ARTICLE IN PRESS GSTM1

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1 GSTM1, GSTP1, prenatal smoke exposure, and atopic dermatitis I-Jen Wang, MD, PhD* ; Yueliang Leon Guo, MD, PhD ; Tien-Jen Lin, MD, MSc ; Pau-Chung Chen, MD, PhD ; and Yu-Nian Wu, MD* Background: The increase in the prevalence of atopic dermatitis (AD) is likely to involve changes in specific environmental exposures among genetically susceptible individuals. Objective: To evaluate the effect of glutathione S-transferase (GST) genotype polymorphisms and prenatal smoke exposure on pediatric AD on the basis of the cord blood cotinine levels. Methods: We conducted a case-control study composed of 34 children with AD and 106 non-ad controls, all of whom were selected from 483 participants in the Taiwan Birth Panel cohort study. Cord blood samples and information on perinatal factors of children were gathered at birth. At 2 years of age, information about the development of AD and environmental exposures was collected. We compared AD with non-ad children for GTM1 and GSTP1 polymorphisms stratified by the cotinine level. Multiple logistic regressions were performed to estimate the association of genotype polymorphisms and cotinine levels with AD. Results: GSTM1 null and GSTP1 Ile/Ile genotypes showed a significant increase in the risk of AD (odds ratio [OR], 3.61; 95% confidence interval [CI], ; and OR, 3.11; 95% CI, ; respectively). In children with a cotinine level less than 0.1 ng/ml, the risk of AD increased for those carrying 2 GSTP1 Ile-105 alleles (OR, 6.63; 95% CI, ). In children a with cotinine level of 0.1 ng/ml or greater, the GSTM1 null genotype was significantly related to AD (OR, 5.21; 95% CI, ). Conclusions: Within groups of children, genetic polymorphisms in GSTM1 and GSTP1 may be responsible for differences in susceptibility to AD with regard to prenatal smoke exposure. Ann Allergy Asthma Immunol. 2010;xx:xxx. INTRODUCTION The prevalence of atopic dermatitis (AD) has increased significantly during the past decade in Taiwan. 1 Generally, such a rapid increase in prevalence cannot be explained solely by genetic factors; environmental factors must also be considered. 2,3 Because the onset of AD occurs relatively early in life, 4 identification of susceptible genes and perinatal environmental factors are of critical importance. According to a recent Taiwan island wide survey, 5 a reported 4.5% of reproductive age women smoke and 59% of nonsmoking women of childbearing ages have been exposed to environmental tobacco smoke (ETS). Exposure to tobacco smoke is Affiliations: * Department of Pediatrics, Taipei Hospital Department of Health, Taipei, Taiwan; Department of Health Risk Management, China Medical University, Taichung, Taiwan; Fu Jen Catholic University, Taipei, Taiwan; Department of Environmental and Occupational Medicine, National Taiwan University Hospital, Taipei, Taiwan; Taipei Medical University, Wan Fang Hospital, Taipei, Taiwan; Institute of Occupational Medicine and Industrial Hygiene, National Taiwan University College of Public Health, Taipei, Taiwan. Disclosures: Authors have nothing to disclose. Drs Wu and Chen contributed equally to this work. Funding Sources: Funding for this study was provided in part by grants from the National Science Council (NSC B MY2) of Taiwan. Received for publication August 18, 2009; Received in revised form April 19, 2010; Accepted for publication April 22, American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved. doi: /j.anai a known risk factor for AD and can modify various aspects of fetal T H 1 and T H 2 cell function. 6-8 Furthermore, a dose-response relationship between cord blood cotinine level and the risk of AD has also been found. 9 However, not all children who have grown up with tobacco exposure develop AD. This unpredictability of disease susceptibility may be related to genetic variability. Glutathione S-transferase (GST), a well-studied phase 2 enzyme, is involved in detoxification of both reactive tobacco metabolic intermediates and reactive oxygen species (ROS). 