Maternal and infant genetic variants, maternal periconceptional use of selective serotonin reuptake inhibitors, and risk of congenital heart defects in offspring: population based studyBMJ 2017; 356 doi: https://doi.org/10.1136/bmj.j832 (Published 06 March 2017) Cite this as: BMJ 2017;356:j832
- Wendy N Nembhard, associate professor1,
- Xinyu Tang, assistant professor2,
- Zhuopei Hu, biostatistician2,
- Stewart MacLeod, assistant professor1,
- Zachary Stowe, professor3,
- Daniel Webber, research associate1,
- the National Birth Defects Prevention Study
- 1Division of Birth Defects Research, Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences and Arkansas Children’s Research Institute, Little Rock, AR, 72202, USA
- 2Division of Biostatistics, Department of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Arkansas Children’s Research Institute, Little Rock, AR, 72202 USA
- 3Department of Psychiatry, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA
- Correspondence to: W Nembhard
- Accepted 27 January 2017
Objective To evaluate whether the association between maternal periconceptional use of selective serotonin reuptake inhibitors (SSRIs) and increased risk of congenital heart defects in offspring is modified by maternal or infant genetic variants in folate, homocysteine, or transsulfuration pathways.
Design Population based study. DNA from mothers, fathers, and infants was genotyped with an Illumina GoldenGate custom single nucleotide polymorphism panel. A hybrid design based on a log linear model was used to calculate relative risks and Bayesian false discovery probabilities (BFDP) to identify polymorphisms associated with congenital heart defects modified by SSRI use.
Data sources Data from the US National Birth Defects Prevention Study on 1180 liveborn infants with congenital heart defects and 1644 controls, born 1997-2008.
Main outcome measures Cases included infants with selected congenital heart defects and control infants had no major defects. SSRI use was obtained from telephone interviews with mothers.
Results For women who reported taking SSRIs periconceptionally, maternal SHMT1 (rs9909104) GG and AGgenotypes were associated with a 5.9 and 2.4 increased risk of select congenital heart defects in offspring, respectively, versus the AA genotype (BFDP=0.69). Compared with the AA genotype, BHMT (rs492842 and rs542852) GG and AG genotypes were associated with twice the riskof congenital heart defects (BFDP=0.74 and 0.79, respectively). MGST1 (rs2075237) CC and ACgenotypes were associated with an increased risk compared with the GG genotype (8.0 and 2.8, respectively; BFDP=0.79). Single nucleotide polymorphism in infant genes in the folate (MTHFS rs12438477), homocysteine (TRDMT1 rs6602178 and GNMT rs11752813) and transsulfuration (GSTP1 rs7941395 and MGST1 rs7294985) pathways were also associated with an increased risk of congenital heart defects.
Conclusions Common maternal or infant genetic variants in folate, homocysteine, or transsulfuration pathways are associated with an increased risk of certain congenital heart defects among children of women taking SSRIs during cardiogenesis.
In the US the prevalence of depression is high in women of childbearing age.1 The prevalence of clinical antenatal depression is 10-15%,2 and 8-13% of pregnant women are prescribed antidepressant drugs.3456 Serotonin selective reuptake inhibitors (SSRIs) are the most common class of antidepressants prescribed during pregnancy.357 Their effectiveness is attributed, in part, to their ability to increase the post-synaptic availability of the neurotransmitter serotonin by inhibiting the serotonin (5-HT) transporter (SERT or 5-HTT), resulting in reduced symptoms of depression among many patients.89101112 5-HT is also a vital signaling molecule in cardiogenesis. Consequently, if present during cardiogenesis, SSRIs could interfere with 5-HT signaling, resulting in several different types of congenital heart defects.911 Infants with such defects have an increased risk of morbidity and mortality.1314151617
Epidemiologic studies have produced inconsistent results regarding the impact of maternal use of SSRIs during embryogenesis. Some studies have reported an increased risk of congenital heart defects,11819202122 while others report no association.1823242526272829303132 Some publications implicate paroxetine253334353637 and fluoxetine2638 as specific SSRIs that are most teratogenic for congenital heart defects, while others do not.1819232426353639 These inconsistent results could be a consequence of a complex relation between similar risk factors, genetics, behaviors, and metabolic pathways for both mental illness and congenital heart defects. Women are prescribed SSRIs for various illnesses, including but not limited to depression, panic disorder, and obsessive compulsive disorder. These psychiatric conditions are associated with poorer health behaviors and obstetric outcomes. For example, women diagnosed with depression are more likely than the general population to have concomitant behaviors and health conditions independently associated with an increased risk of congenital heart defects, including prenatal cigarette smoking, alcohol and/or illicit drug use, poor diet,254041424344 use of other prescription drugs, being overweight/obese, and having diabetes and hypertension.82540454647484950 Moreover, low serum or red blood cell folate5152 and/or raised homocysteine concentrations535455 are associated with an increased risk of both congenital heart defects and depression56575859; whereas, folate supplementation could lower the risk of congenital heart defects,60616263 diminish depressive symptoms,1012 and overcome drug resistant depression.12646566 Polymorphisms in genes involved in the folate metabolic pathway are also associated with an increased risk of congenital heart defects53676869 and depression.707172
