{"paper_id":"fd66b368-a577-4ee1-8d08-f5e9bfc3d604","body_text":"Since publication of the first genome-wide association studies (GWAS) of coronary artery disease (CAD) in 2007, Chr9p21 has emerged as the most significant risk locus associated with this frequent disease ( 1 – 4 ). The region contains a number of strongly interlinked SNPs within a stretch of 58 kilobases (kb) of non-protein-coding DNA. Later, the same haplotype block has been associated with other endpoints of atherosclerosis, such as stroke ( 5 – 11 ), peripheral artery disease ( 12 – 14 ), and also with different types of aneurysms ( 2 ,  8 ,  15 ,  16 ). Due to the availability of large study cohorts and the better resolution of genetic recombination in this region, it has now become clear that associations with other phenotypes at Chr9p21 fall in distinct haplotype blocks not overlapping with the CAD block (Figure  1A ). Closely nearby, and proximal to the CAD locus, GWAS found associations with cancer, such as melanoma, glioma, basal cell carcinoma, and acute lymphoblastic leukemia [see ( 40 ) for review], and also with glaucoma, and diverse proliferative or inflammatory diseases, such as endometriosis of the reproductive tract ( 41 ), periodontitis ( 42 ), and platelet reactivity ( 43 ). The region located distally to the CAD region contains a distinct haplotype block associated with type 2 diabetes ( 44 ,  45 ).\nANRIL and the CAD risk locus at Chr9p21.  (A)  Genomic mapping of SNPs identified in a region ± 300 kb around the top-ranking sentinel SNP rs4977574 based on data of the most recent large CAD GWAS ( 17 ). Chromosome ideogram and zoom-in onto RefSeq transcripts for  ANRIL, CDKN2A, CDKN2B , and  MTAP  ( top ), regional association plot of CAD risk alleles and graph of recombination rate in the locus ( middle ), scaled linkage disequilibrium heatmap (D′) as derived from the 1000Genomes Project dataset (Phase3V5, CEU) ( bottom ). The threshold for significance of GWAS hits is indicated as horizontal dotted line ( p  < 5E-8). Dots for SNPs described in Table  1  are marked in yellow. The suspected core CAD risk region, corresponding to the distal region of  ANRIL , has been defined experimentally through multiple CAD GWAS and is highlighted in red. The physical genomic map and the haplotype map are connected by oblique lines. Note that not all RNA transcripts and isoforms are depicted, and that type 2 diabetes (T2D, highlighted blue) and cancer risk regions (highlighted gray) are shown in simplified forms.  (B)  Model how the genotype at Chr9p21 controls the balance of linear and circular  ANRIL  RNA expression and potential molecular mechanisms of the different  ANRIL  isoforms. Linear  ANRIL  upregulation regulates gene expression  in trans  and pro-adhesive, pro-proliferative, anti-apoptotic cell functions. High levels of  circANRIL  inhibit over-proliferation of vascular cells by controlling rRNA maturation through impairing PES1 function in the PeBoW complex.\nIn the last 10 years, GWAS have been successfully used to increase the number of genetic loci implicated in CAD risk inheritance. The number of CAD risk loci in the genome rose from 56 by 2013 ( 24 ,  46 – 52 ) to 80 by 2015 ( 53 – 56 ), to 243 by 2017 ( 17 ). Concerning the Chr9p21 locus in these studies, the association rose steadily from  p  = 5.40 × 10 −23  (rs4977575) ( 57 ), over  p  = 4.68 −101  (rs4977574) ( 17 ) to  p  = 8.8 × 10 −223  (rs4977574) ( 58 ). In populations of European descent, the allele frequency is very high (0.48), leading to the situation that approximately one-fourth of people are homozygous for the CAD risk alleles. CAD risk SNPs on Chr9p21 have recurrently been shown to have one of the top-ranking effect sizes [allele-specific odds ratio (OR) for CAD > 1.3] ( 3 ,  24 ). Despite the extent of effects, the Chr9p21 risk is independent of classically known CAD risk determinants, such as dyslipidemia, diabetes mellitus, age, and sex.\nThe Chr9p21 region contains at least 5 genes, which are, in part, tightly clustered and overlapping. These include the 3.8 kb long  ANRIL  non-coding RNA, and the tumor suppressors  cyclin dependent kinase inhibitor CDKN2A/p16 INK 4 A ,  CDKN2A/p14 ARF ,  CDKN2B/p15 INK 4 B , and  methylthioadenosine phosphorylase  ( MTAP ) . ANRIL  overlaps in antisense the full length of the  p15  gene body, while sharing a bidirectional promoter with  CDKN2A . Hence, it was also termed  CDKN2B antisense RNA  ( CDKN2B-AS1 ). Only recently, the picture got even more complex: Advances in high-throughput sequencing and adaptions in bioinformatics mapping of RNA reads to reference genomes have revealed that thousands of genes in our genome produce not only mature linear RNA but also 3′-5′ covalently linked circular RNAs (circRNAs) ( 59 ). So far, two studies have shown that a number of circular  ANRIL  ( circANRIL ) isoforms exist, comprised of different exons, whereby a downstream exon is fused to an upstream exon by the enzymatic activity of the spliceosome in a reaction termed “backsplicing” [see ( 60 ,  61 ) for review]. Circularizing exons in  ANRIL  stemmed mostly from middle parts of the lncRNA (Figure  1A ), which are in part also shared by the linear  ANRIL  isoforms.  