CECR1 Research Articles

Deficiency of adenosine deaminase 2 as an unrecognized cause of early-onset stroke and cranial nerve palsy.

The aim of this study is to evaluate the clinical, laboratory, and radiological findings and prognosis of patients with adenosine deaminase 2 deficiency (DADA2) and to highlight the conditions that DADA2 should be considered in the differential diagnosis in patients with neurological findings. A case series of six DADA2 patients was presented in this retrospective, descriptive study. Clinical and laboratory data, treatment protocols, and prognosis of the patients were recorded. A diagnosis of DADA2 was established by ADA2 enzyme activity assay and/or ADA2 gene sequencing. Six patients with DADA2 were included in the study. The median age at symptom onset was 6.5 years (range 3.5-13.5 years). The median time to diagnosis from the initial presentation was 9 (3-72) months. Consanguinity was present in the families of 4 cases. The skin, nervous system, and musculoskeletal system were the most commonly involved systems. Vasculitis mimicking polyarteritis nodosa (PAN) was the predominant phenotype (n=4) in our case series. Four patients with PAN-like features had neurological involvement. Ischemic strokes were found in 3 patients, cranial nerve palsy in 2 patients, and seizures in 2 patients. The CECR1 gene was analyzed in all patients. We analyzed plasma ADA2 enzyme activity only in one patient. Anti-tumor necrosis factor (TNF)-α therapy was initiated. Inflammation was suppressed and remission was achieved in all patients. DADA2 should be considered in patients with PAN-like disease, a history of familial PAN/vasculitis, early-onset strokes/neurological involvement with systemic inflammation. Furthermore, anti-TNF-α therapy appears to be beneficial for the treatment of DADA2.

Adenosine deaminase 2 deficiency with a novel variant of CECR1 gene mutation: Responding to tumor necrosis factor antagonist therapy

Deficiency of Adenosine deaminase 2 (DADA2) syndrome is a chronic, systemic, and inflammatory disorder, characterized by early-onset recurrent strokes, fever, livedo reticularis, and immunodeficiency. We report the case of a 4-year-old child, a product of consanguineous marriage, who presented with three episodes of hemiparesis within 1 year. She also manifested skin discoloration in the form of livedo reticularis. Workup with magnetic resonance imaging (MRI) of the brain revealed acute infarction in the right aspect of the cerebral peduncle and chronic lacunars infarct in the right thalamus with diffusion restriction. Repeated MRI after 5 months revealed diffuse loss of brain volume. The blood workup showed high inflammatory markers and significantly low adenosine deaminase 2 (ADA2) level. After being on corticosteroid and anticoagulant treatments, she suffered from a recurrent episode of cerebral infarction, after which she was commenced on tumor necrosis factor (TNF)-antagonist therapy in addition to monthly fresh plasma infusion. Thereafter, there was no cerebral insult reported for >18 months. The genetic study of the child and her parents revealed a homozygous mutation c. 336C>A, p. (His112Gln) in the CECR1 gene, and her parents were heterozygous for the same variant. This variant was not previously reported in literature. We would suggest to link this novel variant c. 336C>A, p. (His112Gln) of CECR1 gene mutation with the clinical picture, along with the positive response to TNF-antagonist therapy in the era of ADA2 deficiency syndrome.

Identification of co-occurrence in a patient with Dent's disease and ADA2-deficiency by exome sequencing

