ALBERT

Description: Abstract 2242 Background: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic exocrine dysfunction, neurocognitive and skeletal abnormalities, and bone marrow failure. Mutations in SBDS have been shown to cause SDS. From experiments on its yeast ortholog (Haematologica 2010. 95:57-64), SBDS has been implicated in maturation and function of the 60S ribosomal subunit. In particular, subunit maturation in the SDS yeast model was associated with delayed export and accumulation of 60S-like particles in the nucleoplasm. Methods and Results: To clarify its role in human cells, erythroleukemia TF-1 cells were transduced with lentiviral vectors expressing short hairpin RNA (shRNA) against SBDS. Immunoblot assays confirmed approximately 60% knockdown in individual TF-1 cell clones expressing different shRNAs. The growth and hematopoietic colony forming potential of TF-1 knockdown cells were markedly hindered when compared to cells stably transduced with shRNA against a scrambled SBDS sequence. Using Hoechst 33342/Pyronin Y staining and flow cytometry, we also found an increased percentage of knockdown cells retained at the G0/G1 cell cycle phase. To address whether near-complete knockdown of SBDS affected ribosome synthesis as it does in yeast cells, we silenced SBDS in A549 cells. Our data revealed a reduction in polysomes but in contrast to what was observed in yeast, there was no evidence of half-mer polysomes indicative of decreased 60S subunits participating in translation. The absence of half-mers is not unusual in mammalian systems, so to better analyze the effect of SBDS on 60S subunit maturation subunit localization was assessed by co-transfection with a vector expressing a fusion between human RPL29 and enhanced GFP. Preliminary studies indicated a higher percentage of SBDS-depleted cells with nuclear localization of 60S subunits, when compared with normal controls (Fig. 1). Conclusions: Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2237 Background: Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes (IBMFS), characterized by erythroid hypoplasia, congenital anomalies and a cancer predisposition. DBA is caused by ribosomal protein haploinsufficiency, which somehow triggers apoptosis of erythroid precursors, possibly through activation of p53. Some DBA patients show a response to steroid treatment, while others remain transfusion-dependent. While the mechanism of action of steroids in DBA is unclear, recent work has suggested that p53 may antagonize glucocorticoid-induced proliferation of normal erythroid progenitors1. Objective: Our goal was to create a murine embryonic stem (ES) cell model of DBA with a mutation in Rps19 to study the pathophysiology of DBA and to test glucocorticoid responsiveness. Methods: The Rps19-mutated murine embryonic stem cell line, S17-10H1 was created using a gene trap strategy. The ES cells were induced to undergo primary differentiation into embryoid bodies (EBs). Day 9–11 EBs, representing definitive hematopoiesis, were re-plated with hematopoietic cytokines (stem cell factor, interleukin (IL)-3, IL-6 and erythropoietin) in methylcellulose, and secondary differentiated colonies were scored on day 10. S17-10H1 cells were transfected by electroporation with a plasmid vector expressing either wild-type Rps19 cDNA or an empty vector control. Protein and mRNA levels of the tumor suppressor p53 were measured at the ES cell stage. Secondary differentiated hematopoietic colonies were grown with and without glucocorticoids (either dexamethasone at 1nM, 10nM, 100nM, 1μM or hydrocortisone at 50nM, 2.5μM concentration). Results: Western blot analyses confirmed S17-10H1 Rps19-haploinsufficiency. The mutant cell line had reduced EB formation (hematopoietic and non-hematopoietic) following primary differentiation. Significant defects in both erythroid (BFU-E) and myeloid (CFU-GM) formation were found following secondary hematopoietic differentiation of day 9–11 EBs. These defects were specific to Rps19 haploinsufficiency since all defects were rescued by stable transfection of the mutant cell lines with an Rps19-expressing correction vector. However, glucocorticoid treatment was unable to rescue defects in secondary differentiation (hematopoietic colony formation). Finally, we also examined baseline levels of the tumor suppressor p53, which may be induced