Three metabolic pathways implicated in an increased risk of congenital heart defects—folate, homocysteine, and glutathione/transsulfuration pathways—could be related to pathways involved in synthesis of serotonin and other important neurotransmitters that influence depression. L-methylfolate, the biologically active form of folate, regulates the formation of tetrahydrobiopterin (BH4), which is involved in the synthesis of the neurotransmitters, serotonin, dopamine, and norepinephrine.1012 Because of the potential interplay between folate, homocysteine, depression, and congenital heart defects, we hypothesized that the impact of SSRIs on cardiogenesis could be modulated by common maternal or infant genetic variants in folate and homocysteine related pathways. Thus, it is plausible that maternal or infant genetic variants within these pathways augment the adverse effects of SSRIs if key protein or enzyme production is inhibited or amplified. We examined the potential impact of common allelic variants in maternal and infant genes involved in the folate, homocysteine, and glutathione/transsulfuration metabolic pathways on the association between maternal periconceptional use of SSRIs and risk of congenital heart defects in their offspring.
We tested our hypothesis with a case control design using data from the National Birth Defects Prevention Study (NBDPS). This was the largest population based case control study conducted in the US and was designed to investigate the contribution of genetic, environmental, and behavioral factors to the occurrence of major non-syndromic birth defects.7374 Briefly, families with affected children (cases) and control families were identified from population based birth defects surveillance systems in 10 US states, specifically Arkansas, California, Georgia, Iowa, Massachusetts, New Jersey, New York, North Carolina, Texas, and Utah. The study enrolled non-Hispanic white, non-Hispanic black, Hispanic, and Asian women and their families. Our analysis included non-Hispanic white, non-Hispanic black, and Hispanic families of infants with estimated dates of delivery from October 1997 to August 2008.
Our study population consisted of 1180 case families with singleton liveborn infants with a diagnosis of a conotruncal or right or left obstructive heart defect within the first year of life and 1644 control families with singleton liveborn infants without any diagnosed major structural birth defect who provided a genetic sample. Infants with congenital heart defects affected by a known single gene disorder, chromosomal abnormality, or syndrome were excluded. In each surveillance program, trained staff abstracted medical records and actively ascertained cases from hospitals, birthing centers, and other medical facilities. For NBDPS, controls were randomly selected (one control to three cases) by each program from the same underlying population area as case infants from birth certificate records or hospital discharge logs. The number of controls selected each month for the overall study was proportionate to the number of births in the same month in the prior year.74
Ascertainment of infants with congenital heart defects
Cases were identified based on the infant having at least one of the following diagnostic procedures: echocardiograms, surgical reports, cardiac catheterizations, or autopsies. A pediatric cardiologist at each study center then reviewed all information on diagnostic procedures to ensure uniform criteria for diagnoses. Classification of congenital heart defects was done centrally by pediatric cardiologists, using the classification system specifically developed for the NBDPS, which incorporates cardiac phenotype, cardiac complexity, and extra-cardiac anomalies.75 In our study, conotruncal defects included truncus arteriosus, interrupted aortic arch type B, transposition of the great arteries, double outlet right ventricle, conoventricular septal defects, tetralogy of Fallot, and pulmonary atresia with ventricular septal defect.75 Obstructive heart defects (left or right) included hypoplastic left heart syndrome, tricuspid atresia, pulmonary valve atresia, coarctation of the aorta, interrupted aortic arch types A and C, aortic stenosis, pulmonary valve stenosis, and Ebstein’s anomaly.75
Ascertainment of maternal use of SSRIs
After they had given informed consent, mothers of affected children and controls completed a one hour computer assisted telephone interview administered in English or Spanish.7376 During the interview, women were asked about periconceptional (that is, one month before conception through to three months after conception of the index pregnancy) folic acid supplementation and maternal drug use. For our study, we defined periconceptional folic acid supplementation as use for at least two months during the exposure window. SSRI use was defined as any use of the following medications: fluoxetine (Sarafem, Prozac, Prozac Weekly, Selfemra, Rapiflux), fluvoxamine (Luvox, Luvox CR), paroxetine (Pexeva, Brisdelle, Paxil, Paxil CR), escitalopram (Lexapro), citalopram (Celexa), sertraline (Zoloft), and vortioxetine (Brintellix), for any indication during the periconception period.
