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Disease-associated mutations identify a novel region inhuman STING necessary for the control of type I interferonsignalingDOI:10.1016/j.jaci.2016.10.031

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Citation for published version (APA):Melki, I., Rose, Y., Uggenti, C., Van Eyck, L., Frémond, M-L., Kitabayashi, N., Rice, G. I., Jenkinson, E. M., Boulai,A., Jeremiah, N., Gattorno, M., Volpi, S., Sacco, O., Terheggen-Lagro, S. W. J., Tiddens, H. A. W. M., Meyts, I.,Morren, M-A., De Haes, P., Wouters, C., ... Crow, Y. J. (2017). Disease-associated mutations identify a novelregion in human STING necessary for the control of type I interferon signaling. The Journal of allergy and clinicalimmunology. https://doi.org/10.1016/j.jaci.2016.10.031Published in:The Journal of allergy and clinical immunology

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Disease-associated mutations identify a novel region in human STING necessary 1

for the control of type I interferon signaling 2

3

Isabelle Melki, MD,a,b,c,d Yoann Rose, BSc,a,b Carolina Uggenti, PhD,a,b Lien Van 4

Eyck, MD,a,b,l Marie-Louise Frémond, MD,a,b,d Naoki Kitabayashi, BSc,a,b Gillian I 5

Rice, PhD,e Emma M Jenkinson, PhD,e Anaïs Boulai, BSc,a,b Nadia Jeremiah, PhD,b,p 6

Marco Gattorno, MD,f Sefano Volpi, MD, PhD,f Olivero Sacco, MD,g Suzanne W.J. 7

Terheggen-Lagro, MD,h Harm A.W.M. Tiddens, MD, PhD,i,j Isabelle Meyts, MD, 8

PhD,k,l Marie-Anne Morren, MD,l,m Petra De Haes, MD, PhD,l,m Carine Wouters, 9

MD, PhD,d,k,l Eric Legius, MD,n,o Anniek Corveleyn, PhD,o Frederic Rieux-Laucat, 10

PhD,b,p Christine Bodemer, MD, PhD,b,q Isabelle Callebaut, PhD,r Mathieu P Rodero, 11

PhD,a,b Yanick J Crow, MD, PhD,a,b,e 12

13 aINSERM UMR 1163, Laboratory of Neurogenetics and Neuroinflammation, Paris, 14

France 15 bParis Descartes University, Sorbonne-Paris-Cité, Institut Imagine, Paris, France 16 cGeneral Pediatrics, Infectious Disease and Internal Medicine Department, Hôpital 17

Robert Debré, AP-HP, Paris, France 18 dPediatric Hematology-Immunology and Rheumatology Department, Hôpital Necker-19

Enfants Malades, AP-HP, Paris, France 20 eManchester Centre for Genomic Medicine, Institute of Human Development, Faculty 21

of Medical and Human Sciences, Manchester Academic Health Centre, Manchester, 22

United Kingdom 23 fUO Pediatria 2 Reumatologia, G. Gaslini, Genova, Italy 24 gUO Pneumologia, G. Gaslini, Genova, Italy 25 hPaediatric Pulmonologist, Department of Paediatric Pulmonology Emma Children’s 26

Hospital AMC DD, Amsterdam, Netherlands 27 iDepartment of Radiology, Erasmus Medical Center, Rotterdam, Netherlands 28 jDepartment of Paediatrics, Respiratory Medicine and Allergology,Erasmus Medical 29

Center-Sophia Children’s Hospital, Zuid-Holland, Netherlands 30 kDepartment of Pediatrics, University Hospitals Leuven, Leuven, Belgium 31 lDepartment of Immunology and Microbiology, Childhood Immunology, University 32

of Leuven, Leuven, Belgium 33 mDepartment of Dermatology, University Hospitals Leuven, Leuven, Belgium 34

 

nDepartment of Human Genetics, KU Leuven, Leuven, Belgium 35 oClinical Department of Human Genetics, University Hospitals, Leuven, Belgium 36 pINSERM UMR 1163, Laboratory of Immunogenetics of Pediatric Autoimmunity, 37

Paris, France 38 qDermatology Department, National reference center for rare skin diseases MAGEC, 39

Hôpital Necker-Enfants Malades, Paris, France 40 rCNRS UMR7590, Sorbonne Université, Université Pierre et Marie Curie-Paris6–41

MNHN–IRD–IUC, Paris, France 42

43

Corresponding author: Yanick J Crow 44

INSERM UMR 1163, Laboratory of Neurogenetics and Neuroinflammation, Paris, 45

France, 24, boulevard du Montparnasse, 75015 Paris, France. Mail: 46

yanickcrow@mac.com, Phone: +331 42 75 44 51, Fax: +331 44 49 50 70 47

48

Sources of funding: This study was funded by the European Research Council, the 49

National Research Agency (France), the Imagine Institute (France), the Research 50

Foundation Flanders (FWO) and Telethon (Italy). 51

 

Capsule summary 52

53

Three new mutations in TMEM173 are reported in patients presenting STING-54

associated autoinflammation. These mutations confer constitutive activation 55

independent of ligand stimulation, revealing a novel functionally important region of 56

the protein. 57

58

Key words 59

60

Stimulator of interferon genes (STING), TMEM173, interferon, type I 61

interferonopathy, STING associated vasculopathy with onset in infancy (SAVI) 62

 

