A genome-wide survey of CD4(+) lymphocyte regulatory genetic variants identifies novel asthma genes

Abstract

Background: Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing.

Objective: We evaluated 6706 cis-acting expression-associated variants (eSNPs) identified through a genome-wide eQTL survey of CD4(+) lymphocytes for association with asthma.

Methods: eSNPs were tested for association with asthma in 359 asthmatic patients and 846 control subjects from the Childhood Asthma Management Program, with verification by using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by using formaldehyde-assisted isolation of regulatory elements (FAIRE) quantitative PCR and chromatin immunoprecipitation PCR in lung-derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes.

Results: Cis-acting eSNPs demonstrated associations with asthma in both cohorts. We confirmed the previously reported association of ORMDL3/GSDMB variants with asthma (combined P = 2.9 × 10(-8)). Reproducible associations were also observed for eSNPs in 3 additional genes: fatty acid desaturase 2 (FADS2; P = .002), N-acetyl-α-D-galactosaminidase (NAGA; P = .0002), and Factor XIII, A1 (F13A1; P = .0001). Subsequently, we demonstrated that FADS2 mRNA is increased in CD4(+) lymphocytes in asthmatic patients and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity.

Conclusions: Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.

Keywords: Asthma; CD4(+) lymphocytes; expression quantitative trait locus; haplotype; integrative genomics; regulatory variants.

Figure 1

Genome-wide significant expression quantitative trait loci (eQTLs) previously identified in CD4 positive lymphocytes of asthmatic subjects. A. Association of SNP rs4795405 with the expression of ORMDL3. B. Association of SNP rs174611 with FADS2 expression. C. Association of SNP rs2413669 with NAGA expression. D. Association of rs11243081 with F13A1 expression.

Figure 2. SNP-based eQTL analysis across the FADS2 locus

SNP-based eQTL analysis across FADS2, demonstrating association of rs968567 with FADS2 expression (p=10⁻¹⁵, 30% expression variance explained). SNP rs968567 (blue arrow), located in a conserved region of the promoter, demonstrates association with FADS2 expression (p=10⁻¹⁵). SNPs rs174627 (green arrow) and rs174611 (red arrow) are in LD with rs968567 and are associated with FADS2 expression and asthma susceptibility.

Figure 3. Haplotype analysis of the FADS2 locus

(a) Haplotype structure: Haplotype frequencies observed in at least 1% of CAMP subjects are listed to the right of each haplotype sequence. Dots indicate the allele present in haplotype H1 – the most common haplotype. The asthma-associated rs968567 T allele is unique to, and uniquely tags, haplotype 2 (H2, boxed in blue), observed at 18.1% frequency. (b) Haplotype-based eQTL analysis: H2 demonstrates strong association with increased FADS2 expression (p<10⁻¹⁰), while haplotype 1 (H1, frequency 31.4%) is associated with low FADS2 expression. Others (H3-H8) showed intermediate levels of FADS2 expression.

Figure 4. Differential expression of FADS2 in asthmatic subjects compared to non-asthmatic controls in Asthma BRIDGE

Significantly increased expression of FADS2 in peripheral blood CD4+ lymphocytes of asthmatic subjects compared to non-asthmatic control subjects (p=0.003)

Figure 5. Functional annotation of the three asthma associated regions by FAIRE-PCR analysis

Formaldehyde assisted isolation of regulatory enrichment (FAIRE) was performed at three association regions nearby FADS2, NAGA and F13A genes in Beas-2B, A549 and Jurkat cell lines by real-time PCR analysis. The graph depicts the relative FAIRE signal at each locus normalized to input and negative control regions. Each locus contains three SNPs nearby eQTL signals. Mean±SD are from two to four repeats for each SNP. **p<0.01 (unpaired one way t test.)

Figure 6. Enrichment of active histone marks in two asthma associated regions detected by ChIP-PCR analysis

Chromatin immunoprecipitation (ChIP) was performed using H3K4Me1 and H3K27Ac antibodies in Beas-2B, A549 and Jurkat cell lines targeting genomic regions nearby two SNPs rs968567 and rs1801311 in FADS2 and NAGA loci respectively. IgG was used as a negative control. The bars represent the average of a two biological replicates with standard deviations. * p<0.05; ** p<0.01 (paired one way t test).