Congenital heart defects (CHDs) are the most common developmental abnormalities, affecting around 1% of live births1. Aneuploidy causes around 15% of CHDs, with trisomy 21 (also known as Down syndrome) being the most frequent form2. CHDs occur in around 50% of cases of Down syndrome, with an approximately 1,000-fold enrichment of atrioventricular canal (AVC) defects that disrupt the junction between the atria and ventricles3,4. The AVC contains unique myocardial cells that are essential for valvuloseptal development; however, the specific combination of dosage-sensitive genes on chromosome 21 that are responsible for Down syndrome-associated CHDs have remained unknown. Here, using human pluripotent stem cell and mouse models of Down syndrome, we identify HMGN1, a nucleosome-binding epigenetic regulator encoded on chromosome 21, as a key contributor to these defects. Single-cell transcriptomics showed that trisomy 21 shifts human AVC cardiomyocytes towards a ventricular cardiomyocyte state. A CRISPR-activation single-cell RNA droplet sequencing (CROP-seq) screen of chromosome 21 genes expressed during heart development revealed that HMGN1 upregulation mimics this shift, whereas deletion of one HMGN1 allele in trisomic cells restored normal gene expression. In a mouse model of trisomy 21, a similar transcriptional shift of AVC cardiomyocytes was restored by a reduction in Hmgn1 dosage, leading to rescue of valvuloseptal defects. These findings identify HMGN1 as a dosage-sensitive modulator of AVC development and cardiac septation in Down syndrome. This study offers a paradigm for dissecting aneuploidy-associated pathogenesis using isogenic systems to map causal genes in complex genetic syndromes.
