The Challenge of Diagnosing SAVI: Case Studies

Abstract

Background: Stimulator of interferon genes (STING)–associated vasculopathy with onset in infancy (SAVI) was first described in 2014 as a type I interferonopathy resulting from heterozygous mutations in the transmembrane protein 173 (TMEM173) gene. SAVI is characterized by the neonatal onset of systemic inflammation, severe cutaneous vasculopathy, and interstitial lung disease. Janus kinase inhibitors are considered effective therapeutics. We sought to describe 2 patients who were diagnosed with SAVI only at postmortem to increase awareness of this disorder.

Methods: Clinical data were collected, and Sanger sequencing of the TMEM173 gene was performed in 2 patients suspected of SAVI. This article reviews details of these cases and lessons learned from clinical review and postmortem studies.

Results: Two male children shared similar manifestations, including recurrent skin abscesses in winter, skin lesions, and recurrent respiratory tract infections, since birth. Computed tomography of the chest revealed pulmonary fibrosis, but no mutations in relevant genes (including ABCA3 and SFTPC) were discovered in patient 1 (P1). Joint pain was significant in P2 and he was diagnosed with arthritis. Antibiotic treatment yielded little improvement and did not prevent progression. Finally, P1 and P2 died of respiratory and circulatory failure in 2016 and 2012, respectively. In 2018, mutations (P1: c.463G>A, p.V155M; and P2: c.461A>G, p.N154S) in exon 5 of the TMEM173 gene were discovered, confirming the diagnosis of SAVI.

Conclusions: The experience with these 2 patients suggests that SAVI should be considered in children with systemic inflammation, chilblain skin lesions, and pulmonary fibrosis, and TMEM173 gene analysis can be beneficial in the diagnosis of SAVI.

Introduction

Type I interferons (IFNs) are thought to play a critical role in the innate immune response to pathogens, and increases in this response can cause an abnormal immune state named type I interferonopathy.¹ Stimulator of interferon genes (STING)–associated vasculopathy with onset in infancy (SAVI) is a rare type I interferonopathy first described in 2014,² that is, caused by mutations in the transmembrane protein 173 (TMEM173) gene. This gene encodes the STING adaptor protein, and mutations lead to gain of function of STING and overproduction of IFN-β. Type I IFNs act through binding to IFN receptors (IFNRs), which lead to activation of Janus kinases (JAKs) and increased phosphorylation of signal transducer and activator of transcription, ultimately causing a cytokine storm.

SAVI is a disease characterized by early-onset fever and elevated acute-phase reactants (systemic inflammation), severe cutaneous vasculopathy (chilblain-like rashes) resulting in extensive tissue loss, and progression from pulmonary interstitial changes (organ inflammation) to pulmonary fibrosis in the terminal stage. The prevalence of this disease is unknown; only 22 affected individuals^2–11 have been described in the literature. Most of the cases reported so far are sporadic, but 6^3,4 were inherited. To date, a total of 9 gain-of-function mutations have been reported, including V155M,^2–6 N154S,^2,8 V147L,² V147M,⁷ C206Y,⁹ R281Q,⁹ R284G,⁹ R284S,¹⁰ and S102P/P279L (c.304T>C and c.835T>C).¹¹ The onset of SAVI was infancy in 17 reported patients.

Owing to unfamiliarity with this disease, some infants may have been misdiagnosed. In this report, we present the phenotypes of 2 Chinese children diagnosed postmortem with SAVI by TMEM173 gene sequencing and discuss the diagnosis and differential diagnosis, which may contribute to a better understanding of this disease and facilitate early and correct diagnosis.

Materials and Methods

Patients and study approval

Two Chinese boys, ages 8.75 and 6.75 years, respectively, who were suspected of SAVI are presented. The study was approved by the ethics committee of Children's Hospital of Chongqing Medical University. Written and oral informed consent for the publication of case information and images was obtained from the patients' parents.

Genetic analysis

Total mRNA was extracted from fresh, heparinized venous blood samples from the patients using the RNAprep Pure Blood Kit (BioTeke, Beijing, China) and subjected to first-strand complementary DNA (cDNA) synthesis using the PrimeScript RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer's protocol in 2011. cDNA was stored at −80°C in the Biobank Center of Children's Hospital of Chongqing Medical University. The PCR primer TMEM173 was designed according to the human TMEM173 mRNA sequence (NM_198282.4, gene ID 340061), and PCR reagents were purchased from Wuhan Bio-Company. Sanger sequencing was performed on amplified cDNA with the sequencing primers 5′-CACTTGGATGCTTGCCCTCCT-3′ and 5′-ATGAGGCGGCAGTTGTTC-3′ at the optimal annealing temperature of 65.3°C.