10,11 GSTM1 and GSTT1 are highly polymorphic members of the GST gene family, and deficiencies in the enzyme activity are caused by the homozygous deletion of the GST gene. The GSTP1 polymorphism is due to Ile-105 replacement by Val, so there are 3 genotypes at codon 105: Ile/Ile, Ile/Val, and Val/Val. The GSTM1 or GSTT1 null genotype and the variant GSTP1 allele may increase susceptibility to tobacco smoke because of decreased detoxification ability. It has previously been found that GSTP1 and GSTM1 polymorphisms are predictors of childhood asthma. 12 It has also been reported that ETS exposure is associated with wheezing and asthma among GSTM1 null children and that in utero ETS exposure is more important than current ETS exposure. 13 Previous studies have also shown that GSTM1 and GSTP1 genotypes can determine predisposition to AD. 14,15 However, the association of GST genotypes and smoke exposure during pregnancy with AD occurrence is not yet clear. In this study, we compared the genotypic distribution of the GSTM1 and VOLUME xx, MONTH,

2 GSTP1 genes in a case-control study nested within our previous Taiwan Birth Panel cohort study. 16 The effect of GSTM1 and GSTP1 polymorphisms and prenatal smoke exposure on pediatric AD on the basis of the cord blood cotinine levels was also investigated. METHODS Study Population In 2004, considering potential environmental exposures and nationwide representativeness, we recruited consecutive samples of mothers and their neonates from hospitals and obstetric clinics in Taiwan. Pregnant women during the third trimester of pregnancy who had prenatal examination in selected hospitals were invited to join the study and cord blood samples were collected at delivery. The study protocol has been described previously in the Taiwan Birth Panel cohort study. 16 In 2006, we conducted the present genetic case-control study focusing on the 34 children who developed AD with available specimens. Four control subjects from the cohort were matched to each case patient by sex, age (within 5 months), and enrollment time (within 3 months). Finally, only 106 controls fulfilled the criteria and were selected for the study because there were not enough eligible control subjects from the cohort. The institutional review board of the National Taiwan University Hospital approved the study, and written informed consent was obtained from the parents before enrollment in the study. Cases of AD were defined by the following 3 questions from the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire: Has your child ever had an itchy rash which was coming and going for at least 6 months at any time? Has the itchy rash been coming and going over elbows, knees, face, wrists, or generalized (4 or more localizations)? and Has your child ever had atopic dermatitis diagnosed by a doctor? As described elsewhere, a dermatologist examined a subgroup of the participating children, and the combination of answers that resulted in the highest sum of sensitivity and specificity was determined. 17,18 Exclusion criteria include multiple gestation (eg, twins, triplets), inability to answer questions in Chinese, and plans to move out of the area before delivery. Questionnaire Survey The parents were asked by home interview questionnaires at birth for birth year, parental education levels and occupation, family income, parental history of atopic diseases (such as AD, allergic rhinitis, or asthma), alcohol and drug use, diets and supplements (such as multivitamins) during pregnancy, and history of smoke exposure, including ETS exposure and active maternal smoking during pregnancy. From the records of the cooperating hospitals, we gathered neonates health data at birth, such as head circumference, birth body weight, height, weeks of gestation, and type of delivery. When children were 2 years of age, the ISAAC questionnaire was administered and some postnatal exposures, such as duration of breastfeeding and infant formula use, early consumption of egg, wheat, soy bean, or shrimp before 1 year of age, older siblings, furry pets or carpets at home, fungi at house walls, incensing at home, and postnatal ETS exposure, were determined. Laboratory Methods The plasma samples of cord blood were stored at 80 C before analysis. An average of 0.5 ml of plasma