cells (Fig. 2D, 2E). In addition, EBV–B cells from P32 contained little or no GAF-binding complex after stimulation with IFN-γ and IL-27, respectively (Fig. 2F, 2G). ISRE binding activity was abolished in cells from P14 and P16, as shown by assessments of the ISGF3 complex after stimulation with IFN-α2b in EBV–B cells. By contrast, ISRE binding activity after IFN-α stimulation was similar in the cells of P32 and in control cells (Fig. 2H). Thus, c.541 + 1G > A and c.541 + 2dup STAT1 mutations abolished STAT1 expression and both GAS and ISRE DNA-binding activities in the patients’ EBV–B cells, although EBV–B cells carrying p.I648T displayed impairment, but not a total abolition of STAT1 expression, IFN-γ–induced STAT1 phosphorylation and GAS DNA binding and STAT1 phosphorylation after exposure to a higher dose of IFN-α2b.

FIGURE 2.

Impaired STAT1 expression and function in cell lines from patients with AR STAT1 deficiency. (A and B) EBV–B cells from controls (C+), P14 (c.541 + 1G > A), P16 (c.541 + 2dup), P32 (p.I648T), and a patient with known AR complete STAT1 deficiency (STAT1−/−) were stimulated 20 min with 1 × 105 IU/ml IFN-γ or IFN-α2b or with 100 ng/ml IL-27 or were left unstimulated and were then subjected to Western blotting with a specific Ab recognizing the N-terminal (aa 69–169) part of the STAT1α isoform. (A) and (B) show representative results from at least two independent experiments. (C) SV40 fibroblasts from a control (C+) and P19 carrying the homozygous p.S62* variant was stimulated for 20 min with 1 × 105 IU/ml IFN-γ or IFN-α2b or with 100 ng/ml IL-27 or were left unstimulated and were then subjected to Western blotting. (DG) GAS DNA-binding activity in EBV–B cells from patients P2, P5, P11, P14, P16, and P32, with or without stimulation with 1 × 105 IU/ml IFN-γ or 100 ng/ml IL-27 for 30 min. (H) ISRE binding activity in terms of ISGF3 complex formation after the stimulation of EBV–B cells with IFN-α2b for 30 min. These results (D–G) are representative of two independent experiments.

FIGURE 2.

Impaired STAT1 expression and function in cell lines from patients with AR STAT1 deficiency. (A and B) EBV–B cells from controls (C+), P14 (c.541 + 1G > A), P16 (c.541 + 2dup), P32 (p.I648T), and a patient with known AR complete STAT1 deficiency (STAT1−/−) were stimulated 20 min with 1 × 105 IU/ml IFN-γ or IFN-α2b or with 100 ng/ml IL-27 or were left unstimulated and were then subjected to Western blotting with a specific Ab recognizing the N-terminal (aa 69–169) part of the STAT1α isoform. (A) and (B) show representative results from at least two independent experiments. (C) SV40 fibroblasts from a control (C+) and P19 carrying the homozygous p.S62* variant was stimulated for 20 min with 1 × 105 IU/ml IFN-γ or IFN-α2b or with 100 ng/ml IL-27 or were left unstimulated and were then subjected to Western blotting. (DG) GAS DNA-binding activity in EBV–B cells from patients P2, P5, P11, P14, P16, and P32, with or without stimulation with 1 × 105 IU/ml IFN-γ or 100 ng/ml IL-27 for 30 min. (H) ISRE binding activity in terms of ISGF3 complex formation after the stimulation of EBV–B cells with IFN-α2b for 30 min. These results (D–G) are representative of two independent experiments.

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STAT1 induced the transcription of various targeted genes (ISGs) (23). We assessed ISG induction by RT-qPCR after stimulation with IFN-γ, IFN-α2b, IFN-λ1, and IL-27 for 2 h in EBV–B cells or SV40 fibroblasts. We compared the induction of the CXCL10, CXCL9, IFIT1, IRF1, ISG15, and MX1 ISGs after activation of cell lines from patients carrying homozygous STAT1 mutations (c.541 + 1G > A, c.541 + 2dup, c.1757_1758delAG, c.1928insA, p.L600P, and p.I648T) with that in healthy controls. After stimulation with IFN-α2b, the induction of CXCL10, CXCL9, IRF1, ISG15, and MX1 was strongly impaired in EBV–B cells from patients with complete STAT1 deficiency compared with healthy controls (Fig. 3A). EBV–B cells from the patients displayed a strong impairment of the induction of CXCL10, CXCL9, and IRF1 after IFN-γ stimulation compared with the controls (Fig. 3B). The induction of IFIT1 and IRF1 in EBV–B cells from the patients was also impaired after stimulation with IL-27 and IFN-λ, respectively (Fig. 3C, 3D). However, the range of ISGs for which induction was impaired was narrower in EBV–B cells from P32 than in those from patients with complete STAT1 deficiency, with the normal induction of ISG15 after stimulation with IFN-α2b and the normal induction of IFIT1 after stimulation with IFN-α2b and IFN-λ1. ISG induction was also strongly impaired in SV40 fibroblasts from P19 stimulated with IFN-α2b or IFN-γ (Fig. 3E, 3F). These findings highlight the almost complete loss of induction of a broad range of ISGs after stimulation with IFN-α2b, IFN-λ, IFN-γ, and IL-27 in EBV–B cells or SV40 fibroblasts from patients with AR complete STAT1 deficiency, whereas the EBV–B cells from the patient with AR partial STAT1 deficiency displayed an impaired induction of some, but not all ISGs tested at early timepoints.

FIGURE 3.

Impaired ISG response in STAT1-deficient EBV–B cells and SV40 fibroblasts. (A) The fold induction of ISG transcripts was analyzed by RT-qPCR after the treatment, for 2 h, with 1 × 105 IU/ml IFN-α2b, 1 × 105 IU/ml IFN-γ, 100 ng/ml IL-27, or 20 ng/ml IFN-λ1 of EBV–B cells from healthy controls (n = 8) (WT/WT, black dots), five patients with AR complete STAT1 deficiency (red) and one patient with AR partial STAT1 deficiency (blue). The results shown are the fold induction after stimulation with IFN-α2b (A), IFN-γ (B), IL-27 (C), or IFN-λ1 (D) in EBV–B cells or after stimulation with IFN-α2b (E) and IFN-γ (F) in SV40 fibroblasts. Error bars indicate the mean ± SEM from duplicates of two independent experiments performed in triplicate. All RT-qPCR data are shown as fold (2−ΔCt) values relative to GUSB, normalized against nonstimulated conditions. Representative results from two independent experiments performed in triplicate were compared (mean ± SEM).

FIGURE 3.

Impaired ISG response in STAT1-deficient EBV–B cells and SV40 fibroblasts. (A) The fold induction of ISG transcripts was analyzed by RT-qPCR after the treatment, for 2 h, with 1 × 105 IU/ml IFN-α2b, 1 × 105 IU/ml IFN-γ, 100 ng/ml IL-27, or 20 ng/ml IFN-λ1 of EBV–B cells from healthy controls (n = 8) (WT/WT, black dots), five patients with AR complete STAT1 deficiency (red) and one patient with AR partial STAT1 deficiency (blue). The results shown are the fold induction after stimulation with IFN-α2b (A), IFN-γ (B), IL-27 (C), or IFN-λ1 (D) in EBV–B cells or after stimulation with IFN-α2b (E) and IFN-γ (F) in SV40 fibroblasts. Error bars indicate the mean ± SEM from duplicates of two independent experiments performed in triplicate. All RT-qPCR data are shown as fold (2−ΔCt) values relative to GUSB, normalized against nonstimulated conditions. Representative results from two independent experiments performed in triplicate were compared (mean ± SEM).

