A meta-analysis of epigenome-wide association studies on pregnancy vitamin B12 concentrations and offspring DNA methylation

Figures & data

Table 1. Subject characteristics.

	Cohort	N participants	Ancestry	Vitamin B12 concentration (pmol/L), median (95% range)	Gestational age at vitamin B12 sampling (weeks), mean (SD)	Age at DNA methylation measurement^1, mean (SD)
Maternal vitamin B12 concentrations
Meta-analysis	Total	2,420
	GENR	823	European	180.0 (84.0, 425.2)	13.1 (1.7)	40.2 (1.5)
	INMA	372	European	222.5 (138.0, 359.8)	13.5 (1.8)	39.8 (1.4)
	MoBa1	1,007	European	297.3 (150.3, 535.9)	NA^2	39.5 (1.6)
	MoBa2	218	European	294.4 (155.7, 529.5)	NA^2	39.4 (1.6)
Look-up multi-ethnic population	MARBLES	48	Mixed	236.3 (150.8, 438.0)	23.0 (9.4)	39.1 (1.2)
Look-up early childhood (4–7 y)	Total	479
	GENR	284	European	185.5 (84.3, 435.4)	40.2 (1.5)	6.0 (0.4)
	INMA	195	European	224.3 (126.5, 351.8)	39.7 (1.5)	4.5 (0.2)
Look-up late childhood (8–10 y)	Total	482
	GENR	267	European	182.0 (78.0, 434.9)	40.1 (1.5)	9.8 (0.3)
	INMA	215	European	223.6 (139.0, 360.1)	39.9 (1.4)	8.8 (0.6)
Newborn vitamin B12 concentrations
Meta-analysis	Total	1,029
	ALSPAC	81	European	300.0 (120.0, 670.0)	40.2 (1.5)	40.2 (1.5)
	GENR	948	European	306.5 (130.2, 818.1)	39.6 (1.3)	39.6 (1.3)
Look-up early childhood (4–7 y)	Total	417
	ALSPAC	85	European	306.0 (121.3, 665.3)	39.7 (1.2)	7.4 (0.1)
	GENR	332	European	315.5 (135.2, 861.5)	40.2 (1.5)	6.0 (0.4)
Look-up late childhood (8–10 y)	GENR	321	European	302.0 (128.2, 869.9)	40.1 (1.5)	9.8 (0.3)
Look-up adolescence (17 y)	ALSPAC	83	European	306 (120.7, 667.7)	39.6 (1.3)	17.0 (1.0)

Abbreviations: ALSPAC, Avon Longitudinal Study of Parents and Children; GENR, Generation R Study; INMA, Sabadell subcohort of the INfancia y Medio Ambiente (INMA) Project; MARBLES, Markers of Autism Risk Learning Early Signs; MoBa, Norwegian Mother, Father, and Child Cohort Study.

For analyses at birth in weeks gestational age; for analyses in childhood in years.

For MoBa1 and MoBa2, individual data on gestational age at blood sampling (study population median 18, 25th–75th percentile 16–21) weeks were not available. Gestational age at blood sampling was not included as covariate in the models.

Figure 1. Study design. Schematic representation of the analyses of circulating vitamin B12 concentrations during foetal development and epigenome-wide DNA methylation in cord blood.

Abbreviations: adj: adjusted; cis-eQTM: cis-expression quantitative trait methylation; EWAS, epigenome-wide association study

* In this complete-case analysis, cohorts excluded participants with circulating vitamin B12 concentrations outside ± 5 standard deviations (SD) from the mean this complete-case analysis, cohorts excluded participants with circulating vitamin B12 concentrations outside ± 5 standard deviations (SD) from the mean of their study population, all twins, and in case of non-twin siblings, one child was excluded by selecting on completeness of data or, if equal, randomly. We prioritized CpGs with FDR-P-value <0.05 and showed low heterogeneity (I²<50%) for follow-up analyses. Vitamin B12, folate, and homocysteine were measured in either serum or in plasma.

Figure 2. Volcano plots show the directions of associations in epigenome-wide meta-analyses of circulating vitamin B12 concentrations during foetal development.

Abbreviations: B12, vitamin B12; CpG, cytosine-phosphate-guanine site; FDR, false discovery rate; SDS, standard deviation score.

