From genes to phenotype: investigating the link between smad2rs29725537:ca and high take off in mice

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucía, Ministerio de Ciencia e Innovación, Gobierno de España. Introduction and Purpose High take off (HTO) is a congenital anomaly with a prevalence of 0.36% that can cause severe ischemia and sudden cardiac death. The C57Bl/6 (B6) mouse strain shows around 60% incidence of HTO, whereas it is absent in the Balb/c (BC) strain. The aim of this study was to identify the genes responsible for the development of HTO. Methods Following an inheritance study, a genetic linkage study was performed in B6C mice using 134 microsatellites (MIT). After selection of Smad2 as a candidate gene, a genotype-phenotype study was performed by PCR-RFLP genotyping of 293 B6C mice. The process of coronariogenesis was examined in E13.5 and E14.5 mouse embryos of B6 (n = 27) and BC (n = 23) strains with specific markers for the vascular plexuses involved in coronariogenesis (PROX1 and ERG 1/2/3) and for SMAD2. Finally, the SMAD2 and pSMAD2 protein expression levels were analyzed by western blot in ventricle, liver, and kidney of B6C mice. Results The incidence of HTO in B6, BC and B6C mice were concordant with an inheritance pattern mediated by a dominant causal allele and dominant modifier in the BC strain that reduces the incidence of HTO. The genetic linkage study revealed the association of a region between markers MIT-49 and 106 on chromosome 18 with the presence of HTO. This region contained a total of 10 genes, of which Smad2 was selected as a candidate gene because of its functions in aortic root formation. Comparative sequence analysis of Smad2 between the B6 and BC mouse strains uncovered an intronic SNP (Smad2rs29725537:CA or Smad2CA) that predicts and error in the splicing of intron 10-11. A statistically significant association (p0.05) between the Smad2CA allele and the presence of HTO was found. pSMAD2 was detected in embryonic coronary primordia, the subepicardial aortic plexus (SAP) and the primary coronary plexus. Around 60% of B6 examined embryos showed an exacerbated SAP compared to the BC embryos. Additionally, we detected a differential expression of total SMAD2, but not of pSMAD2, in different organs according to the Smad2CA allele genotype (C/C A/C A/A). Conclusions We conclude that: 1) Smad2CA allelic variant is associated with HTO in mice, suggesting it as a major causal factor. 2) Smad2 altered expression may affect the establishment of the embryonic coronary primordia leading to a mispositioned ostium. 3) the Smad2CA allele has a systemic effect on total SMAD2 expression without affecting its phosphorylation level, which suggest a compensatory mechanism of expression that maintain the normal activity of pSMAD2 in most tissues.