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Mycobacterial diseases occurred in 24 of 31 symptomatic patients (one patient was asymptomatic because of preemptive HSCT). About half the patients (n = 14/32) were vaccinated with BCG at birth or shortly thereafter (Table I). The first clinical manifestation, throughout the cohort, was BCG-osis, which was reported in all patients vaccinated with BCG (100%, 11 patients with complete and 3 with partial deficiency), within 4 mo of vaccination. Ten of the eighteen patients not vaccinated with BCG (61.1%) developed disseminated EM infection caused by M. avium (n = 3), M. kansasii (n = 2), M. abscessus (n = 1), M. szulgai (n = 1), M. scrofulaceum (n = 1), or undefined Mycobacterium species (n = 2) (Table I). P22 suffered from both BCG-osis at the age of 11 mo and EM infection caused by M. malmoense at the age of 4 y. No tuberculosis cases were reported in this cohort. Five siblings of three index cases (P9, P10, P12, P13, and P30) were suspected to have AR STAT1 deficiency because they died during the first year of life from suspected BCG-osis or sepsis (Fig. 1A, Table I). The reported cause of the death was BCG-osis with sepsis (P9), sepsis (P10 and P12), severe pneumonia (P13), and fever with hepatosplenomegaly and cytopenia (P30). Patients with AR complete STAT1 deficiency were younger at the onset of the first manifestation of the disease (mean: 3.0 mo, SD: 2.6, range: 1–10) than patients with AR partial STAT1 deficiency (mean: 35.8 mo, SD: 60.8, range: 1–174) (p = 0.036) (Fig. 4A). Mycobacterial infection occurred at younger age in patients with AR complete STAT1 deficiency than in patients with AR partial STAT1 deficiency (mean: 6.3 mo, SD: 5.0 mo, range: 1–18 mo versus mean age: 75.0 mo, SD: 82.0 mo, range: 1–174 mo) (p = 0.114) (Fig. 4B). As expected, BCG-osis occurred before the first EM infection in patients with AR STAT1 deficiency (mean age: 3.6 mo, SD: 2.8 mo, range: 1–10 mo versus mean age: 22.0 mo, SD: 64.4 mo, range: 10–204 mo) (p < 0.0001) (Fig. 4C). The mycobacterial disease involved lymph nodes (n = 14/17 patients, 82.4%), skin (typically maculopapular rash) (n = 8/17, 47.1%), liver and spleen (n = 10/17, 58.8%), lungs (n = 6/17, 35.3%), bones (n = 4/17, 23.5%), CNS (n = 1), or bone marrow (n = 1). A granulomatous reaction, mainly epithelioid, was reported in six patients, with caseation, necrosis, or acid-fast bacillus positivity in some cases. All patients received combined antimycobacterial treatment, including rifampin associated with other drugs in all cases. Three patients received s.c. recombinant human IFN-γ in addition to the antimycobacterial drugs. Overall, 24 (77.4%) symptomatic AR STAT1-deficient patients suffered from 29 different episodes of mycobacterial infection, with 17 episodes reported in 16 patients with AR complete STAT1 deficiency, with a median time to HSCT or death of 12.0 mo (range: 3–80 mo), and 12 such episodes in eight patients with AR partial STAT1 deficiency (median time: 139.5 mo; range: 2–327 mo). Complete recovery with no relapse of mycobacterial infection after treatment occurred in eight AR STAT1-deficient patients (complete deficiency, n = 6/16, and partial deficiency, n = 2/8). However, EM relapse occurred in three patients with AR partial deficiency, with two to three episodes reported, a median time of 1 y after the end of treatment (range 0.4–2 y) (10, 24, 25).

FIGURE 4.

Onset of infectious diseases in AR STAT1 deficiency. (A) Time from onset of the first severe infectious disease to death or HSCT in patients with complete (red line), partial (blue line), or AR complete (C) and partial (P) (black line) STAT1 deficiencies. Age at onset of mycobacterial (B), EM and BCG (C), and viral (D) infections in patients with C (red line) and P (blue line) deficiencies and in patients with both P and C (black line) STAT1 deficiencies. (E and F) Onset of mycobacterial (dashed line) and viral (dotted line) infections in patients with C (red) or P (blue) STAT1 deficiency.

FIGURE 4.

Onset of infectious diseases in AR STAT1 deficiency. (A) Time from onset of the first severe infectious disease to death or HSCT in patients with complete (red line), partial (blue line), or AR complete (C) and partial (P) (black line) STAT1 deficiencies. Age at onset of mycobacterial (B), EM and BCG (C), and viral (D) infections in patients with C (red line) and P (blue line) deficiencies and in patients with both P and C (black line) STAT1 deficiencies. (E and F) Onset of mycobacterial (dashed line) and viral (dotted line) infections in patients with C (red) or P (blue) STAT1 deficiency.

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STAT1 is involved in the responses to both type I and III IFNs (26, 27). Nineteen patients displayed severe viral infections before HSCT or death, with 54 documented episodes in 16 patients: 38 episodes in 11 patients with AR complete STAT1 deficiency and 16 in five patients with AR partial STAT1 deficiency (Table I). Herpesviridae was the most frequent viral family involved, with CMV (n = 11 episodes in seven patients), varicella zoster virus (VZV; n = 6 in six patients), HSV-1 (n = 5 in five patients), and human herpesvirus 6 (HHV-6; n = 4 in three patients) isolated (Table I). The spectrum of HSV-1 infections was broad, from recurrent and fatal herpes simplex encephalitis (HSE; P1) to skin-limited infections (P6). CMV caused interstitial pneumonitis (P6, P15, P19, P24, and P32), hepatitis (P24, P25, and P32), skin infection (P24 and P26), meningitis (P15), nephritis (P24), and enteritis (P32). VZV infections caused chickenpox (P20, P22, P23, and P26) or multiple organ disease (P21 and P27). HHV-6 caused hemophagocytic syndrome and meningoencephalitis (P7 and P20) (3). Based on PCR or serological tests performed before IVIG initiation, only 2 of 11 patients that had encountered EBV reported mild manifestations (infectious mononucleosis in P6 and possible hepatitis with the detection of IgM against CMV and EBV in P25). One patient (P25) developed disseminated adenovirus infection that resolved after brincidofovir treatment (25). All other viral infections cleared spontaneously. They included rhinovirus (n = 4), respiratory syncytial virus (n = 3), enterovirus (n = 3), influenza A (n = 2), rotavirus, metapneumovirus, influenza B virus, coronavirus, and bocavirus (one case each) (Table I). Mean age at onset of the first severe viral infection was 5.6 mo (SD: 4.4 mo, range: 1–12 mo) in patients with AR complete STAT1 deficiency, and 28.8 mo (SD: 40.1 mo, range: 2–96 mo) in patients with AR partial STAT1 deficiency (p = 0.442) (Fig. 4D). Age at onset of the first severe viral infection did not differ from age at onset of the first mycobacterial infection in patients with AR partial (Fig. 4E) or AR complete STAT1 deficiency (Fig. 4F). Half the cohort reported both mycobacterial and documented viral infection (14/32), with eight patients (complete deficiency, n = 5; partial deficiency, n = 3) presenting viral infection before mycobacterial infection. In addition, serological tests and Ab profiling by PhIP-Seq showed that some additional viruses had been spontaneously cleared without causing severe disease (enterovirus, norovirus, parvovirus B19, adenovirus, and rhinovirus) (Fig. 5B).

FIGURE 5.

Serological profiling and PhIP-Seq in patients with AR STAT1 deficiency. (A) Serological tests of patients with AR STAT1 deficiency. Red square indicates a positive serological test, blue squares represent a negative serological test, and a white square represents serological tests that could not be performed. No serological information was available for P1–P4, P8–P10, P15, P18, and P28. P8 and P14 had been on IVIG since birth, and no prior serological tests were performed. #Positive PCR result on blood; *, negative PCR result on blood; §, all the tests for HBs Ag and anti-HBc Abs were negative in all patients. Age corresponds to age at sampling in months. (BD) Antiviral Ab responses to species were tested seropositive by PhIP-Seq based on our stringent cutoff values. (B) Hierarchical clustering of species-specific adjusted score values for patients (P*), pooled plasma used for IVIG therapy, IgG-depleted serum, and a mock immunoprecipitation (IP) sample. IVIG was used as positive and IgG depleted and mock IP as negative controls. (C) Number of species shown in (A) for which a given sample tested seropositive (significant). (D) Principal component (PC) analysis of samples with species-specific score shown in (B).

FIGURE 5.

Serological profiling and PhIP-Seq in patients with AR STAT1 deficiency. (A) Serological tests of patients with AR STAT1 deficiency. Red square indicates a positive serological test, blue squares represent a negative serological test, and a white square represents serological tests that could not be performed. No serological information was available for P1–P4, P8–P10, P15, P18, and P28. P8 and P14 had been on IVIG since birth, and no prior serological tests were performed. #Positive PCR result on blood; *, negative PCR result on blood; §, all the tests for HBs Ag and anti-HBc Abs were negative in all patients. Age corresponds to age at sampling in months. (BD) Antiviral Ab responses to species were tested seropositive by PhIP-Seq based on our stringent cutoff values. (B) Hierarchical clustering of species-specific adjusted score values for patients (P*), pooled plasma used for IVIG therapy, IgG-depleted serum, and a mock immunoprecipitation (IP) sample. IVIG was used as positive and IgG depleted and mock IP as negative controls. (C) Number of species shown in (A) for which a given sample tested seropositive (significant). (D) Principal component (PC) analysis of samples with species-specific score shown in (B).

