Deficiency Of Adenosine Deaminase 2 Patients Research Articles

Early bone marrow alterations in patients with adenosine deaminase 2 deficiency across disease phenotypes and severities

Deficiency of adenosine deaminase 2 (DADA2) is a complex monogenic disease caused by recessive mutations in the ADA2 gene. DADA2 exhibits a broad clinical spectrum encompassing vasculitis, immunodeficiency, and hematological abnormalities. Yet, the impact of DADA2 on the bone marrow (BM) microenvironment is largely unexplored. This study comprehensively examined the BM and peripheral blood of pediatric and adult patients with DADA2 presenting rheumatologic/immunologic symptoms or severe hematological manifestations. Immunophenotyping of hematopoietic stem cells (HSCs), progenitor cells, and mature cell populations was performed for 18 patients with DADA2. We also conducted a characterization of the mesenchymal stromal cells (MSCs). Our study revealed a significant decrease in primitive HSCs and progenitor cells, alongside their reduced clonogenic capacity and multilineage differentiation potential. These BM defects were evident in patients with both severe and non-severe hematological manifestations, including pediatric patients, demonstrating that BM disruption can emerge silently and early on, even in patients who do not show obvious hematological symptoms. Beyond stem cells, there was a reduction in mature cell populations in the BM and peripheral blood, affecting myeloid, erythroid, and lymphoid populations. Furthermore, BM MSCs in DADA2 patients exhibited reduced clonogenic and proliferation capabilities and were more prone to undergo cellular senescence marked by elevated DNA damage. Our exploration into the BM landscape of DADA2 patients sheds light on the critical hematological dimension of the disease and emphasizes the importance of vigilant monitoring, even in the case of subclinical presentation.

ADA2 regulates inflammation and hematopoietic stem cell emergence via the A2bR pathway in zebrafish

Deficiency of adenosine deaminase 2 (DADA2) is an inborn error of immunity caused by loss-of-function mutations in the adenosine deaminase 2 (ADA2) gene. Clinical manifestations of DADA2 include vasculopathy and immuno-hematological abnormalities, culminating in bone marrow failure. A major gap exists in our knowledge of the regulatory functions of ADA2 during inflammation and hematopoiesis, mainly due to the absence of an ADA2 orthologue in rodents. Exploring these mechanisms is essential for understanding disease pathology and developing new treatments. Zebrafish possess two ADA2 orthologues, cecr1a and cecr1b, with the latter showing functional conservation with human ADA2. We establish a cecr1b-loss-of-function zebrafish model that recapitulates the immuno-hematological and vascular manifestations observed in humans. Loss of Cecr1b disrupts hematopoietic stem cell specification, resulting in defective hematopoiesis. This defect is caused by induced inflammation in the vascular endothelium. Blocking inflammation, pharmacological modulation of the A2r pathway, or the administration of the recombinant human ADA2 corrects these defects, providing insights into the mechanistic link between ADA2 deficiency, inflammation and immuno-hematological abnormalities. Our findings open up potential therapeutic avenues for DADA2 patients.

A case series of ten plus one deficiency of adenosine deaminase 2 (DADA2) patients in Iran

BackgroundDeficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive autoinflammatory disease caused by mutations in the ADA2 gene. DADA2 has a broad spectrum of clinical presentations. Apart from systemic manifestations, we can categorize most of the signs and symptoms of DADA2 into the three groups of vasculitis, hematologic abnormalities, and immunologic dysregulations. The most dominant vasculitis features are skin manifestations, mostly in the form of livedo racemosa/reticularis, and early onset ischemic or hemorrhagic strokes. Hypogammaglobulinemia that is found in many cases of DADA2 brings immunodeficiencies into the differential diagnosis. Cytopenia, pure red cell aplasia (PRCA), and bone marrow failure (BMF) are the hematologic abnormalities commonly found in DADA.Case presentationWe introduce eleven patients with DADA2 diagnosis, including two brothers and sisters, one set of twin sisters, and one father and his daughter and son. Ten patients (91%) had consanguineous parents. All the patients manifested livedo racemose/reticularis. Ten patients (91%) reported febrile episodes, and seven (64%) had experienced strokes. Only one patient had hypertension. Two of the patients (11%) presented decreased immunoglobulin levels. One of the patients presented with PRCA. Except for the PRCA patient with G321E mutation, all of our patients delivered G47R mutation, the most common mutation in DADA2 patients. Except for one patient who unfortunately passed away before the diagnosis was made and proper treatment was initiated, the other patients’ symptoms are currently controlled; two of the patients presented with mild symptoms and are now being treated with colchicine, and the eight others responded well to anti-TNFs. The PRCA patient still suffers from hematologic abnormalities and is a candidate for a bone marrow transplant.ConclusionsConsidering the manifestations and the differential diagnoses, DADA2 is not merely a rheumatologic disease, and introducing this disease to hematologists, neurologists, and immunologists is mandatory to initiate prompt and proper treatment. The efficacy of anti-TNFs in resolving the symptoms of DADA2 patients have been proven, but not for those with hematologic manifestations. Similarly, they were effective in controlling the symptoms of our cohort of patients, except for the one patient with cytopenia.

Clinical Symptoms, Laboratory Parameters and Long-Term Follow-up in a National DADA2 Cohort

Deficiency of adenosine deaminase-2 (DADA2) is an autosomal recessive autoinflammatory disease with an extremely variable disease presentation. This paper provides a comprehensive overview of the Dutch DADA2 cohort. We performed a retrospective cohort study in 29 ADA2-deficient patients from 23 families with a median age at inclusion of 26 years. All patients had biallelic pathogenic variants in the ADA2 gene. The most common clinical findings included cutaneous involvement (79.3%), (hepato)splenomegaly (70.8%) and recurrent infections (58.6%). Stroke was observed in 41.4% of the patients. The main laboratory abnormalities were hypogammaglobulinemia and various cytopenias. Patients presented most often with a mixed phenotype involving vasculopathy, immunodeficiency and hematologic manifestations (62.1%). In this cohort, malignancies were reported in eight patients (27.6%), of whom five presented with a hematologic malignancy and two with a basal cell carcinoma. Four patients developed hemophagocytic lymphohistiocytosis (HLH) or an HLH-like episode, of whom three passed away during or shortly after the occurrence of HLH. TNF-inhibitors (TNFi) were effective in treating vasculopathy-associated symptoms and preventing stroke, but were hardly effective in the treatment of hematologic manifestations. Three patients underwent hematopoietic cell transplantation and two of them are doing well with complete resolution of DADA2-related symptoms. The overall mortality in this cohort was 17.2%. In conclusion, this cohort describes the clinical, genetic and laboratory findings of 29 Dutch DADA2 patients. We describe the occurrence of HLH as a life-threatening disease complication and report a relatively high incidence of malignancies and mortality.

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

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. We sought to further delineate disease phenotypes in DADA2 and define the mechanistic basis of ADA2 variants. 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. 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. 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.

Deficiency of adenosine deaminase 2: a challenging differential diagnosis of polyarteritis nodosa.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that was first described in 2014. It is a monogenic disease that is caused by loss-of-function variants in the ADA2 gene. Deficiency of adenosine deaminase 2 involves small- and medium-sized vessels and its clinical presentations include polyarteritis nodosa (PAN)-like features such as livedoid rash, early-onset stroke, hypogammaglobulinemia, hematological abnormalities, and systemic inflammation. Early diagnosis and treatment of DADA2 are crucial as the clinical features could be potentially life-threatening but might be treatable. The first-line treatment of choice in DADA2 is tumor necrosis factor α inhibitors. We aimed to provide an overview of the known pathophysiology, clinical presentations, diagnosis, and treatment of DADA2. A clearer knowledge of DADA2 may help to better diagnose, manage, and improve the clinical outcome of DADA2 patients. However, further studies are required to investigate the genotype-phenotype associations and exact pathophysiology of DADA2.

Adenosine Deaminase 2 Deficiency (DADA2): A Crosstalk Between Innate and Adaptive Immunity.

Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic autoinflammatory disorder presenting with a broad spectrum of clinical manifestations, including immunodeficiency, vasculopathy and hematologic disease. Biallelic mutations in ADA2 gene have been associated with a decreased ADA2 activity, leading to reduction in deamination of adenosine and deoxyadenosine into inosine and deoxyinosine and subsequent accumulation of extracellular adenosine. In the early reports, the pivotal role of innate immunity in DADA2 pathogenic mechanism has been underlined, showing a skewed polarization from the M2 macrophage subtype to the proinflammatory M1 subtype, with an increased production of inflammatory cytokines such as TNF-α. Subsequently, a dysregulation of NETosis, triggered by the excess of extracellular Adenosine, has been implicated in the pathogenesis of DADA2. In the last few years, evidence is piling up that adaptive immunity is profoundly altered in DADA2 patients, encompassing both T and B branches, with a disrupted homeostasis in T-cell subsets and a B-cell skewing defect. Type I/type II IFN pathway upregulation has been proposed as a possible core signature in DADA2 T cells and monocytes but also an increased IFN-β secretion directly from endothelial cells has been described. So far, a unifying clear pathophysiological explanation for the coexistence of systemic inflammation, immunedysregulation and hematological defects is lacking. In this review, we will explore thoroughly the latest understanding regarding DADA2 pathophysiological process, with a particular focus on dysregulation of both innate and adaptive immunity and their interacting role in the development of the disease.

Allogeneic Hematopoietic Cell Transplantation for Patients With Deficiency of Adenosine Deaminase 2 (DADA2): Approaches, Obstacles and Special Considerations.

Deficiency of adenosine deaminase 2 (DADA2) is an inherited autosomal recessive disease characterized by autoinflammation (recurrent fever), vasculopathy (livedo racemosa, polyarteritis nodosa, lacunar ischemic strokes, and intracranial hemorrhages, end organ vasculitis), immunodeficiency, lymphoproliferation, immune cytopenias, and bone marrow failure. Allogeneic hematopoietic cell transplantation (HCT) is curative for DADA2 as it reverses the hematological, immune and vascular phenotype of DADA2. The primary goal of HCT in DADA2, like in other non-malignant diseases, is engraftment with the establishment of normal hematopoiesis and normal immune function. Strategies in selecting a preparative regimen should take into consideration the specific vulnerabilities to endothelial dysfunction and liver toxicity in DADA2 patients. Overcoming an increased risk of graft rejection while minimizing organ toxicity, graft-versus-host disease, and infections can be particularly challenging in DADA2 patients. This review will discuss approaches to HCT in DADA2 patients including disease-specific considerations, barriers to successful engraftment, post-HCT complications, and clinical outcomes of published patients with DADA2 who have undergone HCT to date.

Clinical presentation of children with Deficiency of Adenosine deaminase 2: A case series

Deficiency of Adenosine deaminase 2 (DADA2) is a monogenic inflammatory disease, caused by mutations in ADA2 gene, which encodes an extracellular enzyme acting as a monocyte differentiation factor. DADA2 is first described with the clinical picture resembling polyarteritis nodosa, including livedo racemose, recurrent fever, musculoskeletal complaints. Besides, some patients have cytopenia, lymphoproliferation and mild to moderate immunodeficiency. The most crucial complication of DADA2 is neurological involvement, especially arterial stroke, which necessitates continuous treatment with anti-tumor necrosis factor α (anti-TNFα) treatment for preventing further stroke attacks.Herein, we report 5 DADA2 patients from 5 unrelated families, all had G47R mutation in at least one allele. All patients had livedo racemose, and 4 patients suffered from recurrent fever. Besides, musculoskeletal complaints and gastrointestinal symptoms were present in 4 and 3 patients, respectively. One patient had chronic arthritis and only one patient had a history of recurrent stroke without any sequela. Hematological and immunological involvement occurred in 3 and 4 patients, respectively, whereas only one had significant panhypogammaglobulinemia, requiring replacement therapy. We started etanercept treatment to all patients, which resulted the complete resolution of systemic inflammatory attacks and skin lesions and provided neurologically symptom free during their follow-up. With this report, we emphasize the importance of early referral of the patients with suspected livedo racemose to avoid the delay of DADA2 diagnosis for favorable outcome.

