Regulatory Variants of ATF3, CDH17 and FAM71A are Risk Factors for Diisocyanate Induced Occupational Asthma (DA)

Using Next Generation Sequencing (NGS) we identified non-coding SNPs associated with confirmed DA that might impact gene expression via transcription factor (TF) binding interactions. DNA was collected from 91 diisocyanate exposed workers with confirmed DA. Fourteen loci containing DA associated SNPs were sequenced and compared to 293 subjects from the 1,000 genomic (1KG) data set. 22 top-ranked SNPs associated with DA (X2 p< 1 x 10-3) underwent transcriptomic analysis. Electrophoretic mobility shift assays (EMSA) identified oligonucleotide-protein binding for risk and non-risk SNPs to nuclear extracts of A549, BEAS 2B, and IMR-90 lung cell lines. Oligonucleotide bound proteins were purified by DNA affinity precipitation assays (DAPA) and eluted proteins were identified by mass spectrometry (MS). A luciferase reporter assay, in A549 cells transfected with luciferase reporter constructs, identified allele-dependent mRNA transcription. The 22 top-ranked SNPs identified in non-coding regions included: CDH17 (10), ATF3 (7), FAM71A (2), PITPNC1, TACR1, ZBTB16. EMSA detected A549 nuclear protein binding to 10 of the 22 variants, with 8 displaying preferential binding to non-risk alleles (rs1001304, rs2287231,rs2513789, rs2513791, rs11571537, rs14798008) or risk alleles (rs2513788, rs2513790). MS revealed rs14798008 bound to H1 histones. Four SNPs exhibited allele-dependent increases in gene expression (rs2287231, rs2513789, rs11571537 and rs2446824). We identified 5 potential regulatory allele-dependent SNPs for DA. Four variants exhibited functional activity for dysregulation of gene transcription. The fifth variant, in the promoter region of FAM71A (rs14798008), showed non-risk allele preferential binding to H1 histones, that modulate chromosomal accessibility of regulatory proteins to DNA.