GST polymorphism as a predictive biomarker for modulating the susceptibility to chronic obstructive pulmonary disease: A North Indian study

Abstract Chronic obstructive pulmonary disease (COPD) is commonly characterized by shortness of breath, coughing or expectoration. Smoking is the leading cause of COPD development, but only a small percentage of smokers develop symptoms, implying a genetic component. Glutathione S‐transferase enzymes are responsible for detoxifying cigarette smoke components. The role of glutathione S‐transferase T1 (GSTT1) and glutathione S‐transferase M1 (GSTM1) gene polymorphism was assessed with COPD susceptibility and associated clinical parameters in the North Indian population. This was a cross‐sectional study involving 200 COPD patients and 200 healthy individuals, with peripheral blood sampling and adequate questionnaires. Multiplex PCR was used for genotyping GSTT1 and GSTM1 gene polymorphism. Logistic regression was used to calculate the odds ratio and 95% confidence intervals to assess the COPD risk and GST polymorphisms. The GSTT1 gene deletion rate was higher in COPD cases (34.5%) than in healthy individuals (20.5%). A statistical relationship between the GSTT1(−) null genotype and COPD risk was observed (odds ratio = 2.04, 95% CI = 1.30–3.20, P = 0.0019). After adjusting for covariates like age, sex and smoking status, a significant association was found for GSTT1(−) null genotype and COPD risk (adjusted odds ratio = 2.90, 95% CI = 1.43–5.87, P = 0.003). The GSTT1(−) genotype was also significantly correlated with clinical parameters for COPD risk. Another primary observation was that females with the GSTT1(−) null genotype were more vulnerable to COPD than males with the same gene deletion. The GSTT1(−) null genotype strongly correlates with COPD development, while no association was observed in the GSTM1(−) null genotype in the North Indian population.


INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is the second leading cause of death and disability-adjusted life years (DALYs) in India, according to the Global Burden of Disease report (Salvi & Ghorpade, 2021).It is a significant public health problem and is currently a leading cause of morbidity and mortality worldwide, leading to a considerable rise in the social and economic burden (Lozano et al., 2012;Vos et al., 2012).Tobacco smoke exposure or tobacco smoking is one of the most critical factors leading to COPD, in addition to genetic and environmental factors.
Studies in the past suggest that genetic variations in the enzymes that detoxify cigarette smoke products might be correlated to COPD development.These oxidation-inhibiting enzymes include microsomal epoxide hydrolase, cytochrome p450 1A1 and glutathione S-transferase (GST) (Yim et al., 2000).Genetic variation reduces the production or activity of such oxidation-inhibiting enzymes, and the dynamic balance of oxidation/antioxidation is lost, resulting in oxidative damage (Zhang et al., 2015).GST is one of the most studied genes in various human populations, and GST is involved in the metabolism of endogenous and environmental xenobiotics and plays a role in the susceptibility to COPD (Lakhdar et al., 2011).GST is a detoxifying phase II biotransformation enzyme.It is classified as a cytoplasmic, membrane, mitochondrial or leukotriene C4 synthase.
There are eight mammalian classes of these enzymes represented by the Greek letters α, μ, π, σ, θ, κ, ζ and ω (Nebert & Vasiliou, 2004).The conjugating reaction of reduced glutathione and GSTs eliminates the harmful electrophilic compounds formed by oxidative stress (Rezaei et al., 2013).The enzyme is required for organisms to respond to physiochemical stimulation in the environment and protect cells, proteins and nucleic acids from damage caused by free radicals.According to recent research, GST enzymes bind to various toxic molecules from cigarettes, that is, oxidizing agents or free radicals, which act as substrates for biotransformation metabolism, safeguarding cells from carcinogenic and cytotoxic factors (Sharma et al., 2015;Tang et al., 2013).
The GSTM1 and GSTT1 genes are expressed in the respiratory tract.
Gene deletion is the primary cause of GSTM1 and GSTT1 null alleles (Lakhdar et al., 2010).Individuals with GSTM1 and GSTT1 homozygous null alleles lack the corresponding enzyme function, increasing susceptibility to COPD.COPD susceptibility has been seen to be polygenic.Several studies have found polymorphisms of these genes in people of different ethnicities with COPD.However, the association of GSTM1 and GSTT1 polymorphisms with COPD progression was inconclusive across various nationalities.
The present study was undertaken to investigate the role of GSTT1 and GSTM1 polymorphisms as genetic markers for COPD risk in the North Indian population and evaluate the relationship.

