Diamond Blackfan Anemia Registry Research Articles

GATA-1 Defects in Diamond-Blackfan Anemia: Phenotypic Characterization Points to a Specific Subset of Disease.

Diamond–Blackfan anemia (DBA) is one of the inherited bone marrow failure syndromes marked by erythroid hypoplasia. Underlying variants in ribosomal protein (RP) genes account for 80% of cases, thereby classifying DBA as a ribosomopathy. In addition to RP genes, extremely rare variants in non-RP genes, including GATA1, the master transcription factor in erythropoiesis, have been reported in recent years in patients with a DBA-like phenotype. Subsequently, a pivotal role for GATA-1 in DBA pathophysiology was established by studies showing the impaired translation of GATA1 mRNA downstream of the RP haploinsufficiency. Here, we report on a patient from the Dutch DBA registry, in which we found a novel hemizygous variant in GATA1 (c.220+2T>C), and an Iranian patient with a previously reported variant in the initiation codon of GATA1 (c.2T>C). Although clinical features were concordant with DBA, the bone marrow morphology in both patients was not typical for DBA, showing moderate erythropoietic activity with signs of dyserythropoiesis and dysmegakaryopoiesis. This motivated us to re-evaluate the clinical characteristics of previously reported cases, which resulted in the comprehensive characterization of 18 patients with an inherited GATA-1 defect in exon 2 that is presented in this case-series. In addition, we re-investigated the bone marrow aspirate of one of the previously published cases. Altogether, our observations suggest that DBA caused by GATA1 defects is characterized by distinct phenotypic characteristics, including dyserythropoiesis and dysmegakaryopoiesis, and therefore represents a distinct phenotype within the DBA disease spectrum, which might need specific clinical management.

1011 – INVESTIGATION OF HUMAN BONE MARROW PROGENITORS ELUCIDATES CELLULAR AND MOLECULAR MECHANISMS OF ERYTHROID FAILURE IN DIAMOND-BLACKFAN ANEMIA

Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome caused by monoallelic loss-of-function mutations or deletions in small (RPS) or large (RPL) ribosomal protein genes. The molecular mechanisms that underpin differences in clinical phenotypes are yet to be elucidated and therapeutic options are limited. By combining flow-cytometry, cell-sorting and single-cell clonogenic assays of human bone marrow from healthy donors, we identified the immunophenotypic markers that define early and late erythroid progenitors; the cells that give rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies respectively. We then applied these definitions to bone marrow derived from children with DBA and elucidated two distinct cellular trajectories segregating with RP genotype: almost complete loss of erythroid lineage specification in RPS-DBA versus relative preservation of qualitatively abnormal erythroid progenitors in RPL-DBA. Single-cell studies of DBA hematopoietic stem and progenitor cells showed that this difference is underpinned by erythroid differentiation arrest and reduced GATA1 activity in RPS-DBA versus preserved GATA1 expression in RPL-DBA. We identified a pro-inflammatory milieu in DBA bone marrow accompanied by P53 activation. The compensatory stress erythropoiesis detected was deficient in its hallmark endogenous glucocorticoid-regulated transcriptional program, leading to disordered differentiation in RPL-DBA. Finally, analysis of patients in the UK DBA registry (n=106) showed that RPL genotype is an independent predictor of older age at presentation with anemia and more frequent and durable responses to corticosteroids. In summary, we present unbiased charting at single-cell resolution of erythropoiesis in DBA bone marrow. Our data categorize developmental trajectories and in turn define genotype-phenotype correlations to facilitate precision-based therapeutic approaches in a rare heritable disease. Ribosome dysfunction underlies the pathogenesis of many cancers and heritable ribosomopathies. Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome caused by monoallelic loss-of-function mutations or deletions in small (RPS) or large (RPL) ribosomal protein genes. The molecular mechanisms that underpin differences in clinical phenotypes are yet to be elucidated and therapeutic options are limited. By combining flow-cytometry, cell-sorting and single-cell clonogenic assays of human bone marrow from healthy donors, we identified the immunophenotypic markers that define early and late erythroid progenitors; the cells that give rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies respectively. We then applied these definitions to bone marrow derived from children with DBA and elucidated two distinct cellular trajectories segregating with RP genotype: almost complete loss of erythroid lineage specification in RPS-DBA versus relative preservation of qualitatively abnormal erythroid progenitors in RPL-DBA. Single-cell studies of DBA hematopoietic stem and progenitor cells showed that this difference is underpinned by erythroid differentiation arrest and reduced GATA1 activity in RPS-DBA versus preserved GATA1 expression in RPL-DBA. We identified a pro-inflammatory milieu in DBA bone marrow accompanied by P53 activation. The compensatory stress erythropoiesis detected was deficient in its hallmark endogenous glucocorticoid-regulated transcriptional program, leading to disordered differentiation in RPL-DBA. Finally, analysis of patients in the UK DBA registry (n=106) showed that RPL genotype is an independent predictor of older age at presentation with anemia and more frequent and durable responses to corticosteroids. In summary, we present unbiased charting at single-cell resolution of erythropoiesis in DBA bone marrow. Our data categorize developmental trajectories and in turn define genotype-phenotype correlations to facilitate precision-based therapeutic approaches in a rare heritable disease.

Early Onset Colorectal Cancer: An Emerging Cancer Risk in Patients with Diamond Blackfan Anemia.

