ALBERT

Description: Diamond Blackfan Anemia (DBA) is a rare congenital red cell aplasia, presenting in early infancy. The anemia is characteristically steroid responsive in the majority of cases, but eventually 40% of affected individuals are dependent on long-term transfusion and chelation programmes, underscoring the need for alternative therapies. A report by Abkowitz et al (Blood2002;100:2687) suggests a potential benefit of prolactin, induced indirectly by metoclopramide treatment, in a proportion of patients with steroid refractory DBA. We have therefore investigated the in vitro effect of prolactin on erythropoiesis in DBA, using a 2-phase liquid erythroid culture system in which we have previously demonstrated a severe consistent erythroid defect in DBA, and an enhancing effect of added steroids in both normal and DBA cultures. Peripheral blood mononuclear cells were cultured in serum-free medium containing 50ng/ml IL-3, 100ng/ml SCF, 1μg/ml cyclosporin A in the absence of erythropoietin (EPO) for 7 days (phase I), before transfer of non-adherent cells to phase II culture, with medium as for phase I plus 2U/ml EPO. Erythroid output was expressed as the total number of hemoglobinised cells generated after 7 days in phase II culture per cell transferred from phase I. In the absence of steroids, the addition of prolactin (PRL) 20–200ng/ml to both phases had no effect on erythroid output in normal (n=10) or DBA (n=9) cultures (table) normal (n=10) DBA (n=9) Interaction between prolactin and dexamethasone on erythroid output in normal and DBA cultures (mean±SEM) no PRL PRL 50ng/ml no PRL PRL 50ng/ml no dex 5.31±1.38 4.87±1.39 0.24±0.1 0.18±0.07 dex 10−7M 10.30±1.43 10.44±1.40 1.85±0.84 1.86±0.87 We then studied the potential interaction between PRL and steroids, given their known synergy in lactogenesis. While PRL 50ng/ml had no overall effect on mean erythroid output in the presence of 10−7M dex (table), there was striking variation between cultures. Notably, in 3/10 normal cultures there was an apparent prolactin-induced inhibition of the normal steroid stimulatory effect. A similar phenomenon was observed in DBA cultures, with apparent synergy between 50ng/ml PRL and 10−7M dex in 3/9 (erythroid output in PRL+dex 139%, 141% and 180% of the erythroid output with dex alone), but inhibition in 3/9 (erythroid output in PRL+dex 26%, 62%, 87% of output with dex alone). Interestingly, the inhibitory effect of prolactin in DBA cultures appeared to be both more common and more pronounced at the lower PRL concentration of 20ng/ml, equivalent to the top end of the physiological range. Conversely, the stimulatory effect appeared to be more pronounced at lower concentrations of steroid (10−8M). These observations would be consistent with a dose-dependent enhancement by prolactin of steroid sensitivity, causing a left shift of the bell-shaped steroid dose response curve. The complex dose-dependence between steroids and prolactin may be of relevance to the potential therapeutic effect of metoclopramide, which increases endogenous levels of both cortisol and prolactin. Until the interactions between prolactin and steroids are more completely understood, we would advise caution in using metoclopramide in other than transfusion-dependent and steroid refractory DBA, as our results predict the risk of inhibition of steroid responsiveness in vivo, with exacerbation of anemia.

Description: The erythroid defect in Diamond Blackfan anemia (DBA) is known to be intrinsic to the stem cell, but its molecular pathophysiology remains obscure. Using a 2-phase liquid erythroid culture system, we have demonstrated a consistent defect in DBA, regardless of clinical severity, including 3 first-degree relatives with normal hemoglobin levels but increased erythrocyte adenosine deaminase activity. DBA cultures were indistinguishable from controls until the end of erythropoietin (Epo)–free phase 1, but failed to demonstrate the normal synchronized wave of erythroid expansion and terminal differentiation on exposure to Epo. Dexamethasone increased Epo sensitivity of erythroid progenitor cells, and enhanced erythroid expansion in phase 2 in both normal and DBA cultures. In DBA cultures treated with dexamethasone, Epo sensitivity was comparable to normal, but erythroid expansion remained subnormal. In clonogenic phase 2 cultures, the number of colonies did not significantly differ between normal cultures and DBA, in the presence or absence of dexamethasone, and at both low and high Epo concentrations. However, colonies were markedly smaller in DBA under all conditions. This suggests that the Epo-triggered onset of terminal maturation is intact in DBA, and the defect lies down-stream of the Epo receptor, influencing survival and/or proliferation of erythroid progenitors.

