DNA methylation profiles in sibling pairs discordant for intrauterine exposure to maternal gestational diabetes

Figures & data

Table 1. Characteristics of the study population.

Characteristics of offspring	Siblings exposed to GDM in utero (n = 18)	Siblings not exposed to GDM in utero (n = 18)	P value
Current age (years)	5.8 ± 1.4	9.4 ± 2.5	<0.0001
Sex (male/female)	11/7	10/8	0.999
Maternal age at delivery (years)	34.4 ± 2.5	30.8 ± 2.4	<0.0001
Delivery (NFSD/PSD/C-sec)	6/0/12	5/1/11	0.999
Birth weight (kg)	3.3 ± 0.4	3.2 ± 0.6	0.671
Current BMI Z-score	0.19 ± 0.73	0.31 ± 0.86	0.594
Current HbA1C (%)	5.3 ± 0.3	5.3 ± 0.2	0.326
Characteristics of mothers^*	During GDM pregnancy (n = 18)
Age (years)	34.4 ± 2.5
BMI before pregnancy (kg/m^2)	22.7 ± 2.4
BMI at delivery (kg/m^2)	28.6 ± 8.2
Cigarette smokers	None
Gestational weeks at 50 g OGTT (weeks)	26 ± 2
Glucose levels at 50 g OGTT (mg/dL)	170 ± 26
Gestational weeks at 100 g OGTT (weeks)	28 ± 2
Glucose levels at 100 g OGTT (mg/dL)
0-hour	97 ± 18
0.5-hour	206 ± 40
2-hour	178 ± 39
3-hour	154 ± 34
Insulin treatments	6 treated (33%)

* Maternal data during GDM pregnancy only were available.

Continuous data are presented as mean ± standard deviation and P values were calculated using paired t-test. Categorical data were presented in counts and tested with Pearson's Chi-square test. BMI Z-score were calculated according 2007 Korean National Growth Charts. NFSD, normal full-term spontaneous delivery; PSD, preterm spontaneous delivery; C-sec, Caesarean section.

Figure 1. Differential DNA methylation analysis in sibling pairs discordant for intrauterine exposure to maternal GDM. (A) Volcano plot for the differential DNA methylation analysis of the 465,446 CpG sites. The effect size of GDM exposure on DNA methylation levels at each of the CpG sites is shown in x axis and the significance levels are shown in y axis. Coefficients > 0 refer to increased methylation in the GDM group compared with the non-GDM group. (B) Heatmap of adjusted β values for the top 100 differentially methylated CpG sites associated with intrauterine exposure to maternal GDM. The 36 samples (column-wise) and 100 CpG sites (row-wise) associated with intrauterine exposure to GDM were displayed using unsupervised hierarchical clustering. The upper panel and the lower panel represent sibling pairs and group information (black: GDM group; gray: non-GDM group), respectively. Blue to red gradient correspond to increasing methylation. Manhattan distance measures were used to compute inter-sample distances based on DNA methylation levels. S1-S18 denotes sibling IDs and G and N represent the GDM group and the non-GDM group, respectively.

Table 2. Differential DNA methylation in siblings discordant for their exposure to maternal GDM (q < 0.15).

CpG ID	Position (hg19)	Adjusted differences,*^,† (M-values)	Adjusted differences,*^,† (β values)	Raw β differences	P value^†	q value	P value^‡	Nearby gene	CGI information	Gene property
cg04988367	chr5:10,933,006	1.098	0.039	0.026	2.45 × 10^−7	0.09	3.12 × 10^−4	CTNND2	Open sea	Intergenic
cg08407434	chr20:43,001,691	1.003	0.039	0.013	7.17 × 10^−7	0.109	1.51 × 10^−5	HNF4A	Open sea	Body
cg01617280	chr8:8,745,938	1.094	0.077	0.031	1.08 × 10^−6	0.109	1.74 × 10^−5	MFHAS1	Shelf	Body
cg13911801	chr9:35,860,912	0.981	0.078	0.042	1.46 × 10^−6	0.109	1.36 × 10^−4	LOC92973	Open sea	Body
cg09304293	chr6:163,475,252	1.186	0.066	0.041	1.56 × 10^−6	0.109	2.16 × 10^−4	PACRG	Open sea	Body
cg23109897	chr8:48,871,883	0.927	0.158	0.065	1.90 × 10^−6	0.109	5.79 × 10^−4	PRKDC	Shore	Body
cg06319822	chr16:215,960	−0.652	−0.016	−0.005	2.08 × 10^−6	0.109	2.30 × 10^−4	HBM	Island	TSS200
cg06928695	chr17:6,384,119	0.713	0.086	0.059	2.39 × 10^−6	0.11	1.49 × 10^−4	PITPNM3	Open sea	Body
cg18255813	chr6:7,195,966	1.131	0.029	0.007	3.10 × 10^−6	0.127	3.95 × 10^−4	RREB1	Open sea	Body
cg26952618	chr16:10,912,545	−0.785	−0.124	−0.063	4.34 × 10^−6	0.142	7.63 × 10^−4	FAM18A	Island	5′UTR
cg19565171	chr6:73,935,200	1.17	0.053	0.032	4.58 × 10^−6	0.142	5.16 × 10^−4	KHDC1L	Open sea	TSS200
cg10431340	chr1:161,279,108	1.127	0.063	0.030	4.65 × 10^−6	0.142	2.76 × 10^−4	MPZ	Open sea	Body

