Evolving spectrum of adenosine deaminase (ADA) deficiency: Assessing genotype pathogenicity according to expressed ADA activity of 46 variants.

BACKGROUND: Deficiency of adenosine deaminase (ADA or ADA1) has broad clinical and genetic heterogeneity. Screening techniques can identify asymptomatic infants whose phenotype and prognosis are indeterminate, and who may carry ADA variants of unknown significance. OBJECTIVE: We systematically assessed the pathogenic potential of rare ADA missense variants to better define the relationship of genotype to red blood cell (RBC) total deoxyadenosine nucleotide (dAXP) content and to phenotype. METHODS: We expressed 46 ADA missense variants in the ADA-deficient SØ3834 strain of Escherichia coli and defined genotype categories (GCs) ranked I to IV by increasing expressed ADA activity. We assessed relationships among GC rank, RBC dAXP, and phenotype in 58 reference patients with 50 different genotypes. We used our GC ranking system to benchmark AlphaMissense for predicting variant pathogenicity, and we used a minigene assay to identify exonic splicing variants in ADA exon 9. RESULTS: The 46 missense variants expressed ∼0.001% to ∼70% of wild-type ADA activity (40% had <0.05% of wild-type ADA activity and 50% expressed >1%). RBC dAXP ranged from undetectable to >75% of total adenine nucleotides and correlated well with phenotype. Both RBC dAXP and clinical severity were inversely related to total ADA activity expressed by both inherited variants. Our GC scoring system performed better than AlphaMissense in assessing variant pathogenicity, particularly for less deleterious variants. CONCLUSION: For ADA deficiency, pathogenicity is a continuum and conditional, depending on the total ADA activity contributed by both inherited variants as indicated by GC rank. However, in patients with indeterminate phenotype identified by screening, RBC dAXP measured at diagnosis may have greater prognostic value than GC rank.

Treatment with Elapegademase Restores Immunity in Infants with Adenosine Deaminase Deficient Severe Combined Immunodeficiency.

PURPOSE: Patients with adenosine deaminase 1 deficient severe combined immunodeficiency (ADA-SCID) are initially treated with enzyme replacement therapy (ERT) with polyethylene glycol-modified (PEGylated) ADA while awaiting definitive treatment with hematopoietic stem cell transplant (HSCT) or gene therapy. Beginning in 1990, ERT was performed with PEGylated bovine intestinal ADA (ADAGEN®). In 2019, a PEGylated recombinant bovine ADA (Revcovi®) replaced ADAGEN following studies in older patients previously treated with ADAGEN for many years. There are limited longitudinal data on ERT-naïve newborns treated with Revcovi. METHODS: We report our clinical experience with Revcovi as initial bridge therapy in three newly diagnosed infants with ADA-SCID, along with comprehensive biochemical and immunologic data. RESULTS: Revcovi was initiated at twice weekly dosing (0.2 mg/kg intramuscularly), and monitored by following plasma ADA activity and the concentration of total deoxyadenosine nucleotides (dAXP) in erythrocytes. All patients rapidly achieved a biochemically effective level of plasma ADA activity, and red cell dAXP were eliminated within 2-3 months. Two patients reconstituted B-cells and NK-cells within the first month of ERT, followed by naive T-cells one month later. The third patient reconstituted all lymphocyte subsets within the first month of ERT. One patient experienced declining lymphocyte counts with improvement following Revcovi dose escalation. Two patients developed early, self-resolving thrombocytosis, but no thromboembolic events occurred. CONCLUSION: Revcovi was safe and effective as initial therapy to restore immune function in these newly diagnosed infants with ADA-SCID, however, time course and degree of reconstitution varied. Revcovi dose may need to be optimized based on immune reconstitution, clinical status, and biochemical data.

