case_images: PMC8023226_01
case_images: Three novel variants in STING1 cause SAVI. (A-D) Erythematous lesions of the hands and tapered fingers in Patient 1 (A, B), Patient 2 (C) and Patient 4 (D). (E-G) Erythematous rash on the feet and tapered toes in Patient 1 (E), with similar lesions and tissue loss of the feet in Patient 2 (F) and Patient 4 (G). Patient number is depicted on the bottom right corner of each picture. (H) Pedigree charts. Solid symbols represent affected individuals; open symbols, unaffected individuals. P1-P5: patients, WT: wild type. (I) Exon locations of all SAVI-causing STING1 variants. Open boxes represent non-coding exons, solid boxes represent coding exons, while lines represent introns. Previously reported variants are in black and the three novel variants in red. Transcript diagram was obtained from ENSEMBL (). (J) 28-gene Interferon (IFN) scores from whole blood (WB) and peripheral blood mononuclear cells (PBMCs). Solid symbols indicate WB and open symbols indicate PBMCs. Healthy control (HC) samples included 19 WB and 10 PBMCs. SAVI patients with N154S mutation (n=3) in black, family members with H72N mutation (n=3) in red, and patient with G158A mutation (n=1) in blue. Paired PBMC and WB IFN scores from the same time point are connected by a line. Each patient with SAVI is represented by a different symbol.
case_images: fimmu-12-636225-g001.jpg
case_images: PMC8023226_04_fimmu-12-636225-g001.jpg
case_images: f1
case_images: As expected, interferon scores from peripheral blood mononuclear cell (PBMC) samples of patient 1-4 are much higher than those of healthy controls ( Figure 1J ); samples from patient 5 were not available. Notably, interferon scores from the whole blood samples were negative, which is different from previously reported SAVI mutants including p.N154S ( Figure 1J ). The autoactivation was not due to higher expression of the mutant proteins compared to the WT ( Supplementary Figure S1 ).
case_images: PMC8023226_02
case_images: Three novel variants in STING1 cause SAVI. (A-D) Erythematous lesions of the hands and tapered fingers in Patient 1 (A, B), Patient 2 (C) and Patient 4 (D). (E-G) Erythematous rash on the feet and tapered toes in Patient 1 (E), with similar lesions and tissue loss of the feet in Patient 2 (F) and Patient 4 (G). Patient number is depicted on the bottom right corner of each picture. (H) Pedigree charts. Solid symbols represent affected individuals; open symbols, unaffected individuals. P1-P5: patients, WT: wild type. (I) Exon locations of all SAVI-causing STING1 variants. Open boxes represent non-coding exons, solid boxes represent coding exons, while lines represent introns. Previously reported variants are in black and the three novel variants in red. Transcript diagram was obtained from ENSEMBL (). (J) 28-gene Interferon (IFN) scores from whole blood (WB) and peripheral blood mononuclear cells (PBMCs). Solid symbols indicate WB and open symbols indicate PBMCs. Healthy control (HC) samples included 19 WB and 10 PBMCs. SAVI patients with N154S mutation (n=3) in black, family members with H72N mutation (n=3) in red, and patient with G158A mutation (n=1) in blue. Paired PBMC and WB IFN scores from the same time point are connected by a line. Each patient with SAVI is represented by a different symbol.
case_images: fimmu-12-636225-g001.jpg
case_images: PMC8023226_04_fimmu-12-636225-g001.jpg
case_images: f1
case_images: As expected, interferon scores from peripheral blood mononuclear cell (PBMC) samples of patient 1-4 are much higher than those of healthy controls ( Figure 1J ); samples from patient 5 were not available. Notably, interferon scores from the whole blood samples were negative, which is different from previously reported SAVI mutants including p.N154S ( Figure 1J ). The autoactivation was not due to higher expression of the mutant proteins compared to the WT ( Supplementary Figure S1 ).
case_images: PMC8023226_03
case_images: Three novel variants in STING1 cause SAVI. (A-D) Erythematous lesions of the hands and tapered fingers in Patient 1 (A, B), Patient 2 (C) and Patient 4 (D). (E-G) Erythematous rash on the feet and tapered toes in Patient 1 (E), with similar lesions and tissue loss of the feet in Patient 2 (F) and Patient 4 (G). Patient number is depicted on the bottom right corner of each picture. (H) Pedigree charts. Solid symbols represent affected individuals; open symbols, unaffected individuals. P1-P5: patients, WT: wild type. (I) Exon locations of all SAVI-causing STING1 variants. Open boxes represent non-coding exons, solid boxes represent coding exons, while lines represent introns. Previously reported variants are in black and the three novel variants in red. Transcript diagram was obtained from ENSEMBL (). (J) 28-gene Interferon (IFN) scores from whole blood (WB) and peripheral blood mononuclear cells (PBMCs). Solid symbols indicate WB and open symbols indicate PBMCs. Healthy control (HC) samples included 19 WB and 10 PBMCs. SAVI patients with N154S mutation (n=3) in black, family members with H72N mutation (n=3) in red, and patient with G158A mutation (n=1) in blue. Paired PBMC and WB IFN scores from the same time point are connected by a line. Each patient with SAVI is represented by a different symbol.
