Coronary artery disease susceptibility loci extensively tested in the present Japanese study.

<p>A cororary artery disease (CAD) association study comprises two-tiered sample; the tier-1 was done in 1347 cases and 1.337 controls and the tier-2 done in 3,052 cases and 6.335 controls. Association results from the two tiers were combined by pooling the genotype counts.</p>a<p>rs735396 was genotyped in replacement for rs2259816 in the GWA study panel (<i>r2</i> = 1.000 to rs735396 in HapMap JPT+CHB). Rs735396 is in LD with rs1169300 (<i>r2</i> = 0.783 in HapMap JPT+CHB), which was tested for LDL-C association. The direction of association with CAD risk appears to be opposite to that for increased LDL-C in the <i>HNF1A</i> locus.</p>b<p>Following the previous meta-analysis of CAD association with ApoE genotype (Benett et al. JAMA 2007, ref. 35), CAD risk was compared between E3/E3 individuals and E2 carriers (excluding E2/E4).</p>c<p>Alleles are nominated as those in dbSNP Build 130 mapped on the strand of Human Genome Build 36.3.</p>d<p>Allele frequencies in the Japanese general population from GeMDBJ (<i>n</i> = 964) or HapMap JPT (<i>n</i> = 90; rs662799, rs2259816, rs2303790, and rs7412) or Amagasaki Study panel (rs429358).</p

Cross-population comparison of per-allele effect of SNPs associated with LDL-C (a), HDL-C (b), and TG (c) between the Japanese and European-descent populations.

<p>Effect alleles are defined as those that increase LDL-C or TG or that decrease HDL-C. The effects of each variant on lipid traits are shown by squares, colored in red (Japanese) and blue (Europeans). The gray dotted lines between the red and blue squares represent an identical locus. See details about the individual SNP loci in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385.s004" target="_blank">Table S3</a>.</p

Meta-analysis of CAD association with selected SNPs or variants, including the current and previously reported studies.

<p>Effect sizes of <i>SORT1</i> and <i>APOE</i> variants were heterogeneous between the current study and those previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385-Schunkert1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385-Bennet1" target="_blank">[35]</a>: <i>p</i> =  6.8×10⁻⁴ for <i>SORT1</i>, <i>p</i> = 1.7×10⁻³ for <i>APOE</i> (E2 carriers vs. E3/E3) and <i>p</i> = 0.041 for <i>APOE</i> (E4 carriers <i>vs.</i> E3/E3) by Woolf's test.</p

Correlation of effect sizes for CAD risk and 3 lipid traits–LDL-C (a), HDL-C (b), and TG (c)–at SNPs tested for replication in the current study.

<p>Genetic impacts on lipid level (β in <i>x</i>-axis) and CAD risk (OR in <i>y</i>-axis) are compared for the SNPs that were previously reported to associate with the corresponding (lead) lipid trait in Europeans: 18 SNPs for LDL-C (a), 20 SNPs for HDL-C (b), and 12 SNPs for TG (c), where 3 SNPs at <i>LPL</i> are included in both (b) and (c). The names of SNPs that were genotyped in the tier-2 CAD case-control study panel are denoted in the plots. For the purpose of readability, error bars are not shown at the individual SNP loci in the figure. See details about the individual SNP loci in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046385#pone.0046385.s003" target="_blank">Table S2</a>.</p

Baseline characteristics of participants in the present study.

<p>Plus–minus values are means ± SD.</p><p>Diabetes, hypertension, and dysipidemia were identified as risk factors on the basis of the meeting of diagnostic criteria or the receipt of treatment for these conditions (Note S1).</p><p>In the GWA-scanned panel, 414 individuals were from the Amagasaki Study panel; only the latter panel was included and analyzed in the current replication study.</p

Deletion of CDKAL1 Affects High-Fat Diet–Induced Fat Accumulation and Glucose-Stimulated Insulin Secretion in Mice, Indicating Relevance to Diabetes

<div><h3>Background/Objective</h3><p>The <em>CDKAL1</em> gene is among the best-replicated susceptibility loci for type 2 diabetes, originally identified by genome-wide association studies in humans. To clarify a physiological importance of CDKAL1, we examined effects of a global <em>Cdkal1</em>-null mutation in mice and also evaluated the influence of a <em>CDKAL1</em> risk allele on body mass index (BMI) in Japanese subjects.</p> <h3>Methods</h3><p>In <em>Cdkal1</em>-deficient (<em>Cdkal1</em>^−/−) mice, we performed oral glucose tolerance test, insulin tolerance test, and perfusion experiments with and without high-fat feeding. Based on the findings in mice, we tested genetic association of <em>CDKAL1</em> variants with BMI, as a measure of adiposity, and type 2 diabetes in Japanese.</p> <h3>Principal Findings</h3><p>On a standard diet, <em>Cdkal1</em>^−/− mice were modestly lighter in weight than wild-type littermates without major alterations in glucose metabolism. On a high fat diet, <em>Cdkal1</em>^−/− mice showed significant reduction in fat accumulation (17% reduction in %intraabdominal fat, <em>P</em> = 0.023 vs. wild-type littermates) with less impaired insulin sensitivity at an early stage. High fat feeding did not potentiate insulin secretion in <em>Cdkal1</em>^−/− mice (1.0-fold), contrary to the results in wild-type littermates (1.6-fold, <em>P</em><0.01). Inversely, at a later stage, <em>Cdkal1</em>^−/− mice showed more prominent impairment of insulin sensitivity and glucose tolerance. mRNA expression analysis indicated that <em>Scd1</em> might function as a critical mediator of the altered metabolism in <em>Cdkal1</em>^−/− mice. In accordance with the findings in mice, a nominally significant (<em>P</em><0.05) association between <em>CDKAL1</em> rs4712523 and BMI was replicated in 2 Japanese general populations comprising 5,695 and 12,569 samples; the risk allele for type 2 diabetes was also associated with decreased BMI.</p> <h3>Conclusions</h3><p><em>Cdkal1</em> gene deletion is accompanied by modestly impaired insulin secretion and longitudinal fluctuations in insulin sensitivity during high-fat feeding in mice. CDKAL1 may affect such compensatory mechanisms regulating glucose homeostasis through interaction with diet.</p> </div

