Erythrocyte Adenosine Deaminase Research Articles

Novel Mouse Model That Recapitulates the Hematologic Phenotype of Diamond Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that presents during early childhood and is characterized by macrocytic anemia, congenital malformations, and predisposition to cancer. More than 90% of DBA patients are diagnosed during their first year of life (median age 12 weeks). The pathogenesis of DBA is linked to loss-of-function mutations in genes encoding ribosomal proteins (RP), although mutations in 3 non-RP genes ( GATA1, TSR2, and HEATR3) have been detected. DBA patients present with a high degree of clinical heterogeneity with varying severity and responses to steroid therapy. DBA provides a unique disease model to study how RP deficiency impacts the phenotype and pathogenesis of ribosomopathies. However, one challenge is the lack of animal models that faithfully recapitulate the clinical features of DBA in terms of disease severity and onset age. Heterozygous loss-of-function mutations in RPL11 are found in 5-20% of DBA patients, but previous mouse models carrying Rpl11 haploinsufficiency have only a mild anemia. Here, we report a novel mouse model of DBA with inducible haploinsufficient expression of Rpl11 which results in a severe macrocytic anemia in juvenile mice. Mx1-CreRpl11+/flox experimental mice were generated by cross-breeding homozygous Rpl11 flox/flox and Mx1-cre breeders. Haploinsufficiency of Rpl11 ( Rpl11+/Δ) was induced by intraperitoneal injection of polyinosinic-polycytidylic acidto mice with Mx1-CreRpl11 +/flox on postnatal day 8 and 10. Diseased mice with Rpl11+/Δ showed macrocytic anemia at 2 weeks (wks) post-induction with hemoglobin (Hb) < 10 g/dL, eventually dying of severe anemia (Hb< 2 g/mL) by 30 wks (median survival 27 wks, p<0.001, n=7 )( Figure 1). We observed a 50% decreased expression of Rpl11 in blood neucleated cells from Rpl11+/Δ mice and several characteristic clinical features of DBA. Erythrocyte adenosine deaminase was significantly elevated in the blood of all Rpl11+/Δ mice when compared to wild type (WT) littermates (median 5 vs 1 EU/g Hg, p<0.001, n=6). Similarly, EPO concentrations were significantly elevated in Rpl11+/Δ mice compared to WT mice (median 38752 vs 23 pg/mL, p<0.01, n=6). On postmortem examination, the DBA mice displayed splenomegaly. FACS analysis data demonstrated that the differentiation of erythropoiesis is significantly blocked in peripheral blood, bone marrow (BM), and spleen of Rpl11+/Δ mice compared to WT mice (p<0.05, n = 5). In addition, methylcellulose colony assays revealed that CFU-E colonies were significantly decreased in BM from Rpl11+/Δ mice in comparison to WT mice (median 23.5 vs 40.5 per 0.2 million BM cells in the presence of EPO at a concentration of 1 unit/mL, p<0.05, n=4). To determine whether BM hematopoietic stem cells/progenitor cells from Rpl11+/Δ mice can transfer the DBA phenotype, we transplanted bone marrow cells of Rpl11+/ Δ mice (CD45.2+) into sublethally irradiated WT mice (CD45.1+). Our data demonstrate that the BM cells from Rpl11+/Δ donor mice can induce lethal macrocytic anemia in WT recipient mice. Studies on the molecular mechanisms underlying the dysregulated erythropoiesis in Rpl11+/Δ mice are ongoing. In conclusion, we have successfully generated the first DBA mouse model that recapitulates the hematologic features of DBA patients. This model provides an ideal tool to study the pathogenesis of DBA and other ribosomopathies.

Missense mutation in RPS7 causes Diamond-Blackfan anemia via alteration of erythrocyte metabolism, protein translation and induction of ribosomal stress

Diamond-Blackfan anemia (DBA) is predominantly underlined by mutations in genes encoding ribosomal proteins (RP); however, its etiology remains unexplained in approximately 25 % of patients.We previously reported a novel heterozygous RPS7 mutation hg38 chr2:g.3,580,153G > T p.V134F in one female patient and two asymptomatic family members, in whom mild anemia and increased erythrocyte adenosine deaminase (eADA) activity were detected. We observed that altered erythrocyte metabolism and oxidative stress which may negatively affect the lifespan of erythrocytes distinguishes the patient from her asymptomatic family members. Pathogenicity of the RPS7 p.V134F mutation was extensively validated including molecular defects in protein translational activity and ribosomal stress activation in the cellular model of this variant.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

