Ferravante, Carlo (2022) Next-Generation Sequencing for the diagnosis of canine myocardial disorders: role of pathogens and study of gene expression pathways. [Tesi di dottorato]

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Cardiomyopathy, in clinical medicine, defines the primary myocardial disease triggered by an unknown etiology. The secondary myocardial disease may be described as some often reversible, inflammatory, metabolic, toxic, or infiltrative disease of the myocardium with a known etiology or causative agent. In this context, it is possible to differentiate myocardial disease in forms related to myocarditis, frequently associated with pathogens infection, from other forms secondary to endocrine abnormalities, electrolyte imbalances, trauma and neoplasias. Myocarditis can evolve in dilated cardiomyopathy, with consequent devastating effects on dogs’ health. Despite the problematic clinical conditions that characterize canine myocarditis, few data are present in the veterinary literature. Nonetheless, in recent years, more attention has been focused on this heart muscle disease, probably due to the advances in diagnostic technology and the more frequent use of endomyocardial biopsies that require advanced cardiology division heart failure programs, the technical capabilities and the expertise to appropriately analyze the specimens. Analyzing tissue specimens with more sophisticated methods, rather than routine staining used in light microscopy, may improve the diagnostic yield and clinical and research utility of endomyocardial biopsies. In this context, Next-Generation Sequencing (NGS) may be considered a supreme advantage. This high-throughput technology can be used to achieve a comprehensive and unbiased sequencing of the nucleic acids present in a sample. The sequencing of the whole nucleic acids content of a specimen enables the exploration of both host and exogenous DNA/RNA sequences. The large amount of data produced by the NGS approach, requires comprehensive and user-friendly pipelines for data analysis, that speed up the bioinformatics steps. In this thesis, the transcriptomic responses of the dogs to myocardial disorders were investigated along with the role of pathogens in myocardial disease. The sequencing of total RNA molecules from endomyocardial biopsies of 13 dogs affected with myocardial disorders, was performed using NextSeq 500 Illumina platform. Each endomyocardial biopsy was histologically evaluated, to reveal the presence of inflammation signs. Histological diagnosis of fibrosis was identified in 10/13 animals enrolled in this study. Three dogs with fibrosis showed also the presence of inflammatory infiltrates. In order to discover the presence of pathogens in cardiac tissues, a metagenomic approach was performed on RNA-seq data. Considering the lack of exhaustive and easy-to-use bioinformatics pipelines publicly available for the identification of pathogens, a new software (HOME-BIO (sHOtgun MEtagenomic analysis of BIOlogical entities)) was developed to provide a comprehensive taxonomy profiling of the endomyocardial biopsies. The metagenomic analysis results showed no presence of cardiotropic pathogen commonly related to myocarditis and myocardial damage, associated with a high number of RNA fragments. The expression profiles of enrolled samples were compared with those of the control healthy group, downloaded from public dataset (NCBI BioProject PRJNA78827), in order to identify cardiac tissue genes and gene pathways differentially regulated between the 2 populations. The analysis highlighted genes enriched in the dilated cardiomyopathy disease pathway and involved in cellular energy metabolism, along with altered cardiac structural proteins in affected dogs. Moreover, important results were achieved from the analysis of genes that characterized samples with clear signs of inflammation. In these samples, differentially expressed genes were involved in cardiac altered activity, as well as in structural cell reorganization and metabolism. By evaluating the different pathways and genes involved during myocardial disease, the results revealed molecular and genetic events that could play a key role in the progression of myocardial disorders and in particular may provide important insights into the pathogenesis of dilated cardiomyopathy.

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