Though ADA1 is expressed in all human tissues, maximal expression is noted in lymphocytes and is critically important for development of adaptive immune system (4–6). Deficiency of ADA1 results in severe combined immunodeficiency with profound depletion of T, B, and NK cells due to accumulation of toxic deoxyadenosine nucleotides.

ADA2 is highly expressed in myeloid cells and is secreted by activated macrophages, monocytes and dendritic cells (7–9). ADA2 also interacts with lymphocytes and other leukocytes through adenosine receptors (10). ADA2 plays a significant role in development of hematopoietic and endothelial cells, maintaining balance between M1 and M2 macrophages (7, 9, 11). Pathophysiology of DADA2 is still evolving. In DADA2, monocyte differentiation is skewed towards pro-inflammatory M1 macrophages and results in generation of inflammatory cytokines like interleukin (IL)-6 and tumour necrosis factor (TNF)-α (3). Nihira et al. had performed transcriptomic and proteomic analysis on peripheral blood mononuclear cells in a Japanese cohort of DADA2 patients and reported elevated type II interferon signatures. By network analysis, the authors identified STAT1 gene as pivotal gene in pathogenesis of DADA2. Further, STAT1 phosphorylation in monocytes and B cells following interferon gamma stimulation was significantly higher in patients with DADA2 as compared to controls (12). Watanabe et al. performed single cell RNA sequencing in monocytes (CD14+) from DADA2 patients and healthy controls. They confirmed higher numbers of non-classical monocytes and an up regulation of M1 macrophage markers in DADA2 patients. Thus, authors suggested that high levels of IFNγ may drive the differentiation of monocytes to a M1 phenotype that leads to release of proinflammatory cytokine TNFα (13).

Vasculopathy, affecting the medium and small sized arteries, is the commonest manifestation of DADA2. Clinical manifestations vary from limited cutaneous involvement to severe and fatal systemic vasculitis with multiorgan involvement. Cutaneous involvement includes livedoid rash, erythema nodosum, peripheral gangrene, ulcers and Raynaud’s phenomenon. Most common cutaneous manifestation of DADA2 is livedo racemosa. Skin biopsy often shows extensive neutrophil infiltration predominantly in interstitium, macrophage infiltration, and perivascular T lymphocytes without overt features of vasculitis.

In systemic involvement, central nervous system (CNS) is most commonly involved followed by renal and gastrointestinal (GI) systems. Hallmark of CNS vasculopathy is recurrent ischemic lacunar strokes. Other CNS manifestations include cranial nerve palsy, spastic diplegia, encephalopathy, peripheral neuropathy, sensory neural hearing loss, labyrinthitis, and cerebral atrophy. Although abdominal pain and inflammatory bowel disease are predominant GI manifestations, intestinal perforation and aneurysms in celiac and mesenteric arteries have also been reported (14). Renal involvement in patients with DADA2 is seen in form of renal artery stenosis, renal artery aneurysms, arterial hypertension, and glomerular scarring (1, 15, 16).

The initial reports of DADA2 describe this disorder as a mild immunodeficiency with reduced levels of immunoglobulin (Ig) M. Subsequently, immunological aberrations in DADA2 are being reported in a much greater detail. Up to two-thirds of patients with DADA2 may have decreased levels of either Ig isotypes, while hypogammaglobulinemia has been reported in approximately one fourth of patients (17). Impaired vaccine responses have also been reported (18). Around 10% of these patients present with B cell lymphopenia and low switched memory B cells. Overt immunodeficiency in the form of recurrent infections has been noted in 15–20% of patients (19). Clinical presentation of immunodeficiency phenotype may mimic common variable immunodeficiency (CVID). Therefore, it is prudent to consider differential diagnosis of DADA2 in patients with CVID-like immunodeficiency especially having vasculopathic manifestations (20–22). As recurrent inflammation in DADA2 may inhibit B cell differentiation and function, treatment with anti-inflammatory therapy may be beneficial in improving Ig levels (21). Tissue biopsies can also show the CVID-like phenotype with absent plasma cells (23). Besides CVID-like presentation, clinical features suggestive of a combined immunodeficiency have also been noted in DADA2. These patients have been reported with fungal infections and infections from DNA viruses including molluscum contagiosum, warts, and members of herpes virus family (24). Low numbers of natural killer (NK) cells and T cells have also been reported (18, 25). Overall, lymphopenia is reported in 15% of patients with DADA2 (17). A recent report on DADA2 patients using in-depth immunophenotyping and functional analysis of lymphocytes revealed a multitude of immunological aberrations. These include impaired class switching and differentiation of B cells, reduced memory and regulatory T cells, increased senescent T cells, diminished mucosa associated invariant T cells and invariant NKT cells, and decreased classical monocytes (26). The authors also reported arrest in B cell development in the bone marrow at the pro-B to pre-B cell stage and defect in terminal B cell differentiation. Authors have also shown that healthy heterozygous carrier state for DADA2 showed intermediate values of lymphocyte phenotypes and functions in comparison to DADA2 patients and healthy controls, further highlighting the role of heterozygous state (26). Thus, immune defect in patients with DADA2 deficiency may present with myriad immunological aberrations (26). Immunological abnormalities reported in patients with DADA2 have been summarized in Figure 1 .