Evaluation of subclinical ocular involvement in patients with deficiency of adenosine deaminase 2 (DADA2).

To evaluate changes in the peripapillary, macular, and choroidal microvasculature in the eyes of patients with deficiency of adenosine deaminase 2 (DADA2) and no clinical signs of ocular involvement. The study included 12 eyes of 12 patients with DADA2 and 24 eyes of 24 healthy subjects. The foveal avascular zone (FAZ), macular vessel densities (VDs) in the superficial and deep retinal capillary plexuses, peripapillary VDs, and choroidal thickness were evaluated by optical coherence tomography angiography (OCTA). Measurements were compared between DADA2 patients and healthy controls. The median age was 17 (8-25) years in DADA2 patients and 17.5 (7-23) years in control group at the OCTA visit (p = 0.934). FAZ area did not differ between the groups (p = 0.224). In the superficial capillary plexus, whole-image, foveal, and parafoveal VD values were slightly lower in DADA2 patients than in controls (p = 0.054, p = 0.052, p = 0.117). In the deep capillary plexus, whole-image and parafoveal VD values were significantly lower in DADA2 patients than controls (p = 0.010, p = 0.001). VD in the radial peripapillary capillary plexus was also lower in DADA2 patients, with significantly lower peripapillary VD (p = 0.002). Subfoveal choroidal thickness was significantly higher in patients with DADA2 (p < 0.001). This OCTA study demonstrates that both retinal and choroidal involvement may occur in DADA2 patients before the emergence of evident clinical findings.

Treatment and Outcome in Deficiency of Adenosine Deaminase 2: A Literature Review.

Deficiency of adenosine deaminase 2 (DADA2) is a rare disease with varying phenotypes and disease outcomes. We evaluated the treatment of DADA2 and explored the factors associated with disease outcome. A systemic literature review of DADA2 was conducted. Cases were included if they had documented detailed genotypes, phenotypes, treatment protocols, and outcomes. Patients were categorized as having uncontrolled and controlled disease. Factors associated with disease outcome were analyzed using logistic regression models. The study population comprised 242 DADA2 patients with data on treatment protocols and responses, of whom 17 required no treatment. Tumor necrosis factor a inhibitors (TNFi) were effective in 78.6% (103/131). Hematological abnormalities and increased acute phase reactants are independently associated with the effectiveness of TNFi (OR, 0.21 [95%CI, 0.07-0.661; P=.007] and 9.62 [95%CI, 2.31-40.00; P=.002, respectively). Among the 225 patients requiring active treatment, 157 (69.8%) had controlled disease and 68 (30.2%) uncontrolled disease. Neither age of disease onset nor genotype was associated with disease outcome. Increased acute phase reactant values, constitutional symptoms, neurological symptoms, and treatment with TNFi were independently associated with disease control, while recurrent infections and severe vascular events were the main causes of mortality (10/21 and 6/21, respectively). In patients requiring treatment, symptoms of systemic inflammation and vasculitis and treatment with TNFi are associated with disease control. Recurrent infections and severe vascular events should be treated intensively, as they are the main causes of death. Hematological abnormalities should be monitored, as they decrease the effectiveness of TNFi.

