Occupational Asthma (OA) is the most common occupational lung disorder and diisocyanates the most common causes of OA. Despite many years of research, there are no reliable predictors of risk or susceptibility to diisocyanate asthma (DA). In our current candidate gene association study (NIOSH RO1), we replicated a Korean GWAS study by demonstrating that two SNP variants (rs10762058, rs7088181) of the CTNNA3 gene (coding for ?-catenin) are associated with DA in Caucasian workers. ?-T-catenin, ?- and ?-catenin are cytoplasmic proteins that complex with E-cadherin to form the epithelial junctional complex (EJC), critical in cell-cell adhesion and for maintaining epithelial integrity. In this renewal, we hypothesize that fine mapping of CTNNA3 loci and genotyping other proteins of the EJC will reveal genetic functional variants that define susceptibility for DA among exposed workers. To test this hypothesis, we will use sequence capture and next generation DNA sequencing of 170 kB of the CTNNA3 locus containing rs10762058 and rs7088181 to identify new candidate SNPs with higher allele frequencies in DA+ workers compared to comparator asymptomatic workers (Aim 1).
In Aim 2, we will genotype tagging SNPs (tSNP) within E-cadherin, ?, ? and ? catenin genes with an r2=0.8 and minor allele frequency e 0.1. These and SNPs identified by DNA sequencing will be tested for associations with confirmed DA (n=150) versus two comparator groups of 150 asymptomatic workers (AWs) exposed to methylene diphenyl diisocyanate (MDI) or hexamethylene diisocyanate (HDI). All DA-associated SNPs will be re-genotyped and replicated in a distinct background population of 73 Korean workers with DA and 100 AWs. Finally (Aim 3), the possible functional role of replicable DA associated SNPs will be studied by measuring myc-tagged CTNNA3 protein expression in A549 cells transfected with constructs containing different coding region SNPs. The allele-specific changes in the expression of CTNNA3 will be detected by Western blot analysis using anti-myc antibody. This novel approach can identify functional DA-associated genotypes that will enable identification of exposed workers at highest risk and define safe exposure levels for genetically susceptible workers.
In our published study, we have identified two SNP variants (rs10762058 and rs7088181) of the CTNNA3 locus (T-catenin gene) associated with diisocyanate asthma (DA). In this renewal, next generation DNA sequencing of 170 kB of the CTNNA3 locus containing these candidate SNPs will be performed in workers with DA and asymptomatic exposed workers, as will genotyping of all tagging SNPs of T-catenin and associated junctional protein (i.e., E- cadherin, and catenin) genes. DA associated SNPs will be evaluated for replicability in two separate background populations, and functional effects of replicable SNPs on junctional protein expression will be investigated in A549 epithelial cells transfected with constructs containing coding region SNPs.
|Yucesoy, Berran; Johnson, Victor J; Lummus, Zana L et al. (2014) Genetic variants in the major histocompatibility complex class I and class II genes are associated with diisocyanate-induced Asthma. J Occup Environ Med 56:382-7|
|Bernstein, David I; Kashon, Michael; Lummus, Zana L et al. (2013) CTNNA3 (*-catenin) gene variants are associated with diisocyanate asthma: a replication study in a Caucasian worker population. Toxicol Sci 131:242-6|
|Ouyang, Bin; Bernstein, David I; Lummus, Zana L et al. (2013) Interferon-? promoter is hypermethylated in blood DNA from workers with confirmed diisocyanate asthma. Toxicol Sci 133:218-24|
|Bernstein, David I; Kissling, Grace E; Khurana Hershey, Gurjit et al. (2011) Hexamethylene diisocyanate asthma is associated with genetic polymorphisms of CD14, IL-13, and IL-4 receptor ?. J Allergy Clin Immunol 128:418-20|
|Lummus, Zana L; Wisnewski, Adam V; Bernstein, David I (2011) Pathogenesis and disease mechanisms of occupational asthma. Immunol Allergy Clin North Am 31:699-716, vi|