Association between the N-acetylation genetic polymorphism and bronchial asthma

Abstract

Aims Since polymorphic N-acetyltransferase 2 (NAT2) has been suggested as a susceptibility factor for atopic diseases, the study was undertaken to investigate whether an association exists between acetylation polymorphism and asthma patients with atopy.

Methods The frequencies of NAT2 alleles and genotypes were determined by PCR/RFLP in a total of 210 asthma patients (extrinsic (n = 108) and intrinsic (n = 102) asthmatics) and 240 control subjects. Presence of the NAT2*4 (wild-type) allele defined a NAT2 genotype as rapid and combinations of mutant alleles NAT2*5 A, *5B, *5C, *6 A, and *7B as slow.

Results Genotypes coding for slow acetylation were detected in 70.4, 58.4 and 58.3% of extrinsic asthmatics, but intrinsic asthmatics and control subjects, respectively. The frequency of slow acetylators was higher among extrinsic asthmatics than intrinsic asthmatics, this difference did not reach statistical significance (odds ratio 1.02, 95% confidence interval 0.64, 1.63, P = 0.085). However, we found a relatively moderate, but significantly higher, increased frequency of slow acetylators among extrinsic asthma patients compared with control subjects (odds ratio 1.70, 95% confidence interval 1.04, 2.76, P = 0.042).

Conclusions This study shows an association between acetylation polymorphism and susceptibility to extrinsic asthma, but not to intrinsic asthma, suggesting a minor role of the NAT2 polymorphism in the development of atopic asthma.

Introduction

Interindividual  and  interethnic  differences  in  the ­acetylation capacity of polymorphic arylamine N-­acetyltransferase 2 (NAT2) affects therapeutic efficacy and the occurrence of side-effects of some clinically used drugs [1, 2]. Moreover, NAT2 is involved in the metabolism of carcinogens from environmental, industrial and dietary sources [1, 3]. Therefore, extensive studies have been performed to identify the associations between the acetylation capacity of individuals and their susceptibility to certain malignancies or diseases such as diabetes mellitus [2, 4, 5, 6]. Moreover, it has been suggested that genetic defects in acetylation may be involved in the pathogenesis of allergic diseases and atopy [7–9].

Asthma is characterized by airway inflammation, bronchial hyper-responsiveness and variable airway obstruction. According to disease onset, patient and family history, epidermal prick test, specific IgE levels, and allergen dependency, there are, with wide overlap, two major types of asthma, namely extrinsic or allergic, and intrinsic asthma. The onset of the allergic, allergen-dependent asthma is often in childhood. Patients exhibit positive skin prick tests and elevated specific IgE levels [10].

Previous studies have indicated that the slow acetylation phenotype may be associated with allergic diseases and atopy. Therefore the present study was performed to investigate further whether there is an association between this polymorphism and asthma risk.

Methods

Subjects

The study, performed on 210 patients with bronchial asthma (all of them unrelated Turkish individuals from south-east Anatolia) and 240 control individuals after their written informed consent. The study was approved by the local ethics committee of the University of Gaziantep. Asthma patients were further classified as having extrinsic or intrinsic disease (Table 1). Careful evaluation was performed to exclude patients whose symptoms started before 16 years of age. The subjects were outpatients of the Department of Pulmonology, Sahinbey Hastanesi, Gaziantep, Turkey. The diagnosis was based on medical history, physical examination, lung function tests and chest X-rays, skin ‘prick’ tests, and total immunglobulin E concentrations (analysed in 107 patients). In addition we determined the serum concentrations of α₁-antitrypsin (α₁ AT) in 107 patients to examine whether there is a difference between extrinsic and intrinsic asthmatics and whether the various NAT2 alleles or genotypes are related to the former. Demographic characteristics and laboratory measurements are shown in Table 1. Control individuals were selected from outpatients of other Departments of our Hospital who had no signs of allergy/atopy (according to familial and medical history), of lung diseases, or of diseases that may have an association with the acetylation polymorphism, such as diabetes mellitus and cancer.

Table 1.

Demographic characteristics and laboratory parameters (mean ± s.e. mean) in patients and control subjects.

	Extrinsic (n = 108)	Patients with asthma Intrinsic (n = 102)	Controls (n = 240)	Pa
Age range (median)	20–61 (39)	17–70 (41)	17–73 (43)	NSb
Sex (female/male)	80/28	76/26	174/66	NSb
α1AT (mg dl−1)*	261.2 ± 10.7	270.0 ± 10.9	NDc	NSb
Total IgE (IU ml−1)*	311.2 ± 42.2	107.2 ± 20.7	NDc	< 0.001

^aCalculated by anova

^bnot significant

^cnot determined.

^*Measured in 47 extrinsic and 60 intrinsic asthmatics.

Identification of NAT2 alleles and genotypes

DNA was extracted from a blood sample by a standard phenol/chloroform extraction. 1211 base pair (bp) fragments containing the coding region of the NAT2 gene were amplified by the polymerase chain reaction (PCR). Seven mutation sites were identified by PCR-restriction fragment length polymorphism (RFLP) analysis as described [5, 11] with little modification. Alleles were classified according to Vatsis et al.[12]. The presence of the NAT2*4 (wild-type) allele is associated with the fast acetylator genotype, whereas combinations of the mutant alleles NAT2*5 A, *5B, *5C, *6 A, and *7B produce the slow acetylator genotype.

