Journal of Developmental Origins of Health and Disease (2014), 5(2), 132–141. © Cambridge University Press and the International Society for Developmental Origins of Health and Disease 2014 doi:10.1017/S2040174414000038
ORIGINAL ARTICLE
Placental lipoprotein lipase DNA methylation levels are associated with gestational diabetes mellitus and maternal and cord blood lipid profiles A. A. Houde1,2, J. St-Pierre2,3, M. F. Hivert4,5, J. P. Baillargeon4, P. Perron2,4, D. Gaudet2,6, D. Brisson2,6 and L. Bouchard1,2* 1
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Qc, Canada ECOGENE-21 and Clinical Research Center and Lipid Clinic, Chicoutimi Hospital, Saguenay, Qc, Canada 3 Department of Pediatric, CSSS de Chicoutimi, Saguenay, Qc, Canada 4 Department of Medicine, Division of Endocrinology, Université de Sherbrooke, Sherbrooke, Qc, Canada 5 General Medicine Division, Massachusetts General Hospital, Boston, MA, USA 6 Department of Medicine, Université de Montréal, Montréal, Qc, Canada 2
Placental lipoprotein lipase (LPL) is crucial for placental lipid transfer. Impaired LPL gene expression and activity were reported in pregnancies complicated by gestational diabetes mellitus (GDM) and intra-uterine growth restriction. We hypothesized that placental LPL DNA methylation is altered by maternal metabolic status and could contribute to fetal programming. The objective of this study was thus to assess whether placental LPL DNA methylation is associated with GDM and both maternal and newborn lipid profiles. Placenta biopsies were sampled at delivery from 126 women including 27 women with GDM diagnosed following a post 75 g-oral glucose tolerance test (OGTT) between weeks 24 and 28 of gestation. Placental LPL DNA methylation and expression levels were determined using bisulfite pyrosequencing and quantitative real-time PCR, respectively. DNA methylation levels within LPL proximal promoter region (CpG1) and intron 1 CpG island (CpGs 2 and 3) were lower in placenta of women with GDM. DNA methylation levels at LPL-CpG1 and CpG3 were also negatively correlated with maternal glucose (2-h post OGTT; r = –0.22; P = 0.02) and HDL-cholesterol levels (third trimester of pregnancy; r = –0.20; p = 0.03), respectively. Moreover, we report correlation between LPL-CpG2 DNA methylation and cord blood lipid profile. DNA methylation levels within intron 1 CpG island explained up to 26% (r ⩽ –0.51; P < 0.001) of placental LPL mRNA expression variance. Overall, we showed that maternal metabolic profile is associated with placental LPL DNA methylation dysregulation. Our results suggest that site-specific LPL epipolymorphisms in the placenta are possibly functional and could potentially be involved in determining the future metabolic health of the newborn. Received 10 September 2013; Revised 7 January 2014; Accepted 9 January 2014; First published online 12 February 2014 Key words: fetal programming, HDL-c, hyperglycemia
Introduction Fetal lipid metabolism is crucial for growth and development and highly dependent on maternal lipid supply. The fetus, unable to synthesize sufficient amounts of essential fatty acids, relies on maternal triglyceride-rich lipoproteins and nonesterified fatty acids (NEFA) as sources of free fatty acids (FFA).1 While NEFA are directly transferred to the fetus through diffusion or via placenta fatty acid binding proteins,2 triglycerides (TG) from lipoproteins first need to be hydrolyzed into FFA by placental lipases. Endothelial lipase (LIPG) and lipoprotein lipase (LPL) both contribute to most of the transfer of FFA from maternal lipoproteins to the fetus.3 The LIPG is a phospholipase with a low TG lipase activity, which mainly hydrolyzes phospholipids from high-density lipoproteins (HDL). In contrast, the placental LPL is a TG lipase and *Address for correspondence: L. Bouchard, PhD, MBA, Laboratoire ECOGENE-21, Pavillon des Augustines-AUG-5–06, CHAU régional de Chicoutimi, Saguenay, Canada G1K 7P4. (Email: [email protected])
mainly hydrolyses maternal lipoproteins enriched in TG such as chylomicrons and very low-density lipoproteins (VLDL).4 Impaired placental LPL gene expression and activity were previously associated with gestational conditions characterized by a suboptimal in utero environment and fetal development. Gestational diabetes mellitus (GDM) was associated with lower LPL mRNA levels whereas higher LPL mRNA levels were observed in cases of intra-uterine growth restriction (IUGR).5–7 This suggests that disturbances of placental LPL impair materno-fetal lipid transfer, fetal fat accretion, growth and development.8 Fetal conditions previously associated with increased long-term risk for obesity and metabolic disorders in the newborn.9,10 In line with these observations, we propose that maternal metabolic status could impair placenta’s LPL DNA methylation profile possibly contributing to fetal metabolic programming. DNA methylation is the most stable and studied epigenetic mark. The addition of a methyl group at position 5′ of the pyrimidine ring of the cytosines upstream of a guanine (dinucleotide CpG) is catalyzed by DNA methyltransferases.11
LPL DNA methylation and maternal metabolic profile DNA methylation regulates transcriptional activity and is generally associated with gene expression repression.12 The methylation of cytosines is sensitive to environmental stimuli including the intra-uterine environment.13,14 The placenta methylome has been shown to adapt to the intra-uterine environment to support adequate fetal development and seems to be involved in the regulation of gene expression, placentation, fetal growth and birth weight.15,16 Likewise, we have previously reported alterations in DNA methylation profiles of leptin and adiponectin genes in placenta of newborn exposed, in utero, to maternal hyperglycemia.17,18 This study was conducted to verify whether placental LPL DNA methylation levels are associated with GDM and both maternal and cord blood lipid profile. In addition, we have investigated whether placental LPL gene DNA methylation was correlated with its expression levels. Considering the importance of LPL in materno-fetal lipid metabolism, changes in placental LPL gene expression through DNA methylation variation could impair fetal lipid supply, affect fetal growth and development, and potentially be involved in fetal metabolic programming. Method One hundred twenty-six Caucasian pregnant women were recruited in the Saguenay region. Women aged over 40 years old as well as those that had a positive history of drug or alcohol abuses during the current pregnancy, pre-gestational type 1 or 2 diabetes, polycystic ovary syndrome, hypercholesterolemia, morbid obesity [body mass index (BMI) >40] or those who gave birth prematurely (gestational age < 37 weeks) were excluded. The Chicoutimi Hospital Ethics Committee approved the project. All subjects provided a written informed consent before their inclusion in the study, and all clinical data were de-identified. This project was conducted in accordance with the Declaration of Helsinki. Participants were followed at weeks 10–14 as well as at weeks 24–28 and 36–39. At each visit, anthropometric parameters were measured and recorded by a research nurse according to standard procedures.19 Blood samples were collected at each trimester following a 12-h overnight fast. At delivery, placenta biopsies and cord blood samples were collected. All glucose and insulin concentrations were assessed on fresh serum samples at the Chicoutimi Hospital Clinical Laboratory. Insulin measurements were performed using a radioimmunoassay method (Advia Centaur, Simmens). Maternal blood glucose along with total cholesterol (TC) and TG levels were determined using the enzymatic method on a Beckman analyzer (model CX7; Beckman, Fullerton, CA). VLDL and lowdensity lipoproteins (LDL) in plasma were precipitated with heparin and MnCl2 to isolate HDL particles for cholesterol (HDL-c) measurement. Then, plasma LDL-c concentrations were estimated using the Friedewald formula.20 GDM was diagnosed according to WHO criteria: glycaemia ⩾7.8 mmol/l following the 2-h post 75 g-oral glucose tolerance test (OGTT)