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome, the founding member of a class of disorders known as ribosomopathies. Most cases result from loss of function mutations or deletions in 1 of 23 genes encoding either a small or large subunit-associated ribosomal protein (RP), resulting in RP haploinsufficiency. DBA is characterized by red cell hypoplasia or aplasia, poor linear growth and congenital anomalies. Small case series and case reports demonstrate DBA to be a cancer predisposition syndrome. Recent analyses from the Diamond Blackfan Anemia Registry of North America (DBAR) have quantified the cancer risk in DBA. These studies reveal the most prevalent solid tumor, presenting in young adults and in children and adolescents, to be colorectal cancer (CRC) and osteogenic sarcoma, respectively. Of concern is that these cancers are typically detected at an advanced stage in patients who, because of their constitutional bone marrow failure, may not tolerate full-dose chemotherapy. Thus, the inability to provide optimal therapy contributes to poor outcomes. CRC screening in individuals over the age of 50 years, and now 45 years, has led to early detection and significant improvements in outcomes for non-DBA patients with CRC. These screening and surveillance strategies have been adapted to detect familial early onset CRC. With the recognition of DBA as a moderately penetrant cancer risk syndrome a rational screening and surveillance strategy will be implemented. The downstream molecular events, resulting from RP haploinsufficiency and leading to cancer, are the subject of significant scientific inquiry.

Colorectal cancer screening and surveillance strategy for patients with Diamond Blackfan anemia: Preliminary recommendations from the Diamond Blackfan Anemia Registry.

Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by red cell failure, congenital anomalies, poor linear growth, and cancer predisposition. Two previous analyses from the Diamond Blackfan Anemia Registry have quantified DBA as a cancer predisposition syndrome of moderate cancer penetrance. Patients with DBA have a 4.8-fold higher relative risk of developing cancer with an overall cumulative incidence of 13.7% by age 45 years. The two most prevalent solid tumors are colorectal cancer (CRC) and osteogenic sarcoma. Current and evolving data support the institution of cancer screening and surveillance strategies for CRC in DBA.

Single-Cell Transcriptional Landscapes of Human Bone Marrow Reveal Distinct Erythroid Phenotypes Underpinned By Genotype in Diamond-Blackfan Anemia

Background: Diamond- Blackfan Anemia (DBA) is a rare, heritable ribosomopathy caused by mutations in ribosomal protein large (RPL) and small (RPS) subunit genes. The diagnostic criteria of DBA include presentation in infancy with virtually no mature erythroblasts (EB) on bone marrow (BM) examination, however atypical presentations in later life with milder haematological manifestations are increasingly reported. The cellular and molecular mechanisms underpinning variable clinical phenotypes, and how they relate to genotype, are yet to be elucidated. Furthermore, as many DBA patients do not respond to corticosteroids, understanding the pathological processes contributing to erythroid failure is a prerequisite for developing novel, precision-medicine therapies. Aim: To elucidate phenotypic and functional differences in erythropoiesis in RPS- and RPL-DBA, using primary BM samples from patients. Methods: We performed single-cell transcriptome profiling (scRNAseq), using the 10X Genomics chromium platform, of 45,488 CD34+ Lineage negative (Lin-) BM hematopoietic stem and progenitor cells (HSPC), purified by fluorescence-activated cell sorting. We included six patients with red cell transfusion-dependent DBA (aged 2-19y) with mutations in RPS19 (n=3), RPL11 (n=1) and RPL5 (n=2) and three healthy donors (aged 3-17y). We validated our findings using bulk RNAseq, functional assays, and deep immunophenotyping-based dissection of the haematopoietic architecture of DBA BM ex vivo. Results: High quality sequencing data was obtained for all nine donors; after quality control, 41,415 single cells were carried forward for analysis. Unsupervised clustering analysis and lineage identification revealed two divergent cellular patterns in DBA compared with age-matched control BM that segregated with genotype: a selective loss of erythromegakaryocyte (E/MK) progenitors in RPS-DBA, but relative preservation of the erythroid developmental trajectory in RPL-DBA, at the expense of megakaryopoiesis (Fig 1a). Gene Set Enrichment Analysis (GSEA) of differentially expressed genes between control and DBA HSPC clusters revealed p53-mediated apoptosis, TNFa-, IFNa- and IFNg-mediated inflammatory pathways in DBA EP. Although these pathways were enriched across all HSPC populations irrespective of genotype, they were detected at an earlier stage of the stem cell hierarchy, and more potently, in RPS- versus RPL-EP. Expression of transcriptional targets of the master E/MK transcription factor, GATA1, was significantly upregulated in RPL- versus RPS-DBA EP (Fig 1b), supporting the maintained erythroid program in RPL-DBA HSPC. However, expression of genes denoting erythroid differentiation, including adult haemoglobin (Hb) genes, was significantly elevated in RPL-DBA, suggesting aberrant accelerated differentiation to EB. These findings were confirmed by immunophenotypic examination of patient BM and bulk RNAseq of RPL-EB. Additionally, single-cell clonogenic assays of RPL-DBA EP showed severe qualitative defects. Thus, although erythroid commitment occurs in RPL-DBA, it is coupled with accelerated maturation beyond EP to functionally impaired mature EB, enriched in inflammatory and p53 pathways. Finally, we analysed the clinical characteristics of the 161 patients comprising the U.K DBA registry. In line with the milder erythroid specification defect, patients with RPL-DBA (n=44) presented with anaemia later (P=0.004), and with a higher average Hb concentration (P=0.04), than those with RPS-DBA (n=63). Furthermore, we identified higher corticosteroid responses in RPL-DBA, assessed at 6 months post initiation (P=0.006), consistent with our findings in RPL-DBA of preservation of the cellular EP populations that are targeted by corticosteroids. Impact: In conclusion, a preserved but distinct erythroid developmental trajectory, characterised by accelerated differentiation, underpins a milder haematological phenotype in RPL-DBA. Furthermore, we reveal the first single-cell transcriptomic dataset from haematopoietic cells in a ribosomopathy, uncovering novel cell intrinsic and extrinsic pathogenetic insights into failing erythropoiesis in DBA. Integration of these data with clinical genomics and phenomics provides a paradigm by which single cell approaches can be used to decipher genotype-phenotype relationships in Mendelian genetic disorders. Disclosures Mead: Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau; Celgene/BMS: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding; Abbvie: Consultancy; CTI: Consultancy; Gilead: Consultancy.

