ANRIL Expression Is Associated With Atherosclerosis Risk at Chromosome 9p21
Objective— We tested the hypothesis that expression of transcripts adjacent to the chromosome 9p21 (Chr9p21) locus of coronary artery disease was affected by the genotype at this locus and associated with atherosclerosis risk.
Methods and Results— We replicated the locus for coronary artery disease (P=0.007; OR=1.28) and other manifestations of atherosclerosis such as carotid plaque (P=0.003; OR=1.31) in the Leipzig Heart Study, a cohort of 1134 patients with varying degree of angiographically assessed coronary artery disease. Expression analysis in peripheral blood mononuclear cells (n=1098) revealed that transcripts EU741058 and NR_003529 of antisense noncoding RNA in the INK4 locus (ANRIL) were significantly increased in carriers of the risk haplotype (P=2.1×10−12 and P=1.6×10−5, respectively). In contrast, transcript DQ485454 remained unaffected, suggesting differential expression of ANRIL transcripts at Chr9p21. Results were replicated in whole blood (n=769) and atherosclerotic plaque tissue (n=41). Moreover, expression of ANRIL transcripts was directly correlated with severity of atherosclerosis (EU741058 and NR_003529; P=0.02 and P=0.001, respectively). No consistent association of Chr9p21 or atherosclerosis was found with expression of other genes such as CDKN2A, CDKN2B, C9orf53, and MTAP.
Conclusion— Our data provide robust evidence for an association of ANRIL but not CDKN2A, CDKN2B, C9orf53, and MTAP, with atherosclerosis and Chr9p21 genotype in a large cohort.
Genome-wide association studies have independently led to the identification of a susceptibility locus of coronary artery disease (CAD) on human chromosome (Chr) 9p21.1–3 Until now, the locus has been replicated in a large number of additional studies4–7 and can be considered the most robust genetic marker of atherosclerotic cardiovascular disease.
Despite these advances, the pathophysiology underlying this locus is currently not understood. Chr9p21 is not associated with known cardiovascular risk factors such as hyperlipidemia and hypertension.7 Moreover, no known protein-coding genes reside within the haplotype block except for a long noncoding transcript of unknown function, designated antisense noncoding RNA in the INK4 locus (ANRIL).8 In the absence of annotated genes at the immediate locus, adjacent ones such as the cyclin-dependent kinase inhibitors CDKN2A, CDKN2B, and methyl-thioadenosine phosphorylase (MTAP) have been regarded as potential candidate genes for atherosclerosis susceptibility at Chr9p21. However, the role of these genes in atherogenesis is not clear even though there is a wealth of data about their function in cancer, cell-cycle control, apoptosis, and aging.9–11 In addition, data from recent genome-wide association studies have revealed single nucleotide polymorphisms (SNPs) at Chr9p21 associated with the risk for glioma and skin cancers.12,13
Results from a recent study in T cells isolated from peripheral blood of 170 healthy blood donors indicated that the G allele at SNP rs10757278, which had been associated with increased CAD in previous studies, was associated with decreased expression of INK4/ARF-associated transcripts (CDKN2A, CDKN2B, ARF, and ANRIL) but not with MTAP.14 An effect of Chr9p21 on ANRIL expression was also demonstrated in whole blood in another study of 124 healthy individuals.15 This study looked more closely at expression of different ANRIL transcripts. It found that the sum of expression levels of 2 short ANRIL transcripts (DQ485454 and EU741058) was significantly increased in carriers of the risk haplotype, whereas the same allele was associated with a decrease of expression of the long ANRIL variant (NR_003529). Even though this study also included a subgroup of 42 CAD patients, the association of ANRIL and INK4/ARF-associated transcripts with the severity of atherosclerosis has not been investigated yet.
Thus, the aim of the present study was to investigate whether RNA expression in the Chr9p21 region was affected by the genotype at this locus and whether expression levels were associated with atherosclerosis in a large cohort. Analyses were performed in peripheral blood mononuclear cells (PBMC) available from 1098 patients of the Leipzig Heart Study with angiographically assessed CAD. Findings were replicated in RNA isolated from stabilized whole blood (PAXgene) of a subset of 769 patients. In addition, expression of differentially regulated transcripts was studied in atherosclerotic plaque tissue.