following abortive ribosome assembly resulting from ribosomal protein haploinsufficiency. Although there was no difference in transcription levels of p53 mRNA between mutant and control cells as determined by quantitative PCR, baseline levels of p53 protein were significantly increased in the mutant ES cells as compared to controls. Conclusions: (1) In our model system, Rps19 mutation was associated with a relatively early defect in hematopoietic progenitors, since both erythroid and myeloid (or mixed) colony formation was diminished. (2) Hematopoietic defects in our murine ES cell model of DBA were rescued by Rps19 gene transfer but not by glucocorticoid therapy. (3) We found significant accumulation of baseline p53 protein (but not mRNA) levels in mutant ES cells, suggesting that Rps19 haploinsufficiency leads to inhibition of the p53-degradation pathway. (4) Taken together, these results suggest that p53 may antagonize glucocorticoid-induced proliferation in our Rps19-mutant ES cell model. Experiments to test this hypothesis are in progress. Reference: Ganguli G, Back J, Sengupta S, Wasylyk B. The p53 tumour suppressor inhibits glucocorticoid-induced proliferation of erythroid progenitors. EMBO Rep. 2002;3:569-574. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract LBA-2 Background: 5q- myelodysplastic syndrome is a rare, acquired macrocytic anemia with a female predominance. The bone marrow is characterized by a paucity of erythroid precursors with relatively normal leukocyte and platelet counts and no excess blasts. The mean age at diagnosis is approximately 70 years. The phenotype of 5q deletion has been shown to result from haploinsufficiency of the RPS14 gene. Historically red blood cell transfusions have been the primary treatment; however lenalidomide has recently been effective in ameliorating the anemia with a response rate of 67%. DBA is a rare heritable red cell aplasia which usually presents in infancy. It too is characterized by a bone marrow deficient in erythroid precursors. Mutations or deletions in eleven ribosomal protein (RP) genes, resulting in protein haploinsufficiency, have been reported in 50–60% of patients. To date RPS14 mutations have not been identified in DBA patients. Array Comparative Genomic Hybridization (CGH) has been used to identify large deletions in patients with DBA, but a more sensitive approach was hypothesized to identify additional deletions. Purpose: To address the question of whether chromosomal deletions could be the underlying defect in patients with DBA who did not have mutations in the known RP genes, Single Nucleotide Polymorphism (SNP) genotyping array hybridization was utilized. Methods: Seventy-five patient samples from the DBA Registry (DBAR) underwent resequencing of 80 RP genes. Approximately 40% of the patients had no identifiable mutation. High resolution SNP array genotyping analysis was done on 23 probands who did not have a mutation detected by resequencing. Results: An acquired internal deletion on chromosome 5q involving RPS14 was identified in one of 23 patients with presumed DBA. The patient presented with anemia at 5 10/12 years of age. The hemoglobin was 8.4 g/dl, MCV 108.2 fL, and reticulocyte count 0.4%. The erythrocyte adenosine deaminase (eADA) activity, elevated in 85% of DBA patients, was normal. The bone marrow showed decreased cellularity and megaloblastoid changes in the erythroid series. There were adequate numbers of megakaryocytes with no hypolobulation. Cytogenetics performed at diagnosis in 1991 appeared normal. The patient had no significant family history or congenital anomalies. A diagnosis of non-classical DBA was made. The patient failed a trial of corticosteroids and had remained transfusion-dependent for 19 years. No RP gene mutation was identified by sequencing. SNP array genotyping analysis identified mosaicism in two discrete regions covering ∼17.7 Mb on 5q-, with an estimated 63.7% monosomy and 36.3% disomy in this region. The major region extends from 141.1M to 157.2M (hg18), including all of the 5q- syndrome commonly deleted region (CDR) at 5q33, though it excludes the 5q31 CDR, miR146a, as well as Cdc25C and PPP2Acα, factors for which haploinsufficient expression has previously been suggested to be important in response to lenalidomide. SNP array genotyping from purified populations indicated that