Clonal Hematopoiesis in Patients with Diamond Blackfan Anemia

Background: Diamond Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome characterized by anemia, congenital anomalies and a predisposition to cancer. Patients usually present during infancy or early childhood, but can also be diagnosed as adults. In the vast majority of cases DBA is due to a mutation in a gene encoding a small or large subunit-associated ribosomal protein (RP) leading to RP haploinsufficiency. In a study of 702 patients enrolled in the DBA Registry (DBAR), the observed to expected ratio for acute myeloid leukemia (AML) was 28.8 and for myelodysplastic syndrome (MDS), 352.1 (Vlachos et al, Blood, 2018). The average age of onset for MDS in the DBA cohort was 26 years, compared to 60-70 years in the general population. Evolving clonal hematopoiesis (CH) with age has been observed as a precursor to MDS, with CH rarely observed in individuals younger than 40 years of age. Thus we hypothesized that the young age at the development of MDS in DBA would be presaged by evolving CH. Objective: The primary objective was to perform whole exome sequencing (WES) specifically screening for previously reported somatic mutations in 56 genes associated with CH (Jaiswal et al, NEJM, 2014). Design/Method: A total of 69 samples were analyzed from 65 patients, mostly targeting patients older than 18 years (median age 30 years). Multiple samples were run on patients who had available samples in the DBAR Biorepository to determine rate of acquisition of mutations. 468 age- and sex-matched healthy controls were made available from GeneDx who performed the WES for the study. We used a threshold for variant calling of minimum 5% with a minimum of 2 variant reads. Results: Three of the 65 DBA patients (5%) were found to have somatic mutations in STAG1, U2AF1, SF3B1, and DNMT3A at 8, 20, 41, and 70 years, respectively (Table 1). The patient who was 20 years of age had a sample in the DBAR biorepository from when he was age 8 years which was found to have a different somatic mutation (STAG1) than was found at present (U2AF1). This patient did go on to develop MDS at the age of 21 years. In comparison, of the 468 controls, 4 (0.8 %) had a somatic mutation in SF3B1, LUC7L2, DNMT3A, and LUC7L2 at ages 12, 31, 33 and 40 years, respectively. Conclusion: Patients with DBA show more somatic mutations as compared to controls (p<0.05). This early acquisition of mutations may be the driving force for their developing MDS at an earlier age than that of the general population. Further studies with more sensitive methods are warranted to accurately determine the prevalence of somatic CH mutations and their potential association with the development of myelodysplastic syndrome in these patients. Disclosures No relevant conflicts of interest to declare.

Malignancies and MDS in Patients with Diamond-Blackfan Anemia (DBA) from the Czech National DBA Registry

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<0.05). Median age at diagnosis was 41 years and cumulative incidence of solid tumors/leukemias was approximately 20% at the age of 46 years. Five years later, additional solid tumors were reported, in particular gastrointestinal tumors, and an incidence of MDS was alarming. Our aim was to evaluate cancer incidence within Czech National DBA Registry and characterize underlying molecular pathology. Patients and methods: Czech National DBA Registry currently includes 62 patients. In cooperation with 8 national centers of pediatric and adult hematology, we collected data about patients followed with pre-malignant or malignant condition including a detailed analysis of the course of the disease, treatment and type of developed malignant disorder. In patients with an evolving MDS, a classical cytogenetic analysis, flow cytometry, mutational profile (TruSight Myeloid Sequencing Panel (Illumina) containing 54 genes) and commercial TUNEL assay for detection of apoptotic changes of erythroid cells in the bone marrow were performed. Results: Eight of 62 patients from the Registry (13%) had malignant or pre-malignant condition: four females (6.5%) had solid tumors, 3 males (4.8%) had MDS and one female (1.6%) had multiple myeloma. Age of the onset of these disorders ranged between 25-70 years. Three patients harbored RPL5 mutation, 3 patients RPL11 mutation and 2 patients RPS19 mutation. Cancer incidence was significantly higher within the RPL11 and RPL5 subgroups (p=0.0056, Fisher Exact Test). Two female patients were diagnosed with triple-negative breast carcinoma, both of them during pregnancy. The first patient died despite treatment at the age of 29 years, shortly after delivery of her second child. The second patient is currently undergoing neoadjuvant chemotherapy.One patient developed diffuse large B cell lymphoma (DLBCL), underwent chemotherapy and autologous BMT and is alive in second remission.One patient underwent hemicolectomy for colorectal adenocarcinoma at 53 years of age and is in remission 6 years after the surgery.One patient succumbed to multiple myeloma which evolved from monoclonal gammopathy of unknown significance (MGUS) after 14 years of follow-up.Three male patients developed suspected MDS at the age of 25, 28 and 29 years. Two of them had RPL5 mutation and one harbored RPS19 mutation. None of them had decrease of erythroid cells in the bone marrow, but apoptosis of erythroid progenitors was significantly increased in all cases. All 3 patients had bicytopenia in peripheral blood (anemia, leukopenia) and dysplasia in 2 or 3 hematopoietic lineages in the bone marrow, thus fulfilling criteria for MDS with multilineage dysplasia (MDS-MLD). They had no abnormalities detected by flow cytometry or cytogenetics. The patient with RPS19mut harbored ASXL1 mutation; in patient with RPL5 mut, mutational screening is ongoing. Conclusion: Our results confirmed increased incidence of cancer (13%) in patients with DBA at young age. Our cases of DLBCL and MGUS in DBA are the first published to date. In cases with suspected MDS, cytopenia and dysplastic changes in the bone marrow may either reflect specific features of ribosomopathy, or represent a severe disorder of regulatory mechanisms with propensity to clonal proliferation. International collaboration is required to refine the incidence of malignancies in DBA and to issue consensus guidelines for timely detection of solid tumors, leukemias and MDS. The best approach to DBA patients who developed MDS without harboring any mutation considered prognostically significant in MDS, is another subject of discussion. Support: NV16-32105A Disclosures No relevant conflicts of interest to declare.