Phenotypic characterization of Cdkal1-knockout mice with and without high-fat feeding.

<p>Values are means ± SEM. The number of animals (all males) in each group is shown in parentheses.</p><p>Mice were weaned at 4weeks (wks) of age. For the standard diet (STD) group, all mice were characterized at 30 wks of age. For the high fat diet (HFD) group, the diet was shifted from STD to HFD at 8 wks of age; i.e., mice in the 4 wks of HFD group were 12 wks of age.</p><p>Plasma levels of insulin, glucose, leptin, TNFa, and lipids were measured after 16-hr fast.</p

Cohort-wise BMI association of SNPs genotyped in two general Japanese populations.

<p>Effect sizes are indicated as beta per SD unit of phenotype.Two-tailed P values are shown in the table.</p><p>T2D-risk, fasting plasma glucose (FPG)-increasing and BMI-increasing alleles reported in the previous studies are tested.</p><p>When we arbitrarily categorized the samples into two age groups (age<60 years and age≥60 years), there was no significant inter-age-group difference in BMI association at <i>CDKAL1</i> rs4712523; P = 0.076, β = −2.66 for age<60 years; P = 0.006, β = −3.85 for age≥60 years in the combined samples.</p><p>Part of the samples (414 individuals in the Amagasaki Study panel) were included in the GWA meta-analysis of BMI (ref.35,36), where proxy SNPs (<i>r²</i>>0.78 in HapMap JPT+CHB) were tested for SNP-BMI acid association at <i>CDKAL1</i>. For the purpose of comprehensive evaluation, all the Amagasaki Study samples consecutively-enrolled are included in the present study.</p

Clinical characteristics of study participants.

<p>Values are means (SD) unless otherwise indicated.</p><p>All clinical assessments were performed using uniform standards in each population.</p><p>Blood samples were taken after ≥6 hours fast in the Amagasaki panel; without setting strict fasting condition in the Fukuoka panel.</p>a<p>Since the questionnaire did not differeniate the category of chance drinker from that of current drinker, the corresponding subjects are combined for the category of current drinker in the Fukuoka panel.</p>b<p>HbA1c was measured for 1,288 subjects in the Amagasaki panel; for all participants in the Fukuoka panel.</p>c<p>LDL cholesterol was calculated in the Amagasaki panel using the Friedewald formula, with missing values assigned to individuals with triglycerides >400 mg/dl. Since blood samples were taken without setting strict fasting condition, the values of LDL cholesterol and the prevalence of dyslipidemia are not shown for the Fukuoka panel.</p>d<p>Hypertension is defined when systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg, or taking antihypertensive medication. Diabetes is defined when fasting plasma glucose ≥7.0 mmol/l and/or HbA1c ≥6.5%, or taking blood glucose lowering medication. Dyslipidemia is defined according to the Japan Atherosclerosis Society Guidelines (Teramoto et al. J Atheroscler Thromb 14∶155–158, 2007).</p

Phenotype comparison between wild-type littermates (WT, <i>Cdkal1^+/+</i>) and <i>Cdkal1</i> knockout (KO, <i>Cdkal1</i>^−/−) mice.

<p><b>A:</b> Weight curves from animals on a standard diet [WT (<i>n</i> = 11) vs. KO (<i>n</i> = 12)]. <i>P</i> = 0.15; F (1, 16) = 2.3 by repeated measure ANOVA. <b>B:</b> Weight was measured after 4, 8, 16, and 20 weeks on a high fat diet [WT (<i>n</i> = 21–26) vs. KO (<i>n</i> = 19–25)]. <i>P</i> = 0.18; F (1, 3) = 1.9 by repeated measure ANOVA. <b>C:</b> Plasma glucose (mM) and insulin (ng/ml) were measured; these were used to calculate HOMA-IR [HOMA-IR = G × I (in ng/ml) × 1.16 = G × I (in µIU/ml)/22.5]. %intraabdominal and %subcutaneous fat was calculated by dividing intraabdominal and subcutaneous fat content, measured using CT scan, with body weight. *<i>P</i><0.05, **<i>P</i><0.01.</p