Recent insights into the pathogenesis of Diamond-Blackfan anaemia

Diamond-Blackfan anaemia (DBA) is a congenital anaemia and broad developmental disease that develops soon after birth. The anaemia is due to failure of erythropoiesis, with normal platelet and myeloid lineages, and it can be managed with steroids, blood transfusions, or stem cell transplantation. Normal erythropoiesis after transplantation shows that the defect is intrinsic to an erythroid precursor. DBA is inherited in about 10-20% of cases, and genetic studies have identified mutations in a ribosomal protein gene, RPS19, in 25% of cases; there is evidence for involvement of at least two other genes. In yeast, RPS19 deletion leads to a block in ribosomal RNA biogenesis. The critical question is how mutations in RPS19 lead to the failure of proliferation and differentiation of erythroid progenitors. While this question has not yet been answered, understanding the biology of DBA may provide insight not only into the defect in erythropoisis, but also into the other developmental abnormalities that are present in about 40% of patients, and into the cancer predisposition that is inherent to DBA.     

Clinical and laboratory evidence for a trilineage haematopoietic defect in patients with refractory Diamond-Blackfan anaemia

Diamond-Blackfan anaemia (DBA) is a constitutional pure red cell aplasia presenting in early childhood. In some patients, neutropenia and/or thrombocytopenia have also been observed during the course of the disease. We have followed 28 patients with steroid-refractory DBA for up to 13 years with serial peripheral blood counts and bone marrow (BM) aspirates and biopsies. In 21/28 (75%) patients, moderate to severe generalized BM hypoplasia developed, with overall cellularities ranging from 0% to 30%. Marrow hypoplasia correlated with the development of neutropenia (9/21; 43%) and/or thrombocytopenia (6/21; 29%) in many patients. No patient had either cytogenetic abnormalities or progressed to acute leukaemia, although one 13-year-old developed marked marrow fibrosis and trilineage dysplasia. We used the in vitro long-term culture-initiating cell (LTC-IC) assay to quantify multilineage, primitive haematopoietic progenitors in a representative subset of these patients. LTC-IC assays showed equivalent frequencies of cobblestone area-forming cells (CAFCs) with a mean of 5.42/10(5) cells +/- 1.9 SD and 6.13/10(5) cells +/- 2.6 SD in nine patients and six normal controls respectively. The average clonogenic cell output per LTC-IC, however, was significantly lower in DBA patients (mean 2.16 +/- 1.2 SD vs. 7. 36 +/- 2.7 SD in normal controls, P = 0.0008). Our results suggest that the underlying defect in patients with severe refractory DBA may not be limited to the erythroid lineage, as was evidenced by the development of pancytopenia, bone marrow hypoplasia and reduced clonogenic cell output in LTC-IC assays.     

Deficient RPS19 protein production induces cell cycle arrest in erythroid progenitor cells

The gene encoding ribosomal protein S19 (RPS19) is one of the responsible genes for Diamond-Blackfan anaemia (DBA), a congenital erythroblastopenia. Although haplo-insufficiency of RPS19 has been suggested to be the onset mechanism underlying the pathogenesis of DBA, the sequential mechanism has not been elucidated. In order to analyse the consequences of the missense mutation of RPS19 specific for DBA patients, we made mutated RPS19 expression vectors. Twelve C-terminally Flag-tagged missense mutants were exogenously expressed from retroviral vectors and analysed by Western blot analysis and flow cytometry. When these 12 mutants were expressed in the erythro-leukaemic cell lines K562 and human bone marrow CD34(+) cells, almost all of the mutant proteins (except for G120R) were unstable, and the levels of mutated RPS19 protein were significantly low. To address the effect of deficient RPS19 expression on cell proliferation, RPS19 was downregulated by siRNA. Repressive expression of RPS19 in human CD34(+) cells produced an elevated number of cells at G0 and induced erythroid progenitor-specific defects in BM cells. These results suggest that abnormal ribosomal biogenesis causes inadequate cell cycle arrest in haematopoietic progenitors, and that, subsequently, erythroid progenitors are specifically hampered. These in vitro phenotypes of genetically manipulated CD34(+) cells mimic DBA pathogenesis.     

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.     

