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Chromosome 9p21 and coronary risk – The mystery continues
Atherosclerosis 214 (2011) 257–258
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Invited Commentary
Chromosome 9p21 and coronary risk – The mystery continues Patrick Linsel-Nitschke, Heribert Schunkert ∗ Department of Medicine II, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany
a r t i c l e
i n f o
Article history: Received 1 August 2010 Accepted 5 August 2010 Available online 21 September 2010
The strongest genomic signal for coronary artery disease (CAD) and myocardial infarction (MI) resides at the chromosomal locus 9p21.3 [1–4]. Interestingly, CAD and MI are not the only diseases associated with this locus since aortic and intracerebral aneurysms, peripheral arterial disease, as well as type 2 diabetes mellitus also gave strong signals [5,6]. Moreover, initially the genes expressed at the locus had been implicated in somatic mutations of several cancers [7,8]. Despite impressive association data for this locus little is known about the mechanisms by which the 9p21.3 variants exert their effects on these phenotypes. Given the strong association data that come without functional explanation it may be worthwhile to have a closer look at the genes encoded in the vicinity, the cyclin-dependent kinases 2A and 2B (CDKN2A and CDKN2B also termed p15ink4a and p16ink4b /p14arf ) and methylthioadenosine phosphorylase (MTAP). Even closer to the CAD/MI risk variants is the INK4 locus of the non-coding RNA ANRIL. But here the confusion continues. For example, the linkage disequilibrium (LD) block that associates strongly with CAD/MI is not identical with the one affecting diabetes risk (Fig. 1, [5]). Moreover, at the transcriptional level, variants that profoundly affect expression of coding genes in the region (CDKN2A/CDKN2B and MTAP) do not associate necessarily with CAD/MI risk and vice versa, other variants that associate with CAD/MI risk do not affect expression levels of these genes [9]. By contrast, the expression levels of two out of three different transcripts of the non-coding RNA located in the region (ANRIL) are affected in by the same variants that associate with CAD/MI risk [10]. Thus, the only thing that seems to be clear at the present time is that multiple mechanisms are at work. In this issue of Atherosclerosis Holdt et al. elegantly analyse the expression of the genes encoded at chromosome 9p21.3 in human atherosclerotic plaques [11]. Using immunohistochemical staining the authors demonstrate that CDKN2A/CDKN2B and MTAP are expressed in the smooth muscle cell layer of normal
∗ Corresponding author. Tel.: +49 451 5002501; fax: +49 451 5006437. E-mail address: [email protected] (H. Schunkert). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.08.059
human arteries. Of particular interest is the finding that expression of these genes is also induced in macrophages and foam cells of human atherosclerotic plaques. Expression levels in the atherosclerotic plaque also correlate with markers of lesion instability according to the convincing data by Holdt and colleagues. Indeed, in macrophage-rich inflammatory lesions CDKN2B was induced whereas MTAP was downregulated in such lesions. This observation is supported by in vitro data showing that upon stimulation of cultured cells with TNF␣ downregulation of MTAP can be observed. Most knowledge on the function of CDKN2A, CDKN2B and MTAP relates to tumour-biology, where these proteins are believed to play a central role in controlling cell division and proliferation. Very recently a mouse model with a deletion of the 9p21 synthetic region was reported [12]. In this mouse model a downregulation of CDKN2A and CDKN2B expression went along with an increase in cell proliferation. The fact that only about 50% sequence homology exist between the mouse and human 9p21.3 synthenic region and the finding that there was no measurable effect on atherosclerosis progression in this mouse model have raised doubts whether definitive conclusions can be drawn from this study for humans with the 9p21.3 variants. In line with these doubts recent expression studies in large human cohorts were unable to find any association of the 9p21.3 MI/CAD risk-variant with the expression of CDKN2A, CDKN2B and MTAP [9,10]. It is thus questionable whether these proteins are involved in atherosclerotic plaque formation or progression albeit their expression is clearly shown by Holdt and colleagues. Several groups studying large human samples have detected a significant association of the 9p21.3 genetic variant with the expression of the non-coding RNA ANRIL in several large studies involving human subjects [10,13,14]. Adding to the complexity of the whole picture is the finding that several transcripts of ANRIL exist that differ in their length, with only the two longest ANRIL transcripts showing a significant association with the 9p21.3 risk allele. In the study presented by Holdt and colleagues in the issue of Atherosclerosis a negative correlation between MTAP expression
258
P. Linsel-Nitschke, H. Schunkert / Atherosclerosis 214 (2011) 257–258
Fig. 1. Functional associations at the 9p21 CAD risk locus. The graph depicts the chromosomal location (x-axis) and the p-values for association with CAD (y-axis). SNPs associated with CAD are represented as dots. Transcripts encoded at the 9p21 locus are displayed as gray arrows according to their location. In the lower part of the figure the genes in the region are displayed together with functional data published thus far.