Upper panel: maternal meta-analysis;Lower panel: newborn meta-analysis

The X-axis represents the difference in DNA methylation per SDS increase in circulating vitamin B12 concentrations; the Y-axis represents the −log₁₀(P). The red dotted line and the blue line represent the thresholds below which we considered associations significant using a Bonferroni correction (absolute P-value <1.2 × 10⁻⁷) and FDR-P-value <0.05, respectively, to account for multiple testing. In total, 56.1% of all analysed CpGs in the maternal meta-analysis showed increased methylation in relation to maternal vitamin B12 concentrations during pregnancy. Similarly, 51.4% of all analysed CpGs in the newborn meta-analysis showed increased methylation in relation to higher newborn circulating vitamin B12 concentrations.

Figure 3. Manhattan plots of epigenome-wide meta-analyses of circulating vitamin B12 concentrations during foetal development.

Abbreviations: B12, vitamin B12; CpG, cytosine-phosphate-guanine site; FDR, false discovery rate.

Upper panel: maternal meta-analysis;Lower panel: newborn meta-analysis

The X-axis represents chromosomes; the Y-axis represents the −log₁₀(P). The black dotted line and the red dashed line represent the thresholds below which we considered associations significant using a Bonferroni correction (absolute P-value <1.2 × 10⁻⁷) and FDR-P-value <0.05, respectively, to account for multiple testing. Models were adjusted for maternal confounders during pregnancy (age, education, body mass index, smoking, parity, and gestational age at blood sampling in maternal meta-analysis), child sex, cell-type proportion, and batch.

Table 2. Prioritized CpGs (n = 109) with differential methylation in cord blood in relation to maternal circulating vitamin B12 concentrations during pregnancy¹.