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between the 24th and 28th weeks of gestation. The 126 women were tested between weeks 22.4 and 28.9 (average = 25.7 ± 1 weeks) of gestation. Insulin resistance levels were assessed using HOMA-IR (fasting glucose (mmol/l) × fasting insulin (mU/l)/22.5) at the second trimester of pregnancy.21 Clinical information about the newborn was collected at birth from medical files and included gestational age, sex of the newborn and anthropometric measurements. The gestational age was calculated at the first visit from the date of the last menstrual period and was corrected afterwards, as required, based on the date from the ultrasound scans. Cord blood lipid and glucose levels were assessed as described for the mother. In addition, cord blood C-peptide levels, measured by ELISA, were used as a marker of the newborn’ insulin-secretory activity.22 Because the lipid measurements (HDL-c, TG, TC) were not available for all women, the number of samples (n) provided in Table 2 are lower than 126. The lipid profile was available for 92 newborns out of 126. Placenta tissue sampling Placenta tissues were sampled by well-trained clinicians (MD) within a few minutes after delivery and placental expulsion. Two biopsies of 0.5 cm3 were taken on the fetal side of the placenta near the insertion point of the umbilical cord. The biopsies consisted of the chorionic plate with the chorionic villi. Placental biopsies were washed in PBS 1× (137 mM NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.4) to remove cord/maternal blood, dissected to remove conjunctive tissues and kept in RNALater (Qiagen, Valencia, CA) at −80°C until nucleic acid extraction. DNA and RNA were purified from placenta biopsies using the All Prep DNA/RNA/Protein Mini Kit (Qiagen). RNA quality was assessed with Agilent 2100 Bioanalyzer RNA Nano Chips (Agilent Technologies, Santa Clara, CA). On average, the RNA was considered of good quality (mean RNA integrity number 8.00 ± 0.70). DNA methylation analysis at the LPL gene locus Based on a first epigenome-wide DNA methylation association study (EWAS) [Illumina’s HumanMethylation450 BeadChips (Illumina Inc., San Diego)] conducted with 30 placenta samples of mother diagnosed with GDM and 14 from controls,23 we identified three loci flanking the first exon of the LPL gene that showed DNA methylation variability and potential differences between groups. These three CpGs were retained for DNA methylation analyses in the current study (total n = 126). Cytosine methylation was quantified using sodium bisulfite (NaBis) pyrosequencing (Pyromark Q24, QiagenBiotage).24 Combined with the NaBis DNA treatment, pyrosequencing is a quantitative real-time sequencing technology that allows measuring DNA methylation levels (%) for each cytosine of the covered region. NaBis treatment of DNA (EpiTect Bisulfite Kit, Qiagen) specifically converts unmethylated cytosine into uracil, while the methylated cytosines are
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protected from this transition. Once treated, NaBis-DNA is amplified (Pyromark PCR kit, Qiagen) and pyrosequenced. PCR and sequencing primers for analysis of the LPL CpGs were designed with Pyromark Assay Design (version 2.0.1.15; Qiagen) to cover the three CpGs found to be differentially methylated from our ongoing EWAS of GDM cases-controls (cg16125291, cg08918749 and cg16420199). The designed primers, after bisulfite conversion of DNA, were as follow: LPLCpG1F: 5′-AAGTATAAGTTGGGAYGTAATGTG TG-3′, LPLCpG1R: 5′-CCAAAAAAAAAAAATTTAACTATTA AATTAC-3′ (177 bp), and LPLCpG1seq: 5′-GTGTGTTTTTTTATTTTTATATT GA-3′; LPLCpG2F: 5′-GGAGGGGTTTTGGAATGAAAG-3′, LPLCpG2R: 5′-TTCCCCAAAAAAAACCACAATCRAC CC-3′ (113 bp), and LPLCpG2seq: 5′-GGTTTTGGAATGAAAGG-3′ and; LPLCpG3F: 5′-GTTTTGGGGGTTGAGGTT-3′, LPLCpG3R: 5′-AATTAAACAAACTACCTCCATTAC-3′ (202 bp), and LPLCpG3seq: 5′-CTAAAACATTCTCATTTAAC-3′. LPL expression analysis cDNA was generated from total RNA using a random primer hexamer provided with the High Capacity cDNA Archive Kit from Applied Biosystems (Foster City, CA). Equal amounts of cDNA were run in duplicate and amplified in a 20 µl reaction containing 10 µl of Universal PCR Master Mix (Applied Biosystems). Primers and Taqman probes were obtained from Applied Biosystems (LPL: Hs00173425_m1; Applied Biosystems). The YWHAZ housekeeping gene (endogenous control; YWHAZ: Hs00237047_m1) was amplified in parallel. LPL and YWHAZ amplifications were performed using an Applied Biosystems 7500 Real Time PCR System, as recommended by the manufacturer (Applied Biosystems). The expression of LPL gene was normalized to the expression of the YWHAZ reference gene. The YWHAZ gene has been shown to be suitable for gene expression normalization in placenta.25–27 Placental LPL expression was compared between normal glucose tolerance (NGT) and GDM women using the 2 − ΔΔCt method28 and YWHAZ/LPL Ct ratio were used for correlation analysis. Statistical analyses DNA methylation levels of CpGs at the LPL gene locus and maternal and newborn characteristics were compared between groups of women with or without GDM using the unpaired Student t-test or Pearson’s χ2 test for categorical variable (history of GDM). Kolmogorov-Smirnov test was applied to verify data distribution. The following variables were not normally distributed and were thus log transformed: maternal fasting insulin, LDL-c at first trimester and TG at each trimesters as well as cord blood TG, HDL-c and LDL-c. Pearson’s correlation
coefficient was used to assess the relationship between LPL DNA methylation and dependent variables (mRNA expression, maternal metabolic data, birth and placental weight and cord blood lipid profile). The statistical models were adjusted for the following confounders when applicable: age and BMI of the mother at first trimester of pregnancy, history of gestational diabetes, gestational age at delivery, method of childbirth (vaginal v. caesarean section), newborn’s sex and birth weight. The results remained unchanged after adjusting for parity. Furthermore, as maternal TG concentrations were significantly correlated with maternal HDL-c and 2-h post-OGTT glucose concentrations, the statistical models were also adjusted for TG when applicable. Stepwise multivariate linear regression was used to identify predictors of the variance of placental LPL DNA methylation levels. The input-independent variables were: maternal characteristics (age, BMI and TG at first trimester of pregnancy, glucose 2-h post-OGTT and HDL-c at second trimester of pregnancy, HDL-c at third trimester of pregnancy, history of gestational diabetes), gestational age at delivery and method of childbirth. All the variables with P < 0.05 were retained in the regression model. Results with P-values ⩽0.05 (two-sided) were considered statistically significant. All analyses were performed with SPSS version 20.