CircANRIL  was found not only in many different cell lines, but also in many primary cell types, including vascular smooth muscle cells (VSMCs) and macrophages, as well as in heart and vascular tissue ( 22 ,  36 ).\nA major focus in exploring how risk is effected by Chr9p21 has been on whether genetic variation affected expression of genes at the locus  in cis  (Figure  1A ) or whether it elicited gene expression changes  in trans . Top CAD-associated SNPs lie within the distal parts of long linear  ANRIL  isoforms (Figure  1A ) and several studies have shown that they co-localize with sequences marked by chromatin modifications, RNA polymerase II transcription patterns and DNA motifs characteristic of  bona-fide  transcriptional enhancers ( 19 ,  35 ,  62 – 65 ). Using expression quantitative trait locus (eQTL) analyses in patient samples, several groups have by now investigated if the risk alleles at the locus were associated with the expression of specific target genes  in cis  (cis-eQTLs). Whereas studies investigating  ANRIL  expression have mostly used quantitative PCRs (qPCRs) targeting different exons from the lncRNA, expression of  p14, p15, p16 , or  MTAP  has either been investigated using genome-wide expression arrays or isoform-specific qPCRs. Here, we focus on studies investigating eQTLs in atherosclerosis cohorts but do not cover studies related to other phenotypes, such as cancer, which are reviewed elsewhere ( 66 ).\n\nANRIL  expression at Chr9p21 is complex and at least 20 linear isoforms as well as multiple circular isoforms have been reported [ www.ensembl.org , ( 22 ,  36 ,  39 )]. In principal, linear and circular isoforms can be distinguished by the fact that the latter derive from a backsplice event, where splicing of a downstream exon (e.g., exon 7) to an upstream exon (e.g., to exon 5) can be detected. Backsplicing of ex7-5 was the most common event observed in our own study in peripheral blood monocytes ( 36 ). Concordantly, Burd and colleagues have reported dominant backsplice isoforms spanning ex14-4 in peripheral blood T lymphocytes ( 22 ). In both studies, exon 1 and exons 17-20 were not contained in circularized  ANRIL  (Table  1 ). Thus, for classification reasons, results from studies targeting these exons will be referred to as proximal linear isoforms (containing the first  ANRIL  exons) and long linear isoforms (containing the distal exons 17-20) (Table  1 ). Since both linear and circular  ANRIL  may contain exons from the middle portion of the lncRNA (e.g., exons 4-16), a clear distinction as to whether linear or circular isoforms were investigated cannot be made in cases where these exons were targeted by qPCRs which were non-specific for backsplice junctions (Table  1 ).\nChr9p21 cis-eQTLs in patients.\nCAD-relevant SNPs encoded in Chr9p21 and defined as cis-eQTLs for regulation of ANRIL, p15, p16, p14, and/or MTAP through gene expression profiling with microarrays or targeted qPCR analysis .\nUpregulation of target gene expression (green), downregulation (red), no effect on RNA abundance (gray), effect not determined (n.d.). Effects on different isoforms of ANRIL (linear or circular) is specified when exon-specific qPCR analysis was performed targeting proximal, middle or distal ANRIL exons (ex). In total 23 studies are reviewed here. References: ( 13 ,  17 – 38 ). Note that the percentages (%) of studies showing up- and down-regulation of ANRIL isoforms do not necessarily add up to 100%, because different ANRIL exons were quantified in different studies, not all classes of ANRIL transcripts were analyzed in each study, and because one study can report on both up- and downregulation of different isoforms belonging to the same class of ANRIL transcripts (proximal/middle/distal) .\n(ANRIL linearity determined by PCR forward primer residing in exons 1, 2, or 3) .