A 6-CpG validated methylation risk score model for metabolic syndrome: The HyperGEN and GOLDN studies.

There has been great interest in genetic risk prediction using risk scores in recent years, however, the utility of scores developed in European populations and later applied to non-European populations has not been successful. The goal of this study was to create a methylation risk score (MRS) for metabolic syndrome (MetS), demonstrating the utility of MRS across race groups using cross-sectional data from the Hypertension Genetic Epidemiology Network (HyperGEN, N = 614 African Americans (AA)) and the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN, N = 995 European Americans (EA)). To demonstrate this, we first selected cytosine-guanine dinucleotides (CpG) sites measured on Illumina Methyl450 arrays previously reported to be significantly associated with MetS and/or component conditions in more than one race/ethnic group (CPT1A cg00574958, PHOSPHO1 cg02650017, ABCG1 cg06500161, SREBF1 cg11024682, SOCS3 cg18181703, TXNIP cg19693031). Second, we calculated the parameter estimates for the 6 CpGs in the HyperGEN data (AA) and used the beta estimates as weights to construct a MRS in HyperGEN (AA), which was validated in GOLDN (EA). We performed association analyses using logistic mixed models to test the association between the MRS and MetS, adjusting for covariates. Results showed the MRS was significantly associated with MetS in both populations. In summary, a MRS for MetS was a strong predictor for the condition across two race groups, suggesting MRS may be useful to examine metabolic disease risk or related complications across race/ethnic groups.

Introduction

Genome wide association studies (GWAS) have examined the cumulative effect of novel, single variants on both quantitative trait variance and disease status, by summing up independent risk alleles at each locus weighted by effect size, otherwise known as a genetic risk score (GRS) [1]. While once limited by small sample sizes, GRSs have since benefitted from the growth and development of large-disease based consortia, along with improved methodology which allow for the aggregation of thousands to millions of genetic variants (i.e. polygenic risk scores, PRS), which better inform risk prediction [2]. A major limitation of GRS–including PRS—is that they have been developed and optimized for European-ancestry populations, thus limiting their utility and generalizability in non-European ancestry populations [3, 4]. Overall, GWAS results and GRS have been difficult to replicate across race groups for complex disease. This is in contrast to data on DNA methylation which has demonstrated more successful cross-race replication [3, 5, 6]. Given these observations we aimed to apply statistical approaches common to GRS and PRS to epigenome-wide association (EWAS) data to evaluate if methylation risk score (MRS) may further enhance accuracy for prediction of metabolic syndrome, a cluster of conditions that can increase risk for cardiometabolic diseases. We hypothesized that leveraging existing EWAS data and previously reported associations between cytosine-phosphate-guanine (CpG) sites and metabolic syndrome may help identify persons with elevated risk for MetS.

There is a rich body of literature demonstrating independent associations between DNA methylation loci and MetS and/or conditions related to MetS such as obesity [7-9], insulin resistance (9), and type 2 diabetes [10-12]. In this study, we leverage previously reported CpGs that have been significantly associated with MetS and/or conditions comprising MetS in more than one race/ethnic group: waist circumference [5], triglycerides [13], fasting blood glucose [11], systolic and diastolic blood pressure [14], and HDL cholesterol [15, 16], to construct the MRS. Independent CpGs included were cg00574958 from carnitine palmitoyl transferase 1A (CPT1A), cg02650017 from Phosphoethanolamine/Phosphocholine Phosphatase 1 (PHOSPHO1), cg06500161 from ATP Binding Cassette Subfamily G Member 1 (ABCG1), cg11024682 from Sterol Regulatory Element Binding Transcription Factor 1 (SREBF1), cg18181703 from Suppressor Of Cytokine Signaling 3 (SOCS3), and cg19693031 from Thioredoxin Interacting Protein (TXNIP). Bolstering to the validity of our CpG selection each gene has strong biological plausibility for association with MetS including roles in fatty acid metabolism (CPT1A) [17], lipid homeostasis and metabolism (SREBF1 [18], ABCG1) [19], skeletal endocrine regulation of metabolism (PHOSPHO1) [20], cytokine signaling (SOCS3) [21] and oxidative stress (TXNIP) [22].

