Endothelial Nitric Oxide Synthase (NOS3) Genetic Variants, Maternal Smoking, Vitamin Use, and Risk of Human Orofacial Clefts
Gary M. Shaw1,
David M. Iovannisci2,
Wei Yang1,
Richard H. Finnell3,
Suzan L. Carmichael1,
Suzanne Cheng4 and
Edward J. Lammer2
1 California Birth Defects Monitoring Program, March of Dimes Birth Defects Foundation, Berkeley, CA
2 Children's Hospital Oakland Research Institute, Oakland, CA
3 Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX
4 Department of Human Genetics, Roche Molecular Systems, Inc., Alameda, CA
Reprint requests to Dr. Gary M. Shaw, California Birth Defects Monitoring Program, 1917 Fifth Street, Berkeley, CA 94710 (e-mail: gsh{at}cbdmp.org).
Received for publication March 15, 2005.
Accepted for publication July 7, 2005.
 |
ABSTRACT
|
---|
Orofacial clefts have been associated with maternal cigarette smoking and lack of folic acid supplementation (which results in higher plasma homocysteine concentrations). Because endothelial nitric oxide synthase (NOS3) activity influences homocysteine concentration and because smoking compromises NOS3 activity, genetic variation in NOS3 might interact with smoking and folic acid use in clefting risk. The authors genotyped 244 infants with isolated cleft lip with or without cleft palate (CL/P), 99 with isolated cleft palate, and 588 controls from a California population-based case-control study (19871989 birth cohort) for two NOS3 polymorphisms: A(922)G and G894T. Analyses of gene-only effects for each polymorphism revealed a 60% increased risk of CL/P among NOS3 A(922)G homozygotes (odds ratio (OR) = 1.6, 95% confidence interval (CI): 1.0, 2.6). There was some evidence for higher risk of CL/P with maternal periconceptional smoking in infants with an NOS3 922G allele (for homozygotes, OR = 2.5, 95% CI: 1.2, 5.6) but not in those with an 894T allele. For CL/P risk, odds ratios were over 4 among mothers who smoked, who did not use vitamins, and whose infants had at least one variant allele for each NOS3 polymorphism (for A(922)G, OR = 4.6, 95% CI: 2.1, 10.2; for 894T, OR = 4.4, 95% CI: 1.8, 10.7). No similar patterns were observed for risk of cleft palate.
abnormalities; cleft lip; cleft palate; genetics; nitric-oxide synthase; risk factors; smoking; vitamins
Abbreviations:
NCBI, National Center for Biotechnology Information; NOS3, endothelial nitric oxide synthase; SNP, single nucleotide polymorphism
 |
INTRODUCTION
|
---|
Orofacial clefts are suspected of being etiologically heterogeneous (1
4
). A certain number of orofacial clefts occur as part of recognizable patterns of malformation or have genetic etiologies (5
7
). Epidemiologic studies indicate that increased risks of clefting may be associated with prenatal exposures, such as exposure to cigarette smoke (8
12
), anticonvulsants (13
, 14
), retinoic acid (15
), alcohol (16
18
), agricultural pesticides (19
), or organic solvents (20
, 21
), and with lack of folic acid supplementation during pregnancy (8
, 22
29
).
A promising approach to identifying etiologies of orofacial clefts is exploration of possible gene-environment interactions. Such an approach has been used to explore relations between maternal smoking and gene variants (9
, 12
, 29
35
) and between maternal vitamin use and gene variants (34
, 36
42
). However, we are not aware of any study that has investigated the potential multiple interactions of maternal smoking, maternal vitamin use, and gene variants, which was the central approach used in the current study.