CpG	Chromosome	Gene (UCSC RefGene)	Gene location (UCSC RefGene)	Flagged	Coefficient	Standard error	P-value	FDR-corrected P-value	I^2	Persistence childhood	Overlap with folate hits in previous study^5	Overlap with birth weight hits in previous study ^7	Overlap with gestational age hits in previous study ^9	Overlap with overall cognitive skills hits in previous study ^9	Overlap with nonverbal IQ hits in previous study ^9	eQTM	Transcript Cluster Gene
cg25327343	8	MAL2	Body	N	0.59	0.10	7.49 × 10^−10	2.94 × 10^−4	11.3	Y	N	N	N	N	N	N
cg05665581	10	FAM24A	TSS1500	Y	−0.7	0.12	2.02 × 10^−9	2.94 × 10^−4	0.0	Y	Y	Y	N	N	N	N
cg17900015	17	KRT28	TSS1500	N	−0.62	0.10	2.71 × 10^−9	2.94 × 10^−4	33.5	Y	Y	N	N	N	N	N
cg00200803	20	CDH22	Body	Y	0.64	0.11	3.07 × 10^−9	2.94 × 10^−4	0.0	Y	N	N	N	N	N	N
cg00292513	12	IQSEC3		N	0.69	0.12	3.42 × 10^−9	2.94 × 10^−4	0.0	Y^3	N	N	N	N	N	Y	IQSEC3
cg15908975	7	MIR592		N	−0.8	0.14	1.70 × 10^−8	1.22 × 10^−3	0.0	Y	Y	Y	N	N	N	N
cg08849628	3			N	−0.83	0.15	2.17 × 10^−8	1.33 × 10^−3	0.0	N	N	N	N	N	N	N
cg25396728	12	IQSEC3		N	1.21	0.22	3.27 × 10^−8	1.56 × 10^−3	0.0	Y	N	N	N	N	N	Y	IQSEC3
cg21341928	13	PABPC3		N	0.88	0.16	4.49 × 10^−8	1.83 × 10^−3	0.0	Y	N	N	N	N	N	N
cg19083407	2	PAX8		N	1.31	0.24	4.69 × 10^−8	1.83 × 10^−3	0.0	N	N	N	N	N	N	Y	PAX8
cg12302982	2			N	1.05	0.19	5.52 × 10^−8	1.94 × 10^−3	0.0	N	Y	N	N	N	N	N
cg05282518	14	OR4K2	1stExon	N	−0.79	0.14	5.88 × 10^−8	1.94 × 10^−3	0.0	N	Y	N	N	N	N	N
cg09367432	4			N	−0.27	0.05	7.13 × 10^−8	2.19 × 10^−3	0.0	N	N	N	N	N	N	N
cg07296387	20	CDH22	Body	N	0.78	0.15	1.04 × 10^−7	2.97 × 10^−3	0.0	N	N	N	N	N	N	N
cg14776195	20	DEFB118	TSS1500	Y	−0.63	0.12	1.25 × 10^−7	3.36 × 10^−3	14.4	Y^3	N	N	N	N	N	Y	FRG1BP
cg19149785	19	KLK9		N	0.64	0.12	1.58 × 10^−7	3.99 × 10^−3	0.0	Y^3	N	N	N	N	N	N
cg06468072	11	DSCAML1	Body	N	0.61	0.12	1.94 × 10^−7	4.45 × 10^−3	0.0	N	N	N	N	N	N	N
cg11421509	8			Y	0.9	0.17	2.14 × 10^−7	4.45 × 10^−3	0.0	Y^3	N	N	N	N	N	N
cg03873392	16			Y	−0.58	0.11	2.15 × 10^−7	4.45 × 10^−3	0.0	Y	N	N	N	N	N	N
cg03327325	10			Y	1.17	0.22	2.17 × 10^−7	4.45 × 10^−3	4.7	Y	N	N	N	N	N	N
cg26393629	14			N	1.22	0.24	2.94 × 10^−7	5.75 × 10^−3	48.5	Y^4	N	N	N	N	N	N
cg19474546	14			Y	−0.71	0.14	3.18 × 10^−7	5.94 × 10^−3	30.9	N	N	N	N	N	N	N
cg04634427	15			N	0.95	0.19	3.39 × 10^−7	6.07 × 10^−3	32.5	N	N	N	N	N	N	N
cg14120919	10	CRTAC1	Body	N	0.83	0.16	3.79 × 10^−7	6.28 × 10^−3	0.0	N	N	N	N	N	N	N
cg19529709	22	TOP1P2		N	0.51	0.10	3.81 × 10^−7	6.28 × 10^−3	47.4	Y	N	N	N	N	N	N
cg20581874	14			N	0.82	0.16	4.01 × 10^−7	6.28 × 10^−3	18.2	Y^4	N	N	N	N	N	Y	RNASE6
cg05371552	7	VIPR2	Body	N	1.02	0.20	4.09 × 10^−7	6.28 × 10^−3	0.0	Y^3	N	N	N	N	N	N
cg07594247	2	PAX8		N	1.39	0.27	4.37 × 10^−7	6.32 × 10^−3	0.0	Y	N	N	N	N	N	Y	PAX8/RPL23AP7
cg27181142	19	SIX5	Body	N	0.41	0.08	4.41 × 10^−7	6.32 × 10^−3	0.0	N	N	N	Y	N	N	N
cg17445212	2	PAX8		N	1.16	0.23	5.39 × 10^−7	7.26 × 10^−3	0.0	N	N	N	N	N	N	Y	PAX8/RPL23AP7
cg10678190	16			Y	−0.7	0.14	5.40 × 10^−7	7.26 × 10^−3	30.6	Y^3	N	N	N	N	N	N
cg21482265	2	PAX8		N	1.31	0.26	6.00 × 10^−7	7.61 × 10^−3	0.0	N	N	N	N	N	N	Y	PAX8/RPL23AP7
