Page 1 of 53 Diabetes

Title: Interplay of placental DNA methylation and maternal insulin sensitivity in pregnancy

Short title: Placental epigenetics of insulin sensitivity

Authors: Marie-France Hivert1,2,3; Andres Cardenas4; Catherine Allard5; Myriam Doyon5; Camille E Powe2; Patrick M Catalano6; Patrice Perron3,5; Luigi Bouchard5,7,8

Affiliations

1- Department of Population Medicine, Harvard Medical School and Harvard Pilgrim Health Care Institute, Boston, MA, USA 2- Diabetes Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, MA USA 3- Department of Medicine, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada 4- Division of Environmental Health Sciences, School of Public Health, University of California, Berkeley, Berkeley, CA, USA 5- Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, QC, Canada 6- Mother Infant Research Institute, Department of Obstetrics and Gynecology, Tufts Medical Center, Tufts University School of Medicine and Friedman School of Nutrition and Science Policy, Boston, MA, USA 7- Department of Biochemistry, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, QC, Canada 8- Department of Medical Biology, CIUSSS Saguenay-Lac-Saint-Jean — Hôpital de Chicoutimi, Saguenay, QC, Canada

*Corresponding Author:

Marie-France Hivert, MD Department of Population Medicine Harvard Medical School and Harvard Pilgrim Health Care Institute 401 Park Drive, Suite 401 Boston, MA 02215 Phone: (617) 867-4505; Fax: (617) 867-4845 Email: [email protected]

Word count: 3,996 Number of tables: 1 Number of figures: 2 Supplementary Tables: 11 Supplementary Figures 3

Diabetes Publish Ahead of Print, published online December 27, 2019 Diabetes Page 2 of 53

Abstract:

The placenta participates in maternal insulin sensitivity changes during pregnancy, however mechanisms remain unclear. We investigated associations between maternal insulin sensitivity and placental DNA methylation markers across the genome. We analyzed data from 430 mother- offspring dyads in the Gen3G cohort. All women underwent 75g oral glucose tolerance tests at ~26 weeks of gestation; we used glucose and insulin measures to estimate insulin sensitivity (Matsuda index). At delivery, we collected samples from placenta (fetal side) and measured DNA methylation using IlluminaEPIC arrays. Using linear regression models to quantify associations at 720,077 CpGs, adjusted for maternal age, gravidity, smoking, body mass index, child sex and gestational age at delivery, we identified 188 CpG sites where placental DNA methylation was associated with Matsuda index (P<6.94x10-8). Among genes annotated to these 188 CpGs, we found enrichment in targets for microRNAs, histone modifications, and in parent-of-origin DNA methylation including H19/MIR675 locus (paternally imprinted). We identified 12 known placenta imprinted genes, including KCNQ1. Mendelian Randomization analyses revealed 5 loci where placenta DNA methylation may causally influence maternal insulin sensitivity, including the maternally imprinted gene DLGAP2. Our results suggest that placental DNA methylation is fundamentally linked to the regulation of maternal insulin sensitivity in pregnancy. Page 3 of 53 Diabetes

Introduction

Insulin sensitivity decreases drastically in second half of pregnancy to levels that are similar to

individuals with early type 2 diabetes (T2D).1 It is hypothesized that this dramatic decrease in

insulin sensitivity is meant to help provide nutrients from maternal sources to the growing fetus.

The placenta likely plays a role in this physiologic adaptation, but the exact mechanisms remain

unclear.

The placenta is a unique organ of fetal origin that lies at the maternal and fetal interface with

primary roles to optimize fetal growth, protect the fetus against infections, and produce key

hormones to maintain pregnancy; these hormones profoundly influence maternal physiology. In

its role of nutrient transfer, the placenta responds to both fetal demands and maternal availability

of nutrients, and further adapts to regulate resources allocation. Yet, the ‘maternal-fetal conflict’

theory posit that the mother and the fetus ‘disagree’ on an optimal level of resource allocation

from the mother to the fetus to allow pregnancy to its term, and a healthy baby2. During early

embryogenesis, the trophoectoderm develops to form the placenta with a distinctive epigenetic

process.3 The placenta de-methylation process in early development is characterized by a great

number of genomic regions remaining imprinted from their parent-of-origin. Some investigators

have proposed that the maintenance of the parental origin of imprinted regions in placenta

contributes to the ‘maternal-fetal conflict’ where genes that are expressed from paternal alleles

act to shunt more nutrients to the fetus while expression driven by maternal alleles helps the

mother maintain her resources.2,4 Thus, it is plausible that specific DNA methylation patterns in

the placenta may influence the decline in maternal insulin sensitivity that leads to the transfer of

glucose and other fuels to the fetus. Diabetes Page 4 of 53

Based on these hypotheses, we investigated associations between maternal insulin sensitivity estimated in second trimester and genome-wide DNA methylation in placenta collected at birth in a large prospective pregnancy cohort. We had initially hypothesized that maternal insulin sensitivity could lead to changes in placental DNA methylation (given temporality of our measures) but also tested the possibility that placenta DNA methylation may influence maternal insulin sensitivity. To untangle whether placental DNA methylation is influencing maternal insulin sensitivity or vice versa, we tested potential causality using a bi-directional Mendelian

Randomization (MR) framework. Additionally, we conducted pathway analyses to deepen our understanding of our findings.

Methods

Description of participants

This study is based on mother-child pairs in the Genetics of Glucose regulation in Gestation and

Growth (Gen3G) prospective cohort. We have previously published details of recruitment and phenotypic measurements during pregnancy.5 In brief, we recruited women in the 1st trimester

(V1: 5-14 weeks), inviting all women presenting for their routine prenatal laboratories at the

Centre Hospitalier Universitaire de Sherbrooke (CHUS). We excluded women with diabetes prior to pregnancy (known or discovered at 1st trimester) and non-singleton pregnancies. In addition to blood samples, our trained research staff collected demographics, medical history, and completed standardized anthropometric measurements. We calculated body mass index (BMI) using standard formula (kg/m2).

We followed participating women in the 2nd trimester (V2: 24-30 weeks) and completed similar measurements. After overnight fasting, women completed a 75g oral glucose tolerance testing Page 5 of 53 Diabetes

(OGTT) for gestational diabetes mellitus (GDM) screening. We collected plasma samples at

baseline (fasting) and at 1h and 2h during the OGTT. We measured glucose and insulin at each

OGTT time-point, which allowed us to derive indices of insulin sensitivity.

We followed women until delivery and collected delivery and neonatal outcomes in addition to

cord blood and placenta samples. At birth, trained research staff collected placenta samples (<30

minutes postpartum) based on standardized protocol: a one cm3 of placenta tissue was collected

approximately 5cm from the umbilical cord insertion on both sides of the placenta. Prior studies

have shown high concordance of placental DNA methylation levels at specific loci across

biopsies locations6. Placenta samples were rapidly put in RNALater (Qiagen, USA) and store at

4°C for a ≥24h before to be stored at -800C. For this study, we selected fetal side placenta

samples based on availability of adequate tissue (high quality DNA extraction) and excluded

complications in late pregnancy or delivery (e.g. preeclampsia, chorioamnionitis, stillbirth).

The CHUS ethic board review committee approved this study; all women provided written

consent. For this study, we included only women of European origin (self-reported) who had

consented for genetics investigations.

Bioassays

We measured glucose by the glucose hexokinase method (Roche Diagnostics) immediately after

collection, and insulin using multiplexed particle-based flow cytometric assays (Human Milliplex

map kits, EMD Millipore) from plasma samples previously frozen (-80°C). We estimated insulin

sensitivity using the Matsuda index, given by the following equation: 10,000/ (√[(fasting glucose

× fasting insulin) × (mean glucose during OGTT × mean insulin during OGTT)]).7 We selected Diabetes Page 6 of 53

the Matsuda index over other measures of insulin sensitivity because it has been validated against euglycemic-hyperinsulinemic clamps in pregnant women.7

DNA methylation measurements

We purified DNA from 460 placenta samples using the All Prep DNA/RNA/Protein Mini Kit

(Qiagen, USA). After bisulfite conversion, Illumina Laboratory (San Diego, CA) performed epigenome-wide DNA methylation measurements using the Infinium MethylationEPIC

BeadChip. We imported methylation data into R for preprocessing using minfi. We performed quality control (QC) at the sample level, excluding samples that failed (n=5), mismatched genotype based on paired cord blood data (n=6) or sex (n=1). Our final placenta DNA methylation dataset included 448 samples. We then excluded women because of missing data

(Matsuda or key co-variables). Thus, our final dataset for this study was composed of 430 mother-child pairs (see Supplementary Figure 1), which fully overlap with our prior publication of maternal 2h-glucose associations with placenta DNA methylation8.

We normalized our data as previously described.8 We applied functional normalization9

(FunNorm) and Regression on Correlated Probes10 (RCP) to adjust for technical variability and probe type-bias, respectively. Briefly, FunNorm removes technical variability using control probes from the array and RCP corrects type II probe distributions using the distribution of proximal type I probes to increase precision. We removed CpG probes with SNPs at the target site (4,453), single base extension (5,552) or anywhere within the probe (71,054) with a minor allele frequency of >5%, probes on sex chromosomes (19,129) and previously identified cross- reactive probes (40,448)11 to analyze 720,077 high-quality probes. We used the ComBat function from the sva package to adjust for batches. We logit transformed the β-values to M-values for Page 7 of 53 Diabetes

statistical analyses as they have been shown to be more appropriate, meeting statistical model

assumptions12. However, we also reported effect estimated on the β-value scale to ease

interpretability since it approximates the proportion of methylation (from 0 to 1).

Genotyping

We isolated DNA from maternal buffy coat using the Gentra Puregene Blood Kit (Quiagen,

Mississauga, ON, Canada). We performed genotyping on 598 maternal samples using Illumina

MEGAex arrays. We removed 16 samples with a call rate ≤98% or failed sample QC. All

remaining samples passed additional QC measures (percent heterozygosity, sex concordance,

PCA derived outliers). We removed SNPs that were monomorphic, on sex chromosomes, had

minor allele frequency (MAF)<1% in our sample, HWE (Hardy-Weinburg Equilibrium) p-value

<1x10-8, and insertions/deletions. After the above steps, we had data available on 838,884 SNPs

in 582 women. We performed genotyping imputation using ShapeIT v2.r790 phasing HRC r1.1

2016 reference panel and minimac3 software provided by the Michigan imputation server, which

resulted in a dataset containing a total of 39,183,141 autosomal SNPs for overall population.

Before analyses, we excluded all monomorphic SNPs or with a MAF<0.05.

Statistical analyses

We described participants’ characteristics using median and interquartile range (IQR) for

continuous variables, frequency and percentage for categorical variables. We used natural log (ln)

transformation to obtain a normal distribution of Matsuda index and used ln values for all

analyses. We conducted analyses using R version 3.5.1. Diabetes Page 8 of 53

In 430 mother-infant pairs, we performed an epigenome-wide association study (EWAS) by fitting robust linear regression models using the rlm function for each CpG site. In CpG-by-CpG models, we included DNA methylation (M-values) as the response variable and Matsuda (ln) as the exposure of interest. In Model 1, we adjusted for maternal age, gravidity, smoking during pregnancy, maternal BMI (1st trimester), child sex, and gestational age at delivery. To control for genomic inflation, we used ReFACTor (Model 2), a reference-free method that adjusts for heterogeneity putative to cell-types in EWAS from heterogenous tissues.13 We used the top-10

Principal Components (PCs) estimated from ReFACTor as proxy for placenta cellular heterogeneity as suggested by the scree plot (Supplementary Figure 2). We used Quantile- quantile plots and histograms for the regression P-values to visually inspect genomic inflation (λ) in both models (Supplementary Figure 3). We corrected for multiple testing using Bonferroni with significant P-values<6.94x10-8.

Gene annotation and pathways analyses

First, we annotated CpGs with the R package IlluminaHumanMethylationEPICmanifest.14

Second, we utilized the R package humarray 15 to find the nearest gene based on base pair distance upstream and downstream. We tested for enrichment in biologic pathways using

Enrichr16,17 web platform using only one gene per identified CpG: at each CpG, we prioritized gene annotation from UCSC Reference extracted from the Illumina manifest (except in cases of updated gene names), then used the closest informative gene name from humarray annotation

(priority to coding genes). We focused our attention on TargetScan microRNA 2017, ENCODE

Histone Modification 2015, WikiPathways 2019, Biocarta 2016, GWAS 2019, and dbGAP reported databases in Enrichr16,17. Page 9 of 53 Diabetes

Mendelian randomization (MR) analyses

We conducted MR analyses to untangle direction of effect of associations based on 401 women

with complete data from genotyping arrays, placental DNA methylation, and Matsuda index.

First, we used MR to test if placental DNA methylation levels may influence maternal insulin

sensitivity. We searched for SNPs in cis (within 500kb on each side) at each 188 CpGs identified

in our Model 1 and tested SNP-to-methylation associations to identify genetic instrumental

variables (IVs) in each region. We removed SNPs with a MAF<0.05. We assumed a genetic

additive model and chose the effect allele as associated with greater DNA methylation levels. If

multiple SNPs present in the determined cis region were associated with DNA methylation levels

at the CpG of interest, we used elastic net procedure with the glmnet18 package. We looked at

models with alpha from 0.1 to 1 by steps of 0.1. For each model, lambda was chosen as the value

that gave the minimum mean cross-validated error (lambda.min). Finally, the alpha was chosen

as the value that gave the smallest mean square error (MSE). When there were more than one

SNP remaining in the final model from the elastic net procedure, we built a Genetic Risk Score

(GRS) assuming additive genetic effect and summed the number of risk alleles as a global genetic

IV. If there was only one SNP associated with DNA methylation in the designated in cis region,

we used additive genetic modeling for that one SNP as genetic IV. We tested associations

between the genetic IVs (GRS or individual SNP) and Matsuda index (ln). To compare effect

estimates, we used the Two Stage Least Square (TSLS) that uses the predicted DNA methylation

value by its respective genetic IV as the independent variable in the linear regression to predict

Matsuda index.19,20 We used Durbin-Wu-Hausman to test whether TSLS estimates were

significantly different from the observed estimates. We corrected for the number of CpGs tested

(n=131 with genetic IV available) using false discovery rate (FDR). Diabetes Page 10 of 53

Second, we used prior knowledge of SNPs known to influence insulin sensitivity21. We selected 8

SNPs (See Supplementary Table 1) that were previously established as determinants of fasting insulin in large GWAS (with P<5x10-8 in Meta-Analyses of Glucose and Insulin-related traits

Consortium (MAGIC) datasets)22,23 and were also nominally associated (P<0.05) with Matsuda index in non-pregnant individuals24. We built a GRS assuming additive genetic effect and summed the number of risk alleles. We tested associations between the insulin sensitivity GRS and placenta DNA methylation (in M-values) for the 188 CpGs identified in Model 1, and corrected using FDR.

Data and resource availability: Datasets analyzed during the current study are not publicly available because we did not obtain consent for such public release of epigenetic data from participants. However, data is available from the corresponding author with the appropriate permission from the Gen3G study team upon reasonable request and institutional review boards approval. Summary statistics of EWAS results for Models 1 and 2 are available via https://figshare.com/s/5040ad2ece334944bf34

Results

We presented characteristics of participants in Table 1. At the beginning of pregnancy, women’s median age was 28 years old (IQR=25-31), median BMI was 23.7 kg/m2 (IQR=21.6-27.9), a third were primigravid, and less than 9% reported smoking. Median Matsuda insulin sensitivity index was estimated at 7.72 (IQR=5.69-10.67) and appeared normally distributed after natural log transformation. Page 11 of 53 Diabetes

In Model 1, we found that maternal insulin sensitivity was associated with placental DNA

methylation at 188 CpGs (P-values <6.94x10-8; adjusted for maternal age, gravidity, smoking,

maternal BMI, child sex, and gestational age at delivery). Adding GDM status as covariate had

minimal impact on association estimates at identified CpGs (0.02% to 9.9% changes in beta

coefficients). These 188 individual CpGs were distributed across the genome (Figure 1A); in 14

regions, multiple CpGs were in close genomic vicinity and were annotated to the same gene

(Supplementary Table 2). Among annotated genes, we identified 12 genes known to be imprinted

in the placenta (9 maternally imprinted: SPHKAP, CNTN6, KCNIP4, PODXL, DLGAP2,

KCNQ1, DSCAML1, GPC6, OCA2; and 3 paternally imprinted: H19/MIR675, MCF2L,

LINC01056)25. We also noted that specific microRNAs were listed at 11 loci (Supplementary

Table 2). We examined our findings and found no CpGs overlapping with the list of CpGs that

we had previously identified for associations with maternal 2h-glucose in the same cohort8

despite moderate correlation (r= -0.44) between Matsuda and 2h-glucose. In Model 2, adjusting

bioinformatically for cell-type heterogeneity, we did not find any individual CpGs that reached

Bonferroni (Figure 1B). Examining PCs generated by ReFACTor that reflect the cellular

heterogeneity of tissue samples, we observed that PC1, PC2, and PC5 were strongly associated

with Matsuda index, suggesting that cell-type specific placental DNA methylation profile is

strongly related to maternal insulin sensitivity (Supplementary Table 3).

Mendelian randomization #1 – does placental methylation affect maternal insulin sensitivity?

We identified genetic IVs in 131 of the 188 CpGs identified in Model 1 (Supplementary Table 4).

Specific GRS built with selected cis-SNPs captured respective CpG methylation levels with r2

ranging from 1% to 32%. We found 28 GRS capturing methylation at their respective CpG that

were nominally associated (P<0.05) with Matsuda index: 5 of these were statistically significant Diabetes Page 12 of 53

at FDR<0.05. These 5 GRS represented methylation levels at cg01618245 (CHRNA4), cg12673377 (MICALL2/UNCX), cg24475484 (DLGAP2), cg08099672 (ENTPD2), and cg03699074 (BDP1P). In all 5 cases, higher DNA methylation levels (as represented by GRS) were associated with lower Matsuda index and were in line with the direction of effect detected in our primary observational analyses (Figure 2). TSLS estimates were also all in the same direction as the observed associations and significant (FDR<0.05). In 2 of the 5 CpGs

(cg03699074 at BDP1P and cg24475484 at DLGAP2), the Durbin-Wu-Hausman test suggested that observed associations between DNA methylation and Matsuda index might be confounded.

The fact that MR estimates were larger than the observational estimates suggest that observational estimates were negatively confounded, and that “true” causal effects may be larger than the “observed”.

Mendelian randomization #2 – does maternal insulin sensitivity affect placenta?

The insulin sensitivity GRS build with 8 SNPs captured about 1.5% of Matsuda index variance

(r2=0.015). Among our 188 identified CpGs (Model 1), we did not identify any CpGs at which the insulin sensitivity GRS was associated with placental DNA methylation levels

(Supplementary Table 5).

Pathways analyses

Among transcription pathways databases, we noticed a strong enrichment in TargetScan microRNA database (Supplementary Table 6): we found 34 microRNA target terms with adjusted P-values<0.05 including hsa-miR-3180(-3p), hsa-miR-3196, hsa-miR1538, and hsa- miR-4745-3p being at the top of the list. We also observed enrichment in ENCODE Histone Page 13 of 53 Diabetes

Modifications database, mainly driven by H3K27me3 in variety of tissues and cell types

(Supplementary Table 7).

The top term emerging from the GWAS Catalog (Supplementary Table 8) was ‘DNA

methylation (Parent-of-origin)’ with H19, MIR675, and SEPT5 (P=3.76x10-5; adjusted P=0.017).

Among the top 10 terms from the GWAS Catalog, we also observed “hemoglobin A1c” (driven

by TCF7L2, KCNQ1, and PIEZO1) and “fasting insulin” (driven by TCF7L2, and CNTN6)

which is the most common proxy of insulin resistance in large GWAS analyses26 (both adjusted

P-value >0.05). From dbGAP, we identified 10 terms that reached adjusted P-values<0.05

including ‘Body Mass Index’, and ‘Body Height’ and other cardiovascular traits (triglycerides,

blood pressure – Supplementary Table 9).