Results

Clinical characteristics

Two Chinese boys, ages 8.75 and 6.75 years, respectively, were referred in 2011 because of newborn respiratory tract infection and recurrent skin abscesses beginning shortly after birth. When patient 1 (P1) was 2 months old (the first winter after his birth), the rashes first appeared on his cheeks. He was treated with topical drugs (unidentified) without improvement. However, it appeared to improve as the weather became warmer. The rashes appeared again in the same location every winter and pus, ulcers, and scars eventually formed on the face. The skin lesions progressed to involve the ears and wings of the nose at the age of 5 years. No significant skin abscesses spread to the extremities, trunk, or buttocks.

Telangiectatic skin lesions appeared earlier and more severely in P2 on the cheeks, ears, and nose, from the age of 1 month. The facial infections also occurred every winter and were treated mainly by traditional Chinese medicine. He recovered, but had large deep scars on his face and lesions on his ears and nose wings (Fig. 1). Skin lesions developed on the extremities, trunk, and buttocks, with blisters and skin cracks on the feet.

FIG. 1.

Clinical features of 2 patients diagnosed postmortem with SAVI. Defects over the malar cheeks and lesions on the nose wings in P1 (A) and P2 (F). (B) Defects on the left and right ears of P1. Thoracic deformity (C) and clubbed fingers (D) and toes (E) on P1. Computed tomography indicated mosaic signs (G) and lung fibrosis (H) in P1 in 2012 and 2016, respectively. (I) Interstitial lung changes in P2. P1, patient 1; P2, patient 2; SAVI, stimulator of interferon genes–associated vasculopathy with onset in infancy.

These boys had frequent (at least once per month) upper or lower respiratory tract infections characterized by fever and cough lasting several days. P1 was managed as an outpatient with clinical improvement following intravenous antibiotics (empirically, unidentified) for 2–3 days. He was noted to have pigeon chest and clubbing when he was 2 years old. He occasionally experienced oral ulcers, eczema (extremities and trunk), and mild joint pain of the knees without redness, swelling, or dysfunction. The joint pain was relieved without treatment.

P2 had been diagnosed with pneumonia several times and had been hospitalized twice, at 2 months and 2 years old. Outpatient administration of oral corticosteroids (prednisone 2.5–5 mg/day) was recommended, but with poor compliance and erratic administration, and dexamethasone was administered intravenously during febrile episodes. Fever and cough were partially relieved after antibiotic (orally, cephalosporins) and glucocorticoid treatment. He had no mouth ulcers, but had oral thrush in infancy. Before admission, he had a 5-month history of polyarthralgia with swelling and progressive limitation in range of motion. Intravenous steroids were given during exacerbation episodes with transient symptom relief.

There was no history of seizure, paralysis, otitis, diarrhea, abdominal pain, or bleeding in either child.

Both boys were born to nonconsanguineous parents of Asian ethnicity after an uneventful pregnancy at 37 weeks of gestation, weighing 3 kg (P1) and 2.7 kg (P2). P1 was G1P1. His mother had an elder brother who died in infancy, but no more information was available. P2 was G4P2 with a healthy 17-year-old sister. His mother had 2 unexplained miscarriages (at 2 and 3 months of pregnancy). Neither boy showed omphalitis or umbilical cord discharge delay. Both sets of parents were healthy and denied similar manifestations in family members.

Both children had poor weight gain (116 and 93 cm) and linear growth (17 and 12 kg), pallor, and pectus carinatum deformity. P1 had clubbed fingers and toes, while P2 had only clubbed toes. There were many large scars and defects on their cheeks, ears, and nose wings (Fig. 1). For P2, scars were present on the buttocks, and scattered red maculopapular and pustular eruptions were found on the extremities; he had swollen knee and wrist joints with limited range of motion. Reflexes were normal for both boys.

