Maternal Genetic Polymorphisms of Phase II Metabolic Enzymes and the Risk of Fetal Neural Tube Defects Linlin Wang, Lei Jin, Jufen Liu, Yali Zhang, Yue Yuan, Deqing Yi, and Aiguo Ren*
Background: Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with the risk of fetal neural tube defects (NTDs). Whether maternal genetic variants related to PAH metabolism contribute to the development of fetal NTDs remains unclear. Methods: We conducted a case–control study in a Chinese population to examine the association of selected maternal genetic variants of phase II enzymes involved in the elimination of the metabolic intermediates of these chemicals with fetal NTD risk, and to evaluate possible interaction of the genetic variant and maternal exposure to indoor air pollution from coal combustion and smoking (IAPCC). Blood samples were collected from 534 NTD case mothers and 534 control mothers and assayed for 12 polymorphisms of 5 genes encoding phase II enzymes. Results: We found that the rs9282861 GG genotype of SULT1A1
was associated with an elevated risk of total NTDs (odds ratio [OR] 5 2.12, 95% confidence interval [CI]: 1.49–3.00), compared with the GA genotype. The SULT1A1 rs9282861 variant showed a significant additive interaction with maternal exposure to IAPCC for NTD risk, with a relative excess risk of interaction of 1.20 (95% CI 0.23–2.18), and the OR for the joint effect of high-level IAPCC exposure and the GG genotype was 8.37 (95% CI: 3.63– 19.28). Conclusion: Maternal SULT1A1 polymorphism is associated with the risk of fetal NTDs, and has an additive-scale interaction with maternal IAPCC exposure for NTD risk.
Introduction
be excreted (Timbrell, 2000; Shimada et al., 2004). Usually, the biotransformation in phase II leads to a detoxification process; but in some cases, the opposite occurs (Glatt, 1997). The major phase II metabolic enzymes involved in PAH metabolism are sulfotransferase (SULT), glutathione Stransferase (GST), UDP-glucuronosyltransferase (UGT), epoxide hydrolase (EPHX), and NAD(P)H dehydrogenase (quinone, NQO). Genes that encode these enzyme proteins are polymorphic and the activities of these enzymes exhibit a high variability in human population (Ryberg et al., 1997; Moreno et al., 2005). The change in enzymatic activity due to gene polymorphism may in turn affect maternal metabolism of the xenobiotics, and therefore the chemical environment in which the fetus develops. However, the genetic variants and gene-environment interactions in the pathway of phase II metabolism in relation to the development of fetal NTDs are largely unknown. We hypothesized that maternal genetic variants of the phase II metabolic enzymes could influence the risk of fetal NTDs, and interact with maternal exposure to indoor air pollution from smoking (active or passive) or coal combustion (IAPCC) to increase the susceptibility to NTDs. We examined this hypothesis in a large-scale, case–control study in a population in the Shanxi Province, China. This province has the highest prevalence of NTDs (Li et al., 2006) and the largest emissions of PAHs from both industrial sources and domestic coal use (Xu et al., 2006; Liu et al., 2009).