was spiked with 15 L of 10 ng/ml of deuterium (ISOTEC, Miamisburg, Ohio) labeled cotinine (Sigma, St Louis, Missouri) as an internal standard, 100 L of 5N potassium hydroxide (Sigma), and 3 ml of dichloromethane (Mallinckrodt Baker, Phillipsburg, New Jersey), followed by vigorous shaking for 1 minute and centrifugation at 3,000g for 10 minutes. The clear organic phase was transferred to a tube, and the aqueous phase was repeatedly extracted with dichloromethane. Then, the organic solutions were combined and dried under nitrogen. Each sample was raised in 150 L of 5% methanol (Mallinckrodt Baker) and 20mM ammonium acetate (Merck, Darmstadt, Germany). Finally, 20 L was injected into highperformance liquid chromatography (PerkinElmer, Boston, Massachusetts) coupled to a triple-quadruple tandem mass spectrometer (API 3000TM; Applied Biosystems, Carlsbad, California) to measure the cotinine levels. The column a is Purospher STAR RP-18 column (4 55 mm, 3 m; Merck). The cotinine concentration was determined by comparison of the peak area to a calibration curve, which was constructed by injecting 9 standard cotinine concentrations, ranging from 0.02 to 100 ng/ml and plotted using peak area vs the concentration of cotinine. The genetic polymorphisms of the GSTM1 and GSTT1 genes were recognized by polymerase chain reaction (PCR). 19 The analysis of the GSTM1 and GSTT1 polymorphisms was performed at the same time as the coamplification of the -globin gene. The primer sequences for the GSTM1 forward and reverse primers were 5 -CTG CCC TAC TTG ATT GAT GGG-3 and 5 -CTG GAT TGT AGC AGA TCA TGC-3. The primers of GSTT1 were 5 -TTC CTT ACT GGT CCT CAC ATC TC-3 and 5 -TCA CCG GAT CAT GGC CAG CA-3. A 110-bp fragment of the -globulin gene was used as an internal positive control using primers (5 -ACA CAA CTG TGT TCA CTA GC-3 and 5 -CAA CTT CAT CCA CGT TCA CC-3 ). The PCR reaction was conducted in a total volume of 25 L containing 50 ng of genomic DNA, 0.3 L of 10 pmol of each primer, 1 L of 2.5mM dntps, 0.5 U of AmpliTaq Gold enzyme (PE Applied Biosystems, Foster City, California), and 2.5 L of GeneAmp 10 buffer II (10mM Tris hydrochloride, ph of 8.3, 50mM potassium chloride), and 2 L of 25mM magnesium chloride provided by the manufacturer. Amplifications were performed in a PCR system (Thermal Cycler 2720; Applied Biosystems) with an initial denaturation at 94 C for 5 minutes and then 35 cycles with denaturation at 94 C for 1 minute, annealing at 55 C for 1 minute, and extension at 72 C for 1 minute. The final cycle was terminated at 72 C for 7 minutes. The PCR- 2 ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY

3 amplified fragments were detected by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. GSTP1 was detected by the PCR restriction fragment length polymorphism method. 20 The primer sequences for GSTP1 forward and reverse primers were 5 -GTAGTT TGC CCA AGGTCA AG-3 and 5 -AGC CAC CTG AGG GGT AAG-3. An elevated annealing temperature was applied in the first 15 cycles of PCR to prevent nonspecific priming. After an initial denaturation step 12 minutes at 95 C, 15 cycles of PCR were performed (denaturation for 30 seconds at 95 C, annealing for 30 seconds at 58 C, elongation for 60 seconds at 72 C), followed by 25 cycles of amplification (denaturation for 30 seconds at 95 C, annealing for 30 seconds at 55 C, elongation for 60 seconds at 72 C) and 1 cycle of elongation for 5 minutes at 72 C, yielding a PCR product of 433 bp. A total of 15 L of the PCR product was digested with5uofbsma1 (New England BioLabs, Ipswich, Massachusetts) in a total volume of 25 L of a 37 C water bath overnight for 20 hours. The digested fragments were separated on a 1.5% agarose gel stained with ethidium bromide to visualize the bands. Statistical Analysis Cotinine concentrations were divided into 2 categories: less than 0.1 ng/ml, indicating low ETS exposure, and 0.1 to 9 ng/ml, indicating high ETS exposure, with the lowest level used as the reference category. 