Case Series: Bone Marrow Failure in Teen Siblings with Unique RPS19 Variant

Aplastic anemia occurs when there is a lack of hematopoiesis in the bone marrow leading to peripheral pancytopenia. It is a rare entity and is often idiopathic. However, upon presentation, inherited bone marrow failure syndromes and acquired etiologies must be considered. Investigating causality is particularly important when multiple family members are affected, especially in a short amount of time. It is also essential to identify novel causative genetic variants of bone marrow failure in order to direct treatment in these patients.In our case series, we describe two siblings who presented two weeks apart with severe pancytopenia. The first patient is a 13 year-old non-binary female (prefers pronouns they/them/theirs) who presented from clinic after getting routine labs with white blood cell (WBC) 1.83 x10(3)/mcL, absolute neutrophil count (ANC) 0.16 x10(3)/mcL, hemoglobin (Hgb) 4.8 gm/dL, platelet 13 x10(3)/mcL. Bone marrow biopsy revealed marked hypocellularity (0-10%) with hypoplasia. The second patient is their 16 year-old brother who presented two weeks later with new-onset petechial rash and was found to have WBC 4 x10(3)/mcL, ANC 2.19 x10(3)/mcL, Hgb 6.3 gm/dL, platelet 16 x10(3)/mcL. His bone marrow biopsy demonstrated variable cellularity (10-70%), but after months of transfusion-dependence he met criteria for severe aplastic anemia. Laboratory evaluation for acquired etiologies such as infection was negative. Both patients had a shared medical history of depression briefly treated with fluoxetine, but otherwise no potential medical triggers or environmental exposures were identified. Telomere length analysis was normal and chromosomal breakage analysis was negative. Upon genetic evaluation, both patients were found to have a heterozygous variant of unknown significance of RPS19 (c.-163>T), which substitutes a moderately conserved nucleotide in the noncoding exon 1 in 5‘ untranslated region of RPS19. Although this variant has not been classified as pathogenic, three other variants in the 5‘ untranslated region of RPS19 have been reported in patients with Diamond-Blackfan Anemia (DBA). In contrast to classic DBA, these patients did not present in infancy or early childhood. Likewise, they lacked congenital anomalies or other classic phenotypic characteristics of disease. Lab studies showed normal erythrocyte adenosine deaminase levels, though it should be noted that these levels could not be obtained prior to transfusion in both patients. Given lack of other identified etiologies, however, an inherited bone marrow failure syndrome, possibly due to this variant, was presumed. With suspected but potentially unidentifiable genetic predisposition, matched sibling donor transplant was deferred and both patients underwent a matched unrelated donor bone marrow transplant with reduced-intensity conditioning.With these cases, we aim to share a unique presentation of aplastic anemia that reveals a potentially novel pathogenic variant as well as to provide our approach to medical management in pediatric aplastic anemia in the setting of uncertainty. Identification of other patients with bone marrow failure and this genetic variant will be important to determine its pathogenicity. DisclosuresNo relevant conflicts of interest to declare.

AML-345: Algorithmic Diagnostic Approach and Spectrum of Hematological Malignancies in Patients with Germline Predisposition Syndromes

Context: Germline predisposition syndromes (GPS) are genetic disorders that increase the risk of all types of malignancies, with a growing list of genes associated specifically with hematological malignancies. Although many of these conditions have syndromic features, some GPS patients present without identifiable traits. We have established a hematology GPS clinic that uses an algorithmic approach to diagnose GPS patients. Objective: To illustrate the heterogeneity of GPS and the benefits of an algorithmic approach to identify non-syndromic GPS patients. Design: Medical records were reviewed retrospectively from patients diagnosed with GPS at our institution from 1994 to 2018. Prospectively, we investigated new pediatric and adult patients with GPS from 2017 to 2020 using an algorithmic approach summarized as follows: 1) Evaluation of relevant family history, 2) phenotype-specific functional assays (telomere measurement, breakage assay, erythrocyte adenosine deaminase testing), 3) germline genetic testing through a custom-designed gene panel, and 4) research whole exome sequencing for negative cases. Both a detailed explanation of the algorithm and the methodology for genetic variant interrogation are included in Mangaonkar et al. MC Proc 2019. Setting: This study was carried out at the Division of Hematology at Mayo Clinic. Patients or Other Participants: GPS was investigated in pediatric and adult patients with one or more first-degree relatives with hematological/visceral malignancies, in those with antecedent thrombocytopenia, or in those with specific features indicative of inherited bone marrow failure (IBMF) (short telomere syndromes, GATA2 haploinsufficiency, Fanconi anemia, Schwachman-Diamond syndrome). Interventions: N/A Main Outcomes Measures: The number of patients diagnosed with GPS both retrospectively and prospectively were tallied and compared. Results: A total of 146 individuals were included in the study divided as follows: 28 (19.4%) presented with hematological malignancy with precedent thrombocytopenia, 27 (18.8%) without precedent thrombocytopenia, 74 (51.3%) showed IBMF, and 15 (10.4%) showed general cancer predisposition syndrome. Eighty-five patients were retrospectively included, while 61 were prospectively evaluated through our algorithmic approach. Among retrospective patients, 7.1% (6 patients) did not present syndromic features, while this value increased to 14.7% (9 patients) when using the algorithmic approach. Conclusions: By using an algorithmic approach, we can identify a higher percentage of GPS patients without syndromic features.

High Adenosine Deaminase Level Among Healthy Probands of Diamond Blackfan Anemia (DBA) Cosegregates With the DBA Gene Region on Chromosome 19q13

AbstractPhenotypic characterization of Diamond Blackfan Anemia (DBA) patients and their relatives was performed in 54 families. Complete blood count, fetal hemoglobin level, erythrocyte i antigen expression, and erythrocyte adenosine deaminase (eADA) activities were quantitated in patients and relatives. eADA was elevated in 28 of 34 transfusion-independent DBA patients, whereas persistence of erythrocyte i antigen was noticed in only 10 of 20 DBA patients. High eADA activities were also found in 14 of 149 healthy family members, allowing us to identify an isolated high eADA phenotype in these families. In contrast, increase in erythrocyte i antigen expression, elevated fetal hemoglobin levels, and macrocytosis were much less frequently noted in nonaffected members of the DBA families studied. Importantly, isolated high eADA phenotype was found to be significantly associated with genetic markers on chromosome 19 that segregate with the DBA phenotype. Isolated high eADA phenotype thus seems to reflect a silent phenotype of DBA in affected families. These findings suggest that elevated eADA activity in unaffected individuals needs to be taken into account during genetic assessment of DBA families and could be used for accurate assessment of mode of inheritance.

