arylamine n -acetyltransferase gene polymorphisms: markers for atopic...

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Arylamine N-acetyltransferase gene polymorphisms: markers for atopic asthma, serum IgE and blood eosinophil counts
Jyotsna Batra1, Surendra K Sharma2 & Balaram Ghosh1†
†Author for correspondence1Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007, IndiaTel.: +91 11 2766 2580;Fax: +91 11 2766 7471; +91 11 2741 6489;E-mail: [email protected] India Institute of Medical Sciences, Division of Pulmonary and Critical Care Medicine, Department of Medicine, Delhi-110029, IndiaTel.: +91 11 2658 8500 3303;E-mail: [email protected]

Keywords: atopic asthma, eosinophil counts, genotype, haplotype, NAT2, serum total IgE

10.2217/14622416.7.5.673 © 2

Introduction: Polymorphisms in N-acetyltransferase 2 (NAT2), present on chromosome 8p22, are responsible for the N-acetylation variants, which segregate human populations into rapid, intermediate and slow acetylators and influence the susceptibility towards atopic disorders. We have undertaken a study of the North Indian population to screen for various NAT2 polymorphisms and to investigate their association with atopic asthma and related phenotypes. Methods: First, to establish linkage of the 8p22 region with asthma, 158 families were recruited from North India. Next, a total of 219 unrelated atopic asthmatics and 210 unrelated healthy controls were recruited for case–control disease association studies. Results: A suggestive linkage was observed with microsatellite marker D8S549, 2.6 MB upstream of NAT2. By sequencing the DNA of 40 individuals, the T111C, G191A, A434C and C759T single nucleotide polymorphisms (SNPs) in NAT2 were found to be nonpolymorphic in our population and a pattern of strong linkage disequilibrium was observed among the T341C, C481T and A803G polymorphisms. Thus, a total of 429 individuals were genotyped for the C481T and unlinked C282T polymorphisms. The C481T polymorphism was found to be significantly associated with asthma in our case–control studies at the genotype level (Armitage p = 0.00027). C481T also showed a marginal association with serum total IgE (TsIgE) (p = 0.022). Furthermore, percent blood eosinophil counts were found to be significantly higher in patients carrying the 481T allele (p = 0.0037). Significant association was also detected with respect to the C282T polymorphism and TsIgE (p = 0.008). Moreover, C_T was found to be an important risk (p = 0.001), while C_C was a major protective haplotype (p = 0.0005). The associations remained significant after Bonferroni correction for multiple testing. Conclusion: In summary, the genetic variants of the NAT2 gene do not seem to affect asthma alone, but act as modulators of asthma-related traits, such as serum IgE and blood eosinophil counts, and therefore could serve as genetic markers.

Atopic asthma is characterized by increasedbronchial responsiveness, constriction andmucous hypersecretion in the bronchial wallsin response to a variety of direct and indirectstimuli, leading to the symptoms of cough,wheeziness and shortness of breath [1,2]. Thepathogenesis and etiology of asthma is verycomplex and not fully understood. The occur-rence of asthma and allergy has been suggestedto be induced by exposure to ambient environ-ment/air pollution acting on a specific geneticbackground [3–5]. However, xenobiotics thatcould initiate the development of these diseaseshave yet to be identified. Furthermore, fewstudies have shown an association betweenasthma and polymorphisms of enzymes thatplay an important role in the biotransforma-tion of these exogenous, as well as endogenouscompounds, such as histamine N-methyltrans-ferase, glutathione S-transferase, and so on[6–8]. In addition, polymorphisms of N-acetyl-

transferase (NAT) members have also been sug-gested as one of the susceptibility factors tolung diseases [9–11].

