Genetic variants with gene regulatory effects are associated with diisocyanate-induced asthma - 06/09/18
, Zana L. Lummus, PhD a, Banu Kesavalu, MS a, Jianbo Yao, PhD b, Leah Kottyan, PhD c, d, Daniel Miller, BS c, André Cartier, MD, FAAAAI e, Maria-Jesús Cruz, MD f, Catherine Lemiere, MD e, Xavier Muñoz, MD f, Santiago Quirce, MD, PhD g, Susan Tarlo, MD, FAAAAI h, Joaquin Sastre, MD, PhD, FAAAAI i, Louis Philippe Boulet, MD j, Matthew T. Weirauch, PhD c, d, k, Kenneth Kaufman, PhD c, d, lAbstract |
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 × 10−6 to 6.21 × 10−4), 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.
Le texte complet de cet article est disponible en PDF.Key words : Asthma, bioinformatics, diisocyanate, electrophoretic mobility shift assay, functional genomics, gene regulation, genetic, histone, isocyanate, luciferase assay, next-generation sequencing, occupational asthma, oligonucleotide, single nucleotide polymorphism
Abbreviations used : ATF3, CDH17, CEBPB, ChIP-Seq, DA, DAPA, EMSA, FAM71A, GWAS, MAF, Nano-LC-MS/MS, NGS, OA, SIC, SNP, TACR1, ZBTB16
Plan
| Supported by National Institute for Occupational Safety and Health (NIOSH)/Centers for Disease Control and Prevention (CDC) grant OH008795 (PI: D. I. Bernstein); a National Institutes of Health (NIH) shared instrumentation grant (S10 RR027015-01; PI: K. D. Greis); NIH grant P30 AR070549 for Cincinnati Children's Hospital Medical Center Functional Genomics Core; Cincinnati Children's Hospital Medical Center CpG Award 53553 (PI: M. T. Weirauch); a Lupus Research Institute “Novel Approaches” Award (PI: M. T. Weirauch); and NIH grant R21 HG008186 (PI: M. T. Weirauch). |
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| Disclosure of potential conflict of interest: D. I. Bernstein has received research support from National Institute for Occupational Safety and Health (NIOSH)/Centers for Disease Control and Prevention (CDC) and consultancy fees from the International Isocyanate Institute outside the submitted work. A. Cartier received personal fees and other from AZ Pharma Canada, TEVA Pharma Canada, Sanofi Genzyme Canada, Boehringer Ingelheim Pharma Canada, GlaxoSmithKline Pharma Canada, and IC-EBM Canada outside the submitted work. C. Lemiere received grants from Institut de Recherche Robert Sauvé en santé et sécurité au Travail and grants from the Canadian Institutes of Health Research outside the submitted work. S. Tarlo has received reimbursement from the NIOSH/CDC for this study and support from grants during 2003-2018 (Workplace Safety & Insurance Board, Network of Centres of Research Expertise “Centre of Research Expertise in Occupational Disease [CRE-OD]” and a Verma Grant outside the submitted work and has a patent and book editor royalties with royalties paid. J. Sastre received payment from a National Institutes of Health grant and personal fees from Sanofi, Novartis, AstraZeneca, ALK-Abelló, and LETI outside the submitted work. L. P. Boulet has received payments for participation in multicenter research studies from AIM Therapeutics, Amgen, Asmacure, AstraZeneca, Axikin, GlaxoSmithKline, Hoffman La Roche, Novartis, Ono Pharma, Sanofi, and Takeda; research support from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck, and Takeda; consultancy fees from AstraZeneca, Novartis, and Methapharm; royalties for coauthorship of UpToDate (occupational asthma); grants for production of educational materials from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Merck Frosst, and Novartis; and conference fees from AstraZeneca, GlaxoSmithKline, Merck, Novartis, and Takeda. The rest of the authors declare that they have no relevant conflicts of interest. |
Vol 142 - N° 3
P. 959-969 - septembre 2018 Retour au numéro
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