Table 2.

Top CpG sites from adjusted associations of gestational PFAS concentrations with repeated measures of DNA methylation in the HOME Study (at delivery and at 12 years of age) and Project Viva (at delivery and at 7 years of age).

CpG	Chr	Position	CpG location	Gene	Gene region	FANTOM5 enhancers	ENCODE DNAse hypersensitivity sites	Discovery cohort [$\mathrm{\beta }$ ($p$-value)]	FDR $q$-value	Replication cohort [$\mathrm{\beta }$ ($p$-value)]	Replicateda
PFOA
cg25869005	14	95048198	Open sea	SERPINA5	5′UTR	—	chr14:95047965-95048250	0.101 ($5.87\times {10}^{–9}$)	0.003	NA	—
cg03707973	12	54826237	Open sea	LOC102724050	Body	—	chr12:54826045-54826655	$-0.072$ ($3.34\times {10}^{–8}$)	0.008	NA	—
cg04042784	7	42884749	Open sea	—	—	—	—	0.078 ($3.94\times {10}^{–8}$)	0.008	NA	—
cg09309282	2	236523347	Open sea	AGAP1	Body	—	chr2:236523145-236523410	$-0.051$ ($9.88\times {10}^{–8}$)	0.015	NA	—
cg22971191	13	103719706	Open sea	SLC10A2	Promoter	—	—	0.121 ($1.95\times {10}^{–7}$)	0.023	$-0.026$ (0.435)
cg01787798	1	175474437	Open sea	TNR	5′UTR	—	chr1:175474340-175474655	$-0.088$ ($2.30\times {10}^{–7}$)	0.023	$-0.067$ (0.001)	$\sqrt{}$
cg04977784	5	53972541	Open sea	LOC102467080	Body	chr5:53972314-53972314	chr5:53972220-53972635	0.060 ($5.73\times {10}^{–7}$)	0.043	NA	—
cg14212748	12	53343703	Shore	KRT18	Body	—	chr12:53343445-53343855	$-0.068$ ($6.25\times {10}^{–7}$)	0.043	$-0.010$ (0.492)	—
cg05195288	3	65582834	Open sea	MAGI1	Body	—	—	0.073 ($7.10\times {10}^{–7}$)	0.043	NA	—
cg21821684	15	47686828	Open sea	—	—	—	chr15:47686685-47686890	0.104 ($7.31\times {10}^{–7}$)	0.043	$-0.034$ (0.379)	—
cg16415457	3	23713441	Open sea	—	—	—	chr3:23713340-23713815	$-0.101$ ($9.57\times {10}^{–7}$)	0.048	$-0.082$ (0.007)	$\sqrt{}$
cg03760072	3	133502685	Island	SRPRB	Promoter	—	chr3:133502565-133503275	$-0.130$ ($9.77\times {10}^{–7}$)	0.048	NA	—
PFOS
cg16381104	6	99578913	Open sea	—	—	—	chr6:99578800-99579035	0.060 ($1.82\times {10}^{–8}$)	0.011	NA	—
cg21421331	10	100618466	Open sea	HPSE2	Body	—	chr10:100618360-100618610	0.076 ($1.01\times {10}^{–7}$)	0.033	NA	—
PFHxS
cg02794779	13	100150711	Island	—	—	—	—	$-0.142$ ($1.18\times {10}^{–8}$)	0.007	$-0.046$ (0.106)	—
cg11331322	3	42530661	Open sea	VIPR1	Promoter	—	—	0.086 ($6.48\times {10}^{–8}$)	0.018	NA	—
cg00983482	10	130083707	Open sea	—	—	—	chr10:130082925-130083755	0.082 ($1.16\times {10}^{–7}$)	0.022	$-0.015$ (0.440)	—
cg26477711	12	39518309	Open sea	—	—	—	—	0.088 ($2.10\times {10}^{–7}$)	0.030	NA	—
cg24735489	6	31088352	Open sea	CDSN;PSORS1C1	Promoter;5′UTR	—	chr6:31088120-31088430	$-0.087$ ($3.88\times {10}^{–7}$)	0.042	0.019 (0.227)	—
cg03639671	4	145430689	Open sea	—	—	—	—	0.085 ($4.46\times {10}^{–7}$)	0.042	$-0.039$ (0.087)	—
cg00457165	6	31088366	Open sea	CDSN;PSORS1C1	Promoter;5′UTR	—	chr6:31088120-31088430	$-0.064$ ($5.40\times {10}^{–7}$)	0.043	NA	—
cg00647458	4	139760831	Open sea	—	—	—	—	0.063 ($6.57\times {10}^{–7}$)	0.046	NA	—
PFNA
cg25601695	16	55690178	Island	SLC6A2	5′UTR;Promoter	—	chr16:55689225-55691015	$-0.094$ ($2.87\times {10}^{–9}$)	0.002	NA	—
cg04509825	14	62162178	Island	HIF1A	1stExon;5′UTR	—	chr14:62161865-62162315	$-0.087$ ($3.66\times {10}^{–8}$)	0.008	0.002 (0.807)	—
cg01713535	14	57046409	Shore	C14orf101	Promoter	—	chr14:57046105-57047510	$-0.091$ ($4.50\times {10}^{–8}$)	0.008	0.019 (0.525)	—
cg11506907	11	118972568	Shore	DPAGT1	5′UTR;1stExon	—	chr11:118972040-118973615	$-0.096$ ($5.57\times {10}^{–8}$)	0.008	0.005 (0.672)	—
cg12967001	18	5544089	Island	EPB41L3	Promoter	—	chr18:5543880-5544455	$-0.084$ ($6.59\times {10}^{–8}$)	0.008	$-0.004$ (0.681)	—
cg05792003	1	95582492	Shore	TMEM56	Promoter	—	chr1:95582305-95582570	$-0.091$ ($9.34\times {10}^{–8}$)	0.009	0.002 (0.874)	—
cg24620436	7	4918906	Shelf	RADIL	5′UTR	—	chr7:4918205-4919455	0.209 ($1.06\times {10}^{–7}$)	0.009	0.118 (0.055)	—
cg09861318	3	149530987	Island	RNF13	1stExon;5′UTR	—	chr3:149530185-149531075	$-0.083$ ($1.25\times {10}^{–7}$)	0.009	0.011 (0.544)	—
cg26975787	12	2613778	Open sea	CACNA1C	Body	—	—	$-0.083$ ($4.16\times {10}^{–6}$)	0.029	$-0.045$ (0.010)	$\sqrt{}$
cg24822529	5	134582880	Open sea	—	—	—	chr5:134582625-134582950	$-0.098$ ($7.53\times {10}^{–6}$)	0.033	$-0.062$ (0.005)	$\sqrt{}$
cg19497517	8	145028257	Island	PLEC1	Body;TSS200	—	chr8:145028185-145028535	0.194 ($1.01\times {10}^{–5}$)	0.035	0.073 (0.027)	$\sqrt{}$
cg02842197	17	5389625	Island	DERL2;MIS12	TSS200;TSS1500	—	chr17:5389485-5389890	0.136 ($2.04\times {10}^{–5}$)	0.042	0.048 (0.026)	$\sqrt{}$