The role of vascular inflammation markers in deficiency of adenosine deaminase 2

ObjectivesThe first objective was to assess the role of vascular inflammatory factors in the pathogenesis of deficiency of adenosine deaminase 2 (DADA2) and to compare these markers among DADA2 patients with different phenotypes. We also aimed to investigate differences between DADA2 patients with vasculitic features and classic polyarteritis nodosa (PAN) for the aforementioned markers. MethodsThe study included eighteen DADA2 patients, ten PAN patients, and eight healthy controls. Plasma levels of sST2, sRAGE, Tie-2, sCD40L, Tie-1, sFlt-1, LIGHT, TNF-α, PlGF, IL-6, IL-18, IL-10, MCP-1 were studied by cytometric bead-based multiplex assay panel. ResultsAmong the DADA2 patients, five had hematological manifestations, 13 had vasculitic findings, and accompanying immunological findings were present in seven patients. Nine patients had neurological findings, five of whom had neuropathy. Plasma levels of Tie-1 and sFlt-1 were higher in the overall DADA2 patients compared to healthy controls and PAN patients (p<0.001 and p = 0.004, respectively). DADA2 patients with PAN-like features had higher sRAGE, Tie-2, and TNF-α levels compared to PAN patients (p = 0.013, p = 0.003, and p = 0.001, respectively).In DADA2 patients with hematological findings, plasma IL-18 levels were higher than those with PAN-like phenotype (p = 0.001). Finally, DADA2 patients with neuropathy had higher sRAGE concentrations than patients without neuropathy and healthy controls (p = 0.03 and p = 0.008, respectively). ConclusionsWe suggest that the high plasma IL-18 levels observed in DADA2 patients with hematologic manifestations may be associated with an activated IFNγ pathway, and lack of response to anti-TNF treatment. We identified sRAGE as a potential biomarker of neuropathy in DADA2 patients. Higher concentrations of Tie-1, Tie-2, sFlt-1, sRAGE, and TNF-α distinguished DADA2 patients with PAN-like features from PAN patients.

Validation of the provisional seven-item criteria for the diagnosis of polyarteritis nodosa.

The 1990 American College of Rheumatology (ACR) criteria for the classification of polyarteritis nodosa (PAN) have many pitfalls and performed poorly when used for diagnostic purposes. Recently, a provisional seven-item diagnostic criteria for PAN was proposed. To validate the provisional seven-item diagnostic criteria for PAN in a cohort of PAN patients from a tertiary care centre in India. Clinical details of patients diagnosed as PAN as per the European Medicines Agency algorithm between 2005 and 2020 were collected retrospectively. Age and sex-matched anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) patients were included in the non-PAN group. Patients with a deficiency of adenosine deaminase 2 (DADA2) were included as a separate group. The sensitivity, specificity, positive and negative predictive values (PPV and NPV) for ACR criteria, the Ministry of Health, Labour and Welfare (MHLW) in Japan diagnostic criteria and the seven-item diagnostic criteria were calculated. Thirty-seven PAN, 14 DADA2 and 37 AAV patients were included in the analysis. The sensitivity, specificity, PPV and NPV of the seven-item criteria were 83.7%, 96.8%, 97.3% and 81.1% respectively. For the ACR criteria, sensitivity was 82.9% and specificity was 79.5%. The sensitivity, specificity for MHLW criteria were 77.3% and 90% respectively. The sensitivity and specificity of seven-item criteria for DADA2 patients were 58.8% and 88.2% respectively. There was very poor agreement between the ACR criteria and the seven-item and MHLW criteria and fair agreement between seven-item and MHLW criteria (κ = 0.279). The provisional seven-item criteria for PAN performed well with high specificity and PPV.

Systematic review of childhood-onset polyarteritis nodosa and DADA2

BackgroundDiagnosis of childhood polyarteritis nodosa (PAN) has become challenging after the definition of deficiency of adenosine deaminase 2 (DADA2). We aimed to define the differential features of pediatric PAN and DADA2 patients in our center and in the literature. MethodsThe charts of pediatric PAN and DADA2 patients followed at the Pediatric Rheumatology Unit of Hacettepe University between 2010–2020 were analyzed. A systematic literature review was conducted for articles regarding pediatric PAN or DADA2. ResultsThirty-four pediatric PAN and 18 pediatric DADA2 patients were included. The age at onset was younger, parental consanguinity, livedo reticularis, neurologic involvement (especially strokes), lymphopenia, and hypogammaglobulinemia were more frequent, while thrombocytosis and panniculitis were less frequent in DADA2 patients. The primary treatment was anti-tumor necrosis factor (anti-TNF) in DADA2. For induction treatment, all systemic PAN patients received corticosteroids, and cyclophosphamide (n=11) or mycophenolate mofetil (MMF) (n = 3). Cyclophosphamide was replaced with MMF in nine once remission was confirmed with PVAS. In the literature, 28 articles describing 613 pediatric PAN patients and 26 articles describing 207 pediatric DADA2 patients were identified. Neurologic, gastrointestinal, and cardiac involvements were more frequent in DADA2, while constitutional symptoms and testis involvement were more common in PAN. ConclusionIn a child with PAN-like phenotype, DADA2 should be considered in the presence of young age at disease onset, parental consanguinity, strokes, lymphopenia, and lack of thrombocytosis during active disease. Anti-TNF treatment is indicated for vasculitic DADA2. Cyclophosphamide could be switched to MMF when remission is confirmed with PVAS in severe PAN.

