Allele-Specific Expression of Angiotensinogen in Human Subcutaneous Adipose Tissue

Abstract

The angiotensinogen gene is genetically linked with hypertension, but the mechanistic basis for association of sequence variants in the promoter and coding region of the gene remains unclear. An E-box at position -20 has been hypothesized to control the level of angiotensinogen expression, but its mechanistic importance for angiotensinogen expression in human tissues is uncertain. We developed an allele-specific polymerase chain reaction-based assay to distinguish between angiotensinogen mRNA derived from variants at the -20 position (rs5050) in the angiotensinogen promoter in adipose tissues obtained during surgery. The assay takes advantage of linkage disequilibrium between the rs5050 (located in the promoter) and rs4762 (located in the coding region) single nucleotide polymorphisms. This strategy allowed us to assess the level of allele-specific expression in A-20C heterozygous subjects comparing the relative proportion of each allele with the total, thus eliminating the problem of variability in the level of total angiotensinogen mRNA among subjects. We show that angiotensinogen mRNA derived from the -20C allele is expressed significantly higher than that derived from the -20A allele in subcutaneous adipose tissue, and increased expression correlates with enriched chromatin binding of upstream stimulatory factor-2 to the -20C E-box compared with -20A. This may be depot selective because we were unable to detect these differences in omental adipose. This provides the first data directly comparing expression of angiotensinogen mRNA and differential transcription factor binding derived from 2 variant alleles in human tissue where the ratio of expression of one allele to another can be accurately determined.