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 PCR-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 to the total, thus eliminating the problem of variability in the level of total angiotensinogen mRNA among subjects. We show that 1) angiotensinogen mRNA derived from the −20C allele is expressed significantly higher than that derived from the −20A allele in subcutaneous adipose tissue, and 2) 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 two variant alleles in human tissue where the ratio of expression of one allele to another can be accurately determined.

An Angiotensinogen Gene Polymorphism (rs5050) Is Associated with the Risk of Coronary Artery Aneurysm in Southern Chinese Children with Kawasaki Disease

“…We selected two polymorphisms in the AGT gene, i.e., rs699 (M235T in exon 2) and rs5050 (A-20C in the promoter region), for detection. The selection of the two functional polymorphisms was based on a thorough evaluation of polymorphisms associated with vasculopathy [22, 25–27]. Genomic DNA was extracted from all KD cases and controls blood using the TIANamp Blood DNA Kit (Qiagen, Dusseldorf, Germany).…”

Obesity-Associated Hypertension

“…Analysis of human adipose tissues obtained during surgery revealed a great variability in the total level of angiotensinogen gene expression among subjects (59). This analysis also showed enhanced levels in the subcutaneous adipose tissue of the mRNA derived from a particular allele of the angiotensinogen gene bearing the −20C variant in its promoter.…”

“…This analysis also showed enhanced levels in the subcutaneous adipose tissue of the mRNA derived from a particular allele of the angiotensinogen gene bearing the −20C variant in its promoter. This was associated with enriched binding to the −20C allele of a transcription factor, the upstream stimulatory factor 2, which is essential for angiotensinogen transcriptional regulation (59). Future studies are needed to examine the relevance of these genetic determinants in angiotensinogen gene to obesity and hypertension.…”

The prevalence of unique SNPs in the renin‐angiotensin system highlights the need for pharmacogenetics in Indigenous Australians

“…In other non‐Indigenous Australian populations worldwide there is a higher prevalence of RAS SNPs (associated with over‐expression of RAS proteins) in cardiovascular and other chronic diseases. For example, the polymorphism rs4762 in the angiotensinogen ( AGT ) gene is linked to variants in the promoter region of this gene and are associated with greater AGT expression in human adipose tissue . This variant has been associated with diseases including preeclampsia .…”

“…For example, the polymorphism rs4762 in the angiotensinogen (AGT) gene is linked to variants in the promoter region of this gene and are associated with greater AGT expression in human adipose tissue. 3 This variant has been associated with diseases including preeclampsia. 4 The variant rs4291 in the angiotensin converting enzyme (ACE) gene, which is associated with increased ACE activity, 5 has been linked with hypertension.…”