21,22 For the association between cotinine levels and genetic polymorphisms with AD, odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by conditional logistic regression models. Analyses were performed in both univariate and multivariate models. Next, we tested for gene-environment interaction by adding a product term in the regression model. Potential confounders from the literature reviews, such as infant sex, premature birth, maternal education and occupation, family income, maternal history of atopy, duration of breastfeeding, early consumption of solid food, older siblings, pet raising, postnatal ETS exposure, incense use at home, and fungi on house walls, were taken into consideration. Selection of the confounders who would be left in the model was based on the standard statistical procedures 10% change in point estimate. All hypothesis testing was 2-sided at the significance level of.05 and was performed with SAS statistical software, version 8.2 (SAS Institute Inc, Cary, North Carolina). RESULTS Our study comprised 34 AD case patients and 106 non-ad controls. Table 1 presents the demographic characteristics of the mother, children, and environmental exposures. Maternal education, premature birth, and older siblings were significantly different between the AD case patients and the non-ad controls. Because distributions of the GSTP1 105 Val/Val and Ile/ Val genotypes between children with AD and non-ad controls were similar and the frequency of homozygosity at the GSTP1 Val-105 locus was relatively low, we combined the GSTP1 105 Val/Val and Ile/Val genotypes as in dominant genetic models for the subsequent analyses. The genotype frequencies of GSTM1, GSTT1, and GSTP1 among the AD case patients and non-ad controls are listed in Table 2. The genotype frequencies matched the prediction by the Hardy- Weinberg theorem based on the allele frequencies. After adjusting for potential confounders, GSTM1 null (OR, 3.61; Table 1. Characteristics of the Study Population Characteristic Total (N 140) AD case patients (n 34) Non-AD controls (n 106) Mother Age, mean (SD), y 28.7 (5.3) 27.2 (6.4) 29.4 (4.8) Education, No. (%) a High school and below 71 (50.7) 12 (35.3) 59 (55.7) College and above 69 (49.3) 22 (64.7) 47 (44.3) History of atopy, No. (%) 28 (20.0) 8 (23.5) 20(18.9) Children Male, No. (%) 74 (52.9) 16 (47.1) 58 (54.7) Birth weight, mean (SD), g (459.1) (550.5) (428.6) Premature birth ( 37 weeks), No. (%) a 24 (17.1) 8 (23.5) 16 (15.1) Environmental factors Older siblings ( 2), No. (%) a 76 (54.3) 12 (35.3) 64 (60.4) Duration of breastfeeding ( 6 months), No. (%) 95 (67.9) 25 (73.5) 70 (66.0) Pet raising, No. (%) 38 (27.1) 8 (23.5) 30 (28.3) Postnatal ETS exposure, No. (%) 53 (37.9) 13 (38.2) 40 (37.7) Family income per year (New Taiwan dollars), No. (%) 600, (22.9) 9 (26.5) 23 (21.7) 600,000-1,500, (60.0) 20 (58.8) 64 (60.4) 1,500, (17.1) 5 (14.7) 19 (17.9) Abbreviations: AD, atopic dermatitis; ETS, environmental tobacco smoke. a P.05. VOLUME xx, MONTH,

4 Table 2. Association Between Neonate Genetic Polymorphisms and Atopic Dermatitis Stratified by Cord Blood Cotinine Levels No. (%) of AD case patients (n 34) No. (%) of non-ad controls (n 106) Crude OR (95% CI) Adjusted OR (95% CI) a Total population GSTM1 Present 10 (29.4) 56 (52.8) Null 24 (70.6) 50 (47.2) 2.69 ( ) b 3.61 ( ) b GSTT1 Present 14 (41.2) 50 (47.2) Null 20 (58.8) 56 (52.8) 1.28 ( ) 1.14 ( ) GSTP1 Ile/Val or Val/Val 6 (17.6) 37 (34.9) Ile/Ile 28 (82.4) 69 (65.1) 2.56 ( ) b 3.11 ( ) b Cotinine levels 0.1 ng/ml GSTM1 Present 5 (41.7) 33 (58.9) Null 7 (58.3) 23 (41.1) 2.01 ( ) 1.88 ( ) GSTT1 Present 5 (41.7) 26 (46.4) Null 7 (58.3) 30 (53.6) 1.21 ( ) 1.02 ( ) GSTP1 Ile/Val or Val/Val 2 (16.7) 27 (48.2) Ile/Ile 10 (83.3) 29 (51.8) 4.66 ( ) b 6.63 ( ) b Cotinine levels of ng/ml GSTM1 Present 5 (22.7) 23 (46.0) Null 17 (77.3) 27 (54.0) 2.90 ( ) 5.21 ( ) b GSTT1 Present 9 (40.9) 24 (48.0) Null 13 (59.1) 26 (52.0) 1.33 ( ) 1.28 ( ) GSTP1 Ile/Val or Val/Val 7 (31.8) 23 (46.0) Ile/Ile 15 (68.2) 27 (54.0) 1.81 ( ) 2.61 ( ) Abbreviations: AD, atopic dermatitis; CI, confidence interval; OR, odds ratio; a Model adjusted for maternal history of atopy, maternal education, and postnatal environmental tobacco smoke exposure. b P % CI, ) and GSTP1 Ile/Ile genotypes (OR, 3.11; 95% CI, ) still showed a significant increase in the risk of AD compared with GSTM1 