Diamond-Blackfan Anemia Gene Research Articles

Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia.

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Regulated Expression of GATA1 As a Gene Therapy Cure for Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a bone marrow failure disorder characterized by impaired red blood cell production leading to severe anemia. The only treatment options are regular transfusions, corticosteroids, or bone marrow transplant, and each has variable effectiveness and considerable morbidity. More effective, safer, and permanent cures for DBA are lacking, but few drugs are in active development. DBA is genetically heterogenous, characterized by loss-of-function mutations in one copy of at least 26 different ribosomal protein (RP) genes that alters the mRNA translation of a select group of genes, most notably the hematopoietic master regulator GATA1. Overexpression of GATA1 in hematopoietic stem and progenitor cells (HSPCs) from patients with DBA rescues the erythroid differentiation defects in vitro, but unregulated GATA1 expression in hematopoietic stem cells (HSCs) promotes premature erythroid differentiation at the expense of long-term (LT-) HSC maintenance. Gene therapy is an attractive strategy to achieve a cure for DBA, but a traditional gene therapy approach that overexpresses a functional copy of a mutated gene is not the ideal option, as it would require the development and validation of dozens of gene therapy vectors, each with a copy of one of the mutated DBA genes. Here, we report a unified gene therapy strategy, using developmentally-regulated and highly restricted expression of GATA1 that would be curative in most, if not all, patients with DBA, regardless of the underlying disease-causing mutation. To be a cure for DBA, a GATA1 gene therapy vector must incorporate into the genome of undifferentiated LT-HSCs but only drive robust GATA1 expression in early erythroid progenitors after lineage commitment. To design a vector with lineage-restricted expression, we identified accessible chromatin regions upstream of GATA1 that only gain accessibility in differentiating erythroid cells, but not in upstream HSPCs. Using these selectively accessible regions concatenated together, we constructed a human GATA1 enhancer (hG1E) element and used it to drive GATA1 and/or GFP expression from a lentiviral backbone. To ensure erythroid-restricted expression of the hG1E-GATA1-GFP vector, we infected CD34+ HSCs from healthy human donors and cultured them in conditions that either allowed HSC maintenance or promoted erythrocyte differentiation. We observed very low GFP expression in bulk HSCs and essentially no expression in the subpopulation of immunophenotypic LT-HSCs. However, more than 80% of committed erythroid cells demonstrated high levels of GFP expression, confirming the fidelity of lineage-restricted expression from the hG1E-GATA1 vector. To examine whether lineage-restricted GATA1 expression confers a functional correction of the erythroid maturation block in DBA, we used CRISPR editing to recapitulate RPS19 haploinsufficiency in human HSPCs. In RPS19 edited samples that were infected with a control GFP virus, we observed a progressive depletion over time of RPS19 edits as the cells underwent erythroid differentiation, consistent with the notion that RPS19 editing leads to impaired erythroid differentiation and are selected against. In contrast, RPS19-edited and hG1E-GATA1 treated samples had a more than 10-fold higher preservation of RPS19 edits in the bulk population and in erythroid colonies derived from single progenitor clones, indicating that regulated GATA1 expression could overcome RPS19 haploinsufficiency. To validate the results from the CRISPR model of DBA, we sought to correct the erythroid maturation block in primary HSCs from a DBA patient with a pathogenic mutation in RPL5. There was skewed differentiation of GFP-infected RPL5+/- HSCs away from the erythroid lineage and toward myeloid and megakaryocyte lineages, but regulated GATA1 expression from the hG1E-GATA1 vector reversed that skewing. There was a 10-fold increase in the ratio of erythroid to non-erythroid cells, and a significant expansion of erythroid cells in the hG1E-GATA1 treated patient cells. Preclinical assessment in additional patient samples both in vitro and in vivo is ongoing. Collectively, our preclinical data demonstrate that lineage-restricted expression of GATA1 is sufficient to rescue the impaired erythroid differentiation in DBA regardless of the underlying genetic lesion. These results will set the stage for the first in-human gene therapy trial for DBA.