Comparison of disease phenotypes and mechanistic insight on causal variants in patients with DADA2.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) results in heterogeneous manifestations including systemic vasculitis and red cell aplasia. The basis of different disease phenotypes remains incompletely defined. OBJECTIVE: We sought to further delineate disease phenotypes in DADA2 and define the mechanistic basis of ADA2 variants. METHODS: We analyzed the clinical features and ADA2 variants in 33 patients with DADA2. We compared the transcriptomic profile of 14 patients by bulk RNA sequencing. ADA2 variants were expressed experimentally to determine impact on protein production, trafficking, release, and enzymatic function. RESULTS: Transcriptomic analysis of PBMCs from DADA2 patients with the vasculitis phenotype or pure red cell aplasia phenotype exhibited similar upregulation of TNF, type I interferon, and type II interferon signaling pathways compared with healthy controls. These pathways were also activated in 3 asymptomatic individuals with DADA2. Analysis of ADA2 variants, including 7 novel variants, showed different mechanisms of functional disruption including (1) unstable transcript leading to RNA degradation; (2) impairment of ADA2 secretion because of retention in the endoplasmic reticulum; (3) normal expression and secretion of ADA2 that lacks enzymatic function; and (4) disruption of the N-terminal signal peptide leading to cytoplasmic localization of unglycosylated protein. CONCLUSIONS: Transcriptomic signatures of inflammation are observed in patients with different disease phenotypes, including some asymptomatic individuals. Disease-associated ADA2 variants affect protein function by multiple mechanisms, which may contribute to the clinical heterogeneity of DADA2.

Evaluation and Management of Deficiency of Adenosine Deaminase 2: An International Consensus Statement.

Importance: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited disease characterized by systemic vasculitis, early-onset stroke, bone marrow failure, and/or immunodeficiency affecting both children and adults. DADA2 is among the more common monogenic autoinflammatory diseases, with an estimate of more than 35 000 cases worldwide, but currently, there are no guidelines for diagnostic evaluation or management. Objective: To review the available evidence and develop multidisciplinary consensus statements for the evaluation and management of DADA2. Evidence Review: The DADA2 Consensus Committee developed research questions based on data collected from the International Meetings on DADA2 organized by the DADA2 Foundation in 2016, 2018, and 2020. A comprehensive literature review was performed for articles published prior to 2022. Thirty-two consensus statements were generated using a modified Delphi process, and evidence was graded using the Oxford Center for Evidence-Based Medicine Levels of Evidence. Findings: The DADA2 Consensus Committee, comprising 3 patient representatives and 35 international experts from 18 countries, developed consensus statements for (1) diagnostic testing, (2) screening, (3) clinical and laboratory evaluation, and (4) management of DADA2 based on disease phenotype. Additional consensus statements related to the evaluation and treatment of individuals with DADA2 who are presymptomatic and carriers were generated. Areas with insufficient evidence were identified, and questions for future research were outlined. Conclusions and Relevance: DADA2 is a potentially fatal disease that requires early diagnosis and treatment. By summarizing key evidence and expert opinions, these consensus statements provide a framework to facilitate diagnostic evaluation and management of DADA2.

Long-Term Immune Reconstitution in ADA-Deficient Patients Treated With Elapegademase: A Real-World Experience.

