{"paper_id":"c3f0ddcd-58c7-45c7-a39f-1430dbe31436","body_text":"Atherosclerotic vascular disease (ASVD) is a leading cause of human mortality worldwide  [1] . While there are well-recognized risk factors for ASVD such as tobacco use, obesity and hyperlipidemia, the identification of common genetic variants associated with the disease has proven difficult despite strong evidence that susceptibility is heritable. Recently, multiple unbiased genome-wide wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) on chromosome 9p21 to ASVD and other related conditions ( i.e.  coronary artery disease, stroke, myocardial infarction and aortic aneurysm)  [2] – [12] . These associations have been replicated in multiple independent studies and are not associated with “classical” ASVD risk factors such as hypertension, obesity, tobacco use or lipid levels. While causal variants within 9p21.3 have yet to be identified, the risk associated SNPs cluster together within a 53 kb region roughly 100 kb centromeric to the  INK4/ARF  ( CDKN2a/b ) tumor suppressor locus ( [13]  and  Figure S1 ). Congenic mapping using mice differentially susceptible to ASVD has identified syntenic susceptibility alleles near the murine  Ink4/Arf  locus  [14] , suggesting that this risk interval is conserved in mammals.\nThe  INK4/ARF  locus encodes three archetypal tumor suppressor genes:  p16 INK4a ,  p15 INK4b  and  ARF  ( p14 ARF  in humans,  p19 ARF  in mice), as well as a long non-coding RNA called  A ntisense  N on-coding  R NA in the  I NK4 \n L ocus ( ANRIL  or  CDKN2BAS ). Other than a role for ARF in development of the optic vasculature, all of the  INK4/ARF  proteins are thought to be largely dispensable for normal mammalian development, but play important roles in restraining aberrant proliferation associated with cancer and other disease states (reviewed in:  [15] ). In purified T-cells from healthy individuals, we have shown a pronounced effect of ASVD-associated SNPs at 9p21 on  INK4/ARF  expression, with those harboring the risk alleles demonstrating reduced levels of  p15 INK4b ,  p16 INK4a ,  ARF  and  ANRIL \n [16] . Decreased expression of such anti-proliferative molecules could promote pathologic monocytic or vascular proliferation, thus accelerating ASVD development (reviewed in  [17] ,  [18] ). For example, mice lacking  p16 INK4a  exhibit increased vascular hyperplasia following intra-arterial injury  [19]  and  ARF  deficiency has been implicated in atherosclerotic plaque formation  [20] . Additionally, TGF-β signaling, which induces the expression of  p16 INK4a  and  p15 INK4b , is anti-atherogenic in some settings  [21] – [23] . Most recently, excess proliferation of hematopoietic progenitor cells, which is in part controlled by  p16 INK4a  expression during aging  [24] , has been associated with atherosclerosis in a murine model  [25] . Moreover, targeted deletion of a region syntenic to the ASVD risk interval in mice resulted in severely attenuated expression of  p15 INK4b  and  p16 INK4a \n [26] . Although these results suggest that the ASVD-associated 9p21 SNPs control  INK4/ARF  expression, and that decreased expression of the  INK4/ARF  tumor suppressors may promote ASVD, it is not known how polymorphisms located ∼120 kb away from the locus might influence  INK4/ARF  expression.\nANRIL  was first uncovered in a genetic analysis of familial melanoma patients with neural system tumors  [27] . Based upon EST assembly,  ANRIL  has 19 exons with no identified open reading frame  [27]  ( Figure S2 ). Although cloning a full-length version of the predicted transcript has proven difficult, a growing number of alternatively spliced  ANRIL  transcripts have recently been reported in the literature  [28] ,  [29] . Many of these reports suggest that multiple  ANRIL  isoforms can be expressed in a single cell type. For example, two  ANRIL  variants have been reported in testes, five in HUVECs and three in lung  [27] ,  [28] . Further confounding the study of  ANRIL , the majority of predicted exons are <100 nucleotides in length and many consist entirely of repetitive LINE, SINE and  Alu  elements ( i.e.  exons 7, 8, 12, 14 and 16)  [30] . Without a firm understanding of  ANRIL  structure, deciphering the biological function of this non-coding RNA has become increasingly complicated.\nMore than a decade ago, elegant genetic work by Van Lohuizen, DePinho and colleagues demonstrated that the  Ink4/Arf  locus is potently repressed by Polycolmb group (PcG) complexes  [31] . Such repression appears critical for the persistence and proliferation of somatic stem cells and other self-renewing tissues such as pancreatic beta-cells  [32] ,  [33] . Until recently little progress was made in understanding the biochemical basis for PcG-mediated  INK4/ARF  silencing, however two independent groups have since shown that the PcG complexes, PRC-1 and PRC-2, localize to the  INK4/ARF  locus and repress its activity through the establishment of repressive chromatin modifications such as H3K27 trimethylation  [34] – [36] . Prior work has also shown that long, non-coding RNAs such as  Xist ,  Kcnq1ot1  and  HOTAIR  can repress genes in  cis - or trans -  through interaction with PcG complexes  [37] – [40] . Moreover, dysregulation of  HOTAIR  has been recently implicated in breast cancer progression, suggesting that long, non-coding RNAs play an important role in human disease  [41] . Based on this evidence, we and others postulated that  ANRIL  could play a similar role in PcG-mediated repression of the  INK4/ARF  locus  [16] .\nAgainst this background of prior work, we sought to better determine  ANRIL  structure and expression in relation to ASVD-SNP genotype and  INK4/ARF  expression. Toward that end, we performed comprehensive DNA and RNA analyses of  ANRIL  using RACE and next-generation sequencing in primary and transformed cell lines. We showed that the expression of  ANRIL  isoforms containing exons 1–2 or 4–6 correlated with ASVD-SNP genotype, however those containing exons 18–19 did not. Surprisingly, we also uncovered circular  ANRIL  ( cANRIL ) RNA species whose expression correlated with ASVD-SNP genotype. These new data suggest that the causal variant(s) within 9p21 influence ASVD susceptibility through regulation of  ANRIL  expression and splicing, leading to differential PcG recruitment and  INK4/ARF  repression.