Abstract 63

64

Gain-of-function mutations in TMEM173 encoding STING (stimulator of interferon 65

genes) underlie a recently described type I interferonopathy. We report three 66

individuals variably exhibiting the core features of STING-associated vasculopathy 67

with onset in infancy (SAVI) including systemic inflammation, destructive skin 68

lesions and interstitial lung disease. Molecular and in vitro data demonstrate that the 69

pathology in these patients is due to substitutions at positions 206, 281 and 284 of the 70

human protein. These mutations confer cGAMP-independent constitutive activation 71

of type I interferon signaling through TBK1 (TANK binding kinase). Structural 72

analysis indicates that these three amino acids lie in a discrete region of the protein, 73

thereby implicating a novel cluster of amino acids in STING as functionally important 74

in the regulation of type I interferon signaling. 75

76

Key Messages 77

78

• p.Cys206, p.Arg281 and p.Arg284 are new mutations in TMEM173 identified 79

in patients with STING-associated vasculopathy with onset in infancy (SAVI). 80

• These mutations confer constitutive activation independent of ligand 81

stimulation, thereby revealing a novel, functionally important region of the 82

protein. 83

84

Abbreviations 85

86

3D: Three-dimensional; AMP: Adenosine monophosphate; ANA: Antinuclear 87

antibodies; BSA: Bovine serum albumin; c: Complementary DNA; CBD: cGAMP 88

binding domain; cGAMP: 2'3'-cyclic GMP-AMP; cGAS: Cyclic GMP-AMP 89

synthase; cm: Centimeter; CT: Computed tomography; CTD: C-terminal cyclic 90

dinucleotide-binding domain; DNA: deoxyribonucleic acid; ER: Endoplasmic 91

reticulum; ERGIC: ER-Golgi intermediate compartment; ExAC: Exome Aggregation 92

Consortium; GMP: Cyclic guanosine monophosphate; HEK: Human embryonic 93

kidney; IFN: Interferon; IFNα: Interferon alpha; IFNAR: Interferon alpha receptor; 94

IFNß: Interferon beta; IRF3: Interferon regulatory factor 3; ISGs: Interferon-95

 

stimulated genes; JAK1: Janus kinase 1; Kg: Kilogram; mAb: Monoclonal antibody; 96

MAFFT: Multiple alignment using fast fourier transform; P: Patient; pcDNA: plasmid 97

cDNA; PCR: Polymerase chain reaction; PDB: Protein Data Bank; RCSB: Research 98

Collaboratory for Structural Bioinformatics; RNA: Ribonucleic acid; p: Protein; 99

SAVI: STING associated vasculopathy with onset in infancy; SD: Standard deviation; 100

STAT1: Signal transducer and activator of transcription 1; STING: Stimulator of 101

interferon genes; TBK1: TANK binding kinase; TBS: Tris-buffered saline; Tm: 102

Melting temperature; TMEM173: Transmembrane protein 173; TNF: Tumor necrosis 103

factor; TYK2: Tyrosine kinase 2; WB: Western blot; WT: Wild type. 104

105

 

Introduction 106

107

Gain-of-function mutations in TMEM173 encoding STING (stimulator of interferon 108

genes) have been described to cause an autoinflammatory syndrome1–8 belonging to a 109

recently defined class of disorders referred to as the type I interferonopathies9,10. This 110

phenotype, known as STING associated vasculopathy with onset in infancy (SAVI), 111

is characterized by early-onset systemic inflammation with fever, a severe skin 112

vasculopathy leading in some cases to extensive tissue loss, and interstitial lung 113

disease resulting in pulmonary fibrosis and end-stage respiratory failure1–5,7,8 114

115

To date, twenty-three patients from fifteen families have been reported with gain-of-116

function mutations in STING1–8, a key adaptor molecule in the cytosolic DNA-117

sensing pathway, expressed in various endothelial and epithelial cell types, as well as 118

in haematopoetic cells, such as T cells, macrophages and dendritic cells. Once 119

stimulated, STING induces the transcription of type I interferons (IFN) and the 120

expression of a set of IFN-stimulated genes (ISGs), thus establishing an antiviral 121

state11–13. In all of these published cases the heterozygous substitution involves one of 122

three amino acids at positions 147, 154, 155, and 166, being located either in the 123

linker connecting the transmembrane domain of STING to the cyclic dinucleotide 124

domain (CTD) (p.Val147), or within the N-terminal dimerization region of the CTD 125

(p.Asn154, p.Val155 and p.Gly166)14,15. It has been suggested that these disease-126

associated residues play non-redundant roles in retaining the protein on the 127

endoplasmic reticulum (ER), causing STING to constitutively localize to the ER-128

Golgi intermediate compartment (ERGIC) and activate downstream signaling through 129

the TANK binding kinase (TBK1) - Interferon regulatory factor 3 (IRF3) axis15,16. 130

131

We describe three individuals variably exhibiting the characteristic features of 132

STING-associated autoinflammation. However, in contrast to earlier reports, our data 133

show that the pathology in these patients is due to substitutions at positions 206, 281 134

and 284 of the human protein, thus implicating a novel region of STING as 135

functionally important in the regulation of type I IFN signaling. 136

 

Methods 137

138

Patient and study approval 139

The study was approved by the Comité de Protection des Personnes (ID-140

RCB/EUDRACT: 2014-A01017-40) and undertaken with written informed parental 141

consent. 142

143

Genetic analysis 144

DNA was extracted from whole blood samples using standard methods. Whole-145

exome sequencing was performed on genomic DNA from patient 2 (P2) and her 146

mother using SureSelect Human All Exon kit (Agilent Technologies) for targeted 147

enrichment and Illumina HiSeq2000 for sequencing. Sanger sequencing was 148

performed on DNA from patient 1 (P1), patient 3 (P3) and their parents, and from P2 149

and her mother to confirm the variant found by exome sequencing (Primers in Table 150

EII). Variant frequency data in controls was derived from the Exome Aggregation 151

