The Effect of 9p21.3 Coronary Artery Disease Locus ... - Circulation

DOI: 10.1161/CIRCULATIONAHA.111.064881

The Effect of 9p21.3 Coronary Artery Disease Locus Neighboring Genes on Atherosclerosis in Mice Running title; Kim et al.; Effect of 9p21.3 genes on atherosclerosis

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Juyong Brian Kim, MD, MPH1*; Andres Deluna, MD, MS2†; Imran N. Mungrue, PhD1‡; Christine Vu, BS2; Delila Pouldar, BS1; Mete Civelek, PhD1; Luz Orozco, PhD3; Judy Wu, BS1; Xuping Wang, BS1; Sarada Charugundla, BS1; Lawrence W. Castellani, PhD1; Marta Rusek, BS4; Hieronim Jakobowski, PhD4; Aldons J. Lusis, PhD1,2,3 1

Div of Cardiology, Dept of Medicine; 2Dept of Microbiology, Immunology, and Molecular Genetics; 3Dept of Human Genetics, David Geffen School of Medicine, University ty y of of California, Cali Ca lifo forn rnia Los Angeles, Los Angeles, CA; 4Dept of Microbiology and Molecular Genetics, Genetics cs,, UMDNJ-New UMDN UM DNJDN J-Ne JNew Jersey Medical School, Newark, NJ *Present *P Pre rese sent se nt aaddress: d reess:: Di dd D Div v of Cardiovascular Medicine Medicine, nee, D Dept ept of Medicine Medicine, ne, St Stanford tanfo anfo ford Univ, Palo Alto, CA † ‡

Presentt ad addr address: dres dr ess: D Div iv ooff Ca Card Cardiology, rdiiolo rd iolo logy g ,D gy Dept eptt ooff Me M Medicine, diccinee, Un Univ iv ooff Te T Texas xass Sou xa S Southwestern, outhw wes esttern tern rn, Da Dal Dallas, ll s, TX llas

Present P res esent addr address: drres e s: D Dept ept of Pha ep Pharmacology armac rmac a olog ogy og y & Experimental Exp Ex perrime riment ntaal Therapeutics, The hera rap peuticcs, Sch ooff Me peu Medicine, ediici cine n , Lou L Louisiana ouisiian anaa St State tate U Un iver iv e si sity ty-H ty -Hea ealt ea ltth Sc S ienc ie nces nc es C trr, Ne New w Or Orle leean a s, L A University-Health Sciences Ctr, Orleans, LA

Address Addr Ad dres esss for for Correspondence: Corr Co rres espo pond nden ence ce:: Aldons J. Lusis, Ph.D. Departments of Microbiology, Medicine and Human Genetics UCLA School of Medicine 695 Charles E. Young Drive South MRL 3730 Tel: (310) 825-1359 Fax: (310) 794-7345 E-mail: [email protected] Journal Subject Codes: [4] Acute myocardial infarction; [7] Chronic ischemic heart disease; [134] Pathophysiology; [135] Risk Factors; [143] Gene regulation; [145] Genetically altered mice; [89] Genetics of cardiovascular disease

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Abstract:

Background - The human 9p21.3 chromosome locus has been shown to be an independent risk factor for atherosclerosis in multiple large scale genome-wide association studies, but the underlying mechanism remains unknown. We set out to investigate the potential role of the 9p21.3 locus neighboring genes, including Mtap, the two isoforms of Cdkn2a, p16Ink4a and p19Arf, and Cdkn2b in atherosclerosis using knockout mice models. Methods and Results - Gene targeted mice for neighboring genes, including Mtap, Cdkn2a, Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

