Spectrum of Systemic Auto-Inflammatory Diseases in India: A Multi-Centric Experience

Background: Systemic autoinflammatory diseases (SAID) are rare inherited disorders involving genes regulating innate immune signaling and are characterized by periodic or chronic multi-systemic inflammation. Objective: To describe spectrum of clinical, immunological, molecular features, and outcomes of patients with SAID in India. Methods: Request to share data was sent to multiple centers in India that are involved in care and management of patients with Inborn Errors of Immunity. Six centers provided requisite data that were compiled and analyzed. Results: Data on 107 patients with SAID were collated—of these, 29 patients were excluded due to unavailability of complete information. Twelve patients (15%) had type 1 interferonopathies, 21 (26%) had diseases affecting inflammasomes, 30 patients (41%) had non-inflammasome related conditions and 1five patients (19%) had Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis (PFAPA). Type1 interferonopathies identified in the cohort included patients with Deficiency of Adenosine Deaminase 2 (DADA2) (six patients; five families); STING-associated vasculopathy infantile-onset (SAVI) (three patients, one family); Spondyloenchondro-dysplasia with Immune Dysregulation (SPENCD) (two patients). Diseases affecting inflammasomes include Mevalonate Kinase Deficiency (eight patients); Cryopyrin-Associated Periodic Syndromes (CAPS) (seven patients); NLR Family, Pyrin domain-containing 12 (NLRP12) (two patients); Familial Mediterranean fever (FMF) (two patients); Autoinflammation and PLCG2-associated antibody deficiency and immune dysregulation (APLAID) (two patients). TNF receptor-associated periodic syndrome (TRAPS) (three patients); A20 haploinsufficiency (four patients); Deficiency of Interleukin 1 Receptor Antagonist (DIRA) (two patients) were categorized as non-inflammasome related conditions. There were significant delays in diagnosis Corticosteroids and other immunosuppressive agents were used for treatment as anti-IL-1 drugs and other biological agents were and still are not available in India. Eight (16.3%) patients had so far succumbed to their illness. Conclusions: This is the first nationwide cohort of patients with SAID from India. Clinical manifestations were diverse. Overlapping of clinical features with other relatively common rheumatological disorders often resulted in delays in diagnosis. More nationwide efforts are needed to enhance awareness of SAID among health care professionals and there is an urgent need to make targeted immunotherapies universally available.


INTRODUCTION
Systemic autoinflammatory diseases (SAID) are complex inherited disorders caused by defects in several genes regulating innate immune signaling and are characterized by periodic or chronic multisystem sterile inflammation (1)(2)(3).
The term "autoinflammatory disorders" was coined in 1999 by Daniel Kastner's group when they proposed a new group of immunological diseases (4). The paper described genetic background of familial Hibernian fever, and rechristened it as "TNF receptor-associated periodic syndrome (TRAPS)." It also linked it with previously described mutations in Pyrin (MEFV)gene that causes familial Mediterranean fever (FMF) (4)(5)(6). In 2010, Kastner et al. defined autoinflammatory diseases as "clinical disorders marked by abnormally increased inflammation, mediated predominantly by cells and molecules of the innate immune system with a significant host predisposition" (1,7). Euro fever registry and Pediatric Rheumatology International Trials Organization (PRINTO) have also proposed classification criteria for different hereditary recurrent fever syndromes (8).
SAIDs can be monogenic and polygenic or multifactorial (9,10). Monogenic SAID (e.g., TRAPS, FMF) follow Mendelian inheritance and result from pathogenic variants in a single gene. On the other hand, disorders such as systemic juvenile idiopathic arthritis, Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis (PFAPA) syndrome and Adult-Onset Still Disease have polygenic or multifactorial etiology. The 2019 International Union of Immunological Societies (IUIS) Expert Committee classified monogenic SAID into 3 major groups: Type 1 interferonopathies, defects affecting the inflammasome and noninflammasome-related conditions (11).
Over the last 2 decades due to an increasing awareness and availability of high throughput genetic sequencing techniques, there has been an exponential increase in discovery of genes responsible for SAID (2,12,13). Further, molecular insights of these disorders have provided the basis for new therapeutic interventions leading to improved outcomes and long-term survivals. There is paucity of data on SAID from India with published literature comprising of only anecdotal case reports (14)(15)(16)(17)(18)(19)(20)(21). In this manuscript we describe clinical features, molecular profile, treatment and outcome in patients with monogenic SAID from six centers in our country. This paper reports nationwide cohort on SAID.