BACKGROUND: ADAGEN, a bovine-based enzyme replacement therapy (ERT), has been used to treat adenosine deaminase severe combined immunodeficiency (ADA-SCID). In 2018, ADAGEN was replaced by REVCOVI (elapegademase), a modified bovine recombinant protein. OBJECTIVE: To determine the real-life long-term benefits of REVCOVI in ADA-SCID. METHODS: Data on ERT, infectious and noninfectious complications, and metabolic and immune evaluations were collected from 17 patients with ADA-SCID treated for 6 months or more with REVCOVI. RESULTS: Eleven patients had previously received ADAGEN for 16 to 324 months, whereas 6 patients were ERT-naive. REVCOVI was administered twice weekly at 0.4 mg/kg/wk in ERT-naive patients, whereas patients transitioning to REVCOVI from ADAGEN typically continued at the same frequency and equivalent dosing as ADAGEN, resulting in a significantly lower (P = .007) total REVCOVI dose in the transitioning group. REVCOVI treatment in the ERT-naive group led to the resolution of many clinical and laboratory complications of ADA deficiency, whereas there were no new adverse effects among the transitioning patients. REVCOVI treatment increased plasma ADA activity and decreased dAXP (which included deoxyadenosine mono-, di-, and tri phosphate) among most patients, effects that persisted throughout the 7- to 37-month treatment periods, except in 2 patients with incomplete adherence. Among some patients, after 0.5 to 6 months, injection frequency was reduced to once a week, while maintaining adequate metabolic profiles. All ERT-naive infants treated with REVCOVI demonstrated an increase in the number of CD4+ T and CD19+ B cells, although these counts remained stable but lower than normal in most transitioning patients. CONCLUSIONS: REVCOVI is effective for the management of ADA-SCID.

Case Report: Consistent disease manifestations with a staggered time course in two identical twins affected by adenosine deaminase 2 deficiency.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disease associated with a highly variable clinical presentation, including vasculitis, immunodeficiency, and hematologic manifestations, potentially progressing over time. The present study describes the long-term evolution of the immuno-hematological features and therapeutic challenge of two identical adult twin sisters affected by DADA2. The absence of plasmatic adenosine deaminase 2 (ADA2) activity in both twins suggested the diagnosis of DADA2, then confirmed by genetic analysis. Exon sequencing revealed a missense (p.Leu188Pro) mutation on the paternal ADA2 allele. While, whole genome sequencing identified an unreported deletion (IVS6_IVS7del*) on the maternal allele predicted to produce a transcript missing exon 7. The patients experienced the disease onset during childhood with early strokes (Patient 1 at two years, Patient 2 at eight years of age), subsequently followed by other shared DADA2-associated features, including neutropenia, hypogammaglobulinemia, reduced switched memory B cells, inverted CD4:CD8 ratio, increased naïve T cells, reduced follicular regulatory T cells, the almost complete absence of NK cells, T-large granular cell leukemia, and osteoporosis. Disease evolution differed: clinical manifestations presented several years earlier and were more pronounced in Patient 1 than in Patient 2. Due to G-CSF refractory life-threatening neutropenia, Patient 1 successfully underwent an urgent hematopoietic stem cell transplantation (HSCT) from a 9/10 matched unrelated donor. Patient 2 experienced a similar, although delayed, disease evolution and is currently on anti-TNF therapy and anti-infectious prophylaxis. The unique cases confirmed that heterozygous patients with null ADA2 activity deserve deep investigation for possible structural variants on a single allele. Moreover, this report emphasizes the importance of timely recognizing DADA2 at the onset to allow adequate follow-up and detection of disease progression. Finally, the therapeutic management in these identical twins raises significant concerns as they share a similar phenotype, with a delayed but almost predictable disease evolution in one of them, who could benefit from a prompt definitive treatment like elective allogeneic HSCT. Additional data are required to assess whether the absence of enzymatic activity at diagnosis is associated with hematological involvement and is also predictive of bone marrow dysfunction, encouraging early HSCT to improve functional outcomes.

Outcomes following treatment for ADA-deficient severe combined immunodeficiency: a report from the PIDTC.

