Maternal Smoking, Genetic Variation of Glutathione S-Transferases, and Risk for Orofacial Clefts

Abstract

Background: 

Maternal smoking is a known risk factor for orofacial clefts. We investigated whether risk is greater among offspring who lack the genetic capacity to produce glutathione S-transferase enzymes relevant to detoxification of chemicals in cigarette smoke.

Methods: 

Using a population-based case–control design, we genotyped 423 California infants with an isolated cleft and 294 nonmalformed controls for null variants of the glutathione S-transferases GSTT1 and GSTM1.

Results: 

If a mother smoked during pregnancy and her fetus was homozygous null for GSTT1, the risk of isolated cleft lip with or without cleft palate was tripled (odds ratio = 2.9; 95% confidence interval = 1.2–7.2). For fetuses who were homozygous null for GSTM1 and whose mothers smoked ≥20 cigarettes per day, we found nearly a 7-fold increased risk (6.8; 0.82–57). Combined absence of GSTM1 and GSTT1 enzymes among the offspring of smoking mothers was associated with a nearly 6-fold increased risk for cleft lip (6.3; 1.3–42). A similar increased risk for cleft palate was associated with absence of GSTM1, but not for absence of GSTT1.

Conclusions: 

Maternal smoking during pregnancy increases risks for clefts among fetuses lacking enzymes involved in the detoxification of tobacco-derived chemicals.

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Oral clefts have been associated with smoking during pregnancy.^1–11 The risk to an embryo is presumably related to number of cigarettes smoked, as well as maternoplacental transfer and detoxification. Glutathione S-transferases (GSTs) are important phase II detoxification enzymes, conjugating activated reactive intermediates to make them more water-soluble so that they can be excreted. GSTM1 detoxifies active metabolites of polycyclic aromatic hydrocarbons, whereas GSTT1 catalyzes the S-glutathionation of low-molecular-weight halogenated compounds and reactive epoxides.^12–14 GSTs are expressed in embryonic tissues, suggesting detoxification in utero is important.¹⁵ We hypothesized that a lack of GSTT1 or GSTM1 activity would decrease the biotransformation of teratogenic intermediates originating from maternal tobacco smoking during pregnancy, thus increasing infant susceptibility to orofacial clefts.

METHODS

This research was approved by the California State Committee for the Protection of Human Subjects and Children's Hospital and Research Center Oakland Institutional Review Boards.

Study Population

Details of the population-based case–control study used for these analyses have been described elsewhere.² Preliminary results of genotyping of GSTM1 from a subset of these cases were previously published.¹⁷ Case infants or fetuses with an isolated orofacial cleft (cleft lip with or without palate; or cleft palate) were ascertained by reviewing medical records at all hospitals and genetic centers in a known geographic population base. Eligible were infants (within 1 year of birth) and fetuses diagnosed with an orofacial cleft among the cohort of 552,601 births and fetal deaths that occurred between 1 January 1987 and 31 December 1989 to women residing in most counties in California (metropolitan areas of Los Angeles and San Francisco were excluded). Infants diagnosed with any chromosomal aneusomy were excluded (n = 81). Nonmalformed controls (n = 972), delivered of mothers residing in the same counties as cases, were electronically selected from California vital records using a pseudorandom number.

Questionnaire

We interviewed mothers of cases and controls in English (91%) or Spanish, nearly all by telephone. We excluded women who only spoke languages other than English or Spanish (26 cases and 33 controls) and 3 case mothers who died before interview contact, yielding 863 cases and 939 controls eligible. Interviews were completed an average of 3.5 years after delivery for cases and 3.6 years after delivery for controls. To assess active maternal smoking exposures, women were asked how many cigarettes they smoked daily for the 4-month period beginning 1 month before conception and including the 3 months after conception, as well as for each month during the period. To assess passive smoke exposures in the 4-month period, a woman was asked whether anyone smoked inside her home (including specific questions about paternal smoking), near her at work or school, or while she was commuting to work or school, and whether she regularly frequented (at least once a week) a place such as a restaurant or laundromat where others smokednearby.

Genotyping

A DNA specimen (residual dried blood spots from newborn screening) was identified for 83% of cases and 87% of controls. To minimize the number of samples to be genotyped, the 652 control samples were randomly reduced to 299. Genotyping methods for null polymorphisms of GSTM1 and GSTT1 were adapted from Arand et al¹⁸ and are available with the electronic version of this article.

Statistical Analyses

Odds ratios (ORs) and their 95% confidence intervals (CIs) were used to estimate risks.

RESULTS

Maternal and infant characteristics of cases (n = 437) and controls (n = 299) are available elsewhere.¹⁹ We genotyped 97% (n = 423) of cases and 98% (n = 294) of controls for GSTT1 and GSTM1 null polymorphisms. Among controls, 15% were homozygous null for GSTT1, whereas 50% were homozygous null for GSTM1. These frequencies were similar for each ethnic/racial stratum. Neither risk for cleft lip ± cleft palate nor for cleft palate was increased among infants homozygous null for GSTM1 or homozygous null for GSTT1 compared with reference genotypes (Table 1).

TABLE 1: Risks of GSTT1 and GSTM1 Genotypes Among Isolated Orofacial Cleft Cases and Effects of Maternal Smoking During Pregnancy

Twenty-five percent of control mothers and 35% of case mothers smoked tobacco in the periconceptional period. The overall risk with smoking was the same for cleft lip ± cleft palate (OR = 1.6; 95% CI = 1.1–2.4) and for cleft palate (1.6; 1.1–2.6) (additional raw data available with the electronic version of this article).

