The Relationship Between Glutathione S-Transferase-P1 and Beta-2 Adrenoreceptor Genotypes with Asthmatic Patients in the Turkish Population

Abstract

Background: Individual differences in the activity of enzymes that metabolize xenobiotics can impact health and disease. Beta-2 adrenoreceptor (ADRB2) is a functional G-coupled protein expressed in the vascular endothelium of lungs, alveolar walls, and the ganglions of cholinergic nerves which induces bronchodilation in response to catecholamines. Glutathione S-Transferase-P1 (GSTP1) is a candidate pi class GST gene, which controls pi class glutathione S-transferase activity. Aims: In this study we determined the relationship between the ADRB2 Arg16Gly polymorphism and GSTP1 polymorphisms, involved in bronchodilator response and oxidative stress, respectively, with susceptibility to asthma. Methods: In this study, 129 asthmatic patients and 127 healthy control cases were recruited to determine ADRB2 and GSTP1 genotypes by allele-specific polymerase chain reaction and restriction fragment length polymorphism assays, respectively. Results: The ADRB2 genotype frequencies of the patients and control cases were found to be 10.9% (Arg16Arg), 48.8% (Arg16Gly), and 40.3% (Gly16Gly) and 24.4% (Arg16Arg), 36.2% (Arg16Gly), and 39.4% (Gly16Gly), respectively. GSTP1 genotype frequencies of patients and control cases were found to be 55% (Ile105Ile), 43.4% (Ile105Val), and 1.6% (Val105Val) and 75.6% (Ile105Ile), 22% (Ile105Val), and 2.4% (Val105Val), respectively. In the case of the GSTP1 gene, we found statistically significant differences in the genotype frequency of Ile105Val and the allele frequency of Val105 in the asthmatic group compared with the controls. Moreover, we observed a relationship between allele frequencies and clinical phenotypes including atopia nocturnal dyspnea, and steroid dependency in the asthmatic patients. Conclusion: Our results suggest that the GSTP1 Ile105Val polymorphism may be linked to the severeness of airway dysfunction.

Introduction

Asthma is a chronic inflammatory disease of the airway, in which many cells and cellular elements play a role (Moorman et al., 2013). Personal genetic polymorphisms, which control certain receptors and enzyme systems, have been regarded as being among the etiological factors (Homer and Elias, 2005; Osborne and Deffebach, 2004; Vijverberg et al., 2013).

The β2 adrenoceptor (ADRB2) is a G protein-coupled protein family, which comprises seven transmembrane domain receptors that are widely distributed in the tissues (Johnson, 1998; Busjahn et al., 2000). ADRB2 agonists induce vasodilatation following the activation of receptors, which are present on both endothelium and vascular smooth muscle (Littlejohn et al., 2002). Although four amino acid polymorphisms have been reported within the open reading frame, only two of them are functionally important. These are two common coding SNPs within the amino terminal extracellular domain of the ADRB2 gene, including Arg16Gly and Gln27Glu (Kobilka et al., 1987; Hall, 1996; Littlejohn et al., 2002). Both of them have been found to alter the receptor function significantly. Liggett et al. (2000) detected decreased receptor numbers in Gly16Gly homozygote mutant individuals when they were exposed to endogenous catecholamine. ADRB2 polymorphisms with Arg16Gly and Glu27Gln can alter physiological and pharmacological responses to ADRB2-mediated stimulation (Green et al., 1995). Several studies have examined the ADRB2 gene as a risk factor associated with bronchodilator response (Birbian et al., 2012; Zheng et al., 2012; Karam et al., 2013). Due to the downregulation of beta-2 receptors, the level of bronchodilatator desensitization is significantly high in asthmatic individuals with the Gly16Gly homozygote genotypic feature (Tan et al., 1997; Ioannidis et al., 2005).