cg12889195^2	2	PAX8		N	1.45	0.29	6.02 × 10^−7	7.61 × 10^−3	0.0	N	N	N	N	N	N	Y	PAX8/RPL23AP7
cg07646362	16			N	−0.66	0.13	7.11 × 10^−7	8.73 × 10^−3	0.0	N	N	N	N	N	N	N
cg02679336	10	FANK1	Body	N	−0.3	0.06	8.12 × 10^−7	9.70 × 10^−3	0.0	Y	N	N	N	N	N	N
cg27343456	2			N	0.59	0.12	8.52 × 10^−7	9.75 × 10^−3	0.0	N	Y	N	N	N	N	N
cg06882571	16	GSG1L		N	−0.49	0.10	8.84 × 10^−7	9.75 × 10^−3	0.0	Y	Y	N	N	N	N	N
cg21540359	7			N	0.67	0.14	8.85 × 10^−7	9.75 × 10^−3	0.0	N	N	N	N	N	N	N
cg02614024	15			N	0.47	0.10	9.18 × 10^−7	9.87 × 10^−3	0.0	Y	N	N	N	N	N	N
cg15612221	18			N	1	0.20	9.55 × 10^−7	1.00 × 10^−2	0.0	N	N	N	N	N	N	N
cg02105458	15			N	0.7	0.14	1.07 × 10^−6	1.10 × 10^−2	35.0	N	N	N	N	N	N	N
cg09317502	7	HEPACAM2	TSS1500	Y	−0.71	0.15	1.19 × 10^−6	1.19 × 10^−2	11.4	N	N	N	N	N	N	N
cg22157494	1	KIAA0562		N	0.64	0.13	1.22 × 10^−6	1.19 × 10^−2	0.0	N	N	N	N	N	N	N
cg23504719	10	CRTAC1	Body	N	0.82	0.17	1.28 × 10^−6	1.19 × 10^−2	0.0	N	N	N	N	N	N	N
cg01809217	1	GREM2	Body	N	0.6	0.12	1.28 × 10^−6	1.19 × 10^−2	0.0	N	N	N	N	N	N	N
cg05312960	22	TOP1P2		N	0.43	0.09	1.33 × 10^−6	1.19 × 10^−2	29.7	N	N	N	N	N	N	N
cg25843439	19	OR10H3	1stExon	Y	−0.54	0.11	1.33 × 10^−6	1.19 × 10^−2	25.2	N	N	N	N	N	N	N
cg13315047	12			N	0.7	0.14	1.42 × 10^−6	1.22 × 10^−2	0.0	N	N	N	N	N	N	N
cg03043822	22	TOP1P2		N	0.66	0.14	1.42 × 10^−6	1.22 × 10^−2	33.5	Y	N	N	N	N	N	N
cg23638640	22	TOP1P2		N	0.56	0.12	1.59 × 10^−6	1.34 × 10^−2	0.0	N	N	N	N	N	N	N
cg06324373	10	CRTAC1	Body	Y	0.96	0.20	1.73 × 10^−6	1.42 × 10^−2	0.0	Y^4	N	N	N	N	N	N
cg14605520	9	LHX3		N	0.79	0.17	1.88 × 10^−6	1.43 × 10^−2	25.2	N	N	N	N	N	N	N
cg08097631	14	C14orf48		N	0.51	0.11	1.88 × 10^−6	1.43 × 10^−2	0.0	N	N	N	N	N	N	N
cg01743593	7			N	−0.49	0.10	1.89 × 10^−6	1.43 × 10^−2	20.6	N	Y	N	N	N	N	N
cg01988325	5	MIR449B		N	−0.74	0.16	1.89 × 10^−6	1.43 × 10^−2	0.0	Y	N	N	N	N	N	N
cg10340210	4			Y	−0.6	0.13	1.95 × 10^−6	1.44 × 10^−2	12.3	Y	Y^6	N	N	N	N	N
cg22730830	16	PRSS21		N	0.76	0.16	1.99 × 10^−6	1.44 × 10^−2	0.0	N	Y	N	N	N	N	Y	PRSS21
cg15015426	1	OR10J5	TSS1500	Y	−0.75	0.16	2.01 × 10^−6	1.44 × 10^−2	0.0	Y	N	N	N	N	N	N
cg01925498	1			Y	−0.3	0.06	2.16 × 10^−6	1.49 × 10^−2	0.0	N	N	N	N	N	N	Y	LOC645166
cg11763394	2	PAX8		N	1.27	0.27	2.21 × 10^−6	1.49 × 10^−2	0.0	N	N	N	N	N	N	Y	PAX8/RPL23AP7
cg13174253	8	C8orf33	Body	N	−0.47	0.10	2.24 × 10^−6	1.49 × 10^−2	26.1	Y	N	N	N	N	N	N
cg00534274	4	SLC7A11		N	0.57	0.12	2.25 × 10^−6	1.49 × 10^−2	2.6	N	N	N	N	N	N	N
cg17465112	1			N	0.61	0.13	2.25 × 10^−6	1.49 × 10^−2	0.0	Y	N	N	N	N	N	N
cg16964673	1	KIAA0562		N	0.45	0.10	2.36 × 10^−6	1.54 × 10^−2	2.7	N	N	N	N	N	N	N
cg19480274	20			Y	−0.23	0.05	2.49 × 10^−6	1.60 × 10^−2	0.0	N	N	N	N	N	N	N
cg17234513	15			N	0.74	0.16	3.01 × 10^−6	1.85 × 10^−2	19.6	N	N	N	N	N	N	N
cg24554151	11	PANX1	Body	N	−0.7	0.15	3.04 × 10^−6	1.85 × 10^−2	5.5	Y^3,4	N	N	N	N	N	Y	PANX1
cg03829137	15			N	0.41	0.09	3.07 × 10^−6	1.85 × 10^−2	26.5	N	N	N	N	N	N	N
cg19479373	10	CRTAC1	Body	N	0.67	0.14	3.08 × 10^−6	1.85 × 10^−2	0.0	N	N	N	N	N	N	N
cg04206517	5	ACSL6		N	−0.13	0.03	3.18 × 10^−6	1.87 × 10^−2	42.9	N	N	N	N	N	N	N
cg05086444	7	VIPR2	Body	N	0.61	0.13	3.55 × 10^−6	2.06 × 10^−2	0.0	N	N	N	N	N	N	Y	PTPRN2
