Maternal Smoking During Early Pregnancy, GSTP1 and EPHX1 ...

Maternal Smoking During Early Pregnancy, GSTP1 and EPHX1 Variants, and Risk of Isolated Orofacial Clefts Dorian Ramirez, M.D., Edward J. Lammer, M.D., David M. Iovannisci, Ph.D., Cecile Laurent, Richard H. Finnell, Ph.D., Gary M. Shaw, Dr.P.H.

Objective: To examine the interactions between four fetal xenobiotic metabolizing gene polymorphisms, maternal cigarette smoking, and risk for oral cleft defects. Design and Participants: California population–based case-control study of 431 infants born with isolated orofacial clefts and 299 nonmalformed controls. Main Outcome Measures: Infants were genotyped for functional polymorphisms of the detoxification enzymes microsomal epoxide hydrolase-1 (EPHX1 T→C [Tyr113His], and A→G [His139Arg]), and glutathione-S transferase Pi-1 (GSTP1 A→G [Ile105Val] and C→T [Ala114Val]), and risks for cleft outcomes were measured for gene only and gene-maternal smoking effects. Results: Although smoking was associated with an increased risk for isolated cleft lip ⴞ palate, we found no independent associations of genotypes of EPHX1-codon 113 or GSTP1-codon 105 polymorphisms for either isolated cleft lip ⴞ palate or isolated cleft palate. The heterozygote genotype for the EPHX1codon 139 polymorphism was associated with an increased risk of isolated cleft palate (odds ratio ⴝ 1.6 [95% confidence interval, 1.0 to 2.6]). Infant EPHX1 and GTSP1 polymorphic variants did not appreciably alter the risks for clefts associated with maternal smoking, nor were any EPHX1 combined genotypespecific risks found. Infant genotypes of the GSTP1-codon 105 polymorphism, combined with glutathione-S-transferase-␮-1 null genotypes, did not appreciably alter the risk of orofacial clefts. Conclusions: Our results suggest that genetic variation of the detoxification enzymes EPHX1 and GSTP1 did not increase the risks of orofacial clefting, nor do they influence the risks associated with maternal smoking. KEY WORDS: cleft lip, cleft palate, EPHX1, GSTM1, GSTP1, smoking

Maternal tobacco smoking during pregnancy is associated with a variety of adverse outcomes, including the presence of orofacial cleft defects in newborns. The increased risk is around twofold (Lieff et al., 1999; Beaty et al., 2001; Wyszynski and Wu, 2002). Maternal and fetal xenobiotic metabolizing enzymes may play a crucial role in inactivating teratogens in tobacco smoke. Thus, genetic variants of these enzymes that adversely influence biotransformation of toxins may enhance the risks as-

sociated with smoking during pregnancy. Previously, we demonstrated that deletions of phase II detoxification enzymes glutathione-S-transferase-␮-1 (GSTM1) and glutathione-S-transferase-␶-1 (GSTT1) increased risks of orofacial clefts among infants whose mothers smoked during early pregnancy (Lammer et al., 2005). Further analysis of polymorphisms in xenobiotic metabolizing enzymes may help clarify the potential gene-environment interaction responsible for cleft formation among children born to mothers who smoked during pregnancy. Potential teratogens in tobacco smoke include nicotine, polycyclic aromatic hydrocarbons (PAH), arylamines, N-nitrosamines, and carbon monoxide (Brunnemann and Hoffmann, 1991; Schottenfeld and Fraumeni, 1996). These compounds are absorbed into maternal blood and reach the developing fetus, yet the mechanisms through which tobacco smoke may cause abnormal development remain poorly understood (Hansen et al., 1992; Arnould et al., 1997). The presence of developmental abnormalities in infants whose mothers smoked during pregnancy is likely associated with the level of fetal exposure to these teratogens. Exposures probably are related to the number of cigarettes smoked, rates of placental and fetal transfer, and maternal and fetal metabolic biotransformation.

Dr. Ramirez is Medical Resident, Department of Emergency Medicine, Harbor–UCLA Medical Center, Torrance, California; Dr. Lammer is Associate Scientist and Dr. Iovannisci is Assistant Staff Scientist, Children’s Hospital and Research Center, Oakland, California; Ms. Laurent is Research Analyst, California Birth Defects Monitoring Program, Berkeley, California; Dr. Finnell is Executive Director and President, Texas Institute for Genomic Medicine, Texas A&M University Health Science Center, Houston, Texas; Dr. Shaw is Senior Epidemiologist, California Birth Defects Monitoring Program, Berkeley, California. Submitted March 2006; Accepted August 2006. Address correspondence to: Dr. Edward Lammer, Children’s Hospital and Research Center, 5700 Martin Luther King Jr. Boulevard, Oakland, CA 94609. E-mail [email protected]. DOI: 10.1597/06-011.1 366

