Study of deferiprone-induced agranulocytosis in β-thalassemic patients through a metabolomics approach

ABSTRACT β-Thalassemia is one of the most prevalent forms of congenital blood disorders, characterised by a reduced or absent ability to produce haemoglobin. The mainstay of treatment consists of blood transfusion to maintain the patient’s haemoglobin above 9-10 g/dL. Repeated transfusions result in an excessive accumulation of iron in the body, removal of which is achieved through iron chelating agents. Deferiprone is the first orally bioavailable iron chelator, approved for clinical use in 1997. Considering its potential toxicity, the use of Deferiprone is allowed in Europe only for the treatment of thalassemia major, when Deferoxamine therapy is contraindicated or unappropriated. The main Deferiprone adverse effect is the development of agranulocytosis (in 1–2% of patients). The mechanisms behind this negative effect remain largely unresolved. Currently, only a few metabolomics studies have been performed on neutrophils. Neutrophils are the immune cells forming the major arm of innate immunity. Due to their physiological characteristic, the study of these cells in vitro presents several difficulties meaning that standard protocols for most assays must be optimised. This thesis aimed to establish protocols for the study of the metabolomic profile of human neutrophils in healthy and pathological conditions. In particular, this study aimed to investigate the PMNs (polymorphonuclear leukocytes) metabolic profile of beta thalassemic patients treated with iron chelator Deferiprone, to explore its potential relationship with the onset of agranulocytosis, using a metabolomics approach. Our data demonstrated that analysis of the metabolomic profiles in PMNs with GC-MS, allowed us to identify different metabolites including organic acids, amino acids, fatty acids, and sugars. The results showed a different metabolomic profile between PMNs obtained from patients with Deferiprone-induced agranulocytosis and PMNs of patients without Deferiprone-induced agranulocytosis. Multivariate statistical analysis of GCMS data revealed that the PMNs of patients with Deferiprone-induced agranulocytosis have a metabolic profile characterized by an increase of metabolites directly involved in the metabolic pathways of glutathione synthesis and by a decrease of arachidonic acid stearic acid and inosine. 5 Considering the important physiological roles of these metabolites, our results could shed light on the physiopathological mechanisms of this harmful side effect of DFP treatment. This work is a pilot study that has been only validated in a small independent cohort and, therefore, further confirmation in larger studies is required.