Neural tube defects (NTDs) are among the most common birth defects (March of Dimes Foundation, 2006; Wallingford et al., 2013). The exact causes of NTDs remain unclear although many environmental and genetic factors have been suggested to play a role (Wallingford et al., 2013). Recent epidemiological studies found that maternal exposure to indoor air pollution from coal combustion and occupational polycyclic aromatic hydrocarbons (PAHs) was associated with an increased risk of fetal NTDs (Li et al., 2011; Langlois et al., 2012). In addition, higher levels of PAHs in maternal blood and placental tissue were observed in NTD case mothers than in healthy control mothers (Naufal et al., 2010; Ren et al., 2011). Biotransformation is essential for PAHs to produce their toxicity (World Health Organization, 2000). In the human body, PAHs are metabolized in two phases. In phase I, the original exogenous molecule is added with a functional group; in phase II, the product can be conjugated with functional groups and subsequently the coupled molecule can
Additional Supporting information may be found in the online version of this article. This work was supported in part by grants from the National Natural Science Foundation of China (Grant No. 31071315 and 81202215). Institute of Reproductive and Child Health, Ministry of Health Key Laboratory of Reproductive Health, and Department of Epidemiology and Health Statistics, School of Public Health, Peking University, Beijing, China *Correspondence to: Aiguo Ren, Institute of Reproductive and Child Health, Peking University Health Science Center, Beijing 100191. E-mail address: [email protected] Published online 4 December 2013 in Wiley Online Library (wileyonlinelibrary. com). Doi: 10.1002/bdra.23196
C 2013 Wiley Periodicals, Inc. V
Birth Defects Research (Part A) 100:13–21, 2014. C 2013 Wiley Periodicals, Inc. V
Materials and Methods STUDY POPULATION
A case–control study was conducted in 5 rural counties (Pingding, Xiyang, Taigu, Zezhou, and Shouyang) of Shanxi Province in northern China, where a population-based birth
14
defects surveillance system had been established (Shouyang joined the case–control study since 2010) (Li et al., 2006). The surveillance system monitors major external structural birth defects through active case ascertainment. The overall NTD prevalence was reported as 13.9 in every 1000 births in the population in 2003 (Li et al., 2006). Cases were newborns or terminated fetuses with NTDs, and controls were term healthy newborns without congenital malformations. The controls were matched to cases according to fetal sex, mother’s county of residence and date of mother’s last menstrual period (as close as possible to that of the case mother’s). Although the study was originally designed as a matched case–control study, some blood samples were not available for evaluation because consent could not be obtained from some women, or collecting a blood sample from a control mother matched to a terminated case mother was often delayed because we had to wait till term delivery of the control mother. Therefore, the pairs were broken in the present study. Balance in the matching variables was considered and was adjusted for if imbalance was present. The study protocol was approved by the institutional review board. Informed consent was obtained from all of the mothers. Data on maternal socio-demographic characteristics, reproductive history, lifestyle, active or passive smoking history, periconceptional use of folic acid supplements, and domestic fuel use for cooking and heating were collected through face-to-face interviews within the first week of delivery or pregnancy termination. Dried blood spots (DBSs) were prepared using filter paper from venous blood of women during the period from 2002– 2007. After being dried, these DBSs were kept in sealed plastic bags with desiccants to protect them from dust and moisture and the DBSs were stored at 220 C until analysis. Fresh venous blood samples were collected beginning in 2010 and continuing through 2012; the blood cells were separated and kept at 280 C until analysis. Gene variant analyses were conducted in two stages. In stage 1, we explored possible associations between the selected polymorphisms and the risk of NTDs with the samples collected from 2002 through 2007. At first, 387 DBS samples from NTD mothers (185 anencephaly and 202 spina bifida; encephalocele was not included) and 387 control mothers were randomly selected for DNA extraction. DNA extraction was not successful for six controls; therefore, these six samples were excluded from the analyses. Thus, we genotyped 387 DNA samples from the NTD mothers and 381 control mothers in stage 1. In stage 2, the remaining DBS samples of case and control mothers collected from 2002 through 2007 (27 anencephaly, 25 spina bifida, and 33 encephalocele versus 90 controls, they were not genotyped in stage 1) and the fresh blood samples (20 anencephaly, 35 spina bifida, and 7 encephalocele versus 63 controls) collected from 2010 through 2012 were further genotyped to confirm the positive results of stage 1.
MATERNAL GENETIC VARIANTS AND FETAL NTD RISK
EXPOSURE ASSESSMENT
IAPCC exposure index was calculated by the method of Li (Li et al., 2011) with minor modifications. Smoking, cooking and heating exposure were identified as potential IAPCC exposure sources and were assigned an exposure index for each source according to the mother’s responses. (Detailed information is shown in Method of Exposure assessment of the Supplemental Materials.) The IAPCC exposure index was calculated by summing the exposure values for all individual indoor air pollution sources. The index assumed that the effects of coal smoke and smoking were cumulative across the various exposure sources. LABORATORY ANALYSIS DNA extraction from blood samples.