DNA sample collection
After completing the maternal telephone interview, case and control families were mailed buccal swab kits for collection of maternal, infant, and paternal DNA samples.7376 DNA sample storage and processing methods for the NBDPS have been described in detail elsewhere.7677 We included all cases and controls who had DNA samples available (mother, infant, and father, or any paired combination).
Genotyping and quality control
We selected a custom panel of 1536 single nucleotide polymorphisms from 62 genes involved in the folate, homocysteine, and glutathione/transsulfuration metabolic pathways or DNA synthesis/repair for analysis.78 We performed genotyping using 200 ng of whole genome amplified DNA on Illumina’s customizable GoldenGate platform.79 We uses Puregene DNA purification reagents (Qiagen, Valencia CA) to extract DAN from buccal brush samples. The resultant genomic DNA was quantified with TaqMan RNase P Detection Reagents Kit (Life Technologies, ThermoFisher) and a standard curve of DNA of known DNA concentration.
Genomic DNA extracted from buccal samples was subjected to whole genome amplification. Briefly, 10-15 ng of genomic DNA was amplified with the GenomePlex kit (Sigm Chemical, Saint Louis MO) according to the manufacturer’s protocol. The whole genome amplified product was quantified by the TaqMan RNase P Detection Reagents Kit, and 200 ng was used as input DNA for the Illumina Golden Gate assay. The performance of whole genome amplified DNA in microarray analysis was previously demonstrated by Cunningham and colleagues.80
Initial genotyping calls were generated with GenCall, Illumina’s proprietary algorithm. We found that the quality of genotype clustering varied substantially from one single nucleotide polymorphism to another on the GoldenGate custom platform, whether we used blood or buccal samples. We therefore developed SNPMClust, a bivariate Gaussian model based genotype calling algorithm to complement the default Illumina GenCall algorithm and applied strict quality control measures.81 We tested this method in a pilot study (described in detail by Hobbs and colleagues68) on blood and buccal samples from 94 women. We found a 99.2% concordance rate between whole genome amplified buccal DNA and blood derived DNA among genotype calls and are therefore confident in the genotypes we used.
We genotyped 6416 individual samples and removed 576 samples from analysis because of study ineligibility, low genotype call rates, or high rates of Mendelian inconsistency, resulting in an analytic sample consisting of 5840 individuals from 1180 case and 1644 control families. To ensure high quality genetic data, we applied stringent quality control criteria and excluded single nucleotide polymorphisms with obviously poor clustering behavior, no call rates >10%, Mendelian error rates >5%, minor allele frequencies <5%, or significant deviation from Hardy-Weinberg equilibrium in at least one racial group (P<10−4). After the quality assessment, the analytical data had 872 variants in total.
