Animal and plant protein intake during infancy and childhood DNA methylation: a meta-analysis in the NutriPROGRAM consortium

Figures & data

Table 1. Study-specific descriptives for animal protein analysis.

	Early childhood DNAm measurement		Late childhood DNAm measurement
	CHOP N = 255	Generation R N = 324	CHOP N = 300	Generation R N = 304
E% from animal protein (%)	10.6 (3.2)	8.1 (2.4)	10.7 (3.5)	8.1 (2.4)
Total energy intake (kcal)	894 (169)	1309 (331)	887 (167)	1322 (334)
Age at blood collection (years)	5.6 (0.1)	6.0 (0.2)	11.3 (0.13)	9.78 (0.24)
Age at dietary data collection (months)	11.9 (0.2)	13.8 (1.7)	11.9 (0.2)	14.0 (1.8)
Sex
Boy	120 (47.1%)	154 (47.5%)	147 (49.0%)	157 (51.6%)
Girl	135 (52.9%)	170 (52.5%)	153 (51.0%)	147 (48.4%)
Maternal smoking during pregnancy
No smoking/smoking stopped before 2nd trimester	217 (85.1%)	290 (89.5%)	252 (84.0%)	270 (88.8%)
Sustained smoking into 2nd trimester	38 (14.9%)	34 (10.5%)	48 (16.0%)	34 (11.2%)
Maternal educational level*
Low	45 (17.6%)	82 (25.3%)	51 (17.0%)	88 (28.9%)
High	210 (82.4%)	242 (74.7%)	249 (83.0%)	216 (71.1%)
Maternal BMI (kg/m^2)	23.9 (4.1)	23.1 (3.6)	23.6 (3.8)	23.2 (3.6)
Gestational age at birth (weeks)	39.7 (1.2)	40.3 (1.4)	39.8 (1.2)	40.2 (1.4)
CHOP Study centre
Belgium	43 (16.9%)	−	36 (12.0%)	−
Spain	89 (34.9%)	−	101 (33.7%)	−
Germany	18 (7.1%)	−	42 (14.0%)	−
Italy	105 (41.2%)	−	91 (30.3%)	−
Poland	−	−	30 (10.0%)	−

DNAm= DNA methylation.

Results presented as mean ± SD or N (%).

*In both cohorts, maternal educational level was defined as ‘Low’ if no, primary or secondary school education obtained, and as ‘High’ if college or university degree obtained.

Table 2. Study-specific descriptives for plant protein analysis.

	Early childhood DNAm measurement		Late childhood DNAm measurement
	CHOP N = 255	Generation R N = 322	CHOP N = 300	Generation R N = 302
E% from plant protein (%)	4.04 (1.03)	4.67 (1.31)	3.93 (1.06)	4.76 (1.26)
Total energy intake (kcal)	894 (169)	1311 (331)	887 (167)	1323 (333)
Age at blood collection (years)	5.6 (0.1)	6.0 (0.2)	11.3 (0.13)	9.77 (0.24)
Age at dietary data collection (months)	11.9 (0.2)	13.8 (1.7)	11.9 (0.2)	14.0 (1.8)
Sex
Boy	120 (47.1%)	154 (47.8%)	147 (49.0%)	157 (52.0%)
Girl	135 (52.9%)	168 (52.2%)	153 (51.0%)	145 (48.0%)
Maternal smoking during pregnancy
No smoking/smoking stopped before 2nd trimester	217 (85.1%)	288 (89.4%)	252 (84.0%)	268 (88.7%)
Sustained smoking into 2nd trimester	38 (14.9%)	34 (10.6%)	48 (16.0%)	34 (11.3%)
Maternal educational level*
Low	45 (17.6%)	82 (25.5%)	51 (17.0%)	88 (29.1%)
High	210 (82.4%)	240 (74.6%)	249 (83.0%)	214 (70.9%)
Maternal BMI (kg/m^2)	23.9 (4.1)	23.1 (3.6)	23.6 (3.8)	23.2 (3.6)
Gestational age at birth (weeks)	39.7 (1.2)	40.3 (1.4)	39.8 (1.2)	40.2 (1.4)
CHOP Study centre
Belgium	43 (16.9%)	−	36 (12.0%)	−
Spain	89 (34.9%)	−	101 (33.7%)	−
Germany	18 (7.1%)	−	42 (14.0%)	−
Italy	105 (41.2%)	−	91 (30.3%)	−
Poland	−	−	30 (10.0%)	−

DNAm= DNA methylation.

Results presented as mean ± SD or N (%).

*In both cohorts, maternal educational level was defined as ‘Low’ if no, primary or secondary school education obtained, and as ‘High’ if college or university degree obtained.