Like existing GWAS data existing methylation data can be used to calculate risk scores that could become useful for diagnosis and/or prevention. Building on GRS methods we used a weighted sum approach to create a MRS among African Americans from the Hypertension Genetic Epidemiology Network (HyperGEN), Wevalidated the score in European Americans from Genetics of Lipid Lowering Drugs and Diet Network (GOLDN). We further examined the score’s association with the individual MetS components and stratified by sex.

Methods

Discovery and validation study populations

Data for the discovery phase of this study was obtained from the HyperGEN study. HyperGEN is a cross-sectional study including over 1900 African-Americans from families, which included at least two siblings with hypertension onset before age 60 [23]. The study purpose was to examine possible interactions between genetic and non-genetic determinants of hypertension. In 2015, an ancillary epigenetic study was conducted on stored HyperGEN samples in the upper and lower tertial of echocardiography measured left ventricular mass [24]. After excluding those missing relevant phenotype data as previously described [25] a total 614 participants were included in the analysis. Participants in the HyperGEN and GOLDN studies provided written and oral informed consent to participate in these studies and both studies were approved by the Institutional Review Board at the University of Alabama at Birmingham.

Validation was conducted in GOLDN study [26]. European ancestry families in GOLDN were recruited from the Family Heart Study at two centers, Minneapolis, MN and Salt Lake City, UT to participate in a diet and/or drug intervention. In each case, only families with at least two siblings were recruited and only participants who did not take lipid-lowering agents (pharmaceuticals or nutraceuticals) for at least 4 weeks prior to the initial visit were included. For the present study, 994 GOLDN participants had available methylation data for a validation study of the HyperGEN MRS. Sample characteristics as well as clinical and lifestyle factors were considered in HyperGEN and GOLDN, including blood pressure, antihypertensive and lipid lowering medications, fasting blood glucose, triglycerides, HDL cholesterol, height, weight, and waist circumference have been described [7-9]. We used the published joint harmonized criteria to define MetS in HyperGEN in both studies [27].

DNA methylation and data processing

HyperGEN

The Illumina HumanMethylation450 array was used to analyze DNA extracted from buffy coat obtained from whole blood samples at > 480,000 cytosine-phosphate-guanine (CpG) sites. Briefly, 500 ng of buffy coat DNA was hybridized to the Methyl450 array after bisulfite conversion with EZ DNA kits (Zymo Research, Irvine, CA) prior to standard Illumina amplification, hybridization, and imaging steps. The resulting intensity files were analyzed with Illumina’s GenomeStudio, which generated beta (β) scores (i.e., the proportion of total signal from the methylation-specific probe or color channel) and “detection p values” (probability that the total intensity for a given probe falls within the background signal intensity). Quality control (QC) measures were conducted by removing samples having more than 1% of CpG sites with a detection p value > 0.05, removing CpG sites having more than 5% of samples with a detection p value > 0.01, and individual CpG sites with detection p value > 0.01 set as missing. After these QC filters, 484,366 CpG sites were eligible for analysis. We normalized the β scores using the Subset-quantile Within Array Normalization (SWAN) method in minifi package to correct for differences between batches and the type I and type II assay designs within a single 450K array [28]. Cell count proportions (CD8 T lymphocytes, CD4 T lymphocytes, natural killer cells, B cells, and monocytes) were created using the algorithm developed by Houseman, which predicts underlying cellular composition of each sample from DNA methylation patterns [29].

GOLDN

CD4+ T-cells were isolated from frozen buffy coat samples stored from whole blood peripheral blood collected at the baseline visit (prior to intervention). DNA was extracted using DNeasy kits (Qiagen, Venlo, Netherlands). 500ng of each DNA sample was treated with sodium bisulfite (Zymo Research, Irvine, CA). Normalization was performed on random subsets of 10,000 CpGs per run, with each array of 12 samples used as a "batch." Probes from the Infinium I and II chemistries were separately normalized and β scores for Infinium II probes were then adjusted using the equation derived from fitting a second order polynomial to the observed methylation values across all pairs of probes located <50bp apart (within-chemistry correlations >0.99), where one probe was Infinium I and one was Infinium II. The filtered β scores were normalized using the ComBat R-package.