Mutations in Ribosomal Protein S19 Gene and Diamond Blackfan Anemia: Wide Variations in Phenotypic Expression

AbstractMutations of the ribosomal protein S19 (RPS19) gene were recently identified in 10 patients with Diamond Blackfan anemia (DBA). To determine the prevalence of mutations in this gene in DBA and to begin to define the molecular basis for the observed variable clinical phenotype of this disorder, the genomic sequence of the 6 exons and the 5′ untranslated region of the RPS19 gene was directly assessed in DBA index cases from 172 new families. Mutations affecting the coding sequence of RPS19 or splice sites were found in 34 cases (19.7%), whereas mutations in noncoding regions were found in 8 patients (4.6%). Mutations included nonsense, missense, splice sites, and frameshift mutations. A hot spot for missense mutations was identified between codons 52 and 62 of the RPS19 gene in a new sequence consensus motif W-[YFW]-[YF]-x-R-[AT]-A-[SA]-x-[AL]-R-[HRK]-[ILV]-Y. No correlation between the nature of mutations and the different patterns of clinical expression, including age at presentation, presence of malformations, and therapeutic outcome, could be documented. Moreover, RPS19 mutations were also found in some first-degree relatives presenting only with isolated high erythrocyte adenosine deaminase activity and/or macrocytosis. The lack of a consistent relationship between the nature of the mutations and the clinical phenotype implies that yet unidentified factors modulate the phenotypic expression of the primary genetic defect in families with RPS19 mutations.

A 20-year long term experience of the Italian Diamond-Blackfan Anaemia Registry: RPS and RPL genes, different faces of the same disease?

Diamond-Blackfan anaemia (DBA) is a rare and heterogeneous disease characterised by hypoplastic anaemia, congenital anomalies and a predisposition for malignancies. The aim of this paper is to report the findings from the Italian DBA Registry, and to discuss the Registry's future challenges in tackling this disease. Our 20-year long work allowed the connection of 50 Italian Association of Paediatric Haematology and Oncology (AIEOP) centres and the recruitment of 283 cases. Almost all patients have been characterised at a molecular level (96%, 271/283), finding a causative mutation in 68% (184/271). We confirm the importance of determination of erythrocyte adenosine deaminase activity(eADA) and of ribosomal RNA assay in the diagnostic pipeline and characterisation of a remission state. Patients with mutations in largeribosomal subunitprotein (RPL) genes had a significant correlation with the incidence of malformations, higher eADA levels and more severe outcomes, compared to patients with mutations in small ribosomal subunit protein (RPS) genes. Furthermore, as a consequence of our findings, particularly the incidence of malignancies and the high percentage of patients aged >18years, we stress the importance of collaboration with adult clinicians to guarantee regular multi-specialist follow-up. In conclusion, this study highlights the importance of national registries to increase our understanding and improve management of this complex disease.

Erythrocyte Adenosine Deaminase Levels Are Elevated in Classic Diamond Blackfan Anemia but Not in the 5q- Syndrome

Diamond Blackfan anemia (DBA) is congenital bone marrow failure syndrome characterized by red cell aplasia. It was the first disease linked to ribosomal dysfunction with 70% of patients having haploinsufficiency of a ribosomal protein (RP) gene. More recently, several patients with DBA have been shown to have mutations in GATA-1. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is also characterized by a severe anemia that is caused by heterozygous loss of RPS14 on chromosome 5q. The reciprocal relationship between these two diseases, which are now collectively known as the ribosomopathies, has spurred interest in how we may be able to better understand the pathophysiology of these disorders to broaden therapeutic options, and to improve diagnosis.Erythrocyte adenosine deaminase (eADA) levels have been used for the last three decades in the diagnosis of DBA based on the finding in 1983 that eADA enzyme levels are significantly elevated in patients with DBA. It is now known that more than 75% of DBA patients have elevated eADA levels. The goal of the present study is to measure the levels of eADA in patients with the 5q- syndrome and determine whether they are elevated implying that eADA may be specific to ribosomal haploinsufficiency in general or normal suggesting that elevated eADA may be specific to DBA.Under a Stanford Univeristy approved IRB protocol, we measured eADA levels in patients with the 5q- syndrome who were undergoing routine blood draws for clinical purposes. The patients with Diamond Blackfan anemia are either followed at our center in Stanford University or were previously referred to us for eADA testing with accompanying clinical information. Analysis was performed using SAS 9.3 (SAS Institute, Carey, NC). Continuous variables were presented as means and standard deviations (SD) and analyzed using 2-tailed T-Test.Complete results by mutation type are summarized in Tables 1 and 2.In a disorder with the marked genotypic and phenotypic variability such as Diamond Blackfan anemia (DBA), it is critical to have sensitive tests to aid with the diagnosis. Erythrocyte adenosine deaminase (eADA) levels have long been used as supportive diagnostic criteria. The test has been especially useful in distinguishing DBA from other causes of anemia and other inherited bone marrow failure syndromes. However, the reason for the elevation of eADA in DBA remains unknown.Our goal was to study eADA levels in the 5q- syndrome where there is an acquired ribosomal haploinsufficiency (from loss of RPS14 on chromosome 5q), which is in contrast to the congenital haploinsufficiency seen in DBA. Interestingly, we found that the eADA levels were normal in the 5q- syndrome suggesting that eADA is specific for DBA. We then studied eADA levels in patients with DBA that is not the results of a ribosomal protein (RP) mutation but instead due to mutations in GATA-1. Once again, we found that eADA levels were normal in this cohort suggesting that elevated eADA is specific feature of DBA due to RP mutations. It is interesting to note that patients with RPS19 mutations, who tend to have a milder clinical phenotype, and patients without an identified mutation had lower (but still elevated) levels of eADA when compared to other RP mutations, in particular RPL11. A larger number of patient samples in future studies would improve the statistical power to compare eADA levels between individual RP mutations.This is the first report to examine the value of eADA in non-classical DBA and in another disorder of ribosomal haploinsufficiency, namely del(5q) MDS. In summary, we found that an elevated eADA strongly suggests the diagnosis of classic DBA although a normal eADA does not exclude the diagnosis, particularly in the setting of GATA-1 mutations. There is no utility for using eADA in the diagnosis of the 5q- syndrome despite the connection of the disease with impaired ribosome function. The reason for elevated eADA in classic DBA remains to be defined. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Two Novel Mutations in Ribosomal Proteins in the Czech National Diamond-Blackfan Anemia Registry