Ramirez et al., MATERNAL SMOKING, GSTP1 AND EPHX1, AND CLEFTING

Microsomal epoxide hydrolase-1 (EPHX1) is a phase I enzyme involved in the hydration/detoxification of several tobacco smoke constituents, including PAHs (Morisseau and Hammock, 2005). Two coding region polymorphisms have been identified that produce proteins with altered enzyme activity. A T→C nucleotide transition that is transcribed as EPHX1-codon 113 results in a Tyr113His substitution and leads to decreased enzyme activity. In contrast, the A→G transition that is transcribed as EPHX1-codon 139 results in a His139Arg substitution that enhances enzyme activity (Hassett et al., 1994, 1997; Omiecinski et al., 2000). These alterations, however, exert only modest effects on EPHX1 activity (Omiecinski et al., 2000; Hosagrahara et al., 2004). The glutathione-S transferases (GSTs) are phase II detoxification enzymes that conjugate reactive intermediates formed during the biotransformation of xenobiotic toxins. Eight classes of human GST isoenzymes exist, each exhibiting overlapping, but distinct, substrate specificity (Hayes and Pulford, 1995; Wormhoudt et al., 1999). We, as well as Dutch investigators, recently reported higher risks for orofacial clefts among infants who had homozygous deletions of the GST isoenzymes GSTM1 or GSTT1 and whose mothers smoked during pregnancy (van Rooij et al., 2001). Glutathione-S transferase Pi-1 (GSTP1) is highly expressed early in fetal life and is likely to be involved in the detoxification of constituents of tobacco smoke. The two GSTP1 polymorphisms that we investigated lead to amino acid changes that are within a putative electrophilic binding site of the human GSTP1 protein (Ahmad et al., 1990; Ali-Osman et al., 1997). These variant forms of GSTP1, therefore, are likely to alter its enzymatic activity, although this is not known. This study investigated whether variant forms of infant EPHX1 and GSTP1 genes influenced the risks between maternal tobacco smoking during pregnancy and susceptibility to orofacial clefts. METHODS The research protocols for this study were approved by the California Department of Health Services and Children’s Hospital Oakland Institutional Review Boards. Study Population Details of the population-based case-control study used for these analyses have been described elsewhere (Shaw et al., 1996; Lammer et al., 2005). Liveborn case infants and stillborn fetuses with an orofacial cleft (cleft lip with or without cleft palate [CL/P] or cleft palate [CP]) were ascertained by reviewing medical records at all hospitals and genetic centers in a known geographic population base. Infants and fetuses were eligible if diagnosed within 1 year of delivery from a cohort of 552,601 births and fetal deaths that occurred between January 1, 1987, and December 31, 1989, to women residing in most counties in California (metropolitan areas of Los Angeles and San Francisco were excluded). Diagnostic information

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from medical records, autopsies, and surgical reports of all infants and fetuses with orofacial clefts or similar orofacial anomalies was reviewed in order to restrict eligibility to those infants with CL/P or CP. Infants with diagnoses of holoprosencephaly, bifid uvula, submucous cleft palate, notching of the alveolar ridge or vermillion border of the upper lip were excluded. Infants diagnosed with chromosomal aneuploidy were excluded (n ⫽ 81). There were 892 eligible infants/fetuses. A medical geneticist (E.J.L.) classified each case as isolated or multiple, based on the nature of any accompanying major congenital anomalies. Cases of CL/P and CP were classified as isolated (iCL/P or iCP) if the infants or fetuses had no other major anomaly or had anomalies considered either minor (e.g., low-set ears) or not true malformations (e.g., undescended testicles). Only cases with isolated clefts were included in these analyses. A control infant was potentially eligible if: (1) he or she was born alive during 1987 to 1989; (2) the mother was a resident of a county from which cases were ascertained; and (3) no major structural congenital anomaly was diagnosed prior to the first birthday. Other than being delivered during a similar time period and within the same geographic area, controls were not matched to cases. A total of 972 controls were selected electronically from California vital records using a pseudorandom number generator among all eligible infants (N ⫽ 584,844). Questionnaire Mothers of cases and controls were interviewed in English (91%) or Spanish, nearly all by telephone. Women who only spoke languages other than English or Spanish (26 cases and 33 controls) and 3 case mothers who died prior to interview contact were excluded, yielding 863 cases and 939 controls. Interviews of case mothers were completed an average of 3.5 years after the date of delivery; control mothers were interviewed an average of 3.6 years after delivery. At the beginning of the interview, an interviewer assisted each woman with establishing a 4-month time period that was referred to throughout the interview to elicit information on exposures and events. The 4-month period ranged from 1 month before to 3 months after conception. This encompasses the embryological period of palate and lip formation, including closure, which is complete by approximately 60 days postconception. In addition to the more detailed inquiries about tobacco exposures, the average 40-minute interview elicited information on maternal medical history, use of medications, and use of alcohol or recreational drugs, as well as a wide variety of other exposures in the periconceptional period. To assess active maternal smoking exposures, women were asked how many cigarettes they smoked daily for the 4-month period. The women also were asked how many cigarettes they smoked during each month of the 4-month period. To assess passive smoke exposures during that time frame, a woman was asked whether anyone smoked inside her home (including specific questions about paternal smoking), smoked near her at work or school or while she was

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Cleft Palate–Craniofacial Journal, July 2007, Vol. 44 No. 4

commuting to work or school, and whether she regularly frequented (at least once per week) a place such as a restaurant or laundromat where others smoked nearby.

TABLE 1 Maternal Characteristics of Cases and Controls* (NOTE: Percentages may not add to 100, due to rounding or missing information for some subjects) iCL/P (n ⫽ 305)