Results Characteristics of mothers according to their glucose tolerance status as well as newborns’ gestational age and birth weight are shown in Table 1. At first trimester of pregnancy, women were on average 29 years old, slightly overweight and had normal fasting glucose concentrations and lipid profile. Of the 126 women recruited, 27 had GDM, whereas 99 were normoglycemic (NGT). Among the women with GDM, 16 were treated with a diet only whereas 11 received a diet and insulin treatment. Overall, blood glucose 2-h post-OGTT covered a wide range of values (from 3.80 to 10.80 mmol/l). Mothers with GDM had a lower gestational weight gain. Average birth weight was within the normal weight range in both NGT and GDM groups. Nevertheless, according to Olsen et al.29 intrauterine growth curves, two babies were found to be small for gestational age whereas five babies were large for gestational age (LGA). Among these seven babies, one LGA was born to GDM mothers. Interestingly, newborn exposed to GDM had significantly higher levels of cord blood C-peptide, hence suggesting that they had been exposed to higher glucose concentrations. DNA methylation analysis of the placental LPL gene Placental DNA methylation was measured at 3 CpGs located within the LPL gene locus first identified using an EWAS strategy (Fig. 1). The first CpG (LPL-CpG1) was located in its proximal promoter region, 46 bp upstream from the first exon (Fig. 1). The two other CpGs, CpG2 and CpG3, were located
LPL DNA methylation and maternal metabolic profile
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Table 1. Characteristics of women and newborn according to maternal glucose tolerance status
Women History of GDM Parity 0 1 2 First trimester of pregnancy Age (years) BMI (kg/m2) Fasting glucose (mmol/l) Fasting insulin (mU/l)a Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l)a Second trimester of pregnancy Fasting glucose (mmol/l) Fasting insulin (mmol/l)a 2-h post-OGTT glucose concentration (mmol/l) HOMA-IRa Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l) Third trimester of pregnancy Fasting glucose (mmol/l)a Fasting insulin (mmol/l)a HOMA-IRa Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l) Weight gain between first and third trimester (% of initial weight) Newborn Gestational age (weeks) Birth weight (kg) Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l) C-peptide (pmol/l)
GDM (n = 27)
NGT (n = 99)
9 (33.3%)
4 (4.0%)**
9 (33.3%) 14 (51.9%) 4 (14.8%)
54 (54.5%)† 28 (28.3%)† 17 (17.2%)†
Mean ± S.D. Minimum–maximum
Mean ± S.D.
Minimum–maximum
29.1 ± 3.6 25.7 ± 3.9 4.48 ± 0.44 5.69 ± 4.33 1.16 ± 0.51 4.55 ± 0.60 1.60 ± 0.31 2.31 ± 0.52
20–36 19.6–35.4 3.60–5.30 1.70–16.70 0.60–2.70 3.10–6.00 1.19–2.31 1.20–3.30
28.8 ± 3.9 24.6 ± 4.7 4.41 ± 0.34 5.10 ± 3.67 1.00 ± 0.45† 4.67 ± 0.79 1.56 ± 0.32 2.46 ± 0.69
21–39 16.6–39.7 3.80–5.20 1.00–17.60 0.40–2.70 3.10–7.20 0.81–2.42 1.30–5.10
4.52 ± 0.41 9.19 ± 6.13 8.40 ± 0.61 1.86 ± 1.35 1.88 ± 0.72 5.90 ± 0.75 1.75 ± 0.29 3.24 ± 0.76
3.70–5.60 2.60–27.60 7.80–10.80 0.43–5.89 0.90–3.70 4.70–7.70 1.36–2.44 1.80–4.50
4.28 ± 0.39** 7.03 ± 5.30† 6.22 ± 0.81** 1.33 ± 1.04* 1.72 ± 0.64 6.26 ± 1.12 1.80 ± 0.41 3.62 ± 1.00
3.60–5.70 1.40–38.90 3.80–7.70 0.25–7.09 1.00–4.60 4.10–10.20 0.96–2.74 1.80–7.00
4.19 ± 0.44 7.60 ± 5.22 1.44 ± 1.08 2.58 ± 0.97 6.63 ± 1.10 1.74 ± 0.27 3.61 ± 0.97 13.8 ± 5.2
3.20–5.00 2.00–24.70 0.32–4.83 1.50–6.10 5.10–8.90 1.25–2.24 2.10–5.70 4.0–25.0
4.29 ± 0.63 9.86 ± 7.68* 1.85 ± 1.73† 2.62 ± 0.86 6.82 ± 1.28 1.73 ± 0.42 3.85 ± 1.13 19.5 ± 6.8**
3.60–8.30 3.80–42.20 0.73–9.75 1.10–5.20 4.10–10.80 0.89–2.80 1.70–7.40 2.0–34.0
39.7 ± 1.2 3.45 ± 0.45 0.43 ± 0.23 1.63 ± 0.29 0.61 ± 0.21 0.80 ± 0.19 171.3 ± 86.0
37.4–41.6 2.6–4.6 0.22–0.94 1.20–2.25 0.33–1.06 0.53–1.14 14.8–338.0
39.4 ± 1.1 3.45 ± 0.40 0.40 ± 0.23 1.69 ± 0.41 0.61 ± 0.20 0.87 ± 0.28 125.3 ± 66.8*
37.3–41.6 2.0–4.3 0.23–1.82 0.85–2.89 0.26–1.27 0.39–1.90 1.0–353.9
GDM, gestational diabetes mellitus; NGT, normoglycemic; BMI, body mass index; OGTT, oral glucose tolerance test; HDL, high-density lipoproteins; LDL, low-density lipoproteins. a Geometric mean (value obtained after log10 transformation of the variable). **p ⩽ 0.01;* p ⩽ 0.05; †p ⩽ 0.10.
405 and 925 bp downstream from the first exon, within the LPL intron 1 CpG island. DNA methylation level at LPL-CpG1 locus was not found to be correlated with the methylation levels at CpG2 and CpG3 (r < 0.08). Nevertheless, LPL DNA methylation levels at CpG2 and CpG3 sites
were found partially correlated with each other (r = 0.32; P < 0.001). Average DNA methylation at LPL gene locus was between 34.7% and 39.8% (Fig. 1). Overall, LPL DNA methylation levels were lower in placenta exposed to GDM compared with
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CpG1
CpG2
CpG3
ERE PPARG
Average LPL gene DNA methylation CpG1 (cg16125291): 35.4 ± 17.4%
Promoter
CpG2 (cg08918749): 39.8 ± 16.1%
Exon
CpG3 (cg16420199): 34.7 ± 10.6%
CpG island
Fig. 1. Schematic representation of the LPL gene locus CpG island and localization of the 3 loci epigenotyped. CpG1 is located within the LPL proximal promoter region whereas CpG2 and CpG3 are located downstream from the first exon within the LPL intron 1 CpG island. Transcription factor binding sites were identified using the UCSC Genome Browser (NM_131127)51 software, accessed 12 September 2012.
