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

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.

Comparison of biological effects with albumin adducts of 4,4'-methylenediphenyl diisocyanate in workers.

Lung sensitization and asthma are the main health effects of 4,4'-methylenediphenyl diisocyanate (MDI). Albumin adducts (isocyanate-specific adducts) of MDI might be involved in the etiology of sensitization reactions. Albumin adducts of MDI were analyzed in sera of diisocyanate-exposed worker with and without diisocyanate occupational asthma (DA), as well as in exposed workers with and without diisocyanate-specific IgG antibodies. In DA-positive workers and IgG-positive workers, albumin adducts were significantly higher versus workers without DA and those who were specific IgG negative. The odds ratio to be DA-positive was 57 times larger for workers with adduct levels above 230 fmol/mg. The odds ratio to be IgG-positive was 10 times larger for workers with adduct levels above 113 fmol/mg. Therefore, albumin adducts appear to be a good predictor of the biological effects. The albumin-adduct levels in workers without biological effects were in the range of the adduct levels found in previous studies of healthy MDI-factory and construction site workers.

Genetic variants in TNFα, TGFB1, PTGS1 and PTGS2 genes are associated with diisocyanate-induced asthma.

Diisocyanates are the most common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants in inflammatory response genes (TNFα, IL1α, IL1ß, IL1RN, IL10, TGFB1, ADAM33, ALOX-5, PTGS1, PTGS2 and NAG-1/GDF15) are associated with increased susceptibility to diisocyanate asthma (DA). These genes were selected based on their role in asthmatic inflammatory processes and previously reported associations with asthma phenotypes. The main study population consisted of 237 Caucasian French Canadians from among a larger sample of 280 diisocyanate-exposed workers in two groups: workers with specific inhalation challenge (SIC) confirmed DA (DA(+), n = 95) and asymptomatic exposed workers (AW, n = 142). Genotyping was performed on genomic DNA, using a 5' nuclease PCR assay. After adjusting for potentially confounding variables of age, smoking status and duration of exposure, the PTGS1 rs5788 and TGFB1 rs1800469 single nucleotide polymorphisms (SNP) showed a protective effect under a dominant model (OR = 0.38; 95% CI = 0.17, 0.89 and OR = 0.38; 95% CI = 0.18, 0.74, respectively) while the TNFα rs1800629 SNP was associated with an increased risk of DA (OR = 2.08; 95% CI = 1.03, 4.17). Additionally, the PTGS2 rs20417 variant showed an association with increased risk of DA in a recessive genetic model (OR = 6.40; 95% CI = 1.06, 38.75). These results suggest that genetic variations in TNFα, TGFB1, PTGS1 and PTGS2 genes contribute to DA susceptibility.

N-Acetyltransferase 2 Genotypes Are Associated With Diisocyanate-Induced Asthma.

OBJECTIVE: To investigate whether genetic variants of N-acetyltransferase (NAT) genes are associated with diisocyanate asthma (DA). METHODS: The study population consisted of 354 diisocyanate-exposed workers. Genotyping was performed using a 5'-nuclease polymerase chain reaction assay. RESULTS: The NAT2 rs2410556 and NAT2 rs4271002 variants were significantly associated with DA in the univariate analysis. In the first logistic regression model comparing DA+ and asymptomatic worker groups, the rs2410556 (P = 0.004) and rs4271002 (P < 0.001) single nucleotide polymorphisms and the genotype combination, NAT2 rs4271002*NAT1 rs11777998, showed associations with DA risk (P = 0.014). In the second model comparing DA+ and DA- groups, NAT2 rs4271002 variant and the combined genotype, NAT1 rs8190845*NAT2 rs13277605, were significantly associated with DA risk (P = 0.022, P = 0.036, respectively). CONCLUSIONS: These findings suggest that variations in the NAT2 gene and their interactions contribute to DA susceptibility.

Genome-Wide Association Study Identifies Novel Loci Associated With Diisocyanate-Induced Occupational Asthma.