Consortium (ExAC)17. 152

153

Structural analysis 154

The experimental 3D structures of STING were extracted from the Research 155

Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) and 156

analyzed using Chimera (https://www.cgl.ucsf.edu/chimera/). 157

158

Construct generation 159

Site-directed mutagenesis was performed to generate the desired TMEM173 variants 160

by using the Q5® Site-Directed Mutagenesis Kit (E0554S from NEB) following the 161

manufacturer’s instructions. pMSCV-hygro(+) (Addgene) carrying wild type (WT) 162

TMEM173 gene was used as a template for the PCR reactions18. This plasmid carries 163

the haplotype most frequently recorded in the general population (p.Arg71, p.Gly230, 164

p.Arg232, p.Arg293)19. Mutagenic primer sequences were designed using the NEBase 165

Changer software (http://nebasechanger.neb.com/). Cycling parameters are indicated 166

in Table EI. NEB 5-alpha Competent E. coli were transformed with the newly 167

synthesized pcDNA, and colonies were screened for the presence of the desired 168

variants (primers in Table EII). 169

170

 

Cell culture procedures 171

Human embryonic kidney (HEK) 293T cells were grown in 96-well plates at 37°C in 172

5% CO2 in DMEM (GIBCO #31966-021) supplemented with 10% (v/v) fetal bovine 173

serum (GIBCO #10270), 100 U/ml penicillin and 100 mg/ml streptomycin (GIBCO 174

#15070-063). At 70% confluency, HEK293T cells were co-transfected with 60 ng of 175

vector pMSCV- hygro(+) either empty or encoding the TMEM173 variants, 40 ng of 176

IFNß promoter-driven firefly luciferase reporter plasmid (IFNß-pGL3) and 1.4 ng of 177

constitutively expressed renilla luciferase reporter plasmid (pRL-TK) by using 178

TransIT-293 (Mirus #MIR2700 from Euromedex). 24 hours later, cells were 179

stimulated by transfecting 1.3-12 µg/ml 2’3’-cGAMP_STING ligand (tlrl-nacga23, 180

InvivoGen) using lipofectamine 2000 (#11668027 from Thermo Fischer). 24 hours 181

after cGAMP stimulation, cells were lysed with passive lysis buffer 5X (#E194A 182

from Promega) containing protease and phosphatase inhibitors. One third of the lysate 183

for each condition was used for the luciferase assay, and the remaining material for 184

protein analysis by western blotting (WB). 185

186

Luciferase assay 187

The IFNß-pGL3 plasmid was used to measure IFNß promoter activity, and the renilla 188

reporter control plasmid to normalize for transfection efficiency. Luciferase assays 189

were performed using the Dual-Glo® Luciferase Assay System (E2940 from 190

Promega) following the manufacturer’s protocol. Luminescence was acquired on a 191

FLUOstar OPTIMA microplate reader (BMG LABTECH). Firefly luciferase activity 192

was normalized against renilla luciferase activity. 193

194

Western blot 195

Bolt LDS Sample Buffer (4X) (#B0008 Novex Life Technologies) and Bolt Sample 196

Reducing agent (10X) (#B0009 Novex Life Technologies) were added to protein 197

lysates, and samples were resolved on 4-12% Bis-Tris Plus gels (# NW04122BOX 198

Invitrogen) transferred to nitrocellulose membrane (#IB23002 Invitrogen). When the 199

phosphorylation status of the protein was investigated, membranes were blocked in 5 200

% BSA in TBS, and primary phospho-antibodies were incubated overnight in the 201

blocking solution. Otherwise, membranes were blocked with 5 % milk in TBS and 202

primary antibodies were incubated overnight in the blocking solution. Proteins were 203

 

blotted with monoclonal anti-STING (R&D MAB7169), anti-Phospho IRF3 Rabbit 204

mAb (Ser396, #4947 Cell Signaling), anti-IRF3 Rabbit mAb (#11904 Cell Signaling) 205

and anti-cofilin Rabbit mAb (#5175 Cell Signaling). Primary antibodies were detected 206

with horseradish peroxidase–conjugated secondary antibody (#7074S and 7076S, Cell 207

Signaling). All western blot images were captured and quantified with a ChemiDoc 208

MP imager and Image Lab software (Bio-Rad Laboratories, Hercules, CA) after 209

adding Clarity Western ECL substrate (Biorad). 210

211

STAT1 phosphorylation assay staining 212

Ficolled PBMCs were treated or not with ruxolitinib 1 µM for 45 minutes at 37°C. 213

Cells were fixed and permeabilized according to PerFix EXPOSE Kit from Beckman 214

Coulter (#B26976) recommendations. Cells were stained for pSTAT1 PE-anti-STAT1 215

pY701, and cell surface marker (PE-CyTM 7-CD19, APC-CD3, BV421-CD8) for 1 216

hour at room temperature. Flow cytometry analysis was performed on a Gallios 217

Beckman Coulter flow cytometer. Results were analyzed using Kaluza software v1.3. 218

219

Results 220

221

Clinical presentation (Table I, Fig 1) 222

223

P1 (F1058) is a 7-year-old female born to non-consanguineous parents of white 224

European ethnicity. She was delivered at term weighing 3.2kg. She presented at the 225

age of 3 months with feeding difficulties and respiratory distress necessitating oxygen 226

therapy (Table I). Computed tomography (CT) scan of the lungs at age 1.5 years 227

revealed extensive ground glass abnormalities, and there was interstitial fibrosis on 228

lung biopsy without signs of vasculitis (Fig 1, A). Telangiectatic skin lesions on the 229

cheeks and nose were noted from the age of 4 months (Fig 1, B). She has not 230

experienced any involvement of the digits. Limited skin biopsy did not reveal any 231

sign of vasculitis. Elevated transaminases and hepatomegaly prompted a liver biopsy 232

at age 2 years, when the presence of necrotizing granulomatous hepatitis was 233

suggested (intralobular granulomas characterized by necrotizing zones with mixed 234

inflammatory infiltrates). She has demonstrated no further evidence of liver disease. 235

