STING-associated vasculopathy with onset in infancy: the first case in Bulgaria and review of the literature

Abstract We report a clinical case of a 4-year-and-10-month-old boy whose first symptoms (fever and tachypnoea) were noticed at the age of 6 months. In the upcoming months, skin acral violaceous plaques developed. Failure to thrive and growth impairment were detected. In the journey to find the correct diagnosis, a lot of diseases were suspected. The final diagnosis was suggested after more than 2 years since the first symptoms had appeared. A multi-detector computed tomography of the chest with low radiation dose protocol showed signs of interstitial lung disease (ILD). Next-Generation Sequencing was done, and it revealed a pathogenic heterozygous N154S mutation (NM_198282.4 (Stimulator of IFN genes STING1):c.461A > G (p.Asn154Ser)) in STING1 gene. Treatment with JAK-inhibitor baricitinib was started at the age of 3 years and 10 months. The presence of cutaneous vasculopathy, lung involvement, laboratory signs of inflammation, recurrent fevers and failure to thrive should indicate STING1 gene analysis. STING-associated vasculopathy of infantile-onset (SAVI) is one of the newly identified type I interferonopathies. SAVI is characterized by systemic inflammation via the overproduction of type I IFN due to heterozygous gain-of-function mutations in STING1. The onset of the symptoms of SAVI is usually early in infancy with recurrent fevers accompanied by increased acute phase reactants and ulcerating skin lesions.


Introduction
Interferonopathies comprise an expanding group of monogenic diseases characterised by disturbance of the homeostatic control of interferon (IFN)-mediated immune responses [1]. Stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI) is a type of monogenic interferonopathy that presents early in life. SAVI is an autosomal dominant autoinflammator y disease characterized by neonatal-onset systemic inflammation with a severe cutaneous vasculopathy and interstitial lung disease. SAVI is caused by gain-of-function mutations in STING1 (previously known as TMEM173) which encodes STING -an adaptor molecule which takes part in sensing foreign DNA (bacterial and viral), which leads to the production of type I IFNs as part of the innate immune response [2,3]. The gain-of-function mutations in STING1 lead to constitutive activation of STING, which is associated with upregulation of type I IFN production. The key role of IFN in the pathogenesis of the disease is supported by the fact that immune cells and fibroblasts derived from SAVI patients show constitutive ligand-independent activation of the STING-IFNβ pathway [3]. Peripheral blood analysis shows strong IFN response-gene expression signature and elevated circulating levels of IFN-induced cytokines. Moreover, when skin biopsy is performed small-vessel vasculopathic changes, occlusions and lymphocytic inflammation are being observed [3,4]. STING is widely expressed in alveolar macrophages, bronchial epithelial cells, and alveolar pneumocytes, which may explain the extensive lung pathology seen in patients with SAVI. In addition, STING is also expressed in endothelial cells. Thus, the pathological changes observed on biopsy in the vascular wall are considered to be secondary to both intrinsic defect in endothelial cells and up-regulation of type I IFN [3][4][5]. Molecular confirmation is required to establish a diagnosis of SAVI.
Patients with SAVI usually have neonatal or early infancy onset of systemic inflammation with elevations in erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), a severe cutaneous vasculopathy leading to extensive tissue loss, and major interstitial lung disease (ILD). As reviewed by Eleftheriou et al. 2019, patients with SAVI can have a variety of skin changes -acral violaceous plaques, nodules on the face, nose or ears, distal ulcerative lesions with infarcts, livedo reticularis, Raynaud phenomenon, teleangiectasia, pustulosis [5]. Over time, cutaneous lesions progress to painful ulcerations with eschar formation; sequelae include nasal and ear tissue loss with nasal perforation, nail destructions and digital gangrene necessitating surgical amputation [6]. Disease flares often occur during winter months. The most commonly observed lung involvement is ILD, as well as radiographic evidence of paratracheal adenopathy, varying degrees of lung fibrosis, and pulmonary hypertension. Neurodevelopmental delay and failure to thrive have been also described in SAVI patients. Involvement of musculoskeletal system with myositis and arthritis have also been reported [7]. When skin biopsy is performed signs of neutrophilic inflammatory infiltrate with blood-vessel damage are noticed. Lymphocytic inflammatory infiltrate, leading to interstitial fibrosis and emphysematous changes, is observed also on lung biopsy [3,4].
Treatment options for SAVI patients include JAK 1/2 inhibition. Liu et al. showed that incubation of lymphocytes from patients with SAVI with a JAK inhibitor resulted in reduced levels of STAT1 phosphorylation and a reduction of IFNβ production in fibroblasts activated by cGAMP [3]. Treatment of patients with SAVI with JAK inhibitors has resulted in normalization of inflammatory markers, resolution of cutaneous symptoms and gradual improvement of the lung disease in some [5,8,9].

