Background

Chronic Rhinosinusitis (CRS) accompanied by asthma drastically affects the quality of life and socioeconomic condition of millions of people worldwide [1].The antioxidant system counters oxidative stress by a complex network of phase I and phase II xenobiotic metabolizing enzymes (XMEs). The efficiencies of XMEs also balance pro-inflammatory, anti-inflammatory and mucosal hyperresponsiveness in allergic diseases [2]. CYP1A are the most important phase I biotransforming enzymes that constitute a superfamily of heme proteins responsible for the oxidative metabolism of a vast number of exogenous and endogenous compounds [3]. The Glutathione S-transferase (GST) superfamily consisting of phase II XMEs protect cells from the damage caused by reactive oxygen species and are also involved in the deactivation of allergens. Several families of soluble GSTs referred to as alpha, mu (GSTM), pi (GSTP), theta (GSTT), sigma and kappa have been identified in humans. Homozygosity for a nonfunctional allele (GSTT1-null and GSTM 1 null genotype) results in the loss of enzyme activity [4, 5]. GSTP1 is more abundant in respiratory tissues than other GSTs and plays an important role in neutralising oxidative stress in response to allergen and environmental exposure [5].

Independent studies reported CRS to be associated with single and multiple nucleotide polymorphisms that modulate immunologic responses or airway inflammation to allergy and asthma [1]. Several important gene variants of CYP1A1, CYP1A2 and GST genes have been identified to cause variability in the enzyme activity. The most common gene variants of CYP1A1 are CYP1A1*m1 T3801C rs4646903, CYP1A1*m2 A2455G rs1048943, CYP1A1*m3 T3204C rs1048945 and CYP1A1*m4 C2453A rs1799814; CYP1A2 are CYP1A2*1C G3858A rs2069514, CYP1A2*1E T739G rs2069526 and CYP1A2*1F C163A rs762551 and GSTP1 A313G rs1605, GSTP1 C341T rs1799811, and null variants GSTM1 and GSTT1 [3,4,5]. Umamaheswaran et al., 2014 reported that the frequency of the mutant alleles of XME genes including CYP1A1 and GST genes were significantly different among populations and no studies have been conducted on CYP1A2 polymorphism in South Indian population so far [6]. To the best of our knowledge, no study has related CYP1A1 and CYP1A2 gene polymorphism to allergic CRS and its endotypes. With regard to GST genes for their risk for respiratory disease and allergies, evidence of an association has been suggested but results are not concordant and further investigations were suggested in this direction [7, 8]. Also, studies on the impact of XME genes in the inter-individual susceptibility to the development of asthma in allergic CRS are very meager. In view of the above, the present study aimed to investigate the role of XME gene variants, (Cytochrome P450 1A1, Cytochrome 1A2 and Glutathione S transferase—GSTT, GSTM & GSTP), in the etiology with and without asthma in allergic CRS subjects.

Methods

A case–control study was performed with a total of 826 adult subjects that includes 224 allergic CRS subjects with asthma, 252 allergic CRS subjects without asthma and 350 healthy controls from 2013 to 2020. All the study subjects were diagnosed and confirmed by ENT specialists at MAA ENT Hospital, Somajiguda, Hyderabad based upon the guidelines of European Position Paper on Rhinosinusitis and Nasal Polyps (EP3OS) and Global Initiative for Asthma (GINA) [9, 10]. A special case proforma was prepared as per the Global Allergy and Asthma European Network (GA2LEN) project. Patients with a high level of total IgE > 180 IU (ELISA) and allergic symptoms like nasal discharge, itching, and sneezing more than 5 times per day were classified as allergic CRS patients. Patients with symptoms of allergy, breathlessness and/or a cough were considered as allergic CRS subjects with asthma. The confirmation of asthma in allergic CRS subjects was also made by pulmonary function test. 12% increase in Forced Expiratory Volume 1 (FEV1) after 200 µg of salbutamol inhalation was considered as the cut-off criteria. Healthy age and sex matched subjects from the same geographical location, without any family history of allergy, asthma, and CRS were considered as controls. Cases with other syndromes such as cystic fibrosis, immunodeficiency diseases, immune-compromised conditions, nosocomial infections and allergic fungal sinusitis were excluded from the study. All patients provided informed written consent in accordance with the study protocol, and the study was approved by the institutional ethics committee.