cg05779786	2	AGAP1		N	0.55	0.12	3.83 × 10^−6	2.12 × 10^−2	0.0	Y	N	N	N	N	N	N
cg26536593	1	ZNF670	TSS1500	Y	0.71	0.15	3.84 × 10^−6	2.12 × 10^−2	28.9	Y^3	N	N	N	N	N	N
cg15971980	6			Y	−0.52	0.11	3.90 × 10^−6	2.12 × 10^−2	0.0	Y	Y	Y	N	N	N	N
cg21218093	20	ZNF335	5‘UTR	N	−0.37	0.08	4.01 × 10^−6	2.15 × 10^−2	0.0	Y	N	N	N	N	N	N
cg09807524	11			N	0.39	0.09	4.50 × 10^−6	2.35 × 10^−2	31.1	N	N	N	N	N	N	N
cg11258452	12	SRRM4	Body	N	0.69	0.15	4.54 × 10^−6	2.35 × 10^−2	0.0	N	N	N	N	N	N	N
cg01613965	2	LOC654342	TSS200	Y	−0.34	0.07	4.90 × 10^−6	2.48 × 10^−2	2.8	N	N	N	N	N	N	Y	LOC654342
cg03689146	7			N	0.75	0.17	4.97 × 10^−6	2.49 × 10^−2	0.0	Y	N	N	N	N	N	N
cg01273232	15			N	0.67	0.15	5.35 × 10^−6	2.64 × 10^−2	0.0	Y	N	N	N	N	N	N
cg06178315	1	KIAA0562		N	0.52	0.11	5.48 × 10^−6	2.68 × 10^−2	0.0	N	N	N	N	N	N	N
cg27353899	3	MUC4		N	1.37	0.30	5.68 × 10^−6	2.75 × 10^−2	4.1	Y	N	N	N	N	N	Y	MUC20
cg22809920	20	RBPJL		N	0.42	0.09	5.84 × 10^−6	2.79 × 10^−2	20.6	Y	N	N	N	N	N	N
cg17270081	9	LHX3		N	0.62	0.14	6.11 × 10^−6	2.88 × 10^−2	0.0	N	N	N	N	N	N	N
cg15730180	7			N	−0.47	0.10	6.17 × 10^−6	2.88 × 10^−2	38.8	N	Y^6	N	N	N	N	N
cg04579415	6	TCTE1	Body	Y	−0.32	0.07	6.75 × 10^−6	3.09 × 10^−2	0.0	N	Y^6	N	N	N	N	N
cg27403609	2			N	0.38	0.08	7.03 × 10^−6	3.18 × 10^−2	23.2	N	N	N	N	N	N	N
cg08761659	16	OR1F1	TSS200	Y	−0.41	0.09	7.27 × 10^−6	3.26 × 10^−2	0.0	N	N	N	N	N	N	N
cg06671706	8	CLDN23	1stExon	N	0.71	0.16	7.43 × 10^−6	3.29 × 10^−2	0.0	Y	N	N	N	N	N	Y	RPL23AP7
cg05010260	7	C7orf52	5‘UTR	N	0.49	0.11	7.66 × 10^−6	3.36 × 10^−2	0.0	Y^3,4	N	N	N	N	N	N
cg16991589	13	PABPC3	TSS1500	N	1.13	0.25	7.78 × 10^−6	3.38 × 10^−2	0.0	NA	N	N	N	N	N	N
cg07091678	10	CRTAC1	Body	N	0.68	0.15	7.98 × 10^−6	3.43 × 10^−2	0.0	Y	N	N	N	N	N	N
cg17780447	19			N	0.65	0.14	8.25 × 10^−6	3.51 × 10^−2	0.0	Y^3	N	N	N	N	N	N
cg00074818	8	CLDN23	1stExon	N	0.47	0.11	8.93 × 10^−6	3.76 × 10^−2	0.0	Y	N	N	N	N	N	Y	CLDN23
cg08930881	10	CRTAC1	Body	N	0.61	0.14	9.32 × 10^−6	3.89 × 10^−2	0.0	N	N	N	N	N	N	N
cg13605615	1	DIRAS3	TSS1500	N	0.32	0.07	9.40 × 10^−6	3.89 × 10^−2	0.0	N	N	N	N	N	N	N
cg27539527	7			N	0.56	0.13	9.51 × 10^−6	3.90 × 10^−2	39.6	Y^3	N	N	N	N	N	N
cg26434090	11	DSCAML1	Body	N	0.47	0.11	9.68 × 10^−6	3.93 × 10^−2	0.0	N	N	N	N	N	N	N
cg18914258	4			Y	−0.33	0.07	1.02 × 10^−5	4.05 × 10^−2	0.0	N	N	N	N	N	N	N
cg01232511	16	PRSS21		N	0.83	0.19	1.02 × 10^−5	4.05 × 10^−2	0.0	N	Y	N	N	N	N	Y	PRSS21
cg15574301	5	PDLIM4		N	0.67	0.15	1.03 × 10^−5	4.05 × 10^−2	0.0	N	N	N	N	N	N	N
cg01330954	16			N	−0.48	0.11	1.04 × 10^−5	4.08 × 10^−2	0.0	N	N	N	N	N	N	N
cg21610815	2	PAX8		N	0.41	0.09	1.08 × 10^−5	4.16 × 10^−2	0.0	N	N	N	N	N	N	N
cg07280731	16	PRSS21		N	0.73	0.17	1.08 × 10^−5	4.16 × 10^−2	0.0	Y	N	N	N	N	N	Y	PRSS21
cg00466136	11	NTM	Body	N	0.66	0.15	1.14 × 10^−5	4.29 × 10^−2	0.0	N	N	N	N	N	N	N
cg08935125	16	CLDN6	5‘UTR	N	0.39	0.09	1.14 × 10^−5	4.29 × 10^−2	40.2	N	N	N	N	N	N	N
cg05146852	11	ANO3	Body	N	−0.34	0.08	1.17 × 10^−5	4.29 × 10^−2	0.0	N	N	Y	N	N	N	N
cg04453501	7			Y	0.73	0.17	1.18 × 10^−5	4.29 × 10^−2	0.0	Y	N	N	N	N	N	N
cg09520393	4	DRD5	TSS200	Y	−0.28	0.06	1.18 × 10^−5	4.29 × 10^−2	7.0	N	Y	N	N	N	N	N