Description: Despite excellent long-term results of bone marrow transplantation (BMT) in acquired severe aplastic anemia (SAA), graft-versus-host disease (GVHD) continues to remain a major concern. Approximately 30–40% patients experience chronic GVHD resulting in long-term complications and impaired quality of life. At St. George’s Hospital, we investigated the role of anti-CD52 monoclonal antibodies (MoAb) in transplant protocols for SAA. The CD52 antigen is expressed widely on virtually all lymphocytes including T and B-cells, NK cells, dendritic cells, eosinophils and macrophages but is absent from erythrocytes, platelets and marrow progenitors. Between Aug 1989 and Nov 2003, 33 patients at our centre with acquired SAA underwent BMT from HLA-identical sibling donors using cyclophosphamide (CY) 50 mg/kg x 4 (days −5 to −2) and anti-CD52 MoAb 0.75–1 mg/KgBW as conditioning. Prior to 1999, rat derived anti-CD52 MoAb (Campath-1G) was used. We switched to humanized version of anti-CD52 MoAb (Alemtuzumab) when it became available in 1999. Median age at BMT was 17 yrs (range 4–46). Prior to BMT, 58% were heavily transfused (〉50 transfusions) and 42% had previously failed anti-thymocyte globulin (ATG) based immunosuppressive therapy. Unmanipulated bone marrow was used as source of stem cells in all except one. GVHD prophylaxis was with cyclosporine (CSA) alone in 19 (58%) patients; 14 received anti-CD52 MoAb in addition to CSA. The conditioning regimen was well tolerated without significant acute toxicity. Graft failure was seen in 8 (24%) patients (primary,4; secondary, 4). Graft failure was non-significantly higher in heavily transfused patients and those receiving CSA and anti-CD52 MoAb as GVHD prophylaxis. Of those failing grafts, 4 survived long-term (complete autologous recovery, 2; autotransplant from previous stored marrow, 1; second allograft, 1). Acute grade II-IV GVHD and chronic GVHD was seen in 13% and 4%, respectively. None developed extensive chronic GVHD. Of the 19 recipients positive for cytomegalovirus (CMV), reactivation was seen in 5(26%) with in 100 days. No cases of late CMV reactivation were observed. Six patients died of complications related to BMT at a median of 248 days (range 47–414). With a median follow-up of 59 months, the 5-year survival was 81% (95% C.I. 68–96). There was a non-significant trend towards improved survival in patients transplanted after 1995 (93% vs. 74%). The performance status of all survivors is 100% except one who developed avascular necrosis of hip. We conclude that the conditioning regimen containing CY and anti-CD52 MoAb is well tolerated and efficacious for acquired SAA using HLA-matched sibling donors. The long-term survival with this conditioning appears to be equivalent to current standard conditioning for acquired AA (CY and ATG) and the impact on acute and chronic GVHD is particularly notable. Based on the results of our study we are further investigating the role of Alemtuzumab in the transplant protocols for AA using CSA alone as GVHD prophylaxis.

Description: Improved survival in aplastic anemia (AA) has shown a high incidence of late clonal marrow disorders. To investigate whether accelerated senescence of hematopoietic stem cells might underlie the pathophysiology of myelodysplasia (MDS) or paroxysmal nocturnal hemoglobinuria (PNH) occurring as a late complication of AA, we studied mean telomere length (TRF) in peripheral blood leukocytes from 79 patients with AA, Fanconi anemia, or PNH in comparison with normal controls. TRF lengths in the patient group were significantly shorter for age than normals (P 〈 .0001). Telomere shortening was apparent in both granulocyte and mononuclear cell fractions, suggesting loss at the level of the hematopoietic stem cell. In patients with acquired AA with persistent cytopenias (n = 40), there was significant correlation between telomere loss and disease duration (r = −.685; P 〈 .0001), equivalent to progressive telomere erosion at 216 bp/yr, in addition to the normal age-related loss. In patients who had achieved normal full blood counts (n = 20), the rate of telomere loss had apparently stabilised. There was no apparent association between telomere loss and secondary PNH (n = 13). However, of the 5 patients in the study with TRF less than 5.0 kb, 3 had acquired cytogenetic abnormalities, suggesting that telomere erosion may be relevant to the pathogenesis of MDS in aplastic anemia.