PATIENTS AND METHODS
Centers supported by the Foundation for Primary Immunodeficiency Diseases (FPID), USA, and other institutions involved in care of patients with Inborn Errors of Immunity (IEI)across India were contacted to share details of patients with SAID on a template designed by lead author (DS). Data including demographics, prominent clinical manifestations, laboratory investigations, molecular results, treatment regimens, and long-term outcomes were collated on predesigned Microsoft Excel sheet. Findings of radiology and histopathology were also

DEFINITION OF SAID
Several definitions have been proposed for SAID (4,8,11,22). For the purpose of this study we have used European Society for Immunodeficiencies (ESID) working group definition for the categorization of SAID. ESID has defined "unclassified autoinflammatory diseases" to be characterized by recurrent fever (temperature >38 • C) having occurred on at least six occasions with exclusion of other known infective/inflammatory autoimmune disorders and documented evidence of increased inflammatory markers [erythrocyte sedimentation rate (ESR), C-reactive protein (CRP)], age of onset under 40 years and predominantly but not exclusively with systemic symptoms (23). In the present study all patients who fulfilled ESID working group definition and had molecular confirmation of monogenic SAID were included. Patients with polygenic SAID (e.g., systemic juvenile idiopathic arthritis, chronic non-infectious osteitis) and infantile inflammatory bowel disease were excluded.
All patients were further classified into three subtypes according to 2019 IUIS classification for SAID (11). Coatamer complex one protein alpha subunit (COPA) syndrome was classified as Type 1 interferonopathy (24,25). Patients without molecular confirmation of diagnosis and/or could not be classified in accordance with IUIS classification were also excluded. Some patients included in this series have been reported earlier and these have been duly cited (14,17,19,26,27).

MOLECULAR INVESTIGATIONS
Molecular analysis of patients for PGIMER, Chandigarh was performed at Pediatric Allergy Immunology Laboratory at PGIMER, Chandigarh or in collaboration with international centers, namely Center for Autoinflammatory Diseases and Immunodeficiency, Genoa, Italy (1three patients) and National Institutes of Health (NIH), USA (three patients). Measurement of plasma adenosine deaminase 2 (ADA2) activity in extracts of dried plasma spots was performed in the laboratory of Dr. Michael Hershfield at Duke University School of Medicine, Durham NC, USA (28).

LABORATORY INVESTIGATION AT PGIMER, CHANDIGARH
Molecular analysis of Nucleotide binding oligomerization domain 2 (NOD 2) gene in patients suspected to have Blau syndrome (11/14 patients) and Adenosine deaminase 2(ADA2) gene for Adenosine Deaminase 2 (ADA2) deficiency was performed in-house in Pediatric Immunology Laboratory, Advanced Pediatric Center by Sanger sequencing. Exon-4 of NOD2 gene was amplified using specified oligonucleotide primers and results were analyzed using Codon Code Aligner software (Codon Code Corporation, Massachusetts, USA). Screening of hotspot region (exon 2) of ADA2 gene was also performed in patients clinically suspected to have Deficiency of Adenosine Deaminase 2 (DADA2).
Molecular analysis in most patients at other centers was carried out at commercial laboratories that use targeted gene panel by Next Generation Sequencing (NGS) techniques. Sanger sequencing was used to confirm the variants obtained by NGS.