Patients with co-occurrence of two independent pathologies pose a challenge for clinicians as the phenotype often presents as an unclear syndrome. In these cases, exome sequencing serves as a powerful instrument to determine the underlying genetic causes.Here, we present the case of a 4-year old boy with proteinuria, microhematuria, hypercalciuria, nephrocalcinosis, livedo-like rash, recurrent abdominal pain, anemia and continuously elevated CRP. Single exome sequencing revealed the pathogenic nonsense mutation p.(Arg98*) in the CLCN5 gene causing the X-linked inherited, renal tubular disorder Dent's disease. Furthermore, the two pathogenic and compound heterozygous missense variants p.(Gly47Ala) and p.(Pro251Leu) in the CECR1 gene could be identified. Mutations in the CECR1 gene are associated with a hereditary form of polyarteritis nodosa, called ADA2-deficiency. Both parents were carriers of a single heterozygous variant in CECR1 and the mother was carrier of the CLCN5 variant.This case evidently demonstrates the advantage of whole exome sequencing compared to single gene testing as the pathology in the CECR1 gene might have only been diagnosed after the occurrence of signs of systemic vasculitis like strokes or hemorrhages. Therefore, treatment and prevention can now start early to improve the outcome of these patients.

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. DisclosuresGrosse:Addmedica: Membership on an entity's Board of Directors or advisory committees.

Deficiency of Adenosine Deaminase 2 (DADA2), an Inherited Cause of Polyarteritis Nodosa and a Mimic of Other Systemic Rheumatologic Disorders.

A new autoinflammatory disease, deficiency of adenosine deaminase 2 (DADA2), caused by mutations in the CECR1 gene, was first reported in 2014. This review aims to update progress in defining, treating, and understanding this multi-faceted disorder. DADA2 was first described in patients with systemic inflammation, mild immune deficiency, and vasculopathy manifested as recurrent stroke or polyarteritis nodosa (PAN). More than 125 patients have now been reported, and the phenotype has expanded to include children and adults presenting primarily with pure red cell aplasia (PRCA), or with antibody deficiency. Age of onset and clinical severity vary widely, even among related patients, and are not clearly related to CECR1 genotype. Inflammatory features often respond to anti-TNF agents, but marrow failure and severe immune deficiency may require hematopoietic stem cell transplantation. ADA2 is expressed and secreted by monocytes and macrophages, but its biological function and the pathogenesis of DADA2 are uncertain and will remain an important area of research. Pre-clinical investigation of ADA2 replacement therapy and CECR1-directed gene therapy are warranted, but complicated by the absence of a suitable animal model.

Autoimmune phenotype with type I interferon signature in two brothers with ADA2 deficiency carrying a novel CECR1 mutation

BackgroundLoss-of-function CECR1 mutations cause polyarteritis nodosa (PAN) with childhood onset, an autoinflammatory disorder without significant signs of autoimmunity. Herein we describe the unusual presentation of an autoimmune phenotype with constitutive type I interferon activation in siblings with adenosine deaminase 2 (ADA2) deficiency.Case presentationWe describe two siblings with early-onset recurrent strokes, arthritis, oral ulcers, discoid rash, peripheral vascular occlusive disease and high antinuclear antibody titers. Assessment of interferon signatures in blood revealed constitutive type I interferon activation. Aicardi-Goutières syndrome (AGS) was suspected, but no mutation in the known AGS genes were detected. Whole exome sequencing identified compound heterozygosity for a known and a novel mutation in the CECR1 gene. Functional consequences of the mutations were demonstrated by marked reduction in ADA2 catalytic activity.ConclusionsOur findings demonstrate that ADA2 deficiency can cause an unusual autoimmune phenotype extending the phenotypic spectrum of PAN. Constitutive interferon I activation in patient blood suggests a possible role of type I interferon in disease pathogenesis which may have therapeutic implications.

