Patient 5 is a 28-year-old Caucasian man, who presented from the age of one year with telangiectasias and chilblains lesions of nose, cheeks, hands and feet during winter months. Capillaroscopy revealed capillary dilation and tortuosity. Episodes of recurrent fevers started at age 26 without apparent triggers. His neurodevelopment was normal. He never developed failure to thrive, and denied arthritis, lymphadenopathy, hepatosplenomegaly, or respiratory symptoms. A chest CT performed at age 27 was normal, a CBC revealed lymphopenia. Inflammatory markers were normal and autoantibodies (including ANA, anti-dsDNA, antibodies to extractable nuclear antigens and antineutrophil cytoplasmic antibodies) were negative, serum immunoglobulins were normal. Genetic testing showed a heterozygous STING1 variant. p.F153V, which is not observed in healthy populations from gnomAD database. He is treated with calcium channel blockers.
Most SAVI patients present with an elevated interferon response gene signature recognized as elevated interferon scores. 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 mechanism remains unclear. Further investigation is needed to assess the interferon scores in various cell populations of the blood, and in patients with the same mutation from different kindreds that may become available in the future.
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 autoactivation was not due to higher expression of the mutant proteins compared to the WT ( Supplementary Figure S1 ). The p.F153V and p.H72N mutations remained responsive to ligand stimulation with cGAMP, while the p.G158A mutant was maximally activated at baseline and was not further enhanced by cGAMP stimulation. 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). Their activation mechanism differs and was previously described by different models. The p.G158A mutation is predicted by a rotation model to highly mimic the ligand-bound form of STING thus conferring autoactivation. The model suggests that cGAMP binding on wildtype STING triggers a 180  rotation of the ligand binding "head" of the STING dimer, which allows a closed dimer conformation, "flattening" of the polymer interface and STING polymerization via side-by-side packing. The STING oligomers recruit TBK1 and IRF3 which activate downstream signaling. 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 ). Mutation to the larger Alanine results in "space limitation" that is predicted by the model to "force" a 180  rotation of the ligand-binding head, which mimics the ligand-bound STING conformation thus conferring autoactivation.
The p.R284S and p.R284G highly-autoactivating mutations, however, are not likely to trigger a rotation and are better explained by a polymer interface blocking model. This model suggests that STING is kept inactive via C-terminal tail binding to the polymer interface, which blocks polymerization and prevents autoactivation. As a result, mutating the polymer interface residues to various residues lead to broad autoactivation. However, neither model explains STING autoactivation caused by the p.H72N and p.F153V mutations; both are unlikely to "push" a 180  rotation and are not located in the polymer interface.
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 ). However, neighboring mutations in the transmembrane linker region, p.E68A and p.E69A, resulted in loss-of-function which raised the question how the p.H72N mutation would confer a gain-of-function.
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 ). Mutations to the hydrophobic residue Leucine (L) resulted in a dramatic reduction in the cGAMP response. In contrast, mutations to residues with uncharged polar side chains, including Glutamine (Q) or Phenylalanine (F), resulted in responses similar to wildtype construct, with a maintained dose dependent cGAMP response. These data suggest that the charged and hydrophobic residues may result in improper STING dimer formation, while residues with uncharged polar side chains may maintain the wildtype configuration of the dimer.
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. The polymer interface, which is on the outer surface of the ligand binding head, would also become more flexible, which attenuates its binding to the C-terminal tail thus conferring the autoactivation.
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 ). The autoactivation via a 180  rotation is energy consuming and although likely favored by the p.G158A mutation that would "force" the rotation due to space limitations, it is not a likely mechanism in patients with the p.H72N mutation, which may only allow random rotations that are inefficient in causing autoactivation.
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). These findings are in concordance with a structural study of bacterial cyclic-di-GMP (CDG)-bound STING, which revealed that CDG can activate STING without a rotation and a STING dimer closing. Furthermore, STING binding to TBK1 is quite flexible through its unordered C-terminal tail, which was predicted to occur even in an open dimer conformation.
A presumed "small protrusion" in the polymer interface of unrotated STING was thought to be sufficient to keep STING inactive. However, the aforementioned examples of STING activation in an "unrotated state" (with the protrusion still present) raised questions about the exclusive validity of that model and led to the proposal of an inhibitor that blocked STING polymerization. In fact, the C-terminal tail binding can serve that function and no other inhibitor protein is needed, as autoinhibitory STING dimers can be formed in a cryo-EM structure where only STING is present.
These observations led to the development of an adjusted model of STING activation that accommodates all currently reported STING-causing mutations in exons 3, 5, 6 and 7. 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 ). Ligand binding induces 180  rotation of the ligand binding domain and leads to conformational change in the polymer interface, which results in loss of C-terminal tail binding. This clears the polymer interface and allows polymer formation via side-by-side packing ( Figure 3A ).
In this reconciled model, SAVI-causing mutations can be categorized into 4 classes. 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 ). Mutations in the polymer interface at residues R281, 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 ). A residue with reduced size such as the phenylalanine (F) to Valine (V) (p.F153V) mutation would be predicted to increase the flexibility of the polymer interface, thus relieving the autoinhibition by loosening the C-terminal tail binding. The p.G166E located in the ligand binding pocket constitutes class 4. This mutation confers a very low level of autoactivation and also abolishes cGAMP responsiveness ( Figure 2A ). Its autoactivating potential is not explained by current models, and it is not clear whether this low autoactivation causes SAVI. It is possible that binding of a non-cGAMP ligand is needed to activate STING and cause the disease.
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. However, the patient with the highly-autoactivating mutation, p.G158A, showed an intermediate phenotype, less severe than that of patients who develop interstitial lung disease and peripheral tissue loss including autoamputations. Her clinical picture was characterized by peripheral vasculopathy with nasal septal perforation, mild to moderate lung involvement, and hematologic manifestations (neutropenia). These findings indicate that disease severity in SAVI is influenced by additional factors that include family genetic backgrounds, treatment histories and yet unknown environmental factors such as infections which all may contribute to the variable disease severity which is even seen within the same family. Additional studies of patients from different families with the same mutations would help to address this question.