RESULTS
Data on 107 patients with SAID were collated from various centers in India. Of these, 19 patients had to be excluded as molecular confirmation was not available. Ten patients with variants of unknown significance (VUS) in genes associated with SAIDs were also excluded if found inconsistent with clinical profiles or non-pathogenic based on predictive analysis tools. Remaining 78 patients (Figure 1) included Periodic Fever, Aphthous Stomatitis, Pharyngitis, Adenitis (PFAPA) (

DADA2
Age of onset of symptoms in patients with DADA2 ranged from 5 months to 17 years while age at diagnosis ranged from 9 months to 48 years. All patients with DADA 2 were diagnosed and managed as polyartritis nodosa (PAN). Family history was contributory in three patients (patient no. 2, 3, and 5). Predominant clinical features included fever (4/5), recurrent stroke (3/5), vasculitic rash (3/5), and retinal changes (2/5). Patient one had presented with hypertensive stroke at 3.3 years of aging 1992 and had second episode at the age of 16 years in 2002. The diagnosis of DADA2 was established in 2018 after three decades of follow-up.
Patient no 3 was diagnosed to have central retinal artery occlusion. Inflammatory markers were persistently normal. His sister (patient two) was under treatment and follow up for PAN. She had presented with recurrent abdominal pain with perforation peritonitis and catheter angiography had revealed microaneurysms in mesenteric arteries and renal arteries (Figure 2). In view of family historyDAD2 was suspected and mutation in ADA2 gene was detected. Establishment of diagnosis lead to stoppage of aspirin and commencement of anti-TNF agents.

SAVI
A 10-year-old girl (patient no. 7), previously reported (19) had presented with fever, polyarthritis, and interstitial lung disease (ILD). Initial diagnosis of juvenile idiopathic arthritis with ILD was considered. Younger sibling (patient no. eight) and father (patient no. nine) of index patient also had similar symptoms. Father gave history of gangrene of both lower limbs with amputation of right midfoot and left 2nd toe. Exome sequencing revealed pathogenic variant in Transmembrane protein 173 (TMEM173) gene confirming the diagnosis of SAVI.

SPENCD
A 13-year-old girl (patient no 10) had persistent pyrexia, decreased vision with bilateral optic atrophy, hypertensive stroke, seizures, and proteinuria. Investigations showed hypergammaglobulinemia and positive antinuclear antibodies (ANA) with elevated anti-double stranded DNA (dsDNA) but normal complements. Initial diagnosis of systemic lupus erythematosus was proffered. Renal biopsy revealed IgA nephropathy. Magnetic Resonance Imaging (MRI) brain showed basal ganglia calcifications. Exome sequencing revealed pathogenic variant in Acid phosphatase 5 (ACP 5)gene which was confirmed on Sanger sequencing.
A 4-year-old girl (patient no 11) had presented with bleeding manifestations (skin, mucosal and intracranial bleed) since infancy (Figure 3). She had steroid refractory anemia and thrombocytopenia with no autoantibodies and hypocellular bone marrow. She was later noted to have short stature and metaphyseal dysplasia along with bilateral basal ganglia calcification. Targeted gene panel revealed homozygous nucleotide deletion in exon 1 of ACP5 gene.

COPA Syndrome
The index patient (patient no 12), previously reported (30) was diagnosed to have COPA syndrome when they had presented with rheumatoid factor positive deforming polyarthritis and interstitial lung disease. His father also had arthritis and had succumbed to progressive lung disease.

Cryopyrin-Associated Periodic Syndromes CAPS
In this cohort we report seven patients with CAPS caused by mutations in NLR family pyrin domain containing 3 (NLRP3) gene. All patients had been symptomatic since early infancy but there were significant delays in diagnosis. Age at diagnosis ranged from 15 months to 13 years. Most of these patients were initially diagnosed as JIA. Seven patients had classical phenotype of NOMID with infantile onset of fevers, urticarial rash, arthritis, and progressive deformities with bony overgrowths (Figure 4). Sensory neural hearing loss and headache was found in only1 patient. Of the seven patients, pathogenic variants in NLRP3 gene were identified in four patients (patient no. [21][22][23]27) while no mutation could be identified in patient no 24 on exome sequencing.