She had a gastrostomy fitted at age 2 years because of poor weight gain, and 236

subsequently suffered recurrent skin infections with poor healing around the entry 237

 

site. In light of a continued oxygen requirement and raised inflammatory markers she 238

was treated with immunosuppressants (steroids, methotrexate and anti-TNFα therapy) 239

starting at age 23 months, but with limited efficacy. She remains oxygen dependent 240

with a severe deficit of lung function and markedly limited exercise tolerance. 241

242

P2 (F1125) is a 25-year-old female born to non-consanguineous parents of white 243

European ethnicity. There is no family history of note. She demonstrated intra-uterine 244

growth retardation (birth weight at term -3 SD), with subsequent failure to thrive 245

(adult weight / height of 143 cm and 34 kg) requiring a gastrostomy to be placed by 246

age 1 year until puberty. She suffered recurrent bacterial infections of the upper 247

respiratory tract, leading to a severe septicemia at the age of five months (due to 248

Pseudomonas aeruginosa) and palatal and nasal septum necrosis. Screening for 249

primary immune deficiencies was negative. A widespread livedo was noted from 250

birth, which worsened gradually with an impressive purpuric aspect in patches on 251

both cheeks and buttocks, and a marked dark purple marbling on the limbs (Fig 1, C 252

and D). When last seen at the age of 25 years she had lost a large part of the external 253

nares, and demonstrated extreme acrocyanosis of the hands and feet. She has never 254

experienced fevers, and systemic markers of inflammation and autoantibody titers 255

have been consistently normal until recent antinuclear antibodies (ANA) were 256

detected at a low level (1/160). Lung function is normal. She is not currently treated. 257

258

P3 (F1802) is a 15-year-old male born to non-consanguineous parents of white 259

European ethnicity. A fixed erythema of the cheeks was noted shortly after birth. 260

Telangiectasia of the cheeks, buttocks and limbs were observed for the first time at 261

age 10 years. From the age of 14 years he has suffered recurrent acrocyanosis 262

moderately influenced by cold temperature, with painful acral erosions that heal as 263

atrophic scars on the ear helices and digits. Lesional gluteal skin biopsies revealed 264

dilated capillaries without obvious vasculitis or thrombosis. He has been treated with 265

nifedipin, pentoxyfillin and acetyl salicylic acid, with limited efficacy. He has never 266

experienced fevers or other features of systemic disease. Blood analysis, including 267

markers of inflammation and autoantibody profile, has been consistently normal. 268

Recent chest CT revealed no evidence of interstitial lung disease. Lung function 269

testing and a six minute exercise tolerance test were normal. 270

271

 

Identification of three new variants in STING 272

273

A summary of the molecular data relating to these three patients is given in Figure 2 274

and Table I. A multiple sequence alignment of STING proteins from various species 275

is provided in Supplementary Figure E1. 276

277

P1 (F1058) was observed to carry a c.842G>A (p.Arg281Gln) variant in exon 7 of 278

TMEM173 (Fig 2, A) which has not been recorded on more than 120,000 control 279

alleles on the ExAC database. Both parents were WT at this nucleotide, and 280

microsatellite testing confirmed that the substitution had arisen de novo. The arginine 281

at position 281 is conserved in mouse, but is an alanine in Xenopus tropicalis and a 282

glutamate in Danio rerio. We note a similarly limited degree of conservation of the 283

valine at position 147, substitution of which for a leucine has been previously shown 284

to act as a gain-of-function mutation1. 285

286

P2 (F1125) was observed to carry a c.850A>G (p.Arg284Gly) variant in exon 7 of 287

TMEM173 (Fig 2, A) which has not been recorded on more than 120,000 control 288

alleles on the ExAC database. The mother was WT for this variant. DNA was not 289

available from the father. The arginine is conserved to Nematostella vectensis. 290

291

P3 (F1802) was observed to carry a c.617G>A (p.Cys206Tyr) variant in exon 6 of 292

TMEM173 (Fig 2, A) which has not been recorded on more than 120,000 control 293

alleles on the ExAC database. Both parents were WT at this nucleotide, and 294

microsatellite testing confirmed that the substitution had arisen de novo. The cysteine 295

at this position is generally well conserved, except in Danio rerio where it is replaced 296

by an alanine. 297

298

3D structural analysis reveals a new mutation hotspot in STING 299

300

In order to gain further insight into the effect of the three STING substitutions that we 301

observed in the patients described above, we examined the experimental 3D structures 302

of the CTD of STING (Fig 2, B). p.Cys206 is buried within the 3D structure, whilst 303

p.Arg281 and p.Arg284 lie on the surface of the protein. However, these three amino 304

acids are in close proximity and are aligned, with p.Arg284 located in between 305

 

p.Cys206 and p.Arg281 (Fig 2, C and D). The substitution of the cysteine for a 306

tyrosine is predicted to induce a steric clash, which might locally destabilize the 3D 307

structure of the helix bundle and affect the positioning of the 281 / 284 arginine 308

residues. 309

310

p.Cys206, p.Arg281 and p.Arg284 mutations in STING induce over-activation of 311

IFNß transcription 312

313

In order to determine if these mutations were gain-of-function, we used a luciferase-314

based IFNß transcription reporter assay to measure their impact on type I IFN 315

induction1,15,16. As previously shown, the p.Val155Met and p.Asn154Ser mutants 316

induce reporter activity in the absence of ligand (Fig 2, E)1,2,15. We observed a 317

similarly robust activation with patient-associated substitutions at p.Cys206Tyr, 318

p.Arg281Gln and p.Arg284Gly (Fig 2, E), supporting the hypothesis that these 319

variants are constitutively active. 320

321

To investigate whether these three variants lie within a novel functional cluster in 322

