A novel STING1 variant causes a recessive form of STING-associated vasculopathy with onset in infancy (SAVI)

Abstract

Stimulator of interferon response genes (STING) encoded by stimulator of interferon response cGAMP interactor 1 (STING1), previously known as transmembrane protein 173 (TMEM173) is an important pattern recognition receptor that detects microbial dinucleotides and functions as an adaptor molecule in the cytosolic DNA sensing pathway that binds 2’3’-cyclic GMP-AMP (cGAMP), which is generated when cytosolic DNA activates cyclic GMP-AMP synthase (cGAS).1Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1674) Google Scholar,2Ablasser A. Chen Z.J. cGAS in action: Expanding roles in immunity and inflammation.Science. 2019; 363Crossref PubMed Scopus (352) Google Scholar STING activation stimulates the induction of type I interferons, which activate interferon responses. Gain-of-function (GOF) variants in STING1 lead to autoactivation without ligand binding and cause a rare autoinflammatory disease named STING-associated vasculopathy with onset in infancy (SAVI) (Online Mendelian Inheritance in Man catalog no. 615934).3Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (817) Google Scholar,4Jeremiah N. Neven B. Gentili M. Callebaut I. Maschalidi S. Stolzenberg M.C. et al.Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations.J Clin Invest. 2014; 124: 5516-5520Crossref PubMed Scopus (337) Google Scholar Patients with SAVI present in infancy with the following symptoms: recurrent fevers; cold-induced skin vasculitis that can progress to tissue loss and amputation of fingers and toes; and/or interstitial lung disease, which is the main cause of the mortality that often occurs before patients reach adulthood. So far, all reported cases of SAVI have been caused by autosomal dominant variants, with most of them occurring de novo. We have identified 6 patients from 4 unrelated families, all of whom are of Arabic ethnicity and harbor pathogenic STING1 variants that are disease causing only in homozygosity. The patients had clinical disease suggestive of SAVI and were enrolled into institutional review board–approved protocols, including the National Institutes of Health natural history protocol (NCT02974595). Patient 1, the index patient, presented at 4 weeks of age with a cough and failure to thrive, as well as with a maculopapular violaceous rash with a livedoid appearance (Fig 1, A and B). A chest computed tomography scan showed diffuse bilateral parenchymal opacities. His lung disease progressed despite steroid therapy and short-term treatment with the JAK inhibitor tofacitinib, and he died of respiratory failure at 5 months of age. His older brother (patient 2) died at 18 months of age with chronic cough and failure to thrive. Although genetic testing was not performed, the clinical manifestations and similarities of patient 2 to those of his younger brother strongly suggest that he had SAVI. Patient 3 presented at 3 months of age with recurrent fever, erythematous rash, cough, and dyspnea, ultimately progressing to oxygen dependence. He had a chest computed tomography scan with results consistent with interstitial lung disease and a lung biopsy specimen that showed chronic interstitial pneumonitis with intraalveolar hemorrhage. He is currently taking the JAK inhibitor ruxolitinib. His brother, patient 4, had respiratory symptoms starting at the age of 6 months; he was diagnosed with SAVI at the age of 15 months and was initially treated with steroids, after which baricitinib (a selective JAK1/2 inhibitor) was added to his treatment regimen. Patient 5 presented at the age of 2 months with cough, tachypnea, and recurrent lung infections; she was diagnosed with chronic aspiration pneumonia and had a laryngeal cleft that was repaired at the age of 4.5 years. Because of her lung disease (Fig 1, E), she has required supplemental oxygen since the age of 7 months and bilevel positive airway pressure support while sleeping since the second year of life, when digital clubbing was noticed. She developed pulmonary hypertension by the age of 4 years. At the age of 5 she developed polyarthritis, an erythematous rash over the soles of her feet and clubbing. She began taking baricitinib at the age of 7 years; there was clinical improvement, but her oxygen dependence continued. Patient 6 presented at the age of 8 months with a history of persistent tachypnea and failure to thrive, polyarthritis, intermittent vasculitic rashes, and clubbing (Fig 1, C and D). All of the parents and siblings were asymptomatic with normal inflammatory markers. Additional clinical and laboratory information is available in Table E1 (in this article’s Online Repository at www.jacionline.org). Patients 1, 3, and 4 underwent targeted sequencing of STING1, whereas whole exome sequencing was performed on families 3 and 4. Sequencing of all patients revealed a homozygous STING1 (NM_198282) variant c.841C>T, p.Arg281Trp, p.R281W (Fig 1, F). This variant was present in heterozygosity in only 2 of 282,822 alleles reported in gnomAD (gnomad.broadinstitute.org) and is predicted to be