Anemia with hemoglobin levels of 10.3 g/dL (P1) and 9.4 g/dL (P2) was identified. Both patients had elevated C-reactive protein (CRP) levels of 23 and 50 mg/L and elevated erythrocyte sedimentation rates (ESRs) of 74 and 114 mm/h. The counts of lymphocyte subsets, including CD4⁺ T cells, CD8⁺ T cells, B cells, and natural killer (NK) cells, were normal. Both boys had increased immunoglobulin E (IgE) (316.7 and 292.4 g/L; reference range, <150 g/L) and immunoglobulin A (IgA) (3.66 and 6.18 g/L; reference range, 0.22–2.2 g/L) levels; however, immunoglobulin G (IgG) and immunoglobulin M (IgM) levels were increased in P2 (23.7 and 3.07 g/L; reference ranges, 2.86–16.8 and 0.43–1.63 g/L) and normal in P1 (14.3 and 1.32 g/L). P1 was negative for serum autoantibodies, while P2 was strongly positive (+++) for anticyclic citrullinated peptide antibodies.

No etiological data were available for P1. Sputum culture suggested Haemophilus influenzae that was sensitive to amoxicillin in P2. Adenovirus and coxsackievirus IgM antibodies were positive (++ and ++) for him. Chest X-ray showed bronchopneumonia. Computed tomography of the chest revealed pectus carinatum and pulmonary fibrosis characterized by interstitial changes (Fig. 1).

For P1, pulmonary function tests indicated small airway obstruction without significant bronchodilator response. An echocardiogram performed in 2016 suggested right atrial and right ventricular enlargement as well as severe pulmonary hypertension and pericardial effusion. Abdominal ultrasonography revealed congestive hepatomegaly. For P2, X-ray showed osteoporosis of the long bones and metacarpal–phalangeal epiphysis without signs of destruction. Bronchoscopy indicated endobronchitis and destruction of the nasal septum. Bronchoalveolar lavage revealed an inflammatory infiltrate with numerous lymphocytes. Chest and buttock skin biopsies revealed signs of perivascular lymphocytic and neutrophilic infiltration and hyperplasia.

Genetic analysis and diagnosis

Neutrophil defects such as chronic granulomatous disease (CGD) were initially considered since the major clinical manifestations were recurrent skin abscess with scar formation and recurrent respiratory tract infections beginning early in life. However, the nitroblue tetrazolium (NBT) test results and respiratory burst were normal in both patients. In 2011, no studies had been published regarding this newly reported disease.

For P1, peroxidase staining, Wright's staining, and hematoxylin–eosin staining of blood smears were completed, but produced no further findings. Defects in innate immunity were suspected, but no mutation was found in the myeloid differentiation primary response 88 (MYD88) gene. The ABCA3 and SFTPC genes were analyzed based on the suspicion of interstitial lung disease, but no mutations were found. For P2, arthritis was diagnosed, and naproxen was prescribed to relieve the pain, but the effect was temporary. Prophylactic antibiotic sulfamethoxazole was used in P2 only for 2 weeks. Amoxicillin and clavulanate potassium, even meropenem, were administered to protect against pulmonary infections.

However, the disease progression was not stopped. Over time, there was an evolution of telangiectasia on the nose and cheeks with violaceous scaling, and both boys developed pulmonary fibrosis, ultimately leading to their demise. P2 and P1 died in 2012 and 2016, respectively, of respiratory failure due to irreversible, progressive, chronic lung fibrosis.

The diagnosis confused us for more than 6 years until we read related reports. Because these 2 children harbored a similar phenotypic spectrum, SAVI was finally considered. Sanger sequencing identified heterozygous mutations in the TMEM173 gene in both patients. P1 and P2 were found to harbor c.463G>A (p.V155M) and c.461A>G (p.N154S), respectively, in exon 5 (Fig. 2), and these mutations had already been reported in 2014. Thus, the diagnosis of SAVI was finally confirmed in 2018. We had not sequenced this gene in the parents as we had no samples. Since the patients had an early onset and severe manifestations and their families had no similar phenotypes, we speculated de novo variants.

FIG. 2.

TMEM173 gene sequencing in P1 and P2. (A) P1: c.463G>A, p.V155M. (B) P2: c.461A>G, p.N154S. TMEM173, transmembrane protein 173.

Discussion

Among the 22 previously published cases, 18 showed increased CRP/ESR, and fever was common. Chronic anemia and failure to thrive were also found in most patients. Autoantibodies, especially antinuclear antibodies, are notable, followed by antiphospholipid antibodies and rheumatoid factors. The rare autoantibodies include anti-double-stranded DNA antibody,⁸ antineutrophil cytoplasmic autoantibodies,^4,7 anticardiolipin IgG,⁶ and lupus anticoagulant.^6,8 Distal ulcers and perforations of the nasal septum have been found in approximately half of the reported SAVI patients. In contrast, hyperimmunoglobulinemia, with increased IgG or IgA levels, was unusual.