Numerous recent studies have shown the polymorphisms in the GSTP1 gene coding for enzymes to be associated with altered risk or the outcome of various diseases (Ruscoe et al., 2001; Zhong et al., 2006; Kadioglu et al., 2012; Moormann et al., 2013; Vijverberg et al., 2013). Alteration in the structure, function, or level of expression of GST genes or polymorphisms could alter the ability of the cell to inactivate, which would protect cells against the harmful effects of oxidative stress (Yin et al., 2000; Zhong et al., 2006). Although adult pulmonary epithelial cells are affected by several GST gene isoenzymes, more than 90% of cytosolic GST isoenzyme activity is coded by the GSTP1 gene. A functional sequence variant in GSTP1 at codon 105 (Ile105Val) has been associated with asthma (Fryer et al., 2000; Mapp et al., 2002; Aynacioglu et al., 2004; Lee et al., 2004; Tamer et al., 2004; Islam et al. 2009).

In the present study, the aim is to determine the relationship between asthma and ADRB2 and GSTP1 genetic polymorphisms, which causes significant clinical pathology both in children and adults in the Turkish population.

Methods

Study groups

After receiving the institutional approval and informed consent, asthma patients (n = 129) and healthy controls (n = 127) were recruited in the Turkish population. Control subjects comprised individuals who received no medication and who had no cancer/hereditary disease, asthma in particular, either personally or in the family.

DNA extraction and genotype analysis

Whole blood samples were collected from the study population. All participants completed a questionnaire assisted by a physician, which included all individual information on age, sex, height, weight, occupation, history of cigarette smoking, and alcohol consumption. Whole blood samples (5 mL) were removed through venepuncture into tubes containing EDTA, and samples were stored at -20°C until DNA extraction. Genomic DNA was extracted from whole blood using sodium perchlorate/chloroform extraction.

ADRB2 polymorphism

The ADRB2 alleles were detected by modification of the method described by Martinez et al. (1997). Allele-specific polymerase chain reaction and restriction fragment length polymorphism assays were used to determine the genotypes, respectively. The sequences of the PCR primers were 5′-GCCTTCTTGCTGGCACCCCAT-3′ for the forward and 5′-CAGAGCACATCAATGGAAGTC-3′ for the reverse primer. The underlined base was modified from the reported sequence to create NcoI restriction sites on the PCR product generated from the Gly16 allele, but not from Arg16. A 36 μL PCR reaction was performed using ∼0.2-0.5 μg genomic DNA, 30 ng of each primer, 200 μM of each dNTP, 2.5 mM MgCl₂, and 3.75 U Taq polymerase (Promega) in the 1× PCR buffer supplied by the manufacturer (50 mM KCl, 10 mM Tris-HCl, pH 9,0.1% TritonX-100). Amplification was carried out for 1 cycle by first denaturing at 96°C for 2 min, and then for 35 cycles by denaturing at 96°C for 25 sec, annealing at 58°C for 25 sec, extending at 72°C for 35 sec, and final extention at 72°C for 10 min for 1 cycle. A PCR product of 318 bp was obtained. The same PCR aliquots were used for different restriction enzyme digests to identify the Arg16 and Glu27 SNPs. To detect the Arg16 allele, 10 μL PCR products were digested with 2 U NcoI (NEBiolabs) and 2 U BtsI (NEBiolabs) in 2 μL 1 × NEB4 buffer at 37°C and analyzed by electrophoresis on 3% NuSieve agarose gels, followed by visualization of the bands with ethidium bromide. NcoI cut 18 bp from the 5′ end, and BtsI cut 236 bp from the 5′ end of the Gly16 allele. Glu27 allele was detected by restriction digestion with 0.9 U BbvI (NEBiolabs) in 2 μL 1× NEB2 buffer at 37°C. BbvI digests only Gln27 allele to produce 105 and 63 bp fragments, which are separated from the uncut Glu27 allele on 3% NuSieve agarose gels.