*log₂ fold difference in methylation levels (sibling exposed to GDM – control sibling). Positive values indicate higher DNA methylation levels in the sibling exposed to maternal GDM. ^†Adjusted for age, sex and experimental batch (Model 1) ^‡Adjusted for age, sex, experimental batch, and cell type composition (Model 2). CHR, chromosome; CGI, CpG island; hg19, human genome version 19; UTR, untranslated region; TSS200, within 200 base pair upstream of transcription start site.

Table 3. Gene set enrichment analysis of genes associated with differentially methylated CpG sites.

Gene sets	Number of genes	Enrichment score	Normalized enrichment score	P value	q value
Signal transduction pathways
Regulation of insulin like growth factor transport and uptake  by insulin like growth factor binding proteins (Reactome)	15	0.770	1.538	0.0024	1
TRK receptor signaling mediated by the MAPK pathway (PID)	18	0.740	1.528	0.0049	1
β3 integrin cell surface interactions (PID)	20	0.714	1.483	0.0154	1
Retinoic acid receptors mediated signaling (PID)	15	0.694	1.417	0.0358	0.7638
IGF signaling (INOH)	22	0.673	1.433	0.0219	0.6932
Regulation of RAS family activation (PID)	21	0.665	1.405	0.0379	0.7746
FGF signaling pathway (PID)	23	0.663	1.408	0.0351	0.8159
GPCRs other (WikiPathways)	35	0.634	1.445	0.0199	0.7079
Human thyroid stimulating hormone signaling pathway  (WikiPathways)	39	0.632	1.459	0.011	0.8781
Signaling by FGFR (Reactome)	35	0.597	1.366	0.0382	0.7321
Fibroblast growth factor 1 (NetPath)	40	0.593	1.383	0.0427	0.754
MAPK signaling pathway (WikiPathways)	73	0.547	1.33	0.0427	0.7644
Signaling by GPCR (Reactome)	457	0.446	1.164	0.034	0.8706
Metabolism
Superpathway of conversion of glucose to acetylCoA and  entry into the TCA cycle (HumanCyc)	15	0.711	1.418	0.0379	0.7746
C21 steroid hormone biosynthesis and metabolism (EHMN)	25	0.664	1.449	0.0186	0.8017
Androgen and estrogen biosynthesis and metabolism  (EHMN)	39	0.663	1.54	0.0033	1
Central carbon metabolism in cancer (KEGG)	33	0.652	1.46	0.009	0.9084
Metabolism of nucleotides (Reactome)	29	0.646	1.443	0.0174	0.7035
Selenocysteine synthesis (Reactome)	41	0.623	1.448	0.0154	0.7547
Glycerophospholipid metabolism (KEGG)	36	0.608	1.372	0.0446	0.75
Pi metabolism (Reactome)	31	0.605	1.378	0.0382	0.7201
Biological oxidations (Reactome)	82	0.576	1.399	0.0093	0.7397
Purine metabolism (KEGG)	62	0.562	1.355	0.0295	0.7328
Phospholipid metabolism (Reactome)	83	0.551	1.359	0.0115	0.7141
Metabolism of carbohydrates (Reactome)	119	0.532	1.338	0.0192	0.7502
Metabolism of amino acids and derivatives (Reactome)	143	0.507	1.294	0.0193	0.8099

Gene set enrichment analysis was performed with differentially methylated genes and gene sets from ConsensusPathDB. Gene set names and their source database (shown in parenthesis) with nominal P value lower than 0.05 are listed. FDR q values were presented. PID, pathway interaction database; INOH, integrating network objects with hierarchies; EHMN, Edinburgh

Figure 2. Within sibling pair comparison of DNA methylation levels at cg08407434 and HNF4A gene and correlation with mRNA expression. (A) Within sibling pair differences in covariate-adjusted β values (GDM – non-GDM) at cg08407434 show consistent increased methylation in siblings exposed to GDM. (B) Within sibling pair differences of covariate-adjusted overall methylation levels of all CpG sites across the HNF4A region also show a marginal trend of increased methylation in siblings exposed to GDM (P = 0.053). (C) Overall DNA methylation level of HNF4A showed a negative correlation with mRNA expression albeit non significant (Pearson's coefficient: -0.364, P = 0.068).