0268 : Involvement of LRRFip1 gene and canonical Wnt pathway in Mitral Valve Prolapse (MVP)

Heart valves diseases affect 3% of world population, and surgery is often the only therapeutic mean. A genetic study performed on a family in which several members exhibited a MVP identified a R94G mutation on LRRFip1 gene. LRRFip1 alternative transcription splicing gives rise to five isoforms in humans, three of which are targeted by the mutation (Iso1, 3 and 4). Previous studies only focused on LRRFip1-iso5 that was first described as a transcription factor interacting with positive (Dishevelled) and negative (Flightless-1) regulators of the canonical Wnt β-catenin dependant pathway. As it may participate and regulate crucial events of cardiac valve development and homeostasis involving Wnt pathway, we hypothesised that LRRFip1 could be involved in MVP pathology. We first analysed the expression of LRRFip1 in valves by RNA sequencing and quantitative PCR and showed that LRRFip1- iso1 is expressed in human valves. In mouse, it prevails during embryonic development and then levels down to that other isoforms expression. We thus focused on LRRFip-iso1. Using cell fractionation, we showed a nuclear localization of LRRFip1-iso1 while other isoforms are strictly cytoplasmic. Using luciferase-based Wnt reporter assays and co-IP, we further demonstrated that out of the five isoforms, LRRFip1-iso1 is the strongest interactor of Dvl-1 and Fli-1, and the strongest activator of the canonical Wnt pathway. Although activation requires beta-catenin, it does not involve beta-catenin stabilization nor activation. Using site directed mutagenesis, we mapped the domain responsible for Wnt pathway activation to the 25 amino-acids region surrounding arginine 94 and showed that R94G mutation also decreases Wnt activation. This work demonstrates the involvement of LRRFip1-iso1 in canonical Wnt pathway activation. Taken together, our results suggest a potential role for LRRFip1 in valvulogenesis and/or valve homeostasis regulation that may be impeded by the R94G mutation