Ethical approval
The current study included COPD patients who visited the

Study design
A

DNA extraction and genotyping
Genomic DNA was isolated using standard protein K digestion,

Statistics
Differences in the distributions of demographic characteristics between the COPD cases and healthy individuals were evaluated using the Chi-square (χ 2 ) test for the categorical data and Student's t-test for continuous variables.The Hardy-Weinberg equilibrium theory (p 2 + 2pq + q 2 = 1, where p is the frequency of the wild-type allele and q is the frequency of the variant allele) was used both in COPD cases and healthy individuals to calculate the genotype frequencies of GSTM1 and GSTT1 gene polymorphism using the χ 2 test.Pearson's χ 2 test was used to determine whether there was any significant difference in allele and genotype frequencies between

Demographic and clinical characteristics of COPD patients and healthy controls
The COPD patients and healthy controls had a mean age of 59.3 ± 9.9 (range 41-80) and 47 ± 15.6 (range 21-92) years, respectively.The COPD group consisted of 183 (91.5%) males and 17 (8.5%)females, while the healthy control group included 115 (57.5%) males and 85 (42.5%) females.The mean body mass index (BMI) of COPD cases was 21.7 ± 5.3 kg/m 2 , and the BMI in healthy controls was 25.3 ± 4.7 kg/m 2 .
The mean number of pack-years of smoking in the study was 49.6.
The categorization of the cases as per GOLD severity based on forced expiratory volume in 1 s (FEV1) spirometric readings is given in Table 1.
Shortness of breath was the most common symptom with which the patients presented to us (Table 1).

Distribution and association of GST polymorphism with COPD risk
The GSTT1(−) null genotype prevalence was 34.5% in COPD patients and 20.5% in healthy individuals.When adjusted with covariates such as age, sex and smoking status, a significant association was observed genotype and COPD risk (odds ratio (OR) = 1.0, 95% CI = 0.66−1.50,P = 1) (Table 2).

Association of GST polymorphism with modified Medical Research Council dyspnea score and COPD Assessment Test score for COPD risk
We evaluated the relationship of the modified Medical Research Council (mMRC) dyspnea score with GST polymorphism in both COPD patients and healthy individuals.Two groups were categorized based on the cut-off point of mMRC < 2 and mMRC ≥ 2 as recommended by the GOLD 2011 guidelines.In the first group, that is, mMRC < 2, the GSTT1(−) null genotype was observed in 12 COPD cases (6%) and 41 (20.5%) healthy individuals.The GSTT1(−) null genotype was significantly associated with mMRC in COPD patients (OR = 2.32, 95% CI = 1.05-5.14,P = 0.03).On the contrary the GSTM1(−) null genotype was present in 17 (8.5%)COPD cases and 73 (36.5%) healthy individuals.No significant relationship was found between GSTM1(−) and mMRC in COPD cases (OR = 1.97, 95% CI = 0.92-4.18,P = 0.07) (Table 4).

Association of GST polymorphism with GOLD severity and GOLD 'ABCD' symptom-based assessment for COPD risk
We also assessed the relationship between GST polymorphism and GOLD severity.Subjects were divided into two groups: group A included mild (FEV1 ≥ 80% predicted) and moderate (50% ≤ FEV1 < 80% predicted) COPD cases of airflow obstruction, and group B included severe (30% ≤ FEV1 < 50% predicted) and very severe (FEV1 < 30% predicted) COPD cases of airflow limitation.
However, there was no association between GSTM1(−) and GOLD severity of airway limitation in COPD cases (Table 5).
In group B, the GSTT1(−) null genotype was found in 43 (21.5%)COPD cases and 41 (20.5%) healthy individuals with a two-fold risk of severity in COPD patients, and the association was found to be significant (OR = 2.11, 95% CI = 1.27-3.50,P = 0.003).After adjusting for various covariates like age, sex and smoking status, there was an associated risk between GSTT1(−) null individuals and GOLD severity in COPD patients (AOR = 3.33, 95% CI = 1.42-7.81,P = 0.005) However, no association was found between GSTM1(−) and GOLD severity of airway limitation in COPD cases (Table 5).
GOLD ABCD is a refined evaluation tool that offers symptom burden and exacerbation risk information that can be used to guide treatment.According to GOLD 2011 guidelines, we divided our COPD TA B L E 5 Association of GST polymorphism with GOLD severity for COPD risk and GOLD 'ABCD' symptom-based assessment.showed no significant association (Table 5).