Description: Diamond-Blackfan anemia (DBA), a form of congenital red cell aplasia with marked clinical heterogeneity and increased risk of malignancy, has been associated with mutations in ribosomal protein (RP) gene RPS19 in 25% of probands and in RPS24 or RPS17 in ∼2% of patients. Thus, DBA appears to be a disorder of ribosome synthesis. To test the hypothesis that mutations in other RP genes may also cause DBA, we carried out direct sequencing of candidate RP genes. Genomic DNA samples from 96 unrelated DBA probands (14 familial and 82 sporadic cases) without RPS19 or RPS24 mutations were screened for mutations in RPS3a, RPS13, and RPS16 (previous studies revealed that RPs S19, S24, S3a, S13, and S16 are involved in binding of eIF-2 to the 40S subunit); RP genes L18, L13A, L36, L28, L18A, L40, S5, S9, S11, and S28 (located on chromosome 19); and RP genes, L5, L11, L22, S8, and S27 (on chromosome 1). PCR primers were designed to amplify the coding exons and intron/exon boundaries. We found multiple mutations in two RP genes, L5 and L11. Subsequently we sequenced these two genes in 42 additional DNA samples from DBA probands. In total, we screened 5′UTR, promoter and coding regions, and exon/intron boundaries of RPL5 and RPL11 in 138 DBA unrelated probands. We identified 14 mutations in RPL5 in 138 probands (∼10%), 13 of which are nonsense mutations, deletions or insertions of 1–5 nucleotides causing frameshift and premature termination. One missense mutation, 418G〉A, results in a G140S substitution. We found nine mutations in RPL11 in138 DBA probands (6.5%), including five acceptor or donor splice site mutations (introns 1–4) and four deletions or insertions of 1–4 nucleotides causing frameshifts (codons 32-120). None of these sequence changes were found on the NCBI (http://www.ncbi.nlm.nih.gov/SNP/) or the HapMap (http://www.hapmap.org/) SNP lists. Both genes, as well as RPL23 have recently been demonstrated by others to activate the p53 tumor suppressor protein by inhibiting MDM2-mediated p53 ubiquitination and degradation. Moreover, knockdown of any of these genes by siRNAs markedly reduced p53 induction by the ribosomal biogenesis stressor, actinomycin-D. These findings suggest that DBA patients with mutated L5 and L11 proteins may have inadequate p53 pathway activation and (consistent with clinical observations) be at increased risk for neoplasia. We are currently investigating the role of RPL5 and RPL11 mutations in ribosomal biogenesis and in the p53-mediated cell cycle arrest and apoptosis in DBA patients.

Description: Improved survival in aplastic anemia (AA) has shown a high incidence of late clonal marrow disorders. To investigate whether accelerated senescence of hematopoietic stem cells might underlie the pathophysiology of myelodysplasia (MDS) or paroxysmal nocturnal hemoglobinuria (PNH) occurring as a late complication of AA, we studied mean telomere length (TRF) in peripheral blood leukocytes from 79 patients with AA, Fanconi anemia, or PNH in comparison with normal controls. TRF lengths in the patient group were significantly shorter for age than normals (P 〈 .0001). Telomere shortening was apparent in both granulocyte and mononuclear cell fractions, suggesting loss at the level of the hematopoietic stem cell. In patients with acquired AA with persistent cytopenias (n = 40), there was significant correlation between telomere loss and disease duration (r = −.685; P 〈 .0001), equivalent to progressive telomere erosion at 216 bp/yr, in addition to the normal age-related loss. In patients who had achieved normal full blood counts (n = 20), the rate of telomere loss had apparently stabilised. There was no apparent association between telomere loss and secondary PNH (n = 13). However, of the 5 patients in the study with TRF less than 5.0 kb, 3 had acquired cytogenetic abnormalities, suggesting that telomere erosion may be relevant to the pathogenesis of MDS in aplastic anemia.

Description: Abstract 175 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by anemia usually presenting during infancy or in early childhood, birth defects, and increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, that are present in ∼30–50% of patients. To date, DBA has been associated with mutations in seven ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, L11, and S7 in about ∼43% of patients. To complete our large scale screen of RP genes in a DBA population, we sequenced 49 ribosomal protein genes in our DBA patient cohort of 117 probands. Together with our previous efforts of screening 29 RP genes, this completes the analysis of all known RP genes (excluding RPS4Y on the Y chromosome). Here we report probable mutations in four more RP genes, RPS10, RPS26, RPL19, and RPL26, and variants of unknown significance in six more, RPL9, RPL14, RPL23A, RPL7, RPL35, and RPL3. RPS10 and RPS26 are now confirmed DBA genes as we identified five mutations in RPS10 in five probands and nine mutations in RPS26 in 12 probands. We found single small frameshifting deletions in RPL19 and RPL26 in single DBA families. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knock-down of RPS10 or RPS26 expression with siRNAs, which indicate that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins. This brings to 11, the total number of RP genes mutated in ∼54% of patients with DBA, with nine more genes harboring variants of unknown significance requiring further study. Disclosures: No relevant conflicts of interest to declare.

Description: Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome characterized by anemia, congenital abnormalities, and cancer predisposition. Small ribosomal subunit genes RPS19, RPS24, and RPS17 are mutated in approximately one-third of patients. We used a candidate gene strategy combining high-resolution genomic mapping and gene expression microarray in the analysis of 2 DBA patients with chromosome 3q deletions to identify RPL35A as a potential DBA gene. Sequence analysis of a cohort of DBA probands confirmed involvement RPL35A in DBA. shRNA inhibition shows that Rpl35a is essential for maturation of 28S and 5.8S rRNAs, 60S subunit biogenesis, normal proliferation, and cell survival. Analysis of pre-rRNA processing in primary DBA lymphoblastoid cell lines demonstrated similar alterations of large ribosomal subunit rRNA in both RPL35A-mutated and some RPL35A wild-type patients, suggesting additional large ribosomal subunit gene defects are likely present in some cases of DBA. These data demonstrate that alterations of large ribosomal subunit proteins cause DBA and support the hypothesis that DBA is primarily the result of altered ribosomal function. The results also establish that haploinsufficiency of large ribosomal subunit proteins contributes to bone marrow failure and potentially cancer predisposition.