Corina Lesseur

Global and gene-specific DNA methylation across multiple tissues in early infancy: implications for children's health research

An increasing number of population studies are assessing epigenetic variation in relation to early‐life outcomes in tissues accessible to epidemiologic researchers. Epigenetic mechanisms are highly tissue specific, however, and it is unclear whether the variation observed in one of the tissue types is representative of other sources or whether the variation in DNA methylation is distinct, reflecting potential functional differences across tissues. To assess relations between DNA methylation in various samples from newborns and children in early infancy, we measured promoter or gene‐body DNA methylation in matched term placenta, cord blood, and 3–6 mo saliva samples from 27 unrelated infants enrolled in the Rhode Island Child Health Study. We investigated 7 gene loci (KLF15, NR3C1, LEP, DEPTOR, DDIT4, HSD11B2, and CEBPB) and global methylation, using repetitive region LINE‐1 and ALUYb8 sequences. We observed a great degree of interlocus, intertissue, and interindividual epigenetic variation in most of the analyzed loci. In correlation analyses, only cord blood NR3C1 promoter methylation correlated negatively with methylation in saliva. We conclude that placenta, cord blood, and saliva cannot be used as a substitute for one another to evaluate DNA methylation at these loci during infancy. Each tissue has a unique epigenetic signature that likely reflects their differential functions. Future studies should consider the uniqueness of these features, to improve epigenetic biomarker discovery and translation.—Armstrong, D. A., Lesseur, C., Conradt, E., Lester, B. M., Marsit, C. J. Global and gene‐specific DNA methylation across multiple tissues in early infancy: implications for childrens health research. FASEB J. 28, 2088–2097 (2014). www.fasebj.org

Maternal obesity and gestational diabetes are associated with placental leptin DNA methylation.

OBJECTIVE In this study, we aimed to investigate relationships between maternal prepregnancy obesity and gestational diabetes mellitus and placental leptin DNA methylation. STUDY DESIGN This study comprises data on 535 mother-infant dyads enrolled in the Rhode Island Child Health Study, a prospective cohort study of healthy term pregnancies. Prepregnancy body mass index was calculated from self-reported anthropometric measures and gestational diabetes mellitus diagnoses gathered from inpatient medical records. DNA methylation of the leptin promoter region was assessed in placental tissue collected at birth using quantitative bisulfite pyrosequencing. RESULTS In a multivariable regression analysis adjusted for confounders, infants exposed to gestational diabetes mellitus had higher placental leptin methylation (β = 1.89, P = .04), as did those demonstrating prepregnancy obesity (β = 1.17, P = .06). Using a structural equations model, we observed that gestational diabetes mellitus is a mediator of the effects of prepregnancy obesity on placental leptin DNA methylation (β = 0.81, 95% confidence interval, 0.27-2.71). CONCLUSION Our results suggest that the maternal metabolic status before and during pregnancy can alter placental DNA methylation profile at birth and potentially contribute to metabolic programming of obesity and related conditions.

A case-control study of polymorphisms in xenobiotic and arsenic metabolism genes and arsenic-related bladder cancer in New Hampshire.

Arsenic is associated with bladder cancer risk even at low exposure levels. Genetic variation in enzymes involved in xenobiotic and arsenic metabolism may modulate individual susceptibility to arsenic-related bladder cancer. Through a population-based case-control study in NH (832 cases and 1191 controls), we investigated gene-environment interactions between arsenic metabolic gene polymorphisms and arsenic exposure in relation to bladder cancer risk. Toenail arsenic concentrations were used to classify subjects into low and high exposure groups. Single nucleotide polymorphisms (SNPs) in GSTP1, GSTO2, GSTZ1, AQP3, AS3MT and the deletion status of GSTM1 and GSTT1 were determined. We found evidence of genotype-arsenic interactions in the high exposure group; GSTP1 Ile105Val homozygous individuals had an odds ratio (OR) of 5.4 [95% confidence interval (CI): 1.5-20.2; P for interaction=0.03] and AQP3 Phe130Phe carriers had an OR=2.2 (95% CI: 0.8-6.1; P for interaction=0.10). Bladder cancer risk overall was associated with GSTO2 Asn142Asp (homozygous; OR=1.4; 95% CI: 1.0-1.9; P for trend=0.06) and GSTZ1 Glu32Lys (homozygous; OR=1.3; 95% CI: 0.9-1.8; P for trend=0.06). Our findings suggest that susceptibility to bladder cancer may relate to variation in genes involved in arsenic metabolism and oxidative stress response and potential gene-environment interactions requiring confirmation in other populations.

