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Russian Journal of Genetics, Vol. 38, No. 4, 2002, pp. 439–445. Translated from Genetika, Vol. 38, No. 4, 2002, pp. 539–545.Original Russian Text Copyright © 2002 by Vavilin, Makarova, Lyakhovich, Gavalov.

INTRODUCTION

Bronchial asthma is a complex disease dependingon numerous genetic and environmental factors [1]. Forthe past thirty years, increasing asthma prevalence hasbeen observed worldwide. This unfavorable epidemio-logical phenomenon is believed to be caused bydegraded environment rather than by “asthma genes”,the occurrence of which could not change in such ashort period of time [2, 3]. Thus, it is important to studythe mechanisms and predisposing factors responsiblefor the disease onset under the environmental impact.

From this viewpoint, it seems expedient to study therole xenobiotic-metabolizing enzymes. Along with thechemical agents known to have a direct toxic and otherunfavorable effects on bronchi, the other compoundswere identified which in vivo acquire sensitizing prop-erties upon activation by the xenobiotic-metabolizingenzymes (XMEs) [4, 5]. Polymorphism of many genesof the cytochrome P450 superfamily and the conjugat-ing enzymes suggest that these genes are probablyassociated with predisposition to asthma under unfa-vorable environmental conditions, as is the case withecological (chemical) carcinogenesis [6]. XMEs arealso known to be involved in the metabolism of endoge-nous substrates, and, hence, they participate in sensitiza-tion, inflammation, brochoconstriction and other pro-cesses [7], which is another aspect related to the effect ofXMEs on predisposition to asthma and the clinical picture.

Little is known about the role of XMEs in asthmaetiology and pathogenesis. In the “case–control” stud-

ies, we have recently shown that the valine allele ofcytochrome P4501A1 (

CYP1A1-

Val), as well ashomozygous deletions of the glutathione S-transferasegene, M1 and T1 (null genotypes

GSTM1

- and

GSTT1

-),and

S2

mutation in the gene of arylamine N-acetyl-transferase are the factors of predisposition to bronchialasthma in children. Conversely, the

S1

mutation of the

NAT2

gene proved to be resistance factors [8]. Detect-ing the associations between the genetic markers andcomplex diseases by assessment of disequilibrium intheir distribution between healthy and affected subjectscan be a critical first step toward identification of thegenetic basis of the disease. Further analysis of coseg-regation of the genetic markers and the disease inpatient’s families by using parents and sibs as controlmakes it possible to obtain a more reliable support forassociation between the trait and disease due to mini-mizing stratification of the groups for the external fac-tor, living conditions, etc. [9]. The first approach was acomparison the frequencies of allele

CYP1A1-

Val,mutations

S1

and

S2

of the

NAT2

gene, null genotypes

GSTM1

and

GSTT1

in affected children born toaffected parents and in affected children from familieshaving no patients with bronchial asthma.

MATERIALS AND METHODS

We have examined 100 children with bronchialasthma (68 males and 32 females) aged six to fifteenyears (the average age 10.8 years). In each case history,attention was focused on the following aspects: the

Polymorphic Genes of Xenobiotic-Metabolizing Enzymes Associated with Predisposition to Bronchial Asthma

in Hereditarily Burdened and Nonburdened Children

V. A. Vavilin

1

, S. I. Makarova

1

, V. V. Lyakhovich

1

, and S. M. Gavalov

2

1

Institute of Molecular Biology and Biophysics, Russian Academy of Medical Sciences, Novosibirsk, 630117 Russia;e-mail: drugsmet@cyber.ma.nsc.ru

2

Novosibirsk State Medical Academy, Novosibirsk, 630091 Russia

Received August 21, 2000; in final form, July 24, 2001

Abstract

—The frequencies of the

CYP1A1

valine allele, homozygous deletions of

GSTM1

and

GSTT1

, andtwo point mutations of the

NAT2

gene,

NAT2

:

