Placental HTR2A methylation is associated with infant neurobehavioral outcomes

Abstract

The serotonin receptor, HTR2A, exhibits placental expression and function and can be controlled through DNA methylation. The relationship between methylation of HTR2A in the placenta and neurodevelopmental outcomes, evaluated using the NICU Network Neurobehavioral Scales (NNNS), was assessed in newborn infants (n = 444). HTR2A methylation was significantly higher in males and marginally higher in infants whose mothers reported tobacco use during pregnancy. Controlling for confounding variables, HTR2A methylation was negatively associated with infant quality of movement (p = 0.05) and positively associated with infant attention (p = 0.0001). These results suggest that methylation of the HTR2A gene can be biologically and environmentally modulated and is associated with key measures of neurodevelopment.

Introduction

Increasing evidence suggests that the fetal environment has a strong influence on lifelong physical and mental health by altering programming of the developing brain.¹ In a short window of time during early prenatal development, neural networks are formed and behavioral pathways are programmed, and this programming is vulnerable to environmental influences.² Environmental factors, including maternal stress, mood, and lifestyle, have been linked to altered neurodevelopmental programming and later life behavioral and neurologic outcomes,^3,4 but the molecular mechanisms underlying these relationships remain unclear.⁵

The fetal environment is modulated by the placenta, which produces growth factors, neuropeptides and components of monoamine transporter systems, and has been described as a “third brain” linking the mother and infant.⁶ The placenta is a plastic organ and can quickly adapt to changes from the maternal environment through epigenetic mechanisms. One such mechanism is promoter DNA methylation, which alters the transcription or transcriptional potential of genes.⁷ DNA methylation in the placenta is dynamic⁸ and can be altered based on environmental cues. Such alterations have the potential to alter gene expression, affecting placental function and thereby influencing development.

The serotonin response pathway plays an important role in neurodevelopment by modulating neuronal events, including cell division, migration and cell differentiation.⁹ Modulation of serotonin signaling through maternal use of selective serotonin reuptake inhibitors (SSRIs) has been linked to alterations in neural development in both epidemiologic and mechanistic studies.¹⁰ Alterations to serotonergic tone have also been shown to alter brain development in vivo.¹¹ The fetus develops a full serotonin response pathway before it can produce its own serotonin¹² and relies on maternal- and placental-derived serotonin during early development,¹³ suggesting an important role for this neurotransmitter within the placenta in during early neurodevelopment.

The placenta is emerging as a key mediator of serotonergic signaling, and additional functional elements of this pathway are intact within this transient organ, including the HTR2A gene, which encodes the G-protein coupled, 5-hydroxytryptamine (serotonin) receptor 2A. The HTR2A gene is expressed in both choriocarcinoma and normal human placental tissue¹⁴ and acts as a mitogen.¹⁵ However, the physiologic role of the serotonin receptor in the placenta has yet to be elucidated. The effect of the placental mitogenesis resulting from HTR2A receptor stimulation has been predicted to have an effect on placental implantation,¹⁵ which is a crucial first step in fetal development. Low levels of placental serotonin have been hypothesized to alter the serotonin concentration in the fetal forebrain, leading to mis-wiring of this region and alterations in the serotonergic system.¹⁶ Genetic variation in the HTR2A receptor has been linked to internalizing traits in infant populations, suggesting it plays a role in the development of infant neurobehavioral characteristics.¹⁷ The role of HTR2A in other developmental pathways within the placenta remains unexplored.

Previous studies have analyzed HTR2A DNA methylation in adult populations in relation to poor mental health outcomes. Aberrations in HTR2A promoter methylation in peripheral blood mononuclear cells are associated with chronic fatigue syndrome,¹⁸ and in post mortem brain samples, HTR2A methylation, which was associated with decreased expression, was higher in schizophrenic patients compared with controls.¹⁹ However, no existing study has analyzed methylation of the promoter region of HTR2A in placental tissue or explored the role of DNA methylation in this region in relation to fetal development or newborn neurobehavior. The objective of this study was to analyze the DNA methylation status of the HTR2A promoter region in placental samples from infants involved in an ongoing birth cohort study and to determine the relationship between factors in the fetal environment, methylation and infant neurodevelopment quantified with the validated NICU Network Neurobehavioral Scales (NNNS).