Due to the rare occurrence of SAVI, we report these 2 additional children to highlight the diagnostic challenges and to discuss the clinical course to increase awareness of this disorder. The fatal outcome in the patients presented in this report demonstrates the need not only for early diagnosis but also effective therapeutic strategies. Before accurate recognition of this disease, different phenotypes triggered patients to present at different departments, such as the pneumology, dermatology, and thoracic surgery departments. Due to lung involvement, patients may be diagnosed with idiopathic lung fibrosis or nonspecific pneumonia, even after lung transplant.⁴ Infants with SAVI might go undiagnosed after routine examinations, leading to a substantial delay in the correct diagnosis, as was the case for our patients.

Therefore, it is necessary to focus on the key features defining SAVI, especially chilblain-like rashes, interstitial pneumonia, and elevated acute-phase reactants, and then to analyze the IFN-β signaling pathway and TMEM173 gene. Of the 9 reported mutations that cause SAVI, mutations are frequently detected in exon 5 (of which the V155M and N154S are predominant) in nearly all patients with typical chilblain-like rashes and lung changes; however, 1 patient harboring V155M³ had no skin lesions. Among the few identified mutations, 2 patients with V147L in exon 5² and C206Y in exon 6⁹ did not show a relationship with interstitial lung disease, indicating that not all individuals suffering from SAVI develop rashes and typical pulmonary fibrosis.

Skin lesions are described as chilblain-like lesions with multiple morphological features (livedo reticularis, ulcers, atrophic scars, nodules, and necrosis, etc.) triggered or exacerbated by exposure to cold and localized at regions with insufficient blood supply (zygomatic zones, fingertips, nose, and ears), which is related to capillary involvement. Interstitial lung disease has been present in all but 2 cases.^2,9 The lesions present on computed tomography show a broad spectrum of changes, including ground-glass shadows, uneven inflation, bronchiectasis, and fibrosis. Because of chronic hypoxia, patients may have clubbed fingers or toes, thoracic deformities, or even growth retardation, as did our patients.

The variable presentation and severity of interstitial lung disease in SAVI patients correlate with the TMEM173 haplotype at position 232 and possibly with the local induction of an IFN response¹² domain. Regarding our patients, P2 harbored the wild-type R232 variant of STING, while P1 had a homozygous mutation (c.695G→A, p.R232H), which may be protective.¹² However, paradoxically, P1 also had interstitial lung disease that was not in accordance with earlier reports.¹² These findings may partially explain why P1 had a later onset and less severe symptoms and survived longer. Respiratory failure associated with underlying SAVI-related lung disease was the main cause of death from natural causes for 3 reported deceased patients^2,4,6 and our patients.

However, the exact mechanism underlying the development of interstitial lung disease remains unclear; it may be directly related to local IFN injury.⁴ Nevertheless, research on how STING drives inflammation has implicated both IFN and the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) signaling, which probably participates in immune dysregulation,^10,13 reminiscent of the contributions of cytokines to SAVI. A large number of cytokines in T and B lymphocytes can lead to cell apoptosis,¹⁴ resulting in tissue damage.

In addition, STING is primarily expressed in alveolar macrophages, type 2 pneumocytes, and bronchial epithelial cells, and STING mutations could cause activation of these cells, thereby inducing lesions involving pulmonary epithelial cells. In addition, STING mutants can localize to the Golgi in fibroblasts from affected patients,³ leading to follicular proliferation and fibrosis. However, the role of type I IFN is controversial. STING N153S mice lacking IFR3 also developed lung disease,¹⁵ and Motwani et al. found that V155M mice could not be rescued despite deficiencies in IRF3 and IFNAR.¹⁶

Additionally, a report on STING N153S mice indicated that these mice are more vulnerable to infection and their combined innate and adaptive immunodeficiency leads to virus-induced pulmonary fibrosis.¹⁷ Moreover, lung disease in STING N153S mice develops independently of type I IFN, but T cells are involved,^18,19 potentially implicating viral involvement in causing fibrosis, whereas IFN may act purely as an intermediary with an indirect effect. For example, herpesvirus promotes fibrosis in Th2-biased mouse models lacking IFN or IFNR.²⁰ However, in an hSTING-N154S mouse model, in which STING N154S is expressed under control of the Vav promoter, Martin et al. found that the disease was dependent on IFN/IFNAR1, although no significant lung inflammation was observed.²¹

