Related Articles

Diamond-Blackfan anemia (DBA) is a rare, pure red-cell aplasia that presents during infancy. Approximately 40% of cases are associated with other congenital defects, particularly malformations of the upper limb or craniofacial region. Mutations in the gene coding for the ribosomal protein RPS19 have been identified in 25% of patients with DBA, with resulting impairment of 18S rRNA processing and 40S ribosomal subunit formation. Moreover, mutations in other ribosomal protein coding genes account for about 25% of other DBA cases. Recently, the analysis of mice from which the gene coding for the heme exporter Feline Leukemia Virus subgroup C Receptor (FLVCR1) is deleted suggested that this gene may be involved in the pathogenesis of DBA. FLVCR1-null mice show a phenotype resembling that of DBA patients, including erythroid failure and malformations. Interestingly, some DBA patients have disease linkage to chromosome 1q31, where FLVCR1 is mapped. Moreover, it has been reported that cells from DBA patients express alternatively spliced isoforms of FLVCR1 which encode non-functional proteins. Herein, we review the known roles of RPS19 and FLVCR1 in ribosome function and heme metabolism respectively, and discuss how the deficiency of a ribosomal protein or of a heme exporter may result in the same phenotype.

Diamond-Blackfan Anemia (DBA) is a congenital erythroid aplasia characterized as a normochromic macrocytic anemia with a selective deficiency in red blood cell precursors in otherwise normocelullar bone marrow. DBA is known to be associated with mental retardation and learning disabilities. Although comorbidities with other psychiatric conditions have not been reported in the existing literature, we report in this paper a case of a DBA patient with previously undiagnosed comorbidity of obsessive compulsive disorder (OCD), successfully treated with sertaline 200 mg/day and valproic acid 600 mg/day. This case of comorbid presentation has clinical, therapeutic and pathophysiological implications. Given the difficulty of distinguishing among mental retardation, learning disabilities and OCD and the importance of precocious diagnosis in treating OCD especially since there are treatment methods interfering with anemia symptoms, physicians should adapt an adequate screening tool treating a child with DBA and comorbid mental disorder.

Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia, bone marrow (BM) erythroblastopenia, and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in ~25% of DBA patients; however, the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis, we compared highly purified multipotential, erythroid, and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes, such as TNFRSF10B and FAS, transcriptional control genes, including the erythropoietic transcription factor MYB (encoding c-myb), and translational genes were greatly dysregulated, mostly in diseased erythroid cells. Cancer-related genes, including RAS family oncogenes and tumor suppressor genes, were significantly dysregulated in all diseased progenitors. In addition, our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes, as well as downregulation of genes involved in translation in DBA cells. In conclusion, the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression, which causes complete failure of fetal liver erythropoiesis in knockout mice, suggests a link between RPS19 mutations and reduced erythropoiesis in DBA.

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplastic anemia, characterized by macrocytic anemia, reticulocytopenia, and severely reduced numbers of erythroid precursors in the bone marrow. For more than fifty years, glucocorticoids have remained the main option for pharmacological treatment of DBA. While continuous glucocorticoid administration increases hemoglobin levels in a majority of DBA patients, it also causes severe side effects. There is therefore a great need for more specific and effective treatments to boost or replace the use of glucocorticoids. Over the years, many alternative therapies have been tried out, but most of them have shown to be ineffective. Here we review previous and current attempts to develop such alternative therapies for DBA. We further discuss how emerging knowledge regarding the pathological mechanism in DBA and the therapeutic mechanism of glucocorticoids treatment may reveal novel drug targets for DBA treatment.

