American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 157:215–226 (2011) ARTICLE

Genetic Basis of Susceptibility to Teratogen Induced Birth Defects BOGDAN J. WLODARCZYK,* ANA M. PALACIOS, CLAUDIA J. CHAPA, HUIPING ZHU, TIMOTHY M. GEORGE, AND RICHARD H. FINNELL

Birth defects remain the leading cause of infant death in US. The field of teratology has been focused on the causes and underlying mechanisms of birth defects for decades, yet our understanding of these critical issues remain unacceptably vague. Conclusions from years of animal and human studies made it clear that the vast majority of birth defects have multifactorial origins, with contributions from environmental and genetic factors. The environment comprises not only of the physical, biological, and chemical external environment surrounding the pregnant woman, but it also includes the internal environment of the woman’s body that interact with the developing embryo in a complex fashion. The importance of maternal and embryonic genetic factors consisting of countless genetic variants/mutations that exist within every individual contribute to birth defect susceptibility is only now being more fully appreciated. This great complexity of the genome and its diversity within individuals and populations seems to be the principal reason why the same teratogenic exposure can induce severe malformation in one embryo, while fail to do so to other exposed embryos. As the interaction between genetic and environmental factors has long been recognized as the first ‘‘Principle of Teratology’’ by Wilson and Warkany [1965. Teratology: Principles and techniques. Chicago: University of Chicago Press], it is only recently that the appropriate investigative tools have been developed with which to fully investigate this fundamental principle. The introduction of high throughput technologies like whole genome sequencing or genome-wide association studies are promising to deliver an enormous amount of new data that will shed light on the genomic factors that contribute susceptibility to environmental teratogens. In this review, we attempt to summarize the epidemiological and experimental literature concerning birth defects whose phenotypic expression can be clearly related to the interactions between several select environmental factors and those genetic pathways in which they are most likely to have significant modifying effects. ß 2011 Wiley-Liss, Inc.

KEY WORDS: birth defects; teratogen; genome; mutations; gene–environment interaction How to cite this article: Wlodarczyk B, Palacios A, Chapa C, Zhu H, George TM, Finnell RH. 2011. Genetic basis of susceptibility to teratogen induced birth defects. Am J Med Genet Part C Semin Med Genet 157:215–226.

INTRODUCTION ple organs including: the brain, heart, including inherited (genetic) condi- lungs, liver, bones, intestinal tract, limbs tions, toxic exposure of the embryo Birth defects are abnormalities present at and other structures. These defects and fetus, and as is most often the case, or before birth, and may involve multi- can occur for a variety of reasons for as yet unknown reasons. In the

Bogdan J. Wlodarczyk is an assistant Professor at the Dell Pediatric Research Institute and holds a faculty position in the Department of Nutritional Sciences, The University of Texas at Austin. He is a reproductive toxicologist and uses animal models to study the mechanisms of birth defects. Ana M. Palacios is a physician whose clinical activities and research have focused mainly on clinical genetics and neural tube defects. She has been involved in the creation and consolidation of two independent birth defect surveillance programs in Colombia and Nicaragua. Currently she is a graduate student in Dr. Richard Finnell’s laboratory. Claudia J. Chapa is a physician currently involved in the Medical Genetics research team in Dr. Richard Finnell’s laboratory. Her main research interest is in identifying genetic polymorphisms in patients with birth defects. Huiping Zhu is an assistant Professor at the Dell Pediatric Research Institute, Department of Nutritional Sciences, The University of Texas at Austin. Dr. Zhu has a doctoral degree in epidemiology, and has been working on birth defects etiology, especially gene-environment interactions. Timothy M. George is the Chief of Pediatric Neurosurgery and Neurosciences at the Dell Children’s Medical Center in Austin, Additionally; he is an Adjunct Professor of Cell and Molecular Biology at the University of Texas at Austin and serves as Director of the Pediatric Educational and Translational Research Institute. Dr George serves on Biomedical Engineering Advisory Board and the McComb Business School Health Care Initiative Advisory Board at the University of Texas at Austin. His research focuses on the molecular genetics of neural tube defects. Richard H. Finnell is a Professor in the Department of Nutritional Sciences and in the Department of Chemistry and Biochemistry at the University of Texas at Austin, and serves as the Director of Genomic Research at Dell Children’s Medical Center. A pediatric geneticist, he has a distinguished career researching environmentally induced birth defects. Research in the Finnell Laboratory focuses on the interaction between specific genes and nutritional factors as they influence normal embryonic development and susceptibility to complex birth defects. *Correspondence to: Bogdan Wlodarczyk, Dell Pediatric Research Institute, 1400 Barbara Jordan Blvd., Austin, TX 78723. E-mail: [email protected] DOI 10.1002/ajmg.c.30314 Published online 15 July 2011 in Wiley Online Library (wileyonlinelibrary.com).

