PROJECT SUMMARY Mitral valve prolapse (MVP), a condition affecting 2-3% of the general populace, is typified by irregular thickening of valve leaflets. The histopathological modifications associated with MVP are well known, yet a comprehensive understanding of the specific molecular structures and their impact on the disease's onset remains elusive. Bulk RNA-sequencing has been utilized to investigate gene expression in MVP, although inconsistencies between these data and those derived from traditional IHC and RT-PCR analysis have been observed. This discrepancy may be due to the fact that averaged expression cannot accurately portray heterogeneity across different cellular populations. At present, the study of MVP mitral valve leaflet samples using single-cell approaches is considerably limited. Moreover, spatial omics methodologies, capable of illustrating the spatial heterogeneity within tissue microenvironments, have not been applied to MVP research. To address this knowledge gap, we intend to construct a single-cell and spatial molecular atlas using mitral valve leaflet samples from both MVP patients and healthy contributors. In-depth data analysis will enable us to elucidate the molecular underpinnings of the disease. In this endeavor, we will focus on three specific objectives: 1) we aim to accurately identify cell subpopulations and molecular states in MVP using single-nucleus multi-omics sequencing; 2) we plan to characterize MVP's mitral valve tissue through spatially resolved transcriptomic, epigenomic, and proteomic techniques such as spatial-CITE-seq and spatial ATAC-seq; 3) we will extend our research to construct a molecular atlas of Marfan syndrome MVP utilizing single-cell and spatial omics analysis. In sum, the molecular data and insights gained from this research will facilitate further exploration into the diverse cell populations and tissue microenvironment associated with MVP.