We calculated summary statistics for main study variables: means with standard deviations for continuous variables and frequencies with percentages for categorical variables. We compared the distributions of continuous variables between cases and controls using independent two sample t tests and the proportions of categorical variables using χ2 tests. Log linear models based on the hybrid design82 were used for analysis as we genotyped both triads of case-parents and control-parents; this method is the most optimal and appropriate statistical approach.82 The log linear model was fitted for each single nucleotide polymorphism as a function of: mating types (three possible genotypes for each parent); the interaction between mating types and SSRI exposure; disease status (case/control); maternal genotypes; infant genotypes; the interaction between disease status and maternal genotypes; the interaction between disease status and infant genotypes; the interaction between disease status, maternal genotypes, and SSRI exposure; and the interaction between disease status, infant genotypes, and SSRI exposure. This model specification assessed the interaction between maternal genotype and SSRI exposure and the interaction between infant genotype and SSRI exposure. We assumed a log additive model (multiplicative risk of alleles), in which the genotype was coded as 0, 1, and 2, indicating zero, one, and two copies of the minor allele. Under this assumption, the relative risk of carrying two copies of the allele is the square of the relative risk of carrying one copy of the same allele, with zero copies as the reference.
We also calculated the Bayesian false discovery probability (BFDP)83 for the interaction between maternal genotype and SSRI exposure and the interaction between infant genotype and SSRI exposure using the interaction estimates from the log linear models. This probability balances the relative cost between a false negative and a false positive finding; in the results section, we report associations where probability <0.8 and 95% confidence intervals where α=0.05. Probability <0.8 is widely used to indicate noteworthy findings in candidate gene based association studies with strong prior knowledge that the selected genes are likely to be associated with the disease.848586 We used statistical software R v3.1.0 (R Foundation for Statistical Computing, Vienna) for computing summary statistics, performing tests, and calculating Bayesian false discovery probabilities. LEM87 statistical software was used to fit log linear models with incomplete family genotype data by using the expectation maximization algorithm.888990
The research question and outcome measures were not informed by participant priorities, experiences, or preferences. Participants were not involved in the design, recruitment process, or conduct of this study. Findings from this study and all publications that use NBDPS data are available to participants on www.nbdps.org.
Of 1180 affected infants (cases), 616 had conotruncal defects and 569 had obstructive heart defects. There were no significant differences in sociodemographic or maternal periconceptional characteristics between cases and controls, including folic acid supplementation and prenatal maternal cigarette smoking (tables 1 and 2⇓), with the exception of maternal body mass index (BMI). Case mothers were more likely to be overweight and obese than control mothers (P<0.002). Overall the prevalence of SSRI use during the periconceptional period was relatively low. Only 5% of case and 3% of control mothers reported SSRI use during the relevant period, but the difference was significant (P<0.04). The prevalence of SSRI use did not differ between mothers of infants with a conotruncal or obstructive heart defect. The SSRIs most commonly taken during the periconception period by women in our study were sertraline (38.7%), fluoxetine (27.0%), and paroxetine (21.6%). No one reported using fluvoxamine or vortioxetine (table 3⇓).
Maternal genotype and risk of congenital heart defects
We included 872 single nucleotide polymorphisms within 60 genes in the final analyses. There was a significant (BFDP <0.8) increased risk of congenital heart defects for maternal genotypes for four single nucleotide polymorphisms in three genes—serine hydroxymethyltransferase 1 (soluble) (SHMT1), betaine-homocysteine S-methyltransferase (BHMT), and microsomal glutathione S-transferase 1 (MGST1)—among women taking SSRIs during the periconceptional period (table 4⇓). Overall, the risk of having an infant with a congenital heart defect was higher for women with homozygous minor alleles than for women with only one copy of the minor allele. Among women reporting periconceptional use of SSRIs during pregnancy, those who had one or two minor allele (G) copies of rs9909104 in SHMT1 had increased risks of congenital heart defects in offspring compared with exposed women who carried no copies (relative risk 2.43 (95% confidence interval 1.46 to 4.03) and 5.90 (2.13 to 16.24), respectively). SSRI users who were heterozygous (AG allele) for two single nucleotide polymorphisms on the BHMT gene had an increased risk of a congenital heart defect if they took SSRIs during the periconception period (2.15 (1.33 to 3.46) for rs492842 and 2.06 (1.26 to 3.34) for rs542852) compared with women who did not take SSRIs during the periconception period and had the AA allele. SSRI users who were homozygous for the G allele of the rs492842 and the rs542852 single nucleotide polymorphisms had over four times the risk of having an affected child (4.62, 1.77 to 11.97). The greatest risk of having an affected child was found in women who took SSRIs during the periconception period and had the CC and AC alleles for rs2075237 on gene MGST1 (7.95 (2.50 to 25.40) and 2.82 (1.58 to 5.04), respectively) compared with women who had the AA allele.