Our study focused on endothelial nitric oxide synthase (NOS3), which regulates nitric oxide production and is expressed in human endothelial cells (43
) and mouse embryos (44
). Brown et al. (45
) demonstrated that the NOS3 G894T single nucleotide polymorphism (SNP) was associated with homocysteine concentrations. Some of their study groups comprising persons with the TT genotype showed more than double the homocysteine concentrations of subgroups comprising persons with the GG genotype. These investigators proposed that nitric oxide modulated homocysteine levels via an effect on folate catabolism. It has also been demonstrated that cigarette smoking compromises NOS3 activity (46
). Because risk of clefting has been associated with maternal cigarette smoking and lack of folic acid supplementation (which results in higher plasma homocysteine concentrations), we reasoned that genetic variation in NOS3 might interact with these two exposures. Here we investigated a potential association between clefting risks and NOS3 gene variants and whether the association was modified by maternal cigarette smoking and intake of folic acid supplements during the periconceptional period.
 |
MATERIALS AND METHODS
|
---|
Details on this case-control study have been provided previously (9
, 23
). Included as cases were infants and fetal deaths (
20 weeks' gestation) diagnosed with orofacial clefts within 1 year after delivery among women residing in most California counties. All infants or fetal deaths with delivery occurring between January 1987 and December 1989 (among 552,601 total infants or fetal deaths) were eligible. Case eligibility was determined by one clinical geneticist (E. J. L.) who reviewed detailed diagnostic information from medical records of all hospitals and genetics centers in the surveillance area. Orofacial cleft cases were defined as infants or fetuses born with cleft palate or with cleft lip with or without cleft palate (hereafter called cleft lip/palate) that was confirmed by clinical description, surgical report, or autopsy report. This distinction in phenotype is consistent with embryologic underpinnings (1
). Cases were further classified on the basis of the nature of accompanying congenital anomalies. Cases with no other major anomaly or with anomalies considered minor were classified as isolated. Cases with at least one accompanying major anomaly were classified as multiple. Only isolated cases of cleft palate and cleft lip/palate were considered in these analyses. Infants diagnosed with single gene disorders, trisomies, or Turner's syndrome (45
,X) were excluded.
As controls, 972 infants were randomly selected from all infants born alive in the same geographic area and time period as the cases. Control infants had no major congenital anomalies identified before their first birthday, as defined by the California Birth Defects Monitoring Program (47
).
Telephone interviews were completed with 489 mothers of isolated orofacial cleft cases (85 percent of those eligible) and 734 control mothers (76 percent). Interviews were completed within an average of 3.7 years from the date of delivery for cases and within 3.8 years for controls. Interviews elicited maternal information on medical and reproductive histories and activities associated with various lifestyles. The interviewer assisted each woman in establishing a 4-month periconceptional period, ranging from 1 month before conception to 3 months after conception, that was referred to throughout the interview to elicit information. Women were asked whether they had used vitamin and mineral supplements during this period and which supplements (types or brands) they had used in each month. We divided women into two categories relative to their use of vitamins containing folic acid: 1) "use" was defined as starting vitamin use anytime during the period ranging from 1 month before conception through the end of the third month after conception and 2) "nonuse" was defined as starting vitamin use after the third month from conception (postdating the relevant embryologic timing of the studied phenotypes) or absence of use during pregnancy. For assessment of active maternal smoking exposures, women were asked how many cigarettes they had smoked daily during the 4-month periconceptional period (1 month before conception through the first trimester) and in each month during that period.
Our analyses were restricted to 1) cases and controls whose mothers were interviewed and 2) liveborn case and control infants, because the source of DNA was residual newborn screening blood specimens (filter paper). For the 489 infants with isolated cleft lip/palate or isolated cleft palate, 343 (244 with cleft lip/palate and 99 with cleft palate) had DNA available and were genotyped. Among the 652 control infants for whom DNA was available, 588 were genotyped. All interviews and samples were obtained with approval from the State of California Health and Welfare Agency Committee for the Protection of Human Subjects.
In addition to the G894T SNP, we were able to explore two other NOS3 SNPs that were available to us on a panel containing multiple known SNPs. Thus, case and control infants were genotyped for three NOS3 SNPs: A(922)G (rs1800779), C(690)T (rs3918226), and G894T (E298D) (rs1799983).