Materials and Methods
An expanded Materials and Methods section is provided in the Supplemental Materials (available online at http://atvb.ahajournals.org).
A total of 1134 patients (age, 25–85 years) were recruited from the ongoing Leipzig Heart Study, a cross-sectional study of patients undergoing first diagnostic coronary angiography for suspected coronary heart disease.16 All patients gave written consent to participate in the study. The study has been approved by the Ethics Committee of the Medical Faculty of the University Leipzig (Reg. number 276-2005) and is registered with ClinicalTrials.gov (NCT00497887).
Definition of Atherosclerosis Phenotypes
Coronary angiography was performed in 1101 participants. The percentage narrowing of each coronary artery segment was assessed according to the AHA classification.17 Arteriographic extent of disease was defined as: (1) no CAD, angiographically normal coronary arteries; (2) CAD <50%, wall irregularities, and any stenosis <50%; and (3) CAD ≥50%, any stenosis ≥50% in at least 1 major coronary artery. In addition, degree of occlusion of the left main coronary artery was analyzed.
Carotid ultrasound was performed in 1065 participants according to a modified protocol from the Atherosclerosis Risk in Communities (ARIC) study.18 The ankle–brachial index was determined in all probands. Patients were classified as negative for peripheral artery disease (PAD) when ankle–brachial index ≥0.9 in absence of previous peripheral revascularization. Patients with ankle–brachial index <0.9 or patients with previous peripheral revascularization irrespective of their ankle–brachial index measurement were classified as positive for PAD.
Laboratory and SNP Genotyping
PBMC were isolated using Cell Preparation Tubes (Becton Dickinson). Whole blood RNA was collected using PAXgene tubes (Qiagen). SNPs rs10757274, rs2383206, rs2383207, and rs10757278 previously associated with CAD1,2 and SNP rs10738605 were genotyped in all probands using a homogenous fluorescent method as previously described.19 Sequences of primers and probes are given in Supplemental Table I (available online at http://atvb.ahajournals.org).
RNA Isolation and Quantitative Reverse-Transcription Polymerase Chain Reaction (TaqMan)
Total RNA was extracted from PBMC (n=1098) using TRIzol reagent (Invitrogen). Whole blood RNA (n=769) collected in PAXgene tubes was isolated using the PAXgene Blood RNA Kit (Qiagen). RNA from human carotid (n=20), aortic (n=7), and femoral (n=14) atherosclerotic plaques was also extracted with TRIzol reagent. The utilization of human vascular tissues was approved by the Ethics Committee of the Medical Faculty Carl Gustav Carus of the Technical University Dresden (EK316122008). Quantitative fluorogenic reverse-transcription polymerase chain reaction was performed in an ABI PRISM 7900 Sequence Detection System (384-well plate format; Applied Biosystems). Specific primers and probes for β-actin (BA), MTAP (transcripts ENST00000355696 and ENST00000380172 designated MTAP T1 and MTAP T2, respectively), chromosome 9 open reading frame 53 (C9orf53), CDKN2A transcripts p16INK4a and p14ARF, CDKN2B transcript p15INK4b, ANRIL transcripts NR_003529, Σ NR_003529+DQ485454, and EU741058 were selected to span 2 exons to avoid coamplification of genomic DNA (Figure 1, Supplemental Table II). Absolute copies of DQ485454 were determined by subtraction of NR_003529 copies from Σ NR_003529+DQ485454. Primer concentrations and cycling conditions are given in Supplemental Table III. DNA controls revealed negative results in all experiments. It was necessary to run 13 different plates per assay to accommodate all samples and standard curves. Each of these plates contained identical control samples from a PBMC pool allowing calculation of coefficients of variation, which were 11% for NR_003529, 10% for DQ485454, and 16% for EU741058. All measurements were performed in quadruplicates. mRNA expression levels were normalized to 107 copies of BA as a housekeeping gene. Plate-to-plate variations of transcript expression levels were normalized by quantile normalization.
