Analysis of Genomic DNA Methylation Levels in Human Placenta using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

Background: DNA-methylation is a common epigenetic tool which plays a crucial role in gene regulation and is essential for cell differentiation and embryonic development. The placenta is an important organ where gene activity can be regulated by epigenetic DNA modifications, including DNA methylation. This is of interest as, the placenta is the interface between the fetus and its environment, the mother. Exposure to environmental toxins and nutrition during pregnancy may alter DNA methylation of the placenta and subsequently placental function and as a result the phenotype of the offspring. The aim of this study was to develop a reliable method to quantify DNA methylation in large clinical studies. This will be a tool to analyze the degree of DNA methylation in the human placenta in relationship to clinical readouts. Methods: Liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS) technique was used for the quantification of the 5dmC/dG ratio in placentas from 248 healthy pregnancies. We were able to demonstrate that this method is a reliable and stable way to determine global placental DNA methylation in large clinical trials. Results/Conclusion: The degree of placental DNA methylation seen in our pilot study varies substantially from 2% to 5%. The clinical implications of this variation need to be demonstrated in adequately powered large studies

Association between placental global DNA methylation and blood pressure during human pregnancy

Objective: Gene-specific placental DNA methylation patterns differ between normal pregnancies and pregnancies complicated by hypertension. However, whether global placental DNA methylation is associated with maternal blood pressure remains controversial. Methods: Using multiple linear regression models, we analysed the association between maternal mean arterial pressure (MAP) at the third trimester of pregnancy and global DNA methylation in the placenta in 922 mothers using LC-ESI-MS/MS. To better characterize the contribution of genetic or epigenetic mechanisms, we performed isolated analyses in mothers with and without a family history of hypertension. Results: Mean placental global DNA methylation was 3.00 ± 0.46%. A significant negative correlation between placental global DNA methylation and mean arterial blood pressure (MAP) in the third trimester could be observed (P = 0.023, r = –0.075). This association remained significant after adjusting for confounders. In placenta samples from mothers with a family history of hypertension, mean maternal MAP was higher (86.1 ± 8.1 vs. 84.6 ± 7.5, P < 0.01) and placental global DNA methylation was lower (2.94 ± 0.43 vs. 3.04 ± 0.47, P < 0.01) compared with samples without a family history of hypertension. Furthermore, the significant independent negative correlation between global placental DNA methylation and MAP was only found in mothers without a family history of hypertension. Conclusion: This study showed an independent negative correlation between placental global DNA methylation and maternal MAP in mothers without a family history of hypertension

Ghrelin influences novelty seeking behavior in rodents and men.

Recent discoveries indicate an important role for ghrelin in drug and alcohol reward and an ability of ghrelin to regulate mesolimbic dopamine activity. The role of dopamine in novelty seeking, and the association between this trait and drug and alcohol abuse, led us to hypothesize that ghrelin may influence novelty seeking behavior. To test this possibility we applied several complementary rodent models of novelty seeking behavior, i.e. inescapable novelty-induced locomotor activity (NILA), novelty-induced place preference and novel object exploration, in rats subjected to acute ghrelin receptor (growth hormone secretagogue receptor; GHSR) stimulation or blockade. Furthermore we assessed the possible association between polymorphisms in the genes encoding ghrelin and GHSR and novelty seeking behavior in humans. The rodent studies indicate an important role for ghrelin in a wide range of novelty seeking behaviors. Ghrelin-injected rats exhibited a higher preference for a novel environment and increased novel object exploration. Conversely, those with GHSR blockade drastically reduced their preference for a novel environment and displayed decreased NILA. Importantly, the mesolimbic ventral tegmental area selective GHSR blockade was sufficient to reduce the NILA response indicating that the mesolimbic GHSRs might play an important role in the observed novelty responses. Moreover, in untreated animals, a striking positive correlation between NILA and sucrose reward behavior was detected. Two GHSR single nucleotide polymorphisms (SNPs), rs2948694 and rs495225, were significantly associated with the personality trait novelty seeking, as assessed using the Temperament and Character Inventory (TCI), in human subjects. This study provides the first evidence for a role of ghrelin in novelty seeking behavior in animals and humans, and also points to an association between food reward and novelty seeking in rodents

Plasma ghrelin levels in high and low NILA rats.

<p>A. Total ghrelin levels and B. active ghrelin levels are not different in animals with low (n = 12) vs. high (n = 12) NILA. C. The same rats display a markedly different activity level in the novel environment. Data on the bar graphs represent mean ± SEM. ***P<0.0005.</p

The <i>GHSR</i> SNPs rs2948694 and rs495225 are significantly associated with novelty seeking (p = 0.002 and p = 0.026, respectively).

<p>For both rs2948694 and rs495225, the less common G/G genotype is associated with lower scores on the personality trait novelty seeking.</p

Role of ghrelin in novelty place preference.

<p>A. Ghrelin-treated rats tend to have slightly higher preference for exploring a novel environment. In contrast those that received GHSR antagonist display much lower preference for a novel environment as compared to the more familiar one. B. Ghrelin markedly increases and the GHSR antagonist strikingly decreases exploration of a novel environment in high NILA rats (HLA). C. When only the low NILA (LLA) rats are considered ghrelin does not significantly alter the place preference, however GHSR antagonist is still effective at reducing the preference. ***P<0.0005. Ghrelin alters the relationship between preference for novelty and NILA. D. Novelty place preference (NPP, here time spent exploring the novel environment) is not correlated with locomotor activity during NILA at baseline. E. The two traits become significantly correlated after ghrelin treatment. F. GHSR antagonist does not influence the correlation.</p

Ghrelin induces a significant preference for exploration of a novel object in rats that do not show a preference for a novel object at baseline.

<p>GHSR blockade does not alter this behavior. ***P<0.0005.</p

The analyzed tag SNPs, the genotype frequencies and the p-values for the association tests using linear regression.

<p><i>GHRL, pro-ghrelin gene; GHSR, growth hormone secretagogue receptor gene</i>; β, β-value describing the slope of the curve in the linear regression model; p, p-value using linear regression; p_corrected, p-value corrected for multiple testing using permutation test.</p

Role of the VTA ghrelin and GHSR in the NILA response.

<p>A. Rats that received the VTA directed ghrelin microinjection had an elevated NILA response after 45 min of the NILA test. B. Conversely rats that received a VTA microinjection of the GHSR antagonist JMV2959 displayed a reduced NILA response. C. Rat brain section (right) and equivalent panel from the rat brain atlas (left) showing an example of the VTA microinjection used. *P<0.05. SNR, substantia nigra, reticular part; ml, medial longitudinal fasciculus; fr, fasciculus retroflexus; VTA, ventral tegmental area.</p