High Expression of p53 and Growth Differentiation Factor-15 in Beta-Thalassemia.

Abstract 2130

Beta thalassemia phenotype is exacerbated by marrow expansion, tissue hypoxia, and erythroblast apoptosis caused by globin chain imbalances. The expanded erythroid compartment contributes to iron overload in thalassemia syndromes by inhibiting hepcidin expression. It was shown that growth differentiation factor 15 (GDF15), a member of the transforming growth factor-b super family was elevated in b thalassemia and contributes to hepcidin suppression. p53 plays a key role in the cellular response to stress by activating a subset of genes responsible for cell cycle arrest and apoptosis in tumours. Recently association of P53 with defective erythropoiesis has been demonstrated in Diamond Blackfan anemia. In this study we analyzed the expression of these genes in patients with b thalassemia. We observed a higher expression of p53 and p21 in b thalassemia. Thirty six patients with b thalassemia (b thalassemia major=27; b thalassemia intermedia=9) were included in the study. Diagnosis was based on clinical, haematological and molecular parameters. Healthy volunteers were included as controls (n=28). Serum GDF15 levels were measured using ELISA (Ray Biotech, Inc, Georgia) and ferritin by chemiluminescent immunoassay. Reticulocytes from patients and controls were isolated from peripheral blood using cellulose columns. RNA was extracted by Trizol method and cDNA was synthesised. The relative expression of p53, GDF15 and p21 mRNA was analyzed using actin as the control gene. Approximately 1×10⁷ reticulocytes were used to prepare whole cell lysate for western blot (controls=6, patients=6). As expected serum ferritin (Median-3544ng/ml) and GDF15 levels (Median-6059.87pg/ml) were higher in the patient cohort as compared to controls. Higher expression of p21 and p53 was observed in patients as compared to controls (Table.1). P53 expression strongly correlated with GDF15 expression at the mRNA (r=0.649; p=0.000) and protein level (r=0.406; p=0.049) in b thalassemia major. The p53 expression also significantly correlated with serum ferritin levels (r=0.562; p=0.003) in the thalassemia major cohort. We also assessed the expression of p21 one of the targets of p53 in these samples. Significantly higher expression of p21 was observed in the patients than in controls (Median dCt-8.34 vs 10.78; p=0.000). Western blot analysis carried out in six b-thalassemia major and six controls also showed a higher expression of p53 in the patients as compared to controls. Higher expression of these genes was also observed in b thalassemia intermedia as compared to controls but not different from thalassemia major (Table.1). GDF15 has two p53 response elements in the promoter region and p53 binding activates GDF15 in the erythroid compartment (Osada et.al 2007). P53 may thus contribute to GDF15 elevation in beta thalassemia.