Czech and Slovak Diamond-Blackfan Anemia (DBA) Registry update: Clinical data and novel causative genetic lesions

Diamond-Blackfan anemia (DBA) is a rare congenital erythroid aplasia, underlied by haploinsufficient mutations in genes coding for ribosomal proteins (RP) in approximately 70% of cases. DBA is frequently associated with somatic malformations, endocrine dysfunction and with an increased predisposition to cancer.Here we present clinical and genetic characteristics of 62 patients from 52 families enrolled in the Czech and Slovak DBA Registry. Whole exome sequencing (WES) and array comparative genomic hybridization (aCGH) were employed to identify causative mutations in newly diagnosed patients and in cases with previously unrecognized molecular pathology.RP mutation detection rate was 81% (50/62 patients). This included 8 novel point mutations and 4 large deletions encompassing some of the RP genes. Malignant or predisposing condition developed in 8/62 patients (13%): myelodysplastic syndrome in 3 patients; breast cancer in 2 patients; colorectal cancer plus ocular tumor, diffuse large B-cell lymphoma and multiple myeloma each in one case. These patients exclusively harbored RPL5, RPL11 or RPS19 mutations.Array CGH is beneficial for detection of novel mutations in DBA due to its capacity to detect larger chromosomal aberrations. Despite the importance of genotype-phenotype correlation in DBA, phenotypic differences among family members harboring an identical mutation were observed.

PF345 THE INVOLVEMENT OF OXIDATIVE STRESS, INFLAMMATION, AND DNA DAMAGE RESPONSE IN PATHOPHYSIOLOGY OF DIAMOND‐BLACKFAN ANEMIA

Background:Diamond‐Blackfan anemia (DBA) is a rare congenital bone marrow failure syndrome characterized by erythroid aplasia mostly caused by different pathogenic variants in ribosomal protein (RP) genes. The ribosomal stress and subsequent activation of the p53 tumor suppressor pathway result in increased apoptosis of erythroid precursors and/or induction of cell cycle arrest.Aims:We studied the contribution of p53 pathway, oxidative stress, and inflammation to the DBA pathophysiology.Methods:We analyzed the bone marrow and peripheral blood samples from DBA patients listed in the Czech DBA Registry. We also prepared the RPL5‐ and RPS19‐deficient murine erythroleukemia cells (MEL) by using CRISPR/Cas9 technology. Activation of p53 pathway, oxidative DNA damage, and pro‐inflammatory cytokines were assessed in undifferentiated and differentiated RPL5‐ and RPS19‐deficient cells; the differentiation was induced by dimethyl sulfoxide (DMSO) for at least 48 hours.Results:Created RP‐deficient MEL cells presented the main phenotypic features of DBA patients’ erythroid cells: decreased levels of RPL5 or RPS19 proteins, decreased proliferation capacity, increased apoptosis, and abnormal GATA1 expression (2‐times lower than in control cells) upon differentiation. The increased p53 activation was also confirmed in RPL5‐ and RPS19‐deficient cells consistent with increased p53 expression in the bone marrow of DBA patients (p53+ cells: 8.4% in RPL5‐ and 7.3% in RPS19‐mutant patient vs. 0.4% in normal controls). One of the known inducer of p53 is oxidative stress causing oxidative DNA damage. Indeed, elevated positivity for 8‐oxoguanine (8‐oxoG), a marker of oxidative DNA damage, was detected in differentiated RP‐deficient MEL clones and in DBA patients’ samples in contrast to control samples. It is known, that reactive oxygen species (ROS) are produced under inflammation resulting in ROS‐mediated DNA damage response (DDR). The upregulation of pro‐inflammatory cytokines TNF‐α, IL6, and IL1b was found in RPL5‐ and RPS19‐deficient cells and also in the serum from DBA patients along with the upregulation of other inflammatory cytokines (IL1a, IL4, IL10, IL12, IL17a, INF‐γ, and GM‐CSF). Moreover, elevated pro‐inflammatory cytokines play a role in cellular senescence which was confirmed in RP‐deficient MEL clones by significantly elevated β‐galactosidase activity. In addition, increased ROS levels were detected in all differentiated RPL5‐ and RPS19‐deficient cells and in the serum of all DBA patients with elevated inflammatory cytokines. Finally, the DNA double‐strand breaks can occur as an outcome of ROS production responding by phosphorylation of Ser‐139 residue of the histone variant H2AX (γ‐H2AX). Indeed, higher γ‐H2AX positivity was seen for RP‐deficient MEL clones in comparison with control cells (mean intensity of fluorescence for RPL5‐deficient cells: 2.63 ± 0.34; RPS19‐deficient cells: 3.29 ± 0.41; control cells: 1.02 ± 0.22).Summary/Conclusion:The defective ribosomal biogenesis is associated with abnormal GATA1 expression and augmented apoptosis of erythroid cells. Our results show that the upregulation of inflammatory cytokines, senescence, oxidative stress, increased DNA damage, and activation of DDR signaling are also playing an important role in DBA pathogenesis. Our results also suggest the possibility of using the anti‐oxidative and anti‐inflammation therapy in DBA patients as the additive treatment for moderating DBA phenotype.Grants support: AZV 16–32105A, IGA_LF_2019_006, and by LTAUSA17142 (BK,LL).