\n\nMultiple  ANRIL  isoforms have been proposed based upon the assembly of ESTs and the sequencing of cDNA libraries (See  Figure S2 ). To determine which of these isoforms predominates  in vivo , we performed RNA ligase mediated (RLM)-RACE in cell lines and primary human peripheral blood T-lymphocytes (PBTL). In addition to using the RLM procedure to maximize the detection of mRNA transcripts, we employed a high-fidelity Taq polymerase capable of amplifying complex DNAs such as those containing  SINE ,  LINE  and  Alu  elements. Primers for 3′ and 5′ RACE were designed within exons 1, 2, 4, 6, 9, 13, 16 and 18 of the originally reported transcript,  NR_003529   [27] , but only primers in exons 4 and 6 selectively amplified  ANRIL  sequences (data not shown). These amplicons were cloned and sequenced to verify the resulting DNA sequences. Using exon 4 and 6 primers, we identified multiple  ANRIL  variants, including novel splice isoforms that were not previously reported ( Figure 1A , novel exons shown in blue). We also detected several transcripts with a peculiar, non-colinear exon sequence, most notably in the HeLa and primary PBTL populations (shown in red in  Figure 1A ). Given that no particular  ANRIL  isoform appeared to predominate from our RACE data, we exploited the observation that  ANRIL  RNAs containing exon 15 frequently maintained the canonical exonic structure ( e.g.  15–16–17–18–19). We termed these exons as “distal” because they are located at the 3′ end of  ANRIL , and those prior to exon 15 as “proximal”.\nA, 3′ and 5′ RACE was performed using primers directed against exons 4 and 6 where long stretches of unique sequence were observed (top). The resulting PCR products were cloned and sequenced, revealing several novel exons shown in blue (10a and 13b) and multiple non-colinear species (red). B, Equal quantities of total RNA were harvested from growing cell lines of various tissue types and absolute expression of the indicated transcript was determined. Expression levels are shown in a box-whisker plot on a log 10  scale in 11 of 27 analyzed cell lines which did not harbor homozygous 9p21 deletion. Validated Taqman detection strategies for the indicated  ANRIL  species are shown (top).\nTo examine the absolute abundance of transcripts containing the “proximal” or “distal” exons in a variety of cell types, we developed and validated quantitative Taqman primer-probe sets spanning  ANRIL  exons 1–2, 4–6 and 18–19 (See  Materials and Methods  and  Figure 1B ). We also quantified the expression of other  INK4/ARF  transcripts  (e.g. p15 INK4b ,  p16 INK4a  and  ARF ) since we previously observed a correlation between the expression of  ANRIL4-6  and  p16 INK4a  in PBTLs (r 2  = 0.32, p<0.0001;  [42] ). Importantly, none of these Taqman strategies amplify  ANRIL  exons containing repetitive sequences. Moreover, the products from these assays were gel-purified, cloned and sequenced to verify their identities. Equal amounts of RNA from a panel of 27 primary and transformed cell lines were subjected to reverse transcription using a combination of random hexamer and oligo dT primers. Deletion or methylation of the 9p21 tumor suppressors is a common event in cancer and was observed in 59% of our cell line panel ( Figure S4 ). Even after exclusion of these lines from our analyses, all three  ANRIL  species (exon  1 – 2 ,  4 – 6  and  18 – 19 ) were rare: based on an estimate of 15 pg of total RNA per cell, expression of  ANRIL  species ranged between 0.01 to 1 copy per cell ( Figure 1B ). For comparison,  INK4/ARF  expression is considered quite low in primary cells  [43] ,  [44] , yet  p16 INK4a  and  p15 INK4b  were more abundant than any  ANRIL  species in non-transformed Hs68, HUVEC and IMR90 cell lines (on average 155.9±18.9- and 14.5±5.5-fold, respectively; See  Figure S4 ). These data indicate that  ANRIL  species are readily detectable but non-abundant in a wide variety of cell types.\nTo further characterize 9p21 transcription in a manner unbiased by PCR primer choice, we analyzed publically available, next-generation RNA sequencing (RNA-seq) datasets of oligo dT-primed and reverse transcribed RNA from primary human brain or HeLa cells. ( [45] ; Genbank Short Read Archive Study ID: SRP002274 and ENCODE RNA-seq replicates, respectively). These datasets were solely chosen based upon the high-depth of sequencing provided (>368 million and >110 million reads, respectively), allowing for the study of non-abundant ncRNAs. In accord with the TaqMan-based analyses, reads mapping to  ANRIL  were rare in both the human brain and HeLa datasets ( Figure 2 ). In fact, the peak height of reads mapping to  p16 INK4a  was >40 times higher than that of any  ANRIL  exon, even though brain expresses low levels of p16 INK4a \n [44] ,  [46] . The level of  ANRIL  expression detected in both datasets (maximal peak height of 12) was comparable to that of other long, non-coding RNAs including  HOTAIR  and  Kcnq1ot1  (maximal peak heights of 10 and 18, respectively).\nA, Coverage plots of RNA sequencing reads derived from short read archive study SRP002274  [45] .  Top , Read coverage across all  ANRIL  exons and nearby tumor suppressor genes  p16 INK4a ,  ARF  and  p15 INK4b  is shown.  Bottom , The grey regions in the top panel were graphed on a truncated scale to better depict  ANRIL  coverage. Annotations above the larger peaks show the maximum number of reads mapping to these areas. B, Maximum peak height at each exon (normalized by overall locus coverage) is displayed from three independent samples: SRP002274 (Brain) and two ENCODE RNA-sequencing replicates of the HeLa cell line (HeLa rep 1 and 2). The inset shows all  ANRIL  exons on y-axis with peak height of 150 reads.\nIntriguingly, by both TaqMan ( Figure 1B ) and RNA-seq ( Figure 2B ), we observed a disparity in the number of molecules of each  ANRIL  exon detected, with those containing the ‘central’  ANRIL  (4–12) exons being the least abundant when averaged across the entire locus. The most prevalent  ANRIL  peaks localized to the 5′ and 3′ ends of the transcript ( i.e.  exons 1–3 and 13–19). The relative excess of the more distal exons might be a consequence of 9p21 deletion, as we observed splicing between  MTAP , a gene 100 kb telomeric to the  INK4/ARF  locus, and the 3′ end of  ANRIL  in RNA-seq datasets from cell lines with  INK4/ARF  loss ( i.e.  SUM102 and MCF7, data not shown). Although such  MTAP - ANRIL  fusions have been previously described  [47] , using a highly sensitive Taqman strategy we only detected these fusions in cell lines with 9p21 deletion, and not in any primary cells or lines with two copies of an intact  INK4/ARF  locus (data not shown). Therefore, while exon deletions and/or  MTAP  splicing explain the relative excess transcription of distal  ANRIL  exons in some cancer cell lines harboring 9p21 deletion, these mechanisms do not explain the decreased transcription of exons 4–12 versus exons 1–3, in any cell line. Likewise, somatic deletions and  MTAP  splicing do not explain the uniform decrease of the central exons compared to the proximal and distal exons in cultures of primary cells ( Figure S4 ). Together, these RNA-seq and TaqMan analyses indicate that  ANRIL  is a rare, multi-variant RNA species in which transcripts containing exons 1–3 or 13–19 predominate over those containing exons 4–12. Based on these findings as well as the observation of non-colinear RNA species by RACE ( Figure 1A ), we hypothesized that the reduced level of the central exons (4–12) in mature, polyadenylated RNA may be the result of alternative splicing events in which the central exons of  ANRIL  are frequently skipped.