Background and purpose: Previously, we described the presence of 5 haplotypes (A to E) at the angiotensinogen (AGT) promoter and reported a significant association between the B-haplotype (nucleotide substitutions -6:G-->A and -20:A-->C compared with the wild-type A-haplotype) and magnetic resonance imaging correlates of cerebral small vessel disease (cSVD). The association was independent of hypertension, suggesting a brain-specific effect of this haplotype. In the current study, we investigated transcriptional activities of the 5 promoter haplotypes in astrocytes, the main source of cerebral AGT, and in hepatocytes, the main source of systemic AGT, as well as determined the evolutionary relatedness of the promoter haplotypes.
Methods: Transcriptional activity depending on the haplotypes and the -6:A and -20:C substitutions was measured in transiently transfected A172 and HepG2 cells. We genotyped 5 new single nucleotide polymorphisms (SNPs) at the AGT gene and measured linkage disequilibrium (LD) among SNPs and the promoter haplotypes. An evolution-based haplotype tree was constructed.
Results: The B-haplotype increased transcriptional activity in both cell types. Its effect was stronger in astrocytes than in hepatocytes (2.4+/-0.09-fold, P<0.001 versus 1.6+/-0.06-fold, P=0.014). Importantly, in astrocytes the combination of the -6:A and the -20:C was mandatory for increased activity, whereas in hepatocytes the -20:C on its own was sufficient. Strong LD between the 5 new SNPs and the promoter haplotypes allowed the reconstruction of 9 haplotypes over the AGT gene. Cladistic analyses suggest that the B-haplotype represents an ancient promoter variant.
Conclusions: Combination of the -6:A and -20:C substitutions in the B-haplotype may promote the development of cSVD by enhancing cerebral angiotensinogen expression.