Nine 3-mm disks of each DBS were punched into 1.5-mL tubes using the filter paper puncher. The punched filter paper disks were processed to extract DNA using the QIAamp DNA mini kit according to the manufacturer’s instructions (Qiagen Inc, Valencia, CA). Genomic DNA from blood cells was extracted using RelaxGene Blood DNA extraction Kit (Tiangen Biotech CO.,LTD., Beijing, China). The extracted DNA samples were stored at 220 C till genotyping. SNP selection and genotyping. We selected single nucleotide polymorphisms (SNPs) in the EPHX, GST, NQO, SULT, and UGT genes according to the following criteria: (1) SNPs that have been reported to be significantly associated with various diseases (London et al., 2000; Gu et al., 2007; Jada et al., 2007; Sun et al., 2008) or PAH levels or PAH metabolic product levels in human body (Kim et al., 2007) in previous studies; (2) minor allele frequency (MAF) >0.1 in Chinese Han people. A total of 14 polymorphisms were selected, including the GSTM1 null and GSTT1 null. Two SNPs had to be excluded during the process of primer design because the assays for the two SNPs could not be performed at the same time with the assays for other majority of the candidate SNPs by the MassARRAY genotyping platform. A total of 12 polymorphisms of the EPHX1, EPHX2, GSTM1, GSTT1, GSTP1, NQO1, SULT1A1, and UGT1A1 genes (2 in EPHX1, 4 in UGT1A1 and 1 in each remaining gene) were selected (Supp. Table S1, which is available online). The dual-PCR assay for the GSTM1 and GSTT1 polymorphisms was performed according to Arand’s method (Arand et al., 1996). The primer sequences used for amplifications were as follows: GSTM1, forward primer GAACTCCCTG AAAAGCTAAA GC, reverse primer GTTGGGCTCA AATATACGGT GG; GSTT1, forward primer TTCCTTACTG GTCCTCACAT CTC, reverse primer TCACCGGATC ATGGCCAGCA. The PCR products of GSTM1 and GSTT1 were 215 base pairs and 480 base pairs, respectively. The other SNPs were determined by using the MassARRAY genotyping platform (Sequenom, San Diego, CA) with matrix-assisted laser desorption/ionization time-of-flight
BIRTH DEFECTS RESEARCH (PART A) 100:13–21 (2014)
(MALDI-TOF) mass spectrometry. Genotyping was repeated in 5% of samples for verification and quality control. Quality control testing revealed that genotype data had an error rate
Background: Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with the risk of fetal neural tube defects (NTDs). Whether maternal genetic variants related to PAH metabolism contribute to the development of fetal NTDs remains unclear. Methods: We conducted a case–control study in a Chinese population to examine the association of selected maternal genetic variants of phase II enzymes involved in the elimination of the metabolic intermediates of these chemicals with fetal NTD risk, and to evaluate possible interaction of the genetic variant and maternal exposure to indoor air pollution from coal combustion and smoking (IAPCC). Blood samples were collected from 534 NTD case mothers and 534 control mothers and assayed for 12 polymorphisms of 5 genes encoding phase II enzymes. Results: We found that the rs9282861 GG genotype of SULT1A1
was associated with an elevated risk of total NTDs (odds ratio [OR] 5 2.12, 95% confidence interval [CI]: 1.49–3.00), compared with the GA genotype. The SULT1A1 rs9282861 variant showed a significant additive interaction with maternal exposure to IAPCC for NTD risk, with a relative excess risk of interaction of 1.20 (95% CI 0.23–2.18), and the OR for the joint effect of high-level IAPCC exposure and the GG genotype was 8.37 (95% CI: 3.63– 19.28). Conclusion: Maternal SULT1A1 polymorphism is associated with the risk of fetal NTDs, and has an additive-scale interaction with maternal IAPCC exposure for NTD risk.