50% 41.8% 37.2%
DNA methylation(%)
40%
36.0%
32.5% 28.6%
29.9%
30%
20%
10% GDM
NGT
GDM
NGT
0% P value
GDM
NGT
CpG1
CpG2
CpG3 0.007
0.005
0.007
a
P value
0.023
0.009
0.006
P valueb
0.033
0.013
0.007
P valuec
0.062
0.038
0.011
a
Adjusted for maternal age and BMI at 1st trimester and history of gestational diabetes b Adjusted for maternal age, BMI and TG at 1st trimester and history of gestational diabetes c Adjusted for maternal age, BMI and TG at 1st trimester, history of gestational diabetes, gestationnal age at delivery and method of childbirth
Fig. 2. DNA methylation levels at placental LPL loci according to maternal glucose tolerance status. Mean DNA methylation levels at placental LPL gene CpG1, CpG2 and CpG3 loci in mothers with gestational diabetes mellitus (n = 27) (black) and with normal glucose tolerance (n = 99) (white). Error bars represent the standard error of the mean (n = 126). Mean DNA methylation levels were compared with the Student t-test and adjusted for confounding variables.
those not exposed (Fig. 2). DNA methylation differences remained unchanged for CpG2 and CpG3 after adjusting the statistical models for age, BMI and TG at first trimester of pregnancy and history of gestational diabetes, gestational age at delivery and method of childbirth. Furthermore, DNA methylation differences between the GDM and NGT mothers
remained unchanged at the 3 CpGs when the obese women [BMI > 30 kg/m2 (n = 13)] were removed from the analysis or when the statistical models were adjusted for maternal weight gain between the first and third trimester of pregnancy. The largest DNA methylation difference between groups (9.3%) was found with CpG2 (P = 0.04) (Fig. 2).
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Table 2. Pearson’s correlation coefficient between placental LPL DNA methylation and maternal metabolic profile First trimester of pregnancy Triglycerides [mmol/l (n = 126)]
CpG1 methylation (%) CpG2 methylation (%) CpG3 methylation (%) mRNA levels (AU)
Second trimester of pregnancy 2-h post-OGTT glucose [mmol/l (n = 126)]
Third trimester of pregnancy
HDL-c [mmol/l (n = 120)]
HDL-c [mmol/l (n = 120)]
I
II
I
III
I
III
I
III
–0.16† –0.14 –0.04 0.19*
–0.14 –0.12 –0.03 0.16†
–0.26** –0.12 –0.18* –0.17†
–0.22* –0.05 –0.14 0.10
0.03 –0.04 –0.15† –0.05
–0.02 –0.06 –0.17† 0.01
0.06 –0.12 –0.20* 0.07
–0.01 –0.13 –0.20* 0.11
HDL, high-density lipoproteins. I – adjusted values for age, body mass index (BMI) at first trimester and history of gestational diabetes. II – adjusted values for age, BMI at first trimester, maternal glucose 2-h post oral glucose tolerance test at second trimester, history of gestational diabetes, gestational age at delivery and method of childbirth. III – adjusted values for age, BMI and triglycerides at first trimester, history of gestational diabetes, gestational age at delivery and method of childbirth. **p ⩽ 0.01; *p ⩽ 0.05; †p ⩽ 0.10.
Correlations between LPL DNA methylation levels and maternal metabolic profile To further confirm the initial EWAS findings (that maternal hyperglycemia is negatively associated with LPL DNA methylation), the correlation between placental LPL DNA methylation and maternal glucose levels was tested. We observed that CpG1 and CpG3 DNA methylation levels were negatively correlated with maternal 2-h post-OGTT glucose concentrations (r = − 0.26; P = 0.003; r = − 0.18; P = 0.05) (Table 2). The latter correlation was slightly affected by additional corrections for maternal TG levels at first trimester of pregnancy, gestational age at delivery and method of childbirth and did not remain significant (r = − 0.14; P = 0.13). Nevertheless, the correlation between LPL CpG1 DNA methylation levels and maternal 2-h post-OGTT glucose concentrations remained significant after consideration of all potential confounding variables (r = − 0.22; P = 0.02) and exclusion of the 13 obese mothers. Finding a correlation between LPL DNA methylation and 2-h post-OGTT glucose concentrations thus confirms the initial association results. As LPL is a key player in TG and lipoprotein metabolism, we also tested for correlations between placental DNA methylation at the LPL gene locus and maternal TG, HDL-c and LDL-c concentrations. We found that CpG3 methylation levels were negatively correlated with concentrations of maternal HDL-c at third trimester of pregnancy (r = − 0.20; P = 0.03). A trend for correlation was also observed between CpG3 DNA methylation levels and HDL-c at second trimester of pregnancy (r = − 0.15; P = 0.10) (Table 2). These correlations remained unchanged after adjusting for all the potential confounding variables or when obese women were excluded from the analysis. Maternal TG and LDL-c concentrations were not correlated with placental LPL DNA methylation. Overall, stepwise multivariate linear regression analysis showed that maternal glucose concentrations (2-h postOGTT) (β = − 0.236; P = 0.01) and HDL-c levels (third
trimester of pregnancy) (β = − 0.182; P = 0.05) could explain up to 7.0% (R2adj = 0.070; P = 0.006) of placental DNA methylation variance at LPL-CpG3 locus. Correlations between LPL DNA methylation levels and offspring characteristics To further evaluate the impacts of LPL DNA methylation profile on materno-fetal transport we tested the association between LPL DNA methylation levels and offspring developmental characteristics and lipid profile. Methylation levels at the LPL-CpG2 locus in the placenta was found to be negatively correlated with cord blood HDL-c levels (r = − 0.24; P = 0.03), and TC/HDL-c ratio (r = 0.22; P = 0.04) and tended to be correlated with TG concentrations (r = 0.19; P = 0.08) after adjusting for newborn’s sex, gestational age and method of childbirth. The strength of these three correlations was increased after adjustment for birth weight (r = − 0.27; P = 0.01; r = 0.27; P = 0.01 and r = 0.25; P = 0.02 for HDL-c, TC/HDL-c and TG, respectively) and remained similar after correction for maternal BMI at first trimester of pregnancy, gestational weight gain and maternal metabolic variables throughout pregnancy. Correlation between placental LPL DNA methylation and mRNA expression levels To assess the impacts of DNA methylation on placental LPL gene expression, LPL mRNA levels were quantified. Placental LPL mRNA levels, expressed as arbitrary units and normalized to YWHAZ mRNA levels, were found to be 1.6 fold higher in placenta of women with GDM (1.04 ± 0.012 AU) compare to NGT (1.02 ± 0.006 AU) (P = 0.04) according to the 2 − ΔΔCt method.28 We also found that LPL DNA methylation levels at CpG2 and CpG3, both located within the intronic CpG island, were negatively correlated with placental LPL gene expression (Fig. 3). Nevertheless, DNA methylation at CpG1, located directly within the LPL gene promoter, was not
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(b)
1.2
r =–0.43 p
ORIGINAL ARTICLE
Placental lipoprotein lipase DNA methylation levels are associated with gestational diabetes mellitus and maternal and cord blood lipid profiles A. A. Houde1,2, J. St-Pierre2,3, M. F. Hivert4,5, J. P. Baillargeon4, P. Perron2,4, D. Gaudet2,6, D. Brisson2,6 and L. Bouchard1,2* 1