GST polymorphism

GSTP1 Genotype Analysis: To detect the variants of codon 105 of GSTP1, PCR-RLFP analysis was applied. The Ile105→Val105 substitution in GSTP1 was examined after PCR amplification through the use of primers to exon 5. For GSTP1 PCR amplification, 50-100 ng genomic DNA was used in a total of 25 μL reaction volume containing 10 pmol of each of the primers, PiF2306 (5′-GTA GTT TGC CCA AGG TCA AG-3′) and PiR3800 (5′-AGC CAC CTG AGG GGT AAG-3′), specific for exon 5, 200 μM of dNTP mix, 10× PCR buffer, and 1 U DNA Taq polymerase. The amplification conditions were as follows: initial denaturing step at 94°C for 5 min, followed by 30 cycles of denaturing for 30 sec at 94°C, annealing for 30 sec at 57°C, and extension for 30 sec at 72°C. The reaction was completed with one cycle of extension at 72°C for 7 min. The resulting 176 bp fragment was digested with Bsm AI (New England BioLabs, Inc.) to identify the A-G transition at nucleotide +313. PCR products from the homozygote for the Ile105-encoding allele (GSTP1 Ile105Ile genotype) comprised the undigested 176 bp fragment, whereas products from homozygous Val105-encoding individuals (GSTP1 Val105Val genotype) comprised fragments of 91 and 85 bp. PCR products from heterozygote (GSTP1 Ile105Val genotype) comprised fragments of 176, 91, and 85 bp, expected amplification product of 176 bp.

Statistical analysis

The Mann-Whitney U test was used to compare the descriptive demographic data of two groups. The chi-square test was applied to compare the two groups in terms of sex, smoking, and menopausal situation. Logistic regression analysis was applied to determine the effective risk factors on ADRB2 and GSTP1 genotype frequency. A p-value of <0.05 was considered significant.

Results

Healthy subjects (n = 127) and asthma patients (n = 129) were evaluated to determine ADRB2 and GSTP1 genetic polymorphism in the Turkish population. Table 1 shows individual characters of both patients and controls. Atopic (IgE >100 IU/mL) incidence in asthma patients was 32.55% (42 patients). Allele and genotype frequencies of control and asthma groups are shown in Tables 2 and 3.

Table 1.

Individual Characteristics of Both Patients and Controls

	N (%)		Median(min-max)
Group	Female	Male	Age (year)	Weight (kg)	Length (cm)
Control	84 (66.1%)	43 (33.9%)	38 (18-65)	70 (40-95)	165 (162-190)
Asthma	88 (68.2%)	41 (31.8%)	39 (18-75)	72 (44-106)	160 (143-194)

Table 2.

Allele and Genotype Frequencies of Control Groups

	Genotype frequency			Allele frequency
N (%)	Arg16Arg	Arg16Gly	Gly16Gly	Arg	Gly
Control	31 (24.4%)^a	46 (36.2%)	50 (39.4%)	108 (42.5%)	146 (57.5%)
	Ile105Ile	Val105Ile	Val105Val	Ile	Val
Control	96 (75. 6%)	28 (22%)	3 (2. 4%)	86.6%	13.4%

p < 0.05 (p = 0.011).

Table 3.

Allele and Genotype Frequencies of Asthma Patients

	Genotype frequency			Allele frequency
N (%)	Arg16Arg	Arg16Gly	Gly16Gly	Arg	Gly
Asthma	14 (10.9%)	63 (48.8%)	52 (40.3%)	91 (35.3%)	167 (64.7%)
	Ile105Ile	Val105Ile	Val105Val	Ile	Val
Asthma	71 (55%)	56 (43.4%)^a	2 (1.6%)	76.7%	23.3%^b

p < 0.05 (p = 0.001).

p < 0.01 (p = 0.004).

When the asthma and control groups were compared in terms of ADRB2 genotype frequency, no difference was observed between heterozygote and mutant genotypes, while the homozygote genotype frequency in the control group was significantly high (p = 0.011). No significant difference was found between the Arg and Gly allele frequencies of the two groups. In the asthma group, when individuals who have one or both mutant alleles (115 patients, 89.1%) were compared with those who have no mutant allele (14 patients, 10.9%), individuals with the mutant allele had a significantly higher risk for asthma (p = 0.005; OR = 2.653; CI = 1.335-5.272). When phenotypic features of asthmatic individuals were examined, Arg16 and Gly16 allele frequencies were found as 27.38% and 72.62% in atopic individuals, 21% and 79% in individuals with nocturnal asthma, and 19.23% and 80.73% in individuals with steroid addiction, respectively.