The arylamine NATs are a unique family ofenzymes that participate in the detoxification of aplethora of hydrazine and arylamine drugs [12,13].There are two NAT isoforms, NAT1 and NAT2,which acetylate amino, hydroxyl and sulfhydrylgroups. N-acetylation of molecules is typically adetoxifying reaction, while O-acetylation can pro-duce reactive species [14,15]. In general, NAT2 hashigh affinity for N-acetylation of most aromaticamines and deactivates them, while NAT1 has amajor role in the O-acetylation of N-hydroxy aro-matic amines and leads to the activation of aro-matic amines [16]. NAT1 and NAT2 are located onhuman chromosome 8p22 and share 87% nucle-otide homology in the coding region [17,18]. Boththe enzymes are highly polymorphic. The classicalN-acetylation patterns resulting from variantNAT2 alleles segregate human populations into

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RESEARCH REPORT – Batra, Sharma & Ghosh

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rapid (NAT2*4), intermediate, and slow acetylator(NAT2*5, *6, *7 and *14) phenotypes [19] and hasbeen correlated with various malignancies and dis-eases, such as rheumatoid arthritis and diabetes[20–22]. The presence of NAT2 mRNA in the epi-thelial cells lining of respiratory bronchioles, albeitat low levels, suggests that the enzyme may beimportant in metabolizing inhaled pollutants inthe lungs, and thus the susceptibility to various res-piratory disorders including asthma [23,24]. Previ-ous studies on the possible role of the acetylationpolymorphism in asthma pathogenesis providedambiguous results. Makarova and colleagues havereported the protective effect of slow acetylationgenotypes (*5) in bronchial asthma in the Russianpopulation [25], while Luszawska-Kutrzeba andNacak and colleagues showed a high prevalence ofslow acetylation encoding genotypes in patientswith atopy, and particularly bronchial asthma,from the Polish population and in extrinsic asth-matics from the Turkish population, respectively[26,27]. Slow acetylation genotypes have also beenshown to be an important factor for individual sus-ceptibility to other atopic diseases in the Polishpopulation [28,29]. However, to date no such studieshave been reported from the Indian population.

The effector role of NAT2 in asthma pathologyand the results of the previous genetic studiesprompted us to analyze the association of varioussingle nucleotide polymorphisms (SNPs) andrepeat polymorphisms in and around the NAT2gene with asthma and associated phenotypes in agenetically untapped Indian population.

Patients & methods Subjects In a multicenter-based asthma genetic study pro-gram, a total of 219 asthmatic patients and210 normal healthy controls were recruitedthrough various collaborating hospitals (n = 87from Kalawati Saran Children's Hospital, NewDelhi, India, n = 59 from All India Institute ofMedical Sciences, New Delhi, India, n = 38 fromThe Vallabhbhai Patel Chest Institute [VPCI],Delhi, India and n = 35 from Asthma and AllergyCentre, Mumbai, India) of North and North-WestIndia (Table 1). Approval of the ethics committeesof all the participating centers and hospitals wereobtained. Written consent was obtained from allthe participants for genetic studies, performingskin prick tests and drawing blood samples.

For the family-based analysis, an independentcohort of 158 families ascertained through asth-matic probands was recruited from thecollaborating hospitals and family visits (Table 1).

Study design was such that at least oneatopic/atopic asthmatic child was collected alongwith both the parents. Families were furtherextended wherever other members gave their con-sent to participate. Thus, a total of 538 individualswere recruited with an average family size of 3.41(3–6) individuals per family.

Clinical evaluation of the phenotypesAsthma in the recruited study population wasdefined by clinical history and subsequently vali-dated by interview questions as described previ-ously [30]. Medical data on the diagnosis of asthmaand atopic diseases or other respiratory disorders,their duration, skin problems, types and doses ofmedications and history of tobacco smoking wereobtained by completing a detailed questionnaire.Details of environmental factors, family history ofasthma/atopy, and the geographical region oforigin and migration status were also enquired.

Patients and family members were diagnosedby a physician for asthma on the basis ofNational Asthma Education and Prevention Pro-gram (Expert Panel Report-2) guidelines [30] andwere examined for a self-reported history ofbreathlessness and wheezing. All the probandsmet the following criteria: a positive skin pricktest, a positive response to at least one of the twoquestions (have you ever had attacks of breath-lessness at rest with wheezing? Have you ever hadan asthma attack?), associated with positive val-ues for at least two out of the following threeparameters: presence of bronchial hyper-responsiveness (BHR) (defined as forced expira-tory volume in 1 second [FEV1]/forced vitalcapacity [FVC] below 70% at the time of attackand improvement by bronchodilator), hospitali-zation for asthma in life, or asthma therapy. Theclinical parameters are summarized in Table 1.

A total of 15 common environmental aller-gens with both negative and positive controlswere used for the skin prick test. Atopy wasdefined as a dichotomous variable (yes/no), hav-ing wheal reaction equal to or greater thanhistamine for at least one allergen.