We investigated potential combined influences of infant GST null genotypes and maternal smoking during the periconceptional period.²⁰Table 1 shows a 2-fold higher risk for cleft lip ± cleft palate among smoking mothers whose infants lacked GSTT1 compared with the risk from smoking when the infant had at least one copy of GSTT1. Quantifying smoking as 1 to 19 cigarettes per day or ≥20 cigarettes per day, it was apparent that the increased risk from the fetal null genotype was particularly high (nearly 7-fold) among maternal smokers of ≥20 cigarettes per day, but this risk lacks precision. For isolated cleft palate and GSTT1 null, there was less difference in risks.

For GSTM1, Table 1 shows minimal differences in odds ratios between homozygous null infants and infants who had at least one copy of the gene for each of the cleft groups. Unlike GSTT1, there is little difference in risks for cleft lip ± cleft palate among smoking mothers whose infants lacked GSTM1 compared with the risk from any amount of smoking when the infant had at least one copy of GSTM1. However, mothers who smoked ≥20 cigarettes per day had a nearly 6-fold higher risk for isolated cleft lip ± cleft palate when the fetus was homozygous null for GSTM1. Similarly, the risk for isolated cleft palate was 3 to 4 times higher when the fetus was homozygous null for GSTM1, but these odds ratios were less precise.

Among controls, 7.8% were combined homozygous null for both GSTT1 and GSTM1 compared with 9.1% of cleft lip ± cleft palate and 6.4% of cleft palate cases. Table 1 shows a 4-fold higher risk for cleft lip ± cleft palate for infants who were homozygous null for both GSTs, whereas data for isolated cleft palate were too sparse to draw interpretations. When we investigated the risks associated with absence of either GSTT1 or GSTM1, the results were very similar to those shown in Table 1 (results not shown).

We also analyzed passive smoking exposures, limited to those mothers who did not smoke during the periconceptional period. ORs for cleft lip ± cleft palate from any passive smoke exposure (home or workplace) were 1.5 (95% CI = 0.80–2.9) for infants who were homozygous GSTT1 null compared with 1.3 (0.84–2.0) for infants who had at least one GSTT1 gene. For cleft palate and passive smoking exposure, the results were similar: OR = 1.6 (0.67–3.9) forinfants who were homozygous GSTT1 null and OR = 1.7(0.92–3.0) for infants who had one or more copies of GSTT1. For GSTM1 genotypes, we observed no difference in risks for passive smoking; infants with cleft lip and homozygous null for GSTM1 had an OR = 1.3 (CI 0.77–2.3), whereas those with at least one copy of GSTM1 had an OR = 1.4 (0.83–2.5). For cleft palate and GSTM1 genotypes, we observed a slightly lower risk for passive smoking exposure: infants homozygous null for GSTM1 genotype had an OR = 1.6 (0.71–3.8), whereas those with at least one copy of GSTM1 had an OR = 2.8 (1.2–6.3).

DISCUSSION

We previously reported that maternal cigarette smoking during early pregnancy increased risks for both isolated cleft lip ± cleft palate and isolated cleft palate.² The increased risks were relatively modest (OR = 1.7) and were higher for mothers who smoked ≥20 cigarettes per day. The present study was designed to determine whether null polymorphisms of 2 glutathione S-transferases made the fetus more susceptible to the effects of smoking. Neither GST null polymorphism was an independent risk factor for isolated oral clefts. When we assessed potential interactions between the known smoking risks and GSTT1 and GSTM1 null polymorphisms, however, we found higher risks associated with absence of one or both of these xenobiotic metabolizing enzymes among infants born to smoking mothers. Among infants born to smoking mothers, the risk for cleft lip was doubled for fetuses who were homozygous null for GSTT1 or GSTM1 compared with fetuses who had a least one functional copy of a GST gene. In addition, our findings suggest that for the heaviest smokers, the magnitude of increased risk was nearly 6- and 7-fold for GSTT1 null and GSTM1 null genotypes, respectively—substantially higher than the 1.7-fold risks with smoking in the absence of any genotypic information. Among nonsmoking mothers, we saw no modification of risk for passive exposure to smoking and GST null polymorphisms.

Our findings confirm and extend suggestive results of a smaller Dutch study of 100 nonsyndromic orofacial cleft cases. Van Rooij and colleagues¹⁶ reported a 5-fold increased risk for all clefts combined (OR = 4.9; 95% CI = 0.7–37) among smoking mothers when both mother and infant were GSTT1 null. The frequency of homozygous GSTT1 null genotype among these Dutch mothers (26%) and infants (37%) was higher than the 15% that we observed. The frequencies of GSTT1 null genotypes in U.S. studies range from 15% to 27% for whites, 22% to 29% for blacks, and 10% to 12% for Hispanics.²¹ In the United States, the frequency of GSTM1 null genotype is approximately 51% among western European descendants, 46% among Hispanics, 59% among Asians, and 29% among blacks.²²

Maternal metabolism of toxins in tobacco smoke clearly influences fetal exposures, and without the maternal genotypic information, we have an incomplete picture of all of the components of detoxification in utero. Because embryonic and maternal genotypes are correlated, it is possible that part of the effect we detected may result from the maternal genotype.

ACKNOWLEDGMENTS

We are indebted to George Cunningham and Fred Lorey (Genetic Diseases Branch) for making it possible to access archived bloodspot specimens. We thank Eric Neri and Wei Yang for SAS programming efforts, and Poulina Uddin and Nam Do for laboratory contributions.