Greenway, StevenWeatherald, JasonIqbal, Fatima2024-06-272024-06-272024-06-26Iqbal, F. (2024). Investigating pulmonary vascular disease in patients with long COVID using methylation patterns in cell-free DNA (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.https://hdl.handle.net/1880/119050https://doi.org/10.11575/PRISM/46646Introduction: Coronavirus-19 disease (COVID-19) continues to influence the health and quality of life of Canadians to this day, even after recovering from the initial infection itself. Long COVID is a heterogenous and multi-organ disease that captures a range of symptoms that are prevalent months after infection, including persistent breathlessness (dyspnea for >12 weeks post-infection). Hypoxia and inflammation are important potential mechanisms for long COVID that cause endothelial damage and changes to the pulmonary vasculature which may contribute to unexplained dyspnea. Tissue-specific damage can be characterized using fragments of DNA released into the circulation known as cell-free DNA (cfDNA). Importantly, these fragments retain epigenetic information that can be leveraged to determine the tissue of origin as well as disease-specific methylation changes. Objective: To develop a cfDNA methylation assay to characterize cell-specific damage in PVD groups and delimitate the role of PVD in long COVID. Specific Aims: Aim 1: Identify and validate DMRs for pulmonary cell types. Aim 2: Use Nanopore sequencing to find tissue and disease-specific DMRs. Aim 3: Associate levels of DMRs in patients with PVD and long COVID with clinical presentations. Key Results and Significance: We have validated the specificity of endothelial cell and pulmonary tissue DMRs against a tissue panel to quantify cell-specific injury in patient cfDNA. We have also performed Nanopore sequencing of cfDNA from patients with long COVID, Pulmonary Arterial Hypertension (PAH), and Chronic Thromboembolic Pulmonary Hypertension (CTEPH). We have used this data to demonstrate disease-specific methylation patterning. Our work has also highlighted some gaps to address in order to use the advantages of a PCR-free, Bisulfite-conversion-free and absolute quantification of cfDNA methylation via Nanopore sequencing.enUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.EpigeneticscfDNAPulmonary Arterial Hypertension (PAH)Long COVIDNanoporeBiology--MolecularBiophysics--Medicalmaster thesis