present and GSTP1 Ile/Val and Val/Val (Table 2). The GSTT1 null genotype was positively correlated with the risk of AD but failed to reach statistical significance. We also investigated the distribution of GSTP1 105 and GSTM1 gene polymorphisms in AD patients and non-ad controls by cotinine levels (Table 2). In children with a cotinine level less than 0.1 ng/ml, the risk of AD increased for those carrying 2 GSTP1 Ile-105 alleles (OR, 6.63; 95% CI, ). In children with a cotinine level of 0.1 ng/ml or higher, the GSTM1 null genotype was significantly related to AD (OR, 5.21; 95% CI, ). Subsequently, we tried to assess the interaction effects between the GSTM1 and GSTP1 genotypes and cotinine levels in AD. However, the interpretations were limited. DISCUSSION This study is an interesting contribution to the literature on the potential association among genetic polymorphisms, gestational smoke exposure, and AD in offspring. To the best of our knowledge, the combination effect of GSTM1 and GSTP1 polymorphisms and prenatal smoke exposure on the risk of AD in children based on objective biomarkers has not been previously studied. We found that AD was positively associated with cotinine levels and that the adverse effects of prenatal smoke exposure were largely confined to children carrying GSTM1 null and homozygous GSTP1 Ile-105 genotypes. On the basis of our previous studies, 9 a dose-response relationship between cord blood cotinine levels and the risk of AD development was found. It is possible that fetal ingestion of tobacco smoke products present in the amniotic fluid may have long-term effects on gut immune responses that appeared to be important in allergic sensitization. 23 Therefore, toxins from prenatal smoke exposure can influence sensitization to common antigens, which may also increase the risk of childhood AD in the affected population. Groups with different ethnicity may differ in frequency of genotypes. The frequencies of GSTT1 null and GSTP1 Ile/Ile in our study were significantly higher compared with studies in the white 4 ANNALS OF ALLERGY, ASTHMA & IMMUNOLOGY

5 population (54% vs 25% and 69% vs 40%, respectively) Family income, maternal occupation, duration of breastfeeding, early consumption of solid food, premature birth, older siblings, pet raising, incensing at home, and fungi on house walls were considered potential confounders in our survey. Adjustment for these factors merely resulted in small changes in the effect estimates, and these covariates were not included in our final models. In addition, our study participants were all Asian Taiwanese, and we assumed that they were a rather genetically homogenous population. Therefore, the confounding effect of ethnicity was weak. The polymorphism of GSTM1 and GSTP1 played a role in the occurrence of AD in our study. One possible explanation might be that the GST enzyme was involved in detoxification of both reactive tobacco metabolic intermediates and ROS from tobacco smoke. ROS acted as secondary messengers in the induction of several biological responses, such as the activation of NF- B, the generation of cytokines, and the modulation of signaling pathways. 27 On the other hand, altered antioxidant defenses, lipid peroxidation, and anti-inflammation pathways were important in AD pathogenesis. 28 In human skin, the GST enzyme, serving as a marker of putative oxidative stress, was largely expressed in suprabasal keratinocytes. 29 GST gene polymorphisms were proven to modify the risk of skin lesions among arsenic-exposed individuals and to increase susceptibility to basal cell skin carcinoma. 30,31 Our results confirmed the hypothesis that the individual ability to detoxify ROS, determined by polymorphisms in GSTP1 and GSTM1, contributes to the development of pediatric AD. Studies demonstrating that individuals with decreased antioxidant capacity were at an elevated risk of AD also supported our hypothesis. 