Tissue-specific Leptin promoter DNA methylation is associated with maternal and infant perinatal factors

Leptin a regulator of body weight is involved in reproductive and developmental functions. Leptin promoter DNA methylation (LEP) regulates gene expression in a tissue-specific manner and has been linked to adverse pregnancy outcomes. In non-pathologic human pregnancies, we assessed LEP methylation, genotyped the single nucleotide polymorphism (SNP) rs2167270 in placental (n=81), maternal and cord blood samples (n=60), and examined the association between methylation, genotype, and perinatal factors. Maternal blood LEP methylation was lower in pre-pregnancy obese women (P=0.01). Cord blood LEP methylation was higher in small for gestational age (SGA) (P=4.6×10(-3)) and A/A genotype (P=1.6×10(-4)), lower (-1.47, P=0.03) in infants born to pre-pregnancy obese mothers and correlated (P=0.01) with maternal blood LEP. Gender was associated with placental LEP methylation (P=0.05). These results suggest that LEP epigenetic control may be influenced by perinatal factors including: maternal obesity, infant growth, genotype and gender in a tissue-specific manner and may have multigenerational implications.

Inherent and Benzo[a]pyrene-Induced Differential Aryl Hydrocarbon Receptor Signaling Greatly Affects Life Span, Atherosclerosis, Cardiac Gene Expression, and Body and Heart Growth in Mice

Little is known of the environmental factors that initiate and promote disease. The aryl hydrocarbon receptor (AHR) is a key regulator of xenobiotic metabolism and plays a major role in gene/environment interactions. The AHR has also been demonstrated to carry out critical functions in development and disease. A qualitative investigation into the contribution by the AHR when stimulated to different levels of activity was undertaken to determine whether AHR-regulated gene/environment interactions are an underlying cause of cardiovascular disease. We used two congenic mouse models differing at the Ahr gene, which encodes AHRs with a 10-fold difference in signaling potencies. Benzo[a]pyrene (BaP), a pervasive environmental toxicant, atherogen, and potent agonist for the AHR, was used as the environmental agent for AHR activation. We tested the hypothesis that activation of the AHR of different signaling potencies by BaP would have differential effects on the physiology and pathology of the mouse cardiovascular system. We found that differential AHR signaling from an exposure to BaP caused lethality in mice with the low-affinity AHR, altered the growth rates of the body and several organs, induced atherosclerosis to a greater extent in mice with the high-affinity AHR, and had a huge impact on gene expression of the aorta. Our studies also demonstrated an endogenous role for AHR signaling in regulating heart size. We report a gene/environment interaction linking differential AHR signaling in the mouse to altered aorta gene expression profiles, changes in body and organ growth rates, and atherosclerosis.

Sex-specific associations between placental leptin promoter DNA methylation and infant neurobehavior

BACKGROUND Leptin (LEP) is a hormone central for energy homeostasis and has been implicated in neurodevelopment. This adipokine is produced by the placenta and is epigenetically regulated by promoter DNA methylation. Recent evidence has suggested a role for LEP in behavioral development. In this study, we investigated associations between profiles of human newborn neurobehavior and placental LEP DNA methylation. METHODS We determined LEP promoter methylation in 444 placental samples from healthy term infants and measured LEP gene expression in a random subset of these samples. Infant neurobehavior was assessed with the NICU Network Neurobehavioral Scales (NNNS) and we examined the relationship between LEP promoter methylation and profiles of infant neurobehavior derived from these scores generated using a hierarchical model-based clustering method. RESULTS LEP methylation is negatively correlated with gene expression only in placentas from male infants (r=-0.6, P=0.006). A 10% increase in LEP DNA methylation was associated with membership in a profile of infant neurobehavior marked by increased lethargy and hypotonicity (OR=1.9; 95% CI: 1.07-3.4), and consistently with reduced risk of membership in a profile characterized by decreased lethargy and hypotonicity (OR=0.54; 95% CI: 0.3-0.94) only in male infants (n=223). No statistically significant associations were observed amongst female infants. DISCUSSION These results suggest that increased placental LEP DNA methylation, related to reduced expression, may play a role in human newborn neurodevelopment, particularly in reactivity to various stimuli, but that these effects may be sexually dimorphic.

Placental HTR2A methylation is associated with infant neurobehavioral outcomes

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.

Placental FKBP5 Genetic and Epigenetic Variation Is Associated with Infant Neurobehavioral Outcomes in the RICHS Cohort