The Czech National Diamond-Blackfan Anemia (DBA) Registry currently consists of 51 patients; in 36 of them ribosomal protein (RP) mutations have previously been identified. Targeted sequencing of genes encoding RP, exomes and transcriptomic sequencing and/or array CGH were performed in newly diagnosed DBA patients and in patients without a causative mutation. Two novel mutations in RPS7 and RPL11 were discovered in 5 individuals from two families.Aheterozygous mutation g[3580153G>T] in exon 6 of a gene coding for RPS7, leading to V134F substitution, was found in an 18-year-old female who was transfusion dependent during infancy, responded to steroids and is currently in remission. The identical mutation was detected in the patient’s mother and older sister, who both are asymptomatic with borderline hemoglobin (Hb) levels, mild macrocytosis with no active treatment. A functional study using cellular models revealed that RPS7-deficient MRC-5 fibroblasts transiently expressing RPS7-V134F exhibit altered protein synthesis, defective ribosomal RNA processing, increased nucleolar and ribosomal stress. This was evidenced by overexpression and translocation of p53 protein to the nucleus in RPS7-V134F but not RPS7 wild type transfected cells. These data are consistent with previously reported cellular phenotypes in DBA. In addition, increased levels of erythrocyte adenosine deaminase (e-ADA) were detected in all three family members with V134F RPS7 mutation (patient: 4.2±0.8 IU/g Hb; mother: 3.5±0.2 IU/g Hb; sister: 4.6±0.3 IU/g Hb; reference range: 0.8-2.5 IU/g Hb). Recently it was shown that ribosomal insufficiency increases oxidative stress in red blood cells (RBC) of DBA patients. In this family, increased levels of reactive oxygen species (ROS) were detected by flow cytometry only in patient’s RBC, but not in RBC of her asymptomatic mother and sister. Consistently, only the patient showed increased anti-oxidative defense parameters compared to the controls and her mother and sister. Markedly elevated levels of reduced glutathione (GSH) and glucose 6-phosphate dehydrogenase (G6PD), two essential antioxidants, were detected in patient’s RBC (GSH: 4356±44 mM; G6PD: 10.3±0.6 IU/g Hb) compared to controls (GSH: 2434±454 mM; G6PD: 5.4-7.0 IU/g Hb) and patient’s mother (GSH: 2125 mM; G6PD: 5.3±0.2 IU/g Hb) and sister (GSH: 2450±35 mM; G6PD: 6.5±0.1 IU/g Hb). Concomitant increase (approximately 2.5 times) in the activity of hexokinase (HK) and pyruvate kinase (PK) and in the levels of ATP in patient’s RBC indicates augmented energy metabolism to sustain the integrity of RBC. Flow cytometry of Annexin V binding revealed increased exposure of phosphatidylserines on RBC membrane of the patient compared to controls and her mother and sister suggesting that the tendency towards redox balance restoration and membrane integrity does not completely prevent an enhanced recognition and destruction of patient’s RBC by reticuloendothelial macrophages.The second c.281T>G point mutation in RPL11 was found in a 34-year-old woman with unspecified macrocytic anemia, who developed diffuse large B-cell lymphoma (DLBCL). She was transfusion dependent in infancy, responded to steroids and is currently in the anemia remission and second remission of DLBCL. Identical mutation was detected in her 56-year-old mother, followed for macrocytic anemia and gammopathy.In 10 DBA cases without the causative mutation an array CGH was performed, but no large deletions in RPS19, RPS17, RPS26, RPL5 and RPL11 genes were detected.In summary, we present the first case of a missense mutation in RPS7 as the cause of DBA; all to date published RPS7 mutations clinically associated with DBA were located in splice sites. Similarly, the co-occurrence of DBA and DLBCL has not been previously published. We also emphasize that the phenotype of family members with the same mutations could be different, including silent carriers. All family members should therefore be examined even if asymptomatic. Testing of eADA levels could serve as a sensitive screening method in apparently unaffected family members. Finally we suppose that altered RBC metabolism may affect the lifespan of RBC in DBA and thus contribute to the worsening of the blood condition, especially during infections. The analyses in a large cohort of DBA patients are ongoing.Grant Support: AZV16-32105A, GA15-13732S, MH CZ-DRO FNOL 00098892, NPU LO 1304, IGA UP LF_2016_014. DisclosuresNo relevant conflicts of interest to declare.