Infant genotypes and risk of congenital heart defect
We also observed a significant increased risk of congenital heart defects for infant genotypes (table 4⇑). We observed increased risk in three single nucleotide polymorphisms in three genes involved in the transsulfuration pathway: O-6-methylguanine-DNA methyltransferase (MGMT), glutathione S-transferase pi (GSTP1), and microsomal glutathione S-transferase 1 (MGST1). Infants who had the AG allele in single nucleotide polymorphisms rs11511217 on gene MGMT, rs7941395 on GSTP1, and rs7294985 on MGST1 also had increased risk of congenital heart defects if their mothers took SSRIs during the periconceptional period (relative risks 2.41 (95% confidence interval 1.45 to 4.01), 2.17 (1.38 to 3.41), and 2.47 (1.45 to 4.21), respectively). Infants with periconceptional exposure to SSRIs who had the GG allele on the same single nucleotide polymorphisms had increased risks of congenital heart defects, ranging from 4.71 to 6.10. The greatest risk for having an infant with a congenital heart defect occurred in women who took SSRIs during the periconception period and had an infant with the AA and AC alleles for rs12438477 on 5,10-methenyltetrahydrofolate synthetase (MTHFS) gene (6.76 (2.56 to 17.89) and 2.60 (1.60 to 4.23), respectively) compared with an exposed infant with the CC allele. Increased risks of congenital heart defects were also seen in infants with variants in single nucleotide polymorphisms in two genes involved in the homocysteine pathway, tRNA aspartic acid methyltransferase 1 (TRDMT1) and glycine N-methyltransferase (GNMT). Women had about twice the risk of having an infant with a congenital heart defect if she was exposed to a periconceptional SSRI and had an infant with the allele AC on single nucleotide polymorphism rs66022178 in the TRDMT1 gene or the CG allele on single nucleotide polymorphism rs11752813 in the GNMT gene (1.79 (1.14 to 2.80) and 1.80 (1.15 to 2.80), respectively). For both single nucleotide polymorphisms in the respective genes, the risks were 3.2 (1.30 to 7.84 and 1.32 to 7.84) for the CC alleles.
We conducted subanalyses to determine whether risk varied by subgroup of congenital heart defect (table 5⇓). We did not find any significant risk ratios among conotruncal heart defects (as determined by BFDP) but did find increased risk by maternal and infant genotype for obstructive heart defects among periconceptional SSRI users. The risk was evident only for single nucleotide polymorphisms on the TRDMT1 gene. The risk ratio for women who took an SSRI during the periconception period and had the AG or GG genotype for polymorphism rs2038576 in the TRDMT1 gene was 4.64 (95% confidence interval 2.20 to 9.77) and 21.53 (4.84 to 95.45), respectively) compared with exposed women with the AA genotype. For women taking SSRIs during the periconception period with the AG or GG genotype for polymorphism rs2273735 in the TRDMT1 gene the relative risks were 3.20 (1.67 to 6.10) and 10.24 (2.79 to 37.21) compared with women with the AA genotype exposed to SSRIs.
Similar to our findings for maternal genotypes, increased risk was observed only for single nucleotide polymorphisms in the TRDMT1 gene for infant genotypes. Relative risks ranged from 2.61 to 3.00, and the greatest risk was for infants with the AG on polymorphism rs10795459 (relative risk 3.0, 95% confidence interval 1.63 to 5.51). Risks ranged from 6.81 to 9.00 for the homozygous alleles on the respective single nucleotide polymorphisms.
In this population based study we found that common single nucleotide polymorphisms, related to glutathione and one-carbon metabolism, modified the risk of birth defects such that mothers with specific genotypes and periconceptual SSRI use had more than twice the risk of having an infant with an obstructive or conotruncal heart defect. Likewise, infants with maternal SSRI exposure and specific genotypes had 1.79 to 2.60 times the risk of the included heart defects. A subgroup analysis showed that two maternal and three infant single nucleotide polymorphisms, each within TRDMT1, were strongly associated with development of an obstructive heart defect. In contrast, we found no interaction among genotype, SSRI exposure, and risk of conotruncal heart defects. These findings provide insight into the genetic susceptibility of congenital heart defects to maternal SSRI use around pregnancy and provide support for our effect modification hypothesis.
Strengths and limitations
Strengths of our study include its large multi-ethnic population based sample size, verification of diagnoses, and standardization of classification of congenital heart defects across study centers. Unlike many previous investigations of SSRI use and risk of congenital heart defects, our information on exposure was based on maternal self report, which is a better measure of drugs taken rather than drugs prescribed during the exposure window. Furthermore, the use of the log linear model for our analyses enabled us to assess the simultaneous effects of maternal and infant genotype on risk of congenital heart defects among SSRI users and non-users. This analytic approach also controls for possible population stratification of the infant’s genotype.
Some potential limitations, however, should be considered. Information concerning periconceptional SSRI use could be subject to measurement error and recall bias as women were asked 12-24 months after delivery to report use. We also examined the effects of all SSRIs combined and did not assess the effects of specific SSRIs. Although we examined the use of periconceptional folic acid supplementation we did not assess dietary intake of folate and its role on the association between SSRI use and risk of congenital heart defects in our population. The DNA used in our study was obtained from self collected buccal cheek cell samples, which resulted in variable quality of samples. Although the hybrid design is the most appropriate method for analyzing our trio data, a current limitation of this statistical approach is inability to include potential confounders in the model. Despite our fairly large sample size, only a small number of women took SSRIs during the periconception period. Although the log linear model adjusts for potential confounding by population stratification, we assessed our results only among non-Hispanic white mothers because 85% of SSRI users fell into this ethnic group. Results from this homogenous subgroup were similar to those from the full sample, including significant associations for the maternal single nucleotide polymorphisms on SHMT and BHMT and infant single nucleotide polymorphisms on MTHFS (table 6⇓).
Comparison with previous studies
We found no directly comparable studies when we searched PubMed for studies published in English before 2016 that investigated the association between maternal or infant genotype and risk of congenital heart defects among women taking SSRIs during pregnancy. We have therefore provided comparisons with the broader literature. An earlier study also using data from the NBDPS did not find a significantly increased risk of conotruncal defects (odds ratio 1.3, 95% confidence interval 0.8 to 2.1) among women taking an SSRI during pregnancy.3276 A 2015 study using 1997-2009 data from the NBDPS, however, reported significant associations between maternal use of the SSRIs fluoxetine or paroxetine and atrial septal defects (1.8, 1.1 to 3.0) and right ventricular outflow tract obstruction defects (2.4, 1.4 to 3.9).76 An earlier case control study conducted in the US also reported similar findings.39 A large Norwegian study showed an increased risk of congenital heart defects among infants exposed to any SSRI during early pregnancy (1.15, 1.05 to 1.26) and reported a significant association between SSRI exposure and right ventricular outflow tract obstructions (1.48, 1.15 to 1.89).91 A sibling controlled analysis within the Norwegian study, however, did not find a significant association between SSRI exposure and congenital heart defects. In our study, over half of the women who took SSRIs were taking sertaline or fluoxetine, both of which show little evidence for increased risk of congenital heart defects in empiric studies or meta-analyses of epidemiologic studies,73536 yet we still observed an increased risk of such defects among women and infants with common allelic variants.
Our findings highlighted single nucleotide polymorphisms related to one-carbon metabolism and the transsulfuration pathway. One-carbon metabolism encompasses both the folate and methionine/homocysteine cycles and is integral to cardiac development.92 It supports nutrient metabolism and other processes vital to a developing heart. It supplies methyl groups to DNA, RNA, and chromatin, allowing the body to maintain tight control over gene transcription during cardiogenesis. The transsulfuration pathway is another important component of one-carbon metabolism. One of its most important byproducts is glutathione,929394 an antioxidant that helps the developing heart resist damage from reactive oxygen species and xenobiotic compounds.6895 The single nucleotide polymorphisms reported in this study could serve as markers for nearby functional variants (a genetic variant that alters the function of the gene). Alternatively, they could directly affect protein expression and function. Most of the single nucleotide polymorphisms identified in the results introduce new splice sites and could alter protein function as a result (rs9909104, rs492842, rs542852, rs11511217, rs7294985, rs2038576, rs2273735, rs17464824, rs10904893). The remaining polymorphisms do not seem to directly impact protein function. Instead, they could be marking nearby functional variants.
