Genetic variation in the adiponectin receptor 2 (ADIPOR2) gene is associated with coronary artery disease and increased ADIPOR2 expression in peripheral monocytes

Abstract

Background: Adiponectin is an adipose tissue secreted protein known for its insulin sensitising and anti-atherogenic actions. To this date two adiponectin receptors have been discovered, adiponectin receptor 1 (ADIPOR1) and adiponectin receptor 2 (ADIPOR2). The aim of this study was to investigate the association of ADIPOR2 gene variations with coronary artery disease (CAD).

Methods: Eight common single nucleotide polymorphisms (SNPs) spanning the entire ADIPOR2 locus were chosen to perform association studies with anthropometric and metabolic parameters in a Greek population. They were classified as either CAD (stenosis >50% in at least one main vessel) or non-CAD individuals in accordance with coronary angiography data.Genotyping was performed using a microsphere-based suspension array and the Allele Specific Primer Extension (ASPE) method. Expression of ADIPOR2 protein and mRNA in circulating CD14+ monocytes were determined using flow cytometry and real time Polymerase Chain Reaction assays respectively.

Results: There was a significant difference in the distribution of genotypes of polymorphism rs767870 of ADIPOR2 between CAD and non-CAD individuals (p = 0.017). Furthermore, heterozygotes of the rs767870 polymorphism had significantly lower Flow Mediated Dilatation (FMD) values, higher values of Intima-Media Thickness (IMT) and increased ADIPOR2 protein levels in peripheral monocytes, compared to homozygotes of the minor allele after adjustment for age, sex, waist to hip ratio and HOMA.

Conclusions: Our findings suggest that variants of ADIPOR2 could be a determinant for atherosclerosis independent of insulin resistance status, possibly by affecting ADIPOR2 protein levels.

Figure 1

Schematic representation of the ADIPOR2 gene. The polymorphisms studied are represented by vertical arrows. Empty boxes denote exons and black boxes denote sequences translated into protein. +1 represents the ATG translation start site.

Figure 2

Representative PCR reactions showing the PCR specific bands for all polymorphisms studied of the ADIPOR2 (A). Detection of the PCR specific bands for polymorphism rs767870, lanes 2 and 3 show the PCR specific bands amplified from human genomic DNA from two CAD individuals, while lanes 4 and 5 show the PCR bands amplified from human genomic DNA from two non-CAD individuals. (B) Detection of the PCR specific bands for polymorphisms: rs10773980 (lane 1), rs1029629 (lane 2), rs16928751 (lane 3), I290I (lane 4), rs9805042 (lane 5), rs12342 (lane 6) and rs1044771 (lane 7). Arrows show the base pair size of the various PCR products. M: ΦX174-HaeIII digest

Figure 3

Diagrammatic representation of: (A) the genotyping results for rs767870 polymorphism of ADIPOR2 using the Allele Specific Primer Extension assay and the xMAP technology and (B) the detection of the relative ADIPOR2 mRNA levels using the real-time PCR assay. A. Bars represent the two alleles of the rs767870 polymorphism (G allele: blue bars, A allele: orange bars). The results show three different individuals: on the left, an individual homozygote for the A allele (AA genotype), in the centre, a heterozygote carrying both alleles (AG genotype) and on the right side, a homozygote for the G allele (GG genotype). B. A quantification analysis screen from a typical real-time PCR run, showing the fluorescence acquisition for the ADIPOR2 (green and black curves) and the β-actin (red and pink curves) mRNA levels on the y axis and the number of cycles on the x axis. Results are shown for two different individuals, a CAD (green and red curves) and a non-CAD individual (black and pink curves).

Figure 4

Genotype effects of the SNP 767870 of ADIPOR2 on the: (A) IMTbulb measurement, (B) FMD measurement and (C) ADIPOR2 protein levels from CD14+ monocytes. Data are means ± SEM. Differences between genotype groups were analysed by ANOVA adjusted for age, sex, BMI, WHR and HOMA (A and B, p = 0.04 and p = 0.002 respectively) and ANOVA adjusted for age, sex, WHR and HOMA (C, p = 0.05) * p < 0.05 vs GG homozygotes §p < 0.05 vs AG heterozygotes.