STING, we went on to mutate other residues spatially close to the amino acids at 206, 323

281 and 284. Specifically, we changed p.Asp205 to p.Asp205Tyr and p.Asp205His, 324

p.Leu265 to p.Leu265Tyr, p.Phe269 to p.Phe269Tyr, p.Gln273 to p.Gln273Ala and 325

p.Gln273Leu, and p.Glu282 to p.Glu282Ala and p.Glu282Asp (Fig 3, A and B). All 326

these amino acids are exposed at the surface of the protein, except p.Leu265 which is 327

buried and in contact with p.Cys206. In contrast to our patient-associated variants, 328

substitution of any of these residues, except for the aspartate at position 205, behaved 329

in a manner similar to WT (Fig 3, C and Table EIII). In contrast, substitution of the 330

p.Asp205 resulted in an upregulation of IFNß reporter activity as for the gain-of-331

function variants (Fig 3, C and Table EIII). 332

333

Altogether, these results suggest the identification of a novel region implicated in 334

STING regulation, involving at least four amino acids. The fact that all of the 335

different substitutions tested at these residues (two, four, three and three at p.Asp205, 336

p.Cys206, p.Arg281 and p.Arg284 respectively) result in increased IFNß activity 337

indicates that subtle conformational changes are sufficient to disrupt the normal 338

control of the pathway. The specificity of this effect was emphasized by the fact that 339

 

four other amino acids, p.Leu265, p.Phe269, p.Gln273 and p.Glu282, located in close 340

proximity in the same tertiary structure did not induce a gain-of-function when 341

substituted. 342

343

STING gain-of-function induced by p.Cys206Tyr, p.Arg281Gln and 344

p.Arg284Gly is independent of cGAMP binding 345

346

While all previously described STING-associated mutations are located close to the 347

cGAMP binding site, the three novel mutations described here lie on the surface of 348

the protein, without direct contact with this binding site (Fig 2, B). In order to 349

determine if the gain of function observed in association with our new mutations 350

resulted from ligand independent constitutive activation, or reflected an increased 351

sensitivity to low levels of endogenous cGAMP, we generated double mutants 352

involving the arginine at 232 which is essential for cGAMP binding14,15. For every 353

previously and newly described mutation we observed a similarly robust upregulation 354

of signaling, suggesting that despite clearly distinct localization in the tertiary 355

structure they all confer ligand-independent constitutive activation (Fig 4, A). 356

357

Furthermore, using an antibody against phosphorylated residue p.Ser396 of IRF3, we 358

recorded increased phosphorylation of IRF3 in cells expressing the three patient-359

associated mutations, comparable with the previously described p.Val155Met. 360

Consistent with our luciferase results, this increased IRF3 phosphorylation was not 361

impaired by introducing a second mutation at p.Arg232Ala, which abrogates cGAMP 362

binding and IRF3 phosphorylation in the WT state (Fig 4, B and E2, A and D). 363

364

STING gain-of-function induced by p.Cys206Tyr, p.Arg281Gln and 365

p.Arg284Gly is independent of the membrane fusion pathway 366

367

Recently, it has been reported that a STING-dependent but cGAS-independent 368

pathway can induce type I IFN expression in response to membrane fusion of 369

enveloped RNA viruses20–22. In this model, the arginine at 169 in human STING was 370

shown to be important for stimulation of IFN expression by liposomes but not 371

cGAMP, demonstrating that activation by these two stimuli can be mechanistically 372

separated. p.Arg169 is surface exposed, located just outside the STING dimerization 373

 

domain and facing away from the cGAMP-binding pocket. To determine if the 374

arginine at position 169 was crucial in the constitutive activation that we observed 375

with our patient-associated mutations, we substituted an alanine for the wild-type 376

arginine at 169 (p.Arg169Ala) and expressed the double-mutants in our in vitro 377

system. We observed no effect of the 169 substitution with mutations at 206, 281 and 378

284, or with the previously described mutations at 154 and 155, indicating that human 379

disease-associated constitutive activation of STING is not dependent on this 380

alternative pathway (Fig 5, A). A similar result was obtained with the double mutant 381

p.Asp205His and p.Arg169Ala (Fig E2, B and E). Consistent with these data, 382

constitutively activated constructs bearing the second mutation p.Arg169Ala 383

demonstrated phosphorylated IRF3 at p.Ser396 in the basal state (Fig 5, B and E2, B 384

and E). 385

386

Constitutive activation of newly identified disease-causing STING mutants is 387

dependent on IRF3 phosphorylation 388

389

Phosphorylation of STING at p.Ser366 by TBK1 is critical for the recruitment and 390

activation of IRF3 by STING and the subsequent activation of IFN signaling23,24. To 391

investigate whether SAVI-associated variants were dependent on TBK1-mediated 392

STING phosphorylation and IRF3 recruitment, we introduced a mutation of serine to 393

alanine at position 366 (p.Ser366Ala) into our mutant constructs. In all cases, this 394

substitution led to an abolition of IFNß activation despite similar expression of single 395

and double STING mutants (Fig 6, A and B). This was associated with loss of IRF3 396

phosphorylation, indicating that patient-associated mutations are dependent on 397

phosphorylation at residue 366 for subsequent downstream activation of IRF3 (Fig 6, 398

B and Fig E2, C and D) and transcription of ISGs. 399

400

STING constitutive activation leads to increased phosphorylation of STAT1 in T 401

and B cells of P3 and normalize after ruxolitinib treatment in vitro 402

403

Type I interferons bind to a specific IFNα receptor (IFNAR), thereby activating Janus 404

kinase 1 (JAK1) and Tyrosine kinase 2 (TYK2), which then induce phosphorylation 405

of Signal transducer and activator of transcription 1 (STAT1). We investigated 406