Diisocyanates, reactive chemicals used to produce polyurethane products, are the most common causes of occupational asthma. The aim of this study is to identify susceptibility gene variants that could contribute to the pathogenesis of diisocyanate asthma (DA) using a Genome-Wide Association Study (GWAS) approach. Genome-wide single nucleotide polymorphism (SNP) genotyping was performed in 74 diisocyanate-exposed workers with DA and 824 healthy controls using Omni-2.5 and Omni-5 SNP microarrays. We identified 11 SNPs that exceeded genome-wide significance; the strongest association was for the rs12913832 SNP located on chromosome 15, which has been mapped to the HERC2 gene (p = 6.94 × 10(-14)). Strong associations were also found for SNPs near the ODZ3 and CDH17 genes on chromosomes 4 and 8 (rs908084, p = 8.59 × 10(-9) and rs2514805, p = 1.22 × 10(-8), respectively). We also prioritized 38 SNPs with suggestive genome-wide significance (p < 1 × 10(-6)). Among them, 17 SNPs map to the PITPNC1, ACMSD, ZBTB16, ODZ3, and CDH17 gene loci. Functional genomics data indicate that 2 of the suggestive SNPs (rs2446823 and rs2446824) are located within putative binding sites for the CCAAT/Enhancer Binding Protein (CEBP) and Hepatocyte Nuclear Factor 4, Alpha transcription factors (TFs), respectively. This study identified SNPs mapping to the HERC2, CDH17, and ODZ3 genes as potential susceptibility loci for DA. Pathway analysis indicated that these genes are associated with antigen processing and presentation, and other immune pathways. Overlap of 2 suggestive SNPs with likely TF binding sites suggests possible roles in disruption of gene regulation. These results provide new insights into the genetic architecture of DA and serve as a basis for future functional and mechanistic studies.

Genetic variants in the major histocompatibility complex class I and class II genes are associated with diisocyanate-induced Asthma.

OBJECTIVE: To investigate the association between single nucleotide polymorphisms (SNPs) located across the major histocompatibility complex and susceptibility to diisocyanate-induced asthma (DA). METHODS: The study population consisted of 140 diisocyanate-exposed workers. Genotyping was performed using the Illumina GoldenGate major histocompatibility complex panels. RESULTS: The HLA-E rs1573294 and HLA-DPB1 rs928976 SNPs were associated with an increased risk of DA under dominant (odds ratio [OR], 6.27; 95% confidence interval [CI], 2.37 to 16.6; OR, 2.79, 95% CI, 0.99 to 7.81, respectively) and recessive genetic models (OR, 6.27, 95% CI, 1.63 to 24.13; OR, 10.10, 95% CI, 3.16 to 32.33, respectively). The HLA-B rs1811197, HLA-DOA rs3128935, and HLA-DQA2 rs7773955 SNPs conferred an increased risk of DA in a dominant model (OR, 7.64, 95% CI, 2.25 to 26.00; OR, 19.69, 95% CI, 2.89 to 135.25; OR, 8.43, 95% CI, 3.03 to 23.48, respectively). CONCLUSION: These results suggest that genetic variations within HLA genes play a role in DA risk.

Interferon-γ promoter is hypermethylated in blood DNA from workers with confirmed diisocyanate asthma.

Risk factors have not been identified that determine susceptibility for development of diisocyanate-induced occupational asthma (DA). We hypothesized that diisocyanate (DI) exposure could modify gene promoter regions regulating transcription of cytokine mediators and thereby influence expression of DA. A cross-sectional study was designed to investigate the promoter methylation status of candidate genes in DI-exposed workers. Subjects consisted of 131 workers in three groups: 40 cases with DA confirmed by a positive specific inhalation challenge (SIC) (DA+), 41 exposed workers with lower respiratory symptoms and negative SIC (DA-), and 50 asymptomatic exposed workers (AWs). We studied four candidate genes (GSTM1, DUSP22, IFN-γ, and IL-4) for which altered promoter methylation has been previously investigated for relationships with a variety of other environmental exposures. Methylation status was determined using methylation-specific quantitative PCR performed on genomic DNA extracted from whole blood. Results showed that relative methylation of IFN-γ promoter was significantly increased in DA+ in comparison with both comparator groups (DA- and AW), and it exhibited good sensitivity (77.5%) and specificity (80%) for identifying DA workers in a multivariate predictive model after adjusting for type of DI exposure, smoking status, methacholine PC20, and gender. IL-4 promoter was slightly less methylated only in DA+ compared with AW among nonsmoking workers. Both GSTM1 and DUSP22 promoter methylations were found not associated with DA. Our finding suggests that exposure to occupational chemicals could play a heretofore undefined mechanistic role via epigenetic modification of specific genes in the promoter region.

CTNNA3 (α-catenin) gene variants are associated with diisocyanate asthma: a replication study in a Caucasian worker population.

