Detection of pleiotropy through a Phenome-wide association study (PheWAS) of epidemiologic data as part of the Environmental Architecture for Genes Linked to Environment (EAGLE) study.

We performed a Phenome-wide association study (PheWAS) utilizing diverse genotypic and phenotypic data existing across multiple populations in the National Health and Nutrition Examination Surveys (NHANES), conducted by the Centers for Disease Control and Prevention (CDC), and accessed by the Epidemiological Architecture for Genes Linked to Environment (EAGLE) study. We calculated comprehensive tests of association in Genetic NHANES using 80 SNPs and 1,008 phenotypes (grouped into 184 phenotype classes), stratified by race-ethnicity. Genetic NHANES includes three surveys (NHANES III, 1999-2000, and 2001-2002) and three race-ethnicities: non-Hispanic whites (n = 6,634), non-Hispanic blacks (n = 3,458), and Mexican Americans (n = 3,950). We identified 69 PheWAS associations replicating across surveys for the same SNP, phenotype-class, direction of effect, and race-ethnicity at p0.01, and sample size >200. Of these 69 PheWAS associations, 39 replicated previously reported SNP-phenotype associations, 9 were related to previously reported associations, and 21 were novel associations. Fourteen results had the same direction of effect across more than one race-ethnicity: one result was novel, 11 replicated previously reported associations, and two were related to previously reported results. Thirteen SNPs showed evidence of pleiotropy. We further explored results with gene-based biological networks, contrasting the direction of effect for pleiotropic associations across phenotypes. One PheWAS result was ABCG2 missense SNP rs2231142, associated with uric acid levels in both non-Hispanic whites and Mexican Americans, protoporphyrin levels in non-Hispanic whites and Mexican Americans, and blood pressure levels in Mexican Americans. Another example was SNP rs1800588 near LIPC, significantly associated with the novel phenotypes of folate levels (Mexican Americans), vitamin E levels (non-Hispanic whites) and triglyceride levels (non-Hispanic whites), and replication for cholesterol levels. The results of this PheWAS show the utility of this approach for exposing more of the complex genetic architecture underlying multiple traits, through generating novel hypotheses for future research

Study population characteristics.

<p>Abbreviations: non-Hispanic white (NHW), non-Hispanic black (NHB), Mexican American (MA).</p><p>*χ² = 35.95; p<0.0001.</p><p>Study population characteristics.</p

Potentially pleiotropic results.

<p>These are the PheWAS-significant results of this study with more than one distinct phenotype-class associated with the same SNP. This is a plot of SNP-phenotype associations observed in both NHANES III and Continuous NHANES with p-value <0.01, for SNPs with an allele frequency >0.01, and a sample size >200, for the same race-ethnicity, phenotype-class, and direction of effect. Plotted are results where the significant SNP-phenotype association matches a previously reported SNP-Phenotype association. The first column indicates the chromosome and bp location of the SNP. The second column indicates the SNP ID, the associated phenotype-class, the self-reported race-ethnicity (NHW  =  Non-Hispanic Whites, NHB  =  Non-Hispanic Blacks, or MA  =  Mexican Americans), and the coded-allele. The next column contains a colored box if association results were available for natural log transformed NHANES III phenotypes (NHANES III ln+1), un-transformed NHANES III phenotypes (NHANES III), or natural log transformed Continuous NHANES phenotypes (Continuous NHANES ln+1) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004678#s4" target="_blank">methods</a> for more details on phenotype transformation), or untransformed Continuous NHANES phenotypes. The next column indicates the p-value for each association, and the triangle direction indicates whether the association had a positive (triangle pointed to the left) or negative direction of effect (triangle pointed to the right). The following columns indicate magnitude of the effect (beta), the coded allele frequency (CAF), and the sample size for the association.</p

Overview of the approach for this study.

<p>Genotypic and phenotypic data were collected in NHANES III and Continuous NHANES. The phenotypes for the two studies were matched into phenotype classes. Comprehensive associations were calculated for the genotypes and phenotypes for each survey independently. The results that were found in both surveys, with p<0.01, for the same phenotype-class, and race-ethnicity, and same direction of effect, were maintained for further inspection in this study.</p

Using PheWAS results, Biofilter, and Cytoscape to explore gene-gene connections with NetPath.

<p>We used Biofilter to annotate the SNPs of this PheWAS with gene information. We then mapped the genes to concomitant pathways or other gene groupings through GO, KEGG, and NetPath. This is one example for the results for Mexican Americans and annotation with NetPath. The pink diamonds are associated phenotypes of this PheWAS, the green hexagons are SNPs, blue boxes are genes, and circles are biological connections that link genes together, in this case the two genes are in the same TGF NetPath biological pathway. Thus, we see that in the PheWAS results, the <i>LPL</i> SNP rs328 had a significant association with HDL cholesterol levels, and <i>FADS1</i> rs17547 association with Ferritin levels, and both genes are found in the TGF beta receptor pathway.</p

Sun plot of (p<0.01) results for <i>LIPC</i> rs1800588, coded allele T.