Heterozygote GSTP1 genotype frequencies in the asthma group were statistically higher than those of the control group (p = 0.001). When allele frequencies of both groups were compared, no difference was detected in Ile105 allele frequency, while Val105 allele frequency in the asthma group was significantly higher (p = 0.004). In the comparison of individuals who had both mutant alleles (Val105Val) or one mutant allele (Ile105Val) with individuals who had no mutant allele in both groups, a statistical difference was observed between the asthma and control groups (p = 0.001; OR = 2.53; 95% CI = 1.484-4.312). It was found that individuals who had one mutant allele had 2.71 times higher risk for asthmatic disease than individuals with two or no mutant alleles (Table 4).

Table 4.

GSTP1 Genotype Frequency of Asthma and Control Groups

Genotype	Val105Ile	Val105Val+Ile105Ile
Control (n = 127)	28 (22%)	99 (78%)
Asthma (n = 129)	56 (43.4%)^a	73 (56.6%)

p < 0.05 (p = 0.001).

OR = 2.712; 95% CI = 1.572-4.679.

Forty-two (35.55%) of the asthma patients were atopic. Among these, 54.8% (n = 23) were Ile105Ile, while 45.2% (n = 19) were Ile105Val. Ile105 and Val105 allele frequencies in atopic patients were 77.38% and 22.62%, respectively. Fifty-seven (44.18%) of the asthma patients had nocturnal dispnea; among those 56.15% (n = 32) were Ile105Ile, 42.10% (n = 24) were Ile105Val, and 1.75% (n = 1) were Val105Val. Ile105 and Val105 allele frequencies in nocturnal dispnea patients were 77.19% and 22.81%, respectively. Thirty-seven (30.23%) of the asthma patients had steroid addiction; among those 59.46% (n = 22) were Ile105Ile, 37.84% (n = 14) were Ile105Val, and 2.8% (n = 1) were Val105Val (Table 5).

Table 5.

Allele and Genotype Frequencies of Subphenotypes of Asthma Patients

N (%)		Atopy	Nocturnal dipnea	Steroid addiction
Total asthma patients		42 (35.55%)	57 (44.18%)	37 (30.23%)
Genotype frequency	Ile105Ile	23 (54.8%)	32 (56.15%)	22 (59.46%)
	Ile105Val	19 (45.2%)	24 (42.10%)	14 (37.84%)
	Val105Val	—	1 (1.75%)	1 (2.8%)
Allele frequency	Ile105	77.38%	77.19%
	Val105	22.62%	22.81%

Discussion

In the present study regarding ADRB2, the allele frequencies of Arg16 and Gly16 of the healthy Turkish population were determined as similar to those of Aynacıoğlu et al. (1999) and Caucasian populations. Asthmatic individuals of different populations have been examined in terms of ADRB2 genetic polymorphism. Arg16 and Gly16 allele frequencies have been reported as 38% and 62% (Martinez et al., 1997) in a Spanish population; 40.7% and 59.3% (Holloway et al., 2000) in a New Zeland population; 36.7% and 63.3% (Lipworth et al., 1999) in an English population; 33% and 67% (Dewar et al., 1997) in an American English population, and 46.7% and 53.3% (Yim et al., 2002) in a Korean population, respectively. In general, the reported allele frequencies for Arg16 and Gly16 in the Caucasian, African American, and Asian asthmatic populations were 0.39, 0.50, and 0.40, respectively, while for Gln27, the reported frequencies were 0.57, 0.73, and 0.80, respectively (Dewar et al., 1997; Lipworth et al., 1999; Holloway et al., 2000; Yim et al., 2002).

The Arg16Gly and Gln27Glu polymorphisms cause differential agonist-stimulated downregulation of the receptor in transfected cell systems, including human airway smooth muscle cells (Green et al., 1995). Many previous studies have investigated possible associations between asthma and polymorphisms in the coding region of the ADRB2 gene, particularly the Arg16Gly and Gln27Glu SNPs (Tan et al., 1997; Ioannidis et al., 2005; Birbian et al., 2012; Zheng et al., 2012). However, diverse results are found in the literature regarding asthma and its relationship with ADRB2 polymorphism (Bouzigon et al., 2010; Tamari et al., 2011).

