ALBERT

Description: Introduction Germline mutations in GATA2 were recently identified as causative for several overlapping syndromes: MonoMAC (monocytopenia, mycobacterial infections), DCML (dendritic cells, monocytes, B and NK cells deficiency), Emberger syndrome (lymphedema, sensorineural deafness, multiple warts) and familiar myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Of note, GATA2 mutations were also found in children and young adults with “primary” MDS. Aplastic anemia (AA) constitutes an important differential diagnosis to pediatric MDS, particularly in patients with normal cytogenetics. Because of heterogeneous phenotype of GATA2 mutated patients, defining a set of typical findings would help in their earlier identification and understanding the natural course of the disease. Therefore we aimed to analyze monocytes and lymphocyte subpopulations with the emphasis on B cell lineage by flow cytometry (FC) and polymerase chain reaction (PCR) in all pediatric patients with GATA2 mutation diagnosed in the Czech Republic. Patients and methods Eleven pediatric patients were found to harbor GATA2 mutations in the Czech Republic so far. Three mutations were intronic. There was a clear male predominance (9/11). In 7 patients the disease manifested with MDS in childhood, 2 female patients were followed for immunodeficiency and developed MDS in adulthood. One another patient was diagnosed with interstitial lung disease and chronic EBV infection. His brother, carrying the same mutation, has mild neutropenia. Bone marrow (BM) and peripheral blood (PB) samples were analyzed by FC. The level of intronRSS-Kde recombination excision circles (KREC) and T-cell receptor excision circles (TREC) for assessment of proliferation history of B and T cells was examined by PCR. The control group comprised 26 GATA2 wild-type MDS (“other MDS”) patients and 36 AA patients. Results Disturbance of B cell compartment was the most frequently observed anomaly in the patients with GATA2 mutation. We observed a decrease of absolute and relative B cell numbers in PB and BM (n=9/11). In BM there was a decrease of immature CD10pos B cells (n=10) with proportional increase of plasma cells. Peripheral blood B cell immunophenotype was shifted towards memory B cells (n=5/7). Presence of normal B cell precursors CD19pos10pos34pos in BM was observed only in 1 patient in part of follow-up samples. Atypical malignant B lymphoblasts were present in another patient, whose MDS quickly progressed to AML with a clear switch to B lymphoid phenotype. Despite significantly reduced number of B cells the levels of IgG were normal in majority of patients. Only 2 patients had IgG hypogammaglobuliemia, in one patient with chronic active EBV infection IgG hypergammaglobulinemia was present. Slightly decreased IgA level was present in 6 patients. Although B cell numbers in other MDS control patients were significantly lower compared to AA, still the decrease was less prominent in comparison with GATA2. The decrease of immature and naive B cells in patients with GATA2 mutation was reflected in very low level of KREC in PB and BM. Stored newborn dry blood spots from 4 patients were evaluated for TREC and KREC numbers. Strikingly, only one patient had negative KREC levels (the youngest patient from our cohort with MDS diagnosed at age 4). The remaining 3 patients had normal TREC and KREC levels at birth. Thus, the deterioration of de novo production of B cells occurred supposedly postnatally in most patients. Low KREC levels were also present in some patients with other MDS (n=5). Relative monocytopenia was found in 2 patients, low NK cells were present in 6 patients. T cells were mostly of naive non-activated phenotype. Conclusions Changes in B cell compartment are the most characteristic feature in patients with GATA2 mutation. Decreased number of B cells together with a shift towards mature phenotype and decreased level of KREC reflect history of substantial B cell proliferation in an environment of impaired production. This process appears to happen postnatally and resemble normal ageing process, which is accelerated due to progenitor cell impairment. Immunophenotyping is a useful tool in identifying patients for GATA2 sequencing. Supported by GAUK 802214, IGA NT/14534-3, NT/13462-4, UNCE 204012, GAČR P301/10/1877 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2104 Mutations inactivating the divalent metal transporter 1 (DMT1) cause impaired erythroid iron utilization and lead to the development of hypochromic microcytic anemia associated with ineffective erythropoiesis (IE). The anemia can be ameliorated with high-dose erythropoietin (EPO) therapy (Pospisilova D, et al. Blood. 2006. 108:404–5). In contrast to β-thalassemia mouse model with dramatically elevated EPO levels which were proposed to protect erythroid cells from apoptosis (Libani IV, et al. Blood. 