\nWe observed non-colinear RNA species in RACE analysis of several cell lines and primary cells ( Figure 1A ). We considered the possibility that this represented alterations of the germline DNA sequence ( e.g.  duplications or transversions) but found no evidence for this using BreakDancer  [48]  to analyze next-generation DNA sequencing from 10 individuals. Given that cryptic germline DNA alterations did not appear to explain these non-colinear forms, we considered that non-canonical RNA splicing events such as  trans- splicing or re-splicing of RNA lariats might occur as part of  ANRIL  processing. In the latter case, the splice sites of certain consecutive exons are not recognized by the splicing machinery, resulting in the inclusion of exonic sequences within the RNA lariat  [49] . Internal splicing of this exon-containing lariat structure may then occur, leading to the production of exon-only RNA circles in which non-consecutive exon junctions are generated by splicing across the branch site ( e.g . exon 13–14–4–5).\nTo determine the structure of such non-colinear  ANRIL  RNAs, we designed and validated a Taqman strategy using outward facing primers to detect transcripts containing the non-colinear exon 14-5 junction ( Figure 3A ). This detection scheme was chosen based upon preliminary PCR studies wherein amplification of the 14-5 splice junction predominated over that of the exon 14-4 junction. As lariat or circular RNAs would not be polyadenylated, we assayed the amount of each  ANRIL  species detected in RNA samples reverse transcribed with random hexamers (HEX), oligo dT (dT) or both ( Figure 3B ). As would be expected for polyadenylated RNAs, conversion of  ANRIL1-2 ,  ANRIL18-19 ,  p16 INK4a  and  p15 INK4b  into cDNA was efficiently accomplished with either oligo dT or HEX primers alone. Conversely,  ANRIL4-6  and  14-5  were effectively primed with HEX but not oligo dT, confirming that these transcripts were not polyadenylated.\nA, Schematic representation of the  ANRIL14-5  Taqman detection strategy wherein the probe (red) spans the exon 5-exon 14 boundary amplified with outward facing primers. B, Expression of the indicated transcripts was quantified in cDNA from Hs68 cells made using the indicate primers (-RT: no reverse transcriptase, H+dT: an equal mix of random hexamers and oligo dT, dT: oligo dT alone, and HEX: random hexamers alone). Error bars represent the standard deviation for three replicates. C, Total RNA harvested from growing Hs68 (top) and IMR90 (bottom) cells was incubated with our without RNase R, purified and reverse transcribed. The indicated transcripts were quantified in ‘B’. D, The average fold enrichment by RNase R for each transcript is shown on a log 10  scale.\nTo further examine the structure of these non-canonical transcripts, we determined transcript sensitivity to the RNA exonuclease, RNAse R. RNAse R specifically digests both structured and non-structured linear RNAs, but spares RNA circles and lariats  [50] . Using equal amounts of RNA from normal and immortalized human fibroblasts (IMR90 and Hs68, respectively) treated with or without RNAse R, we generated cDNA and conducted TaqMan analysis for  ANRIL  expression. As expected for linear species, RNAse R treatment caused a marked reduction in the number of  p16 INK4a  and  p15 INK4b  transcripts detected (4.5- and 8.4-fold decrease, respectively), demonstrating that these coding transcripts are predominantly linear ( Figure 3C and 3D ). In contrast, we observed a 6-fold enrichment of  ANRIL14-5  molecules with RNAse R treatment, confirming their circular nature. We were surprised to find that  ANRIL4-6  expression also exhibited RNAse R-dependent enrichment in both cell lines.  ANRIL1-2  levels decreased consistent with a predominantly linear species, and  ANRIL18-19  demonstrated an intermediate behavior consistent with a mix of linear and circular forms ( Figure 3C and 3D ). Together, these data provide evidence that  ANRIL4-6  and  14-5  are predominantly contained within non-polyadenylated, circular (or lariat)  ANRIL  ( cANRIL ) transcripts.\nBased upon analysis of non-sequential cDNA and EST sequences, stable RNA circles have been hypothesized to represent <1% of the total transciptome  [51] . To determine the ubiquity of  cANRIL  RNAs, we employed the Taqman-based strategy shown in  Figure 3A . Using this method, we observed amplification of  ANRIL14-5  in 16 of 20  ANRIL  expressing primary cultures and cell lines ( Figure S5 ). The finding of  cANRIL  in diverse cell types is perhaps surprising given the low abundance of  ANRIL  transcripts and that RNA lariats are usually unstable intermediates which undergo rapid degradation. These data suggest that  cANRIL  is a stable, naturally occurring, circular RNA species produced in most  INK4/ARF  expressing cells.\nTo better define the structure of  cANRIL  RNAs, we used outward-facing primers located in five  ANRIL  exons to amplify cDNA produced using a mix of random hexamers and oligo dT ( Figure 4A ). In Hs68 and IM90 cells, PCR products specific to 9p21 were detected using primers sets targeting exons 4 and 6. These products were more efficiently amplified using cDNAs from RNase R-treated samples, suggesting that they arose from circular RNAs. In contrast, PCR products specific to chromosome 9 were not detected using primers internal to exons 1 and 18, and only detected in one instance with primers internal to exon 16, suggesting these exons were not commonly included in stable, circular  ANRIL  species. To determine the contents of the circular transcripts containing  ANRIL  exons 4 and 6, we cloned and sequenced the observed PCR products. The resulting sequences predominantly included  ANRIL  exons 4 to14. Several exons were never observed in the looped structures ( i.e.  exons 1, 2, 3, 8, 9, 11, and 12) ( Figure 4B and 4C ). Novel sequences were also discovered within the products, most notably, regions intronic to the previously described  ANRIL  transcript ( i.e.  parts of intron 3). These data explain the paucity of expression of  ANRIL  exons 4 to 14 as determined by Taqman and RNA-seq ( Figure 1B  and  Figure 2B ), suggesting that  ANRIL  processing may involve exon skipping events which preferentially incorporate the central exons into lariats which may then be internally spliced to form  cANRIL .