p19Arf, and Cdkn2b, were each bred to mice carrying the human APO*E3 Leiden transgene which sensitizes the mice for atherosclerotic lesions through elevated plasma ch cholesterol. hol oles este es tero te rol. ro l We l. found that the mice heterozygous for Mtap developed larger lesion compared to wild-type mice (49623±21650 (Mean±SD); with 496 9623 23±2 23 ±216 ±2 1 50 16 5 vs. vs. 18899±9604 18899±9604 μm2/section (M Mea ean± n SD); p=0.01), w itth si ssimilar milar morphology as wild The wild d type mice. mice mi ce.. T he Mtap Mtap heterozygous het eter eroz er o yg oz ygou o s mice ou micee demonstrated dem monst sttraated changes cha hang ng ges es in in metabolic meta taabo boli licc and li a d an methylation CD4+ The meeth hyl y at a ionn pr pprofiles offiles es aand ndd CD D4+ ce D4+ cell lll ccounts. ounts. nts. T he Cdkn2a he Cdkn Cd kn2a 2a knockout knocckoout mice micce had had d smaller sma m lller lesions lessio ons compared significant differences comp co mpar mp ared ar ed to to wild-type wild wi ld-t -typ typ ypee and and heterozygous hete he tero te rozy ro zygo zy gous go us mice mic icee and and there ther th eree were er were nno o si sign gnif gn ific if ican ic antt di an diff ffer ff eren er ence en cess in llesion ce esio es ionn io size in p19Arf and Cdkn2b mutants as compared to wild type. We observed extensive, tissuespecific compensatory regulation of the Cdkn2a and Cdkn2b genes among the various knockout mice, making the effects on atherosclerosis difficult to interpret. Conclusions - Mtap plays a protective role against atherosclerosis, whereas Cdkn2a appears to be modestly proatherogenic. However, no relation was found between the 9p21 genotype and the transcription of 9p21 neighboring genes in primary human aortic vascular cells in vitro. There is extensive compensatory regulation in the highly conserved 9p21 orthologous region in mice. Key words: atherosclerosis; coronary artery disease; 9p21 risk locus; Gene targeted mice; Methylthioadenosine phosphorylase 2


The 9p21.3 region of the genome has been identified as the locus with strongest association to coronary artery disease (CAD) and myocardial infarction (MI) in multiple independent large scale genome-wide association studies (GWAS).1-3 The locus is within a 58kb region that is devoid of protein coding genes, suggestive of a regulatory function (Figure 1). Interestingly, the neighboring genes in the region include well-known tumor suppressor genes, including CDKN2A and CDKN2B.4-6 The CDKN2A locus encodes a cyclin-dependent protein kinase (CDK) inhibitory protein (CKI), p16INK4A, and a p53-regulatory protein, p19ARF. The Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

CDKN2B gene encodes another CKI, p15INK4B. Another gene in the region is methylthioadenosine phosphorylase (MTAP), which encodes a ubiquitously expressed metabolic enzyme S-methyl-5’-thioadenosine phosphorylase7 that processes the polyaminee bi bios biosynthesis o yn ynth thes th esiis es is byproduct in the methionine salvage pathway. Loss or inactivation of MTAP has frequently be been een observed obs bser erve er v d inn a number number of different human tumors, tum umoors, and it has been um b en be n shown shown ho to have a tumor suppressive uppressive ppp role ro ole inn a mice m ce model. mi mod odel el.8 Mu Multiple ult ltip iple ip le studies stu tu udi dies es demonstrated dem emon o st on stra rate ra tedd a potential te pote po tent te nttia i l role ro ole l for for ccell elll cy ccycle clee re cl rregulatory gulato gul lato ory m mechanisms echannism ech ni ms iin n atherosclerosi atherosclerosis siis pr pprogression. ogre og ress re ssio ss i n. Pr P Previously, evio ev ious io u ly y, th thee ma mast master stter ttumor um mor ssuppressor uppr up pres pr e so sorr ge gene ne pp53 53 3 hhas as bbeen e n ee implicated in the development of atherosclerosis in apolipoprotein E (ApoE)-null mice9, 10, affecting both cell proliferation and apoptosis within the atheroma. Another tumor suppressor gene, p21Waf1, was also shown to increase the atheroma size in ApoE-null mice11, whereas the tumor suppressor p27Kip1 was shown to protect against atherosclerosis.12 Correlations of the 9p21 locus SNP genotype to differential expression of the neighboring genes have been observed in several studies with inconsistent findings.13-15 A knockout (KO) mouse model involving the entire region orthologous to the 9p21.3 CAD locus showed significant decreases in the expressed levels of Cdkn2a and Cdkn2b, and increased proliferation