Genotyping was accomplished in a manner similar to that of Cheng et al. (48
) using a multilocus sequence-specific hybridization assay developed by Roche Molecular Systems, Inc. (Alameda, California). Briefly, multiplex polymerase chain reaction with a blend of biotinylated primer pairs is used to amplify each polymorphic site. Biotin-tagged amplification products are hybridized to a linear array of immobilized oligonucleotide probes specific for each allele under stringent conditions. Chromogenic reagents are used to visualize the biotin-tagged amplicons that remain hybridized.
After color development, arrays were manually scored and genotypes were interpreted by two observers. When the observers' interpretations were discrepant, the samples were reassayed. All genotyping was performed blinded to the subjects' case/control status. Genotypic frequencies for each NOS3 SNP were evaluated for Hardy-Weinberg equilibrium among the controls, both overall and in each of the three racial/ethnic groups studied (non-Hispanic White, Hispanic, or other). Each SNP showed distributions consistent with Hardy-Weinberg expectations. The frequency of homozygosity for the C(690)T SNP was very lowone infant with cleft lip/palate and three control infants. Thus, we did not include this SNP in further analyses. The observed allele frequencies associated with the other two SNPs were consistent with allele frequencies reported in the National Center for Biotechnology Information (NCBI) database (www.ncbi.nlm.nih.gov). That is, for G894T, we observed the frequency of the T allele to be 0.26 among controls as compared with 0.24 in the NCBI database. For the A(922)G SNP, we observed the frequency of the G allele to be 0.31 as compared with 0.30 in the NCBI database.
Logistic regression was used to estimate odds ratios and 95 percent confidence intervals for each NOS3 genotype and for each cleft phenotype (cleft lip/palate or cleft palate). Infants with homozygous variant genotypes or heterozygous variant genotypes were compared with infants with homozygous wild-type genotypes for estimation of gene-only effects and gene-smoking effects. To investigate gene-smoking-vitamin combination effects, we used a dominant genetic model for analyses. That is, infants whose genotypes were either homozygous variant or heterozygous were combined and compared with infants whose genotypes were wild-type. In specified analyses, risk estimates were adjusted for maternal race/ethnicity (non-Hispanic White, Hispanic White, or other). Frequencies of the covariates maternal smoking, maternal vitamin use, and maternal race/ethnicity are displayed in table 1.
View this table:
[in this window]
[in a new window]
|
TABLE 1. Characteristics of case and control mothers in a study of risk factors for isolated orofacial clefts, California, 19871989
|
|
 |
RESULTS
|
---|
Analyses investigating gene-only effects of each NOS3 SNP revealed a 60 percent increased risk of cleft lip/palate among A(922)G homozygotes (table 2). Risks of this magnitude were not observed for cleft palate (table 2). We also performed analyses comparing persons who were homozygous variant for either NOS3 SNP with persons who were homozygous wild-type for both NOS3 SNPs. These analyses yielded odds ratios of 1.2 (95 percent confidence interval: 0.8, 1.9) for cleft lip/palate and 0.5 (95 percent confidence interval: 0.2, 1.1) for cleft palate.
View this table:
[in this window]
[in a new window]
|
TABLE 2. Risks of isolated orofacial clefts associated with single nucleotide polymorphisms in the endothelial nitric oxide synthase (NOS3) gene among California infants relative to nonmalformed population-based controls, 19871989
|
|
We investigated potential modification of risk between NOS3 SNPs and maternal cigarette smoking. Results (table 3) showed some evidence for higher risk of cleft lip/palate in infants whose mothers smoked cigarettes periconceptionally and who had the A(922)G SNP but not in those who had the G894T SNP. However, these results did not provide statistical evidence for heterogeneity; that is, p values associated with the interaction model term for gene variant x maternal smoking were 0.3 for A(922)G and 0.8 for G894T. We did not observe such gene-smoking effects on risk of cleft palate (table 4). We extended these analyses by comparing persons who were homozygous variant for either NOS3 SNP with those who were homozygous wild-type for both NOS3 SNPs in combination with maternal smoking. These analyses did not produce results markedly different from those displayed in tables 3 and 4 (data not shown).