DISCUSSION
The case-control research approach was used in this experimental investigation; our COPD patients had a mean age of 59.3 ± 9.9 years, while healthy individuals had a mean age of 47 ± 15.6 years.Similar results were obtained by Dimov et al. (2008), with a mean age of 67 years for COPD cases and 57 years for healthy individuals (Dimov et al., 2008).Gaspar et al. (2004) reported a mean age of 64.3 years for COPD cases and 31.9 years for healthy individuals (Gaspar et al., 2004).
The mean BMI was 21.7 kg/m 2 for COPD cases and 25.3 kg/m 2 for healthy individuals.A study by Arja et al. (2013)  The frequency of null genotypes for GSTT1 and GSTM1 varies among ethnic groups.The variation in gene polymorphisms may be related to different metabolizing enzyme activities and dominant functional enzymes against oxidative stress in different races (Cheng et al., 2004).
Ethnic differences in COPD prevalence are difficult to distinguish from environmental parameters (Yim et al., 2000).A study suggested that 60% of Asians, 40% of Africans and 20% of Caucasians show GSTT1 enzyme deficiency (Zhang et al., 2014).Similar results were obtained from our study, which showed the prevalence of the GSTT1(−) genotype to be 34.null genotype is a critical factor in developing COPD; however, a few studies have yielded contradictory results (Dimov et al., 2008;Gaspar et al., 2004;Hemimi, 2008;Yim et al., 2000;Žuntar et al., 2014).
The combined effect of the GSTM1(−) null and GSTT1(−) null genotype was a two-fold COPD risk, but the association was insignificant, which was similar to the investigation conducted by Mehrotra et al. (2010).The combinatorial association of GSTM1(+) and GSTT1(−) null genotypes increased the susceptibility to COPD by two-fold.
Per our understanding, our study is the first to report the correlation of GST polymorphism with mMRC grading and CAT score.The null genotype of GSTT1 showed a strong association with higher mMRC and CAT scores.It thus shows that individuals lacking the GSTT1 gene are more likely to experience escalating breathlessness and higher CAT scores.
The GSTT1(−) null allele was found to be associated with both males and females for COPD susceptibility.Females with the GSTT1(−) null genotype were at a higher risk of developing COPD than males with the same gene deletion.Women smokers were roughly 50% more likely than men to develop COPD in a systematic review despite smoking fewer cigarettes (Gan et al., 2006).
A strong correlation between GST polymorphism and GOLD severity was found for the GSTT1(−) null genotype.Research done by Ahmad et al. (2016) proposed that the prevalence of the GSTM1(−) null genotype was greater in patients with severe COPD (Ahmad et al., 2016).
A strong association of the GSTT1(−) null allele with the GOLD B category for susceptibility to COPD was found.This suggests that the patients having this allele have higher symptom severity but lower cross-sectional study was conducted on 200 COPD patients and 200 healthy controls presenting to the outpatient department of Pulmonary Medicine, Government Medical College, Patiala, Punjab, India.After the clinical history and physical examination, they were subjected to spirometry and were classified according to the clinical criteria of COPD set down in the Global Strategy for Obstructive Lung Disease (GOLD) 2019 guidelines (Gruffydd-Jones, 2012).Blood samples of all 400 individuals were collected in EDTA-coated tubes and stored at −20 • C for extraction of DNA.
phenol-chloroform extraction and ethanol precipitation from whole blood samples.The presence of genomic DNA was checked on 0.8% agarose gel.Multiplex PCR amplifies multiple targets in a single set of reaction conditions using multiple primers.The PCR mixture of 20 μL comprised 1× PCR buffer, 100μg/ml bovine serum albumin, 0.5 μM forward primers and 0.5 μM reverse primers of both GSTM1 and GSTT1, 0.3 μM of forward and reverse primer of Albumin, 0.2 μM dNTPs, 3 U/μL of Taq polymerase and 300 ng of DNA.An optimal combination of annealing temperature and buffer concentration was maintained, as is necessary for multiplex PCR to obtain amplified products.The sequence of primers used was as follows: for GSTM1, forward: 5′-GAACTCCCTGAAAAGCTAAAGC-3′, and reverse: 5′-GTTGGGCTCAAATATACGGTGG-3′; forGSTT1, forward: 5′ TTCCTTACTGGTCCTCACATCTC-3′, and reverse: 5′-TCACCGGATCATGGCCAGCA-3′; and for the albumin gene, forward: 5′-GCCCTCTGCTAACAAGTCCTAC-3′, and reverse: 5′-CCCTAAAAAGAAAATCGCCAATC-3′.The PCR was done as follows: initial 95 • C for 5 min, 30 cycles of denaturation (94 • C, 1 min), annealing (59 • C, 1 min) and elongation (72 • C, 1 min), and a final elongation step at 72 • C for 5 min.The PCR product was then subjected to 2.0% agarose gel electrophoresis.A band of size 480 bp indicated the GSTT1 allele, and that of 215 bp indicated the GSTM1 allele.The absence of both 215 bp and 480 bp bands indicated the null genotype.Albumin was an internal control indicated by a band of size 312 bp (Figure 1).