Genotyping Genomic DNA was obtained from residual dried blood spots on newborn screening specimens (Guthrie filter papers) collected from all liveborn children in California and stored by the Genetic Diseases Branch, Health and Human Services. A DNA specimen was identified for 83% of eligible cases and 87% of eligible controls for whom maternal interviews also were available. Reasons for the inability to identify a specimen included (1) no sample remained on the filter paper; (2) filter paper could not be located; and (3) information on the child was insufficient to adequately match records. To minimize the number of samples to be genotyped, the control samples were reduced randomly to 299. DNA was extracted from the residual newborn screening filter papers using a modification of the salting out method (Iovannisci, 2000) and was resuspended in 20 ␮L of TE buffer. Filter paper samples contained approximately 5 ␮L of whole blood. Results from genotyping of GSTM1 and GSTT1 null polymorphisms from this study population were published previously (Hartsfield et al., 2001; Lammer et al., 2005). Each sample underwent whole genome amplification before an aliquot was taken for genotyping. Whole-genome amplification was performed in 50-␮L reactions using an established method, but with 3 ␮L of DyNAzyme EXT DNA polymerase (New England Biolabs, Ipswich, MA) per reaction (Zheng et al., 2001). This modified whole-genome amplification protocol greatly reduces the frequency of allelic dropouts reported by others to an undetectable level using a multilocus genotyping assay we have employed extensively for the genotyping analysis of other blood spot–derived DNAs (D. Iovannisci, unpublished results). TaqMan assays were performed using 1 ␮L of the resulting whole genome amplified–DNA using the ABI H7900 Sequence Analysis System (http://docs. appliedbiosystems.com/pebiodocs/00103857.pdf). In order to reduce confusion, we used the codon nomenclature for the four polymorphisms that was used in previous publications, rather than using the nucleotide nomenclature. The polymorphisms for GSTP1-codon 105 (rs1695) and GSTP1-codon 114 (rs1138272) were typed using polymerase chain reaction (PCR) primers (Proligo, Boulder, CO) and probes (Applied Biosystems, Foster City, CA) as shown in Table 1. Real-time PCR for the GSTP1 polymorphisms consisted of a hold at 95⬚C for 10 minutes and 50 cycles of 20 seconds at 95⬚C and 1 minute at 67⬚C. The GSTP1 primers and probes were described previously (Kelada et al., 2003). The EPHX1 real-time PCR consisted of a hold at 95⬚C for 10 minutes and 50 (EPHX1-codon 113; rs1051740) or 55 cycles (EPHX1-codon 139; rs2234922) of 15 seconds at 92⬚C and 1 minute at 60⬚C. For the GSTP1 Ile105Val (rs947894) genotyping, the forward and reverse primers were: 5⬘-GGCAGCCCTGGTGGAC AT-3⬘ and 5⬘-AGCCCCCAGTGCCCAAC-3⬘, respectively.

Race/ethnicity Hispanic Non-Hispanic/White Black Asian Other Age (y) ⬍20 20–24 25–29 30–34 35–39 ⬎39

iCP (n ⫽ 126)

Controls (n ⫽ 299)

90 189 8 6 12

(29.5%) (62.0%) (2.6%) (2.0%) (3.9%)

25 88 6 2 5

(19.8%) (69.8%) (4.8%) (1.6%) (4.0%)

89 175 10 6 19

(29.8%) (58.5%) (3.3%) (2.0%) (6.4%)

43 82 93 54 25 5

(14.1%) (26.9%) (30.5%) (17.7%) (8.2%) (1.6%)

19 25 49 21 11 1

(15.1%) (19.8%) (38.9%) (16.7%) (8.7%) (0.8%)

29 68 98 78 24 1

(9.7%) (22.7%) (32.8%) (26.1%) (8.0%) (0.3%)

* iCL/P ⫽ isolated cleft lip with or without cleft palate; iCP ⫽ isolated cleft palate.

The wild-type probe used was 5⬘-TCCGCTGCAAATAC ATCTCCCTCATCTAC-3⬘ and was 5⬘ labeled with 6FAM娂 fluorescent dye and 3⬘ conjugated with TAMRA娂 quencher dye. The variant probe, 5⬘-CCGCTGCAAATACGTCTCCC TCATCTA-3⬘, was 5⬘ labeled with VIC娂 fluorescent dye and 3⬘ conjugated with TAMRA娂 quencher dye. For GSTP1 Ala114Val (rs1799811) genotyping, the forward and reverse primers were 5⬘-AGGGATGAGAGTAGGATGATACAT-3⬘ and 5⬘-GGGACAGCAGGGTCTCAAAA-3⬘, respectively. The wild-type and mutant probes, respectively, were 5⬘-CC TTGCCCGCCTCCTGCC-3⬘, 5⬘ labeled with 6FAM娂 fluorescent dye and 3⬘ conjugated with TAMRA娂 quencher dye, and 5⬘-CATCCTTGCCCACCTCCTGCC-3⬘, 5⬘ labeled with TET娂 fluorescent dye and 3⬘ conjugated with TAMRA娂 quencher dye. For the EPHX1 Tyr113His genotype, the forward and reverse primers were 5⬘-ACTGGAAGAAGCAG GTGGAGATT-3⬘ and 5⬘-CGTTTTGCAAACATACCT TCAATC-3⬘, respectively. The wild-type probe used was 5⬘-AACAGACACCCTCACT-3⬘, and was 5⬘ labeled with VIC娂 fluorescent dye and 3⬘ conjugated with MGBNFQ娂 quencher dye. The variant probe, 5⬘-CTCAACAGATAC CC-3⬘, was 5⬘ labeled with 6FAM娂 fluorescent dye and 3⬘ conjugated with MGBNFQ娂 quencher dye. For EPHX1 His139Arg genotyping, the forward and reverse primers were 5⬘-ACATCCACTTCATCCACGTGAA-3⬘ and 5⬘-CCAGCCG TGCACCATCA-3⬘, respectively. The wild-type and mutant probes, respectively, were 5⬘-AGGCCGTACCCCGA-3⬘, 5⬘ labeled with VIC娂 fluorescent dye and 3⬘ conjugated with MGBNFQ娂 quencher dye, and 5⬘-AGGCCATACCCCG-3⬘, 5⬘ labeled with 6FAM娂 fluorescent dye and 3⬘ conjugated with MGBNFQ娂 quencher dye. All genotyping was performed while blinded to case/control status. Statistical Analyses Odds ratios (ORs) and their 95% confidence intervals (CIs) were used to estimate risks. We estimated the risk of iCL/P or iCP associated with genotypes for GSTP1-codon 105, GSTP1-


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TABLE 2 Risks for Orofacial Clefts Related to EPHX1 and GSTP1 Genotypes* iCL/P OR (95% CI)

No.