Recently, a genome-wide association study (GWAS) conducted in Korean subjects identified four CTNNA3 (alpha-T catenin) single nucleotide polymorphisms (SNPs) (rs10762058, rs7088181, rs1786929, and rs4378283) associated with diisocyanate-induced occupational asthma (DA). The CTNNA3 gene codes for a cadherin involved in formation of stretch-resistant cell-cell adhesions. We conducted a candidate gene association study to replicate these findings in Caucasian workers. Genotyping was performed on DNA using a 5' nuclease PCR assay collected from 410 diisocyanate-exposed and predominantly Canadian workers including 132 workers with DA confirmed by a specific inhalation challenge (DA+); 131 symptomatic workers in whom DA was excluded by a negative challenge (DA-); and 147 hexamethylene diisocyanate-exposed asymptomatic workers (AWs). As in the Korean study, highly linked CTNNA3 rs7088181 and rs10762058 SNPs (but not rs4378283 and rs1786929) were significantly associated with DA+ when compared with AWs but not in comparison with DA- workers (p ≤ 0.05). After adjusting for potentially confounding variables of age, smoking status, and duration of exposure, minor allele homozygotes of rs7088181 and rs10762058 SNPs were at increased risk for DA compared with AWs (OR = 9.05 [95% CI: 1.69, 48.54] and OR = 6.82 [95% CI: 1.65, 28.24], respectively). In conclusion, we replicated results from the only reported GWAS study of DA demonstrating an association between two closely linked CTNNA3 gene SNPs and DA. These findings lend further support to the clinical relevance of these genotypes in predicting susceptibility to DA and the potential importance of catenins in the disease process.

Genetic variants in antioxidant genes are associated with diisocyanate-induced asthma.

Diisocyanates are a common cause of occupational asthma, but risk factors are not well defined. A case-control study was conducted to investigate whether genetic variants of antioxidant defense genes, glutathione S-transferases (GSTM1, GSTT1, GSTM3, GSTP1), manganese superoxide dismutase (SOD2), and microsomal epoxide hydrolase (EPHX1) are associated with increased susceptibility to diisocyanate-induced asthma (DA). The main study population consisted of 353 Caucasian French-Canadians from among a larger sample of 410 diisocyanate-exposed workers in three groups: workers with specific inhalation challenge (SIC) confirmed DA (DA(+), n = 95); symptomatic diisocyanate workers with a negative SIC (DA(-), n = 116); and asymptomatic exposed workers (AW, n = 142). Genotyping was performed on genomic DNA, using a 5'-nuclease PCR assay. The SOD2 rs4880, GSTP1 rs1695, and EPHX1 rs2740171 variants were significantly associated with DA in both univariate and multivariate analyses. In the first logistic regression model comparing DA(+) and DA(-) groups, SOD2 rs4880, GSTM1 (null), GSTP1 rs762803, and EPHX1 rs2854450 variants were associated with DA (p = 0.004, p = 0.047, p = 0.021, p <0.001, respectively). Genotype combinations GSTT1*GSTP1 rs762803, GSTM1*EPHX1 rs2854450, EPHX1 rs2740168*EPHX1 rs1051741, and GSTP1 rs762803*EPHX1 rs2740168 were also associated with DA in this model (p = 0.027, p = 0.002, p = 0.045, p = 0.044, respectively). The GSTP1 rs1695 and EPHX1 rs1051741 and rs2740171 variants showed an association with DA in the second model comparing DA(+) and AW groups (p = 0.040, p = 0.019, p = 0.002, respectively). The GSTM3 rs110913*EPHX1 rs1051741 genotype combination was also associated with DA under this model (p = 0.042). The results suggest that variations in SOD2, GST, and EPHX1 genes and their interactions contribute to DA susceptibility.

Pathogenesis and disease mechanisms of occupational asthma.

Occupational asthma (OA) is one of the most common forms of work-related lung disease in all industrialized nations. The clinical management of patients with OA depends on an understanding of the multifactorial pathogenetic mechanisms that can contribute to this disease. This article discusses the various immunologic and nonimmunologic mechanisms and genetic susceptibility factors that drive the inflammatory processes of OA.

A case of progesterone-induced anaphylaxis, cyclic urticaria/angioedema, and autoimmune dermatitis.

OBJECTIVE: Women have exhibited anaphylaxis, urticaria/angioedema, and autoimmune progesterone dermatitis (APD) coinciding with the progesterone premenstrual rise. We report a detailed immunological evaluation of such a woman responsive to a gonadotropin hormone-releasing agonist (GHRA). METHODS: Skin testing, enzyme-linked immunosorbent assays (ELISAs), leukocyte histamine release (LHR), and inhibition assays were performed to demonstrate progesterone immunoresponsiveness. RESULTS: Serum specific-progesterone immunoglobulin G (IgG) and IgE were detected initially and disappeared 6 months after GHRA treatment. Dose-response LHR using patient basophils was observed for different hormones but after 3 months persisted only for 5ß-pregnanediol. Preincubation with mouse antiprogesterone monoclonal antibody (PmAb) or mifepristone, a progesterone inhibitor, over a range of doses inhibited specific progesterone-induced LHR. Experiments with varying progesterone concentrations and a fixed dose of anti-IgE resulted in 100% LHR at a concentration as low as 0.016 nmol/mL, which, without anti-IgE, failed to release histamine. CONCLUSIONS: This is the first report of combined recurrent anaphylaxis, cyclic urticaria/angioedema, and APD induced by immunoresponsiveness to progesterone.