In Biocarta, the top pathway was ‘Role of PPAR-gamma Coactivators in Obesity and

Thermogenesis’ with MED1 and RXRA leading the emergence of this pathway (Supplementary

Table 10). The top pathways emerging from WikiPathways (Supplementary Table 11) were

“Adipogenesis” (6 out 130 genes: MEF2A, WWTR1, MBNL1, RXRA, GATA4, PRLR) and “Genes

targeted by miRNAs in adipocytes” driven by KCNQ1 and HCN2 (2 out 13 genes), yet neither

pathway had adjusted P-values<0.05.

Discussion

Our results suggest that placental DNA methylation is fundamentally linked to maternal insulin

sensitivity regulation. Using MR, we identified 5 loci where placental DNA methylation may be

modulating the pregnancy-associated decrease in insulin sensitivity. To our knowledge, this is the Diabetes Page 14 of 53

first study to suggest that placental DNA methylation may causally influence maternal insulin sensitivity. Other identified loci are within known placenta-specific imprinted regions, consistent with the theory of maternal-fetal conflict. In addition, our assessment of cellular heterogeneity showed that the two first components of overall placenta DNA methylation profile are strongly associated with maternal insulin sensitivity. On one hand, it is possible that insulin sensitivity influences cell repertoire in the placenta as well as cell lineage commitment early during development. This cellular model has been termed as Polycreodism, or systematic variability in cell fate, which is relevant during embryonic development27. Our MR #2 analyses did not support this direction, but our IV for insulin sensitivity was limited (r2=0.015). On the other hand, placental DNA methylation at delivery might also reflect DNA methylation stability across gestation at some loci. Despite the well known global increase in placenta DNA methylation throughout gestation, Novakovic et al. showed that substantial changes in methylation (β ≥0.2) were observed in 954 CpG sites between 1st and 3rd trimester, and in only 157 CpG sites between

2nd and 3rd trimester (out of >26,000 CpG sites)28. Moreover, Schroeder et al. demonstrated that

Partially Methylated Domains are common in placenta (37% of placental genome) and stable across gestation29. Furthermore, by definition, imprinted loci remain stably methylated during fecundation and throughout in utero development4.

MicroRNAs are suspected to have key roles in placenta development and function30. Many of our findings implicated microRNAs as a potential link between placental DNA methylation and maternal insulin sensitivity. First, among the 188 identified CpGs, 11 were annotated to a microRNA as one of the closest gene. Second, TargetScan MicroRNA showed that our list of genes was greatly enriched for targets of multiple human microRNAs. In addition, we identified

CpGs near genomic imprinted regions that contain microRNAs known to play an important role Page 15 of 53 Diabetes

in placenta, for example at the H19/MIR675 and DIO3OS loci. DIO3OS is located near the

placenta-specific microRNA cluster on Chr14q32 known as C14MC in the imprinted Dlk1-Dio3

domain. DIO3 is paternally imprinted during fetal development, suggesting that DIO3OS is a

noncoding gene that may have a role in maintaining monoallelic maternal expression of DIO331.

The locus H19/miR-675 is paternally imprinted and thus maternally expressed30. miR-675 is a

highly conserved miRNA, located in the first exon of H19 and is specifically expressed by the

placenta, with expression rising as the gestation advance32. Putative role of miR-675 is to limit

placental growth, likely via reducing the expression of IGF1R (main receptor of IGF2 key

placental growth factor)32.

It is notable that many imprinted genes are predominantly or solely expressed in the placenta30.

The different parental origin of DNA methylation patterns led to the “maternal-fetal conflict”

hypothesis whereby paternal expression should favor fetal growth by deriving more maternal

resources, while the maternally expressed genes should act to conserve maternal resources. We

identified 3 loci at which the annotated gene is a known paternally imprinted (maternally

expressed) gene in the placenta (including H19/mir675) and 9 loci at which the annotated gene is

maternally imprinted (paternally expressed) in placental tissue25 including KCNQ1. Our MR

investigations suggested that methylation levels at cg24073146 in KCNQ1 could causally

influence maternal insulin sensitivity, yet non-significant after accounting for multiple testing.

Loss of maternal-specific methylation of KCNQ1 causes Beckwith-Wiedemann syndrome,

characterized by prenatal overgrowth and hypoglycemia in infancy33. During normal fetal

development, fetal pancreas shows monoallelic expression of KCNQ1 suggesting an important

role of imprinting at this locus during pancreatic development, while adult pancreas show bi-

allelic expression34. Genetic variants at KCNQ1 are well-established T2D risk variants with Diabetes Page 16 of 53

evidence of parental-of-origin effect where the transmission of maternal allele shows a very strong association for risk of T2D in comparison to paternal transmission35.

Another identified CpG (cg24475484) is located within a known placenta-specific maternally imprinted region annotated to DLGAP2. DLGAP2 is bi-allelically expressed in the brain, but only paternally expressed in the testis36 and was differentially methylated in spermatozoal DNA of infertility studies37. Our MR analyses supported that methylation at cg24475484 (DLGAP2) causally influence maternal insulin sensitivity (FDR<0.05), in line with the “maternal-fetal conflict” hypothesis where the paternally expressed gene would reduce maternal insulin sensitivity to drive more nutrients towards the fetus.

Based on our MR analyses, we found that placenta DNA methylation levels at some identified

CpGs may be causal in influencing maternal insulin sensitivity: 4 loci passed FDR significance threshold in addition to cg24475484 (DLGAP2). These CpGs were not located near genes with known placental function; this highlights the importance of agnostic approaches to discover new biologic candidates. Some of these loci deserve further functional studies in placenta and/or other relevant tissues. For example, cg08099672 is located near ENTPD2 expressed by the placenta, in addition to ovaries and testis, nervous system, and islets of Langerhans29. The placenta expresses

MICALL2 (located ~80kb of cg12673377) and the protein is also highly detected in pancreas, adrenal, and stomach tissues38. One intriguing finding is cg01618245, located near CHRNA4, that encodes for Cholinergic Receptor, Nicotinic, Alpha 4, associated with epilepsy and nicotine addiction31. It is noteworthy that two microRNAs (MIR3674 and MIR596) are located nearby cg24475484 at DLGAP2 (less than 150kb apart) and the MIR4326 is located ~72kb from Page 17 of 53 Diabetes

cg01618245 at CHRNA4 and again implicating placental microRNAs as potential biologic

mediators of pregnancy-associated changes in maternal insulin sensitivity.

Our pathway analyses highlighted many genes and pathways involved in adipose tissue

regulation. From the WikiPathways, Adipogenesis39 emerged from genes such as MEF2A,

WWTR1, MBNL1, RXRA, GATA4, and notably PRLR that encodes for the prolactin receptor.

MED1 and RXRA (+/- MEF2A) also highlighted the role of PPAR-gamma co-activators in

obesity and thermogenesis (BioCarta) and Energy Metabolism (Wiki) with PPARGC1A central to

this pathway40. We also identified two probes annotated to PRDM16, a key regulator of brown

adipose tissue differentiation. Our group previously demonstrated in candidate gene studies that

maternal hyperglycemia is associated with placental DNA methylation at PRDM16 and

PPARGC1A 41; our current analyses using agnostic approach validate our previous findings. On

the other hand, none of the 188 identified CpGs in the current analyses overlapped with loci

identified in our prior EWAS of maternal 2h-glucose and placenta DNA methylation8. The

absence of overlapping findings may be due to different biological phenomena, or that we would

need a larger sample size to observe associations between placenta DNA methylation and both of

these two moderately correlated glycemic traits.

Strengths and Limitations:

Among our strengths, we have investigated a large number of placenta samples using the most

comprehensive DNA methylation array covering >720,000 CpGs across the genome, in a

prospective cohort of pregnant women with well-characterized phenotypes, including a validated

measure of insulin sensitivity during pregnancy. We were able to account for many potential

confounders, and applied a MR approach to test potential causality. Despite our attempts at Diabetes Page 18 of 53

untangling direction of effect, we found only a handful CpGs from which MR supported causality. It is likely that #2 MR analyses were limited in power given that our IV captured only

1.5% of the variance in Matsuda, resulting into a weak IV – one of the major limitation of our

MR analyses in this direction. After adjusting for heterogeneity using ReFACTor, none of the

CpGs reached Bonferonni, so it is possible that our signals from Model 1 reflect placental cell- specific DNA methylation. Top ReFACToR PCs, reflecting cell-type heterogeneity, were associated with insulin sensitivity; this might suggest that early DNA methylation programming might be driving a distinctive repertoire of cells in the placenta, also known as Polycreodism27.

We feel this is highly biologically relevant and future studies should investigate if specific cells are responsible for the signals we found and potential causal biological effect on maternal insulin sensitivity. Finally, our cohort is composed of women of European descent, thus may not be generalizable to other ethnicities.

Conclusions

In summary, our findings support a placental DNA methylation signature fundamentally linked to maternal insulin sensitivity. We identified CpGs at which our MR investigations supported that placental DNA methylation has a causal influence on maternal insulin sensitivity. The enrichment in microRNAs targets and identification of specific microRNAs add to recent literature implicating microRNAs in placenta biology, either as paracrine or endocrine actors. Stimulation of insulin responsive cells (adipocytes, hepatocytes, myocytes) or trophoblasts by exposure to identified microRNAs could reveal potential functions. Finally, the identification of both maternally and paternally imprinted genes is in line with the ‘maternal-fetal conflict’ hypothesis, yet also suggests that imprinted genes from both parents regulate of maternal insulin sensitivity during pregnancy. Page 19 of 53 Diabetes

Acknowledgments: This work was supported by American Diabetes Association accelerator

award #1-15-ACE-26 (M.F.H), Fonds de recherche du Québec en santé (FRSQ) #20697 (M.F.H);

Canadian Institute of Health Research #MOP 115071 (M.F.H), and Diabète Québec grants (P.P.

and L.B.). LB is a senior research scholar from the FRSQ. LB, MFH and PP are members of the

FRSQ-funded CRCHUS. C.E.P. is supported by career development awards from the National

Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (K23DK113218) and the Robert

Wood Johnson Foundation’s Harold Amos Medical Faculty Development Program (74256). The

authors declare no competing financial interests. None of the author had any conflict of interest to

declare.

Author Contributions: MFH conceived the original study design and analyses plan, supervised

the analyses, interpreted the results, and wrote the manuscript. MD contributed to samples and

data collection. AC and CA carried analyses, contributed to interpretation of data, reviewed and

critically edited the manuscript. MD, CEP, PMC, PP, and LB reviewed and critically edited the

manuscript. All authors approved the final version.

Guarantor: Marie-France Hivert is the guarantor of this work and takes responsibility for the

content of the article

Prior Presentation. The results were previously presented at the Scientific Sessions of the

American Diabetes Association in June 2018. Diabetes Page 20 of 53

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18. Friedman J, Hastie T, Tibshirani R. Regularization Paths for Generalized Linear Models via Coordinate Descent. Journal of statistical software. 2010;33(1):1-22. 19. Lawlor DA, Harbord RM, Sterne JA, Timpson N, Davey Smith G. Mendelian randomization: using genes as instruments for making causal inferences in epidemiology. Statistics in medicine. 2008;27(8):1133-1163. 20. Angrist JD, Imbens GW. Two-Stage Least Squares Estimation of Average Causal Effects in Models with Variable Treatment Intensity. Journal of the American Statistical Association. 1995;90(430):431-442. 21. Powe CE, Nodzenski M, Talbot O, et al. Genetic Determinants of Glycemic Traits and the Risk of Gestational Diabetes Mellitus. Diabetes. 2018;67(12):2703-2709. 22. Manning AK, Hivert MF, Scott RA, et al. A genome-wide approach accounting for body mass index identifies genetic variants influencing fasting glycemic traits and insulin resistance. Nat Genet. 2012;44(6):659-669. 23. Scott RA, Lagou V, Welch RP, et al. Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways. Nat Genet. 2012;44(9):991-1005. 24. Prokopenko I, Poon W, Magi R, et al. A central role for GRB10 in regulation of islet function in man. PLoS Genet. 2014;10(4):e1004235. 25. Sanchez-Delgado M, Court F, Vidal E, et al. Human Oocyte-Derived Methylation Differences Persist in the Placenta Revealing Widespread Transient Imprinting. PLoS Genet. 2016;12(11):e1006427. 26. Manning AK, Hivert MF, Scott RA, et al. A genome-wide approach accounting for body mass index identifies genetic variants influencing fasting glycemic traits and insulin resistance. Nature genetics. 2012;44(6):659-669. 27. Lappalainen T, Greally JM. Associating cellular epigenetic models with human phenotypes. Nature reviews Genetics. 2017;18(7):441-451. 28. Novakovic B, Yuen RK, Gordon L, et al. Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/stochastic factors. BMC Genomics. 2011;12:529. 29. Schroeder DI, Blair JD, Lott P, et al. The human placenta methylome. Proc Natl Acad Sci U S A. 2013;110(15):6037-6042. 30. Malnou EC, Umlauf D, Mouysset M, Cavaille J. Imprinted MicroRNA Gene Clusters in the Evolution, Development, and Functions of Mammalian Placenta. Frontiers in genetics. 2018;9:706. 31. Hernandez A, Martinez ME, Croteau W, St Germain DL. Complex organization and structure of sense and antisense transcripts expressed from the DIO3 gene imprinted locus. Genomics. 2004;83(3):413-424. 32. Keniry A, Oxley D, Monnier P, et al. The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r. Nature cell biology. 2012;14(7):659-665. 33. Lee MP, DeBaun MR, Mitsuya K, et al. Loss of imprinting of a paternally expressed transcript, with antisense orientation to KVLQT1, occurs frequently in Beckwith- Wiedemann syndrome and is independent of insulin-like growth factor II imprinting. Proc Natl Acad Sci U S A. 1999;96(9):5203-5208. 34. Travers ME, Mackay DJ, Dekker Nitert M, et al. Insights into the molecular mechanism for type 2 diabetes susceptibility at the KCNQ1 locus from temporal changes in imprinting status in human islets. Diabetes. 2013;62(3):987-992. Diabetes Page 22 of 53

35. Lyssenko V, Groop L, Prasad RB. Genetics of Type 2 Diabetes: It Matters From Which Parent We Inherit the Risk. Rev Diabet Stud. 2015;12(3-4):233-242. 36. DLGAP2 Gene. 2019; https://www.genecards.org/cgi- bin/carddisp.pl?gene=DLGAP2&keywords=DLGAP2, 2019. 37. Sujit KM, Sarkar S, Singh V, et al. Genome-wide differential methylation analyses identifies methylation signatures of male infertility. Hum Reprod. 2018;33(12):2256- 2267. 38. MICALL2 Gene. https://www.genecards.org/cgi- bin/carddisp.pl?gene=MICALL2&keywords=MICALL2, 2019. 39. Mary-Elizabeth Patti KH, Nathan Salomonis, Martijn van Iersel, et al. Adipogenesis (Homo sapiens). 2018; https://www.wikipathways.org/index.php/Pathway:WP236, 2019. 40. Madeline Kosho AP, Kristina Hanspers, Egon Willighagen, et al. Energy Metabolism. 2017; https://www.wikipathways.org/index.php/Pathway:WP1541, 2019. 41. Cote S, Gagne-Ouellet V, Guay SP, et al. PPARGC1alpha gene DNA methylation variations in human placenta mediate the link between maternal hyperglycemia and leptin levels in newborns. Clin Epigenetics. 2016;8:72. Page 23 of 53 Diabetes

Table 1: Characteristics of Gen3G mother-child pairs included in maternal insulin sensitivity epigenome-wide association study of placenta

Characteristics Median [IQR] or n (%) N=430 Mothers Age (years) 28 [25; 31] Ethnicity (% European descent) 430 (100.0%) Gravid status (% first pregnancy) 142 (33.0%) Parity (% first term pregnancy) 218 (50.7%) Smoking (% smoking in early pregnancy) 36 (8.4%) Body mass index in early pregnancy (kg/m2) 23.7 [21.6 ; 27.9] Waist circumference at first trimester (cm) 23.7 [21.6; 27.9]

Blood pressure at 2nd trimester visit (systolic/diastolic mmHg) 107/67 [100/63; 112/72] Oral glucose tolerance test (2nd trimester) Fasting glucose (mmol/L) 4.2 [3.9; 4.4] 1h-glucose (mmol/L) 7.1 [6; 8.2] 2h-glucose (mmol/L) 5.7 [4.8; 6.6] Insulin sensitivity - Matsuda index (Raw) 7.72 [5.69; 10.67] Insulin sensitivity - Matsuda index (Natual Log Transformed) 2.04 [1.74; 2.37] Gestational Diabetes Mellitus * (% yes) 37 (8.6%) Children Gestational age at birth (weeks) 39.7 [38.9; 40.4] Child sex (% female) 204 (47.4%) Birth weight (kg) 3.44 [3.17; 3.71] Large-for-Gestational Age (% >90th percentile) 31 (7.2%) Placental weight (g) 542 [467 ; 642]

* Gestational Diabetes Mellitus was diagnosed according to IADPSG Diabetes Page 24 of 53

Figure Legends:

Figure 1: Manhattan plots representing the results of the epigenome-wide association analyses between maternal insulin sensitivity (Matsuda index, ln transformed) and placenta methylation

(in M-values). Panel A: Model 1 adjusted for maternal age, gravid status, smoking, maternal

BMI, sex, and gestational age at delivery (Genomic Inflation= 2.884). The horizontal line indicates the Bonferonni level of statistical significance (P-values < 6.94x10-8). Panel B: Model 2 adjusted for maternal age, gravid status, smoking, maternal BMI, sex, and gestational age at delivery and 10 Principal Components from ReFACTor (Genomic Inflation= 1.158).