Erythropoiesis failure due to RPS19 deficiency is independent of an activated Tp53 response in a zebrafish model of Diamond-Blackfan anaemia

Diamond-Blackfan anaemia (DBA) is a cancer-prone genetic disorder characterized by pure red-cell aplasia and associated physical deformities. The ribosomal protein S19 gene (RPS19) is the most frequently mutated gene in DBA (~25%). TP53-mediated cell cycle arrest and/or apoptosis in erythroid cells have been suggested to be major factors for DBA development, but it is not clear why mutations in the ubiquitously expressed RPS19 gene specifically affect erythropoiesis. Previously, we showed that RPS19 deficiency in zebrafish recapitulates the erythropoietic and developmental phenotypes of DBA, including defective erythropoiesis with severe anaemia. In this study, we analysed the simultaneous loss-of-function of RPS19 and Tp53 in zebrafish to investigate the role of Tp53 in the erythroid and morphological defects associated with RPS19 deficiency. Co-inhibition of Tp53 activity rescued the morphological abnormalities, but did not alleviate erythroid aplasia in RPS19-deficient zebrafish. In addition, knockdown of two other RP genes, rps3a and rpl36a, which result in severe morphological abnormalities but only mild erythroid defects, also elicited an activated Tp53 response. These results suggest that a Tp53-independent but RPS19-dependent pathway could be responsible for defective erythropoiesis in RPS19-deficient zebrafish.     

Familial transient erythroblastopenia of childhood is associated with the chromosome 19q13.2 region but not caused by mutations in coding sequences of the ribosomal protein S19 (RPS19) gene

Transient erythroblastopenia of childhood (TEC) is a rare condition, which at onset may be difficult to distinguish from Diamond-Blackfan anaemia (DBA). We have previously shown that mutations in the ribosomal protein S19 gene (RPS19) cause DBA. In order to clarify whether TEC and DBA are allelic, we investigated the segregation of markers spanning the RPS19 gene region on chromosome 19q13.2 and performed sequence analysis of all exons in the RPS19 gene in seven TEC sibling pairs. Linkage analysis supported allelism for TEC and DBA at the RPS19 gene locus and implies molecular mechanisms other than structural mutations in the RPS19 gene.     

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.     

Evidence of parvovirus B19 infection in patients of pre-eclampsia and eclampsia with dyserythropoietic anaemia

Parvovirus B19 (PVB19) infection can induce transient anaemia in patients with increased erythropoiesis. However, the dynamic change within the bone marrow after PVB19 infection is not well understood. Increased erythropoiesis is a physiological phenomenon in puerperital women. Nevertheless, anaemia as a result of PVB19 infection in puerperital women has never been reported. We report one patient with eclampsia and two patients with pre-eclampsia who had transient, severe anaemia during the puerperital period because of PVB19 infection. Viral genomes were detected in the peripheral blood during the anaemic period by polymerase chain reaction and became undetectable after the anaemia was resolved. Viral genomes and protein could also be detected in bone marrow by in situ hybridization and immunohistochemical staining, respectively. Serial aspiration cytology of bone marrow showed severe dysplastic change involving erythroid precursors with a few apoptotic cells at the initial onset of anaemia, markedly increased apoptotic cells that was confirmed by the increased expression of activated caspase 3, around the nadir of anaemia, and a normal marrow picture without features of apoptosis after recovery from anaemia. Our data indicates that PVB19 infection can induce transient, severe dyserythropoietic anaemia in puerperital women with pre-eclampsia/eclampsia and the pathogenetic mechanism may probably involve the induction of apoptosis following PVB19 infection.     

Telomere length and somatic mutations in correlation with response to immunosuppressive treatment in aplastic anaemia

We investigated the frequencies of cytogenetic aberrations and somatic mutations of prognostic relevance in 393 patients with aplastic anaemia (AA). Clonality was determined by G‐banding/fluorescence in situ hybridization (FISH) (n = 245), and targeted capture sequencing was performed for 88 haematopoiesis‐related genes (n = 70). The telomere length (TL) of bone marrow nucleated cells was measured at the single cell level by FISH (n = 135). Eighteen (4·6%) patients showed disease progression, and monosomy 7 (50·0%) was the most predominant cytogenetic evolution at disease transformation. One third of patients (32·9%) presented at least 1 mutation; the most frequently mutated genes were NOTCH1, NF1, SCRIB, BCOR and DNMT3A. The patient group with clonal changes (30·7%) showed an adverse response to immunosuppressive treatment (IST), compared to the non‐clonal group, but this finding did not show statistical significance. The TL of AA patients was significantly shorter than normal control and patients with clonal changes showed significantly shorter TLs. Patients with TL>5·9 showed a higher response rate to IST (P = 0·048). In conclusion, the patients with clonal changes or TL attrition showed a poor response to IST. Shorter TL can be used not only as a biomarker, but also as a predictive marker for treatment response to IST.     