NLRP12
Variants in NLRP12 gene were identified t in two patients (patient no 28,29). Both children were symptomatic since early infancy. Patient no. 28 had presented with recurrent episodes of fever, and infections (skin and subcutaneous abscess, diarrhea, meningitis, pneumonia), arthritis, sensorineural hearing loss and hepatosplenomegaly while patient no. 29 in addition had urticarial rash, pustular skin lesions, and lymphadenopathy. Heterozygous mutation in exon 3 in NLR family pyrin domain containing12 (NLRP12) gene was identified. Corticosteroids were used for treatment in patient 28 and is currently well.

FMF
Clinical profile of patients (Patient no 30, 31) with variants in MEFV gene is summarized in Table 2. Patient no. 30 had presented with periodic fever, rash, and abdominal pain. Targeted panel revealed variants of unknown significance in MEFV gene and Phospholipase C Gamma 2 (PLCG 2) gene. The patient is doing well on colchicine. Nine months old boy (patient 31) had presented with recurrent oral ulcers. In view of family history of oral ulceration exome sequencing was performed. Heerozygyous issensense mutation in MEFV gene was identified. Symptomatic improvement has been noted after initiation of colchicine.

APLAID
Clinical profiles of patients no. 32, 33 with PLCG2 variants are summarized in Table 2. Patient no. 32 had erythematous macular

TRAPS
Patient no. 34 had recurrent episodes of fever lasting for 2-3 weeks every 3-4 months with rash since 18 months of age. These f episodes were associated with pain abdomen, myalgias, arthritis, periorbital edema (Figure 6), and subcutaneous swellings. She had received multiple courses of antimicrobials in view of marked polymorphonuclear leukocytosis. Her father was also symptomatic and used to have fever and intermittent subcutaneous swelling and rash. Father was diagnosed to have acute rheumatic fever in childhood. In view of periodic fever, with systemic manifestations and family history suggestive of autosomal dominant disorder, diagnosis of TRAPS was proffered and confirmed on exome sequencing. She was initially managed with corticosteroids followed by injection etanercept. She remains well at follow up.
A 45 years old female (patient no. 35) who had been under follow-up of Dermatology services was diagnosed to have pustular psoriasis since early adolescence. She would have intermittent flares with rash, fever and arthritis. I Her disease was refractory to methotrexate, cyclosporin and corticosteroids. In view of recurrent episodes of fevers, markedly elevated inflammatory parameters, sterile neutrophilic infiltrates on skin biopsy, a possibility of autoinflammatory disease was considered. Whole exome sequencing revealed variant in TNF Receptor Superfamily Member 1A(TNFRSF1A) mutation. She could not be initiated on biological agents due to financial constraints and succumbed to her illness 2 years after diagnosis was established.
A 10-year-old boy (patient no 36) had presented with high grade fever without focus. He reported having febrile episodes lasting for 3-4 weeks with variable afebrile periods since early childhood. These episodes were associated with rash over the trunk and limbs, myalgia and limp, abdominal pain, vomiting, and periorbital swelling. The inflammatory parameters were elevated and targeted panel for autoinflammatory diseases confirmed the diagnosis of TRAPS. The patient demonstrated partial response to etanercept which was changed to tocilizumab to which he responded well.

PAPA Syndrome
A 4-years old girl (patient no 39) had been unwell for 2.5 years when she presented with periodic fevers associated with painful oral ulcers, abdominal pain with hematochezia and colitis. Over the years, she developed multiple pyoderma gangrenosum lesion over extremities, angle of mouth and gluteal region that caused complete destruction of left cheek and lower lip. The lesion were difficult to heal and resulted in fistulae formation. She was initially suspected to have inflammatory bowel disease and oral prednisolone and azathioprine were initiated. Injection infliximab (3 doses) were also commenced. There was partial response in skin lesions and colitis initially. However, lesions reoccurred, and she succumbed to her illness.