Figure 2: Mendelian Randomization supporting direction of effect at 5 loci where placenta DNA methylation may influence maternal insulin sensitivity: A) cg01618245 (CHRNA4), B) cg12673377 (MICALL2/UNCX), C) cg24475484 (DLGAP2), D) cg08099672 (ENTPD2), and E) cg03699074 (BDP1P). In each panel, the a arrow indicates the association between genetic IV representing the fetal placental DNA methylation levels at CpG site (using GRS from maternal genotypes), the b arrow indicates the association with the build genetic IV and Matsuda index; and c arrow (with β and SE below) is the observed association between methylation levels at the

CpG and Matsuda index (reverse of original EWAS, to allow comparison of betas). TSLS estimates, SE, and Durbin-Wu-Hausman test P-values are presented under each c observed estimates. All estimates are unadjusted (no covariates); the adjusted P-values for b association results are FDR-adjusted for number of tests performed (n=131 with a genetic IV available). Page 25 of 53 Diabetes

Figure 1A: Manhattan plots representing the results of the epigenome-wide association analyses between maternal insulin sensitivity (Matsuda index, ln transformed) and placenta methylation (in M-values). Panel A: Model 1 adjusted for maternal age, gravid status, smoking, maternal BMI, sex, and gestational age at delivery (Genomic Inflation= 2.884). The horizontal line indicates the Bonferonni level of statistical significance (P-values < 6.94x10-8). Diabetes Page 26 of 53

Figure 1B: Manhattan plots representing the results of the epigenome-wide association analyses between maternal insulin sensitivity (Matsuda index, ln transformed) and placenta methylation (in M-values). Panel B: Model 2 adjusted for maternal age, gravid status, smoking, maternal BMI, sex, and gestational age at delivery and 10 Principal Components from ReFACTor (Genomic Inflation= 1.158). Page 27 of 53 Diabetes

Figure 2 A

GRS elastic net for cg01618245 (CHRNA4) β= -0.022 SE=0.007 β=0.113 Adjusted P=0.03 b SE=0.012 a R2= 0.19

Methylation at c Matsuda cg01618245 (ln-transformed) (M-values) β obs= -0.106 SE=0.025

β TSLS= -0,195 SE= 0,059 Durbin-Wu-Hausman test P= 0.096 Diabetes Page 28 of 53

Figure 2 B

GRS elastic net for cg12673377 (MICALL2/UNCX) β= -0.015 SE=0.004 β=0.062 Adjusted P=0.03 b SE=0.005 a R2= 0.25

Methylation at c Matsuda cg12673377 (ln-transformed) (M-values) β obs= -0.150 SE=0.033

β TSLS= -0,238 SE= 0,073 Durbin-Wu-Hausman test P= 0.167 Page 29 of 53 Diabetes

Figure 2 C

GRS elastic net for cg24475484 (DLGAP2) β= -0.046 SE=0.014 β=0.189 Adjusted P=0.03 b SE=0.025 a R2= 0.12

Methylation at c Matsuda cg24475484 (ln-transformed) (M-values) β obs= -0.103 SE=0.025

β TSLS= -0,242 SE= 0,074 Durbin-Wu-Hausman test P=0.047 Diabetes Page 30 of 53

Figure 2 D

GRS elastic net for cg08099672 (ENTPD2) β= -0.034 SE=0.010 β=0.095 Adjusted P=0.03 b SE=0.013 a R2= 0.11

Methylation at c Matsuda cg08099672 (ln-transformed) (M-values) β obs= -0.156 SE=0.036

β TSLS= -0,342 SE= 0,107 Durbin-Wu-Hausman test P=0.065 Page 31 of 53 Diabetes

Figure 2 E

GRS elastic net for cg12658750 (BDP1P) β= -0.079 SE=0.023 β=0.169 Adjusted P=0.03 b SE=0.028 a R2= 0.08

Methylation at c Matsuda cg12658750 (ln-transformed) (M-values) β obs= -0.150 SE=0.039

β TSLS= -0,448 SE= 0,142 Durbin-Wu-Hausman test P=0.029 Diabetes Page 32 of 53

Supplementary Figure 1 : Participants flow chart included in current study

1024 participating women initially recruited in Gen3G (first trimester)

898 participating women with a visit at 2nd trimester (856 with complette OGTT and 822 with Matsuda index)

854 mother-child pairs with data at delivery

694 mother-child pairs with placenta collected at delivery

460 placenta samples sent to Illumina

430 participants included in the present study (passed DNAm QC and Matsuda available) Page 33 of 53 Diabetes

203x101mm (600 x 600 DPI) Diabetes Page 34 of 53

304x152mm (200 x 200 DPI) Page 35 of 53 Diabetes

304x152mm (200 x 200 DPI) Diabetes Page 36 of 53

Supplementary Table 1: Single Nucleotide Polymorphisms (SNPs) used to create genetic risk score (GRS) of insulin sensitivity initial association in Association in Gen3G SNPs chr position annotated gene Prokenpenko et al. with Matsuda (ln) Beta beta (SE) rs1260326 2:27730940 GCKR -0.067 -0.02307 (0.03824) rs35767 12:102875569 IGF1 -0.080 0.01581 (0.05446) rs459193 5:55806751 ANKRD55/MAP3K1 -0.053 -0.09932 (0.04122) rs4846565 1:219722104 LYPLAL1 -0.044 -0.09431 (0.04130) rs4865796 5:53272664 ARL15 -0.071 -0.01121 (0.04271) rs6822892 4:157734675 PDGFC -0.093 -0.006239 (0.040982) rs7607980 2:165551201 COBLL1 -0.120 -0.09199 (0.05452) rs1801282 3:12393125 PPARG -0.084 -0.10657 (0.06494) Page 37 of 53 Diabetes

Supplementary Table 2: Results from epigenome-wide analyses of maternal insulin sensitivity (Matsuda, ln-transformed) on placental DNA methylation (model adjusted for maternal age in years, gravid status [1 vs >1], smoking (yes/no in pregnancy), maternal body mass index [in kg/m2], child sex [m/f], and gestational age at birth in weeks) Illumina association with insulin sensitivity association with insulin sensitivity annotation Humarray annotation M-Value, Natural Log(Matsuda) Beta-Value, Natural Log(Matsuda) Known Methylation imprinted position position Mean Beta- Relation to UCSC Ref Closest gene Closest gene genes in CpG chr (build 37) (cytoband) value CpG Island Gene Name on the right on the left placenta* coefficient SE p-value coefficient SE p-value cg00918900 1 1057335 p36.33 0.039 OpenSea MIR200B C1orf159 -0.522 0.084 5.55E-10 -0.011 0.002 2.70E-09 cg16140890 1 1082406 p36.33 0.116 OpenSea MIR200B C1orf159 -0.487 0.09 6.79E-08 -0.027 0.005 1.04E-07 cg21778193 1 1098070 p36.33 0.271 Island MIR200B C1orf159 -0.472 0.087 5.35E-08 -0.061 0.011 6.85E-09 cg14839558 1 1874488 p36.33 0.252 N_Shore C1orf222 C1orf222 -0.476 0.088 5.67E-08 -0.057 0.010 2.45E-08 cg04131502 1 2845583 p36.32 0.076 N_Shore ACTRT2 TTC34 -0.389 0.071 4.96E-08 -0.016 0.003 1.83E-07 cg11837181 1 3001002 p36.32 0.131 S_Shore PRDM16 PRDM16 PRDM16 -0.413 0.076 5.18E-08 -0.028 0.005 1.11E-07 cg07686906 1 3353825 p36.32 0.226 S_Shelf PRDM16 PRDM16 PRDM16 -0.351 0.057 5.75E-10 -0.040 0.006 7.12E-10 cg14494450 1 4190881 p36.32 0.106 N_Shore AJAP1 LINC01134 -0.425 0.077 3.32E-08 -0.023 0.004 8.96E-08 cg09073467 1 6155515 p36.31 0.193 OpenSea KCNAB2 KCNAB2 KCNAB2 -0.283 0.052 6.90E-08 -0.029 0.005 3.19E-08 cg00389386 1 6188136 p36.31 0.168 Island CHD5 CHD5 CHD5 -0.308 0.056 4.33E-08 -0.028 0.005 1.53E-08 cg00386725 1 18617791 p36.13 0.099 OpenSea IGSF21 IGSF21 IGSF21 -0.329 0.059 2.26E-08 -0.019 0.003 1.12E-08 cg12011250 1 30198037 p35.3 0.196 OpenSea MATN1 PTPRU -0.452 0.078 6.74E-09 -0.043 0.008 1.59E-08 cg26178126 1 37740931 p34.3 0.174 OpenSea RPS29P6 RNA5SP43 -0.422 0.069 9.46E-10 -0.038 0.006 1.15E-09 cg07203271 1 53926112 p32.3 0.196 Island DMRTB1 DMRTB1 DMRTB1 -0.394 0.072 4.68E-08 -0.040 0.007 3.20E-08 cg24064348 1 54181174 p32.3 0.238 OpenSea GLIS1 GLIS1 GLIS1 -0.201 0.035 1.09E-08 -0.025 0.004 5.29E-09 cg08491066 1 156030669 q22 0.13 OpenSea RAB25 RAB25 LAMTOR2 -0.318 0.059 6.63E-08 -0.022 0.004 6.84E-08 cg04847540 1 182754070 q25.3 0.055 OpenSea NPL RGS8 -0.437 0.079 2.59E-08 -0.011 0.002 9.29E-07 cg20659588 1 237963267 q43 0.165 OpenSea RYR2 RYR2 RYR2 -0.433 0.075 8.42E-09 -0.036 0.006 2.75E-08 cg21307723 1 245215135 q44 0.807 OpenSea EFCAB2 EFCAB2 RNU6-999P 0.294 0.053 3.95E-08 0.029 0.006 1.42E-07 cg07865738 2 3649447 p25.3 0.093 OpenSea COLEC11 COLEC11 COLEC11 -0.560 0.099 1.80E-08 -0.024 0.005 1.59E-07 cg05444304 2 31442675 p23.1 0.934 OpenSea RNA5SP90 CAPN14 0.306 0.055 2.12E-08 0.011 0.002 4.86E-08 cg04140215 2 60540175 p16.1 0.159 OpenSea RNU1-32P RNA5SP94 -0.432 0.073 2.82E-09 -0.035 0.006 7.64E-09 cg00460471 2 61437605 p15 0.176 OpenSea USP34 USP34 AHSA2 -0.442 0.078 1.52E-08 -0.038 0.007 5.41E-08 cg04293688 2 75466188 p12 0.113 OpenSea GAPDHP57 TACR1 -0.420 0.077 4.79E-08 -0.026 0.005 1.07E-07 cg02384870 2 216161208 q35 0.2 OpenSea ATIC ABCA12 -0.636 0.102 5.39E-10 -0.059 0.010 1.61E-09 SPHKAP cg16819051 2 229173235 q36.3 0.143 OpenSea RPL7L1P10 SPHKAP (maternal) -0.382 0.071 6.38E-08 -0.027 0.005 1.08E-07 SPHKAP cg07728373 2 229266790 q36.3 0.087 OpenSea RPL7L1P10 SPHKAP (maternal) -0.647 0.11 4.65E-09 -0.024 0.004 4.03E-08 CNTN6 cg16857113 3 1133865 p26.3 0.052 OpenSea CNTN6 CNTN6 RN7SL120P (maternal) -0.422 0.075 1.86E-08 -0.011 0.002 6.94E-07 cg11294782 3 10487409 p25.3 0.065 OpenSea ATP2B2 ATP2B2-IT1 MIR885 -0.293 0.051 1.21E-08 -0.011 0.002 1.94E-08 cg25721832 3 81913557 p12.2 0.085 OpenSea RPL7AP23 GBE1 -0.351 0.061 7.75E-09 -0.016 0.003 2.75E-08 cg06751809 3 105047227 q13.11 0.562 OpenSea ALCAM TUBBP11 -0.726 0.117 5.89E-10 -0.114 0.018 1.30E-10 cg08389995 3 131645295 q22.1 0.085 OpenSea CPNE4 MIR5704 CPNE4 -0.533 0.097 4.06E-08 -0.021 0.005 5.27E-06 cg08058801 3 147974120 q24 0.072 OpenSea RPL38P1 HNRNPA1P20 -0.509 0.093 3.79E-08 -0.017 0.003 6.42E-08 cg16853336 3 149396490 q25.1 0.29 OpenSea WWTR1 WWTR1 WWTR1-AS1 -0.244 0.042 9.01E-09 -0.033 0.006 1.23E-08 cg18861231 3 152057943 q25.1 0.079 OpenSea MBNL1 TMEM14E TMEM14E -0.445 0.081 3.57E-08 -0.017 0.003 2.95E-07 cg00188627 3 159557779 q25.33 0.915 OpenSea SCHIP1 SCHIP1 SCHIP1 0.384 0.07 5.01E-08 0.016 0.003 1.25E-06 cg25285919 3 188312215 q28 0.088 OpenSea LPP MIR28 LPP-AS1 -0.395 0.073 6.49E-08 -0.019 0.004 1.99E-07 KCNIP4 cg20018106 4 21768009 p15.2 0.391 OpenSea KCNIP4 KCNIP4 KCNIP4 (maternal) -0.526 0.097 6.74E-08 -0.083 0.015 1.14E-08 cg17756860 4 110510374 q25 0.102 OpenSea CCDC109B CCDC109B CCDC109B -0.430 0.076 1.39E-08 -0.022 0.004 3.84E-07 cg18650968 4 176340837 q34.2 0.049 OpenSea TSEN2P1 ADAM29 -0.444 0.075 2.74E-09 -0.011 0.002 5.42E-07 cg02287371 5 1246274 p15.33 0.057 Island SLC6A18 SLC6A18 SLC6A18 -0.497 0.09 3.31E-08 -0.016 0.003 1.50E-07 cg25314607 5 2148308 p15.33 0.096 OpenSea IRX2 IRX4 -0.418 0.074 1.34E-08 -0.021 0.004 2.29E-08 cg14757962 5 2176287 p15.33 0.088 N_Shelf IRX2 IRX4 -0.450 0.083 6.71E-08 -0.021 0.004 4.42E-08 cg13480617 5 35230725 p13.2 0.026 OpenSea PRLR PRLR PRLR -0.247 0.044 1.60E-08 -0.004 0.001 2.17E-07 cg23561197 5 125852064 q23.2 0.084 OpenSea ALDH7A1 GRAMD3 -0.388 0.07 3.66E-08 -0.017 0.003 1.80E-08 cg26849382 5 140901022 q31.3 0.929 OpenSea DIAPH1 DIAPH1 DIAPH1 0.317 0.059 6.15E-08 0.010 0.002 2.61E-07 cg17587481 5 152603672 q33.1 0.158 OpenSea GRIA1 NMUR2 -0.557 0.102 4.23E-08 -0.043 0.008 1.68E-07 cg18682781 5 176277687 q35.2 0.15 OpenSea UNC5A HK3 UNC5A -0.469 0.08 3.82E-09 -0.036 0.006 9.41E-09 cg06077821 5 180048893 q35.3 0.168 Island FLT4 FLT4 FLT4 -0.416 0.064 7.54E-11 -0.036 0.006 1.32E-10 cg00078259 6 2656232 p25.2 0.128 OpenSea MYLK4 C6orf195 -0.451 0.083 5.89E-08 -0.028 0.006 4.78E-07 cg14886500 6 23447204 p22.3 0.066 OpenSea SPTLC1P2 RNU6-1060P -0.504 0.09 2.54E-08 -0.015 0.003 3.24E-07 cg27652925 6 31740951 p21.33 0.47 OpenSea C6orf27 VWA7 VWA7 -0.225 0.042 6.85E-08 -0.039 0.007 7.28E-08 cg01530082 6 32784327 p21.32 0.178 OpenSea HLA-DOB HLA-DOB HLA-DOB -0.447 0.081 2.93E-08 -0.040 0.007 2.79E-08 cg00045690 6 32825897 p21.32 0.197 S_Shelf PSMB9 PSMB9 PSMB9 -0.558 0.099 1.93E-08 -0.049 0.010 2.08E-07 cg01948226 6 32829208 p21.32 0.098 OpenSea PPP1R2P1 PSMB9 -0.455 0.077 4.27E-09 -0.023 0.004 2.58E-09 cg12954267 6 33093109 p21.32 0.13 OpenSea HLA-DPB2 HLA-DPA3 HLA-DPB2 -0.467 0.078 2.56E-09 -0.029 0.005 1.10E-08 cg17226458 6 33780022 p21.31 0.149 OpenSea LINC01016 MLN -0.307 0.055 2.79E-08 -0.024 0.005 5.73E-08 cg17538538 6 106825021 q21 0.921 OpenSea RNA5SP211 ATG5 0.311 0.057 4.64E-08 0.013 0.003 1.44E-07 cg12673377 7 1388271 p22.3 0.127 OpenSea MICALL2 UNCX -0.363 0.067 6.60E-08 -0.025 0.005 8.50E-08 Diabetes Page 38 of 53

cg26037026 7 1641922 p22.3 0.26 Island TFAMP1 PSMG3-AS1 -0.349 0.065 6.71E-08 -0.044 0.008 1.80E-07 cg26225794 7 27717723 p15.2 0.05 OpenSea TAX1BP1 HIBADH -0.335 0.061 3.70E-08 -0.009 0.002 8.68E-07 cg11138231 7 99475824 q22.1 0.33 OpenSea OR2AE1 TRIM4 OR2AE1 -0.564 0.102 3.01E-08 -0.077 0.014 4.91E-08 cg08582801 7 99588335 q22.1 0.117 OpenSea ZKSCAN1 AZGP1P1 -0.255 0.046 3.48E-08 -0.018 0.003 1.60E-07 PODXL cg25903144 7 131609806 q32.3 0.097 OpenSea LOC101928782PLXNA4 PODXL (maternal) -0.520 0.093 2.27E-08 -0.023 0.004 1.81E-07 cg06981471 7 142982076 q34 0.116 N_Shelf TMEM139 TMEM139 TMEM139 -0.304 0.055 3.73E-08 -0.018 0.003 2.05E-07 cg18616558 7 158798592 q36.3 0.163 Island LINC00689 WDR60 -0.697 0.122 1.25E-08 -0.051 0.009 6.11E-08 DLGAP2 cg24475484 8 1625395 p23.3 0.061 OpenSea DLGAP2 DLGAP2 DLGAP2 (maternal) -0.414 0.072 7.35E-09 -0.013 0.002 6.68E-09 cg16513326 8 10877312 p23.1 0.143 S_Shelf XKR6 MIR598 XKR6 -0.385 0.071 5.23E-08 -0.028 0.005 1.66E-07 cg15952840 8 11451312 p23.1 0.038 OpenSea GATA4 LINC00208 -0.409 0.075 5.32E-08 -0.008 0.002 7.70E-07 cg14204490 8 33330967 p12 0.032 S_Shore FUT10 TTI2 FUT10 -0.217 0.039 3.32E-08 -0.004 0.001 3.03E-08 cg05202700 8 38258250 p11.23 0.29 OpenSea LETM2 LETM2 LETM2 -0.446 0.083 6.94E-08 -0.058 0.011 3.43E-08 cg24848724 8 142578640 q24.3 0.208 OpenSea MIR4472-1 MROH5 -0.580 0.105 3.19E-08 -0.055 0.011 2.01E-07 cg03710412 8 142938779 q24.3 0.149 OpenSea MIR4472-1 MROH5 -0.494 0.09 3.38E-08 -0.037 0.007 5.36E-08 cg09851596 8 143545214 q24.3 0.213 N_Shore BAI1 BAI1 BAI1 -0.279 0.051 4.05E-08 -0.031 0.006 2.50E-08 cg04535454 8 144035192 q24.3 0.186 OpenSea CDC42P3 CYP11B2 -0.345 0.064 5.61E-08 -0.032 0.006 4.06E-08 cg13022891 9 5817706 p24.1 0.168 OpenSea ERMP1 ERMP1 ERMP1 -0.304 0.055 4.21E-08 -0.027 0.005 9.05E-08 cg05775586 9 28889354 p21.1 0.177 OpenSea MIR873 RBMXP2 MIR873 -0.502 0.085 2.84E-09 -0.045 0.008 3.35E-09 cg05851077 9 29802712 p21.1 0.223 OpenSea RBMXP2 MIR873 -0.790 0.129 8.76E-10 -0.075 0.013 1.29E-08 cg03275961 9 90386329 q21.33 0.112 OpenSea CTSL3P CTSL3P CTSL -0.353 0.063 2.72E-08 -0.022 0.004 4.20E-08 cg06830529 9 124960107 q33.2 0.19 OpenSea MORN5 MORN5 MORN5 -0.363 0.062 5.64E-09 -0.035 0.006 1.04E-08 cg05389237 9 125314173 q33.2 0.134 OpenSea OR1N2 OR1N2 OR1N1 -0.541 0.09 2.10E-09 -0.034 0.006 4.19E-09 cg03510150 9 130690744 q34.11 0.077 S_Shore PIP5KL1 PIP5KL1 PIP5KL1 -0.225 0.04 1.43E-08 -0.011 0.002 3.89E-08 cg21927431 9 134678366 q34.13 0.147 OpenSea MED27 EIF4A1P3 -0.358 0.063 1.20E-08 -0.027 0.005 5.05E-08 cg13449257 9 137355128 q34.2 0.301 N_Shore COL5A1 RXRA -0.378 0.069 4.81E-08 -0.052 0.009 1.73E-08 cg17224410 9 137852661 q34.3 0.171 OpenSea OLFM1 FCN1 -0.404 0.074 4.91E-08 -0.035 0.006 4.27E-08 cg13463516 9 138632121 q34.3 0.231 S_Shelf KCNT1 KCNT1 KCNT1 -0.368 0.064 8.37E-09 -0.043 0.008 1.35E-08 cg12760575 9 139158654 q34.3 0.528 N_Shore GPSM1 QSOX2 -0.306 0.054 1.75E-08 -0.052 0.009 1.42E-08 cg08099672 9 139944162 q34.3 0.092 S_Shore ENTPD2 ENTPD2 ENTPD2 -0.396 0.071 2.00E-08 -0.021 0.004 2.72E-08 cg14440140 9 140177567 q34.3 0.078 S_Shore NRARP TOR4A -0.375 0.069 4.79E-08 -0.015 0.003 5.09E-08 cg14361947 9 140780551 q34.3 0.178 S_Shelf CACNA1B CACNA1B CACNA1B -0.218 0.04 4.68E-08 -0.021 0.004 9.75E-08 cg08511578 10 1403938 p15.3 0.133 N_Shore ADARB2 ADARB2-AS1 ADARB2 -0.507 0.094 6.00E-08 -0.033 0.006 8.32E-08 cg20112984 10 1447183 p15.3 0.087 OpenSea ADARB2 ADARB2-AS1 ADARB2 -0.469 0.082 1.02E-08 -0.020 0.004 3.71E-08 cg02831474 10 1783879 p15.3 0.065 S_Shelf LINC00700 ADARB2 -0.613 0.094 7.64E-11 -0.019 0.003 3.86E-09 cg12697796 10 49954016 q11.23 0.157 OpenSea WDFY4 WDFY4 WDFY4 -0.311 0.058 6.23E-08 -0.027 0.005 4.85E-08 cg08149223 10 50507130 q11.23 0.111 OpenSea C10orf71 C10orf71 C10orf71-AS1 -0.285 0.05 1.01E-08 -0.018 0.003 2.41E-08 cg18233010 10 114911413 q25.3 0.92 OpenSea TCF7L2 TCF7L2 TCF7L2 0.246 0.044 1.78E-08 0.012 0.002 3.32E-07 cg23521973 10 125117429 q26.13 0.089 OpenSea GPR26 BUB3 -0.432 0.077 1.90E-08 -0.021 0.004 4.09E-08 cg00403246 10 134794753 q26.3 0.18 S_Shelf GPR123 LINC01168 -0.248 0.045 4.40E-08 -0.024 0.005 1.05E-07 cg16608656 11 1082797 p15.5 0.121 N_Shore MUC2 MUC2 MUC2 -0.458 0.083 3.07E-08 -0.028 0.005 1.04E-07 cg03568769 11 1800837 p15.5 0.108 OpenSea RPL36AP39 CTSD -0.400 0.071 1.51E-08 -0.023 0.004 2.34E-08 H19 cg17985533 11 2019116 p15.5 0.136 N_Shore H19;MIR675 H19 H19 (paternal) -0.265 0.048 3.44E-08 -0.020 0.004 1.01E-07 H19 cg09701145 11 2019436 p15.5 0.226 N_Shore MIR675;H19 H19 H19 (paternal) -0.367 0.064 9.76E-09 -0.042 0.007 3.79E-09 KCNQ1 cg24073146 11 2543204 p15.5 0.177 OpenSea KCNQ1 KCNQ1 KCNQ1 (maternal) -0.291 0.054 6.07E-08 -0.027 0.005 4.92E-08 cg00875983 11 3240213 p15.4 0.349 S_Shore MRGPRG MRGPRG-AS1 MRGPRG -0.310 0.056 2.43E-08 -0.047 0.008 2.13E-08 cg15575356 11 47616757 p11.2 0.188 S_Shore C1QTNF4 MTCH2 C1QTNF4 -0.274 0.049 2.54E-08 -0.027 0.005 6.17E-08 cg12161743 11 76750531 q13.5 0.138 N_Shore B3GNT6 B3GNT6 B3GNT6 -0.412 0.076 6.35E-08 -0.031 0.006 7.25E-08 DSCAML1 cg01124546 11 117515332 q23.3 0.243 OpenSea DSCAML1 FXYD2 DSCAML1 (maternal) -0.284 0.053 6.13E-08 -0.035 0.007 7.59E-08 cg13625403 11 120008510 q23.3 0.249 Island TRIM29 TRIM29 TRIM29 -0.511 0.095 6.66E-08 -0.059 0.011 1.97E-07 cg23519631 12 72690355 q21.1 0.185 OpenSea TRHDE TRHDE TRHDE-AS1 -0.378 0.07 5.68E-08 -0.036 0.007 2.03E-07 cg23536293 12 99524459 q23.1 0.113 OpenSea ANKS1B ANKS1B RNA5SP366 -0.614 0.113 5.63E-08 -0.033 0.007 2.71E-06 cg08737919 12 108840669 q23.3 0.073 OpenSea FICD CMKLR1 -0.333 0.06 2.41E-08 -0.012 0.002 8.65E-08 cg13116906 12 119552989 q24.23 0.055 OpenSea SRRM4 SRRM4 SRRM4 -0.412 0.07 4.37E-09 -0.010 0.002 5.23E-08 cg06343669 12 132690782 q24.33 0.126 S_Shore GALNT9 GALNT9 GALNT9 -0.472 0.082 1.05E-08 -0.029 0.005 5.05E-08 cg04907218 12 132826672 q24.33 0.173 N_Shore GALNT9 GALNT9 GALNT9 -0.427 0.076 1.88E-08 -0.039 0.007 5.20E-09 cg02688867 12 133019854 q24.33 0.39 Island MUC8 GALNT9 -0.380 0.07 6.06E-08 -0.060 0.011 9.19E-08 cg18617770 12 133035939 q24.33 0.229 OpenSea MUC8 GALNT9 -0.329 0.06 3.73E-08 -0.038 0.007 6.02E-08 cg07435932 13 19648633 q12.11 0.095 OpenSea RNA5SP24 PHF2P2 -0.532 0.096 3.12E-08 -0.024 0.004 6.21E-08 cg18042910 13 19662609 q12.11 0.71 OpenSea RNA5SP24 PHF2P2 -0.462 0.083 2.37E-08 -0.062 0.011 2.44E-08 cg23359229 13 29976597 q12.3 0.049 OpenSea MTUS2 MTUS2-AS1 GAPDHP69 -0.437 0.081 5.87E-08 -0.011 0.002 1.20E-06 cg06066032 13 48369175 q14.2 0.156 OpenSea LINC00444 RPL27AP8 -0.528 0.09 4.05E-09 -0.039 0.007 2.23E-08 cg07080246 13 63306618 q21.31 0.347 OpenSea LINC00448 LINC00376 LINC00448 -0.636 0.117 6.04E-08 -0.086 0.016 7.21E-08 GPC6 cg13827123 13 93971495 q31.3 0.164 OpenSea GPC6 HNRNPA1P29 GPC6 (maternal) -0.534 0.094 1.29E-08 -0.041 0.007 1.87E-08 MCF2L cg24857040 13 113565214 q34 0.128 OpenSea MCF2L MCF2L (paternal) -0.412 0.076 6.66E-08 -0.028 0.005 1.00E-07 Page 39 of 53 Diabetes

