Objective: We aim to better understand the genetic etiology of congenital heart disease (CHD) through the identification and functional testing of rare, damaging variants identified by exome sequencing (ES) of multiple affected individuals in families with recurrent CHD.

Methods: ES was performed on three family members with bicuspid aortic valve (BAV). Variants common to all three patients were filtered and ranked by predicted damaging scores and minor allele frequency using the Churchill pipeline. Likely disease-causing candidate variants were studied in cell based assays for evidence of functional changes. A NRP2b variant was tested by introducing it into the coding sequence in an expression construct that was transfected into HUVECs. A wound healing assay was performed on transfected cells to determine whether the variant affects the protein's ability to regulate cell migration. A variant in PLXNA2 was transfected into COS7 cells to test its ability to mediate semaphorin signaling in a cell collapse assay.

Results: Rare, damaging variants in both NRP2b and PLXNA2 were found in all three affected family members. Neuropilins and plexins heterodimerize to form receptors that bind semaphorins to mediate signaling that regulates cell migration, including migration of neural crest cells into the embryonic heart, where they contribute to the valve development. In a wound healing assay, cells expressing the variant NRP2b transgene showed significantly slower migration in response to VEGF than cells expressing the wild type protein. In a cell collapse assay, the PLXNA2 variant did not show a significant difference from wild type PLXNA2.

Conclusions: The identification of potentially damaging variants in two genes that may function in the same signaling pathway suggests a possible digenic inheritance for BAV in this family. We are currently crossing PlxnA2 knockout mice to Nrp2b knockout mice to generate double knockout animals that will be examined for heart valve defects.