Molecular and functional characterization of ANKRD1, a candidate gene for total anomalous pulmonary venous return.

Total Anomalous Pulmonary Venous Return (TAPVR, OMIM %106700) is a rare congenital heart defect in which the pulmonary veins fail to enter the left atrium and drain instead into the right atrium or one of its venous tributaries. Previously, a TAPVR patient bearing a t(10;21) de novo balanced translocation was found in our laboratory and the ANKRD1/CARP gene, mapping on chromosome 10 about 130 Kb proximally to the breakpoint, was defined as a good TAPVR candidate gene. Significantly, we found highly increased ANKRD1 expression levels in lymphoblastoid cell lines derived from both the translocation-bearing proband and a second independent sporadic TAPVR patient, suggesting that disruption of the normal ANKRD1 expression pattern is associated with TAPVR. Moreover, an ANKRD1 non-conservative missense mutation was found in a third sporadic TAPVR patient. Such mutation was subsequently found to confer an increased stability to the ANKRD1 protein in vitro and to affect its function as a repressor of cardiac-specific gene expression. The aim of this doctoral project was to characterize at the molecular and functional level the ANKRD1 gene, in order to provide new evidences for its involvement in TAPVR pathogenesis. First, ANKRD1 function as a negative regulator of cardiac genes was further investigated, and its role as a repressor of Nkx2.5 and Pitx2cmediated activation of the cardiac-specific ANF promoter was demonstrated. Moreover, in situ hybridization assays revealed that the expression pattern of the murine Ankrd1 gene strongly overlaps with that of both Nkx2.5 and Pitx2 genes, supporting both the occurrence of a functional interaction between these genes and a role for ANKRD1 in TAPVR pathogenesis. Co-immunoprecipitation assays added a further support for this hypothesis, by showing a physical interaction between the ANKRD1 and Nkx2.5 proteins. The intracellular pathways involved in ANKRD1 turnover were also investigated. ANKRD1 was shown to behave as a short-lived protein in vivo, with the PEST motif playing a crucial role in its turnover rate. Moreover, intracellular degradation of ANKRD1 was found to be carried out mainly by the ubiquitin proteasome system, through several independent degron elements. These data defined for the first time both the intracellular pathway responsible for ANKRD1 degradation and the functional relevance of the PEST motif in this process. Finally, ANKRD1-overexpressing transgenic mice were generated in the attempt to develop an animal model for TAPVR. Several independent transgenic lines are currently under investigation in order to better define the role of ANKRD1 in heart development.