\nA, cDNA generated in the presence (+R) or absence (-R) of RNase R as in  Figure 3C  was subjected to PCR using outward facing primers within the same exon as depicted (left) and separated by gel electrophoresis. B and C, The PCR products in ‘A’ were purified, cloned and sequenced. The resulting sequences are shown for each exon pair.\nWe have previously shown  [42]  a correlation between expression of the coding  INK4/ARF  transcripts and  ANRIL4-6 . We now recognize that the latter is largely contained in circular RNA species ( Figure 3B and 3C ). Next, we investigated correlations between other, newly identified  ANRIL  isoforms and the coding  INK4/ARF  transcripts ( Figure 5A  and  Figure S6 ). We performed Taqman-based gene expression analyses in 106 primary human peripheral blood T-lymphocyte (PBTL) samples from two previously published cohorts  [16] ,  [42] . Remarkably,  cANRIL  expression ( ANRIL14-5  and  ANRIL4-6 ) was observed in all PBTL samples. Expression of  ANRIL18-19  did not correlate with any other  ANRIL  or  INK4/ARF  transcript ( Figure 5A ). In contrast, a strong correlation was observed between the expression of  ANRIL4-6  and all of the  INK4/ARF  tumor suppressors (p<1*10 −15  for all pair-wise comparisons). Significant associations (p<0.05) were also observed between  ANRIL1-2  and  ARF , and between  ANRIL14-5  and  p16 INK4a .\nA, Taqman anlaysis of  ANRIL  and  INK4/ARF  transcripts was conducted and normalized as described in  Materials and Methods . Correlations between  ANRIL  and  INK4/ARF  transcripts in 106 primary peripheral blood T-lymphocytes (PBTLs) were determined using linear regression. The R value for each pair-wise comparison is shown, with those achieving significance (p<0.05) depicted in blue. Due to limitations in sample availability,  ANRIL 1-2  and  ANRIL 14-5  levels were determined in only a subset of individuals (n = 94 and 98, respectively). B, The relative expression of  ANRIL1-2 ,  14-5  and  18-19  normalized as in ‘A’ is plotted versus rs10757278 genotype. p-values were determined by a two-sided t-test.\nWe have also demonstrated an effect of a replicated SNP associated with atherosclerosis (rs10757278) on the expression of  p15 INK4b ,  p16 INK4a ,  ARF  and  ANRIL4-6  in PBTLs  [16] . We therefore examined whether expression of other  ANRIL  isoforms correlated with ASVD SNP genotype ( Figure 5B ). While no significant correlation between  ANRIL18-19  with SNP genotype was noted, expression of  ANRIL1-2  and  ANRIL14-5  showed significantly decreased expression in individuals harboring the risk (G) allele (p<0.0001 and p<0.04, respectively). In aggregate, these data demonstrate that expression of both circular and linear transcripts containing the proximal but not distal  ANRIL  exons correlates with ASVD-SNP genotype and expression of the coding  INK4/ARF  transcripts.\nWe performed next-generation DNA sequencing of the ASVD risk interval to identify polymorphisms that might influence  ANRIL  expression or splicing. To enhance detection of rare variants, we pooled DNA from 5 individuals of Asian and European descent who were homozygous for either the A or G allele of rs10757278. To increase the chance that the analyzed DNAs would harbor causal variants that influence  INK4/ARF  expression, we selected individuals based on their age-adjusted expression of  p16 INK4a  ( i.e.  AA donors with higher than average expression, GG donors with lower than average expression  [16] ). We performed sequence capture using an Illumina tiling array on the AA vs. GG pooled samples. The tiling array was designed to bind all non-repetitive human chromosome 9 regions from 22,054,888 to 22,134,171 bp, a region chosen as it contains the previously identified ASVD and type 2 diabetes mellitus risk intervals ( Figure 6A ). The 9p21 enriched DNA was sequenced using both Roche 454 (400 bp reads) and Illumina GAII (35 bp reads) technologies. The resulting sequences were aligned to the UCSC reference genome (hg18) using three separate alignment algorithms: gsMapper, BWA and SOAP  [52] ,  [53] . As shown in  Figure 6B , SOAP identified the highest number of polymorphisms within each pooled sample; however, many of these were not recapitulated using other mapping techniques. Thus, for further analysis we focused our efforts on polymorphisms which were identified by at least two mapping algorithms. Employing these criteria, we discovered 101 SNPs that differed from the reference genome within our samples: 11 in the AA pool, 64 in the GG pool, and 26 in both ( Figure 6C ). As expected, more differences from the reference genome were identified in the GG sample, as the reference genome harbors the A allele, and most SNPs in the captured region are in moderate to strong linkage disequilibrium with rs10757278 ( Figure S1 ). Therefore, using next generation sequencing of captured DNA from 10 informative individuals biased to harbor causal variants, the chosen sequencing approach found 75 (11+64) SNPs that differed between the two pooled samples.\nA, The region captured using DNA sequence capture technology is shown on the ‘Tiling’ track. The ‘Unique’ track shows the Duke 35 bp Uniqueness information as provided in the UCSC Genome Browser. The bar at the top of the figure represents the 53 kb risk interval previously defined by Broadbent  et al. \n [13] . B, Venn diagrams depicting SNP calls for the AA (left) and GG (right) samples using three different algorithms. C, Using the UCSC Genome Browser, SNPs identified by next-generation DNA sequencing are depicted across the captured region of 9p21. The ‘Discovered’ track shows the polymophisms identified by two or more algorithms in either the pooled AA or GG sample. SNPs unique to each genotype are shown below. The ‘Unique Splice’ track depicts the location of the 4 SNPs, unique to the GG sample, which modify  cis -acting splice regulation sites (See also  Table S1 ).