Defining The Role of PRPF8 in Congenital Heart Diseases

Abstract

Congenital heart disease (CHD) is the most common birth abnormality, impacting over 1% of live births worldwide and representing a major cause of infant mortality and morbidity. Despite advances in genetic studies identifying several causal variants, the molecular mechanisms behind many congenital heart defects (CHDs) remain insufficiently elucidated. We have discovered a mutant mouse line with laterality and cilia defects accompanied by cardiac abnormalities caused by a missense mutation in the spliceosome gene Prpf8. The role of PRPF8 in congenital heart defects has not been investigated prior to this thesis research. In this project four PRPF8 missense variants: V250M, P372L, T589M and M1730T, that have been found in CHD patients, were studied to determine if PRPF8 function is altered by these variants. These PRPF8 variants were predicted that may cause aberrant splicing and/or altered biochemical interactions between PRPF8 and the other spliceosomal proteins, leading to developmental heart malformations likely due to aberrant splicing or expression of cardiac genes. PRPF8 variants were investigated using multiple bioinformatics prediction tools to predict the impact of PRPF8 variants found in human CHD patients on protein function. Moreover, PRPF8 variant protein-protein interactions and protein stability were assessed. We also generated the analogous missense variants in yeast Prp8 to study their effect during splicing. Moreover, a Prpf8 mouse model was used to evaluate the expression of Prpf8 in cardiac and neural crest cell markers. Our bioinformatics analysis revealed some of these variants were predicted to be damaging and affect protein functions while others were not. Significantly, although the most highly predicted damaging variant, PRPF8 T589M, did not interfere with PRPF8 interactions with its binding partners, EFTUD2 and PPIL2, it, and other PRPF8 variants, adversely impacted PRPF8 protein stability. This suggests a specific mechanism in which decreased protein stability, rather than a loss of physical interactions, accounts for the impact of these mutations. Our splicing reporter assay findings indicate that all examined mutations impair PRP8's splicing efficiency, causing remarkable gene expression alteration and splicing defects. The Prpf8 mutant mouse displays reduced neural crest marker expression, suggesting a vital role for PRPF8 in cardiac development via the regulation of neural crest cell development. Novel insights gained from this work may aid our ability to understand the role of PRPF8 in CHD and its links with cardiac defects, informing diagnostic and therapeutic strategies.