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J Allergy Clin Immunol. 2018 Sep;142(3):959-969. doi: 10.1016/j.jaci.2018.06.022. Epub 2018 Jun 30.

Genetic variants with gene regulatory effects are associated with diisocyanate-induced asthma.

Author information

1
Division of Immunology, Allergy and Rheumatology, University of Cincinnati College of Medicine, Cincinnati, Ohio. Electronic address: bernstdd@ucmail.uc.edu.
2
Division of Immunology, Allergy and Rheumatology, University of Cincinnati College of Medicine, Cincinnati, Ohio.
3
Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, WV.
4
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio.
5
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
6
Department of Medicine, Université de Montréal, and Respiratory Division, Hôpital du Sacré-Coeur de Montréal, Montreal, Quebec, Canada.
7
Respiratory Department, Hospital Vall D'Hebron, Barcelona and CIBER de Enfermedades Respiratorias CIBERES, Madrid, Spain.
8
Department of Allergy, Hospital La Paz-IdiPAZ and CIBER de Enfermedades Respiratorias CIBERES, Madrid, Spain.
9
Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
10
Department of Allergy, Fundación Jiménez Díaz and CIBER de Enfermedades Respiratorias CIBERES, Madrid, Spain.
11
Institut Universitaire de Cardiology et de Pneumologie de Quebec, Université Laval, Hôpital Laval, Sainte-Foy, Quebec, Canada.
12
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
13
Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; Cincinnati VA Medical Center, Cincinnati, Ohio.

Abstract

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.

KEYWORDS:

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

PMID:
29969634
DOI:
10.1016/j.jaci.2018.06.022
[Indexed for MEDLINE]

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