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Qc, Canada ECOGENE-21 and Clinical Research Center and Lipid Clinic, Chicoutimi Hospital, Saguenay, Qc, Canada 3 Department of Pediatric, CSSS de Chicoutimi, Saguenay, Qc, Canada 4 Department of Medicine, Division of Endocrinology, Université de Sherbrooke, Sherbrooke, Qc, Canada 5 General Medicine Division, Massachusetts General Hospital, Boston, MA, USA 6 Department of Medicine, Université de Montréal, Montréal, Qc, Canada 2
Placental lipoprotein lipase (LPL) is crucial for placental lipid transfer. Impaired LPL gene expression and activity were reported in pregnancies complicated by gestational diabetes mellitus (GDM) and intra-uterine growth restriction. We hypothesized that placental LPL DNA methylation is altered by maternal metabolic status and could contribute to fetal programming. The objective of this study was thus to assess whether placental LPL DNA methylation is associated with GDM and both maternal and newborn lipid profiles. Placenta biopsies were sampled at delivery from 126 women including 27 women with GDM diagnosed following a post 75 g-oral glucose tolerance test (OGTT) between weeks 24 and 28 of gestation. Placental LPL DNA methylation and expression levels were determined using bisulfite pyrosequencing and quantitative real-time PCR, respectively. DNA methylation levels within LPL proximal promoter region (CpG1) and intron 1 CpG island (CpGs 2 and 3) were lower in placenta of women with GDM. DNA methylation levels at LPL-CpG1 and CpG3 were also negatively correlated with maternal glucose (2-h post OGTT; r = –0.22; P = 0.02) and HDL-cholesterol levels (third trimester of pregnancy; r = –0.20; p = 0.03), respectively. Moreover, we report correlation between LPL-CpG2 DNA methylation and cord blood lipid profile. DNA methylation levels within intron 1 CpG island explained up to 26% (r ⩽ –0.51; P < 0.001) of placental LPL mRNA expression variance. Overall, we showed that maternal metabolic profile is associated with placental LPL DNA methylation dysregulation. Our results suggest that site-specific LPL epipolymorphisms in the placenta are possibly functional and could potentially be involved in determining the future metabolic health of the newborn. Received 10 September 2013; Revised 7 January 2014; Accepted 9 January 2014; First published online 12 February 2014 Key words: fetal programming, HDL-c, hyperglycemia
Introduction Fetal lipid metabolism is crucial for growth and development and highly dependent on maternal lipid supply. The fetus, unable to synthesize sufficient amounts of essential fatty acids, relies on maternal triglyceride-rich lipoproteins and nonesterified fatty acids (NEFA) as sources of free fatty acids (FFA).1 While NEFA are directly transferred to the fetus through diffusion or via placenta fatty acid binding proteins,2 triglycerides (TG) from lipoproteins first need to be hydrolyzed into FFA by placental lipases. Endothelial lipase (LIPG) and lipoprotein lipase (LPL) both contribute to most of the transfer of FFA from maternal lipoproteins to the fetus.3 The LIPG is a phospholipase with a low TG lipase activity, which mainly hydrolyzes phospholipids from high-density lipoproteins (HDL). In contrast, the placental LPL is a TG lipase and *Address for correspondence: L. Bouchard, PhD, MBA, Laboratoire ECOGENE-21, Pavillon des Augustines-AUG-5–06, CHAU régional de Chicoutimi, Saguenay, Canada G1K 7P4. (Email: [email protected])
mainly hydrolyses maternal lipoproteins enriched in TG such as chylomicrons and very low-density lipoproteins (VLDL).4 Impaired placental LPL gene expression and activity were previously associated with gestational conditions characterized by a suboptimal in utero environment and fetal development. Gestational diabetes mellitus (GDM) was associated with lower LPL mRNA levels whereas higher LPL mRNA levels were observed in cases of intra-uterine growth restriction (IUGR).5–7 This suggests that disturbances of placental LPL impair materno-fetal lipid transfer, fetal fat accretion, growth and development.8 Fetal conditions previously associated with increased long-term risk for obesity and metabolic disorders in the newborn.9,10 In line with these observations, we propose that maternal metabolic status could impair placenta’s LPL DNA methylation profile possibly contributing to fetal metabolic programming. DNA methylation is the most stable and studied epigenetic mark. The addition of a methyl group at position 5′ of the pyrimidine ring of the cytosines upstream of a guanine (dinucleotide CpG) is catalyzed by DNA methyltransferases.11
LPL DNA methylation and maternal metabolic profile DNA methylation regulates transcriptional activity and is generally associated with gene expression repression.12 The methylation of cytosines is sensitive to environmental stimuli including the intra-uterine environment.13,14 The placenta methylome has been shown to adapt to the intra-uterine environment to support adequate fetal development and seems to be involved in the regulation of gene expression, placentation, fetal growth and birth weight.15,16 Likewise, we have previously reported alterations in DNA methylation profiles of leptin and adiponectin genes in placenta of newborn exposed, in utero, to maternal hyperglycemia.17,18 This study was conducted to verify whether placental LPL DNA methylation levels are associated with GDM and both maternal and cord blood lipid profile. In addition, we have investigated whether placental LPL gene DNA methylation was correlated with its expression levels. Considering the importance of LPL in materno-fetal lipid metabolism, changes in placental LPL gene expression through DNA methylation variation could impair fetal lipid supply, affect fetal growth and development, and potentially be involved in fetal metabolic programming. Method One hundred twenty-six Caucasian pregnant women were recruited in the Saguenay region. Women aged over 40 years old as well as those that had a positive history of drug or alcohol abuses during the current pregnancy, pre-gestational type 1 or 2 diabetes, polycystic ovary syndrome, hypercholesterolemia, morbid obesity [body mass index (BMI) >40] or those who gave birth prematurely (gestational age < 37 weeks) were excluded. The Chicoutimi Hospital Ethics Committee approved the project. All subjects provided a written informed consent before their inclusion in the study, and all clinical data were de-identified. This project was conducted in accordance with the Declaration of Helsinki. Participants were followed at weeks 10–14 as well as at weeks 24–28 and 36–39. At each visit, anthropometric parameters were measured and recorded by a research nurse according to standard procedures.19 Blood samples were collected at each trimester following a 12-h overnight fast. At delivery, placenta biopsies and cord blood samples were collected. All glucose and insulin concentrations were assessed on fresh serum samples at the Chicoutimi Hospital Clinical Laboratory. Insulin measurements were performed using a radioimmunoassay method (Advia Centaur, Simmens). Maternal blood glucose along with total cholesterol (TC) and TG levels were determined using the enzymatic method on a Beckman analyzer (model CX7; Beckman, Fullerton, CA). VLDL and lowdensity lipoproteins (LDL) in plasma were precipitated with heparin and MnCl2 to isolate HDL particles for cholesterol (HDL-c) measurement. Then, plasma LDL-c concentrations were estimated using the Friedewald formula.20 GDM was diagnosed according to WHO criteria: glycaemia ⩾7.8 mmol/l following the 2-h post 75 g-oral glucose tolerance test (OGTT)
