Expression quantitative trait locus fine mapping of the 17q12-21 asthma locus in African American children: a genetic association and gene expression study

Abstract

Background: African ancestry is associated with a higher prevalence and greater severity of asthma than European ancestries, yet genetic studies of the most common locus associated with childhood-onset asthma, 17q12-21, in African Americans have been inconclusive. The aim of this study was to leverage both the phenotyping of the Children's Respiratory and Environmental Workgroup (CREW) birth cohort consortium, and the reduced linkage disequilibrium in African Americans, to fine map the 17q12-21 locus.

Methods: We first did a genetic association study and meta-analysis using 17q12-21 tag single-nucleotide polymorphisms (SNPs) for childhood-onset asthma in 1613 European American and 870 African American children from the CREW consortium. Nine tag SNPs were selected based on linkage disequilibrium patterns at 17q12-21 and their association with asthma, considering the effect allele under an additive model (0, 1, or 2 effect alleles). Results were meta-analysed with publicly available summary data from the EVE consortium (on 4303 European American and 3034 African American individuals) for seven of the nine SNPs of interest. Subsequently, we tested for expression quantitative trait loci (eQTLs) among the SNPs associated with childhood-onset asthma and the expression of 17q12-21 genes in resting peripheral blood mononuclear cells (PBMCs) from 85 African American CREW children and in upper airway epithelial cells from 246 African American CREW children; and in lower airway epithelial cells from 44 European American and 72 African American adults from a case-control study of asthma genetic risk in Chicago (IL, USA).

Findings: 17q12-21 SNPs were broadly associated with asthma in European Americans. Only two SNPs (rs2305480 in gasdermin-B [GSDMB] and rs8076131 in ORMDL sphingolipid biosynthesis regulator 3 [ORMDL3]) were associated with asthma in African Americans, at a Bonferroni-corrected threshold of p<0·0055 (for rs2305480_G, odds ratio [OR] 1·36 [95% CI 1·12-1·65], p=0·0014; and for rs8076131_A, OR 1·37 [1·13-1·67], p=0·0010). In upper airway epithelial cells from African American children, genotype at rs2305480 was the most significant eQTL for GSDMB (eQTL effect size [β] 1·35 [95% CI 1·25-1·46], p<0·0001), and to a lesser extent showed an eQTL effect for post-GPI attachment to proteins phospholipase 3 (β 1·15 [1·08-1·22], p<0·0001). No SNPs were eQTLs for ORMDL3. By contrast, in PBMCs, the five core SNPs were associated only with expression of GSDMB and ORMDL3. Genotype at rs12936231 (in zona pellucida binding protein 2) showed the strongest associations across both genes (for GSDMB, eQTLβ 1·24 [1·15-1·32], p<0·0001; and for ORMDL3 (β 1·19 [1·12-1·24], p<0·0001). The eQTL effects of rs2305480 on GSDMB expression were replicated in lower airway cells from African American adults (β 1·29 [1·15-1·44], p<0·0001).

Interpretation: Our study suggests that SNPs regulating GSDMB expression in airway epithelial cells have a major role in childhood-onset asthma, whereas SNPs regulating the expression levels of 17q12-21 genes in resting blood cells are not central to asthma risk. Our genetic and gene expression data in African Americans and European Americans indicated GSDMB to be the leading candidate gene at this important asthma locus.

Funding: National Institutes of Health, Office of the Director.

Figure 1. Asthma association studies and LD nine SNPs at the 17q12–21 locus in CREW European American and African American children.