case_images: fimmu-12-636225-g001.jpg
case_images: PMC8023226_04_fimmu-12-636225-g001.jpg
case_images: f1
case_images: As expected, interferon scores from peripheral blood mononuclear cell (PBMC) samples of patient 1-4 are much higher than those of healthy controls ( Figure 1J ); samples from patient 5 were not available. Notably, interferon scores from the whole blood samples were negative, which is different from previously reported SAVI mutants including p.N154S ( Figure 1J ). The autoactivation was not due to higher expression of the mutant proteins compared to the WT ( Supplementary Figure S1 ).
case_images: PMC8023226_04
case_images: Three novel variants in STING1 cause SAVI. (A-D) Erythematous lesions of the hands and tapered fingers in Patient 1 (A, B), Patient 2 (C) and Patient 4 (D). (E-G) Erythematous rash on the feet and tapered toes in Patient 1 (E), with similar lesions and tissue loss of the feet in Patient 2 (F) and Patient 4 (G). Patient number is depicted on the bottom right corner of each picture. (H) Pedigree charts. Solid symbols represent affected individuals; open symbols, unaffected individuals. P1-P5: patients, WT: wild type. (I) Exon locations of all SAVI-causing STING1 variants. Open boxes represent non-coding exons, solid boxes represent coding exons, while lines represent introns. Previously reported variants are in black and the three novel variants in red. Transcript diagram was obtained from ENSEMBL (). (J) 28-gene Interferon (IFN) scores from whole blood (WB) and peripheral blood mononuclear cells (PBMCs). Solid symbols indicate WB and open symbols indicate PBMCs. Healthy control (HC) samples included 19 WB and 10 PBMCs. SAVI patients with N154S mutation (n=3) in black, family members with H72N mutation (n=3) in red, and patient with G158A mutation (n=1) in blue. Paired PBMC and WB IFN scores from the same time point are connected by a line. Each patient with SAVI is represented by a different symbol.
case_images: fimmu-12-636225-g001.jpg
case_images: PMC8023226_04_fimmu-12-636225-g001.jpg
case_images: f1
case_images: As expected, interferon scores from peripheral blood mononuclear cell (PBMC) samples of patient 1-4 are much higher than those of healthy controls ( Figure 1J ); samples from patient 5 were not available. Notably, interferon scores from the whole blood samples were negative, which is different from previously reported SAVI mutants including p.N154S ( Figure 1J ). The autoactivation was not due to higher expression of the mutant proteins compared to the WT ( Supplementary Figure S1 ).
case_images: H72N, G158A, F153V variants lead to autoactivation. (A) Novel STING variants lead to strong STING autoactivation as measured by IFNB1 reporter activation in 293T cells. 50 pg of various STING constructs were transfected. The three novel SAVI-causing variants are labeled in blue, and other H72 variants are labeled in green. The SAVI-causing variants are shown in the order of autoactivating potential. *An asterisk above the white bar designating 0 mug/ml cGAMP indicates significant change compared (p<0.0001) to wildtype (WT) non-treated, by ordinary one-way ANOVA Dunnett's multiple comparisons tests. An asterisk above the black bar designating 20 mug/ml cGAMP indicates significant change (p<0.003) of 20 mug/ml cGAMP treated cells compared to the respective non-treated cells; by two-way ANOVA, Bonferroni's multiple comparisons test. Data are presented as mean+-SD of duplicates. Similar results were obtained in independent experiments. (B) Ribbon structural model of apo STING dimer, with the two subunits colored in yellow and green. Location of SAVI-causing variants are highlighted with spheres, with the three novel variants in red, variants in the connector helix loop region in blue, p.G166E variants in the ligand-binding pocket in cyan, and variants in the polymer interface in purple. The major mutation region is enlarged on the right, showing how the F153 phenyl ring buttress the polymer interface. (C) Structure model of H72 compared to N72 indicates increased dimer flexibility of p.H72N mutation, which confers gain-of-function. The two STING monomers forming a dimer are in green and gold/purple respectively.