Results

Demographic characteristics of the 444 infants that were studied are shown in Table 1 (categorical variables) and Table 2 (continuous variables). All infants were at or near term gestation, with a mean gestational age of 39 weeks. There was a slightly higher proportion of females compared with males (51% vs. 49%). Based on medical record reports, nearly 5% of mothers reported tobacco use during pregnancy, 2% reported alcohol use, 13% of mothers reported anxiety during pregnancy and 14% reported depression. These data are consistent with the prevalence of these exposures and conditions in other cohorts.²⁰

Table 1. Categorical characteristics of study sample and their association with HTR2A promoter methylation.

	N	%	HTR2A methylation: mean (SD)	p
Infant gender				0.0005
Female	228	51	35.72 (8.13)
Male	216	49	38.63 (9.31)
Infant race				0.79
White	291	66	37.06 (8.86)
Nonwhite	145	33	37.39 (8.93)
Unknown	8	1	35.34 (6.85)
Maternal education				0.25
Less than 11th grade	37	8	38.69(11.19)
High school graduate	85	19	38.11 (7.82)
Junior college or equivalent	114	26	37.46 (9.65)
College graduate	144	31	36.68 (8.03)
Post-graduate education	62	14	35.31 (8.76)
Unknown	2	<1
rs6311				0.57
AA	83	19	36.3 (6.41)
AG	215	48	37.16 (9.06)
GG	146	33	37.59 (9.67)
Birth weight group				0.57
SGA	95	21	37.46 (8.86)
AGA	225	51	36.71 (8.01)
LGA	124	28	37.67 (10.18)
Depression pregnancy				0.86
Yes	61	14	37.01 (7.45)
No	374	84	37.19 (9.06)
NA	9	2
Anxiety/OCD/panic pregnancy				0.49
Yes	57	13	36.57 (6.37)
No	377	85	37.25 (9.18)
NA	10	2
Alcohol pregnancy				0.5
Yes	7	2	42.71 (11.99)
No	434	97	37.10 (8.81)
NA	3	<1
Tobacco pregnancy				0.088
Yes	22	5	41.28 (11.21)
No	414	93	36.94 (8.65)
NA	8	2

SD, standard deviation.

Table 2. Continuous clinical characteristics, NNNS descriptive information and correlations with HTR2A promoter methylation.

	N	Mean	Median	SD	Min	Max	Pearsons correlation coefficient with HTR2A mean methylation	p
Infant continuous variables
HTR2A mean methylation	444	37.14	36.44	8.83	12.97	89.85
Birth weight (g)	444	3461	3450	685.62	1705	5465	<0.01	0.94
Gestational age (weeks)	444	39.05	39	0.96	37	41	<0.01	0.84
Maternal age (y)	442	29.13	30	5.63	18	40	−0.09	0.06
Infant NNNS scores
Habituation*	210	7.19	7.5	1.29	2	9	−0.05	0.51
Attention†	336	4.1	4	1.26	1.57	7.57	0.18	0.0008
Handling	375	0.35	0.25	0.24	0	1	−0.022	0.67
Quality of movement†	374	4.08	4.17	0.65	1.8	5.5	−0.12	0.02
Stress abstinence	375	0.18	0.18	0.07	0	0.41	−0.06	0.21
Arousal	375	4.16	4.14	0.81	1.86	6.33	0.04	0.42

^*Scale requires infants to be in a sleep state at beginning of assessment. ^†Scales require a minimum number of items. SD, standard deviation.