ß 2011 Wiley-Liss, Inc. 216 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

United States, 1 out of every 33 babies is variants from multiple genes have been Finnell et al., 2004; Graham and Shaw, born with a major birth defect (http:// linked to NTDs risk individually with 2005; Shi et al., 2008]. One commonly www.nbdpn.org/docs/US_2010_C.pdf). rather small effect [van der Put et al., cited birth defect-oriented example of a The most common types of birth defects 1995; Ou et al., 1996; Volcik et al., GxE interaction is the effect of maternal include congenital heart defects (CHDs) 2003a,b; Zhu et al., 2003, 2005, 2006, smoking and an important detoxifica- and neural tube defects (NTDs), and 2007; van der Linden et al., 2006, 2007; tion gene, CYP1A1, on risk for oral these defects can be very serious, often Parle-McDermott et al., 2007; David- clefts in the exposed embryos [van Rooij life-threatening. Another group of son et al., 2008], leading us to believe et al., 2001; Shi et al., 2007]. This is common birth defects, craniofacial that these small effects interact with consistent with the extreme complexity malformations, include cleft lip with or select environmental factors to create a of the developmental processes during without cleft palate (CL/P) and cleft more significant developmental disequi- early embryogenesis. palate only (CPO), while less commonly librium beyond which these genetic Biological interactions and their life-threatening, they require compre- factors could have produced individual- role in etiological mechanisms require hensive medical and surgical attention. ly, disrupting normal development, and further functional studies [Ottman, Currently, about 70% of the causes resulting in the abnormal phenotype 1990]. Herein we will review both of birth defects are unknown. The [Prescott et al., 2001; Murray, 2002; human and animal studies on the genetic etiology of the majority of birth defects basis of environmentally induced birth whose causes are unclear is believed to defects. Inherited diseases caused by involve both environmental and genetic For example, more than chromosomal abnormalities, as well as factors. The list of environmental factors defects caused by single genes are not that have been associated with birth 240 spontaneous and within the scope of this review. defects is long and remarkably varied genetically modified mouse [Blom et al., 2006]. Some examples models of NTDs exist, with 205 FOLATE PATHWAY GENES include: maternal smoking [Shi et al., AND NTDs 2007; Shea and Steiner, 2008; Suarez of them representing specific et al., 2008, 2011], organic solvents It has been demonstrated that folic [Brender and Suarez, 1990; Brender genes. These models provided acid supplementation prevents NTDs et al., 2002], environmental toxicants abundant candidate genes for [Smithells et al., 1980; Czeizel, 2000; Au like pesticides, nitrates, and heavy metals human studies; however, very et al., 2010; Blencowe et al., 2010; [White et al., 1988; Sever, 1995; Shaw Obican et al., 2010; Collins et al., 2011] et al., 1999b], inadequate intake of few mutations have been and other birth defects such as cono- nutrients [Shaw et al., 1999a; Carmi- found in these genes providing truncal heart defects [Botto et al., 2003b; chael et al., 2003], maternal fever/ Bailey and Berry, 2005]. Folate metab- influenza [Shaw et al., 1998a; Lundberg evidence as having a major role olism is a complex process involving a et al., 2003], maternal obesity and for human NTDs. Instead, large number of enzymatic reactions, diabetes [Watkins and Botto, 2001; variants from multiple genes producing a variety of intermediates that Watkins et al., 2003; Waller et al., function in vital physiological processes 2007]. On the other hand, there is an have been linked to NTDs risk such as nucleotide synthesis, cell divi- abundance of supportive evidence that individually with rather small sion, and tissue growth. Perturbation of complex birth defects have a genetic this complex process may result in frank component [Campbell et al., 1986; effect, leading us to believe that folate deficiency and/or accumulation Shaw et al., 1994; Blom et al., 2006]. these small effects interact with of homocysteine. Animal studies have One important source of scientific generated several hypotheses regarding evidence supporting a genetic contribu- select environmental factors the mechanisms underlying the preven- tion to the etiology of birth defects to create a more significant tive effect of folic acid. Inactivation of Folr1 Folr2 comes from studies involving animal developmental disequilibrium folate pathway genes such as , , models. For example, more than 240 or Mthfr renders the developing embryo spontaneous and genetically modified beyond which these genetic susceptible to an increased risk of NTDs mouse models of NTDs exist, with 205 factors could have produced following in utero exposure to specific of them representing specific genes teratogens. Human studies exploring the [Harris and Juriloff, 2010]. These mod- individually, disrupting interaction between folate pathway els provided abundant candidate genes normal development, genes and teratogens on NTDs risk have for human studies; however, very few and resulting in the been limited by the insufficiency of mutations have been found in these statistical power due to small sample size. genes providing evidence as having a abnormal phenotype. There have been reports of possible major role for human NTDs. Instead, interactions between the MTHFR ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 217