Serum samples from both cohorts were analyzedfor total immunoglobulin E (IgE) levels using anenzyme immunoassay based on the sandwich tech-nique (Bethyl Laboratories Inc., TX, USA) asdescribed [5], except for few individuals (<10%)where sera were not available. All IgE values wereconverted to log-normal scale for analysis.

Healthy volunteers (referred to as normal con-trols) were recruited from the general population(case-matched geographical area) who answered

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negatively to a screening questionnaire forrespiratory symptoms and on the basis of the cri-teria of having no symptoms or history of allergicdiseases. Individuals having a history of smokingand parasitic/helminthic infestations wereexcluded from the study. Skin prick and pulmo-nary function tests were also performed, whereverconsent was obtained.

Genomic DNA preparationDNA was isolated from peripheral white bloodcells using the modified salting out method, andwas stored at -20°C until further analysis [30].

Genotyping of microsatellites around NAT2Two repeat markers flanking the NAT2 gene;D8S549 (2.6 MB upstream) and D8S258(3.5 MB downstream) were chosen from ABILinkage Mapping set V2.0 (Applied Biosystems,CA, USA) and were validated for distribution inour study population by means of a polymerasechain reaction (PCR) using hexachlorofluorescein(HEX)-labeled oligos (according to the manufac-turer’s instructions). Fragment lengths were deter-mined using the GeneMapper® Software version3.7 (Applied Biosystems).

Identification of NAT2 polymorphisms & genotypingTo screen for the sequence variation in NAT2, a895 base pair (bp) fragment containing the

coding region of NAT2 was amplified using theprimers and cycling conditions described byDoll and colleagues [31], with slight modifica-tions. PCR products from 40 individuals weresequenced on an ABI 3100 capillary sequencer(Applied Biosystems) by BigDye® terminator kitV 3.1 using both forward and reverse primers.Sequences were aligned and analyzed usingSeqMan™ II (DNASTAR, WI, USA).

The 282C/T and 481C/T polymorphismswere investigated in the study population bydigestion with FokI and KpnI, respectively [31].These fragments were subjected to electrophoresison 2% agarose gel and visualized with an ultra-violet (UV) transilluminator following ethidiumbromide staining.

Data analysisTo test for linkage with the genetic markers(microsatellite repeats) flanking NAT2, familydata was analyzed for the transmission of repeatpolymorphisms using the TDT/sTDT programv1.1 [101]. Linkage disequilibrium between all theidentified SNPs was evaluated using Haploview[32]. An association analysis, based on thecase–control design, was performed for eachpolymorphism (C282T and C481T) using theArmitage trend test, following the guidelinesprovided by Sasieni [33] as implemented in theprogram FINETTI [102]. The Armitage trendtest based on the genotypes remains valid even if

Table 1. Demographic profile of the patient and the control groups in case–control study and probands of families in linkage study.

Patients (n = 219) Controls (n = 210) Probands (n = 158)

Native place* North India North India North India

Mean age (years) 20.07 (± 16.4) 27.47 (± 9.95) 12.9 (± 9.6)

Sex ratio (F:M) 46:54 32:68 36:64

Familial history of asthma/atopy†

All None All

Smoking history¶ None None None

% reversibility from baseline FEV1 (after β2-agonist usage)

>15% ND >15%

Log10-mean serum total IgE

(IU/ml)

2.94 (± 0.65) 2.33 (± 0.61) 2.97 (± 0.65)

Self-reported history of allergies All None All

*Patients and controls were recruited from various collaborating hospitals in Delhi (North India) and 16% from Mumbai (North-west India).†Control individuals were subjected to a questionnaire so as to eliminate all individuals having atopic disorders or family history of atopic disorders. Only those patients who have at least one first-degree relative affected with atopy and/or asthma were included for the study.¶Patients and controls known to have experienced smoking in the past, or suffering from parasitic infections, were excluded from the study. Parenthesis contains the values for standard deviation (SD). ‘ND’ denotes that the test was not done. FEV1: Forced expiratory volume in 1 second; IgE: Immunoglobulin E.