\n(ANRIL circularity determined by PCR primers detecting backsplicing between exons 10-2, 5-intron3, 6-intron3, 6-4, 7-4, 14-4, 10-4, 12-4, 13-4, 14-4, 16-4, 6-5, 7-5, 8-5, 10-5, 14-5 16-5, 19-5, 7-6, 10-6, 14-6, 16-13 * , 16-15) ( 22 ,  36 ,  39 ) .\n(cis-eQTL locating in enhancer element, but with unspecified direction of effect on expression) .\nAs one of the first studies on Chr9p21, Jarinova et al. have shown that  ANRIL  expression was induced by the CAD risk SNP rs1333049 in peripheral blood monocytes (PBMCs). No significant effects on  CDKN2A  or on  CDKN2B  were recorded in that study ( 19 ). Over the years, comparable quantifications of these genes followed in whole blood, peripheral blood T lymphocytes, lymphoblastoid cells lines, aortic smooth muscle cells (SMCs) and in different tissue samples that are known to have a role in atherosclerosis. For example, vascular tissues such as carotid atherosclerotic plaque samples, samples from aorta, mammary artery, and from the heart ventricles have been analyzed, but also tissues like subcutaneous or omental fat have been used (Table  1 ). Of the 23 cis-eQTL studies conducted in the Chr9p21 CAD region to date, 16 investigated different isoforms of  ANRIL , out of which 10 used assays targeting proximal  ANRIL  exons, 8 used assays targeting the middle region, 6 used assays targeting downstream linear  ANRIL  exons, and two investigated backsplices contained in  circANRIL  (Table  1 ). Complicating a clear-cut interpretation, in the different studies, different risk genotypes were used to indicate risk haplotypes. The expression of  CDKN2A  and of  CDKN2B  was investigated in 18 studies and  MTAP  in 10 studies (Table  1 ).\nOverall, 80% of the studies investigating  ANRIL  expression found an association with the Chr9p21 genotype. Here, a trend toward higher expression of the proximal and distal exons contained in linear  ANRIL  in patients carrying the CAD-risk allele was observed (7 of 10 and 3 of 6 studies). In contrast, circular  ANRIL  was downregulated in the two published studies in patients carrying the Chr9p21 risk haplotype. No clear tendency was observed when assays targeting the middle region of  ANRIL  were used (Table  1 ). This is likely explained by the fact that these assays target both, linear and circular,  ANRIL  isoforms, which seem to be inversely regulated. With respect to the tumor suppressor genes contained at the Chr9p21 locus, 78 and 67% of the studies failed to find an association of  CDKN2A  and  CDKN2B  with Chr9p21, respectively. When reporting an association, specifically  CDKN2B  was down-regulated in the majority of studies (94%), yet its expression was not always anticorrelating with  ANRIL  expression ( 19 ,  21 ,  22 ,  29 ).  MTAP  expression was not associated with the Chr9p21 genotype in any of the published studies. Overall, the picture emerges that circular  ANRIL  and  CDKN2B  tend to be down-regulated in patients carrying the risk allele, whereas linear  ANRIL  isoforms tend to be inversely regulated (Figure  1B ). It is currently unclear, why expression of  p15  or of  p14  and  p16  were in many cases positively correlated with  ANRIL  ( 19 ,  21 ,  22 ,  27 ,  29 ,  32 ,  34 ,  65 ). Also,  MTAP , which was not associated with Chr9p21 (Table  1 ), was in some conditions anticorrelating to  ANRIL , but not in all cases or contexts ( 20 ,  34 ,  67 ). SNPs in  ANRIL  can hypothetically affect enhancers in both directions, either by disrupting transcription factor binding sites in open chromatin ( 68 ) or by increasing enhancer activity through yet unknown primary effects ( 24 ,  65 ).\nIn summary, many studies document  cis -eQTLs for  ANRIL  or, separately, for  CDKN2B  ( 35 ). Throughout, from the existing data, it can be concluded that these effects are cell-type specific and combinatorial. Of note, many studies have investigated only very small cohorts and those, simultaneously testing both  ANRIL  and  CDKN2B  in larger cohorts (>1000 samples) identified much stronger effects of Chr9p21 on  ANRIL  than on  CDKN2B  ( 13 ,  33 ,  36 ). This observation might be explained by the haplotype block structure of the region, where effects of CAD lead SNPs are located within  ANRIL  but bleed through due to linkage disequilibrium, resulting in more subtle concomitant effects on  CDKN2B  expression. Another possibility is that the Chr9p21 genotype impacts transcription enhancers at the locus which contact and activate gene promoters affecting CAD. The consequences of such contacts would not be expected to be captured through traditional non-allelic RNA expression analysis. In fact, when allelic expression control through 3D-enhancer looping was specifically measured in a separate study in human coronary aortic SMCs ( 64 ), physical contacts of CAD variant-containing enhancers in the locus and the promoters of  CDKN2A, CDKN2B , and  ANRIL  were corroborated.