C677T polymorphism and maternal When the Folr2 knockout mice, NTDs and some CHDs [Kallen, 1998; smoking [Hobbs et al., 2006], BHMT which do not present with any patho- Grewal et al., 2008], the vast majority of and obesity, as well as transcobalamin II logical phenotype even in the nullizy- studies have reported an increased risk of (TCII) and maternal alcohol use [Hobbs gous state, were administered sodium adverse pregnancy outcomes including: et al., 2010], on CHD risk. Blanton et al. arsenate, the treatment was significantly birth defects, preterm delivery, sponta- / [2011] recently explored a number of more teratogenic to Folr2 mice than neous abortion, growth restriction, or þ/þ folate pathway genes (including folate to wildtype (Folr2 ) mice. To further negative neurodevelopmental outcomes transport genes) as risk factors for non- investigate how additional environmen- in the exposed offspring of women who syndromic CLP, and found possible tal factors (potentially teratogenic) can smoked [Shea and Steiner, 2008; Suarez interactions between polymorphisms in modulate this gene–teratogen interac- et al., 2008, 2011; Shaw et al., 2009], or FOLR1 and FOLR2 and maternal tion, the Folr2 knockout mice were fed a were breathing in second hand smoke smoking. folate deficient diet during the pericon- [Venners et al., 2004; Meeker et al., Data from animal studies concern- ceptional period and throughout the 2007; Li et al., 2008; Suarez et al., 2008, ing the possible involvement of folate pregnancy and the arsenate was admin- 2011]. Association between maternal pathway genes in teratogen-induced istered during the period of neural tube cigarette smoking and non-syndromic birth defects remain equivocal. In our closure. In this experiment, the terato- orofacial clefts (NSOFC) has been / laboratory,we have treated four different genic effect observed in Folr2 was observed in a number of epidemiological knockout mouse strains (Rfc1, Folr1, considerably exacerbated by the low studies [Lorente et al., 2000; Little et al., Folr2, and Mthfr) that are involved folate diet (exencephaly rate increased 2004; Shaw et al., 2009]. þ/þ in folate transport or metabolism with from 40 to 64%), whereas in Folr2 Hwang et al. [1995] was the first to sodium arsenate, a known mouse neural mice, the rate of NTDs did not change suggest a possible interaction between tube teratogen [Wlodarczyk et al., 2001, [Wlodarczyk et al., 2001]. These results the transforming growth factor alpha 2006a; Spiegelstein et al., 2005]. The illustrate that the gene–teratogen inter- (TGFa) Taq1 variant and maternal Rfc1 knockout mice treated with 40 mg/ actions can be further complicated when smoking on NSOFC [Hwang et al., kg of sodium arsenate, failed to show more than one teratogen is considered. 1995]. TGFa is a secretory protein that any genotype dependent differences Some teratogens can act in a synergistic binds to the epidermal growth factor in teratogenic outcomes. The rates of manner in individuals with ‘‘sensitive’’ receptor. It plays important roles in cell early embryonic loss (resorptions) and genotype, as exemplified by arsenic and migration and differentiation, and is / exencephaly (NTDs) among fetuses folate deficiency in Folr2 nullizygous also a potent promoter of angiogenesis from treated wildtype and heterozygous mice. [Kumar et al., 1995]. Hwang reported Rfc1 dams did not differ significantly. The results following the treatment that TGFa Taq1 variant was associated However, there was a substantial differ- of Mthfr knockout mice in utero with with a higher risk of non-syndromic ence seen in arsenic embryotoxicity arsenic were somewhat similar to those cleft lip with or without cleft palate in þ/ between heterozygous Rfc1 and observed in the Rfc1 mice. There was no mothers who smoked 20 or more þ/ heterozygous Folr1 dams, another difference in teratogenic response to cigarettes/day. Infants carrying the less folate transport mutant mouse line arsenic when the pregnancy outcomes common C2 allele who were exposed to described in this study [Spiegelstein in dams of different Mthfr genotypes (þ/ maternal smoking of 10 or less cigarettes þ/ et al., 2005]. In Rfc1 mice treated þ, þ/, and /) were compared. per day showed a 6.16-fold increase in with arsenic, 12–22% of the embryos However the Mthfr mutant mice were the risk for CPO [95% confidence þ/ were resorbed, whereas in Folr1 significantly more sensitive to arsenic interval (CI): 1.09–34.7], while the þ/ mice embryolethality