damaging by PolyPhen-2 and deleterious by the scale-invariant feature transform algorithm (SIFT), and it has a Combined Annotation-Dependent Depletion (CADD)-PHRED-scaled score of 26.4. This variant has not been reported previously in patients with SAVI (https://infevers.umai-montpellier.fr). All of the parents, as well as several of the unaffected siblings, were heterozygous carriers of this variant (Fig 1, F). A variant affecting the same amino acid residue but mutated to a glutamine (c.842G>A, p.Arg281Gln, p.R281Q) has previously been described to cause an autosomal dominant form of SAVI5Melki I. Rose Y. Uggenti C. Van Eyck L. Fremond M.L. Kitabayashi N. et al.Disease-associated mutations identify a novel region in human STING necessary for the control of type I interferon signaling.J Allergy Clin Immunol. 2017; 140: 543-552.e5Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar (Fig 1, G). A strikingly high interferon response gene signature and systemic inflammation in patients with SAVI has led to their designation as having an autoinflammatory interferonopathy.6Kim H. de Jesus A.A. Brooks S.R. Liu Y. Huang Y. VanTries R. et al.Development of a validated interferon score using nanostring technology.J Interferon Cytokine Res. 2018; 38: 171-185Crossref PubMed Scopus (76) Google Scholar A standardized interferon response gene score was elevated only in patients who were homozygous for the p.R281W variant and who had clinical disease, and not in the siblings and parents who were heterozygous carriers of the variant or in healthy controls (Fig 1, H). Transfection of HEK293T cells with the STING1 construct containing the R281W mutation led to activation of IFNB1 luciferase reporter without ligand binding (see Fig E1 in this article’s Online Repository at www.jacionline.org), indicating that p.R281W is a pathogenic GOF variant. The mutant remained responsive to cGAMP (see Fig E1), suggesting no effect on ligand binding. Disease-causing SAVI variants3Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (817) Google Scholar cluster in 2 areas. The cryoelectron microscopy structure model of STING7Shang G. Zhang C. Chen Z.J. Bai X.C. Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567: 389-393Crossref PubMed Scopus (69) Google Scholar has mapped the class 1 variants, p.N154S, p.V155M, and p.V147L to the connector helix loop (Fig 1, G) which controls a cGAMP ligand–induced 180o rotation of the ligand-binding area of the STING dimer that induces polymerization and activation. The class 3 variants, including p.R281Q, p.R284G5Melki I. Rose Y. Uggenti C. Van Eyck L. Fremond M.L. Kitabayashi N. et al.Disease-associated mutations identify a novel region in human STING necessary for the control of type I interferon signaling.J Allergy Clin Immunol. 2017; 140: 543-552.e5Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar and p.R284S,8Konno H. Chinn I.K. Hong D. Orange J.S. Lupski J.R. Mendoza A. et al.Pro-inflammation associated with a gain-of-function mutation (R284S) in the innate immune sensor STING.Cell Rep. 2018; 23: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar are located at the polymerization interface7Shang G. Zhang C. Chen Z.J. Bai X.C. Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567: 389-393Crossref PubMed Scopus (69) Google Scholar (Fig 1, G) and have been postulated to bind a C-terminal tail that might prevent polymerization and autoactivation.9Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Concordant with previous reports, we hypothesized that the p.R281W variant confers a GOF that is weaker than the heterozygous SAVI variants. To test this possibility, we transfected HEK293T cells with either wild-type or mutant STING constructs including R281W at increasing concentrations and assessed IFNB1 reporter activity (Fig 2, A). At equal transfection efficiency, consistent with the homozygosity requirement at this location, R281W mutant constructs exhibited lower autoactivation than R281Q constructs did but were similar to mutant constructs with the pathogenic V155M variant. This suggests differential autoactivation thresholds for disease-causing class 1 variants at the connector helix loop (ie, V155M) versus class 3 variants at the polymer interface (ie, R281 and R284). In line with this hypothesis, pathogenic variants at the class 3 residues that cause disease in heterozygosity, including R281Q, R284S, and R284G, are all more autoactivating than V155M in the transfection model (Fig 2, A and B), with R284S, R284G, and G158A constituting the top 3 autoactivating SAVI variants (Fig 2, B). Although class 1 disease-causing SAVI variants are GOF variants, mutating these residues into charged amino acids (eg, V155R3Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (817) Google Scholar and G158E8Konno H. Chinn I.K. Hong D. Orange J.S. Lupski J.R. Mendoza A. et al.Pro-inflammation associated with a gain-of-function mutation (R284S) in the innate immune sensor STING.Cell Rep. 2018; 23: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) leads to loss of function, likely through destabilizing the STING dimer structure. In contrast, none