Reanalysing genomic data by normalized coverage values uncovers CNVs in bone marrow failure gene panels

Inherited bone marrow failure syndromes (IBMFSs) are genetically heterogeneous disorders with cytopenia. Many IBMFSs also feature physical malformations and an increased risk of cancer. Point mutations can be identified in about half of patients. Copy number variation (CNVs) have been reported; however, the frequency and spectrum of CNVs are unknown. Unfortunately, current genome-wide methods have major limitations since they may miss small CNVs or may have low sensitivity due to low read depths. Herein, we aimed to determine whether reanalysis of NGS panel data by normalized coverage value could identify CNVs and characterize them. To address this aim, DNA from IBMFS patients was analyzed by a NGS panel assay of known IBMFS genes. After analysis for point mutations, heterozygous and homozygous CNVs were searched by normalized read coverage ratios and specific thresholds. Of the 258 tested patients, 91 were found to have pathogenic point variants. NGS sample data from 165 patients without pathogenic point mutations were re-analyzed for CNVs; 10 patients were found to have deletions. Diamond Blackfan anemia genes most commonly exhibited heterozygous deletions, and included RPS19, RPL11, and RPL5. A diagnosis of GATA2-related disorder was made in a patient with myelodysplastic syndrome who was found to have a heterozygous GATA2 deletion. Importantly, homozygous FANCA deletion were detected in a patient who could not be previously assigned a specific syndromic diagnosis. Lastly, we identified compound heterozygousity for deletions and pathogenic point variants in RBM8A and PARN genes. All deletions were validated by orthogonal methods. We conclude that careful analysis of normalized coverage values can detect CNVs in NGS panels and should be considered as a standard practice prior to do further investigations.

De Novo Point Mutation of RPL31 in Diamond-Blackfan Anaemia: A Story from Patient to Zebrafish

Diamond Blackfan anemia (DBA) is a rare bone marrow failure disorder that usually manifests as severe macrocytic anemia and erythroid hypoplasia ,often accompanied by various physical malformations and increased frequency of cancer. The disease are associated with known ribosomal protein gene and rarely GATA1 mutations in most of DBA cases, with the genetic etiology unclear in the remaining patients. Here, we present one patient with Diamond-Blackfan anaemia who lacked documented mutations involving known DBA genes by screening.Whole-exome sequencing (WES) was performed and we identified a single missense mutation in RPL31.The missense variant c.254G>T is predicted to give rise to a gene product with substitution p. Arg85Leu ,which is regarded as causative because the unaffected parents and sister of the patient do not possess the mutation,implying that this mutation was de novo. Previous studies showed that nearly all of the causative variants of RP genes observed in DBA are loss-of function mutations and RP haploinsufficiency accounts for most cases of DBA. Particularly, The coding region of RPL31 shares 89.6% amino acid identities with its zebrafish ortholog, which is highly conserved between these two species.All of above led us to explore the effects of RPL31 deficiency on morphology and erythropoietic status during embryonic development. Therefore, morpholino antisense oligonucleotides (MOs) targeting zebrafish rpl31, orthologs of human RPL31,was injected at a proper concentration into one-cell-stage zebrafish embryos to mimic RPL31 haploinsufficiency in DBA. The specificity of the rpl31-MO was valitated by western blot detection.Compared with wild-type embryos, rpl31 deficiency embryos showed developmental abnormalities such as pericardial edema, microcephaly ,delayed pigmentation and bent tail. We also performed haemoglobin staining at 48 hr post fertilization (48hpf) and found a significant decrease of erythrocyte production in the cardial vein of the morphants. Meanwhile,the number of lcr+red blood cells was reduced about 50% in lcr:GFP transgenic embryos at 48 hpf by fluorescence microscopy and 3dpf by flow cytometry when compared to control embryos.However,the myeloid and lymphoid lineages were preserved as analyzed by using lyz:dsRed transgenic line and by observing expression of rag1,respectively. Lots of evidence showed that many other ribosomal protein genes deficiency can up-regulate p53 network,resulting in consequent cell cycle arrest or apoptosis.so we want to investigate the connection between RPL31 and p53 signaling in erythropoiesis.However,p53 null can not completely rescue the erythroid defect phenotype when we injected rpl31-MO into p53-/- embryos,suggesting alternative pathways may be involved in this process. In summary, we reported a brand new point mutation in RPL31 gene and we phonecopied loss of function of RPL31 observed in the DBA patient in vivo by constructing a RPL31 deficiency zebrafish model,indicating preliminarily that the loss of function mutation in RPL31 is a candidate responsible for DBA.However,underlying molecular mechanisms and reasons of the specific erythroid impairments in RPL31 deficiency are still needed to be clarified.we will focused on these in the future. Disclosures No relevant conflicts of interest to declare.

