Functional sequence variants of intergenic long noncoding RNA on chromosome 17q21 are associated with asthma

Abstract

Background: The genetic and molecular basis of asthma remains unclear and its gene-environment interaction is still enigmatic. In the present study, we aimed to identify asthma-causing genetic variants and their interactions with the environment.

Methods: We performed case-control genome-wide association studies on individuals of Han Chinese descent from the Taiwan Biobank (n=4877 cases, n=98 218 controls) to identify asthma susceptibility loci, validated in a hospital-based population of subjects (n=2595). The 10-15-year exposures to cumulative ambient particulate matter with a diameter of <2.5 μm (PM_2.5) and polycyclic aromatic hydrocarbons (PAHs) were assessed for gene-environment relationships. The function of the newly identified long noncoding RNA lncZPBP2-3 and its interactions with PM_2.5 and PAH exposure were analysed using RNA immunoprecipitation, RNA pulldown, reverse transcription quantitative PCR and Western blotting.

Results: Chromosome 17q12-21 was found to be a significant risk region, encompassing variants of lncZPBP2-3 and its neighbouring genes, which interacted with increasing exposure to PM_2.5 and adsorbed PAHs. The expression of lncZPBP2-3 was elevated, correlating with the expression of its neighbouring genes, in the peripheral blood of patients with asthma compared to that in controls. Unlike non-risk lncZPBP2-3, the risk variant of lncZPBP2-3 disrupted transcriptional suppression of the risk locus via its interaction with the transcription insulator CCCTC-binding factor, concomitant with the higher expression levels of neighbouring genes in individuals with the risk genotype.

Conclusion: A functional variant of lncRNA, lncZPBP2-3, was significantly associated with asthma and is inducible by environmental PAH, suggesting a potentially novel genetic and molecular mechanism of asthma.

Overview of the study. 95% CI: 95% confidence interval; AHR: aryl hydrocarbon receptor; aOR: adjusted odds ratio; ChIP: chromatin immunoprecipitation; CTCF: CCCTC-binding factor; GSDMB: gasdermin B; lncRNA: long noncoding RNA; OR: odds ratio; ORMDL3: ORMDL sphingolipid biosynthesis regulator 3; PAH: polycyclic aromatic hydrocarbon; PBMC: peripheral blood mononuclear cell; PM_2.5: particulate matter with a diameter of <2.5 μm; PRC2: polycomb repressive complex 2; RT-qPCR: reverse transcription quantitative PCR; SNP: single nucleotide polymorphism; XRE: xenobiotic response element; ZPBP2: zona pellucida binding protein 2. ^#: models were adjusted for age, sex and principal components 1–10. **: p-value of Wald chi-square <0.005; ***: p-value of Wald chi-square <0.0001.

FIGURE 1

Overall study design. We performed a case–control genome-wide association study (GWAS) on individuals of Han Chinese descent and identified an asthma susceptibility locus in the 17q21 region. We then examined the interaction between this locus and the 10–15-year cumulative exposure to particulate matter with a diameter of <2.5 μm (PM_2.5) and polycyclic aromatic hydrocarbons (PAHs) for each individual to assess gene–environment interaction. Further, with a combination of human cohorts and cell models, we found the sequence variants of a novel long noncoding RNA (lncRNA), lncZPBP2-3, at 17q21 locus, inducible by environmental PAHs, were significantly associated with asthma, with significant gene–environment interaction and able to differentially regulate the expression of neighbouring genes, including gasdermin B, via epigenetic mechanisms. aOR: adjusted odds ratio; chr: chromosome; PBMC: peripheral blood mononuclear cell; SNP: single nucleotide polymorphism. ^#: models were adjusted for age, sex and principal components 1–10. **: p-value of Wald chi-square <0.005; ***: p-value of Wald chi-square <0.0001.

FIGURE 2

Genome-wide association study (GWAS) reveals a novel locus for asthma located at chromosome 17q12-21. a) Manhattan plots showing GWAS results for the first cohort (n=2559 asthma cases and 51 174 controls, upper portion) and the second cohort (n=2318 cases and 47 044 controls, lower portion) cohort. The y-axes show the −log10(p-values) from the GWASs. The x-axis shows the position of each single nucleotide polymorphism (SNP) along the 22 autosomes. The red line indicates a genome-wide significance threshold of 5×10⁻⁸. b) Common haplotypes at the 17q12-q21 locus with four genes and their relative locations at the extended locus (chr17: chr17: 39 865 000–39 930 000; build hg38). The core region haplotypes, consisting of five SNPs, are displayed along with their respective positions relative to each gene. The frequencies of these haplotypes, the allelic composition of the five variants, and the outcomes of logistic regression analysis for haplotypes with frequencies ≥0.01 are presented. 95% CI: 95% confidence interval; chr: chromosome; GSDMB: gasdermin B; HLA: human leukocyte antigen; lnc: long noncoding; OR: odds ratio; ORMDL3: ORMDL sphingolipid biosynthesis regulator 3; ZPBP2-3: zona pellucida binding protein 2-3.