\nGiven the finding that  cANRIL  expression correlates with rs10757278 genotype, we sought to determine if any of the identified SNPs might influence  ANRIL  splicing. For this analysis, we restricted the SNP list to include only those within 200 bp of an  ANRIL  intron-exon boundary, as genetic alterations in this region have the highest potential to influence RNA splicing  [54] . Of the 75 SNPs present in only one of the two pooled samples, four were within 200 bp of an  ANRIL  intron-exon boundary ( Figure 6C ,  Table S1 ). Using previously described prediction algorithms  [54] ,  [55] , we determined the likelihood of these variants near intron-exon boundaries to change  cis -regulatory splice elements including exon splicing enhancers (ESEs) and silencers (ESSs) as well as intronic splicing enhancers (ISEs) and silencers (ISSs). A score of -1 indicates that the minor allele destroys one  cis -element and +1 indicates that the minor allele creates one  cis -element ( Table S1 ). ESEs and ISEs favor exon recognition by the spliceosome when they occur in exons or introns, respectively. However, ISSs and ESSs promote exon skipping irrespective of position  [56] . A pair of SNPs (rs34660702 and NOVEL) 10 bp apart near the start of exon 12 were predicted to alter ANRIL splicing, but the effects of these SNPs worked in opposite directions, and both exhibited low frequencies in the CEU population ( Figure 6C  and  Table S1 ). More provocatively, we also observed two SNPs (rs7341786 and rs7341791) in strong linkage disequalibrium with rs10757278 and which bracketed exon 15. These SNPs demonstrated high minor allele (CC and GG, respectively) frequencies and with the major allele of both SNPs enhancing the “exon-ness” of exon 15. These results indentify common, linked SNPs whose major allele is likely to inhibit skipping of exon 15 and thereby promote the production of  cANRIL  species ending in exon 14 in individuals homozygous for the A (protective) allele of rs10757278.\nDue to the small number of pooled individuals for sequencing (n = 5/group), we also sought to identify additional, rare polymorphisms with the potential to modulate  ANRIL  splicing. We analyzed all SNPs reported in the HapMap database in the chromosome 9 region 22,054,888 to 22,134,171 within 200 bp of  ANRIL  intron-exon boundaries for their potential to alter RNA splicing in  cis- . Using this method, we identified 31 additional SNPs in HapMap that modified ESE, ESS, ISE or ISS sequences ( Table S1 ). Notably, the majority of these variants were rare and almost never reported in the CEU population  [57] . Therefore, among all of the SNPs examined from the ASVD risk interval, rs7341786 and rs7341791 appear most likely to influence  ANRIL  splicing. These SNPs are also in very strong linkage disequilibrium with the ASVD-associated SNP, rs10757278 (r 2 >0.96, based upon the nearest database SNPs in CEU), and therefore would be expected to correlate with  ANRIL  and  INK4/ARF  expression. However, it is important to note that other classes of germline variants ( e.g.  complex indels or alterations in repetitive elements) would not have been identified by our sequencing strategy, and therefore we are unable to exclude a role for other such variants in  ANRIL  expression and/or splicing.\n\nMotivated by the role of other long, non-coding RNAs in PcG repression, we investigated whether  ANRIL  transcription and/or structure was SNP-dependent. In primary and cultured cell lines, we used RACE and RNA-seq to identify novel  ANRIL  variants ( Figure 1  and  Figure 2 ). Intriguingly, the central  ANRIL  exons were underrepresented in these data ( Figure 1B  and  Figure 2B ). This observation, along with non-colinear products detected by RACE, led to the unexpected discovery of multiple circular RNAs emanating from the  ANRIL  locus. Sequencing of these circular species showed non-sequential linkages between various  ANRIL  exons, especially those from the central portion of the transcript ( e.g.  exons 4-14) ( Figure 4B and 4C ). Expression of both circular and linear  ANRIL  isoforms proximal to the  INK4/ARF  locus strongly correlated with  INK4/ARF  transcription and the ASVD risk genotype at rs10757278 ( Figure 5  and  Figure S6 ). In contrast, distal  ANRIL  variants containing exons 18 and 19 were expressed in a genotype-independent manner and did not correlate with the levels of any  INK4/ARF  transcript. Using next-generation sequencing to genotype captured DNA from the ASVD risk interval, we identified a common pair of linked SNPs near exon 15 predicted to influence  ANRIL  splicing ( Figure 6  and  Table S1 ). Together, these findings suggest that common polymorphisms in the ASVD risk interval could modulate  ANRIL  transcription and/or splicing, thereby influencing PcG-mediated  INK4/ARF  repression and atherosclerosis susceptibility.\nMultiple  ANRIL  isoforms have been reported in the literature and EST databases, with some exhibiting differential expression patterns and SNP associations ( Figure S2  and  [28] ,  [29] ). Using RACE, RNA-seq and sensitive quantitative real-time PCR techniques, we now show that no single  ANRIL  species predominates  in vivo , and that splicing to  MTAP  does not occur in cells with an intact 9p21 locus. Moreover, our analyses identified new  ANRIL  exons and variants, uncovering a novel group of circular  ANRIL  ( cANRIL ) species ( Figure 4 ). Using independent approaches, we found that all  ANRIL  exons were expressed at very low levels, orders of magnitude lower than even the relatively rare  INK4/ARF  tumor suppressors,  p15 INK4b  and  p16 INK4a  ( Figure 1B ,  Figure 2B , and  Figure S4 ). This low level of expression is comparable to what we observed for other regulatory non-coding RNAs ( i.e. HOTAIR  and  Kcnq1ot1 ) associated with PcG-mediated repression.\nThe discovery of non-colinear  ANRIL  species whose expression correlated with  INK4/ARF  transcription suggested that alternative splicing events might modify  ANRIL  structure leading to changes in PcG-mediated  INK4/ARF  repression. There are two major mechanisms by which non-colinear RNAs are thought to arise:  trans- splicing and exon skipping  [58] . During  trans- splicing, a Y-branched RNA structure is formed that is sensitive to RNase R digestion  [50] . In contrast, exon skipping events generate large lariat structures, which can then undergo  cis -splicing to create RNase R-resistant circular RNAs. To confirm the circular nature of the  ANRIL  species, we showed that transcripts containing  ANRIL4-6  and  14-5  were resistant to RNase R degradation, were not polyadenylated and could be PCR amplified using sets of outward facing primers ( Figure 3  and  Figure 4 ). Therefore,  cANRIL  species appear to result from exon skipping events occurring during RNA splicing.\nBased on these observations, we propose a model suggesting that common polymorphisms in 9p21.3 modifiy  INK4/ARF  gene expression through  cis -regulation of  ANRIL  transcription and/or splicing ( Figure 7 ). In turn, such changes in  ANRIL  levels or structure influence the ability of these ncRNAs to repress the  INK4/ARF  locus. Specifically, changes in PcG-mediated repression of  p15 INK4b ,  p16 INK4a  and/or  ARF  occur, altering potentially atherogenic cellular proliferation and ASVD risk as previously suggested  [16] – [20] ,  [26] .\nPcG complexes (e.g. PRC-1) are targeted to the coding INK4/ARF locus by  ANRIL , and modulate its repression. Nascent  ANRIL  transcripts are spliced to produce circular ANRIL species ( cANRIL ). Causal variants in the ASVD risk interval modulate  ANRIL  transcription or splicing to influence  INK4/ARF  expression. See discussion for further description.