Total IgE, α₁AT and prick tests

Total IgE was determined by Immulite®[Diagnostic Products Corporation (DPC), CA, USA] which is a chemiluminescent enzyme-labelled qualitative immunoassay technique (normal range 1.0–183 IU ml⁻¹). α₁ AT concentrations were determined using an end point Behring nephelometer® (Mannheim, Germany) with an original kit (normal range 140.0–320.0 mg dl⁻¹). We used Stallargenes-Pasteur allergen extracts including Dermatophagoides pteronyssinus, Alternaria, Cladosporium, cat epithelia, Olea europea, Parietaria officinalis and Phleum pratense for the prick test. We used a phenolated glycerol-saline solution as a negative control and a histamine solution of 1 mg ml⁻¹ as a positive control. The results of the prick test were considered to be positive if the diameters of the indurations produced by histamine and the allergens were the same.

Statistics

Comparisons of sex, age, α₁AT and IgE concentrations between groups were analysed by one-way analysis of variance (anova). Fisher's two-sided exact test was used to compare the NAT2 genotype and allele frequencies between patient and control groups.

Results

Table 1 shows the serum concentrations of IgE and α₁ AT in extrinsic (n = 47) and intrinsic (n = 60) asthmatics. Total IgE concentrations were found to be significantly higher in extrinsic asthma patients (P < 0.001), whereas the concentrations of α₁ AT were similar in both groups.

The NAT2*5B allele was the most common among both patient groups and controls. However the difference in distinct allele frequencies was not statistically significant between groups. In extrinsic asthmatics, intrinsic asthmatics and control subjects the prevalence of slow acetylation genotypes was 70.4, 58.4, and 58.3%, respectively ( Figure 1). There was no statistically significant difference  in  the  prevalence  of  slow  acetylators  be­-tween extrinsic and intrinsic asthmatics (odds ratio 1.66, 95% confidence interval (CI) 0.94, 2.94, P = 0.085) or between all asthmatics and control subjects (odds ratio 1.31, CI 0.90, 1.92, P = 0.17). However, a moderate, but significant over-representation of slow acetylators was found among extrinsic asthmatics compared with control subjects (odds ratio 1.70, CI 1.04, 2.76, P = 0.042).

Figure 1.

Prevalencies of the NAT2 slow (▪) (the sum of *5 A/*6 A,*5B/*5C,*5B/*6 A,*5B/*7B,*5C/*5C,*5C/*6 A,*5C/*7B,*6 A/*6 A, and*6 A/*7B genotypes) and rapid () (the sum of *4/*4, *4/*5B, *4/*5C, *4/*6 A, and*4/*7B genotypes) acetylators in extrinsic asthmatics (EA), intrinsic asthmatics (IA), and control subjects (CS).

Discussion

Interindividual differences in the generation of reactive metabolites, due to genetic polymorphisms of xenobiotic-metabolizing enzymes, including NAT2, may influence formation of protein adducts, which in turn may result in a different susceptibility to chemically induced allergy and autoimmunity [13]. Two previous studies concluded that slow acetylation is associated with a predisposition to atopic disease [7, 8]. In both studies patients with atopic disease had an increased frequency of mutated NAT2 alleles. Zielinska et al. found that the prevalence of slow acetylators in the patient and control group was 91 and 62%, respectively [7]. Similarly, Gawronska-Szklarz et al. reported an over-representation of slow acetylators and found no homozygous rapid acetylators (NAT2*4/*4) among patients with atopic disease [8]. In a subgroup consisting of 15 patients with atopic asthma, the slow acetylation genotype was also over-represented [8]. In a subgroup of patients with extrinsic asthma (n = 7), phenotyped for NAT2, only slow acetylators were observed [14], although a predominance of rapid acetylators (83%) was detected among patients with pollinosis and mixed asthma. Therefore, the authors of these studies concluded that bacterial or viral infections may alter hepatic enzyme activity or modulate the sensitivity of the bronchi to external stimuli. In contrast, Schnuch et al.[15] found a significantly increased proportion of rapid acetylators in contact-allergic patients phenotyped and genotyped for NAT2 and suggested that acetylation may enhance contact sensitization or that NAT2 status may be a genetic marker for contact sensitizability. Overall, these findings suggest that the putative relationship between NAT2 polymorphism and allergy/atopy may be affected by the aetiology of the allergy.

In the present study, although the distribution of NAT2 genotype frequencies was not different in extrinsic asthmatics compared with intrinsic asthmatics, we found an association between NAT2 slow acetylation genotype and extrinsic bronchial asthma i.e. in patients with an atopic characteristic. Although the distribution of some drug metabolizing enzyme polymorphisms are different in the Turkish population [16] compared with other Caucasian populations, the prevalence of NAT2 genotypes [17] are very similar to that in Middle European populations [11, 18], including Poland. One explanation of the relatively ‘low’ frequency of slow acetylators among extrinsic asthmatics compared with those found in the two previous studies in this patient group may be due to the different ages studied. Although, there is no evidence of a sex difference in the distribution of NAT2 alleles and genotypes, the female/male ratio in allergic diseases may change significantly from a predominance of males in childhood to a higher proportion of females in adulthood [19]. Another possibility could be differences in environmental factors that lead to allergic diseases within populations [20]. Recently, Yilmaz et al.[21] reported that the allergens that cause allergic reactions in adults, but not in children, are significantly different in Turkey than other parts of Europe.

In conclusion, we have found an association between the NAT2 slow acetylation genotype and extrinsic asthma. Further studies are necessary to find out whether environmental differences within populations may influence this putative association.

This study was supported by a grant of the Research Foundation of University of Gaziantep, Turkey (Grant no. TF 97.11).