A Small Nucleolar RNA Linked to Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia associated with haploinsufficiency of more than 15 ribosomal protein genes. We identified in the French registry a DBA patient exhibiting a large heterozygous deletion in ribosomal protein gene RPSA, making it a new candidate as a DBA-associated gene. The deletion was confirmed by qPCR using specific intron/exon primers that could discriminate the RPSA gene from pseudo-genes. Consistent loss of function of this allele, RPSA mRNAs were underrepresented in erythroid cultures and in lymphoblastoid cells (LCLs) derived from the patient. Additionally, an important decrease in erythroid proliferation was observed in erythroid progenitor and precursor cells derived from the patient's bone marrow CD34+ cells, together with a delay in erythroid differentiation, the activation of the p53 pathway, and a G0/G1 cell cycle arrest, which are regular features of DBA patient cells. Similar to other RP genes linked to DBA, interruption of RPSA expression in zebrafish embryos strongly affected development of the head and erythroid cell differentiation, supporting its relevance as a candidate DBA gene. Northern blot analysis also revealed impaired pre-ribosomal RNA (pre-rRNA) processing in patient LCLs compared to controls. But unexpectedly, the observed pre-rRNA profile was clearly different from that observed in HeLa cells upon RPSA knockdown and suggested a dysfunction of pre-rRNA processing upstream of that produced by a shortage of RPSA. We noticed that the RPSA gene hosts the intron-encoded H/ACA snoRNAs snoRA6 and snoRA62, the latter being included in the deletion. Q-RT-PCR analysis of patient cells showed decreased levels of snoRA62 when compared to controls and to other RP-mutated DBA patients. Strikingly, knocking out the snoRNA62 coding sequence in human haploid HAP1 cells with Crispr/Cas9 resulted in a pre-rRNA maturation defect similar to that observed in the RPSA+/mut DBA patient, thereby revealing a role for snoRA62 in human pre-rRNA processing. These data strongly suggest that, in addition to RPSA haploinsufficiency, the primum movens of DBA in this particular patient includes the deletion of the intronic snoRA62 hosted in the RPSA gene. To our knowledge, this is the first time that a snoRNA is linked to DBA. We will discuss this ongoing work in light of our most recent results in cells and zebrafish models. DisclosuresNo relevant conflicts of interest to declare.

Molecular analysis and genotype-phenotype correlation of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) features hypoplastic anemia and congenital malformations, largely caused by mutations in various ribosomal proteins. The aim of this study was to characterize the spectrum of genetic lesions causing DBA and identify genotypes that correlate with phenotypes of clinical significance. Seventy-four patients with DBA from across Canada were included. Nucleotide-level mutations or large deletions were identified in 10 ribosomal genes in 45 cases. The RPS19 mutation group was associated with higher requirement for chronic treatment for anemia than other DBA groups. Patients with RPS19 mutations, however, were more likely to maintain long-term corticosteroid response without requirement for further chronic transfusions. Conversely, patients with RPL11 mutations were less likely to need chronic treatment. Birth defects, including cardiac, skeletal, hand, cleft lip or palate and genitourinary malformations, also varied among the various genetic groups. Patients with RPS19 mutations had the fewest number of defects, while patients with RPL5 had the greatest number of birth defects. This is the first study to show differences between DBA genetic groups with regards to treatment. Previously unreported differences in the rate and types of birth defects were also identified. These data allow better patient counseling, a more personalized monitoring plan, and may also suggest differential functions of DBA genes on ribosome and extra-ribosomal functions.