Three specific CpGs, selected based on a prior study¹⁸ were analyzed. These CpGs are located 1439, 1420 and 1224 bp upstream of the HTR2A gene (Chr 7: 28415063-28414801). The presence of the first CpG was dependent on the SNP rs6311, an A/G polymorphism with a minor allele frequency of 0.43 in this population. Because of the prevalence of this SNP and since the A allele would lead to loss of the potential site of methylation (CA vs. CG) in a substantial proportion of the population, we did not include this CpG in further analysis. The mean methylation of the two remaining CpGs within this region was 37.1% (range 13.0–89.9%, SD = 8.8%), and there was no difference in the mean methylation of the remaining sites by genotype at rs6311 (A/A = 36.3%, A/G = 37.2%, G/G = 37.6%, p = 0.57, ANOVA).

Males had a statistically significantly higher mean methylation within this region (38.6% compared with 35.7% in females, p = 0.0005). Psychosocial factors that could affect HTR2A mean methylation were also examined, and the mean methylation by these factors is shown in Table 1. Medically reported anxiety or depression during pregnancy had no significant relationship with HTR2A promoter methylation. We also observed no significant difference in methylation among those who reported using alcohol during pregnancy. There was a marginally significant relationship between smoking and HTR2A promoter methylation; smokers had a 4.3% higher mean methylation (41.3% vs. 36.9%).

We observed no significant correlations between HTR2A methylation and the continuous characteristics shown in Table 2. NNNS summary scores are missing for some infants due to the nature of the examination, which requires infants to be in a certain state for some assessments. We identified a significant positive correlation between methylation and infant attention score (p = 0.0008) and a significant negative correlation between methylation and quality of movement (p = 0.02), but no correlations with the other NNNS summary scores examined.

We fit linear regression models to examine the relationship between HTR2A promoter methylation and infant attention and quality of movement NNNS scores (Table 3) controlled for potential confounders (maternal education, gestational age, birth weight, gender and smoking status). Controlling for confounders, the level of significance was attenuated (adjusted p = 0.05) for the relationship between methylation and quality of movement, although the effect estimate was not affected. We observed an enhancement in the statistical significance between HTR2A methylation and infant attention score when controlling for the same confounding variables, while the effect estimate remained the same (adjusted p = 0.0001, β = 0.03). In unadjusted models, male infants had a negative, but not statistically significant, association with attention scores compared with females (β = −0.2, p = 0.15); however, in the adjusted model, this relationship became stronger and statistically significant (β = −0.28, p = 0.05). Gestational age also became significant in the fully adjusted attention model (p = 0.02).

Table 3. Linear regression models for the association of mean HTR2A methylation and infant attention and quality of movement.

	Quality of movement						Attention
	Unadjusted			Adjusted			Unadjusted			Adjusted
	(β) Estimate	SE	p	(β) Estimate	SE	p	(β) Estimate	SE	p	(β) Estimate	SE	p
HTR2A mean Methylation	−0.01	0	0.02	−0.01	<0.001	0.05	0.03	0.01	0.008	0.03	0.01	0.001
Infant race
Non-white	Ref.
Unreported	0.02	0.25	0.95	−0.04	0.26	0.89	−0.22	0.49	0.65	−0.12	0.49	0.81
White	0	0.07	0.99	−0.04	0.08	0.6	−0.18	0.15	0.24	−0.14	0.15	0.36
Maternal Education
Less than 11th grade	−0.06	0.13	0.66	−0.03	0.14	0.83	0.18	0.28	0.51	0.07	0.28	0.8
High school grad	−0.08	0.09	0.39	−0.08	0.1	0.44	−0.01	0.2	0.95	−0.03	0.2	0.9
Junior college grad	0.02	0.09	0.84	0.01	0.09	0.93	−0.1	0.18	0.6	−0.13	0.18	0.47
College grad	Ref.
Post-college grad	0.12	0.11	0.26	0.11	0.11	0.33	0.08	0.22	0.73	0.18	0.22	0.42
Gestational age (weeks)	0.03	0.03	0.38	0.02	0.04	0.51	0.13	0.07	0.08	0.18	0.07	0.02
Birth weight (g)	<0.001	<0.001	0.2	<0.001	<0.001	0.29	<0.001	<0.001	0.2	<0.001	<0.001	0.15
Gender
Female	Ref.
Male	−0.03	0.07	0.65	−0.01	0.07	0.92	−0.2	0.14	0.15	−0.28	0.14	0.05
Tobacco use during pregnancy
No	Ref.
Unknown	0.36	0.25	0.14	0.38	0.25	0.13	−0.03	0.48	0.95	−0.17	0.48	0.72
Yes	−0.17	0.17	0.31	−0.09	0.17	0.62	−0.35	0.33	0.3	−0.5	0.34	0.14

