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Glutathione S-Transferase M1 Genotype (GSTM1) Plus Prenatal Exposure to Smoke as Risk Factors for Pediatric Asthma

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Glutathione S-Transferase M1 Genotype (GSTM1) Plus Prenatal Exposure to Smoke as Risk Factors for Pediatric Asthma
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    The Egyptian Journal of Medical Human Genetics, Vol. 6, No. 2, Nov. 2005. 183   Glutathione S  -Transferase M1 Genotype (GSTM1) Plus Prenatal Exposure to Smoke as Risk Factors for Pediatric Asthma Maged A.A.F. Ibrahim,* Malak A. Shaheen,* Nasser A. Elhawary,** Rasha A. Abdel Fattah* * Pediatrics Department, Faculty of Medicine, and Medical Genetic Center, **AinShams University, Cairo Abstract: Pediatric asthma is considered a complex multifactorial disease, with an obvious genetic predisposition and the possible involvement of noxious environmental factors. Glutathione S  -transferase genes are known as risk factors predisposing to some environ-mentally induced diseases.   This study has examined the hypothesis that glu-tathione   S  -transferase ( GSTM1 ) geno-type may play a role in asthma and wheezing   occurrence among those exposed to tobacco smoke.   Genomic DNA samples isolated from 35   asth-matic children and 35 healthy children were amplified using the flanking GSTM1  primer set premixed with the internal set. Asthmatic children show-ed a significant high prevalence of the GSTM1 -null genotype (odds ratio   [OR] 2.2, 95% confidence interval [CI] 1.4-3.4). Among GSTM1 -null children, in utero   smoke exposure was associated   with increased prevalence of asthma (OR 3.7, 95% CI 1.9-7.3).   The inter-mediate electrophilic metabolites, aris-ing in the first phase of detoxification of tobacco smoke, are not utilized by GST enzyme in asthmatic children. These intermediate metabolites may therefore attack cells and provoke oxidative stress, which contribute to the pathogenesis of asthma. Our findings indicate that there are im-portant   long-term effects of in utero   smoke exposure in a genetically susc-ptible   group of children (genetic-environmental interaction). Introduction: Asthma affects nearly 14 million people worldwide and has been stea-dily increasing in frequency for the past 50 years. (1) Although environme-ntal factors clearly influence the onset, progression, and severity of this dise-ase, family and twin studies indicate that genetic variation also influences susceptibility. (2,3)  Although a rapid rise in childhood asthma prevalence sug-gests a role for environmental factors in the aetiology of this evolving epide-mic, it is logistic that genetics also in-fluence the occurrence of asthma. (4-6)  The evidence that both genes and en-vironment play etiologic roles sugges-ts that the increase in asthma occur-rence is likely to involve changes in specific exposures among the popula-tion of genetically susceptible individu-als. (7,8)  The full spectrum of exposures and susceptibility genes involved in the pathogenesis of asthma and wheezing have yet to be establish-ed (6,9)  Tobacco smoke is an exposure    GSTM1  genotype plus prenatal exposure to smoke as risk factors for pediatric asthma, Ibrahim et al. 184 of interest among children, a group with high prevalence of asthma and increased sensitivity to air pollut-ants. (10-16)  An extensive body of evidence indicates that involuntary to-bacco smoke exposure increases the prevalence of wheezing, cough, and phlegm, and that childhood household environmental tobacco smoke (ETS) exposures cause exacerbations in asthma. (10-15)  Fetal exposure to mater-nal smoking may contribute to the occurrence of asthma and wheezing; however, the evidence for independ-ent effects of in utero   exposure on the occurrence is still emerging . (6,10–15)  The glutathione S-transferases(GST EC 2.5.1.18) are a family of proteins that detoxify chemical carcinogens, ei-ther by binding them directly or by co-njugating glutathione to reactive elec-trophilic sites. (17)  The nonmicrosomal glutathione transferases have been grouped into four classes based on protein sequence similarity, namely, alpha ( α ), mu (  ), kappa ( κ ), pi ( π ), and theta ( θ ), sigma ( σ ) (http://www.omim.new.html). The maj-or liver classmu isoenzyme is found only in about half the adult population. This enzyme is encoded at the GSTM1  locus (OMIM #138350) on ch-romosome 1p13.3, which displays fur tightly clustered alleles GSTM1 , GSTM2  , GSTM3   and GSTM4  . (18)  These genes are spaced approximately 20 kb apart and are arranged as 5'- GSTM4- GSTM2  - GSTM1 - GSTM5  -3'. Individuals who are homozygous GSTM1 -null lack a GST-activity against the substrate trans  -stilbene oxide in their peripheral blood. (19,20)  It has been reported that the GSTM1 -null deletion has been associated with lung cancer (21)  and adenocarcinoma of the colon. (22,23)  Although GSTM1  has the potential to explain a substantial portion   of asthma occurrence at the population level, its role in asthma   pathogenesis has not been extensively investigated. To date, there have no preceded studies   to estimate