Genotyping

Approximately 2 ml of whole blood was used for DNA extraction using salting out method [11]. Genotyping of SNPs of CYP1A1 C3801T rs4646903, CYP1A1 C2453A rs1799814, CYP1A1 A2455G rs1048943, CYP1A2 G3858A rs2069514, CYP1A2 C163A rs762551, CYP1A2 T739G rs2069526, GSTP1 A313G rs1605 and GSTP1 C341T rs1799811were performed by PCR–RFLP method and GSTM1 and GSTT1 null variants was done using the multiplex PCR [7, 8, 12,13,14,15,16,17]. PCR was carried out in a reaction mixture containing 1U of thermostable Taq DNA polymerase in a final volume of 25 μl. The purified DNA was used for RFLP using restriction enzymes (Fermentas, United Kingdom). The restriction digest products were run through 1.5–4% agarose gel electrophoresis with ethidium bromide and photographed under ultraviolet light. The PCR conditions, specific forward primer and reverse primer used and RFLP conditions are given in Table 1. Random PCR–RFLP retesting of approximately 10% of the samples per SNP was performed to find the concordance with the initial genotyping results. The genotyping results were scored by two independent investigators who had no idea whether the sample came from the cases or the control group.

Table 1 Primers, restriction enzymes and PCR–RFLP conditions for the CYP1A1, CYP1A2 and GST gene variants studied
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Statistical analysis

Using SPSS (version 21; IBM Corporation, Armonk, NY, USA) the genotype and allele frequencies of patients and controls were compared. Using an electronic calculator, genotype frequency distributions were tested for agreement with the Hardy–Weinberg equilibrium (HWE) [http://www.genes.org.uk/software/hardy-weinberg.html]. Statistical significance of the differences in frequency of alleles, genotypes and genotypic models of inheritance; dominant (wild homozygotes vs. heterozygotes plus variant homozygotes), recessive (wild homozygotes plus heterozygotes vs. variant homozygotes) and co-dominant (wild homozygotes vs. heterozygotes vs. variant homozygotes), were carried out using logistic regression analysis for all the SNPs individually. The Haploview software (version 4.1 Broad Institute, Cambridge) was used to create linkage disequilibrium (LD) plots [18]. The THESIAS software (version 3.1, INSERM U525, Paris, France) was used to generate haplotype frequencies and to perform logistic regression analysis on the haplotypes [19]. p < 0.05 was considered statistically significant. In the cumulative effect analysis of risk alleles for each SNP, the genotypes were coded as 0, 1, or 2 indicating the number of allergic CRS and asthma risk alleles. The unweighted cumulative genetic risk score (CGRS) of an individual is the total count of disease alleles from all SNPs obtained by adding coded genotypes. Benjamini–Hochberg adjustment procedure was applied to correct for multiple testing by setting false discovery rate (FDR) < 0.25 as the threshold for significance [20]. G*Power software (version 3.1, Universitat Dusseldorf, Germany) was used to calculate the study's power for individual SNPs.

Results

Male preponderance of 61.9% was seen in the aCRS and aCRS with asthma subjects under study. The mean age of the allergic CRS subjects without asthma [38.7 ± 13.30 years (18–65 years] was low when compared to allergic CRS subjects with asthma [41.53 ± 14.87 years (18–81 years)]. With regard to the mean age of onset of the disease for aCRS subjects was 21 ± 10.38 years whereas aCRS with asthma had early predisposition at mean age of 16 ± 10.05 years. The mean duration of the disease in aCRS with asthma (8.9 ± 3.65 years) was longer when compared to aCRS (3.50 ± 2.27 years). Increased frequency of smoking was seen in aCRS with asthma (25.6%) when compared to aCRS (14.7%). The frequency of aCRS subjects with asthma was seen to be high (73.3%) in urban population, while in aCRS it was 61.6% (Table 2). With respect to socio economic status, aCRS subjects with (20.1%) were mostly belonged to low socio economic status.

Table 2 Demographic of subjects with allergic CRS, allergic CRS with asthma and controls
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Hardy–Weinberg equilibrium test

Genotype frequencies for all the SNPs under study were in HWE in allergic CRS cases and controls (all p > 0.05, data not shown).