reached 82% of teratogenicity than were the Rfc1 or risk was 8.69-fold higher (95%CI: / the conceptuses. Despite the fact that Folr2 knockout mice [Wlodarczyk 1.57–47.8) if the mothers smoked these two mutant mouse strains were not and Finnell, unpublished data]. more than 10 cigarettes/day. compared directly in that study, this is More recently in a population- still a compelling example that genetic based case control study in California, variants can deeply modulate the out- MATERNAL EXPOSURE TO Shaw et al. [1998b] observed an elevated come to a teratogen exposure. It should CIGARETTE SMOKE risk associated with the same TGFa be also noted, that only wildtype and Taq1 C2 allele in smoking mothers, heterozygous animals were used in this Embryonic exposure to cigarette smoke resulting in an OR of 6.1 (95%CI: 1.1– study, since Rfc1 and Folr1 homozygous (first-hand and/or passive) is a known 36.1) for cleft lip with or without cleft mutations are embryolethal. Having one risk factor for structural defects such as palate (CL/P), and an OR of 9 (95%CI: active copy of these genes is apparently orofacial clefts [Lie et al., 2008; Shi et al., 1.4–61.9) for CPO. It is noteworthy that sufficient for viability and it appears that 2008] and gastroschisis [Feldkamp et al., when analyzing interactions, the ORs, the heterozygous mice can compensate 2008]. Although there are a few studies although elevated, often have wide for their genetic deficit in folate metab- suggesting that maternal exposure to CIs, indicating the rather imprecise risk olism [Spiegelstein et al., 2005]. cigarette smoking is ‘‘protective’’ against estimations. 218 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

A meta-analysis examining five Maternal smoking has been identi- been associated with birth defects that case–control studies found a significant fied as a risk factor for defects other than are not typically considered to be a part overall smoking effect for CL/P craniofacial, including CHD [Malik of the FAS. For example, one study (OR ¼ 1.64, 95%CI ¼ 1.33–2.01) and et al., 2008; Kuciene and Dulskiene, found that women who consumed CPO (OR ¼ 1.42, 95%CI ¼ 1.06– 2010] and gastroschisis [Lammer et al., alcohol at least once a week had a 2.1- 1.90). There was no evidence of in- 2008; Chabra et al., 2011]. In a popula- fold increased risk (95%CI: 1.1, 4.0) of creased risk for CPO if the infant carried tion-based case–control study, women having a child affected with an NTD the C2 allele among non-smoking who smoked and carried a TCII—776 [Grewal et al., 2008]. mothers. However, if the mother CG or GG genotype—were 1.8 times Studies on attempting to under- reported smoking and the infant carried more likely to have a CHD-affected stand the genetic basis of susceptibility the C2 allele, there was an overall fetus than were women who smoked and to the etiology of this condition have increased risk for CP, with an OR of carried a CC genotype. The CHDs not been successful. Although a recent 1.95 (95%CI: 1.22–3.10). Based on were: non-syndromic septal, conotrun- population-based case–control study this meta-analysis, the TGFa genotype cal, right or left-sided obstructive heart reported that variants in the alcohol failed to demonstrate an increased risk defects [Hobbs et al., 2010]. TCII is dehydrogenase 1C (ADH1C) gene may for CL/P, regardless of maternal smok- an important enzymatic transporter of modify the association between alcohol ing status. The authors concluded that cobalamin from the intestinal lumen to and oral clefts, it did not extend to gene–environment interactions between the bloodstream, and eventually to target the whole pattern of FAS dysmorphic the infant’s TGFa Taq1 polymorphism tissues, suggesting a role for vitamin B12 features [Boyles et al., 2010]. Alcohol and maternal smoking were limited to in the etiology of this group of defects dehydrogenase (ADH) is an enzyme that CPO [Zeiger et al., 2005]. [Quadros et al., 1999]. Torfset al. [2006] oxidizes ethanol to form acetaldehyde Folic acid is an essential B vitamin assessed the interaction between mater- during its metabolism. Functional SNPs required for both amino and nucleic acid nal smoking and selected candidate in ADH1C gene are associated with a biosynthesis. It also participates in the genes in a case–control study of 57 cases reduced ratio of alcohol oxidation. In one-carbon unit transfers, a key step of gastroschisis and 506 controls. Thirty- this study, mothers who consumed five in biomethylation reactions. Quickly two genes involved in angiogenesis, or more alcoholic drinks per sitting proliferating cells of a rapidly growing blood vessel integrity, inflammation, during the first trimester of pregnancy embryo/fetus have a high demand for wound repair, and dermal/epidermal had an elevated risk for a malformed folate; therefore adequate supply of folic strength were interrogated for variants. infant with an oral cleft (OR of 2.6, acid is especially important for women Of these genes, NOS3 glu298asp, 95%CI: 1.4–4.7). This increased