Prediction of cardiac transcription networks based on molecular data and complex clinical phenotypes.

We present an integrative approach combining sophisticated techniques to construct cardiac gene regulatory networks based on correlated gene expression and optimized prediction of transcription factor binding sites. We analyze transcription levels of a comprehensive set of 42 genes in biopsies derived from hearts of a cohort of 190 patients as well as healthy individuals. To precisely describe the variety of heart malformations observed in the patients, we delineate a detailed phenotype ontology that allows description of observed clinical characteristics as well as the definition of informative meta-phenotypes. Based on the expression data obtained by real-time PCR we identify specific disease associated transcription profiles by applying linear models. Furthermore, genes that show highly correlated expression patterns are depicted. By predicting binding sites on promoter settings optimized using a cardiac specific chromatin immunoprecipitation data set, we reveal regulatory dependencies. Several of the found interactions have been previously described in literature, demonstrating that the approach is a versatile tool to predict regulatory networks.

Characterization of TBX20 in human hearts and its regulation by TFAP2.

The T-box family of transcription factors has been shown to have major impact on human development and disease. In animal studies Tbx20 is essential for the development of the atrioventricular channel, the outflow tract and valves, suggesting its potential causative role for the development of Tetralogy of Fallot (TOF) in humans. In the presented study, we analyzed TBX20 in cardiac biopsies derived from patients with TOF, ventricular septal defects (VSDs) and normal hearts. Mutation analysis did not reveal any disease causing sequence variation, however, TBX20 is significantly upregulated in tissue samples of patients with TOF, but not VSD. In depth analysis of TBX20 transcripts lead to the identification of two new exons 3' to the known TBX20 message resembling the mouse variant Tbx20a, as well as an extended 5'UTR. Functional analysis of the human TBX20 promoter revealed a 100 bp region that contains strong activating elements. Within this core promoter region we recognized functional binding sites for TFAP2 transcription factors and identified TFAP2 as repressors of the TBX20 gene in vitro and in vivo. Moreover, decreased TFAP2C levels in cardiac biopsies of TOF patients underline the biological significance of the pathway described. In summary, we provide first insights into the regulation of TBX20 and show its potential for human congenital heart diseases.