S1

(C

481

T)

and

S2

(G

590

A)

, were compared in healthychildren and children having bronchial asthma. The

S1

mutation was associated with resistance, and all of theother traits, with predisposition to the disease. In families of patients with diseased progenitors and in those withhealthy progenitors, the estimates of the asthma risk were similar. In both groups, parameters of the trait asso-ciation with the disease depended on passive smoking. At passive smoking, a trend to an overrepresentation(high odds ratio, OR) of the

GSTM1

null genotype and

S2

mutation of the

NAT2

gene was observed, whereasthe odds ratio of the

GSTT1

null genotype decreased, and those of the

CYP1A1

and

S1

mutation of the

NAT2

gene remained unchanged. The highest OR = 36.25 (

P

< 0.01) was characteristic of the

GSTT1

null genotypein nonsmoking hereditary burdened patients. The results obtained suggest an important role of xenobiotic-metabolizing enzymes in development of bronchial asthma.

HUMAN GENETICS

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et al

.

presence of patients with bronchial asthma in the fam-ily, unfavorable environmental factors associated withresidence and parent occupation, and smoking of par-ents and children. The affected children, whose parentshad bronchial asthma, constituted the group of heredi-tary burdened patients (30 subjects). The group ofhereditary nonburdened patients (65 subjects) com-prised affected children whose nearest progenitors didnot suffer from asthma. Five children who had asthmapatients among brothers and sisters of their parentswere excluded from analysis. In both groups, the pas-sive smokers (PS) were distinguished. These werethose patients whose relatives or neighbors were smok-ing in the apartment. The children not exposed totobacco smoke were referred to as nonsmoking. Thecontrol group comprised 104 children including58 males (55.8%) and 46 females (44.2%) aged 4 to14 years (average age 8.2 years). The children consti-tuting the control group met the major criterion: theydid not show any signs of sensitization. The controlgroup comprised 74 passive smokers and 30 nonsmok-ing children (71.2 and 28.8%, respectively). All thechildren examined were Caucasoids, which excludedthe influence of the ethnic factor on the polymorphictrait distribution in the groups.

The onset and progression of the bronchial asthmaare known to be age- and sex-dependent [2]. In ourstudy, the significant differences in these traits betweenthe groups of diseased and healthy subjects were elim-inated, as well as between the nonsmoking subjects andPS within and between the groups.

DNA was isolated from the whole blood samples asdescribed by Kunkel [10]. Genotyping was performedusing polymerase chain reaction (PCR). Isoleucine–valine polymorphism of

CYP1A1

, and the

GSTM1

and

GSTT

null genotypes was analyzed as described byHayashi

et al.

[11], Zhong

et al.

[12], and Pemble

et al.

[13], respectively. Analysis of restriction fragmentlength polymorphism was used to identify mutations inthe

NAT2

gene. To amplify the 547-bp regions of the

NAT2

gene, we used primers described by Gil et Lech-ner [14]. The

S1

mutations (a cluster of alleles

NAT2*5A, B, D, F

;

NAT2*11

,

NAT2*12C

, and

NAT2*14C

) [15] was identified by DNA hydrolysiswith restriction endonuclease

Kpn

I. The

S2

mutation(a cluster of alleles

NAT2*5E, NAT2*6A, B, C, D,NAT2*14D

) [15] was identified by DNA hydrolysiswith restriction endonuclease

Taq

I [14].