Large Granular Lymphocyte Infiltration in the Bone Marrow in Children and Young Adults May Suggest Primary Immune Deficiency

Large granular lymphocyte (LGL) leukemia is a group of rare lymphoproliferative disorders which involve inappropriate clonal expansion of either cytotoxic T-lymphocytes (CTLs) or natural killer cells (NK). The usual age at onset is between 40 to 60 years. We here describe four very young patients showing LGL infiltration in the bone marrow (BM) and all diagnosed with primary immune deficiency.Patient 1 is a 13-year old male who presented with gingivostomatitis, but no severe infections, autoimmune disease or allergies. He had persistent leukopenia (1.9x109/L), neutropenia (<0.05x109/L), thrombocytopenia (60-80x109/L) and mild anemia. The BM showed myeloid maturation arrest and infiltration of LGL cells with NK cell phenotype (CD3-, CD2+, CD16+, CD56-/+, CD57-, CD4-, CD8-) at 50% of the BM cellularity. In peripheral blood (PB) T, B and NK cell counts were normal. However, low levels of monocytes (0.09x109/L), normal levels of monocytoid (2.7x106/L) and low levels of plasmacytoid (0.1x106/L) dendritic cells (DC) were detected. While germline whole exome sequencing (WES) did not identify mutations likely to cause the disease, targeted sequencing of GATA2 mRNA showed uniallelic GATA2 expression, confirming haploinsufficiency.Patient 2 is a 15-year old female who has neonatal diabetes, autoimmune desquamative interstitial pulmonary disease, autoimmune enteropathy, exocrine pancreatic insufficiency and delayed growth. She developed transfusion-dependent, Coombs-negative anemia at age of 15. In the PB, 50% of the lymphocytes were LGLs. BM showed pure red cell aplasia plus LGL infiltration of TCRgd positive T cells (CD3+, CD2+, CD5+, CD8-/+, CD4-/+, CD57+) at 43% of BM cellularity. Clonal TCRg rearrangement was detected. Hypogammaglobulinemia presented at age of 7. T, B and NK cell counts were low normal, but DCs were non-existent. WES and functional studies revealed a germline gain-of-function mutation of STAT3 gene.Patient 3 is 42-year old female who has had severe infections (bacterial, viral and protozoan) starting at the age of 4 months. From age of 9, autoimmune manifestations occurred with immune thrombocytopenia, neutropenia and anemia, and intensively positive rheumatoid factor. Since age of 17, CD8+ LGLs were detected in PB reaching level of 50% of lymphocytes. In the BM, CD3+CD5-CD56- T -lymphocyte infiltration with clonal TCRd rearrangement was detected. In her 30ies she developed pulmonal arterial hypertension and hypogammaglobulinemia (0.8 g/L). She has low counts of B cells (0.02x109/L), and monocytoid (1.9x106/L) and plasmacytoid (0) DCs. WES identified a homozygous germline missense mutation in CECR1 gene, leading to total loss-of-function of the encoded adenosine deaminase 2 (ADA2) protein.Patient 4 presented with waxing and waning skin nodules from age of 10 years, but no severe infections or allergies. At age of 26, she was diagnosed with hemolytic anemia with spherocytosis, splenomegaly and cholelithiasis. Five years later remarkable hepatomegaly, and clinical autoimmune manifestations with SLE-type skin lesions, autoimmune hemolytic anemia and iritis were observed. Complement C3 (0.17 g/l) and C4 (<0.02 g/L) and NK cell (0.081) levels were low, but CD3, CD8 and CD4 counts and IgM and IgG levels were increased. T-cell infiltration was shown in the skin nodules and liver. BM showed mildly decreased myelo- and erythropoiesis and T cell infiltration (CD3+, CD8+, CD7+, CD45RO+, BCL2+, CD56-) up to 70% of the BM cellularity consistent with LGL infiltration. WES identified the same germline missense mutation in CECR1 gene as seen in patient 3, and a functional analysis showed no ADA2 activity.We conclude that LGL lymphoproliferation in young patients is often a sign of underlying primary immunodeficiency, highlighting the need for detailed genetic studies in these cases.Table 1PatientAge at LGLLGL in PB(109/L)LGL immune-phenotypeClonalityBM LGL infiltrationClinicalPID1130.38NKNA40%N,THepatomegalyGATA2 deficiency2151.79CTLTCRg43%AutoimmunityAHepatosplenomegalySTAT3 hyperactivation3171,49CTLTCRdNAN,T, AAutoimmunityPulmonary arterial hypertensionADA2 deficiency431NACTLTCRg weak40%AutoimmunityHepatosplenomegalyHepatopulmonal syndromeADA2 deficiencyA, anemia; N, neutropenia, CTL, cytotoxic T cell; LGL, large granular lymphocyte; NK, natural killer cell; PID, primary immune deficiency; TCR, T cell receptor; T, thrombocytopenia DisclosuresMustjoki:Signe and Ane Gyllenberg Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Siitonen:Pzizer: Other: charges of EAHAD congress 2015; Novartis: Other: charges of EHA congress 2015. Saarela:Roche: Honoraria.