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of primary smooth muscle cells (SMC) and mouse embryonic fibroblasts (MEF), although an effect on atherosclerosis in vivo was not demonstrated.16 Mice deficient in the p19Arf gene were found to have increased atherosclerotic lesions in an ApoE null background with significant attenuation of apoptosis in lesions as well as in cultured primary macrophages and vascular smooth muscle cells.17 However, to date no observation regarding atherosclerotic phenotype has been made involving the other neighboring genes. We set out to survey the 9p21.3 orthologous region using knockout mice models to Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

systematically address the role of the neighboring protein-coding genes in atherosclerosis. We chose the APOE*3 Leiden sensitizing model because it is dominant, simplifying the construction of the models, and also because it exhibits relatively modest elevations of choles stero tero ol,, m oree or cholesterol, more ealistically modeling the human disease than other widely used models. realistically

Me Met th thods Methods De eta t il iled e m ed ethhod et hods ccan an bee fo foun un nd in tthe he ssupplemental he up ppl plem emen em e tal en tal mate m ateeri riaal. Pr P imer imer erss uused seed ed ffor o tthe or hee ggenotyping enootyypin en ypin ng Detailed methods found material. Primers and qPCR ass sssay ayss ar aree li llisted s ed st d iin n su upp p le leme ment me ntal nt al table tab a le 1. 1. assays supplemental Mice All mouse protocols were approved by the UCLA Animal Review Committee. APOE*3-Leiden transgenic mice were maintained on a C57BL/6 background were obtained from TNO (Leiden, Netherlands)18 and re-derived at the UCLA Division of Laboratory Animal Medicine. The Cdkn2a KO mice4, and the p19Arf KO mice5 were obtained from the National Cancer Institute (NCI) Mice Repository, and re-derived at UCLA. The Cdkn2a KO mice were generated by targeted knock-out of the exons 2 and 3 of the Cdkn2a gene, which are shared by both isoforms of the Cdkn2a gene, p16Ink4a and p19Arf. For the p19Arf KO mice, the alternate reading frame

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of p19Arf gene was selectively mutated and hence the expression of p16Ink4a isoform was maintained (Figure 1). Both strains were created on a mixed background of 129/Sv and C57BL/6 then backcrossed to the C57BL/6 for more than 5 generations. The Cdkn2b KO mice were a generous gift from the Licia Selleri’s lab at Cornell University. They were originally derived from the Barbacid lab in Spain.6 For the Cdkn2b KO mice, the second exon of Cdkn2b gene was replaced with a Neor cassette using 129/Sv DNA then transfected to CJ7 ES cells (Figure 1). The ES cells were then injected into C57BL/6 Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

blastocysts and subsequently bred to C57BL/6 mice for more than 5 generations. The KO mice for each strain were initially bred to an APOE*3 Leiden mice to generate F1 mice heterozygous for the mutation. F1 mice were mated with each other whe heeree one n ooff th ne thee where pair carried the Leiden transgene. The F2 generation resulted in homozygous knock-out (KO), wild wi ld d-t -tyype ype mice micce (WT), mi (WT WT), and heterozygous mice (Het). (H Het). Only femalee mice micce carrying ca wild-type the APOE*3 L eid den transgene transgeenee were wer eree selected sele sele lect cted ct ed fo or th tthee aatherosclerosis therrossclero rosis st ro tud udyy. Leiden for study. Gt(RRK081)Byg G t(RRK081 RRK 1)By ) g Th M app hheterozygous eteero et erozyg ozy ou ouss mi mice ce w erre dderived eri rive ri v d at ve at UCLA UCL CLA A with w th wi h ES ES cells cellls (Mtap (Mt Mtap apGt(R ) Thee Mt Mtap were

obtained from m th he Mu M tant ta n M o se R ou e io eg ona nall Re Reso s ur so urce ce C ente en terr (M te (MMR MRRC MR RC)) at U RC CD avis av is. Briefly, a is the Mutant Mouse Regional Resource Center (MMRRC) UC Davis. gene-trap vector encoding the En2 splice acceptor site fused to ȕ-galactosidase/neo fusion gene (ȕ-geo) was inserted between exon 3 and 4 of the mouse MTAP locus. These mice were maintained on a CBA/Ca background. Mice that are homozygous for the MTAP mutation are embryonic lethal, and hence the heterozygotes were mated with APOE*3 Leiden mice, and resulting wildtype and heterozygote female mice carrying the APOE*3 Leiden transgene were selected for the atherosclerosis study. Diet A custom diet consisting of 1% cholesterol and 33kcal% fat from mostly cocoa butter was