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A subset of inborn errors of type I and type III IFN immunity confers intrinsic susceptibility to some live viral attenuated vaccines (LAVs) (28). These inborn errors include AR IFNAR1, IFNAR2, STAT1, STAT2, IRF7, and IRF9 deficiencies (3, 2934). All five patients with AR complete STAT1 deficiency (P7, P20, P21, P22, and P23) vaccinated with measles, mumps, and rubella (MMR) and/or VZV LAVs developed related adverse reactions (Table I, II). P7 presented an episode of hemophagocytic syndrome with encephalopathy 1 wk after MMR vaccination at 13 mo of age (3). P20 developed vaccine-stain varicella (chickenpox) at 11 mo of age (skin culture positive for the VZV vaccine strain). At 13 mo of age, he developed a fever and skin rash after MMR vaccination that was suspected to be caused by measles vaccine strain (not tested). P21 suffered from disseminated VZV disease and multiple organ failure and obstructive hydrocephalus ∼10 d after vaccination with both the MMR and VZV vaccines. The VZV vaccine strain was detected in cerebrospinal fluid (CSF) and skin. P22 and P23 developed vaccine-strain varicella (chickenpox) 15 d after vaccination (7, 13). By contrast, three patients with AR partial STAT1 deficiency (P25, P26, and P29) received the MMR vaccine with no reported related adverse events. P5 was also vaccinated with oral poliovirus type 2 vaccine (4), and P24 received oral rotavirus vaccination, with no complications in either case. None of the patients reported rubella live attenuated viral vaccine-induced granulomatous disease.

Hemophagocytosis secondary to severe infections has rarely been reported in patients with defects of IFN-γ immunity (3538). Seven patients with AR STAT1 deficiency presented nine episodes of secondary hemophagocytic syndrome (Table II). The reported triggers were live vaccines (BCG, n = 2; MMR, n = 1; VZV, n = 2), other infectious pathogens (HHV-6, n = 2; Candida parapsilosis, n = 1), or unidentified (n = 1). Mean age at onset was 10.7 mo (SD: 8.3 mo, range: 3–25 mo), and this condition was the first manifestation of the disease in two patients. Relapses occurred in two patients (P7 and P14), 5 and 3 mo, respectively, after the first episode. All episodes of hemophagocytic syndrome fulfilled the HLH-04 criteria (12) (Table II). During these nine episodes, patients displayed fever (n = 9/9, 100%), splenomegaly (n = 5/8, 62.5%), anemia, hemoglobin < 9 g/dl (n = 6/7, 85.7%), thrombocytopenia, platelets < 100 × 109/l (n = 9/9, n = 100%), hyperferritinemia > 500 μg/l (n = 8/8, 100%), hypertriglyceridemia ≥ 3 mmol/l (n = 3/7, 50.0%), hypofibrinogenemia < 1.5 g/l (n = 6/8, 75.0%), an increase in sCD25 levels (n = 5/5, 100%), and hemophagocytosis (n = 5/6, 71.4%). Other manifestation included hepatitis, skin rash, coagulopathy, and encephalopathy. NK cell degranulation and cytotoxicity were normal in three of the four patients tested. In addition to antimicrobial treatment, patients received IVIG (n = 2), the HLH-04 protocol (12) (n = 1), or steroids alone (n = 4). P30 died during a hemophagocytic episode in a context of uncontrolled BCG-osis (Table II). P11 developed fatal hemophagocytic syndrome during the posttransplantation period, following Metapneumovirus infection.

Table II.

Clinical and immunological profile of hemophagocytosis episodes in patients with AR STAT1 deficiency

PatientP7P15P20P21P22P23P31
DeficiencyCompleteCompleteCompleteCompleteCompleteCompletePartial
Recurrence of hemophagocytosisYesYesNoNoNoNoNo
Age at onset (months)813371725144
Trigger HHV-6 MMR vaccine Suspected BCG vaccine Candida parapsilosis HHV-6 VZV and MMR vaccine unknown pathogen VZV vaccine and Parainfluenza virus Suspected BCG vaccine 
HLH-04 criteria (12Yes (5/8) Yes (7/8) Yes (5/8) Yes (6/8) Yes (5/5) No (5/6) Yes (5/7) Yes (6/8) Yes (6/8) 
Fever ≥38.5°C Yes Yes Yes Yes Yes Yes Yes Yes Yes 
Splenomegaly No Yes Yes Yes — Yes No No Yes 
Hemoglobin (g/dl) 7.8 6.2 7.6 7.7 Severe pancytopenia — 12.3 5.9 8.3 
Platelets (109/l) 22 68 84 25 Severe pancytopenia 22 10 
Neutrophils (109/l) NA 2.9 22 12 Severe pancytopenia 18 4.9 Normal 4.9 
Triglyceride (mmol/l) NA 2.6 2.8 3.5 — 3.0 2.1 4.9 2.2 
Ferritin (μg/l) 2,467 4,960 2,800 5,500 — 1,858 8,404 22,935 51,455 
Fibrinogen (g/l) 0.8 1.1 0.8 0.5 — 0.48 4.21 <0.7 2.1 
Hemophagocytosis (site) No (bone marrow) Yes (bone marrow) — Yes (bone marrow) — Yes (bone marrow) No Yes (bone marrow) Yes (liver) 
NK cell activity Normal (cytotoxicity and degranulation) Normal (cytotoxicity and degranulation) — — Decreased to absent — — Normal (degranulation) — 
sCD25 (>2,400 IU/ml) 37,000 IU/ml > 50,000 IU/ml — — — — 8,270 IU/ml 15,718 IU/ml >5,000 IU/ml 
Others Hepatomegaly, exanthema, hepatitis, hyperbilirubinemia, coagulopathy Hepatomegaly, rash, diarrhea, encephalopathy (ataxia) Hepatitis Hepatitis Meningoencephalitis, respiratory failure, hyponatremia, and hypertension Hepatitis, encephalitis, AKI — Liver failure — 
Treatment IVIG Dexamethasone — — — Ceftriaxone, fluconazole, acyclovir, IVIG Dexamethasone Steroids HLH-04 protocol (VP16, dexamethasone, ciclosporin A) 
HLH outcome Resolution Resolution, then HSCT Resolution Resolution — Resolution Resolution Resolution, then HSCT Death 
PatientP7P15P20P21P22P23P31
DeficiencyCompleteCompleteCompleteCompleteCompleteCompletePartial
Recurrence of hemophagocytosisYesYesNoNoNoNoNo
Age at onset (months)813371725144
Trigger HHV-6 MMR vaccine Suspected BCG vaccine Candida parapsilosis HHV-6 VZV and MMR vaccine unknown pathogen VZV vaccine and Parainfluenza virus Suspected BCG vaccine 
HLH-04 criteria (12Yes (5/8) Yes (7/8) Yes (5/8) Yes (6/8) Yes (5/5) No (5/6) Yes (5/7) Yes (6/8) Yes (6/8) 
Fever ≥38.5°C Yes Yes Yes Yes Yes Yes Yes Yes Yes 
Splenomegaly No Yes Yes Yes — Yes No No Yes 
Hemoglobin (g/dl) 7.8 6.2 7.6 7.7 Severe pancytopenia — 12.3 5.9 8.3 
Platelets (109/l) 22 68 84 25 Severe pancytopenia 22 10 
Neutrophils (109/l) NA 2.9 22 12 Severe pancytopenia 18 4.9 Normal 4.9 
Triglyceride (mmol/l) NA 2.6 2.8 3.5 — 3.0 2.1 4.9 2.2 
Ferritin (μg/l) 2,467 4,960 2,800 5,500 — 1,858 8,404 22,935 51,455 
Fibrinogen (g/l) 0.8 1.1 0.8 0.5 — 0.48 4.21 <0.7 2.1 
Hemophagocytosis (site) No (bone marrow) Yes (bone marrow) — Yes (bone marrow) — Yes (bone marrow) No Yes (bone marrow) Yes (liver) 
NK cell activity Normal (cytotoxicity and degranulation) Normal (cytotoxicity and degranulation) — — Decreased to absent — — Normal (degranulation) — 
sCD25 (>2,400 IU/ml) 37,000 IU/ml > 50,000 IU/ml — — — — 8,270 IU/ml 15,718 IU/ml >5,000 IU/ml 
Others Hepatomegaly, exanthema, hepatitis, hyperbilirubinemia, coagulopathy Hepatomegaly, rash, diarrhea, encephalopathy (ataxia) Hepatitis Hepatitis Meningoencephalitis, respiratory failure, hyponatremia, and hypertension Hepatitis, encephalitis, AKI — Liver failure — 
Treatment IVIG Dexamethasone — — — Ceftriaxone, fluconazole, acyclovir, IVIG Dexamethasone Steroids HLH-04 protocol (VP16, dexamethasone, ciclosporin A) 
HLH outcome Resolution Resolution, then HSCT Resolution Resolution — Resolution Resolution Resolution, then HSCT Death 

AKI, acute kidney injury; —, unavailable.