risk during pregnancy. Cigarette smoking ICAM1 gly241arg, and NPPA T2238C was evident only in mothers or children has been documented to lower folate showed a strong interaction with mater- who carried ADH1C haplotype that was and increase homocysteine blood levels nal smoking. Women who smoked and associated with slow alcohol metabolism [Ozerol et al., 2004; Jauniaux and carried one or two of the aforemen- (OR of 3.0, 95%CI: 1.4–6.8) [Boyles Burton, 2007]. High level of homocys- tioned variants were at a significantly et al., 2010]. There was no evidence of teine during pregnancy might affect higher risk of having an infant with alcohol-related risk when both mother important developmental processes gastroschisis, when compared to women and infant carried only the rapid meta- such as neural crest cell motility and with wildtype alleles who did not smoke bolism ADH1C variant (OR of 0.9, migration [Brauer and Tierney, 2004]. [NOS3 OR ¼ 5.2 (95%CI: 2.4–11.4); 95%CI: 0.2–4.1). Hobbs et al. [2010] Additionally, high level of homocysteine ICAM1 OR ¼ 5.2 (95%CI: 2.1–12.7); reported a possible gene–environment and its conversion to homocysteine– and NPPA OR ¼ 6.4 (95%CI: 2.8– interaction in infants with CHDs ex- thiolactone, have been associated with 14.6)] [Torfs et al., 2006]. posed in utero to alcohol. Women who post-translational modification of the consumed alcohol while pregnant and folate receptors. As a result, creation of were carriers of the TCII—776 CG or neoantigens might be inducing a patho- MATERNAL ALCOHOL GG genotype—were 1.7 times more logical maternal immune response that (ETHANOL) EXPOSURE likely to have a CHD-affected fetus, than ultimately will impair folic acid uptake were women who drank and carried a [Jakubowski et al., 2009]. In a recently Fetal alcohol syndrome (FAS) was CC genotype. published study, Blanton et al. [2011] initially described in the French medical analyzed 89 single nucleotide polymor- literature by Lemoine et al. [1968]. phisms (SNPs) in fourteen folate metab- Subsequently, Jones and collaborators MATERNAL olism-related genes in non-Hispanic were the first to systematically define the HYPERGLYCEMIA/ white and Hispanic NSCLP families. association between maternal alcohol GLUCOSE METABOLISM Evidence was documented for interac- abuse and the pattern of birth defects tions with smoking, and SNPs in the associated with the FAS [Jones et al., An extensive epidemiological literature FOLR1 and FOLR2 genes. 1973]. Maternal alcohol use has also indicates that maternal diabetes is a ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 219 strong risk factor for birth defects transcriptional programming in devel- and perinatal mortality [Mills et al., There is sufficient evidence to oping embryos by down-regulating a 1979; Myrianthopoulos and Melnick, suggest that compromised large group of transcriptional factors 1987; Reece and Homko, 1993; Waller [Pavlinkova et al., 2009; Salbaum and et al., 1994; Shaw et al., 1996, 2000; maternal glucose control Kappen, 2010]. Werler et al., 1996; Hendricks et al., predisposes embryos to Studies have shown that the in- 2001; Eriksson et al., 2003; Honein creased level of oxygen free radicals in et al., 2003; Watkins et al., 2003; developmental anomalies, embryos of diabetic rats are contributing Anderson et al., 2005]. Maternal meta- even among non-diabetic to the observed malformations, and that bolic syndrome, obesity and pre-preg- women. Maternal diabetes, the removal of these reactive oxygen nant weight are also associated with risk species reduces the likelihood of fetuses of central nervous system birth defects obesity, and metabolic expressing the diabetic embryopathy [Waller et al., 1994; Shaw et al., 1996; syndrome create a compromised [Eriksson and Borg, 1991; Hagay et al., Watkins et al., 1996; Werler et al., 1996; 1995; Eriksson and Siman, 1996; Sivan Kallen, 1998; Hendricks et al., 2001; in utero environment for et al., 1996; Wentzel et al., 1997; Fine Ray et al., 2007; Correa et al., 2008] as the developing embryo. The et al., 1999]. Increased glucose delivery well as other birth defects such as CHDs, to embryos, or activation of pathways omphalocele and instances where the genetic basis underlying the that are stimulated by high glucose such child presents with multiple anomalies susceptibility to abnormal as the hexosamine biosynthetic pathway [Watkinsand Botto, 2001; Watkins et al., glucose metabolism induced or hypoxia, increase oxidative stress in 2003]. Gestational diabetes mellitus embryos. Blocking these pathways, or (GDM) may also be associated with birth defects involves both providing antioxidants such as reduced increased risk of central nervous system maternal and embryonic glutathione or vitamin E, suppress birth defects [Anderson et al., 2005]; the adverse effects of excess glucose however, the increased risk may in fact gene variants. [Loeken, 2005, 2006]. Impaired embry- be due to undiagnosed type 2 diabetes. onic gene expression resulting from Therefore, GDM has not been recog- oxidative