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prepared from Research Diets, Inc (product #D10042101). The mice were put on diet between 6 to 8 weeks of age, then kept on the diet for 16 weeks prior to being sacrificed for specimen collection. Global metabolic profiling assay 100ug of freshly extracted liver tissue was flash frozen and sent to Metabolon, Inc. (Durham, NC) for extraction and analysis.19 The platform for sample analysis has been described in detail.20 Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

Global methylation pattern analysis We obtained genomic DNA from liver tissue from Mtap Het and WT male mice of 332-weeks 2 we week ekss of age and used Reduced Representation Bisulfite Sequencing (RRBS) to examinee ap approximately ppr prox oxim ox imat im atel at elyy el 1% of the genome, comprised off sequences enriched in CpG.21 To determine sites that were difffe di fferen erential ally ly y met ethy hyla l te tedd be betw wee eenn th he tw twoo sa ssamples, mple mp les,, we co cons nstruccte tedd a co conf nfiden ence ce iinterval n errvaal fo nt fforr th the differentially methylated between the constructed confidence percent ppeerccent methylation methyl y attio on level leeveel off each each site site using using the thhe binomial biinom inomia iaal distribution disttribu di utiion (binofit (bi bino noffitt in MATLAB).We MATLA LAB).. We called callled ssites ites it es as di diff differentially ffer ff eren er enti en tial ti ally al ly m methylated ethy et hyla hy late t d be betw between tweeen tw een th thee tw twoo samp ssamples amp mple less if tthe he ppercent ercent erce nt m methylation ethy et hyyla lati tion ti on llevel eveel ev of o eeach acch sample s mp sa mple l was le was outside out utsi side de of of th thee 95% 9 % confidence 95 conf co nffid den ence ce interval. int nter erva val. l. Statistical Analysis Two-group comparisons were made using Welch’s modified t-test if met normality by the Kolmogorov-Smirnov test and further evaluated by both bootstrapping and permutations22 at the alpha=0.05 level if the Welch’s test was significant (See supplemental methods). All threegroup comparisons were performed using the non-parametric Kruskal-Wallis ANOVA test, followed by a pairwise comparison using Dunn’s post test if found to have a significant KruskalWallis test.

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Results Baseline characteristics of mice The baseline characteristics, including age, weight, fasting plasma lipids, fasting glucose, and body weight composition, are shown in Table 1 for all groups from each strain studied. There were no statistically significant differences in weight, lipid profile, and body composition (by nuclear magnetic resonance, NMR) results between the groups within each strain. A significant difference was observed in the fasting glucose levels in the Cdkn2a and p19Arf mutants as Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

compared to their respective wild-type groups (P value 0.001 and 0.007, respectively). Effect of Mtap gene modulation on atherosclerosis Mtap WT and Mtap Het mice on an APOE*3 Leiden background were compared ed d ffor o aaortic or orti or ticc ti lesion esion development and other characteristics (Mtap KO are not viable). No significant differences (Figure di ifffer eren ence en cees in the hee baseline baseline weight or lipid levelss were we observed (Fi F gu Fi ure 22), ), and other baseline characteristics composition, were ch harac arra teristicss including incllud in din i g fasting fast fa stin st ingg glucose, in gluc gl ucoose, insulin uc inssullin llevels evelss aand nd bbody odyy ffat od at co ompossit itio ion, n, w eree al er also so comparable Mtap mRNA expression was co omp mpar arab ar able lee ((Table Tab Ta blee 11). ). As ). A expected exp xpec xp ecte ec tedd the te th he Mt Mta ap m ap RNA RN A ex xpr preessi sion si on n llevel evel ev ell w as aapproximately ppro pp roxi xima xi mattelly ma ly hhalved alvved ve inn the heterozygous heteroz ozyg ygou yg ouus mice m ce mi c compared com ompaare redd to to the the wild-type w ld wi ld-t -typ ypee mice. yp mice mi ce.. A significant ce sign g iffic ican antt increase an incr in c ea cr ease se in in the t e aortic th sinus lesion size was observed in the heterozygotes compared to the Mtap WT mice (Figure 2). No significant difference in the expression level of the two Cdkn2a isoforms, p16Ink4a and p19Arf, or Cdkn2b was seen between the two groups (Supplemental Figure 1). The sections obtained from the aortic sinus were subjected to immuno-staining with antibodies against CD68. Staining showed the lesions to contain >90% CD68+ cells in both Mtap WT and Mtap Het mice (Figure 2). Minority of cells in the lesions stained for SM-Actin and Mason's trichrome and there was no difference in the staining pattern between the WT and Het mice (data not shown).