lymphocytes were 〉95% normal, while the myeloid cells were 〉95% 5q-. CD34+ cells showed a marked decrease in both myeloid and erythroid colony formation. Patient fibroblasts were normal and neither of the parents have 5q abnormalities by SNP analysis. Although the deletion was not identified in 1991, the 46,XX,der(5)del(5)(q15q22)del(5)(q32q33) deletion was detected on high resolution karyotyping in a post-SNP array genotyping marrow sample. Haploinsufficiency of RPS14 was confirmed by quantitative RT-PCR. After a trial of lenalidomide, complicated by Grade 4 neutropenia and Grade 3 thrombocytopenia, the patient has a reticulocyte count of 7.4% (from a previous baseline of

Description: Abstract 4430 Background: Diamond Blackfan anemia is a rare heritable red cell aplasia which usually presents in infancy but can also be diagnosed in childhood and even adulthood. Mutations or deletions in eleven ribosomal protein (RP) genes, resulting in protein haplo-insufficiency have been reported in about 54% of the patients. The 5q- syndrome is an acquired myelodysplastic syndrome (MDS) characterized by a similar erythroid failure. Another RP gene included in the 5q deleted region, RPS14, has been identified as a causal gene in 5q- MDS but has not been reported in DBA. Purpose: Array Comparative Genomic Hybridization has been used to identify large deletions in patients with DBA. This report demonstrates the use of Single Nucleotide Polymorphism (SNP) genotyping array hybridization to identify a patient, previously thought to have DBA, as having a 5q- deletion consistent with 5q- syndrome. Method: Seventy-five patient samples from the Diamond Blackfan Anemia Registry of North America, a patient database of now 608 patients designed to better understand the biology and epidemiology of DBA, underwent resequencing of 80 RP genes. Approximately 40% of the patients had no identifiable mutation. High resolution SNP array genotyping analysis was done on 23 probands from this cohort who did not have a mutation detected in either the resequencing project and/or the targeted sequencing efforts lead by Gazda and colleagues. Result: An acquired internal deletion on chromosome 5q involving RPS14 was identified in one patient with presumed DBA. The patient presented at 5 years 10 months of age with anemia noted on a routine blood count. The hemoglobin was 8.4 grams/dl, MCV 108.2 fL, and reticulocyte count 0.4%. The eADA was normal. The bone marrow showed decreased cellularity and megaloblastic changes in the erythroid series. There were adequate numbers of megakaryocytes with no hypolobulation. The cytogenetics performed at diagnosis in 1991 were reported as normal. The patient had no significant family history of anemia and was found to have no congenital physical anomalies. A diagnosis of non-classical DBA was presumed and the patient failed a trial of corticosteroids. At present the patient has marrow red cell aplasia and is on a chronic transfusion schedule. SNP array genotyping analysis identified mosaicism in two discrete regions covering ~17.7 Mb on 5q-, with an estimated 63.7% monosomy and 36.3% disomy in this region. The major region extends from 141.1M to 157.2M (hg18), including all of the 5q- syndrome commonly deleted region (CDR) at 5q33 though it excludes the 5q31 CDR associated with AML and more aggressive MDS as well as miR146a, a factor recently postulated to play a role in 5q- MDS. SNP array genotyping from purified peripheral blood populations indicated that lymphocytes were greater than 95% normal, while the myeloid cells were greater than 95% 5q-. CD34+ cells obtained from this patient showed a marked decrease in both myeloid and erythroid colony formation when compared with normal cells. Patient fibroblasts were normal and neither of the parents have any 5q anomalies by SNP array genotyping. Although the deletion was not identified in 1991 at the time of the diagnosis, the 46,XX,der(5)del(5)(q15q22)del(5)(q32q33) deletion was able to be detected on high resolution karyotyping in a post-SNP array genotyping marrow sample. Haploinsufficiency of RPS14 was confirmed by quantitative RT-PCR. Conclusion: Patients with non-classical DBA may have unique acquired 5q deletions with RPS14 haploinsufficiency. A search for other acquired somatic mutations or deletions in patients with DBA, in particular non-classical cases, is underway. SNP array genotyping is an essential diagnostic tool in this search. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 942 Background: Emerging evidence suggests that microRNAs (miRNAs) are critical in cancer and adult leukemia by functioning as tumor suppressors and/or oncogenes. Zhang et al identified 32 pediatric acute myeloid leukemia (AML)-specific miRNA patterns by analysis of bone marrow (BM) samples (1). They also established potential miRNAs as biomarkers for predicting CNS-relapse in pediatric acute lymphocytic leukemia (ALL). Altered miRNA expression disrupts normal hematopoiesis and might play a role in niche-induced oncogenesis. Dysfunction of mesenchymal stromal cells induces formation of myeloid sarcomas that infiltrate in the surrounding tissues (2). Previously, we described that mesenchymal stem cells (MSC) of the BM microenvironment participate in leukemic stem cell regulation in an in vivo model of the childhood AML stem cell niche(3). These human MSC niches, created in ectopic bioengineered 3D scaffolds, supported leukemogenesis in NOD/SCID mice. Pediatric AML engrafted at 1 month in the MSC-coated scaffolds in the mice and was retained in the niche up to 4 months, after which distant seeding to murine BM, liver and spleen occurred. The bioengineered niche created a sanctuary for quiescent leukemia cells and at 4 months the AML cells exited the niche and spread hematogenously, mimicking leukemia relapse. Analysis of miRNA patterns in our leukemia niche model could provide novel directions for individual risk-adapted therapy in childhood leukemia. Objective: To analyze miRNA expression patterns of pediatric AML after exposure to the niche microenvironment at different time points. Design and Method: miRNAs were obtained from primary CD34+ selected AML cells at (d0) Day0= no niche exposure, (1Mo) 1 month =niche engraftment, (4Mo) 4 months=hematogenous spread with leukemic exit from the niche. miRNAs were isolated from single cell suspensions with the mirVana miRNA isolation kit (Ambion) and analyzed on an Ilumina MicroRNA Expression Profiling single Beadchip (#RNA probes = 1145). Results: 498/1145 miRNAs expression profiles were selected with a detection p value 〈 0.00001. Out of 498 miRNAs expressed in the leukemic niche model, 23 were previously described as AML-specific miRNAs (2). 10/23 miRNAs were significantly upregulated and 13/23 were downregulated. Pediatric AML-specific miRNAs – miR100 and miR125b had high expression profile at baseline, but were down regulated upon contact with the niche. AML miR195 and miR193a had low expression at baseline, but miR195 was upregulated on engraftment while miR193a only upregulated at the time of hematogenous spread (niche exit). CNS-relapse in ALL might represent a physiological mechanism of leukemic exit of dormant cells from the niche sanctuary. Consistent with this notion, the same expression profile that was found in CNS-relapse in ALL patients (miR198 up – miR551a downregulated) was seen in our model when AML cells became invasive and exited the niche at 4 months. Conclusion: (1) Zhang et al. 2009, PloSOne, 4, p1 (2) Raaijmakers et al. 2010, Nature, 464, p852 (3) Vaiselbuh et al, 2010, Tissue Eng Part C Methods, June 29 (ePub) Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 877 Background: Diamond Blackfan anemia (DBA), a rare inherited bone marrow failure syndrome, is characterized mainly by erythroid hypoplasia but is also associated with congenital anomalies, short stature and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS17, RPS19, RPS24, RPL5, RPL11, RPL35a), which renders erythroid precursors highly sensitive to death by apoptosis. The ontogeny and basis of the hematopoietic defect are unclear. The typical presentation of anemia occurs at 2–3 months of age, although there are rare cases of hydrops fetalis. Marked phenotypic variations exist among members of the same family and also between subsets of patients with different mutations. Methods: We studied in vitro hematopoietic differentiation of two murine embryonic stem (ES) cell lines: YHC074, Rps19 mutant with the pGT0Lxf gene trap vector inserted in intron 3 of Rps19, and D050B12, Rpl5 mutant with the FlipRosaβgeo gene trap vector inserted in intron 3 of Rpl5. Wild-type parental cell lines were used as controls. For primary differentiation and