28,32 The GSTM1 null genotype eliminated enzyme activity and may increase susceptibility to oxidative stress in the skin. 22 In our study, the risk of AD increased for those with homozygote deletion of the GSTM1 gene. The positive relationship between the GSTM1 null genotype and AD did not reach statistical significance in the low-exposure group (cotinine level 0.1 ng/ml). In contrast, it was significantly associated with AD in the high-exposure group (cotinine level 0.1 ng/ml). In Russian populations, researchers found that individuals with the GSTM1 null genotype were at higher risk of developing AD. 14 It has also been reported that arsenic skin lesions were associated with oxidative stress in those who lacked a functional GSTM1 detoxification enzyme. 33 GSTM1 may be especially important during the fetal period because the effects of a given level of tobacco-related toxins were greater in the fetus compared with the mother. 34 It followed that variation in the function of this enzyme regulated the amount of smoke-related toxins that affected the development of AD. We also observed that homozygous GSTP1 Ile-105 may increase the risk of AD. People with GSTP1 Ile-105 homozygotes were reported to have elevation of IgE and histamine levels in nasal lavage fluid after challenging with diesel exhaust particles and allergens. 35 Although homozygous GSTP1 Ile-105 was positively associated with AD, it was not statistically significant in children with a cotinine level of 0.1 ng/ml or greater in our study. This phenomenon could be explained by evolution of a diversified pathogenic pathway that caused either low- or high-dose environmental stimulation. In addition, GSTP1 was not the enzyme directly involved in cotinine metabolism, although we stratified our study participants by cotinine levels to represent the amount of smoke exposure. 36 The small sample size limited the statistical power of our study to detect effects caused by multifactorial interactions. However, we found different associations of GSTP1 and GSTM1 with AD, when the sample was stratified by cotinine level. This may enhance the plausibility of a potential biological interaction between GST enzymes and smoke exposure in the detoxification process. The strength of our study was that we used the objective biomarker for smoke exposures. Assessing smoke exposures by cotinine, a sensitive quantitative biomarker of tobacco smoke, can reduce the recall bias of exposure assessment from questionnaires. 37 Cotinine appeared to be the most adequate biomarker of fetal exposure to smoking at the end of pregnancy, distinguishing not only active smoking from passive smoking but also exposure to ETS from nonexposure. Although in our study the cotinine measurement taken from cord blood samples was a single measurement at delivery, it has been reported that cotinine levels remain fairly constant and at near steady-state values throughout the day, and the smoking habits investigated in our questionnaire were relatively stable over time. 6 Therefore, the exposure measurement was valid in our study. Another possible limitation of our study is that we did not obtain genotype data from all study participants, which could in theory result in selection bias. However, participants who provided genotype data showed no systematic differences compared with those without such data with respect to various demographic characteristics (data not shown). Finally, our study used questionnaire data to ascertain AD prevalence, which may be considered less accurate than standard diagnostic criteria used by a dermatologist. That said, the ISAAC questionnaire itself has been validated against clinical examination in other large, prospective cohort studies. 17,18 In conclusion, we found adverse effects of prenatal smoke exposure in children carrying the GSTM1 null and GSTP1 Ile/Ile genotypes. Variations of the GSTM1 and GSTP1 polymorphisms may contribute to diverse detoxification ability. Additional research is warranted to explore the different enzyme activity after prenatal or postnatal tobacco smoke exposure among genetically susceptible children. ACKNOWLEDGMENT We appreciate the support from Professor Wu-Shiun Hsieh and Kuen-Yuh Wu from National Taiwan University for data collection and laboratory analysis. The original manuscript was awarded the Taiwan Young Physician Award for Medichem VOLUME xx, MONTH,

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