To detect genotype association with bronchialasthma (BA) the odds ratio (OR) was determined,which indicates how many times higher is the likeli-hood that a subject with a definite genotype may fall illwith asthma than remain healthy:

OR = (A/B)/(C/D)

,where

A

is the number (percent) of subjects with thegiven genotype in the group of patients (“case”);

C

isthe same value in the group of healthy subjects (“con-trol”) [16];

B

and

D

are the numbers (percent) of sub-jects that do not express the given genotype among

patients and healthy subjects, respectively. In the“case–control” experiments, the odds ratio is in a sensesimilar to the relative risk index in the cohort studies.When calculated from comparison of frequencies innonsmoking patients and healthy children, or, con-versely, in diseased and healthy PS, the ORs are indic-ative of the effect of genotype on the BA risk thoughunder different environmental conditions. The resultsobtained were treated by using the Epilnfo 6 computerprogram. The

χ

2

analysis with Yates corrections wasused to determine significant differences in frequenciesof the studied traits between the group of patients andthe corresponding control group. When less than fiveobservations were available in the reference group, thetwo-tailed Fisher’s test was used.

RESULTS AND DISCUSSION

The results of genotyping are shown in Tables 1 and 2.In the control sample, the frequency of the genotypesstudied was similar to that observed in populations ofadult Caucasoids in West Europe and North America[12, 17, 18–21]. In the patient group, the frequency ofthe

CYP1A1

Ile/Val genotype and that of the

GSTM1

and

GSTT1

null genotypes were higher than in control,whereas the frequency of

S1

mutation in the

NAT2

genewas lower. The OR values indicate that the first threegenotypes were predisposing to bronchial asthma, andthe effect of

GSTT1

– genotype as risk factor was signif-icantly higher than that of the

GSTM1

–. The

S2

muta-tion of the

NAT2

gene was not a BA risk factor. Notethat the presence of

S1

mutation of the

NAT2

gene is aBA resistance factor, whereas the absence of this muta-tion is a factor predisposing to BA.

Association between the combinations of genotypesand asthma were also analyzed, because interactionbetween traits is commonly observed in vivo. Table 3shows that the effects of the

S1

mutation were superiorto those of the

S2.

All combinations including the

S1

mutation were associated with resistance to asthma.Note that the effect of the

S2

as a risk factor wasobserved in the absence of

S1

mutation, which was notrevealed by analysis of individual genotypes. In combi-nations including the glutathione S-transferase geno-type, the

GSTT1

gene dominated: irrespective of the

GSTM1

genotype, the

GSTT1

null allele was the BArisk factor, whereas the

GSTT1

plus genotype was aresistance factor.

In both hereditarily burdened and nonburdened chil-dren, the

GSTT1

null genotype was significantly asso-ciated with predisposition to asthma, whereas the

S1

mutation was associated with resistance to the disease(Table 4).

Despite the high OR values, the

CYP1A1

Ile/Valassociation with predisposition to asthma was not sta-tistically significant because of the small number ofobservations. In both groups, the

GSTM1

– genotypewas also associated with BA risk, though to a lesser

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POLYMORPHIC GENES OF XENOBIOTIC-METABOLIZING ENZYMES 441

extent than with the

CYP1A1

Ile/Val and

GSTT1

– geno-types.

Thus, the hereditary burden does not necessarilylead to the effect of the XME genes as asthma risk fac-tors, and the genes studied were similarly associatedwith asthma in hereditarily burdened and nonburdenedpatients.

To evaluate the role of the traits studied in patho-genic effects of the environmental factors, associationof the genotypes with predisposition to asthma wasstudied in both groups of patients separately in non-smoking subjects and passive smokers (Table 4). The

results obtained suggest that the effects of traits as riskfactors differed in the nonsmoking and PS. In passivesmokers, the effects of

S2

mutation and the

GSTM1

–genotype as risk factors were more pronounced, as wellas the protective effect of a combination including

S1

mutation of the

NAT2

gene and the

GSTT1

and

GSTM1

plus genotypes. Conversely, the risk estimates of the

GSTT1

– genotype were higher in the nonsmoking sub-jects.