Novel and Known Ribosomal Causes of Diamond-Blackfan Anemia Identified through Comprehensive Genomic Characterization

Introduction: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by erythroid hypoplasia. It is associated with a number of congenital anomalies and a high risk of developing specific cancers. DBA is caused by germline mutations or deletions in genes affecting ribosomal biogenesis and function, with autosomal dominant or X-linked recessive patterns of inheritance. The most commonly mutated gene is RPS19, seen in approximately 25% of patients. About 45% of DBA families have no known disease-causing pathogenic variant.Methods: Affected and unaffected individuals from families with DBA were ascertained through the IRB-approved NCI IBMFS retrospective/prospective cohort study (ClinicalTrials.gov Identifier: NCT00027274). Study participants completed detailed family and medical history questionnaires, medical records were reviewed, and a subset of families underwent clinical evaluations at the NIH Clinical Center. DBA patients enrolled prior to 2014 underwent routine clinical mutation testing for the established DBA genes; beginning in 2014, DBA patient samples (buccal and blood DNA) were evaluated by whole exome sequencing (WES) for mutation identification. We incorporated WES with deletion analyses and copy number variant (CNV) assessment to uncover the genetic changes causative of DBA. Deletion analyses performed included SNP genotyping and array comparative genomic hybridization. Functional effects of the genetic variants were proven by pre-rRNA processing defect analysis by Northern blot. Controls for functional studies were healthy mutation-negative individuals from the IBMFS study.Results: Genetic testing information was available in 61 of the 87 families with DBA enrolled in the IBMFS study. Thirty-five of the 61 families did not have a known genetic cause at enrollment. Our combined approach of WES, deletion and CNV analyses identified the causative pathogenic variant in 18 of the 35 (51%) uncharacterized DBA families. We discovered pathogenic variants in two previously undescribed genes in two DBA families. One family had a nonsynonymous variant (p.K77N) in RPL35; the second family had a nonsynonymous variant (p. L51S) in RPL18. Both of these variants result in characteristic pre-rRNA processing defects. Our analyses also uncovered germline mosaic deletions in known DBA genes in both buccal and blood cells of two patients from two different families. One was a 1.8 Mb mosaic deletion in chromosome 15 including RPS17; the other was a large 2.5 Mb mosaic deletion on chromosome 3 including RPL35A. In addition to these findings, we found variants in previously known DBA-associated ribosomal genes in 14 of the 35 families.We further evaluated the genomic characteristics of the entire DBA cohort. Pathogenic variants in ribosomal DBA genes were found in a total of 44 of the 61 families (72%) on whom genetic testing information and/or biospecimens were available. RPS19 was the most frequently mutated gene and accounted for 36% of families, followed by RPL35A and RPS26, accounting for 14% and 11% each, respectively. Notably, 30% of the variation in disease-causing genes in our cohort was due to a single copy or mosaic gene deletion. We had complete parental testing and inheritance information on 23 (52%) of the 44 families whose gene was identified. Ten of the 23 (43%) had an inherited mutation and 13 (57%) had a de novo change in the causative gene (both parents were negative for the affected child's disease-associated mutation). At this time, 17 of 61 families tested (28%) do not have a characterized disease-associated mutation.Conclusion: This efficient comprehensive genomic approach was the basis for our discovery of two novel causes of DBA, characterization of ribosomal gene deletions not previously described to be disease-associated, and of DBA-associated germline mosaicism. We identified the disease-associated mutations in 51% (18 of 35) of our families without a known genetic cause of DBA. A total of 74% (44 of 61) of our families are now genetically characterized. Our comprehensive approach appears to provide more genomic information than other methods since pathogenic variants of DBA genes have been reported previously in about 55% of DBA patients. DisclosuresNo relevant conflicts of interest to declare.

Molecular Analysis of Diamond Blackfan Anemia and Genotype-Phenotype Correlation: Experience from the Canadian Inherited Marrow Failure Registry