Tobacco use during pregnancy had moderate correlations with methylation within our cohort, and is known to effect NNNS outcomes.²¹ Therefore, we examined whether use of tobacco during pregnancy modified the relationship between HTR2A methylation and the NNNS attention score. Multiplicative interaction between tobacco use during pregnancy and methylation was assessed by fitting the linear model with an interaction term and showed a trend toward statistical significance (p = 0.07, likelihood ratio test).

Discussion

Increasing evidence suggests that the intrauterine environment can modulate neurodevelopment through alteration of the function of the placenta by epigenetic mechanisms.^22-25 In this study, we demonstrated that there is variability in the extent of DNA methylation of the promoter region for the HTR2A gene, which is associated with alterations in gene expression.¹⁹ This receptor has been noted to serve a mitogenic role in placental cells, and there may be additional physiologic roles for this receptor and general serotonergic tone within the placenta during development, particularly when this tissue acts as a source of serotonin. This study provides evidence from a human population study that links DNA methylation at this promoter and infant neurobehavior, and provides impetus for further analysis to elucidate the mechanism of placental HTR2A gene expression within the placenta and its role in neurodevelopment.

Specifically, we observed a significant positive correlation between HTR2A promoter methylation and the NNNS attention score. For each increase by 1% in HTR2A methylation, we note an increase in attention of 0.03, which appears only moderate in effect. Yet, the range of methylation in this population was large, from 13% to nearly 90%, so a change across this range would represent an increase of greater than 2 in the infant attention score, which represents 25% of the full range of scores. The infant attention score characterizes the infant’s quality of attention and ability to track and follow auditory and visual stimuli. Prior studies have shown that infants with higher attention scores generally scored higher on the Bayley’s developmental scores, and were more prepared to start school.²⁶ However, limited evidence suggests that high levels of infant attention may also be maladaptive. This increased attention could result in increased anxiety and difficulties with focusing on a task later in life.²⁷ The tipping point where high levels of attention could go from a positive adaptive quality to a maladaptive quality remains unclear at this time.

After correcting for confounding variables, we observed a negative relationship between HTR2A promoter methylation and infant quality of movement scores, with a 1% increase in methylation associated with a reduction of 0.01 on the quality of movement score. Again, across the full range of methylation, this could represent a change of 0.7, or nearly 20% of the full range, which would represent a substantial reduction. This summary score describes the infant’s smoothness of motor control, characterized by a lack of tremors. Having poor muscle tone at birth may predict non-optimal motor development later in life. Prior studies in at-risk infants have linked poor quality of movement with lower scores on the Psychomotor Developmental Index component of the Bayley scales at 2 y old.²⁶ Since the serotonergic tone of the fetal environment is an important part of fetal development and guides the formation of fetal brain, alterations to the serotonergic pathway during early development, including the placental HTR2A, could alter the amount and timing of serotonin reaching the infant during gestation, thus altering the optimal development of the nervous system. Additional mechanistic studies are needed to fully inform the physiologic role of this receptor in the placenta and its role in neurodevelopment.