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MTAP deficiency affects metabolic pathways, global methylation dosage and peripheral Tcell composition We examined the global metabolomics profiles of liver samples from a group of Mtap Het mice compared to WT controls (n=3 each), using the Metabolon, Inc platform. Supplemental Table 2 lists the metabolites assayed in these samples, and raw expression values in each sample. Mtap Het mice generally had trends toward lower levels of metabolites involved in the methionine processing pathway including S-adenosylmethionine (SAM), SDownloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

adenosylhomocysteine (SAH), 5-methythioadenosine (MTA). Plasmas from Mtap WT and Mtap Het mice were assessed for total plasma homocysteine levels. However, no significant difference was found between the two groups (WT 4.64r0.55PM vs. Het 4.43r0.45PM, 4.43r0. 0..455PM, MeanrSD). Interestingly, taurine, a cysteine metabolite was significantly decreased (0.6 fold) in the Het mice he Mt Mtap a H e mic et icee co ccompared mpared to WT mice. Given th thee role role le off MTA MT TA as a a major majoor methyl metthyl donor, dono or, r, w wee com ccompared ompa mpared ed th the he glo gglobal lobal obal m methylation e hyla et lati tioon patt pa pattern ttter ernn between betw be twee tw eenn the the WT and and n Het Hett mice mice ice at a single-base sin ingl glegl e-ba ebaase m methylome ethy et hylo hy lome lo me llevel. eveel. ev el Th T There eree wa er wass a sign ssignificant ign gnif ifiican if ican nt genome wide methylation levels difference in n th thee ge geno nome no m w me idee % me id meth th hyl y at atio io on le leve vels ve ls ---- 55 5565 65 ssites ites it es hhad ad ssignificant igni ig nifi ni f caant ddifference fi iffe if ference in fe methylation level on binomial distribution test, with over 2000 sites showing greater than 20% change in methylation levels (Supplemental Figure 2). We also compared complete blood counts using the HemaTrue Hematology analyzer device (Heska) in Mtap-het compared to controls (n=3 vs. 3). We noted no changes in counts of red blood cells, hemoglobin content, platelets, and white blood cells (WBC), including lymphocytes, monocytes, and granulocytes (Data not shown). Further analysis of the WBC population using FACS analysis of the peripheral blood showed that there was a significant decrease in the level of CD4+ T-cells in the MTAP Het mice compared to the WT mice

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(11.8r4.0 vs. 32.9r13.2 %CD4+CD3+ leukocytes, p20 fold) in its absence as previously reported17, but also an increase in Cdkn2b (>3 fold). Given the complexity inherent in this locus and modulation of the different neighboring genes, the effect on atherosclerosis of these individual genes is difficult to interpret. By inference, these findings suggest a mechanism to protect the critical function of this region. The high functional redundancy in this locus also points to the likelihood that the 9p21 risk locus Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

affects a cis-regulatory element of this region that is critical for compensatory mechanism activated in response to stress. ntial allly y iinvolve nvol nv olve ol v ve The mechanism of this compensatory regulation is unclear, but cann poten potentially changes in methylation patterns, histone modification, and common binding motif to a ran nsc scri ript ptio tio ionn fa ffactor. cttor or.. It is interesting that the mu uta tatiion status of the hee Cdkn2a, Cddkn kn22a, p19Arf, f and Cdkn2b transcription mutation gennes ne affected d tthe he Mtap Mtaap Mt ap ggene enee expr en eexpression xprresssi sion on n lev vells ass w ell, l, w hereas th her he vice he vice vversa errsa sa is is not not true. trruee. The The he genes levels well, whereas the promoter prom pr omot om oter ot er rregion egiion eg ion off Mtap Mtap p is is relatively reela lati tive ti velly ve ly distant dissta tannt nt from fro rom m tthe he pr prom promoter omotter om e rregions eg gio ionns ns ooff Cdkn2a Cdkn Cd k 2a and kn and d Cdkn2b, Cdkn Cdkn n2b, b, aand nd tthis hiss ma hi mayy su ugg g es est a transtrran anss- effect effe ef fect fe ct such succh as a ttranscription ransscr ra crip ipti ip tion ti on ffactor acto ac torr me to medi d ated over r at about 200 kkb, suggest mediated overexpression as a more likely mechanism of this modulation. There were no statistically significant differences in the Cdkn2a, Cdkn2b genes between Mtap wild type and heterozygotes, which allowed a direct comparison of the effect of Mtap on atherosclerosis. In summary, we identified the MTAP gene as a novel regulator of atherosclerotic lesion development potentially by modulating the methionine and cysteine processing pathways, the Tlymphocytes, and the global methylation pattern. We also observed significant compensatory regulation of this region, which corroborates previous findings of dense regulatory components inherent to this locus.47 Because of this compensatory regulation, the effects of the other genes in