Erythrocyte glutathione is a novel biomarker of Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia with mutations in ribosomal protein (RP) genes. Elevated activity of erythrocyte adenosine deaminase (eADA) has been utilized as a biomarker of DBA. We examined erythrocyte reduced glutathione (GSH) as well as eADA in 22 patients in 18 DBA families, in whom RP gene mutations had been identified. Simultaneous evaluation of both eADA and GSH demonstrated that all examined DBA patients showed elevated values of either eADA or GSH, whereas presence of both eADA and GSH elevation was able to distinguish DBA patients from 34 normal controls and 14 unaffected members of the DBA families. Furthermore, a support vector machines analysis using both eADA and GSH levels yielded a formula to differentiate DBA from both normal controls and non-DBA family members. To confirm the usefulness of the formula, we analyzed additional 7 patients diagnosed by the clinical criteria. Although eADA showed within normal values in 3 patients, all of these patients were diagnosed as ‘DBA’ by use of the formula. Because extensive analysis of the RP genes failed to detect no causative mutation in approximately 40% of clinically diagnosed DBA patients, GSH may be useful an additional biomarker for diagnosis of DBA.

Adenosine Deaminase 2 Deficiency As a Cause of Pure Red Cell Aplasia Mimicking Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited pure red cell aplasia. Most cases present in the first year of life with elevation in erythrocyte adenosine deaminase (eADA) and frequently with increased mean corpuscular volume (MCV) and hemoglobin F (hgb F). Approximately 70 percent of cases are due to a mutation in one of several ribosomal protein (RP) genes or in GATA1, whereas the remaining cases are genetically uncharacterized. Here we report a child born with severe anemia and diagnosed with DBA at 2 months of age. His bone marrow was normocellular with a paucity of erythroid progenitors and scattered lymphocytes. An eADA level was not obtained prior to the first red cell transfusion. He was red cell transfusion dependent and his anemia did not respond to a steroid trial. His 4 year old sister, who had normal hemoglobin, MCV, hgb F, and eADA measurements, served as his HLA identical donor for hematopoietic stem cell transplantation (HSCT). HSCT resulted in 100% donor chimerism, but red cell engraftment was not achieved. He subsequently underwent a mismatched unrelated HSCT with trilineage engraftment.Ten years later, at the age of 14 years, the sister presented with profound hypoproductive normocytic anemia. The bone marrow showed absence of erythroid precursors and presence of lymphoid aggregates. Findings of immunodeficiency included numerous cutaneous warts, recurrent aphthous ulcers, Epstein Barr virus (EBV) reactivation, low IgM, and low numbers and percentages of CD19+ and CD3-56+16+ lymphocytes. The anemia and reticulocytopenia persisted despite resolution of EBV reactivation. Upon her presentation, levels of iron, ferritin, transferrin saturation, and liver transaminases were elevated. A liver biopsy obtained after transfusion of a total of 60 ml/kg packed red blood cells demonstrated 4.8 mg Fe/g dry liver weight with stage 2 portal fibrosis.Targeted DNA sequencing studies performed on the affected sister were negative for single nucleotide variants in any of 12 RP genes previously implicated in DBA and a genome wide chromosome microarray was normal. Whole exome analysis of her and her parents demonstrated that she carried compound heterozygous variants in CECR1 (cat eye syndrome chromosome region, candidate 1). The variant p.R169Q had been previously reported as pathogenic, while the p.G358R variant was of uncertain significance. These variants are present at frequencies of 4.9X10-4 and 2.6X10-5 in the Exome Aggregation Consortium database, respectively. Analysis of buccal swab DNA of the proband showed the same biallelic variants. An unaffected 16-year-old sibling had a normal genotype. CECR1 encodes adenosine deaminase 2 (ADA2) and ADA2 levels in the plasma of the affected sister were markedly low, consistent with a deficiency state.CECR1 is highly expressed in cells of myeloid origin and ADA2 is a secreted protein implicated in macrophage differentiation and proliferation. Deficiency of ADA2 (DADA2) results in aberrant monocyte differentiation favoring M1 over M2 macrophages, thereby resulting in a proinflammatory state. Recent descriptions of patients with DADA2 due to CECR1 mutations reported a spectrum of phenotypes including intermittent fevers, lacunar stroke in childhood, livedoid rash, polyarteritis nodosa, and immunodeficiency with B lymphopenia and low IgM levels. Our cases are similar to the report of one of two brothers, homozygous for CECR1 p.R169Q, described by van Montfrans, et al,. (NEJM, 2014). The eldest was given a diagnosis of atypical DBA (refractory pure red cell aplasia) in infancy and underwent a HSCT from his asymptomatic, HLA identical brother. This HSCT resulted in non-engraftment, necessitating a subsequent unrelated donor HSCT. The younger sibling donor later developed hepatosplenomegaly, profound lymphopenia, and evidence of an inflammatory state.Together, these three cases support pure red cell aplasia as a presentation of DADA2 and that this may be confused with DBA, particularly when manifest in infancy. We propose DADA2 should be considered in patients with genetically uncharacterized DBA. Differentiating features to suggest DADA2 may include normal eADA, MCV, and hgb F levels and findings of associated immunodeficiency. Additionally, the macrophage activation due to DADA2 may have played a role in the iron overload observed in our second patient prior to any red cell transfusion. DisclosuresNo relevant conflicts of interest to declare.