cg11586089 14 66644923 q23.3 0.149 OpenSea LINC00238 YBX1P1 -0.423 0.073 6.64E-09 -0.033 0.006 9.51E-09 cg13257690 14 101908752 q32.31 0.236 N_Shore DIO3OS LINC00524 -0.277 0.051 5.75E-08 -0.034 0.006 3.80E-08 cg11416433 15 25429261 q11.2 0.182 OpenSea SNORD115-8 SNORD115-8 SNORD115-6 -0.316 0.053 2.15E-09 -0.031 0.005 1.26E-09 OCA2 cg17905524 15 28305412 q13.1 0.104 OpenSea OCA2 OCA2 OCA2 (maternal) -0.398 0.069 6.98E-09 -0.021 0.004 2.75E-08 cg05347757 15 51121020 q21.2 0.106 OpenSea AP4E1 SPPL2A -0.425 0.078 4.30E-08 -0.021 0.004 5.32E-07 cg25380707 15 86660946 q25.3 0.089 OpenSea LINC01584 RNA5SP400 MIR548AP -0.519 0.087 2.78E-09 -0.022 0.004 7.83E-09 cg01898225 15 99975141 q26.3 0.118 OpenSea MEF2A LRRC28 -0.346 0.062 2.30E-08 -0.023 0.004 9.45E-09 cg08792976 16 760528 p13.3 0.118 S_Shelf METRN FBXL16 -0.436 0.077 1.51E-08 -0.027 0.005 3.98E-08 cg21396983 16 1275423 p13.3 0.146 OpenSea TPSG1 TPSB2 TPSG1 -0.481 0.083 7.39E-09 -0.035 0.006 7.43E-09 cg16444062 16 2770298 p13.3 0.144 Island PRSS27 PRSS27 PRSS27 -0.529 0.098 6.81E-08 -0.038 0.007 9.03E-08 cg10964639 16 24043296 p12.2 0.141 OpenSea PRKCB PRKCB PRKCB -0.404 0.075 6.26E-08 -0.028 0.005 3.62E-08 cg08474716 16 27079640 p12.1 0.141 OpenSea C16orf82 C16orf82 C16orf82 -0.523 0.09 6.57E-09 -0.035 0.006 3.11E-08 cg08239663 16 52688569 q12.2 0.092 OpenSea CHD9 CASC16 -0.521 0.095 3.74E-08 -0.023 0.005 3.30E-07 cg00781180 16 73984002 q22.3 0.07 OpenSea PSMD7 RPSAP56 -0.441 0.078 1.73E-08 -0.016 0.003 1.41E-07 cg01615861 16 85513978 q24.1 0.382 OpenSea GSE1 MIR5093 -0.161 0.029 4.03E-08 -0.026 0.005 2.60E-08 cg06429120 16 86309214 q24.1 0.422 OpenSea LINC01081 LINC01081 LINC01081 -0.682 0.124 3.51E-08 -0.103 0.019 4.50E-08 cg13254480 16 86513248 q24.1 0.099 OpenSea FENDRR FENDRR FENDRR -0.639 0.118 6.09E-08 -0.028 0.006 2.57E-07 cg08204714 16 87726461 q24.2 0.266 S_Shelf JPH3 KLHDC4 JPH3 -0.265 0.049 5.32E-08 -0.034 0.006 7.84E-08 cg09640571 16 88388040 q24.2 0.225 OpenSea ZNF469 BANP -0.254 0.046 4.33E-08 -0.029 0.005 8.94E-08 cg01095004 16 88454883 q24.2 0.103 S_Shore ZNF469 BANP -0.518 0.093 2.18E-08 -0.028 0.005 6.55E-08 cg03699074 16 88849875 q24.3 0.891 N_Shore FAM38A PIEZO1 PIEZO1 0.422 0.077 3.74E-08 0.022 0.004 2.62E-07 cg08372350 16 88943191 q24.3 0.282 N_Shore CBFA2T3 CBFA2T3 CBFA2T3 -0.452 0.078 5.69E-09 -0.059 0.010 1.65E-09 cg10877514 17 292539 p13.3 0.097 S_Shore FAM101B FAM101B FAM101B -0.474 0.082 7.87E-09 -0.022 0.004 3.02E-08 cg20612875 17 1094308 p13.3 0.319 OpenSea ABR ABR -0.269 0.049 5.27E-08 -0.040 0.007 9.94E-08 cg16340422 17 17110120 p11.2 0.073 Island PLD6 FLCN PLD6 -0.394 0.068 6.58E-09 -0.015 0.003 3.23E-07 cg05790369 17 33202007 q12 0.185 OpenSea CCT6B TMEM132E -0.443 0.079 1.83E-08 -0.042 0.008 5.24E-08 cg00903007 17 34068171 q12 0.271 Island RASL10B RASL10B RASL10B -0.252 0.042 2.34E-09 -0.033 0.005 1.25E-09 cg08357744 17 37559321 q12 0.082 S_Shore FBXL20 MED1 FBXL20 -0.528 0.095 2.48E-08 -0.019 0.004 1.40E-06 cg27410679 17 43866278 q21.31 0.116 S_Shelf CRHR1 CRHR1 CRHR1 -0.479 0.088 5.91E-08 -0.028 0.005 7.82E-08 cg14759277 17 48674158 q21.33 0.255 OpenSea CACNA1G CACNA1G CACNA1G -0.342 0.063 6.40E-08 -0.043 0.008 1.17E-07 cg18450810 17 51111066 q22 0.376 OpenSea RPS2P48 C17orf112 -0.454 0.082 3.34E-08 -0.073 0.013 6.30E-08 cg10883064 17 60757346 q23.2 0.152 Island MRC2 RNU6-446P MRC2 -0.432 0.076 1.54E-08 -0.035 0.006 2.39E-08 cg02110836 17 71953083 q25.1 0.221 S_Shelf RPL38 LINC00469 -0.226 0.039 8.26E-09 -0.026 0.004 3.85E-09 cg21347874 17 77122733 q25.3 0.06 OpenSea HRNBP3 RBFOX3 RBFOX3 -0.471 0.086 4.51E-08 -0.015 0.003 6.04E-07 cg15867652 17 79011277 q25.3 0.081 S_Shore BAIAP2 BAIAP2 BAIAP2 -0.502 0.093 6.32E-08 -0.018 0.004 3.23E-06 cg11086223 18 11487579 p11.21 0.197 OpenSea LINC01255 NPIPB1P PIEZO2 -0.353 0.064 4.12E-08 -0.036 0.007 7.13E-08 cg18216439 18 35132882 q12.2 0.088 OpenSea CELF4 CELF4 CELF4 -0.397 0.073 5.80E-08 -0.018 0.004 2.55E-07 cg27332583 18 35150602 q12.2 0.145 S_Shelf MIR4318 CELF4 -0.294 0.054 4.72E-08 -0.023 0.004 4.77E-08 cg12658750 18 75499382 q23 0.08 OpenSea RNA5SP461 BDP1P -0.327 0.057 8.97E-09 -0.015 0.003 1.43E-08 cg06495184 19 406256 p13.3 0.228 Island C2CD4C C2CD4C C2CD4C -0.383 0.063 1.46E-09 -0.044 0.007 5.12E-09 cg26238771 19 592158 p13.3 0.131 S_Shore HCN2 HCN2 HCN2 -0.382 0.071 6.23E-08 -0.026 0.005 2.83E-07 cg20623853 19 1734365 p13.3 0.092 Island ONECUT3 TCF3 -0.401 0.074 5.80E-08 -0.020 0.004 3.17E-07 cg18108190 19 2727822 p13.3 0.1 N_Shelf SLC39A3 DIRAS1 -0.406 0.074 4.56E-08 -0.021 0.004 7.34E-08 cg12193237 19 3907913 p13.3 0.251 Island ATCAY RN7SL202P ATCAY -0.393 0.07 1.98E-08 -0.047 0.009 4.04E-08 cg11855710 19 29768180 q12 0.063 OpenSea VSTM2B RN7SL340P -0.402 0.072 2.25E-08 -0.013 0.003 2.16E-07 cg24738497 19 36005395 q13.12 0.055 OpenSea DMKN SBSN DMKN -0.371 0.064 5.35E-09 -0.012 0.002 4.34E-09 cg09181711 19 51327196 q13.33 0.126 N_Shelf KLK1 KLK15 KLK1 -0.497 0.083 2.23E-09 -0.031 0.006 2.55E-08 cg18184975 20 3644192 p13 0.093 S_Shelf GFRA4 ADAM33 GFRA4 -0.387 0.072 6.33E-08 -0.020 0.004 2.01E-07 cg11177743 20 21545361 p11.22 0.133 OpenSea PAX1 NKX2-2 -0.376 0.068 3.66E-08 -0.026 0.005 1.60E-07 cg01216882 20 31804277 q11.21 0.205 OpenSea C20orf71 BPIFA3 BPIFA4P -0.407 0.072 1.82E-08 -0.041 0.007 7.15E-09 cg11765907 20 43103949 q13.12 0.182 N_Shore TTPAL TTPAL RPL37AP1 -0.258 0.046 2.04E-08 -0.025 0.005 3.97E-08 cg01864178 20 44662890 q13.12 0.31 S_Shore SLC12A5 SLC12A5 SLC12A5 -0.450 0.081 2.70E-08 -0.061 0.012 1.37E-07 cg22509138 20 44809026 q13.12 0.123 S_Shelf CDH22 CDH22 CDH22 -0.468 0.085 3.21E-08 -0.027 0.005 1.16E-07 cg09319487 20 56771178 q13.32 0.196 OpenSea ANKRD60 C20orf85 -0.399 0.072 2.63E-08 -0.040 0.007 5.35E-08 cg09063835 20 58181392 q13.32 0.143 S_Shore PHACTR3 PHACTR3 PHACTR3 -0.490 0.09 5.79E-08 -0.034 0.007 2.18E-07 LINC01056 cg05808230 20 61726456 q13.33 0.178 OpenSea HAR1B LINC01056 (paternal) -0.426 0.074 1.02E-08 -0.038 0.007 6.16E-09 cg01618245 20 61990279 q13.33 0.1 N_Shore CHRNA4 CHRNA4 CHRNA4 -0.523 0.095 3.66E-08 -0.025 0.005 2.38E-07 cg24600306 20 61990671 q13.33 0.162 N_Shore CHRNA4 CHRNA4 CHRNA4 -0.277 0.051 5.78E-08 -0.024 0.004 5.37E-08 cg27625886 20 62060443 q13.33 0.129 S_Shore KCNQ2 KCNQ2 KCNQ2 -0.514 0.088 4.93E-09 -0.033 0.006 4.33E-08 cg19450046 21 45712938 q22.3 0.304 N_Shore AIRE AIRE AIRE -0.366 0.066 3.27E-08 -0.051 0.009 5.78E-08 cg00046877 22 19702664 q11.21 0.182 S_Shore SEPT5 SEPT5 SEPT5 -0.211 0.037 1.39E-08 -0.021 0.004 2.47E-08 cg23016281 22 27616620 q12.1 0.102 OpenSea MN1 RNU6-1066P -0.588 0.097 1.28E-09 -0.031 0.006 6.80E-07 cg00373987 22 37868968 q13.1 0.253 OpenSea MFNG MFNG MFNG -0.423 0.077 4.96E-08 -0.049 0.009 1.69E-07 cg13377560 22 43860820 q13.2 0.146 OpenSea MPPED1 MPPED1 MPPED1 -0.367 0.068 6.08E-08 -0.027 0.005 1.87E-07 cg11821795 22 47611604 q13.31 0.064 S_Shelf LINC00898 TBC1D22A -0.437 0.081 6.78E-08 -0.013 0.003 3.88E-07

*ref: Sanchez-Delgado M, Court F, Vidal E, et al. Human Oocyte-Derived Methylation Differences Persist in the Placenta Revealing Widespread Transient Imprinting. PLoS genetics. 2016;12(11):e1006427 Diabetes Page 40 of 53

Supplementary Table 3: ReFACtor PCs and associations with Mastuda index and co-variables Pval_PC1 Pval_PC2 Pval_PC3 Pval_PC4 Pval_PC5 Pval_PC6 Pval_PC7 Pval_PC8 Pval_PC9 Pval_PC10 Matsuda index <0.0001 0.011 0.495 0.123 0.021 0.068 0.603 0.822 0.505 0.932 Maternal age 0.202 0.080 0.715 0.642 0.726 0.034 0.225 0.614 0.432 0.163 Maternal BMI (V1) 0.314 0.700 0.407 0.570 0.756 0.433 0.801 0.367 0.971 0.193 Gravid status 0.635 0.338 0.289 0.351 0.598 0.212 0.146 0.350 0.299 0.307 Maternal Smoking 0.539 0.828 0.007 0.342 0.459 0.664 0.227 0.148 0.109 0.257 Gestational age at birth 0.289 0.736 <0.0001 0.344 0.090 0.585 0.003 0.180 0.744 0.694

Child sex 0.834 0.578 0.469 0.073 0.398 0.140 0.580 0.883 0.441 0.007 P-values from correlations if continuous variables, and from Kruskal-Wallis if categorical variables Page 41 of 53 Diabetes

Supplementary Table 4: Results from Mendelian Randomization analyses, building genetic instrumental variables (GRS or single SNP) for DNA methylation at 188 CpGs identified in Model 1 using cis-SNPs within 500kb around each CpG site linear regression ln(matsuda) GRS information on association with CpG and elastic net statistics (N = 418) linear regression ln(matsuda) ~ GRS (N = 401) TSLS results (N = 401) ~ CpG (N = 401)