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.

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.

Myelodysplastic Syndrome and Gastrointestinal Carcinomas Characterize the Cancer Risk in Diamond Blackfan Anemia: A Report from the Diamond Blackfan Anemia Registry

▪Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome (IBMFS) characterized by red cell aplasia and congenital anomalies. In our 2012 report the predisposition to cancer was quantified for the first time by the DBA Registry of North America (DBAR), the largest established DBA patient cohort with prospective follow-up since 1991. Five years from the original analysis, this update of the cancer incidence in patients with DBA reveals additional solid tumors, in particular gastrointestinal tumors, and an alarming incidence of myelodysplastic syndrome (MDS).In this study we report the types, ages, outcomes and hazard rates in DBA patients with neoplasia enrolled in the DBAR. The patients/families and their physicians completed a detailed questionnaire. Information was verified through medical records and telephone interviews. The ratio of observed to expected (O/E) cancers was computed using SEER data, and the cumulative incidence and hazard rate of hematopoietic stem cell transplant (HSCT), acute myeloid leukemia (AML), solid tumors (ST), and MDS by age were calculated.There were 702 patients with 12,376 person-years of follow-up. Twenty-three had solid tumors, 3 had acute myeloid leukemia (AML), and 8 had MDS. The median age at diagnosis of a solid tumor for the patients who had not received a bone marrow transplant was 35 years (range, 11-63). There were 9 gastrointestinal carcinomas (7 colon, 1 gastroesophageal, and 1 esophageal), 4 osteogenic sarcoma, 2 breast cancer and 2 squamous cell carcinomas (vaginal and oral) and 9 others, with 3 patients having more than one cancer. Cancer incidence in DBA was significantly elevated with an observed-to-expected (O/E) ratio for all cancers combined of 4.75, (95% confidence interval 3.15-6.86); significant O/E ratios were 352 for MDS, 45 for colon carcinoma, excluding rectum, 42 for osteogenic sarcoma, and 29 for AML. Although the sample size is small it appears that the cancer risk by genotype correlates only with the relative incidence of that genotype in the DBAR cohort (RPS19, 7; RPL11, 2; RPL5, 2; RPL35a, 3; and RPS17, 1). By their mid-40s, 24% had received a HSCT, 23% had died, 2% had AML, and 12% had developed a ST; 61% had died or experienced a serious adverse event. The median overall survival was 51 years and the actuarial risk of MDS in the absence of any competing risks was 50% by age 30 years.Cancer risks in DBA appear to have reached those seen in dyskeratosis congenita (DC) and are still lower than in Fanconi anemia (FA). Furthermore, the type of solid tumors in DBA (gastrointestinal carcinoma and osteogenic sarcoma) vs. FA (head and neck squamous cell carcinoma) are quite different suggesting different mechanisms of carcinogenesis from nucleolar stress vs. genomic instability. Importantly the risk of MDS in DBA is elevated, as it is in FA and DC. This report also confirms the observation that there was no specific association of cancer type or frequency with genotype. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2457: Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma

Abstract Background: Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by red cell aplasia and congenital anomalies, notably skeletal defects. DBA is known in the majority of cases to be caused by haploinsufficiency of one of the small or large ribosomal protein (rp) subunits. In addition, there is a cancer predisposition in DBA. Indeed, somatic and germline rp mutations have been associated with a number of malignancies. In DBA patients one of the common malignancies is osteogenic sarcoma (OS). It is proposed that selective pressure caused by faulty translation may lead to clonal expansion of cells with acquired interdicting mutations leading to cancer. Purpose: To quantitate the incidence of cancer in patients with DBA and to examine osteoblast formation in vitro using a mouse embryonic stem (mES) cell model haploinsufficient for ribosomal protein Rps19 to identify putative bone defects that may promote tumorigenesis. Methods: The DBA Registry of North America (DBAR), the largest established patient cohort with prospective follow-up since 1991, was interrogated to ascertain the incidence of cancer in DBA. For in vitro studies, mES cells were differentiated towards osteoblasts using 1,25-dihydroxy-vitamin D3, dexamethasone, ascorbic acid and β-glycerophosphate for 10 days. qRT-PCR and histologic stains (alizarin red, alcian blue and alkaline phosphatase) were used to assess osteoblast production in wild type and Rps19+/- cultures. Results: The DBAR reported the first quantitative assessment of cancer incidence in DBA. The significantly elevated observed to expected ratios were 287, 36, 33 and 28 respectively, for myelodysplastic syndrome (MDS), colon carcinoma, OS and acute myeloid leukemia (AML). As of Nov 2015 (N = 751), with a median patient age of 20 years, 11 patients with gastrointestinal carcinoma, 5 with OS, 2 with AML and 8 with MDS are reported to the DBAR. The incidence of gastrointestinal carcinoma and OS is highly significant in this young population. We elected to study OS due to the skeletal defects and poor growth encountered in DBA patients. Using mES cells induced to differentiation towards osteoblasts, we observed reduced mineralization and therefore, bone formation, by alizarin red staining in Rps19+/- cells. Furthermore, rp haplo-insufficient cultures displayed a significant increase in alcian blue staining, a marker for chondrogenesis; these results suggest that regulation of osteochondrogenic potential was altered. In support of this phenotype, qRT-PCR analyses revealed that Rps19 haploinsufficiency induced abnormal expression of the master transcription factors and p53 involved in osteoblast and chondrocyte development. Conclusions: The most common cancers in DBA are AML, OS and adenocarcinoma of the colon. With regards to OS, we are now creating a conditional Rps19 CRISPR murine model, in order to clarify the contribution of both rp and p53 in the etiology of OS. Citation Format: Lionel Blanc, Brian Dulmovits, Julien Papoin, Jessica Kang, Eva Atsidaftos, Jeffrey M. Lipton, Adrianna Vlachos. Ribosomal protein haploinsufficiency in Diamond-Blackfan anemia potentially leads to osteogenic sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2457.