FIGURE 3

Upregulated expression of long noncoding zona pellucida binding protein 2-3 (lncZPBP2-3), gasdermin B (GSDMB), ZPBP2 and ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) in patients with asthma versus controls. a) Relative mRNA expression levels of lncZPBP2-3, ZPBP2, GSDMB and ORMDL3 in peripheral blood mononuclear cells of controls (n=44) and patients with asthma (n=60) as determined by reverse transcription quantitative PCR (RT-qPCR). Correlation between the lncZPBP2-3 expression levels and GSDMB, ZPBP2 or ORMDL3 expression levels in b) patients with asthma or c) controls. Relative mRNA expression levels of GSDMB, ZPBP2 and ORMDL3 in THP-1-derived macrophages with d) lncZPBP2-3 knockdown (lncZPBP2-3 small interfering RNA (siRNA)) or e) lncZPBP2-3 overexpression as detected by RT-qPCR. In panel d, the labels 1 and 2 represent two distinct siRNA sequences targeting the same gene to ensure knockdown specificity/reproducibility. n=3 in each group. Data are shown as the mean±sem and statistical significance was determined by one-way ANOVA followed by Bonferroni correction for multiple comparisons. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 4

The expression of long noncoding RNA (lncRNA) is inducible by the polycyclic aromatic hydrocarbon (PAH)–aryl hydrocarbon receptor (AHR) axis and required for the expression of its neighbouring genes. a) The mRNA expression levels of lncRNA zona pellucida binding protein 2-3 (lncZPBP2-3), gasdermin B (GSDMB), zona pellucida binding protein 2-3 (ZPBP2), ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1) in THP-1-derived macrophages treated with indeno-(1,2,3-cd)-pyrene (IP) were measured by reverse transcription quantitative PCR (RT-qPCR). b) The mRNA expression levels of GSDMB, ZPBP2, ORMDL3 and CYP1B1 in AHR or lncZPBP2-3-knockdown THP-1-derived macrophages with IP treatment were measured by RT-qPCR. n=3 in each group. Data are shown as the mean±sem and statistical significance was determined by ANOVA followed by Bonferroni correction for multiple comparisons. siRNA: small interfering RNA. *: p<0.05, **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 5

Aryl hydrocarbon receptor (AHR) targets xenobiotic response element (XRE) sequence in the promoter region of long noncoding zona pellucida binding protein 2-3 (lncZPBP2-3). a) Chromatin immunoprecipitation (ChIP) assay of the AHR-binding levels at the lncZPBP2-3 promoter region in control, indeno-(1,2,3-cd)-pyrene (IP)-treated THP-1-derived macrophages, which were probed with anti-AHR antibodies and IgG isotype control (supplementary material). b) ChIP assay of the CCCTC-binding factor (CTCF)-binding levels at the 17q12-21 region in control, THP-1-derived macrophages with overexpression (O/E) of lncZPBP2-3, which were probed with anti-CTCF antibodies, and IgG isotype control (supplementary material). c) RNA immunoprecipitation assay of CTCF and lncZPBP2-3 interaction. U1 small nuclear RNA (U1 snRNA) served as a nonspecific control. d) ChIP assay of the CTCF-binding levels at the 17q12-21 region in control, IP-treated THP-1-derived macrophages. n=3 in each group. Data are shown as the mean±sem and statistical significance was determined by Bonferroni correction for multiple comparisons. TSS: transcription start site. **: p<0.01; ***: p<0.001; ****: p<0.0001.

FIGURE 6

Risk versus non-risk long noncoding zona pellucida binding protein 2-3 (lncZPBP2-3) interaction with CCCTC-binding factor (CTCF) complex. a) THP-1-derived macrophages overexpressing risk or non-risk lncZPBP2-3 variants were examined for the interaction of CTCF and lncZPBP2-3 in vitro by RNA immunoprecipitation and reverse transcription quantitative PCR (RT-qPCR). b) Chromatin immunoprecipitation assay of the CTCF-binding levels at the 17q12-21 region in control, THP-1-derived macrophages with lncZPBP2-3 or lncZPBP2-3 mutant (mut) expression and IgG isotype control. n=3 in each group. Data are shown as the mean±sem and statistical significance determined by ANOVA followed by Bonferroni's multiple comparisons test. c) Relative mRNA expression levels of gasdermin B (GSDMB) and ZPBP2 in peripheral blood mononuclear cells of controls and patients with asthma with different lncZPBP2-3 genotypes as determined by RT-qPCR. Macrophage M2-associated genes were evaluated by RT-qPCR in THP-1-derived macrophages with d) lncZPBP2-3 knockdown, e) GSDMB knockdown or f) ZPBP2 knockdown. n=3 in each group. Data are shown as the mean±sem and statistical significance was determined by ANOVA followed by Bonferroni correction for multiple comparisons. CD206: cluster of differentiation 206; CLEC7A: C-type lectin domain containing 7A; IL-4: interleukin 4; siRNA: small interfering RNA. ns: nonsignificant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.