Background/Objectives: Diamond Blackfan anemia (DBA) is an inherited disorder characterized by chronic hypoproductive anemia, physical malformations, and an increased risk of malignancies. At least 12 DBA genes have been identified, which include various ribosomal protein genes and the transcription factor GATA1. The aims of our study were (1) to identify the mutation spectrum of DBA patients, utilizing a cohort of patients enrolled on the Canadian Inherited Marrow Failure Registry (CIMFR) and (2) to determine whether specific hematological abnormalities, malformations, and outcomes are associated with specific mutations.Methods: Patients were enrolled on the CIMFR, which is a multicenter cohort study of inherited bone marrow failure syndromes (IBMFS). Genetic testing was performed using one or more of the following tests: Sanger sequencing, next generation sequencing (NGS) DBA gene panel, a comprehensive NGS IBMFS gene panel developed in our laboratory, or comparative genetic hybridization (CGH). Severity of the hematological disease was dichotomized according to a patient's requirement for chronic treatment: those who were maintained on corticosteroids, blood transfusions, or received a hematopoietic stem cell transplantation were considered to have a more severe phenotype than those who did not require hematological treatment. Chi-square tests with a Fisher's exact test correction were used to compare genetic groups with at least 5 patients on observed phenotypes.Results: 71 patients with DBA have been enrolled in our registry. A causal mutation has been identified in 36 of these patients, with the following rates: RPS19 (n=11), RPL11 (n=7), RPL5 (n=6), RPS26 (n=5), RPL35a (n=2), RPS24 (n=2), and one of each RPS7, RPS29, RPS17. Remarkably, a substantial number of patients in our population-based cohort (19.4%) had mild hematological phenotype requiring no therapy. Patients with RPL11 mutations tended to have a less severe DBA phenotype, while patients with RPS19 mutations tended to have a more severe phenotype (P =0.04). In terms of non-hematological malformations, we found no differences in cardiac, stature and craniofacial malformations across the groups compared (all p>0.1). However, patients with RPL5 mutations had significantly more hand malformations (P =0.02), and patients with RPS26 mutations had more genitourinary malformations (P =0.04). To control for the impact of mutation severity on the observed phenotype, we compared the prevalence of mutations that are predicted to result in truncated or lack of protein from the respective allele (large copy-number variation, nonsense, or indel frameshift) to mutations that are predicted to be hypomorphic or affect function (splicing, indel/inframe and, missense) between mutation categories. There were no differences among genetic groups in the severity of their mutations (P =0.58).Conclusions: Mutations in a wide spectrum of ribosomal protein genes underlie DBA cases in Canada, which approximate those observed by other registries in Western countries. Patients with DBA caused by RPL11 mutations tended to have a milder hematological phenotype, while patients with RPS19 mutation tended to have a more severe phenotype. Mutations in RPS26 and RPL5 are associated with genitourinary and hand malformations, respectively. Our findings may help improve counseling of DBA patients and their family. Future studies are needed to replicate our results and determine whether these findings can help personalize care. DisclosuresLipton:Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

A Novel Conditional Knockout of the Diamond Blackfan Anemia Gene Rpl11 Shows Failure of Erythropoiesis, a Marked Increase in BFU-E Progenitors By Phenotype That Proliferate Poorly in Culture, and Activation of p53 Target Genes

Diamond Blackman Anemia (DBA) is a severe erythroid hypoplasia that usually presents during the first year of life. Most cases are due to heterozygous mutations in ribosomal protein genes including RPS19, RPL5 and RPL11. Ribosomal stress-induced p53 activation is thought to play a major role in DBA, leading to apoptosis and/or cell cycle arrest in the erythroid lineage. This response is dependent on accumulation of RPL5 and RPL11 subunits that bind and sequester MDM2, the ubiquitin ligase that naturally degrades p53. Thus, it is difficult to understand how this pathway can operate in the presence RPL5 or RPL11 mutations. Here we show that Rpl11 haploinsufficiency in mice leads to severe anemia characteristic of DBA and, surprisingly, p53 activation in the bone marrow.We created a conditional Rpl11 mouse with loxP sites that flank exon 2. These animals were crossed with MxCre heterozygotes, and treated with pIpC. After three weeks, Rpl11+/- animals became severely anemic, with increased MCV, decreased reticulocyte counts and normal leukocyte and platelet counts. Sacrifice of survivors after 4 months showed increased spleen weight due to the murine stress erythroid response. FACS analysis of bone marrow cells showed a marked increase in BFU-E and relative decrease in CFU-E in Rpl11+/- animals. However, methylcellulose colony forming assays revealed a decrease in large BFU-E colonies from Rpl11+/- animals, coupled with an increase in smaller, atypical BFU-E colonies, which fail to proliferate at a normal rate. To identify transcriptional signatures associated with severe anemia in Rpl11+/- animals, we sequenced cDNA libraries from total BM of induced Rpl11+/- mice and identified 508 differentially expressed genes relative to controls. Unexpectedly, p53 pathway genes were significantly upregulated in Rpl11+/- mice, which may suggest that Rpl5 accumulation alone or with haploinsufficient amounts of Rpl11 can sequester MDM2, or that p53 activation occurs through an alternative mechanism. In conclusion, we created a unique genetic heterozygous Rpl11 conditional knockout mouse that recapitulates the severe macrocytic anemia of DBA. We wish to use this model to identify novel molecular mechanisms of pathogenesis and test emerging therapies. DisclosuresEbert:Genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy; H3 Biomedicine: Consultancy.