The Gly16 polymorphism appears to be highly labile in recombinant and endogenously expressing cells, and compared with the Arg16 receptor, displays a marked decrease in expression in the presence of catecholamine (Hoit et al., 2000; Snieder et al., 2002). The Gln27Gln and Gln27Glu genotypes were indirectly related to the occurrence of asthma. Reinforcing this finding, elevated serum IgE levels have been found in patients carrying the Arg16 and Gln27 homozygous genotypes (Woszczek et al., 2005). Martinez et al. (1997) showed that individuals with the Gly16Gly genotypic feature have 5.3 times lower agonistic response than individuals with Arg16Arg and 2.3 times lower agonistic response than individuals with the Arg16Gly genotypic feature. Thus, excessive bronchial airway response strengthened by many stimuli has been claimed as a risk factor in asthma development. Accompanying ADRB2 Gly16Gly polymorphism was detected in asthma cases with increased sensitivity of the airway against histamine as well as asthma cases with airway obstruction. The Gly16 allele has been shown to play a role in asthma development, particularly in females (Lipworth et al., 1999). Similarly, in a study on an Italian population, a significant relationship was found between Gly16 polymorphism and airway sensitivity, which was examined as a physiologic marker in asthma (D'amato et al., 1999). In a Chinese population study, significantly higher bronchodilator response was observed in patients with the homozygous genotype, Arg16Arg, compared with those patients with the homozygous genotype, Gly16Gly (Qiu et al., 2010). The Gly16Gly genotype frequency (54%) of severe asthma patients in the New Zeland population was found to be 1.91 times higher compared with the control group (38%) (Holloway et al., 2000). Turki et al. (1995) reported the Gly16 allele frequency of severe asthma patients with nocturnal attacks as 3.8 times higher. In an article of Paiva et al. (2014), it is shown that the Gln27Glu and Arg16Gly polymorphisms of the ADRB2 gene play an important role in asthma prevalence and severity. There are studies that claim that there is no relationship between ADRB2 polymorphism and phenotypic features of asthma, such as total IgE, skin test positiveness against aeroallergens, bronchial insensitivity against agents, and functional respiratory tests. The present study is the first to investigate ADRB2 polymorphism in healthy and asthmatic individuals in the Turkish population. When asthma and control groups were compared in terms of genotype frequency, no difference was observed in Arg16 and Gly16 allele frequencies, while Arg16Arg genotype frequency in the control group was significantly higher compared with the asthmatic individuals.

In studies regarding GSTP1 genetic polymorphism in healthy subjects, different allele and genotype frequencies were obtained in white, black, and Asian races. Ile105 and Val105 allele frequencies according to populations were, respectively, as follows: 78.3% and 21.7% in the Taiwanese population (Lee et al., 2005); 79.3% and 20.7% in the Korean population (Yim et al., 2002); 52.3% and 47.7% in the English population (Freyer et al., 2000); 63% and 37% in the American population (Gilliland et al., 2002); and 68.9% and 31.1% in the German population (Nickel et al., 2005). Ile105 and Val105 allele frequencies in the Turkish population were examined in different studies. Ile105 and Val105 allele frequencies found were as follows, respectively: 57% and 43% (Calikoglu et al., 2006); 69.2% and 30.8% (Aynacioglu et al., 2004); 72.7 and 27.3% (Tamer et al., 2004); 82.23% and 17.77% (Toruner et al., 2001); 69% and 31% (Gonul et al., 2012); and 66.2% and 33.8% (Karahalil et al., 2015). Previous studies on the Turkish population presented various results. Regarding these differences in the results, it may be concluded that studies about GSTP1 polymorphism on the Turkish population should continue. Our results on the healthy individuals of the control group are consistent with those of Toruner et al. (2001).

Biochemical studies have demonstrated a lower thermal stability of GSTP1 Val105 compared with GSTP1 Ile105 genotypes and also a lower conjugating activity of Val105 homozygous compared with Ile105 homozygous, with Ile105Val heterozygous displayed intermediate activity (Watson et al., 1998). Moreover, cytotoxic products derived from reactive oxygen species also play an important role in the mobilization of arachidonic acid after the production of proinflammatory eicosanoids (Hayes and Strange, 2000). It was detected in recent studies that high activity GSTP1 genotypes develop airway sensitivity and predisposition to asthma by causing personal differences in the detoxification activity of epithelial cells against the effects of ROS (Freyer et al., 2000; Spiteri et al., 2000; Hemmingsen et al., 2001; Lee et al., 2004). When individuals with asthma in different populations were investigated, Ile105 and Val105 allele frequencies were as follows: 71.6% and 28.4% in the English population (Freyer et al., 2000); 69.3% and 30.7% in the German population (Nickel et al., 2005); 86.6% and 13.4% in the Taiwanese population (Lee et al., 2005); and 84.3% and 15.7% in the Korean population (Yim et al., 2002). Ile105 and Val105 allele frequencies of asthmatic individuals in the Turkish population were examined in two different studies and found to be 74% and 26% (Aynacioglu et al., 1999) and 55% and 45% (Tamer et al., 2004), respectively. The results of our study are consistent with those of Aynacıoğlu et al. (2004).