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this locus, Cdkn2a, p19Arf, and Cdkn2b, were difficult to assess. However, despite this regulation, an effect of Cdkn2a on atherosclerosis was observed. Further studies are needed to define the regulation of the 9p21 genes, and the mechanisms by which these genes modulate an effect on atherosclerosis.

Funding Sources: The project was funded by the NIH grants HL095154, HL030568 (to A.J.L.) and HL094709-03 (to I.N.M.). Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

Facility that is supported by National Institutes of Health awards CA-16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA Conflict of Interest Disclosures: None. Refe fere renc re nces nc es:: es References: 1.. M c herson cP on R P rtsseml rt mlid idis diss A Kava vasl va slar sl ar N Ste t wa w rt A, A, Roberts Robe Ro b rt be rtss R, C oxx D R, H inds in d D ds A, A, McPherson R,, Pe Pertsemlidis A,, Ka Kavaslar N,, Stewart Cox DR, Hinds DA, Pennacchio P Pe nn nnacchio LA, Tybjaerg-Hansen Tybja jaaerrg-H Hansen Han n A, A, Folsom Fo m AR, AR R, Boerwinkle Boer erwi winnklle E, wi E, Hobbs Hobbbs HH, HH H, Cohen Cohe henn JC. he JC C. A common co omm mon o all allele lel elee oon n cchromosome hrom hr om mosoome 9 associated asso as soci ciaated ed d with wit ithh coronary coroona nary ry hea heart eart rtt ddisease. isease se.. Science. Sccieenc ncee. 2007;316:1488-1491. 2007 20 07;3 07 ;316 ;3 16:1 16 :148 :1 4888--14 1491 91.. 91 Erdmann Hall Hengstenberg C,, Ma Mangino Mayer B,, Di Dixon RJ,, Meitinge Meitinger 2. Samani NJ NJ, Er Erdm dman dm annn J, H an a l AS al AS, He Heng ngst sten st enbe en berg be r C rg Mang ngin inoo M, in M, M ayer ay er B D xo on RJ R geer T, Braund P, Wichmann HE, Barrett JH, Konig IR, Stevens SE, Szymczak S, Tregouet DA, Iles MM, Pahlke F, Pollard H, Lieb W, Cambien F, Fischer M, Ouwehand W, Blankenberg S, Balmforth AJ, Baessler A, Ball SG, Strom TM, Braenne I, Gieger C, Deloukas P, Tobin MD, Ziegler A, Thompson JR, Schunkert H. Genomewide association analysis of coronary artery disease. N Engl J Med. 2007;357:443-453. 3. Helgadottir A, Thorleifsson G, Manolescu A, Gretarsdottir S, Blondal T, Jonasdottir A, Sigurdsson A, Baker A, Palsson A, Masson G, Gudbjartsson DF, Magnusson KP, Andersen K, Levey AI, Backman VM, Matthiasdottir S, Jonsdottir T, Palsson S, Einarsdottir H, Gunnarsdottir S, Gylfason A, Vaccarino V, Hooper WC, Reilly MP, Granger CB, Austin H, Rader DJ, Shah SH, Quyyumi AA, Gulcher JR, Thorgeirsson G, Thorsteinsdottir U, Kong A, Stefansson K. A common variant on chromosome 9p21 affects the risk of myocardial infarction. Science. 2007;316:1491-1493. 4. Serrano M, Lee H, Chin L, Cordon-Cardo C, Beach D, DePinho RA. Role of the ink4a locus in tumor suppression and cell mortality. Cell. 1996;85:27-37.