<p>This SNP was previously associated HDL-C in European-descent populations. Significant associations (p<0.01) are plotted clockwise with the most significant value result at the top. The length of the each line corresponds to the –log(p-value) of each result, with the longest line representing the most significant result for this SNP, meeting our PheWAS replication criteria for inclusion. Study, transformed (LN +1) or untransformed (none) phenotype description, self-reported race-ethnicity, and direction of effect are listed for each association. This SNP was associated with a number of phenotypes including folate in Mexican Americans (MA), total cholesterol in non-Hispanic whites (NHW), triglyceride levels in non-Hispanic whites, and vitamin E levels in non-Hispanic whites.</p

Using PheWAS results, Biofilter, and Cytoscape to explore gene-gene connections with KEGG connections.

<p>The <i>LIPC</i> SNP rs1800588 was associated with folate levels, the <i>LPL</i> SNP rs328 was associated with HDL cholesterol, and both of these genes are in the glycerolipid metabolism KEGG pathway in Mexican Americans. The <i>APOA5</i> SNP rs135506, associated with triglyceride levels in our study, shares the PPAR signaling pathway along with <i>LPL</i>.</p

Replicating results for PheWAS.

<p>This is a plot of SNP-phenotype associations observed in both NHANES III and Continuous NHANES with p-value <0.01, for SNPs with an allele frequency >0.01, and a sample size >200, for the same race-ethnicity, phenotype-class, and direction of effect. Plotted are results where the significant SNP-phenotype association matches a previously reported SNP-Phenotype association. The first column indicates the chromosome and base pair location of the SNP. The second column indicates the SNP ID, the associated phenotype-class, the self-reported race-ethnicity (NHW  =  Non-Hispanic Whites, NHB  =  Non-Hispanic Blacks, or MA  =  Mexican Americans), and the coded-allele. The next column contains a colored box if association results were available for natural log transformed Continuous NHANES (Continuous NHANES ln+1), un-transformed Continuous NHANES phenotypes, NHANES III untransformed phenotypes (NHANES III), or transformed NHANES III phenotypes (NHANES III ln+1) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004678#s4" target="_blank">methods</a> for more details on phenotype transformation). The next column indicates the p-value for each association, and the triangle direction indicates whether the association had a positive (triangle pointed to the left) or negative direction of effect (triangle pointed to the right). The following columns indicate magnitude of the effect (beta), the coded allele frequency (CAF), and the sample size for the association.</p

Using PheWAS results, Biofilter, and Cytoscape to explore gene-gene connections with GO biological processes.

<p>Three SNPs were associated with uric acid levels in Mexican Americans: rs2231142, rs7442295, rs685911 (green hexagons). One of the SNPs is within the gene <i>ABCG2</i>, and the other two SNPs are within <i>SLC2A9</i> (blue boxes). Both <i>ABCG2</i> and <i>SLC2A9</i> are found within the GO biological process “urate metabolic process”, a collection of the gene products involved in the chemical reactions and pathways involving urate. This was also found for non-Hispanic whites.</p

Related results for PheWAS.

<p>This is a plot of SNP-phenotype associations observed in both NHANES III and Continuous NHANES with p-value <0.01, for SNPs with an allele frequency >0.01, and a sample size >200, for the same race-ethnicity, phenotype-class, and direction of effect. Plotted are results where the significant SNP-phenotype association is closely related to the phenotype of a previously reported SNP-Phenotype association. The first column indicates the chromosome and bp location of the SNP. The second column indicates the SNP ID, the associated phenotype-class, the self-reported race-ethnicity (NHW  =  Non-Hispanic Whites, NHB  =  Non-Hispanic Blacks, or MA  =  Mexican Americans), and the coded-allele. The next column contains a colored box if association results were available for natural log transformed Continuous NHANES phenotypes (Continuous NHANES ln+1), un-transformed Continuous NHANES phenotypes, NHANES III untransformed phenotypes (NHANES III), or transformed NHANES III phenotypes (NHANES III ln+1) (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004678#s4" target="_blank">methods</a> for more details on phenotype transformation). The next column indicates the p-value for each association, and the triangle direction indicates whether the association had a positive (triangle pointed to the left) or negative direction of effect (triangle pointed to the right). The following columns indicate magnitude of the effect (beta), the coded allele frequency (CAF), and the sample size for the association.</p