ANRIL expression and atherosclerosis severity at chromosome 9p21

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1 University Leipzig Institute of Laboratory Medicine, Clinical Chemistry and Molecular Genetics ANRIL expression and atherosclerosis severity at chromosome 9p21 Lesca Miriam Holdt, Frank Beutner, Markus Scholz, Stephan Gielen, Gabor Gäbel, Gerhard Schuler, Joachim Thiery, Daniel Teupser ESC Congress Stockholm, Sept. 1 st 2010

2 Chr9p21 Locus of Atherosclerosis Susceptibility

3 Genetic variation at Chr9p21? Atherosclerosis

4 No Effect on Atherosclerosis in Mice Deficient for Chr9p21 Syntenic Region (Mouse Chr4) ~50% sequence homology in syntenic region between mice and men Visel et al., Nature 2010

5 Leipzig-Heart Study Cross-sectional cohort study of patients undergoing first diagnostic coronary angiography Current analysis: n=1140 Recruitment: n=2061 (June 22, 2010) Goal: 4000 patients until 2012 Inclusion criteria: Assignment to coronary angiography with clinical signs of CAD or typical non-invasive diagnostic testing (ECG, exercise stress test) age: years Exclusion criteria: History of PCI, CABG Acute myocardial infarction (NSTEMI, STEMI) Severe valvular disease Significant co-morbidity (tumor etc.)

6 Determination of Atherosclerotic Burden Carotid Ultrasound (Plaque) Plaque CAD PAD Coronary Angiography (CAD) Ankle Brachial Index (PAD) *CAD Coronary artery disease *Plaque- in carotid arteries *PAD- Peripheral artery disease

7 Replication of Chr9p21 in the Leipzig-Heart Study P-Value OR 95% CI Plaque CAD LMCA PAD Plaque- Plaque in carotid artery CAD- coronary artery disease LMCA- Left main coronary artery disease PAD- Peripheral artery disease Holdt et al., ATVB 2010

8 Transcripts in the Chr9p21 Region MTAP CDKN2A/B ANRIL DQ EU NR_ Chr9p21 locus CEU ANRIL antisense non-coding RNA at the INK4 locus CDKN2A Cyclin dependent kinase inhibitor 2A CDKN2B Cyclin dependent kinase inhibitor 2B MTAP Methylthioadenosine phosphorylase

9 Does Chr9p21 influence RNA expression at the locus? Is RNA expression correlated with the extent of atherosclerosis?

10 Transcript Expression in PBMC of the Leipzig-Heart Study Blood drawn in CPT-Vacutainer (n=1140) Separation of mononuclear cells Quantitative RT-PCR Association of transcript expression with Chr9p21 genotype and atherosclerosis

11 ANRIL Expression in PBMC ANRIL Transcripts PBMC (n=1098) ANRIL NR ANRIL DQ ANRIL EU Holdt et al., ATVB 2010

12 Confirmation of Expression Differences ANRIL Transcripts PBMC (n=1098) ANRIL NR ANRIL DQ ANRIL EU Whole Blood (n=769)

13 ANRIL Expression and Atherosclerosis ANRIL NR ANRIL DQ ANRIL EU Holdt et al., ATVB 2010

14 ANRIL Expression in Human Plaques ANRIL NR ANRIL DQ ANRIL EU Holdt et al., ATVB 2010

15 Potential Mechanisms at Chr9p21 Cis regulation ANRIL Trans regulation ANRIL Independent regulation Neighbouring transcripts at Chr9p21 Neighbouring transcripts at Chr9p21? Atherosclerosis Atherosclerosis Atherosclerosis

16 Potential Mechanisms at Chr9p21 Cis regulation ANRIL Trans regulation ANRIL Independent regulation Neighbouring transcripts at Chr9p21 Neighbouring transcripts at Chr9p21? Atherosclerosis Atherosclerosis Atherosclerosis

17 Transcripts in the Chr9p21 Region MTAP CDKN2A/B ANRIL DQ EU NR_ Chr9p21 locus CEU ANRIL antisense non-coding RNA at the INK4 locus CDKN2A Cyclin dependent kinase inhibitor 2A CDKN2B Cyclin dependent kinase inhibitor 2B MTAP Methylthioadenosine phosphorylase

18 Expression of Chr9p21 Transcripts in Human Plaques Scale bar 50µm Holdt et al., Atherosclerosis; Epub June 23, 2010

19 Correlation of Gene Expression with TNFa and CD68 mrna Expression in Human Plaques Holdt et al., Atherosclerosis; Epub June 23, 2010

20 Copies p14 ARF / 10 7 copies BA Copies MTAP/ 10 7 copies BA Copies p15 INK4b / 10 7 copies BA Copies p16 INK4a / 10 7 copies BA Lack of Association of Protein-coding Gene Expression with 9p21 in Atherosclerotic Plaque CDKN2B (p15 INK4b ) CDKN2A (p16 INK4a ) A H G A H G CDKN2A (p14 ARF ) MTAP A H G A H G Holdt et al., Atherosclerosis; Epub June 23, 2010

21 Lack of Association of Protein-coding Gene Expression with 9p21 in the Leipzig-Heart Study PBMC (n=1098) Whole Blood (n=769) Gene Transcript Effect (%) P-value 95% CI CDKN2B p15 INK4b CDKN2A p16 INK4a 0.09 n.s CDKN2A p14 ARF 2.23 n.s MTAP MTAP n.s CDKN2B p15 INK4b n.s Holdt et al., ATVB 2010

22 Potential Mechanisms at Chr9p21 Cis regulation ANRIL Trans regulation ANRIL Independent regulation Neighbouring transcripts at Chr9p21 Neighbouring transcripts at Chr9p21? Atherosclerosis Atherosclerosis Atherosclerosis

23 Summary Chr9p21 is the strongest genetic marker of human atherosclerosis and could successfully be replicated in the Leipzig-Heart Study (n=1140, OR 1.31). The CAD risk haplotype was significantly associated with increased ANRIL NR and ANRIL EU expression in PBMC and whole blood. Increased ANRIL expression was associated with increased atherosclerosis. Neighbouring protein-coding genes were expressed in human atherosclerotic plaques as shown by immunohistochemical and mrna expression analyses. Expression of protein-coding genes was not correlated with the genotype at Chr9p21 nor with atherosclerosis severity. These data speak for an influence of ANRIL on atherosclerosis susceptibility through trans-regulation of so far not identified genes.

24 Institute of Clinical Chemistry, Laboratory Medicine and Molecular Genetics, University Hospital Leipzig Daniel Teupser Frank Beutner Annegret Schink Kristina Sass Claudia Gebhardt Franziska Jeromin Anika Stahringer Wolfgang Wilfert Joachim Thiery Leipzig Heart Centre, University Leipzig Stephan Gielen Gerhard Schuler Department of Visceral, Thoracic, and Vascular Surgery, University Hospital Carl- Gustav-Carus Dresden Gábor Gäbel Hendrik Bergert Institute for Medical Informatics, Statistics and Epidemiology, University Leipzig Markus Scholz

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