CgG candidate selection

The following 6 CpGs were used to construct our MRS: cg00574958 within CPT1A, cg02650017 within PHOSPHO1, cg06500161 within ABCG1, cg11024682 within SREBF1, cg18181703 within SOCS3, and cg19693031 within TXNIP. To select the CpGs for the score, first we started with results from GOLDN (European Ancestry) and HyperGEN (African Ancestry) EWAS, which found replicated association of CPT1A and ABCG1 with MetS, respectively. In those studies, no other genes were statistically significant in the discovery cohort and replicated externally. However, prior work in GOLDN and HyperGEN showed robust associations between cg00574958 (CPT1A) and lipids/BMI and between cg06500161 (ABCG1) and fasting glucose/insulin. To expand our score beyond GOLDN and HyperGEN findings, we included the CpG sites in PHOSPHO1, SREBF1, TXNIP and SOCS2 which—together with ABCG1 –comprised the type 2 diabetes methylation risk score published by Chambers, et al [10]. Those additional CpGs were selected due to their association with multiple MetS component traits across at least two race groups in multiple cohorts. Helping to further validate our gene selection a more recent EWAS of MetS reported replicated association for three of our 6 CpGs sites in TXNIP, ABCG1, and SREBF1 [30]. Additional references for these CpGs and their biological relevance are included in Table 1.

Table 1

CPG selection literature review.

Gene	Trait(s)	Function	Race/Ethnicity groups	Direction of Association (MetS and/or Diabetes)
ATP Binding Cassette sub-family G member 1 (ABCG1)	Lipids [10, 12, 16, 30, 47, 48], diabetes-related [10–12], adiposity [5, 30], incident CHD [49]	Macrophage, cholesterol, and phospholipid transport	Europeans [10–12, 47–49], African Americans [5], Asians [10]	+(23/12)
Carnitine Palmatoyl Transferase 1A (CPT1A)	Lipids [16, 47, 48, 50], Blood pressure [14], adiposity [5, 51], metabolic syndrome [52], adiponectin [53]	Fatty acid oxidation	Europeans [14, 47, 48, 50–52, 54], African Americans [5, 14, 53], Hispanic/Latino [14]	-(36)_
Phosphatase, orphan 1 (PHOSPHO1)	Lipids [10, 12, 48], diabetes-related [12], adiposity [12]	Glycerophospholipid biosynthesis and metabolism	Europeans [10, 12, 48], Asians [10]	-(23)
Suppressor of cytokine signaling 3 (SOCS3)	Lipids [12], diabetes-related, [10], adiposity [12, 55], metabolic syndrome [56]	Regulates cytokine or hormone signaling, inhibits STAT3 activation	Europeans [10, 55, 56], Asians [10]	-(23)
Sterol regulatory element binding transcription factor 1 (SREBF-1)	Lipids, [16, 47, 48], diabetes-related,[10, 30], adiposity [57]	Lipid metabolism and homeostasis	Europeans [10, 12, 47, 48, 57], Asians [10]	+ (23)
Thioredoxin-interacting protein (TXNIP)	Lipids [48], diabetes-related, [10, 30]	Required for the maturation of natural killer cells, suppresses tumor growth	Europeans [10, 12, 48], Asians [10]	-(23)

Methylation risk score model building

A logistic mixed model was used to test the association between methylation at each candidate CpG site and MetS in HyperGEN. We adjusted for age, sex, study site, and estimated blood cell counts as fixed effects, and family structure as a random effect. Parallel models were implemented in GOLDN except methylation principal components replaced the estimated blood cell counts to adjust for cell type impurity (GOLDN was of a single cell type). To calculate the MRS, Z-values from the candidate CpG HyperGEN models described above (shown in Table 2) were utilized as weights and multiplied by the CpG-values and the product was summed to generate a risk score for each sample ((Z1* cpg1 beta score) + (Z2* cpg2 beta score)+ … (Z6* cpg6 beta score)). The mean and standard deviation of the score in HyperGEN was calculated. Each participants MRS was then standardized by subtracting the mean and dividing by the standard deviation. We used a parallel approach to calculate the standardized MRS in GOLDN by using GOLDN CpG values weighted by the HyperGEN Z-values.

Table 2

Baseline characteristics of HyperGEN and GOLDN study participants.