Figure 1. Q-Q, Manhattan and volcano plots for associations between infant animal protein intake (E%) and early and late childhood DNA methylation. Significance line in Manhattan and volcano plots is set to represent the Bonferroni-corrected p-value threshold of P < 1.1 × 10¯⁷.

Figure 2. Q-Q, Manhattan and volcano plots for associations between infant plant protein intake (E%) and early and late childhood DNA methylation. Significance line in Manhattan and volcano plots is set to represent the Bonferroni-corrected p-value threshold of P < 1.1 × 10¯⁷.

Table 3. Meta-analysis results for associations of infant animal protein intake (E%) and early childhood whole blood DNA methylation levels at P < 1 × 10¯^5..

CpG	Effect^a	SE^a	P value	FDR	Direction^b	I^2	Polymorphic	mQTL^c	Chr	Position	Relation to Island	Nearest Gene	Gene Region
cg02751838	−0.10	0.02	1.25E–06	0.12	- -	48.7	Yes	Yes	16	75148330	N_Shore	LDHD	Body
cg19657814	0.04	0.01	1.29E–06	0.12	+ +	13.4	No	Yes	19	47137444	N_Shore	GNG8	TSS1500
cg14183329	0.06	0.01	1.53E–06	0.12	+ +	53.9	Yes	Yes	7	131242962	Island	PODXL	TSS1500
cg11780382	−0.08	0.02	1.55E–06	0.12	- -	54.8	No	Yes	6	24719812	N_Shore	C6orf62	-
cg18177414	0.19	0.04	1.90E–06	0.12	+ +	73	No	Yes	7	149389929	Island	KRBA1	-
cg24044478	0.17	0.04	1.94E–06	0.12	+ +	0	No	Yes	8	145035191	OpenSea	PLEC	Body
cg04730047	0.24	0.05	2.09E–06	0.12	+ +	0	No	Yes	3	99979355	N_Shore	TBC1D23	Body
cg20469139	0.30	0.06	2.64E–06	0.12	+ +	0	No	Yes	17	29297458	N_Shore	RNF135	TSS1500
cg27639457	0.20	0.04	2.76E–06	0.12	+ +	0	No	Yes	5	11384753	Island	CTNND2	Body
cg05226685	0.15	0.03	3.46E–06	0.14	+ +	0	Yes	No	8	30010237	N_Shelf	MIR548O2	-
cg10667167	0.10	0.02	3.97E–06	0.15	+ +	38.3	No	No	19	12662910	S_Shore	ZNF564	TSS1500
cg08785524	0.24	0.05	6.49E–06	0.21	+ +	0	No	No	11	29340853	OpenSea	KCNA4	-
cg04723723	0.09	0.02	7.50E–06	0.21	+ +	0	No	No	1	67966270	OpenSea	SERBP1	-
cg08468732	0.14	0.03	7.54E–06	0.21	+ +	0	No	Yes	2	95722029	OpenSea	MAL	-
cg23991274	−0.10	0.02	7.61E–06	0.21	- -	68.3	No	No	4	71860600	S_Shore	DCK	Body
cg08756033	0.31	0.07	9.65E–06	0.22	+ +	85.5	Yes	Yes	13	31480128	N_Shore	TEX26-AS1	TSS200

^aEffect sizes and SE are presented as a percent change in DNA methylation per 1 E% increase in energy from animal protein intake.

^bStudies are arranged from left to right as follows: CHOP then Generation R.

^cmethylation quantitative trait loci (mQTL) utilized from GoDMC database.

EWAS model was adjusted for total energy intake, age at blood collection, sex, maternal educational level, gestational age, pre-pregnancy BMI, maternal smoking during pregnancy, technical covariates, cell counts.

Table 4. Meta-analysis results for associations of infant animal protein intake (E%) and late childhood whole blood DNA methylation levels at P < 1 × 10¯^5..