2008. 112:875–885), DMT1-mutant mice (mk/mk) have only 2.8-fold higher EPO levels when compared to the wild-type littermates. This corresponds to 2-fold elevation of serum EPO above the normal range for DMT-1-mutant patient before initiation of EPO therapy. Different mechanisms may therefore drive IE in anemia due to DMT1 mutation. In this study we analyzed the bases for the clinical success of high-dose EPO supplementation in a DMT1-mutant patient and mk/mk mice. EPO administration significantly increased hemoglobin levels (7.4 g/dL to 9.1 g/dL for the patient and 7.5±0.6 to 9.5±0.4 g/dL for mk/mk mice) and partially ameliorated IE. Colony forming assay using patient's cells showed significantly improved in vitro growth of post-treatment DMT1-mutant burst-forming unit erythroid (BFU-E) progenitors when compared to pre-treatment BFU-Es. In addition, the reduced plating efficiency and colony-forming capacity of pre-treatment DMT1-mutant BFU-Es can be corrected by the addition of the broad spectrum caspase inhibitor z-VAD-fmk to the cultures. This indicates involvement of caspase-dependent apoptosis in the defective survival of pre-treatment BFU-E progenitors and in their impaired capacity to form erythroid colonies. TUNEL assay on patient's bone marrow smears showed markedly decreased rate of apoptosis (from 4% to 1.5% of TUNEL-positive erythroblasts) after EPO supplementation. No profound changes in erythroblast maturation were noted in post-treatment bone marrow with the exception of additional expansion of polychromatophilic pool suggesting that inhibition of apoptosis rather than increased differentiation of DMT1-mutant erythroid cells predominantly accounts for amelioration of anemia and IE. In accordance with the patient's results, EPO administration to mk/mk mice did not alter the distribution of erythroblasts of different maturation stages. On the other hand, augmented STAT5 activation and enhanced expression of anti-apoptotic proteins BCL-XL and MCL-1 was detected in EPO treated mice. This correlated with decreased number of erythroid Ter119+ precursors undergoing apoptosis in EPO treated mk/mk bone marrow (12.4±2.3% to 5.4±0.9%) and spleen (7.3±0.7% to 3.1±0.9%). EPO supplementation also significantly reduced susceptibility of mk/mk erythrocytes to undergo stress-induced death that could reflect increased eryptosis (apoptosis of DMT1-mutant erythrocytes) in vivo and protective effect of EPO. Low to undetectable expression of hepcidin in mk/mk liver could be attributed to 16-fold increase in GDF15 expression in the bone marrow; the expression of TWSG1 was comparable to wild-type littermates. Also patient's urinary hepcidin is low (55.3 ng/mg creatinine; normal range 71–1762), however, in contrast to mk/mk mice and β-thalassemia patients the suppression of hepcidin seems to be only partly mediated by GDF15 as patient's GDF15 plasma levels are only 1.9-fold higher (548.4 pg/mL) in comparison to gender- and age-matched controls (288.4±56.9 pg/mL). These results indicate that mouse models may not fully mimic the human disease and suggest existence of additional bone marrow-derived regulator of hepcidin expression. In summary we present the bases for the clinically approved success of EPO treatment under condition of iron-deprived erythropoiesis. We conclude that EPO-driven signaling rescues the survival defect of DMT1-mutant erythroid cells. Grant support: Czech Grant Agency, grants No. P305/10/P210 and P305/11/1745; Internal Grant of Palacky University Olomouc (LF_2011_011), and Ministry of Health Czech Republic Grant NS10281-3/2009. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5307 Background: Thalassemias are rare disorders in Middle Europe. However, as a result of historical and recent migration, thalassemias became common cause of congenital anemia in the Czech and Slovak populations. Abnormal hemoglobin variants and red-cell enzymopathies are rare cause of congenital anemia in this region. The aim of this work was to update the original reports of this research published almost two decades ago (Indrak et al., Hum Genet 1992; 88:399–404, Xu et al., Blood 1995; 85:257–63, Lenzner et al., Blood 1997; 89:1793–9). We assessed the frequency and spectrum of β-globin gene mutations in the patients with clinical symptoms of β-thalassemia or δ,β-thalassemia, the α-globin gene status in the patients with clinical symptoms of α-thalassemia, and we characterized red cell enzymopathies on molecular level in the Czech and Slovak populations. Patients and methods: Nearly 390 cases with clinical symptoms of thalassemia or hereditary nonspherocytic hemolytic anemia from several centers of Czech and Slovak Republic were analyzed. Hematological parameters, hemoglobin electrophoresis and enzyme activities were measured by standard procedures. Genomic DNA was used for