A) Pairwise LD between (see also appendix p 12). The top panel shows the location of genes at the locus, with the relative positions of the nine SNPs genotyped in the CREW children shown by vertical lines. The lower panel shows the LD plots in each population. The r² value for pairs of SNPs are shown within the diamond shapes for all values <1.0. SNPs. Genes in the proximal region shown in blue, in the core region shown in orange, and in the distal region in green, as previously described. *rs3894194 was replaced in this study with rs8069202, which is in high LD with rs3894194 in both Europeans (r²=0.984) and African Americans (r²=0.953) (1000 Genomes). PGAP3, post-GPI attachment to proteins 3; ERBB2, erb-b2 receptor tyrosine kinase 2; MEN1, multiple endocrine neoplasia type 1; GRB7, growth factor receptor-bound protein 7; IKZF3, IKAROS family zinc finger 3; ZPBP2, zona pellucida binding protein 2; GSDMB, gasdermin B; ORMDL3, ORM1-like 3; LRRC3C, leucine-rich repeat-containing protein 3C; GSDMA, gasdermin A. B) Forest plots of association results in European Americans (EuAm) and African Americans (AfAm). Asthma odds ratio estimates (filled squares) and 95% confidence intervals (horizontal bars) are shown for the AfAm and EuAm samples in the CREW, EVE and CREW+EVE meta analyses. The size of the squares is proportional to the square root of the sample size. The bolded SNPs are the two that are associated with asthma in the AfAm meta-analysis (Bonferroni P <0.0055.

Figure 1. Asthma association studies and LD nine SNPs at the 17q12–21 locus in CREW European American and African American children.

A) Pairwise LD between (see also appendix p 12). The top panel shows the location of genes at the locus, with the relative positions of the nine SNPs genotyped in the CREW children shown by vertical lines. The lower panel shows the LD plots in each population. The r² value for pairs of SNPs are shown within the diamond shapes for all values <1.0. SNPs. Genes in the proximal region shown in blue, in the core region shown in orange, and in the distal region in green, as previously described. *rs3894194 was replaced in this study with rs8069202, which is in high LD with rs3894194 in both Europeans (r²=0.984) and African Americans (r²=0.953) (1000 Genomes). PGAP3, post-GPI attachment to proteins 3; ERBB2, erb-b2 receptor tyrosine kinase 2; MEN1, multiple endocrine neoplasia type 1; GRB7, growth factor receptor-bound protein 7; IKZF3, IKAROS family zinc finger 3; ZPBP2, zona pellucida binding protein 2; GSDMB, gasdermin B; ORMDL3, ORM1-like 3; LRRC3C, leucine-rich repeat-containing protein 3C; GSDMA, gasdermin A. B) Forest plots of association results in European Americans (EuAm) and African Americans (AfAm). Asthma odds ratio estimates (filled squares) and 95% confidence intervals (horizontal bars) are shown for the AfAm and EuAm samples in the CREW, EVE and CREW+EVE meta analyses. The size of the squares is proportional to the square root of the sample size. The bolded SNPs are the two that are associated with asthma in the AfAm meta-analysis (Bonferroni P <0.0055.

Figure 2.. Associations between five core region SNPs and expression levels of genes at the extended 17q12–21 locus in African American children in A) PBMCs (n=85) and B) upper airway epithelial cells (n=246).

The relative location of genes at this locus are shown in the upper panel. ZPBP2 and LRRC3C were not detected as expressed in these cells. SNPs and the genes in which they reside are shown above each panel. Gene names are shown on the x-axes; normalized gene expression in counts per million are shown on the y-axis (see Methods). At each SNP, blue bars correspond to homozygotes for the non-effect (non-risk) alleles, black bars to heterozygotes, and red bars to homozygotes for the effect (risk) alleles. For SNP rs2305480, the AA and AG genotypes were combined in PBMCs because there was only one homozygote for the A allele. See Table 3 for beta values (95% CIs) and exact P-values. *P < 0.05, **P < 0.01, ***P ≤ 0.0012

Figure 3.. eQTL studies of five core region SNPs and expression levels of GSDMB and ORMDL3 in lower airway (bronchial) epithelial cells from European American (n=44) and African American (n=72) adults.

Upper panel: eQTL results with five core SNPs. The effect sizes (beta) reflect the expected increase in expression associated with each copy of the asthma risk allele. Bonferroni corrected P - value threshold (5 SNPs × 2 genes) = 0.0055. Lower panel: Boxplots showing associations between rs2305480 genotype and expression levels of GSDMB and ORMDL3.