Transcriptome Analysis of Erythroid Cells Cultured from Diamond Blackfan Anemia Patients with Ribosomal and GATA1 Mutations Reveals Dysregulation of Inflammatory Response Genes

Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by red cell aplasia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have identifiable heterozygous gene mutations or deletions in ribosomal protein genes. Additionally, mutations in GATA1, a key transcription factor in erythropoiesis, have been demonstrated in DBA patients (Sankaran VG et al. JCI. 122: 2439-43, 2012; Ludwig LS et al. Nat Med 20(7):748-53, 2014; Klar J et al. Br J Haem 166(6): 949-51, 2014). Despite our knowledge of the molecular pathology, the mechanism underlying the erythroid failure in DBA is not well understood, largely due to the inherent difficulties in studying primary erythroid cells from DBA patients. Furthermore, DBA patients with GATA1 mutations have not been well characterized and it remains unclear whether the pathogenic mechanisms of GATA1 DBA are similar to that of ribosomal DBA.We designed a two-step, 14 day in vitro culture system to generate erythroid cells from CD34+ stem/progenitor cells isolated from <10ml of peripheral blood (PB) collected from patients enrolled in the DBA Registry of North America. Patients with RPL5, RPS17(n=2), RPS24 and RPL35a, GATA1 mutations (n=2), and patients without identifiable mutations (n=3) were studied and compared to healthy control PB CD34+ cells. At day 14, we routinely obtain at least 10x fewer CD235+ erythroid cells (proerythroblasts and basophilic erythroblasts) in DBA cultures vs. controls (1x106 vs. 1x107 from 1x104 CD34+ cells). Further, a 2-7 day delay in the acquisition of CD235 was observed.Gene expression analyses was performed by Affymetrix GeneChip Human Gene ST Arrays and RNASeq to analyze protein coding and long non-coding RNA transcripts in cells from day 14 of erythroid cultures. Ingenuity pathway analysis (IPA) of the dysregulated genes between patients with ribosomal mutations, GATA1 mutations and controls revealed that many of the genes dysregulated in DBA were involved in multiple leukocyte migration and inflammatory signaling pathways, including the IL8, IL1R1, CXCR4, ICAM3, MPO, TNFSF10, and TLR4 genes with IL6, TNF, and lipopolysaccharide as top upstream regulators. Notably, the dysregulated genes in GATA1 patient cells largely overlapped that of the DBA patients with ribosomal mutations, including disruption of the leukocyte migration and inflammatory response genes. Patients with GATA1 and ribosomal protein mutations shared a number of dysregulated erythroid genes including AHSP, FAM132B, HEMGN, and TRIM10, however GATA1 patient cells showed GATA1 as the top upstream regulator and additionally showed dysregulation of heme biosynthesis pathway genes, including the ALAS2, FECH, CPOX, PPOX, and UROS.Few studies have looked at the pathogenesis of DBA in patients with GATA1 mutations. The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of control erythroid cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In cells from the patients with the GATA1 mutation, only GATA1s protein was expressed and the level of GATA1s exceeded the combined level of both GATA1 isoforms in control cells. Erythroid cells from a patient with an RPL5 mutation showed GATA1 protein levels comparable to controls, indicating the DBA phenotype of the RPL5 patient is not due to altered translation of full length GATA1. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing.In summary, our transcriptome analyses have revealed novel insights into the molecular pathogenesis of ribosomal and GATA1-mutated DBA. Significant dysregulation of inflammatory gene pathways in DBA patients with both ribosomal protein and GATA1 mutations were observed, suggesting a shared pathological mechanism. Furthermore, the heme biosynthesis pathway was uniquely disrupted in patients with GATA1 mutations. Further investigating the inflammatory pathways in DBA may reveal novel targets for therapeutic development. DisclosuresNo relevant conflicts of interest to declare.

Endocrine Dysfunction in Diamond-Blackfan Anemia (DBA): A Report from the DBA Registry (DBAR).