Note: All models were adjusted for child age and sex, annual household income (median income of each category), maternal race/ethnicity (non-Hispanic white, other) and smoking during pregnancy (yes, no), and cell type composition. CpG sites were annotated to a promoter region if they were assigned toTSS200 or TSS1500. Data were complete for all variables. PFAS concentrations below the LOD were replaced with the LOD divided by the square root of 2. We analyzed longitudinal associations between ${\log }_2\text{-transformed}$ PFAS concentrations and repeated DNA methylation measures using a generalized estimating equations (GEE) approach with an identity link function, a working independence correlation matrix and normal errors. $\mathrm{\beta }$ can be interpreted as the differences in the logits of DNA methylation proportions across the two visits per 2-fold increase in gestational PFAS concentrations. Statistically significant CpG sites were defined as having an FDR $q<0.05$ (PFOA, PFOS, and PFHxS) or $<0.01$ (PFNA). —, not annotated to any known gene; 5′UTR, between the TSS and the ATG start site; 1stExon, the first exon of the gene; Chr, chromosome; CpG, cytosine–guanine dinucleotide; Body, between the ATG and stop codon; FDR, false discovery rate; HOME, Health Outcomes and Measures of the Environment; LOD, limit of detection; NA, not available in 450K microarray; PFAS, perfluoroalkyl substances; PFHxS, perfluorohexane sulfonate; PFNA, perfluorononanoate; PFOA, perfluorooctanoate; PFOS, perfluorooctane sulfonate; TSS, transcription start site; TSS200, 200 bases from TSS; TSS1500, 1,500 bases from TSS, UTR, untranslated region.

^a CpGs with a $p<0.05$ and the same direction of associations observed in the HOME Study were considered as successful replications.