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Table 2. Transmissio(D8S549, 2.6 MB upflanking NAT2 in 15asthmatic probands

Allele T

D8S549*

1

2

3

4

D8S258§

1

2

3

4

5

6

*Overall p = 0.012, df = 3.§ Overall p = 0.23, df = 5.NAT2: N-acetyltransferase

Hardy–Weinberg equilibrium (HWE) does nothold. Still, for both polymorphisms, we testedwhether the genotype distribution in the con-trols was in HWE using FINETTI. Two locihaplotypes of each individual were inferred andanalyzed using the algorithm developed byStephens and colleagues [34], as implemented inPHASE v 2.1 [103]. Differences in haplotype fre-quencies were also compared in the two groupsusing the Monte Carlo approach, by performingrepeated simulations (1 million in this case) asimplemented in CLUMP v 2.2 [35]. For serumtotal IgE levels and blood eosinophil counts, lin-ear trends and means were tested using analysisof variance (ANOVA). Bonferroni correctionwas applied to correct for loss of significance dueto multiple testing; considering the associationof two NAT2 SNPs: C282T and C481T, withasthma phenotype as the primary variable, α isadjusted to α/2= 0.025.

ResultsLinkage of NAT2 flanking microsatellites with asthma binary traitA total of four and six alleles for the two NAT2flanking genetic markers, D8S549 andD8S258, respectively, were found to be presentin our study population (Table 2). In a test forlinkage with these repeat markers, we evaluatedthe transmission of the marker alleles from aheterozygous parent to an affected offspring. Atransmission distortion of D8S549 alleles was

observed, providing evidence for a significantlinkage of this marker to asthma phenotype(degrees of freedom [df ] = 3, p = 0.012). Thislinkage analysis provided us the necessary sup-porting evidence to study the role of NAT2 inasthma and related phenotypes usingcase–control association studies.

Resequencing of NAT2 gene & linkage disequilibriumWe sequenced the 895 bp coding region of theNAT2 gene in 40 individuals irrespective of theirdisease status. The earlier reported T111C,G191A, A434C and C759T SNPs were notfound to be polymorphic in our sequencingcohort (Figure 1A). A845 and G857A were notchecked due to technical difficulties. Pairwiselinkage disequilibrium (LD) among the otherpolymorphisms, C282T, T341C, C481T,G590A and A803G, were calculated. A haplo-type block consisting of four SNPs was identifiedand a pattern of strong linkage disequilibriumwas observed among the T341C, C481T andA803G polymorphisms (Figure 1B). The most fre-quent C282T and C481T polymorphisms wereselected for further genotyping.

Association of NAT2 SNPs with asthma binary traitGenotype and allele frequencies of the C282Tand C481T polymorphisms in cases and con-trols are shown in Table 3. Both the poly-morphisms were found to be in HWE in thecontrol population. The C481T polymorphismwas found to be significantly associated withasthma in our case–control studies at the geno-typic level (p = 0.00027). The CC genotype wasfound to be more frequent in controls in com-parison with the asthmatics (p < 0.000001,OR = 0.35, 95% CI = 0.224–0.554).

Association of NAT2 with serum total IgE & % blood eosinophil countsLog transformed serum total IgE (TsIgE) levelswere found to follow a normal distribution. Ithas been observed that the total serum IgE levelsin our control group are comparatively higherthan that of western populations. As India is atropical country, this difference is expected dueto exposure to helminthic/parasitic infestation atsome early stage of their life, although at thetime of sample collection the individuals had nosuch infestations. When the cases and controlswere compared with respect to log serum totalIgE values, a highly significant difference was

n disequilibrium test of the marker alleles stream and D8S258, 3.5 MB downstream) 8 families ascertained through .

ransmitted Nontransmitted χ2

62 74 1.059

98 67 5.824

60 77 2.109

0 2 2

10 18 2.286

80 70 0.667

70 62 0.485

62 65 0.071

19 23 0.381

0 3 3

2; df: Degrees of freedom.




Table 3. Allele and gpolymorphisms stud

Allele/genotype

Pati(N =

C282T

C 228 (5

T 208 (4

C481T

C 210 (4

T 228 (5

C282T

CC 58 (2

CT 112 (5

TT 48 (2

C481T

CC 39 (1

CT 132 (6

TT 48 (2

The level of significance αa

§Armitage’s trend test p va‡Test was not done for theNumbers in parentheses inin bold.NAT2: N-acetyltransferase

obtained (F-ratio = 31.87, df = 1, p < 0.0001).Similarly, patients were found to have highabsolute eosinophil counts (694.15 ± 309.15).