	HyperGEN N = 614			GOLDN N = 995
	MetS -	MetS +	P*	MetS -	MetS+	P*
	n = 346	n = 268		n = 602	n = 393
Sex
Female (%)	61.85	72.76	0.014	56.81	45.29	<0.001
Age	45.67 ±11.02	51.66 ±10.35	<0.0001	44.01 ±16.03	56.16 ±13.88	<0.0001
High WC
WC (cm)	96.56 ±16.91	112.40 ±16.55	<0.0001	89.35 ±13.35	107.57 ±13.89	<0.0001
Elevated BP
SBP (mmHg)	127.59 ±23.72	136.03 ±22.83	<0.0001	110.65 ±14.17	123.21 ±17.54	<0.001
DBP (mmHg)	75.48 ±13.25	75.25±11.84	<0.0001	66.09 ±8.46	71.39 ±9.78	<0.0001
Elevated Triglycerides
	77.00 ±35.82	125.50 ±134.61	<0.0001	91.44 ±61.22	176.92 ±107.2	<0.0001
Reduced
HDL
Cholesterol	58.75 ±15.52	46.93 ±12.45	<0.0001	49.54 ±13.22	39.22 ±11.44	<0.0001
Elevated FG
FG	89.00 ±31.22	110.00 ±69.88	<0.0001	94.0 ±10.34	105.0 ±18.91	<0.0001
MetS MRS	-0.38 ±0.95	0.31 ±0.94	<0.0001	-0.30 ±0.93	0.47 ±0.92	<0.0001
Metabolic Components**
0	57	0		174	0
1	115	0		214	0
2	169	0		214	0
3	0	149		0	187
4	0	77		0	138
5	0	42		0	68

Abbreviations: MetS = Metabolic Syndrome, WC = Waist Circumference, BP = Blood Pressure, SBP = systolic blood pressure, DBP = diastolic blood pressure, HDL = high-density lipoprotein, FG = Fasting glucose.

Thresholds: (1) Waist circumference (≥ 88 cm for women and ≥ 102 cm for men), (2) elevated triglycerides (≥ 150 mg/dL) or on treatment for dyslipidemia (statin and/or fibric acid derivative), (3) reduced high-density lipoprotein (HDL) cholesterol (< 40 mg/dL in men and < 50 mg/dL in women) or on treatment for dyslipidemia (statin and/or fibric acid derivative), (4) elevated blood pressure (systolic ≥ 130 and/or diastolic ≥ 85 mmHg) or antihypertensive drug treatment in a patient with a history of hypertension), and (5) elevated fasting glucose (≥ 100 mg/dL) or drug treatment for elevated glucose.

*Significance determined using chi-square test for categorical, t-test for continuous, or kruskal test for non-parametric continuous variables with 95% CI.

**Metabolic components are high waist circumference, elevated triglycerides, reduced HDL cholesterol, elevated blood pressure, and elevated fasting glucose.

***There are 5 individuals with NA for 1–2 components making the total 341 rather than 346 for MetS- in HyperGEN.

Methylation risk score model performance testing

We compared both HyperGEN and GOLDN characteristics between individuals with (MetS+) and without (MetS-) metabolic syndrome. Significance of these characteristics were calculated using a simple t test for continuous traits and a chi-square test for binary traits. We then tested the association between the standardized MRS and MetS (outcome) using a logistic mixed model in HyperGEN adjusting for age, sex, study site, 4 ancestry principal components, estimated blood cell counts as fixed effects, and family id as a random effect. We conducted a 100,000-permutation test to evaluate statistical significance of the relationship between the MRS and MetS in HyperGEN. In GOLDN we used a logistic mixed model to test the association between the GOLDN standardized MRS and MetS adjusting for age, sex, study site, and methylation PCs as fixed effects, and family id as a random effect. Secondary analysis of the score with individual MetS components and by sex were carried out in each study.

Results

Study population characteristics

Demographic characteristics of the HyperGEN (N = 614) and GOLDN (N = 995) populations—with and without MetS—are presented in Table 2. The majority of participants were female in both HyperGEN (66.61%) and GOLDN (52.26%), with an overall mean age of 49 and 50 years, respectively. Participants with MetS (MetS+) were older compared to those without (MetS-) (HyperGEN: 52 ± 10 years and 46 ±11 years, and GOLDN: 56 ±13 and 44 ±16, respectively) and more likely to be male in GOLDN and female in HyperGEN, respectively. In HyperGEN 56.2% of participants with MetS had 3 of the 5 (i.e. WC, BP, TG, HDL and FG) possible components (N = 149) and fewer had 4 (N = 77, 28.7%) and 5 (N = 42, 15.7%) components, while in GOLDN, 47.5% of individuals met 3 out of 5 components (N = 187), 35.1% had 4 of 5 (N = 138) and 17.3% had 5 of 5 (N = 68) components.