\nTaken together, these data suggest that genetic variation within the core 9p21 CAD region relates to differential expression not only of  ANRIL , but in specific cells or conditions, also of the  CDKN2A/B  tumor suppressors encoded in the locus. While either of these factors could potentially increase cell proliferation, or lead to unscheduled senescence, or elicit out of context inflammatory signaling, as far as based on work with cells  in vitro , no study in humans or in mouse models has been able to decisively implicate a downstream effector pathway  in vivo .\n\nAs opposed to  cis  effects, two eQTL studies have so far detected modest and tissue-selective differential expression of dozens of genes associated with Chr9p21 genotype with genome-wide significance ( 19 ,  27 ). Affected genes were from a broad range of classes ( AVPR2, PEAK1, FBLN1, KALRN, DAZL, STAU2, HLA-DQA1, BTNL8, PLEKHA6, TDGF1 ) in whole blood ( 19 ) and different, non-overlapping gene sets linked to tissue wounding, cell migration and inflammatory response, when analyzing heart tissue, plaques, aortas, and arteries ( 27 ).\nOther, and in part, larger studies in vascular tissue ( 20 ), peripheral blood mononuclear cells (PBMC,  n  = 2280) ( 33 ) and in blood monocytes ( n  = 1490) ( 23 ) reported no significant expression association.\nThough not directly comparable, another study showed that in macrophages cultured  in vitro  under stress-bearing IFNγ and LPS stimulation, the CAD risk genotype led to differential up- and downregulation of target genes outside the Chr9p21 locus and yet distinct from the previously mentioned studies ( IL1B, IL12B, CASP5, CCL8, MT1A, MT1E, MUCL1, TNIP3, VCAN, ENPP2, NDP, CD163)  ( 30 ). Also  ANRIL  knockdown in cultured cell lines ( 69 – 72 ) and overexpression of linear  ANRIL  affected the expression of non-overlapping gene sets in the genome  in trans  ( 33 ,  36 ).\nHow  ANRIL  exerts  trans -regulation is not known, and despite a study that showed a physical interaction of  ANRIL  with promoters of target genes ( 33 ), this role is likely not a classical function as enhancer RNA [eRNA ( 73 )], because it involved both up-and down-regulated genes, and was suggested to involve sequence homology ( 33 ). In the case of  ANRIL, trans -regulation of target genes was ascribed to an ALU motif in both  ANRIL  and the target gene promoters ( 33 ). Similarly, an independent study found that  ANRIL  did not only silence its targets, but unexpectedly also upregulated target genes: For example, proinflammatory interleukins  IL6/8  were found to be co-stimulated by  ANRIL  and YY1, a transcription-regulating factor that bound to the  ANRIL RNA , especially in the context of TNFα/NFκB signaling ( 70 ). Therefore, opposite to what could have been expected from the reported physical interaction of  ANRIL  with proteins from the repressive Polycomb group complexes ( 74 ),  ANRIL  might be an activator, at least for some  trans -regulated genes ( 33 ,  70 ) (see chapter 4 for details). Whether  circANRIL , beyond regulating rRNA maturation, is involved in primary transcriptional control, alone or via impacting linear  ANRIL 's function, is not known ( 36 ). Nevertheless, it is interesting to note that  circANRIL  isoforms linked to CAD are produced from exons located in the middle of the  ANRIL  gene ( 22 ,  36 ), and as such do not include the ALU motif, which is important for gene  trans -regulation by linear  ANRIL  and is located more distally in the gene ( 33 ). Thus, variation in  ANRIL  RNA at the molecular level (linear vs. circular) might impose a fundamental alteration in  ANRIL  effector function, while not offering any explanation  per se  on how linear  ANRIL  regulates genes, as scaffold for promoter-activating complexes, or as decoy/inhibitor of repressive chromatin-modifying complexes. Conservatively speaking, it seems possible that Chr9p21 CAD risk genotypes affects genomic expression both  in cis  and  in trans , and linear  ANRIL  RNA may be one, but not the sole, important effector molecule for how the Chr9p21 locus transduces such effects (Figure  1B ).