In hereditarily burdened patients, the risk estimateswere higher in both groups. The only exception wascombination

GSTM1+/GSTT1+/S2 in the NAT2.Our results suggest that the polymorphic genes stud-

ied are the candidate asthma genes having moderateeffects. Note that at present, this gene species isassumed to be of particular importance in studying thenature of complex diseases [22]. The role of the XMEgenes in asthma is probably similar to that in carcino-genesis. As shown by the epidemiological studies, onlyin 5% of cases, the inherited genetic factors areinvolved in malignancy, whereas in 95% of cases, inter-action between environmental carcinogenic agents andgenetic factors leads to an acquired susceptibility [6].Due to specific features of the XME superfamily (themultiple forms, overlapping substrate specificity, andinducible synthesis) the disturbed functioning of indi-vidual xenobiotic-metabolizing enzymes may beimproved at the expense of other enzymatic activities,which is a factor accounting for the low disease riskassociated with these enzymes under normal environ-mental conditions. The XME gene polymorphism is

Table 1. Frequencies of the XME-gene mutant alleles in pa-tients with bronchial asthma and control children from No-vosibirsk

Allele or mutationFrequency

patients control

CYP1A1-Val 0.062 0.005

GSTM1– 0.307 0.234

GSTT1– 0.140** 0.054

S1 0.374*** 0.562

S2 0.337 0.345

Note: For the genotypes CYP1A1, GSTM1, and GSTT1, n = 100(patients) and n = 104 (control); for the S1 and S2 mutationsof the NAT2 gene, n = 95 (patients) and n = 97 (control).* P < 0.05; ** P < 0.01; *** P < 0.001.

Table 2. Association of the XME genotypes with predisposition to asthma

Trait

Number (percent) of the trait carriers

Odds ratio (95% confidence interval)patients# control#

n % n %

CYP1A1 Ile/Ile 88 88 99 95.2

CYP1A1 Ile/Val 12 12 5 4.8 2.7(0.84–10.13)

GSTM1– 52 52 44 42.3 1.48(0.72–2.67)

GSTM1+ 48 48 60 57.7

GSTT1– 26** 26 12 10.5 2.69(1.20–6.11)

GSTT1+ 74 74 92 89.5

Homozygotes for S1 17 17.9 26 26.8 0.60(0.28–1.25)

Heterozygotes for S1 37* 38.9 55 56.7 0.49(0.26–0.90)

The presence of S1 54*** 56.8 81 83.5 0.26(0.13–0.54)

The absence of S1 41*** 43.2 16 16.5 3.79(1.87–7.98)

Homozygotes for S2 12 12.6 13 13.4 0.93(0.37–2.34)

Heterozygotes for S2 40 42.1 41 42.3 0.99(0.54–1.83)

The presence of S2 52 54.7 54 55.7 0.96(0.52–1.77)

The absence of S2 43 45.3 43 44.3 1.04(0.56–1.91)# For the genotypes CYP1A1, GSTM1, and GSTT1, n = 100 (patients) and n = 104 (control); for the NAT2 gene, n = 95 (patients) and n = 97

(control); * P < 0.05; ** P < 0.01; *** P < 0.001.

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widespread in Caucasoid population (40 to 60% ofslow acetylators [21], 40 to 60% and 10 to 20% of car-riers for homozygous deletion in the GSTM1 andGSTT1, respectively [12, 17, 18], and 6.5 to 7% of car-riers for the CYP1A1Ile/Val genotype [19, 20, 23]) and,therefore, the role of this enzymatic system isincreased, under severe environment caused by toxify-ing/detoxifying imbalance of xenobiotics and increasedformation of conjugated antigens, which results inimportant changes in asthma epidemiology. Variousenvironmental factors including NO2, SO2, ozone,tobacco smoke and diesel exhaust may increase sensiti-zation [24]. The two last factors contain many compo-nents that are metabolized in vivo through the pathwaysleading to formation of toxigenic products. Epidemio-logical analysis for the prevalence of occupational