Table 3 shows the 6 candidate CpG association results for MetS in HyperGEN and GOLDN. With the exception of cg02650017 in PHOSPHO1, which was not significant in either study, the direction of association of the CpG with MetS was consistent between GOLDN and HyperGEN with at least marginal significance. Only cg18181703 in SOC3S was not associated with MetS in GOLDN. Both CPT1A cg00574958 and ABCG1 cg06500161 were strongly associated with MetS in both studies (P<0.0001). Finally, the direction of association for CPT1A, ABCG1, SOCS3, TXNIP and SREBF1 was consistent with that reported in the literature for MetS and/or diabetes (Table 1).

Table 3

Single CPG and MetS association results for GOLDN and HyperGEN.

	Genes	CpG site	Estimate	SE	Z-score	P-value
HyperGEN* Participants (N = 614)	CPT1A	cg00574958	-0.495	0.113	-4.396	1.10E-05
	PHOSPHO1	cg02650017	-0.133	0.101	-1.312	0.189
	ABCG1	cg06500161	0.550	0.113	4.865	1.15E-06
	SREBF1	cg11024682	0.570	0.119	4.777	1.78E-06
	SOCS3	cg18181703	-0.204	0.095	-2.153	0.031
	TXNIP	cg19693031	-0.402	0.102	-3.939	8.18E-05
GOLDN** Participants (N = 994)	CPT1A	cg00574958	-0.852	0.113	-7.539	4.72E-14
	PHOSPHO1	cg02650017	0.062	0.097	0.632	0.527
	ABCG1	cg06500161	0.394	0.099	3.966	7.31E-05
	SREBF1	cg11024682	0.270	0.119	2.269	0.023
	SOCS3	cg18181703	-0.059	0.095	-0.623	0.533
	TXNIP	cg19693031	-0.267	0.092	-2.915	0.004

*HyperGEN model adjusted for age, sex, study site, 4 ancestry principle components, estimated blood cell counts (CD8T cells, CD4T cells, Natural Killer cells, B-cells, Monocyte cells) as fixed effects, and family id as a random effect.

**GOLDN model adjusted for age, sex, study site, 4 methylation principle components, and family id as a random effect.

Risk score discovery and validation

Fig 1 shows the normal distribution of the standardized MRS in the GOLDN cohort. Results from association analyses of the MRS with MetS after adjustment for covariates in HyperGEN and GOLDN are presented in Table 4. In HyperGEN, the MRS was significantly associated with MetS (permutation test p<0.0001), with each standard deviation (SD) increase in the score associated with 2.25 higher odds of having MetS (OR = 2.25; 95% CI: 1.79–2.86). The MetS and MRS relationship was also significant in GOLDN where similarly, a 1 SD increase in the score was associated with 2.45 higher odds of having MetS (OR = 2.45; 95% CI: 2.02–3.00). Results for the individual components show the score was most strongly associated with waist circumference, triglycerides and glucose in each study (Table 4). Results stratified by sex show showed the score was generally consistent by gender though slightly more significant in females. We also tested the interaction between MetS and sex, however that interaction was not significant in either HyperGEN or GOLDN.

Fig 1

GOLDN MetS-score distribution, N = 994.

Table 4

Cohort-specific association of the Methylation Risk Score (MRS) with MetS and MetS components.

Outcomes	HyperGEN*	P	GOLDN**	P
MetS	2.25 (1.80–2.86)	6.36E-10	2.46 (2.03–3.0)	2.0E-16
MetS (M only)	1.67(1.21–2.33)	2.10E-3	2.16 (1.65–2.84)	2.49E-8
MetS (F only)	2.34 (1.78–3.02)	3.29E-10	2.75 (2.06–3.67)	7.53E-12
Elevated BP	1.36 (1.12–1.68)	2.52E-3	1.66 (1.35–2.04)	1.65E-6
High WC	1.81 (1.46–2.25)	6.16E-8	1.86 (1.56–2.23)	7.49E-12
Low HDL	1.24 (1.04–1.49)	0.013	1.35 (1.15–1.59)	2.50E-4
Elevated TG	2.10 (1.61–2.53)	1.54E-9	2.42 (2.00–2.94)	2.00E-16
Elevated FG	2.21 (1.80–2.73)	5.42E-14	1.81 (1.50–2.17)	2.14E-10

