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Oral clefts, smoking & variation toxin biotransformation

Institution: Children's Hospital Oakland Research Institute
Investigator(s): Edward Lammer, M.D.
Award Cycle: 1997 (Cycle 6) Grant #: 6RT-0360 Award: $677,128
Subject Area: Epidemiology
Award Type: Research Project Awards

Initial Award Abstract
Smoking during pregnancy has been identified as a risk factor for orofacial clefts (cleft lip or cleft palate) in several studies, but not in others. Recently, we completed a large research study of parental smoking during pregnancy and risk for birth defects, supported by two TRDRP grants. California infants who participated were born during 1987-1989 and had either an oro-facial cleft or one of three other selected major birth defects (spina bifida, a heart malformation, or absence of all, or some part, of a limb). We found that about 24% of pregnant California women smoke during pregnancy--this makes smoking one of the most common environmental exposures during pregnancy, so that even small risks may have major implications because so many pregnancies would be at risk. The 24% percent figure is comparable to the frequency of smoking during pregnancy reported from other parts of the U.S. We found that if mothers smoked during early pregnancy, they doubled their risk of delivering an infant with a cleft malformation. The more cigarettes that were smoked per day, the higher risk that the baby would be born with a cleft. The highest risks associated with parental smoking, however, occurred among those infants who inherited an uncommon form of an embryonic facial growth factor, transforming growth factor- (TGF). Only about 5% of the general population has this particular uncommon form of TGF. Other researchers had noticed that a higher percentage (15%) of children who had a cleft had inherited this form of TGF. Our results suggest that the reason why this uncommon form of TFG is seen more frequently among infants with clefts is because the combination of having a mother who smokes during pregnancy, and the baby having the uncommon form of TGF, makes the chance that the baby develops a cleft much higher.

The purpose of the proposed research is to look for other genetic factors that may influence a fetus’ susceptibility to risk of a smoking-induced cleft. We propose to study the influence of genetic variation of six different enzymes that detoxify chemicals in tobacco smoke on the risk for orofacial clefts when mothers smoke during pregnancy. Our hypothesis is that if a fetus inherits an unusual form of an enzyme that detoxifies chemicals unusually slowly, he/she may be at higher risk to develop a birth defect because smoke-related toxins will remain in its blood and tissues longer, and at a higher concentration than in a fetus who inherits the normal form of the enzyme.

Each enzyme that we plan to assess has several genetic forms, and for each enzyme, at least one of these genetic forms has been associated with higher risks for smoking-induced cancers compared to people who smoke and who have the common forms of the enzymes. The proposed research program makes use of smoking data and DNA samples from our completed, TRDRP-supported research on orofacial clefts, and support is sought only for adding analyses for the six genes that encode the detoxifying enzymes. The proposed research represents a unique opportunity to investigate the interplay between parental tobacco smoking during pregnancy and fetal variation of several enzymes that detoxify chemicals in tobacco smoke, and how this interaction determines the risk that the fetus will develop an orofacial cleft malformation.

Final Report
Several studies have identified maternal tobacco smoking during pregnancy as a risk factor for orofacial clefts, but this finding has been inconsistent. Supported by TRDRP grants 1RT466 and 3RT-0413, we completed a large case-control study of California infants with orofacial clefts, with a focus on risks from both maternal and paternal smoking. We found two-fold increased risks for each major subgroup of clefts, including isolated cleft palate and isolated cleft lip  cleft palate, when mothers smoked during early pregnancy. Risks were dose related; that is, risks were higher for infants whose mothers smoked 20 cigarettes/day. The aims of the ongoing research are to investigate whether the fetus’s ability to metabolize foreign chemical affects risk for clefts when a mother smokes during pregnancy. To do this, we plan to analyze the influence of genetic variation of 6 metabolizing enzyme (proteins involved in biotransforming toxins so that they can be excreted from the body), upon the risk for orofacial clefts when mothers smoke during pregnancy. Our hypothesis is that smoke-related chemicals may be developmental toxicants for embryos that have unusually low, or unusually high, enzyme activity for one or more of this group of metabolizing enzymes.

