Elise Vangeel

DNA methylation in imprinted genes IGF2 and GNASXL is associated with prenatal maternal stress

Epigenetic regulation of imprinted genes during embryonic development is influenced by the prenatal environment. Our aim was to examine the effect of maternal emotional stress and cortisol levels during pregnancy on methylation of imprinted genes, insulin‐like growth factor 2 (IGF2) and guanine nucleotide‐binding protein, alpha stimulating extra‐large (GNASXL), using umbilical cord blood DNA. Maternal depressed mood (Edinburgh Depression Scale; EDS), pregnancy‐related anxiety questionnaire (PRAQ) and cortisol day profiles were assessed throughout pregnancy. At birth, a cord blood sample (n = 80) was taken to study DNA methylation of IGF2 DMR0 (differentially methylated region), IGF2 anti‐sense (IGF2AS) and GNASXL using Sequenom EpiTYPER. Linear mixed models were used to examine the relationship between DNA methylation and maternal stress, while correcting for confounders. We also studied the association of DNA methylation with the child ponderal index at birth. We found a cytosine–guanine dinucleotide (CpG)‐specific association of PRAQ subscales with IGF2 DMR0 (CpG5, P < 0.0001) and GNASXL (CpG11, P = 0.0003), while IGF2AS was associated with maternal EDS scores (CpG33, P = 0.0003) and cortisol levels (CpG33, P = 0.0006; CpG37‐38, P = 0.0005). However, there was no association of methylation with ponderal index at birth. In conclusion, maternal stress during pregnancy, as defined by cortisol measurements, EDS and PRAQ scores, is associated with DNA methylation of imprinted genes IGF2 and GNASXL. Our results provide further evidence that prenatal adversity can influence imprinted gene methylation, although future studies are needed to unravel the exact mechanisms.

Chronic Fatigue Syndrome and DNA Hypomethylation of the Glucocorticoid Receptor Gene Promoter 1F Region: Associations With Hypothalamic-Pituitary-Adrenal Axis Hypofunction and Childhood Trauma

Objectives Chronic fatigue syndrome (CFS) has been associated with hypothalamic-pituitary-adrenal axis hypofunction and enhanced glucocorticoid receptor (GR) sensitivity. In addition, childhood trauma is considered a major risk factor for the syndrome. This study examines DNA methylation of the GR gene (NR3C1) in CFS and associations with childhood sexual and physical trauma. Methods Quantification of DNA methylation within the 1F promoter region of NR3C1 was performed in 76 female patients (46 with no/mild and 30 with moderate/severe childhood trauma) and 19 healthy controls by using Sequenom EpiTYPER. Further, we examined the association of NR3C1-1F promoter methylation with the outcomes of the low-dose (0.5 mg) dexamethasone/corticotropin-releasing factor test in a subset of the study population. Mann-Whitney U tests and Spearman correlations were used for statistical analyses. Results Overall NR3C1-1F DNA methylation was lower in patients with CFS than in controls. After cytosine guanine dinucleotide (CpG)-specific analysis, CpG_1.5 remained significant after Bonferroni correction (adjusted p = .0014). Within the CFS group, overall methylation (&rgr; = 0.477, p = .016) and selective CpG units (CpG_1.5: &rgr; = 0.538, p = .007; CpG_12.13: &rgr; = 0.448, p = .025) were positively correlated with salivary cortisol after dexamethasone administration. There was no significant difference in NR3C1-1F methylation between traumatized and nontraumatized patients. Conclusions We found evidence of NR3C1 promoter hypomethylation in female patients with CFS and the functional relevance of these differences was consistent with the hypothalamic-pituitary-adrenalaxis hypofunction hypothesis (GR hypersuppression). However, we found no evidence of an additional effect of childhood trauma on CFS via alterations in NR3C1 methylation.

DNA methylation analysis of Homeobox genes implicates HOXB7 hypomethylation as risk factor for neural tube defects.

Neural tube defects (NTDs) are common birth defects of complex etiology. Though family- and population-based studies have confirmed a genetic component, the responsible genes for NTDs are still largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, epigenetic factors, such as DNA methylation, are predicted to be involved in NTDs. Homeobox (HOX) genes play a role in spinal cord development and are tightly regulated in a spatiotemporal and collinear manner, partly by epigenetic modifications. We have quantified DNA methylation for the different HOX genes by subtracting values from a genome-wide methylation analysis using leukocyte DNA from 10 myelomeningocele (MMC) patients and 6 healthy controls. From the 1575 CpGs profiled for the 4 HOX clusters, 26 CpGs were differentially methylated (P-value < 0.05; β-difference > 0.05) between MMC patients and controls. Seventy-seven percent of these CpGs were located in the HOXA and HOXB clusters, with the most profound difference for 3 CpGs within the HOXB7 gene body. A validation case-control study including 83 MMC patients and 30 unrelated healthy controls confirmed a significant association between MMC and HOXB7 hypomethylation (-14.4%; 95% CI: 11.9–16.9%; P-value < 0.0001) independent of the MTHFR 667C>T genotype. Significant HOXB7 hypomethylation was also present in 12 unaffected siblings, each related to a MMC patient, suggestive of an epigenetic change induced by the mother. The inclusion of a neural tube formation model using zebrafish showed that Hoxb7a overexpression but not depletion resulted in deformed body axes with dysmorphic neural tube formation. Our results implicate HOXB7 hypomethylation as risk factor for NTDs and highlight the importance for future genome-wide DNA methylation analyses without preselecting candidate pathways.