P_value P_value N of SNPs N SNPs N of adj for adj for within N of SNPs without SNPs multiple multiple CPG chr pos Estimate Std.Error P.Value r.squaredFstat ±500kb MAF<0.05 duplicate in GRS estimate SE P_value Rsquare testing estimate SE P_value testing estimate SE P_value cg12658750 18 75499382 0.169 0.028 3.37E-09 0.081 36.5 16935 3291 1811 5 -0.079 0.023 0.0008 0.028 0.03 -0.448 0.142 0.0017 0.046 -0.150 0.039 1.45E-04 cg24475484 8 1625395 0.189 0.025 2.81E-13 0.120 57.0 25101 4134 2769 10 -0.046 0.014 0.0009 0.027 0.03 -0.242 0.074 0.0012 0.046 -0.103 0.025 5.09E-05 cg01618245 20 61990279 0.113 0.012 3.20E-20 0.185 94.3 18168 2970 1608 34 -0.022 0.007 0.0010 0.027 0.03 -0.195 0.059 0.0011 0.046 -0.106 0.025 3.31E-05 cg08099672 9 139944162 0.095 0.013 2.76E-12 0.111 51.9 17133 1692 1077 13 -0.034 0.010 0.0012 0.026 0.03 -0.342 0.107 0.0015 0.046 -0.156 0.036 1.97E-05 cg12673377 7 1388271 0.062 0.005 2.37E-27 0.246 135.8 20418 2694 1672 51 -0.015 0.004 0.0012 0.026 0.03 -0.238 0.073 0.0012 0.046 -0.150 0.036 3.07E-05 cg03699074 16 88849875 0.095 0.014 2.21E-11 0.102 47.3 21611 2782 1905 17 0.022 0.007 0.0030 0.022 0.07 0.224 0.078 0.0044 0.10 0.075 0.025 3.13E-03 cg24073146 11 2543204 0.108 0.017 1.35E-09 0.085 38.5 17447 2473 1698 6 -0.046 0.016 0.0039 0.021 0.07 -0.416 0.150 0.006 0.11 -0.140 0.042 8.89E-04 cg08511578 10 1403938 0.106 0.024 1.50E-05 0.044 19.2 17754 2647 1657 4 -0.034 0.013 0.0079 0.018 0.13 -0.327 0.134 0.015 0.16 -0.098 0.025 8.29E-05 cg11765907 20 43103949 0.047 0.005 1.50E-16 0.151 74.2 14001 1783 1041 27 -0.016 0.006 0.010 0.016 0.15 -0.324 0.125 0.010 0.16 -0.187 0.050 1.95E-04 cg02384870 2 216161208 0.175 0.033 1.53E-07 0.064 28.5 14134 1835 1063 5 -0.042 0.017 0.014 0.015 0.17 -0.243 0.100 0.016 0.16 -0.112 0.024 4.84E-06 cg21396983 16 1275423 0.079 0.009 1.39E-16 0.152 74.3 21550 2710 1738 24 -0.015 0.006 0.016 0.015 0.17 -0.182 0.074 0.014 0.16 -0.144 0.030 1.97E-06 cg09640571 16 88388040 0.031 0.004 1.75E-12 0.113 52.9 21289 3220 2169 27 -0.011 0.005 0.016 0.014 0.17 -0.356 0.147 0.016 0.16 -0.211 0.049 1.71E-05 cg24857040 13 113565214 0.079 0.008 5.26E-21 0.192 98.7 17276 2194 1324 26 -0.013 0.006 0.018 0.014 0.17 -0.169 0.071 0.017 0.16 -0.126 0.032 7.73E-05 cg20612875 17 1094308 0.035 0.003 4.26E-22 0.201 104.9 18826 2792 1917 39 -0.009 0.004 0.020 0.014 0.17 -0.252 0.106 0.017 0.16 -0.224 0.049 7.78E-06 cg23521973 10 125117429 0.079 0.011 7.45E-13 0.116 54.8 15015 2399 1336 17 -0.017 0.007 0.020 0.014 0.17 -0.212 0.090 0.019 0.16 -0.138 0.031 1.13E-05 cg14440140 9 140177567 0.081 0.008 8.24E-21 0.190 97.6 16195 1470 921 34 -0.013 0.006 0.025 0.012 0.19 -0.154 0.068 0.024 0.19 -0.109 0.030 3.10E-04 cg06429120 16 86309214 0.155 0.013 9.85E-28 0.249 138.1 22860 3698 2812 36 -0.015 0.007 0.027 0.012 0.19 -0.097 0.043 0.023 0.19 -0.101 0.021 1.86E-06 cg00460471 2 61437605 0.102 0.024 3.10E-05 0.041 17.7 13349 1511 814 3 -0.032 0.014 0.028 0.012 0.19 -0.319 0.151 0.035 0.21 -0.118 0.028 3.55E-05 cg02110836 17 71953083 0.057 0.005 2.91E-24 0.220 117.4 17019 2437 1629 30 -0.015 0.007 0.028 0.012 0.19 -0.265 0.119 0.026 0.19 -0.227 0.056 5.57E-05 cg06077821 5 180048893 0.047 0.005 2.68E-19 0.176 89.1 16657 2554 1497 34 -0.008 0.004 0.031 0.012 0.20 -0.178 0.081 0.028 0.19 -0.146 0.034 2.43E-05 cg00875983 11 3240213 0.071 0.008 1.34E-18 0.170 85.3 17171 2667 1744 20 -0.017 0.008 0.033 0.011 0.21 -0.237 0.109 0.031 0.20 -0.176 0.045 1.16E-04 cg14361947 9 140780551 0.056 0.007 5.95E-15 0.136 65.7 12059 978 679 24 -0.016 0.008 0.041 0.010 0.22 -0.295 0.144 0.041 0.21 -0.161 0.052 2.02E-03 cg00386725 1 18617791 0.095 0.012 6.12E-15 0.136 65.6 16267 2471 1494 12 -0.024 0.012 0.041 0.010 0.22 -0.248 0.120 0.038 0.21 -0.185 0.044 3.53E-05 cg10964639 16 24043296 0.059 0.008 4.62E-13 0.118 55.9 14736 2182 1222 24 -0.011 0.005 0.041 0.010 0.22 -0.182 0.088 0.039 0.21 -0.122 0.031 9.72E-05 cg11855710 19 29768180 0.148 0.021 1.85E-11 0.103 47.7 17654 3015 1361 10 -0.029 0.014 0.043 0.010 0.22 -0.199 0.096 0.039 0.21 -0.150 0.031 2.42E-06 cg12954267 6 33093109 0.092 0.026 4.69E-04 0.029 12.4 18517 6412 2328 3 -0.028 0.014 0.045 0.010 0.22 -0.321 0.174 0.07 0.27 -0.089 0.025 5.30E-04 cg00389386 1 6188136 0.062 0.007 8.31E-17 0.154 75.5 16477 1981 1136 22 -0.014 0.007 0.045 0.010 0.22 -0.234 0.115 0.043 0.21 -0.174 0.044 1.06E-04 cg21347874 17 77122733 0.083 0.012 6.99E-11 0.097 44.8 16022 2400 1721 13 -0.016 0.008 0.047 0.010 0.22 -0.188 0.093 0.044 0.21 -0.127 0.029 1.26E-05 cg27625886 20 62060443 0.135 0.015 8.29E-17 0.154 75.6 18081 2937 1609 17 -0.018 0.009 0.05 0.009 0.23 -0.128 0.064 0.048 0.23 -0.108 0.026 3.90E-05 cg11586089 14 66644923 0.090 0.010 2.11E-17 0.159 78.8 13997 1665 708 35 -0.013 0.007 0.06 0.009 0.25 -0.147 0.077 0.06 0.25 -0.110 0.030 2.96E-04 cg16340422 17 17110120 0.137 0.020 6.55E-11 0.098 45.0 13630 1744 1022 10 -0.025 0.013 0.06 0.009 0.26 -0.187 0.098 0.06 0.25 -0.124 0.029 2.89E-05 cg18861231 3 152057943 0.035 0.007 3.48E-07 0.061 26.8 13018 1777 676 18 -0.007 0.004 0.06 0.009 0.26 -0.195 0.106 0.07 0.27 -0.079 0.025 1.53E-03 cg03568769 11 1800837 0.074 0.005 1.28E-36 0.319 195.0 17582 2953 1759 66 -0.008 0.004 0.07 0.008 0.27 -0.104 0.057 0.07 0.27 -0.117 0.032 3.12E-04 cg12760575 9 139158654 0.110 0.013 5.14E-16 0.146 71.3 17109 1972 1300 15 -0.020 0.011 0.07 0.008 0.28 -0.185 0.102 0.07 0.27 -0.151 0.039 1.47E-04 cg26037026 7 1641922 0.194 0.055 0.000489 0.029 12.4 20490 2519 1493 1 -0.078 0.045 0.08 0.008 0.30 -0.406 0.241 0.09 0.30 -0.143 0.039 2.39E-04 cg14839558 1 1874488 0.141 0.017 3.66E-15 0.138 66.8 17112 1789 1113 19 -0.018 0.010 0.08 0.008 0.30 -0.133 0.075 0.08 0.29 -0.104 0.027 1.38E-04 cg24600306 20 61990671 0.070 0.007 5.23E-19 0.174 87.5 18172 2970 1608 24 -0.013 0.008 0.08 0.007 0.30 -0.193 0.110 0.08 0.29 -0.174 0.046 1.91E-04 cg16853336 3 149396490 0.040 0.006 6.82E-11 0.097 44.9 13981 2115 1079 19 -0.011 0.006 0.09 0.007 0.30 -0.270 0.157 0.09 0.30 -0.174 0.050 5.04E-04 cg08149223 10 50507130 0.043 0.007 1.56E-09 0.084 38.1 15782 2177 1029 16 -0.012 0.007 0.09 0.007 0.30 -0.273 0.159 0.09 0.30 -0.171 0.047 2.86E-04 cg00781180 16 73984002 0.218 0.037 4.82E-09 0.079 35.8 15775 1655 1261 6 -0.038 0.023 0.09 0.007 0.30 -0.177 0.103 0.09 0.30 -0.111 0.028 1.11E-04 cg16444062 16 2770298 0.236 0.053 1.03E-05 0.046 19.9 15567 1480 949 3 -0.047 0.029 0.10 0.007 0.32 -0.192 0.117 0.10 0.31 -0.105 0.026 4.92E-05 cg24738497 19 36005395 0.130 0.024 5.27E-08 0.069 30.7 14689 2074 1243 4 -0.028 0.017 0.10 0.007 0.32 -0.206 0.124 0.10 0.31 -0.149 0.034 1.20E-05 cg17756860 4 110510374 0.122 0.034 0.00034286 0.030 13.0 13975 1499 674 4 -0.032 0.020 0.11 0.007 0.32 -0.264 0.163 0.11 0.31 -0.128 0.028 6.14E-06 cg08737919 12 108840669 0.207 0.034 2.47E-09 0.082 37.2 14629 2021 914 6 -0.034 0.021 0.11 0.006 0.32 -0.163 0.101 0.11 0.31 -0.095 0.030 1.37E-03 Diabetes Page 42 of 53

cg12193237 19 3907913 0.048 0.005 8.51E-18 0.163 80.9 16921 2133 1364 36 -0.007 0.004 0.11 0.006 0.32 -0.147 0.090 0.10 0.31 -0.143 0.036 8.65E-05 cg07435932 13 19648633 0.381 0.127 0.00280574 0.021 9.0 16939 2652 1044 1 -0.102 0.065 0.12 0.006 0.34 -0.283 0.192 0.14 0.36 -0.091 0.025 2.94E-04 cg13449257 9 137355128 0.055 0.009 1.52E-08 0.074 33.3 18196 2544 1691 14 -0.011 0.007 0.13 0.006 0.36 -0.191 0.125 0.13 0.36 -0.145 0.034 2.35E-05 cg02287371 5 1246274 0.157 0.037 2.90E-05 0.041 17.9 17997 2289 1564 3 -0.033 0.022 0.13 0.006 0.36 -0.208 0.139 0.14 0.36 -0.097 0.028 4.76E-04 cg06751809 3 105047227 0.106 0.019 2.81E-08 0.072 32.0 14927 2475 943 13 -0.012 0.008 0.13 0.006 0.36 -0.115 0.075 0.13 0.36 -0.086 0.020 3.58E-05 cg01948226 6 32829208 0.092 0.015 1.24E-09 0.085 38.6 19534 8426 2926 10 -0.014 0.010 0.14 0.005 0.37 -0.159 0.106 0.13 0.36 -0.127 0.030 2.87E-05 cg00046877 22 19702664 0.065 0.009 2.83E-12 0.111 51.8 15502 2141 1221 12 -0.016 0.011 0.15 0.005 0.38 -0.248 0.168 0.14 0.36 -0.209 0.057 2.84E-04 cg01864178 20 44662890 0.087 0.014 1.25E-09 0.085 38.6 14114 1927 859 15 -0.012 0.009 0.16 0.005 0.38 -0.148 0.102 0.15 0.37 -0.129 0.029 1.16E-05 cg18682781 5 176277687 0.190 0.042 8.45E-06 0.047 20.3 14377 1242 712 3 -0.035 0.025 0.16 0.005 0.38 -0.181 0.127 0.16 0.37 -0.117 0.028 3.03E-05 cg13463516 9 138632121 0.035 0.005 5.61E-11 0.098 45.3 18674 2377 1670 24 -0.006 0.004 0.17 0.005 0.38 -0.163 0.115 0.16 0.37 -0.147 0.036 5.41E-05 cg09319487 20 56771178 0.148 0.012 4.59E-28 0.252 140.1 15943 2235 1345 28 -0.012 0.009 0.17 0.005 0.38 -0.083 0.060 0.16 0.37 -0.101 0.030 9.53E-04 cg08204714 16 87726461 0.201 0.064 0.00182003 0.023 9.8 21918 2969 2053 1 -0.093 0.067 0.17 0.005 0.38 -0.475 0.355 0.18 0.37 -0.180 0.049 2.58E-04 cg24064348 1 54181174 0.056 0.010 3.99E-08 0.070 31.3 13794 1697 881 8 -0.017 0.012 0.17 0.005 0.38 -0.310 0.224 0.17 0.37 -0.215 0.056 1.65E-04 cg18216439 18 35132882 0.209 0.065 0.0013874 0.024 10.4 13311 1742 916 1 -0.055 0.041 0.18 0.004 0.38 -0.280 0.212 0.19 0.37 -0.128 0.030 2.65E-05 cg04535454 8 144035192 0.049 0.009 2.17E-08 0.073 32.6 17157 2585 1321 16 -0.009 0.006 0.18 0.004 0.38 -0.175 0.129 0.18 0.37 -0.134 0.034 1.02E-04 cg08582801 7 99588335 0.041 0.009 6.73E-06 0.048 20.8 11898 913 363 7 -0.014 0.011 0.19 0.004 0.38 -0.332 0.247 0.18 0.37 -0.232 0.056 3.89E-05 cg27652925 6 31740951 0.049 0.010 1.90E-06 0.053 23.3 15655 5253 2035 8 -0.015 0.011 0.19 0.004 0.38 -0.307 0.230 0.18 0.37 -0.198 0.052 1.73E-04 cg09073467 1 6155515 0.073 0.006 1.34E-26 0.240 131.2 16527 2041 1179 36 -0.009 0.007 0.19 0.004 0.38 -0.126 0.095 0.18 0.37 -0.185 0.046 8.07E-05 cg08239663 16 52688569 0.210 0.044 2.29E-06 0.052 23.0 15429 2052 995 5 -0.028 0.022 0.19 0.004 0.38 -0.139 0.105 0.19 0.37 -0.092 0.023 1.05E-04 cg26225794 7 27717723 0.148 0.043 0.00074188 0.027 11.6 14250 1939 985 2 0.040 0.030 0.19 0.004 0.38 0.241 0.207 0.24 0.41 -0.121 0.033 3.24E-04 cg04293688 2 75466188 0.058 0.007 2.15E-14 0.131 62.8 15363 2334 1330 27 -0.006 0.005 0.19 0.004 0.38 -0.109 0.082 0.19 0.37 -0.115 0.029 1.11E-04 cg05202700 8 38258250 0.125 0.022 2.64E-08 0.072 32.2 12658 1326 661 8 -0.016 0.013 0.19 0.004 0.38 -0.125 0.095 0.19 0.37 -0.103 0.026 1.15E-04 cg15867652 17 79011277 0.119 0.012 9.22E-22 0.198 102.9 18385 2927 1635 23 -0.009 0.007 0.20 0.004 0.38 -0.075 0.057 0.19 0.37 -0.104 0.026 6.38E-05 cg00903007 17 34068171 0.020 0.002 2.67E-15 0.140 67.5 13632 1860 892 52 -0.004 0.003 0.20 0.004 0.38 -0.193 0.147 0.19 0.37 -0.215 0.054 9.47E-05 cg13377560 22 43860820 0.071 0.010 3.25E-12 0.110 51.5 16164 2593 1472 18 -0.010 0.008 0.21 0.004 0.39 -0.129 0.101 0.20 0.38 -0.142 0.036 8.46E-05 cg01095004 16 88454883 0.130 0.018 2.07E-12 0.112 52.5 21315 3131 2093 12 -0.014 0.011 0.21 0.004 0.39 -0.107 0.084 0.20 0.38 -0.105 0.028 2.43E-04 cg24848724 8 142578640 0.095 0.009 2.93E-21 0.194 100.1 16785 2282 1389 32 -0.007 0.005 0.21 0.004 0.39 -0.073 0.058 0.20 0.38 -0.088 0.025 3.51E-04 cg01898225 15 99975141 0.062 0.006 2.03E-19 0.178 89.8 15697 2792 1539 29 -0.007 0.006 0.22 0.004 0.39 -0.114 0.090 0.21 0.38 -0.179 0.038 2.86E-06 cg20018106 4 21768009 0.058 0.011 6.20E-08 0.068 30.4 16045 2280 975 13 -0.007 0.006 0.22 0.004 0.39 -0.123 0.097 0.21 0.38 -0.121 0.026 5.69E-06 cg18450810 17 51111066 0.214 0.045 3.11E-06 0.051 22.4 14484 2171 635 4 -0.034 0.027 0.22 0.004 0.39 -0.157 0.127 0.21 0.38 -0.104 0.028 2.82E-04 cg25285919 3 188312215 0.080 0.011 5.43E-13 0.118 55.5 14200 1596 1007 14 -0.009 0.007 0.22 0.004 0.39 -0.114 0.092 0.21 0.38 -0.114 0.031 2.51E-04 cg18184975 20 3644192 0.076 0.014 4.39E-08 0.070 31.1 14390 2558 1274 10 -0.011 0.009 0.23 0.004 0.39 -0.153 0.124 0.22 0.38 -0.138 0.032 2.40E-05 cg04131502 1 2845583 0.107 0.017 4.55E-10 0.089 40.8 17240 2079 1306 9 -0.014 0.012 0.25 0.003 0.42 -0.134 0.113 0.24 0.41 -0.136 0.033 6.26E-05 cg26849382 5 140901022 0.223 0.081 0.00651404 0.018 7.5 13286 1785 904 1 0.049 0.042 0.25 0.003 0.42 0.213 0.191 0.27 0.43 0.060 0.025 1.83E-02 cg16819051 2 229173235 0.189 0.063 0.00303083 0.021 8.9 14534 2218 867 1 -0.045 0.040 0.26 0.003 0.42 -0.235 0.208 0.26 0.43 -0.112 0.030 1.86E-04 cg25721832 3 81913557 0.089 0.015 2.94E-09 0.081 36.8 12811 1396 484 17 -0.012 0.010 0.27 0.003 0.43 -0.132 0.118 0.26 0.43 -0.101 0.033 2.13E-03 cg22509138 20 44809026 0.357 0.119 0.00283299 0.021 9.0 14092 1752 957 1 0.070 0.064 0.27 0.003 0.43 0.194 0.201 0.33 0.50 -0.107 0.026 3.62E-05 cg05808230 20 61726456 0.095 0.011 1.17E-15 0.143 69.4 18272 2767 1736 19 -0.009 0.008 0.27 0.003 0.43 -0.092 0.083 0.27 0.43 -0.131 0.032 4.96E-05 cg09063835 20 58181392 0.152 0.020 2.26E-13 0.121 57.5 15305 1716 1164 13 -0.013 0.012 0.28 0.003 0.43 -0.088 0.079 0.27 0.43 -0.117 0.027 1.78E-05 cg06066032 13 48369175 0.370 0.116 0.00154941 0.024 10.2 14559 2328 815 1 0.061 0.059 0.30 0.003 0.47 0.185 0.203 0.36 0.53 -0.098 0.025 7.18E-05 cg05389237 9 125314173 0.135 0.019 2.83E-12 0.111 51.9 13529 1515 817 17 -0.010 0.010 0.30 0.003 0.47 -0.078 0.074 0.29 0.46 -0.096 0.024 1.04E-04 cg17538538 6 106825021 0.131 0.026 8.85E-07 0.056 24.9 14420 2498 1195 4 0.020 0.021 0.33 0.002 0.50 0.151 0.152 0.32 0.49 0.133 0.037 3.46E-04 cg23519631 12 72690355 0.111 0.022 7.26E-07 0.057 25.3 12956 1472 563 8 -0.016 0.016 0.33 0.002 0.50 -0.144 0.144 0.32 0.49 -0.164 0.034 1.82E-06 cg08792976 16 760528 0.103 0.011 1.48E-19 0.179 90.6 22023 2572 1696 24 -0.008 0.008 0.34 0.002 0.50 -0.076 0.078 0.33 0.50 -0.137 0.033 3.27E-05 cg20623853 19 1734365 0.040 0.008 2.88E-07 0.061 27.2 17950 1888 1390 13 -0.006 0.006 0.34 0.002 0.50 -0.132 0.135 0.33 0.50 -0.148 0.036 5.67E-05 cg18042910 13 19662609 0.053 0.007 1.31E-13 0.124 58.7 17061 2665 1048 44 -0.004 0.004 0.37 0.002 0.53 -0.068 0.074 0.36 0.53 -0.107 0.026 4.78E-05 cg14204490 8 33330967 0.035 0.006 3.81E-10 0.090 41.2 13877 1541 509 27 0.006 0.007 0.37 0.002 0.53 0.176 0.204 0.39 0.55 -0.222 0.056 8.23E-05 cg01124546 11 117515332 0.065 0.009 5.51E-12 0.108 50.4 15525 2294 1396 13 -0.009 0.010 0.38 0.002 0.54 -0.135 0.150 0.37 0.53 -0.198 0.048 4.04E-05 cg00188627 3 159557779 0.133 0.022 1.50E-09 0.084 38.2 14587 1437 649 8 0.012 0.014 0.40 0.002 0.56 0.089 0.104 0.39 0.55 0.105 0.030 6.05E-04 cg03510150 9 130690744 0.058 0.008 5.78E-13 0.117 55.4 13396 1284 730 17 -0.007 0.009 0.42 0.002 0.59 -0.119 0.145 0.41 0.58 -0.200 0.050 8.62E-05 cg09701145 11 2019436 0.085 0.011 7.50E-13 0.116 54.8 17726 2869 1731 12 0.008 0.010 0.45 0.001 0.61 0.088 0.118 0.46 0.63 -0.174 0.039 1.03E-05 cg05775586 9 28889354 0.034 0.005 1.50E-12 0.113 53.3 15740 2741 1083 55 -0.002 0.003 0.47 0.001 0.64 -0.054 0.074 0.47 0.64 -0.098 0.026 1.85E-04 Page 43 of 53 Diabetes