Genetic variant analysis

CYP1A1 and CYP1A2 gene variant analysis

In the present study CYP1A1*2A or m1 (3798 T > C) rs4646903, CYP1A1*2A, or m2 2455 A > G rs1048943 and CYP1A1*2C or m3 2453 C > A rs1799814 have been analyzed by PCR–RFLP (Table 1). The CYP1A13798T > C, in the co-dominant model (TC vs TT + CC), were found to be significantly associated with increased risk of in only aCRS subjects (OR 1.43, 95% CI 1.03–1.98) when compared to controls. With regard to CYP1A1 2455 A > G and CYP1A1*m4 2453C > A polymorphism no significant association was seen in the aCRS subjects with and without asthma when compared to controls. The frequency of 'G' allele (OR 2.11, p ≤ 0.0001; OR 1.73, p = 0.0009) and GG genotype (OR 5.17, p ≤ 0.001, OR 4.95, p = 0.002) of CYP1A2 rs2069526 was found to show increased risk in allergic CRS with and without asthma when compared to controls. In the overdominant model, the frequency of TG genotype of CYP1A2 rs2069526 was found to be predominant in allergic CRS subjects with asthma (OR 2.26, p = 0.03) when compared to allergic CRS without asthma (OR 1.73, p = 0.0009) and controls (Table 3).

Table 3 Allelic and Genotypic association of CYP1A1, CYP1A2 and GST gene variants in cases with allergic CRS, and allergic CRS with asthma and controls
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GSTP1, GSTT1 and GSTM1 gene variant analysis

In the present study, two SNPs of GSTP (GSTP A313G & GSTP C341T) have been analyzed by PCR–RFLP and deletion polymorphism of GSTT1 and GSTM1 has been identified by multiplex PCR. The GG genotype of GSTP1 rs1605 was seen to be strongly associated with allergic CRS with asthma (OR 4.75, p ≤ 0.001) while CT genotype of GSTP1 rs1799811 polymorphism showed significant protective role only in allergic CRS without asthma (OR 0.48, p-value ≤ 0.001) (Table 3).

The GSTT1 and GSTM1 heterozygous null genotypes were 11.4% and 17.1% in controls, 13.5% and 25.8% in allergic CRS without asthma and 10.4% and 25.2% in allergic CRS with asthma. The double null genotype (i.e., the absence of both alleles) of GSTM1 and GSTT1 polymorphism was 11.3% and 7.5 in allergic CRS cases with and without asthma respectively while it was 5.4% in controls. GSTM1 null was found to be statistically significant in allergic CRS with asthma (OR 1.82, p = 0.006) and without asthma (OR 1.87, p = 0.003) subjects when compared with controls. GSTM1 and GSTT1 double null genotypes was significant only in allergic CRS with asthma (OR 2.58, p = 0.004). The allelic and genotypic frequency distribution, as well as their associated risk with allergic CRS and asthma, is shown in Table 4.

Table 4 Interaction of GSTM and GSTT genes in cases with allergic CRS, and allergic CRS with asthma and controls
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Haplotype analysis in CRS phenotypes

In the present study, haplotypes were constructed based on three polymorphic sites of CYP1A1 gene, three polymorphic sites of CYP1A2 gene on Chr 15 and two polymorphic sites of GSTP1 rs1605 & GSTP1 rs1799811 situated on Chr11 in the study subjects and controls. Haplotype G-G-C at CYP1A2 G3858A rs2069514, T739G rs2069526, and C163A rs762551 has increased the risk of asthma in allergic CRS subjects (OR 5.53, p-value = 0.003) while A-T haplotype of GSTP1 rs1605 & GSTP1 rs1799811 (OR 0.27, p = 0.045) and T-G-C of CYP1A1 rs4646903, rs1048943and rs1799814 had a protective effect in allergic CRS subjects without asthma (OR 0.11, p = 0.045) (Table 5).

Table 5 Haplotype Frequencies of CYP1A1, CYP1A2 and GSTP genes in cases with allergic CRS, and allergic CRS with asthma and controls
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Linkage disequilibrium analysis in CRS phenotypes

It is well understood that associations between alleles at different loci can aid in the identification of disease susceptibility genes. In the present study, linkage disequilibrium among the SNPs of CYP1A2 rs2069514, rs2069526 & rs762551is high (D’ = 99) in allergic CRS with asthma. CYP1A1 rs4646903, rs1048943 and rs1799814 (D’ = 99) were linked in allergic CRS cases without asthma. Further, CYP1A2 gene variants were also linked with exonic variants of CYP1A1 rs1048943 and rs1799814 in allergic CRS with and without asthma (Fig. 1). The total number of risk alleles present in allergic CRS was more when compared to controls. Risk contributed by the more than 6 risk alleles in allergic CRS subjects with asthma was high (OR 11.25, p = 0.004) when compared to allergic CRS without asthma (OR 5.35, p = 0.056) (Table 6).