Since increased serum total IgE levels andperipheral eosinophil counts are the major charac-teristics of atopic asthma, the genetic effects ofNAT2 polymorphisms were also tested on thesetwo traits. Significant association was detectedwith respect to the C282T polymorphism andTsIgE (F-ratio = 4.9, df = 2, p = 0.008). Post hocTukey test analysis showed a significant difference(p < 0.05) of CT vs TT (contrastdifference = 0.25, Tukey 95% CI = 0.062 to0.439). C481T also showed a marginal associa-tion with TsIgE (F-ratio = 3.8, df = 2, p = 0.022)but not in post hoc Tukey test. Furthermore, mean% blood eosinophil counts were found to be sig-nificantly higher in patients carrying the 481Tallele in comparison with the patients carrying481C (F-ratio = 8.8, df = 1, p = 0.0037; Table 4).Homozygous mutants (TT) had the highest meanpercentage of eosinophils followed by hetero-zygous (CT) and then the homozygous wild type(F-ratio = 4.2, df = 2, p = 0.02; Table 4). Post hocTukey test also showed the CC vs CT differenceto be significant (contrast difference = -2.17,Tukey 95% CI= -4.09 to -0.25). The investiga-tions for eosinophil counts were performed withinasthma patients (case-only association study).

Association of NAT2 haplotypes with asthma, TsIgE & % blood eosinophil countsSince on the basis of C282T and C481T SNPswe can not distinguish clearly between slow andfast acetylators, we constructed two locus haplo-types using PHASE. Four haplotypes were gen-erated (Figure 2) and Clump22 was used with1 million Monte-Carlo simulations for associa-tion analysis with asthma binary trait. Haplotypefrequencies showed highly significant differencesbetween the cases and controls (Normal T1χ2 = 17.91, df = 3, p = 0.0004 and maximum χ2

from clumped 2 × 2 table [T4] = 12.53,p = 0.002).

Individually, the haplotype C_T was most fre-quent in the patient population (Figure 2). Theodds of patients, rather than the controls, havingC_T haplotype was 1.6 with 95%CI = (1.2–2.1) (Likelihood χ2 = 10.65, df = 1,p = 0.001). These results remained significanteven after Bonferroni correction. On the otherhand, the haplotype distribution analysis in thepatients and controls identified C_C as the mostfrequent haplotype in the control population(Figure 2). The odds of patients rather than con-trols having the C_C haplotype was 0.52 with95% CI = (0.36–0.76) (Likelihood χ2 = 11.87,df = 1, p = 0.0005).

Furthermore, when analysis was performedwith respect to log TsIgE levels and % bloodeosinophil count, the risk haplotype C_T wassignificantly associated with TsIgE level (Table 4).The individuals with this haplotype had highermean log TsIgE level as compared with the indi-viduals with other haplotypes (F-ratio = 9.2,df = 1, p = 0.0025). Similarly, the mean % bloodeosinophil counts was also higher in the individ-uals carrying the C_T haplotype (F-ratio = 5.4,df = 1, p = 0.021). On the other hand, the pro-tective haplotype C_C was associated with lowermean % blood eosinophil counts(F-ratio = 7.95, df = 1, p = 0.0056), but notwith lower mean TsIgE level (F-ratio = 0.2,df = 1, p = 0.65) (Table 4).

DiscussionAtopic (extrinsic) asthma is characterized by fam-ily history, positive skin prick test, elevated spe-cific IgE levels and high blood eosinophil counts[1,2]. Interindividual differences in their suscepti-bility to atopy from industrial or occupationalchemicals, certain therapeutic drugs, or cancero-genic and heterocyclic amines depends upon theirmetabolic activities toward these foreign sub-stances [5,36]. The process of acetylation by variouspolymorphic NATs may impart a prominent role

enotype frequencies for C282T and C481T ied in the NAT2 gene.

ents219)

Controls(N = 210)

P-value§ Oddsratio§

2.29) 211 (50.24) ND‡ -

7.71) 209 (49.76)

7.95) 251(60.05) ND‡ -

2.05) 167 (39.95)

6.61) 58 (27.62) 0.55 1.086

1.38) 95 (45.24)

2.02) 57 (27.14)

7.81) 80 (38.28) 0.00027 0.606

0.27) 91 (43.54)

1.92) 38 (18.18)

dj = 0.025 (after correction for the multiple testing).

lues and OR as adapted from [33].

allelic association with asthma.dicate the frequency (%). Significant values are shown

2.

