Diamond-Blackfan Anemia Phenotype Caused By Deficiency of Adenosine Deaminase 2

Diamond-Blackfan anemia (DBA) is a prototypic ribosomopathy and remains the most common cause of congenital pure red cell aplasia (PRCA). In 2/3 of patients, ribosomal protein haploinsufficiency is disease-causing, while in remaining 1/3 the genetic etiology is unknown. Recently, deficiency of ADA2 (DADA2) due to biallelic CECR1 -mutations was reported in patients with systemic autoinflammatory disease presenting with early onset vasculopathy, strokes, antibody deficiency, and in some cases variable cytopenias. Based on the clinical findings in an ADA2-deficient patient with PRCA resembling DBA, we aimed to define the prevalence and clinical picture of DADA2 within DBA patient cohorts. Patients enrolled in the national observational DBA registry in Germany were evaluated for the presence of mutations in CECR1 gene; additional nonconsecutive patients from the French and Turkish registries within the European DBA (EuroDBA) consortium were part of this study. Functional studies included profiling of polysomes and pre-rRNAs in patient-derived EBV-cell lines, CECR1 RT-PCR, measurements of autophagy and apoptosis, and analysis of erythropoiesis in zebrafish embryos. Systematic mutational and copy number analysis had identified typical ribosomal haploinsufficiency in 169/242 patients (70%). Out of 73 remaining patients, full CECR1 -sequencing was accomplished in 68 cases, of which 4 (6%) carried biallelic CECR1 -mutations. Additional 3 patients with biallelic CECR1 -mutations and DBA phenotype were referred from Germany (the index PRCA case), France and Turkey. In contrast to typical autoinflammatory DADA2 (caused by missense biallelic CECR1 -mutations) all patients studied here had at least one CECR1 -allele affected by truncating/stop-gain/deletion mutation leading to mRNA degradation in patient cells. Low or missing ADA2 enzyme activity in plasma confirmed DADA2, while erythrocyte ADA (eADA) levels and MCV were normal. Transfusion-dependent hypoproliferative anemia developed at a median age of 5 weeks (birth-14 years), while hypogammaglobulinemia developed in all cases either initially or during disease course. Notably, a transient hematologic response to steroids was achieved in 5/7 patients, but no improvement was observed in 2 patients treated with TNF-inhibitor; all patients at one point became heavily transfusion-dependent. Systemic vasculitis or cerebral complications were not observed in our cohort. At the last follow-up, 6/7 patients were alive; 3 had successfully undergone hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning and 1 patient had died due to septic shock. Next, we addressed the question if ribosome biogenesis is affected in ADA2-deficient patient cells. Using pre-rRNA maturation assays, polysome profiling and Western blots we established that ribosome biogenesis is normal in DADA2-related PRCA and there is no increase of TP53 stabilization over basal levels in patient LCLs. Analysis of CECR1 -morpholino zebrafish embryos revealed early anemia with lethal phenotype. Although there was no evidence for extrinsic (e.g. immune-mediated) pathomechanisms in our patients, it remains to be answered if CECR1 loss directly affects erythroid development. Finally, the association between elevated levels of eADA (=ADA1) specific to classical DBA and decreased ADA2 enzyme levels in DADA2-related PRCA remains obscure. In summary, DADA2 can phenotypically mimic DBA and thus extends the spectrum of congenital PRCA. Ribosome synthesis seems not to be affected by CECR1 mutations. DADA2 should be considered in patients with DBA-like phenotype but with normal eADA/MCV and hypogammaglobulinemia, allowing for early stratification aimed at HSCT in affected individuals.