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome. The mainstays of treatment involve chronic red cell transfusions, long-term glucocorticoid therapy, and stem cell transplantation. Systematic data concerning endocrine function in DBA are limited. We studied patients in the DBA Registry (DBAR) of North America to assess the prevalence of various endocrinopathies. In a pilot study, retrospective data were collected for 12 patients with DBA. Subsequently, patients with DBA aged 1-39 years were recruited prospectively. Combined, 57 patients were studied; 38 chronically transfused, 12 glucocorticoid-dependent, and seven in remission. Data were collected on anthropometric measurements, systematic screening of pituitary, thyroid, parathyroid, adrenal, pancreatic, and gonadal function, and ferritin levels. Descriptive statistics were tabulated and group differences were assessed. Fifty-three percent of patients had ≥ 1 endocrine disorder, including adrenal insufficiency (32%), hypogonadism (29%), hypothyroidism (14%), growth hormone dysfunction (7%), diabetes mellitus (2%), and/or diabetes insipidus (2%). Ten of the 33 patients with available heights had height standard deviation less than -2. Low 25-hydroxy vitamin D (25(OH)D) levels were present in 50%. A small proportion also had osteopenia, osteoporosis, or hypercalciuria. Most with adrenal insufficiency were glucocorticoid dependent; other endocrinopathies were more common in chronically transfused patients. Endocrine dysfunction is common in DBA, as early as the teenage years. Although prevalence is highest in transfused patients, patients taking glucocorticoids or in remission also have endocrine dysfunction. Longitudinal studies are needed to better understand the etiology and true prevalence of these disorders.

Growth hormone improves short stature in children with Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA), an inherited marrow failure syndrome, has severe hypoplastic anemia in infancy and association with aplastic anemia, MDS/leukemia, and other malignancies. Short stature is present in most patients. Isolated cases have demonstrated improved growth on growth hormone (GH) therapy. GH treatment data were obtained from 19 children with DBA (6 at our site and 13 from Genentech). Control data from 44 non-GH treated children were provided by Diamond Blackfan Anemia Registry. Annual growth velocity (GV) and height-for-age Z-scores (HAZ) were compared between groups and for up to 4y of GH treatment. Constructed DBA-specific male and female height-for-age charts for non-GH treated patients revealed short stature compared to CDC norms. GH-treated patients had significantly lower HAZ prior to treatment initiation compared to non-GH-treated controls. Among GH-treated patients, GV significantly improved in the first two years relative to pre-treatment. HAZ significantly improved in each of 4y of GH therapy compared to baseline. After 2y of therapy, HAZ for GH-treated patients were not significantly different from controls, demonstrating successful catch-up growth. GH treatment in children with DBA improves both GV and HAZ during treatment sustained for up to 4y. Very short children with DBA can be treated successfully with GH to restore stature to levels comparable to less affected patients. DBA height charts are useful tools for assessing age-specific growth in this typically short population. Careful consideration of individualized benefit of GH therapy versus risk is important in view of long-term underlying ∼5% malignancy risk in DBA.

Deleterious Consequences of Diamond Blackfan Anemia on Reproductive Health and Pregnancy Outcomes: A Report from the Diamond Blackfan Anemia Registry (DBAR)

Background: Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by red cell aplasia, birth anomalies, and a predisposition to cancer. Due to a primarily autosomal dominant mode of inheritance, DBA affects males and females at a ratio of 1:1. Treatment consists of corticosteroid administration and chronic red cell transfusion therapy and in some cases hematopoietic stem cell transplantation. Current clinical observations suggest that women with DBA may experience delayed puberty, irregular menstrual cycles, and decreased fertility. Women who do become pregnant may sustain a higher than average risk of pregnancy complications, including miscarriage, placental abruption, and stillbirth, and others of vascular-placental origin (Faivre et al. Haematologica, 2006). Anecdotal reports suggest that DBA women experience changes to treatment response and remission status during periods characterized by hormonal fluctuations; namely, puberty and pregnancy. The etiology of these complications is unclear.Methods: Questionnaires were sent to females greater than 15 years of age (n=224) participating in the Diamond Blackfan Anemia Registry of North America (DBAR), a comprehensive database of 720 patients who are enrolled after obtaining informed consent. The questionnaires ask about menstrual and gynecologic health and pregnancies, focusing on complications, treatment requirements, and outcomes. We grouped patients by steroid and/or transfusion dependence status and/or remission before age 12 to analyze the effects of treatment on menarche. The patients were regrouped based on the majority of their treatment to evaluate for gynecologic health and pregnancy complications. The Fisher’s exact test was used to examine associations between variables. When compared to the general population, normal values were obtained from a variety of sources including the American Academy of Pediatrics, the National Institutes of Aging and the March of Dimes.Results and Conclusions: We reviewed results from 84 women aged 15 to 62 years (median age 28.6 years). Menarche was delayed in both steroid dependent and transfusion dependent girls, with 39.5% and 77.8%, respectively experiencing menarche at age 15 or later (vs 2% normal). Transfusion dependent girls were significantly more likely to have delayed menarche compared to the steroid dependent and remission groups (p<.005). Additionally, transfusion dependent females were significantly more likely to experience premature ovarian failure, compared to those who were either steroid dependent or in remission (32.1% vs 8.6%; p<.025). Of those who report entering menopause, 75% stopped by age 40 or younger (vs average, 51 years). Women who are transfusion dependent were more likely to report having taken medication to regulate their menstrual cycle, as compared to steroid dependent and remission groups (48.1% vs 22.9%; p<.06). Of the 50 pregnancies reported in 23 women, 30 resulted in live births (60%). Of the total live births, 43.3% were preterm (vs 11.5% normal). Interestingly, steroid dependent women as well as those in remission had the highest percentage of preterm deliveries at 66.7% and 63.6%, respectively. 26% of the pregnancies resulted in miscarriage, and 4.3% resulted in stillbirth (vs normal, 15% and 0.6%, respectively) and 4.3% of pregnancies were complicated by placental abruption (vs normal, <1%). During pregnancy, 52.2% of women reported requiring transfusions; specifically, 100% of the women who were steroid dependent at conception required transfusions during pregnancy. 7.4% of women in the study had a hysterectomy, half of whom were under age 40. We also noted an increased incidence of endocrinopathies: transfusion dependent women reported a significantly higher prevalence of thyroid disorders compared to the steroid dependent group (42.9% vs 11.1%; p<.03). Additionally, there was a higher prevalence of diabetes in the transfusion group as compared to the steroid dependent group (14.3% vs 0%, p=NS). These findings suggest an important role of iron overload in menstrual abnormalities, reproductive issues and endocrinopathies in DBA women. Pregnancy complications are in excess of those seen in the normal population. Future analyses will determine whether there is a correlation between menstrual abnormalities and/or pregnancy complications and specific DBA genotypes. DisclosuresNo relevant conflicts of interest to declare.