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a specific decrease of erythroid precursors. The disease is also associated with growth retardation, congenital malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. We show herein that primary fibroblast from DBA patients with truncating mutations in RPS19 or in RPS24 have a marked reduction in proliferative capacity. Mutant fibroblasts are associated with extended cell cycles and normal levels of p53 when compared to w.t. cells. RPS19 mutant fibroblasts accumulate in the G1 phase, whereas the RPS24 mutant cells show an altered progression in the S phase resulting in reduced levels in the G2/M phase. RPS19 deficient cells exhibit reduced levels of Cyclin-E, CDK2 and retinoblastoma (Rb) protein supporting a cell cycle arrest in the G1 phase. In contrast, RPS24 deficient cells show increased levels of the cell cycle inhibitor p21 and a seemingly opposing increase in Cyclin-E, CDK4 and CDK6. In combination, our results show that RPS19 and RPS24 insufficient fibroblasts have an impaired growth caused by distinct blockages in the cell cycle. We suggest this proliferative constraint to be an important contributing mechanism for the complex extra-hematological features observed in DBA.

Despite significant improvements in our understanding of the pathophysiology of Diamond Blackfan anemia (DBA), there have been few advances in therapy. The cornerstones of treatment remain corticosteroids, chronic red cell transfusions, and hematopoietic stem cell transplantation, each of which is fraught with complications. In this article, we will review the history of therapies that have been offered to patients with DBA, summarize the current standard of care including management of side effects, and discuss novel therapeutics that are being developed in the context of the research into the roles of ribosomal haploinsufficiency and p53 activation in Diamond Blackfan anemia.

Diamond-Blackfan Anemia (DBA) is a red cell aplasia characterized with physical abnormalities. The incidence of the disease is reported to be five to seven (5–7) cases per million births in Europe and 4–5 per million live births in the UK and Netherlands with equal sex ratio. It was first recognized in 1938 but an exact pathophysiology of the disease has not been described yet. These abnormalities are well known, however, detailed oral and dental conditions related with the disease have not been described previously. We herein presented two cases of DBA together with oral and dental findings. Our study is first to report the gingival status of the patients with a complete investigation of any orthodontic or dental abnormalities in these kind of patients. A careful follow up and preventive therapies should not be missed in these kind of patients.

Background

The Ribosomal protein S19 gene locus (RPS19) has been linked to two kinds of red cell aplasia, Diamond-Blackfan Anemia (DBA) and Transient Erythroblastopenia in Childhood (TEC). Mutations in RPS19 coding sequences have been found in 25% of DBA patients, but not in TEC patients. It has been suggested that non-coding RPS19 sequence variants contribute to the considerable clinical variability in red cell aplasia. We therefore aimed at identifying non-coding variations associated with DBA or TEC phenotypes.

Methodology/Principal Findings

We targeted a region of 19'980 bp encompassing the RPS19 gene in a cohort of 89 DBA and TEC patients for resequencing. We provide here a catalog of the considerable, previously unrecognized degree of variation in this region. We identified 73 variations (65 SNPs, 8 indels) that all are located outside of the RPS19 open reading frame, and of which 67.1% are classified as novel. We hypothesize that specific alleles in non-coding regions of RPS19 could alter the binding of regulatory proteins or transcription factors. Therefore, we carried out an extensive analysis to identify transcription factor binding sites (TFBS). A series of putative interaction sites coincide with detected variants. Sixteen of the corresponding transcription factors are of particular interest, as they are housekeeping genes or show a direct link to hematopoiesis, tumorigenesis or leukemia (e.g. GATA-1/2, PU.1, MZF-1).

Conclusions

Specific alleles at predicted TFBSs may alter the expression of RPS19, modify an important interaction between transcription factors with overlapping TFBS or remove an important stimulus for hematopoiesis. We suggest that the detected interactions are of importance for hematopoiesis and could provide new insights into individual response to treatment.