CpG	Effect^a	SE^a	P value	FDR	Direction^b	I^2	Polymorphic	mQTL^c	Chr	Position	Relation to Island	Nearest Gene	Gene Region
cg21300373	0.16	0.03	4.27E–08	0.018	+ +	0	No	Yes	4	165304540	Island	MARCHF1	TSS200
cg10633363	−0.05	0.01	1.09E–07	0.022	- -	50.6	No	Yes	17	46703854	Island	HOXB9	TSS200
cg10167561	−0.06	0.01	1.13E–06	0.122	- -	0	Yes	No	13	22244956	Island	FGF9	TSS1500
cg15680470	0.17	0.04	1.18E–06	0.122	+ +	49.5	Yes	No	8	54938360	S_Shelf	TCEA1	-
cg09463900	0.05	0.01	1.90E–06	0.157	+ +	0	No	No	19	12939587	Island	RTBDN	Body
cg27026673	−0.10	0.02	2.85E–06	0.180	- -	29.9	No	Yes	2	39664343	Island	MAP4K3	TSS200
cg16516295	0.23	0.05	3.25E–06	0.180	+ +	0	No	Yes	3	129147846	S_Shore	EFCAB12	TSS1500
cg26730050	−0.20	0.04	4.04E–06	0.180	- -	27.2	No	Yes	13	112655007	OpenSea	SOX1	-
cg06993367	−0.11	0.02	4.36E–06	0.180	- -	0	No	Yes	16	982031	S_Shore	LMF1	Body
cg06516150	0.06	0.01	4.68E–06	0.180	+ +	0	No	Yes	12	109592528	Island	ACACB	Body
cg04621866	−0.10	0.02	4.97E–06	0.180	- -	0	No	No	8	144567481	N_Shore	ZC3H3	Body
cg13982505	−0.10	0.02	5.20E–06	0.180	- -	40.5	No	Yes	2	233642094	OpenSea	GIGYF2	TSS1500, Body
cg02288564	0.05	0.01	6.22E–06	0.182	+ +	0	No	No	19	49934404	Island	SLC17A7	Body
cg12140668	0.04	0.01	6.46E–06	0.182	+ -	84.5	No	No	1	197744252	Island	DENND1B	Body
cg15355952	0.24	0.05	6.60E–06	0.182	+ +	0	No	Yes	5	36662829	OpenSea	SLC1A3	Body
cg05965863	0.12	0.03	8.90E–06	0.222	+ +	0	No	Yes	3	137480182	Island	SOX14	-
cg21074631	−0.04	0.01	9.12E–06	0.222	- -	0	No	No	13	50422213	Island	CTAGE10P	-

^aEffect sizes and SE are presented as a percent change in DNA methylation per 1 E% increase in energy from animal protein intake.

^bStudies are arranged from left to right as follows: CHOP then Generation R.

^cmethylation quantitative trait loci (mQTL) utilized from GoDMC database.

EWAS model was adjusted for total energy intake, age at blood collection, sex, maternal educational level, gestational age, pre-pregnancy BMI, maternal smoking during pregnancy, technical covariates, cell counts.

Table 5. Meta-analysis results for associations of infant plant protein intake (E%) and early childhood whole blood DNA methylation levels at P < 1 × 10¯^5..

CpG	Effect^a	SE^a	P value	FDR	Direction^b	I^2	Polymorphic	mQTL^c	Chr	Position	Relation to Island	Nearest Gene	Gene Region
cg15407373	−0.003	0.0005	2.13E–07	0.047	- -	95.6	No	Yes	18	74800029	Island	MBP	Body
cg25973293	−0.003	0.0006	2.26E–07	0.047	- -	5.2	No	Yes	1	1061647	OpenSea	C1orf159	-
cg15473904	0.019	0.0038	5.62E–07	0.078	+ +	73.3	No	Yes	9	140446993	S_Shore	MRPL41	3’UTR
cg23235135	−0.004	0.0008	9.22E–07	0.096	- -	39.0	No	Yes	2	189850571	OpenSea	COL3A1	Body
cg03460239	0.002	0.0005	1.73E–06	0.113	+ +	71.8	No	No	11	124311153	OpenSea	OR8B8	TSS200
cg13920278	−0005	0.0010	1.98E–06	0.113	- -	0	No	Yes	9	93852285	OpenSea	LOC100129316	-
cg03870270	0.002	0.0003	2.08E–06	0.113	+ +	0	No	No	3	15643045	Island	HACL1	1stExon; TSS1500
cg14784944	−0003	0.0006	2.30E–06	0.113	- -	0	No	Yes	7	4855785	N_Shore	RADIL	Body
cg13558954	0.006	0.0013	2.45E–06	0.113	+ -	75.4	Yes	No	9	99521386	OpenSea	ZNF510	Body
cg18861311	−0.002	0.0003	2.92E–06	0.117	- -	54.4	No	Yes	10	102747418	Island	TWNK	1stExon; TSS200
cg23290217	−0.001	0.0003	3.09E–06	0.117	- -	0	No	Yes	19	4909290	Island	UHRF1	TSS1500
cg05040429	0.002	0.0004	4.05E–06	0.140	+ +	78.7	No	No	8	119633844	N_Shore	SAMD12	Body
cg01233720	−0.008	0.0016	4.45E–06	0.142	- -	0	Yes	Yes	5	60456037	N_Shore	SMIM15	5’UTR
cg23272399	−0.002	0.0004	6.32E–06	0.180	- -	81.1	No	Yes	1	53068579	Island	GPX7	Body
cg26753088	0.002	0.0004	6.74E–06	0.180	+ +	75.0	No	Yes	19	1293582	N_Shore	EFNA2	Body
cg02006854	−0.002	0.0004	7.09E–06	0.180	- -	0	Yes	No	2	239542460	Island	LINC01107	-
cg18459618	−0.004	0.0010	7.38E–06	0.180	- -	78.6	No	Yes	17	29297478	N_Shore	RNF135	TSS1500
cg06747907	0.004	0.0009	9.22E–06	0.213	+ +	0	No	Yes	1	16091100	OpenSea	FBLIM1	TSS1500; 1stExon
cg19685229	0.006	0.0014	9.94E–06	0.216	+ +	0	No	No	12	116587387	OpenSea	MED13L	TSS1500; Body

