In search of genetic modifiers that explain the phenotypic variability in SMAD3-related aortopathy

Abstract

Abstract Introduction Loeys-Dietz Syndrome (LDS) is an autosomal dominant connective tissue disorder presenting with thoracic aortic aneurysm and dissection (TAAD). Remarkably, some LDS patients remain cardiovascularly unaffected throughout life, while others carrying the exact same genetic variant die early because of an aortic dissection. We hypothesize that genetic modifiers are the basis of this observation. Objectives Identify genetic modifiers that explain the variability in LDS-related aortopathy. Generate a patient-specific iPSC-vascular smooth muscle cell (VSMC) model to allow functional validation of genetic modifiers. Material and Methods We have access to a large LDS family segregating a pathogenic SMAD3 (p.Arg287Gln) variant. Identification of candidate modifiers in this family encompasses genome-wide SNP-based linkage analysis (n=19, available mutation carriers) and WGS (3 affected (AMC) and 4 unaffected mutation carriers (UMC)). Subsequent functional validation involves CRISPR/Cas-based modifier correction in iPSC-VSMCs of an affected variant carrier, which are created using the CytoTune iPS 2.0 Kit and the Granata et al. VSMC differentiation protocols. Results Linkage analysis suggests the presence of an aggravating modifier at chr2 (LOD: 2.68). The region of 6 Mb contains 19 protein coding genes of which the TNFAIP6 gene was ToppGene prioritized. There were no exonic variants in any of the genes that segregate with affection status at MAF 0.01%. However, we are currently adapting the the filtering strategy and will look into more common variants, MAF 10% for exonic and MAF 5% for intronic variants. iPSCs of an AMC and its isogenic control were created. They express core pluripotency markers and possess trilineage differentiation potential in the absence of the Sendai vectors. SNP array confirmed the genomic stability and identity of the cells. Conclusion and Future Work The obtained data suggest the presence of an aggravating modifier at chr2. Further analyses are ongoing to pinpoint the exact modifier variant explaining the linkage signal. Subsequently, the identified modifier(s) will be modelled in an in-vitro created iPSC-VSMC model.