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in the biotransformation of these xenobiotics intohaptens inducing immunologic reactions. Due togenetic polymorphisms in NAT genes, subjectscan be classified as ‘slow’ or ‘rapid’ acetylatorsaccording to how fast their bodies metabolize iso-niazid, sulphamethazine or procain amide.Although there is a wide variation in acetylationcapacities, there exists a bimodal/trimodal fre-quency distribution showing high concordancebetween acetylator phenotype and NAT2 genemutations [16,19]. Therefore, extensive geneticassociation studies have been performed to iden-tify the correlation between acetylation capacity ofindividuals and their susceptibility to adverse drugeffects, certain malignancies and also to diseasessuch as rheumatoid arthritis, diabetes mellitus andvarious atopic or allergic disorders [20–22,25–29,37].Other supportive evidences have shown thatacetylation may influence the process of inactiva-tion of excessive biogenic amines [38,39], includinghistamine, a major player in all allergic andinflammatory reactions including asthma. Endoand colleagues have found the accumulation ofacetylated histamine derivatives near the receptorsites, serving as the physiologic reserve [40]. Fur-thermore, histamine receptor (H2 receptor) antag-onists, which decrease the release of histamine,also decrease the NAT2 activity at the receptor

site, confirming its role in regulation of histaminerelease [41]. However, previous studies on the pos-sible role of the acetylation polymorphism inasthma pathogenesis are inconsistent. Therefore,in the present study, after confirming the sugges-tive linkage of atopic asthma at 8p22 in a family-based transmission disequilibrium test, we investi-gated whether the genetic polymorphisms ofNAT2 play a role in susceptibility to atopicasthma in the North Indian population. Althoughapproximately 30 allelic variants of NAT2 havebeen described, the genotyping of closely spacedmarkers yields highly redundant data owing to thestrong intermarker linkage disequilibrium (LD) inthis gene: testing them all is expensive and oftenunnecessary. Moreover, as suggested by Sabbaghand colleagues, only one or two SNPs would beenough to obtain a good predictive capacity withno or only a modest reduction in power relative todirect assays of all common markers to determinethe acetylation status of an individual [42]. Afteridentifying five SNPs in the NAT2 gene, wedecided to study two of them, C481T and C282,in 429 individuals. Our result shows that theC481T polymorphism was associated with a sig-nificantly increased risk of atopic asthma, whilethe C282T SNP does not contribute much in thedisease pathogenesis. Notably, both of the allelic

Table 4. Log10 TsIgE levels and % blood eosinophil counts with respect to different genotypes and haplotypes of NAT2 gene. Numbers in parentheses indicate ± standard error.

Locus Number Log serumtotal IgE

levels (±SE)

Overallp-value

Number % bloodeosinophil

counts (±SE)

Overallp-value

C282T

CC 98 2.58 (± 0.06) 20 7.77 (± 0.67)

CT 159 2.68 (± 0.05) 0.008 33 6.79 (± 0.52) 0.19

TT 85 2.43 (± 0.06) 16 8.71 (± 0.76)

C481T

CC 100 2.50 (± 0.06) 21 6.47 (± 0.64)

CT 173 2.68 (± 0.05) 0.022 40 8.64 (± 0.47) 0.020

TT 69 2.49 (± 0.07) 9 8.95 (± 0.99)

Haplotype*

C_C 111 2.58 (± 0.06) 23 6.06 (± 0.61)

C_T 243 2.70 (± 0.04) 0.015 48 8.44 (± 0.42) 0.0039

T_C 256 2.56 (± 0.04) 57 7.38 (± 0.39)

T_T 74 2.47 (± 0.07) 6 9.76 (± 1.19)