View this table:
[in this window]
[in a new window]
|
TABLE 3. Risks of isolated cleft lip with or without cleft palate associated with single nucleotide polymorphisms in the endothelial nitric oxide synthase (NOS3) gene in combination with maternal cigarette smoking* among California infants relative to nonmalformed population-based controls, 19871989
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 4. Risks of isolated cleft palate associated with single nucleotide polymorphisms in the endothelial nitric oxide synthase (NOS3) gene in combination with maternal cigarette smoking* among California infants relative to nonmalformed population-based controls, 19871989
|
|
Gene-smoking effects on risks of cleft lip/palate and cleft palate were further explored in combination with maternal periconceptional vitamin use. For these analyses, we used a dominant genetic model; that is, infants whose genotypes were either homozygous variant or heterozygous were combined and compared with infants whose genotypes were wild-type. These combinations were made because of small numbers of available cases or controls for some comparisons and because of the direction of the results revealed in tables 3 and 4. The results (shown in table 5) indicate higher risks (odds ratios >4) of cleft lip/palate in infants whose mothers smoked cigarettes, whose mothers did not use vitamins periconceptionally, and who had at least one variant allele for either of the two NOS3 SNPs. These results, however, did not provide statistical evidence for heterogeneity; that is, p values associated with the interaction model term for gene variant x maternal smoking x maternal vitamin use were 0.2 for A(922)G and 0.5 for G894T. No such consistent risk pattern was observed for risk of cleft palate (table 6). The estimated risks displayed in tables 5 and 6 were not substantially different after results were adjusted for the potentially confounding effects of maternal race/ethnic background (data not shown).
View this table:
[in this window]
[in a new window]
|
TABLE 5. Risks of isolated cleft lip with or without cleft palate associated with single nucleotide polymorphisms in the endothelial nitric oxide synthase (NOS3) gene in combination with maternal cigarette smoking* and vitamin use among California infants relative to nonmalformed population-based controls, 19871989
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 6. Risks of isolated cleft palate associated with single nucleotide polymorphisms in the endothelial nitric oxide synthase (NOS3) gene in combination with maternal cigarette smoking* and vitamin use among California infants relative to nonmalformed population-based controls, 19871989
|
|
We explored whether the A(922)G and G894T SNPs were in linkage disequilibrium. We observed modest evidence for linkage disequilibrium (D' = 0.47, p < 104). Because the similar findings observed for each SNP could have been a function of an overlapping haplotype, we estimated risks associated with each combination of A(922)G and G894T genotypes, relative to infants with wild-type genotypes for both. The observed results did not provide results substantially different from those observed for the two SNPs analyzed separately (data not shown).
 |
DISCUSSION
|
---|
We hypothesized that a potential association between clefting risks and NOS3 gene variants could be modified by maternal cigarette smoking and vitamin supplement intake during the periconceptional period. Our population-based study of California infants revealed sizable increased risks of cleft lip/palate from the combination between a NOS3 variant SNP and maternal smoking. This risk was further modified by lack of maternal vitamin use. No such pattern, however, was consistently observed for risk of cleft palate.
Several studies have identified an association between maternal smoking during the periconceptional period and delivery of infants with orofacial clefts (8
12
). There are several lines of evidence suggesting that folate-homocysteine metabolism is implicated in the risk of orofacial clefts. These include 1) reduced risks of orofacial clefts in infants whose mothers took folic acid vitamin supplements periconceptionally (8
, 22
29
) and 2) higher postnatal plasma homocysteine concentrations in mothers who deliver infants with clefts than in mothers who deliver nonmalformed infants (49
). However, the underlying explanations for the associations between smoking and clefting and between folate-homocysteine metabolism and clefting are unknown. Lack of knowledge about these associations, coupled with recent information about NOS3 being involved in folate-homocysteine metabolism and NOS3 activity being compromised by smoking, motivated us to explore the combined effects of these exposures and gene variants. Moreover, Plachta et al. (50
) recently observed that different levels of endogenous nitric oxide in different time periods influenced the balance between cell cycle progression and programmed cell death in the developing neural plate of chick embryoscells that contribute to facial development.
