Intronic ANG II type 2 receptor gene polymorphism 1675 G/A modulates receptor protein expression but not mRNA splicing

The X-linked ANG II type 2 receptor (AT2) is supposed to be involved in cardiovascular disorders. Two studies associated the A allele of the AT2 gene polymorphism (PM) 1675 G/A with left ventricular hypertrophy in men and coronary ischemia in women. Because the PM is located in the short intron 1 of the AT2 gene within a sequence motif similar to the splice branch site consensus, we tested whether it might affect pre-mRNA splicing and/or modulate AT2 receptor expression. We first analyzed the AT2 mRNA splice pattern by RT-PCR in myocardial samples from 12 explanted human hearts and compared it with the respective genotypes. All 12 patients, 10 hemizygous males (7 A, 3 G allele carriers) and 2 homozygous females (2 G/G allele carriers), exhibited the same myocardial AT2 splice pattern with a relative abundance of transcript exon 1/2/3 compared with exon 1/3. Next, AT2 minigene constructs were cloned from both alleles, comprising the core promoter and the complete transcribed region up to the translation start codon, upstream of a luciferase reporter gene. These constructs were transfected into human (HT1080) and rat (PC12W) cell lines and rat vascular smooth muscle cells, and luciferase activities were assessed, as well as the splice patterns of the chimeric AT2/luciferase mRNAs. In all transfected cell types, the mRNA expressed from the AT2 constructs was spliced like the endogenous myocardial AT2 mRNA. However, luciferase activities driven by the G allele construct were significantly higher than those expressed from the A allele. Taken together, these data indicate that individuals carrying the G allele may express higher levels of AT2 receptor protein, which may be protective during the development of ventricular hypertrophy and coronary ischemia.