Mitochondrial Genome Changes As a Measure Of Iron-Induced Mitochondrial Stress In Transfusion-Dependent Thalassemia
Authors: Gomez Esteban , Calloway Cassandra , Lee Sang Hoon , Kim Jay , Dhillon Navpreet , Gildengorin Ginny , Lal Ashutosh 
Published: October 2019

Abstract

Introduction
Iron overload is a consequence of chronic blood transfusions to treat severe forms of thalassemia. While patient outcomes have improved with advances in iron overload assessment and chelation therapy, iron toxicity continues to be the primary determinant of morbidity. Mitochondrial dysfunction is of central importance in iron overload. However, clinical assessment of mitochondrial damage has not been feasible because current methods require invasive tissue sampling.

Methods
Novel bioassays were used to assess changes in the mitochondrial genome from a small amount of peripheral blood. We studied the relationship between mitochondrial DNA (mtDNA), mitochondrial respiratory complexes, and clinical measures of iron overload. Real-time PCR-based assays were used to measure mtDNA copy number per nuclear DNA copy (mtDNA:nDNA) and the frequency of the mtDNA 4977-bp deletion (ΔmtDNA4977). The quantity of mitochondrial respiratory complex I and IV in blood was measured using ELISA and expressed as a percentage of quantity in HepG2 cells.

Results
44 individuals with thalassemia (median age 34.9 years, range 14 to 52.4 years) and 27 healthy controls (median age 22 years, range 19 to 39 years) were studied in this analysis. The median liver iron concentration (LIC) in thalassemia was 11.0 mg/g dry-weight (2.3-59.4 mg/g dry-weight), while the median ferritin was 2220 mcg/L (308-9470 mcg/L). The median cardiac T2* was 28.7 ms (6.5-45.8 ms).
The mtDNA:nDNA was estimated and the median mtDNA copy number was 249.8 (87.6-738.3) in thalassemia, compared with 225.4 (101.9-348.7) in controls. The mtDNA copy number was significantly elevated in thalassemia (p=0.033, Mann-Whitney test). The mtDNA copy number increased with age in thalassemia (r=0.45, p=0.005), but no correlation with age was observed in the control group (r=0.06, p=n.s.). Myocardial iron deposition influenced the mtDNA copy number as evidenced by an inverse relationship with T2* (r= -0.45, p=0.006). The frequency of the ΔmtDNA4977deletion was analyzed in a subset of subjects and was found to be 8-fold higher in thalassemia versus controls (p=0.012). The median quantity of complex IV in thalassemia was 3.4% (0 to 104.2) compared with 2.5% (range 0 to 18.67) in controls. The median complex I activity in thalassemia was 6.3% (range 0 to 161.4) compared with 17.86% (range 0 to 55.0) in controls. These differences were not statistically significantly between the two groups. Higher values of mtDNA copy number were associated with increased quantity of complex IV (r=0.515, p=0.10) in thalassemia, but not in controls. No correlation of mtDNA copy number was observed with complex I activity. LIC, ferritin, and platelet count did not influence the mtDNA:nDNA ratios.

Interpretation
Alterations in mtDNA can be measured in blood samples of patients with thalassemia and iron overload. Increases in mitochondrial genome copy number and ΔmtDNA4977 deletion frequency may be a reflection of iron-induced mitochondrial stress. This hypothesis is supported by an increase in mtDNA copy number with age in thalassemia, but not in controls. The relation between mtDNA changes and myocardial iron suggests that this assay may possess physiological relevance. These preliminary results support the potential application of these non-invasive assays for the assessment of iron-induced organ damage.

Disclosures:
No relevant conflicts of interest to declare.