ABSTRACT The 22q11.2 deletion syndrome (22q11.2DS; DiGeorge syndrome) is a rare birth defect disorder affecting 1/4000 live births. This condition is fully penetrant, but expressivity varies, providing the opportunity to identify genetic modifiers. Our main interest is to understand the developmental and genetic basis of congenital heart disease (CHD) in 22q11.2DS patients by taking human and animal model genetic approaches. Approximately 60% of patients with 22q11.2DS have CHD. Among affected patients, most have cardiac outflow tract (OFT) defects, thereby disrupting the formation of the aorta and pulmonary trunk during embryogenesis. The malformations in patients vary from mild to severe and approximately half require surgery for survival after birth. Typical findings include tetralogy of Fallot, persistent truncus arteriosus or right sided aortic arch with a ventricular septal defect. The OFT derives from cells that migrate from the embryonic pharyngeal apparatus to the heart. The TBX1 gene, encoding a T-box transcription factor is expressed in the pharyngeal apparatus. Our hypothesis is that haploinsufficiency of TBX1, mapping to the critical deleted region on 22q11.2, along with modifiers elsewhere in the genome contribute to the overall phenotype in patients. To identify genetic modifiers of CHD, we obtained whole genome sequence (WGS) from 1,182 subjects with 22q11.2DS. Recently we were awarded a contract from NIH CIDR for WGS of 895 additional subjects with DNA in hand. We further initiated a collaboration with the G2MH Network to obtain WGS for another 500, all at no cost to this program, for a total of WGS from 2,577 subjects with 22q11.2DS. Our goal for Aim 1 is to investigate rare single nucleotide coding, non-coding and structural variants to identify genetic modifiers of CHD. We propose to take a gene-set based approach focusing on genes in the TBX1 network identified from our recent single cell transcriptomic experiments on control and Tbx1 loss of function mouse embryos. We will also investigate gene-sets used for genetic studies of sporadic CHD in the general population to determine whether modifiers for 22q11.2DS may also serve as risk factors for general CHD. We are utilizing a novel Bayesian prioritization approach to weight genes in gene-sets based upon their functional importance. As preliminary data we identified chromatin regulatory genes that might increase risk to CHD in subjects 22q11.2DS and for sporadic CHD. In Aim 2, we will perform functional and mechanistic studies of genes discovered in this program using mouse models. We will further perform biological validation studies of DNA variants identified in this program using gene editing in the zebrafish model system. This program will elucidate the molecular pathogenesis of CHD in 22q11.2DS that might also be risk factors for sporadic CHD in the general population. Understanding these should improve diagnostics and help in the future, for understanding the basis of varying outcomes in affected patients.

NARRATIVE This project will take human and animal model genetic approaches to understand the basis of phenotypic variability of congenital heart disease among 22q11.2 deletion syndrome patients. We will analyze whole genome sequence from subjects with 22q11.2 deletion syndrome with/out congenital heart defects and validate discoveries in mouse and zebrafish models. Some of the genes studied may also be risk factors for sporadic congenital heart disease in the general population.