Anti-tumour necrosis factor treatment for the prevention of ischaemic events in patients with deficiency of adenosine deaminase 2 (DADA2).

To evaluate the impact of anti-Tumour Necrosis Factor-α (anti-TNF) treatment on the occurrence of vasculitic ischaemic events in patients with deficiency of adenosine deaminase 2 (DADA2). A retrospective analysis of DADA2 patients referred from six centres to Great Ormond Street Hospital for Children was conducted. Ischaemic events, vasculitic disease activity, biochemical, immunological, and radiological features were compared, before and after anti-TNF treatment. A total of 31 patients with genetically confirmed DADA2 were included in the study. The median duration of active disease activity prior to anti-TNF treatment was 73 months (inter-quartile range [IQR] 27.5-133.5 months). Twenty seven/31 patients received anti-TNF treatment for a median of 32 months (IQR 12.0-71.5 months). The median event rate of central nervous system (CNS) and non-CNS ischemic events before anti-TNF treatment was 2.37 per 100 patient-months (IQR 1.25-3.63); compared with 0.00 per 100 patient-months (IQR 0.0-0.0) post-treatment (p< 0.0001). Paediatric vasculitis activity score (PVAS) was also significantly reduced: median score of 20/63 (IQR 13.0-25.8/63) pre-treatment vs. 2/63 (IQR 0.0-3.8/63) following anti-TNF treatment (p< 0.0001), with mild livedoid rash being the main persisting feature. Anti-TNF treatment was not effective for severe immunodeficiency or bone marrow failure, which required haematopoietic stem cell transplantation (HSCT). Anti-TNF treatment significantly reduced the incidence of ischaemic events and other vasculitic manifestations of DADA2, but was not effective for immunodeficiency or bone marrow failure.

A monogenic autoinflammatory disease with fatal vasculitis: deficiency of adenosine deaminase 2.

To recap the expanding clinical spectrum, genotype-phenotype associations and treatment options in the light of recently published articles regarding the deficiency of adenosine deaminase 2 (DADA2). Whole-exome sequencing enabled novel clinical phenotypes associated with ADA2 mutations. Since its discovery, the phenotypic spectrum of DADA2 has substantially expanded to cover Diamond-Blackfan anaemia, cytopenia and immunodeficiency syndromes. In addition to elevated TNF alpha levels, increased levels of interferon-stimulated genes were also detected in patients with DADA2. Given the absence of clinical trials until now, no standard treatment strategy exists for DADA2. Currently, anti-TNF alpha agents are the mainstay of treatment, based on the data both from the initial two reports and from subsequent studies. However, it is still unclear how to manage asymptomatic patients with ADA2 mutation and/or with absent ADA2 activity and what is the optimal duration of anti-TNF therapy. Among a total of 206 DADA2 patients described so far, the overall mortality was found as 8.3%. Biallelic homozygous G47R mutations were mostly associated with a vascular phenotype, whereas patients with homozygous R169Q mutations seem to display a mixed clinical phenotype including vascular, haematological and immunological manifestations. HSCT should be reserved as a curative treatment option for DADA2 patients unresponsive to the anti-TNF therapy, as it carries a significant morbidity.