OR (95% CI)

No.

Frequency†

Putative Enzyme Activity Toward the PAH BPDE

EPHX1-codon 113 polymorphism TT 136 1.1 (0.6–2.1) CT 137 1.1 (0.6–2.1) CC 27 Reference

55 53 18

0.69 (0.3–1.4) 0.66 (0.3–1.4) Reference

132 132 30

44% 44% 10%

↓

↓

EPHX1-codon 139 polymorphism AA 209 Reference AG 83 1.1 (0.8–1.7) GG 12 1.1 (0.4–2.8)

77 43 6

Reference 1.6 (1.0–2.6) 1.5 (0.4–4.6)

210 73 11

70% 24% 4%

↓

↓

Genotypes

No.

iCP

iCL/P

Controls

iCP

Controls

Hypothesized Effect on Toxin Levels

OR (95% CI)

No.

OR (95% CI)

No.

Frequency*

Putative Enzyme Activity

Hypothesized Effect on Toxin Levels

GSTP1-codon 105 polymorphism AA 120 0.7 (0.4–1.3) AG 132 0.7 (0.4–1.2) GG 50 Reference

43 68 14

1.0 (0.5–2.1) 1.4 (0.7–2.9) Reference

122 135 38

41% 45% 13%

↑

↓

117 9 0

N/D N/D N/D

274 21 2

92% 7% —

↑

↓

Genotypes

No.

GSTP1-codon 114 polymorphism CC 273 CT 29 TT 1

N/D N/D N/D

* N/D ⫽ Odds ratios and confidence intervals were not calculated when there were less than five cases and controls for a single genotype; PAH ⫽ polycyclic aromatic hydrocarbon; BPDE ⫽ benzo(a)pyrene diol epoxides; iCL/P ⫽ isolated cleft lip with or without cleft palate; iCP ⫽ isolated cleft palate. † Percentages may not add up to 100, because of rounding or missing information for some subjects.

codon 114, EPHX1-codon 113, and EPHX1-codon 139 polymorphisms. Risks for these two phenotypic groups and four polymorphisms in the presence or absence of maternal smoking were estimated also. Because the polymorphisms differentially influenced enzyme activity, the wild-type was not treated as the reference genotype for every analysis. Reference group selections were based on the putative influence on enzymatic activity conferred by each genotype (Table 2). The presence of a C allele for EPHX1-codon 113 or a G for EPHX1-codon 139 reduces EPHX1 enzyme activity. Homozygosity for these alleles was chosen as the reference genotypes, because we hypothesized that lowered EPHX1 enzyme activity would decrease the concentration of potentially deleterious reactive electrophilic intermediates formed during metabolism of some tobacco constituent PAHs (Hassett et al., 1994; Hassett et al., 1997; Omiecinski et al., 2000). For GSTP1, the GG genotype was selected as the reference genotype for the codon 105 polymorphism, because the G allele is associated with increased GSTP1 activity, leading to more rapid detoxification of reactive intermediates through their conjugation (Hu et al., 1997). RESULTS Among the 430 cases, male infants represented 63% of the cleft lips and 41% of the cleft palates; 44% of the 299 controls were male infants. Table 1 shows selected maternal characteristics of the case and control mothers. Twenty-five percent of control mothers smoked tobacco in the periconceptional period, compared with 35% of case mothers. For iCL/P, the OR

for any amount of maternal smoking was 1.6 (95% CI, 1.1 to 2.4). For iCP, the OR for any smoking during early pregnancy was also 1.6 (95% CI, 1.1 to 2.6) (Lammer et al., 2005). Ninety-nine percent of cases and controls were genotyped successfully; genotypic frequencies are shown in Table 2. There were a few ethnic-specific differences in genotype frequencies. For the EPHX1-codon 113 polymorphism, TT homozygosity was more frequent among Hispanic infants (17%) than among white non-Hispanic (5%), but heterozygote proportions were similar. The A allele frequency for the EPHX1codon 139 polymorphism was slightly lower for white nonHispanics (0.81) than white Hispanics (0.92). For the GSTP1codon 105 polymorphism, GG genotype was more common among white Hispanics (21%) than white non-Hispanics (9%). The T allele for GSTP1-codon 114 polymorphism was rare (0.04) for all ethnic groups. Observed genotypic proportions among controls were consistent with Hardy-Weinberg expectations for EPXH1-codon 113, EPHX1-codon 139, and GSTP1-codon 105 polymorphisms (␹2 ⫽ 0.13, 2.00, and 0.01, respectively). However, genotypes for the rare GSTP1-codon 114 polymorphism were not (␹2 ⫽ 4.5), although our genotype frequencies are similar to those of previous reports (Watson et al., 1998; Park et al., 1999). We examined whether genotypes of EPHX1-codon 113 influenced risk of clefting from maternal smoking during pregnancy (Table 3). Infants with TT or TC genotypes and any maternal smoking exposure had approximately twofold higher risks of iCL/P. There were too few CC cases and controls whose mothers smoked to make inferences about whether this risk was related to the amount of maternal smoking (see Table 3). For risk of iCP and maternal smoking, a twofold increased

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TABLE 3 Risks for Oral Clefts, Epoxide Hydrolase Polymorphism, and Maternal Smoking* Genotypes of EPHX1-Codon 113 Polymorphism No.

OR (95% CI)

No.