The GSTP1 Ile105Ile genotype frequency increased correspondingly with airway reactivity in the population, while there was a decrease in the genotype frequency of Val105Val and Ile105Val individuals. A prominent relationship was encountered between GSTP1 genotype and asthma. GSTP1 Val105Val genotype frequency was reported to have a six times lower asthma risk compared with Ile105Ile genotype frequency (Martinez et al., 1992; Martinez et al., 1997; Freyer et al., 2000; Hayes and Strange, 2000). Melén et al. (2008) found an interaction effect on allergic sensitization in children with the GSTP1 polymorphism when exposed to traffic-NOx during their first year of life. However, no statistically significant relationship was found when nonasthmatic nonatopic, atopic nonasthmatic, and atopic asthmatic individuals in the same population were evaluated (Hemmingsen et al., 2001). When only individuals with atopic airway pathology in the English population were evaluated, individuals with Val105Val genotype showed lower genotype frequency compared with nonatopic control individuals (Spiteri et al., 2000). No relationship was reported in different studies on white races regarding GSTP1 polymorphism in healthy individuals as well as asthmatics (Nickel et al., 2005). In the Asian population, GSTP1 polymorphism did not show difference in asthma patients who were not exposed to air pollution, while individuals who have Ile105 allele and were exposed to severe air pollution showed 5.52 times higher asthma risk compared with those who have Val105 allele (Lee et al., 2004). In the Turkish population, Val105Val genotype frequencies in asthmatic and healthy individuals were reported as 3.8% and 12.1% (Aynacioglu et al., 2004) and 22.8% and 7.8% (Tamer et al., 2004), respectively. Individuals with Val105Val GSTP1 genotype frequency had 0.29 times lower asthma risk compared with Ile105Val and Ile105Ile individuals. Genotype frequency of asthmatic individuals with GSTP1 Val105Val was found to be 3.68 times higher than the control group. In our study, 129 asthmatic and 127 healthy individuals were evaluated. We did not encounter any difference in the Ile105Ile and Val105Val genotype frequencies of the control and asthmatic groups, but found that the Ile105Val genotype frequency of the asthmatic group was statistically higher than the control group. When allele frequencies of both groups were compared, the asthma risk for individuals who have mutant allele was 2.53 times higher compared with individuals who do not have the mutant allele.

In conclusion, a consensus has not been reached regarding the relationship between ADRB2 genetic variation and asthma. Overall, ADRB2 Arg16Gly polymorphism is related to downregulation in the receptor, nocturnal asthma, and severeness of asthma. Because of the fact that GSTP1 Val105Val genotype frequency shows lower incidence particularly in atopic subjects, individual GSTP1 genotype feature was linked to the severeness of airway dysfunction. We did not observe any significant difference between cases and controls in the cases of the ADRB2 gene, whereas we found a statistically significant difference in the genotype frequency of Ile105Val and the allele frequency of Val105 in the asthmatic group compared with controls. In this context, our study has several strengths and limitations: our sample size may be considered small and there is no control for environmental factors. The population may involve different regions with specific genotype combinations associated with risk, which may also be associated with a peculiar environmental factor.

Footnotes

Acknowledgment

This study was approved by the Ethics Committee of Ankara Training and Research Hospital, Ministry of Health, Ankara, No. 0191/1232.

Author Disclosure Statement

All authors have read and approved the final manuscript and have no direct and indirect commercial financial incentive associated with publishing this article. The article is not under consideration by another journal and has not been previously published. Authors declare no conflicts of interest.

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