Susceptibility, in particular mycobacterial, and viral infectious diseases is frequently reported in patients with AR STAT1 deficiency. However, other sporadic infections have also been reported in this cohort. The pyogenic bacterial infections identified were caused by Staphylococcus aureus (n = 3 patients), Klebsiella pneumoniae (n = 3), S. haemolyticus (n = 2), Corynebacterium spp. (n = 1), Bacillus circulans (n = 1), Pseudomonas aeruginosa (n = 1), and Escherichia coli (n = 1) (Table I). Recurrent/disseminated Salmonella group D (n = 2) and Shigella sonnei (n = 1) diseases occurred in patients with AR partial STAT1 deficiency (P19 and P20). Recurrent or severe episodes of uncharacterized infections (P2, P3, P6, P9, P10, P11, P13, P21, and P28) or pneumonia (P6, P11, P12, P16, P17, P21, P22, P25, P26, and P29) occurred in 10 patients. Five (n = 3 complete STAT1 deficiency; n = 2 partial STAT1 deficiency) patients with AR STAT1 deficiency displayed invasive fungal infections before transplantation, associated in all cases, with catheter use in intensive care and concomitant infection, but with no other identified risk, such as neutropenia or immunosuppressive treatment. Candida (n = 5 episodes in four patients) was the genus most frequently isolated, with the detection of C. parapsilosis (n = 4, from bronchoalveolar lavage [BAL] and/or a central venous line) and C. albicans (n = 1, from BAL and a central venous line). Aspergillus fumigatus Ags were detected in BAL from P6 during recurrent episodes of respiratory distress (Table I). Mean age at onset of the first fungal infection was 24.2 mo (SD: 40.2 mo, range: 4–96 mo). None of the patients displayed chronic mucocutaneous candidiasis. No parasitic infections were reported in this cohort. In addition to the episodes of hemophagocytic syndrome reported in some patients, P5 developed severe inflammatory granulomatous hepatitis in a context of BCG-osis requiring immunosuppressive drugs, and two patients developed atypical Kawasaki disease triggered by an unknown pathogen (P7 and P22). No autoimmune diseases or malignancies were reported in this cohort.

Laboratory studies showed normal counts for circulating myeloid subsets (polymorphonuclear cells and monocytes; data not shown) and normal levels of circulating lymphocytes, in terms of both counts and percentages (CD3+, CD4+, and CD8+ T cells and B and NK cells) in the 19 patients tested. Lymphocyte proliferation in response to mitogens (n = 6) and dihydrorhodamine and/or NBT staining of neutrophils (n = 8) were normal. Serum IgG, IgA, and IgM levels were normal or high; total IgE level was high in 6 of 14 patients tested (range: 125–10,814 kU/l). Most of the patients tested had IgG Abs against EBV (n = 11), CMV (n = 9), VZV (n = 7), and HSV (HSV-1; n = 4) (Fig. 5A). All serological tests for HIV and hepatitis C virus were negative. Other serological tests yielded positive results for Parvovirus B19 (n = 4). Five patients were vaccinated against hepatitis A virus (P7, P19, P23, P24, and P29), and eight were vaccinated against hepatitis B virus, but none developed adverse reactions after these vaccinations. We also performed broad serological profiling to determine the history of exposure of P5, P15, P18, P19, P25–P27, and P32 to viruses in the PhIP-Seq assay, which can be used to assess virus-specific Abs in humans in an unbiased and high-throughput manner (Fig. 5B–D). In addition to Herpesviridae viruses, we detected immunization against viruses typically implicated in respiratory or gastrointestinal infections (enterovirus, norovirus, adenovirus, and rhinovirus) (Fig. 5B). We then evaluated IFN-γ, IL-12p40, and IL-12p70 secretion in whole blood from local controls, travel controls, and patients with AR partial (n = 6) or AR compete (n = 1, P5) STAT1 deficiency, in the presence and absence of stimulation with BCG, BCG+IL-12 or BCG, or BCG+IFN-γ, respectively. After stimulation, IFN-γ secretion was similar to that in controls (Supplemental Fig. 4A–C). However, after stimulation, IL-12p40 secretion was impaired and IL-12p70 production was undetectable, as previously reported in P24 with AR partial STAT1 deficiency (8).

Twelve patients (eleven patients with AR complete deficiency and one patient with AR partial STAT1 deficiency) underwent allogeneic HSCT (twelve HSCT interventions in total, one per patient), including five cases already published (37, 13) (Table III). HSCT was performed before the confirmation of the molecular diagnosis in P5. P8 underwent preemptive HSCT at the age of 5 mo, before the development of clinical manifestations, following molecular diagnosis in her affected brother P7 (3). Mean age at HSCT was 25.2 mo (SD: 20.6 mo, range: 5–70 mo). The grafts were obtained from matched related donors (n = 4), matched unrelated donors (n = 2), mismatched unrelated donors (n = 3), or haploidentical related donors (n = 3). The survivors received cells from mismatched unrelated donors (n = 3) or matched unrelated donors (n = 1) or cells from haploidentical donors (n = 3). The reported stem cell sources were bone marrow (n = 7), peripheral blood stem cells (n = 1) or unrelated cord blood (n = 1) or were not reported (n = 3; P15, P20, and P24). P8 and P11 received TCR-αβ/CD19–depleted grafts. Reduced-intensity conditioning regimens were used in nine cases (six of whom survived). These regimens were based on fludarabine (Flu), busulfan (Bu)/Flu/alemtuzumab (n = 2), Flu/melphalan/alemtuzumab (n = 1), Bu/Flu (n = 1), Bu/Flu/thiotepa/antithymocyte globulin (ATG; n = 1), Flu/treosulfan/thiotepa/ATG (n = 3), or Flu/Bu/ATG/TBI (n = 1). A myeloablative conditioning regimen was used in three cases (all of whom survived). This regimen was based on Bu/cyclophosphamide/ATG (n = 1) or Bu/Flu/ATG (n = 2). Median time to engraftment was 15 d (range: 11–24 d), but engraftment was considered likely to be incomplete in P5 (5, 6). Infections were observed before transplantation in six patients: BCG or EM infection (n = 4, including two cases of uncontrolled disease), CMV disease (n = 2), HSV (n = 1), and HHV-6 disease (n = 3). Two patients had CMV viremia before transplantation. Infections occurred after transplantation in eight cases (Table III). One patient developed EBV posttransplant lymphoproliferative disease, with systemic disease and a fatal outcome (day +70, P5), and another patient displayed EBV reactivation (day +35, P20). Graft-versus-host disease (GVHD) prophylaxis was administered to all but one patient. Acute GVHD occurred in 10 of 12 patients and involved skin (n = 10, including 4 with grades III–IV), gut (n = 5, including 4 with grades III–IV), and lung (n = 1) (Table III). Transplant-related mortality was 30.8%, with four deaths (P5, P11, P20, and P23) 91, 96, 78, and 63 d after HSCT, due to multiple organ failure and mycobacterial infection (n = 2), acute respiratory distress syndrome secondary to Metapneumovirus infection (n = 1), and CMV (n = 1), respectively. None of the eight surviving patients (P6–8, P15, P19, P22, P24, and P32) developed viral or mycobacterial infection after engraftment, with a median follow-up time of 45.7 mo after the procedure (range: 17–1,401 mo). Full donor chimerism was observed in eight of ten surviving patients. P7 displayed mixed chimerism and received a CD34-selected stem cell boost (month +48), which failed to improve engraftment. No secondary graft failure was reported in any of the patients.

Table III.