generation of embryoid bodies (EBs), ES cells were cultured in serum-supplemented methylcellulose medium containing stem cell factor (SCF). After 7 days, the cultures were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 12 for hematopoietic percentage. For secondary differentiation into definitive hematopoietic colonies, day 10 EBs were disrupted, and individual cells were suspended in serum-supplemented methylcellulose medium containing SCF, IL-3, Il-6 and epo. Definitive hematopoietic colonies were counted on day 10. Primitive erythropoiesis differentiation assays were performed by disruption of day 4 EBs, followed by suspension of cells in methylcellulose medium containing plasma-derived serum and epo. Primitive erythropoiesis colonies were counted on day 7. Results: We confirmed haploinsufficient expression (∼50% wild type) of Rps19 in YHC074 and Rpl5 protein in D050B12 by Western blot analysis. By polysome analysis, we found a selective reduction in the 40S subunit peak in the Rps19 mutant cell line and in the 60S subunit peak in the Rpl5 mutant cell line. Both types of mutants produced a significantly decreased number of EBs, particularly hematopoietic EBs, compared to parental cell lines. EB size was not compromised in the Rps19 mutant cell line, while Rpl5 mutant ES cells produced significantly smaller EBs, compared to its parental cells. Upon differentiation of cells to definitive hematopoietic colonies, both Rps19 and Rpl5 mutants showed a similar reduction in the erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation ratio. Primitive erythropoiesis was conserved in the Rps19 mutant (Figure 1. 1, top panel). By contrast, the Rpl5 mutant demonstrated a severe primitive erythropoiesis defect (Figure 1. 1, bottom panel). For confirmation of these results in an isogenic background, we stably transfected YHC074 ES cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones expressed Rps19 at the wild-type level. Upon differentiation of a chosen clone, we demonstrated correction of the EB defect and the definitive erythropoiesis defect, suggesting that the hematopoietic differentiation defects seen are directly related to levels of Rps19 protein. We are currently working on correction of the D050B12 ES cells in a similar manner. Conclusion: Murine ES cell lines with Rps19 and Rpl5 mutations exhibit ribosomal protein haploinsufficiency, demonstrate respective ribosome assembly defects, and recapitulate the major DBA hematopoietic differentiation defect. In addition, a unique defect in primitive erythropoiesis in the Rpl5 mutant ES cell line suggests that the Rpl5 mutation in this mouse strain affects early-stage embryogenesis, a finding which may offer insight into the ontogeny of DBA hematopoiesis and may offer an explanation for phenotypic variations seen in patients (such as hydrops fetalis). Disclosures: No relevant conflicts of interest to declare.

Description: In this paper we review microstructural and petrofabric work carried out on the Moine Thrust zone and overlying thrust nappes. Our review is primarily historical, and starts with contributions made by both ‘amateur’ and ‘professional’ geologists from the 1880s through to the early 1920s during, and immediately following, the original field-mapping of the Moine Thrust zone by the Geological Survey. From the 1920s to the early 1950s contributions were first dominated by Geological Survey work on the microstructural and metamorphic transition between the thrust zone mylonites and the overlying Moine metasedimentary rocks. Subsequent university-based quartz petrofabric work, primarily focused on the Moines, would ultimately lead to the ‘Moine Petrofabric Controversy’ that ran from the late 1940s to the early 1960s. The later stages of this controversy overlapped, from the early 1950s–mid 1960s, with a phase of microstructural and quartz petrofabric work that concentrated on the thrust zone mylonites and immediately overlying Moine Schists. Our review concludes with an overview of microstructural, petrofabric and related strain analyses undertaken since the early 1970s, both within the Moine Thrust zone and its immediate foreland and in the overlying higher grade thrust sheets. Throughout our review we emphasize and track the changing tectonic interpretations that have been placed on available microstructural and petrofabric data.