High Incidence of Thalassemia Traits Among Myelodysplastic Syndrome Patients May Mask Macrocytic Anemia but Not the Enhanced Proteasome Proteolytic Activity

PV and KT as well as VP and MA contributed equally

Chronic anemia is a prominent feature of myelodysplastic syndromes (MDS). Anemia, mainly macrocytic, red cell (RBC) transfusion dependency,and changes in RBC characteristics are linked to negative clinical outcomes or hemolysis in MDS patients. Both acquired RBC defects of unknown genetic basis and inherited RBC defects predisposing to myeloid dysplasia have been reported.

This study examined RBC physiology in MDS patients focusing on hemolysis and proteasome proteolytic activity (PPA) to identify new peripheral signatures of the disease and their association with risk groups, recombinant erythropoietin (rEPO)treatment, and co-existent thalassemia traits. The analysis included blood samples from 70 untransfused patients with low risk (LR, n=59) or high risk (HR, n=11) MDS, and 21 healthy controls. Twenty-six LR patients were rEPO-treated. Elevated serum ferritin was found in 28% of patients. Despite an estimated 8.0% thalassemia carrier frequency in Greece, 20% of patients were thalassemia carriers. Thalassemia carriers had microcytic RBCs and reticulocytes, higher RDW and HDW, lower MCH, and trends towards lower PLT counts compared to non-carriers.

No significant average hemolysis rate was observed, although 14% had high cell-free hemoglobin (Hb) levels (>4x control), consistent with previous reports. Over 35% of untreated MDS exhibited abnormal osmotic hemolysis, excluding thalassemia carriers with osmotically resistant RBCs. Osmotic resistance correlated with higher bone marrow (BM) blast percentages in LR patients but lower anemia in HR patients. Intracellular ROS levels were normal, but high methemoglobin was found in LR MDS thalassemia carriers. Pathological membrane-bound hemichromes, intracellular calcium, phosphatidylserine (PS) surface exposure, and uric acid-independent plasma antioxidant capacity were detected in HR patients, with trends towards higher erythroid dysplasia and serum iron/ferritin levels compared to LR patients. In univariate analyses, BM blast percentage and cellularity significantly correlated with PS exposure and intracellular calcium stress (r 0.478, p<0.01). Hb oxidation inversely correlated with Hb concentration, but positively with the oxidative hemolysis and the HDW index.

PPA and its distribution in MDS RBCs were assessed using fluorogenic substrates for all three enzymatic activities and a pan-PPA inhibitor. PPA was significantly increased in the cytosol of MDS RBCs, especially in HR patients (9326 ± 3862 vs. 5515 ± 960 RFU in control), and in the membrane of HR cells, with no significant effects from thalassemia traits. PPA significantly correlated(p<0.01) with anemia and cellular fragility metrics.

rEPO-treated patients had similar anemia levels to untreated LRs, but higher reticulocyte count and plasma antioxidant capacity (UA-dependent). Despite improved cytopenias, more RBC disturbances were evident regarding RDW, HDW, PS+ RBC%, membrane-associated PPA (especially in osmotically resistant samples), and trends towards increased intracellular ROS and membrane-bound hemichromes. Thalassemia carriers under rEPO treatment had higher RBC counts, HDW index, osmotic and mechanical resistance, and trends towards higher metHb/oxyHb ratios compared to non-carriers.

The higher incidence of thalassemia traits in MDS requires investigation in larger cohorts to confirm thalassemia as a predisposing factor. Acquired thalassemia and RBC defects need further study, particularly regarding osmotic hemolysis and Hb oxidation, to develop targeted treatments. Enhanced PPA appears to be a signature of MDS, correlating with clinical severity and rEPO therapy. Ineffective erythropoiesis with low erythroid precursor apoptosis due to BM blast expansion in HR or due to erythropoiesis stimulation in rEPO-treated LR patients, leads to similar RBC stress phenotypes.