Thus, a common pathogenetic theme that can be hypothesized from the observed results and the available literature points toward elevated homocysteine concentrations. Evidence exists that the NOS3 894TT genotype is associated with elevated serum homocysteine levels in comparison with the GG genotype (45
). Lower folate intake is associated with elevated plasma homocysteine (51
), as is smoking (52
). Our observation of the largest risks for clefting among infants whose mothers smoked, whose mothers did not take vitamin supplements, and who carried genetic variation in NOS3 is consistent with this pathogenetic theme. Nevertheless, NOS3 participates in other biologic pathways, including control of vascular tone (53
), and in pregnant rats, inhibition of nitric oxide results in hypertension and fetal growth retardation (54
). The functional significance of these other pathways for our observed results is unknown.
Some additional evidence in support of our findings associating NOS3 variation and risk of birth defects can be found in a recent study by Brown et al. (55
). These investigators observed a modestly elevated risk of spina bifida, another neural-crest-cell anomaly, among infants who were heterozygous for the NOS3 G894T SNP (55
).
To our knowledge, our study is the first to report this association. Therefore, these analyses need to be replicated before a stronger inference can be drawn. Our results should be considered relative to some limitations as well. First, the functional significance of both NOS3 SNPs studied here has not been fully established. Second, there may be unaccounted genetic diversity that extends beyond the three NOS3 SNPs included in this study; that is, the observations we made could reflect associations with an unmeasured genetic marker that is in linkage disequilibrium with the studied SNPs. Third, many of the associations identified in this study were statistically imprecise. Moreover, because so many analytic comparisons were made, some might be expected to have revealed associations by chance alone.
This study had several strengths. It was relatively large and was among the first to investigate the effects of "gene-environment-environment" interactions on risk of birth defects. Furthermore, case infants with orofacial clefts were identified using a population-based registry system with systematic review of case eligibility. Control infants were randomly identified from birth files and therefore provided a population-based sample of livebirths from the same study base as the case infants. The analyses adequately accounted for the potentially confounding influence of maternal race/ethnicity. Lastly, this study revealed some important clues for further investigation that may help fill the data gap about the underlying process by which folic acid facilitates a reduced risk of human birth defects.
 |
ACKNOWLEDGMENTS
|
---|
This work was partially supported by the National Institutes of Health (grant DE12898); the Centers for Disease Control and Prevention (Centers of Excellence Award U50/CCU913241); the California Tobacco-Related Diseases Research Program (grants 6RT0360, 1RT466, and 3RT0413); and the Environmental Protection Agency (Science to Achieve Results (STAR) Program grant 82829201).
The authors are grateful to Drs. G. Cunningham and F. Lorey for making newborn specimens available for genotyping experiments; Dr. L. Mitchell for comments on earlier versions of the manuscript; A. Silbergleit for work on developing the genotyping assays; and E. Lloyd, B. Cohen, and C. Haun for laboratory technical assistance.
Although this research was partially funded by the Environmental Protection Agency (EPA), it was not subjected to any EPA review and therefore does not necessarily reflect the views of the EPA. No official endorsement should be inferred.
Dr. Suzanne Cheng is employed by Roche Molecular Systems, Inc. (Alameda, California), which provided noncommercial genotyping reagents for this study under a research collaboration.
 |
References
|
---|
- Fogh-Anderson P. Genetic and non-genetic factors in the etiology of facial clefts. Scand J Plastic Reconstr Surg 1967;1:229.
