The Interaction Between Prpf8 and Dzip1 and its Role in Mitral Valve Prolapse

Mitral Valve Prolapse (MVP) is one of the most common cardiac diseases affecting 1 in 40 individuals worldwide. Functionally, MVP is the abnormal billowing of one or both of the mitral valve leaflets into the left atrium during ventricular systole. Structurally, the valves experience an accumulation of proteoglycans, an increase of collagen and hyperplasia. An echocardiogram can be used to diagnose the disease as well as determine the severity of it. MVP can cause several underlying effects such as regurgitation, arrhythmias and even sudden cardiac death in severe cases. Although there is very little known about the cause of the disease, recent discoveries have identified various genetic associations with MVP. This is the basis by which we structure this proposal. A linkage analysis was conducted on a large family with non-syndromic MVP to find a common cause for the disease. Through this linkage analysis a specific region of genes was identified, in particular, a cilia gene, DZIP1 was found along with the point mutation in the gene. Through expression, knockout studies and biochemical approaches we have gained a better understanding of what DZIP1 and the mutation of DZIP1S14R/+ are doing during cardiac development. Currently, little is known about how mutations in the cilia gene can influence the development of MVP. To address this question, a two-hybrid screening was completed, in which DZIP1 wild type plasmid and mutant DZIP1 plasmid were used to determine DZIP1\u27s binding partners. A pre-mRNA processing factor protein named Prpf8, which is a fundamental component of the spliceosome, was found to interact with the wild type DZIP1 plasmid but was the only protein found to not interact with the mutant DZIP1 plasmid. Additionally, the previous GWAS completed on patients with MVP identified a single nucleotide polymorphism (SNP) in close proximity to the PRPF8 locus, which led us to hypothesize that this SNP could function as an enhancer to regulate Prpf8 expression. Here we present data that shows that Prpf8 is expressed in the mitral valves at various embryonic and postnatal stages, and that it co-localizes with DZIP1. Verification of the two-hybrid screening result was tested through co-immunoprecipitation from protein collected from wild type (Dzip1+/+) and mutant (Dzip1S14R/+) adult mouse tail fibroblasts. Furthermore, through collaborators at INSERM, chromatin conformation capture experiments on chromosome 17 (where PRPF8 is located) determined four potential regulatory sequences that are in close proximity to PRPF8\u27s transcription start site. With the given data, luciferase assays indicated sequences with enhancer function. The studies presented will establish a broad mechanism by which DZIP1 mutations regulate RNA splicing, resulting in myxomatous valves and MVP in patients