Clinical phenotypes determining either cardiac or peripheral atherosclerotic burden (CAD, left main coronary artery disease, Plaque, PAD) were adjusted to major risk factors of atherosclerosis. Covariables for all phenotypes analyzed in the current study are given in Supplemental Table IV. Haplotypes were inferred using fastphase 1.2.20 Heat maps of linkage disequilibrium were constructed using Haploview 4.1.21
For association analysis, probability values for the additive model are given. Association analyses were performed in 1006 patients showing no recombination in haplotypes. Normalized transcripts were adjusted for age and monocyte/lymphocyte counts relative to the total number of white blood cells (Supplemental Table V). For genotype-specific means, arithmetic means and standard errors are reported. All association analyses were performed using the statistical software package R 2.8.0 (www.r-project.org).22
Association of Chr9p21 Locus With Atherosclerosis in Different Vascular Beds
Studies were performed in 1134 participants of the Leipzig Heart Study, a cohort of patients with different degrees of CAD, carotid atherosclerosis, and PAD. To confirm the Chr9p21 atherosclerosis locus,1,2 we genotyped SNPs rs10757274, rs2383206, rs2383207, and rs10757278. The 4 SNPs were highly correlated (Supplemental Figure I, available online at http://atvb.ahajournals.org) and used to construct haplotypes.
Patients were grouped according to their Chr9p21 haplotype and no differences in demographic or clinical characteristics were observed except for a higher frequency of statin treatment in patients carrying the Chr9p21 risk allele (P=0.0059; Table 1).
As shown in Table 2, we found significant associations of different atherosclerosis phenotypes with Chr9p21. Numbers of subjects in the different clinical subgroups and their Chr9p21 haplotypes are given in Supplemental Table VI. The GGGG haplotype was significantly associated with CAD and left main coronary artery disease. The most significant association with Chr9p21 was found for plaque in the carotid artery (P=0.0026; OR, 1.31), whereas no association was found for carotid intima-media thickness, a measure of preclinical atherosclerosis (data not shown). A novel finding was the association of Chr9p21 with PAD (P=0.037; OR, 1.36). Taken together, these data confirmed the Chr9p21 locus for multiple atherosclerosis phenotypes available from patients in the Leipzig Heart Study.
Chr9p21 Affects ANRIL Expression in PBMC
The core haplotype block at the Chr9p21 locus encodes no protein-coding genes. However, the region contains a long noncoding transcript of unknown function, designated ANRIL. Because ANRIL might affect expression of protein-coding genes, we investigated whether it was differentially regulated by Chr9p21 in PBMC from patients of the Leipzig Heart Study. We used specific quantitative reverse-transcription polymerase chain reaction assays to determine three annotated ANRIL transcripts, NR_003529, DQ485454, and EU741058. We found that expression of EU741058 and NR_003529 was significantly associated with the Chr9p21 genotype (P=2.1×10−12 and P=1.6×10−5, respectively; Table 2). Carriers of the GGGG risk haplotype showed 23.3% increased expression of EU741058 and 12.6% increased expression of NR_003529. In contrast, expression of DQ485454 remained unaffected. This finding indicated differential regulation of ANRIL transcripts, which was also reflected by the highly significant association of the EU741058/DQ485454 and NR_003529/DQ485454 ratios with the Chr9p21 genotype (P=2.4×10−20 and P=4.3× 10−6; Table 2). Because gene expression in PBMC might be altered by medication and disease status, we also investigated ANRIL expression in PBMC from a subgroup of individuals free of CAD and PAD, and without use of statin medication. Despite the smaller sample size, we could replicate the association of ANRIL transcripts and ratios of expression with similar effect sizes (Supplemental Tables VII, VIII).
Validation of Chr9p21 Effect on ANRIL Expression in Whole Blood
To validate Chr9p21 effects on ANRIL expression in PBMC, we next determined expression levels in whole blood. Samples were collected from the same patients with a separate set of blood sampling tubes (PAXgene), allowing instant preservation of whole blood RNA. We could replicate the association of the Chr9p21 genotype with EU741058 and NR_003529 expression (P=2.3×10−10 and P=0.0098, respectively). DQ485454 was not significantly associated with the Chr9p21 genotype and even showed a slightly inverse effect resulting in highly significant associations of the Chr9p21 haplotype with EU741058/DQ485454 and NR_003529/DQ485454 ratios (P=5.3×10−14 and P=4.9×10−6, respectively; Table 2). Comparable results were found in a subgroup of individuals free of CAD and PAD, and without use of statin medication (Supplemental Tables VII, VIII). Taken together, these data confirmed the results seen in PBMC.