Disrupted Expression of the Splicing Regulators Tra2-Beta and SRSF1 and Subsequent Aberrant Splicing of FLVCR1 Transcript As an Underlying Mechanism for the Erythropoietic Failure Observed in Diamond-Blackfan Anemia

Diamond Blackfan Anemia (DBA) is a rare inherited anemia caused by disruption of erythroid progenitor cell development at the burst-forming and colony-forming units. Approximately 50% of DBA patients have additional non-hematopoietic abnormalities including heart defects and short stature. Sixteen genes encoding specific ribosomal proteins (RPs) and the transcription factor GATA1 have been found mutated in approximately 65% of DBA patients, with the RPS19 gene being mutated in 25% of patients. Because RPs are components of the ribosome that is critical for protein synthesis, the identification of RP mutations suggest a translation deficiency as a mechanism for erythroid progenitor failure. However, erythropoietic failure in DBA may arise by causes other non-translational functions of the DBA genes. We have previously reported that inhibiting the heme exporter protein FLVCR1 in human hematopoietic stem cells and progenitors (HSC/Ps), is sufficient to specifically block erythropoiesis, which mimics the hematological features observed in DBA. Moreover, we reported aberrant splicing of exons (E) 2 and 3 of the FLVCR1 transcript in DBA erythroid cells that resulted in disruption of FLVCR1 protein expression and function. We observed aberrant FLVCR1 splicing in erythroid cells from patients with RPS19 and RPL5 mutations and from typical DBA patients whose genetic background has not been known at that time; thus, suggesting FLVCR1 dysfunction as central for the DBA erythropoietic failure.In the present work, we obtained evidence suggesting that DBA pathogenesis is caused by abnormality in the cellular splicing machinery. We performed In silico analysis of the binding motifs of seven SR proteins on each of the FLVCR1 exons. We found binding motifs for TRA2-β, SRSF1 and 9G8 on E2 and/or E3, the two FLVCR1 exons that are skipped in DBA erythroid cells and in RPS19-disrupted K562 cells. We focused on expression analysis of TRA2B and SRSF1 in RPS19-reduced K562 cells. Expression of both TRA2B and SRSF1 mRNA were significantly disrupted in RPS19-disrupted K562 cells, which correlated with disrupted expression of their respective proteins. To test the specificity of disrupted SR splicing regulators and aberrant FLVCR1 splicing, we analyzed TRA2-β, and SRSF1 protein expression, and FLVCR1 aberrant splicing, in K562 cells disrupted in SBDS gene. The SBDS gene product is critical for ribosome biogenesis and the gene is mutated in 90% of patients with Shwachman-Diamond Syndrome. Interestingly, K562 cells down-regulated in SBDS showed normal levels of TRA2-β and SRSF1 expression, and no FLVCR1 aberrant splicing was observed. Importantly, erythroid differentiation potential of the DBA cell model can be rescued by expression of exogenous Tra2-β cDNA or FLVCR1 cDNA.Altogether our data suggests that mutations in the ribosome protein genes in DBA result in down-regulation of the splicing factors, TRA2-β and SRSF1, which impair normal FLVCR1 splicing and consequently erythropoiesis failure. DisclosuresNo relevant conflicts of interest to declare.

An SCF-FBXW7 Ubiquitin Ligase Mediated Feedback Loop Facilitates GATA Factor Switching and Reinforces Commitment to Terminal Erythroid Maturation