PCR-sequencing analysis. Results: We identified 22 β-thalassemia mutations in more than 260 heterozygotes; most of the mutations were of Mediterranean origin. The newly discovered insertion of transposable element L1 into the HBB gene represents a novel etiology of β-thalassemia due to a silencing effect of repressive chromatin associated with retrotransposon insertion. The list of abnormal hemoglobins now contains 14 β-globin variants, involving Heinz body hemolytic anemia variant Hb Hana (β63(E7) His-Asn), phenotype of which was worsened by concomitant partial glutathione reductase deficiency (Mojzikova et al., Blood Cells Mol Dis 2010; 45:219–22). Several G6PD and PK variants were described in the Czech and Slovak populations; the G6PD variants include G6PD Olomouc, G6PD Varnsdorf and G6PD Praha. Recently, we identified a new frameshift mutation c. 1553delG (p. Arg518fs) at the homozygous state in exon 11 of the PKLR gene of the pediatric patient who suffered from transfusion dependent hemolytic anemia with Hb=9.4 g/dL, Ret=4.5%. His red cells PK activity was 4.52 IU/gHb (normal range 13–17 IU/gHb). The mutation occurs in C domain of PK-R subunit containing the binding site for fructose-1,6-bisphosphate. The patient's extremely elevated level of growth differentiation factor 15 (GDF15, 3577 pg/mL, healthy controls 231–345 pg/mL) could explain hereditary hemochromatosis and signs of iron overload in this patient. Conclusions: In the Czech and Slovak populations, hemoglobinopathies and red-cell enzymopathies appear to be an uncommon disorder, which, however, must be considered as the prevailing cause of congenital anemia. Most of the thalassemia patients were heterozygous, manifesting thalassemia minor. Most of the hemoglobin variants were described in single families, some of them originated locally. Among hemolytic anemias due to red-cell enzymopathies is the most frequent PK deficiency. This work was supported by grants NT11208, NS10281 (Ministry of Health Czech Republic), MSM6198959205 (Ministry of Education, Youth and Sports) and student projects LF_2011_006 and LF_2011_011 of the Palacky University. Disclosures: No relevant conflicts of interest to declare.

Description: Background: RPL35A, a gene encoding a large ribosomal subunit protein located at the telomeric end of chromosome 3q (3q29-qter) is essential for rRNA processing, ribosomal biogenesis, cell proliferation, and apoptosis, and accounts for a subset of patients with Diamond Blackfan anemia (DBA). Reported pathogenic RPL35A mutations include single-nucleotide variants (SNVs), small insertion/deletions (indels), and large contiguous gene deletions associated with 3q29 microdeletion syndrome. 3q29 deletion syndrome is an overlapping syndrome that consists of developmental and intellectual disability with or without dysmorphic features, and other congenital anomalies but no anemia or cytopenia. The clinical phenotype and disease severity of patients with RPL35A-related DBA may be influenced by other genes deleted within 3q29 and be different in patients with large deletions compared to those with SNVs or small indels. Objectives: To determine whether DBA patients with large deletions of the 3q29 region have a more severe disease phenotype than those with SNVs or small indels in RPL35A, and whether other genes deleted within the 3q29 region might contribute to some of the features. Methods and Results: We identified 40 patients in a multi-institutional, international collaborative study of patients with DBA with RPL35A haploinsufficiency: 21 had deletion of RPL35A as part of 3q29 contiguous gene deletion, ranging in size from 0.012 Mb to 11 Mb in 16 patients; the extent of the deletion beyond RPL35A in either direction was unknown in 5 patients. Nineteen patients had SNVs or small indels (7 missense, 1 nonsense, 3 splice site, 6 indels and 2 unclear pathogenicity). Thirty-nine of 40 patients had severe anemia, 32 had neutropenia at some time and 3 had thrombocytopenia. Compared to the patients with SNVs or small indels, a significantly higher proportion of patients with RPL35A haploinsufficiency due to 3q29 deletion had steroid-resistant anemia (17 vs 7; p=0.009), severe chronic or intermittent neutropenia requiring treatment with G-CSF (7 vs 0; p=0.009), and humoral and/or cellular immunodeficiency (7 vs 1; p=0.046) diagnosed in some patients due to recurrent infections requiring hospitalizations (10 vs 2; p=0.03). Learning difficulties (12 vs 2; p=0.003), craniofacial abnormalities (11 vs 3; p=0.02), skeletal and limb defects (9 vs 2; p=0.03) or multiple physical anomalies (≥3) were also more frequent in patients with large deletions than in those with SNVs or indels (11 vs 3; p=0.02). Microcephaly (28%), short stature (33%), cardiac defects (28%) and/or urogenital abnormalities (23%) were equally distributed. The potential genes of