Abstract Body (Do not enter title and authors here): Background: Thoracic aortic aneurysm and dissection (TAAD) is a rare but devastating disorder that affects more than 13,000 people per year in the United States. Genetic testing can help guide early detection and therapy, but despite identification of numerous causal loci, the genetic architecture of TAAD remains poorly understood. There is a need for further investigation to identify and confirm new causal genes to improve screening for TAAD. We hypothesize that genetic testing of the most phenotypically severe cases in pediatric patients will improve the yield of novel gene identification.

Methods: We identified an extreme case of TAA in a 6-month-old patient at Boston Children’s Hospital and performed whole-exome sequencing on DNA samples collected from the proband and unaffected parents. We generated a clonal EMILIN1 knockout (KO) human aortic vascular smooth muscle cell (VSMC) model using CRISPR/Cas9 to assess the effect of complete EMILIN1 loss on gene expression, protein level, and critical VSMC functions including contractility, migration, and proliferation by collagen disk assay, scratch assay, and Ki67 staining respectively.

Results: Whole exome sequencing in a pediatric patient with an extreme, progressive TAA phenotype (4.0cm, Z-score +15) identified compound heterozygous nonsense mutations in EMILIN1, each inherited from one parent, as candidate causal variants. These variants (c.664C>T and c.1606C>T) were validated by Sanger sequencing. The EMILIN1 KO VSMCs demonstrated marked alterations in expression of genes encoding contractile apparatus and extracellular matrix proteins. Functional tests also revealed increased contractility (p=0.0374), decreased proliferation (p<0.0001), and decreased migration (p<0.0001) of EMILIN1 KO cells.

Conclusion: Whole exome sequencing in a 6-month-old patient with a severe presentation of TAA identified likely pathogenic compound heterozygous mutations in the EMILIN1 gene. Complete EMILIN1 loss-of-function in VSMCs results in cellular phenotypes comparable to other pathogenic TAA mutations.