Fig. 1
figure 1

Linkage disequilibrium (LD) plots of CYP1A1 and CYP1A2 genes variants in cases and controls. Pattern of Linkage Disequilibrium (LD) in CYP1A region using the Four Gamete Rule implemented in HaploView software program. Standard color scheme of Haploview was applied to display LD. The different shades of gray indicate different D values. The darker the grey shading, the larger the ׀D’׀. D’ × 100 are shown in each cell. D’ values of 100 are taken as the strongest and the value will get displayed

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Table 6 Distribution of cumulative risk alleles of CYP1A1, CYP1A2 and GSTP genes in cases with allergic CRS, and allergic CRS with asthma and controls
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Discussion

Genetic studies are promising and may offer insights into the pathophysiology of CRS, asthma and allergy, a strong and consistent association has not been established so far. Earlier, need for studies pertaining to clinically relevant phenotypes of airway diseases have been suggested. However, a few sporadic studies have reported that phenotypic manifestations of CRS depend on a complex interplay between multiple genes of the innate and adaptive immunity [21,22,23,24]. Further, the reports indicate the oxidative stress caused by environmental factors such as air pollutants, inhalant, and food allergens activates inflammatory cells, bronchial epithelial cells, and endothelial cells and lead to host susceptibility to the development of asthma, sinonasal inflammation and allergic symptoms [25,26,27]. Hence, it was pertinent to understand the genes and their interactions involved in oxidative stress leading to CRS phenotypes.

Genetic variants of phase I and II xenobiotic metabolism genes might change the enzymatic activity and the kinetics of reactions involved in detoxification of numerous toxic compounds and lead to oxidative stress [27, 28]. Pollutants in the environment, which frequently coexist with allergens, may synergistically elicit allergic inflammation and aryl hydrocarbon receptor (AhR) activation [29]. The aryl hydrocarbon receptor (AhR) regulates the expression of CYP1A1 and 1A2 and maintains the homeostasis by increasing the clearance of metabolic substrates such as PAHs and heterocyclic aromatic amines/amides and is involved in the pathogenesis and exacerbation of allergic and inflammatory diseases such as bronchitis, asthma, and chronic obstructive pulmonary disease (COPD) [5, 30]. As XMEs share overlapping substrate specificities it is suggested to analyze the gene gene variants simultaneously for better correlation with clinical outcome [31]. The present study on XME gene variants (CYP1A1, CYP1A2, GSTP1, GSTM1 and GSTT1) revealed significant association with asthma in allergic CRS.

CYP1A1 and CYP1A2 genes are highly inducible by a number of environmental factors including diet and exhibit variations in expression caused by genetic and epigenetic mechanisms [32,33,34,35,36]. Vrzal et al., (2004) and Congiu et al., (2009) reported that under pathophysiological conditions, such as inflammation processes, the level and activity of CYP1A2 is decreased [37, 38]. The CYP1A gene cluster was discovered on chromosome 15q24.1, with a close relationship between the CYP1A1 and 1A2 genes. CYP1A1 and 1A2 are highly inducible at both mRNA and protein levels by a number of environmental factors such as chemicals, drugs, smoking, and several dietary factors and may lead to the development of respiratory diseases [3]. Human CYP1A2 is a key hepatic metabolising enzyme, accounting for roughly 13% of all CYP proteins that metabolise a variety of drugs, natural substances, and other compounds. According to a recent pathway-based analysis in human liver samples, CYP1A2 genetic variation may account for catalytic activity, protein expression, and mRNA levels [4, 5].

CYP1A1 T3801C rs4646903 located in the 3′ non-coding region is reported to influence gene function and CYP1A1 rs1048943 and CYP1A1 rs1799814 located in exon 7 resulted in elevated enzymatic activity. CYP1A2 rs762551 polymorphism was also reported to increase enzyme activity and inducibility while CYP1A2 rs2069514 and CYP1A2 rs2069526 gene variants were associated with the decreased CYP1A2 activity [4, 6]. Findings of the present study have revealed a significant association of CYP1A2 rs2069526 in allergic CRS subjects with and without asthma. Further, the haplotype analysis also revealed that G-G-C combination of CYP1A2 rs2069514, rs2069526 and rs762551 to increase the risk of asthma (5.5 folds) in allergic CRS subjects which might be due to decreased enzymatic activity. Hence, promoter variants of CYP1A2 seem to play a vital role in the development of asthma in allergic CRS subjects. With regard to CYP1A1 rs4646903, rs1048943 rs1799814 variants, no association was observed with asthma in allergic CRS subjects. Similarly, studies carried out on Caucasians, Japanese and Serbians also failed to confirm its role in COPD [35,36,37,38]. However, earlier studies on Japanese and Indian populations have shown CYP1A1 rs1048943 variant to be associated with COPD [39, 40] indicating discrepancy in the role of CYPIA1 polymorphism in respiratory disorders.