the association of GSTM1  genotype and in utero   expo-sure to   maternal smoking with asthma occurrence among the Egyptian child-ren. The aim of this study was to in-vestigate the effects of GSTM1  geno-type and involuntary tobacco smoke (lifetime tobacco smoke exposure his-tories including in utero   exposure)   on the occurrence of asthma and wheez-ing during childhood. Subjects and Methods: Subjects: Thirty-five children with known bron-chial asthma status recruited   to Chest Clinic at Pediatric Hospital, AinShams University. Their age ranged from 1−8 y with mean age of 5±2.2 y. They included 23 males (66%) and 12 females (34%). The asthmatic child-ren were classified according to the criteria of Global Initiative for Asthma (GINA) guidelines (14)  into 4 groups; mild intermittent asthma (8 cases), mild persistent asthma: (8 cases), mo-derate persistent asthma (9 cases) and severe persistent asthma (10 cas-es). In addition, there were 35 healthy subjects served as controls with ages ranged from 2-8 y and mean value of 5.82±2.0 y. They included 18 males (51%) and 17 females (49%).   At study entry, a parent   or guardian of each participating child provided    The Egyptian Journal of Medical Human Genetics, Vol. 6, No. 2, Nov. 2005. 185   written informed   consent and comple-ted a self-administered questionnaire on demographics,   medical and family health history, indoor air exposures, and   household characteristics. Tobacco smoke exposure:  Exposure to household environ-mental tobacco smoke (ETS) and ex-posure to maternal smoking in    utero   was defined using the responses from the questionnaire   completed by paren-ts or guardians. Household smoking was defined   as daily smoking inside the house by anyone living there. Information   was collected about the current and past household smoking status   of each participant's mother, father, other adult household   membe-rs, and regular household visitors. In utero   exposure to   maternal smoking (yes or no) was assigned by respon-ses to the   question "Did your child's biological mother smoke while she   was pregnant with your child? (Include time when she was pregnant   but did not yet know she was.)"   Genotyping analysis: Genomic DNA was isolated from EDTA-peripheral leukocytes using FlexiGene DNA extraction kit from QIAGEN Inc. (Valencia, CA). In some cases, DNA was prepared in situ by gentle scraping the buccal mucosa for 30 s using using the MasterAmp™ DNA extraction kit from Epicentre (Madison, WI). For GSTM1  genoty-ping assay, the template DNA was amplified using polymerase chain reaction (PCR). The sequences (24) of the GSTM1  oligonucleotide primer set (P1, P2) and the internal control prim-er set (P1, P3) were prepared (Meta-bion, GmbH). Amplification of GSTM1  gene was carried out by mixing 50 ng of the isolated DNA, 2.5    l of 10X PCR buffer (final concentration: 50 mM KCl, 10 mM Tris-HCl pH 8.4, 1.5 mM MgCl 2 ), 200  M dGTP, dATP, dCTP, dTTP, 1.25 ml of 5% DMSO, 25 ng of primers P1 and P2, and 50 ng of P3 and 0.5 U of Taq   DNA poly-merase (Bioron, GmbH), to a final vol-ume of 25  l. A total of 30 cycles with denaturation at 94°C for 60 s, anneal-ing at 52°C for 60 s, and extenstion at 72°C for 60 s was performed. An initi-al denaturing was carried out at 95°C and a final extension stage at 72°C. The PCR product was separated on a 3% NuSieve agarose (BMA) premix-ed with ethidium bromide, and photo-graphed. The presence of the 230-bp band (P1 & P3 set) in addition to the 157-bp band (internal control) repre-sented the active (+) allele, whilst the absence of the 230-bp band repre-sented the null (−) allele. Data analysis:  The individual effects of GSTM1  genotype and   two types of tobacco smoke exposure ( in utero   or current ETS   exposure) on respiratory outcom-es were assessed. Logistic regression was used to assess the relationship between   asthma and wheezing out-comes, GSTM1  genotype, ETS, and in utero    exposure to maternal smok-ing. All odds ratios (ORs) were ad- justed   for town of residence, age, grade, sex, family history   of asthma, atopy, and gestational age. Data were analyzed using PC-compatible soft-ware SPSS (ver. 10). Comparisons of variables between different groups were done using Chi ( χ ) square test. For all tests, p< 0.05 considered signi-ficant.    GSTM1  genotype plus prenatal exposure to smoke as risk factors for pediatric asthma, Ibrahim et al. 186 Results: The frequency of glutathione S  -transferase M1 ( GSTM1 )-null allele was documented in 23/35 (66%) of asthmatic children and present in only 4/35 (11%) of the control group with highly significant p< 0.001 (Table 1). The frequency of GSTM1 -null genotype in asthmatic cases regard-ing the grade of asthma was 1/8 of cases with mild intermittent asthma, 6/8 of cases with mild persistent, 7/9 of cases with moderate persistent asthma and 9/10 of cases with severe persistent asthma and had significant p< 0.05. Table 1. GSTM1  genotype in asthmat-ic children and controls Asthma children (No=35)   Controls (No= 35)   Signific-ance   GSTM1 gene   No. (%)   No. (%)   Active   12 (34) 31 (89) χ  = 21.8   Null allele 23 (66) 4 (11) p< 0.001 OR 2.2, 95% CI 1.4–3.4  Regarding intrauterine tobacco sm-oke exposure 17/35 of asthmatic cas-es had a history of intrauterine tobac-co smoke exposure from smoking mo-ther (active or passive), 5/17 (29.4%) of them had the active GSTM1 geno-type while 12/17 had the null-allele (70.6%). However, among the healthy controls, 12/35 had a previous history of intrauterine tobacco smoke expo-sure and all of them (100%) had the active gene with highly significant p< 0.001 (Table 2). Regarding positive history of ETS exposure among asthmatic cases 27/35 had a positive history, and 10/27 (37%) of them had the active GSTM1  genotype while 17/35 (63%) had the null allele. Nevertheless, am-ong the controls 18/35 of the healthy controls had a positive family history of passive smoking exposure and all of them (100%) had the protective active gene with a significant p< 0.05 (Table 3). Table 2 .   History of intra-uterine to-bacco smoke exposure among cases and   controls   Asthma children (No= 17)   Controls (No= 12)   Signific-ance   Positive  in utero tobacco smoke exposure   No. (%)   No. (%)   Active   5 (29.4) 12 (100) χ  = 14.4   Null allele 12 (70.6) 0 (0) p< 0.001(OR 3.7, 95% CI 1.9–7.3) Children who had the GSTM1 -null genotype were at the greatest   risk for adverse respiratory health effects when exposed to   maternal smoking in utero.  The effects of in utero   exposure   to maternal smoking on both current and lifetime asthma and   wheezing outcomes were largest, and in gener-al, restricted to   children with GSTM1 -null genotype (Table 1 and 2). Furth-  Table 3. Asthmatic children and controls with positive history of ETS Asthma children(No= 27)   Controls (No= 18)   Signific-ance   Positive ETS Exposure   No. (%)   No. (%)   Active   10 (37) 18 (100) χ  = 6Null allele 17 (63) 0 (0) p< 0.05 (OR 1.8, 95% CI 1.3–2.5)    The Egyptian Journal of Medical Human Genetics, Vol. 6, No. 2, Nov. 2005. 187   ermore, effects between   the in utero   exposure and GSTM1 -null genotype group appeared   to be largest for wheezing outcomes with the greatest severity. Discussion: The GSTM1 -null allele appears to result from homologous unequal cros-sing-over between the 2 highly identi-cal 4.2-kb repeat regions that flank the GSTM1  gene. This had resulting in a 15-kb deletion that includes the entire GSTM1  gene. (18)  In this study, we reported that the GSTM1  genotype modifies the effects of fetal tobacco   smoke exposure on childhood asthma and wheezing. The adverse   effects of in utero   exposure to maternal smoking on a broad   range of asthma and whe-ezing outcomes were largely restrict-ed   to children with GSTM1 -null geno-type. Our findings indicate   that there are important long-term effects of in utero   exposure   in a genetically susce-ptible group of children.   A growing body of evidence sup-ports an independent effect of   in utero   exposure to maternal smoking on wheezing and asthma   occurrence dur-ing childhood. (10,11,13)  Although   varia-tion in susceptibility for some of the adverse effects   of tobacco smoke is well established among adults, less is known   about the factors that influence susceptibility to prenatal   tobacco smo-ke exposure. (6,11,14-16)  Although it   is clear that in utero   exposure has direct effects on normal   development consi-stent with its effects on birth weight, genetic   variation may also contribute to wheezing and asthma. This could be interpreted by the larger effects among the children having family pre-disposition for asthma. (25)  Based   on our findings, GSTM1  genotype may be an important susceptibility   factor for childhood asthma after exposures during the fetal   period. Because in utero   exposure has adverse effects beyond   wheezing and asthma occur-ence, the joint effects of GSTM1  and   in utero   exposure on other health outcomes warrants additional   study.   Studies of neonates show   that ma-ternal tobacco smoke exposure during the in utero   period   is associated with increased bronchial hyperreactivity (BHR), especially in those with a   fa-mily history of asthma. (26)  Animal stud-ies also suggest that   exposure during the period of lung development leads to BHR. (27)  Chronically increased BHR from in utero   exposure may contribute   to persistent wheezing and increased asthma predisposition and   diagno-sis. (28)  Furthermore, in utero   exposure may affect   the development and maturation of the pulmonary immune system. (29)  Inappropriate persistence of a TH2-dominant response pattern   appears to increase likelihood of aller-gic sensitization upon   sufficient expo-sure to a variety of common anti-gens. (24)  It   is also possible that fetal ingestion of tobacco smoke products   present in the amniotic fluid may have long-term effects on   gut immune responses that appear to be important in allergic   sensitization. (15)  Based on these findings, it is biologically   plau-sible that toxins from in utero   expo-sure to maternal smoking   influence sensitization to common antigens, inflammation, decreased   lung func-tion, and increased BHR with variable
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