cg00373987 22 37868968 0.228 0.056 6.16E-05 0.038 16.4 15697 2077 1308 2 -0.027 0.039 0.49 0.001 0.65 -0.121 0.169 0.48 0.64 -0.144 0.033 1.38E-05 cg12161743 11 76750531 0.055 0.007 2.42E-14 0.131 62.5 13871 1984 1045 23 -0.004 0.005 0.49 0.001 0.65 -0.065 0.093 0.48 0.64 -0.147 0.033 1.38E-05 cg13827123 13 93971495 0.313 0.105 0.00304799 0.021 8.9 14374 2257 949 1 0.036 0.054 0.50 0.001 0.66 0.116 0.185 0.53 0.68 -0.100 0.024 5.07E-05 cg08491066 1 156030669 0.101 0.019 1.11E-07 0.066 29.2 11201 951 464 7 -0.010 0.016 0.51 0.001 0.66 -0.104 0.153 0.50 0.66 -0.157 0.038 5.39E-05 cg15575356 11 47616757 0.137 0.032 1.70E-05 0.044 18.9 11864 1306 471 4 -0.020 0.032 0.52 0.001 0.68 -0.152 0.231 0.51 0.67 -0.201 0.047 2.78E-05 cg00078259 6 2656232 0.148 0.020 2.76E-13 0.121 57.0 16279 2501 1372 11 -0.008 0.012 0.53 0.001 0.68 -0.051 0.080 0.52 0.68 -0.105 0.027 1.50E-04 cg16140890 1 1082406 0.144 0.032 1.06E-05 0.046 19.9 16090 1384 843 5 0.010 0.017 0.56 0.001 0.72 0.068 0.122 0.58 0.72 -0.093 0.025 2.64E-04 cg21927431 9 134678366 0.173 0.028 2.39E-09 0.082 37.2 14813 1885 1114 6 -0.012 0.021 0.58 0.001 0.73 -0.076 0.136 0.58 0.72 -0.127 0.034 2.49E-04 cg20659588 1 237963267 0.262 0.054 1.77E-06 0.053 23.5 15158 2388 1277 3 -0.018 0.034 0.59 0.001 0.73 -0.073 0.132 0.58 0.72 -0.125 0.029 2.48E-05 cg13022891 9 5817706 0.054 0.006 2.68E-17 0.158 78.2 16090 1838 1205 38 -0.003 0.005 0.59 0.001 0.73 -0.054 0.098 0.58 0.72 -0.148 0.038 1.43E-04 cg25903144 7 131609806 0.086 0.017 3.43E-07 0.061 26.9 15819 2225 1281 10 -0.004 0.008 0.59 0.001 0.73 -0.052 0.095 0.59 0.72 -0.083 0.023 4.29E-04 cg27410679 17 43866278 0.068 0.010 3.00E-11 0.101 46.7 12078 2955 549 28 -0.003 0.006 0.61 0.001 0.73 -0.041 0.080 0.61 0.73 -0.108 0.026 5.00E-05 cg10883064 17 60757346 0.085 0.014 1.18E-09 0.085 38.8 12442 1144 568 20 -0.004 0.009 0.62 0.001 0.73 -0.050 0.098 0.61 0.73 -0.110 0.030 2.51E-04 cg11837181 1 3001002 0.101 0.026 0.00015292 0.034 14.6 17515 2132 1321 3 -0.009 0.018 0.62 0.001 0.73 -0.092 0.180 0.61 0.73 -0.127 0.033 1.51E-04 cg17985533 11 2019116 0.038 0.003 8.78E-38 0.328 202.9 17722 2867 1729 108 -0.001 0.003 0.62 0.001 0.73 -0.038 0.076 0.62 0.73 -0.175 0.043 5.97E-05 cg21778193 1 1098070 0.189 0.067 0.00483001 0.019 8.0 16295 1400 851 1 -0.018 0.040 0.66 0.000 0.77 -0.087 0.192 0.65 0.76 -0.119 0.029 4.25E-05 cg05851077 9 29802712 0.146 0.033 1.68E-05 0.044 19.0 16112 2731 1143 5 -0.006 0.013 0.67 0.000 0.77 -0.039 0.091 0.67 0.77 -0.084 0.019 1.04E-05 cg06981471 7 142982076 0.125 0.026 1.98E-06 0.053 23.3 10365 1303 535 5 0.008 0.020 0.67 0.000 0.77 0.064 0.155 0.68 0.77 -0.127 0.036 4.27E-04 cg12011250 1 30198037 0.065 0.008 3.19E-16 0.148 72.4 13795 2188 1217 29 -0.002 0.005 0.69 0.000 0.78 -0.033 0.080 0.68 0.77 -0.120 0.030 6.05E-05 cg07728373 2 229266790 0.077 0.016 1.82E-06 0.053 23.4 14340 2115 872 11 -0.003 0.007 0.69 0.000 0.78 -0.039 0.096 0.68 0.77 -0.108 0.021 5.53E-07 cg16608656 11 1082797 0.103 0.033 0.00173057 0.023 9.9 16848 2415 1343 2 -0.008 0.020 0.70 0.000 0.78 -0.072 0.184 0.69 0.78 -0.112 0.029 1.47E-04 cg13625403 11 120008510 0.057 0.012 3.76E-06 0.050 22.0 15102 2093 1233 9 0.003 0.007 0.71 0.000 0.79 0.047 0.129 0.71 0.79 -0.117 0.028 4.48E-05 cg00918900 1 1057335 0.087 0.012 1.22E-12 0.114 53.7 15892 1367 827 21 -0.003 0.007 0.71 0.000 0.79 -0.031 0.083 0.71 0.79 -0.124 0.029 1.83E-05 cg00403246 10 134794753 0.096 0.023 3.36E-05 0.041 17.6 18305 1964 1252 2 0.009 0.025 0.73 0.000 0.79 0.090 0.263 0.73 0.80 -0.188 0.051 2.46E-04 cg11138231 7 99475824 0.111 0.017 4.91E-11 0.099 45.6 12236 880 344 20 -0.003 0.008 0.76 0.000 0.83 -0.022 0.073 0.76 0.82 -0.095 0.023 4.48E-05 cg08474716 16 27079640 0.206 0.029 4.42E-12 0.109 50.9 16195 2472 1481 8 -0.004 0.016 0.78 0.000 0.84 -0.022 0.078 0.78 0.83 -0.092 0.025 2.08E-04 cg07203271 1 53926112 0.145 0.024 2.03E-09 0.083 37.6 14665 2120 967 9 -0.004 0.015 0.78 0.000 0.84 -0.028 0.101 0.78 0.83 -0.104 0.029 4.27E-04 cg25380707 15 86660946 0.252 0.080 0.00180737 0.023 9.9 17604 2562 1510 1 -0.010 0.040 0.81 0.000 0.85 -0.043 0.176 0.81 0.85 -0.086 0.024 2.83E-04 cg17905524 15 28305412 0.106 0.014 1.08E-12 0.115 54.0 11317 1447 661 17 -0.002 0.010 0.82 0.000 0.86 -0.021 0.094 0.82 0.86 -0.127 0.030 2.77E-05 cg18616558 7 158798592 0.080 0.014 1.49E-08 0.074 33.4 14247 1918 902 20 -0.001 0.006 0.92 0.000 0.95 -0.008 0.073 0.92 0.95 -0.071 0.020 4.52E-04 cg04140215 2 60540175 0.210 0.075 0.00563409 0.018 7.7 15077 1764 1102 1 0.004 0.050 0.94 0.000 0.96 0.021 0.265 0.94 0.97 -0.119 0.031 1.48E-04 cg26238771 19 592158 0.117 0.011 3.43E-23 0.211 111.2 17193 2642 2036 20 0.000 0.009 0.98 0.000 0.99 -0.002 0.080 0.98 0.99 -0.139 0.036 1.21E-04 cg05347757 15 51121020 0.103 0.034 0.00251697 0.022 9.2 13001 1995 637 3 0.000 0.018 0.98 0.000 0.99 -0.004 0.173 0.98 0.99 -0.083 0.025 1.10E-03 cg02688867 12 133019854 0.180 0.055 0.0012479 0.025 10.6 16623 2355 1394 2 -0.001 0.033 0.98 0.000 0.99 -0.004 0.179 0.98 0.99 -0.100 0.028 4.92E-04 cg15952840 8 11451312 0.159 0.035 5.66E-06 0.048 21.1 21931 3018 2084 3 0.000 0.022 0.99 0.000 0.99 0.003 0.145 0.99 0.99 -0.128 0.030 2.32E-05 cg00045690 6 32825897 19525 8434 2923 0 -0.086 0.022 1.12E-04 cg01216882 20 31804277 14128 1327 702 0 -0.137 0.031 9.94E-06 cg01530082 6 32784327 19703 8668 2934 0 -0.107 0.026 4.05E-05 cg01615861 16 85513978 21138 2881 2231 0 -0.283 0.080 4.45E-04 cg02831474 10 1783879 18860 3144 1828 0 -0.113 0.024 3.12E-06 cg03275961 9 90386329 15207 2428 1223 0 -0.161 0.040 5.57E-05 cg03710412 8 142938779 17500 2608 1446 0 -0.091 0.026 4.85E-04 cg04847540 1 182754070 13816 1976 812 0 -0.112 0.028 7.64E-05 cg04907218 12 132826672 16467 2098 1300 0 -0.118 0.030 9.09E-05 cg05444304 2 31442675 15973 2123 1458 0 0.133 0.039 6.39E-04 cg05790369 17 33202007 14788 2007 1086 0 -0.115 0.030 1.90E-04 cg06343669 12 132690782 16014 2044 1320 0 -0.117 0.028 2.72E-05 cg06495184 19 406256 13720 2023 1579 0 -0.142 0.035 5.34E-05 cg06830529 9 124960107 14549 1938 1035 0 -0.139 0.037 1.73E-04 cg07080246 13 63306618 13919 2164 705 0 -0.076 0.020 1.22E-04 cg07686906 1 3353825 18958 2698 1695 0 -0.150 0.037 5.70E-05 cg07865738 2 3649447 15986 2592 1436 0 -0.095 0.024 6.57E-05 Diabetes Page 44 of 53

cg08058801 3 147974120 14142 1723 693 0 -0.114 0.026 1.13E-05 cg08357744 17 37559321 13259 1346 609 0 -0.084 0.023 2.31E-04 cg08372350 16 88943191 22238 2702 1867 0 -0.107 0.028 1.59E-04 cg08389995 3 131645295 14602 2835 1125 0 -0.083 0.023 3.49E-04 cg09181711 19 51327196 16264 2335 1602 0 -0.136 0.029 2.61E-06 cg09851596 8 143545214 18124 2792 1362 0 -0.177 0.044 7.97E-05 cg10877514 17 292539 12641 1872 1292 0 -0.105 0.028 1.79E-04 cg11086223 18 11487579 16274 2395 1276 0 -0.135 0.034 6.79E-05 cg11177743 20 21545361 13270 1160 444 0 -0.138 0.033 3.15E-05 cg11294782 3 10487409 15223 2264 1314 0 -0.152 0.040 1.69E-04 cg11416433 15 25429261 16557 1988 1141 0 -0.201 0.044 6.72E-06 cg11821795 22 47611604 19073 2440 1408 0 -0.109 0.027 4.59E-05 cg12697796 10 49954016 15686 2331 1086 0 -0.179 0.041 2.03E-05 cg13116906 12 119552989 14166 2319 1181 0 -0.105 0.027 1.44E-04 cg13254480 16 86513248 23316 3570 2722 0 -0.073 0.020 2.91E-04 cg13257690 14 101908752 15386 2170 1265 0 -0.178 0.046 1.17E-04 cg13480617 5 35230725 14639 2107 978 0 -0.121 0.038 1.49E-03 cg14494450 1 4190881 15856 2958 1567 0 -0.116 0.027 2.07E-05 cg14757962 5 2176287 19312 3591 2168 0 -0.108 0.030 3.06E-04 cg14759277 17 48674158 13790 1755 1128 0 -0.164 0.041 7.62E-05 cg14886500 6 23447204 17748 2646 1490 0 -0.080 0.023 7.30E-04 cg16513326 8 10877312 21573 3120 2033 0 -0.111 0.032 5.03E-04 cg16857113 3 1133865 18339 2963 1779 0 -0.114 0.029 1.12E-04 cg17224410 9 137852661 19804 2883 1965 0 -0.123 0.033 2.00E-04 cg17226458 6 33780022 16101 2493 1395 0 -0.145 0.039 2.71E-04 cg17587481 5 152603672 14222 2224 764 0 -0.103 0.024 1.78E-05 cg18108190 19 2727822 18307 2844 1847 0 -0.142 0.033 2.05E-05 cg18233010 10 114911413 14248 1705 1135 0 0.170 0.045 2.13E-04 cg18617770 12 133035939 16438 2356 1404 0 -0.160 0.037 2.04E-05 cg18650968 4 176340837 14564 1524 726 0 -0.100 0.028 4.36E-04 cg19450046 21 45712938 18589 2432 1382 0 -0.138 0.036 1.66E-04 cg20112984 10 1447183 18134 2767 1723 0 -0.103 0.027 2.03E-04 cg21307723 1 245215135 15619 2326 1412 0 0.143 0.038 2.22E-04 cg23016281 22 27616620 17311 3036 1986 0 -0.092 0.025 2.60E-04 cg23359229 13 29976597 15238 2418 1020 0 -0.110 0.026 2.82E-05 cg23536293 12 99524459 13533 1605 925 0 -0.071 0.021 6.41E-04 cg23561197 5 125852064 15210 2226 1108 0 -0.106 0.030 4.06E-04 cg25314607 5 2148308 19252 3620 2202 0 -0.122 0.030 6.85E-05 cg26178126 1 37740931 14242 2023 983 0 -0.125 0.031 6.10E-05 cg27332583 18 35150602 13359 1749 916 0 -0.175 0.041 2.12E-05 Page 45 of 53 Diabetes

Supplementary Table 5: Results from Mendelian randomization analyses using GRS representing insulin sensitivity (8 SNPs) associations with methylation levels at CpG sites identified in Model 1 Maternal GRS8 (raw) association with placenta methylation CPG chr pos estimate SE P_value Rsquare cg12161743 chr11 76750531 0.048 0.022 0.03 0.0112 cg13625403 chr11 120008510 -0.041 0.026 0.12 0.0059 cg04293688 chr2 75466188 -0.039 0.026 0.13 0.0056 cg01216882 chr20 31804277 0.035 0.024 0.15 0.0051 cg13254480 chr16 86513248 0.053 0.038 0.16 0.0047 cg26225794 chr7 27717723 -0.030 0.023 0.19 0.0042 cg13022891 chr9 5817706 -0.024 0.019 0.22 0.0036 cg06830529 chr9 124960107 0.025 0.021 0.23 0.0034 cg26178126 chr1 37740931 0.029 0.024 0.24 0.0034 cg09063835 chr20 58181392 0.032 0.027 0.25 0.0032 cg23519631 chr12 72690355 0.026 0.022 0.25 0.0032 cg16853336 chr3 149396490 -0.016 0.015 0.29 0.0026 cg07080246 chr13 63306618 -0.038 0.038 0.32 0.0023 cg12760575 chr9 139158654 -0.019 0.019 0.33 0.0023 cg07435932 chr13 19648633 0.030 0.031 0.33 0.0023 cg06343669 chr12 132690782 -0.025 0.027 0.36 0.0020 cg24848724 chr8 142578640 0.028 0.031 0.36 0.0020 cg18616558 chr7 158798592 0.033 0.037 0.37 0.0019 cg18617770 chr12 133035939 -0.018 0.020 0.38 0.0019 cg07728373 chr2 229266790 -0.031 0.035 0.38 0.0018 cg03275961 chr9 90386329 0.016 0.019 0.40 0.0017 cg16340422 chr17 17110120 0.021 0.026 0.41 0.0016 cg00903007 chr17 34068171 -0.011 0.014 0.42 0.0016 cg06077821 chr5 180048893 -0.017 0.022 0.43 0.0015 cg12011250 chr1 30198037 -0.020 0.025 0.43 0.0015 cg09640571 chr16 88388040 0.012 0.015 0.43 0.0015 cg11821795 chr22 47611604 0.021 0.028 0.46 0.0013 cg09181711 chr19 51327196 -0.018 0.026 0.48 0.0012 cg24738497 chr19 36005395 -0.016 0.022 0.48 0.0012 cg17587481 chr5 152603672 -0.021 0.031 0.50 0.0011 cg00460471 chr2 61437605 -0.018 0.027 0.50 0.0011 cg18216439 chr18 35132882 0.017 0.025 0.50 0.0011 cg14440140 chr9 140177567 0.017 0.025 0.51 0.0010 cg12697796 chr10 49954016 0.012 0.018 0.51 0.0010 cg00386725 chr1 18617791 0.011 0.017 0.52 0.0010 cg05790369 chr17 33202007 0.016 0.025 0.52 0.0010 cg27410679 chr17 43866278 0.018 0.029 0.53 0.0010 cg08792976 chr16 760528 -0.014 0.023 0.53 0.0009 cg10877514 chr17 292539 0.017 0.027 0.53 0.0009 cg18450810 chr17 51111066 0.016 0.026 0.53 0.0009 cg06066032 chr13 48369175 0.019 0.031 0.54 0.0009 cg18861231 chr3 152057943 -0.018 0.030 0.54 0.0009 cg10964639 chr16 24043296 0.014 0.024 0.55 0.0008 cg02384870 chr2 216161208 0.018 0.031 0.56 0.0008 cg17226458 chr6 33780022 0.011 0.019 0.56 0.0008 cg00373987 chr22 37868968 -0.013 0.023 0.57 0.0008 cg01864178 chr20 44662890 -0.014 0.026 0.58 0.0008 cg24073146 chr11 2543204 -0.010 0.018 0.58 0.0007 cg24475484 chr8 1625395 0.016 0.030 0.58 0.0007 cg27652925 chr6 31740951 0.008 0.014 0.59 0.0007 cg23536293 chr12 99524459 0.020 0.037 0.60 0.0007 cg05808230 chr20 61726456 0.012 0.023 0.60 0.0007 cg21396983 chr16 1275423 0.013 0.025 0.61 0.0006 cg25380707 chr15 86660946 0.016 0.032 0.62 0.0006 cg09701145 chr11 2019436 -0.010 0.019 0.62 0.0006 cg08058801 chr3 147974120 0.015 0.029 0.62 0.0006 cg15952840 chr8 11451312 0.012 0.025 0.62 0.0006 cg08582801 chr7 99588335 0.007 0.013 0.62 0.0006 cg00918900 chr1 1057335 0.012 0.026 0.63 0.0005 cg12658750 chr18 75499382 0.009 0.019 0.63 0.0005 cg15575356 chr11 47616757 -0.008 0.016 0.64 0.0005 cg20659588 chr1 237963267 -0.012 0.025 0.64 0.0005 cg02287371 chr5 1246274 -0.013 0.027 0.64 0.0005 cg16140890 chr1 1082406 0.014 0.030 0.65 0.0005 cg26849382 chr5 140901022 0.013 0.030 0.65 0.0005 cg03710412 chr8 142938779 -0.013 0.029 0.66 0.0005 cg20623853 chr19 1734365 0.009 0.021 0.66 0.0005 cg01124546 chr11 117515332 -0.007 0.016 0.66 0.0005 cg26238771 chr19 592158 0.009 0.021 0.66 0.0005 cg08389995 chr3 131645295 -0.014 0.033 0.66 0.0005 cg13257690 chr14 101908752 0.007 0.016 0.67 0.0004 cg17756860 chr4 110510374 -0.011 0.027 0.67 0.0004 Diabetes Page 46 of 53