- Prescott NJ, Winter RM, Malcolm S. Nonsyndromic cleft lip and palate: complex genetics and environmental effects. Ann Hum Genet 2001;65:50515.[CrossRef][ISI][Medline]
- Murray JC. Face facts: genes, environment, and clefts. Am J Hum Genet 1995;57:22732.[ISI][Medline]
- Murray JC. Gene/environment causes of cleft lip and/or palate. Clin Genet 2002;61:24856.[CrossRef][ISI][Medline]
- Gorlin RJ, Cohen MM, Levin LS. Syndromes of the head and neck. 3rd ed. New York, NY: Oxford University Press, 1990.
- Spritz RA. The genetics and epigenetics of orofacial clefts. Curr Opin Pediatr 2001;13:55660.[CrossRef][ISI][Medline]
- Shaw GM, Carmichael SL, Yang W, et al. Congenital malformations in births with orofacial clefts among 3.6 million California births, 19831997. Am J Med Genet 2004;125:2506.
- Khoury MJ, Gomez-Farias M, Mulinare J. Does maternal cigarette smoking during pregnancy cause cleft lip and palate in offspring? Am J Dis Child 1989;143:3337.[ISI][Medline]
- Shaw GM, Wasserman CR, Lammer EJ, et al. Orofacial clefts, parental cigarette smoking, and transforming growth factor-alpha gene variants. Am J Hum Genet 1996;58:55161.[ISI][Medline]
- Wyszynski DF, Duffy DL, Beaty TH. Maternal cigarette smoking and oral clefts: a meta-analysis. Cleft Palate Craniofac J 1997;34:20610.[CrossRef][ISI][Medline]
- Lieff S, Olshan AF, Werler M, et al. Maternal cigarette smoking during pregnancy and risk of oral clefts in newborns. Am J Epidemiol 1999;150:68394.[Abstract]
- Christensen K, Olsen J, Norgaard-Pedersen B, et al. Oral clefts, transforming growth factor alpha gene variants, and maternal smoking: a population-based case-control study in Denmark, 19911994. Am J Epidemiol 1999;149:24855.[Abstract]
- Speidel BD, Meadow SR. Maternal epilepsy and abnormalities of the foetus and newborn. Lancet 1972;2:83943.[ISI][Medline]
- Shaw GM, Wasserman CR, O'Malley CD, et al. Orofacial clefts and maternal anticonvulsant use. Reprod Toxicol 1995;85:71013.
- Lammer EJ, Chen DT, Hoar RM, et al. Retinoic acid embryopathy. N Engl J Med 1985;313:83741.[Abstract]
- Clarren SK, Smith DW. The fetal alcohol syndrome. N Engl J Med 1978;298:10637.[ISI][Medline]
- Laumon B, Martin JL, Bertucat I, et al. Exposure to organic solvents during pregnancy and oral clefts: a case-control study. Repro Toxicol 1996;10:1519.[CrossRef][ISI][Medline]
- Shaw GM, Lammer EJ. Maternal periconceptional alcohol consumption and risk for orofacial clefts. J Pediatr 1999;134:298303.[CrossRef][ISI][Medline]
- Nurminen T, Rantala K, Kurppa K, et al. Agricultural work during pregnancy and selected structural malformations in Finland. Epidemiology 1995;6:2330.[ISI][Medline]
- Holmberg PC, Hernberg S, Kurppa K, et al. Oral clefts and organic solvent exposure during pregnancy. Int Arch Occup Environ Health 1982;50:3716.[CrossRef][ISI][Medline]
- Cordier S, Bergeret A, Goujard J, et al. Congenital malformations and maternal occupational exposure to glycol ethers. Epidemiology 1997;8:35563.[CrossRef][ISI][Medline]
- Tolarova M. Periconceptional supplementation with vitamins and folic acid to prevent recurrence of cleft lip. (Letter). Lancet 1982;2:217.