Abbreviations: B12, vitamin B12; eQTM: cis-expression quantitative trait methylation; CpG, cytosine-phosphate-guanine site; FDR, false discovery rate; UCSC RefGene, University of California Santa Cruz Reference Gene, SDS, standard deviation score.

1. Coefficients and standard errors represent the percentage change in DNA methylation per SDS increase in vitamin B12. One SDS vitamin B12 corresponded to a weighed mean of 88.8 pmol/L. The prioritized CpGs included those CpGs with FDR-corrected P-value<0.05 and little evidence for heterogeneity (I² < 50%).

2. In bold, the CpG with the largest effect size.

3. Bonferroni-significant in childhood look-up (P-value<0.05/108 tests; i.e., P-value<4.63 × 10–4).

4. Bonferroni-significant in childhood look-up in both early and late childhood (P-value<0.05/108 tests; i.e., P-value<4.63 × 10–4).

5. Look-up in results of meta-analysis of circulating folate concentrations during pregnancy and epigenome-wide cord blood DNA methylation (13).

6. Not epigenome-wide significant (P_FDR>0.05) after adjustment for circulating maternal folate concentrations.

7. Look-up in results of meta-analysis of birth weight and epigenome-wide cord blood DNA methylation (35).

9. Look-up in results of meta-analyses of overall cognitive skills and nonverbal IQ and epigenome-wide cord blood DNA methylation (37).