STING constitutive activation in CD4, CD8 and CD19 cells from P3, and observed 407

 

increased levels of STAT1 phosphorylation compared to a control (Fig 7). Consistent 408

with published data, this upregulation normalized after 45 minutes of treatment with 409

ruxolitinib in vitro (Fig 7)1,5. 410

411

Discussion 412

413

Since its identification in 2008 as an adaptor molecule in the cytosolic DNA-sensing 414

pathway, STING has emerged as a central player in antiviral immunity, 415

autoinflammation and cancer11,13. Thus, an understanding of the mechanism of action 416

and control of STING in health and disease is of considerable scientific and medical 417

importance. 418

419

Here we describe three novel variants seen in association with a phenotype consistent 420

with previously described cases of STING-associated autoinflammation. In two of 421

these patients we were able to show that the amino acid substitutions arose de novo, 422

whilst in the third the mother was WT but DNA was unavailable from the father. 423

These substitutions, involving evolutionarily well-conserved residues, have not been 424

described in publically available databases comprising more than 120,000 alleles. As 425

for other molecularly confirmed cases of STING-associated autoinflammation, all 426

three substitutions were associated with the in vitro induction of IFNß reporter 427

activity. Considering these observations, it is very likely that the variants we describe 428

represent pathogenic gain-of-function mutations. 429

430

In contrast to all previously published cases of type I interferonopathy related to 431

mutations in STING, the three variants reported here do not lie in the linker region 432

connecting the N-terminal transmembrane domain of STING to the CTD or in the 433

first a-helix (a5) of the CTD involved in dimerization. The function of the arginine 434

residues at 281 and 284 is currently undefined. However, these residues are exposed 435

at the surface of the molecule, so that they are unlikely to involve the intramolecular 436

interaction of the STING dimer. A recent survey of STING mutations in the COSMIC 437

database led to the demonstration that a substitution of the arginine at 284 to 438

methionine (p.Arg284Met) was able to induce an IFNß reporter to a significantly 439

greater extent than WT STING25. Alternative substitutions of arginine 284 for lysine 440

or threonine (p.Arg284Lys, p.Arg284Thr) also represented hyperactive mutants. The 441

 

results of these experiments were further supported by demonstrating increased IFNß 442

in tissue culture supernatants, and an enhanced ability to inhibit viral replication. Of 443

note, the mutation identified in P2 affects this same residue, substituting the arginine 444

for a glycine. Thus, multiple types of amino acid substitution at 284 confer a gain-of-445

function, indicating a high degree of specificity for the function of an arginine at this 446

position. 447

448

Similar to the arginine at 284, we found that different substitutions of the arginine at 449

281 and the cysteine at 206 also resulted in constitutive activation. It is of note that 450

these three amino acids are aligned at the 3D level, with the arginine at 284 located in 451

between the cysteine at 206 and the arginine at 281. The substitution of the cysteine 452

for a tyrosine at 206 is predicted to lead to steric hindrance and locally disturb the 453

fold, which might affect the positioning of the arginine at 284. However, substituting 454

other less bulky amino acids at 206 had the same effect, highlighting the specificity of 455

the WT cysteine at this position. 456

457

In order to define the extent of a putative functionally important region involving the 458

amino acids that we identified through human disease genetics, we went on to mutate 459

a selection of other residues predicted to lie in close proximity with these disease-460

associated mutations - specifically, p.Leu265, p.Phe269, p.Gln273 and p.Glu282. We 461

also derived a number of conservative and non-conservative substitutions at Asp205, 462

which lies in contact with the p.Arg284. Substitutions at these residues behaved in a 463

similar manner to WT protein, except for those at position 205, where we observed 464

enhanced signaling in our in vitro assay similar to the patient-associated mutations. Of 465

note, constitutive activation has been described previously at the equivalent residue in 466

murine STING12,26. 467

468

In regards of cGAMP-dependency, the involvement of a cGAMP-independent 469

signaling pathway and TBK1 downstream signaling, our data did not suggest any 470

difference in the behavior of variants at 206, 281 and 284 compared to previously 471

described disease-associated mutations at 147, 154 and 155. Recently, Dobbs et al. 472

suggested that the latter mutations may disrupt the retention of STING in the ER, 473

resulting in translocation to the ERGIC and activation of TBK1 / IRF3 in the absence 474

of cGAMP binding15. Furthermore, these mutations appeared to result in continued 475

 

activation, presumed due to a failure of STING degradation. Our data clearly 476

implicate a novel region of STING as important in type I IFN signaling, leading us to 477

speculate that the surface-exposed arginine residues at 281 and 284 may also be 478

involved in ER retention, or in binding to a negative regulator of STING25. 479

480

Our results indicate that mutations at residues 206, 281 and 284 of human STING are 481

responsible for the disabling autoinflammation observed in the three patients that we 482

describe. Given the favorable effects seen with JAK1/2 inhibition in other patients 483

with STING gain-of-function mutations, these patients may similarly benefit from 484

such a therapeutic approach5. Alternative potential treatment strategies might include 485

ER exit blocking agents, palmitoylation inhibitors or TBK1 antagonists15,27–29. 486

Considering that a p.Ser366Ala substitution abrogated the constitutive activation seen 487

with every patient-associated mutation tested in our in vitro model, TBK1 blockade 488

might be relevant to all SAVI patients. 489

490

A major aspect of the pathology due to mutations in TMEM173 appears to relate to a 491

dysfunction of endothelium consequent upon an upregulation of type I IFN signaling. 492