Changes in Adenosine Metabolism in Asthma. A Study on Adenosine, 5&#39-NT, Adenosine Deaminase and Its Isoenzyme Levels in Serum, Lymphocytes and Erythrocytes

Background: Adenosine deaminase (ADA) and 5'-nucleotidase (5'-NT) play a crucial role in adenosine metabolism in healthy individuals. Adenosine is an inflammatory mediator of asthma. Changes in adenosine metabolism and role of ADA and 5'-NT in regulating adenosine level in asthmatics and correlation of these changes with severity of asthma are not clearly understood. Methods: In this study, we screened 5217 patients, of which 2416 were diagnosed with asthma. Further, of 2416 asthmatics, only 45 patients who strictly fulfilled the selection criteria were enrolled in the study. The patients were classified into mild, moderate and severe persistent groups; each group consisted of fifteen patients. Fifteen healthy subjects served as controls. Adenosine levels and activities of 5'-NT, total ADA, ADA1 and ADA2 in serum, lymphocytes and erythrocytes were determined. The data were analysed statistically and p vice versa.

De NovoRPS20 Mutations in Diamond Blackfan Anemia

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by pure red blood cell aplasia, variable congenital anomalies and increased risk of malignancy. Approximately 50-60% of cases are due to germline, heterozygous mutations in 1 of 13 genes that encode components of either the small (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26 and RPS29) or large (RPL5, RPL11, RPL15, RPL26, RPL31 and RPL35A) ribosomal subunits. The mutations may be either inherited or de novo. Consistent with DBA being a ribosomopathy, defects in ribosome assembly, altered ribosomal RNA processing and nucleolar stress are observed in cells of patients with DBA and ribosomal protein gene mutations. Mutations in GATA1, a hematopoietic transcription factor, have also been reported in rare X-linked recessive cases.Whole exome sequencing was independently carried out on two white/Hispanic probands with genetically uncharacterized DBA (BMF92 and NCI-62-1). Both presented with transfusion-dependent anemia during the first week of life. Bone marrow evaluations revealed marked erythroid hypoplasia characteristic of DBA. One of the probands (BMF92) experienced a spontaneous remission of his anemia toward the end of the first year of life, possibly related to corticosteroids administered for bronchiolitis. Subsequently, his erythrocyte adenosine deaminase(eADA) level was measured and elevated [10.5 units (mol/min/gm hg); reference range 0.42-3.5 units]. BMF92’s hypoplastic anemia later recurred and was found to be steroid refractory. NCI-62-1 is a male, now 12 years of age and red cell transfusion dependent. BMF92 and NCI-62-1 are of normal stature and have no documented congenital anomalies, although both have experienced chronic colitis. In both cases, family history for DBA was negative and parental eADA levels were within normal limits.Whole exome sequencing of peripheral blood DNA, validated by Sanger or Ion Torrent targeted sequencing, identified heterozygous variants of unknown significance in the ribosomal protein gene RPS20. Parental studies revealed both variants to be de novo. The variants were mutated at the same genomic position, but resulted in different amino acid substitutions in RPS20 [hg19 chr8 56985758, c.251A>T, p.I84N (BMF92) and 56985758, c.251A>C, p.I84S (NCI-62-1)]. Buccal swab analysis performed in BMF92 confirmed the presence of the variant. Both variants were novel, based on inspection of several databases, including dbSNP, 1000 Genomes, NHLBI Exome Sequencing Project, clinical WES data at the Whole Genome Laboratory at Baylor College of Medicine, Kaviar, Human Genome Mutation Database and ClinVar, totally well over 10,000 individuals as well as the COSMIC database, which reports somatic mutations in cancer. In silicoanalyses were consistent with the variants being damaging and disease causing (PolyPhen-2, SIFT and Mutation Taster), affecting highly evolutionarily conserved residues (GERP, PhyloP and Sitewise likelihood-ratio score) and decreasing the stability of the protein structure (MUpro and I-Mutant2.0).The above data strongly suggest that the RPS20 variants in these cases are disease causing. A recent report implicated a germline RPS20 truncating mutation in a four-generation pedigree with familial nonpolyposis colorectal carcinoma; however, the mutation carriers were reported to not manifest features of DBA (Nieminen et al., Gastroenterology 2014). This raises the likelihood of allele-specific effects. Functional assays of our probands’ mutations, including quantification of RPS20 protein and ribosomal RNA precursor steady state levels in patient versus control lymphoblastoid cell lines as well as analysis of the impact of mutant RPS20 protein expression on p53 and downstream targets are underway to determine their contribution to the DBA phenotype. DisclosuresNo relevant conflicts of interest to declare.

