Investigating non-coding genetic variation to enhance understanding of individual cardiovascular disease risk

Introduction
Linking genotypes to phenotypes enhances our understanding of complex human diseases, including cardiovascular disease. While some consequences of genetic variation are well understood, this is not true for the non-coding genome. Parts of the non-coding genome are transcribed into non-coding RNAs (ncRNAs), which play crucial roles in regulating gene expression, including via the formation of RNA-DNA interactions at gene regulatory sites. Many disease-relevant sites of genetic variation map to ncRNAs. This project explores whether the disruption of RNA-DNA interactions by genetic variants underlies some non-coding genotype-phenotype relationships with relevance to cardiovascular disease. Identification of contributary variants would enhance our understanding of the molecular pathogenesis of cardiovascular disease, allow more accurate stratification of individual cardiovascular disease risk, and have the potential to lead to novel therapies targeting the non-coding genome and transcriptome.

Methods & Results
Known disease-linked single nucleotide variations available from the NHGRI/EBI GWAS Catalog were mapped to respective genomic features. Regions of non-coding RNAs predicted to be involved in RNA-DNA interactions were found to have a higher rate of disease-relevant genetic variation than protein-coding exon regions, indicating the potential importance of non-coding RNAs to the development of disease. By computationally modelling formation of RNA-DNA interactions by almost 500 wild-type and variant RNA sequences, a repertoire more than 13,000 predicted gene regulatory variation-sensitive ncRNA-DNA interactions was assembled, where sequence variation in the ncRNA is predicted to abrogate the interaction.

To establish the functional importance of these interactions in a cardiovascular disease-relevant context, RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq) was conducted to identify gene regulatory RNA-DNA interactions in control endothelial cells and endothelial cells stimulated to undergo endothelial-to-mesenchymal transition (EndoMT). EndoMT is a dynamic, pathologically important process which has been observed to take place in disease settings including atherosclerosis and myocardial infarction. From these data, ncRNAs whose gene regulatory DNA interactions change between control and disease-like conditions could be found, such as CLDN10-AS1, which displays a complete loss of DNA interactions in EndoMT. Perturbation of candidate RNAs identified as being variation-sensitive and dynamically chromatin-associated will be used to assess the functional consequences of abrogated RNA-DNA interactions on the induction and progression of EndoMT, with promising candidates having the potential to be taken forward into in vivo models of atherosclerosis and myocardial infarction.

Conclusion
This work shows that non-coding genetic variation has the potential to modulate disease-relevant gene regulatory programs relevant to cardiovascular disease. Through computational and molecular methodologies, we will prioritize candidate variant ncRNAs which could be used to further enhance our understanding of cardiovascular disease risk in the future.