Communities near toluene diisocyanate sources: an investigation of exposure and health.

Toluene diisocyanate (TDI) is a well-known cause of occupational asthma, but we know little about the potential for exposure and health effects among residents who live near facilities that release TDI. In the mid-1990's, the North Carolina Department of Health and Human Services and the Agency for Toxic Substances and Disease Registry investigated exposures to TDI and health outcomes in one community, which left some unanswered questions. This cross-sectional study evaluated the potential associations between living near a TDI source and the prevalence of three variables: asthma or asthma-like respiratory symptoms, antibodies specific to TDI, and verifiable levels of TDI in residential air. Results among North Carolina residents living near such facilities (five target communities) were compared with the results from residents living further away (five comparison communities). Overall, the prevalence of reporting either asthma or asthma-like respiratory symptoms was higher (odds ratio = 1.60; 95% confidence interval = 0.97-2.54) among residents in target communities than those in comparison communities. However, this difference was not statistically significant. Symptom prevalence varied greatly among the community populations. The prevalence of respiratory symptoms was higher near facilities with historically higher TDI emissions. Among the 351 participants who provided blood samples, only one had immunoglobulin G specific antibodies to TDI. This participant lived in a target area and may have had non-occupational exposure. TDI was detected at an extremely low level (1 ppt) in one of the 45 air samples from target communities. One ppt is one-tenth the EPA reference concentration. Overall, air sample and antibody test results are not consistent with recent or ongoing exposure to TDI.

Influence of dog ownership and high endotoxin on wheezing and atopy during infancy.

BACKGROUND: Increased exposure to microbial products early in life may protect from development of atopic disorders in childhood. Few studies have examined the relationship of endotoxin exposure and pet ownership on atopy and wheezing during infancy. OBJECTIVE: Evaluate relationships among high endotoxin exposure, pet ownership, atopy, and wheezing in high-risk infants. METHODS: Infants (n = 532; mean age, 12.5 +/- 0.8 months) with at least 1 parent with confirmed atopy were recruited. A complete medical history and skin prick testing to foods and aeroallergens were performed at age 1 year. House dust samples were analyzed for endotoxin. RESULTS: Prevalences of wheezing were not independently associated with dog or cat ownership or endotoxin levels. Percutaneous reactivity to at least 1 allergen was observed in 28.6% of infants. Univariate analyses showed significant associations of any wheezing, recurrent wheezing, and recurrent wheezing with an event with daycare attendance, number of siblings, respiratory infections, maternal smoking, and history of parental asthma. Logistic regression adjusting for the latter variables showed that recurrent wheezing (odds ratio, 0.4; 95% CI, 0.1-0.9) as well as 2 other wheeze outcomes were significantly reduced in homes with high endotoxin exposure in the presence of 2 or more dogs. CONCLUSION: Pet ownership or endotoxin did not independently modify aeroallergen sensitization or wheezing during infancy. However, high endotoxin exposure in the presence of multiple dogs was associated with reduced wheezing in infants. CLINICAL IMPLICATIONS: A home environment with many dogs and high levels of endotoxin may be conducive to reduced wheezing in infancy.

Advances in methods used in evaluation of occupational asthma.

PURPOSE OF REVIEW: Diagnosing occupational asthma (OA) is a complex undertaking, the primary goal of which is to demonstrate a causal relation between exposure to a specific agent encountered at work and asthmatic responses. Recent development or refinement of diagnostic tools may improve the diagnostic accuracy, which may have important economic and social consequences for both employers and workers. RECENT FINDINGS: Although specific inhalation challenge (SIC) testing is the gold standard for diagnosis of OA, these tests are not widely available in many countries. Thus, new less invasive techniques used in the measurement of airway inflammation, such as exhaled nitric oxide and induced sputum are highlighted as are recent developments in both in vivo and in vitro immunologic testing. SUMMARY: Although new perspectives are being evaluated, the diagnosis of occupational asthma still relies mostly on specific inhalation challenge. Further studies are required to confirm the utility of these new techniques in the diagnosis of OA.