and 7.5% (15) in healthy individuals.A strong correlation between the GSTT1(−) null genotype and COPD risk was found among females (OR = 3.26, 95% CI = 1.07-9.96,P = 0.03).A seven-fold increase in COPD susceptibility in females with the GSTT1(−) null genotype was observed after adjusting it with factors like age and smoking status (AOR = 7.11, 95% CI = 1.90-26.64,P = 0.003) (Table cases into four groups designated A, B, C and D. We evaluated the relationship between GST polymorphism and GOLD 'ABCD' symptombased assessment.In category A, 11 (5.5%)COPD cases and 41 (20.5%) healthy individuals had the GSTT1(−) null genotype.A significant correlation was observed between GSTT1(−) and GOLD A category among COPD cases (OR = 2.36, 95% CI = 1.03-5.40,P = 0.04).In category B, the GSTT1(−) null genotype was present in 53 (26.5 %) COPD cases and 41 (20.5%) healthy individuals.A significant association was found between GSTT1(−) and GOLD B category with two-fold risk in patients harbouring the GSTT1(−) null genotype (OR = 2.2, CI = 1.36-3.57,P = 0.001).A strong correlation of GSTT1(−) with the GOLD B category was observed after adjusting for parameters like age, sex and smoking status with a five-fold increase in COPD risk (AOR = 5.06, 95% CI = 2.11-12.11,P = 0.0003).On the other hand, GSTT1(−) showed no correlation with group C and D among COPD cases.Furthermore, the GSTM1(−) null genotype and any of the groups mentioned above Our lungs are subjected to atmospheric pollution, irritants, noxious particles, allergens and pathogens daily, which can trigger inflammatory responses and the production of endogenous oxidants.This may further result in chronic inflammation, tissue injury and remodelling.Inhaled xenobiotic compounds are rapidly absorbed and metabolized by the phase I and II enzymes critical in detoxification, including the glutathione-conjugating enzymes GSTs (Van de Wetering 5% among COPD cases compared to 20.5% among healthy individuals.Nevzorova et al. (2012), Kukkonen et al. (2011), Mehrotra et al. (2010) and Thakur et al. (2011) also reported similar results.Our research arch emphasizes the fact that the GSTT1(−) exacerbation risk.No other study has made this correlation before, and more data are needed to get in-depth knowledge of this association.A significant correlation between GSTT1(−) and COPD duration was found, indicating that patients with a longer duration of COPD have lost the activity of the GSTT1 gene.With the rapidly increasing number of COPD cases and high mortality rates worldwide, greater attention should be paid to treating and preventing the disorder.Currently, no medication has shown the best results against the pulmonary damage caused by COPD.Genetic markers can play a massive role in detecting diseases early and preventing them from progressing to severe stages.Our research has made one such attempt to find a suitable genetic marker for COPD in North Indian patients.According to our findings, the GSTT1(−) null genotype can be used as a genetic biomarker.GSTT1 deletion was also linked to increased risk for developing COPD and linked to higher GOLD grading.A more extensive population study can clarify the relationship between the homozygous null GSTT1 gene and other xenobiotic enzymes as risk factors for COPD development and pathogenesis.

Highlights • What is the central question of this study?
Demographic and clinical characteristics among COPD patients and healthy controls.Distribution and association of GST polymorphism with COPD risk.
TA B L E 1OR, crude odds ratio; AOR, adjusted odds ratio, evaluated by unconditional logistic regression and adjusted for age, sex and smoking.P-values in bold indicate statistical significance (P < 0.05).between GSTT1(−) null genotype and COPD risk (adjusted odds ratio (AOR) = 2.90, 95% CI = 1.43-5.87,P = 0.003).On the contrary, the GSTM1(−) null genotype was present in 36.5% of COPD cases and 36.5% of controls.No association was found between the GSTM1(−) Association of GST polymorphism and COPD based on sex.OR, crude odds ratio; AOR, adjusted odds ratio, evaluated by unconditional logistic regression and adjusted for age, sex and smoking.P < 0.05 was considered to be statistically significant.
Association of GST polymorphism with mMRC and CAT score for COPD risk.