Loss-of-Function Mutations in CECR1, Encoding Adenosine Deaminase 2,Cause Systemic Vasculopathy with Fever and Early Onset Strokes. Q. Zhou1, A. Zavialov2, M. Boehm3, J. Chae1, M. Hershfield4, R. Sood5, S. Burgess6, A. Zavialov2, D. Chin1, C. Toro7, R. Lee8, M. Quezado9, A. Ombrello1, D. Stone1, I. Aksentijevich1, D. Kastner1 1) Inflammatory Disease Section, NHGRI, Bethesda, USA; 2) Turku Centre for Biotechnology, University of Turku, Turku, Finland; 3) Laboratory of Cardiovascular Regenerative Medicine, NHLBI, Bethesda, USA; 4) Department of Medicine, Duke University Medical Center, Durham, USA; 5) Zebrafish Core, NHGRI, Bethesda, USA; 6) Developmental Genomics Section, NHGRI, Bethesda, USA; 7) NIH Undiagnosed Diseases Program, NIH, Bethesda, USA; 8) Translational Surgical Pathology Section, NCI, Bethesda, USA; 9) General Surgical Pathology Section, NCI, Bethesda, USA.

   We recently observed 5 unrelated patients with fevers, systemic inflammation, livedo reticularis, vasculopathy, and early-onset recurrent ischemic strokes. We performed exome sequencing on affected patients and their unaffected parents. The 5 patients shared 3 missense mutations in CECR1, encoding adenosine deaminase 2 (ADA2), with the genotypes A109D/Y453C, Y453C/G47A, G47A/H112Q, R169Q/Y453C, and R169Q/28kb genomic deletion encompassing the 5UTR and first exon of CECR1.All mutations are either novel or present at low frequency (0.001) in several large databases, consistent with the recessive inheritance. The Y453C mutation was present in 2/13004 alleles in an NHLBI database. Both alleles are found in 2 affected siblings who suffered from late-onset ischemic stroke, indicating that heterozygous mutations in ADA2 might be associated with susceptibility to adult stroke. Computer modeling based on the crystal structure of the human ADA2 suggests that CECR1 mutations either disrupt protein stability or impair ADA2 enzyme activity. All patients had at least a 10-fold reduction in serum and plasma concentrations of ADA2, and reduced ADA2-specific adenosine deaminase activity. Western blots showed a decrease in protein expression in supernatants of cultured patients cells. ADA2 is homologous to ADA1, which is mutated in some patients with SCID. In contrast to ADA1, ADA2 is expressed predominantly in myeloid cells and is a secreted protein, and its affinity for adenosine is much less than ADA1. Animal models suggest that ADA2 is the prototype for a family of growth factors (ADGFs).Although there is no mouse homolog of CECR1, there are 2 zebrafish homologs, Cecr1a and Cecr1b. Using morpholino technology to knock down the expression of the ADA2 homologs, we observed intracranial hemorrhages in approximately 50% of the zebrafish embryos harboring the knockdown construct, relative to 3% in controls. Immunohistochemical studies of endothelial cells from patients skin biopsies demonstrate a diffuse systemic vasculopathy characterized by impaired endothelial integrity, endothelial cellular activation, and a perivascular infiltrate of CD8 T-cells and CD163-positive macrophages. ADA2 is not expressed in the endothelial cells. Our data suggest that ADA2 may be necessary for vascular integrity in the developing zebrafish as an endothelial cell-extrinsic growth factor, and that the near absence of functional ADA2 in patients may lead to strokes by a similar mechanism.

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