PM1043: Using Single Cell RNA Sequencing to Understand the Link Between Congenital Heart Disease and down Syndrome

Background: Down Syndrome (DS), a common genetic disorder, is the result of a full or partial 3rd copy of chromosome 21. Along with mental disability and characteristic physical features, 40-60% of DS patients suffer from Congenital Heart Defects (CHDs). Common forms of CHD associated with DS are atrioventricular septal defects (AVSD) and outflow tract (OFT) defects. However, the mechanistic link connecting an additional copy of chromosome 21 in DS to CHD is poorly understood. The purpose of our work is to better underscore which molecular pathways critical for cardiogenesis are disrupted in DS, and determine how genes on chromosome 21 are connected to these pathways.

Methods: Human Induced Pluripotent Stem Cells (iPSC) from a mosaic DS cell line were obtained. iPSCs were differentiated into beating cardiomyocytes through Wnt signaling modulation, using the STEMDiff cardiomyocyte differentiation kit. Using 10x genomics single-cell RNA sequencing technology, trisomic and control iPSC cardiomyocytes were compared at day 10 and 20 of differentiation, and differences in gene expression were analyzed at single cell resolution. Using an analysis pipeline in the programming language ‘R’, cell populations of interest (atrioventricular canal and outflow tract cells) were identified via quantification of expression of known marker genes.

Results: Single cell analysis revealed increased expression levels of chromosome 21 genes in DS cardiomyocytes compared to euploid controls. A subpopulation of cardiomyocytes was identified as atrioventricular cells based on expression levels of RSPO3, BMP2, and TBX2, genes highly expressed in this region of the heart. Of particular interest, cardiomyocytes from the DS line were found to have dysregulated transcription in genes implicated in AVC myocardium formation, including downregulation of TBX2 and TBX3. Expression levels of genes implicated in formation of chamber myocardium, such as HAND1, MYH7, TBX20, NPPA, MYL3, and MEF2C were also disrupted.

Conclusion: Disruptions in expression of chromosome 21 genes play an important role in the increased incidence of CHD seen in patients with Trisomy 21. Although this may be a direct effect of dysregulation of genes on this chromosome, it is more likely that chromosome 21 gene dysregulation serves as an intermediary disruptor in non-chromosome 21 gene expression, specifically in genes that are crucial for cardiogenesis. The decreased expression of TBX2 and TBX3 is particularly notable as these genes repress chamber myocardial genes, and help maintain the formation of the atrioventricular endocardial cushion cells.
Future work will focus on in vivo studies using a partial trisomy mouse model (Dp1Tyb mouse). Expression of candidate chromosome 21 genes will be modulated, and CHD prevalence related to a given gene disruption will be determined histologically. Providing a mechanistic link between Trisomy 21 and CHD will allow for the potential to provide new targets for the therapeutic treatment of patients with CHD in the setting of DS.