Abstract Body (Do not enter title and authors here): Background and aims. Tetralogy of Fallot (TOF; EC: 1565/2019) is the most common cyanotic congenital heart defect with a world-wide incidence of 0.02–0.04%. The pathophysiological mechanisms driven by variable genetic background are poorly understood. We applied the single-nuclei multiome profiling, composed of ATAC and RNA-seq from the same nuclei, of right ventricular samples of children with TOF. The final aim was to define cell types and characterize gene regulation states in different cell clusters including activation and maturation.
Methods. Right ventricular samples were prospectively gathered during routinely performed cardiac surgery for TOF correction (EC: 1565/2019). In total, six paediatric patient samples were processed, and on average ~1,500 nuclei were sequenced per sample. Data was analysed using Scanpy to cluster and annotate cells, pycisTopic to identify cell stats and cis-regulatory topics, and SCENIC+ for enhancer-driven gene Regulatory network (eRegulon) construction.
Results. Through manual annotation, using published markers, we identified cell types including cardiomyocyte, fibroblast, endothelial, pericyte, immune and neuronal cells. From this, we observed cell type specific expression, with overexpression of decorin in fibroblasts, PTPRC in immune cells, and neurexin genes in neuronal cells found. Differential expression analysis highlighted patient specific patterns of expression, of which gene set enrichment showed enrichment for genes associated with actomyosin structure organisation, dilated cardiomyopathy and cardiac muscle contraction. Integration of the gene expression data with the ATAC-seq data, across all patients, allowed for the construction of eRegulons networks. In detail, in the cardiomyocyte-annotated clusters, we identified enrichment of MEF2, MEIS2 and EPAS1 family enhancers with a central role of LIM domain binding 3 protein (LDB3, associated with cytoskeletal actin and muscle alpha-actin binding) (Fig. 1).
Conclusion. Single nuclei multiome profiling of paediatric right ventricular TOF samples identified several patient specific patterns of expression and regulatory networks, related to the development of cyanotic congenital heart disease, and sheds light to the potential drivers of TOF.