and ANRIL transcript levels was observed. However, this correlation could only be demonstrated for the shortest transcript of ANRIL, which in turn is not regulated by the 9p21.3 genetic variant. In conclusion, the induction of CDKN2A, CDKN2B and MTAP in human atherosclerotic plaques raises the possibility of their involvement in plaque formation and composition. What is clearly missing for the time being is a clear link between the 9p21.3 genetic risk variants in human populations and disturbed expression or function of respective gene products. So far the pieces of the puzzle do not yet fit together and the missing link, if existing, has yet to be discovered. Therefore, at this time, the mystery of the 9p21.3 locus remains wide open. References [1] Samani NJ, Erdmann J, Hall AS, et al. Genomewide association analysis of coronary artery disease. N Engl J Med 2007;357:443–53. [2] Schunkert H, Gotz A, Braund P, et al. Repeated replication and a prospective meta-analysis of the association between chromosome 9p21.3 and coronary artery disease. Circulation 2008. [3] McPherson R, Pertsemlidis A, Kavaslar N, et al. A common allele on chromosome 9 associated with coronary heart disease. Science 2007;316:1488–91.
[4] Helgadottir A, Thorleifsson G, Manolescu A, et al. A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science 2007;316:1491–3. [5] Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 2007;447:661–78. [6] Saxena R, Voight BF, Lyssenko V, et al. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science 2007;316:1331–6. [7] Caldas C, Hahn SA, da Costa LT, et al. Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet 1994;8:27–32. [8] Cannon-Albright LA, Goldgar DE, Meyer LJ, et al. Assignment of a locus for familial melanoma, MLM, to chromosome 9p13–p22. Science 1992;258:1080–1. [9] Zeller T, Wild P, Szymczak S, et al. Genetics and beyond – the transcriptome of human monocytes and disease susceptibility. PLoS One 2010;5:e10693. [10] Holdt LM, Beutnr F, Scholz M, et al. ANRIL expression is associated with atherosclerosis risk at chromosome 9p21. Atheroscler Thromb Vasc Biol 2010;30:620–7. [11] Holdt LM, Sass K, Gäbel G, et al. Expression of Chr9p21 genes CDKN2B (p15INK4b), CDKN2A (p16INK4a, p14ARF) and MTAP in human atherosclerotic plaque. Atherosclerosis 2011;214:264–70. [12] Visel A, Zhu Y, May D, et al. Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 2010;464:409–12. [13] Folkersen L, Kyriakou T, Goel A, et al. Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants. PLoS One 2009;4:e7677. [14] Cunnington MS, Koref MS, Mayosi BM, Burn J, Keavney BD. Chromosome 9p21 SNPs associated with multiple disease phenotypes correlate with anril expression. PLoS Genet 2010;6:e1000899.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Invited Commentary
Chromosome 9p21 and coronary risk – The mystery continues Patrick Linsel-Nitschke, Heribert Schunkert ∗ Department of Medicine II, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany
a r t i c l e
i n f o
Article history: Received 1 August 2010 Accepted 5 August 2010 Available online 21 September 2010
The strongest genomic signal for coronary artery disease (CAD) and myocardial infarction (MI) resides at the chromosomal locus 9p21.3 [1–4]. Interestingly, CAD and MI are not the only diseases associated with this locus since aortic and intracerebral aneurysms, peripheral arterial disease, as well as type 2 diabetes mellitus also gave strong signals [5,6]. Moreover, initially the genes expressed at the locus had been implicated in somatic mutations of several cancers [7,8]. Despite impressive association data for this locus little is known about the mechanisms by which the 9p21.3 variants exert their effects on these phenotypes. Given the strong association data that come without functional explanation it may be worthwhile to have a closer look at the genes encoded in the vicinity, the cyclin-dependent kinases 2A and 2B (CDKN2A and CDKN2B also termed p15ink4a and p16ink4b /p14arf ) and methylthioadenosine phosphorylase (MTAP). Even closer to the CAD/MI risk variants is the INK4 locus of the non-coding RNA ANRIL. But here the confusion continues. For example, the linkage disequilibrium (LD) block that associates strongly with CAD/MI is not identical with the one affecting diabetes risk (Fig. 1, [5]). Moreover, at the transcriptional level, variants that profoundly affect expression of coding genes in the region (CDKN2A/CDKN2B and MTAP) do not associate necessarily with CAD/MI risk and vice versa, other variants that associate with CAD/MI risk do not affect expression levels of these genes [9]. By contrast, the expression levels of two out of three different transcripts of the non-coding RNA located in the region (ANRIL) are affected in by the same variants that associate with CAD/MI risk [10]. Thus, the only thing that seems to be clear at the present time is that multiple mechanisms are at work. In this issue of Atherosclerosis Holdt et al. elegantly analyse the expression of the genes encoded at chromosome 9p21.3 in human atherosclerotic plaques [11]. Using immunohistochemical staining the authors demonstrate that CDKN2A/CDKN2B and MTAP are expressed in the smooth muscle cell layer of normal
∗ Corresponding author. Tel.: +49 451 5002501; fax: +49 451 5006437. E-mail address: [email protected] (H. Schunkert). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.08.059