The GSTM1, GSTT1, and GSTP1 genes are mapped to chromosome 1p13.3, 22q11.23 and 11q13 respectively. The GSTP1 A313G and C341T gene variants leads to the substitution of Ile105Val and Ala114Val amino acids located near the substrate-binding site and alter catalytic activity of the GSTP1 [4, 5]. GSTP plays an important role in neutralizing oxidative stress in response to environmental and allergen exposures. GSTP1 is more abundant in alveoli, alveolar macrophages, and respiratory bronchioles, and it may play an important role in lung detoxification [41, 42]. Missense mutations in the GSTP1 gene (Ile105Val and Ala114Val) can result in decreased detoxification of airway irritants, which increases inflammation and oxidative stress and causes airway dysfunction [43]. Previous research indicates that patients with the GSTP1 rs1605 AA genotype can quickly eliminate reactive oxygen species and have lower levels of oxidative DNA damage [41]. A protective effect of the Val105 GSTP1 rs1605 polymorphism in allergic inflammation has been reported in Korean population [44]. No correlation was observed between GSTP1 rs1605 polymorphism and CRS with and without nasal polyps and COPD [44,45,46,47,48,49]. Interestingly, in the present study, the GSTP1 rs1605 Val105 allele showed a significant risk of asthma in allergic CRS subjects. Similarly, studies on COPD in Indian, Tunisian and Russian populations also reveled 105Val allele as a significant risk factor for COPD [40,41,42].

The inability of the GSTM1 null genotype to detoxify polycyclic aromatic hydrocarbons has been linked to lung cellular and tissue damage caused by an excess of oxidants and free radicals [41]. According to Cheng et al. (2006), GSTM1-null genotype is an independent risk factor for developing severe COPD [44]. The present study, also finds GSTM1 to show a strong significant association in both allergic CRS subjects with and without asthma but could not find any association of GSTT 1 null genotype. The study is also in agreement with the studies carried out by Arbag et al., 2006 in the Turkish population and Fruth et al., 2011 in German population which did not find any significant association of GSTT1 null genotype with and without nasal polyposis in CRS [45, 46]. However, a significant protective effect of GSTT1 null genotype was reported in allergic rhinitis patients in a study conducted by Iorio et al., (2012) while Mak et al., (2007) reported GSTM1 null genotype to be protective in the development of atopic asthma [50, 51]. Combined deletion variants of the GSTM1 and GSTT1 genes were found to be a risk factor for the development of asthma in children and adults, as well as a risk factor for the development of nasal polyposis and hyposmia in allergic individuals [52]. Similarly, in the present study the GSTM1/GSTT1 double null genotype was found to be significantly associated with asthma in allergic CRS subjects. Also, risk contributed by double null genotype was higher (2.8 vs 1.9 folds) than the risk attributed by GSTM1 null indicating its additive contribution for risk of asthma in allergic CRS subjects. However, no association between GSTM1 and GSTT1 double null genotype was observed with allergy, CRS, asthma, and COPD [45, 46, 53].

Interactions of several XME genes were reported to be associated in the present study with risk of asthma indicating the polygenic basis of asthma [54]. The results of cumulative genetic risk allele score predicted a high number of risk alleles in both the allergic subgroups when compared to controls indicating genetic susceptibility to allergic CRS with and without asthma. The cumulative risk contributed by risk alleles in allergic CRS with asthma was higher when compared to CRS without asthma. Also, the absence of protective alleles and allelic combinations might have promoted for the development of asthma. It is also reported that each locus could also be in linkage disequilibrium with an unknown casual gene(s) [45]. Further, it is reported that inter-individual differences due to genetic variation, linkage disequilibrium (LD), gene–gene and gene-environment interactions may be responsible for the complexity of allergic phenotypes. Further, the LD analysis in the present study has also revealed close linkage between CYP1A1 and CYP1A2 which is in agreement with earlier studies in Asian population [55,56,57,58]. However, LD of these gene variants varied between the allergic CRS subjects with and without asthma which might contribute to the altered enzyme activity.

Conclusion

The allelic, genotypic, haplotype frequencies and linkage disequilibrium of CYP1A2 gene are the first to be reported in South Indian population and allergic CRS in particular. Findings of the present study revealed a significant association of CYP1A2 and GST gene variants with asthma in allergic CRS individuals. Future studies are warranted to delineate on the functional analyses of these genes and gene–gene and gene environment interactions leading to asthma in allergic CRS.