stress and consequent apopto- nized as a risk factor due to inconsistent Early human embryos do not sis or disturbed organogenesis, may findings [Janssen et al., 1996; Savona- have pancreatic function until the be a general mechanism to explain Ventura and Gatt, 2004; Mitanchez, development of b-cells, usually after the features observed in the diabetic 2010]. In vivo and in vitro studies using gestational week 7. Maternal hypergly- embryopathy [Kambe et al., 1996; Wu animal models suggested that hyper- cemia forces an increased glucose flux et al., 1996; Phelan et al., 1997; Fine glycemia is a major teratogen capable into embryonic cells through glucose et al., 1999; Pani et al., 2002a,b; King of inducing malformations in diabetic transporters during early organogenesis; and Loeken, 2004; Loeken, 2006; animal models, while other associated therefore, the embryonic cells suffer Dheen et al., 2009]. For example, factors such as hyperinsulinemia [Hen- from an increased metabolic overload oxidative stress induced by excess em- dricks et al., 2001], ketone bodies, which is directed at their mitochondria, bryonic glucose metabolism inhibits the branched amino acids, and triglycerides leading to increased formation of reac- expression of Pax3, a critical develop- may also exert some adverse effects on tive oxygen and oxidative stress [Eriks- mental gene, leading to the appearance the developing embryos [Styrud and son et al., 2000; Wentzel et al., 2001; of birth defects [Morgan et al., 2008; Eriksson, 1992; Suzuki et al., 1996; Beemster et al., 2002]. Hyperglycemia Zabihi and Loeken, 2010]. Reece et al., 1996a; Eriksson et al., causes a myo-inositol deficiency with a PAX3 gene encodes a transcription 2000; Zhao and Reece, 2005]. There competitive inhibition of ambient glu- factor required for normal neural tube is sufficient evidence to suggest that cose, which might have been associated closure. Murine embryos with a non- compromised maternal glucose control with a diminished phosphoinositide functional Pax3 gene exhibit open predisposes embryos to developmental signal transduction. Dietary supplemen- NTDs, exencephaly and spina bifida, anomalies, even among non-diabetic tation of deficient substrates for example, with 100% penetrance [Pani et al., women [Groenen et al., 2003; Shaw arachidonic acid or myo-inositol [Gold- 2002a; Wlodarczyk et al., 2006b; Burren et al., 2003]. Maternal diabetes, obesity, man et al., 1985; Reece et al., 1988, et al., 2008]. PAX3 protein is required and metabolic syndrome create a com- 1996b; Baker et al., 1990; Khandelwal during neural tube development to promised in utero environment for the et al., 1998], either in vitro or in vivo, has suppress p53-dependent cell death and developing embryo. The genetic basis been shown to reduce the incidence of consequent failure to close the neural underlying the susceptibility to abnor- diabetes-related malformations in the tube. PAX3 decreases steady-state levels mal glucose metabolism induced birth offspring of diabetic pregnant animals. of the p53 tumor-suppressor protein, defects involves both maternal and Gene expression profiling studies such that when PAX3 is deficient, p53 embryonic gene variants. showed that maternal diabetes alters protein increases, leading to increased 220 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE neuroepithelial apoptosis which may including: intrauterine growth retarda- About 3–10% of these infants present occur prior to the completion of neural tion (IUGR), caudal regression, anen- with birth defects, and approximately tube closure. Supplementation with cephaly with absence of the head, 11–20% of them exhibit neurodevelop- folic acid or 5-methyltetrahydrofolate microphthalmia, and micrognathia, mental impairment, with or without rescues the normal phenotype in spon- which are similar to those defects associated structural abnormalities [Fin- taneous Pax3 mutant (Sp/Sp) embryos observed in the diabetic embryopathy nell, 1991; Kaneko et al., 1999; Dean [Wlodarczyk et al., 2006b]. A recent [Heilig et al., 2003]. A recent study et al., 2002; Mortensen et al., 2003; mouse study suggested the presence of a reported that one silent variant, Pro196, Adab et al., 2004; Gaily et al., 2004; gene–environment interaction between may be associated with risk of spina Vinten et al., 2005]. Intrauterine expo- embryonic folate levels and the Pax3 bifida by affecting the transcription and sure to valproate or to multiple AEDs genotype. Folate deficiency does not spicing of the SLC2A1 (alias: GLUT1) seems to represent the highest malfor- cause NTDs in wildtype mice, but gene [Davidson et al., 2008]. mation risk [Breen and Davenport, causes a significant increase in cranial A recent report suggested that 2006; Pennell, 2006; Veiby et al., NTDs among mutant embryos that GLUT2 may be a risk factor involved 2009]. Irrespective of which AED was carry an intragenic deletion in the Pax3 in NTD occurrence in diabetic preg- studied, the results consistently showed gene (Sp2H). Control treatments, in