Analysis of Deficiency of Adenosine Deaminase 2 Pathogenesis Based on Single Cell RNA Sequencing of Monocytes

Deficiency of adenosine deaminase 2 (DADA2) is a rare autosomal recessive disease caused by loss-of-function mutations in the ADA2 gene. DADA2 typically presents in childhood and is characterized by vasculopathy, stroke, inflammation, and immunodeficiency as well as hematologic manifestations, such as bone marrow failure and lymphoproliferation. The ADA2 protein is predominantly expressed in stimulated monocytes, dendritic cells and macrophages. ADA2 increases in the setting of inflammation and/or infection conditions. ADA2 has been reported to have a critical role in maintaining the balance between M1 (pro-inflammatory) and M2 (anti-inflammatory) macrophages. Macrophages of DADA2 patients are polarized towards M1 subset., DADA2 pathogenesis is not well characterized. To elucidate molecular mechanisms in DADA2 deficiency, we analyzed a gene expression profile of CD14+ monocytes derived from peripheral blood using single cell RNA sequencing (scRNA-seq). Blood was collected from DADA2 patients and age- and sex-matched healthy donors; all patients were studied in a registered research protocol (clinicaltrials.gov NCT00071045). Samples were obtained from 14 DADA2 patients and 6 healthy donors; median age of the DADA2 patients was 23 years old (range, 5 - 57 years). Among the 14 patients, 7 had hematological phenotypes: 5 lymphopenia, 3 neutropenia, 3 thrombocytopenia, and 2 with hypocellular bone marrow histology. Low serum immunoglobulins and cutaneous findings were frequent. Nine of the 14 patients had been treated with TNF inhibitors (etanercept and adalimumab). Mutations were distributed throughout the ADA2 gene; although two siblings had the same mutation, even they showed poor genotype-phenotype correlation. Monocytes were isolated by immunomagnetic positive selection with the EasySep™ positive CD14 selection kit Ⅱ, then subjected to scRNA-seq using Single Cell 3' Reagent Kits v2 (10X Genomics). Libraries for scRNA-seq were sequenced on the HiSeq-3000 instrument. Based on scRNA-seq data, we could classify monocytes into three populations by conventional flow cytometric criteria using cell surface protein expression imputed from scRNA-seq: CD14++CD16- classical, CD14++CD16+ intermediate, and CD14+CD16++ nonclassical monocytes (Figure A). CD16 expression was higher in DADA2 patients than in healthy donors (Figure B). A proportion of nonclassical monocytes among total monocytes were significantly higher in DADA2 patients compared to healthy donors (Figure C). On comparison of gene expression of each monocyte subtypes in DADA2 patients with that of healthy donors, there were 215, 237, and 267 differentially expressed upregulated genes in classical, intermediate, and nonclassical monocytes, respectively (at a threshold avg_logFC &gt; 0.2). Approximately 35% of upregulated genes were overlapped among the three monocyte subtypes of DADA2 patients, including immune response genes such as IFITM1, IFITM2, IFITM3, and C3AR1 (Figure D). Common gene pathways were associated with immune function, such as interferon alpha/beta signaling and interferon gamma signaling. Specific genes to classical and intermediate monocytes were less than 10% of all the upregulated genes. Distinctively, the NF-κB pathway was upregulated in nonclassical monocytes, this might contribute to the pathogenesis of DADA2 as inflammatory disease. Overall, each monocyte subtype of DADA2 patients showed upregulation of immune response gene sets compared to controls. DADA2 patients have increased numbers of nonclassical monocytes which may contribute the immune dysregulation and increased inflammation observed in the disease. Figure Disclosures No relevant conflicts of interest to declare.

A Monogenic Disease with a Variety of Phenotypes: Deficiency of Adenosine Deaminase 2.

Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive autoinflammatory disorder associated with ADA2 mutations. We aimed to investigate the characteristics and ADA2 enzyme activities of patients with DADA2 compared to non-DADA2 patients. This is a descriptive study of 24 patients with DADA2 who were admitted to the Adult and Pediatric Rheumatology, Pediatric Haematology, and Pediatric Immunology Departments of Hacettepe University. All ADA2 exons were screened by Sanger sequencing. Serum ADA2 enzyme activity was measured by modified spectrophotometric method. Twenty-four patients with DADA2 were included: 14 with polyarteritis nodosa (PAN)-like phenotype (Group 1); 9 with Diamond-Blackfan anemia (DBA)-like features, and 1 with immunodeficiency (Group 2). Fourteen PAN-like DADA2 patients did not have the typical thrombocytosis seen in classic PAN. Inflammatory attacks were evident only in Group 1 patients. Serum ADA2 activity was low in all patients with DADA2 except one, who was tested after hematopoietic stem cell transplantation. There was no significant difference in ADA2 activities between PAN-like and DBA-like patients. In DADA2 patients with one ADA2 mutation, serum ADA2 activities were as low as those of patients with homozygote DADA2. ADA2 activities were normal in non-DADA2 patients. ADA2 mutations were affecting the dimerization domain in Group 1 patients and the catalytic domain in Group 2 patients. We suggest assessing ADA2 activity along with genetic analysis because there are patients with one ADA2 mutation and absent enzyme activity. Our data suggest a possible genotype-phenotype correlation in which dimerization domain mutations are associated with PAN-like phenotype, and catalytic domain mutations are associated with hematological manifestations.