\n\nAnother piece of evidence for a functional role of  ANRIL  in determining CAD risk stems from correlation analysis with disease features in patient cohorts. Aside of the genetic association,  ANRIL  levels were often increased in CAD patients, and not only in atherosclerotic plaque tissue, but also in circulating PBMCs or whole blood. Here, linear  ANRIL  levels were positively correlated with the severity of atherosclerosis ( 13 ,  29 ,  75 ) whereas  circANRIL  was anticorrelated ( 36 ) (Figure  1B ). Thus, while the genotype of Chr9p21 determines the production of atherogenic (linear) over antiatherogenic  ANRIL  RNA species (circular), CAD and peripheral artery disease-dependent changes may additionally feed into  ANRIL  regulation. For  CDKN2B , two studies reported a correlation of the expression with atherosclerosis severity ( 34 ,  76 ), where the direction of the correlation (downregulation in plaques) was consistent from what could be expected from the association results. But another study reported increased  p16 INK 4 A  levels to positively correlate with inflammation markers in plaques instead of anticorrelation ( 25 ). Together, results from association as well as correlation analyses have etablished  ANRIL  lncRNA as prime candidate at the Chr9p21 locus.\n\nANRIL  belongs to the group of long non-coding RNAs and as such has been suggested to act as a molecular scaffold of chromatin-modifying complexes that control gene expression through modifying histone tails. Specifically , ANRIL  was found to physically interact with the CBX7 protein inside the PRC1 Polycomb complex, one of the major gene repression complexes in cells ( 74 ). Knockdown of members of this Polycomb group complex led to increased expression of the  CDKN2A  and  CDKN2B  tumor suppressors in the Chr9p21 locus. Also, ongoing RNA polymerase II transcription was important for the association of the Polycomb proteins with the locus, indicative of the importance of RNA for recruitment. It was concluded that  ANRIL 's function may be, at least in part, to repress the  CDKN2A  and  CDKN2B  tumor suppressors. As a consequence increased  ANRIL  levels are thought to promote overproliferation and to be incompatible with senescence onset, a major function of  CDKN2A/B . As described in chapter 2, other work has shown that recruitment of the Polycomb complexes may account also for how  ANRIL  regulates genes  in trans  on a genome-wide level: Overexpression of linear  ANRIL  isoforms in cultured cells was found to promote pro-atherogenic cell functions, such as proliferation and reduced apoptosis, and to trigger the differential expression of hundreds of genes, in this case without affecting  CDKN2A/B  suppressors. Results from that study therefore questioned whether  ANRIL  regulated these tumor suppressor genes  in cis  at all ( 36 ,  77 ).\nHow does circular  ANRIL , whose abundances is reduced in CAD patients, fit into this model? Both in human peripheral blood T-lymphocytes, as well as in PBMCs, whole blood and endatherectomy plaque tissue,  circANRIL  isoforms were found to be downregulated in samples from CAD patients carrying the Chr9p21 risk allele ( 22 ,  36 ). In an initial model, it was suggested that the production of  circANRIL  from central  ANRIL  exons would shorten the linear  ANRIL  lncRNA and, thereby, impaired linear  ANRIL's  function in epigenetic control of target genes ( 22 ). In a second study, a more primary role was found for  circANRIL  that was, furthermore, independent of linear  ANRIL  ( 36 ). Here,  circANRIL  was found to be 10-fold more abundant than linear  ANRIL . Mass-spectrometric analysis of proteins interacting with  circANRIL  showed that it bound to PES1 protein, a member of the evolutionarily conserved PeBoW complex. This complex is essential for proper rRNA-processing, that is the excision of RNA spacer elements from pre-ribosomal rRNA precursors.  CircANRIL  inhibited the activity of the PeBoW complex, as deduced from the accumulation of unsufficiently processed (and non-functional) 26S and 32S pre-rRNA intermediates when  circANRIL  was overexpressed ( 36 ). A deficit in rRNA maturation caused nucleolar stress and p53 activation, culminating in inhibition of cell proliferation and in an increase in apoptosis. Notably, the observed functions of  circANRIL  were inverse to that of linear  ANRIL  and, as shown by genomic knockout of linear  ANRIL  exons, independent from the presence of these lncRNA isoforms. Thus, experimental evidence from expression analysis  in vivo  and from genetic experiments both indicated that  circANRIL  was anti-atherogenic. Together, linear  ANRIL  confers overproliferation, and circular  ANRIL  protects from overproliferation, suggesting that the genotype of Chr9p21 is important to determine the balance of linear and circular  ANRIL  levels in SMCs and macrophages, and that a dominance of linear  ANRIL  in this ratio, even when small, over decades skews for CAD ( 36 ) (Figure  1B ).