Differential response to anakinra and adalimumab in a patient with DADA2 syndrome

Deficiency of adenosine deiminase 2 (DADA2) syndrome is a recently described autosomal recessively inherited autoinflammatory disorder associated with missense mutations in CECR1 gene. Clinical manifestations include early onset stroke, livedoid vascular changes, and a vasculopathy mimicking classical polyarteritis nodosa (cPAN) characterized by microaneurysms and associated inflammatory findings. We herein describe a male patient with homozygous G47R mutation in the CECR1 gene, whose inflammatory findings did not respond to immunosuppressive treatments and recombinant IL-1 receptor antagonist, anakinra injections, but controlled with adalimumab.

Identification of three ADA2 deficiency families with novel CECR1 mutations

Adenosine desaminase 2 deficiency (DADA2) is a rare monogenic autoinflammatory disease, presenting with systemic inflammation, vascular pathology with possible strokes, and mild immunodeficiency. Recently, the CECR1 gene was found to be responsible for this disease with an autosomal recessive inheritance.

CECR1 p.Gly47Arg mutations are not increased in frequency in Turkish Behçet's disease patients compared with healthy controls

Question In Behcet’s disease (BD), vasculitis involving blood vessels of nearly all sizes and types may underlie the diverse tissue and organ involvement. Loss of function mutations in the CECR1 gene (encoding adenosine deaminase 2) have recently been shown to cause a recessive genetic disease, deficiency of adenine deaminase 2 (DADA2). Patients with DADA2 exhibit systemic vasculopathy characterized by intermittent fevers, skin rash, and neurovascular manifestations along with other features that can lead to a diagnosis of polyarteritis nodosa. Patients homozygous for the CECR1 p.Gly47Arg mutation are reported in two nonconsanguineous Turkish families and this mutation is found at low frequency in the Turkish population. We therefore attempted to determine whether some BD cases may be explained by adenosine deaminase 2 deficiency and whether this mutation contributes to BD risk in patients of Turkish ancestry.

Prevalence of CECR1 mutations in pediatric patients with polyarteritis nodosa, livedo reticularis and/or stroke

Methods Pediatric patients of Caucasian Italian origin with the following diseases/manifestations were included in the study: i) histologically confirmed polyartiritis nodosa (PAN) or cutaneous polyartiritis nodosa (cPAN), ii) persistent livedo reticularis with elevation of acute phase reactants, iii) ischemic or hemorrhagic strokes with systemic inflammation. Direct sequencing of CECR1 gene (exons 1-9) was performed with Sanger analysis.

A case with febrile attacks and vasculopathy associated with ADA2 and MEFV gene mutations

