Methylated circulating DNA as a biomarker for cardiac fibrosis progression and mortality in aortic stenosis patients
by Leon Kanwischer
Date of Examination:2024-08-15
Date of issue:2024-07-15
Advisor:Prof. Dr. Elisabeth Zeisberg
Referee:Prof. Dr. Elisabeth Zeisberg
Referee:Prof. Dr. André Fischer
Persistent Address:
http://dx.doi.org/10.53846/goediss-10612
Files in this item
Name:Kanwischer_Leon_Dissertation_online.pdf
Size:1.43Mb
Format:PDF
This file will be freely accessible after 2025-08-15.
Abstract
English
Aortic stenosis is one of the most common cardiac diseases in the elderly and leads to cardiac hypertrophy and fibrosis, and consecutively to heart failure. Currently, there is no causal treatment available instead of surgical or transcatheter aortic valve replacement. As existing guidelines recommend intervention only for symptomatic patients with severe aortic stenosis, no biomarker for risk-stratification in aortic stenosis patients is available by now. Increased insight into the influence of epigenetic mechanisms in the pathogenesis of cardiac diseases has identified four genes (ACAP3, CHRD, GADD45G, IRX3) that exhibited a crucial change in both expression and methylation. Since circulating DNA already serves as a biomarker in other medical disciplines, it was the aim to investigate the promoter methylation of the identified genes in the peripheral blood and assess their association with other clinical parameters, especially mortality. To first assess the gene expression in the myocardium and establish the promoter methylation analysis, a AT-II-treated mouse model was used. RT-qPCR analysis showed a decreased expression for alle genes and increased methylation for Gadd45g and Irx3. To further investigate the impact of promoter hypermethylation on the reduction of gene expression, the GADD45G and IRX3 promoter were inserted into a vector, treated by in-vitro methylation, and then transfected to HEK-293 cells. The methylated samples showed significantly reduced luciferase activity, respectively gene expression. Afterwards, the promoter methylation of the candidate genes of cf-DNA in the peripheral blood of 100 patients who received TAVI-intervention was assessed using MeDIP and RT-qPCR. The methylation levels were statistically analyzed with clinical parameters of those patients and especially IRX3 showed interesting results regarding the cardiac function and mortality, as a low IRX3 promoter methylation was associated with an increased amount of cardiac fibrosis and the average promoter methylation of IRX3 in the group of deaths was significantly decreased. To further analyze these findings, Kaplan-Meier survival analyzes were performed and showed a significantly higher all-cause mortality for patients with decreased IRX3 promoter methylation and an insignificant trend for cardiac mortality. Additionally, multivariate survival analyzes were performed, where lower promoter methylation of IRX3 was again associated with a higher all-cause and cardiac mortality and showed its superiority over other parameters. In order to investigate the ability of the promoter methylation of IRX3 to reflect the gene expression in the myocardium, immunohistochemical staining of myocardial tissue was performed. Due to a lack of myocardial tissue samples of all participants, only ten samples were analyzed. Coherently to the previous results, a low IRX3 promoter methylation level was associated with a high gene expression in the myocardium. From all these results, it could be concluded that IRX3 might play an important role in the pathogenesis of aortic stenosis and its development towards heart failure. Because of very little research on the field of IRX3 in the adult heart, the concrete function remains unclear. Since other biomarkers for identifying the right timepoint for intervention in aortic stenosis patients are still under investigation and other methods, such as risk scores, show several limitations, the promoter methylation level of IRX3 in the peripheral blood could be a candidate biomarker to identify those patients who benefit most from intervention. Overall, out of the group of candidate genes, IRX3 showed a strong association between promoter methylation and gene expression, but most important showed an involvement in survival and mortality of aortic stenosis patients after TAVI. Therefore, it is a promising approach for further research, which is urgently needed to discover its role in the adult heart and confirm the importance of our findings.
Keywords: Aortic stenosis; Cardiac fibrosis; Epigenetics; DNA methylation; Biomarker; ACAP3; CHRD; GADD45G; IRX3