Association of ANRIL Expression With Atherosclerosis
We next tested whether ANRIL expression levels were directly correlated with the severity of atherosclerosis in patients of the Leipzig Heart Study. Expression levels of EU741058 and NR_003529 but not of DQ485454 were significantly increased in patients with high atherosclerotic plaque burden in the carotid arteries, which was the phenotype showing the strongest association with Chr9p21 in the Leipzig Heart Study (P=0.019 and P=0.001, respectively; Figure 2A, C, E). To better understand these findings in relation to the association of ANRIL with the Chr9p21 genotype, we also performed a breakdown of ANRIL expression with atherosclerosis severity and haplotype (Supplemental Tables IX, X). These data suggest that NR_003529 was associated with the Chr9p21 genotype and also increased with atherosclerosis severity, whereas EU741058 was predominantly associated with the genotype and to a lesser extent with atherosclerosis. To further elucidate the potential role of ANRIL in atherosclerosis, we also tested the effect of Chr9p21 on ANRIL expression in human atherosclerotic plaques. We found an increase of EU741058 and NR_003529 expression in plaques from carriers of the GGGG risk haplotype and a concomitant reduction of DQ485454 expression, which was similar to the effects seen in whole blood but statistically significant (Figure 2B, D, F).
Expression of Neighboring Genes at Chr9p21
One potential mechanism underlying the effect of the Chr9p21 region on atherosclerosis susceptibility was that either ANRIL or regulatory elements at the locus might affect expression of neighboring genes. To this end, we systematically analyzed expression of annotated transcripts in the 400 kb region surrounding the core Chr9p21 haplotype block (Supplemental Figure II). The mRNA levels of MTAP transcripts, C9orf53, CDKN2A transcripts p16INK4a and p14ARF, and CDKN2B transcript p15INK4b were determined in PBMC from all patients included in the study. As shown in Figure 3, expression levels were highly correlated. Correlations were particularly high between ANRIL transcripts NR_003529, DQ485454, and EU741058 (Figure 3A). These findings were replicated in whole blood (Figure 3B).
We then tested the association of MTAP, C9orf53, p16INK4a, p14ARF and p15INK4b with Chr9p21 in PBMC and found that only p15INK4b expression was associated with the Chr9p21 genotype (P=0.0076, Table 2). The GGGG risk haplotype was associated with increased p15INK4b expression (7% increase/allele; Table 2, Figure 4A). However, this association could not be replicated in whole blood, where, on the contrary, the GGGG risk haplotype was associated with a slight but not significant decrease of p15INK4b expression (3.8% decrease/allele; Table 2, Figure 4B). Moreover, there was no significant association of p15INK4b expression with severity of atherosclerotic plaque burden (Figure 4C), nor did we see an association of p15INK4b expression with the Chr9p21 genotype in atherosclerotic plaque specimens (Figure 4D). None of the other investigated transcripts showed significant associations with any of the atherosclerosis phenotypes (data not shown). Taken together, these data argue against a role of differential expression of genes surrounding the Chr9p21 region on atherosclerosis, except ANRIL, at least in PBMC and whole blood.
We provide evidence for a differential regulation of the noncoding RNA ANRIL by the Chr9p21 locus and show for the first time to our knowledge that increased expression of ANRIL transcripts was correlated with atherosclerosis susceptibility. Moreover, the risk allele at Chr9p21 was significantly associated with increased expression of transcripts EU741058 and NR_003529 but not with DQ485454, suggesting a role for differential expression or transcript stability. Expression analysis of neighboring genes in the Chr9p21 region (MTAP, C9orf53, p16INK4a, p14ARF, and p15INK4b) revealed that these genes were not correlated with the severity of atherosclerosis or robustly associated with the genotype at the locus.