cg01530082 chr6 32784327 -0.012 0.029 0.67 0.0004 cg00045690 chr6 32825897 0.014 0.034 0.67 0.0004 cg14839558 chr1 1874488 0.012 0.028 0.67 0.0004 cg25314607 chr5 2148308 0.011 0.025 0.67 0.0004 cg00781180 chr16 73984002 -0.011 0.026 0.67 0.0004 cg08474716 chr16 27079640 0.013 0.031 0.67 0.0004 cg13480617 chr5 35230725 0.008 0.020 0.67 0.0004 cg11416433 chr15 25429261 0.007 0.017 0.67 0.0004 cg17538538 chr6 106825021 0.009 0.020 0.67 0.0004 cg05775586 chr9 28889354 -0.012 0.029 0.68 0.0004 cg08737919 chr12 108840669 0.010 0.026 0.69 0.0004 cg01618245 chr20 61990279 0.012 0.030 0.69 0.0004 cg21307723 chr1 245215135 0.008 0.019 0.69 0.0004 cg20018106 chr4 21768009 -0.011 0.028 0.70 0.0004 cg02831474 chr10 1783879 0.012 0.031 0.70 0.0004 cg04535454 chr8 144035192 0.008 0.022 0.70 0.0004 cg08491066 chr1 156030669 0.007 0.019 0.70 0.0003 cg12673377 chr7 1388271 0.008 0.021 0.70 0.0003 cg07203271 chr1 53926112 -0.010 0.026 0.71 0.0003 cg09319487 chr20 56771178 0.009 0.025 0.71 0.0003 cg23359229 chr13 29976597 0.011 0.029 0.71 0.0003 cg23521973 chr10 125117429 0.009 0.024 0.72 0.0003 cg01948226 chr6 32829208 0.009 0.025 0.72 0.0003 cg18184975 chr20 3644192 0.008 0.023 0.72 0.0003 cg13116906 chr12 119552989 0.010 0.027 0.73 0.0003 cg04131502 chr1 2845583 0.008 0.023 0.73 0.0003 cg04907218 chr12 132826672 -0.009 0.025 0.74 0.0003 cg21778193 chr1 1098070 0.009 0.026 0.74 0.0003 cg11855710 chr19 29768180 -0.008 0.024 0.75 0.0002 cg26037026 chr7 1641922 -0.006 0.020 0.75 0.0002 cg25903144 chr7 131609806 0.010 0.033 0.76 0.0002 cg13377560 chr22 43860820 -0.006 0.021 0.76 0.0002 cg16444062 chr16 2770298 0.009 0.029 0.76 0.0002 cg27625886 chr20 62060443 -0.008 0.029 0.77 0.0002 cg01898225 chr15 99975141 -0.006 0.019 0.77 0.0002 cg07865738 chr2 3649447 -0.009 0.032 0.78 0.0002 cg23561197 chr5 125852064 -0.007 0.025 0.79 0.0002 cg14361947 chr9 140780551 0.004 0.015 0.79 0.0002 cg08099672 chr9 139944162 -0.005 0.021 0.79 0.0002 cg08357744 chr17 37559321 0.008 0.034 0.80 0.0002 cg03568769 chr11 1800837 0.006 0.023 0.80 0.0001 cg16857113 chr3 1133865 -0.006 0.026 0.81 0.0001 cg18682781 chr5 176277687 0.007 0.027 0.81 0.0001 cg25721832 chr3 81913557 -0.006 0.023 0.81 0.0001 cg27332583 chr18 35150602 -0.004 0.019 0.81 0.0001 cg11294782 chr3 10487409 0.004 0.019 0.82 0.0001 cg08511578 chr10 1403938 -0.007 0.030 0.82 0.0001 cg07686906 chr1 3353825 0.005 0.020 0.82 0.0001 cg23016281 chr22 27616620 -0.007 0.030 0.83 0.0001 cg08149223 chr10 50507130 -0.004 0.016 0.83 0.0001 cg24600306 chr20 61990671 0.004 0.016 0.83 0.0001 cg02688867 chr12 133019854 -0.006 0.026 0.83 0.0001 cg05444304 chr2 31442675 0.004 0.020 0.83 0.0001 cg05389237 chr9 125314173 0.006 0.031 0.84 0.0001 cg17985533 chr11 2019116 0.003 0.017 0.84 0.0001 cg00389386 chr1 6188136 0.003 0.017 0.84 0.0001 cg13827123 chr13 93971495 -0.006 0.031 0.85 0.0001 cg17905524 chr15 28305412 0.005 0.025 0.85 0.0001 cg11177743 chr20 21545361 0.004 0.023 0.86 0.0001 cg00046877 chr22 19702664 0.002 0.013 0.86 0.0001 cg11138231 chr7 99475824 0.006 0.033 0.86 0.0001 cg06981471 chr7 142982076 -0.004 0.021 0.86 0.0001 cg14757962 chr5 2176287 -0.004 0.025 0.87 0.0001 cg00078259 chr6 2656232 0.004 0.027 0.87 0.0001 cg00188627 chr3 159557779 0.004 0.025 0.88 0.0001 cg08372350 chr16 88943191 -0.004 0.027 0.88 0.0001 cg14886500 chr6 23447204 -0.005 0.032 0.88 0.0001 cg06751809 chr3 105047227 0.005 0.037 0.88 0.0001 cg17224410 chr9 137852661 -0.003 0.023 0.89 0.0000 cg13449257 chr9 137355128 0.003 0.022 0.89 0.0000 cg18650968 chr4 176340837 -0.004 0.027 0.89 0.0000 cg06429120 chr16 86309214 0.005 0.036 0.89 0.0000 cg24857040 chr13 113565214 -0.003 0.024 0.89 0.0000 cg16513326 chr8 10877312 -0.003 0.024 0.89 0.0000 cg20112984 chr10 1447183 -0.004 0.028 0.90 0.0000 cg16608656 chr11 1082797 -0.003 0.026 0.90 0.0000 Page 47 of 53 Diabetes

cg19450046 chr21 45712938 -0.003 0.021 0.90 0.0000 cg09073467 chr1 6155515 -0.002 0.016 0.91 0.0000 cg08204714 chr16 87726461 -0.002 0.015 0.91 0.0000 cg14494450 chr1 4190881 -0.003 0.028 0.92 0.0000 cg00403246 chr10 134794753 0.002 0.015 0.92 0.0000 cg21927431 chr9 134678366 -0.002 0.022 0.92 0.0000 cg03510150 chr9 130690744 -0.002 0.015 0.92 0.0000 cg14204490 chr8 33330967 0.001 0.013 0.92 0.0000 cg11086223 chr18 11487579 0.002 0.022 0.93 0.0000 cg21347874 chr17 77122733 0.002 0.026 0.93 0.0000 cg20612875 chr17 1094308 0.001 0.015 0.94 0.0000 cg14759277 chr17 48674158 -0.001 0.018 0.94 0.0000 cg05851077 chr9 29802712 0.003 0.040 0.94 0.0000 cg05202700 chr8 38258250 -0.002 0.028 0.94 0.0000 cg11837181 chr1 3001002 -0.002 0.023 0.94 0.0000 cg04847540 chr1 182754070 -0.002 0.027 0.95 0.0000 cg08239663 chr16 52688569 -0.002 0.032 0.95 0.0000 cg13463516 chr9 138632121 -0.001 0.021 0.95 0.0000 cg22509138 chr20 44809026 -0.002 0.029 0.95 0.0000 cg04140215 chr2 60540175 0.001 0.024 0.96 0.0000 cg10883064 chr17 60757346 -0.001 0.025 0.96 0.0000 cg15867652 chr17 79011277 -0.002 0.029 0.96 0.0000 cg01095004 chr16 88454883 0.001 0.027 0.96 0.0000 cg09851596 chr8 143545214 0.001 0.017 0.97 0.0000 cg24064348 chr1 54181174 0.000 0.013 0.97 0.0000 cg18042910 chr13 19662609 0.001 0.029 0.98 0.0000 cg01615861 chr16 85513978 0.000 0.009 0.98 0.0000 cg00875983 chr11 3240213 0.000 0.016 0.98 0.0000 cg06495184 chr19 406256 -0.001 0.021 0.98 0.0000 cg18233010 chr10 114911413 0.000 0.017 0.98 0.0000 cg12193237 chr19 3907913 0.000 0.021 0.98 0.0000 cg16819051 chr2 229173235 0.000 0.025 0.99 0.0000 cg11765907 chr20 43103949 0.000 0.015 0.99 0.0000 cg25285919 chr3 188312215 0.000 0.024 0.99 0.0000 cg11586089 chr14 66644923 0.000 0.025 0.99 0.0000 cg03699074 chr16 88849875 0.000 0.030 0.99 0.0000 cg18108190 chr19 2727822 0.000 0.023 0.99 0.0000 cg05347757 chr15 51121020 0.000 0.030 1.00 0.0000 cg12954267 chr6 33093109 0.000 0.030 1.00 0.0000 cg02110836 chr17 71953083 0.000 0.013 1.00 0.0000 Diabetes Page 48 of 53

Supplementary Table 6: Results from enrichment in TargetScan microRNA 2017 database (178 unique genes from annotation to CpG identified in Model 1) Adjusted Old Adjusted Combined Term Overlap P-value P-value Old P-value P-value Z-score Score FCN1;TOR4A;CHRNA4;CHD5;GATA4;PLD6;CRHR1;C1QTNF4;MRC2;ABR;PIP5KL1;PRDMGenes 16;CTSD;GPC6;TBC1D22A;SLC39A3;NKX2- hsa-miR-1538 28/1027 1.194E-07 4.05E-05 1.236E-09 4.1885E-07 -1.924 30.67 FCN1;TOR4A;CHRNA4;CHD5;GATA4;PLD6;CRHR1;C1QTNF4;MRC2;ABR;PIP5KL1;PRDM2;SRRM4;SLC12A5;RBFOX3;PRKCB;RASL10B;IRX4;MTUS2;KCNAB2;BAIAP2;C1ORF159; 16;CTSD;GPC6;TBC1D22A;SLC39A3;NKX2- hsa-miR-4745-3p 28/1027 1.194E-07 4.05E-05 1.236E-09 4.1885E-07 -1.917 30.56 TOR4A;RYR2;CHRNA4;FLT4;CELF4;CHD5;ADARB2;MYLK4;MRC2;IGSF21;ABR;ATCAY;M2;SRRM4;SLC12A5;RBFOX3;PRKCB;RASL10B;IRX4;MTUS2;KCNAB2;BAIAP2;C1ORF159; ROH5;MCF2L;PRDM16;ZNF469;QSOX2;TBC1D22A;GALNT9;SLC39A3;NKX2- hsa-miR-3180 32/1475 2.206E-06 0.000299 1.538E-08 2.0856E-06 -2.443 31.82 2;ANKS1B;ENTPD2;SLC12A5;TPSG1;MTUS2;KCNAB2;BAIAP2;CBFA2T3;SEPT5;KCNQ1;HTOR4A;RYR2;CHRNA4;FLT4;CELF4;CHD5;ADARB2;MYLK4;MRC2;IGSF21;ABR;ATCAY;M ROH5;MCF2L;PRDM16;ZNF469;QSOX2;TBC1D22A;GALNT9;SLC39A3;NKX2- hsa-miR-3180-3p 32/1475 2.206E-06 0.000299 1.538E-08 2.0856E-06 -2.438 31.75 2;ANKS1B;ENTPD2;SLC12A5;TPSG1;MTUS2;KCNAB2;BAIAP2;CBFA2T3;SEPT5;KCNQ1;HTOR4A;RYR2;CHRNA4;FLT4;CELF4;CHD5;ADARB2;MYLK4;MRC2;IGSF21;ABR;ATCAY;M ROH5;MCF2L;PRDM16;ZNF469;QSOX2;TBC1D22A;GALNT9;SLC39A3;NKX2- hsa-miR-3196 32/1475 2.206E-06 0.000299 1.538E-08 2.0856E-06 -2.433 31.68 2;ANKS1B;ENTPD2;SLC12A5;TPSG1;MTUS2;KCNAB2;BAIAP2;CBFA2T3;SEPT5;KCNQ1;H KCNT1;ATCAY;C1ORF159;CHRNA4;TMEM139;PRDM16;PLD6;BAIAP2;CBFA2T3;LPP;SLC hsa-miR-3186-3p 11/270 3.152E-05 0.003562 5.718E-06 0.00043072 -1.241 12.86 39A3TOR4A;SRRM4;SLC12A5;UNC5A;CHRNA4;IRX4;CELF4;C2CD4C;KCNAB2;BAIAP2;AJAP1; ABR;RXRA;KCNT1;JPH3;ATCAY;B3GNT6;MFNG;CDH22;PRDM16;PIEZO1;HCN2;GALNT9 hsa-miR-4707-5p 24/1113 5.295E-05 0.004906 1.543E-06 0.00017431 -1.926 18.96 MEF2A;FCN1;TOR4A;SRRM4;KLK1;UNC5A;RBFOX3;CHRNA4;RASL10B;CHD5;GATA4;BA;SLC39A3 IAP2;CBFA2T3;LPP;ATCAY;TRIM29;KCNQ1;EFCAB2;QSOX2;DLGAP2;HCN2;GALNT9;SLC hsa-miR-1268 23/1055 6.513E-05 0.004906 2.207E-06 0.00018702 -1.882 18.14 MEF2A;FCN1;TOR4A;SRRM4;KLK1;UNC5A;RBFOX3;CHRNA4;RASL10B;CHD5;GATA4;BA39A3 IAP2;CBFA2T3;LPP;ATCAY;TRIM29;KCNQ1;EFCAB2;QSOX2;DLGAP2;HCN2;GALNT9;SLC hsa-miR-1268b 23/1055 6.513E-05 0.004906 2.207E-06 0.00018702 -1.877 18.09 39A3 RBFOX3;CHRNA4;MTUS2;GATA4;KCNAB2;ADARB2;CBFA2T3;SEPT5;MYLK4;C16ORF82; hsa-miR-1250 15/528 7.953E-05 0.005392 8.034E-06 0.0004952 -1.347 12.72 MPPED1;MCF2L;EFCAB2;ZNF469;HCN2 GRIA1;SLC12A5;PRKCB;CHRNA4;RASL10B;GBE1;FUT10;FLT4;CACNA1B;GATA4;ADARB hsa-miR-4462 19/814 0.0001229 0.006944 7.411E-06 0.0004952 -1.614 14.54 2;JPH3;MPPED1;MROH5;MCF2L;TBC1D22A;PLXNA4;GALNT9;SLC39A3 TOR4A;RASL10B;ATP2B2;SEPT5;ATCAY;TRIM29;MCF2L;EFCAB2;KCNQ2;PRDM16;TTPA hsa-miR-3621 14/487 0.0001222 0.006944 1.446E-05 0.0007815 -1.296 11.67 L;TBC1D22A;PLXNA4;GALNT9 MEF2A;GRIA1;RYR2;MTUS2;GATA4;ADARB2;LPP;C1ORF159;PRDM16;GSE1;BUB3;DLG hsa-miR-3676 16/629 0.0001649 0.007987 1.506E-05 0.0007815 -1.450 12.63 AP2;GPC6;TBC1D22A;PLXNA4;GALNT9 TCF7L2;SLC12A5;CHRNA4;IRX4;ATP2B2;KCNAB2;CBFA2T3;LPP;SEPT5;GLIS1;AJAP1;AB hsa-miR-191 15/561 0.000155 0.007987 1.614E-05 0.0007815 -1.339 11.75 R;RXRA;PRDM16;PLXNA4 MEF2A;FCN1;TOR4A;SLC12A5;IRX4;CELF4;CHD5;MTUS2;C2CD4C;PLD6;ADARB2;GLIS1; hsa-miR-4467 18/803 0.0003049 0.013781 2.244E-05 0.00101408 -1.601 12.96 RXRA;PIEZO1;DLGAP2;HCN2;GALNT9;NKX2-2 TOR4A;CHRNA4;CACNA1B;CELF4;C2CD4C;NPL;ADARB2;CBFA2T3;ABR;RXRA;KCNT1;JP hsa-miR-3193 16/695 0.0004984 0.019826 4.896E-05 0.00176735 -1.432 10.89 H3;GSE1;DLGAP2;DSCAML1;TBC1D22A ENTPD2;RBFOX3;IRX4;CHD5;C2CD4C;BAIAP2;LPP;CACNA1G;MYLK4;RXRA;PRDM16;ZN hsa-miR-339-3p 13/497 0.0005264 0.019826 7.614E-05 0.00222858 -1.226 9.26 F469;HCN2 WWTR1;RXRA;KCNT1;RASL10B;PRDM16;MTUS2;QSOX2;PLD6;CBFA2T3;SLC39A3;ANK hsa-miR-574-3p 12/431 0.0004986 0.019826 8.217E-05 0.00222858 -1.172 8.91 S1B;MYLK4 CHRNA4;CACNA1B;C2CD4C;KCNAB2;BAIAP2;GLIS1;CRHR1;CAPN14;C1ORF159;B3GNT hsa-miR-3177-3p 16/713 0.000656 0.023411 6.581E-05 0.00210085 -1.459 10.69 6;PIP5KL1;MCF2L;TTPAL;ALDH7A1;PLXNA4;SLC39A3 TOR4A;SLC12A5;RBFOX3;RASL10B;FLT4;BAIAP2;CBFA2T3;LPP;MRC2;ABR;RXRA;MCF2 hsa-miR-3178 17/789 0.0007014 0.023776 6.263E-05 0.00210085 -1.561 11.34 L;DLGAP2;TBC1D22A;PLXNA4;HCN2;GALNT9GRIA1;TOR4A;PTPRU;TCF7L2;UNC5A;RBFOX3;CHRNA4;CHD5;BAIAP2;CBFA2T3;LPP;RX RA;ATCAY;C10ORF71;C1ORF159;B3GNT6;PIP5KL1;PRDM16;QSOX2;DLGAP2;PLXNA4;A hsa-miR-564 22/1188 0.0008977 0.0279 4.579E-05 0.00176735 -1.966 13.79 NKS1B ATCAY;PRKCB;CHRNA4;MCF2L;PRDM16;MTUS2;DSCAML1;LPP;PLXNA4;SLC39A3;NKX hsa-miR-1306 11/398 0.0009053 0.0279 0.0001748 0.00408772 -1.199 8.40 2-2 MEF2A;GRIA1;UNC5A;PRKCB;FLT4;CHD5;KCNAB2;PLD6;AJAP1;RXRA;C1ORF159;MICA hsa-miR-615-3p 13/531 0.0009684 0.028547 0.0001449 0.00353192 -1.273 8.83 LL2;GSE1 TOR4A;RBFOX3;CHRNA4;FLT4;IRX4;C2CD4C;KCNAB2;BAIAP2;LPP;SEPT5;C1QTNF4;ATC hsa-miR-4707-3p 21/1145 0.0013297 0.037069 7.995E-05 0.00222858 -1.884 12.48 AY;C1ORF159;TMEM139;MCF2L;KCNQ2;PRDM16;QSOX2;TTPAL;TBC1D22A;HCN2 hsa-miR-4665-3p 10/356 0.0013669 0.037069 0.0003065 0.00593688 -1.093 7.21 TOR4A;RYR2;CHRNA4;FLT4;CACNA1B;C2CD4C;ADARB2;LPP;GLIS1;ABR;RXRA;TRIM29;ABR;SLC12A5;UNC5A;FLT4;KCNQ2;PRDM16;CELF4;GSE1;DLGAP2;AJAP1 PRDM16;DLGAP2;TBC1D22A;PLXNA4;SLC39A3;SRRM4;ENTPD2;SLC12A5;SLC6A18;RA hsa-miR-1908 29/1849 0.0018737 0.043806 4.953E-05 0.00176735 -3.059 19.21 TOR4A;RYR2;CHRNA4;FLT4;CACNA1B;C2CD4C;ADARB2;LPP;GLIS1;ABR;RXRA;TRIM29;SL10B;ATP2B2;KCNAB2;BAIAP2;CBFA2T3;SEPT5;B3GNT6;HCN2 PRDM16;DLGAP2;TBC1D22A;PLXNA4;SLC39A3;SRRM4;ENTPD2;SLC12A5;SLC6A18;RA hsa-miR-663 29/1849 0.0018737 0.043806 4.953E-05 0.00176735 -3.051 19.16 SL10B;ATP2B2;KCNAB2;BAIAP2;CBFA2T3;SEPT5;B3GNT6;HCN2 SLC12A5;CHD5;C2CD4C;ADARB2;PRLR;MYLK4;JPH3;C1ORF159;EFCAB2;DLGAP2;PLXN hsa-miR-3131 13/569 0.0017953 0.043806 0.0002797 0.00557854 -1.195 7.56 A4;SLC39A3;ANKS1B SLC12A5;CHD5;C2CD4C;ADARB2;PRLR;MYLK4;JPH3;C1ORF159;EFCAB2;DLGAP2;PLXN hsa-miR-611 13/569 0.0017953 0.043806 0.0002797 0.00557854 -1.187 7.51 A4;SLC39A3;ANKS1BGRIA1;OCA2;CHRNA4;USP34;AP4E1;PRSS27;GATA4;TRHDE;LPP;RXRA;ERMP1;MCF2L; ZNF469;GPC6;TBC1D22A;PLXNA4;MEF2A;PRKCB;RASL10B;MTUS2;ATP2B2;KCNAB2;B mmu-miR-767 28/1784 0.0022365 0.049 6.817E-05 0.00210085 -2.893 17.65 AIAP2;PRLR;CAPN14;DIAPH1;TTPAL;XKR6 SRRM4;RBFOX3;CHRNA4;FUT10;FLT4;CACNA1B;LPP;MYLK4;ATCAY;ALCAM;C1ORF159 hsa-miR-4638-5p 21/1196 0.0022568 0.049 0.0001459 0.00353192 -1.911 11.65 ;MPPED1;B3GNT6;KCNQ2;GSE1;CTSD;DLGAP2;TBC1D22A;HCN2;GALNT9;SLC39A3 TOR4A;KCNAB2;ADARB2;LPP;CRHR1;RXRA;KCNT1;ATCAY;C1ORF159;MFNG;TMEM139 hsa-miR-4738-5p 17/887 0.0024572 0.049 0.0002486 0.00526688 -1.544 9.28 ;KCNQ2;PRDM16;DMRTB1;DLGAP2;WDFY4;GALNT9 hsa-miR-4431 10/383 0.0023355 0.049 0.0005356 0.00864691 -1.040 6.30 TOR4A;SRRM4;CDH22;PRDM16;GSE1;ADARB2;LPP;PLXNA4;NKX2-2;AJAP1 hsa-miR-937 6/152 0.0024265 0.049 0.0009714 0.01197923 -0.335 2.02 SLC12A5;MCF2L;PRDM16;IRX4;C2CD4C;CBFA2T3 Page 49 of 53 Diabetes