interest near RPL35A that may be associated with immune dysregulation and/or neutropenia are RNF168, TFRC, PAK2, PIGZ, DLG1 and LMLN. One or more of these genes were deleted in at least 6 of 7 patients with neutropenia or immunodeficiency. Eight of 9 patients with malformations involving extremities, skeleton and ribs had deletions of TCTEX1D2 which is associated with rib/thoracic dysplasia and polydactyly. The genes of interest deleted in patients with developmental delay and learning disabilities included PAK2 and DLG1 in 9 patients, as well as RNF168, PPP1R2, TNK2 and q29 KIAA0226 in 8 of 12 patients in whom the extent of the deletion was known. Summary and Conclusion: Patients with DBA due to RPL35A contiguous gene deletions are clearly different from those with SNVs or small indels and have increased frequency of steroid-resistant transfusion-dependent anemia, severe neutropenia, immunodeficiency, learning/developmental delay, and craniofacial/skeletal/limb anomalies. Distinction of this subtype of DBA with RPL35A haploinsufficiency due to 3q29 contiguous gene deletion is relevant to their management, and evaluations should include a work-up for immunodeficiency. Further studies are needed to determine whether the complex phenotypes and severe disease manifestations in these patients are solely due to RPL35A haploinsufficiency or to the potential effect of other genes deleted in the 3q29 region. Genotype-phenotype characterization and comparison of DBA patients with 3q29 deletion with those due to deletions in other ribosomal protein genes may determine similarities or differences in disease phenotypes related to large gene deletions versus the influence of multigenic contiguous deletions. Supported by: AZV 16-32105A Disclosures Kattamis: Novartis: Consultancy, Honoraria; CELGENE: Consultancy, Honoraria; ApoPharma: Honoraria; Vifor Pharma: Consultancy. Niemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: Key Points PK deficiency manifests a broad spectrum in anemia severity that moderately improves after splenectomy. Close attention to monitoring for iron overload, gallstones, and other complications is recommended in all patients with PK deficiency.

Description: Introduction: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by macrocytic anemia, reticulocytopenia, selective deficiency of erythroid precursors, presence of congenital anomalies and an increased risk of cancer. DBA is caused by germline mutations of genes coding for ribosomal proteins (RP). Interestingly, somatic RP mutations have also been found in several malignant diseases (T-ALL, CLL, Hodgkin lymphoma, myelodysplastic syndrome (MDS) and AML). The only representative overview of malignancies in DBA was published in 2012 based on the data from the North American DBA Registry. The malignancies developed in 3% of patients (MDS and AML in 4 patients and solid tumors in 15 patients). The observed-to-expected ratio for all cancers combined was 5.4 (p

Description: Background and aim: Immune thrombocytopenia (ITP) is the most common bleeding condition in children. Its prognosis is mostly superior, however, severe refractory disease remains diagnostic and therapeutic challenge. Low platelet counts (

Description: Introduction: Pyruvate kinase (PK) deficiency causes a defect in the glycolytic pathway, leading to a hereditary hemolytic anemia. Management is supportive and consists of splenectomy, transfusions, and chelation therapy. Aim: To better understand the comorbidity and complication profile of adults with PK deficiency, and the extent to which transfusion frequency contributes, the objectives of this study were to (1) quantify the prevalence of comorbidities and complications according to transfusion history and (2) compare the types and rates of select comorbidities and complications with the general population. Methods: Data were obtained from the enrollment survey of the PK Deficiency Natural History Study (NHS), a longitudinal, retrospective and prospective cohort study in which clinical, laboratory, transfusion, and radiologic data were collected; all participants were confirmed to have 2 mutations in the PKLR gene. Patients (n=122) were eligible for this analysis if they were ≥18 years of age and had sufficient data on transfusion history to enable classification into 1 of 3 cohorts: "Ever Regularly Transfused" (ERT, defined as ≥6 transfusions in any 12-month period), "Never Regularly Transfused" (NRT, defined as having ≥1 lifetime transfusion but never having 〉4 transfusions in any 12-month period), or "Never Transfused" (NT). To contextualize the findings, the frequencies of select conditions were compared with an age- and gender-matched cohort of individuals from the insured, general US population who did not have any hemolytic anemia diagnoses and had ≥5 years of continuous enrollment in the Truven MarketScan