Table 3. Prioritized CpGs (n = 7) with differential methylation in cord blood in relation to newborn circulating vitamin B12 concentrations sampled in cord blood at birth.¹.

CpG	Chromosome	Gene (UCSC RefGene)	Gene location (UCSC RefGene)	Flagged	Coefficient	Standard error	P-value	FDR-corrected P-value	I^2	Persistence childhood	Overlap with folate hits in previous study ^5	Overlap with birth weight hits in previous study ^6	Overlap with gestational age hits in previous study ^7	Overlap with overall cognitive skills hits in previous study ^8	Overlap with nonverbal IQ hits in previous study ^8	eQTM
cg13863764	1	PTCHD2	Body	N	−0.94	0.18	1.12 × 10^−7	2.96 × 10^−2	0	Y^3,4	N	N	N	N	N	N
cg00658405	19	ADAMTSL5	Body	N	0.81	0.16	2.79 × 10^−7	2.96 × 10^−2	0	N	N	N	N	N	N	N
cg08243619^2	1	PTCHD2	Body	N	−1.09	0.21	2.85 × 10^−7	2.96 × 10^−2	10.2	Y	N	N	N	Y	Y	N
cg24371425	1	PTCHD2	Body	N	−1.03	0.21	9.36 × 10^−7	4.64 × 10^−2	0	Y^3	N	Y	N	Y	N	N
cg02615136	3	ARIH2	Body	N	−0.26	0.05	9.93 × 10^−7	4.64 × 10^−2	0	N	N	N	N	N	N	N
cg04096723	1	RERE		N	−0.28	0.06	1.09 × 10^−6	4.64 × 10^−2	0	N	N	N	N	N	N	N
cg09573658	1	PTCHD2	Body	N	−0.75	0.15	1.12 × 10^−6	4.64 × 10^−2	43.4	Y^3	N	N	N	N	N	N

Abbreviations: B12, vitamin B12; eQTM: cis-expression quantitative trait methylation; CpG, cytosine-phosphate-guanine site; FDR, false discovery rate; UCSC RefGene, University of California Santa Cruz Reference Gene, SDS, standard deviation score.

1. Coefficients and standard errors represent the percentage change in DNA methylation per SDS increase in vitamin B12. One SDS vitamin B12 corresponded to a weighed mean of 171.2 pmol/L. The prioritized CpGs included those CpGs with FDR-corrected P-value<0.05 and little evidence for heterogeneity (I2 < 50%).

2. In bold, the CpG with the largest effect size.

3. Bonferroni significant in childhood look-up (P-value<0.05/7 tests; i.e., P-value<0.007).

4. Bonferroni-significant in childhood look-up, in both early and late childhood (P-value<0.05/7 tests; i.e., P-value<0.007). No persistence of newborn vitamin B12-related methylation was observed into adolescence at this significance level.

5. Look-up in results of meta-analysis of circulating folate concentrations during pregnancy and epigenome-wide cord blood DNA methylation (13).

6. Look-up in results of meta-analysis of birth weight and epigenome-wide cord blood DNA methylation (35).

7. Look-up in results of meta-analysis of gestational age and epigenome-wide cord blood DNA methylation (36).

8. Look-up in results of meta-analyses of overall cognitive skills and nonverbal IQ and epigenome-wide cord blood DNA methylation (37).

Data availability statement

Analysis plan and R code for cohort-specific analyses and meta-analyses are available via https://github.com/GiuliettaMonasso/PACE-B12-meta-analysis-of-EWAS.

The dataset(s) supporting the conclusions of this article is available in the [Zenodo repository]. All further relevant data supporting the key findings of this study are available within the article and its Supplementary Information files or from the corresponding author upon reasonable request and subject to the study-specific data access procedures. Requests for access to the individual-level data for ALSPAC can be directed to GCS: [email protected]. Requests for access to the individual-level data for GENR can be directed to JFF: [email protected]. Requests for access to the individual-level data for INMA can be directed to MB: [email protected]. Requests for access to the individual-level data for MARBLES can be directed to RJS: [email protected]. Requests for access to the individual-level data for MoBa1 and MoBa2 can be directed to SEH: [email protected].