INK4/ARF transcript expression is associated with chromosome 9p21 variants linked to atherosclerosis

Abstract

Background: Genome-wide association studies (GWAS) have linked common single nucleotide polymorphisms (SNPs) on chromosome 9p21 near the INK4/ARF (CDKN2A/B) tumor suppressor locus with risk of atherosclerotic diseases and type 2 diabetes mellitus. To explore the mechanism of this association, we investigated whether expression of proximate transcripts (p16(INK4a), p15(INK4b), ARF, ANRIL and MTAP) correlate with genotype of representative 9p21 SNPs.

Methodology/principal findings: We analyzed expression of 9p21 transcripts in purified peripheral blood T-cells (PBTL) from 170 healthy donors. Samples were genotyped for six selected disease-related SNPs spanning the INK4/ARF locus. Correlations among these variables were determined by univariate and multivariate analysis. Significantly reduced expression of all INK4/ARF transcripts (p15(INK4b), p16(INK4a), ARF and ANRIL) was found in PBTL of individuals harboring a common SNP (rs10757278) associated with increased risk of coronary artery disease, stroke and aortic aneurysm. Expression of MTAP was not influenced by rs10757278 genotype. No association of any these transcripts was noted with five other tested 9p21 SNPs.

Conclusions/significance: Genotypes of rs10757278 linked to increased risk of atherosclerotic diseases are also associated with decreased expression in PBTL of the INK4/ARF locus, which encodes three related anti-proliferative transcripts of known importance in tumor suppression and aging.

Figure 1. The human INK4/ARF locus and CAD associated SNPs.

(I) INK4/ARF-associated transcripts and tested SNPs are shown as well as the location of MTAP, another gene at 9p21. Blue colored boxes indicate exons 2 and 3 shared by p16^INK4a and ARF. The location of 9p21 SNPs (II) and linkage disequilibrium (LD) coefficient values (III) from HapMap CEU (Utah residents with ancestry from northern and western Europe) are shown. The LD plot of D′/LOD was generated using Haploview software . The strength of LD increases from white to blue to red: white (disequilibrium coefficient (D′) <1 and LOD score <2); blue (D′ = 1 and LOD score <2); pink (D′<1 and LOD score ≥2); and bright red (D′ = 1 and LOD score ≥2). (IV) The correlations with CAD and other age-associated conditions of the indicated SNPs are indicated by corresponding colors.

Figure 2. Reduced transcription of the INK4/ARF locus is associated with the atherosclerosis risk allele of rs10757278.

(A) The risk allele (G) of rs10757278 is associated with reduced transcription of all four INK4/ARF-associated transcripts. Adjusted p-values (t-test) comparing mean AA vs. GG expression are shown: *p<0.001; **p<0.0005; ***p<0.0001. See also Table S2 for details of ANOVA and Kruskal-Wallis comparisons. Error bars indicate SEM. (B) Expression of genes within INK4/ARF locus is associated with the risk allele of rs10757278 throughout chronological age. *p<0.01 for overall slope comparison; intercepts are not compared due to significant difference in slopes. **p>0.05 for overall slope comparison and p<0.0001 for overall intercept comparison.