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Table 1. Baseline Characteristics of All Strains. Downloaded from http://circ.ahajournals.org/ by guest on November 5, 2017

Mtap Variables N Age, median (wks) Weight (gm)

Cdkn2a

p19Arf

Cdkn2b

WT

Het

WT

Het

KO

WT

Het

KO

WT

Het

KO

5

7

11

16

6

8

11

9

9

15

13

23.1

23.1

21.7

22.3

23

23.3

22.7

24.9

22.6

22.3

22.0

23.7 ±1.5

23.2 ±1.7

21.7

21.9

21.8

24.6

24.8

23.8

22.4

20.6

21.1

Lipids (mg/dL) Total cholesterol LDL

954±384 1013.3±148 584±350 821±360 884 ±147 334±350

603±68 508±68

385±249 276±243

663±326 525±324

540±264 417±296

718±344 330±139 330 30±1 ± 39 464±108 ±1 464± 46 4±10 4± 108 10 8 445 4445±112 455 587±319 220±123 358±115 342±107 342

72±12

60±5

66±7

61±8

53±12

64±9

59±12

71±20

63±8

82±12

74±16 74

TG

303±187 30 03± 3 1887

349±142

310±130

167±73

277±35 277 777±3 ±35 5

371±143

319±147

297±251 297± 29 7 251

109±71

119±33

145±32 145

UC

3343±158 34 3±15 158 8

387±64

180±114

209±37

123±88 12 23± ±888

294±158

215±107 215±1 ± 07 ±1

2257±152 57±152 57

85±48

130±38

127±40 127

FFA

49±11 49 49±1 ±1 11

72±24 72 72±2 ±24 ±2 4

54±9 54 ± ±9

553±8 3 8 3±

49±6 49 4 ± ±6

449±11 9±11 9± 11

57±7 57± 7±7 7

44±10 44 4 4±10 0

41±16 41±1 41 ±16 ±1 6

50±10 50±1 0±1 ±10 0

56±15 56

Glucose ose os s

194±78 194± 78

2222±45 2 ±4 22 ± 5

133±24 13 1 3±24 24

1182±28 82±28 2

147*±28 14 1 7*±2 ± 8 ±2

148 14 48 ±4 ±41 1

±50 197 9 ±5 ± 0

118*±37 11 1 8*±3 ±37 37

120±42 12 0± ±422

1127±31 27±31 27 1

141±39 141

Insulin n

160±52 160± 16 52 52

128±51 128 2 ±5 ± 1

-

-

-

-

-

-

-

-

-

2.2±0.5 2.2± 2±0 2± 0.5 5

22.0±0.5 .0± 0±0 0± 0.5 5

22.1±0.7 .1 1

HDL

Weight Body Bo dy W eigh ei ghtt Composition gh Comp mpos pos osit itiion it ion by NMR NMR (gm) (gm gm) m) Fat

33.3±0.6 .3± 3±0 3± 0.6 6

33.2±0.8 .2± 2±0 2± 0.8 8

44.2±0.9 .2± 2±0 2± 0.9 9

44.5±1.1 .5± 5±1 5± 1.1 1

44.4±1.0 .4± 4±1 4± 1.0 0

66.0±1.1 .0± 0±1 0± 1.1 1

66.8±1.5 .8± 8±1 8± 1.5 5

55.3±3.5 .3± 3±3 3± 3.5 5

Muscle le

221.7±1.7 21 .77±1.77

221.6±1.7 1.6± 6±1. 17

19.2±0.8 19 1 .2±0 2±0.88

119.5±1.1 9.5± 5±1. 5± 11

19.2±1.7 19 1 9.2± 2 1.7 7

20.9±1.4 20 0.9 9±1. 14

220.9±1.5 20 .9±1.5 15

14.7±0.7 119.9±3.1 9.9± 9±3. 9± 3 1 15.2±1.4 15.2±1 2±1 1.44 14.9±1.0 14 .7 .7

Free water

0.4±0.5

0.5±0.5

0.4±0.2

0.5±0.2

0.6±0.3

0.4±0.3

0.4±0.2

0.8±0.7

0.5±0.2

0.4±0.2

0.4±0.2

0.13

0.13

0.18

0.18

0.18

0.22

0.24

0.19

0.21

0.21

0.23

%Fat

Mean ± SD for each box unless otherwise mentioned; LDL was calculated based as LDL § TC- HDL - TG/5. Welch’s modified t-test was used to compare the MTAP group; 1-way ANOVA with Kruskal-Wallis test was used to compare all other groups. Insulin levels were only measured for the MTAP mice. *A significant difference existed between the three groups (p