Hematopoietic stem cell transplantation (HSCT) procedure and outcomes of AR STAT1 deficient patients

Patient
P5P6P7P8P11P15P19P20P22P23P24P32
DeficiencyCompleteCompleteCompleteCompleteCompleteCompleteCompleteCompleteCompleteCompleteNAPartial
Age at transplantation (months) 55 14 17 33 20 70 17 17 41 
Year of transplantation 2005 2009 2015 2016 2014 2014 2019 2017 2019 2018 2019 2018 
Donor type MRD MMUD MUD Haploidentical TCR-αß/ CD19- depleted MRD Haploidentical TCR-αß/CD19-depleted Haploidentical MMUD MUD MRD MMUD MMUD 
 
HLA compatibility 10/10 08/10 10/10 05/10 10/10 05/10 NA 09/10 08/08 10/10 09/10 04/06 
Graft type BM BM BM PBSC BM NA BM NA BM BM NA Cord blood 
CMV status (Recipient/Donor) Neg./NA Pos./NA Neg./Neg. Neg./Pos. NA/NA Pos./Pos. Pos./Neg. NA/NA Neg./Neg. Pos./Pos. Pos./Pos. Pos./Pos. 
Total nucleated cell received (×108/kg) 9.0 18.7 NA NA NA 7.49 NA NA 5.3 5.3 NA 0.63 
CD34+ cells received (×106/kg) NA 3.93 4.91 12.01 16.2 10.18 10 NA 7.3 4.9 38.5 0.29 
Conditioning regimen RIC MAC RIC RIC RIC RIC RIC RIC RIC MAC RIC MAC 
Drugs and doses of conditioning regimen Flu 30 mg/kg × 5 d, Mel 140 mg/m2, Al 0.2 mg/kg × 5 d Bu 5 mg/kg × 4 d, Cy 50 mg/kg × 4 d, ATG 2.5 mg/kg × 4 d Adjusted Bu 14 mg/kg (target AUC 60 mg × h/l), Flu 180 mg/m2, Al 2.5 mg/kg Treosulfan 36 g/m2, Flu 160 mg/m2, Thiotepa 10 mg/kg, ATG 7.5 mg/kg Standard regimen dose Treosulfan 36 g/m2, Flu 150 mg/kg, Thiotepa 10 mg/kg, ATG 10 mg/kg, rituximab 100 mg NA Bu 0.8 mg/kg test dose, then 5 mg/kg × 2 d, Flu 30 mg/m2 × 6 d, Thiotepa 5 mg/kg × 1 d, ATG 2 mg/kg × 4 d Adjusted Bu 16 mg/kg (targeted AUC 50 mg × h/l), Flu 180 mg/m2, ATG 5 mg/kg, TBI 5.4 Gy Adjusted Bu 4.8 mg/kg × 4 d (targeted AUC, 81 mg × h/l), Flu 40 mg/m2 × 4 d, ATG 15 mg/kg × 3 d, Rituximab 375 mg/m2/d on day −1 Adjusted Bu (targeted AUC 60 mg × h/l) × 3 d, Flu 45 mg/m2 × 4 d, Al 0.2 mg/kg × 3 d Adjusted Bu 4.9 mg/kg × 4 d (target AUC 90 ±5 mg × h/l), Flu 160 mg/m2, ATG 10 mg/kg 
GVHD prophylaxis Prednisolone and tacrolimus Cyclosporine A Cyclosporine A MMF Cyclosporine A No Post- transplantation Cyclophosphamide and MMF Cyclosporine A and MMF Methotrexate and tacrolimus Cyclosporine A and MMF Cyclosporine A, MMF, prednisolone Cyclosporin A and prednisone 
Pretransplant infectious status Uncontrolled BCG-osis CMV viremia Recurrent HHV-6 encephalitis No No CMV disease CMV viremia, Multifocal Mycobacterial osteomyelitis, Candida tropicalis, norovirus, rhinovirus, metapneumovirus and bocavirus HHV-6 meningoencephalitis and viremia, disseminated M. avium infection Osteomyelitis by M. malmoense Virus-related hemophagocytosis HSV, CMV, HHV-6 CMV viremia, M. avium 
Post-transplant infections Rhinovirus, fulminant EBV/PTLD with multiorgan failurea EBV reactivation No No Metapneumovirus- related ARDS and refractory HLHa E. coli sepsis, K. pneumoniae, CMV viremia Rhinovirus, norovirus EBV/PTLD, HHV-6 viremia, CLABSI, Candida parapsilosis, Candida albicans, C. neoformans, disseminated M kansasiia No CMV-related ARDSa No CMV reactivation, rhinovirus/enterovirus, CLABSI 
Time to engraftment Poor engraftment Day +15 Day +14 (neutrophils) Day +11 (neutrophils) Day +24 (neutrophils) Day +11 (leucocytes, platelets) Day +20 Days +12–14 Day +15 Day +14 (neutrophils) Day +16 (leucocytes, platelets) Day +15 
Acute GVHD Localization (grade) Skin (NA) Skin and gut (grade III) Skin, gut (grade III) Skin (grade II) Skin and lung (NA) No Skin (II) Skin and gut (grade III–IV)a Skin (grade I) No Skin (grade I) Skin and gut (grade III) 
Chronic GVHD No No Skin Skin No No No No No No Skin Lung 
Others transplant issues — AIHA (day+10), PRES (day+35), TTP (day+53), keratoconjunctivitis (day+20), chronic lymphedema (day+300) Progressive worsening of pulmonary function, recurrent lung infections. Lung transplantation considered Progressive bronchiectasis, recurrent lung infections — No Meningoencephalitis with seizure and focal deficit; demyelinating lesions found in brain biopsy VODa with severe fluid overload and GI bleed, TMA No No ITP AIHA, arterial hypertension 
Donor chimerism (time after transplantation) Full donor chimerism (100%, NA) Full donor chimerism (100%, year +13) Chimerism favoring donor (95%) (month +4); myeloid 9%, CD19 21%, CD3 44%, whole blood 24% (month +39); CD34 selected stem cell boost (month +48) with stable chimerism (month+52) Full donor chimerism (100%, year+2) Single tandem repeats: lymphoid 39% and myeloid 31% donor cells (NA) Planned at day+30 Full donor chimerism in T lymphocytes and myeloid cells (100%, months +7) Full donor chimerism (>98%, day +14) Full donor chimerism (donor >95%, day +121) Chimerism favoring donor (90-95%) (day +18) Full donor chimerism (NA) Full donor chimerism (NA) 
Outcome (age and posttransplant time at last FU) Died (aged 11 mo, day +91) Alive (aged 15.5 y, year +11) Alive (aged 6 y, month +62) Alive (aged 4 y, month +46) Died (21 mo, day +96) Alive (2 y, month +19) Alive (4 y, month +17) Died (22 mo day +78) Alive (6 y, month +10) Died (aged 20 mo, day +63) Alive (aged 2.6 y, month +13) Alive (5 y, month +19) 
Patient
P5P6P7P8P11P15P19P20P22P23P24P32
DeficiencyCompleteCompleteCompleteCompleteCompleteCompleteCompleteCompleteCompleteCompleteNAPartial
Age at transplantation (months) 55 14 17 33 20 70 17 17 41 
Year of transplantation 2005 2009 2015 2016 2014 2014 2019 2017 2019 2018 2019 2018 
Donor type MRD MMUD MUD Haploidentical TCR-αß/ CD19- depleted MRD Haploidentical TCR-αß/CD19-depleted Haploidentical MMUD MUD MRD MMUD MMUD 
 