OR (95% CI)

No smoking TT TC CC

76 97 21

0.9 (0.5–1.9) 1.0 (0.5–2.1) Reference

32 40 12

Any smoking TT TC CC

61 39 6

2.3 (1.0–5.1) 1.8 (0.8–4.1) 1.1 (0.3–5.0)

1–9 cigs/day TT TC CC

21 15 2

1.3 (0.5–3.2) 1.4 (0.5–4.2) 0.5 (0–3.2)

10⫹ cigs/day TT TC CC

40 24 4

2.2 (0.9–5.2) 2.1 (0.8–5.7) 4.6 (0.4–234.3)

TABLE 5 Risks of Oral Clefts, Genotypes of GSTP1-Codon 105 Polymorphism, and Maternal Smoking*

No.

Genotypes GSTP1-Codon 105 Polymorphism No.

OR (95% CI)

No.

OR (95% CI)

No.

0.7 (0.3–1.7) 0.8 (0.l–1.8) Reference

93 106 24

No smoking AA AG GG

75 86 34

0.7 (0.4–1.4) 0.7 (0.4–1.4) Reference

30 41 12

0.8 (0.4–2.0) 1.0 (0.4–2.4) Reference

90 102 30

21 15 6

1.1 (0.4–2.8) 1.2 (0.4–3.3) 2.0 (0.4–9.2)

30 25 6

Any smoking AA AG GG

46 46 15

1.0 (0.5–1.9) 1.2 (0.6–2.4) 1.7 (0.6–5.2)

13 27 2

1.0 (0.4–2.9) 2.0 (0.8–5.1) 0.6 (0.1–3.9)

42 34 8

8 3 3

0.8 (0.3–2.8) 0.5 (0–2.4) 1.2 (0.2–7.4)

19 12 5

1–9 cigs/day AA AG GG

18 14 6

0.8 (0.3–1.9) 1.0 (0.4–2.6) 1.3 (0.3–7.0)

4 8 2

0.5 (0.1–2.0) 1.5 (0.4–5.3) 1.3 (0.1–10.1)

20 13 4

13 12 3

1.2 (0.4–3.7) 1.7 (0.5–5.5) N/D†

21 13 1

10⫹ cigs/day AA AG GG

28 32 9

0.7 (0.8–5.2) 1.3 (0.6–3.0) 2.0 (0.5–9.7)

9 19 0

1.0 (0.5–0.4) 2.3 (0.8–6.3) N/D†

12 21 4

iCP

iCL/P

Control

iCP

iCL/P

Controls

* iCL/P ⫽ isolated cleft lip with or without cleft palate; iCP ⫽ isolated cleft palate. † N/D ⫽ Odds ratio was not calculated if there were fewer than five cases ⫹ controls for a particular genotype.

* iCL/P ⫽ isolated cleft lip with or without cleft palate; iCP ⫽ isolated cleft palate. † N/D ⫽ Odds ratio (OR) was not calculated if there were fewer than five cases ⫹ controls for a particular genotype.

risk was found for the CC genotype, but this estimate lacked precision and was not statistically significant. For the EPHX1 genotypes, risks for iCL/P or iCP were not increased (Table 4). For iCP, both GG and AG genotypes of EPHX1-codon 139 polymorphism showed increased risks compared with the reference AA genotype. For GSTP1-codon 105, no substantially elevated risks were observed. For GSTP1-codon 114, risks could not be estimated, owing to small numbers.

Table 4 shows the results of the combined influence of EPHX1 genotypes and maternal smoking, whereas Table 5 shows the results for GSTP1. For iCL/P and for iCP, genotypes of EPHX1 and GSTP1-codon 105 did not appreciably alter risks associated with maternal smoking. There were too few cases and controls with A alleles of GSTP1-codon 114 to permit any combined analyses with maternal smoking. We also analyzed the combined influence of genotypes from EPHX1codon 113 and EPHX1-codon 139 and maternal smoking (Table 6), considering T at codon 113 and A at codon 139 as the reference combined genotype. The results did not demonstrate an increased risk for either type of oral cleft for any particular combination of genotypes. The combined influence of the presence of null deletions of GSTM1, genotypes of GSTP1-codon 105, and maternal smoking was investigated (Table 7). Among infants born to smoking mothers, combined GSTM1 null and AA genotype of GSTP1codon 105 showed a 4.3-fold increase in the risk of iCL/P

TABLE 4 Risks of Oral Clefts, Genotypes of EPHX1-Codon 139 Polymorphism, and Maternal Smoking* Genotypes EPHX1-Codon 139 Polymorphism No.

iCP

iCL/P

Controls

OR (95% CI)

No.

OR (95% CI)

No.

No smoking AA AG GG

142 45 10

Reference 1.0 (0.6–1.7) 1.4 (0.5–4.3)

50 30 4

Reference 1.9 (1.1–3.5) 1.6 (0.35–6.42)

164 51 8

Any smoking AA AG GG

67 38 2

1.6 (1.0–2.6) 2.0 (1.1–3.7) 0.8 (0.6–6.8)

27 13 2

1.9 (1.0–3.4) 1.9 (0.8–4.4) 2.2 (0.2–19.6)

47 22 3

1–9 cigs/day AA AG GG

23 14 1

1.1 (0.6–2.1) 1.6 (0.6–4.2) N/D†

12 2 0

1.6 (0.7–3.7) 0.7 (0.1–3.2) N/D

24 10 2

10⫹ cigs/day AA AG GG

44 24 1

2.2 (1.2–4.0) 2.3 (1.1–5.3) N/D

15 11 2

2.1 (1.0–4.6) 3.0 (1.1–8.0) N/D

23 12 1

* iCL/P ⫽ isolated cleft lip with or without cleft palate; iCP ⫽ isolated cleft palate. † N/D ⫽ Odds ratio (OR) was not calculated if there were fewer than five cases ⫹ controls for a particular genotype.

