Additional file 1: of Long-term outdoor air pollution and DNA methylation in circulating monocytes: results from the Multi-Ethnic Study of Atherosclerosis (MESA)

Supplemental Material. (DOCX 39 kb

Summary of subjects, methods and analyses.

<p>a. Number of SNP pairs for which interaction testing was performed - may not equal the number of possible pair-wise tests [n*(n−1)/2] because some pairs were captured in previous Analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041730#pone.0041730.s001" target="_blank">File S1</a> Supporting <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041730#pone-0041730-g002" target="_blank">Figure 2</a>), and some tests were not feasible due to low allele frequencies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041730#pone.0041730.s001" target="_blank">File S1</a> Section 3.3). b. Significance threshold computed using permutations under the null hypothesis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041730#pone.0041730.s001" target="_blank">File S1</a> Section 3.4) c. SNP pairs with p-value for interaction within 3 orders of magnitude of the significance threshold for each Analysis were brought forward for validation in the WTCCC sample; the numbers of SNP pairs for which data were available in the WTCCC study are shown. <i>LDL</i>, concentration of LDL cholesterol; <i>HDL</i>, concentration of HDL cholesterol; <i>TG</i>, triglyceride concentration; <i>BP</i>, blood pressure; <i>CH</i>, carbohydrate metabolism (loci associated with risk of Type II diabetes and related phenotypes, such as fasting glucose concentration); <i>SMK</i>, smoking; <i>OB</i>, obesity; <i>small LDL</i>, concentration of small atherogenic LDL particles; <i>Lp(a)</i>, plasma levels of lipoprotein(a); <i>CHD</i>, risk of coronary heart disease; <i>MI</i>, risk of myocardial infarction.</p

Results of gene-gene interaction search among CVRF SNPs (Analysis 1).

<p><i>Panel A</i>. Plot of the top result (arrow) from Analysis 1 against the distribution of the top results from 10,000 permutations under the null hypothesis (dotted line). The permuted top results are expected to follow a beta-distribution (solid line, parameters obtained from permuted top results), the 95^th percentile of which was taken as the significance level required to obtain a Type II error of 0.05 (arrow). <i>Inset</i>: While the significance level computed in Analysis 1 (dashed black line) was estimated using 10,000 null permutations, this estimate was found to stabilize rapidly with increasing number of permutations (black points) and to change little after 100–200 permutations. Consequently, we progressively reduced the number of permutations used to estimate the significance level in subsequent Analyses. <i>Panel B</i>. Quantile-quantile plot showing rank-ordered observed results (black points) from 29,161 tests in Analysis 1 (<i>y-axis</i>) against expected results (<i>x-axis</i>) estimated from 10,000 permutations under the null hypothesis (randomized phenotype). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041730#pone.0041730.s001" target="_blank">File S1</a> Section 3.5 for computation methods. The shaded area corresponds to the 95%CI of the permuted expected results. The 95%CI of a normal distribution is indicated by the dotted lines. <i>Panel C</i>. Estimation of the interaction effect sizes this analysis has 80% power to detect across a range of MAF under an additive × additive interaction model. The heights of the vertical bars correspond to the effect size (OR) detectable for a typical pair of SNPs whose MAFs are as indicated on the horizontal axes.</p

Comparison of effect sizes for early AMD from this study versus published effect estimates for late AMD.

a<p>Superscript shows reference for the largest study reporting genome-wide association of the relevant SNP with late AMD, from which the “Late AMD” effect estimates were derived:</p>1<p>Chen et al, 2010 ¹¹.</p>2<p>Yu et al, 2011 ¹⁵.</p>3<p>Klein et al, 2005 ¹².</p>4<p>Kopplin et al, 2010 ¹³.</p>5<p>Arakawa et al, 2011 ¹⁰.</p>6<p>Neale et al, 2010 ¹⁴.</p>b<p>NCBI Human Genome Build 36.3 coordinates;</p>c<p>Effective allele;</p>d<p>Frequency of the effective allele;</p>e<p>Summary meta-analysis regression coefficient, indicating the overall, estimated change in log(odds) associated with each additional copy of the effective allele;</p>f<p>Estimated odds ratio and 95% confidence interval for each additional copy of the effective allele, based on fixed-effects meta-analysis of European-ancestry cohorts;</p>g<p><i>P</i>-value associated with the estimated OR;</p>h<p>NR: not reported;</p>i<p><i>P</i>-value from test of heterogeneity of regression coefficients between early and advanced AMD. The threshold for study-wise significance was 0.0036, after accounting for multiple tests. Significant results are shown in bold. Heterogeneity could not be assessed for SNPs with no published confidence interval for the late AMD effect estimate;</p>j<p>Ratio of regression coefficient for advanced vs early AMD, formulated as Beta_adv/Beta_early.</p><p><i>Notes:</i> This study did not have data and could not assess association for additional published SNPs rs4711751 in <i>VEGFA</i> and rs11200638 in <i>HTRA1</i>.</p