The explanation for variable expression observed between, and indeed within 493

families, will require further study, with such observations likely reflecting 494

differential environmental exposures and / or modifying genetic factors. Indeed, 495

whilst the cGAS-STING cytosolic DNA signaling pathway is essential for the 496

induction of an effective anti-viral response, human genetics is teaching us about the 497

risk of inflammatory disease associated with variation in this system. Polymorphisms 498

in STING, and other molecules involved in cytosolic nucleic acid recognition, can 499

confer differences in signaling to a type I IFN response - a non-binary situation where 500

a balance is struck between anti-viral priming and the risk of autoinflammation19,30. 501

Elucidation of the mechanisms of STING regulation is thus of great importance in 502

understanding both microbial pathogenesis and inflammation. The findings presented 503

here suggest a previously unappreciated aspect of the control of STING in this 504

context. 505

 

Acknowledgements 506

Y.J.C. acknowledges the European Research Council (GA 309449: Fellowship), and a 507

state subsidy managed by the National Research Agency (France) under the 508

"Investments for the Future" program bearing the reference ANR-10-IAHU-01. FRL 509

acknowledges a state subsidy managed by the National Research agency referenced as 510

Lumugene (ANR-14-CE14-0026-01). IM acknowledges the Programme Santé-511

Sciences MD/PhD of Imagine Institute. S.V. and M.G. gratefully acknowledge the 512

financial support of Telethon, Italy (Grant no. # GGP15241A). L.V.E is funded by a 513

research grant from the Research Foundation Flanders (FWO). We thank Nicolas 514

Manel for WT STING, p.Val155Met STING and IFNß-pGL3 plasmids. The authors 515

would like to thank the Exome Aggregation Consortium and the groups that provided 516

exome variant data for comparison. A full list of contributing groups can be found at 517

http://exac.broadinstitute.org/about. 518

519

Conflict of interest 520

The authors declare no conflict of interest. 521

 

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601

 

Figure Legends 602

603

TABLE I. Clinical and molecular data. 604

ANA: Antinuclear Antibodies; ANCA: Anti-neutrophilic cytoplasmic antibodies; F: 605

Female; M: Male 606

607

FIG 1. Clinical and radiological phenotypes. A. High resolution chest CT scan of 608

P1 demonstrating evolution of bilateral alveolar and interstitial disease with ground-609

glass lesions and interlobular septa thickening (at ages 3, 5 and 8). Improvement of 610

interstitial lung disease between ages 3 and 5 years was observed after oral steroid 611

treatment. B. Telangiectatic skin lesions on the cheeks and nose of P1. C. Purpuric 612

aspect to patches on both cheeks of P2 at age 1.5 years. D. Widespread livedo and 613

marked dark purple marbling on the limbs of P2 at age 1.5 years. y: years. 614

615

FIG 2. Constitutively activated mutations of TMEM173. A. Schematic 616

representation of the TMEM173 protein highlighting the four transmembrane domains 617

(TM1, 2, 3 and 4), the cGAMP binding domain (CBD) and the C terminal tail domain 618

(CTD), as well as four phosphorylation sites (P) at p.S345, p.S358, p.S366 and 619

p.S379. In black, previously described mutations in exon 5 (p.V147L; p.V147M; 620

p.N154S; p.V155M). In red, 3 novel mutations in exons 6 and 7 (p.C206Y: P3; 621

p.R281Q: P1; p.R284G: P2). B. Ribbon representation of the 3D structure of the 622

STING CTD dimer in complex with cyclic-di-GMP (PDB reference 4ef4), indicating 623

the position of the three novel amino acids found mutated in this study, as well as 624

amino acids previously reported with mutations and involved in the CTD dimer 625

interface, at the bottom of the ligand binding site. C. Focus on the 3D aligned 626

positions of p.C206, p.R281 and p.R284. D. Surface representation of the 3D 627

structure of the STING CTD dimer (orthogonal view relative to panel B), showing the 628

accessibility of the three amino acids with patient-associated substitutions (p.C206, 629

p.R281 and p.R284). E. IFNß-Luc reporter assay. HEK293T cells were transfected 630

with IFNß-Firefly Luc, Renilla Luciferase and indicated human STING plasmids 631

(EV: Empty vector; WT: Wild type STING; p.N154S p.V155M; p.C206Y; p.R281Q; 632

p.R284G) and stimulated with increasing doses of cGAMP (none; 4 µg/µl; 12 µg/µl). 633

Luciferase activity was determined by Dual-luc assay 24 hours post-stimulation. 634

Mean and SDs shown, representative of 5 different experiments in triplicate. 635

 

636

FIG 3. Analysis of substitutions conferring constitutive activation of STING. A. 637

and B. Localization of amino acids on surface and ribbon representations of the 638

STING dimer 3D structure (PDB reference 4ef4)31. Colored amino acids in which 639

mutations confer constitutive activation: p.D205 in green, p.C206 in yellow, p.R281 640

in red and p.R284 in blue. Amino acids where substitutions did not confer constitutive 641

activation are shown in grey. C. IFNß-Luc reporter assay. HEK293T cells were 642

transfected with IFNß-Firefly Luc, Renilla Luciferase and indicated human STING 643

plasmid (EV: Empty vector; WT: Wild type STING; p.N154A; p.V155M; p.D205Y 644

or H; p.C206Y, F, L or S; p.R281Q, K or V; p.R284G, H or K) and stimulated with 645

increasing doses of cGAMP (none; 4 µg/µl; 12 µg/µl). Luciferase activity was 646

determined by Dual-luc assay 24 hour post-stimulation. Mean and SDs shown, 647

representative of 3 different experiments in triplicate. 648

649

FIG 4. Constitutive activation conferred by p.C206Y, p.R281Q and p.R284G is 650

independent of cGAMP binding. A. IFNß-Luc reporter assay. HEK293T cells were 651

transfected with IFNß-Firefly Luc, Renilla Luciferase and indicated human STING 652

plasmid (EV: Empty vector; WT: Wild type STING; p.V155M; p.C206Y; p.R281Q; 653

p.R284G with or without a second mutation p.R232A) and stimulated with increasing 654

doses of cGAMP (none; 4 µg/µl; 12 µg/µl). Luciferase activity was determined by 655