case_images: fimmu-12-636225-g002.jpg
case_images: PMC8023226_04_fimmu-12-636225-g002.jpg
case_images: f2
case_images: To assess the autoactivating potential of the 3 novel variants, constructs expressing mutant or wild type (WT) STING were transfected into HEK293T cells and IFNB1 firefly luciferase reporter activation was measured ( Figure 2A ). All 3 mutant constructs showed autoactivation in the absence of ligand ( Figure 2A ). The constitutive auto-activation of p.G158A was even higher than WT construct activated with 20 microg/mL of cGAMP ligand ( Figure 2A ). The p.G158A mutation in the connector helix loop increased STING activation more than 13-fold, which is comparable to two other "highly-autoactivating" mutations, p.R284S and p.R284G, in the polymer interface ( Figure 2A , see also  Figure 2B  for the residue location). Consistent with this model, the p.G158 residue that is mutated in patient 1 is situated in a closely packed location of the apo STING dimer, where only Glycine, the smallest residue, can be accommodated ( Figure 2B ). While the previously reported SAVI-causing mutations are distributed around the dimer crossing-over region, the disease-causing p.H72N variant identified in patients 2-4 is the first SAVI-causing mutation in the transmembrane linker region of STING ( Figure 2B ) that, together with the N-terminal tail, forms a supporting arm which restrains the ligand-binding domain ( Figure 2B ). To interrogate how residue p.H72 might control STING activation, we generated various mutant constructs at the p.H72 position and assessed their autoactivating potential and their response to cGAMP stimulation in activating interferon activity ( Figure 2A ). Mutations to charged residues, including Lysine (K), Arginine (R), or Aspartic acid (D), led to a complete loss-of-function with no autoactivation and loss of a cGAMP response ( Figure 2A ). We hypothesized that residues with uncharged polar side chains at position p.H72 may maintain the "supporting arm" structure to restrain the ligand-binding head, and we generated structural models to investigate the effect of the mutation on its interactions with neighboring residues ( Figure 2C ). Structural modeling of the domain in fact confirms that residue p.H72 is essential in forming hydrogen bonds with residues 12 and 13 on the N-terminal tail of the second STING monomer within the STING dimer ( Figure 2C , left), which stabilizes a "supporting arm". If mutated to Asparagine (N), these hydrogen bonds cannot form firmly ( Figure 2C , right), which would result in a "looser support" of the ligand binding head and increased head flexibility. These findings highlight an essential role of hydrogen bonds at the p.H72 location: consistent with our observations, only polar residues can stabilize the supporting arm via hydrogen bonds, while both charged and hydrophobic residues fail to form hydrogen bonds and results in loss-of-function ( Figure 2A ). In fact, rotation of the ligand-binding head is also an unlikely consequence of the highly-autoactivating polymer interface mutations ( Figure 2A ), as these mutations are located in the dimer surface and should not cause a rotation of the ligand-biding head ( Figures 2A, B , purple labeled). R284, C206, and G207, constitute the second class, which directly abolishes binding to the autoinhibitory C-terminal tail and allows side-by-side packing without a 180  rotation ( Figure 3C ); p.H72N, p.F153V, p.V147L, and p.V147M constitute a third class which relieve the constraint on the polymer interface and autoactivate without a rotation. Residues H72 and V147 are part of the supporting arm which stabilizes the ligand-binding head and preserves the C-terminal tail binding to the polymer interface. Residue p.F153 lies right beneath the polymer interface to constrain it and prevents STING polymerization ( Figure 2B ). This mutation confers a very low level of autoactivation and also abolishes cGAMP responsiveness ( Figure 2A ). Patients with mutations that were of lower auto-activating potential p.G166E, p.F153V, p.H72N ( Figure 2A ), showed milder disease with less severe or no lung manifestations, which is in contrast to the p.N154S, p.V155M mutations.
case_images: A hypothetical STING activation model reconciles current structural models. (A) Wild type apo STING forms a dimer in open conformation, with C-terminal tail blocking the polymer interface and preventing polymerization. cGAMP binding leads to a 180  rotation and a closed dimer, which also changes the polymer interface conformation and subsequently loses C-terminal tail blocking. This leads to STING polymerization via side-by-side packing and TBK1 recruitment. (B) SAVI mutant G158A "pushes" the formation of a closed dimer, which mimics ligand-bound STING and confers autoactivation in the absence of ligand binding. (C) Some other SAVI mutants like R284G, R284S, H72N, F153V directly affect the polymer interface conformation, which leads to release of C-terminal tail binding. The STING dimer remains in the open state, but the polymer interfaces are free of C-terminal tail blocking, which allows polymerization and autoactivation.
case_images: fimmu-12-636225-g003.jpg
case_images: PMC8023226_04_fimmu-12-636225-g003.jpg
case_images: f3
case_images: We hypothesize that STING is kept inactive by the C-terminal tail blocking of the polymer interface, which prevents polymerization via side-by-side packing ( Figure 3A ). This clears the polymer interface and allows polymer formation via side-by-side packing ( Figure 3A ). The p.G158A variant and possibly the p.N154S and p.V155M variants, which are all located in the tightly packed connector region, mimic the ligand-bound STING dimer by favoring a "rotated" conformation ( Figure 3B ). R284, C206, and G207, constitute the second class, which directly abolishes binding to the autoinhibitory C-terminal tail and allows side-by-side packing without a 180  rotation ( Figure 3C ); p.H72N, p.F153V, p.V147L, and p.V147M constitute a third class which relieve the constraint on the polymer interface and autoactivate without a rotation. Residues H72 and V147 are part of the supporting arm which stabilizes the ligand-binding head and preserves the C-terminal tail binding to the polymer interface. Residue p.F153 lies right beneath the polymer interface to constrain it and prevents STING polymerization ( Figure 2B ).