- Shaw GM, Lammer EJ, Wasserman CR, et al. Risks of orofacial clefts in children born to women using multivitamins containing folic acid periconceptionally. Lancet 1995;345:3936.
- Itikala PR, Watkins ML, Mulinare J, et al. Maternal multivitamin use and orofacial clefts in offspring. Teratology 2001;63:7986.[CrossRef][ISI][Medline]
- Loffredo LC, Souza JM, Freitas JA, et al. Oral clefts and vitamin supplementation. Cleft Palate Craniofac J 2001;38:7683.[CrossRef][ISI][Medline]
- Czeizel AE, Hirschberg J. Orofacial clefts in Hungary. Epidemiological and genetic data, primary prevention. Folia Phoniatr Logop 1997;49:11116.[ISI][Medline]
- Werler MM, Hayes C, Louik C, et al. Multivitamin supplementation and risk of birth defects. Am J Epidemiol 1999;150:67582.[Abstract]
- Czeizel AE, Toth M, Rockenbauer M. Population-based case control study of folic acid supplementation during pregnancy. Teratology 1996;53:34551.[CrossRef][ISI][Medline]
- Munger RG. Maternal nutrition and oral clefts. In: Wyzsynski DF, ed. Cleft lip and palate: from origin to treatment. New York, NY: Oxford University Press, 2002:17092.
- Lammer EJ, Shaw GM, Iovannisci DM, et al. Maternal smoking and the risk of orofacial clefts: susceptibility with NAT1 and NAT2 polymorphisms. Epidemiology 2004;15:1506.[CrossRef][ISI][Medline]
- Hwang SJ, Beaty TH, Panny SR, et al. Association study of transforming growth factor alpha (TGF-
) Taq1 polymorphism and oral clefts: indication of gene-environment interaction in a population-based sample of infants with birth defects. Am J Epidemiol 1995;141:62936.[Abstract] - Beaty TH, Maestri NE, Hetmanski JB, et al. Testing for interaction between maternal smoking and TGFA genotype among oral cleft cases born in Maryland 19921996. Cleft Palate Craniofac J 1997;34:44754.[CrossRef][ISI][Medline]
- Romitti PA, Lidral AC, Munger RG, et al. Candidate genes for nonsyndromic cleft lip and palate and maternal cigarette smoking and alcohol consumption: evaluation of genotype-environment interactions from a population-based case-control study of orofacial clefts. Teratology 1999;59:3950.[CrossRef][ISI][Medline]
- Jugessur A, Lie RT, Wilcox AJ, et al. Cleft palate, transforming growth factor alpha gene variants, and maternal exposures: assessing gene-environment interactions in case-parent triads. Genet Epidemiol 2003;25:36774.[CrossRef][ISI][Medline]
- van Rooij IA, Wegerif MJ, Roelofs HM, et al. Smoking, genetic polymorphisms in biotransformation enzymes, and nonsyndromic oral clefting: a gene-environment interaction. Epidemiology 2001;12:5027.[CrossRef][ISI][Medline]
- Shaw GM, Wasserman CR, Murray JC, et al. Infant TGF-alpha genotype, orofacial clefts, and maternal periconceptional multivitamin use. Cleft Palate Craniofac J 1998;35:36670.[CrossRef][ISI][Medline]
- Shaw GM, Rozen R, Finnell RH, et al. Infant C677T mutation in MTHFR, maternal periconceptional vitamin use, and cleft lip. Am J Genet 1998;80:1968.[CrossRef][ISI]
- Shaw GM, Finnell RH, Todoroff K, et al. Maternal vitamin risk, infant C677T mutation in MTHFR and isolated cleft palate risk. Am J Med Genet 1999;85:845.[CrossRef][ISI][Medline]
- Shaw GM, Zhu H, Lammer EJ, et al. Genetic variation of infant reduced folate carrier (A80G) and risk of orofacial and conotruncal heart defects. Am J Epidemiol 2003;158:74752.[Abstract/Free Full Text]
- Mills JL, Kirke PN, Molloy AM, et al. Methylenetetrahydrofolate reductase thermolabile variant and oral clefts. Am J Med Genet 1999;86:714.[CrossRef][ISI][Medline]
- Gaspar DA, Pavanello RC, Zatz M, et al. Role of the C677T polymorphism at the MTHFR gene on risk to nonsyndromic cleft lip with/without cleft palate: results from a case-control study in Brazil. Am J Med Genet 1999;87:1979.[CrossRef][ISI][Medline]
- Lammer EJ, Shaw GM, Iovannisci DM, et al. Periconceptional multivitamin intake during early pregnancy, genetic variation of acetyl-N-transferase 1 (NAT1), and risk for orofacial clefts. Birth Defects Res A Clin Mol Teratol 2004;70:84652.[CrossRef][ISI][Medline]
- Bird IM, Zhang L, Magness RR. Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function. Am J Physiol Regul Integr Comp Physiol 2003;284:24558.