TABLE 6 Frequencies of Combined Genotypes of EPHX1-Codon 113 and 139 Polymorphisms Among Cases and Controls* EPHX1 Combined Genotypes

113(TT)_139(AA) 113(TT)_139(AG) 113(TT)_139(GG) 113(TC)_139(AA) 113(TC)_139(AG) 113(TC)_139(GG) 113(CC)_139(AA) 113(CC)_139(AG) 113(CC)_139(GG)

iCL/P

iCP

Control

No.

No.

No.

94 38 5 92 39 6 22 5 0

29 20 4 34 19 2 14 4 0

90 36 7 93 35 3 27 2 1

* iCL/P ⫽ isolated cleft lip with or without cleft palate; iCP ⫽ isolated cleft palate.


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TABLE 7 Risks of Oral Clefts, Combined GSTM1 Null and GSTP1-Codon 105 Genotypes, and Maternal Smoking* iCP

Control

Genotypes GSTP1-Codon 105 Polymorphism

No.

(95% CI)

No.

(95% CI)

No.

No smoking N N N ⫹ ⫹ ⫹

AA AG GG AA AG GG

34 38 17 39 47 15

0.8 (0.3–2.0) 0.8 (0.3–1.9) 1.3 (0.4–4.0) 0.9 (0.4–2.4) 1.0 (0.4–2.5) Reference

14 13 7 15 27 5

1.0 (0.3–4.2) 0.8 (0.2–3.3) 1.6 (0.3–7.9) 1.1 (0.3–4.5) 1.8 (0.5–6.8) Reference

44 52 14 44 49 16

Any smoking N N N ⫹ ⫹ ⫹

AA AG GG AA AG GG

20 13 7 17 24 5

4.3 2.0 2.5 1.4 1.3 1.8

11 8 1 2 19 1

1.9 (0.5–8.2) 2.0 (0.4–9.5) N/D‡ 0.5 (0.0–3.7) 3.0 (0.8–12.5) N/D

5 7 3 13 20 3

1 to 9 cig/day N N N ⫹ ⫹ ⫹

AA AG GG AA AG GG

0 0 0 9 6 3

N/D N/D N/D 1.6 (0.4–6.9) 1.1 (0.2–5.0) N/D

3 2 1 1 6 1

0.7 (0.1–4.3) 1.1 (0.1–7.2) N/D 0.5 (0–6.6) 3.2 (0.6–18.8) N/D

14 6 2 6 6 2

10⫹ cig/day N N N ⫹ ⫹ ⫹

AA AG GG AA AG GG

20 13 7 8 18 2

4.3 (1.1–17.9) 2.0 (0.5–7.5) 2.5 (0.5–17.3) 1.2 (0.3–5.0) 1.4 (0.5–4.2) N/D

8 6 0 1 13 0

5.1 (0.9–29.9) 2.7 (0.5–15.5) N/D 0.5 (0–5.5) 3.0 (0.7–13.2) N/D

5 7 3 7 14 1

GSTM1 Genotypes†

iCL/P

(1.1–17.9) (0.5–7.5) (0.5–17.3) (0.5–4.3) (0.5–3.6) (0.3–12.3)

* iCL/P ⫽ isolated cleft lip with or without cleft palate; iCP ⫽ isolated cleft palate. † N ⫽ null deletion of both copies; ⫹ ⫽ at least one copy present. ‡ N/D ⫽ Odds ratios and confidence intervals were not calculated when there were less than five cases and controls for a single genotype.

(95% CI, 1.1 to 17.9), compared with those infants with the reference genotype and whose mothers did not smoke. Other GSTM1 and GSTP1 combined genotypes did not show an association with maternal smoking and iCL/P. No similar association between GSTM1/GSTP1 combined genotypes and maternal smoking was seen for iCP. DISCUSSION In this study population, we previously found that maternal cigarette smoking during early pregnancy was associated with modestly increased risks (OR ⫽ 1.7) for both iCL/P and iCP (Shaw et al., 1996). The objectives of the current analyses were to investigate whether risks associated with maternal smoking are modified by genetic variants of several enzymes known to be involved in biotransformation of tobacco toxins. Little evidence that these four polymorphisms modify the effects of maternal smoking on clefting risks was found. Cancer researchers have found suggestive gene-gene interactions between polymorphisms of GSTP1-codon 105 and GSTM1 null genotype on risk of lung cancer among smokers. We did not find that the combined GST genotypes appreciably altered risks for clefts beyond the increased risk described for infant GSTM1 null genotype and smoking during pregnancy, al-

though the sample size and thus power to detect such an interaction was modest (Lammer et al., 2005). The current study is among the first to examine GSTP1 and EPHX1 polymorphisms among children born with orofacial clefts. Strengths of this study include its large number of cases and controls, its population-based study design, the systematic collection of data for maternal and passive smoking exposures, and the highly efficient real-time PCR approach used for genotyping. A high percentage of eligible cases and controls was genotyped successfully, increasing the likelihood that this study population is representative of California births during this time interval. Despite the large size of this case-control study, some analyses were limited by the relatively low frequency of infant homozygosity for uncommon alleles EPHX1 and GSTP1, as well as by the rarity of the variant allele at GSTP1-codon 114. Another limitation of this study was the lack of information on maternal genotypes. Maternal metabolism clearly influences the delivery of toxins from tobacco to the developing embryo. Lacking information on maternal genotypes, we have an incomplete picture of the genetic components that contribute to detoxification during embryogenesis. If maternal metabolism is much more critical than fetal detoxification of tobacco toxins, then it will be more difficult to demonstrate that fetal ge-