asthma showed that 0.2 to 0.5% of the young adultsbecome asthmatics because of their occupation [25].There is evidence of the association between bladdercancer and preceding asthma or tuberculosis in Koreansexpressing the GSTM1– and GSTT1– genotypes andfast acetylator phenotype [26]. Examination of passivesmokers in the present study provided the results indi-cating that there is a direct gene–environment interac-tion with regard to the GSTM1–, GSTT1– genes, and S2mutation of the NAT2 gene. According to Khoury andJames classification for the types of gene–environmentinteraction [27], the GSTM1– gene and S2 mutation ofthe NAT2 gene can be assigned to the genetic risk fac-tors interacting with the environmental risk factor toenhance the effect of the latter. The effect of theGSTT1– risk factor is attenuated in passive smokers.

Table 3. Association of genotype combinations with predisposition to asthma

Combination of traitsNumber of subjects expressing the given genotype

Odds ratio (95% confidence interval)patients# (N = 100) control# (N = 104)

The absence of both S1 and S2 9 3 3.28 (0.78–19.33)

The presence of only S1 34 41 0.76 (0.41–1.42)

The presence of only S2 33 13** 3.44 (1.59–7.55)

The presence of both S1 and S2 12 40*** 0.39 (0.18–0.71)

GSTM1– and GSTT1– 11 5 2.45 (0.75–9.3)

GSTM1+ and GSTT1– 15 7* 2.45 (0.88–6.99)

GSTM1– and GSTT1+ 41 39 1.16 (0.63–2.11)

GSTM1+ and GSTT1+ 33 53*** 0.47*** (0.26–0.87)

* P < 0.05; ** P < 0.01; *** P < 0.001; for NAT2, n = 95 (patients) and n = 97 (control).

Table 4. Association of the XME genotypes with predisposition to asthma in hereditary burdened and nonburdened patients

Traits

Number (percent) of subjects with the given traits Odds ratio (95% confidence interval)

control hereditarily burdened patients (N = 30)

hereditarily nonbur-dened patients (N = 65)

hereditarilyburdened patients

hereditarilynonburdened patients

CYP1A1Ile/Val 5 (4.8) 4 (13.8) 8 (12.3) 3.85 3.51

(p = 0.074) (p = 0.06) (0.66–21.86) (0.89–16.51)

GSTM1– 44 (42.3) 18 (58.6) 33 (50.8) 2.05 1.41

(p = 0.087) (p = 0.28) (0.83–5.08) (0.72–2.75)

GSTT1– 12 (10.5) 10 (31) 15 (23.1) 3.83 2.3

(p = 0.0096) (p = 0.046) (1.31–11.24) (0.93–5.73)

S1 in NAT2 81 (83.5) 14 (48.3) 38 (58.5) 0.17 0.27

(p = 0.0001) (p = 0.00069) (0.06–0.46) (0.12–0.6)

S2 in NAT2 54 (55.7) 16 (55.2) 34 (52.3) 0.96 0.92

(p = 0.914) (p = 0.787) (0.39–2.34) (0.47–1.8)

RUSSIAN JOURNAL OF GENETICS Vol. 38 No. 4 2002

POLYMORPHIC GENES OF XENOBIOTIC-METABOLIZING ENZYMES 443

The S1 mutation of the NAT2 gene has a protectiveeffect.

Nevertheless, the hereditary diseases associatedwith the XME genes has been described. Some of themwere accounted for by a defective enzyme function inmetabolism of the endogenous substrate. Thus, the Cri-gler–Najjar syndrome, congenital familial non-hemolytic jaundice, is caused by a decreased activity ofthe UDP-glucuronosyltransferase 1A [28], whereas21α-hydroxylase deficiency accounts for the congeni-tal adrenal hyperplasia [29]. In patients with primarycongenital glaucoma caused by a mutation in theCYP1B1 gene, the mutant CYP1B1 protein is assumedto affect the processes of growth and differentiationduring embryonic development [30].