Adenosine deaminase (ADA) deficiency causes ∼13% of cases of severe combined immune deficiency (SCID). Treatments include enzyme replacement therapy (ERT), hematopoietic cell transplant (HCT), and gene therapy (GT). We evaluated 131 patients with ADA-SCID diagnosed between 1982 and 2017 who were enrolled in the Primary Immune Deficiency Treatment Consortium SCID studies. Baseline clinical, immunologic, genetic characteristics, and treatment outcomes were analyzed. First definitive cellular therapy (FDCT) included 56 receiving HCT without preceding ERT (HCT); 31 HCT preceded by ERT (ERT-HCT); and 33 GT preceded by ERT (ERT-GT). Five-year event-free survival (EFS, alive, no need for further ERT or cellular therapy) was 49.5% (HCT), 73% (ERT-HCT), and 75.3% (ERT-GT; P < .01). Overall survival (OS) at 5 years after FDCT was 72.5% (HCT), 79.6% (ERT-HCT), and 100% (ERT-GT; P = .01). Five-year OS was superior for patients undergoing HCT at <3.5 months of age (91.6% vs 68% if ≥3.5 months, P = .02). Active infection at the time of HCT (regardless of ERT) decreased 5-year EFS (33.1% vs 68.2%, P < .01) and OS (64.7% vs 82.3%, P = .02). Five-year EFS (90.5%) and OS (100%) were best for matched sibling and matched family donors (MSD/MFD). For patients treated after the year 2000 and without active infection at the time of FDCT, no difference in 5-year EFS or OS was found between HCT using a variety of transplant approaches and ERT-GT. This suggests alternative donor HCT may be considered when MSD/MFD HCT and GT are not available, particularly when newborn screening identifies patients with ADA-SCID soon after birth and before the onset of infections. This trial was registered at www.clinicaltrials.gov as #NCT01186913 and #NCT01346150.

Polyethylene Glycol-Like Brush Polymer Conjugate of a Protein Drug Does Not Induce an Antipolymer Immune Response and Has Enhanced Pharmacokinetics than Its Polyethylene Glycol Counterpart.

Protein therapeutics, except for antibodies, have a short plasma half-life and poor stability in circulation. Covalent coupling of polyethylene glycol (PEG) to protein drugs addresses this limitation. However, unlike previously thought, PEG is immunogenic. In addition to induced PEG antibodies, ≈70% of the US population has pre-existing anti-PEG antibodies. Both induced and preexisting anti-PEG antibodies result in accelerated drug clearance, reduced clinical efficacy, and severe hypersensitivity reactions that have limited the clinical utility of uricase, an enzyme drug for treatment for refractory gout that is decorated with a PEG corona. Here, the authors synthesize a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) conjugate of uricase that decorates the protein with multiple polymer chains to create a corona to solve these problems. The resulting uricase-POEGMA is well-defined, has high bioactivity, and outperforms its PEG counterparts in its pharmacokinetics (PK). Furthermore, the conjugate does not induce anti-POEGMA antibodies and is not recognized by anti-PEG antibodies. These findings suggest that POEGMA conjugation may provide a solution to the immunogenicity and antigenicity limitations of PEG while improving upon its PK benefits. These results transcend uricase and can be applied to other PEGylated therapeutics and the broader class of biologics with suboptimal PK.

Comprehensive analysis of ADA2 genetic variants and estimation of carrier frequency driven by a function-based approach.

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease caused by deleterious ADA2 variants. The frequency of these variants in the general population, and hence the expected disease prevalence, remain unknown. OBJECTIVE: We aimed to characterize the functional impact and carrier frequency of ADA2 variants. METHODS: We performed functional studies and in silico analysis on 163 ADA2 variants, including DADA2-associated variants and population variants identified in the Genome Aggregation Database. We estimated the carrier rate using the aggregate frequency of deleterious variants. RESULTS: Functional studies of ADA2 variants revealed that 77 (91%) of 85 of DADA2-associated variants reduced ADA2 enzymatic function by >75%. Analysis of 100 ADA2 variants in the database showed a full spectrum of impact on ADA2 function, rather than a dichotomy of benign versus deleterious variants. We found several in silico algorithms that effectively predicted the impact of ADA2 variants with high sensitivity and specificity, and confirmed a correlation between the residual function of ADA2 variants in vitro and the plasma ADA2 activity of individuals carrying these variants (n = 45; r = 0.649; P < .0001). Using <25% residual enzymatic activity as the cutoff to define potential pathogenicity, integration of our results with the database population data revealed an estimated carrier frequency of at least 1 in 236 individuals, corresponding to an expected DADA2 disease prevalence of ~1 in 222,000 individuals. CONCLUSIONS: Functional annotation guides the interpretation of ADA2 variants to create a framework that enables estimation of DADA2 carrier frequency and disease prevalence.