372


notypes modify an association between smoking and oral cleft occurrence. Thus, it is possible that little evidence for interaction was observed, because maternal biotransformation is so much more important than embryo-fetal, at least regarding lip and palate closure. It will be important in future investigations to include genotyping of both maternal and infant subjects, when feasible. Another limitation, reporting bias, could arise if control mothers underreported their smoking. This seems unlikely, however, because the frequency of smoking among control mothers was comparable to that of previously published investigations and because recall of chronic exposures is thought to be substantially better than that of episodic exposure events during pregnancy. Self-reported cigarette use has been shown to be adequate compared with biochemical validation (Patrick et al., 1994). Without a plausible motive for case mothers to overreport smoking behavior, this also seems unlikely to have occurred. EPHX1, also known as microsomal epoxide hydrolase, hydrolyzes reactive epoxide intermediates (Armstrong, 1987; Fretland and Omiecinski, 2000). This reaction generally is regarded as detoxifying because its products are often less reactive and more easily excreted than the original epoxide intermediate. Depending on the parent compound, however, the reaction may lead to either detoxification or bioactivation, the latter resulting from further oxidation and the production of ‘‘bay-region’’ diol epoxides. These resulting epoxides are highly reactive and potentially more deleterious than the original intermediate (Oesch, 1980; Wormhoudt et al., 1999). EPHX1 plays an important role in the metabolism of PAHs, a group of compounds that are considered a major procarcinogenic element of tobacco smoke (Oesch, 1973). Variations in epoxide hydrolase activity may be an important mediator of teratogen levels following exposure to tobacco smoke or other environmental toxicants. The A allele of EPHX1-codon 139 and C allele of codon 113 are each associated with reduced EPHX1 activity, based on their metabolism of PAHs; thus, homozygotes for each polymorphism were treated as the reference genotype for our analyses. This study aimed to investigate two critical polymorphisms of EPHX1, because of the integral role this enzyme plays in metabolizing potentially carcinogenic components of tobacco smoke. GSTP1 is a phase II detoxification enzyme and its activity often is measured by its conjugation of benzo[a]pyrene diol epoxide through the formation of inert glutathione derivatives (Robertson et al., 1986). The proteins encoded by the G allele of the GSTP1-codon 105 polymorphism and the T allele of the codon 114 polymorphism each increases enzyme activity, which we hypothesized would decrease toxin levels reaching embryos; hence the choice of GG as the reference genotype for the GSTP1-codon 105 polymorphism in Table 3, even though G is the less common allele (Hu et al., 1997; Sundberg et al., 1998; Hu et al., 1999). GSTP1 appears to be the predominant GST present in the early human embryos and placenta, and this was a major reason for choosing to investigate this enzyme (Raijmakers et al., 2001). In conclusion, little evidence that several polymorphic var-

iants of EPHX1 and GSTP1 modify risks between maternal smoking during pregnancy and occurrence of orofacial clefts was found. Acknowledgments. Dr. Ramirez is a recipient of a Doris Duke Clinical Research Fellowship. The authors would like to thank Kenneth Beckman, Ph.D., for his invaluable guidance with the TaqMan assays. This research was supported by the Cigarette and Tobacco Surtax Fund of the California TobaccoRelated Diseases Research Program, University of California, grants 6RT-0360 and 13RT-0109. This research also was supported by funds from the National Institutes of Health, grants DE13613 and HD39084; and from the Centers for Disease Control and Prevention, Centers of Excellence Award U50/ CCU913241.

REFERENCES Ahmad H, Wilson DE, Fritz RR, Singh SV, Medh RD, Nagle GT, Awasthi YC, Kurosky A. Primary and secondary structural analyses of glutathione Stransferase pi from human placenta. Arch Biochem Biophys. 1990;278:398– 408. Ali-Osman F, Akande O, Antoun G, Mao JX, Buolamwini J. Molecular cloning, characterization, and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase pi gene variants. Evidence for differential catalytic activity of the encoded proteins. J Biol Chem. 1997;272: 10004–10012. Armstrong RN. Enzyme-catalyzed detoxication reactions: mechanisms and stereochemistry. CRC Crit Rev Biochem. 1987;22:39–88. Arnould JP, Verhoest P, Bach V, Libert JP, Belegaud J. Detection of benzo[a]pyrene-DNA adducts in human placenta and umbilical cord blood. Hum Exp Toxicol. 1997;16:716–721. Beaty TH, Wang H, Hetmanski JB, Fan YT, Zeiger JS, Liang KY, Chiu YF, Vanderkolk CA, Seifert KC, Wulfsberg EA, Raymond G, Panny SR, McIntosh I. A case-control study of nonsyndromic oral clefts in Maryland. Ann Epidemiol. 2001;11:434–442. Brunnemann KD, Hoffmann D. Analytical studies on tobacco-specific N-nitrosamines in tobacco and tobacco smoke. Crit Rev Toxicol. 1991;21:235–240. Fretland AJ, Omiecinski CJ. Epoxide hydrolases: biochemistry and molecular biology. Chem Biol Interact. 2000;129:41–59. Hansen C, Sorensen LD, Asmussen I, Autrup H. Transplacental exposure to tobacco smoke in human-adduct formation in placenta and umbilical cord blood vessels. Teratog Carcinog Mutagen. 1992;12:51–60. Hartsfield JK Jr, Hickman TA, Everett ET, Shaw GM, Lammer EJ, Finnell RH. Analysis of the EPHX1 113 polymorphism and GSTM1 homozygous null polymorphism and oral clefting associated with maternal smoking. Am J Med Genet. 2001;102:21–24. Hassett C, Aicher L, Sidhu JS, Omiecinski CJ. Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants. Hum Mol Genet. 1994;3:421–428. Hassett C, Lin J, Carty CL, Laurenzana EM, Omiecinski CJ. Human hepatic microsomal epoxide hydrolase: comparative analysis of polymorphic expression. Arch Biochem Biophys. 1997;337:275–283. Hayes JD, Pulford DJ. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol. 1995;30:445–600. Hosagrahara VP, Rettie AE, Hassett C, Omiecinski CJ. Functional analysis of human microsomal epoxide hydrolase genetic variants. Chem Biol Interact. 2004;150:149–159. Hu X, Herzog C, Zimniak P, Singh SV. Differential protection against benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced DNA damage in HepG2 cells stably transfected with allelic variants of pi class human glutathione S-transferase. Cancer Res. 1999;59:2358–2362. Hu X, O’Donnell R, Srivastava SK, Xia H, Zimniak P, Nanduri B, Bleicher RJ, Awasthi S, Awasthi YC, Ji X, Singh SV. Active site architecture of polymorphic forms of human glutathione S-transferase P1-1 accounts for their enantioselectivity and disparate activity in the glutathione conjugation