Understanding the Role of Erythrocyte Adenosine Deaminase in the Ribosomopathies

Diamond Blackfan anemia (DBA) is congenital bone marrow failure syndrome characterized by red cell aplasia and a predisposition to cancer. It was the first disease linked to ribosomal dysfunction with 70% of patients having haploinsufficiency of a ribosomal protein (RP) gene with RPS19 being the most frequently mutated. The 5q- syndrome is a subtype of myelodysplastic syndrome (MDS) also characterized by a severe anemia that is caused by heterozygous loss of RPS14 on chromosome 5q. These patients also have a predisposition to cancer. The reciprocal relationship between these two diseases, which are now collectively known as the ribosomopathies, has spurred interest in how we may be able to better understand the pathophysiology of these ribosomopathies to broaden therapeutic options, and to improve diagnosis.Erythrocyte adenosine deaminase (eADA) levels have been used for last three decades in the diagnosis of Diamond Blackfan anemia based on the finding in 1983 that eADA enzyme levels are significantly elevated in patients with DBA. It is now known the eADA levels are elevated in >75% of patients with DBA. Furthermore, it has a sensitivity of 84%, specificity of 95% and positive and negative predictive values of 91% for the diagnosis of DBA compared with other inherited bone marrow failure syndromes. Our goal was to study the levels of eADA in patients with the 5q- syndrome to determine whether they were elevated (suggesting that eADA may be specific to ribosomal haploinsufficiency in general) or normal (suggesting that eADA may be specific to DBA).Under a Stanford approved IRB, we are testing adult patients with the 5q syndrome who have not been transfused in the previous 3 months (which would affect the results of the test) and who have a confirmed deletion of the 5q- region by cytogenetics. To date, all of the 4 MDS samples, sent to us from collaborating hematologists, have had a normal eADA level. These findings suggest that elevation of eADA may be specific to DBA and not a general feature of ribosomal haploinsufficiency although our sample size is small and we plan to test additional samples. DisclosuresNo relevant conflicts of interest to declare.

Diamond Blackfan anemia: A Cheshire cat of hematology

Diamond Blackfan anemia: A Cheshire cat of hematology

Index of Suspicion

The reader is encouraged to write possible diagnoses for each case before turning to the discussion. We invite readers to contribute case presentations and discussions. Please inquire first by contacting Dr. Deepak Kamat at DKamat@med.wayne.edu. An 18-year-old male with a 3-year history of alcohol abuse is referred from a rehabilitation center for fever, nausea, and nonbloody diarrhea for 3 days. Six weeks ago, he enrolled in an alcohol rehabilitation program and stopped drinking alcohol. Three weeks later, he noticed yellowing of his eyes and skin, difficulty sleeping, and dyspnea. On examination, his temperature is 100.6°F (38.1°C), blood pressure is 99/62 mm Hg, pulse is 124 beats per minute, and respiratory rate is 24 breaths per minute. He is slow to answer questions. There is no asterixis. He appears thin and jaundiced with enlarged parotid glands. Decreased breath sounds are noted on the right. A tender, enlarged liver and mild abdominal distension are noted. Trace bilateral lower-extremity edema is present. No stigmata of chronic liver disease are found. Laboratory findings reveal the following: white blood cell count, 25,600/μL (25.6 × 109/L) (87% neutrophils); hemoglobin, 8.2 g/dL (82 g/L); mean corpuscular volume, 106 fL; platelets, 40 × 103/μL (40 × 109/L); sodium, 135 mEq/L (135 mmol/L); potassium, 3.5 mEq/L (3.5 mmol/L); chloride, 106 mEq/L (106 mmol/L); bicarbonate, 15 mEq/L (15 mmol/L); blood urea nitrogen, 24 mg/dL (8.6 mmol/L); creatinine, 1.01 mg/dL (89 μmol/L); glucose, 118 mg/dL (6.6 mmol/L); alkaline phosphatase, 182 U/L; aspartate aminotransferase (AST), 194 U/L; alanine aminotransferase (ALT), 71 U/L; total bilirubin, 11.8 mg/dL (201.8 μmol/L); direct bilirubin, 7.3 mg/dL (124.9 μmol/L); albumin, 2.7 g/dL (27 g/L); prothrombin time, 19.2 seconds; and international normalized ratio, 1.4. Ethanol and acetaminophen were not detected in the serum. A chest radiograph reveals a right pleural effusion …

Syzygium cumini is more effective in preventing the increase of erythrocytic ADA activity than phenolic compounds under hyperglycemic conditions in vitro

Syzygium cumini (S. cumini) is a plant known for its antidiabetic properties. The aim of this study was to evaluate the effect of Sc aqueous leaf extract (ASc) on adenosine deaminase (ADA) activity in erythrocytes (RBCs) exposed to high glucose concentrations (30mM) in vitro. We also investigated the effects of the main phenolic compounds found in ASc (gallic acid, rutin, and chlorogenic acid) and the effects of insulin, caffeine, and dipyridamole, which are substances involved in the adenosine metabolism, on ADA activity in vitro. Blood samples were obtained from healthy volunteers and a suspension of RBCs was used for the determination of ADA activity. The results showed that: (1) the effect of ASc on ADA activity was more significant than the combination of phenolic compounds; (2) insulin, caffeine, or dipyridamole prevented high glucose increase of ADA activity at doses as low as 50μU/mL, 25μM, and 1μM, respectively; (3) the inhibitory effect caused by ASc on erythrocyte ADA activity remained practically the same after the combination of the extract with insulin or caffeine; (4) when RBCs were exposed to ASc plus dipyridamole, this chemical attenuated the effect of ASc on ADA activity, suggesting an antagonism or a competition with ASc by the same site of action. Therefore, ASc was more effective in preventing the increase in ADA activity than phenolic compounds, suggesting that ASc may collaborate to improve endothelial dysfunction, antioxidant, anti-inflammatory, and antithrombotic properties of adenosine by affecting its metabolism. The results of this study help to provide evidence of the empirically supported benefits of the use of S. cumini in diabetes.

