Genetic Variants with Gene Regulatory Effects are Associated with Diisocyanate Asthma

Publication date: Available online 30 June 2018
Source:Journal of Allergy and Clinical Immunology
Author(s): David I. Bernstein, Zana L. Lummus, Banu Kesavalu, Jianbo Yao, Leah Kottyan, Daniel Miller, André Cartier, Maria-Jesús Cruz, Catherine Lemiere, Xavier Muñoz, Santiago Quirce, Susan Tarlo, Joaquin Sastre, Louis Philippe Boulet, Matthew T. Weirauch, Kenneth Kaufman
BackgroundIsocyanates are major causes of occupational asthma but susceptibility and mechanisms of Diisocyanate Asthma (DA) remain uncertain.ObjectiveThe aim of this study is to identify DA-associated functional genetic variants via next generation sequencing (NGS), bioinformatics, and functional assays.MethodsNGS 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.ResultsNGS 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.ConclusionsWe 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.

Teaser

This study advances knowledge of genetic mechanisms of occupational asthma by identification of a role for regulatory genetic variants associated with diisocyanate-induced asthma.


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