There are several mechanisms that could account for the results. Variants in two genes, SHMT1 and MTHFS, within the folate pathway were associated with an increased risk of congenital heart defects among women with SSRI exposure during early pregnancy. Both genes encode proteins that catalyze the conversion of folate metabolites into 5,10-methylenetetrahydrofolate, a folate pathway intermediate that provides methyl groups for biosynthesis of thymidylate and purines.96 Neither SHMT1 nor MTHFS participate in the synthesis or turnover of 5-HT (serotonin), and it seems unlikely that these two single nucleotide polymorphisms interact directly with 5-HT during cardiac development. We hypothesize that variants within these genes sensitize an embryo to alterations in 5-HT, a critical cardiac signaling molecule.9798 SHMT1 and MTHFS are both vital to embryonic development, and defects in SHMT1 can impair hippocampal neurogenesis99 and cause neural tube defects in mouse models.100 The polymorphism identified in SHMT1 (rs9909104) is associated with increased risk of ovarian cancer,101 and a second polymorphisms (rs669340) within SHMT1 is associated with development of neural tube defect.102 Meanwhile, mice lacking the MTHFS gene do not survive past the embryonic stage, and mice with only one functional allele showed a 50% reduction in de novo purine synthesis.103 MTHFS rs12438477 was associated with conotruncal heart defects in one of our previous studies.68 Our present study identified an association between rs12438477 within MTHFS and an increased risk of congenital heart defects among infants exposed to SSRIs in pregnancy.
Allelic variants in three genes involved in the homocysteine pathway—BHMT, GNMT, and TRDMT1—were also associated with increased risk of congenital heart defects in the presence of periconceptional SSRIs. BHMT facilitates homocysteine re-methylation into methionine.104 The enzyme encoded by GNMT is critical in regulating cellular concentration of S-adenosyl methionine (AdoMet).105 TRDMT1 (formerly DNMT2) encodes a methyltransferase that stabilizes aspartic acid and transfer RNA and supports protein synthesis.106 Deletion of BHMT in mice leads to major perturbations in choline, homocysteine, and AdoMet. In humans, polymorphisms in BHMT were associated with decreased circulating concentrations of the antioxidant glutathione.68 Excess homocysteine is thought to cause endothelial damage in adults107and is associated with increased risk of congenital heart defects in newborns.10108109 Evidence from animal models suggests that homocysteine might increase risk of congenital heart defects either by increasing oxidative stress110 or by binding to N-methyl-D-aspartate (NMDA) receptors, resulting in abnormal cardiac neural crest cell migration and differentiation.111
We found three genes involved in the transsulfuration pathway—MGST1, MGMT, and GSTP—that were associated with an increased risk of congenital heart defects among women taking SSRIs. MGST1 is involved in “conjugation of glutathione to electrophiles and the reduction of lipid hydroperoxides” (RefSeq Accession NM_145792.2). This reaction is one of the mechanisms by which glutathione, a product of the transsulfuration pathway, protects intracellular membranes from oxidative stress. The GST gene family produces enzymes involved in detoxification by catalyzing the conjugation of many hydrophobic and electrophilic compounds with reduced glutathione. The GSTP1 gene is believed to catalyze the detoxification of xenobiotics (chemical compounds—as drugs, pesticides, or carcinogens—that are foreign to a living organism)112 and byproducts of oxidative stress; they could play a role in susceptibility to cancer and other diseases, as this gene is often upregulated in neoplastic cells (RefSeq Accession NM_000853.3).
