title: Genetic variants at the 9p21 locus contribute to atherosclerosis through modulation of ANRIL and CDKN2A/B.
abstract: Genome-wide association studies (GWAS) have identified genetic variants contributing to the risk of cardiovascular disease (CVD) at the chromosome 9p21 locus. The CVD-associated region is adjacent to the two cyclin dependent kinase inhibitors (CDKN)2A and 2B and the last exons of the non-coding RNA, ANRIL. It is still not clear which of or how these transcripts are involved in the pathogenesis of atherosclerosis.
  
  
  OBJECTIVE
  We assessed the hypothesis that 9p21 locus polymorphisms influence the expression of the transcripts in the region (ANRIL, CDKN2A/B) and that these transcripts contribute to atherogenesis through the modulation of proliferation in VSMC.
  
  
  METHODS
  We genotyped 18 SNPs (r(2)<0.8 and MAF>0.05) across the region of interest: CDKN2A/B and ANRIL, encompassing the CVD-associated region. RNA and DNA were extracted from the blood of 57 volunteers (69-72 years old). Carotid ultrasound was performed in 56 subjects. CDKN2A/B and ANRIL (exons 1-2 and 17-18) expression was measured employing RT-PCR. Gene expression and cell growth were evaluated in cultured VSMC after the siRNA-mediated knock-down of ANRIL.
  
  
  RESULTS
  The risk alleles for atherosclerosis-related phenotypes were consistently associated with a lower expression of ANRIL when evaluating exons 1-2. Common carotid artery stenosis was associated with a significantly lower (P<0.01) expression of ANRIL (exons 1-2). ANRIL knock-down in VSMC caused significant variation in expression of CDKN2A/B (P<0.05) and reduction of cell growth (P<0.05) in vitro.
  
  
  CONCLUSIONS
  Disease-associated SNPs at the 9p21 locus predominantly affect the expression of ANRIL. Overall, our results suggest that several CVD-associated SNPs in the 9p21 locus affect the expression of ANRIL, which, in turn modulate cell growth, possibly via CDKN2A/B regulation.
authors.affiliation: Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Japan.
authors.forename: Ada
authors.initials: A
authors.lastname: Congrains
authors.forename: Kei
authors.initials: K
authors.lastname: Kamide
authors.forename: Ryousuke
authors.initials: R
authors.lastname: Oguro
authors.forename: Osamu
authors.initials: O
authors.lastname: Yasuda
authors.forename: Keishi
authors.initials: K
authors.lastname: Miyata
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Yamamoto
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kawai
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kusunoki
authors.forename: Hiroko
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Takeya
authors.forename: Koichi
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Miyuki
authors.initials: M
authors.lastname: Onishi
authors.forename: Ken
authors.initials: K
authors.lastname: Sugimoto
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Katsuya
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Awata
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebe
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Gondo
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Oike
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ohishi
authors.forename: Hiromi
authors.initials: H
authors.lastname: Rakugi
mesh_terms: D000368:Aged; D000818:Animals; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D050197:Atherosclerosis / Q000000981:diagnostic imaging / Q000235:genetics* / Q000473:pathology; D017536:Carotid Artery, Common / Q000000981:diagnostic imaging / Q000378:metabolism; D059168:Carotid Intima-Media Thickness; D016893:Carotid Stenosis / Q000000981:diagnostic imaging / Q000235:genetics* / Q000473:pathology; D049109:Cell Proliferation; D002478:Cells, Cultured; D002899:Chromosomes, Human, Pair 9; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000235:genetics* / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D006801:Humans; D007564:Japan; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D034622:RNA Interference; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000235:genetics* / Q000378:metabolism
chemical_list: D001057:Apolipoproteins E; C561518:CDKN2B antisense RNA, human; C493966:CDKN2B protein, human; C417743:Cdkn2a protein, mouse; C493968:Cdkn2b protein, mouse; D050762:Cyclin-Dependent Kinase Inhibitor p15; D019941:Cyclin-Dependent Kinase Inhibitor p16; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated