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Genetic susceptibility to teratogens: State of the art
Reproductive Toxicology 34 (2012) 186–191
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Genetic susceptibility to teratogens: State of the art Matteo Cassina, Leonardo Salviati, Elena Di Gianantonio, Maurizio Clementi ∗ Teratology Information Service, Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Padova, Italy
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Article history: Received 9 March 2012 Received in revised form 8 May 2012 Accepted 10 May 2012 Available online 1 June 2012 Keywords: Pregnancy Genetics Teratogenicity Birth defects
a b s t r a c t There is evidence that the susceptibility to the teratogenic effect of drugs within human populations varies extremely from one individual to another, even after identical exposures. One of the factors that may explain these interindividual differences is the genetic makeup in the pharmacokinetics and pharmacodynamics of the respective drugs. In fact, both maternal and embryonic/fetal genotypes can affect placental transport, absorption, metabolism, distribution and receptor binding of an agent, influencing its teratogenicity. We have reviewed the literature and commented on the reported correlations between genetic factors and drug-induced birth defects. There is still a clear lack of knowledge regarding this issue and the available data are often conflicting. However, the identification of specific polymorphisms associated with predisposition to teratogenesis may allow in the future the development of personalized non-teratogenic therapies for pregnant women. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Teratogenesis mechanisms in general are still largely unknown. Environment plays a definite role, but the phenotype (i.e. functional and/or morphological defect) is the result of the interaction between the prenatal exposure and the genetic background of the mother and the fetus. Epidemiologic studies have demonstrated that not all embryos/fetuses exposed to a teratogen develop anomalies and the only explanation why one fetus exposed has congenital malformations and another one does not is the genetic (maternal and/or fetal) variation [1]. Genetic susceptibility to teratogenesis was hypothesized since 1959 when Wilson developed the principles to understand the effects of teratogenic agents on the developing organism [2]. The following epidemiological data and laboratory experiments have produced further evidence that, given an exposure to a known teratogen in the sensitive period, both in humans and animals, only a variable proportion of embryos/fetuses develops congenital anomalies. The mechanism of teratogenesis is a complex one, determined by the interaction between environmental factors and genetic variations in two individuals, the mother and the child, sharing the same exposure but having different genetic background and epigenetic phenomena [1,3,4]. Moreover, it has been suggested that also paternal exposure to environmental agents may have
∗ Corresponding author at: Genetica Clinica ed Epidemiologica, Dipartimento di Salute della Donna e del Bambino, Via Giustiniani 3, 35128 Padova, Italy. Tel.: +39 0498213572; fax: +39 0498211425. E-mail address: [email protected] (M. Clementi). 0890-6238/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2012.05.004
teratogenic effects in the offspring, via epigenetic remodeling during the gametogenesis [5]. The analysis of the pharmacokinetics of the drugs as well as the development in knowledge of pharmacogenetics will help in understanding the mechanism of teratogenesis. At present few data are available in the literature. 2. Animal studies Animal models have been extensively used to assess the teratogenic potential of novel pharmacological agents or of other compounds. They include mammalian and non mammalian species. Mammalian species include rodents (mice, rats, rabbits and guinea pigs) and larger animals (pigs, sheep, primates), while non mammalian models include the chick, zebrafish, and also non-vertebrates such as Caenorhabditis elegans and Drosophila. In general simpler organisms are much cheaper, and easy to manipulate genetically, while larger animals reproduce more faithfully the human pathology. In some cases simplicity is an advantage, for example chick embryos lack the placenta, thus allow to eliminate placental and maternal confounding factors in teratogenesis studies. In the past decade the advances in functional genomics and in proteomics have opened exciting new possibilities in the field of experimental teratology. The complete genomic sequence has been determined for an increasing number of animal species, in parallel commercial tools to study gene expression (i.e. microarray chips) have become widely available and much less expensive. Moreover, genetic manipulation of animals is also much simpler and cheaper than in the past. Consortia have generated thousands of embryonic stem cells lines targeted for specific genes, and it has become
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relatively easy to modulate gene expression in zebrafish embryos [6–9]. In parallel, novel separation techniques coupled to mass spectrometry allow the precise identification of individual proteins and also to perform high throughput screenings to identify specific protein expression profiles, which integrate the information gained through gene expression arrays because they may analyze post translational modifications and regulation mechanisms at the protein level. Zebrafish has gained significant importance as a model for experimental teratology in the last decade because it grows rapidly and it has a transparent body that allows simple observation of developmental defects, also of internal organs, even in the live embryo [10]. Moreover zebrafish is particularly suitable for functional studies because it is very simple to manipulate genetically its embryos. It is possible to knockdown expression of specific genes by microinjection of fertilized eggs with specific morpholino oligonucleotides. Overexpression of specific genes can be achieved similarly, microinjecting the fertilized eggs with specific expression vectors or in vitro transcribed mRNA. Overall, these technologies have allowed the identification of the precise molecular targets of teratogens, and the definition of the specific cellular pathways that are perturbed by these substances; in some cases it has been possible to identify the specific genetic factors conferring susceptibility (or protection) to specific compounds in animals. Moreover a novel field of study, focusing on the epigenetic modifications occurring in response to specific environmental factors such as the diet has flourished in the past years. We will present a few examples of how animal models in conjunction with these novel genomic and proteomic technologies have impacted the field of experimental teratology. A good example of the integrated use of different approaches is the discovery of cereblon (CRBN) as a primary target of thalidomide toxicity. Ito and colleagues [11] developed an affinity based technique combined with mass spectrometry that allowed them to identify CRBN as the protein that physically interacts with thalidomide. The results were confirmed by showing that knockdown of CRBN in zebrafish embryos recapitulated the effect of thalidomide, and that an engineered version of CRBN which retains its activity, but does not bind thalidomide, protects both chick and zebrafish from the effect of thalidomide when expressed in embryos. CRBN encodes a component of the ubiquitin-ligase complex which regulates expression of fibroblast growth factor FGF8, which is essential for limb development [12]. However it should be kept in mind that most teratogens have pleiotropic effects; for example there is considerable evidence that thalidomide causes also an increase in oxidative stress which activates the NF-B and bone morphogenic proteins signaling with a downstream perturbation of PTEN and Akt pathways [13,14]. It has been shown that in many species the susceptibility to some specific teratogens is strain specific. This characteristic has been exploited in different studies using both a classical genomic based approach or novel high throughput proteomic techniques in order to identify the genetic determinants of this phenomenon. For example, the aryl hydrocarbon receptor (AHR) is well known to be an important factor mediating the developmental cardiac toxicity of dioxin-like compounds (DLC) [15]. However few data are available on the other downstream factors involved in the process. Waits and Nebert [16] have employed two zebrafish strains which display a 40-fold difference in the susceptibility to DLC. By crossing them and examining the recombinant generation using genome wide quantitative trait loci mapping, they identified at least 12 candidate genes related to DLC susceptibility, confirming that this is a multifactorial trait. Among these were genes encoding the AHR 2, PTEN, calpain 1, cardiac troponin T, phospholipase C gamma, and beta-enolase, indicating that different (and apparently unrelated) cellular pathways are involved in the process.
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In fact, knockdown of AHR2 protects zebrafish from the effect of tetrachlorodibenzo-p-dioxin [17]. Other morpholino-knockdown experiments in zebrafish have shown that cytochrome P450 1C genes are also involved in the pathogenesis of these defects [18]. Another example is the use of a proteomic approach to study the susceptibility to cadmium-induced limb teratogenicity. Chen and coworkers [19] have compared the C57BL/6N mouse strain which is susceptible to cadmium with the SWV/Fnn strain which is resistant. Their objective was to identify differentially displayed proteins in two target tissues of cadmium teratogenesis. They employed an approach based on two-dimensional gel electrophoresis, coupled to mass-spectrometry, which allowed them to identify a set of 38 proteins that are differentially expressed in the two strains. Fourteen of these proteins are involved in the unfolded protein response process and seven in the control of actin polymerization, an important event in chondrogenesis, suggesting a role of these two cellular pathways in cadmium toxicity. Classical gene expression profiling studies using microarrays have been extensively used to characterize specific gene signatures in cells, tissues or whole embryos after exposures to different teratogens (DLC, valproate, alcohol, arsenide and cadmium, etc.) [20–23]. In general these signatures are enriched for developmentally related genes. Moreover, gene expression profiling in embryonic or pluripotent stem cells, has been used as a tool to screen the teratogenic potential of different compounds [24]. A still open issue concerns how the information gained through animal models can be translated to humans. In fact, drug metabolism may be different, moreover some teratogens act in a very specific time frame during development and it is difficult to compare the pregnancy of rodents (which lasts few days) with that of humans; finally, some specific genes may not be present in some species. Nevertheless, identifying the specific targets of teratogens and of the cellular pathways that perturbed is important in order to identify specific candidate genes for human association studies. In fact, the relative paucity of patients makes genome-wide association studies impractical. 2.1. Environmental epigenetics Epigenetics has been recently hypothesized to be a possible mechanism of mediating teratogenesis. Functional variation between cells within the same individual is mediated by epigenetic modifications to the genome which are heritable through multiple cell divisions but do not involve a change in primary DNA sequence. Some of the most widely recognized epigenetic mechanisms include covalent modifications of DNA and histones, as well as small non-coding RNAs [25,26]. Prenatal epigenetic modifications are under the control of several growth factors, hormones, and other signaling molecules [27,28]; it has been demonstrated that exogenous environmental stimuli (for example in vitro culture techniques, alcohol, endocrine disruptors, and dietary supplements such as folic acid, vitamin B12, choline, and betaine) can alter the epigenetic constitution [29–32]. Several animal models have been developed to study the effects of specific environmental agents such as diet, toxic compounds, and, in the case of in vitro fertilization, the components of culture media for embryos, on DNA methylation patterns, or on other features such as histone acetylation and chromatin structure. To date most studies have focused on DNA methylation patterns. Rodents have been useful particularly for maternal nutrition based epigenetic studies, because they reach sexual maturity within six to eight weeks from delivery, they exhibit similar placentation to humans, and they have a short gestational period [4]. These characteristics allow to perform transgenerational studies in a relatively short timeframe, which would be impossible using larger
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animals. A number of specific genetic markers have been identified in mice, such agouti viable yellow (Avy ) or axin 1 fused (Axin1Fu ) mice [4]. In these animals the methylation pattern at specific and well-characterized loci determines precise and clearly demonstrable phenotypic effects (coat color in Avy mice and the morphology of the tail in Axin1Fu animals). These models allow to test the effect of different maternal dietary conditions on the offspring [32]. Interestingly in the case of Avy mice the color of the coat correlates with the susceptibility to develop diabetes and obesity [4]. Using Axin1Fu mice it has been possible to demonstrate transgenerational effects. Parents with the abnormal tails were more likely to generate offspring with the same defect compared to the animals with normal tails. One limitation is that neither the Axin1Fu nor Avy alleles have equivalents in humans and therefore it is not clear yet how the information gained through these models can be translated to humans. Experiments with A/WySn mice are of particular interest; in fact the A/WySn mouse strain, with 15–20% penetrance of cleft lip and palate (CLP), is an animal model for human multifactorial CLP. The CLP is due to the insertion of an intracisternal A-particle (IAP) transposon in the Wnt9b gene; it has been observed that the embryos with unmethylated IAP tended to have lower levels of Wnt9b transcript and were more likely to have the cleft lip phenotype. Moreover, the methylation level of the IAP in Wnt9b depended on the genotype of another gene, named clf2. Exogenous environmental factors may also modify the methylation of the transposon in Wnt9b and thereafter the risk of CLP [33]. Prenatal alcohol exposure has been demonstrated to cause fetal alcohol spectrum disorders (FASD) and embryo/fetal epigenetic disruptions have been suggested to be one of the pathogenetic mechanisms of the syndrome. In fact alcohol is known to affect one-carbon metabolism, the primary source of methyl donors in DNA-transmethylation reactions. Experiments with mice have demonstrated that alcohol exposure during gestation can result in global hypomethylation of fetal DNA [29] and locus specific hypermethylation [34]. Zhou et al. [35] observed that short alcohol exposures equivalent to binge drinking (6 h) delay the DNA methylation program in the dorsal neural tube at the hindbrain, forebrain, and truncal (heart) level of the neural tube; longer exposures (44 h) like chronic drinking delay both the ventral and dorsal neural tube at multiple levels along the neural tube axis. In addition, site-specific hypomethylation of male gametes has been observed in mice chronically exposed to alcohol [36]. Valproic acid (VPA) has been shown to cause a variety of fetal abnormalities in humans and animals. VPA is a direct inhibitor of histone deacetylases and several studies have suggested that VPA-induced epigenetic modifications involving histone acetylation, histone methylation, and DNA methylation, may be a possible mechanism of mediating teratogenesis [37–39]. Recent researches have suggested that also the carcinogenic and teratogenic activity of heavy metals may be associated with epigenetic mechanisms [40]. For instance, chronic exposure to cadmium is associated with elevated DNA methyltransferase activities and global DNA hypermethylation. Moreover, the carcinogenic effects of nickel seem to be associated largely with epigenetic rather than genetic changes. Chronic hyperglycemia in diabetic pregnant women has been associated with an increased risk of birth defects and long-term health consequences in the offspring. It has been suggested that the maternal hyperglycemia alters the status of the embryonic epigenome and has been linked to higher levels of histone acetylation. In fact, excess glucose can lead to a high availability of acetyl-CoA, which is the direct enzymatic substrate for histone acetylation [41]. All these examples suggest that epigenetic alterations may play a role in drug-induced teratogenesis. However a causal relationship
between congenital malformations and the epigenetic alterations observed in cases of maternal diabetes or prenatal exposure to alcohol, valproic acid and heavy metals, still has to be clearly demonstrated.
3. Human studies Although the interaction between teratogens and the maternal/fetal genotype is much more easily investigated in experimental animals than in humans, there are some studies suggesting that different human genetic polymorphisms are associated with a different susceptibility to the teratogen-induced damage. Teratogenicity may be related to both maternal and fetal genetic variants affecting the pharmacokinetics (absorption, distribution, metabolism, excretion, placental transport) and the receptor binding of an agent. However, until now there has been very limited success in identifying genetic variants predisposing to druginduced birth defects. Several published studies focus on the relationship between metabolizing enzymes activity and fetal alcohol syndrome. The importance of genetic factors in determining susceptibility to fetal alcohol syndrome has been hypothesized sine the 1970s, when Christoffel and Salafsky [42] reported a significant discordance in the outcome of a twin pregnancy. A later study involving 16 alcohol-exposed twin pairs, confirmed that concordance for alcohol-induced birth defects is higher among monozygotic twins than among dizygotic twins [43]. Ethanol is mainly oxidized to acetaldehyde by the enzyme alcohol dehydrogenase (ADH) or the cytochrome P450 2E1 (CYP2E1); acetaldehyde is subsequently oxidized to acetate by the enzyme aldehyde dehydrogenase (ALDH). Genetic polymorphisms of these enzymes are known. Viljoen et al. [44] examined the allele frequency of the ADH1B variants in children with fetal alcohol syndrome and their mothers within a mixed ancestry population in the Western Cape Province of South Africa: the ADH1B*2 allele frequency was statistically lower in affected children and their mothers with respect to controls, suggesting a protective role of the ADH1B*2 allele. Moreover, three studies have been conducted within the African American population and two of those showed ADH1B*3 to be potentially protective for alcohol-induced teratogenesis. In a prospective study, McCarver et al. [45] noted that presence of at least one copy of ADH1B*3 genetic variant in drinking African American women was protective for both their offspring’s neurobehavioral development and intrauterine growth. Das et al. [46] extended this study evaluating the facial morphology of the children and observed that the absence of the ADH1B*3 in both mothers and children was associated with a significant reduction of the palpebral fissure length, inner canthal distance, and nasal bridge to mouth length. In another prospective study, Jacobson et al. [47] observed that among the children whose mothers lacked ADH1B*3 allele, prenatal alcohol exposure was associated with smaller head circumference at birth and poorer neurodevelopmental outcomes. On the contrary, Stoler et al. [48] concluded that the absence of the ADH1B*3 allele was protective for the fetal outcome of AfricanAmerican women. A relationship between teratogenicity and maternal/fetal genetic variants affecting metabolism has been reported for phenytoin, caffeine and cigarette smoking. Azzato et al. [49] recruited 155 women (174 pregnancies) who used phenytoin throughout their pregnancy and evaluated the correlation between major craniofacial abnormalities in the offspring and four maternal genetic polymorphisms of two metabolizing enzymes: the CYP2C9 and the microsomal epoxide hydrolase (EPHX1). The selected polymorphisms were: EPHX1 H139R, which is associated with an increased
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enzymatic activity, and CYP2C9 R144C, CYP2C9 I395L and EPHX1 Y113H, which are associated with slow activity. The authors concluded that both maternal EPHX1 polymorphisms are associated with major craniofacial abnormalities in the offspring; such association was not observed with the CYP2C9 polymorphisms. Maternal caffeine consumption has been associated with and increased risk of spontaneous abortion, but the results of the epidemiological studies are inconsistent. Signorello et al. [50] performed a case–control study enrolling 101 cases and 953 controls and observed that an increased caffeine intake is a risk factor for pregnancy loss for women with high activity of CYP1A2, the enzyme that primarily metabolizes caffeine. A previous case–control study by Fenster et al. [51] had failed to detect such association. However, both studies did not genotyped women enrolled in the study and CYP1A2 phenotype and activity were based on the measurement of various caffeine metabolites in urine. Sata et al. [52] performed a case–control study to evaluate the relationship of daily caffeine intake, CYP1A2*1F genotype and recurrent spontaneous abortions. Fifty-eight women with at least two recurrent pregnancy losses and 147 fertile controls were enrolled in the study. The risk for recurrent abortion was significantly increased only among women who had homozygous CYP1A2*1F genotype, which is thought to represent a CYP1A2 high inducibility genotype [53]. The authors also noted a dosage effect of daily caffeine intake and women who ingested daily 300 mg or more caffeine had the highest risk (OR 5.23). Cigarette smoking during pregnancy is also associated with low birth weight and preterm delivery; moreover some studies observed an increased risk of birth defects including orofacial clefts, limb reduction defects, congenital foot deformities, urinary tract abnormalities and craniosynostosis. CYP1A1, glutathione Stransferases (GSTT1 and GSTM1), and N-acetyltransferase 2 (NAT2) are enzymes related to the metabolism of polycyclic aromatic hydrocarbons, nitrosamines, aromatic and heterocyclic amines present in cigarette smoke. Several studies have evaluated the correlation between polymorphisms of these enzymes and the fetal outcome, but conflicting results have been published. In fact, although Wang et al. [54] reported an increased risk of low birth weight in infants of mothers carrying certain polymorphisms of CYP1A1, GSTT1 and GSTM1 genes, a more recent study failed to detect such associations [55]. Shi et al. [56] and Chevrier et al. [57] reported a decreased prevalence of oral clefts in infants carrying specific variants of CYP1A1; van Rooij et al. [58] failed to find such association evaluating the maternal genotype. Moreover, while deletions in GSTT1 were associated with an increase of fetal oral clefts [56,58], deletions in GSTM1 and variant alleles of NAT2 and EPHX1 were not associated [56,57,59,60]. The susceptibility to the teratogenic effect is related to genetic variants affecting not only the pharmacokinetics but also the mechanisms of action of drugs. There is some evidence that specific variants of genes involved in folate/homocysteine metabolism may influence the risk of neural tube defects (NTD) in fetuses exposed to folate-antagonists. In particular, a common polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene, 677C>T, seems to be associated with an increased risk of NTD [61] and to exacerbate the teratogenic effects of antiepileptic drugs. Dean et al. [62] observed that epileptic mothers of children who were diagnosed with fetal anticonvulsant syndrome were more likely than epileptic mothers of unaffected children to be homozygous for the MTHFR 677C>T polymorphism. Kini et al. [63] studied the MTHFR genotype and rate of major malformations in 187 mother–child pairs where the mothers had epilepsy and in 236 matched control pairs. They observed that the rate of major birth defects was increased in the offspring of mothers who were heterozygous or homozygous for the 677C>T polymorphism, amongst antiepileptic drugs-exposed cases
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(especially valproic acid); no association between the child’s MTHFR genotype and the rate of malformation was observed. However the effect of valproic acid on the rate of birth defects was much higher than the effect of the maternal genotype. Dean et al. [64] recruited 200 mothers who had taken antiepileptic drugs during pregnancy and studied the genotype of MTHFR and other folate pathway genes in each parent–child triad. The five polymorphisms analyzed were MTHFR 677C>T, MTHFR 1298A>C, cytosolic serine hydroxymethyl transferase (SHMT1) 1420C>T, methionine synthase (MTR) 2756A>G and methionine synthase reductase (MTRR) 66A>G. In this study they observed that the risk of having a child with congenital malformation or fetal anticonvulsant syndrome was three to four times higher for mothers who were homozygous for the MTHFR 677C>T polymorphism compared with mothers not carrying this polymorphism. They also suggested that the risk of fetal anticonvulsant syndrome or neurodevelopmental disorders may be influenced by the child’s MTR and MTRR genotypes. Finally, a relationship between teratogenicity of serotonin reuptake inhibitors (SRI) and genetic polymorphisms of their receptor has been postulated. SLC6A4 gene codes for the serotonin reuptake transporter that is blocked by SRI and plays a key role in the regulation of serotoninergic neurotransmission. A polymorphism in the regulatory region of the gene has been shown to alter SLC6A4 transcription and activity. The short (s) variant is associated with approximately 50% reduction in serotonin reuptake compared with the long (l) variant. Oberlander et al. [65] investigated whether SLC6A4 genotype influences the risk for adverse outcomes in neonates prenatally exposed to SRI. Neonatal outcomes in 37 prenatally SRI exposed neonates was compared with those of 47 non-exposed neonates and the genotype for SLC6A4 was determined. This study gave conflicting results and variable relationships between polymorphisms and specific outcomes were observed. Moreover, although the infant SLC6A4 genotype seems to moderate the adverse neonatal outcomes in SRI-exposed infants, the authors concluded that, beyond apparent gene–medication interactions, other mechanisms probably contribute to the outcomes. 4. Conclusions The role of genetic predisposition in the drug related mechanisms of teratogenesis is postulated, but the available data are very scarce and often conflicting. Experiments with animal models are fundamental for the identification of the molecular targets of teratogens, the definition of specific cellular pathways that are perturbed by these substances and the detection of genetic variants conferring susceptibility (or protection) to specific compounds. However, epidemiological human studies are necessary to confirm the results obtained from animal studies and data from prospective series, such as cohorts collected from Teratology Information Services, are a possible source for future molecular or expression studies. The identification of specific polymorphisms associated with predisposition to teratogenesis can allow the development of patient-tailored therapies. In this hypothesis, pregnant women affected by chronic or acute diseases will be in the future prescribed with personalized non-teratogenic therapies. Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] Polifka JE, Friedman JM. Medical genetics. 1. Clinical teratology in the age of genomics. Canadian Medical Association Journal 2002;167:265–73.
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Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox
Genetic susceptibility to teratogens: State of the art Matteo Cassina, Leonardo Salviati, Elena Di Gianantonio, Maurizio Clementi ∗ Teratology Information Service, Clinical Genetics Unit, Department of Woman and Child Health, University of Padova, Padova, Italy
a r t i c l e
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Article history: Received 9 March 2012 Received in revised form 8 May 2012 Accepted 10 May 2012 Available online 1 June 2012 Keywords: Pregnancy Genetics Teratogenicity Birth defects
a b s t r a c t There is evidence that the susceptibility to the teratogenic effect of drugs within human populations varies extremely from one individual to another, even after identical exposures. One of the factors that may explain these interindividual differences is the genetic makeup in the pharmacokinetics and pharmacodynamics of the respective drugs. In fact, both maternal and embryonic/fetal genotypes can affect placental transport, absorption, metabolism, distribution and receptor binding of an agent, influencing its teratogenicity. We have reviewed the literature and commented on the reported correlations between genetic factors and drug-induced birth defects. There is still a clear lack of knowledge regarding this issue and the available data are often conflicting. However, the identification of specific polymorphisms associated with predisposition to teratogenesis may allow in the future the development of personalized non-teratogenic therapies for pregnant women. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Teratogenesis mechanisms in general are still largely unknown. Environment plays a definite role, but the phenotype (i.e. functional and/or morphological defect) is the result of the interaction between the prenatal exposure and the genetic background of the mother and the fetus. Epidemiologic studies have demonstrated that not all embryos/fetuses exposed to a teratogen develop anomalies and the only explanation why one fetus exposed has congenital malformations and another one does not is the genetic (maternal and/or fetal) variation [1]. Genetic susceptibility to teratogenesis was hypothesized since 1959 when Wilson developed the principles to understand the effects of teratogenic agents on the developing organism [2]. The following epidemiological data and laboratory experiments have produced further evidence that, given an exposure to a known teratogen in the sensitive period, both in humans and animals, only a variable proportion of embryos/fetuses develops congenital anomalies. The mechanism of teratogenesis is a complex one, determined by the interaction between environmental factors and genetic variations in two individuals, the mother and the child, sharing the same exposure but having different genetic background and epigenetic phenomena [1,3,4]. Moreover, it has been suggested that also paternal exposure to environmental agents may have
∗ Corresponding author at: Genetica Clinica ed Epidemiologica, Dipartimento di Salute della Donna e del Bambino, Via Giustiniani 3, 35128 Padova, Italy. Tel.: +39 0498213572; fax: +39 0498211425. E-mail address: [email protected] (M. Clementi). 0890-6238/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.reprotox.2012.05.004
teratogenic effects in the offspring, via epigenetic remodeling during the gametogenesis [5]. The analysis of the pharmacokinetics of the drugs as well as the development in knowledge of pharmacogenetics will help in understanding the mechanism of teratogenesis. At present few data are available in the literature. 2. Animal studies Animal models have been extensively used to assess the teratogenic potential of novel pharmacological agents or of other compounds. They include mammalian and non mammalian species. Mammalian species include rodents (mice, rats, rabbits and guinea pigs) and larger animals (pigs, sheep, primates), while non mammalian models include the chick, zebrafish, and also non-vertebrates such as Caenorhabditis elegans and Drosophila. In general simpler organisms are much cheaper, and easy to manipulate genetically, while larger animals reproduce more faithfully the human pathology. In some cases simplicity is an advantage, for example chick embryos lack the placenta, thus allow to eliminate placental and maternal confounding factors in teratogenesis studies. In the past decade the advances in functional genomics and in proteomics have opened exciting new possibilities in the field of experimental teratology. The complete genomic sequence has been determined for an increasing number of animal species, in parallel commercial tools to study gene expression (i.e. microarray chips) have become widely available and much less expensive. Moreover, genetic manipulation of animals is also much simpler and cheaper than in the past. Consortia have generated thousands of embryonic stem cells lines targeted for specific genes, and it has become
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relatively easy to modulate gene expression in zebrafish embryos [6–9]. In parallel, novel separation techniques coupled to mass spectrometry allow the precise identification of individual proteins and also to perform high throughput screenings to identify specific protein expression profiles, which integrate the information gained through gene expression arrays because they may analyze post translational modifications and regulation mechanisms at the protein level. Zebrafish has gained significant importance as a model for experimental teratology in the last decade because it grows rapidly and it has a transparent body that allows simple observation of developmental defects, also of internal organs, even in the live embryo [10]. Moreover zebrafish is particularly suitable for functional studies because it is very simple to manipulate genetically its embryos. It is possible to knockdown expression of specific genes by microinjection of fertilized eggs with specific morpholino oligonucleotides. Overexpression of specific genes can be achieved similarly, microinjecting the fertilized eggs with specific expression vectors or in vitro transcribed mRNA. Overall, these technologies have allowed the identification of the precise molecular targets of teratogens, and the definition of the specific cellular pathways that are perturbed by these substances; in some cases it has been possible to identify the specific genetic factors conferring susceptibility (or protection) to specific compounds in animals. Moreover a novel field of study, focusing on the epigenetic modifications occurring in response to specific environmental factors such as the diet has flourished in the past years. We will present a few examples of how animal models in conjunction with these novel genomic and proteomic technologies have impacted the field of experimental teratology. A good example of the integrated use of different approaches is the discovery of cereblon (CRBN) as a primary target of thalidomide toxicity. Ito and colleagues [11] developed an affinity based technique combined with mass spectrometry that allowed them to identify CRBN as the protein that physically interacts with thalidomide. The results were confirmed by showing that knockdown of CRBN in zebrafish embryos recapitulated the effect of thalidomide, and that an engineered version of CRBN which retains its activity, but does not bind thalidomide, protects both chick and zebrafish from the effect of thalidomide when expressed in embryos. CRBN encodes a component of the ubiquitin-ligase complex which regulates expression of fibroblast growth factor FGF8, which is essential for limb development [12]. However it should be kept in mind that most teratogens have pleiotropic effects; for example there is considerable evidence that thalidomide causes also an increase in oxidative stress which activates the NF-B and bone morphogenic proteins signaling with a downstream perturbation of PTEN and Akt pathways [13,14]. It has been shown that in many species the susceptibility to some specific teratogens is strain specific. This characteristic has been exploited in different studies using both a classical genomic based approach or novel high throughput proteomic techniques in order to identify the genetic determinants of this phenomenon. For example, the aryl hydrocarbon receptor (AHR) is well known to be an important factor mediating the developmental cardiac toxicity of dioxin-like compounds (DLC) [15]. However few data are available on the other downstream factors involved in the process. Waits and Nebert [16] have employed two zebrafish strains which display a 40-fold difference in the susceptibility to DLC. By crossing them and examining the recombinant generation using genome wide quantitative trait loci mapping, they identified at least 12 candidate genes related to DLC susceptibility, confirming that this is a multifactorial trait. Among these were genes encoding the AHR 2, PTEN, calpain 1, cardiac troponin T, phospholipase C gamma, and beta-enolase, indicating that different (and apparently unrelated) cellular pathways are involved in the process.
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In fact, knockdown of AHR2 protects zebrafish from the effect of tetrachlorodibenzo-p-dioxin [17]. Other morpholino-knockdown experiments in zebrafish have shown that cytochrome P450 1C genes are also involved in the pathogenesis of these defects [18]. Another example is the use of a proteomic approach to study the susceptibility to cadmium-induced limb teratogenicity. Chen and coworkers [19] have compared the C57BL/6N mouse strain which is susceptible to cadmium with the SWV/Fnn strain which is resistant. Their objective was to identify differentially displayed proteins in two target tissues of cadmium teratogenesis. They employed an approach based on two-dimensional gel electrophoresis, coupled to mass-spectrometry, which allowed them to identify a set of 38 proteins that are differentially expressed in the two strains. Fourteen of these proteins are involved in the unfolded protein response process and seven in the control of actin polymerization, an important event in chondrogenesis, suggesting a role of these two cellular pathways in cadmium toxicity. Classical gene expression profiling studies using microarrays have been extensively used to characterize specific gene signatures in cells, tissues or whole embryos after exposures to different teratogens (DLC, valproate, alcohol, arsenide and cadmium, etc.) [20–23]. In general these signatures are enriched for developmentally related genes. Moreover, gene expression profiling in embryonic or pluripotent stem cells, has been used as a tool to screen the teratogenic potential of different compounds [24]. A still open issue concerns how the information gained through animal models can be translated to humans. In fact, drug metabolism may be different, moreover some teratogens act in a very specific time frame during development and it is difficult to compare the pregnancy of rodents (which lasts few days) with that of humans; finally, some specific genes may not be present in some species. Nevertheless, identifying the specific targets of teratogens and of the cellular pathways that perturbed is important in order to identify specific candidate genes for human association studies. In fact, the relative paucity of patients makes genome-wide association studies impractical. 2.1. Environmental epigenetics Epigenetics has been recently hypothesized to be a possible mechanism of mediating teratogenesis. Functional variation between cells within the same individual is mediated by epigenetic modifications to the genome which are heritable through multiple cell divisions but do not involve a change in primary DNA sequence. Some of the most widely recognized epigenetic mechanisms include covalent modifications of DNA and histones, as well as small non-coding RNAs [25,26]. Prenatal epigenetic modifications are under the control of several growth factors, hormones, and other signaling molecules [27,28]; it has been demonstrated that exogenous environmental stimuli (for example in vitro culture techniques, alcohol, endocrine disruptors, and dietary supplements such as folic acid, vitamin B12, choline, and betaine) can alter the epigenetic constitution [29–32]. Several animal models have been developed to study the effects of specific environmental agents such as diet, toxic compounds, and, in the case of in vitro fertilization, the components of culture media for embryos, on DNA methylation patterns, or on other features such as histone acetylation and chromatin structure. To date most studies have focused on DNA methylation patterns. Rodents have been useful particularly for maternal nutrition based epigenetic studies, because they reach sexual maturity within six to eight weeks from delivery, they exhibit similar placentation to humans, and they have a short gestational period [4]. These characteristics allow to perform transgenerational studies in a relatively short timeframe, which would be impossible using larger
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animals. A number of specific genetic markers have been identified in mice, such agouti viable yellow (Avy ) or axin 1 fused (Axin1Fu ) mice [4]. In these animals the methylation pattern at specific and well-characterized loci determines precise and clearly demonstrable phenotypic effects (coat color in Avy mice and the morphology of the tail in Axin1Fu animals). These models allow to test the effect of different maternal dietary conditions on the offspring [32]. Interestingly in the case of Avy mice the color of the coat correlates with the susceptibility to develop diabetes and obesity [4]. Using Axin1Fu mice it has been possible to demonstrate transgenerational effects. Parents with the abnormal tails were more likely to generate offspring with the same defect compared to the animals with normal tails. One limitation is that neither the Axin1Fu nor Avy alleles have equivalents in humans and therefore it is not clear yet how the information gained through these models can be translated to humans. Experiments with A/WySn mice are of particular interest; in fact the A/WySn mouse strain, with 15–20% penetrance of cleft lip and palate (CLP), is an animal model for human multifactorial CLP. The CLP is due to the insertion of an intracisternal A-particle (IAP) transposon in the Wnt9b gene; it has been observed that the embryos with unmethylated IAP tended to have lower levels of Wnt9b transcript and were more likely to have the cleft lip phenotype. Moreover, the methylation level of the IAP in Wnt9b depended on the genotype of another gene, named clf2. Exogenous environmental factors may also modify the methylation of the transposon in Wnt9b and thereafter the risk of CLP [33]. Prenatal alcohol exposure has been demonstrated to cause fetal alcohol spectrum disorders (FASD) and embryo/fetal epigenetic disruptions have been suggested to be one of the pathogenetic mechanisms of the syndrome. In fact alcohol is known to affect one-carbon metabolism, the primary source of methyl donors in DNA-transmethylation reactions. Experiments with mice have demonstrated that alcohol exposure during gestation can result in global hypomethylation of fetal DNA [29] and locus specific hypermethylation [34]. Zhou et al. [35] observed that short alcohol exposures equivalent to binge drinking (6 h) delay the DNA methylation program in the dorsal neural tube at the hindbrain, forebrain, and truncal (heart) level of the neural tube; longer exposures (44 h) like chronic drinking delay both the ventral and dorsal neural tube at multiple levels along the neural tube axis. In addition, site-specific hypomethylation of male gametes has been observed in mice chronically exposed to alcohol [36]. Valproic acid (VPA) has been shown to cause a variety of fetal abnormalities in humans and animals. VPA is a direct inhibitor of histone deacetylases and several studies have suggested that VPA-induced epigenetic modifications involving histone acetylation, histone methylation, and DNA methylation, may be a possible mechanism of mediating teratogenesis [37–39]. Recent researches have suggested that also the carcinogenic and teratogenic activity of heavy metals may be associated with epigenetic mechanisms [40]. For instance, chronic exposure to cadmium is associated with elevated DNA methyltransferase activities and global DNA hypermethylation. Moreover, the carcinogenic effects of nickel seem to be associated largely with epigenetic rather than genetic changes. Chronic hyperglycemia in diabetic pregnant women has been associated with an increased risk of birth defects and long-term health consequences in the offspring. It has been suggested that the maternal hyperglycemia alters the status of the embryonic epigenome and has been linked to higher levels of histone acetylation. In fact, excess glucose can lead to a high availability of acetyl-CoA, which is the direct enzymatic substrate for histone acetylation [41]. All these examples suggest that epigenetic alterations may play a role in drug-induced teratogenesis. However a causal relationship
between congenital malformations and the epigenetic alterations observed in cases of maternal diabetes or prenatal exposure to alcohol, valproic acid and heavy metals, still has to be clearly demonstrated.
3. Human studies Although the interaction between teratogens and the maternal/fetal genotype is much more easily investigated in experimental animals than in humans, there are some studies suggesting that different human genetic polymorphisms are associated with a different susceptibility to the teratogen-induced damage. Teratogenicity may be related to both maternal and fetal genetic variants affecting the pharmacokinetics (absorption, distribution, metabolism, excretion, placental transport) and the receptor binding of an agent. However, until now there has been very limited success in identifying genetic variants predisposing to druginduced birth defects. Several published studies focus on the relationship between metabolizing enzymes activity and fetal alcohol syndrome. The importance of genetic factors in determining susceptibility to fetal alcohol syndrome has been hypothesized sine the 1970s, when Christoffel and Salafsky [42] reported a significant discordance in the outcome of a twin pregnancy. A later study involving 16 alcohol-exposed twin pairs, confirmed that concordance for alcohol-induced birth defects is higher among monozygotic twins than among dizygotic twins [43]. Ethanol is mainly oxidized to acetaldehyde by the enzyme alcohol dehydrogenase (ADH) or the cytochrome P450 2E1 (CYP2E1); acetaldehyde is subsequently oxidized to acetate by the enzyme aldehyde dehydrogenase (ALDH). Genetic polymorphisms of these enzymes are known. Viljoen et al. [44] examined the allele frequency of the ADH1B variants in children with fetal alcohol syndrome and their mothers within a mixed ancestry population in the Western Cape Province of South Africa: the ADH1B*2 allele frequency was statistically lower in affected children and their mothers with respect to controls, suggesting a protective role of the ADH1B*2 allele. Moreover, three studies have been conducted within the African American population and two of those showed ADH1B*3 to be potentially protective for alcohol-induced teratogenesis. In a prospective study, McCarver et al. [45] noted that presence of at least one copy of ADH1B*3 genetic variant in drinking African American women was protective for both their offspring’s neurobehavioral development and intrauterine growth. Das et al. [46] extended this study evaluating the facial morphology of the children and observed that the absence of the ADH1B*3 in both mothers and children was associated with a significant reduction of the palpebral fissure length, inner canthal distance, and nasal bridge to mouth length. In another prospective study, Jacobson et al. [47] observed that among the children whose mothers lacked ADH1B*3 allele, prenatal alcohol exposure was associated with smaller head circumference at birth and poorer neurodevelopmental outcomes. On the contrary, Stoler et al. [48] concluded that the absence of the ADH1B*3 allele was protective for the fetal outcome of AfricanAmerican women. A relationship between teratogenicity and maternal/fetal genetic variants affecting metabolism has been reported for phenytoin, caffeine and cigarette smoking. Azzato et al. [49] recruited 155 women (174 pregnancies) who used phenytoin throughout their pregnancy and evaluated the correlation between major craniofacial abnormalities in the offspring and four maternal genetic polymorphisms of two metabolizing enzymes: the CYP2C9 and the microsomal epoxide hydrolase (EPHX1). The selected polymorphisms were: EPHX1 H139R, which is associated with an increased
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enzymatic activity, and CYP2C9 R144C, CYP2C9 I395L and EPHX1 Y113H, which are associated with slow activity. The authors concluded that both maternal EPHX1 polymorphisms are associated with major craniofacial abnormalities in the offspring; such association was not observed with the CYP2C9 polymorphisms. Maternal caffeine consumption has been associated with and increased risk of spontaneous abortion, but the results of the epidemiological studies are inconsistent. Signorello et al. [50] performed a case–control study enrolling 101 cases and 953 controls and observed that an increased caffeine intake is a risk factor for pregnancy loss for women with high activity of CYP1A2, the enzyme that primarily metabolizes caffeine. A previous case–control study by Fenster et al. [51] had failed to detect such association. However, both studies did not genotyped women enrolled in the study and CYP1A2 phenotype and activity were based on the measurement of various caffeine metabolites in urine. Sata et al. [52] performed a case–control study to evaluate the relationship of daily caffeine intake, CYP1A2*1F genotype and recurrent spontaneous abortions. Fifty-eight women with at least two recurrent pregnancy losses and 147 fertile controls were enrolled in the study. The risk for recurrent abortion was significantly increased only among women who had homozygous CYP1A2*1F genotype, which is thought to represent a CYP1A2 high inducibility genotype [53]. The authors also noted a dosage effect of daily caffeine intake and women who ingested daily 300 mg or more caffeine had the highest risk (OR 5.23). Cigarette smoking during pregnancy is also associated with low birth weight and preterm delivery; moreover some studies observed an increased risk of birth defects including orofacial clefts, limb reduction defects, congenital foot deformities, urinary tract abnormalities and craniosynostosis. CYP1A1, glutathione Stransferases (GSTT1 and GSTM1), and N-acetyltransferase 2 (NAT2) are enzymes related to the metabolism of polycyclic aromatic hydrocarbons, nitrosamines, aromatic and heterocyclic amines present in cigarette smoke. Several studies have evaluated the correlation between polymorphisms of these enzymes and the fetal outcome, but conflicting results have been published. In fact, although Wang et al. [54] reported an increased risk of low birth weight in infants of mothers carrying certain polymorphisms of CYP1A1, GSTT1 and GSTM1 genes, a more recent study failed to detect such associations [55]. Shi et al. [56] and Chevrier et al. [57] reported a decreased prevalence of oral clefts in infants carrying specific variants of CYP1A1; van Rooij et al. [58] failed to find such association evaluating the maternal genotype. Moreover, while deletions in GSTT1 were associated with an increase of fetal oral clefts [56,58], deletions in GSTM1 and variant alleles of NAT2 and EPHX1 were not associated [56,57,59,60]. The susceptibility to the teratogenic effect is related to genetic variants affecting not only the pharmacokinetics but also the mechanisms of action of drugs. There is some evidence that specific variants of genes involved in folate/homocysteine metabolism may influence the risk of neural tube defects (NTD) in fetuses exposed to folate-antagonists. In particular, a common polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene, 677C>T, seems to be associated with an increased risk of NTD [61] and to exacerbate the teratogenic effects of antiepileptic drugs. Dean et al. [62] observed that epileptic mothers of children who were diagnosed with fetal anticonvulsant syndrome were more likely than epileptic mothers of unaffected children to be homozygous for the MTHFR 677C>T polymorphism. Kini et al. [63] studied the MTHFR genotype and rate of major malformations in 187 mother–child pairs where the mothers had epilepsy and in 236 matched control pairs. They observed that the rate of major birth defects was increased in the offspring of mothers who were heterozygous or homozygous for the 677C>T polymorphism, amongst antiepileptic drugs-exposed cases
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(especially valproic acid); no association between the child’s MTHFR genotype and the rate of malformation was observed. However the effect of valproic acid on the rate of birth defects was much higher than the effect of the maternal genotype. Dean et al. [64] recruited 200 mothers who had taken antiepileptic drugs during pregnancy and studied the genotype of MTHFR and other folate pathway genes in each parent–child triad. The five polymorphisms analyzed were MTHFR 677C>T, MTHFR 1298A>C, cytosolic serine hydroxymethyl transferase (SHMT1) 1420C>T, methionine synthase (MTR) 2756A>G and methionine synthase reductase (MTRR) 66A>G. In this study they observed that the risk of having a child with congenital malformation or fetal anticonvulsant syndrome was three to four times higher for mothers who were homozygous for the MTHFR 677C>T polymorphism compared with mothers not carrying this polymorphism. They also suggested that the risk of fetal anticonvulsant syndrome or neurodevelopmental disorders may be influenced by the child’s MTR and MTRR genotypes. Finally, a relationship between teratogenicity of serotonin reuptake inhibitors (SRI) and genetic polymorphisms of their receptor has been postulated. SLC6A4 gene codes for the serotonin reuptake transporter that is blocked by SRI and plays a key role in the regulation of serotoninergic neurotransmission. A polymorphism in the regulatory region of the gene has been shown to alter SLC6A4 transcription and activity. The short (s) variant is associated with approximately 50% reduction in serotonin reuptake compared with the long (l) variant. Oberlander et al. [65] investigated whether SLC6A4 genotype influences the risk for adverse outcomes in neonates prenatally exposed to SRI. Neonatal outcomes in 37 prenatally SRI exposed neonates was compared with those of 47 non-exposed neonates and the genotype for SLC6A4 was determined. This study gave conflicting results and variable relationships between polymorphisms and specific outcomes were observed. Moreover, although the infant SLC6A4 genotype seems to moderate the adverse neonatal outcomes in SRI-exposed infants, the authors concluded that, beyond apparent gene–medication interactions, other mechanisms probably contribute to the outcomes. 4. Conclusions The role of genetic predisposition in the drug related mechanisms of teratogenesis is postulated, but the available data are very scarce and often conflicting. Experiments with animal models are fundamental for the identification of the molecular targets of teratogens, the definition of specific cellular pathways that are perturbed by these substances and the detection of genetic variants conferring susceptibility (or protection) to specific compounds. However, epidemiological human studies are necessary to confirm the results obtained from animal studies and data from prospective series, such as cohorts collected from Teratology Information Services, are a possible source for future molecular or expression studies. The identification of specific polymorphisms associated with predisposition to teratogenesis can allow the development of patient-tailored therapies. In this hypothesis, pregnant women affected by chronic or acute diseases will be in the future prescribed with personalized non-teratogenic therapies. Conflict of interest statement The authors declare that there are no conflicts of interest. References [1] Polifka JE, Friedman JM. Medical genetics. 1. Clinical teratology in the age of genomics. Canadian Medical Association Journal 2002;167:265–73.
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