Case presentation
We herein report the clinical case of a boy at the age of 4 years and 10 months, whose initial symptoms began at 6 months of age when during an episode of rotavirus gastroenteritis, microcytic hypochromic anaemia (Hb 8.0 g/dL) and tachypnoea were observed. During the following months, the child developed recurrent skin infections and acral violaceous plaques appeared over the cheeks and the fingers (Figure 1).
At the age of 15 months, during a fever episode with tachypnoea, chest X-ray was performed and revealed bilateral coarse areas of increased opacity in middle and upper lung regions ( Figure 2).
The patient had a history of loose stools, and failure to thrive was noted (weight of 7 kg, below the 5th percentile for age of 15 months and gender). Cystic fibrosis was excluded. The X-ray changes, tachypnoea and anaemic syndrome were indications to perform bronchoalveolar lavage (BAL) with rigid bronchoscope, which showed presence of hemosiderin-laden macrophages. Based on the BAL findings, a diagnosis of idiopathic hemosiderosis was made, so the child was started on corticosteroids. Corticosteroid treatment provided partial benefit. When the corticosteroid dosage was being tapered, a worsening in the child's condition was observed with a progression of the chest X-ray changes. Clinical follow-up established episodes of recurrent fevers. Ulcerative skin lesions were observed, as well ( Figure 3). Because of frequent pyodermia and pustulosis due to Staphylococcus aureus, the humoral immunity (high IgG of 17.57 g/L and high IgA of 4.89 g/L) and cellular immunity was investigated. Low CD3 + CD4+ absolute (332 cells/µL) and relative (10%) cells number and high CD3 + CD8+ (1892 cells/ µL; 40%) cells with an index of 0.18 were detected. Multiplex cytokine panel showed Th1 related inflammation: very high serum levels of IFN-ɣ (32.08 pg/mL), interleukin (IL)-12p70 (6.86 pg/mL), IL-2 (18.20 pg/mL), IL-6 (78.21 pg/mL), tumour necrosis factor-α (25.21 pg/ mL), granulocyte-macrophage colony-stimulating factor (13.93 pg/mL) and IL-18 (55.97 pg/mL), while the concentrations of IL-13, IL-1β, IL-4 and IL-5 were normal. The immunology panel (antinuclear antibodies, anti-double strained DNA, antineutrophil cytoplasmic antibodies, anti-glomerular basement membrane) was negative; however, anti-deamidated gliadin peptide IgG antibodies were positive and gluten-free diet was recommended.
At presentation at the Pediatric Rheumatology Department, the child was 2 years and 6 months of age with a weight and height below the 5th percentile. Acral lesions over the cheeks, ears, fingers and feet were observed ( Figure 4). Tachypnoea and finger clubbing were noticed. Laboratory parameters revealed persisting anaemia (hemoglobin 9 g/dL), elevated ESR (40 mm/h) and CRP (10 mg/L). Plain chest radiography was the first-line diagnostic imaging modality of choice. However, it could not properly guide us in this case. Therefore, a multi-detector computed tomography of the chest with a low-radiation dose protocol was selected to present and depict more accurately the central airway, lung parenchyma and mediastinal abnormalities. A wide range of parenchymal abnormalities, affecting both lungs without segmental or lobar predilection were observed: fine reticular opacities, diffuse ground-glass opacities, solid nodules, cystic airspaces and honeycombing in subpleural areas ( Figure 5). No indirect signs of pulmonary hypertension were present when echocardiography was performed.
Based on the clinical, laboratory and imaging findings, a genetic test to confirm SAVI was performed. Next-generation sequencing (NGS; Illumina TruSight One Sequencing panel) revealed pathogenic heterozygous N154S mutation (NM_198282.4(STING1): c.461A > G (p.Asn154Ser)) in STING1 gene. The diagnosis of SAVI was confirmed. Treatment with JAK-inhibitor baricitinib (8 mg/day) was started at the   age of 3 years and 10 months. For a period of one year, there was a significant improvement in terms of vasculitis flares, with no development of new lesions and improved status of the existing ones. Baricitinib treatment led to normalization of the respiratory rate and increase in the physical capacity and exercise tolerance of the patient. Methylprednisolone dose was gradually weaned during the baricitinib dose escalation, from baseline of 0.9 mg/kg/daily to 0.29 mg/kg/ daily. Catch-up growth was not observed, as the patient gained 1.7 centimetres for one year and is still below the 5th percentile for height and weight. After the initiation of baricitinib the patient required inpatient care only once due to an episode of Campylobacter infection. The drug is overall well tolerated.

