Abstract
Ischaemic heart disease is a major cause of death worldwide and a leading cause of mortality and morbidity in New Zealand. Older adults and those of Māori and Pacific ancestry are particularly affected. Ischaemic heart disease accounts for over half of all cardiovascular disease mortality and, again, rates are more than twice as high among Māori than non-Māori. Ischaemic heart disease can lead to myocardial infarction (heart attack) which, if not fatal, can then lead to heart failure, a complex, multifactorial disease characterised by neurohormonal signalling and remodelling of the heart. Currently the natriuretic peptides are the international gold standard for diganosing heart failure and are also excellent prognostic markers in patients with heart failure. However, there is still a clinical need for early biomarkers of myocardial ischaemia (to identify people at risk of myocardial infarction) and to identify patients at risk of developing heart failure before detrimental remodelling has occurred.
As sequencing technologies have evolved there has been intense research in the fields of circulating cell free DNA and RNA, especially non-coding RNA. As RNA is actively transcribed, it has the advantage of providing a ‘real time’ insight into the disease status of an individual. Recent discoveries have highlighted the regulatory roles and diseases associated with non-coding RNAs, including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs). LncRNAs have been demonstrated to have mutliple functional roles both within the nucleus and cytoplasm such as chromatin remodelling, histone modification, transcription factor recruitment, formation of subnuclear structures and control of mRNA translation and decay. CircRNA, a relative newcomer, has also been demonstrated to have functional roles such as sequestering miRNAs, binding proteins and even coding for peptides. There is great excitment for the potential utility of circRNAs as biomarkers as, due to their circular structure, they are more resistant to degradation in the circulation than their linear RNA counterparts.
The overall aim of this thesis was to identify non-coding RNAs associated with ischaemic heart disease. To address this aim, a bioinformatics pipeline was developed to identify mRNAs, lncRNAs including putative novel lncRNAs, and circRNAs using short-read RNA Sequencing (RNA-Seq) data. This pipeline was tested and validated with publicly available data and used to screen for candidate mRNA and lncRNA biomarkers associated with ischaemic heart disease in human heart tissue. A whole genome network correlation approach identified several promising candidate biomarkers for myocardial ischaemia including several novel lncRNAs, which were validated with long-read Nanopore sequencing in independent samples. The sub-cellular localisation of three promising lncRNAs candidates (two annotated lncRNAs, one novel lncRNA) was identified using the in-situ hybridisation assay, RNAscope®. Next, an RNA-Seq protocol was developed to detect mRNAs, lncRNAs and circRNAs in human plasma. This protocol was applied to plasma from patients with ischaemic heart disease and healthy controls to screen for candidate mRNA, lncRNA and circRNA biomarkers for progression from ischaemic heart disease to heart failure. Although candidate biomarkers for disease progression could not be detected in these patients several additional lncRNA candidates for the presence of ischaemic heart disease were identified.
In summary, this study has established a bioinformatics pipeline and methodology for identifying and validating putative novel lncRNAs and circRNAs in human tissue and plasma. This work has identified several promising candidate lncRNA biomarkers for ischaemic heart disease, which, if validated, may provide early diagnostic information in high-risk patients.
The pipeline is freely available to download at https://github.com/zoeward-nz/PhD