Summary

Diamond Blackfan anemia (DBA) is a rare, genetically and clinically heterogeneous, inherited red cell aplasia. Classical DBA presents during the first year of life, affecting about 7 per million live births. However, as genes mutated in DBA have been discovered, non-classical cases with less distinct phenotypes are being described in adults as well as children. In caring for these patients it is often difficult to have a clear understanding of the treatment options and their outcomes because of the lack of complete information on the natural history of the disease. The purpose of this document is to review the criteria for diagnosis, evaluate the available treatment options, including corticosteroid and transfusion therapies and stem cell transplantation, and propose a plan for optimizing patient care. Congenital anomalies, mode of inheritance, cancer predisposition, and pregnancy in DBA are also reviewed. Evidence-based conclusions will be made when possible; however, as in many rare diseases, the data are often anecdotal and the recommendations are based upon the best judgment of experienced clinicians. The recommendations regarding the diagnosis and management described in this report are the result of deliberations and discussions at an international consensus conference.

Diamond-Blackfan anemia (DBA) is congenital pure red-cell anemia due to a differentiation block in erythroid precursors. The disease is commonly caused by mutations in genes for ribosomal proteins. Despite the identification of disease causal genes, the disease pathogenesis is not completely elucidated. The ribosomal abnormalities are assumed to inhibit globin translation which may lead to excess free heme, stimulating a generation of free radicals and thereby damaging the precursors. We studied the effect of hemin (heme chloride) on cultured human erythroid precursors and found that contrary to aforementioned hypothesis, although hemin moderately stimulated free radicals, it did not cause apoptosis or necrosis. In erythroid precursors derived from DBA patients, hemin significantly stimulated growth and hemoglobinization. Thus, heme toxicity is unlikely to play a role in the pathophysiology of most DBA cases. Moreover, its beneficial effect in culture suggests a therapeutic potential.

Diamond-Blackfan anemia (DBA) is a hypoplastic anemia characterized by impaired production of red blood cells, with approximately half of all cases attributed to ribosomal protein gene mutations. We performed exome sequencing on two siblings who had no known pathogenic mutations for DBA and identified a mutation in the gene encoding the hematopoietic transcription factor GATA1. This mutation, which occurred at a splice site of the GATA1 gene, impaired production of the full-length form of the protein. We further identified an additional patient carrying a distinct mutation at the same splice site of the GATA1 gene. These findings provide insight into the pathogenesis of DBA, showing that the reduction in erythropoiesis associated with the disease can arise from causes other than defects in ribosomal protein genes. These results also illustrate the multifactorial role of GATA1 in human hematopoiesis.

Purpose

The inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders that share the inability of the bone marrow to produce an adequate number of blood cells. The four most frequent syndromes are Fanconi anemia (FA), dyskeratosis congenita (DC), Diamond-Blackfan anemia (DBA) and Shwachman-Diamond syndrome (SDS). All four syndromes have been associated with various physical abnormalities. As part of a genotype/phenotype/cancer susceptibility study, we determined the prevalence of ophthalmic manifestations in these four syndromes.

Design

Cross-sectional study of a patient cohort.

Participants

Seventy-five patients with an IBMFS and 121 of their first degree relatives were seen in the National Eye Institute, National Institutes of Health from 2001 to 2007. The patient group included 22 FA, 28 DC, 19 DBA and six SDS.

Methods

Every participant underwent a complete ophthalmic evaluation, as well as digital facial photography with an adhesive paper ruler on the patient's forehead for an internal measure of scale. Interpupillary distance (IPD), inner canthal distance (ICD), outer canthal distance (OCD), palpebral fissure length (PFL) and corneal diameter (CD) were measured. Thirteen of the 22 patients with FA underwent axial length (AL) measurements by A-scan ultrasonography.

Main Outcome Measures

Type and prevalence of ophthalmic manifestations.

Results

Ninety-five percent of patients with FA had at least one abnormal parameter, and 25% at least four abnormal parameters. Eighty-two percent had small palpebral fissures, 69% simple microphthalmia, 64% small OCD, 55% microcornea, 28% ptosis, and six percent epicanthal folds. In patients with DC, abnormalities of the lacrimal drainage system (29%) were the most prevalent findings, followed by retinal abnormalities (pigmentary changes, retinal neovascularization, retinal detachment, exudative retinopathy) in 21%, cicatricial entropion with trichiasis and blepharitis in seven percent each, sparse eyelashes and congenital cataract in three and a half percent each. No significant ophthalmic abnormalities were seen in patients with DBA or SDS.