Dual-luc assay 24 hours post-stimulation. Mean and SDs shown, representative of 4 656

different experiments in triplicate. B. Immunoblot (IB) analysis of STING, 657

phosphorylated IRF3 (p.S396), total IRF3 and cofilin in whole cell lysates of 658

HEK293T cells transfected with empty vector (EV), wild type (WT) human STING 659

with or without cGAMP stimulation, p.V155M, p.C206Y, p.R281Q and p.R284G 660

(unstimulated), either alone or in combination with a second mutation at p.R232A. Of 661

note, in HEK293T cells transfected with WT plasmids, the amount of STING was 662

greater at basal state than upon stimulation with cGAMP, which is consistent with a 663

model of STING degradation, possibly through an autophagic-mediated mechanism24. 664

665

FIG 5. Constitutive activation conferred by p.C206Y, p.R281Q and p.R284G 666

mutants is independent of non-canonical pathway STING engagement. A. IFNß-667

Luc reporter assay. HEK293T cells were transfected with IFNß-Firefly Luc, Renilla 668

Luciferase, and indicated human STING plasmid (EV: Empty vector; WT: Wild type 669

 

STING; p.N154S; p.V155M; p.C206Y; p.R281Q; p.R284G with or without a second 670

mutation p.R169A) and stimulated with increasing doses of cGAMP (none; 4 µg/µl; 671

12 µg/µl). Luciferase activity was determined by Dual-luc assay 24 hours post-672

stimulation. Mean and SDs shown, representative of 4 different experiments in 673

triplicate. B. Immunoblot (IB) analysis of STING, IRF3 phosphorylated at p.S396, 674

total IRF3 and cofilin in whole cell lysate of HEK293T cells transfected with empty 675

vector (EV), wild type (WT) human STING with or without cGAMP stimulation, 676

p.N154S, p.V155M, p.C206Y, p.R281Q and p.R284G unstimulated, with or without 677

double mutant p.R169A. 678

679

FIG 6. Activation of newly identified disease-causing STING mutants relies on 680

IRF3 phopshorylation. A. IFNß-Luc reporter assay. HEK293T cells were 681

transfected with IFNß-Firefly Luc, Renilla Luciferase, and indicated human STING 682

plasmid (EV: Empty vector; WT: Wild type STING; p.V155M; p.C206Y; p.R281Q; 683

p.R284G with or without a second mutation p.S366A) and stimulated with cGAMP 684

(12 µg/µl) or not. Luciferase activity was determined by Dual-luc assay 24 hours 685

post-stimulation. Mean and SDs shown, representative of 3 different experiments in 686

triplicate. B. Immunoblot (IB) analysis of STING, IRF3 phosphorylated at p.S396, 687

total IRF3 and cofilin in whole cell lysate of HEK293T cells transfected with empty 688

vector (EV), wild type (WT) human STING with or without cGAMP stimulation, 689

p.V155M, p.C206Y, p.R281Q and p.R284G unstimulated, with or without a second 690

mutation p.S366A. 691

692

FIG 7. STAT1 phosphorylation status in CD4, CD8 and CD19 cells before and 693

after treatment with ruxolitinib in vitro. Increased basal STAT1 phosphorylation 694

was observed in CD4, CD8 and CD19 cells from P3, which normalized after in vitro 695

treatment with the JAK1/2 blocker ruxolitinib. 696

697

698

TABLE EI. Primer sequences used for site directed mutagenesis. 699

700

TABLE EII. TMEM173 primer sequences. 701

702

TABLE EIII. Summary data of substitutions of STING tested in vitro. WT: Wild 703

 

type. 704

705

FIG E1. Multiple sequence alignment of STING proteins. Sequences are 706

designated using UniProt identifiers. The regular secondary structures, as observed 707

from experimental 3D structure (pdb 4ef4) are reported at the top. MAFFT and 708

ESPript were used for building and rendering of the multiple alignment32,33. BOVIN: 709

Bovine; XENTR: Xenopus tropicalis; DANRE: Danio rerio, NEMVE: Nematostella 710

vectensis. 711

712

FIG E2. Double mutant p.D205H with p.R232A, p.R169A or p.S366A. A, B and 713

C. IFNß-Luc reporter assay. HEK293T cells were transfected with IFNß-Firefly Luc, 714

Renilla Luciferase, and indicated human STING plasmid (EV: Empty vector; WT: 715

Wild type STING; p.D205H with or without a second mutation p.R232A, p.R169A or 716

p.S366A respectively) and stimulated with increasing doses of cGAMP (none; 4 717

µg/µl; 12 µg/µl). Luciferase activity was determined by Dual-luc assay 24 hours post-718

stimulation. Mean and SDs shown, representative of 2 different experiments in 719

triplicate. D and E. Immunoblot (IB) analysis of STING, IRF3 phosphorylated at 720

p.S396, total IRF3 and cofilin in whole cell lysate of transfected HEK293T 721

transfected with wild type (WT) human STING with or without cGAMP stimulation 722

and p.D205H unstimulated, with or without double mutant p.R232A, p.S366A or 723

p.R169A. 724

* Uncharacterized band. Of note, in HEK293T cells transfected with WT plasmids, 725

the amount of STING was greater at basal state than upon stimulation with cGAMP, 726

which is consistent with a model of STING degradation, possibly through an 727

autophagic-mediated mechanism24. 728