Supplementary Table 7: Results from enrichment in ENCODE Histone Modification 2015 database (178 unique genes from annotation to CpG identified in Model 1) Old Adjusted Old P- Adjusted Combined Term Overlap P-value P-value value P-value Z-score Score Genes

PTPRU;RYR2;CHRNA4;ONECUT3;CACNA1B;CELF4;CHD5;GATA4;ADARB2;TRHDE;GLIS1;CRHR1;CACNA1G;C1QTNF4;MRC2;IGSF21;RX RA;JPH3;ALCAM;MROH5;CDH22;PRDM16;DMRTB1;PHACTR3;MICALL2;DLGAP2;PLXNA4;NKX2-2; ANKS1B;DIO3OS;SLC12A5;UNC5A; H3K27me3_ES-Bruce4_mm9 48/2000 9.6E-11 3.9E-08 9.34E-20 3.81E-17 -1.75 40.27 RBFOX3;PRKCB;RASL10B;CCDC109B;IRX4;ATP2B2;TACR1;BAIAP2;SEPT5;AJAP1;FAM101B;MPPED1;KCNQ1;KCNQ2;DSCAML1;HCN2 PTPRU;RYR2;CHRNA4;ONECUT3;FLT4;CELF4;DMKN;CRHR1;CACNA1G;C1QTNF4;IGSF21;JPH3;MROH5;MCF2L;CDH22;DMRTB1;BAI1; DLGAP2;PLXNA4;GALNT9;SLC12A5;UNC5A;RBFOX3;RASL10B;ATP2B2;MIR200B;TACR1;CBFA2T3;GFRA4;PSMB9;MPPED1;KCNQ1;KC H3K27me3_C2C12_mm9 34/1269 6.5E-09 1.3E-06 3.02E-15 6.16E-13 -1.75 33.09 NQ2;HCN2

PTPRU;RYR2;CHRNA4;ONECUT3;C2CD4C;DMKN;GLIS1;CRHR1;CACNA1G;C1QTNF4;KCNT1;MUC2;JPH3;MROH5;MCF2L;CDH22;BAI1; H3K27me3_myocyte_mm9 28/1183 2E-06 0.00027 2.12E-11 1.44E-09 -1.72 22.53 DLGAP2;PLXNA4;GALNT9;NKX2-2;ENTPD2;RBFOX3;RASL10B;CBFA2T3;GFRA4;MPPED1;HCN2 PTPRU;RYR2;CHRNA4;ONECUT3;CACNA1B;CHD5;GATA4;C2CD4C;NPL;TRHDE;CRHR1;C1QTNF4;IGSF21;KCNT1;JPH3;MROH5;PRDM1 6;PHACTR3;BAI1;DLGAP2;PLXNA4;GALNT9;NKX2-2;ANKS1B;WWTR1;SLC12A5;UNC5A;RBFOX3;PRKCB;RASL10B; IRX4;ATP2B2; H3K27me3_small intestine_mm9 38/2000 5.1E-06 0.00052 2E-12 1.81E-10 -1.61 19.67 AJAP1;MPPED1;KCNQ2;ANKRD60;DSCAML1;HCN2 GRIA1;OCA2;RYR2;CHRNA4;CPNE4;AIRE;FLT4;CACNA1B;CHD5;GATA4;C2CD4C;NPL;ADARB2;CACNA1G;C1QTNF4;IGSF21;KCNT1;JPH 3;ATCAY;C10ORF71;MCF2L;PRDM16;PHACTR3;BAI1;PLXNA4;GALNT9;NKX2-2;ANKS1B;SRRM4;ENTPD2;SPHKAP;TPSG1; PRKCB; H3K27me3_kidney epithelial cell_hg19 40/2217 9.5E-06 0.00077 2.22E-12 1.81E-10 -1.65 19.08 KCNIP4;IRX4;TACR1;GFRA4;AJAP1;C16ORF82;HAR1B

PTPRU;RYR2;ONECUT3;SCHIP1;CACNA1B;CELF4;CHD5;GATA4;C2CD4C;TRHDE;CRHR1;CACNA1G;C1QTNF4;IGSF21;JPH3;CDH22;PRD H3K27me3_kidney_mm9 32/1675 3E-05 0.00169 1.41E-10 7.19E-09 -1.68 17.54 M16;BAI1;DLGAP2;NKX2-2;DIO3OS;SRRM4;RBFOX3;PRKCB;CCDC109B;KCNIP4;MTUS2;GFRA4;KCNQ2;ANKRD60;DSCAML1;HCN2

GRIA1;OCA2;RYR2;CPNE4;ONECUT3;FLT4;CACNA1B;CELF4;CHD5;C2CD4C;ADARB2;TRHDE;CACNA1G;C1QTNF4;IGSF21;JPH3;ATCAY; C10ORF71;MCF2L;CDH22;PHACTR3;GPC6;PLXNA4;ANKS1B;SRRM4;SPHKAP;PRKCB;IRX4;ATP2B2;TACR1;PRLR;GFRA4;AJAP1;MPPED1 H3K27me3_bronchial epithelial cell_hg19 37/2082 3.1E-05 0.00169 2.84E-11 1.66E-09 -1.61 16.67 ;KCNQ2;GPR123;DSCAML1 PTPRU;RYR2;ONECUT3;FLT4;CELF4;CHD5;DMKN;MYLK4;C1QTNF4;KCNT1;JPH3;ATCAY;OR2AE1;MCF2L;PHACTR3;BAI1;GALNT9;ANKS 1B;SRRM4;ENTPD2;UNC5A;PRKCB;AZGP1P1;TACR1;PRLR;OR1N2;GFRA4;MPPED1;KCNQ1;KCNQ2;GPR123;DSCAML1;GRIA1;OCA2;C HRNA4;AIRE;CACNA1B;C2CD4C;NPL;TRHDE;GLIS1;IGSF21;CDH22;PRDM16;GPC6;NKX2-2;RASL10B;KCNIP4;IRX4 ;ATP2B2;HCN2; H3K27me3_endothelial cell of umbilical vein_hg19 52/3393 3.3E-05 0.00169 1.98E-13 2.69E-11 -1.53 15.76 HAR1B

PTPRU;CHRNA4;FUT10;GATA4;C2CD4C;NPL;PLD6;TRHDE;CRHR1;C1QTNF4;MRC2;IGSF21;KCNT1;ATCAY;ALCAM;MROH5;CDH22;MIC ALL2;GALNT9;NKX2-2;ANKS1B;WWTR1;ENTPD2;RBFOX3;RASL10B;MTUS2;ATP2B2;SEPT5;KCNQ1;KCNQ2; ANKRD60;DSCAML1; H3K27me3_spleen_mm9 34/2000 0.00016 0.00745 6.82E-10 3.09E-08 -1.57 13.66 HCN2;XKR6

OCA2;RYR2;CPNE4;FLT4;CELF4;CHD5;C2CD4C;CACNA1G;C1QTNF4;IGSF21;ABR;JPH3;ATCAY;MCF2L;CDH22;PRDM16;RPL38;PLXNA4; H3K27me3_BJ_hg19 34/2090 0.00037 0.01527 2.08E-09 8.47E-08 -1.53 12.09 GALNT9;NKX2-2;ANKS1B;ENTPD2;UNC5A;PRKCB;KCNIP4;ATP2B2;TACR1;PRLR;GFRA4;AJAP1;MPPED1;KCNQ1;KCNQ2;GPR123 Diabetes Page 50 of 53

Supplementary Table 8: Results from enrichment in GWAS Catalog 2019 database (178 unique genes from annotation to CpG identified in Model 1) Old Adjusted Old P- Adjusted Combine Term Overlap P-value P-value value P-value Z-score d Score Genes DNA methylation (parent-of-origin) 3-8 3.76E-05 0.017 4.48E-05 0.020 -4.55 46.41 H19;MIR675;SEPT5 End-stage renal disease 2-7 0.001606 0.207 0.001515 0.119 -3.41 21.91 MBNL1;DLGAP2 Aging (time to event) 2-8 0.002129 0.207 0.001886 0.119 -3.42 21.01 GRIA1;ATCAY Disc degeneration (lumbar) 2-9 0.002721 0.207 0.002295 0.119 -2.81 16.62 GPC6;PSMB9 Asparaginase hypersensitivity in acute lymphoblastic leukemia 2-9 0.002721 0.207 0.002295 0.119 -2.61 15.40 GRIA1;CACNA1B Hair color 4-61 0.002132 0.207 0.000905 0.119 -1.30 7.99 OCA2;PIEZO1;ZNF469;CBFA2T3 Fasting blood insulin 2-11 0.004109 0.219 0.003227 0.130 -2.39 13.12 TCF7L2;CNTN6 Joint mobility (Beighton score) 3-35 0.00368 0.219 0.002013 0.119 -1.72 9.62 PIEZO1;CRHR1;XKR6 Hemoglobin A1c levels 3-37 0.004312 0.219 0.002335 0.119 -1.58 8.62 TCF7L2;KCNQ1;PIEZO1 Cutaneous squamous cell carcinoma 2-14 0.006681 0.253 0.004901 0.132 -2.66 13.33 OCA2;LPP Page 51 of 53 Diabetes

Supplementary Table 9: Results from enrichment in dbGAP (accessed 08/2019) database (178 unique genes from annotation to CpG identified in Model 1)

Old Adjusted Old P- Adjusted Combined Term Overlap P-value P-value value P-value Z-score Score Genes GRIA1;RYR2;PRKCB;CACNA1B;TRHDE;IGSF21;ATIC;BPIFA3;WDR60;CDH22;BAI1 Body Height 14/385 9.49E-06 0.001 0.0002 0.02702 -2.36 27.29 ;QSOX2;ANKRD60;XKR6 RYR2;MBNL1;RBFOX3;CPNE4;FUT10;USP34;KCNIP4;ADARB2;LPP;GLIS1;KCNT1; Body Mass Index 13/437 0.000153 0.008 0.0026 0.11918 -2.45 21.52 WDFY4;ANKS1B Triglycerides 9/244 0.000348 0.013 0.0033 0.11918 -2.29 18.22 RBFOX3;CPNE4;AIRE;GBE1;KCNIP4;DLGAP2;DSCAML1;LPP;XKR6 RYR2;MPPED1;BPIFA3;CNTN6;KCNIP4;PHACTR3;MTUS2;ATP2B2;DSCAML1;GP Blood Pressure 12/454 0.000786 0.022 0.0095 0.14918 -2.30 16.44 C6;PRLR;PLXNA4 Electrocardiography 5/87 0.001082 0.024 0.005 0.13639 -1.96 13.37 RYR2;CPNE4;KCNQ1;ANKS1B;HLA-DPB2 Arthritis, Rheumatoid 4/59 0.001885 0.035 0.0069 0.14918 -1.80 11.28 PRKCB;AIRE;PHACTR3;TRHDE Blood Pressure Determination 4/64 0.002543 0.040 0.009 0.14918 -1.88 11.24 TCF7L2;KCNIP4;ADARB2;PLXNA4

Echocardiography 8/273 0.003158 0.040 0.0197 0.19685 -1.97 11.36 CNTN6;CPNE4;SCHIP1;KCNIP4;ADARB2;GPC6;PLXNA4;GLIS1 Body Weight 7/216 0.003272 0.040 0.0178 0.19616 -1.97 11.25 RBFOX3;BPIFA3;CPNE4;WDR60;FUT10;USP34;LPP Elbow 4/72 0.003898 0.043 0.0133 0.18237 -1.68 9.30 CCDC109B;PRDM16;KCNIP4;ADARB2 Diabetes Page 52 of 53

Supplementary Table 10: Results from enrichment in BioCarta 2016 database (178 unique genes from annotation to CpG identified in Model 1)

Old Adjusted Old P- Adjusted Combined Term Overlap P-value P-value value P-value Z-score Score Genes Role of PPAR-gamma Coactivators in Obesity and Thermogenesis_Homo sapiens_h_ppargPathway 2/9 0.003 0.158 0.004 0.256 -1.58 9.33 MED1;RXRA Mechanism of Gene Regulation by Peroxisome Proliferators via PPARa_Homo sapiens_h_pparaPathway 3/52 0.011 0.285 0.013 0.314 -1.49 6.70 MED1;RXRA;PRKCB Control of Gene Expression by Vitamin D Receptor_Homo sapiens_h_vdrPathway 2/27 0.024 0.285 0.030 0.314 -1.39 5.20 MED1;RXRA VEGF, Hypoxia, and Angiogenesis_Homo sapiens_h_vegfPathway 2/30 0.029 0.285 0.036 0.314 -1.55 5.48 PRKCB;FLT4 Phospholipids as signalling intermediaries_Homo sapiens_h_edg1Pathway 2/33 0.035 0.285 0.042 0.314 -1.56 5.23 SPHKAP;PRKCB Angiotensin II mediated activation of JNK Pathway via Pyk2 dependent signaling_Homo sapiens_h_At1rPathway 2/34 0.037 0.285 0.044 0.314 -1.69 5.58 MEF2A;PRKCB Basic mechanism of action of PPARa, PPARb(d) and PPARg and effects on gene expression_Homo sapiens_h_pparPathway 1/5 0.044 0.285 0.056 0.314 1.76 -5.52 RXRA Actions of Nitric Oxide in the Heart_Homo sapiens_h_no1Pathway 2/42 0.054 0.285 0.064 0.314 -1.14 3.33 RYR2;FLT4 Signal Dependent Regulation of Myogenesis by Corepressor MITR_Homo sapiens_h_MITRPathway 1/8 0.069 0.285 0.083 0.314 0.71 -1.89 MEF2A CBL mediated ligand-induced downregulation of EGF receptors_Homo sapiens_h_cblPathway 1/8 0.069 0.285 0.083 0.314 0.77 -2.07 PRKCB Page 53 of 53 Diabetes

Supplementary Table 11: Results from enrichment in WikiPathways 2019 database (178 unique genes from annotation to CpG identified in Model 1) Old Adjusted Old P- Adjusted Combined Term Overlap P-value P-value value P-value Z-score Score Genes Adipogenesis WP236 6/130 0.001 0.150 0.001 0.172 -1.08 7.37 MEF2A;WWTR1;MBNL1;RXRA;GATA4;PRLR Genes targeted by miRNAs in adipocytes WP1992 2/13 0.006 0.263 0.008 0.338 -2.98 15.39 KCNQ1;HCN2 Serotonin and anxiety-related events WP3944 2/13 0.006 0.263 0.008 0.338 -2.91 15.00 PRKCB;CRHR1 Energy Metabolism WP1541 3/47 0.008 0.289 0.010 0.338 -2.11 10.10 MEF2A;MED1;RXRA Pathways Regulating Hippo Signaling WP4540 4/98 0.011 0.311 0.013 0.338 -1.22 5.47 TCF7L2;PRKCB;FLT4;CDH22 Sudden Infant Death Syndrome (SIDS) Susceptibility Pathways WP706 5/158 0.014 0.311 0.015 0.338 -1.03 4.44 RYR2;CHRNA4;KCNQ1;TACR1;NKX2-2 Physiological and Pathological Hypertrophy of the Heart WP1528 2/25 0.021 0.404 0.024 0.468 -2.10 8.14 PRKCB;GATA4 miRs in Muscle Cell Differentiation WP2012 2/29 0.027 0.416 0.031 0.472 -2.48 8.92 MEF2A;PRKCB Non-genomic actions of 1,25 dihydroxyvitamin D3 WP4341 3/71 0.025 0.416 0.028 0.472 -1.09 4.02 MED1;RXRA;PRKCB Cerebral Organic Acidurias, including diseases WP4519 1/7 0.061 0.513 0.069 0.529 -3.50 9.81 ALDH7A1