Normal IgH Repertoire Diversity in an Infant with ADA Deficiency After Gene Therapy.

PURPOSE: Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) through an accumulation of toxic metabolites within lymphocytes. Recently, ADA deficiency has been successfully treated using lentiviral-transduced autologous CD34+ cells carrying the ADA gene. T and B cell function appears to be fully restored, but in many patients' B cell numbers remain low, and assessments of the immunoglobulin heavy (IgHV) repertoire following gene therapy are lacking. METHODS: We performed deep sequencing of IgHV repertoire in peripheral blood lymphocytes from a child following lentivirus-based gene therapy for ADA deficiency and compared to the IgHV repertoire in healthy infants and adults. RESULTS: After gene therapy, Ig diversity increased over time as evidenced by V, D, and J gene usage, N-additions, CDR3 length, extent of somatic hypermutation, and Ig class switching. There was the emergence of predominant IgHM, IgHG, and IgHA CDR3 lengths after gene therapy indicating successful oligoclonal expansion in response to antigens. This provides proof of concept for the feasibility and utility of molecular monitoring in following B cell reconstitution following gene therapy for ADA deficiency. CONCLUSION: Based on deep sequencing, gene therapy resulted in an IgHV repertoire with molecular diversity similar to healthy infants.

Long-term outcomes after gene therapy for adenosine deaminase severe combined immune deficiency.

Patients lacking functional adenosine deaminase activity have severe combined immunodeficiency (ADA SCID), which can be treated with ADA enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT), or autologous HSCT with gene-corrected cells (gene therapy [GT]). A cohort of 10 ADA SCID patients, aged 3 months to 15 years, underwent GT in a phase 2 clinical trial between 2009 and 2012. Autologous bone marrow CD34+ cells were transduced ex vivo with the MND (myeloproliferative sarcoma virus, negative control region deleted, dl587rev primer binding site)-ADA gammaretroviral vector (gRV) and infused following busulfan reduced-intensity conditioning. These patients were monitored in a long-term follow-up protocol over 8 to 11 years. Nine of 10 patients have sufficient immune reconstitution to protect against serious infections and have not needed to resume ERT or proceed to secondary allogeneic HSCT. ERT was restarted 6 months after GT in the oldest patient who had no evidence of benefit from GT. Four of 9 evaluable patients with the highest gene marking and B-cell numbers remain off immunoglobulin replacement therapy and responded to vaccines. There were broad ranges of responses in normalization of ADA enzyme activity and adenine metabolites in blood cells and levels of cellular and humoral immune reconstitution. Outcomes were generally better in younger patients and those receiving higher doses of gene-marked CD34+ cells. No patient experienced a leukoproliferative event after GT, despite persisting prominent clones with vector integrations adjacent to proto-oncogenes. These long-term findings demonstrate enduring efficacy of GT for ADA SCID but also highlight risks of genotoxicity with gRVs. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

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.

Deficiency of Adenosine Deaminase 2 in Adults and Children: Experience From India.

