Mitochondrial Damage in Hemoglobinopathies: The Role of Cell-Free mtDNA as a Potential Biomarker

Abstract

Background/Objectives: In hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), mitochondrial dysfunction has been implicated in chronic oxidative stress and tissue damage. Circulating cell-free mitochondrial DNA (cf-mtDNA) may reflect mitochondrial injury and serve as a biomarker of disease severity and progression. Methods: This cross-sectional study included four groups: three patient groups—β-thalassemia (n = 36), SCD (n = 11), and genetically confirmed mitochondrial disease (positive control, n = 13)—and one healthy control group (n = 14). Venous blood was collected in EDTA tubes, and plasma was separated by sequential centrifugation. cf-mtDNA was extracted from 200 µL plasma using the Quick-cfDNA™ Serum & Plasma Kit (Zymo Research, Irvine, CA, USA). cf-mtDNA was assessed by qPCR targeting ND1 and COI and analyzed by relative quantification using the 2−ΔΔCt method (Q3 Real-Time PCR System, LongGene). Each sample was run in triplicate. Results: cf-mtDNA levels were significantly higher in the β-thalassemia, SCD, and mitochondrial disease groups compared to healthy controls (p < 0.001). No significant correlations were observed between cf-mtDNA levels and hemoglobin or oxygenation parameters. Conclusions: Elevated relative cf-mtDNA abundance in hemoglobinopathy patients suggests mitochondrial damage, possibly associated with chronic hypoxia and oxidative stress. cf-mtDNA may represent a promising noninvasive biomarker, warranting further longitudinal investigation to evaluate its potential clinical utility.