- Nishikimi A, Matsukawa T, Hoshino K, et al. Localization of nitric oxide synthase activity in unfertilized oocytes and fertilized embryos during preimplantation development in mice. Reproduction 2001;122:95763.[Abstract/Free Full Text]
- Brown KS, Kluijtmans LA, Young IS, et al. Genetic evidence that nitric oxide modulates homocysteine: the NOS3 894TT genotype is a risk factor for hyperhomocysteinemia. Arterioscler Thromb Vasc Biol 2003;23:101420.[Abstract/Free Full Text]
- Lowe ER, Everett AC, Lee AJ, et al. Time dependent inhibition and tetrahydrobiopterin depletion of endothelial NO-synthase caused by cigarettes. Drug Metab Dispos 2005;33:1318.[Abstract/Free Full Text]
- Croen LA, Shaw GM, Jensvold NJ, et al. Birth defects monitoring in California: a resource for epidemiological research. Paediatr Perinat Epidemiol 1991;5:4237.[Medline]
- Cheng S, Grow MA, Pallaud C, et al. A multilocus genotyping assay for candidate markers of cardiovascular disease risk. Genome Res 1999;9:93649.[Abstract/Free Full Text]
- Wong WY, Eskes TK, Kuijpers-Jagtman AM, et al. Nonsyndromic orofacial clefts: association with maternal hyperhomocysteinemia. Teratology 1999;60:2537.[CrossRef][ISI][Medline]
- Plachta N, Traister A, Weil M. Nitric oxide is involved in establishing the balance between cell cycle progression and cell death in the developing neural tube. Exp Cell Res 2003;288:35462.[CrossRef][ISI][Medline]
- Selhub J, Jacques PF, Bostom AG, et al. Relationship between plasma homocysteine and vitamin status in the Framingham study population. Impact of folic acid fortification. Public Health Rev 2000;28:11745.[Medline]
- Bates CJ, Mansoor MA, Gregory J, et al. Correlates of plasma homocysteine, cysteine and cysteinyl-glycine in respondents in the British National Diet and Nutrition Survey of young people aged 418 years, and a comparison with the survey of people aged 65 years and over. Br J Nutr 2002;87:719.[CrossRef][ISI][Medline]
- Kugiyama K, Ohgushi M, Motoyama T, et al. Nitric oxide-mediated flow-dependent dilation is impaired in coronary arteries in patients with coronary spastic angina. J Am Coll Cardiol 1997;30:9206.[Abstract]
- Yallampalli C, Garfield RE. Inhibition of nitric oxide synthesis in rats during pregnancy produces signs similar to those of preeclampsia. Am J Obstet Gynecol 1993;169:131620.[ISI][Medline]
- Brown KS, Cook M, Hoess K, et al. Evidence that the risk of spina bifida is influenced by genetic variation at the NOS3 locus. Birth Defects Res A Clin Mol Teratol 2004;70:1016.[CrossRef][ISI][Medline]