HLA compatibility 10/10 08/10 10/10 05/10 10/10 05/10 NA 09/10 08/08 10/10 09/10 04/06 
Graft type BM BM BM PBSC BM NA BM NA BM BM NA Cord blood 
CMV status (Recipient/Donor) Neg./NA Pos./NA Neg./Neg. Neg./Pos. NA/NA Pos./Pos. Pos./Neg. NA/NA Neg./Neg. Pos./Pos. Pos./Pos. Pos./Pos. 
Total nucleated cell received (×108/kg) 9.0 18.7 NA NA NA 7.49 NA NA 5.3 5.3 NA 0.63 
CD34+ cells received (×106/kg) NA 3.93 4.91 12.01 16.2 10.18 10 NA 7.3 4.9 38.5 0.29 
Conditioning regimen RIC MAC RIC RIC RIC RIC RIC RIC RIC MAC RIC MAC 
Drugs and doses of conditioning regimen Flu 30 mg/kg × 5 d, Mel 140 mg/m2, Al 0.2 mg/kg × 5 d Bu 5 mg/kg × 4 d, Cy 50 mg/kg × 4 d, ATG 2.5 mg/kg × 4 d Adjusted Bu 14 mg/kg (target AUC 60 mg × h/l), Flu 180 mg/m2, Al 2.5 mg/kg Treosulfan 36 g/m2, Flu 160 mg/m2, Thiotepa 10 mg/kg, ATG 7.5 mg/kg Standard regimen dose Treosulfan 36 g/m2, Flu 150 mg/kg, Thiotepa 10 mg/kg, ATG 10 mg/kg, rituximab 100 mg NA Bu 0.8 mg/kg test dose, then 5 mg/kg × 2 d, Flu 30 mg/m2 × 6 d, Thiotepa 5 mg/kg × 1 d, ATG 2 mg/kg × 4 d Adjusted Bu 16 mg/kg (targeted AUC 50 mg × h/l), Flu 180 mg/m2, ATG 5 mg/kg, TBI 5.4 Gy Adjusted Bu 4.8 mg/kg × 4 d (targeted AUC, 81 mg × h/l), Flu 40 mg/m2 × 4 d, ATG 15 mg/kg × 3 d, Rituximab 375 mg/m2/d on day −1 Adjusted Bu (targeted AUC 60 mg × h/l) × 3 d, Flu 45 mg/m2 × 4 d, Al 0.2 mg/kg × 3 d Adjusted Bu 4.9 mg/kg × 4 d (target AUC 90 ±5 mg × h/l), Flu 160 mg/m2, ATG 10 mg/kg 
GVHD prophylaxis Prednisolone and tacrolimus Cyclosporine A Cyclosporine A MMF Cyclosporine A No Post- transplantation Cyclophosphamide and MMF Cyclosporine A and MMF Methotrexate and tacrolimus Cyclosporine A and MMF Cyclosporine A, MMF, prednisolone Cyclosporin A and prednisone 
Pretransplant infectious status Uncontrolled BCG-osis CMV viremia Recurrent HHV-6 encephalitis No No CMV disease CMV viremia, Multifocal Mycobacterial osteomyelitis, Candida tropicalis, norovirus, rhinovirus, metapneumovirus and bocavirus HHV-6 meningoencephalitis and viremia, disseminated M. avium infection Osteomyelitis by M. malmoense Virus-related hemophagocytosis HSV, CMV, HHV-6 CMV viremia, M. avium 
Post-transplant infections Rhinovirus, fulminant EBV/PTLD with multiorgan failurea EBV reactivation No No Metapneumovirus- related ARDS and refractory HLHa E. coli sepsis, K. pneumoniae, CMV viremia Rhinovirus, norovirus EBV/PTLD, HHV-6 viremia, CLABSI, Candida parapsilosis, Candida albicans, C. neoformans, disseminated M kansasiia No CMV-related ARDSa No CMV reactivation, rhinovirus/enterovirus, CLABSI 
Time to engraftment Poor engraftment Day +15 Day +14 (neutrophils) Day +11 (neutrophils) Day +24 (neutrophils) Day +11 (leucocytes, platelets) Day +20 Days +12–14 Day +15 Day +14 (neutrophils) Day +16 (leucocytes, platelets) Day +15 
Acute GVHD Localization (grade) Skin (NA) Skin and gut (grade III) Skin, gut (grade III) Skin (grade II) Skin and lung (NA) No Skin (II) Skin and gut (grade III–IV)a Skin (grade I) No Skin (grade I) Skin and gut (grade III) 
Chronic GVHD No No Skin Skin No No No No No No Skin Lung 
Others transplant issues — AIHA (day+10), PRES (day+35), TTP (day+53), keratoconjunctivitis (day+20), chronic lymphedema (day+300) Progressive worsening of pulmonary function, recurrent lung infections. Lung transplantation considered Progressive bronchiectasis, recurrent lung infections — No Meningoencephalitis with seizure and focal deficit; demyelinating lesions found in brain biopsy VODa with severe fluid overload and GI bleed, TMA No No ITP AIHA, arterial hypertension 
Donor chimerism (time after transplantation) Full donor chimerism (100%, NA) Full donor chimerism (100%, year +13) Chimerism favoring donor (95%) (month +4); myeloid 9%, CD19 21%, CD3 44%, whole blood 24% (month +39); CD34 selected stem cell boost (month +48) with stable chimerism (month+52) Full donor chimerism (100%, year+2) Single tandem repeats: lymphoid 39% and myeloid 31% donor cells (NA) Planned at day+30 Full donor chimerism in T lymphocytes and myeloid cells (100%, months +7) Full donor chimerism (>98%, day +14) Full donor chimerism (donor >95%, day +121) Chimerism favoring donor (90-95%) (day +18) Full donor chimerism (NA) Full donor chimerism (NA) 
Outcome (age and posttransplant time at last FU) Died (aged 11 mo, day +91) Alive (aged 15.5 y, year +11) Alive (aged 6 y, month +62) Alive (aged 4 y, month +46) Died (21 mo, day +96) Alive (2 y, month +19) Alive (4 y, month +17) Died (22 mo day +78) Alive (6 y, month +10) Died (aged 20 mo, day +63) Alive (aged 2.6 y, month +13) Alive (5 y, month +19) 
a

Cause of death.

Al, alemtuzumab; AIHA, autoimmune hemolytic anemia; ARDS, acute respiratory distress syndrome; ATG, antithymocyte globulin; Bu, busulfan; tAUC, targeted area under the curve; BM, bone marrow; BO, bronchiolitis obliterans; C, complete; CLABSI, central line-associated bloodstream infection; Cy, cyclophosphamide; FU, follow-up; GI, gastrointestinal; ITP, immune thrombocytopenia; MAC, myeloablative conditioning; Mel, melphalan; MMF, mycofenolate mofetil; MMUD, mismatched unrelated donor; MRD, matched related donor; MUD, matched unrelated donor; NA, not available; P, partial; PBSC, peripheral blood stem cells; PRES, posterior reversible encephalopathy syndrome; RIC, reduced intensity conditioning; SCIG, subcutaneous immunoglobulins; TBI, total body irradiation; TMA, thrombotic microangiopathy; TTP, thrombotic thrombocytopenic purpura; VOD, veno-occlusive disease.

In total, 21 patients (64%) from 13 different kindreds died, including 15 of the 28 (54%) patients with genetically confirmed deficiencies after a median cohort follow-up of 20.5 mo (range: 2–337 mo). Median overall survival was 1.8 y and was shorter in patients with AR complete STAT1 deficiency (1.3 y) than in those with AR partial STAT1 deficiency (not reached) (p = 0.16) (Fig. 6A). At 24 mo, 69% of patients with AR complete STAT1 deficiency and 25.0% of those with AR partial STAT1 deficiency had died. Median age at death was 7.5 mo (SD: 7.3 mo, range: 2–22 mo) in the 18 patients with AR complete deficiency who died, and 5.0 mo (SD 18.8) in the three patients with AR partial STAT1 deficiency who died at 36, 2 and 5 mo of age, respectively. The cause of death was uncontrolled infection (BCG, n = 9; HSV-1, n = 1; nondocumented, n = 7) or transplant-related mortality (n = 4). Death following the progression of mycobacterial infection on treatment occurred in seven patients with AR complete STAT1 deficiency and two with AR partial STAT1 deficiency. The only documented cause of fatal viral infection was HSE in P1. Eight of the thirteen surviving patients followed for a median of 81 mo (range: 31–337 mo) have AR complete STAT1 deficiency, and five have AR partial STAT1 deficiency. Seven of the eight survivors with AR complete STAT1 deficiency underwent HSCT (P6–P8, P15, P19, P22, and P24), and the remaining patient is 4 y old and currently awaiting transplantation. In this group, survival was longer for patients undergoing HSCT than for those who did not undergo transplantation (median survival not reached versus 6 mo, p < 0.0001). Overall survival after HSCT was 64% after a median follow-up time of 54 mo (SD: 50.3 mo, range: 11–195 mo) (Fig. 6B).

FIGURE 6.

Survival in AR STAT1 deficiency. (A) Overall survival among patients with AR complete (red line), partial (blue line), or either complete or partial (black) STAT1 deficiency. (B) Overall survival in patients with AR complete STAT1 deficiency that received (n = 11, red line) or not (n = 7, blue line) HSCT. P11 was not included in this analysis because HSCT was performed before any manifestations of the disease.

FIGURE 6.

Survival in AR STAT1 deficiency. (A) Overall survival among patients with AR complete (red line), partial (blue line), or either complete or partial (black) STAT1 deficiency. (B) Overall survival in patients with AR complete STAT1 deficiency that received (n = 11, red line) or not (n = 7, blue line) HSCT. P11 was not included in this analysis because HSCT was performed before any manifestations of the disease.