of 7beta,8alpha-dihydroxy-9alpha,10alpha-ox y-7,8,9,10-tetrahydrobenzo[a]pyrene. Biochem Biophys Res Commun. 1997;235:424–428. Iovannisci DM. Highly efficient recovery of DNA from dried blood using the MasterPure娂 Complete DNA and RNA Purification Kit. Epicentre Forum. 2000;7:6–8. Kelada SN, Stapleton PL, Farin FM, Bammler TK, Eaton DL, Smith-Weller T, Franklin GM, Swanson PD, Longstreth WT, Checkoway H. Glutathione Stransferase M1, T1, and P1 polymorphisms and Parkinson’s disease. Neurosci Lett. 2003;337:5–8. Lammer EJ, Shaw GM, Iovannisci DM, Finnell RH. Maternal smoking during pregnancy, genetic variation of glutathione-S-transferases, and risk for orofacial clefts. Epidemiology. 2005;16:698–701. Lieff S, Olshan AF, Werler M, Strauss RP, Smith J, Mitchell A. Maternal cigarette smoking during pregnancy and risk of oral clefts in newborns. Am J Epidemiol. 1999;150:683–694. Morisseau C, Hammock BD. Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu Rev Pharmacol Toxicol. 2005;45:311–333. Oesch F. Mammalian epoxide hydrases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica. 1973;3:305–340. Oesch F. Microsomal epoxide hydrolase. In: Jakoby WB, ed. Enzymatic Basis of Detoxication. Vol. 2. New York: Academic Press; 1980:278. Omiecinski CJ, Hassett C, Hosagrahara V. Epoxide hydrolase—polymorphism and role in toxicology. Toxicol Lett. 2000;112–113:365–370. Park JY, Schantz SP, Stern JC, Kaur T, Lazarus P. Association between glutathione S-transferase pi genetic polymorphisms and oral cancer risk. Pharmacogenetics. 1999;9:497–504. Patrick DL, Cheadle A, Thompson DC, Diehr P, Koepsell T, Kinne S. The validity of self-reported smoking: a review and meta-analysis. Am J Public Health. 1994;84:1086–1093. Raijmakers MT, Steegers EA, Peters WH. Glutathione S-transferases and thiol concentrations in embryonic and early fetal tissues. Hum Reprod. 2001;16: 2445–2450.

373

Robertson IG, Guthenberg C, Mannervik B, Jernstrom B. Differences in stereoselectivity and catalytic efficiency of three human glutathione transferases in the conjugation of glutathione with 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Cancer Res. 1986;46: 2220–2224. Schottenfeld D, Fraumeni JF. Cancer Epidemiology and Prevention. New York: Oxford University Press; 1996. Shaw GM, Wasserman CR, Lammer EJ, O’Malley CD, Murray JC, Basart AM, Tolarova MM. Orofacial clefts, parental cigarette smoking, and transforming growth factor—alpha gene variants. Am J Hum Genet. 1996;58:551–561. Sundberg K, Johansson AS, Stenberg G, Widersten M, Seidel A, Mannervik B, Jernstrom B. Differences in the catalytic efficiencies of allelic variants of glutathione transferase P1-1 towards carcinogenic diol epoxides of polycyclic aromatic hydrocarbons. Carcinogenesis. 1998;19:433–436. van Rooij IA, Wegerif MJ, Roelofs HM, Peters WH, Kuijpers-Jagtman AM, Zielhuis GA, Merkus HM, Steegers-Theunissen RP. Smoking, genetic polymorphisms in biotransformation enzymes, and nonsyndromic oral clefting: a gene-environment interaction. Epidemiology. 2001;12:502–507. Watson MA, Stewart RK, Smith GB, Massey TE, Bell DA. Human glutathione S-transferase P1 polymorphisms: relationship to lung tissue enzyme activity and population frequency distribution. Carcinogenesis. 1998;19:275–280. Wormhoudt LW, Commandeur JN, Vermeulen NP. Genetic polymorphisms of human N-acetyltransferase, cytochrome P450, glutathione-S-transferase, and epoxide hydrolase enzymes: relevance to xenobiotic metabolism and toxicity. Crit Rev Toxicol. 1999;29:59–124. Wyszynski DF, Wu T. Use of U.S. birth certificate data to estimate the risk of maternal cigarette smoking for oral clefting. Cleft Palate Craniofac J. 2002; 39:188–192. Zheng S, Ma X, Buffler PA, Smith MT, Wiencke JK. Whole genome amplification increases the efficiency and validity of buccal cell genotyping in pediatric populations. Cancer Epidemiol Biomarkers Prev. 2001;10:697– 700.