The analysis with TsIgE levels was done without considering individual’s disease status, while for % blood eosinophil counts, analysis was done in patient samples only.The level of significance αadj=0.025 (after correction for the multiple testing)*The haplotypes were constructed using C282T and C481T polymorphisms in NAT2 gene. IgE: Immunoglobulin E; NAT2: N-acetyltransferase 2; SE: Standard error; TsIgE: Serum total IgE.




variants do not lead to any amino acid change.However, C481T was always found in concord-ance with the T341C and A803G SNPs in ourinitial analysis (D’=1). The T to C change at 341causes an Ile to Thr at amino acid 114, while A toG at 803 causes a Lys to Arg at amino acid 268.Matthew and colleagues have shown that the341T to C SNP present in NAT2*5 clusters wassufficient to decrease NAT2 immunoreactive pro-tein levels [43]. These results were further clarifiedin our haplotype analysis, where we found that theC_C is an important protective haplotype and isnegatively associated with asthma, while C_T is amajor risk haplotyope positively associated withasthma. These results further showed the impor-tance of C481T in determining the risk status ofan individual as both risk and protective haplo-type have C at position 282. Moreover, C481Twas also associated with % blood eosinophilcounts and IgE. It is to be noted that although Tallele is associated with asthma, carriers of CT het-erozygous had highest mean serum total IgE. Oneof the reasons for this observation could be thatC481T influences the IgE trait when present withsome other polymorphism. Interestingly, C282Talso showed a significant association with serumtotal IgE levels, an important feature of atopy.

Thus, we found that NAT2 variants could con-tribute to atopic asthma and atopy as well. Ourresults support the findings of earlier associationstudies, where protective effect of fast acetylationgenotypes over slow acetylation has been shown inasthma and atopy [26–29].

Since asthma is a complex disorder, we con-ducted a case–control study that could providebetter directions on promising loci [30]. Theseleads can be further tested using a well-controlledfamily-based study. In this study, we haverecruited only those patients who have at least onefirst-degree relative affected with atopy and/orasthma. As the case and control groups were age,sex, ethnically and geographically matched, it isvery unlikely that our results could be false posi-tive. Moreover, we have already demonstrated asuggestive linkage of a marker upstream of NAT2,and also, in our preliminary family-based associa-tion test, we have found a significant associationof the C481T with atopic asthma (p = 0.03; datanot shown). These results added further confi-dence to our results. Nevertheless, it would beinteresting to study the other SNPs, G590A,A845C and G857A, in our study population, ashigh heterozygosity has been reported in a studyfrom the South Indian population [44].

Figure 1. The schematic representation of the NAT2 gene on chromosome 8p22 with common SNP locations and the flanking microsatellite markers.

The SNPs identified in our study population are marked in bold. Pair-wise linkage disequilibrium (LD) for all two-way comparisons among the five polymorphisms identified in the NAT2 gene by sequencing the DNA of 40 individuals as calculated using Haploview. MAF: Minor allele frequency; NAT2: N-acetyltransferase; SNP: Single nucleotide polymorphism.

50 100 150 200 250

(T111C)

Gln64(191A)

(C282T)

Thr114(T341C)

Pro145(T434C)

(C481T)Gln197(G590A)

(C759T)

Arg268(A803G)

Thr282(A845C)

Gln286(G857A)

D8S258

3.0 MB2.6 MB

D8S549

A.

B. NAT2MAF C282T T341C C481T G590A A803G

NAT2

C282T

T341C

C481T

G590A

A803G

0.488

0.2

0.2

0.38

0.21

r2

0.26

0.26

0.51

0.28

1.00

0.88

0.59

0.85

1.00

1.00

0.59

0.85

0.92

1.00

1.00

0.17

1.00

1.00

1.00

1.00

D’

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To summarize, this is the first study from theIndian subcontinent, identifying the association ofthe C481T polymorphism in the NAT2 gene withasthma, serum IgE and blood eosinophil counts,while showing that C282T may be an importantdeterminant of atopy. Thus, we propose that thefast acetylators (for example, CC genotype at 481)could have protective advantage over the slowacetylators against asthma susceptibility, and there-fore could serve as a genetic marker.