^aEffect sizes and SE are presented as a percent change in DNA methylation per 1 E% increase in energy from animal protein intake.

^bStudies are arranged from left to right as follows: CHOP then Generation R.

^cmethylation quantitative trait loci (mQTL) utilized from GoDMC database.

EWAS model was adjusted for total energy intake, age at blood collection, sex, maternal educational level, gestational age, pre-pregnancy BMI, maternal smoking during pregnancy, technical covariates, cell counts.

Table 6. Meta-analysis results for associations of infant plant protein intake (E%) and late childhood whole blood DNA methylation levels at P < 1 × 10¯^5..

CpG	Effect^a	SE^a	P value	FDR	Direction^b	I^2	Polymorphic	mQTL^c	Chr	Position	Relation to Island	Nearest Gene	Gene Region
cg06623197	−0.005	0.001	2,39E–07	0.050	- -	85.1	No	Yes	22	30400763	OpenSea	MTMR3	Body
cg16739865	0.001	0.000	2,43E–07	0.050	+ +	70.6	No	No	12	3186281	Island	TSPAN9	TSS1500
cg18106898	−0.010	0.002	7,79E–07	0.108	- -	0	No	Yes	15	74229671	OpenSea	LOXL1	Body
cg26954671	−0.004	0.001	1,97E–06	0.188	- -	0	Yes	No	8	22223465	N_Shore	SLC39A14	TSS1500
cg03368634	0.002	0.000	2,42E–06	0.188	+ +	0	No	No	16	3824553	OpenSea	CREBBP	Body
cg06333135	−0.002	0.000	2,84E–06	0.188	- -	24.2	No	No	3	47460006	Island	SCAP	Body
cg20272155	0.006	0.001	3,17E–06	0.188	+ +	38	No	Yes	11	1769462	Island	MOB2	Body; TSS200
cg01104489	−0.004	0.001	4,84E–06	0.204	- -	27.2	No	Yes	1	3072235	Island	PRDM16	Body
cg04450606	0.004	0.001	5,08E–06	0.204	+ +	54.4	Yes	No	1	1,53E + 08	OpenSea	PGLYRP3	TSS200
cg12198729	−0.001	0.000	5,28E–06	0.204	- -	15.6	No	No	10	1,03E + 08	Island	BTRC	TSS200; TSS200
cg01668281	−0.008	0.002	5,41E–06	0.204	- -	0	No	Yes	21	37915281	OpenSea	CLDN14	5’UTR; TSS1500
cg24353466	0.001	0.000	6,42E–06	0.222	+ +	0	No	No	3	1,5E + 08	Island	RNF13	1stExon; 5’UTR
cg21953251	−0.003	0.001	8,13E–06	0.245	- -	51.5	Yes	No	5	766985	OpenSea	ZDHHC11	-
cg15209277	−0.002	0.000	8,29E–06	0.245	- -	0	No	No	1	64472035	OpenSea	ROR1	Body

^aEffect sizes and SE are presented as a percent change in DNA methylation per 1 E% increase in energy from animal protein intake.

^bStudies are arranged from left to right as follows: CHOP then Generation R.

^cmethylation quantitative trait loci (mQTL) utilized from GoDMC database.

EWAS model was adjusted for total energy intake, age at blood collection, sex, maternal educational level, gestational age, pre-pregnancy BMI, maternal smoking during pregnancy, technical covariates, cell counts.

Data availability statement

The databases used here in this study were exclusively available for the authors to access and use after an administrative permission. All relevant data and analysis code used in supporting the findings in this research article can be made available upon a reasonable request. Full EWAS results for all models can be found at: https://doi.org/10.5281/zenodo.8375454.