of the 6 different amino acid mutations at position R281 caused loss of function (Fig 2, B [green labels]). In fact, all of the mutations except for R281A, which is similar to the wild type, led to autoactivation. Maintenance of a positive charge at residue R281 (by mutating into lysine [K] or histidine [H]) was insufficient to keep STING inactive at the basal state. Moreover, mutations into hydrophobic amino acids such as tryptophan (W), leucine (L), or methionine (M), or into negatively charged amino acids (including glutamic acid [E]), cause autoactivation (Fig 2, A and B). The autoactivation was not due to differential protein expression in the transfection assay (see Fig E2 in this article’s Online Repository at www.jacionline.org). The R281 and R284 mutations that were assessed remained responsive to cGAMP stimulation (Fig 2, B and see Fig E1), indicating no effect on ligand binding. The broad autoactivation of the R281 and R284 mutations was consistent with the model of polymer interface binding to an inhibitor to suppress STING autoactivation.9Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Although this potential inhibitor is not addressed in the 180o rotation model of the connector helix loop mutations, it is quite likely that the 180o rotation also leads to loss of inhibitor binding on the polymer interface, thus allowing side-by-side packing. Our data thus suggest a possible common mechanism to reconcile the 2 previously reported structural models (Fig 2, C and see also the Discussion section for detailed description of the model shown in Fig E3 in this article’s Online Repository at www.jacionline.org), which is supported by similar superactivating mutations in both the connector helix loop (G158A) and the polymer interface (R281E, R284G, and R284S). In summary, we have reported 6 patients from 4 unrelated families with recessively inherited STING1 variants and characteristic clinical features of SAVI. Our data support the notion that the disease-causing homozygous p.R281W variant causes GOF but is weaker in activating STING than the previously reported heterozygous variants, and it requires biallelic mutations to constitutively activate STING. We have demonstrated a critical role of residue R281 in maintaining STING in an inactive state, likely without affecting dimer conformation. Our data unveil limitations of the current structural models that cannot explain the superactivating potential of the R281 and R284 mutations and raise questions as to whether class 3 residues represent an autoinhibitory domain or a binding site for an external inhibitor of STING. Considering the critical function of R281 and the nearby region, novel therapeutic options may arise from high-throughput screens of drugs that bind to this area and inhibit polymerization. We thank the patients and their families for participating in this study. All patients were enrolled into the institutional review board–approved National Institutes of Health natural history protocol Studies of the Natural History, Pathogenesis, and Outcome of Autoinflammatory Diseases (NOMID/CAPS, DIRA, CANDLE, SAVI, NLRC4-MAS, Still’s-like and Autoinflammatory for their in with the of HEK293T cells were with STING constructs and an IFNB1 luciferase reporter construct with catalog no. in with catalog no. with a transfection for 1 well of of IFNB1 luciferase reporter construct was with 5 of of and of STING constructs in a of as The were with the containing 5 of and of and at for to the HEK293T cells were by and in to a of The transfection was to the following the after which of HEK293T cells was added to To the was and and to of the by of at to the cells to The were to in a with for The luciferase assay was by using the catalog no. with of luciferase cGAMP of cGAMP was with of and added to 1 well a containing of cells at 6 after transfection for a of Transfection was as described except that were catalog no. and and cells were added in 4 than in the of STING construct and of IFNB1 luciferase reporter construct were into HEK293T cells in were at after transfection and were with 1 of after which of assay of of and 1 of catalog no. 1 of catalog no. 1 of inhibitor catalog no. and of catalog no. was The was on for to the The was into a for 15 and at at for The was to a concentrations were by using a protein assay catalog no. with as a All were to the same with with catalog no. and catalog no. and at for of protein was in catalog no. and was in acid catalog by using catalog no. at for of catalog no. was to the before the The were to catalog no. with the catalog the protocol for with a A and for 7 The were and with with catalog no. as detailed the were in of and at for 1 were for 5 3 with of and by in of catalog no. in of and at with The was with for 3 by 1 of at in of containing 6 of catalog no. and of catalog no. 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Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567: 389-393Crossref PubMed Scopus Google S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus Google Scholar The cryoelectron microscopy structure model suggests that cGAMP binding to the STING dimer causes a rotation of the ligand-binding domain a connector helix This the polymerization and side-by-side of STING into G. Zhang C. Chen Z.J. Bai X.C. Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567: 389-393Crossref PubMed Scopus Google Scholar that as a for and C. G. X. Zhang X. Bai X.C. Chen Z.J. of STING binding with and by 2019; 567: PubMed Scopus Google Scholar of residues in the connector helix loop are to as class 1 SAVI mutations and and (see Fig 1, in the mutations a of the STING dimer structure into an conformation. This cannot explain the mechanism of autoactivation of class 3 mutations in the polymer interface, including p.R281Q, and which are not predicted to of the STING structural model that residues in the polymer interface bind a C-terminal which STING S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus Google Scholar The class 3 SAVI mutations might the to bind the C-terminal tail or an inhibitor is shown in the and cause autoactivation. the the 2 both at a in the polymer interface and STING activation. Our data in suggest a possible common mechanism to reconcile the 2 previously reported structural The broad autoactivation of R281 and R284 mutations strongly suggests that the polymer interface binds to an inhibitor to suppress STING autoactivation. the of this cGAMP binding to STING may not the polymer interface but may also lead to loss of inhibitor binding as a of the structural in the polymerization interface (in the top Fig G. Zhang C. Chen Z.J. Bai X.C. Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567: 389-393Crossref PubMed Scopus Google L. Chen X. Chen Z.J. GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon PubMed Scopus Google M. Y. et is the second by cyclic GMP-AMP Full Text Full Text PDF PubMed Scopus Google M. et innate immune DNA sensor cGAS a cyclic that activates human STING.Cell Rep. Full Text Full Text PDF PubMed Scopus Google A. M. G. I. et a cyclic second that activates PubMed Scopus Google X. H. Zhang X. L. Chen C. et GMP-AMP containing is an ligand for Full Text Full Text PDF PubMed Scopus Google S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus Google Scholar the bind to the 2 dimer on the and are shown in This 180o rotation and STING polymerization. STING with class 1 mutations the STING structure and inhibitor which leads to activation (in the of Fig the of connector helix loop is by a Our model likely the autoactivation for class 3 mutations than for class 1 the of the current structural is located in the dimer interface, and mutations into a methionine the the an dimer G. Zhang C. Chen Z.J. Bai X.C. Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567: 389-393Crossref PubMed Scopus Google Scholar This can in the which is supported by the that V155M mutations are disease causing in heterozygosity and a lower autoactivation than class 3 mutations, including R281Q, R284S, and (see also Fig 2, A and B). In contrast, for class 3 mutations in the polymer interface by a in the of Fig only the are to the inhibitor on both of the STING dimer (Fig with this hypothesis, the class 3 mutations either homozygosity to be disease causing (eg, or to be to cause SAVI in heterozygosity (eg, R281Q, R284S, and structural of the class 3 mutations are required to test this and of STING activation. Considering the of the STING C-terminal tail as an autoinhibitory S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus Google Scholar and of STING binding including Yang X. N. et of STING protein at the by Immunol. PubMed Scopus Google Scholar and S. J.S. B. et sensor regulates the type I interferon response by the adaptor STING at the Immunol. 2019; PubMed Scopus Google Scholar it to be whether these represent STING or form a expression of transfected constructs in HEK293T cells by constructs were transfected into HEK293T and STING1 and were in and in the same disease models for class 1 and class 3 The 2 of the STING dimer are shown in and of mutation are by are shown as a The model that a inhibitor is to the polymer interface of the wild-type STING which the polymer to the binding domain of STING or mutations in the connector region cause a rotation of the ligand-binding domain the connector helix which leads to the of the This polymer side-by-side and STING activation. in the polymer interface lead to of inhibitor which causes STING autoactivation. with from et G. Zhang C. Chen Z.J. Bai X.C. Zhang X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP-AMP.Nature. 2019; 567: 389-393Crossref PubMed Scopus Google and laboratory and of patients with homozygous STING1 c.841C>T, at clinical laboratory bilateral parenchymal to at age 5 at age 18 lung disease, failure to thrive, positive and and with with a lung disease and failure to thrive, positive and with baricitinib with a pulmonary and and pulmonary and with baricitinib with a failure to thrive, elevated and genetic 5 had a heterozygous mutation in but with normal with baricitinib with a 3 computed not Patient 5 had a heterozygous mutation in but with normal in a 3 computed not

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