Overlap of significantly associated rSNPs identified by ASE and GTE.

<p>The percentage of overlapping rSNPs detected by allele-specific expression (ASE) and genotype expression (GTE) analysis is plotted for varying numbers of samples. The top 9536 SNPs from the GTE analysis are compared with the top 38203 SNPs from the ASE analysis, which corresponds to a Bonferroni threshold of p = 0.05 for a GTE sample size of 395 and an ASE sample size of 188. The p-value cut-offs were adapted so that the same SNP top-list sizes were obtained at all sample sizes for both GTE (p-value of 1.17E-7, 1.06E-4, 1.93E-3, 6.12E-3 for n = 395, n = 188, n = 95, and n = 50 respectively) and ASE (p-value of 8.06E-8, 9.35E-5, 4.90E-3 for n = 188, n = 95, and n = 50 respectively). The vertical axes show the percentage of SNPs in the top-lists detected by both GTE and ASE analysis and the horizontal axes show the number of samples analyzed using GTE and ASE, respectively. The percentage overlap is calculated by dividing the number of overlaps with the number of top SNPs in the GTE analysis. In (A), each line shows the effect on the number of overlapping SNPs detected by ASE analysis of a specific sample size when the sample size in GTE analysis was increased. In (B), each line shows the effect on the number of overlapping rSNPs detected by GTE analysis of a specific sample size when the samples size in ASE analysis is increased.</p

KERA is expressed in atherosclerotic but not in non-diseased arteries.

<p><b>A</b>, KERA is not expressed in tonsil tissue; <b>B</b>: KERA is expressed in corneal tissue <b>C</b>: KERA is not expressed in mammary artery. <b>D</b>: KERA is expressed in arterial segments obtained from a patient with the <i>KERA</i> mutation.</p

The ability of ASE and GTE analysis to detect significantly associated rSNPs at different MAF.

<p>Fractions of rSNPs are shown for different minor allele frequencies (MAF) with significant association signals according to a Bonferroni-corrected p-value of 0.05. Each data point underlying the curves represents the fraction of significant associations within a 1% MAF bin. Sliding 5% MAF window averages are plotted for different sample sizes analyzed by ASE and GTE. Both methods detect a lower fraction of low frequency rSNPs, compared to the fraction of all the SNPs at the same frequency (black line). The ASE method detects a higher fraction of the SNPs (solid lines) with a MAF <15% than GTE (dashed lines) regardless of sample size except for the largest GTE sample set.</p

Characteristics of the core pedigree included in linkage analysis.

<p>Age is expressed as mean ± standard deviation and data are expressed as number (N). Lipid values are expressed as median with interquartile range (IQR). Pedigree members with an event use lipid lowering medication. LDL = low density lipoprotein; HDL = high density lipoprotein.</p

Co-localization of KERA with CXCL1 and CD3 positive type I helper T lymphocytes was assessed in human plaque segments.

<p><b>A</b>: triple stain with KERA (blue), CXCL1 (red) and CD3 positive cells (brown). <b>B</b>: Spectral Imaging of triple staining. <b>C</b>: The yellow staining demonstrates that these cells are positive for all three components.</p

Clinical characteristics of the core pedigree included in linkage analysis.

<p>AMI = Acute Myocardial Infarction; AP = Angina Pectoris; PTCA = Percutaneous Transluminal Coronary Angioplasty; CABG = Coronary Artery Bypass Graft; CVA = Cerebrovascular Accident. Simva = Simvastatin, Rosuva = Rosuvastatin, Prava = Pravastatin and Atorva = Atorvastatin. BMI = body mass index (kg/cm²).</p>†<p> = index case. Subjects were considered smokers if they were current smokers or when they quitted smoking within the last 5 years.</p

Structures obtained from molecular dynamic simulations of the mutant p.Ser307Cys KERA protein.

<p>Molecular dynamic simulations were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098289#pone.0098289.s001" target="_blank">File S1</a>. The residue Cys303 is highlighted in yellow, Cys343 in green, Ser307 in cyan and Cys307 residue in blue. <b>A</b>, Structure of wild-type KERA. <b>C</b> Structure of the KERA mutant p.Ser307Cys. <b>B</b>, A detailed view of the C-terminal part of wild type KERA highlighting the Cys303–Cys343 disulphide bond. <b>D</b>, Possible structural effects of the substitution of a serine for a cysteine at residue 307 showing a favourable Cys303–Cys307 disulphide bond. Consequently, Cys343 is available for binding with cysteine residues of other proteins.</p

Expression of KERA in atherosclerotic tissue in <i>Apoe^−/−</i> mice after induction of atherosclerosis by collar placement.

<p><b>A–B</b>, Early (week = 2, A) and advanced (week = 8, B) atherosclerotic tissue from murine carotid arteries were stained for KERA (brown) and hematoxylin (blue). While present mainly near endothelial cells in early lesion, KERA is predominantly present in the matrix of the plaque at more advanced lesions. <b>C–D</b>, KERA expression overtime in <i>Apoe^−/−</i> mice with collar placement show significant correlation with plaque size (r² = 0.69, P<0.0001).</p

Schematic overview of the gene discovery strategy.

<p>Schematic overview of the gene discovery strategy.</p

Seven identified SNPs compared to previously published loci.

a<p>The Effect allele refers to a positive effect direction in the discovery stage for the trait and gender, the SNP was selected for;</p>b<p>Gene near this SNP which was published previously from sex-combined analyses.</p><p>The seven SNPs with sex difference are considered to depict a known locus, if the index SNP is close to a published top SNP (<1 cM). These include four of the previously reported sexually dimorphic WHR loci (Heid et al., Nat Genet 2010).</p