Adverse maternal environments can lead to increased fetal exposure to maternal cortisol, which can cause infant neurobehavioral deficits. The placenta regulates fetal cortisol exposure and response, and placental DNA methylation can influence this function. FK506 binding protein (FKBP5) is a negative regulator of cortisol response, FKBP5 methylation has been linked to brain morphology and mental disorder risk, and genetic variation of FKBP5 was associated with post-traumatic stress disorder in adults. We hypothesized that placental FKBP5 methylation and genetic variation contribute to gene expression control, and are associated with infant neurodevelopmental outcomes assessed using the Neonatal Intensive Care Unit (NICU) Network Neurobehavioral Scales (NNNS). In 509 infants enrolled in the Rhode Island Child Health Study, placental FKBP5 methylation was measured at intron 7 using quantitative bisulfite pyrosequencing. Placental FKBP5 mRNA was measured in a subset of 61 infants by quantitative PCR, and the SNP rs1360780 was genotyped using a quantitative allelic discrimination assay. Relationships between methylation, expression and NNNS scores were examined using linear models adjusted for confounding variables, then logistic models were created to determine the influence of methylation on membership in high risk groups of infants. FKBP5 methylation was negatively associated with expression (P = 0.08, r = −0.22); infants with the TT genotype had higher expression than individuals with CC and CT genotypes (P = 0.06), and those with CC genotype displayed a negative relationship between methylation and expression (P = 0.06, r = −0.43). Infants in the highest quartile of FKBP5 methylation had increased risk of NNNS high arousal compared to infants in the lowest quartile (OR 2.22, CI 1.07–4.61). TT genotype infants had increased odds of high NNNS stress abstinence (OR 1.98, CI 0.92–4.26). Placental FKBP5 methylation reduces expression in a genotype specific fashion, and genetic variation supersedes this effect. These genetic and epigenetic differences in expression may alter the placenta’s ability to modulate cortisol response and exposure, leading to altered neurobehavioral outcomes.

Examining the joint contribution of placental NR3C1 and HSD11B2 methylation for infant neurobehavior

Infant neurobehavior, a potential sentinel of future mental and behavioral morbidity characterized in part by reflex symmetry, excitability and habituation to stimuli, is influenced by aspects of the intrauterine environment partially through epigenetic alterations of genes involved in the stress response. DNA methylation of two related cortisol response genes, the glucocorticoid receptor (NR3C1), a nuclear receptor to which cortisol binds, and 11-beta hydroxysteroid dehydrogenase (HSD11B2), the enzyme responsible for conversion of cortisol into inactive cortisone, independently associate with infant neurobehavior. Although these factors are part of a common cortisol regulation pathway, the combined effect of DNA methylation of these factors on infant neurobehavior has not been characterized. Therefore, we conducted an examination of the joint contribution of NR3C1 and HSD11B2 DNA methylation on infant neurobehavior. Among 372 healthy term newborns, we tested the interaction between placental NR3C1 and HSD11B2 DNA methylation in association with neurobehavior as assessed with the validated NICU Network Neurobehavioral Scales. Controlling for confounders, interactions between DNA methylation of these genes were detected for distinct domains of neurobehavior (habituation, excitability, asymmetrical reflexes). Moreover, different patterns of DNA methylation across the cortisol regulation pathway associated with different neurobehavioral phenotypes. Those with low NR3C1 methylation but high HSD11B2 methylation had lower excitability scores; those with high NR3C1 methylation but low HSD11B2 methylation had more asymmetrical reflexes; those with high DNA methylation across the entire pathway had higher habituation scores. These results suggest that epigenetic alterations across the cortisol regulation pathway may contribute to different neurobehavioral phenotypes, likely though varying degrees of glucocorticoid exposure during gestation. While the postnatal environment may continue to affect neurobehavioral risk, this study provides novel insights into the molecular basis for fetal origins of mental conditions.

Distinct patterns of DNA methylation in conventional adenomas involving the right and left colon.

Recent studies have shown two distinct non-CIMP methylation clusters in colorectal cancer, raising the possibility that DNA methylation, involving non-CIMP genes, may play a role in the conventional adenoma–carcinoma pathway. A total of 135 adenomas (65 left colon and 70 right colon) were profiled for epigenome-wide DNA methylation using the Illumina HumanMethylation450 BeadChip. A principal components analysis was performed to examine the association between variability in DNA methylation and adenoma location. Linear regression and linear mixed effects models were used to identify locus-specific differential DNA methylation in adenomas of right and left colon. A significant association was present between the first principal component and adenoma location (P=0.007), even after adjustment for subject age and gender (P=0.009). A total of 168 CpG sites were differentially methylated between right- and left-colon adenomas and these loci demonstrated enrichment of homeobox genes (P=3.0 × 10−12). None of the 168 probes were associated with CIMP genes. Among CpG loci with the largest difference in methylation between right- and left-colon adenomas, probes associated with PRAC (prostate cancer susceptibility candidate) gene showed hypermethylation in right-colon adenomas whereas those associated with CDX2 (caudal type homeobox transcription factor 2) showed hypermethylation in left-colon adenomas. A subgroup of left-colon adenomas enriched for current smokers (OR=6.1, P=0.004) exhibited a methylation profile similar to right-colon adenomas. In summary, our results indicate distinct patterns of DNA methylation, independent of CIMP genes, in adenomas of the right and left colon.