Variance explained by the SNP (black bar). Each panel represents a

Variance explained by the SNP (black bar). Each panel represents a different cohort: Laval (n = 392), UBC (n = 287), Groningen (n = 342). The eQTL p-values were 5.6610261, 2.8610251 and 3.8610255, respectively. doi:10.1371/journal.pone.0070220.gRefining COPD Susceptibility Loci with Lung eQTLsFigure 3. Linkage disequilibrium plot of selected SNPs on the 4q22 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrates the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2 = 1) and white indicates perfect equilibrium (r2 = 0). The genotypes are from the 1000 Genome Project interim phase 1 release (2010/11/23). Red rectangles are SNPs previously associated with COPD (Table 2). Blue rectangles are the most significant eQTL-SNPs for the four regulated genes found on 4q22 (Figure 1). The other illustrated SNPs were order 301353-96-8 genotyped in our study and in LD (r2.0.5) with COPD SNPs. doi:10.1371/journal.pone.0070220.gexpression of genes located in this predefined 19q13 locus. The gene most significantly regulated by rs7937 was NUMBL (p = 0.0187). Three SNPs were regulating the expression of EGLN2, a gene previously associated with COPD. The most significant association with EGLN2 was with rs4803369 (p = 8.961027). Rs4803369 is located at 13,274 bp away from rs7937 and is in modest LD (r2 = 0.33) with the latter. The eQTL results for rs4803369-EGLN2 from the three cohorts are illustrated in Figure 9. This eQTL was significant and had the same direction of effect in all 3 cohorts.DiscussionThe goal of this study was to identify causal variants and genes within susceptibility loci associated with COPD. GWAS have indicated four loci associated with this disease as defined by lung function measures [9?1]. However, GWAS could not fully revealed the genetic mechanisms mediating the risk within these loci. In this study, we used genotypes and expression values of a large number of lung samples derived from three independent populations to identify eQTLs. Our analyses were centered on three loci previously associated with COPD: 4q22 (FAM13A), 4q31 (HHIP) and 19q13 (RAB4B, EGLN2, MIA, CYP2A6). We identified genetic variants influencing gene expression at each locus and replicated findings in two independent cohorts. The first study to identify an association between the 4q22 locus and COPD was published in 2010 [19]. Three other studiesconfirmed an association between this locus and COPD/lung function [10,11,20]. In this study, we found 91 eQTLs on 4q22 in the discovery cohort and 61 of them were replicated in both replication sets. The majority of the SNPs were located in introns (n = 30) and intergenic Title Loaded From File regions (n = 27). Other SNPs were located in the 39 UTR (n = 4) and upstream of the gene (n = 2). Only one missense SNP was found to regulate the expression of MMRN1. Lung eQTLs on 4q22 were found and validated with four genes: PPM1K, SNCA, PPM1 and GPRIN3 genes. A SNP located in SNCA (rs2035268) has been associated with accelerated FEV1/ FVC decline [21]. Three SNPs in our dataset were in perfect LD with the rs2035268: rs3889917, rs7684637 and rs3775461. However, none of those SNPs were significantly associated with the expression level of a gene. The best r2 between one of our significant eQTL-SNP and rs2035268 was 0.047. No SNP previously associated with CO.Variance explained by the SNP (black bar). Each panel represents a different cohort: Laval (n = 392), UBC (n = 287), Groningen (n = 342). The eQTL p-values were 5.6610261, 2.8610251 and 3.8610255, respectively. doi:10.1371/journal.pone.0070220.gRefining COPD Susceptibility Loci with Lung eQTLsFigure 3. Linkage disequilibrium plot of selected SNPs on the 4q22 locus in the 1000 Genome Project. The white horizontal bar on the upper part of the figure illustrates the location of SNPs on a physical scale. LD values (r2) are indicated in each box. The color of the squares illustrates the strength of pairwise r2 values on a black and white scale where black indicates perfect LD (r2 = 1) and white indicates perfect equilibrium (r2 = 0). The genotypes are from the 1000 Genome Project interim phase 1 release (2010/11/23). Red rectangles are SNPs previously associated with COPD (Table 2). Blue rectangles are the most significant eQTL-SNPs for the four regulated genes found on 4q22 (Figure 1). The other illustrated SNPs were genotyped in our study and in LD (r2.0.5) with COPD SNPs. doi:10.1371/journal.pone.0070220.gexpression of genes located in this predefined 19q13 locus. The gene most significantly regulated by rs7937 was NUMBL (p = 0.0187). Three SNPs were regulating the expression of EGLN2, a gene previously associated with COPD. The most significant association with EGLN2 was with rs4803369 (p = 8.961027). Rs4803369 is located at 13,274 bp away from rs7937 and is in modest LD (r2 = 0.33) with the latter. The eQTL results for rs4803369-EGLN2 from the three cohorts are illustrated in Figure 9. This eQTL was significant and had the same direction of effect in all 3 cohorts.DiscussionThe goal of this study was to identify causal variants and genes within susceptibility loci associated with COPD. GWAS have indicated four loci associated with this disease as defined by lung function measures [9?1]. However, GWAS could not fully revealed the genetic mechanisms mediating the risk within these loci. In this study, we used genotypes and expression values of a large number of lung samples derived from three independent populations to identify eQTLs. Our analyses were centered on three loci previously associated with COPD: 4q22 (FAM13A), 4q31 (HHIP) and 19q13 (RAB4B, EGLN2, MIA, CYP2A6). We identified genetic variants influencing gene expression at each locus and replicated findings in two independent cohorts. The first study to identify an association between the 4q22 locus and COPD was published in 2010 [19]. Three other studiesconfirmed an association between this locus and COPD/lung function [10,11,20]. In this study, we found 91 eQTLs on 4q22 in the discovery cohort and 61 of them were replicated in both replication sets. The majority of the SNPs were located in introns (n = 30) and intergenic regions (n = 27). Other SNPs were located in the 39 UTR (n = 4) and upstream of the gene (n = 2). Only one missense SNP was found to regulate the expression of MMRN1. Lung eQTLs on 4q22 were found and validated with four genes: PPM1K, SNCA, PPM1 and GPRIN3 genes. A SNP located in SNCA (rs2035268) has been associated with accelerated FEV1/ FVC decline [21]. Three SNPs in our dataset were in perfect LD with the rs2035268: rs3889917, rs7684637 and rs3775461. However, none of those SNPs were significantly associated with the expression level of a gene. The best r2 between one of our significant eQTL-SNP and rs2035268 was 0.047. No SNP previously associated with CO.