human arteries. Of particular interest is the finding that expression of these genes is also induced in macrophages and foam cells of human atherosclerotic plaques. Expression levels in the atherosclerotic plaque also correlate with markers of lesion instability according to the convincing data by Holdt and colleagues. Indeed, in macrophage-rich inflammatory lesions CDKN2B was induced whereas MTAP was downregulated in such lesions. This observation is supported by in vitro data showing that upon stimulation of cultured cells with TNF␣ downregulation of MTAP can be observed. Most knowledge on the function of CDKN2A, CDKN2B and MTAP relates to tumour-biology, where these proteins are believed to play a central role in controlling cell division and proliferation. Very recently a mouse model with a deletion of the 9p21 synthetic region was reported [12]. In this mouse model a downregulation of CDKN2A and CDKN2B expression went along with an increase in cell proliferation. The fact that only about 50% sequence homology exist between the mouse and human 9p21.3 synthenic region and the finding that there was no measurable effect on atherosclerosis progression in this mouse model have raised doubts whether definitive conclusions can be drawn from this study for humans with the 9p21.3 variants. In line with these doubts recent expression studies in large human cohorts were unable to find any association of the 9p21.3 MI/CAD risk-variant with the expression of CDKN2A, CDKN2B and MTAP [9,10]. It is thus questionable whether these proteins are involved in atherosclerotic plaque formation or progression albeit their expression is clearly shown by Holdt and colleagues. Several groups studying large human samples have detected a significant association of the 9p21.3 genetic variant with the expression of the non-coding RNA ANRIL in several large studies involving human subjects [10,13,14]. Adding to the complexity of the whole picture is the finding that several transcripts of ANRIL exist that differ in their length, with only the two longest ANRIL transcripts showing a significant association with the 9p21.3 risk allele. In the study presented by Holdt and colleagues in the issue of Atherosclerosis a negative correlation between MTAP expression
258
P. Linsel-Nitschke, H. Schunkert / Atherosclerosis 214 (2011) 257–258
Fig. 1. Functional associations at the 9p21 CAD risk locus. The graph depicts the chromosomal location (x-axis) and the p-values for association with CAD (y-axis). SNPs associated with CAD are represented as dots. Transcripts encoded at the 9p21 locus are displayed as gray arrows according to their location. In the lower part of the figure the genes in the region are displayed together with functional data published thus far.
and ANRIL transcript levels was observed. However, this correlation could only be demonstrated for the shortest transcript of ANRIL, which in turn is not regulated by the 9p21.3 genetic variant. In conclusion, the induction of CDKN2A, CDKN2B and MTAP in human atherosclerotic plaques raises the possibility of their involvement in plaque formation and composition. What is clearly missing for the time being is a clear link between the 9p21.3 genetic risk variants in human populations and disturbed expression or function of respective gene products. So far the pieces of the puzzle do not yet fit together and the missing link, if existing, has yet to be discovered. Therefore, at this time, the mystery of the 9p21.3 locus remains wide open. References [1] Samani NJ, Erdmann J, Hall AS, et al. Genomewide association analysis of coronary artery disease. N Engl J Med 2007;357:443–53. [2] Schunkert H, Gotz A, Braund P, et al. Repeated replication and a prospective meta-analysis of the association between chromosome 9p21.3 and coronary artery disease. Circulation 2008. [3] McPherson R, Pertsemlidis A, Kavaslar N, et al. A common allele on chromosome 9 associated with coronary heart disease. Science 2007;316:1488–91.
[4] Helgadottir A, Thorleifsson G, Manolescu A, et al. A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science 2007;316:1491–3. [5] Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 2007;447:661–78. [6] Saxena R, Voight BF, Lyssenko V, et al. Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science 2007;316:1331–6. [7] Caldas C, Hahn SA, da Costa LT, et al. Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet 1994;8:27–32. [8] Cannon-Albright LA, Goldgar DE, Meyer LJ, et al. Assignment of a locus for familial melanoma, MLM, to chromosome 9p13–p22. Science 1992;258:1080–1. [9] Zeller T, Wild P, Szymczak S, et al. Genetics and beyond – the transcriptome of human monocytes and disease susceptibility. PLoS One 2010;5:e10693. [10] Holdt LM, Beutnr F, Scholz M, et al. ANRIL expression is associated with atherosclerosis risk at chromosome 9p21. Atheroscler Thromb Vasc Biol 2010;30:620–7. [11] Holdt LM, Sass K, Gäbel G, et al. Expression of Chr9p21 genes CDKN2B (p15INK4b), CDKN2A (p16INK4a, p14ARF) and MTAP in human atherosclerotic plaque. Atherosclerosis 2011;214:264–70. [12] Visel A, Zhu Y, May D, et al. Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 2010;464:409–12. [13] Folkersen L, Kyriakou T, Goel A, et al. Relationship between CAD risk genotype in the chromosome 9p21 locus and gene expression. Identification of eight new ANRIL splice variants. PLoS One 2009;4:e7677. [14] Cunnington MS, Koref MS, Mayosi BM, Burn J, Keavney BD. Chromosome 9p21 SNPs associated with multiple disease phenotypes correlate with anril expression. PLoS Genet 2010;6:e1000899.