Dysfunction in either the transsulfuration pathway or the homocysteine pathways can lead to an increased level of oxidative stress.110 Oxidative stress can in turn decrease the bioavailability of 5,6,7,8-tetrahydrobiopterin (BH4),113 a critical precursor for 5-HT (serotonin). Increased oxidative stress in the presence of 5-HT can also promote the formation of harmful free radicals, which could damage developing cardiac tissue.114115 Although the teratogenic effects of SSRIs on the fetal heart are postulated to occur through interference of 5-HT signaling during cardiogenesis,11 the specific role of the common variants and/or genes is poorly understood in these three metabolic pathways in the association between depression, metabolism of SSRIs, and increased risk of congenital heart defects. These genes, however, have previously been investigated for their role in risk of congenital heart defects. Hobbs and colleagues reported a 1.8 increased risk of congenital heart defects among obese women carrying the 742G>A polymorphism in BHMT,116 and Mitchell and colleagues also showed a similar pattern of increased risk for left sided of congenital heart defects for the same polymorphism, though not significant.117 Li and colleagues found significant main effects for different fetal polymorphisms in the MGMT and MTHFS genes.118 Zhu and colleagues investigated rs9909104 on SHMT1 for its association with increased risk of congenital heart defects but found no association.119
These complex relations could partially explain the conflicting epidemiologic literature concerning the teratogenicity of SSRI use during the periconception period as studies that do show an association do not distinguish between pharmaceutical effects and the underlying effects of depression and its associated behaviors and conditions. Several investigators posited confounding by indication to explain the reported associations in the literature.4120 One large US epidemiologic study that restricted the study population to depressed women (as opposed to adjusting for depression) to account for potential confounding by indication and associated behaviors found no association between prenatal SSRI exposure and risk of congenital heart defects.4 A nationwide study in Denmark found an increased risk of such defects for women taking SSRIs during pregnancy and for women who stopped taking SSRIs six months before conception of the index pregnancy,92 which also supports the potential confounding effect of maternal illness. Although recent cohort studies conducted in the US4 and the UK121 reported significant associations between SSRI use (paroxetine) and congenital heart defects, the associations were not significant in fully adjusted models restricted to women with depression. We hypothesized that some women might have an increased genetic susceptibility to possible adverse effects of SSRIs on the developing heart. Polymorphisms in genes involved in folate metabolism are associated with increased risk of congenital heart defects6878116122123124125126127128 and depression.7071129 Thus, we postulate that some women who take SSRIs during cardiogenesis could be at higher risk if they have genetic polymorphisms in other folate metabolism related genes.
Future directions and conclusions
Our results provide initial evidence of a modifying effect of common maternal and infant variants in genes that encode for critical enzymes in the folate, homocysteine, and glutathione/transulfuration pathways on the association between maternal periconceptional SSRI use and risk of congenital heart defects in their offspring. Given the widespread use of SSRIs among women for various conditions, further exploration is warranted of the complex association between risk of congenital heart defects and folate supplementation, periconceptional SSRI use, and variants in folate, homocysteine, and glutathione/transsulfuration pathways among women with psychiatric illness.
What is already known on this topic
Epidemiologic studies have produced inconsistent results regarding the impact of the maternal use of SSRIs during embryogenesis on congenital heart defects
These inconsistent results could be a consequence of a complex relation between competing risk factors, behaviors, and metabolic pathways for both mental illness and congenital heart defects
Low serum or red blood cell folate and/or raised homocysteine concentrations are associated with an increased risk of both congenital heart defects and depression; whereas folate supplementation could lower the risk, diminish depressive symptoms, and overcome drug resistant depression
What this study adds
Common allelic variants in maternal and infant genes involved in the folate, homocysteine, and glutathione/transulfuration metabolic pathways can modify the association between maternal periconceptional use of SSRIs and risk of congenital heart defects
We thank the families who participated in the National Birth Defects Prevention Study and made this research study possible. We also thank the Centers for Birth Defects Research and Prevention in California, Georgia, Iowa, and Texas for generously contributing their DNA samples for these analyses and Laura Rakes for her tremendous assistance in preparing this manuscript.
Contributors: WNN, ZS, and DW designed the study. XT and ZH did the statistical analysis. SMacL developed the genotyping. All authors developed the manuscript. WNN is guarantor.
Funding: The study received funding from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (#5R01HD039054-12), the CDC National Center on Birth Defects and Developmental Disabilities (#5U01DD000491-05), and the Arkansas Biosciences Institute. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention. The study funders played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. The researchers are independent from the funders, and all authors, external and internal, had full access to all of the data (including statistical reports and tables) in the study and can take responsibility for the integrity of the data and the accuracy of the data analysis.
Competing interests: All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: no financial relationships with any organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.
Ethical approval: This study was approved by the University of Arkansas for Medical Sciences institutional review board, and the National Birth Defects Prevention Study (NBDPS) was approved by the institutional review boards at the Centers for Disease Control and Prevention and all NBDPS institutions. All study participants provided written informed consent.
Data sharing: No additional data available.
Transparency: The lead author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.
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