The GATA family transcription factors GATA2 and GATA1 play reciprocal roles during terminal erythroid and megakaryocytic maturation. GATA2 is expressed early in hematopoiesis and is required for early progenitor cell proliferation and survival. It must be down regulated in order for terminal maturation to occur. In contrast, GATA1 increases in expression during erythropoiesis and megakaryopoiesis, and is required for terminal maturation of these lineages. During this process, GATA1 displaces GATA2 at a large number of chromatin loci, typically leading to repression of early progenitor genes and activation of terminal maturation genes. This “GATA factor switch” is facilitated by the considerably shorter half-life of GATA2 (~30 min) compared to GATA1 (>4-6 hours). GATA2’s short half-life is mediated by ubiquitin-proteosomal degradation mechanisms. Yet the ubiquitin complex(es) responsible for GATA2-specific clearance has not been identified. In this study, we show that the Skip1-Cullin-Fbox (SCF) substrate recognition factor Fbxw7 is involved in GATA2 ubiquitin-mediated degradation. Proteomic and co-immunoprecipitation experiments show physical association of Fbxw7 with GATA2, but not GATA1. CRISPR/Cas9 deletion of Fbxw7 in G1ER4 cells results in elevated GATA2 steady-state protein levels, a prolonged GATA2 half-life, delayed GATA2 clearance upon estradiol induction, and impaired erythroid terminal maturation. Importantly, Fbxw7 mRNA and protein levels normally increase during terminal erythroid maturation and this occurs in a GATA1 dependent manner. We identified a key cis-regulatory element upstream of the Fbxw7 gene that is occupied by GATA2 during early erythropoiesis, but becomes bound by GATA1/TAL1 during late erythroid maturation in induced G1-ER4 cells and human CD34+ in vitro differentiated erythroblasts. This is accompanied by the acquisition of active enhancer histone marks at this site. Deletion of this regulatory element in G1ER4 cells blocks the GATA1 dependent increase in Fbxw7 mRNA transcript levels during erythroid cell maturation. Lastly, we identified a family with congenital hypoplastic anemia (lacking mutations in all known Diamond Blackfan Anemia genes) who harbor a germline Fbxw7missense mutation. Collectively, these findings identify GATA2 as a novel Fbxw7 substrate and uncover an ubiquitin-mediated post-transcriptional GATA factor feedback loop that reinforces GATA factor switching and commitment to terminal erythroid maturation. DisclosuresCantor:Amgen: Membership on an entity’s Board of Directors or advisory committees.

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.

Exploiting pre-rRNA processing in Diamond Blackfan anemia gene discovery and diagnosis.

Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene.

Diamond–Blackfan anemia with mandibulofacial dystostosis is heterogeneous, including the novel DBA genes TSR2 and RPS28

Patients with physical findings suggestive of Treacher Collins syndrome (TCS) or mandibulofacial dysostosis (MFD) and macrocytic anemia diagnostic of Diamond-Blackfan anemia (DBA) have been reported. Disease-causing genes have been identified for TCS and other MFDs. Mutations in several ribosomal protein genes and the transcription factor GATA1 result in DBA. However, no disease-causing mutation had been identified in the reported patients with the combination of TCS/MFD and DBA phenotype, and we hypothesized that pathogenic mutations in a single gene could be identified using whole exome analysis. We studied probands from six unrelated families. Combining exome analysis and Sanger sequencing, we identified likely pathogenic mutations in 5/6 families. Two mutations in unrelated families were seen in RPS26, the known DBA10 gene. One variant was predicted to affect mRNA splicing, and the other to lead to protein truncation. In another family a likely pathogenic X-linked mutation affecting a highly conserved residue was found in TSR2, which encodes a direct binding partner of RPS26. De novo mutations affecting the RPS28 start codon were found in two unrelated probands, identifying RPS28 as a novel disease gene. We conclude that the phenotype combining features of TCS with DBA is genetically heterogeneous. Each of the pathogenic variants identified is predicted to impede ribosome biogenesis, which in turn could result in altered cell growth and proliferation, causing abnormal embryologic development, defective erythropoiesis and reduced growth. The phenotype combining TCS/MFD and DBA is highly variable, overlaps with DBA and lies within the phenotypic spectrum of ribosomopathies. © 2014 Wiley Periodicals, Inc.