Case A patient with juvenile onset recurrent febrile attacks associated with familial polyarteritis nodosa (PAN) and who carries CECR1 and MEFVgene mutations is described. The index case, a 23 year old male patient with recurrent attacks of fever and arthritis since the age of 7 was diagnosed initially as Familial Mediterranean Fever (FMF). A beneficial response to treatment with colchicine was observed. A year later he developed livedo reticularis and nodular dermal lesions compatible with cutaneous PAN. He was treated with prednisolone and azathioprine. Arthralgia, fever and dermal lesions regressed and he was in remission until he developed anemia and macrocytosis a year later and azathioprine was stopped. Due to the activation of his skin vasculitis anakinra 100mg/day was instituted. The beneficial response obtained with anakinra was lost when he discontinued the treatment. A family history revealed a brother two years older than himself who also had livedo reticularis, Raynaud’s phenomena, fever and arthritis since the age of 8, diagnosed as PAN before the index case and died at age 22 because of gut perforation secondary to acute mesenteric ischemia. With the probable diagnosis of ADA2, the index patient was analyzed for CECR1 gene mutations on chromosome 22q11.1. After amplification of the exons 2, 4, 5, 6 and 9 on PCR, DNA sequencing analysis was performed. A homozygous c.139G-A transition in exon 2, resulting in a gly47-to-arg (G47R) substitution at a highly conserved residue in the dimerization domain was identified in this patient who was known to be heterozygous for M694V and R202Q mutations of the MEFV gene. His vasculitic lesions responded to Infliximab after the fourth infusion together with daily colchicine treatment.

B cells characterization in ADA2 Deficiency patients

ADA2 deficiency, a recently described disease, is characterized by systemic vasculopathy and episodes of strokes. The defect is due to a loss of function mutation of CECR1 gene, codifying for Adenosine Deaminase 2 protein. This protein regulates the catabolism of extracellular adenosine, which we have recently shown is an important regulator of Class Switch Recombination in B lymphocytes. Accordingly DADA2 patients can present hypogammaglobulinemia.

The invertebrate growth factor/CECR1 subfamily of adenosine deaminase proteins

Adenosine deaminase (ADA) catalyzes the hydrolysis of adenosine to inosine. Its lack determines severe combined immunodeficiency in mammals, possibly due to accumulation of extracellular adenosine, which induces apoptosis in lymphocytes (Franco et al., 1998). Thus, presence of normal levels of ADA leads to normal growth and proliferation of lymphocytes. Several vertebrate and microbial ADA amino-acid sequences are known, with substantial similarity to each other. On the other hand, there are invertebrate growth factors as well as a candidate gene for the human cat eye syndrome (CECR1) (Riazi et al., 2000. Genomics 64, 277–285), which share substantial similarity to each other, and also to ADA. In this study, we report the expression and ADA enzymatic activity of a cDNA from the salivary glands of Lutzomyialongipalpis, a blood-sucking insect, with substantial similarity to insect growth factors and to human CECR1. We also demonstrate the existence of a subfamily of the adenosine deaminase family characterized by their unique amino-terminal region. Both Drosophilamelanogaster and humans have both types of adenosine deaminases. Results indicate that these invertebrate proteins previously annotated as growth factors, as well as the human CECR1 gene product, may exert their actions through adenosine depletion. The different roles played by each type of adenosine deaminase in humans and Drosophila remains to be fully investigated.

The Human Homolog of Insect-Derived Growth Factor, CECR1, Is a Candidate Gene for Features of Cat Eye Syndrome

Cat eye syndrome (CES) is a developmental disorder with multiple organ involvement, associated with the duplication of a 2-Mb region of 22q11.2. Using exon trapping and genomic sequence analysis, we have isolated and characterized a gene, CECR1, that maps to this critical region. The protein encoded by CECR1 is similar to previously identified novel growth factors: IDGF from Sarcophaga peregrina (flesh fly) and MDGF from Aplysia californica (sea hare). The CECR1 gene is alternatively spliced and expressed in numerous tissues, with most abundant expression in human adult heart, lung, lymphoblasts, and placenta as well as fetal lung, liver, and kidney. In situ hybridization of a human embryo shows specific expression in the outflow tract and atrium of the developing heart, the VII/VIII cranial nerve ganglion, and the notochord. The location of this gene in the CES critical region and its embryonic expression suggest that the overexpression of CECR1 may be responsible for at least some features of CES, particularly the heart defects.