While smoking was independently associated with risk of both cleft lip ± cleft palate and cleft palate alone, no such independent associations were found for N-acetyl transferase 1 (NAT1) 1088 or 1095 genotypes, nor for NAT2 acetylator status. Both infant NAT1 1088 and 1095 polymorphisms strongly increased the association of smoking with risk of cleft. This increased risk was largely contributed by women who smoked less than a pack of cigarettes/day (odds ratio [95% confidence interval] among infants with NAT1 1088 genotype A/A vs. T/T, 4.4 [1.1-25.2] and with NAT1 1095 genotype A/A vs. C/C, 6.4 [1.3-61]. The increased risk for orofacial clefts among mothers who smoked a pack or more/day was unaltered by infant NAT1 polymorphisms. Infant NAT2 acetylator status did not appreciably alter risk for either type of cleft associated with maternal smoking. These results suggest that maternal smoking during pregnancy increases risk for orofacial clefts among the greatest smokers, and among lesser smokers whose fetuses have a variant for an enzyme that detoxifies aromatic amines.

Null genotypes for glutathione-S-transferase M1 (GSTM1) and T1 (GSTT1) were not independent risk factors for orofacial clefts. Compared to non-smoking mothers whose infants had at least one functional copy of the GSTM1 gene, however, we found an increased risk (odds ratio = 1.6) for an oral cleft among smoking mothers whose infants were homozygous null for GSTM1. For GSTT1 homozygous null genotype, we found an even higher risk. Smoking mothers whose infants were homozygous null for GSTT1 had an odds ratio of 2.9 for oral cleft compared to the same reference group.

We found significant interactions between maternal smoking during pregnancy and genetic variation of two enzymatic pathways that probably lessen the developing embryo’s ability to detoxify chemicals found in tobacco smoke. Our results represent one of the first examples of a gene-environment interaction as the cause of a common structural malformation in humans. This may help to better understand why smoking during pregnancy imparts adverse effects on some babies, but not others. Our observations may lead to an improved understanding of why some people are more susceptible to other smoking-induced diseases as well.

Infant TGF-alpha genotype, orofacial clefts, and maternal periconceptional multivitamin use
Periodical: Cleft Palate-Craniofacial Journal Index Medicus:
Authors: Shaw GM, Wasserman CR, Murray JC, Lammer EJ ART
Yr: 1998 Vol: 35 Nbr: Abs: Pg: 366-370

Infant C677T mutation in MTHFR, maternal periconceptional vitamin use, and cleft lip
Periodical: American Journal of Medical Genetics Index Medicus:
Authors: Shaw GM, Rozen R, Finnell RH, Todoroff K, Lammer EJ ART
Yr: 1998 Vol: 80 Nbr: Abs: Pg: 196-198

Neural tube and craniofacial defects with special emphasis on folate pathway genes
Periodical: Critical Reviews in Oral Biology and Medicine Index Medicus:
Authors: Finnell RH, Greer KA, Barber RC, Piedrahita JA, Shaw GM, Lammer EJ ART
Yr: 1998 Vol: 9 Nbr: Abs: Pg: 38-53

Maternal periconceptional alcohol consumption and risk for orofacial clefts
Periodical: Journal of Pediatrics Index Medicus:
Authors: Shaw GM, Lammer EJ ART
Yr: 1999 Vol: 134 Nbr: Abs: Pg: 298-303

Maternal vitamin use, infant C677T mutation in MTHFR, and isolated cleft palate risk
Periodical: American Journal of Medical Genetics Index Medicus:
Authors: Shaw GM, Todoroff K, Finnell RH, Rozen R, Lammer EJ ART
Yr: 1999 Vol: 85 Nbr: Abs: Pg: 84-85