Functional Consequences Of RPS29 Germline Mutations In Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red blood cell aplasia, variable physical anomalies, and increased risks of cancer. Approximately half of DBA cases have a germline mutation or deletion in a ribosomal protein gene (i.e., RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, or RPS26), or in GATA1, an X-linked hematopoietic transcription factor. The inheritance of mutated ribosomal protein genes is autosomal dominant (AD), but de novo germline mutations may also occur. We previously reported a novel nonsynonymous ribosomal protein S29 (RPS29) mutation, I31F, which segregated with DBA in a large multi-case family (NCI-193). We now present functional data that link this RPS29 mutation to the DBA phenotype and report an additional large multi-case DBA family (NCI-38) with another novel RPS29 mutation.Individuals in this study are participants in the IRB-approved longitudinal cohort IBMFS study at the NCI. We have identified a second large family with DBA, NCI-38, in which the male proband had steroid-responsive anemia as a child, elevated erythrocyte adenosine deaminase (eADA = 1.26 IU/g Hb, normal <0.96), and has been in remission for approximately 20 years. Two unaffected male half-siblings have no symptoms of DBA, and 1 female half-sibling has only a borderline elevated eADA (1.00 IU/g Hb). The proband's mother is an asymptomatic obligate carrier because her full brother and two half-siblings have DBA or an unspecified bone marrow failure; her eADA is normal (0.8 IU/g Hb). We performed whole-exome sequencing using Nimblegen v2 capture arrays and paired-end sequencing on the Illumina HiSeqTM. After applying quality control filters, removing variants present in publicly available databases (1000Genomes, ESP, Kaviar, and dbSNP), and applying an AD inheritance model, we identified an I50T mutation in RPS29 that tracked with AD inheritance in this family (NCI-38). The sibling with elevated eADA and the mother also carried this RPS29 mutation while the two asymptomatic healthy siblings were mutation negative. I50T, like I31F, occurs in a very highly evolutionarily conserved region of the RPS29 protein and in silico prediction programs (i.e., SIFT, Polyphen 2, Condel, MutationTaster) predict this variant to be deleterious.The original DBA family, NCI-193, has AD DBA due to the heterozygous I31F mutation in RPS29. We used quantitative rtPCR to determine RPS29 expression in the proband with the I31F mutation compared with a healthy individual with wildtype RPS29. The proband demonstrated haploinsufficiency of RPS29 mRNA, with levels consistent with approximately 50% expression compared with wildtype RPS29 values. Zebrafish are ideal for modeling ribosomal protein knockdown because hematopoietic regulation is conserved with mammals, they are amenable to high throughput in vivo genetics, and ribosomal proteins can be knocked down in the embryo with morpholino technology. The rps29-/- mutant zebrafish displays many aspects of the DBA phenotype including significant defects in red blood cell development, shown by a decrease in hemoglobin levels. We used this as a model to determine if the RPS29 mutation identified in our DBA cases could rescue the hemoglobin defect. The rps29-/- zebrafish embryos were injected with 50pg of either wildtype or the mutated (I31F) human RPS29 RNA. The wildtype human RPS29 RNA rescued the hemoglobin phenotype (increased hemoglobin levels) of the rps29-/- zebrafish embryos, whereas the mutated human RPS29 RNA could not (no increase in hemoglobin levels; P <0.01).In summary, germline mutations in RPS29 can cause AD DBA. RPS29 is an essential protein in the 40s small subunit of the ribosome, important for ribosomal RNA processing and ribosome biogenesis. Using functional assays, we show for the first time that the mutant RPS29 in our DBA cases results in haploinsufficiency of RPS29. The patient-associated I31F RPS29 mutation failed to rescue the defective hematopoiesis in the rps29-/- mutant zebrafish DBA model and provides further evidence that RPS29 is a DBA-associated gene. Disclosures:Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Evaluation of acetylcholinesterase and adenosine deaminase activities in brain and erythrocytes and proinflammatory cytokine levels in rats submitted to neonatal hypoxia-ischemia model

Perinatal hypoxic-ischemic (HI) brain injury is a common problem with severe neurologic sequelae. The definitive brain injury is a consequence of pathophysiological mechanisms that begin at the moment of HI insult and may extend for days or weeks. In this context, the inflammatory response and the formation of reactive oxygen species seem to play a key role during evolution of brain damage after injury. Thus, the aim of this study was to describe the chronological sequence of acetylcholinesterase (AChE) activity and the lipid peroxidation changes in the cerebral cortex using the classic model of neonatal HI. Furthermore, the erythrocyte AChE and adenosine deaminase (ADA) activities as well as the serum levels of proinflammatory cytokines were assessed. We observed that neonatal HI caused an increase of lipid peroxidation immediately after HI insult, which remained for several days afterward. There was a time-related change in the AChE activity in the cerebral cortex and the same was observed in erythrocyte AChE and ADA activities. In addition, immediately after HI, ADA activity showed a strong positive correlation with all proinflammatory cytokines assessed. Together, these findings may help the understanding of some mechanism related to the pathophysiology of neonatal HI, therefore highlighting the putative therapeutic targets to minimize brain injury and enhance recovery.