Adenosine Deaminase Two and Immunoglobulin M Accurately Differentiate Adult Sneddon’s Syndrome of Unknown Cause

Background: The association that exists between livedo reticularis (LR) and stroke is known as Sneddon’s syndrome (SnS). The disorder is classified as primary SnS (PSnS), if the cause remains unknown and secondary SnS. The condition is rare and it occurs mainly sporadically. In 2014, 2 independent teams described a new genetic disorder with childhood-onset, which was called deficiency of adenosine deaminase 2 (DADA2), characterized by recurrent fevers and vascular pathologic features that included LR and stroke. All the patients carried recessively inherited mutations in cat eye syndrome chromosome region candidate 1 gene (CECR1), encoding the adenosine deaminase 2 (ADA2) protein. Genetic testing is the standard for the diagnosis of DADA2. However, the diagnostic accuracy of more affordable laboratorial analysis in CECR1-mutated individuals remains to be established. We aim to determine whether plasma ADA2 activity and serum immunoglobulin M (IgM) levels can distinguish (1) DADA2 from other adult patients within the SnS spectrum, and (2) healthy CECR1 heterozygous (HHZ) from healthy controls (HC). Methods: ADA2 activity in plasma and serum IgM concentrations was measured in adult patients within the SnS spectrum, healthy first-degree relatives and HC. Genetic results were used as the reference standard. The primary outcome measures were sensitivity and specificity derived from receiver operating curve analysis. Results: A total of 73 participants were included in the study: 26 patients with PSnS with no CECR1 mutation (PSnS), 6 bi-allelic (DADA2 patients) and 7 HHZ CECR1 mutations and 34 HC. Plasma ADA2 activity and serum IgM levels were significantly lower in DADA2 patients than in PSnS. With the use of the best indexes, plasma ADA2 activity differentiated PSnS from DADA2 with a sensitivity and specificity of 100.0% and HHZ from HC with a sensitivity of 97.1% and specificity of 85.7%. Serum IgM levels also differentiated PSnS from DADA2 with a sensitivity of 85.2% and specificity of 83.3%. Conclusion: Serum IgM levels might be used as a triage tool and plasma ADA2 activity performs perfectly as a diagnostic test for DADA2 in adult patients within the SnS spectrum. ADA2 activity in plasma also reliably distinguishes HHZ from HC.

Vasculopathy, Immunodeficiency, and Bone Marrow Failure: The Intriguing Syndrome Caused by Deficiency of Adenosine Deaminase 2.

Deficiency of adenosine deaminase 2 (DADA2) is a monogenic form of systemic vasculopathy that often presents during early childhood. Linked to biallelic mutations in ADA2 (previously CECR1), DADA2 was initially described as a syndrome of recurrent fever, livedo racemosa, early-onset strokes, and peripheral vasculopathy that resembles polyarteritis nodosum. However, the wide spectrum of clinical findings and heterogeneity of disease, even among family members with identical mutations, is increasingly recognized. Evidence of systemic inflammation and vasculopathy is not uniformly present in DADA2 patients and some can remain asymptomatic through adulthood. Humoral immunodeficiency characterized by low immunoglobulin levels and increased risk of infection is another common feature of DADA2. Variable cytopenias including pure red cell aplasia that mimics Diamond-Blackfan anemia can also be primary manifestations of DADA2. How defects in a single gene translate into these heterogeneous presentations remains to be answered. In this review, we will summarize lessons learned from the pleiotropic clinical manifestations of DADA2.