Specialeforsvar: Caroline Bang Løppke

Supervisors
Prof. Søren Tvorup Christensen (Section for Cell Biology and Physiology, BIO-UCPH)
Prof. Lars Allan Larsen (Department of Cellular and Molecular Medicine, Panum Institute, UCPH)

External examiner
Prof. Steen Gammeltoft

Abstract
Congenital heart defects (CHDs) account for nearly one-third of all major congenital anomalies and are the leading cause of premature deaths in newborns. Consequently, there is an increasing demand for a better understanding of disease etiology and improved genetic counseling. Unfortunately, the link between genotype and phenotype is not easily established and in most patients the cause of the disease is unknown. Genetic analysis in CHD patients coupled with studies in model organisms have contributed greatly to the discovery of new candidate disease genes and provided researchers with valuable information unveiling the understanding of CHD complexity and heart development. By merging of data from different national registries, we identified a Danish family with recurrent cases of CHD. Whole exome sequencing combined with linkage analysis revealed two missense mutations, p. S181L and p. R646Q, within PLEKHA6 that segregated with the disease in the family, thereby classifying PLEKHA6 as a novel candidate gene in CHD. Furthermore, CRISPR-Cas9-mediated knockdown of plekha6 in zebrafish resulted in a clear cardiac phenotype and established a role of PLEKHA6 in heart development. The function of PLEKHA6 was further analyzed in vitro using the P19.CL6 cell model for cardiomyogenesis. We here show that both mRNA and protein levels of PLEKHA6 increase during cardiomyocyte differentiation and that the protein localizes at the plasma membrane during the end stages of differentiation. Furthermore, knockdown of PLEKHA6 in these cells resulted in an earlier onset of cardiomyogenesis, suggesting a potential role of the protein as a negative regulator of cardiomyocyte differentiation. Expression of PLEKHA6-GFP fusion proteins in other model cell systems confirmed localization of PLEKHA6 to the plasma membrane and more specifically to E-cadherin junctional complexes. These results suggest a role of PLEKHA6 in cell polarity processes such as collective cell migration, although preliminary analyses with migration assays of cells expressing either WT or mutant fusion proteins were inconclusive as to the role of the protein in cell polarity and migration. In conclusion, we provide novel information on the etiology of CHD, although further investigations are required for understanding the molecular and cellular mechanisms by which PLEKHA6 regulates heart development, and when defective leads to CHD.