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We report in this study an international cohort of 32 patients with AR STAT1 deficiency from 20 unrelated kindreds and 13 countries. Sixteen STAT1 variants were found to be LOF, and five were hypomorphic. The patients’ cells displayed abolished or impaired IFN-γ/IFN-II and type I/III IFN responses, potentially accounting for susceptibility to mycobacteria and viruses, respectively, against which these cytokines play a crucial role (summarized in the Table IV). Mycobacterial infection was the principal infection observed in patients with AR STAT1 deficiency (77%), as reported in other MSMDs caused by impaired responses to IFN-γ, including AD partial STAT1, IFN-γR1, and IFN-γR2 deficiencies and AR partial IFN-γR1, IFN-γR2, and JAK1 deficiencies (3947). The penetrance of clinical manifestations after BCG vaccination was complete in patients with STAT1 deficiency. Typically, mycobacterial infections began before 1.5 y in patients with AR complete STAT1 deficiency, as reported for patients with AR complete IFN-γR1 or IFN-γR2 deficiencies, in whom penetrance is almost complete and prognosis poor (∼50% mortality) (41, 44). By contrast, AR and AD partial IFN-γR1 and IFN-γR2 deficiencies and AR partial JAK1 deficiency had a milder clinical phenotype, with a later disease onset, and a better prognosis; they could be received a long-term antibiotic treatment and resembled partial AR STAT1 deficiency (40, 41, 4547). Overall, only 8 of the 32 patients with AR STAT1 deficiency (complete in all these cases) did not suffer from mycobacterial disease, but follow-up was short for these patients, who either died early or underwent HSCT (range: 2–14 mo). In addition, four of the eight patients with EM disease followed for more than 2 y after their first mycobacterial episode presented a relapse within 2 y of stopping treatment. This suggests that it may be wise to continue antimycobacterial treatment in the long term for patients for whom HSCT is not considered because of milder disease. We also report seven patients with AR STAT1 deficiency (complete in six of these patients) who developed secondary hemophagocytic syndrome, in which IFN-γ is thought to play a major pathophysiological role. In addition, seven additional MSMD patients developed episodes of hemophagocytic syndrome triggered by mycobacterial infection caused by impaired or abolished responses to IFN-γ (29, 3538) or IL-12/23 (36). Overall, these findings provide additional evidence to suggest that hemophagocytic syndrome can develop independently of IFN-γ–STAT1 immunity in humans (35). The mechanism underlying the uncontrolled state of hyperinflammation in these patients remains unclear, as most of these patients have preserved NK cell cytotoxicity and degranulation.

Table IV.

Summary of the functional studies in the patients’ cells

Patient (Kindred)MutationDeficiencySTAT1 ExpressionSTAT1 PhosphorylationGAS and ISRE DNA BindingISG Induction
P1 (A) c.1757_1758delAG/1757_1758delAG Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P2 (B) p.L600P/L600P Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P5 (C) c.1928insA/1928insA Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P6 (D) p.Q124H/Q124H Complete Impaired (T cells and EBV-B cells) Impaired after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells 
P7 (E) c.88delA/88delA Complete NT Abolished (monocytes) NT NT 
P11 (F) c.1757_1758delAG/1757_1758delAG Complete Abolished (EBV-B cells) Abolished (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) NT 
P14 (G) c.541 + 1G>A/541 + 1G>A Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P16 (H) c.541 + 2dup/541 + 2dup Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P17 (I) c.769dup/769dup Complete NT NT NT NT 
P19 (J) p.S62*/S62* Complete Abolished (SV40 fibroblasts) Abolished after IFN-α and IFN-γ (SV40 fibroblasts) NT Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (SV40 fibroblasts) 
P20 (K) p.Q9*/E625* Complete NT NT NT NT 
P21 (L) c.693_696del/693_696del Complete NT NT NT NT 
P22 (M) c.542-8G>A/128 + 2T>G Complete Abolished (PBMCs and SV40 fibroblasts) Abolished after IFN-α and IFN-γ (PBMCs and SV40 fibroblasts) Abolished GAS after IFN-γ (SV40 fibroblasts) Strongly impaired after IFN-α and IFN-γ (monocytes) 
P23 (N) c.1011_1012delAG/1011_1012delAG Complete Abolished (PBMCs) NT NT Strongly impaired after IFN-α and IFN-γ (PBMCs) 
P24 (O) p.E618*/E618* Complete NT NT NT NT 
P25 (P) p.P696S/P696S Partial Impaired (EBV-B cells) Impaired after IFN-α and IFN-γ (EBV-B cells) Impaired GAS after IFN-γ and IL-27; impaired ISRE after IFN-α (EBV-B cells) Impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P27 (Q) p.K201N/K201N Partial Impaired (EBV-B cells) Impaired after IFN-α and IFN-γ (EBV-B cells) Impaired GAS after IFN-γ and IL-27; impaired ISRE after IFN-α (EBV-B cells) Impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P29 (R) p.A46T/K211R Partial Impaired (EBV-B cells) Impaired after IFN-α and IFN-γ (PBMCs) NT NT 
P31 (S) p.L407R/L407R Partial NT NT NT NT 
P32 (T) p.I648T/I648T Partial Normal (EBV-B cells) Impaired after IFN-γ, IFN-α and IL-27(EBV-B cells) Abolished GAS after IFN-γ and IL-27; normal ISRE after IFN-α (EBV-B cells) Impaired after IFN-α, IFN-γ and IL-27 (EBV-B cells) 
Patient (Kindred)MutationDeficiencySTAT1 ExpressionSTAT1 PhosphorylationGAS and ISRE DNA BindingISG Induction
P1 (A) c.1757_1758delAG/1757_1758delAG Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P2 (B) p.L600P/L600P Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P5 (C) c.1928insA/1928insA Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P6 (D) p.Q124H/Q124H Complete Impaired (T cells and EBV-B cells) Impaired after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells 
P7 (E) c.88delA/88delA Complete NT Abolished (monocytes) NT NT 
P11 (F) c.1757_1758delAG/1757_1758delAG Complete Abolished (EBV-B cells) Abolished (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) NT 
P14 (G) c.541 + 1G>A/541 + 1G>A Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P16 (H) c.541 + 2dup/541 + 2dup Complete Abolished (EBV-B cells) Abolished after IFN-α and IFN-γ (EBV-B cells) Abolished GAS after IFN-γ and IL-27; abolished ISRE after IFN-α (EBV-B cells) Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P17 (I) c.769dup/769dup Complete NT NT NT NT 
P19 (J) p.S62*/S62* Complete Abolished (SV40 fibroblasts) Abolished after IFN-α and IFN-γ (SV40 fibroblasts) NT Strongly impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (SV40 fibroblasts) 
P20 (K) p.Q9*/E625* Complete NT NT NT NT 
P21 (L) c.693_696del/693_696del Complete NT NT NT NT 
P22 (M) c.542-8G>A/128 + 2T>G Complete Abolished (PBMCs and SV40 fibroblasts) Abolished after IFN-α and IFN-γ (PBMCs and SV40 fibroblasts) Abolished GAS after IFN-γ (SV40 fibroblasts) Strongly impaired after IFN-α and IFN-γ (monocytes) 
P23 (N) c.1011_1012delAG/1011_1012delAG Complete Abolished (PBMCs) NT NT Strongly impaired after IFN-α and IFN-γ (PBMCs) 
P24 (O) p.E618*/E618* Complete NT NT NT NT 
P25 (P) p.P696S/P696S Partial Impaired (EBV-B cells) Impaired after IFN-α and IFN-γ (EBV-B cells) Impaired GAS after IFN-γ and IL-27; impaired ISRE after IFN-α (EBV-B cells) Impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P27 (Q) p.K201N/K201N Partial Impaired (EBV-B cells) Impaired after IFN-α and IFN-γ (EBV-B cells) Impaired GAS after IFN-γ and IL-27; impaired ISRE after IFN-α (EBV-B cells) Impaired after IFN-α, IFN-γ, IL-27 and IFN-λ (EBV-B cells) 
P29 (R) p.A46T/K211R Partial Impaired (EBV-B cells) Impaired after IFN-α and IFN-γ (PBMCs) NT NT 
P31 (S) p.L407R/L407R Partial NT NT NT NT 
P32 (T) p.I648T/I648T Partial Normal (EBV-B cells) Impaired after IFN-γ, IFN-α and IL-27(EBV-B cells) Abolished GAS after IFN-γ and IL-27; normal ISRE after IFN-α (EBV-B cells) Impaired after IFN-α, IFN-γ and IL-27 (EBV-B cells) 

This table summarizes functional results obtained in cell lines (EBV-B cells, SV40 fibroblasts) or primary cells (PBMCs, monocytes).

GAS, IFN-γ activation site; ISRE, IFN-stimulated response element; NT, not tested; PBMC, peripheral blood mononuclear cell.