Methylome analysis for spina bifida shows SOX18 hypomethylation as a risk factor with evidence for a complex (epi)genetic interplay to affect neural tube development

BackgroundNeural tube defects (NTDs) are severe congenital malformations that arise from failure of neurulation during early embryonic development. The molecular basis underlying most human NTDs still remains largely unknown. Based on the hypothesis that folic acid prevents NTDs by stimulating methylation reactions, DNA methylation changes could play a role in NTDs. We performed a methylome analysis for patients with myelomeningocele (MMC). Using a candidate CpG analysis for HOX genes, a significant association between HOXB7 hypomethylation and MMC was found.MethodsIn the current study, we analyzed leukocyte methylome data of ten patients with MMC and six controls using Illumina Methylation Analyzer and WateRmelon R-packages and performed validation studies using larger MMC and control cohorts with Sequenom EpiTYPER.ResultsThe methylome analysis showed 75 CpGs in 45 genes that are significantly differentially methylated in MMC patients. CpG-specific methylation differences were next replicated for the top six candidate genes ABAT, CNTNAP1, SLC1A6, SNED1, SOX18, and TEPP but only for the SOX18 locus a significant overall hypomethylation was observed (P value = 0.0003). Chemically induced DNA demethylation in HEK cells resulted in SOX18 hypomethylation and increased expression. Injection of sox18 mRNA in zebrafish resulted in abnormal neural tube formation. Quantification of DNA methylation for the SOX18 locus was also determined for five families where parents had normal methylation values compared to significant lower values for both the MMC as their non-affected child. SOX18 methylation studies were performed for a MMC patient with a paternally inherited chromosomal deletion that includes BMP4. The patient showed extreme SOX18 hypomethylation similar to his healthy mother while his father had normal methylation values.ConclusionsThis is the first genome-wide methylation study in leukocytes for patients with NTDs. We report SOX18 as a novel MMC risk gene but our findings also suggest that SOX18 hypomethylation must interplay with environmental and (epi)genetic factors to cause NTDs. Further studies are needed that combine methylome data with next-generation sequencing approaches to unravel NTD etiology.

Epigenome-Wide Analysis Of Methylation And Perceived Parenting In Adolescents And Its Correlation With Depressive Symptoms Over Time

Background Adolescents are continuously exposed to their parents. Previous research has shown that parenting can affect adolescents’ wellbeing. Prior research has also argued that adolescents’ impaired wellbeing can influence their performance over time. We present a study with an Illumina 450k array comparing methylation in adolescents reporting either perceived supportive-guiding or punishing-neglecting parenting at T0 and how these methylation differences are correlated with depressive symptoms over time. Methods Following a cluster analysis with a 6 cluster solution, 45 Belgian adolescents (Mage (SD) = 13.88 (0.90) at T0; 48% boys) from the STRATEGIES dataset (n= 1116) were randomly selected from the two most extreme clusters: perceived supportive-guiding parenting qnd punishing-neglecting. Perceived parenting was measured with the Leuven Adolescent Perceived Parenting Schale (LAPPS) and the Parental Behavior Scale (PBS). Methylation was measured with an Illumina Infinium HumanMethylation450 BeadChip. 1 individual was excluded after quality control (RnBeads, R), resulting in 44 adolescents for analysis. DMRs were identified using DMRcate (R) and comb-p (Python). We corrected for gender, batch, cell types (Horvath), and hidden stratification. The accuracy of the 450k-array was verified with Sequenom Epityper (min. r=.46, max. r= .97; p Results Only DMRs overlapping between the top 20 of DMRcate, ranked by “minpval” and comb-p at the e-2 (14 DMRs) and e-3 level (28 DMRs), were taken into account. Despite the major statistical differences in the two approaches, 13 DMRs overlapped between DMRcate and comb-p at the e-3 level. 4 additional DMRs overlapped when adding the e-2 level. Regions are annotated to the genes PEX10, ASCL2, KCNQ1, GPR19, DLL3, HDAC4, RFPL2, PPT2, ACAT2, KIF25, HOXA11, PTPRN2, and SCRIB. For the most significant CpG per region, only three CpGs were correlated with depressive symptoms at T2: cg13306335 in PEX10 (r= .47, p = .0014), cg05171197 in HDAC4 (r= .33, p = .021) and cg13417420 in GPR19 (r= .33, p= .030), with PEX10 surviving Bonferroni correction for 17 tests (p Discussion Despite our limited sample size, we show that two statistically different methods overlap highly in the regions they identify to be significantly different based on perceived parenting in this adolescent sample. Furthermore, we show that for PEX10 the methylation at the most significant CpG is more strongly correlated with depressive symptoms two years later (T2) than the cluster at T0 and depressive symptoms at T2. This raises the question if parenting in adolescents can affect methylation at T0, which may secondarily predispose some adolescents to depressive symptoms over time. More research in a larger and independent sample is needed to validate this preliminary hypothesis.