Conclusions

Syndrome-specific ocular findings are associated with FA and DC and may antedate diagnosis of the specific syndrome. Early recognition of these abnormalities is important for optimal management.

Synopsis

Diamond Blackfan anemia (DBA) is a genetically and clinically heterogeneous disorder characterized by erythroid failure, congenital anomalies and a predisposition to cancer. Faulty ribosome biogenesis, resulting in pro-apoptotic erythropoiesis leading to erythroid failure, is hypothesized to be the underlying defect. The genes identified to date that are mutated in DBA all encode ribosomal proteins associated with either the small (RPS) or large (RPL) subunit and in these cases haploinsufficiency gives rise to the disease. Extraordinarily robust laboratory and clinical investigations have recently led to demonstrable improvements in clinical care for patients with DBA.

Mutations in numerous genes encoding ribosomal proteins (RPs) occur in 50%–70% of individuals with Diamond-Blackfan anemia (DBA), establishing the disease as a ribosomopathy. As described in this issue of JCI, Sankaran, Gazda, and colleagues used genome-wide exome sequencing to study DBA patients with no detectable mutations in RP genes. They identified two unrelated pedigrees in which the disease is associated with mutations in GATA1, which encodes an essential hematopoietic transcription factor with no known mechanistic links to ribosomes. These findings ignite an interesting and potentially emotional debate on how we define DBA and whether the term should be restricted to pure ribosomopathies. More generally, the work reflects the powerful knowledge and controversies arising from the deluge of data generated by new genetic technologies that are being used to analyze human diseases.

Purpose: Diamond–Blackfan anaemia (DBA) is a rare pure congenital red cell aplasia, usually presenting in infancy or early childhood. The literature suggests a predisposition to haemopoietic malignancy but in addition solid tumours have been reported, with five cases of osteosarcoma described.

Patient: A sixth case of a 12-year-old girl with DBA who developed an osteosarcoma of the distal femur is presented.

Results: She was treated with methotrexate followed by tumour excision and distal femoral replacement. The patient is currently alive with multiple pulmonary metastases.

Discussion: We discuss the association between the administration of growth hormone and future development of malignancy in patients with DBA.

Diamond-Blackfan anaemia (DBA) is a constitutional red blood cell hypoplasia which may be associated with a variety of developmental abnormalities. A gene for DBA was recently mapped to chromosome 19q13.2 and subsequently cloned. Analysis of 19q marker alleles in DNA of sporadic DBA cases showed de novo microdeletions in three patients also presenting with mental retardation. We have studied one of these patients and characterised the deletion by fluorescence in situ hybridisation (FISH) to extended DNA fibres. The deletion was shown to be continuous over a 3.2 Mb region and the fibre-FISH analysis showed both chromosomal breakpoints. In combination, the clinical and molecular findings suggest a contiguous gene syndrome with a gene locus for mental retardation and, probably, skeletal malformations included in the deletion.


Keywords: chromosome 19q13; microdeletion syndrome; fibre-FISH

Diamond Blackfan anemia (DBA) is a lineage-selective inherited bone-marrow failure syndrome characterized primarily by anemia and physical malformations. Recent advances in identifying the genetic abnormalities underlying DBA have demonstrated involvement of genes encoding both large (RPL) and small (RPS) ribosomal subunit proteins, including mutations of RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26 in 50–60% of affected patients. Despite significant progress, identification of gene abnormalities in the remaining patients remains an important question since present data suggests that mutations in other members of the ribosomal protein gene complement do not explain those cases without an identified genetic lesion in these genes. Genetic studies have also raised new questions with the recognition of substantial variability in the manifestations of DBA, ranging from ribosomal protein mutations in otherwise asymptomatic individuals to those with classic severe red-cell aplasia with characteristic malformations, at times within the same kindred. In this review, we summarize the genetic basis of DBA and discuss mechanisms by which the phenotype of DBA might be modified.

