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Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice


Sequence polymorphisms in a 58-kilobase (kb) interval on chromosome 9p21 confer a markedly increased risk of coronary artery disease (CAD), the leading cause of death worldwide1,2. The variants have a substantial effect on the epidemiology of CAD and other life-threatening vascular conditions because nearly one-quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein-coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70-kb non-coding interval on mouse chromosome 4 affects cardiac expression of neighbouring genes, as well as proliferation properties of vascular cells. Chr4Δ70kb/Δ70kb mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the non-coding interval, Cdkn2a and Cdkn2b, is severely reduced in chr4Δ70kb/Δ70kb mice, indicating that distant-acting gene regulatory functions are located in the non-coding CAD risk interval. Allele-specific expression of Cdkn2b transcripts in heterozygous mice showed that the deletion affects expression through a cis-acting mechanism. Primary cultures of chr4Δ70kb/Δ70kb aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval has a pivotal role in regulation of cardiac Cdkn2a/b expression, and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation.

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Figure 1: Deletion of the non-coding region orthologous to the 58-kb CAD risk interval on human chromosome 9p21.
Figure 2: Deletion of the CAD risk interval affects expression of neighbouring genes Cdkn2a and Cdkn2b.
Figure 3: Deletion of the CAD risk interval affects gene expression through a cis -regulatory mechanism.
Figure 4: Deletion of the CAD risk interval disrupts normal dynamics of cellular proliferation and senescence.

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We thank G. Owens for critical suggestions and discussion, T. Ley for providing vector pTURBO-Cre, and D. Lee for help with plasma lipid analysis. L.A.P., E.M.R. and J.C.C. were supported by the National Heart, Lung, and Blood Institute, D.M. and C.A. by EMBO long-term fellowships and L.A.P. by the National Human Genome Research Institute. Research was conducted at the E.O. Lawrence Berkeley National Laboratory and performed under Department of Energy Contract DE-AC02-05CH11231, University of California. Plasma lipid analysis at the University of Cincinnati Mouse Metabolic Phenotyping Center was supported by MMPC DK59630. All animal work was reviewed and approved by the LBNL Animal Welfare and Research Committee.

Author Contributions A.V. and L.A.P. wrote the manuscript. All authors contributed to data collection and analysis and provided comments on the manuscript.

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Correspondence to Len A. Pennacchio.

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Visel, A., Zhu, Y., May, D. et al. Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice. Nature 464, 409–412 (2010).

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