Involvement of Smad2 Allelic Variants in Murine Coronary Artery High Take‐off Development

Abstract

The abnormal origin of a coronary artery from the ascending aorta, above the sinotubular junction or high take-off (HTO) is a rare congenital anomaly, which is associated with sudden cardiac death in approximately 1.5% of cases. HTO has been reported in 62.8% of mice from the C57Bl/6 (B6) strain, while it is absent in the Balb/c (BC) mouse strain. We have observed that the incidences of HTO in hybrid mice are intermediate between those of the parental animals (F1: 26.32%; F1xF1: 20%; F1xB6: 46.34%; F1xBC: 9.80%). In addition, we found that these incidences fit with a mode of inheritance consisting in the influence of a major causal dominant allele, present in the B6 strain, and a modifier dominant allele in the BC strain that reduces by half the incidence of HTO. In order to identify chromosomal regions including genes responsible for HTO development, we have carried out a genetic linkage study through the generation of a congenic strain, using microsatellite genetic markers. A region of 2,699,738 bp in chromosome 18 between the markers MIT-49 and MIT-106 was found statistically associated with HTO occurrence. This chromosomal region included a total of 9 genes (Smad2, Skor2, Ier3pi, Hdhd2, Katnal2, Pias2, St8sia5, Loxhd1, Rnf165). Among these, Smad2 was selected as a candidate gene, due to its multiple biological functions related to cardiovascular system development, specifically to aortic root formation and coronariogenesis. The comparative analysis of the Smad2 sequence between B6 and BC mouse strains revealed the presence of an intronic SNP (Smad2rs29725537:C>A or Smad2C>A) located in the 5’ splicing zone of intron 10-11. Sequence analysis predicted a possible error in Smad2 10-11 intron splicing caused by the B6 missense variant (Smad2C>A). After genotyping a total of 104 B6xBC F2 to F5 hybrid individuals of the developing congenic strain, by means of PCR-RFLP using Smad2 specific primers, we found a statistically significant (p<0.05) association between the allele Smad2C>A and HTO occurrence. We conclude that HTO is an inherited trait in mice, probably caused by a dominant allele and a dominant modifier allele. Our genotype-phenotype association study points to the Smad2rs29725537:C>A allele as the main causal factor for HTO in mice. Further studies are required to 1) confirm the role of the Smad2C>A SNP in murine HTO development; 2) check whether human HTO development is also influenced by the Smad2C>A allele; and 3) investigate the possible involvement of the Smad2C>A allele in the development of additional traits, particularly those differentially expressed between mice of the B6 and BC strains.