Fanconi anaemia group A (FANCA) mutations in Israeli non-Ashkenazi Jewish patients

Fanconi anaemia (FA) is a genetically heterogeneous disease with at least eight complementation groups (A-H). In the present study, we investigated the molecular basis of the disease in 13 unrelated Israeli Jewish (non-Ashkenazi) patients with FA. All 43 exons of the Fanconi anaemia A (FANCA) gene were amplified from genomic DNA and screened for mutations by single-strand conformation polymorphism and DNA sequencing. We identified four ethnic-specific mutations: (1) 2172-2173insG (exon 24), the first 'Moroccan mutation': (2) 4275delT (exon 43), the second 'Moroccan mutation'; (3) 890-893del (exon 10), the 'Tunisian mutation'; and (4) 2574C > G (S858R), the 'Indian mutation'. The tetranucleotide CCTG motif, previously identified as a mutation hotspot in FANCA and other human genes, was found in the vicinity of 2172-2173insG and 890-893del. According to our study, the four mutations account for the majority (88%) of the FANCA alleles in the Israeli Jewish (non-Ashkenazi) FA population. A screening of 300 Moroccan Jews identified three carriers of the first 'Moroccan mutation', but we did not find any carrier of the second 'Moroccan mutation' among 140 Moroccan Jews, nor any carrier of the 'Tunisian mutation' among 50 Tunisian Jews. Two 'Indian mutation' carriers were identified among 53 Indian Jews. All carriers within each ethnic group had the same haplotype, suggesting a common founder for each mutation.     

Relative response of patients with myelodysplastic syndromes and other transfusion-dependent anaemias to deferasirox (ICL670): a 1-yr prospective study

Objectives/methods:  This 1-yr prospective phase II trial evaluated the efficacy of deferasirox in regularly transfused patients aged 3–81 yrs with myelodysplastic syndromes (MDS; n  = 47), Diamond–Blackfan anaemia (DBA; n  = 30), other rare anaemias ( n  = 22) or β-thalassaemia ( n  = 85). Dosage was determined by baseline liver iron concentration (LIC).
Results:  In patients with baseline LIC ≥7 mg Fe/g dry weight, deferasirox initiated at 20 or 30 mg/kg/d produced statistically significant decreases in LIC ( P  < 0.001); these decreases were greatest in MDS and least in DBA. As chelation efficiency and iron excretion did not differ significantly between disease groups, the differences in LIC changes are consistent with mean transfusional iron intake (least in MDS: 0.28 ± 0.14 mg/kg/d; greatest in DBA: 0.4 ± 0.11 mg/kg/d). Overall, LIC changes were dependent on dose ( P  < 0.001) and transfusional iron intake ( P  < 0.01), but not statistically different between disease groups. Changes in serum ferritin and LIC were correlated irrespective of disease group ( r  = 0.59), supporting the potential use of serum ferritin for monitoring deferasirox therapy. Deferasirox had a safety profile compatible with long-term use. There were no disease-specific safety/tolerability effects: the most common adverse events were gastrointestinal disturbances, skin rash and non-progressive serum creatinine increases.
Conclusions:  Deferasirox is effective for reducing iron burden with a defined, clinically manageable safety profile in patients with various transfusion-dependent anaemias. There were no disease-specific adverse events. Once differences in transfusional iron intake are accounted for, dose-dependent changes in LIC or serum ferritin are similar in MDS and other disease groups.     

G6PD gene mutations in India producing drug-induced haemolytic anaemia

We report 17 cases of glucose-6-phosphate dehydrogenase (G6PD) deficiency with drug-induced haemolytic anaemia. In most cases the drug involved was an antimalarial. However, we also found two cases in which other drugs could have been responsible for the haemolysis. The degree of severity of haemolysis differed in the individuals and most required multiple transfusions.     

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.