The results obtained in our study show that associa-tion between the genotypes studied and predispositionto asthma is observed in children born to healthy par-ents and depends on such an environmental factor aspassive smoking. This suggests that XME participate inasthma development through formation of the“acquired susceptibility”. At the same time, the highOR values for the GSTT1– and S2, a strong protectiveeffect of the GSTT1+ and S1 mutation of the NAT2gene, and increased OR values in the group of thehereditary burdened patients suggest also anotherexplanation. The disturbed XME functioning mayaffect the development of the immune system and for-mation of bronchial reactivity during intrauterinedevelopment and in early childhood. Analysis of famil-ial inheritance of the polymorphic XME genes in fami-

Table 5. Association of the genotypes with predisposition to asthma in nonsmoking and PS groups of hereditary burdenedand nonburdened patients

Combination of traits

Odds ratio

hereditarily burdened patients hereditarily nonburdened patients

nonsmoking PS nonsmoking PS

CYP1A1Ile/Val 4.0 3.84 4.48 3.04

GSTM1– 0.98 1.75 1.01 0.72

GSTT1– 36.25** 1.59 6.96# 2.1#

S1 mutation in NAT2 0.17 0.17** 0.27 0.26**

S2 mutation in NAT2 0.19 1.16 0.52 0.64

Neither S1* nor S2 present in NAT2 3.86 1.67 4.32 2.36

Only S1 in the NAT2 1.33 0.56 0.42 1.3

Only S2 in the NAT2 1 6.2** 2.65 2.95#

Both S1 and S2 present in NAT2 0.117 0.16# 0.86 0.2**

GSTM1– /GSTT1– 17.4* 2.68 3.35 1.0

GSTM1+/GSTT1– 9.67 0.99 3.35 2.14*

GSTM1–/GSTT1+ 0.27 2.34 1.16 1.1

GSTM1+/GSTT1+ 0.18# 0.36# 0.37# 0.34*

GSTM1– /GSTT1– and only S1 9.33 1.48 2.07 3.15

GSTM1+/GSTT1– and only S1 9.24 1.48 0.97 2.1

GSTM1+/GSTT1– and only S2 4.14 3.14 3.35 2.06

GSTM1– /GSTT1+ and only S1 No case among patients 0.84 0.53 1.21

GSTM1– /GSTT1+ and only S2 4.14 5.79** 0.97 1.24

GSTM1– /GSTT1+ /S1 in NAT2/S2in NAT2

No case among patients 0.31 1.23 0.42

GSTM1+ /GSTT1+ and only S1 0.43 0.9 0.22# 0.37

GSTM1+ /GSTT1+ and only S2 1.04 2.13 0.96 5.74*

GSTM1+ /GSTT1+ /S1 in NAT2/S2 in NAT2 No case among patients 0.16# 0.48 0.11*

Note: Significant differences between control and the group: * P < 0.05; ** P < 0.01; # P ≤ 0.1.

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VAVILIN et al.

lies of patients with bronchial asthma would be helpfulin distinguishing between the two above probabilities.

The following conclusions can be inferred from theresults obtained:

(1) the CYP1A1-Val allele, S2 mutation in the NAT2gene, the absence of the S1 mutation in the latter gene,and both GSTM1– and GSTT1– genotypes are associ-ated with predisposition to bronchial asthma in chil-dren;

(2) The same associations were not necessarilyobserved in the hereditary burdened patients, thoughthe risk estimates in the latter were higher.

ACKNOWLEDGMENTS

This work was supported by the State Research Pro-gram “Russian Population Health,” direction 06.04(project no. 06.04.01.04).

The authors thank to O.A. Ryabova, an assistant ofthe Department of Pediatrics, Faculty of PhysicianImprovement, Novosibirsk State Medical Academy, forperforming the clinical studies. We are grateful forgenotyping GSTM1, GSTT1, and CYP1A1 to O.B. Cha-sovnikova and N.I. Gutkina (Institute of MolecularBiology and Biophysics, Siberian Division, RussianAcademy of Medical Sciences).

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