OutlookAsthma is a complex genetic disorder with aheterogeneous phenotype, largely attributed tothe interactions between many genes andbetween these genes and the environment.Numerous loci and candidate genes have beenreported to show linkage and association ofasthma and the asthma-associated phenotypes,atopy, elevated IgE levels, and bronchial hyper-responsiveness to alleles of microsatellite mark-ers and SNPs. The chromosomal locus 8p22 hasbeen linked to total IgE levels in French asth-matic families [45]. We have also found a sugges-tive association of D8S549 with atopic asthmain 158 asthmatic families. Not only that, wehave established the association of NAT2genetic variants with asthma, IgE and eosi-nophil counts. NAT2 is present approximately2.5 MB downstream of the microsatellitemarker D8S549. Other genes, such as NAT1,tumor necrosis factor receptor superfamily,member 10d precursor (DCR2) and 10 a(APO2), defensin, α 1 preproprotein (DEFA1),fibroblast growth factor receptor 1 isoform 1precursor (FGFR1) and so on, which could have

an effector role in asthma pathogenesis arepresent near this locus linked to asthma. Itwould be interesting to study the genetic associ-ation of these genes with asthma and associatedphenotypes in the future. Further detailed stud-ies on these genes would help us in better under-standing the complex network of underlyingpathways involved in asthma etiology.

NAT2 is an important enzyme involved in themetabolism and biotransformation of manyxenobiotics. Thus, our results of its associationwith asthma and the growing incidence of aller-gic diseases over the last 30–40 years coincidingwith environmental deterioration necessitateevaluation of the role of xenobiotic transforma-tion in the pathogenesis and etiology of thesedisorders. NAT1, GST, and cytochrome P450could be other important candidate genes in thisrespect. However, as asthma is a complex disor-der, a single gene effect is often not easy to dem-onstrate. Therefore, searching for susceptibilitygenes for asthma requires a thorough under-standing of gene–gene and gene–environmentinteractions. Such studies will have a valuablepredictive magnitude in asthma susceptibility.

AcknowledgmentsThe authors acknowledge the Council of Scientific and Indus-trial Research (CSIR, Task Force project-SMM0006), Gov-ernment of India for financial assistance. Jyotsna Batra wishesto thank CSIR for her fellowship.The authors wish to thankall participating clinicians; VK Vijayan, PV Niphadkar,V Kumar and volunteers for helping in this study. The authorsalso wish to thank Rajshekhar Chatterjee, A Kumar, U Mab-alirajan and Deepti Maan for their technical assistance. Theauthors hereby declare no competing interests.

Figure 2. Frequency distribution of the haplotypes of the NAT2 gene using C282T and C481T SNPs in the patients and the unrelated controls.

NAT2: N-acetyltransferase; SNP: Single nucleotide polymorphism.

0

5

10

15

20

25

30

35

40

45

C_C C_T T_C T_T

Haplotypes: C282T_C481T

*

**%

Fre

qu

ency

Case

Control

** *

p = 0.0005

p = 0.001

Pharmacogenomics



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Highlights

• The chromosomal locus 8p22 has been shown to be linked to total immunoglobulin E (IgE) levels in the French Epidemiological study of the Genetics and Environment of Asthma (EGEA) asthmatic families.

• N-acetyltransferase (NAT2) is an important gene present at the 8p22 region, involved in the metabolism and the biotransformation of various xenobiotics and biogenic amines including histamine, an important precursor of allergic reactions.

• The aim of our study was to investigate the linkage of NAT2 flanking markers with asthma and to study the association of NAT2 genetic variants with asthma, serum total IgE and eosinophil counts.

• In our study, D8S549 showed a suggestive linkage with asthma (p = 0.012) in 158 nuclear asthmatic families from North India, suggesting the presence of a gene with minor contribution in asthma in its vicinity.

• Five single nucleotide polymorphisms in NAT2 previously identified were found to be polymorphic and were also found to be in strong linkage disequilibrium (LD) with each other after resequencing NAT2 gene fragment in our population.

• A study cohort of 219 atopic asthmatic unrelated patients and 210 normal healthy controls were recruited for the population based association study after proper clinical phenotyping.

• C481T was found to be associated with asthma, log10 serum total IgE and percentage of peripheral blood eosinophil counts, while C282T was significantly associated with log10 serum total IgE.

• We have also identified the risk and protective haplotypes associated with asthma, IgE levels and eosinophil counts.• We conclude that fast acetylators (CC at 481) have protective advantage over the slow acetylators against asthma and atopy

susceptibility and could serve as a genetic marker.

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