Periodic Reporting for period 3 - ROSALIND (Investigating fibROmuscular dysplasia and spontaneous coronary Artery dissection using genetic and functionaL genomics to decipher the origIN of two female specific cardiovascular Diseases) Berichtszeitraum: 2020-03-01 bis 2021-08-31 Zusammenfassung vom Kontext und den Gesamtzielen des Projekts Cardiovascular disease (CVD) is under-diagnosed and under-investigated specifically in women. Clinical presentation of CVD is often different in women and the aetiology of some diseases is potentially triggered by specific female sex environmental factors (e.g hormonal cycles and pregnancy) and could result in distinct pathophysiology from men. This may apply to fibromuscular dysplasia (FMD) and spontaneous coronary artery dissection (SCAD), two devastating arterial diseases that share clinical features, which are non-atherosclerotic stenosis of medium-size arteries (renal and/or cerebrovascular arteries in FMD, coronary artery in SCAD) and an age of onset under 50, in addition to a high proportion of female patients (75-90%). In addition, genetic predisposing factors may interact with the female-specific context and disturb the artery structure and/or function resulting in a female-specific propensity to these CVDs. The ROSALIND project aims to: 1) decipher the genetic basis of FMD and SCAD using genome-wide association in case-control cohorts; 2) Examine the functional significance of the genetic susceptibility variants at confirmed loci and their targeted genes using high throughput NGS-based genomic methods and 3) explore the link of genes in FMD susceptibility loci with vascular function by the analyses of engineered cell lines and total expression in human renal arteries. This project will provide an unprecedented resource of genetics, gene expression and functional genomics data that will be instrumental to guide the uncovering of new genes and mechanisms involved in FMD and SCAD. This project will help better understand the pathophysiology and shed light on novel and promising therapeutic targets for the non-atherosclerotic arterial stenosis that characterize these female CVDs. Arbeit, die ab Beginn des Projekts bis zum Ende des durch den Bericht erfassten Berichtszeitraums geleistet wurde, und die wichtigsten bis dahin erzielten Ergebnisse Our project has advanced toward the identification of genetic causes of FMD and SCAD. We first validated the PHACTR1 association with SCAD through the largest genetic study on SCAD ever conducted. We investigated the association between rs9349379 and SCAD, to assess whether, at this locus, SCAD is genetically closer to FMD, given their clinical overlap, or to MI. In the largest case-control study conducted so far for SCAD (>1,000 patients), we reported a highly significant association with SCAD that is independent of FMD status in SCAD patients (Adlam et al JACC 2019). We also followed-up the search for highly penetrant variations in FMD from exome sequencing data performed in siblings and found that the prostacyclin (IP) receptor gene (PTGIR), is likely to be enriched for loss-of-function variations in >1,300 FMD patients. Prostacyclin signalling was described to regulate SMCs proliferation and migration, as well as the fibrotic transformation of fibroblasts, several mechanisms suspected to participate in the physiopathology of FMD. We also demonstrated these variants to impair the receptor function in vitro and found several of these variations in some SCAD patients from a >800 patients cohort supporting further the genetic and molecular link between FMD and SCAD (Georges et al., ASHG Meeting 2019). We also generated genome-wide genotyping for >1200 FMD, SCAD and control individuals and now have access to 2 case-control studies by collaboration. SCAD genetics has taken an important position in the project with the identification of several case-control studies that are not available to us after the collaboration for the PHACTR1 study (Adlam JACC 2019). A full GWAS is ongoing specifically for SCAD. We have also generated genome-wide open chromatin profiles in several cell lines using the ATAC-Seq method. We have generated data in endothelial, smooth muscle cells and dermal fibroblasts. We have advanced the optimization of 4C analyses and generated genomic interaction experiments in preliminary association loci obtained from the French FMD and SCAD GWASs. Finally, we have initiated the optimization of iPSC differentiation into SMCs to study the enhancers activities, gene expression and effects of knockout on differentiation, migration and secretion of the extracellular matrix components of cells. Fortschritte, die über den aktuellen Stand der Technik hinausgehen und voraussichtliche potenzielle Auswirkungen (einschließlich der bis dato erzielten sozioökonomischen Auswirkungen und weiter gefassten gesellschaftlichen Auswirkungen des Projekts) In this project, we are now planning to perform an extensive genetic investigation not only for FMD but also for SCAD through a full GWAS and functional exploration at SCAD loci. The initial plan was to validate FMD genes in SCAD, and we did this through 2 studies: PHACTR1 (Adlam JACC 2019) and PTGIR (Georges et al in preparation). We have now made major advances in collecting patients’ data and identifying new collaborators who gave us access to DNA from a total of more than 1000 SCAD patients, which was unexpected given the neglected feature of SCAD. This is partially due to an increase in awareness about the disease that our work is fueling every time we publish a new genetic study. There is now a clear interest from the cardiology community to understand the genetics and physiopathology of SCAD, and my group is a major contributor to this field. In the cellular part, we have decided to create SMC models from iPSCs, which is a new technical strategy that I have decided to adopt in order to go beyond the limitations of primary cell lines planned initially. We found that the transition from pluripotency to SMC will provide us with more data about gene regulation and function at FMD and SCAD loci. We also plan to create iPSCs to endothelial cells, the current focus on SMCs comes from the current candidate genes PHACTR1 and a new one under validation at the genetics level.