ADA2 activity can be measured by spectrophotometry or LC-MS/MS based assay in serum/plasma/tissue-culture supernatant or dried plasma spot respectively. It quantifies the adenosine-dependent generation of ammonia in the presence of erythro-9-Amino-β-hexylα-methyl-9H-purine-9-ethanol hydrochloride (EHNA), a selective inhibitor of ADA1. It is essential to perform ADA enzyme activity in addition to genetic analysis since heterozygous carriers can present with decreased enzymatic activity and clinical manifestations (38). Newer cost-effective and rapid methods for estimation of ADA2 enzyme activity may serve as a screening tool for ordering genetic testing in patients with DADA2. Utilizing such techniques, Cafaro et al. noted all patients with variant-proven DADA2 to have ADA2 enzyme activity of ≤0.06 mU/mL (60).

Genetic sequencing remains the mainstay of genetic diagnosis of DADA2. Next-generation sequencing (NGS) including whole-exome sequencing is increasingly being utilized to diagnose DADA2, even when assays for ADA2 enzyme activity are not readily available (61). Given the pleiotropic manifestations of DADA2, it is important to include ADA2 gene in various customized NGS panels used for evaluation of haematological, immunological, and rheumatological disorders. As the most common disease variants are found in exon-2 (p.G47R, p.G47A) followed by exon 3 and 4 ( Supplementary Table ), Sanger sequencing can also be employed upfront for evaluation. Few ADA2 pathogenic variants (homozygous 800 bp duplication in exon 7), however, may not be detected by such strategies. Other techniques such as Multiplex Ligation-dependent Probe Amplification (MLPA) in combination with long-read polymerase chain reaction (PCR) sequencing need to be employed in such scenarios (55). Deep RNA sequencing can be used to evaluate the impact of novel splice variants (62).

Corticosteroids are widely used in acute phase of the disease; however, patients often show only a modest response. Disease flares are common while tapering steroids. A steroid-refractory course has also been described (2, 30, 43). TNF blockade is the therapy of choice for vasculitic and inflammatory manifestations. Etanercept and adalimumab are the commonly employed TNF inhibitors. In resource constrained settings, thalidomide may be used as an alternative due to its anti-TNF activity. Carosi et al. have reported the effectiveness of thalidomide in controlling disease activity in 7 patients (14). IL-6 blockers (tocilizumab) are also effective in controlling inflammation; however, recurrence of stroke has been noted (12, 23, 54, 63, 64). Tocilizumab has been used successfully in the patient with Castleman disease and DADA2. IL-1 blockade has not been noted to be of significant therapeutic benefit in patients with DADA2 (65).

In patients with hypogammaglobulinemia, immunoglobulin replacement therapy and antibiotic prophylaxis (21, 35) are the usual treatment modalities. Hematological manifestations are usually refractory to glucocorticoids (30). Other immunosuppressive drugs (azathioprine, mycophenolate mofetil, cyclosporine and anti-thymocyte globulin) have shown variable response in conditions like PRCA and other hematological phenotypes (35, 66). Rituximab has been shown to result in a favorable response in patients with autoimmune cytopenias (32). Mild manifestations (e.g. lymphopenia) may respond to TNF-blockers (66–68); however, TNF-blockade is ineffective for treatment of severe hematological manifestations (e.g. bone marrow failure and PRCA/DBA) (33, 35).

HSCT is the definitive treatment for hematological and immunological manifestations of DADA2. Hashem et al. have recently collated the multicentric experience of HSCT in 30 patients of DADA2 who underwent total 38 HSCTs. Indications for HSCT were bone marrow failure syndromes, autoimmune cytopenia, lymphoproliferation (benign or malignant) and immunodeficiency phenotypes. Overall survival after 2 years of follow-up was 97% and HSCT resolved the hematological phenotypes in all patients (33). Plasma ADA2 activity may be restored to normal as early as 2 weeks post-transplant. HSCT may also benefit vasculopathic manifestations (33).