ANRIL is a large noncoding transcript in the core region of the Chr9p21 haplotypic block.4,8 Its first exon is located in the promoter of the CDKN2A gene, overlapping the 2 exons of CDKN2B (Supplemental Figure II). Two recently published studies have investigated the association of ANRIL transcripts in blood cells with the Chr9p21 genotype.14,15 The study by Liu et al14 measured expression of transcripts NR_003529 and DQ485454 in T cells of 170 healthy subjects and found a significant decrease associated with the risk allele, which is at odds with the increase of NR_003529 expression and lack of effect of DQ485454 expression in our study. However, it should be noted that Liu et al did not perform transcript-specific quantitative reverse-transcription polymerase chain reaction and only investigated the sum of ANRIL transcripts; thus, a potential increase of NR_003529 expression would have gone undetected. In addition, we identified a SNP (rs10738605) colocalizing with their 3′ TaqMan primer that led to significantly increased expression measurements in individuals carrying the G allele perfectly matching the primer sequence (Figure 1, Supplemental Figure IIIA, B). After redesigning the assay, no significant association of NR_003529 and DQ485454 transcript expression was found with rs10738605 or with Chr9p21 (Supplemental Figure IIIC, D), confirming that the assay by Liu et al overestimated ANRIL expression in carriers of the risk haplotype. In the study by Jarinova et al,15 the sum of EU741058 and DQ485454 was significantly increased in carriers of the risk allele. In our study, the sum of EU741058 and DQ485454 was unaffected (data not shown). Jarinova et al15 also reported a significant association of the risk allele with increased EU741058 expression and decreased NR_003529 (the nomenclature DQ485453 was used in that study) expression, which only partially corroborates our results in which expression levels of both EU741058 and NR_003529 were increased. Possible reasons for divergent results between the study by Jarinova et al and our study might be attributable to the small sample size (n=120 vs n=1098 in our study) and the use of samples from healthy individuals as opposed to individuals with clinically suspected CAD. The latter possibility was ruled out in our study in a subanalysis of individuals free of atherosclerosis and without medication, which confirmed the results obtained in our complete cohort (Supplemental Tables VII, VIII).
Another finding in our study was the highly significant association of increased EU741058/DQ485454 and NR_003529/DQ485454 ratios with the risk haplotype (Table 2). These associations were robustly replicated in whole blood, suggesting a regulation of ANRIL in cis (Table 2). This finding indicated that EU741058 and NR_003529 were either transcribed more frequently or spliced more efficiently in patients carrying the Chr9p21 risk haplotype. Another possible explanation for these findings might be differences in the rate of decay of the 2 ANRIL transcripts. The genetic basis for the Chr9p21 effect on differential expression of ANRIL is presently unknown. Bioinformatic analyses indicated the presence of genetic variants colocalizing with potential transcription factor binding sites within the core haplotype block (data not shown). However, differential promoter activation was deemed unlikely because the first exons are shared between ANRIL transcripts. In addition, genome-wide association studies have identified the strongest associations with CAD and other atherosclerosis end points for SNPs mapping to the 3′ end of ANRIL.1,2,7 Thus, other mechanisms influencing transcript stability or differential splicing appear to be more likely. Because not all sequence variants in the region have been mapped, complete sequencing will be necessary for systematic identification of potentially causative mutations.
A novel finding of the present study was the association of ANRIL expression with the severity of atherosclerosis. This association was strongest for carotid plaque, particularly with transcript NR_003529 and to a lesser extent with transcript EU741058 (Figure 2). Subgroup analyses (Supplemental Tables IX, X) indicated that NR_003529 was associated with Chr9p21 genotype and severity of atherosclerosis, whereas EU741058 was predominantly associated with Chr9p21 genotype but to a smaller extent with atherosclerosis severity. However, larger numbers of samples are needed to provide a definite answer whether the association of ANRIL with atherosclerosis is mainly caused through the association of ANRIL with the 9p21 genotype or is to some extent independent.