Diamond Blackfan anemia: A Cheshire cat of hematology

Diamond Blackfan anemia: A Cheshire cat of hematology

Ribosomal protein mutations in Korean patients with Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by hypoproliferative anemia, associated physical malformations and a predisposition to cancer. DBA has been associated with mutations and deletions in the large and small ribosomal protein genes, and genetic aberrations have been detected in ∼50–60% of patients. In this study, nine Korean DBA patients were screened for mutations in eight known DBA genes (RPS19, RPS24, RPS17, RPS10, RPS26, RPL35A, RPL5 and RPL11) using the direct sequencing method. Mutations in RPS19, RPS26 and RPS17 were detected in four, two and one patient, respectively. Among the mutations detected in RPS19, two mutations were novel (c.26T>A, c.357-2A>G). For the mutation-negative cases, array-CGH analysis was performed to identify copy-number variations, and no deletions involving the known DBA gene regions were identified. The relative mRNA expression of RPS19 estimated using real-time quantitative PCR analysis revealed two- to fourfold reductions in RPS19 mRNA expression in three patients with RPS19 mutations, and p53 protein expression analysis by immunohistochemistry showed variable but significant nuclear staining in the DBA patients. In conclusion, heterozygous mutations in the known DBA genes RPS19, RPS26 and RPS17 were detected in seven out of nine Korean DBA patients. Among these patients, RPS19 was the most frequently mutated gene. In addition, decreased RPS19 mRNA expression and p53 overexpression were observed in the Korean DBA patients, which supports the hypothesis that haploinsufficiency and p53 hyperactivation represent a central pathway underlying the pathogenesis of DBA.

Translation of branched-chain aminotransferase-1 transcripts is impaired in cells haploinsufficient for ribosomal protein genes

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome linked to mutations in ribosomal protein (RP) genes that result in the impaired proliferation of hematopoietic progenitor cells. The etiology of DBA is not completely understood; however, the ribosomal nature of the genes involved has led to speculation that these mutations may alter the landscape of messenger RNA (mRNA) translation. Here, we performed comparative microarray analysis of polysomal mRNA transcripts isolated from lymphoblastoid cell lines derived from DBA patients carrying various haploinsufficient mutations in either RPS19 or RPL11. Different spectrums of changes were observed depending on the mutant gene, with large differences found in RPS19 cells and very few in RPL11 cells. However, we find that the small number of altered transcripts in RPL11 overlap for the most part with those altered in RPS19 cells. We show specifically that levels of branched-chain aminotransferase-1 (BCAT1) transcripts are significantly decreased on the polysomes of both RPS19 and RPL11 cells and that translation of BCAT1 protein is especially impaired in cells with small RP gene mutations, and we provide evidence that this effect may be due in part to the unusually long 5'UTR of the BCAT1 transcript. The BCAT1 enzyme carries out the final step in the biosynthesis and the first step of degradation of the branched-chain amino acids leucine, isoleucine, and valine. Interestingly, several animal models of DBA have reported that leucine ameliorates the anemia phenotypes generated by RPS19 loss. Our study suggests that RP mutations affect the synthesis of specific proteins involved in regulating amino acid levels that are important for maintaining the normal proliferative capacity of hematopoietic cells.

Loss of GATA‐1 full length as a cause of Diamond–Blackfan anemia phenotype

Mutations in the hematopoietic transcription factor GATA-1 alter the proliferation/differentiation of hemopoietic progenitors. Mutations in exon 2 interfere with the synthesis of the full-length isoform of GATA-1 and lead to the production of a shortened isoform, GATA-1s. These mutations have been found in patients with Diamond-Blackfan anemia (DBA), a congenital erythroid aplasia typically caused by mutations in genes encoding ribosomal proteins. We sequenced GATA-1 in 23 patients that were negative for mutations in the most frequently mutated DBA genes. One patient showed a c.2T > C mutation in the initiation codon leading to the loss of the full-length GATA-1 isoform.

Mutation of the Diamond-Blackfan Anemia Gene Rps7 in Mouse Results in Morphological and Neuroanatomical Phenotypes

The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7Mtu and Rps7Zma) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.

A Novel Mutation of Ribosomal Protein S10 Gene in a Japanese Patient With Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA) is an inherited bone marrow disease. The condition is characterized by anemia that usually presents during infancy or early childhood and congenital malformation. Several reports show that DBA is associated with mutations in the ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7. Recently, 5 and 12 patients with mutations in RPS10 and RPS26, respectively, were identified in a cohort of 117 DBA probands. Therefore, we screened the DBA patients who were negative for mutations in these DBA genes for mutations in RPS10 and RPS26. The present case report describes the identification of the first Japanese DBA patient with a novel mutation in RPS10.