PROJECT SUMMARY Down syndrome (DS) is the most common chromosomal abnormality affecting one in every 700 live births. Up to 50% of individuals born with DS, also known as trisomy 21 (T21), are born with a congenital heart disease (CHD). Despite the high frequency of DS-CHD, the exact mechanisms of DS-related CHD remain unknown. In this Supplement to the Parent R01 HL130020-5, we propose to leverage our established iPSC-based experimental models to understand the genetic basis of DS-CHD. We hypothesize that an interaction of genes between T21 and non-T21 mutations causes CHD in DS. Patients. In Aim 1, we will perform whole genome- sequencing (WGS) in DS patients with or without CHD to identify single nucleotide variants (SNV) associated with DS-CHD. In Aim 2, we will validate the pathogenicity of SNVs on development of CHDs in the presence of T21. We will use CRISPR/Cas9 to correct or insert the SNVs to iPSCs from control and DS patients to establish a genotype-phenotype relationship between SNVs and CHDs. Completing the aims of this supplement will likely increase our understanding of DS-related CHD as well as broaden the overall impact of the parent R01 award. In summary, this INCLUDE Administrative Supplement proposal will create novel opportunities to build a massive functional and sequencing database as well as iPSC biorepository that will be broadly shared with multi- disciplinary investigators. The combination of deep clinical phenotyping, the use of iPSC, CRISPR/Cas9 and multi-omic approaches will undoubtedly help to move this field forward and better understand DS-related cardiac pathologies.

PROJECT NARRATIVE Down syndrome (DS) is a common cause of congenital heart disease (CHD), yet its mechanism is poorly understood due in part to lack of suitable experimental models that can recapitulate the complexity of genotype- phenotype relationship. By using human induced pluripotent stem cells (iPSC), whole genome sequencing, CRISPR/Cas9 and bioinformatics strategy, we propose to examine the genetic interactions that cause CHD in DS patients. The results obtained here may provide us with important clues for pathogenesis of DS-related CHD as well as the development of disease-specific intervention strategies.