Part One Preliminary Study On The Relationship Between Iron Metabolism And Ineffective Erythropoiesis In Myelodysplastic Syndromes Part Two Abstract Clinical Importance Of SF3B1Mutations In Chinese With Myelodysplastic Syndromes With Ring Sideroblasts

[Background] Though prolonged red blood cells (RBC) transfusion therapy appears the main contributor to iron overload, many patients appear to develop iron overload at an early stage of the disease before the onset of transfusions. It has been postulated that an altered production of hepcidin, the recently discovered key hormone regulating iron homeostasis may play a role in this regard. Growth differentiation factor15(GDF-15), a protein produced by erythroid precursors, has been proposed to be a major hepcidin suppressor in B-thalassemia, but data in MDS with ineffective erythropoiesis are less conclusive.[Object] The present study was aimed to investigate the relationship between ineffective erythropiesis and iron metabolism and the effect of iron overload on the proliferative capacity of bone marrow progenitor cells in MDS patients who had not received any transfusion.[Methods]①The concentrations of GDF15and hepcidin in serum of patients with MDS were measured by ELISA.②The mRNA expressions of HIF-la and Bcl-xL in bone marrow mononuclear cell were detected by Real time PCR.③The expression of ROS and ratio of apoptosis in CD71+cells were detected by FACS.Then we analyze the correlation between serum ferritin levels and the ROS, also the correlation between the ROS and ratio of apoptosis in their CD71+cells.④K562cell lines were cultured with feric citrate (FAC) at different concentrations and for different time to create iron overload model, confirmed by the detection of cellular labile iron pool(LIP). The changes of ROS, apoptosis rate and the protein expression of mitochondrial apoptosis pathway were detected by FACS and Western-blot among controls, NAC pretreated and FAC groups.[Results]①Compared with healthy controls, MDS patients without transfusion had higher concentrations of SF, EPO, hepcidin and GDF15(P<0.001), but significantly lower hepcidin to ferritin ratio (P=0.005).②GDF15concentrations significant positively correlated with percent of bone marrow erythroblasts (P<0.001), sTfR (P=0.018), ISAT (P=0.038) and negatively correlated with TRF(P=0.008).③The hepcidin to ferritin ratio showed a significant variability across different MDS subtypes (P=0.011), with the lowest values in patients with refractory anemia with ringed sideroblasts (RARS) who appeared as the most iron overloaded with highest level of SF and ISAT among different MDS subtypes (P=0.028and P=0.004).The level of hepcidin negatively correlated with EPO concentrations and HIF-la mRNA levels (P=0.022and P=0.048). There was no statistically significant correlation between serum ferritin, serum iron with hepcidin levels (P=0.873and P=0.702).The hepcidin to ferritin ratio negatively correlated with EPO concentration, and GDF15levels (P=0.014and P<0.001). Subjects with higher levels of EPO (≥200mu/ml) or GDF15(≥5000ng/ml) had significantly lower hepcidin to ferritin ratio than patients with lower concentrations of EPO (<200mu/ml)、GDF15(<5000ng/ml) The hepcidin to ferritin ratio was independently associated with GDF15concentration and WHO subtype in multivariable analysis(Β=-0.292, P=0.029and Β=-0.390, P=0.006).④Patients with lower levels of ferritin (<500ng/ml) showed significantly higher numbers of BFU-E than patients with elevated ferritin levels (≥500ng/ml)(P=0.007). However, there were no differences for CFU-GM (P=0.190). A correlation was detected between ROS and SF levels (r=0.739, P=0.001), also positive correlation between ROS level and their apoptotic rates was found in erythroid CD71+cells (r=0.600, P=0.018).⑤The model of iron overload was established by adding lmM FAC for24hours.The apoptotic rates and ROS level of FAC treated cell was significantly higher than the controls, while NAC pretreated group had lower apoptotic rates and ROS level than the FAC groups.Western-blot analysis showed increased expression of Bax and cleaved caspase-3, while decreased expression of Bcl-2among controls, NAC pretreated and FAC groups.[Conclusion]①Iron overload occurs in MDS patients even without transfusion, hepcidin concentrations are inappropriately low considering the severe iron overload.②High levels of GDF15is a feature of ineffective erythropiesis in MDS.③GDF15is among the erythroid factors down-regulating hepcidin and contributing to iron overload in conditions of dyserythropoiesis in MDS. ④Iron overload impairs proliferation of erythroid progenitors cells and induce apoptosis in erythroid CD71+cells from patients with MDS. The higher rate of apoptosis in erythroid CD71+cells may be responsible for increasing level of ROS in MDS.⑤In vitro study demonstrates that apoptosis rate can be ameliorated by incubation with NAC. [Object] To determine the frequency of SF3B1mutation and evaluate clinical and prognostic implications in China.[Methods] A retrospective study on147MDS-RS cases was performed according to2008World Health Organization (WHO) diagnostic criteria.144RCMD patients were enrolled as controls.To screen for SF3B1mutations, we amplified genomic DNA corresponding to the mutation hotspots of SF3B1(exons13-15) by PCR using the primers SF3B1. The clinical and prognostic implications were compared among the patients with gene mutation and those with wild type.[Results](DSF3B1mutation state was determined in104Chinese with MDS-RS. SF3B1mutations were found in55subjects (53%) including25of39with refractory anemia and RS (RARS),26of45(58%) of those with refractory cytopenia with multi-lineage dysplasia and RS (RCMD-RS),3of6with refractory anemia with excess blasts-1-RS (RAEB1-RS) and1of14with RAEB2-RS, The proportion of subjects with SF3B1mutations was significantly higher in the RARS and RCMD-RS cohorts than the other MDS-RS categories (P=0.001and P=0.005). All missense mutations leading to amino acid substitutions clustered in exons14and15②There were significant correlations between SF3B1mutation state and platelet levels (P=0.007), mean RBC corpuscular volume (MCV;(P<0.001), proportion of RS (P<0.001) and percent bone marrow erythroblasts (P=0.012) and myeloblasts (P=0.044).③Survival analyses were conducted for140MDS-RS and141RCMD subjects with a median follow-up of22mo (range,1-102mo).Subjects with SF3B1mutation had better survival (median,63mo(95%CI,49-77mo) vs25mo (95%CI,20-30mo);P<0.001).Multivariate analyses using a Cox proportional hazards regression model including sex, age, SF3B1mutation state, hemoglobin concentration, absolute neutrophil level, platelet level, MCV, international prognostic scoring system (IPSS) cytogenetics category, WHO morphologic category and treatment showed SF3B1mutation state to independently predict survival.[Conclusion]①Our study confirms the high incidence of SF3B1mutations inMDS-RS,especially in RARS and RCMDRS patients.②There was strong correlation with SF3B1mutation and the clinical features in MDS-RS patients.③SF3B1mutation is independently correlated with better survival in multivariate analysis.