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between the 24th and 28th weeks of gestation. The 126 women were tested between weeks 22.4 and 28.9 (average = 25.7 ± 1 weeks) of gestation. Insulin resistance levels were assessed using HOMA-IR (fasting glucose (mmol/l) × fasting insulin (mU/l)/22.5) at the second trimester of pregnancy.21 Clinical information about the newborn was collected at birth from medical files and included gestational age, sex of the newborn and anthropometric measurements. The gestational age was calculated at the first visit from the date of the last menstrual period and was corrected afterwards, as required, based on the date from the ultrasound scans. Cord blood lipid and glucose levels were assessed as described for the mother. In addition, cord blood C-peptide levels, measured by ELISA, were used as a marker of the newborn’ insulin-secretory activity.22 Because the lipid measurements (HDL-c, TG, TC) were not available for all women, the number of samples (n) provided in Table 2 are lower than 126. The lipid profile was available for 92 newborns out of 126. Placenta tissue sampling Placenta tissues were sampled by well-trained clinicians (MD) within a few minutes after delivery and placental expulsion. Two biopsies of 0.5 cm3 were taken on the fetal side of the placenta near the insertion point of the umbilical cord. The biopsies consisted of the chorionic plate with the chorionic villi. Placental biopsies were washed in PBS 1× (137 mM NaCl, 2.7 mM KCl, 10 mM NaH2PO4, 1.8 mM KH2PO4, pH 7.4) to remove cord/maternal blood, dissected to remove conjunctive tissues and kept in RNALater (Qiagen, Valencia, CA) at −80°C until nucleic acid extraction. DNA and RNA were purified from placenta biopsies using the All Prep DNA/RNA/Protein Mini Kit (Qiagen). RNA quality was assessed with Agilent 2100 Bioanalyzer RNA Nano Chips (Agilent Technologies, Santa Clara, CA). On average, the RNA was considered of good quality (mean RNA integrity number 8.00 ± 0.70). DNA methylation analysis at the LPL gene locus Based on a first epigenome-wide DNA methylation association study (EWAS) [Illumina’s HumanMethylation450 BeadChips (Illumina Inc., San Diego)] conducted with 30 placenta samples of mother diagnosed with GDM and 14 from controls,23 we identified three loci flanking the first exon of the LPL gene that showed DNA methylation variability and potential differences between groups. These three CpGs were retained for DNA methylation analyses in the current study (total n = 126). Cytosine methylation was quantified using sodium bisulfite (NaBis) pyrosequencing (Pyromark Q24, QiagenBiotage).24 Combined with the NaBis DNA treatment, pyrosequencing is a quantitative real-time sequencing technology that allows measuring DNA methylation levels (%) for each cytosine of the covered region. NaBis treatment of DNA (EpiTect Bisulfite Kit, Qiagen) specifically converts unmethylated cytosine into uracil, while the methylated cytosines are
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protected from this transition. Once treated, NaBis-DNA is amplified (Pyromark PCR kit, Qiagen) and pyrosequenced. PCR and sequencing primers for analysis of the LPL CpGs were designed with Pyromark Assay Design (version 2.0.1.15; Qiagen) to cover the three CpGs found to be differentially methylated from our ongoing EWAS of GDM cases-controls (cg16125291, cg08918749 and cg16420199). The designed primers, after bisulfite conversion of DNA, were as follow: LPLCpG1F: 5′-AAGTATAAGTTGGGAYGTAATGTG TG-3′, LPLCpG1R: 5′-CCAAAAAAAAAAAATTTAACTATTA AATTAC-3′ (177 bp), and LPLCpG1seq: 5′-GTGTGTTTTTTTATTTTTATATT GA-3′; LPLCpG2F: 5′-GGAGGGGTTTTGGAATGAAAG-3′, LPLCpG2R: 5′-TTCCCCAAAAAAAACCACAATCRAC CC-3′ (113 bp), and LPLCpG2seq: 5′-GGTTTTGGAATGAAAGG-3′ and; LPLCpG3F: 5′-GTTTTGGGGGTTGAGGTT-3′, LPLCpG3R: 5′-AATTAAACAAACTACCTCCATTAC-3′ (202 bp), and LPLCpG3seq: 5′-CTAAAACATTCTCATTTAAC-3′. LPL expression analysis cDNA was generated from total RNA using a random primer hexamer provided with the High Capacity cDNA Archive Kit from Applied Biosystems (Foster City, CA). Equal amounts of cDNA were run in duplicate and amplified in a 20 µl reaction containing 10 µl of Universal PCR Master Mix (Applied Biosystems). Primers and Taqman probes were obtained from Applied Biosystems (LPL: Hs00173425_m1; Applied Biosystems). The YWHAZ housekeeping gene (endogenous control; YWHAZ: Hs00237047_m1) was amplified in parallel. LPL and YWHAZ amplifications were performed using an Applied Biosystems 7500 Real Time PCR System, as recommended by the manufacturer (Applied Biosystems). The expression of LPL gene was normalized to the expression of the YWHAZ reference gene. The YWHAZ gene has been shown to be suitable for gene expression normalization in placenta.25–27 Placental LPL expression was compared between normal glucose tolerance (NGT) and GDM women using the 2 − ΔΔCt method28 and YWHAZ/LPL Ct ratio were used for correlation analysis. Statistical analyses DNA methylation levels of CpGs at the LPL gene locus and maternal and newborn characteristics were compared between groups of women with or without GDM using the unpaired Student t-test or Pearson’s χ2 test for categorical variable (history of GDM). Kolmogorov-Smirnov test was applied to verify data distribution. The following variables were not normally distributed and were thus log transformed: maternal fasting insulin, LDL-c at first trimester and TG at each trimesters as well as cord blood TG, HDL-c and LDL-c. Pearson’s correlation
coefficient was used to assess the relationship between LPL DNA methylation and dependent variables (mRNA expression, maternal metabolic data, birth and placental weight and cord blood lipid profile). The statistical models were adjusted for the following confounders when applicable: age and BMI of the mother at first trimester of pregnancy, history of gestational diabetes, gestational age at delivery, method of childbirth (vaginal v. caesarean section), newborn’s sex and birth weight. The results remained unchanged after adjusting for parity. Furthermore, as maternal TG concentrations were significantly correlated with maternal HDL-c and 2-h post-OGTT glucose concentrations, the statistical models were also adjusted for TG when applicable. Stepwise multivariate linear regression was used to identify predictors of the variance of placental LPL DNA methylation levels. The input-independent variables were: maternal characteristics (age, BMI and TG at first trimester of pregnancy, glucose 2-h post-OGTT and HDL-c at second trimester of pregnancy, HDL-c at third trimester of pregnancy, history of gestational diabetes), gestational age at delivery and method of childbirth. All the variables with P < 0.05 were retained in the regression model. Results with P-values ⩽0.05 (two-sided) were considered statistically significant. All analyses were performed with SPSS version 20.