A20 Haploinsufficiency
Patient 40 was 2 years old when she had presented with recurrent oral ulcers and genital ulcers (Figure 7). She had colitis, refectory ulcers requiring repeated hospitalization. Markers of inflammation were elevated, and Human Leucocyte Antigen 51 (HLA B 51) allele was detected. Considering a possibility of Bechet's disease, she was commenced on corticosteroid and azathioprine. At 5 years, she was readmitted with persistent  headache, blurring of vision and relapse of oro-genital ulcers. She had papilledema and magnetic resonance imaging (MRI) of brain revealed type 2 Arnold Chiari malformation. When younger brother also developed recurrent oral ulcers, genetic studies were performed in the index patent and targeted panel revealed a novel variant in Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) gene ( Table 3). She is doing well on follow up. Mother is carrier for the same variant while the younger sibling does not carry this variant. Patient 41 had also presented with early onset inflammatory bowel disease and oral ulcers and a novel variant in TNFAIP3) gene was found.

TRNT1 Deficiency
Patient no. 44, had presented with recurrent fever, and diarrhea. Two elder brothers earlier had died. He was evaluated for primary immune deficiency and was noted to have pan hypogammaglobulinemia. A provisional diagnosis of X-linked agammaglobulinemia was made and intravenous immunoglobulin replacement initiated. Exome sequencing revealed a compound heterozygous mutation in exon 2 of TRNT1 gene. Similarly, patient no. 48 also had recurrent infections, bronchiectasis and hypogammaglobulinemia. CARD 14: Patient 46had early onset difficult to treat psoriasis and was found to have mutations in CARD 14 gene.

