Abstract

BACKGROUND:
Isocyanates are major causes of occupational asthma but susceptibility and mechanisms of Diisocyanate Asthma (DA) remain uncertain.

OBJECTIVE:
The aim of this study is to identify DA-associated functional genetic variants via 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 to 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 (R) and non-risk (NR) oligonucleotides were tested for binding of nuclear extracts from A549, BEAS 2B, and IMR-90 lung cell lines by electrophoretic mobility shift assays (EMSA). 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 x10-6 - 6.21x10-4), of which 129 were located in non-coding regions. The 21 SNPs prioritized by functional genomic data sets were in or proximal to five genes: CDH17 (n=10), ATF3 (n=7), FAM71A (n=2), TACR1 (n=1), and ZBTB16 (n=1). EMSA 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 Western blot.

CONCLUSIONS:
We identified five DA associated potential regulatory SNPs. Four variants exhibited effects on gene regulation (ATF rs11571537, CDH17 rs2446824, rs2513789, and TACR1 rs2287231). A fifth variant (FAM71A rs147978008) showed non-risk 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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