Heterozygous mutations in the ribosomal protein S19 (RPS19) gene are associated with Diamond-Blackfan anemia (DBA). The mechanism by which RPS19 mediates anemia are still unclear, as well as the regulation of RPS19 expression. We show herein that RPS19 binds specifically to the 5′ untranslated region of its own mRNA with an equilibrium binding constant (KD) of 4.1±1.9 nM. We investigated the mRNA binding properties of two mutant RPS19 proteins (W52R and R62W) identified in DBA patients. We observed a significant increase in KD for both proteins (16.1±2.1 and 14.5±4.9 nM, respectively), indicating a reduced RNA binding capability (p<0.05). We suggest that the binding of RPS19 to its mRNA has a regulatory function and hypothesize that the weaker RNA binding of mutant rRPS19 may have implications for the pathophysiological mechanisms in DBA.

Background

Diamond-Blackfan anemia (DBA) is a lineage specific and congenital erythroblastopenia. The disease is associated with mutations in genes encoding ribosomal proteins resulting in perturbed ribosomal subunit biosynthesis. The RPS19 gene is mutated in approximately 25% of DBA patients and a variety of coding mutations have been described, all presumably leading to haploinsufficiency. A subset of patients carries rare polymorphic sequence variants within the 5′untranslated region (5′UTR) of RPS19. The functional significance of these variants remains unclear.

Methodology/Principal Findings

We analyzed the distribution of transcriptional start sites (TSS) for RPS19 mRNAs in testis and K562 cells. Twenty-nine novel RPS19 transcripts were identified with different 5′UTR length. Quantification of expressed w.t. 5′UTR variants revealed that a short 5′UTR correlates with high levels of RPS19. The total levels of RPS19 transcripts showed a broad variation between tissues. We also expressed three polymorphic RPS19 5′UTR variants identified in DBA patients. The sequence variants include two insertions (c.-147_-146insGCCA and c.-147_-146insAGCC) and one deletion (c.-144_-141delTTTC). The three 5′UTR polymorphisms are associated with a 20–30% reduction in RPS19 protein levels when compared to the wild-type (w.t.) 5′UTR of corresponding length.

Conclusions

The RPS19 gene uses a broad range of TSS and a short 5′UTR is associated with increased levels of RPS19. Comparisons between tissues showed a broad variation in the total amount of RPS19 mRNA and in the distribution of TSS used. Furthermore, our results indicate that rare polymorphic 5′UTR variants reduce RPS19 protein levels with implications for Diamond-Blackfan anemia.

Diamond–Blackfan anemia (DBA) is a rare congenital disease linked to mutations in the ribosomal protein genes rps19, rps24 and rps17. It belongs to the emerging class of ribosomal disorders. To understand the impact of DBA mutations on RPS19 function, we have solved the crystal structure of RPS19 from Pyrococcus abyssi. The protein forms a five α-helix bundle organized around a central amphipathic α-helix, which corresponds to the DBA mutation hot spot. From the structure, we classify DBA mutations relative to their respective impact on protein folding (class I) or on surface properties (class II). Class II mutations cluster into two conserved basic patches. In vivo analysis in yeast demonstrates an essential role for class II residues in the incorporation into pre-40S ribosomal particles. This data indicate that missense mutations in DBA primarily affect the capacity of the protein to be incorporated into pre-ribosomes, thus blocking maturation of the pre-40S particles.