Taken together, the patterns of associations between HTR2A promoter methylation and NNNS scores noted in this study, with a positive relationship identified with attention and negative with quality of movement may seem counterintuitive. It is important to note that these are two independent developmental domains covering cognitive and motor control. The positive correlation between methylation and attention may represent the fetus adapting to some levels of increased stress in utero and thus preparing itself for that environment following birth. The slight reduction of movement quality with methylation we observed could suggest that this domain may be less adaptable to the environment. This pattern is similar to what has been observed within the same infant population examining methylation of the HSD11B2 promoter region.²³ This data supports the “predictive response model,” which reveals that some level of in utero stress may provide the fetus with adaptive advantages later in life.^28,29 For example, low levels of maternal stress have recently been specifically linked to higher developmental scores in animal studies.³⁰ Further research is needed to elucidate the point at which adaptation is no longer possible and that these epigenetic modifications become maladaptive, as these would be the infants most critical to target for intervention.

We observed significant differences in the extent of methylation of HTR2A by sex. This may suggest that sex hormones or regulators of sex determination in utero may alter the mechanisms by which methylation patterns are set in the placenta at specific regions, or that there are differential pressures for methylation of this receptor in males. In the adjusted model of infant attention, we noted that males had a significant negative relationship with attention compared with females, and that this is independent of HTR2A promoter methylation. This work suggests that gender may have not only an influence on HTR2A methylation but also an independent role on infant neurobehavior through other mechanisms

We did not observe a statistically significant relationship between HTR2A promoter methylation and a history of tobacco or alcohol use but did note that tobacco use during pregnancy was associated with a marginally significant increase in HTR2A promoter methylation. The lack of statistical significance for this association is likely due to the limited power of this study to detect a smoking effect (post-hoc Power = 0.61). Our study was not specifically designed to interrogate these questions but suggests that the relationship between HTR2A promoter methylation and tobacco use should be examined in studies better powered to assess this relationship.

The cohort used in this examination provides a unique perspective of the developmental origins of adult disease theory, as this is a cohort of healthy, term infants with no overt pathology at birth. We were able to observe moderate modulations in infant neurobehavior that are comparable to reported measurement norms.³¹ Prior studies analyzing components of the Barker hypothesis have focused on infants developing in more extreme environmental conditions,²⁷ or from mothers with high-risk pregnancies.^3,32 This study suggests that there is a level of epigenetic variability even among healthy infants and that this may result from more prevalent environmental conditions and stressors that are most likely experienced by the population at large.

Overall, these results suggests potential importance of the HTR2A receptor in the placenta on fetal development and will provide an impetus for future work in this area to further explore the physiology of the serotonin response pathway within the placenta and its role on development. Future studies examining additional genes involved in the development of the HPA axis may also reveal the relationship of epigenetic changes within the placenta and neurodevelopment.

Materials and Methods

Study population

The infants from this study are participants in the ongoing Rhode Island Child Health Study (RICHS), which enrolls viable mother infant pairs from Women and Infants Hospital of Rhode Island,²³ and represents all infants enrolled in the study from September 2010 until July 2012 with neurodevelopmental assessments. Exclusion criteria for this study include life-threatening health complications of mother, congenital or chromosomal abnormalities of the infant, or maternal age less than 18. Anthropomorphic and clinical data about both the mother and child were collected from medical chart information, and mothers were subjected to an interviewer-administered questionnaire to obtain information about lifestyle, demographics and exposure histories. The neurodevelopmental status of the infants was assessed via the NICU Network Neurobehavioral Scales (NNNS), which was administered by certified psychometrists. Individual components of the NNNS test were compiled into a series of 13 summary scores,³³ and for these analyses we focused on the six summary scores (habituation, attention, stress-abstinence, quality of movement, handling and self-regulation) found to be most sensitive in prior studies.^26,34 All patients provided written informed consent for participation under protocols approved by the institutional review boards at Women and Infants Hospital and Dartmouth College.