Regional association results for chromosome 19 SNPs in the <i>PVRL2</i>/<i>APOE</i>/<i>TOMM40</i> gene cluster.

<p>The index, associated SNP is named and shown as a purple diamond (rs2075650: <i>P</i> = 1.1×10⁻⁶).</p

Population characteristics for the individual cohorts.

<p>Population characteristics for the individual cohorts.</p

Results for SNPs showing suggestive evidence of association (<i>P</i>&lt;1×10⁻⁵) in the primary (European-ancestry) meta-analysis of early AMD.

<p>Where multiple correlated SNPs in the same gene/region showed similar association evidence, the most strongly associated SNP is shown.</p>a<p>NCBI Human Genome Build 36.3 coordinates;</p>b<p>Effective allele;</p>c<p>Frequency of the effective allele;</p>d<p>Estimated odds ratio and 95% confidence interval for the effect of each additional copy of the effective allele, based on the fixed-effects, inverse variance-weighted meta-analysis of European-ancestry cohorts;</p>e<p><i>P</i>-value associated with the estimated OR;</p>f<p>Heterogeneity <i>I²</i> statistic;</p>g<p>Heterogeneity <i>P</i>-value, based on Cochran’s Q statistic;</p>h<p>within a 500 kb genomic region centred on the associated SNP.</p

Comparison of estimated effect sizes for early versus advanced AMD for published SNPs showing genome-wide significant association with AMD.

a<p>Superscript shows reference for the largest study reporting genome-wide association of the relevant SNP with AMD:</p>1<p>Chen et al, 2010 ¹¹.</p>2<p>Yu et al, 2011 ¹⁵.</p>3<p>Klein et al, 2005 ¹².</p>4<p>Kopplin et al, 2010 ¹³.</p>5<p>Arakawa et al, 2011 ¹⁰.</p>6<p>Neale et al, 2010 ¹⁴.</p>b<p>NCBI Human Genome Build 36.3 coordinates;</p>c<p>Effective allele;</p>d<p>Frequency of the effective allele;</p>e<p>Summary meta-analysis regression coefficient, indicating the overall, estimated change in log(odds) associated with each additional copy of the effective allele;</p>f<p>Estimated odds ratio and 95% confidence interval for each additional copy of the effective allele, based on fixed-effects meta-analysis of European-ancestry cohorts;</p>g<p><i>P</i>-value associated with the estimated OR;</p>h<p>Heterogeneity <i>P</i>-value, based on Cochran’s Q statistic;</p>i<p><i>P</i>-value from test of heterogeneity of regression coefficients between early and advanced AMD. The threshold for study-wise significance was 0.0024, after accounting for multiple tests. Significant results are shown in bold;</p>j<p>Ratio of regression coefficient for advanced vs early AMD, formulated as Beta_adv/Beta_early.</p><p><i>Notes:</i> This study did not have data and could not assess association for additional published SNPs rs4711751 in <i>VEGFA</i> and rs11200638 in <i>HTRA1</i>.</p

Regional association results for chromosome 11 SNPs in the tyrosinase precursor (<i>TYR</i>) gene.

<p>The index, associated SNP is named and shown as a purple diamond (rs621313: <i>P</i> = 3.5×10⁻⁶).</p

Regional association plot.

<p>The figures display–log10 p-values for SNPs within the locus of highest significance for the analysis of fish consumption (top panel) and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) consumption (bottom panel). The degree of linkage disequilibrium (r²) is displayed in gradients of gray from dark (low linkage) to light (high linkage).</p