OBJECTIVE: Deficiency of adenosine deaminase 2 (DADA2) is a potentially fatal monogenic syndrome characterized by variable manifestations of systemic vasculitis, bone marrow failure, and immunodeficiency. Most cases are diagnosed by pediatric care providers, given the typical early age of disease onset. This study was undertaken to describe the clinical phenotypes and treatment response both in adults and in children with DADA2 in India. METHODS: A retrospective analysis of pediatric and adult patients with DADA2 diagnosed at various rheumatology centers across India was conducted. Clinical characteristics, diagnostic findings, and treatment responses were analyzed in all subjects. RESULTS: In total, 33 cases of DADA2 were confirmed in this cohort between April 2017 and March 2020. Unlike previous studies, nearly one-half of the confirmed cases presented during adulthood. All symptomatic patients exhibited features of vasculitis, whereas constitutional symptoms and anemia were more common in pediatric patients. Cutaneous and neurologic involvement were common, and 18 subjects had experienced at least one stroke. In addition, the clinical spectrum of DADA2 was expanded by recognition of novel features in these patients, including pancreatic infarction, focal myocarditis, and diffuse alveolar hemorrhage. Treatment with tumor necrosis factor inhibitors (TNFi) was initiated in 25 patients. All of the identified disease manifestations showed marked improvement after initiation of TNFi, and disease remission was achieved in 19 patients. Two cases were complicated by tuberculosis infection, and 2 deaths were reported. CONCLUSION: This report presents the first case series of patients with DADA2 from India, diagnosed by adult and pediatric care providers. The findings raise awareness of this syndrome, particularly with regard to its presentation in adults.

Morbidity in an adenosine deaminase-deficient patient during 27 years of enzyme replacement therapy.

INTRODUCTION: Adenosine deaminase (ADA) deficiency causes severe immunodeficiency that is lethal in infancy. Enzyme replacement therapy (ERT) can improve the metabolic, immune and non-immune abnormalities in patients prior to transplantation, however, its benefits over extended periods are not well characterized. We describe a 28-year-old female who received ERT for 27 years. She suffered from EBV negative B cell lymphoma of the hip at 14 years of age and Guillian-Barre Syndrome 2 years later. At 22 years of age, she experienced a gastrointestinal infection with Mycobacterium genavense. At 26 years of age, lymphoma reoccurred with multiple liver lesions followed by Mycobacterium genavense infection with dissemination to the brain. Throughout this period, ADA activity in the plasma was within the therapeutic range. Repeated evaluations demonstrated very low lymphocyte counts and impaired T cell function. CONCLUSIONS: ERT might be insufficient to maintain normal immunity over extended periods in some ADA-deficient patients.

Adenosine deaminase 2 as a biomarker of macrophage activation syndrome in systemic juvenile idiopathic arthritis.

OBJECTIVE: Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (sJIA) characterised by a vicious cycle of immune amplification that can culminate in overwhelming inflammation and multiorgan failure. The clinical features of MAS overlap with those of active sJIA, complicating early diagnosis and treatment. We evaluated adenosine deaminase 2 (ADA2), a protein of unknown function released principally by monocytes and macrophages, as a novel biomarker of MAS. METHODS: We established age-based normal ranges of peripheral blood ADA2 activity in 324 healthy children and adults. We compared these ranges with 173 children with inflammatory and immune-mediated diseases, including systemic and non-systemic JIA, Kawasaki disease, paediatric systemic lupus erythematosus and juvenile dermatomyositis. RESULTS: ADA2 elevation beyond the upper limit of normal in children was largely restricted to sJIA with concomitant MAS, a finding confirmed in a validation cohort of sJIA patients with inactive disease, active sJIA without MAS or sJIA with MAS. ADA2 activity strongly correlated with MAS biomarkers including ferritin, interleukin (IL)-18 and the interferon (IFN)-γ-inducible chemokine CXCL9 but displayed minimal association with the inflammatory markers C reactive protein and erythrocyte sedimentation rate. Correspondingly, ADA2 paralleled disease activity based on serial measurements in patients with recurrent MAS episodes. IL-18 and IFN-γ elicited ADA2 production by peripheral blood mononuclear cells, and ADA2 was abundant in MAS haemophagocytes. CONCLUSIONS: These findings collectively identify the utility of plasma ADA2 activity as a biomarker of MAS and lend further support to a pivotal role of macrophage activation in this condition.