Genomewide association analysis of coronary artery disease

Background - Modern genotyping platforms permit a systematic search for inherited components of complex diseases. We performed a joint analysis of two genomewide association studies of coronary artery disease. Methods - We first identified chromosomal loci that were strongly associated with coronary artery disease in the Wellcome Trust Case Control Consortium (WTCCC) study (which involved 1926 case subjects with coronary artery disease and 2938 controls) and looked for replication in the German MI [Myocardial Infarction] Family Study (which involved 875 case subjects with myocardial infarction and 1644 controls). Data on other single-nucleotide polymorphisms (SNPs) that were significantly associated with coronary artery disease in either study (P<0.001) were then combined to identify additional loci with a high probability of true association. Genotyping in both studies was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix). Results - Of thousands of chromosomal loci studied, the same locus had the strongest association with coronary artery disease in both the WTCCC and the German studies: chromosome 9p21.3 (SNP, rs1333049) (P=1.80x10–14 and P=3.40x10–6, respectively). Overall, the WTCCC study revealed nine loci that were strongly associated with coronary artery disease (P80%) of a true association: chromosomes 1p13.3 (rs599839), 1q41 (rs17465637), 10q11.21 (rs501120), and 15q22.33 (rs17228212). Conclusions - We identified several genetic loci that, individually and in aggregate, substantially affect the risk of development of coronary artery disease

Prussian blue staining for detection of iron deposits.

<p>The staining was performed on paraffin-embedded sections of 28 days-old flies. No iron deposits were detected in the medulla or retina of either controls or double RNAi flies. Clear iron inclusions were present in paraffin-embedded sections of mouse spleen (positive control).</p

Alignment of the fly C19orf12 orthologs (CG3740, CG11671) with human C19orf12 protein.

<p>Proteins have been aligned using Clustal 2.1 multiple alignment tool. Stars (*) indicate identities and dots indicate a higher (:) and a lower (.) degree of similarity. The two <i>D. melanogaster</i> proteins are 63% and 55% similar to C19orf12 and share 72% similarity with each other. The transmembrane domains predicted by PolyPhobius are marked in yellow.</p

Detection of vacuoles in ultrathin Epon plastic sections.

<p>A) Horizontal head sections from 28 days-old control (w¹¹¹⁸) and down-regulated (<i>CG3740</i>; <i>CG11671</i> RNAi) flies. B) Number and size of vacuoles have been quantified with Image J running the applications “Find Edges” and “Analyze Particles” on thresholded 8-bit pictures. Particle size was imposed bigger than 20 pixels² and with a circularity factor between 0,5 and 1. Detected vacuoles were displayed using “Overlay Mask”. Data were manually validated to exclude artifacts. Brains analyzed for each fly strain: n = 3. Scale bar: 100 µm.</p

Summary of significant mtSNPs.

<p>Genomic position in base pairs (bp), alleles, rs_number, and type of mutation are based on the NCBI dbSNP GRCh38 human genome assembly (rCRS, GeneBank ID J01415.2). Alleles are given in terms of major→minor allele. An estimated effect size (β_SNP)<0 indicates that the minor allele increases BMI. Nominal p-values and adjusted p-values are provided.</p

Bang test in young (1 day-old) and aged (1 month-old) control (w¹¹¹⁸ and <i>act</i>-GAL4/+) and double RNAi males.

<p>A) Flies have been vortexed twice (Bang I and II) with 10 minutes in between and the time needed to upright recorded. A) Representative trajectories are reported for aged control (green), double RNAi (red) and for double heterozygous (blue) flies.</p

Expression of <i>CG3740</i> and <i>CG11671</i> in head, thorax and abdomen of <i>D. melanogaster</i>.

<p>The analysis has been performed on total RNA extracted from adult w¹¹¹⁸ flies using the same number of males and females (n = 4). The <i>Ribosomal Protein 49</i> has been used as endogenous control to normalize <i>CG3740</i> and <i>CG11671</i> expression level. The level of <i>CG11671</i> in the abdomen has been set to 1 and the expression of <i>CG3740</i> and <i>CG11671</i> in the other tissues expressed relatively.</p

Expression of <i>CG3740</i> and <i>CG11671</i> in heads from 10 days-old flies.

<p>The <i>Ribosomal Protein 49</i> has been used as endogenous control to normalize <i>CG3740</i> and <i>CG11671</i> expression level. (<b>A</b>) <i>CG3740</i> and <i>CG11671</i> have been measured in single RNAi flies (<i>CG3740</i> RNAi and <i>CG11671</i> RNAi) and in double RNAi flies (<i>CG3740</i>; <i>CG11671</i> RNAi) using the same number of males and females (n = 4). The level of <i>CG3740</i> and <i>CG11671</i> in control flies has been set to 1 and the expression of both genes in down-regulated flies expressed relatively. (<b>B</b>) <i>CG3740</i> and <i>CG11671</i> have been measured in single heterozygous deletion flies (<i>Df(1)</i>BSC589/+ and <i>Df(3L)</i>BSC579/+) and in double heterozygous deletion flies (<i>Df(1)</i>BSC589/+; <i>Df(3L)</i>BSC579/+) using female flies (n = 4). The level of <i>CG3740</i> and <i>CG11671</i> in control flies has been set to 1 and the expression of both genes in heterozygous deletion flies expressed relatively. Data refer to the average of three independent experiments ± SD.</p

Coverage of the mitochondrial genome provided by each genotyping chip.

<p>Each vertical bar stands for one mtSNP. The x-axis represents the mitochondrial genome, displaying the position and relative size of each of the 13 major mitochondrial genes, 12S and 16S rRNAs, hypervariable region 1 (HVRI), hypervariable region 2 (HVR II) as well as the position of the 22 tRNAs.</p

Mitochondrial genome-wide <i>P</i> values after adjusting for multiple testing.

<p>On the y axis, adjusted p-values transformed into the negative of the base 10 logarithm, −log₁₀(p-value), are shown. The x-axis represents the mitochondrial genome, displaying the position and relative size of each of the 13 major mitochondrial genes, 12S and 16S rRNAs, hypervariable region 1 (HVRI), hypervariable region 2 (HVR II) as well as the position of the 22 tRNAs (gray). The dashed lines show the critical values of the pointwise significance level corresponding to an FDR of 0.05.</p