Sample collection, nucleic acid extraction and bisulfite modification

For each subject, 3 samples from each of the 4 quadrants (totaling approximately 10 g of tissue) were excised from the maternal side of the placenta, 2 cm from the umbilical cord insertion site, and free of maternal decidua. The samples were placed immediately in RNAlater (Qiagen, 76104) and stored at 4 °C. At least 72 h later, samples were removed from RNAlater, blotted dry, snap frozen in liquid nitrogen, pulverized to powder using a liquid nitrogen cooled stainless steel mortar and pestle and stored at −80 °C. DNA was extracted from 20–30 mg of placental powder using the QIAamp DNA Mini Kit (Qiagen, 51306) and was quantified using an ND-1000 spectrophotometer (Nanodrop), and 500 ng of DNA from each sample was bisulfite modified using the EZ-DNA methylation column kit (Zymo Research) and stored at −20 °C before analysis.

Bisulfite pyrosequencing and DNA methylation analysis

Our analysis of the HTR2A promoter region was based on differential methylation patterns examined in a previous study.¹⁸ A 276 bp. region of the HTR2A promoter region (Chr 7: 28415063-28414801) was amplified from bisulfite modified DNA using the following primers: HTR2A-F, ′5-GATATAAATATTGTTGGTTTTGGATGGA-3′ and HTR2A-R, ′5-biotin-ACTACAAAATAACAACAACCAAAAA-3′ (IDT Inc.), using the pyromark PCR kit (Qiagen, 203443). Cycling conditions were as follows: 95 °C for 9 min, followed by 40 cycles of 95 °C for 30 sec, 58 °C for 1 min and 72 °C for 1 min, followed by a final extension of 5 min at 72 °C.

PCR products were sequenced following a serial pyrosequencing protocol using a PyroMark MD system (Qiagen) and the following sequencing primers: HTR2A SEQ1 5′-GTTTTGGATGGAAGTG-3′, HTR2A Seq 2: 5′-GGGAGAAGAAAAAGTTTG-3′. The first sequencing primer was designed to quantify methylation at two CpG sites, and the second sequencing primer for one CpG site. Each sequencing run contained no template and genomic DNA negative controls, as well as a methylated and unmethylated control samples (Epitect, 59655), and each pyrosequencing assay contained a bisulfite conversion control to assess conversion efficiency. All samples examined demonstrated >95% conversion. Genotyping of the rs6311 SNP (A/G) was assessed through visual inspection of the site on the pyrogram resulting from the first pyrosequencing assay.

Statistical analysis

Although methylation data was obtained from 3 CpG sites, we excluded data on the first, as the presence of the site was dependent on the genotype of rs6311 (a A/G SNP). The mean of the remaining two sites was used in all analyses. The methylation data conformed to a normality assumption, so bivariate associations between methylation and continuous variables were assessed with the Pearson correlation and between methylation and categorical variables with Student’s t test or ANOVA, as appropriate. Generalized linear models were used to model the association between methylation and NNNS summary scores, which were controlled for confounders including maternal education as a marker of socioeconomic status, gestational age, birth weight, gender and smoking status. The rs6311 genotype was included in a separate model, but it had no significant effect on either attention or quality of movement scores, and so was removed to allow for a more parsimonious model. The presence of interactions, including with gender and maternal pregnancy tobacco use, were assessed through likelihood ratio tests. Power calculations were performed in PS (Open source: biostat.mc.vanderbilt.edu/wiki/Main/PowerSampleSize). Data was analyzed in R v2.15.2 and p < 0.05 was considered statistically significant.

Glossary

Abbreviations:

HTR2A

gene encoding 5-hydroxytryptamine (serotonin) receptor 2A

NNNS

Neonatal Intensive Care Unit Network Neurobehavioral Scale

RICHS

Rhode Island Child Health Study

HSD11B2

gene encoding hydroxysteroid (11- β) dehydrogenase 2

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.