Introduction
be excreted (Timbrell, 2000; Shimada et al., 2004). Usually, the biotransformation in phase II leads to a detoxification process; but in some cases, the opposite occurs (Glatt, 1997). The major phase II metabolic enzymes involved in PAH metabolism are sulfotransferase (SULT), glutathione Stransferase (GST), UDP-glucuronosyltransferase (UGT), epoxide hydrolase (EPHX), and NAD(P)H dehydrogenase (quinone, NQO). Genes that encode these enzyme proteins are polymorphic and the activities of these enzymes exhibit a high variability in human population (Ryberg et al., 1997; Moreno et al., 2005). The change in enzymatic activity due to gene polymorphism may in turn affect maternal metabolism of the xenobiotics, and therefore the chemical environment in which the fetus develops. However, the genetic variants and gene-environment interactions in the pathway of phase II metabolism in relation to the development of fetal NTDs are largely unknown. We hypothesized that maternal genetic variants of the phase II metabolic enzymes could influence the risk of fetal NTDs, and interact with maternal exposure to indoor air pollution from smoking (active or passive) or coal combustion (IAPCC) to increase the susceptibility to NTDs. We examined this hypothesis in a large-scale, case–control study in a population in the Shanxi Province, China. This province has the highest prevalence of NTDs (Li et al., 2006) and the largest emissions of PAHs from both industrial sources and domestic coal use (Xu et al., 2006; Liu et al., 2009).
Neural tube defects (NTDs) are among the most common birth defects (March of Dimes Foundation, 2006; Wallingford et al., 2013). The exact causes of NTDs remain unclear although many environmental and genetic factors have been suggested to play a role (Wallingford et al., 2013). Recent epidemiological studies found that maternal exposure to indoor air pollution from coal combustion and occupational polycyclic aromatic hydrocarbons (PAHs) was associated with an increased risk of fetal NTDs (Li et al., 2011; Langlois et al., 2012). In addition, higher levels of PAHs in maternal blood and placental tissue were observed in NTD case mothers than in healthy control mothers (Naufal et al., 2010; Ren et al., 2011). Biotransformation is essential for PAHs to produce their toxicity (World Health Organization, 2000). In the human body, PAHs are metabolized in two phases. In phase I, the original exogenous molecule is added with a functional group; in phase II, the product can be conjugated with functional groups and subsequently the coupled molecule can
Additional Supporting information may be found in the online version of this article. This work was supported in part by grants from the National Natural Science Foundation of China (Grant No. 31071315 and 81202215). Institute of Reproductive and Child Health, Ministry of Health Key Laboratory of Reproductive Health, and Department of Epidemiology and Health Statistics, School of Public Health, Peking University, Beijing, China *Correspondence to: Aiguo Ren, Institute of Reproductive and Child Health, Peking University Health Science Center, Beijing 100191. E-mail address: [email protected] Published online 4 December 2013 in Wiley Online Library (wileyonlinelibrary. com). Doi: 10.1002/bdra.23196
C 2013 Wiley Periodicals, Inc. V
Birth Defects Research (Part A) 100:13–21, 2014. C 2013 Wiley Periodicals, Inc. V
Materials and Methods STUDY POPULATION
A case–control study was conducted in 5 rural counties (Pingding, Xiyang, Taigu, Zezhou, and Shouyang) of Shanxi Province in northern China, where a population-based birth
14
defects surveillance system had been established (Shouyang joined the case–control study since 2010) (Li et al., 2006). The surveillance system monitors major external structural birth defects through active case ascertainment. The overall NTD prevalence was reported as 13.9 in every 1000 births in the population in 2003 (Li et al., 2006). Cases were newborns or terminated fetuses with NTDs, and controls were term healthy newborns without congenital malformations. The controls were matched to cases according to fetal sex, mother’s county of residence and date of mother’s last menstrual period (as close as possible to that of the case mother’s). Although the study was originally designed as a matched case–control study, some blood samples were not available for evaluation because consent could not be obtained from some women, or collecting a blood sample from a control mother matched to a terminated case mother was often delayed because we had to wait till term delivery of the control mother. Therefore, the pairs were broken in the present study. Balance in the matching variables was considered and was adjusted for if imbalance was present. The study protocol was approved by the institutional review board. Informed consent was obtained from all of the mothers. Data on maternal socio-demographic characteristics, reproductive history, lifestyle, active or passive smoking history, periconceptional use of folic acid supplements, and domestic fuel use for cooking and heating were collected through face-to-face interviews within the first week of delivery or pregnancy termination. Dried blood spots (DBSs) were prepared using filter paper from venous blood of women during the period from 2002– 2007. After being dried, these DBSs were kept in sealed plastic bags with desiccants to protect them from dust and moisture and the DBSs were stored at 220 C until analysis. Fresh venous blood samples were collected beginning in 2010 and continuing through 2012; the blood cells were separated and kept at 280 C until analysis. Gene variant analyses were conducted in two stages. In stage 1, we explored possible associations between the selected polymorphisms and the risk of NTDs with the samples collected from 2002 through 2007. At first, 387 DBS samples from NTD mothers (185 anencephaly and 202 spina bifida; encephalocele was not included) and 387 control mothers were randomly selected for DNA extraction. DNA extraction was not successful for six controls; therefore, these six samples were excluded from the analyses. Thus, we genotyped 387 DNA samples from the NTD mothers and 381 control mothers in stage 1. In stage 2, the remaining DBS samples of case and control mothers collected from 2002 through 2007 (27 anencephaly, 25 spina bifida, and 33 encephalocele versus 90 controls, they were not genotyped in stage 1) and the fresh blood samples (20 anencephaly, 35 spina bifida, and 7 encephalocele versus 63 controls) collected from 2010 through 2012 were further genotyped to confirm the positive results of stage 1.