Discussion
The presence of cutaneous vasculopathy, lung involvement, laboratory signs of inflammation, recurrent fevers and failure to thrive should prompt STING1 gene testing. In the presented clinical case, it took more than 2 years for the diagnosis to be made since the first clinical symptoms appeared. The first signs of tachypnoea were noticed at 6 months of age. During the following months skin lesions were observedacral violaceous plaques, pustular rash due to S. aureus skin infection and ulcerative skin lesions. Recurrent fevers, failure to thrive and growth impairment were detected. The lung CT revealed signs of ILD. In the journey to find the correct diagnosis, a lot of differential diagnoses were suspected and subsequently excluded, i.e. autoimmune anaemia, cystic fibrosis, hemosiderosis, coeliac disease, HIV, primary immune deficiency, Heiner Syndrome, Bloom Syndrome, Lane Hamilton Syndrome, Acrodermatitis enteropatica. The final diagnosis was suggested for the first time when the child was 2 years and 6 months of age and was consulted by paediatric rheumatologists. It took additional 6 months for the genetic studies to be performed. The exact mutation (c.461 A > G; p.Asn154Ser), confirming SAVI was detected when the child was 3 years old. Treatment with JAK-inhibitor baricitinb was initiated when the child was 3 years and 10 months old. At that time the JAK-inhibitors were drugs of compassionate use for children in Bulgaria.

