An epigenome-wide association meta-analysis of prenatal maternal stress in neonates: A model approach for replication

Figures & data

Table 1. Sample characteristics.

	Generation R (n = 912)	ALSPAC (n = 828)
Child characteristics
Sex, % girls	48.4	48.9
Gestational age, weeks	40.2 (1.43)	39.6 (1.50)
Maternal characteristics
Age, years	31.7 (4.13)	29.56 (4.39)
PMSE score, log transformed	0.15 (0.11)	0.25 (0.14)
Smoking during pregnancy, %
never	77.5	87.1
until pregnancy was confirmed	7.7	3.4
continued	14.8	9.5

* Values represent mean (standard deviation), unless otherwise specified.

Table 2. Summary of the number of CpGs and their proportional distribution according to mean methylation (β-value) density in the Generation R and ALSPAC samples.

	Mean β-value in cord blood
Generation R	<0.3	≥ 0.3 to 0.7	≥ 0.7	Total
CpGs (n)	181,765	51,296	202,952	436,013
CpGs as proportion of total array (%)	42	11	47	100
ALSPAC
CpGs (n)	187,967	69,082	220,655	477,704
CpGs as proportion of total array (%)	39	15	46	100

Table 3. Top 10 CpG probes (meta P < 1.00e-04) derived from the EWAS meta-analysis of prenatal maternal stress exposure in neonates, sorted by ascending meta P-value (n_total = 1,740).

			Generation R (n = 912)		ALSPAC (n = 828)		Meta-analysis (ntotal = 1,740)
Probe name	Chromosome	Position	Effect (SE)	P	Effect (SE)	P	Effect (SE)	Direction^*	Meta P	Het P	Nearest gene (s)
cg13529437	6	43607635	−0.04 (0.009)	3.31e-06	−0.03 (0.016)	1.15e-01	−0.04 (0.007)	—	1.00e-06	.47	MAD2L1BP
cg04129946	2	201753996	0.02 (0.006)	2.63e-04	0.02 (0.007)	9.71e-03	0.02 (0.004)	++	7.59e-06	.73	PPIL3;NIF3L1
cg20959676	1	23696021	0.01 (0.003)	6.54e-03	0.02 (0.005)	1.01e-04	0.01 (0.003)	++	8.67e-06	.09	C1orf213;ZNF436
cg17631424	4	69312514	−0.06 (0.016)	2.64e-04	−0.05 (0.022)	1.53e-02	−0.06 (0.013)	—	1.13e-05	.81	TMPRSS11E
cg19459675	4	166249239	0.02 (0.005)	7.99e-04	0.02 (0.006)	6.72e-03	0.02 (0.004)	++	1.59e-05	.76	SC4MOL
cg12947485	4	25310668	−0.04 (0.010)	6.78e-04	−0.04 (0.015)	5.84e-03	−0.04 (0.009)	—	1.71e-05	.75	NA
cg20011562	6	12749352	0.01 (0.003)	2.78e-02	0.01 (0.004)	9.98e-05	0.01 (0.002)	++	1.85e-05	.18	PHACTR1
cg02644494	19	6412686	0.02 (0.008)	2.30e-03	0.03 (0.009)	2.77e-03	0.03 (0.006)	++	1.88e-05	.82	PVRL1
cg01686933	11	119596104	−0.02 (0.008)	6.49e-03	−0.03 (0.008)	1.11e-03	−0.02 (0.006)	—	2.12e-05	.81	NA
cg00409356	5	1879525	0.05 (0.012)	5.86e-05	0.02 (0.013)	5.75e-02	0.04 (0.009)	++	2.17e-05	.17	IRX4

* input order: the Avon Longitudinal Study of Parents and Children (ALSPAC) Study, the Generation R Study.

The prenatal maternal stress exposure score was not standardized and findings cannot be directly compared across ALSPAC and the Generation R Study.

NA: not available.

Figure 1. Manhattan plot of the EWAS meta-analysis of the PMSE score in cord blood (n_total = 1,740). The x-axis represents the autosomal (1–22) and sex (X,Y) chromosomes and the y-axis shows the –log₁₀(P). The red line indicates the Bonferroni-corrected epigenome-wide threshold (P = 1.00e-07).

Figure 2. Quantile-quantile (QQ) plot illustrating probability values from the EWAS meta-analysis of the PMSE score in cord blood (n_total = 1,740). The red line indicates the distribution under the null hypothesis and the shaded area indicates the 95% confidence band.

Table 4. Pathway analysis of top CpG probes (meta P < 1.00e-04) derived from the EWAS meta-analysis of prenatal maternal stress exposure in neonates (n_total = 1,740).

Pathway tool	Output
PANTHER GO-slim Molecular Function	Expected ngenes	Observed ngenes	Fold enrichment	Pathway enrichment P
Methyltransferase activity	0.26	4	>5	0.002^$
DAVID	Count	Coverage	Enrichment score	Pathway enrichment P
Methyltransferase activity	5	1.1%	2.46	0.06^*
GeneMANIA			Coverage	Pathway enrichment P
Methyltransferase activity			7/87	4.76e-04^*

^$ PANTHER reports the Bonferroni method

* DAVID and GeneMANIA report the Benjamini-Hochberg (false-discovery rate, FDR) method.

Table 5. Top Differential Methylated Regions (DMRs) associated with prenatal maternal stress exposure in the Generation R (n =912) and ALSPAC (n = 828) samples.

Cluster Number	n CpGs	Chromosome	Start Position	End Position	PGeneration R	PALSPAC	Nearest gene(s)
90946	3	20	62948037	62948235	3.75e-06	0.72	MYT1;LINC00266-1;CICP4
135488	11	7	133811808	133812369	1.27e-05	0.52	LRGUK
66361	15	17	73974861	73976089	3.55e-04	0.63	ACOX1; C17orf106