Results Characteristics of mothers according to their glucose tolerance status as well as newborns’ gestational age and birth weight are shown in Table 1. At first trimester of pregnancy, women were on average 29 years old, slightly overweight and had normal fasting glucose concentrations and lipid profile. Of the 126 women recruited, 27 had GDM, whereas 99 were normoglycemic (NGT). Among the women with GDM, 16 were treated with a diet only whereas 11 received a diet and insulin treatment. Overall, blood glucose 2-h post-OGTT covered a wide range of values (from 3.80 to 10.80 mmol/l). Mothers with GDM had a lower gestational weight gain. Average birth weight was within the normal weight range in both NGT and GDM groups. Nevertheless, according to Olsen et al.29 intrauterine growth curves, two babies were found to be small for gestational age whereas five babies were large for gestational age (LGA). Among these seven babies, one LGA was born to GDM mothers. Interestingly, newborn exposed to GDM had significantly higher levels of cord blood C-peptide, hence suggesting that they had been exposed to higher glucose concentrations. DNA methylation analysis of the placental LPL gene Placental DNA methylation was measured at 3 CpGs located within the LPL gene locus first identified using an EWAS strategy (Fig. 1). The first CpG (LPL-CpG1) was located in its proximal promoter region, 46 bp upstream from the first exon (Fig. 1). The two other CpGs, CpG2 and CpG3, were located
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Table 1. Characteristics of women and newborn according to maternal glucose tolerance status
Women History of GDM Parity 0 1 2 First trimester of pregnancy Age (years) BMI (kg/m2) Fasting glucose (mmol/l) Fasting insulin (mU/l)a Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l)a Second trimester of pregnancy Fasting glucose (mmol/l) Fasting insulin (mmol/l)a 2-h post-OGTT glucose concentration (mmol/l) HOMA-IRa Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l) Third trimester of pregnancy Fasting glucose (mmol/l)a Fasting insulin (mmol/l)a HOMA-IRa Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l) Weight gain between first and third trimester (% of initial weight) Newborn Gestational age (weeks) Birth weight (kg) Triglycerides (mmol/l)a Total cholesterol (mmol/l) HDL-c (mmol/l) LDL-c (mmol/l) C-peptide (pmol/l)
GDM (n = 27)
NGT (n = 99)
9 (33.3%)
4 (4.0%)**
9 (33.3%) 14 (51.9%) 4 (14.8%)
54 (54.5%)† 28 (28.3%)† 17 (17.2%)†
Mean ± S.D. Minimum–maximum
Mean ± S.D.
Minimum–maximum
29.1 ± 3.6 25.7 ± 3.9 4.48 ± 0.44 5.69 ± 4.33 1.16 ± 0.51 4.55 ± 0.60 1.60 ± 0.31 2.31 ± 0.52
20–36 19.6–35.4 3.60–5.30 1.70–16.70 0.60–2.70 3.10–6.00 1.19–2.31 1.20–3.30
28.8 ± 3.9 24.6 ± 4.7 4.41 ± 0.34 5.10 ± 3.67 1.00 ± 0.45† 4.67 ± 0.79 1.56 ± 0.32 2.46 ± 0.69
21–39 16.6–39.7 3.80–5.20 1.00–17.60 0.40–2.70 3.10–7.20 0.81–2.42 1.30–5.10
4.52 ± 0.41 9.19 ± 6.13 8.40 ± 0.61 1.86 ± 1.35 1.88 ± 0.72 5.90 ± 0.75 1.75 ± 0.29 3.24 ± 0.76
3.70–5.60 2.60–27.60 7.80–10.80 0.43–5.89 0.90–3.70 4.70–7.70 1.36–2.44 1.80–4.50
4.28 ± 0.39** 7.03 ± 5.30† 6.22 ± 0.81** 1.33 ± 1.04* 1.72 ± 0.64 6.26 ± 1.12 1.80 ± 0.41 3.62 ± 1.00
3.60–5.70 1.40–38.90 3.80–7.70 0.25–7.09 1.00–4.60 4.10–10.20 0.96–2.74 1.80–7.00
4.19 ± 0.44 7.60 ± 5.22 1.44 ± 1.08 2.58 ± 0.97 6.63 ± 1.10 1.74 ± 0.27 3.61 ± 0.97 13.8 ± 5.2
3.20–5.00 2.00–24.70 0.32–4.83 1.50–6.10 5.10–8.90 1.25–2.24 2.10–5.70 4.0–25.0
4.29 ± 0.63 9.86 ± 7.68* 1.85 ± 1.73† 2.62 ± 0.86 6.82 ± 1.28 1.73 ± 0.42 3.85 ± 1.13 19.5 ± 6.8**
3.60–8.30 3.80–42.20 0.73–9.75 1.10–5.20 4.10–10.80 0.89–2.80 1.70–7.40 2.0–34.0
39.7 ± 1.2 3.45 ± 0.45 0.43 ± 0.23 1.63 ± 0.29 0.61 ± 0.21 0.80 ± 0.19 171.3 ± 86.0
37.4–41.6 2.6–4.6 0.22–0.94 1.20–2.25 0.33–1.06 0.53–1.14 14.8–338.0
39.4 ± 1.1 3.45 ± 0.40 0.40 ± 0.23 1.69 ± 0.41 0.61 ± 0.20 0.87 ± 0.28 125.3 ± 66.8*
37.3–41.6 2.0–4.3 0.23–1.82 0.85–2.89 0.26–1.27 0.39–1.90 1.0–353.9
GDM, gestational diabetes mellitus; NGT, normoglycemic; BMI, body mass index; OGTT, oral glucose tolerance test; HDL, high-density lipoproteins; LDL, low-density lipoproteins. a Geometric mean (value obtained after log10 transformation of the variable). **p ⩽ 0.01;* p ⩽ 0.05; †p ⩽ 0.10.
405 and 925 bp downstream from the first exon, within the LPL intron 1 CpG island. DNA methylation level at LPL-CpG1 locus was not found to be correlated with the methylation levels at CpG2 and CpG3 (r < 0.08). Nevertheless, LPL DNA methylation levels at CpG2 and CpG3 sites
were found partially correlated with each other (r = 0.32; P < 0.001). Average DNA methylation at LPL gene locus was between 34.7% and 39.8% (Fig. 1). Overall, LPL DNA methylation levels were lower in placenta exposed to GDM compared with
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CpG1
CpG2
CpG3
ERE PPARG
Average LPL gene DNA methylation CpG1 (cg16125291): 35.4 ± 17.4%
Promoter
CpG2 (cg08918749): 39.8 ± 16.1%
Exon
CpG3 (cg16420199): 34.7 ± 10.6%
CpG island
Fig. 1. Schematic representation of the LPL gene locus CpG island and localization of the 3 loci epigenotyped. CpG1 is located within the LPL proximal promoter region whereas CpG2 and CpG3 are located downstream from the first exon within the LPL intron 1 CpG island. Transcription factor binding sites were identified using the UCSC Genome Browser (NM_131127)51 software, accessed 12 September 2012.