Diamond Blackfan anemia is a genetic syndrome characterized by red blood cell aplasiain association with developmental abnormalities such as growth retardation, orofacial, hand or limb malformations, urogenital anomalies and heart defects. The only known cause is heterozygosity for mutations in genes encoding ribosomal proteins. Understanding how defective ribosome biogenesis and function, important for all cells, causes defects in erythropoiesis and tissue-specific phenotypes during development is paramount to the evolution of effective treatment protocols. Here, we discuss how animal models based on mammals, insects and fish replicate genetic or developmental aspects of DBA and have led to the identification of pathways and candidate molecules that are important in the pathogenesis of the disease. A recurring theme in many of these models suggests that defective ribosome biogenesis induces a p53-dependent cell cycle checkpoint in cells that require high levels of ribosome production and leads to cell type-specific, whole animal phenotypes.

Summary

Fanconi anaemia (FA), dyskeratosis congenita (DC), Diamond-Blackfan anaemia (DBA), and Shwachman-Diamond syndrome (SDS) comprise major inherited bone marrow failure syndromes (IBMFS). Adverse events include severe bone marrow failure (BMF), myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML), and solid tumours (ST). The natural history of FA is well characterised; hazard rates in the other syndromes have not yet been quantified. An open cohort was established at the National Cancer Institute (NCI) in 2002. Patients enrolled prior to December, 2007 were followed up to December, 2008. Diagnoses were confirmed with standard tests. Age-associated risks of adverse events were calculated. Most patients in each syndrome survived to young adulthood. Patients with FA had earlier onset of cancers, need for stem cell transplant, and death; followed by DC; DBA and SDS were mildest. While FA and DC patients had markedly increased risks of cancer, AML and MDS, there were no cases of leukaemia in DBA or SDS patients. The NCI cohort provides the first direct quantitative comparison of timing and magnitude of cancer risk in the IBMFS. The findings demonstrate that both FA and DC are major cancer susceptibility syndromes. The IBMFS, historically considered paediatric disorders, have important management implications for physicians treating adult patients.

Background

Congenital diaphragmatic hernia (CDH) can occur in isolation or in association with other abnormalities. We hypothesized that some cases of non-isolated CDH are caused by novel genomic disorders.

Methods and Results

In a cohort of >12,000 patients referred for array comparative genomic hybridization testing, we identified three individuals—two of whom had CDH—with deletions involving a ~2.3 Mb region on chromosome 15q25.2. Two additional patients with deletions of this region have been reported, including a fetus with CDH. Clinical data from these patients suggest that recurrent deletions of 15q25.2 are associated with an increased risk of developing CDH, cognitive deficits, cryptorchidism, short stature, and possibly Diamond-Blackfan anemia (DBA). Although no known CDH-associated genes are located on 15q25.2, four genes in this region—CPEB1, AP3B2, HOMER2 and HDGFRP3—have been implicated in CNS development/function and may contribute to the cognitive deficits seen in deletion patients. Deletions of RPS17 may also predispose individuals with 15q25.2 deletions to DBA and associated anomalies.

Conclusions

Individuals with recurrent deletions of 15q25.2 are at increased risk for CDH and other birth defects. A high index of suspicion should exist for the development of cognitive defects, anemia, and DBA-associated malignancies in these individuals.

Ribosomal protein S19 (RPS19) is mutated in patients with Diamond-Blackfan anemia (DBA). We hypothesized that decreased levels of RPS19 lead to a coordinated down-regulation of other ribosomal (r-)proteins at the subunit level. We show that siRNA knock-down of RPS19 results in a relative decrease of small subunit r-proteins (S20, S21 and S24) when compared to large subunit r-proteins (L3, L9, L30 and L38). This correlates with a relative decrease in 18S rRNA with respect to 28S rRNA. The r-protein mRNA levels remain relatively unchanged indicating a post-transcriptional regulation of r-proteins at the level of subunit formation.

Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia (DBA), a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells (HSCs) in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29-/- embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes up-regulated in the rps29 mutant are enriched for genes up-regulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in HSCs and red blood cell development.