In Vitro Analysis of Erythroid Cells Derived from the Culture of Diamond Blackfan Anemia Patient CD34+ Cells

Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have heterozygous mutations or deletions in ribosomal protein genes. Additionally, mutations in the GATA1 gene, which encodes the GATA1 erythroid transcription factor, have been demonstrated in two DBA patients (Sankaran VG et al. J Clin Invest. 122: 2439-43, 2012). The genetic cause of DBA in the remaining 35% of patients is unknown. Despite our knowledge of the genotypes, the mechanism underlying the erythroid failure in DBA is not completely understood. This is largely due to the inability to study primary erythroid cells from DBA patients. To begin to delineate the mechanisms regulating erythroid differentiation in DBA, we developed an in vitro culture system starting with CD34+ progenitor cells isolated from approximately 20 ml of DBA patient peripheral blood collected prior to transfusion at the DBA Registry of North America. Using this system, we have characterized DBA patients with mutations in large (RPL5) and small (RPS17) subunit ribosomal protein genes, one individual with a mutation in the GATA1 gene, and several patients with unknown mutations. At the end of the culture, we routinely obtained 6x107 CD235+ erythroid cells from an initial population of 5x104 control CD34+ progenitor cells. In contrast to control cells, cells from the DBA patients exhibited a significantly reduced growth rate and generated approximately 100-fold fewer CD235+ erythroid cells, with a two day delay in the acquisition of the CD235 marker. Using flow cytometry, we isolated populations of CD41-, CD44+, CD235+ erythroid cells from both control and patient cell cultures from which we extracted RNA. This allowed the first time comparison of mRNA expression in DBA erythroid cells. Protein coding and long non-coding RNA transcripts were compared using Affymetrix GeneChip Human Gene ST Arrays and RNASeq. Compared to controls, CD235+ cells from the patient with the RPL5 mutation showed decreased levels of the GAS5 (growth arrest) and NOP56 (large ribosomal subunit assembly) mRNAs, as well as small nucleolar RNAs. Ingenuity Pathway Analysis (IPA) identified the tRNA charging and RNA Polymerase II assembly pathways as significantly perturbed in this patient.The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of CD235+ control cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In the patient cells with the GATA1 mutation, only the GATA1s protein was expressed and exceeded the combined level of both GATA1 isoforms in the controls. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing. The microarray and RNASeq expression profiles of the patient with the GATA1 mutation differed significantly from controls and from that of the patient with the RPL5 mutation. Many known GATA1 target genes including SLC4A1, AHSP, and TRIM10 were down regulated in the CD235+ erythroid cells of the patient with the GATA1mutation compared to control cells, clearly indicating that these genes depend on full length GATA1 for activation. IPA identified the heme biosynthesis pathway as significantly perturbed in this patient and GATA1 as the top regulator.In summary, we have shown that DBA patient cells show decreased proliferative and erythroid differentiation capabilities in vitro. RNA transcript analysis in DBA patient cells has revealed significant differences between DBA patients with a ribosomal protein gene mutation and a mutation in the GATA1 gene. These data delineate multiple signaling pathways or mechanisms involved in DBA and erythroid differentiation. Finally, we have demonstrated that many GATA1 target genes depend on full length GATA1 for activation. DisclosuresNo relevant conflicts of interest to declare.

Clinical phenotype and genetic analysis of RPS19, RPL5, and RPL11 genes in Greek patients with Diamond Blackfan Anemia.

Diamond Blackfan Anemia (DBA) is a rare congenital, bone marrow failure syndrome characterized by normochromic macrocytic anemia, reticulocytopenia and absence or insufficiency of erythroid precursors in normocellular bone marrow, frequently associated with somatic malformations. Here, we present our findings from the study of 17 patients recorded in the Greek DBA registry. Clinical evaluation of patients and data collection was performed followed by the molecular analysis of RPS19, RPL5, and RPL11 genes. Mutation screening included PCR amplification, ECMA analysis, and direct sequencing. Congenital anomalies were observed in 71% of the patients. Six patients (35.2%) were found to carry mutations on either the RPS19 gene (three patients,) or the RPL5 gene (three patients). Mutations c.C390G (p.Y130X) and c.197_198insA (p.Y66X) detected in the RPL5 gene were novel. No mutations at the RPL11 gene were identified in Greek patients with DBA. The clinical course of the patients was similar to previous reports. The occurrence of thyroid carcinoma in an adult patient with DBA is the first to be reported in DBA.

Diamond Blackfan anemia: A Cheshire cat of hematology

Diamond Blackfan anemia: A Cheshire cat of hematology