50% 41.8% 37.2%
DNA methylation(%)
40%
36.0%
32.5% 28.6%
29.9%
30%
20%
10% GDM
NGT
GDM
NGT
0% P value
GDM
NGT
CpG1
CpG2
CpG3 0.007
0.005
0.007
a
P value
0.023
0.009
0.006
P valueb
0.033
0.013
0.007
P valuec
0.062
0.038
0.011
a
Adjusted for maternal age and BMI at 1st trimester and history of gestational diabetes b Adjusted for maternal age, BMI and TG at 1st trimester and history of gestational diabetes c Adjusted for maternal age, BMI and TG at 1st trimester, history of gestational diabetes, gestationnal age at delivery and method of childbirth
Fig. 2. DNA methylation levels at placental LPL loci according to maternal glucose tolerance status. Mean DNA methylation levels at placental LPL gene CpG1, CpG2 and CpG3 loci in mothers with gestational diabetes mellitus (n = 27) (black) and with normal glucose tolerance (n = 99) (white). Error bars represent the standard error of the mean (n = 126). Mean DNA methylation levels were compared with the Student t-test and adjusted for confounding variables.
those not exposed (Fig. 2). DNA methylation differences remained unchanged for CpG2 and CpG3 after adjusting the statistical models for age, BMI and TG at first trimester of pregnancy and history of gestational diabetes, gestational age at delivery and method of childbirth. Furthermore, DNA methylation differences between the GDM and NGT mothers
remained unchanged at the 3 CpGs when the obese women [BMI > 30 kg/m2 (n = 13)] were removed from the analysis or when the statistical models were adjusted for maternal weight gain between the first and third trimester of pregnancy. The largest DNA methylation difference between groups (9.3%) was found with CpG2 (P = 0.04) (Fig. 2).
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Table 2. Pearson’s correlation coefficient between placental LPL DNA methylation and maternal metabolic profile First trimester of pregnancy Triglycerides [mmol/l (n = 126)]
CpG1 methylation (%) CpG2 methylation (%) CpG3 methylation (%) mRNA levels (AU)
Second trimester of pregnancy 2-h post-OGTT glucose [mmol/l (n = 126)]
Third trimester of pregnancy
HDL-c [mmol/l (n = 120)]
HDL-c [mmol/l (n = 120)]
I
II
I
III
I
III
I
III
–0.16† –0.14 –0.04 0.19*
–0.14 –0.12 –0.03 0.16†
–0.26** –0.12 –0.18* –0.17†
–0.22* –0.05 –0.14 0.10
0.03 –0.04 –0.15† –0.05
–0.02 –0.06 –0.17† 0.01
0.06 –0.12 –0.20* 0.07
–0.01 –0.13 –0.20* 0.11
HDL, high-density lipoproteins. I – adjusted values for age, body mass index (BMI) at first trimester and history of gestational diabetes. II – adjusted values for age, BMI at first trimester, maternal glucose 2-h post oral glucose tolerance test at second trimester, history of gestational diabetes, gestational age at delivery and method of childbirth. III – adjusted values for age, BMI and triglycerides at first trimester, history of gestational diabetes, gestational age at delivery and method of childbirth. **p ⩽ 0.01; *p ⩽ 0.05; †p ⩽ 0.10.
Correlations between LPL DNA methylation levels and maternal metabolic profile To further confirm the initial EWAS findings (that maternal hyperglycemia is negatively associated with LPL DNA methylation), the correlation between placental LPL DNA methylation and maternal glucose levels was tested. We observed that CpG1 and CpG3 DNA methylation levels were negatively correlated with maternal 2-h post-OGTT glucose concentrations (r = − 0.26; P = 0.003; r = − 0.18; P = 0.05) (Table 2). The latter correlation was slightly affected by additional corrections for maternal TG levels at first trimester of pregnancy, gestational age at delivery and method of childbirth and did not remain significant (r = − 0.14; P = 0.13). Nevertheless, the correlation between LPL CpG1 DNA methylation levels and maternal 2-h post-OGTT glucose concentrations remained significant after consideration of all potential confounding variables (r = − 0.22; P = 0.02) and exclusion of the 13 obese mothers. Finding a correlation between LPL DNA methylation and 2-h post-OGTT glucose concentrations thus confirms the initial association results. As LPL is a key player in TG and lipoprotein metabolism, we also tested for correlations between placental DNA methylation at the LPL gene locus and maternal TG, HDL-c and LDL-c concentrations. We found that CpG3 methylation levels were negatively correlated with concentrations of maternal HDL-c at third trimester of pregnancy (r = − 0.20; P = 0.03). A trend for correlation was also observed between CpG3 DNA methylation levels and HDL-c at second trimester of pregnancy (r = − 0.15; P = 0.10) (Table 2). These correlations remained unchanged after adjusting for all the potential confounding variables or when obese women were excluded from the analysis. Maternal TG and LDL-c concentrations were not correlated with placental LPL DNA methylation. Overall, stepwise multivariate linear regression analysis showed that maternal glucose concentrations (2-h postOGTT) (β = − 0.236; P = 0.01) and HDL-c levels (third
trimester of pregnancy) (β = − 0.182; P = 0.05) could explain up to 7.0% (R2adj = 0.070; P = 0.006) of placental DNA methylation variance at LPL-CpG3 locus. Correlations between LPL DNA methylation levels and offspring characteristics To further evaluate the impacts of LPL DNA methylation profile on materno-fetal transport we tested the association between LPL DNA methylation levels and offspring developmental characteristics and lipid profile. Methylation levels at the LPL-CpG2 locus in the placenta was found to be negatively correlated with cord blood HDL-c levels (r = − 0.24; P = 0.03), and TC/HDL-c ratio (r = 0.22; P = 0.04) and tended to be correlated with TG concentrations (r = 0.19; P = 0.08) after adjusting for newborn’s sex, gestational age and method of childbirth. The strength of these three correlations was increased after adjustment for birth weight (r = − 0.27; P = 0.01; r = 0.27; P = 0.01 and r = 0.25; P = 0.02 for HDL-c, TC/HDL-c and TG, respectively) and remained similar after correction for maternal BMI at first trimester of pregnancy, gestational weight gain and maternal metabolic variables throughout pregnancy. Correlation between placental LPL DNA methylation and mRNA expression levels To assess the impacts of DNA methylation on placental LPL gene expression, LPL mRNA levels were quantified. Placental LPL mRNA levels, expressed as arbitrary units and normalized to YWHAZ mRNA levels, were found to be 1.6 fold higher in placenta of women with GDM (1.04 ± 0.012 AU) compare to NGT (1.02 ± 0.006 AU) (P = 0.04) according to the 2 − ΔΔCt method.28 We also found that LPL DNA methylation levels at CpG2 and CpG3, both located within the intronic CpG island, were negatively correlated with placental LPL gene expression (Fig. 3). Nevertheless, DNA methylation at CpG1, located directly within the LPL gene promoter, was not
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(b)
1.2
r =–0.43 p
