Diamond Blackfan anaemia in the UK: clinical and genetic heterogeneity

A detailed family study was undertaken of patients notified to the UK Diamond Blackfan Anaemia (DBA) Registry. RPS19 mutations were detected in 16 of 104 families, including two patients with deletions detected by intragenic loss of heterozygosity of tightly linked polymorphisms. In two further cases, polymorphisms were used to determine the parental allele of origin of RPS19 point mutations. A review of clinical details of patients with mutations and patients in the literature having identical or equivalent mutations revealed evidence for a genotype:phenotype correlation with respect to the prevalence of physical anomalies, and the occurrence of mild or variable haematological severity. Nine of 60 patients had a known family history of DBA. Haematological abnormalities, including raised red cell adenosine deaminase activity, were found in first-degree relatives of 16 of 51 (31%) of patients not previously considered to have familial DBA. Results of both parents and any siblings were normal in only 35 of 60 (58%) of cases, who were therefore assumed to have sporadic de novo DBA. The classical inheritance pattern for DBA is autosomal dominant; however, 12 of 60 families (20%) had more than one affected child despite normal results in both parents. These results have important implications for genetic counselling, and for the selection of potential sibling bone marrow donors.     

Ribosomal RNA analysis in the diagnosis of Diamond‐Blackfan Anaemia

Diamond‐Blackfan anaemia (DBA) is an inherited disease characterized by pure erythroid aplasia that has been tagged as a ‘ribosomopathy’. We report a multi‐centre study focused on the analysis of rRNA processing of 53 Italian DBA patients using capillary electrophoresis analysis of rRNA maturation of the 40S and 60S ribosomal subunits. The ratio of 28S/18S rRNA was higher in patients with mutated ribosomal proteins (RPs) of the small ribosomal subunit. In contrast, patients with mutated RPs of the large ribosomal subunit (RPLs) had a lower 28S/18S ratio. The assay reported here would be amenable for development as a diagnostic tool.     

Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference

Diamond Blackfan anaemia (DBA) is a rare, genetically and clinically heterogeneous, inherited red cell aplasia. Classical DBA affects about seven per million live births and presents during the first year of life. However, as mutated genes have been discovered in DBA, 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.     

Target enrichment and high‐throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond‐Blackfan anaemia

Diamond‐Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50–60% of cases. The remaining 40–50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high‐throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high‐throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA.     

Loss of function mutations in RPL27 and RPS27 identified by whole‐exome sequencing in Diamond‐Blackfan anaemia

Diamond‐Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond‐Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond‐Blackfan anaemia, we performed whole‐exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond‐Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond‐Blackfan anaemia. In vitro knockdown of gene expression disturbed pre‐ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond‐Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond‐Blackfan anaemia.     

Haematopoietic stem cell transplantation for Diamond Blackfan anaemia: a report from the Italian Association of Paediatric Haematology and Oncology Registry

Allogeneic haematopoietic stem cell transplantation (HSCT) is the only curative option for patients with Diamond Blackfan anaemia (DBA). We report the transplantation outcome of 30 Italian DBA patients referred to the Italian Association of Paediatric Haematology and Oncology Registry between 1990 and 2012. This is one of the largest national registry cohorts of transplanted DBA patients. Most patients (83%) were allografted after 2000. A matched sibling donor was employed in 16 patients (53%), the remaining 14 patients (47%) were transplanted from matched unrelated donors. Twenty‐eight of the 30 patients engrafted. One patient died at day +6 due to veno‐occlusive disease without achieving neutrophil recovery and another patient remained transfusion‐dependent despite the presence of a full donor chimerism. The 5‐year overall survival and transplant‐related mortality was 74·4% and 25·6%, respectively. Patients younger than 10 years as well as those transplanted after 2000 showed a significantly higher overall survival and a significantly lower risk of transplant‐related mortality. No difference between donor type was observed. Our data suggest that allogeneic HSCT from a related or unrelated donor was a reasonable alternative to transfusion therapy in young and well chelated DBA patients.     

In vitro progenitor analysis in a Diamond Blackfan anaemia patient who responded once but not twice to interleukin-3 therapy, using short-term and long-term cultures and c-kit analysis

Interleukin-3 (IL-3) therapy as a treatment for Diamond Blackfan anaemia (DBA) patients has been largely disappointing despite early hope it would be suitable for stimulating arrested erythropoiesis. Initial hope came from in vitro discoveries that IL-3 (+EPO) generated well-haemoglobinized BFU-E colonies in some patients, but was soon tempered by the realization that in vitro and in vivo IL-3 response did not, in the majority of cases, correlate. Nevertheless in vitro testing has been the main focus in analysing the abnormality in the stem and progenitor cell compartment in DBA. Here we report in vitro analysis of a DBA patient who responded once to IL-3 therapy, but not a second time following relapse, using short-term culture, long-term culture and c-kit analysis. Progenitor numbers before and after the first therapy were in the high normal range, but after relapse were much reduced below normal levels. Long-term cultures suggested some arrested progenitors had been reactivated into normal cycle by the first therapy, but may not have been replaced by more immature progenitors. c-kit analysis revealed increased expression in all tested cell populations. These results imply that the first IL-3 therapy reactivated some erythroid progenitors but left the progenitor pool depleted when more immature cells remained arrested.     

Successful treatment of Diamond-Blackfan anemia with metoclopramide

Diamond-Blackfan anemia (DBA) is a congenital anemia characterized by a low reticulocyte count, the absence or severe reduction of hemoglobin-containing cells in the bone marrow, and normal megakaryocytic and granulocytic differentiation. Although the anemia may initially respond to corticosteroid therapy, many patients require lifelong red blood cell (RBC) transfusion, leading to infectious complications and iron overload. Metoclopramide has recently been used to treat DBA. Treatment with metoclopramide induces the release of prolactin from the pituitary and stimulates erythropoiesis. For these reasons, we used metoclopramide to treat a 20-year-old man with DBA refractory to low and high doses of corticosteroids, cyclosporin A, and tacrolimus (FK506). The hemoglobin and hematocrit slowly increased, and he has remained asymptomatic and transfusion-independent for 8 months. Metoclopramide therapy should be considered in patients with refractory DBA before treatment-related complications develop.     

Glucocorticoids improve erythroid progenitor maintenance and dampen Trp53 response in a mouse model of Diamond–Blackfan anaemia

Diamond–Blackfan anaemia (DBA) is a rare congenital disease causing severe anaemia and progressive bone marrow failure. The majority of patients carry mutations in ribosomal proteins, which leads to depletion of erythroid progenitors in the bone marrow. As many as 40% of all DBA patients receive glucocorticoids to alleviate their anaemia. However, despite their use in DBA treatment for more than half a century, the therapeutic mechanisms of glucocorticoids remain largely unknown. Therefore we sought to study disease specific effects of glucocorticoid treatment using a ribosomal protein s19 (Rps19) deficient mouse model of DBA. This study determines for the first time that a mouse model of DBA can respond to glucocorticoid treatment, similar to DBA patients. Our results demonstrate that glucocorticoid treatment reduces apoptosis, rescues erythroid progenitor depletion and premature differentiation of erythroid cells. Furthermore, glucocorticoids prevent Trp53 activation in Rps19‐deficient cells‐ in a disease‐specific manner. Dissecting the therapeutic mechanisms behind glucocorticoid treatment of DBA provides indispensible insight into DBA pathogenesis. Identifying mechanisms important for DBA treatment also enables development of more disease‐specific treatments of DBA.     

Anaemia of prematurity: Pathophysiology and treatment

Most infants with birth weight < 1.0 kg are given multiple red blood cell (RBC) transfusions within the first few weeks of life. The anaemia of prematurity is caused by untimely birth occurring before placental iron transport and fetal erythropoiesis are complete, by phlebotomy blood losses taken for laboratory testing, by low plasma levels of erythropoietin due to both diminished production and accelerated catabolism, by rapid body growth and need for commensurate increase in red cell volume/mass, and by disorders causing RBC losses due to bleeding and/or hemolysis. RBC transfusions are the mainstay of therapy with recombinant human erythropoietin largely unused because it fails to substantially diminish RBC transfusion needs — despite exerting substantial erythropoietic effects on neonatal marrow.     

Anaemia in rheumatoid arthritis: pathogenesis,diagnosis and treatment

Summary The pathogenesis, diagnosis and treatment of the anaemia of chronic disorders (ACD) in rheumatoid arthritis (RA) were reviewed. Causes of anaemia other than ACD frequently present in RA. Decreased iron absorption was shown to be the result of active RA rather than a cause of ACD or iron deficiency. It has been hypothesized that bone morrow iron availability decreases due to decreased iron release by the mononuclear phagocyte system or that the anaemia in ACD is due to ineffective erythropoiesis; these remain controversial theories. Studies considering a decreased erythropoietin responsiveness have not produced consistent results. Erythroid colony growth is suppressed in vitro by interleukins and tumour necrosis factor but their role in vivo in ACD is unknown. The diagnosis of ACD is made by exclusion. Iron deficiencyis detected by transferrin, ferritin, and cellular indices after adaption of their normal values. Treatment of the anaemia consists merely of antirheumatic treatment. Iron administration is counterproductive since iron chelators or exogenous erythropoietin administration might increase erythropoiesis.     

Hepcidin and Cytokines in Anaemia

Hepcidin is a cytokine-induced antibacterial protein which is produced in the liver, circulates in the blood, and is excreted in the urine. It is a major regulator of iron balance in the intestinal mucosa, and appears to have a significant role in the pathogenesis of haemochromatosis and related disorders. Hepcidin appears to be a major contributor to the hypoferraemia associated with inflammation. Serum ferritin concentration is strongly correlated with hepcidin protein levels in either urine or serum, and certain patients with hepatic adenomas exhibit a microcytic, hypoferraemic hepcidin-dependent anaemia. For these reasons, it has been proposed that hepcidin is a primary factor in the pathogenesis of the anaemia of chronic disease (ACD), a cytokine-mediated anaemia commonly encountered in clinical practice and characterized by hypoferraemia with adequate reticuloendothelial iron stores. However, the pathogenetic basis of ACD is not entirely due to changes in iron metabolism, but also involves abnormalities in red cell survival and the erythropoietic response to anaemia. In this review, the evidence for involvement of hepcidin as a major mediator of ACD is evaluated. Hepcidin appears to be a major factor in the systemic iron abnormalities seen in ACD; whether it contributes to the other aspects of the pathogenesis of the syndrome requires further investigation.     

Chronic parvovirus infection and G6PD deficiency masquerading as Diamond-Blackfan anemia

Bone marrow aplasia due to parvovirus B19 infections is usually mild and self-limited. In patients with hereditary or acquired hemolytic anemias, B19 infection can cause a severe and life-threatening anemia due to the shortened half-life of red cells, but here, too, the transient nature of the infection soon remits the symptoms. Chronic infections with parvovirus are more characteristically associated with immunodeficiency states. We report here a case of B19-induced anemia in a patient with G6PD deficiency and hypogammaglobulinemia which was mislabeled as Diamond-Blackfan anemia until the use of modern laboratory techniques allowed a correct diagnosis to be made.