DNA methylation provides insight into intergenerational risk for preterm birth in African Americans

Figures & data

Table 1. Demographics table for maternal samples

Phenotype	PTB (N=16) Mean ± SD	TB (N=24) Mean ± SD	P-value*
Maternal Age	24.69 ± 4.7	24.13 ± 6.2	NS
Weeks Gestation	30.2 ± 3.6	39.9 ± 0.4	<0.0001
Birthweight, grams	1555.8 ± 717.9	3309.7 ± 353.7	<0.0001
Birthweight percentile	38.9 ± 30.6	46.2 ± 26.7	NS
Parity	1.2 ± 1.4	1.4 ± 1.7	NS
BMI	29.93 ± 8.4	26.46 ± 5.9	NS
Household Income
<15 K	(10) 66.7%	(11) 45.8%	NS
15–30 K	(2) 13.3%	(7) 29.2%	NS
>30 K	(3) 20%	(6) 25%	NS
Smoking	(4) 25%	(5) 21%	NS
Married	(5) 33.3%	(5) 20.8%	NS
Employed	(1) 7.1%	(6) 42.9%	NS
Chorioamnionitis	(9) 56%	NA	NS
Granulocytes	74.7 ± 9.2	69.6 ± 7.8	NS
Monocytes	9.8 ± 3.7	10.7 ± 3.2	NS

* NS indicates that the P-value is non-significant (P > 0.05).

Figure 1. Manhattan plot of the relationship between maternal and fetal DNA methylation. The x-axis represents the position of each CpG site by chromosome. The y-axis represents the negative log₁₀ of the P-value for the association between maternal and fetal methylation for each CpG site. The red line indicates experiment-wide significance based on a false discovery rate of 5% such that the 5,171 CpG sites above this line are significantly correlated in leukocytes from a mother and her fetus.

Figure 2. Distribution of maternal DNA methylation for CpG sites. The x-axis represents the standard deviation (SD) of each CpG site's methylation (β) values in maternal leukocytes. The y-axis indicates the proportion of CpG sites in bins determined by SD. Black represents CpG sites that may be attributed to genetic variation while gray represents CpG sites that cannot be attributed to genetic variation. Graph (A) depicts the distribution of correlated CpG sites (n = 5,171). Graph (B) depicts the distribution of all CpG sites (n = 479,808).

Figure 3. Correlated CpG sites and their enrichment in meQTLs. The x-axis represents increasing statistical significance of genotype-methylation associations (cis-meQTLs). The –log₁₀ is used to represent the statistical threshold (α level) used to define meQTLs (i.e., 1 is equivalent to P < 0.1, and 2 is equivalent to P < 0.01, etc.). The y-axis represents the odds ratios comparing the probability that a correlated CpG site associates with a genotype (i.e., is an meQTL) with greater likelihood than an uncorrelated CpG site. The vertical lines represent the confidence interval for each odds ratio.

Table 2. Enrichment for correlation analysis of maternal and fetal blood

	Correlated	Not Correlated	P-value
CpG Islands	17.1%	31.3%	< 2.2 × 10^−16
Shores	21.9%	23.2%	1.5 × 10^−6
Shelves	12.1%	9.7%	9.0 × 10^−9
Open Sea	49.0%	35.7%	< 2.2 × 10^−16
Promoter	17.3%	25.2%	< 2.2 × 10^−16
5′UTR	6.0%	8.9%	9.9 × 10^−13
1^st Exon	2.4%	4.7%	8.9 × 10^−15
Gene Body	37.0%	33.2%	7.2 × 10^−9
3′UTR	4.9%	3.6%	1.2 × 10^−6
Intragenic	32.4%	24.3%	< 2.2 × 10^−16
PTB	6.5%	3.8%	<2.2 × 10^−16

Table 3. Pathway analysis of CpG sites in genes that correlate in maternal-fetal pairs

	KEGG ID	Enrichment P-value	Enrichment P-value	Enrichment P-value
		Correlated N=2639	Genetic N=1985	Non-genetic N=831
Metabolic
Type 1 diabetes mellitus	04940	0.006	0.018	NS
Fructose and mannose metabolism	00051	0.028	0.043	NS
Ether lipid metabolism	00565	0.034	0.017	NS
Type 2 diabetes mellitus	04930	0.038		NS
Glycolysis/Gluconeogenesis	00010	0.045	NS	NS
Cardiovascular
Arrhythmogenic right ventricular cardiomyopathy	05412	0.004	0.031	0.012
Hypertrophic cardiomyopathy	05410	0.013	NS	0.022
Dilated cardiomyopathy	05414	0.015		0.033
Viral myocarditis	05416	0.020	0.041	NS
Immune
Natural killer cell mediated cytotoxicity	04650	0.013	0.018	NS
Fc gamma R-mediated phagocytosis	04666	0.021	0.021	NS
Graft-vs.-host disease	05332	0.026	0.011	NS
Allograft rejection	05330	0.040	NS	NS
Other
Tight Junction	04530	9.8 × 10^−4	0.001	NS
Endocytosis	04144	0.001	0.006	0.036
Focal adhesion	04510	0.012	0.011	0.034
Cell adhesion molecules	04514	0.021	0.030	NS
ECM-receptor interaction	04512	NS	NS	0.020
Regulation of actin cytoskeleton	04810	NS	NS	0.028

The enrichment P-value indicates whether genes with correlated CpG sites are more likely to occur in the indicated biological pathway. The group of correlated CpGs is also stratified by whether or not they can be attributed to genetic variation. NS indicates that the P-value is not significant.

Figure 4. Association of MICB methylation and expression in PTB and TB pairs. (A) Association between cg06284756 in maternal leukocytes and PTB. The x-axis represents PTB status where 0 denotes term birth and 1 denotes PTB. The y-axis represents the methylation levels (β values) for cg06284756. (B) Correlation between maternal methylation (x-axis) and fetal methylation (y-axis) for cg06284756. Open circles represent maternal-fetal pairs that are preterm and closed circles represent pairs that are term. The dashed line represents correlation in PTB pairs, and the solid line represents correlation in term birth (TB) pairs. (C) Correlation between maternal leukocyte MICB expression (x-axis) and fetal leukocyte MICB expression (y-axis).