Patients with AR STAT1 deficiency are particularly prone to severe viral infections with herpesviruses the most frequently identified. Indeed, almost all the patients with AR STAT1 deficiency that had encountered α-(HSV-1/2, VZV) or β-(CMV, HHV-6) herpesviruses reported severe manifestations. By contrast, only one of the 11 patients that had encountered EBV reported a related manifestation (infectious mononucleosis), whereas another developed posttransplant EBV– posttransplant lymphoproliferative disease, and none of the patients appeared to be susceptible to HHV-8. Patients with AR STAT1 deficiency differ from patients with other IEIs of type I and/or III IFN immunity in having a wider and more severe pattern of viral infections and a clear susceptibility to herpesviruses. Indeed, severe herpesvirus infection has not been reported in patients with biallelic JAK1, IRF9, IRF7, or IL10R mutations (34, 42, 4852). Five of the ten patients with complete TYK2 deficiency described in this study suffered from mild viral disease (mainly HSV and VZV) (5355). Severe infections due to weakly virulent viruses have been reported in patients with AR complete STAT2 deficiency, a few of whom have suffered from severe herpesvirus infections (32, 33). Patients with AR IFNAR1, IFNAR2, IRF7, and IRF9 deficiencies have an even narrower susceptibility to viruses, restricted to LAVs and isolated influenza pneumonitis and HSE (for IFNAR1 and IRF9) (2931, 49, 56). Deficiencies of IFNAR1, IFNAR2, IRF3, and IRF7 can also underlie severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (57). No patients with AR STAT1 deficiency and SARS-CoV-2 infection have yet been reported, but our findings strongly suggest that these patients may be at high risk of developing life-threatening COVID-19. Overall, these findings suggest that STAT1 plays a crucial role in defenses against α- and β-herpesviruses but is probably redundant against γ-herpesviruses (EBV and HHV-8).

Adverse reactions to LAVs have been reported in five patients with AR complete STAT1 deficiency (3, 28). An adverse reaction to MMR vaccine was reported in all six MMR-vaccinated patients with AR STAT2 deficiency, one patient with AR IRF7 deficiency, two of three patients with AR IRF9 deficiency, one of two patients with AR IFNAR1 deficiency, and one with AR IFNRA2 deficiency (28, 3034). A severe adverse reaction to yellow fever vaccine was reported in one patient with AR IFNAR1 deficiency and one patient with AR IRF9 deficiency (31). Vaccine-strain VZV infections are extremely rare and have been reported only in the context of severe adaptive immunity deficiency (5863), in one patient with AR complete IFN-γR1 deficiency (64) and exceptional cases in otherwise healthy children (6568). The three MMR-vaccinated patients with AR partial STAT1 deficiency and the one patient with AR partial IFNAR1/complete IFN-γR2 deficiency and residual STAT1-dependent signaling did not develop adverse reactions (29). These findings highlight the essential role of STAT1-dependent IFN signaling in antiviral immunity in natura. They also provide strong evidence that IEIs of IFN type I/III constitute a major cause of life-threatening reactions to LAVs and suggest that functional type I IFN–dependent ISGF3 complex formation is essential for host defense against measles and VZV.

Finally, we report the procedure and clinical outcome for 12 patients who underwent HSCT. Seven of the eleven patients with AR complete STAT1 deficiency who underwent HSCT survived. All of the others died of infection by the age of 22 mo. Despite the use of various regimens and donor types, HSCT was associated with a considerable risk, as four of the seven patients with pretransplant viral infections died. HSCT remains the only curative treatment available for AR complete STAT1 deficiency and severe forms of AR partial STAT1 deficiency. None of the nine surviving patients who have undergone HSCT suffered from severe viral infection after engraftment, with a follow-up of up to 11 y after the procedure. HSCT may have corrected the genetic defect only in the hematopoietic cells, with no correction in nonhematopoietic cells (5). This aspect is of particular interest because nonhematopoietic cells may play a major role in brain and lung antiviral immunity, as reported for neurons and oligodendrocytes in the brain, and for plasmacytoid dendritic cells and pulmonary epithelial cells in the lung (69, 70). In addition, three of the nine HSCT survivors are currently receiving IVIG supplementation, which may have conferred additional antiviral immunity, as reported in patients with AR STAT2 deficiency (32, 71). Chronic lung disease is accelerated after viral clearance in mice with Stat1-deficient lung epithelial cells, suggesting an intrinsic role of STAT1 in epithelial cell defenses (72, 73). Two patients developed progressive lung disease, with non-GVHD bronchiectasis and bronchiolitis obliterans, 4 and 2 y after transplantation while on IVIG treatment. This finding suggests that STAT1 type I/III–dependent immunity in hematopoietic cells may be sufficient to prevent severe viral infection in patients with AR STAT1 deficiency. However, the impact of the STAT1 cell–intrinsic defect in nonhematopoietic cells in the brain and lung remains uncertain (70). Overall, the description of this series of patients with AR STAT1 deficiency should help to improve the genetic diagnosis of this group of severe IEIs, making it possible to implement adequate treatment strategies.

We thank the patients and the relatives and physicians of the patients. We also thank Yelena Nemirovskaya, Dana Li, Christine Rivalain, and Lazaro Lorenzo-Diaz for administrative support.

This work was supported in part by National Institute of Allergy and Infectious Diseases Grant R37AI095983, the National Center for Research Resources, National Center for Advancing Sciences of the National Institutes of Health Grant 8UL1TR000043, The Rockefeller University, the St. Giles Foundation, INSERM, the University of Paris, Laboratoire d’Excellence Integrative - Biology of Emerging Infectious Diseases (ANR-10-LABX-62-IBEID), and the Agence Nationale de la Recherche under Investments for the Future Grant ANR-10-IAHU-01 and GENMSMD (ANR-16-CE17.0005-01). T.L.V. was supported by the French Ministry of Health and Clermont-Ferrand University Année Recherche program. A.N.-P. was supported by a National Council of Science and Technology (CONACYT) national Ph.D. fellowship. V.J.-J. was supported by LABEIX-IBEID. J.R. was supported by INSERM. T.L.V. and J.R. are supported by the Imagine Institute M.D.-Ph.D. program with the support of the Bettencourt-Schueller Foundation. A.-L.N. is supported by the Imagine Institute international Ph.D. program with the support of the Bettencourt-Schueller Foundation. S.O. was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research 16H05355 and 19H03620, Promotion of Joint International Research Grant 18KK0228, and the Practical Research Project for Rare/Intractable Diseases of the Japan Agency for Medical Research and Development. F.H. was funded by the German Federal Ministry of Education and Research (BMBF, 01GM1910C).

Most of the experiments were performed by T.L.V. under the supervision of J.B. and J.-L.C. M.B. and L. Abel provided the founder effect data. A.E.-S., A.S., B.A.S., H.E.G., G.E.E., P.T., M.C., Y.A.T., S.E.B., I.M., M.H.A., T.C., R.F., P.D.A., R.B., L. Alsina, M.D., H.A.-M., I.H., C.S., F.H., L.-M.K., A.D.-M., N.A.K., M.A.H., S.H.A., and S.A.-M. took care of the patients and participated in data collection. F.R. performed viral serological tests, N.M. and T.K. performed phage immunoprecipitation sequencing analysis. V.J.-J., A.E.-S., A.N.-P., M.R., A.-L.N., C.O.-Q., J.R., and S.B.-D. contributed new reagents/analytical tools, and M.T., S.N., S.S., F.S., and S.O. made the STAT1 plasmids and performed experiments in the overexpression system. J.B. and T.L.V. recorded the clinical data and created the figures. T.L.V., J.L.-C., and J.B. wrote the paper. All authors commented on and discussed the paper.

The online version of this article contains supplemental material.

Abbreviations used in this article

AR

autosomal recessive

ATG

antithymocyte globulin

BAL

bronchoalveolar lavage

BCG

bacillus Calmette–Guérin

Bu

busulfan

CADD

combined annotation-dependent depletion

EM

environmental mycobacteria

Flu

fludarabine

GAF

IFN-γ activated factor

GAS

IFN-γ activation sequence

GVHD

graft-versus-host disease

HHV-6

human herpesvirus 6

HLH

hemophagocytic lymphohistiocytosis

HSCT

hematopoietic stem cell transplantation

HSE

herpes simplex encephalitis

IEI

inborn error of immunity

ISGF3

IFN-stimulated gene factor 3

ISRE

IFN-sensitive response element

LAV

live viral attenuated vaccine

LOF

loss-of-function

MMR

measles, mumps, and rubella

MRCA

most recent common ancestor

MSC

mutation significance cutoff

MSMD

Mendelian susceptibility to mycobacterial disease

PhIP-Seq

phage immunoprecipitation sequencing

RT-qPCR

quantitative real-time PCR

VZV

varicella zoster virus

WES

whole-exome sequencing

WT

wild-type

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The authors have no financial conflicts of interest.

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