Journal
JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY
Volume 142, Issue 3, Pages 959-969Publisher
MOSBY-ELSEVIER
DOI: 10.1016/j.jaci.2018.06.022
Keywords
Asthma; bioinformatics; diisocyanate; electrophoretic mobility shift assay; functional genomics; gene regulation; genetic; his tone; isocyanate; luciferase assay; next-generation sequencing; occupational asthma; oligonucleotide; single nucleotide polymorphism
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Funding
- National Institute for Occupational Safety and Health (NIOSH)/Centers for Disease Control and Prevention (CDC) grant [OH008795]
- National Institutes of Health (NIH) shared instrumentation grant [S10 RR027015-01]
- NIH [P30 AR070549, R21 HG008186]
- Cincinnati Children's Hospital Medical Center CpG Award [53553]
- Lupus Research Institute Novel Approaches Award
- NATIONAL HUMAN GENOME RESEARCH INSTITUTE [R21HG008186] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES [P30AR070549] Funding Source: NIH RePORTER
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Background: Isocyanates are major causes of occupational asthma, but susceptibility and mechanisms of diisocyanate-induced asthma (DA) remain uncertain. Objective: The aim of this study was to identify DA-associated functional genetic variants through next-generation sequencing (NGS), bioinformatics, and functional assays. Methods: NGS was performed in 91 workers with DA. Fourteen loci with known DA-associated single nucleotide polymorphisms (SNPs) were sequenced and compared with data from 238 unexposed subjects. Ranking of DA-associated SNPs based on their likelihood to affect gene regulatory mechanisms in the lung yielded 21 prioritized SNPs. Risk and nonrisk oligonucleotides were tested for binding of nuclear extracts from A549, BEAS-2B, and IMR-90 lung cell lines by using electrophoretic mobility shift assays. DNA constructs were cloned into a pGL3 promoter vector for luciferase gene reporter assays. Results: NGS detected 130 risk variants associated with DA (3.1 x 10-6 to 6.21 X 10-i), 129 of which were located in noncoding regions. The 21 SNPs prioritized by using functional genomic data sets were in or proximal to 5 genes: cadherin 17 (CDH17; n = 10), activating transcription factor 3 (ATF3; n = 7), family with sequence similarity, member A (FAM71A; n = 2), tachykinin receptor 1 (TACR1; n = 1), and zinc finger and BTB domain-containing protein 16 (ZBTB16; n = 1). Electrophoretic mobility shift assays detected allele-dependent nuclear protein binding in A549 cells for 8 of 21 variants. In the luciferase assay 4 of the 21 SNPs exhibited allele-dependent changes in gene expression. DNA affinity precipitation and mass spectroscopy of rs147978008 revealed allele-dependent binding of H1 histones, which was confirmed by using Western blotting. Conclusions: We identified 5 DA-associated potential regulatory SNPs. Four variants exhibited effects on gene regulation (ATF rs11571537, CDH17 rs2446824 and rs2513789, and TACR1 rs2287231). A fifth variant (FAM71A rs147978008) showed nonrisk allele preferential binding to H1 histones. These results demonstrate that many DA-associated genetic variants likely act by modulating gene regulation.
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