nancies [Li et al., 2007]. In animal that not all exposed fetuses were born which intermediate levels of folate are models, maternal diabetes and hypergly- with birth defects [German et al., 1970; supplied, failed to induce NTDs, sug- cemia do not down-regulate Glut1 and Jones et al., 1989; Dansky and Finnell, gesting that NTD risk is related to Glut2 expression in the embryos and 1991]. These observations indicate that embryonic folate concentration, not visceral yolk sac during the critical in most instances, AEDs alone are maternal blood folate concentration. periods of organogenesis, as they do in insufficient to induce teratogenic effects This study suggested that folate deficien- pre-implantation embryos; therefore, in exposed fetuses. The observed birth cy increases the risk of NTDs in embryonic glucose transport continues defects are multifactorial in their origin genetically predisposed Splotch embry- and results in increased glucose flux into and beside exposure to AEDs, some os, probably via embryonic growth the cells [Maeda et al., 1993; Takao et al., other interacting components, most retardation [Burren et al., 2008]. Var- 1993; Trocino et al., 1994; Li et al., likely genetic, must be involved in order iants in human PAX3 gene potentially 2007]. Inactivation of Glut2 gene in a to predispose the developing embryo to associated with risk of spina bifida have mouse strain protects embryos from express an abnormal phenotype follow- been suggested [Lu et al., 2007]; there- maternal diabetes-induced NTDs [Li ing the in utero AED exposure. It seems fore, interaction between these variants et al., 2007]. The interactions between and environmental factors including human SLC2A2 (alias: GLUT2) and folic acid use and maternal hyperglyce- maternal glucose metabolism; however, mia merit evaluation. have not yet been evaluated. Intrauterine exposure to Glucose is transported across the valproate or to multiple AEDs lipid bi-layers of cell membranes by a EXPOSURE TO family of structurally related membrane ANTIEPILEPTIC DRUGS seems to represent the highest spanning glycoproteins called glucose transporters [Joost and Thorens, 2001; Epilepsy is the most common neuro- malformation risk. Irrespective Joost et al., 2002]. Erythrocyte-type logic disorder requiring medical treat- of which AED was studied, the glucose transporter (GLUT1) is the ment in pregnant women [Hvas et al., results consistently showed that major type of glucose transporter 2000]. Antiepileptic drugs (AEDs) have expressed during early organogenesis long been recognized as having a not all exposed fetuses were [Maeda et al., 1993; Matsumoto et al., teratogenic potential capable of disrupt- born with birth defects. These 1995]. Consistent with the high demand ing normal development in exposed for glucose, GLUT1 protein is expressed infants. Several epidemiological studies observations indicate that in at a high level in the embryonic tissues indicated that infants of mothers treated most instances, AEDs alone undergoing differentiation, mainly in with AEDs during pregnancy have a are insufficient to induce the neural tube, while expression de- two- to threefold higher risk of having clined as gestational age progressed birth defects when compared to the teratogenic effects in exposed [Shepard et al., 1970; Tanimura and general population [Holmes et al., 2001; fetuses. The observed birth Shepard, 1970; Akazawa et al., 1989; Perucca, 2005]. In the USA annually Matsumoto et al., 1995]. Homozygous almost 25,000 children are born to defects are multifactorial Glut1 knockout mice die in utero women with epilepsy and most of them in their origin and beside [Ohtsuki et al., 2006]. Glut1-deficient require AEDs such as valproic acid, mice generated by antisense-Glut1 carbamazepine, phenytoin, phenobarbi- exposure to AEDs, some other exhibit developmental malformations tal, or lamotrigine [Meador et al., 2008]. interacting components, most ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 221 likely genetic, must be involved pine, comparing to a control group of slight strain dependent variation in healthy children [Vurucu et al., 2008]. timing of neural tube closure, as evi- in order to predispose the Based on this finding, one can speculate denced by the study which failed to developing embryo to express an that VPA lowers an already limited produce at least moderately high level supply of folate in pregnant MTHFR of NTDs in C57 mice treated at abnormal phenotype following 677TT homozygous women, pushing various timepoints during pregnancy the in utero AED exposure. the developing embryo beyond the [Beck, 1999]. The observed strain spe- threshold of normal development and cific sensitivity to VPA was also con- ultimately leading to NTDs. Insight firmed in an in vitro study where 6–8 from the animal studies demonstrating somite stage mouse embryos of two that either the mother or the embryo that the teratogenicity of VPA can be different strains; SWV and C57BL/6J, carry genetic factors determining sus- prevented/diminished by folic