administrative claims database. The NHS reported lifetime prevalence rates, whereas rates obtained from the MarketScan data were based on diagnosis and procedure codes over varying look-back periods; therefore, to minimize bias, we limited PK deficiency vs. general population comparisons to (1) chronic conditions that require lifetime management and would thus still be recorded in claims data years after initial diagnosis, and/or (2) conditions for which a diagnosis/procedure date was available in the NHS and could be matched in time to the average 8-year look-back period for the general population. Frequencies were compared across mutually exclusive cohorts using Fisher's exact 2-tailed tests of significance. Results: ERT (n=65), NRT (n=30), and NT patients (n=27) had a mean age of 34.2, 39.5, and 37.2 years at enrollment, respectively (not significant [ns]), with 46.2%, 56.7%, and 59.3%, respectively, being male (ns). ERT patients trended toward being more likely than NT patients to be Amish and have the homozygous R479H splice variant (30.8% vs 11.1% [p=0.064]) but were significantly less likely to have a missense/missense PKLR genotype (32.3% vs 70.4% [p=0.001]). Compared with the general population, patients with PK deficiency had significantly higher rates of splenectomy, cholecystectomy, osteoporosis, liver cirrhosis, pulmonary hypertension, and current prophylactic antibiotic and anticoagulant use (Table). Rates of splenectomy, cholecystectomy, and osteoporosis were significantly higher in patients with PK deficiency, regardless of transfusion cohort, and both ERT and NRT patients had significantly higher rates of liver cirrhosis than individuals from the general population. A gradient was seen across transfusion cohorts for other conditions. Notably, 83.1% of ERT patients, 50.0% of NRT patients, and 25.9% of NT patients had a history of liver iron overload. ERT patients were also significantly more likely than NRT and NT patients to have had a splenectomy, cholecystectomy, and/or thrombosis, and to currently use prophylactic antibiotics. Findings were consistent when the analysis was restricted to non-Amish patients with PK deficiency. Conclusions: Patients with PK deficiency have higher rates of select comorbidities and complications than age- and gender-matched individuals who do not have PK deficiency. Even patients with PK deficiency who have never been transfused are at increased risk of complications of the disease and its treatment. Disclosures Boscoe: Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Yan:Agios Pharmaceuticals, Inc.: Consultancy. Hedgeman:IBM Watson Health: Employment. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. Al-Samkari:Agios: Consultancy, Research Funding; Dova: Consultancy, Research Funding; Moderna: Consultancy. Barcellini:Incyte: Consultancy; Alexion: Consultancy, Speakers Bureau; Agios Pharmaceuticals, Inc.: Consultancy; Novartis: Speakers Bureau; Apellis: Consultancy; bioverativ: Consultancy. Eber:Agios Pharmaceuticals, Inc.: Consultancy. Glader:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chonat:Alexion: Other: advisory board; Agios Pharmaceuticals, Inc.: Other: advisory board. Rothman:Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Kuo:Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy. Kwiatkowski:Agios: Consultancy; bluebird bio, Inc.: Consultancy, Research Funding; Imara: Consultancy; Apopharma: Research Funding; Novartis: Research Funding; Celgene: Consultancy; Terumo: Research Funding. Ravindranath:Agios Pharmaceuticals, Inc.: Other: I am site PI on several Agios-sponsored studies, Research Funding. Neufeld:Octapharma, Shire Pharmaceuticals (Baxalta), Novo Nordisk, Celgene, NHLBI/NIH: Research Funding; Octapharma, Agios, Acceleron, Grifols, Pfizer, CSL Behring, Shire Pharmaceuticals (Baxalta), Novo Nordisk, ApoPharma, Genentech, Novartis, Bayer Healthcare: Consultancy; Octapharma: Other: study investigator, NuProtect study (Octapharma-sponsored). Holzhauer:Agios Pharmaceuticals, Inc.: Consultancy. Verhovsek:Sickle Cell Disease Association of Canada: Membership on an entity's Board of Directors or advisory committees, Research Funding; Canadian Haemoglobinopathy Association: Membership on an entity's Board of Directors or advisory committees; Vertex: Consultancy; Sickle Cell Awareness Group of Ontario: Membership on an entity's Board of Directors or advisory committees. Kunz:Novartis: Membership on an entity's Board of Directors or advisory committees. Sheth:Apopharma: Other: Clinical trial DSMB; Celgene: Consultancy; CRSPR/Vertex: Other: Clinical Trial Steering committee. Despotovic:Novartis: Research Funding; Dova: Honoraria. Grace:Agios Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

Description: Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site–mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.