MATERNAL GENETIC VARIANTS AND FETAL NTD RISK
EXPOSURE ASSESSMENT
IAPCC exposure index was calculated by the method of Li (Li et al., 2011) with minor modifications. Smoking, cooking and heating exposure were identified as potential IAPCC exposure sources and were assigned an exposure index for each source according to the mother’s responses. (Detailed information is shown in Method of Exposure assessment of the Supplemental Materials.) The IAPCC exposure index was calculated by summing the exposure values for all individual indoor air pollution sources. The index assumed that the effects of coal smoke and smoking were cumulative across the various exposure sources. LABORATORY ANALYSIS DNA extraction from blood samples.
Nine 3-mm disks of each DBS were punched into 1.5-mL tubes using the filter paper puncher. The punched filter paper disks were processed to extract DNA using the QIAamp DNA mini kit according to the manufacturer’s instructions (Qiagen Inc, Valencia, CA). Genomic DNA from blood cells was extracted using RelaxGene Blood DNA extraction Kit (Tiangen Biotech CO.,LTD., Beijing, China). The extracted DNA samples were stored at 220 C till genotyping. SNP selection and genotyping. We selected single nucleotide polymorphisms (SNPs) in the EPHX, GST, NQO, SULT, and UGT genes according to the following criteria: (1) SNPs that have been reported to be significantly associated with various diseases (London et al., 2000; Gu et al., 2007; Jada et al., 2007; Sun et al., 2008) or PAH levels or PAH metabolic product levels in human body (Kim et al., 2007) in previous studies; (2) minor allele frequency (MAF) >0.1 in Chinese Han people. A total of 14 polymorphisms were selected, including the GSTM1 null and GSTT1 null. Two SNPs had to be excluded during the process of primer design because the assays for the two SNPs could not be performed at the same time with the assays for other majority of the candidate SNPs by the MassARRAY genotyping platform. A total of 12 polymorphisms of the EPHX1, EPHX2, GSTM1, GSTT1, GSTP1, NQO1, SULT1A1, and UGT1A1 genes (2 in EPHX1, 4 in UGT1A1 and 1 in each remaining gene) were selected (Supp. Table S1, which is available online). The dual-PCR assay for the GSTM1 and GSTT1 polymorphisms was performed according to Arand’s method (Arand et al., 1996). The primer sequences used for amplifications were as follows: GSTM1, forward primer GAACTCCCTG AAAAGCTAAA GC, reverse primer GTTGGGCTCA AATATACGGT GG; GSTT1, forward primer TTCCTTACTG GTCCTCACAT CTC, reverse primer TCACCGGATC ATGGCCAGCA. The PCR products of GSTM1 and GSTT1 were 215 base pairs and 480 base pairs, respectively. The other SNPs were determined by using the MassARRAY genotyping platform (Sequenom, San Diego, CA) with matrix-assisted laser desorption/ionization time-of-flight
BIRTH DEFECTS RESEARCH (PART A) 100:13–21 (2014)
(MALDI-TOF) mass spectrometry. Genotyping was repeated in 5% of samples for verification and quality control. Quality control testing revealed that genotype data had an error rate