acid sup- were cultured in the serum supple- ceptibility to AED induced adverse plementation tend to provide biological mented with VPA for 48 hr. As in the effects. Epidemiological study showed plausibility to this hypothesis [Padma- in vivo studies, the SWV embryos were that the recurrence risk of NTDs among nabhan and Shafiullah, 2003; Dawson more likely to have open NTDs than pregnant women who were exposed to et al., 2006]. Unfortunately, there is no were the C57BL/6J embryos. Addition- VPAwas higher than the occurrence risk concrete evidence that supplemental ally, the minimum lethal dose of VPA of NTD in women exposed to VPA, folic acid protects against NTDs in was also significantly lower for SWV suggesting possible genetic contribu- humans [Ornoy, 2009]. On the other cultured embryos [Naruse et al., 1988]. tions to VPA sensitivity [Dansky and hand, rather surprising results were Results of this study point out that Finnell, 1991]. Other studies concern- reported from studies on Mthfr knockout besides variation in the maternal genes þ/þ ing NTDs demonstrated that these mice treated with VPA. Mthfr wild- that interact with environmental factors, defects are more common if there was type mice turned out to be more the embryonic genetic component þ/ an existing family history of NTDs sensitive than Mthfr heterozygotes alone can modulate the teratogenic [Delgado-Escueta and Janz, 1992; Malm when treated with VPA. There were no outcome. et al., 2002]. cases of exencephaly in the fetuses from Complex birth defects are known þ/ Methylenetetrahydrofolate reduc- Mthfr dams, whereas 21% of fetuses to have multifactorial etiologies and the þ/þ tase (MTHFR) is an enzyme that from Mthfr had NTDs. Further combination of genetic susceptibility as plays a key role in folate metabolism. analysis revealed that VPA lowered well as the dose and the time of exposure It catalyzes the conversion of 5,10- homocysteine plasma level and increased to certain environmental compounds methylenetetrahydrofolate (5,10-MTHF) the Mthfr expression which resulted in (e.g., drugs and other toxic xenobiotics) to 5-methyltetrahydrofolate (5-MTHF), lower teratogenicity in Mthfr deficient appear to account for significant varia- which is the main methyl donor in the mice [Roy et al., 2008]. Much of our tion in the human response. Both homocysteine to methionine remethy- understanding of how genetic factors maternal and fetal genotypes can affect lation cycle. Studies of MTHFR in modulate teratogenic action of drugs or placental transport, absorption, metabo- human revealed a common polymor- other chemicals comes from studies on lism, distribution, and receptor binding phism (677C!T substitution) in this animal models. Results of VPA terato- of the drug, influencing its teratogenici- gene that is associated with hyperhomo- genicity studies in mice clearly demon- ty [Hall et al., 1997; Spiegelstein et al., cysteinemia and a slightly increased risk strated a strong genetic component 2003]. Polymorphisms in maternal and for birth defects [Botto and Yang, 2000]. involved in determining sensitivity to fetal genes involved in xenobiotic de- Epileptic mothers taking AEDs who the induction of NTDs. Different strains toxification may modify risks of adverse were MTHFR 677T homozygotes were of mice reacted very differently to VPA pregnancy outcome in response to found to have a three- to fourfold higher treatment during pregnancy as evi- maternal use of AED. Many drugs and risk for having a child with the fetal denced by the presence of fetuses with endogenous compounds need to be anticonvulsant syndrome, compared exencephaly, a form of NTD equivalent metabolized in order to be activated or with 677CC homozygous mothers re- of human anencephaly [Finnell, 1991; inactivated and ultimately excreted. The ceiving AED treatment [Deen et al., Hall et al., 1997; Malm et al., 2002]. enzymes responsible for these reactions 1999; Kini et al., 2007; Dean et al., More specifically SWV/Fnn, AKR/J, belong to the extensive cytochrome 2008]. This syndrome consists of a C3H/HeJ, and P/J mice turned out to P450 (CYPs) gene family. The human characteristic pattern of neurodevelop- be the most sensitive; LM/Bc had an genome contains 115 cytochrome P450 mental impairment, facial features, and intermediate sensitivity, while DBA and genes, of which at least 57 are functional. major birth defects including NTDs, C57BL/6J were highly resistant [Azar- Among CYP families 1–3 (responsible orofacial clefts, heart, and renal defects. bayjani and Danielsson, 1998; Faiella for drug metabolism), there are 22 dif- It appears that there are significantly et al., 2000; Spiegelstein et al., 2003; ferent P450 isoforms and a high degree lower plasma folate concentrations in Wiltse, 2005]. These differences in of polymorphism has been identified children taking VPA and carbamaze- sensitivity cannot be explained by the [Ingelman-Sundberg and Sim, 2010; 222 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE

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