\nPrior studies provide evidence for this model. For example, we and others have described an effect of ASVD-genotype on  ANRIL  transcription  [16] ,  [30] ,  [59] ,  [60] . Moreover, meticulous allele-specific expression analyses by Cunnington  et al.  suggested that the  cis -effects of 9p21 SNPs were stronger for  ANRIL  than for the coding  INK4/ARF  transcripts (20% vs. <8%) [60] . In contrast, the effect of these SNPs on expression of the coding  INK4/ARF  transcripts predominantly occurred in  trans -. Such findings are consistent with our model whereby causal variants directly influence  ANRIL  structure in  cis -, thereby controlling repression of the  INK4/ARF  locus, possibly in  trans - ( Figure 7 ). Likewise, recent work has supported a role for ncRNAs in  INK4/ARF  repression. Knockdown of the RNA helicase, Mov10, led to  INK4/ARF  deregulation, suggesting a role for RNA metabolism in regulation of the locus  [61] . Supporting these data, Yap  et al . recently demonstrated that the 5′ end of  ANRIL  contains stem-loop structures capable of binding CBX7, a member of the PRC-1 complex  [62] . Disruption of this interaction led to premature senescence marked by increased  INK4/ARF  expression. Therefore, strong evidence supports the notion that  ANRIL  expression correlates with ASVD-SNP genotype and that PcG-mediated repression of the  INK4/ARF  locus is modulated by this expression.\nThe outstanding question of this model is the biochemical mechanism whereby causal variants at 9p21.3 located more than 100 kb distant from the proximal exons of  ANRIL  ( Figure S1 ) modulate  ANRIL  expression and/or structure. Our data are consistent with a “transcriptional model” wherein distal  cis -regulatory elements within the ASVD risk interval directly influence  ANRIL  transcription. Jarinova  et al.  have identified an enhancer sequence within  ANRIL  intron 17, which in heterologous reporter assays, enhanced transcription in a genotype-specific manner  [30] . Consistent with this view, we also observed a significant effect of ASVD-SNP genotype on the expression of the linear  ANRIL1-2  transcript ( Figure 5B ). As such, the correlation of  cANRIL  expression with ASVD-SNP genotype would presumably reflect passive production of these circular species in the setting of increased or decreased in total  ANRIL  transcription. However, this model would not easily explain the  positive  correlation we observed between the expression of coding  INK4/ARF  transcripts and proximal  ANRIL  exons ( Figure 5A  and  Figure S6 ), as the 5′end of  ANRIL  is reported to foster  INK4/ARF  repression  [62] .\nAlternatively, we believe our present data are more consistent with a “splicing model” in which causal variants in the ASVD risk interval influence  ANRIL  splicing by regulating exon skipping. Provocatively, the ASVD risk interval includes exon 15 where the termination of most exon skipping events that produce  cANRIL  occur ( Figure 4B and 4C ). Using next-generation sequencing of this region in individuals biased to harbor causal variants, we identified exon 15 SNPs (rs7341786 and rs7341791) which are in strong linkage disequilibrium with the ASVD-associated SNP rs10757278 (r 2 >0.96;  Table S1 ). These polymorphisms have high minor allele frequencies in our sample and the CEU population and are predicted to alter  ANRIL  splicing ( Figure 6 ,  Table S1 ). In particular, the major allele (‘A’) of rs7341786, detected only in individuals homozygous for the A-allele at rs10757278, is predicted to increase the strength of exon 15 as a splice acceptor, which would promote the expression of spliced RNA circles ending in exon 14, as observed ( Figure 4B and 4C ).\nFor the splicing model to explain the observed  positive  correlation between the expression of  ANRIL  and the coding  INK4/ARF  transcripts, distinct species of  ANRIL  would need to differ in their ability to repress the  INK4/ARF  locus. Supporting this possibility, PcG-mediated repression of the murine  Kcnq1  locus is dependent upon the length of the long, ncRNA,  Kcnq1ot1 \n [63] ,  [64] . Specifically, longer  Kcnq1ot1  transcripts are associated with increased PcG recruitment and Kcnq1 silencing potential. Therefore, shorter  ANRIL  variants ( i.e.  those lacking exons 4–16) generated by exon skipping events may also be less efficient at repressing the  INK4/ARF  locus. Important predictions of the splicing model are that  cANRIL  expression (reflecting  ANRIL  splicing) should correlate with  INK4/ARF  expression (which it does,  Figure 5A  and  Figure S6 ), that individuals homozygous for the ‘A’ allele of rs7341786 (and rs10757278) should exhibit increased production of  cANRIL  species containing exon 14 but not exon 15 (which they do, see  Figure 4B and 4C ) and that these individuals with increased propensity for splicing should exhibit de-repressed  INK4/ARF  expression (which they do,  [16] ).\nA caveat to the splicing model is that the identified exon 15 SNPs need not be the true causal variant(s) influencing  ANRIL  splicing. It is possible that other polymorphisms not detected by our sequencing strategy could regulate  ANRIL  splicing. In particular, the sequencing strategy employed would not find differences between the pooled samples in repetitive regions, such as the large  LINE  element in the 12 th  intron of  ANRIL  ( Figure 6A ).  ANRIL  harbors several  LINE  and  SINE  elements, and such repetitive motifs have been reported to modulate RNA splicing in other systems  [65] ,  [66] . Therefore, while the exon 15 SNPs appear to be prime candidates to regulate  ANRIL  splicing, a variety of other classes of polymorphisms could also influence splicing and would not have been observed by the chosen sequencing approach.\nImportantly, the splicing and transcriptional models are not mutually exclusive. A single causal variant may influence both processes or there may be multiple causal variants that influence either process within the ASVD risk interval. Finally, while we believe the correlation of  cANRIL  expression and ASVD-SNP genotype is most likely explained by an effect of common 9p21 polymorphisms on transcription and/or splicing, a third possibility also exists. While circular RNA byproducts of exon skipping have generally been regarded as inconsequential, circular RNAs with catalytic activities ( e.g.  group I and some group II introns) are well described in bacteria, lower eukaryotes, plants  [67] . In addition, some viroids and the hepatitis delta satellite virus have circular RNA genomes  [68] ,  [69] . Although we are not aware of any endogenously produced circular RNA with discrete function in mammals, clearly circular RNAs species can possess independent functions in non-mammalian species, and we remain open to the possibility that  cANRIL  itself can directly participate in  INK4/ARF  regulation.