\nWhether suppressing linear  ANRIL  or boosting circularization is sufficient to protect from atherosclerotic cues  in vivo  is matter of ongoing research. The fact that  ANRIL  RNA is not conserved beyond primates complicates the functional  in vivo  analysis of the Chr9p21 locus. So far, insight on how CAD is controlled by Chr9p21 through genetic modeling in mouse mutants is fragmented. The genetic elements of Chr9p21 and their relative positioning are overall syntenically conserved in mouse chromosome 4. So far, only one study has investigated, if deletion of a 70 kb long portion of mouse Chr4 corresponding to the CAD haplotype block in humans had an effect on atherosclerosis  in vivo  ( 78 ). This region contains a multi-exon lncRNA,  AK148321 , which is, however, likely not corresponding to human  ANRIL . Mutant mice ( 78 ) developed tumors, reminiscent of tumorigenesis associated with mutation in the Chr9p21 region. But despite some metabolic changes in the mutant mice and enhanced platelet activation, no significant change in atherosclerotic fatty lesion formation was observed ( 78 ), putting in question the validity of this mouse model for studying  ANRIL -driven atherogenesis. On the other hand, the mutants did develop more vascular aneurysms ( 79 ), supporting that some aspects of CAD were indeed contained in the noncoding mouse sequence.\nOverall, the picture is not yet fully clear. While the genetic data from mice support the importance of individual noncoding genetic elements and of some of the protein-coding tumor suppressors for regulation of atherosclerosis and other CAD entities, whether the lncRNA encoded in the locus regulates CAD mechanistically via epigenetically regulating the neighboring tumor suppressors  in cis  has not been determined. Nevertheless, mouse genetics remains an interesting research avenue to explore some aspects of Chr9p21 biology, at least relating to aneurysm, cancer, and glaucoma formation.\n\nStarting from a GWAS signal for CAD in a “gene desert” on Chr9p21 in 2007, research in the last decade has firmly established this region as strongest genetic factor of human atherosclerosis and has contributed to a better understanding of the underlying pathophysiology. The picture has emerged that one of the major routes how this locus controls atherosclerosis risk is through regulating the expression of the lncRNA  ANRIL in cis , where the risk allele leads to high levels of linear  ANRIL  but decreases circular  ANRIL  expression. Linear  ANRIL  has been established as molecular scaffold guiding epigenetic protein complexes and promoting pro-atherogentic cells functions. On the contrary, circularization shifts  ANRIL's  function toward controlling ribosomal RNA processing and controlling protein translation thereby promoting athero-protection (Figure  1B ). The molecular mechanisms of how the ratio of linear and circular  ANRIL  is controlled by the genotype at the locus are currently not resolved and it will be important to determine which gene regulatory elements within the  ANRIL  gene are disturbed by causal CAD risk SNPs. Experimentally exploring details of the molecular effector mechanisms for linear  ANRIL  and for circular  ANRIL  will be paramount, but this task will not be trivial because linear and circular  ANRIL  isoforms always co-exist and in part share the same sequence. Not last, more nuanced relations between Chr9p21 genotype and gene expression output can be expected to be found in the future if, for example, analyses were to take into account cell type-specific and context (stress, inflammation, senescence)-specific effects, aspect that whole tissue expression profiling is currently missing. Additionally, although it is early days, measuring the levels of  circANRIL/linear ANRIL , might offer a prognostic value and help improve CAD risk stratification or allow to better monitor treatment response or disease recurrence.Yet, since  circANRIL  levels are reduced in plaque tissue, and since  circANRIL  has been found to be anti-atherogenic with or without co-existing linear  ANRIL , increasing  circANRIL  abundance in patients could also be of therapeutic relevance. Expressing  circANRIL  levels in the cells of the vasculature in CAD disease models might, therefore, be a promising next step to exploit the accumulated knowledge on the Chr9p21 CAD risk locus.\n\nAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.","source_license":"CC-BY-4.0","license_restricted":false}