Differential diagnosis of interstitial lung disease/lung fibrosis

Most patients with SAVI have accompanying pulmonary lesions. The onset of pulmonary changes is early in life, but these lesions are often too mild to recognize early and gradually worsen as the disease progresses. In addition to SAVI, many other diseases are characterized by early-onset pulmonary lesions, including some primary immunodeficiency diseases (PIDDs), cystic fibrosis (CF), and certain disorders of surfactant metabolism. However, the interstitial changes dominant in SAVI do not exactly match the inflammation in PIDDs, and PIDDs in children may result in recurrent pulmonary infections with pulmonary consolidation, nodules, or granulomas rather than diffuse interstitial changes. For example, patients with CGD may have granuloma formation and recurrent perianal abscesses that can be identified with NBT and DHR123.

The typical pulmonary appearance of SAVI, with respiratory failure early in life and transition to chronic interstitial lung disease, can be similar in CF, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Patients with CF are more susceptible to chronic bacterial infections, especially Pseudomonas aeruginosa and Staphylococcus aureus infections. Due to mucus retention, CF can manifest in multiple organ systems, such as the pancreas, liver, sweat glands, and vas deferens, in addition to the lungs. Deficient trypsin secretion and fatty diarrhea are other prominent features of CF. Immunoreactive trypsinogen measurements and sweat chloride tests are considered screening methods.

In another large series of childhood interstitial lung disease (chILD), congenital surfactant deficiency was quite prevalent, and the most frequent variants were SFTPC and ABCA3 mutations. These mutations have been identified equally across a wide spectrum of lung diseases, from respiratory distress syndrome at birth or in infancy to mild symptoms such as chronic interstitial lung disease.^22–24 SFTPC and ABCA3 mutations are present in alveolar type II pneumocytes in chILD, as in SAVI, but the mechanisms are not identical. The onset of chILD in patients with SFTPC or ABCA3 mutations may be even earlier (neonates) and more serious because of the direct effects of surfactant protein defects; conversely, the onset of SAVI may be later and more insidious due to the continued effect of IFN signaling.

However, it is typically difficult to distinguish these diseases based on pulmonary manifestations or radiological findings, even with apparent evidence supporting interstitial lung disease, so genetic testing should be considered when similar symptoms are present. Before diagnosis, the SFTPC and ABCA3 genes in P1 were sequenced, but no corresponding mutations were found.

PIDDs, CF, and SFTPC or ABCA3 mutations are all differential diagnoses for SAVI; however, most patients with these other disorders did not develop chilblain-like rashes, which could be one diagnostic indicator. In other words, a diagnosis of SAVI should be considered for patients who present with pulmonary interstitial disease and chilblain-like rashes.

Treatment

IFN/IFNR and JAK inhibitors are currently considered effective therapeutics for SAVI.^6,25 JAK inhibitors block signaling downstream of an array of cytokine receptors, including IFNR. It is not known whether the blockade of IFN signaling is the mechanism by which JAK inhibitors show some efficacy in SAVI. Additionally, the efficacy of hematopoietic stem cell transplantation has not been demonstrated. Traditional treatments, such as steroids, have limited efficacy.¹³ P2 received dexamethasone, but did not experience significant relief. Regrettably, our patients did not receive timely treatment due to the lack of timely diagnosis.

Conclusions

The 2 reported cases reinforce the manifestations of SAVI, a rare type I interferonopathy characterized by chilblain rashes and interstitial lung disease. The fatal outcome of our patients highlights the severity of this disease and the necessity for effective treatments. To minimize misdiagnosis, it is necessary to make a differential diagnosis with other interstitial-related lung diseases to further reveal the underlying genetic cause based on clinical suspicion. The pathogenesis of SAVI and critical role of IFN/IFNR in promoting fibrosis require more exploration, and IFN and JAK inhibitors are the current therapies.

Author Disclosure Statement

Authors declare that they have no conflicts of interest or financial ties to disclose.

Author Contribution

Y.C. performed experiments, collected the data, and prepared the manuscript. L.J. designed the study, supervised the study, and revised the manuscript. The authors approved the final version to be published.

Funding Information

No funding was received for this article.