\nIn summary, this work links ASVD-genotype to  ANRIL  structure and  INK4/ARF  regulation, providing evidence for what we believe is a first association between endogenous circular RNA expression and a mammalian phenotype (ASVD). Even were  cANRIL  expression merely a marker of exon skipping with no specific biologic function, we believe its expression may be a useful marker of  ANRIL  isoform selection, which our findings suggest is of pathogenic relevance to ASVD susceptibility. We believe this work has implications beyond ASVD, as altered  ANRIL  splicing could influence  INK4/ARF  expression, explaining the association of other nearby 9p21 SNPs with a variety of non-ASVD phenotypes in humans including longevity, type II diabetes, endometriosis and several tumors types  [70] – [77] .\n\nResearch involving human subjects was approved by the University of North Carolina Institutional Review Board and all participants provided informed, written consent.\nWM266-4, UACC 257, A2058, A375, SUM-149, RPMI-8322 and telomerized Hs68 cells were obtained and grown as previously described  [78] – [80] . MDA-MB-468, MDA-MB-436, MDA-MB-231, MCF7, BT-474, BT-549, T-47D, COLO 205, T84, LoVo, LS 174T, SW480, LS1034, HeLa, HUVEC, IMR90, Ramos, Raji, Jurkat and U-87 cells were originally obtained from ATCC and cultured as suggested.\nCD3 positive T-cells were isolated from human peripheral blood samples as previously described  [42] . RNA was generated from proliferating cell lines and isolated human T-cells using the RNAeasy system (Qiagen Inc., Valencia, CA). 3′ and 5′ RACE was performed as described in the Firstchoice RLM-RACE manual (Ambion Inc., Austin, TX). This procedure is optimized for the detection of rare transcripts and provides additional steps to improve the specificity of mRNA amplification. Gene-specific primers were designed within  ANRIL  exons 4 and 6 as shown in  Figure 1A  and  Table S2 ; RACE primers for other  ANRIL  exons tested did not amplify chromosome 9 specific products. All PCR reactions were conducted using SuperTaq-Plus (Ambion) in a Bio-Rad DNA Engine thermocycler. SuperTaq-Plus is a high fidelity, long range polymerase with the capability to amplify complex DNAs such as repetitive  SINE ,  LINE  and  Alu  elements. Cycling conditions for 5′RACE were: 94°C 3 min, 34×[94°C 30 s, 60°C 30 s, 68°C 3 min], 68°C 5 min (inner reaction) and 94°C 3 min, 34×[94°C 30 s, 62°C 30 s, 68°C 3 min], 68°C 5 min (outer reaction). Cycling conditions for 3′RACE were: 94°C 3 min, 34× [94°C 30 s, 57 or 60°C 30 s, 68°C 3 min] (outer reaction) and 94°C 3 min, 34×[94°C 30 s, 60°C 30 s, 68°C 3 min] (inner reaction). Cloning of the resulting PCR products was conducted using the TOPO-Blunt cloning kit (Invitrogen). Sequencing of the resulting clones was conducted using both M13F and M13R primers.\nRNA was isolated using the Qiagen RNAeasy Kit and subjected to reverse transcription using the ImPromII reverse transcription system (Promega, Madison, WI). RNA samples from breast and colorectal cell lines were kindly provided by Drs. N. Mitin and J. Yeh (UNC). Taqman primer and probe sets for the detection of  ANRIL  exons 1–2, 4–6, 14–5, and 18–19 as well as those for  p15 INK4b ,  p16 INK4a ,  ARF  and 18S rRNA are described in  Table S2 . The  ANRIL  primer sets were designed to span at least one intron and were shown to have high specificity with linear amplification efficiencies between 88 and 94% ( Figure S3 ). Final primer and probe concentrations were 900 and 250 nM, respectively. Products from the  ANRIL  1–2, 4–6, 14–5 and 18–19 qRT-PCR reactions were cloned separately into the pBluntII-TOPO vector (Invitrogen) and verified. Real-time PCR was carried out in triplicate on an ABI 7900HT thermocyler.\nFor relative expression studies, differentials were first calculated between each sample and the average 18S cycle threshold (Ct) for the entire experiment. Transcript Ct values were normalized using the equation: Expression  =  Lower limit of detection (37 or 40) - (Ct  target  – Ct  18S differential ) and plotted on a log 2  scale. The relative expression of  p16 INK4a ,  p15 INK4b ,  ARF  and  ANRIL4-6  in PBTLs has been previously reported  [42] ; in this work, these same samples were reanalyzed for  ANRIL1-2 ,  ANRIL14-5  and  ANRIL18-19  expression as shown in  Figure 5  and  Figure S6 . In  Figure 5  and  Figure S6 , data was corrected to account for batch effects between the pilot and verification datasets previously described  [42] . To determine the absolute number of transcripts present, a standard curve of five dilutions was generated for each experimental plate using known amounts of linearized plasmid containing the target sequence. The number of molecules detected in each sample was calculated using the equation: #Molecules  =  10∧((Ct – y-intercept)/slope). Primer efficiencies were calculated using the equation: Efficiency  =  (10∧(-1/slope))-1. Multiple comparison plots and statistical analysis appearing in  Figure 5A  and  Figure S6  was performed using the gpairs function of the R YaleToolkit library.\nA total of 368,846,235 reads generated on the Illumina platform (study SRP002274) were downloaded from the NCBI Short Read Archive. The reads were first screened for unique 20mers deriving from chromosome 9∶21,700,000–22,300,000 using the UCSC genome bowser Duke uniqueness mapability table. The resulting reads were mapped using the TopHat spliced aligner (PMID: 19289445) to the reference human genome (hg18). The resulting coverage plot was imported into the UCSC genome browser for display. Also analyzed were two independent CalTech ENCODE mRNA-seq datasets ( http://genome.ucsc.edu/cgi-bin/hgTrackUi?g=wgEncodeCaltechRnaSeq ) from HeLa cells. These datasets were chosen because they lacked deletion of 9p21 and showed the highest level of  ANRIL  expression of any publically available RNA-seq data we analyzed. For  Figure 2B , the peak number of reads at a given base (SRP002274 data) or peak reads per kilobase mapping (CalTech HeLa data) were quantified and scaled relative to the average exonic coverage.\nTotal RNA was isolated from proliferating Hs68 and IMR90 cells using a Qiagen RNAeasy kit. Equal amounts of RNA (43–50 µg, depending on the experiment) were incubated with or without 40 U of Rnase R ( RNR07250 , Epicentre Biotechnologies) for 2.5 hours at 37°C. The resulting RNA was purified using the RNAeasy column and quantified. Equal amounts of RNA were then subjected to reverse transcription using the ImPromII reverse transcription system and a mixture of random hexamer and oligo dT primers (Promega, Madison, WI). Transcripts were quantified using Taqman-based real-time PCR.