What might be a role of ANRIL in atherogenesis? ANRIL is a large noncoding antisense RNA and thus might influence gene expression of other protein-coding genes through mechanisms such as RNA interference.23 This hypothesis is particularly appealing because the Chr9p21 region partially overlaps the INK4/ARF tumor-suppressor locus. It is well-known that genes at the locus, namely p16INK4a, p15INK4b, and p14ARF, are regulated in a coordinated manner.24 These genes are strongly related to cell-cycle control, a process also important in atherosclerotic lesion development.25 Evidence for a potential role of ANRIL on gene regulation at the INK4/ARF locus was provided by the transcription of ANRIL in antisense direction and its overlap, particularly with CDKN2B (p15INK4b).8 Correlation analysis of annotated transcripts in the 400 kb region surrounding the core haplotypic block confirmed a high degree of correlation between transcripts. As expected, correlations were particularly high for different transcripts of the same gene, such as ANRIL (EU741058, NR_003529 and DQ485454), MTAP (T1 and T2), and CDKN2A (p16INK4a and p14ARF; Figure 4). We also found a good correlation of ANRIL transcripts with CDKN2B (p15INK4b), especially in whole blood (Figure 4B), a finding also reported by Jarinova et al.15 These authors suggested that CDKN2B (p15INK4b) therefore might be directly affected by ANRIL.15 However, this hypothesis has not been proven in that article, and our data show that p15INK4b was associated with the genotype at Chr9p21 only in PBMC but not in whole blood (Table 2). In addition, expression of none of the other genes at Chr9p21 was associated with the Chr9p21 genotype. Moreover, no gene except ANRIL was associated with atherosclerosis phenotypes. Taken together, these data speak against modulation of mRNA expression of neighboring genes at the INK4/ARF locus by ANRIL as a causal mechanism for atherosclerosis susceptibility at this locus, at least in PBMC and whole blood. As noted for other long noncoding RNA,26 ANRIL might affect gene expression in trans, and further genome-wide expression analysis will be necessary to test this hypothesis.
Because cis-regulated transcripts are frequently regulated in a coordinated manner,27 one could speculate that ANRIL might also be differentially expressed in other tissues relevant to atherogenesis. Furthermore, one could speculate that other not yet annotated transcript variants of ANRIL might also play a role in atherosclerosis. Broadbent et al4 have shown that ANRIL is expressed in cells that play a role in atherogenesis such as human coronary artery smooth muscle cells, macrophages, and vascular endothelial cells. However, no association analysis of ANRIL expression with the respective genotype of the samples was performed in that study. Here, we show that ANRIL was also expressed in human atherosclerotic plaques (Figure 2) in which we also observed a significantly higher EU741058 expression and EU741058/DQ485454 ratio in individuals carrying the 9p21 risk haplotype.
At this point it is not clear whether differential expression of ANRIL at Chr9p21 and its association with atherosclerosis provide a functional link. Alternatively, ANRIL might only be a bystander of a completely different effect of the Chr9p21 locus that still needs to be determined. This notion is supported by the relatively low expression levels and relative expression differences of ANRIL transcripts NR_003529 and EU741058. Nevertheless, our data provide, for the first time to our knowledge, evidence for an association of ANRIL expression with atherosclerosis. This was strengthened by the finding that ANRIL was differentially regulated by Chr9p21 in human atherosclerotic plaques. In addition, the fact that only EU741058 and NR_003529 but not DQ485454 were associated with Chr9p21 suggests that differential splicing or transcript stability might play a role in atherosclerosis susceptibility. Taken together, our data provide robust evidence for an association of ANRIL, but not CDKN2A, CDKN2B, C9orf53, and MTAP, with atherosclerosis and Chr9p21 genotype in a large cohort. Further studies are needed to determine whether ANRIL expression is a marker or modulator of atherosclerosis susceptibility at Chr9p21.
The authors thank Christa Döring, Manuela Fritzsche, Franziska Jeromin, Kerstin Kothe, Marlies Oehlert, Annegret Schink, Katja Tautz, Claudia Weise, and Wolfgang Wilfert for their excellent technical assistance.
Sources of Funding
This work was supported by a grant from the Roland-Ernst-Foundation (to D.T.), a grant from the National Genome Research Network (NGFNplus to D.T. and J.T.), a grant from the Medical Faculty, University Leipzig (LIPIGENETICS to D.T., M.S., and J.T.), a grant from the Medical Faculty, University Leipzig (to L.M.H.), and a grant from the Medical Faculty, University Leipzig (to F.B.). M.S. was funded by the German Federal Ministry for Education and Research (01KN0702).
Received September 1, 2009; revision accepted December 8, 2009.
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