DISCUSSION
SAID were first recognized in 1999 (4). Over the last two decades, knowledge and recognition of SAID has grown at an unprecedented speed and there have been a plethora of publications on this subject (3,12,13,22). Significant improvement in understanding of genetic and pathogenic mechanisms of SAIDs has resulted in remarkable progress in their management. However, the darta from India is limited (14-18, 26, 27, 33-40). There is no national registry for SAID and there is lack of knowledge on nationwide burden of these diseases. This manuscript is the first attempt to collate data from various centers involved in care of patients with SAID and highlight diagnostic difficulties, treatment, and outcomes of such patients in India.
SAID display wide range of clinical manifestations and can affect almost every organ system. They are uncommon and difficult to diagnose clinically. Overlapping clinical features of these disorders with other relatively common rheumatological disorders often lead to delayin diagnosis. At Chandigarh, we diagnosed our first child with NOMID in 2005. This child was being managed as systemic onset JIA for over 10 years. Similarly, patients with Blau syndrome were initially treated as JIA with uveitis, and patients with DADA2 as PAN. Other initial diagnosis included systemic lupus erythematosus, inflammatory bowel disease and Bechet's disease. With improved awareness amongst internists and pediatrician along with availability of affordable diagnostic techniques, these syndromes are now being suspected and diagnosed early.
Diagnosis of most SAID is based on clinical suspicion, family history and demonstration of elevated inflammatory parameters (ESR, CRP, serum amyloid A protein). Distinct interferon signatures and cytokine patterns may be helpful biomarkers to stratify and monitor patients. However, interpretation and standardization of these tests is difficult. Despite expansion of various laboratory investigations, these investigations are not yet available for clinical use in India. Genetic analysis is needed in all patients for confirmation of diagnosis. In the past, most genetic studies were performed in collaboration with international centers. In the last 5 years, molecular diagnostic techniques were established at PGIMER Chandigarh and National Institute of Immunohematology Mumbai, which are Indian Council of Medical Research (ICMR) recognized Centers for Advanced Research (CAR) in Primary Immune Deficiency Diseases. However, diagnostic facilities are still limited. At PGIMER, among SAID, we can perform Sanger sequencing for ADA2 and NOD2 genes. In recent years, NGS based targeted autoinflammatory panels are available in commercial laboratories, albeit expensive. Interpretation of data and functional validation of variants of unknown significance (VUS) detected remains a challenge.
Management of SAID is aimed at suppression of systemic inflammation. Colchicine and glucocorticoids have been traditionally used to treat SAID. However, with improved knowledge and understanding of pathogenic mechanisms of autoinflammation and availability of specific targeted immunotherapies, treatment strategies have been completely revolutionized (41,42). Anti-IL-1 drugs (anakinra, canakinumab, and rilonacept), have become standard of care for most    inflammasomopathies. These agents successfully control inflammation and improve growth and quality of life. Other biologic agents used include anti-TNF drugs (etanercept), anti-IL-6 drugs (tocilizumab), and Janus kinase inhibitors (tofacitinib, baricitinib, and ruxolitinib). Treatment of SAID is extremely challenging in resource constrained settings. Anti-IL-1 drugs are not readily available in India and other developing countries. These drugs have to be imported on a "named-patient-basis" and are exorbitantly expensive. Some biosimilar molecules like anti TNF (adalimumab, infliximab) and anti IL6 (tocilizumab) are available alternative therapies. Though these molecules are cost cheaper in India when compared to Western countries, yet they remain well beyond the scope of average Indian family. Thus, corticosteroids and other conventional immunosuppressive agents still form the main stay of therapy. Patients often require higher doses as the disease progresses and they often develop corticosteroid related side effects. Off late, hematopoietic stem cell transplant (HSCT) is also emerging as a curative option for some SAID (43,44). However, none of our patients received HSCT. AID related morbidity and mortality continues to be high. In our cohort (8/49) have died at the time of analysis due to non-availability of treatment and development of complications. Amyloidosis had already developed in four patients at the time of diagnosis and it remains an important cause of death.
There are several limitations of this study. It is a case-based record review report from major primary immunodeficiency diseases centers across the country. Data from various individual rheumatology units could not be collated. Moreover, shared data were not uniform from all centers. Patients with unclassified SAID and patients in whom molecular diagnosis could not be established were excluded from the study. This is a first comprehensive multicentric report of patients with SAID from India. Varied clinical and molecular spectrum has been reported. Considerable delays in diagnosis were recognized. Application of NGS based targeted panels and whole exome sequencing has helped in identifying known as well as novel gene defects. Establishment of diagnosis in a patient enabled early diagnosis FIGURE 7 | (A,B) Oral cavity erythema and ulceration and genital ulcers in child with A20 haploinsufficiency ( Table 3; Patient no 40). (C) MRI Brain T2 weighted sagittal section images demonstrating type 2 Arnold Chiari malformation ( Table 3; Patient no 40). (D) MR venography of brain demonstrated normal flow and no evidence of cerebral sinus venous thrombosis ( Table 3; Patient no 40). of other family members and provided an opportunity for prenatal diagnosis. Although, ability to diagnose SAID has improved, non-availability of expensive immunotherapies remains a major drawback. In India corticosteroids and conventional immunosuppressive agents continue to remain corner stones for treatment. Lack of availability of targeted immunotherapies for treatment prevents the initiation of effective treatments that can change patients' lives. SAID continue to result in significant morbidity and mortality.
To conclude, more efforts are needed to enhance awareness of autoinflammatory diseases among health care professionals and there is an urgent need to make life saving drugs universally available.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institute Ethics Committee, PGIMER, Chandigarh (Ref No: INT/IEC/2021/SPL-264). Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements. Written informed consent was obtained from the minor(s)' legal guardian/next of kin for the publication of any potentially identifiable images or data included in this article.