\nPCR primers pointing in opposite directions on  ANRIL  exons 1, 4, 6, 13, 16 and 18 were designed using Primer3 software and analyzed for hairpins using Netprimer (Premier Biosoft and  http://frodo.wi.mit.edu/primer3/ ) ( Table S2 ). PCR reactions were conducted using cDNA representing 15 ng of mock or Rnase R-treated RNA. Reactions were performed using Apex Hot Start Taq DNA polymerase and Buffer 2 (Genesee Scientific) in a Bio-Rad DNA Engine thermocycler. The cycling conditions were as follows: 95°C 15 min, 40×[94°C 30 s, 59°C 30 s, 72°C 1 min], 72°C 2 min. The resulting PCR products were cloned into the TOPO-Blunt cloning kit (Invitrogen) and sequenced using M13F and M13R primers.\nGenomic DNA was generated from T-cells of healthy human volunteers of known rs10757278 genotype, age and  p16 INK4a  expression status using the Qiagen Peripheral Blood DNA isolation kit. For sequence capture, 21 ug of DNA was pooled from five individuals homozygous for the G-allele of rs10757278 and another five individuals homozygous for the A-allele. Samples were sent to NimbleGen for sequence capture using a tiled array spanning human chromosome 9 (22,054,888-22,134,171). The resulting amplified DNA fragments were analyzed at the UNC Genome Analysis Facility using both Illumina GAII and Roche 454 technology. For sequencing on the Illumina platform, DNA was randomly sheered and appropriate adapters ligated. Resulting sequences were aligned to the entire human genome (hg18) using MAQ, SOAP, and gsMapper software  [52] ,  [53] . MAQ was run with default settings and output was translated into BAM format. SOAP alignment was performed allowing up to 10 gap bases and 2 mismatches and also translated into BAM format. Mapping with gsMapper was performed with default settings. SNP calls from MAQ and SOAP were generated using the pileup function of the SAMtools library  [81] . Calls were culled to include only those SNPs appearing in ≥20 percent of reads.\n\nPolymorphisms within the  INK4/ARF  locus linked to age-related diseases. A, Schematic diagram of the 9p21 locus depicting the  INK4/ARF  tumor suppressors,  ANRIL  and the ASVD risk interval. The captured (“tiling”) region for next generation DNA sequencing is indicated. B, The localization of SNPs linked in the literature to age-related diseases including is shown. T2D- type II diabetes, CAD- coronary artery disease, MI-myocardial infarction, AD-Alzheimer's disease C, A heatmap depicting the SNP linkage disequilibrium (D′/LOD) was generated from the Hapmap CEU population using Haploview software. The strength of linkage disequilibrium 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 red (D′ = 1 and LOD score ≥2). The heatmap can be aligned to the depicted 9p21 image as shown in the diagram above.\n(1.37 MB TIF)\nClick here for additional data file.\nSchematic of previously reported  ANRIL  variants. All Ensembl (blue) and GenBank (black) records for  ANRIL  ( CDKN2BAS ) are shown. Some sequences are derived from cDNA sequencing whereas others were inferred by EST assembly.\n(0.64 MB TIF)\nClick here for additional data file.\nEfficiency curves for Taqman probe strategies. Cloned cDNAs or PCR products corresponding to the Taqman target sequences were linearized and quantified. For each realtime PCR run, a standard curve of 5 independent dilutions was run in triplicate. Primer efficiency was calculated using the formula: Efficiency  = 10∧(-1/slope)-1. Shown are representative graphs from individual experiments.\n(0.52 MB TIF)\nClick here for additional data file.\nExpression of 9p21 transcripts in transformed and non-transformed cell lines. As described in  Figure 1B , RNA was harvested, reverse transcribed and quantitative real-time PCR performed. Bars represent the log10 of the average number of molecules detected. The error bars denote the standard deviation between three replicates. The letter ‘D’ denotes deletion events previously reported in the literature. ‘M’ indicates gene methylation. Breast cancer are shown in pink, colorectal cancers in blue, melanomas in black, hematological malignancies in green, cervical carcinomas in turquoise, glioblastomas in gold and nontransformed cells in red.\n(0.58 MB TIF)\nClick here for additional data file.\nANRIL14-5  expression is detected in a wide variety of cell types. cDNA generated as described in  Figure 1B  was assayed for  ANRIL14-5  expression using the Taqman strategy shown in  Figure 3A . Bars represent the log10 of the average number of molecules detected. The error bars denote the standard deviation between three replicates. Cell lines are color coded as in  Figure S4 .\n(0.50 MB TIF)\nClick here for additional data file.\nCorrelation of  ANRIL  and  INK4/ARF  expression in primary peripheral blood T-lymphocytes. Diagram depicting the correlations between 9p21 transcripts in primary peripheral blood T-lymphocytes from 106 patients. Taqman anlaysis of  ANRIL  and  INK4/ARF  transcripts was conducted and normalized as described in  Materials and Methods . Data for  ANRIL4-6 ,  p16 INK4a ,  p15 INK4b  and  ARF  expression were previously reported  [42] . Scatter plots, below the diagonal, show the relationships between all pairs of transcripts on a log2 scale. Linear regression is depicted in red. Boxes above the diagonal list and are color coded by r-value. A star (*) indicates significant associations (p<0.05). Histograms along the diagonal show the distribution of expression for each transcript assayed. Due to limitations in sample availability,  ANRIL 1-2  and  ANRIL 14-5  levels were not determined for several individuals as indicated (n = 94 and 98, respectively).\n(1.55 MB TIF)\nClick here for additional data file.\nSplice site analysis of polymorphisms in the ASVD risk interval near  ANRIL  exon-intron boundaries. SNPs within 200 bp of an  ANRIL  inton-exon boundary were analyzed for their effects on putative exon splicing enhancer (ESE), exon splicing silencer (ESS), intron splicing enhancer (ISE), and intron splicing silencer (ISS) sequences as described ( [54] ,  [55]  and Z. Wang unpublished data). A score of -1 indicates that the minor allele destroys one  cis- element and +1 indicates that the minor allele creates one  cis -element. SNPs identified as unique to the AA or GG samples using sequence capture are shown in blue. Those identified in the HapMap database are depicted in black. The position of each intronic SNP relative to the nearest  ANRIL  exon is given under the ‘ ANRIL  Position’ column. Exonic SNPs in this column, list the exon in which they occur. If available, the minor allele frequency from Utah residents with ancestry from northern and western Europe (HapMap3, CEU) is given in the ‘CEU Minor Allele’ column. H- Hapmap3; GG- individuals homozygous for the ‘G’ allele at rs10757278.\n(0.59 MB TIF)\nClick here for additional data file.\nPrimers used for RACE, Taqman, and PCR analysis.\n(0.67 MB TIF)\nClick here for additional data file.","source_license":"CC-BY-4.0","license_restricted":false}