Literature review
SAVI is one of the newly identified type I interferonopathies. The term 'type I interferonopathy' entered the medical dictionary in 2011 [2]. It was 2014 when SAVI was first described by Liu et al. [3]. The prevalence of this disease is unknown. The authors of the largest cohort state that SAVI is rare -with 52 patients in 37 families reported up to the moment of publishing the report [10].
STING, also known as transmembrane protein 173 (TMEM173) is an endoplasmic reticulum-associated membrane protein which plays an essential role in innate immunity. It induces the production of interferons against viral DNA infection and the expression of IFN-related genes by activating an immune response through STING signalling [11]. STING is encoded by the STING1 gene. SAVI is characterized by systemic inflammation via the overproduction of type I IFN due to heterozygote gain-of-function mutations in STING1. Type I IFNs act through binding to IFN receptors, which lead to activation of Janus kinases (JAKs) and increased phosphorylation of signal transducer and activator of transcription, ultimately causing a cytokine storm [12]. Autoactivating variants in STING1 in exons 3, 4, 5, 6, 7 and 8 are so far described in Infevers -a registry for mutations in hereditary autoinflammatory disorders [13]. The total current number of sequence variants in STING1 is nineteen. Most cases are due to de novo mutations; however, familial cases have been reported [14][15][16]. The mutation that was found in our patient, N154S mutation (c.461 A > G; p.Asn154Ser), has been identified in other patients with SAVI [3,6,9,12,17]. N154S mutation has been found in 4 of the 6 patients who were described for the first time in 2014 [3]. The same mutation was detected in 4 of the 21 patients described in the largest cohort of SAVI patients reported by Fremond et al. (Table 1) [10].
The original description of SAVI by Liu et al. is in terms of pulmonary, cutaneous and vascular manifestations [3]. Liu et al. reported six unrelated cases of SAVI in children. Four patients presented within the first 8 weeks of life with skin lesions on the extremities. Two of the patients presented with tachypnoea in the perinatal period. More recently it has been recognized that the associated phenotype is highly variable and the initially described phenotype was subsequently expanded. Munoz et al. [21] in their description of a boy with skin vasculopathy, recurrent fevers, ILD and failure to thrive and a diagnosis of SAVI 7 years after the onset of the disease, emphasize the differential diagnosis with childhood granulomatosis with polyangiitis. König et al. [14] report a four-generation kindred with five family members affected with chilblain lupus in whom a heterozygous gain-of-function mutation of STING causing constitutive type I IFN activation was identified. Moreover, the authors showed that treatment of two affected family members with JAK inhibitors suppresses type I IFN activation. In addition, Clarke et al. [15] describe a three-generation kindred and highlights that SAVI may present as ILD with early onset juvenile idiopathic arthritis.
Furthermore, Wang et al. [7] revealed an atypical phenotype of a familial case series of SAVI patients with predominant ILD manifestations, absence of cutaneous lesions, and poor response to JAK inhibition with ruxolitinib. This report adds to three previously published cases of SAVI patients without skin vasculopathy [16,18,20]. The typical cutaneous manifestations in SAVI patients are described as chilblain-like lesions with multiple morphological features (acral violaceous plaques, nodules on face, nose or ear, distal ulcerative lesions with infarcts, livedo reticularis, teleangiectasia, pustulosis) triggered or exacerbated by exposure to cold and localized at acral regions (fingers, toes, ears, nose). Prominent erythematous plaques on cheeks, resembling malar rash and painful ulcerations with eschar formation on cold-sensitive areas are the characteristic features of SAVI [6].
The largest cohort of SAVI patients published by Marie-Louise Fermond et al. [10] describes the core features of 21 patients with SAVI. Genetic diagnosis was made at a median age of 8.5 years (1.25 − 65 years), and retrospectively in 3 patients who died at the age of 29, 34 and 11 years, respectively. Median age of disease onset was 8.5 months (birth − 13 years) with most patients being symptomatic from infancy but late onset in adulthood occurred in one patient. Skin vasculopathy was detected in 18 patients, whereas ILD was observed in all of the 21 patients included in the report. Nine patients required supplemental oxygen therapy at onset or during the course of the disease, of whom 6 progressed to end-stage respiratory failure in adolescence or early adulthood. The key features revealed via lung CT were ground-glass opacities with crazy-paving pattern, and cysts. Frequently, the described CT lesions had an asymmetrical pattern. Neurological manifestations are not a core feature of SAVI in comparison to other monogenic interferonopathies. However, when brain CT scan was performed, calcification of basal ganglia was observed in 3 patients. In seven patients, polyarthritis developed with rheumatoid factor being positive in six of them.
SAVI is minimally responsive to conventional immunosuppressive therapies, with steroids displaying limited effect in SAVI [22]. In SAVI pathogenesis, mutations in SAVI1 cause constitutive activation of STING resulting in increased IFN-β [3]. Based on our current understanding on the molecular pathogenesis of type I interferonopathies, targeting the JAK/signal transducers and activators of transcription pathway to inhibit the pathogenic type I IFN response might be therapeutically effective in SAVI patients [23]. JAK-inhibitors are promising treatment in SAVI patients. In 2016 Fremond et al. [8] reported improvement of cutaneous and pulmonary phenotype of the disease under JAK inhibition with ruxolitinib in 3 children with SAVI. This experience was confirmed by further reports using JAK inhibitors in SAVI patients [9,17]. In a recently published article by Sanchez et al. [19], 18 patients with interferonopathies were treated with JAK inhibition with baricitinib. The patients with SAVI included in the report were 4 and the diary score improvement criteria was fulfilled by 3 of the 4 SAVI patients. Baricitinib treatment prevented the progression of spontaneous amputation and the development of gangrene. Baricitinib also stabilized ILD. In the 4 patients with SAVI reported by Sanchez et al. [19] the vasculitis flares improved but still occurred, albeit with reduced duration and severity. The most common observed adverse events in relation to JAK-inhibitor treatment were upper respiratory tract infections, gastroenteritis and BK viremia. Moreover, the largest cohort describes long-term follow-up in 8 children and confirms the clinical benefits of JAK inhibition with ruxolitinib in SAVI unless the treatment is started early in the course of the disease [10]. The best response was observed in cases when the treatment was started early. Tolerance towards ruxolitinib was overall good with the authors emphasizing the risk of respiratory infections in patients with poor lung function. Given the paucity of long-term outcome data on newly available treatments, monitoring of disease activity, and development of organ-specific and treatment-related complications is essential [24]. Recently, the 2021 European Alliance of Associations for Rheumatology/American College of Rheumatology published points to consider for diagnosis and management of autoinflammatory type I interferonopathies [24]. The aim was to address the unmet need and provide guidance for healthcare professionals involved in the care of patients with the recently characterized type I interferonopathies, including SAVI.

Conclusions
SAVI is a disease characterized by early-onset fever, signs of systemic inflammation with elevated acute-phase reactants, and severe cutaneous vasculopathy are hallmark features of SAVI. The severe cutaneous involvement might result in extensive tissue loss. Moreover, patients with SAVI might develop pulmonary fibrosis due to progression of ILD. The complexity of SAVI requires a multidisciplinary team to manage these patients. Early diagnosis and treatment are essential in preventing progressive organ injury and early mortality.

Ethics statement
Informed consent to publish has been obtained from the patient's parents.

Data availability
The data that support the findings of this study are available upon reasonable request from the corresponding author, [MG]. The data are not publicly available due to their containing information that could compromise the privacy of participants.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.