*HyperGEN model adjusted for age, sex, study site, 4 ancestry principle components, estimated blood cell counts (CD8T cells, CD4T cells, Natural Killer cells, B-cells, Monocyte cells) as fixed effects, and family id as a random effect. (N = 205 M ales (M), 409 Females (F))

**GOLDN model adjusted for age, sex, study site, 4 methylation principle components, and family id as a random effect. (N = 476 Males(M), 520 Females(F))

MetS Components: elevated waist circumference/WC (≥ 88 cm for women and ≥ 102 cm for men), elevated triglycerides/TG (≥ 150 mg/dL) or on treatment for dyslipidemia (statin and/or fibric acid derivative), low high-density lipoprotein/HDL cholesterol (< 40 mg/dL in men and < 50 mg/dL in women) or on treatment for dyslipidemia (statin and/or fibric acid derivative), elevated blood pressure/BP (systolic ≥ 130 and/or diastolic ≥ 85 mmHg) or antihypertensive drug treatment in a patient with a history of hypertension), and elevated fasting glucose/FG (≥ 100 mg/dL) or drug treatment for elevated glucose.

Discussion

Signatures of DNA methylation associated with cardiometabolic diseases have not been widely tested for their utility in generating genomic risk scores. With better replication results across external groups and even by race these CpGs may prove useful for evaluating disease risk. Here, we introduce a six-CpG methylation risk score estimate for MetS that was consistent in two independent populations (HyperGEN and GOLDN). Overall, the successful performance of this MRS in two different racial populations, provides promise for future exploration of MRSs for complex disease prediction.

A substantial number of studies support the role of DNA methylation in MetS and its components. However, unlike studies of single nucleotide polymorphisms (SNPs) that have extensively considered the utility of GRS and PRS (noting many limitations, especially with respect to race), relatively few publications have included MRS [3, 31]. For instance, Hamilton et al, reported a positive association between an epigenetic BMI risk score and higher BMI (R2 = 0.1) in the Lothian Birth Cohort [32]. MRS for arterial stiffness measurements have been reported using data from the REGICOR and Framingham studies. In that study, two different MRS (based on alternate analytical approaches) were directly associated with arterial distensibility coefficient and inversely with pulse wave velocity [33]. Braun and others constructed a MRS in the Rotterdam study for HDL and triglycerides, finding that HDL-C levels decreased as quartiles of MRS increase, while triglyceride levels increased from the first quartile to the second quartile but remained similar for the third quartile and the fourth quartile of the MRS [34]. In another EWAS for BMI, a MRS constructed from the findings predicted future development of type 2 diabetes [35]. Finally, in the study of type 2 diabetes (~2000 Asian Indians and ~1000 Europeans) the Chambers et al score was associated with developing type 2 diabetes (RR of type 2 diabetes incidence 1·41 per 1 SD change in methylation score; p = 1·3 × 10−26) [10]. Along with our study these findings suggest promise in the use of methylation scores for metabolic disease risk prediction.

Generation of genomic risk scores is an evolving field. Some approaches focus on biological candidates and/or statistically significant markers for inclusion, while others include hundreds of thousands or more markers capturing small polygenic effects [22, 36]. We selected a limited number of CpGs that had strong statistical association with relevant traits and replication in at least two race groups. Each of the CpGs was highly biologically relevant being associated with fatty acid metabolism (CPT1A), reverse cholesterol transport particularly HDL (ABCG1), oxidative stress mediation (TXNIP), phosphatase activity involving glycerophospholipid biosynthesis (PHOSPHO1), inhibition of cytokine signal transduction by binding to tyrosine kinase inhibitors including insulin and leptin receptors (SOCS3), and sterol biosynthesis (SREBF1) (Table 1). Other studies noted above took a similar approach to construct methylation risk scores using statistically significant findings (20, 23) or a combination of statistical significance and the literature to select a short list of CpGs (21).

MetS is strongly associated with risk for developing future diabetes and atherosclerotic and nonatherosclerotic cardiovascular disease (CVD). Though beyond the scope of this cross-sectional work, CpG sites associated with MetS may be the cause or consequence of the condition (or features of the condition). Therefore, a MetS MRS score may capture information about cumulative exposure to risk trait features. Importantly, this information could improve upon existing risk algorithms (constructed from clinical, demographic and lifestyle factors) used to predict future cardiometabolic disease. For instance, in a study set in the Bogalusa Heart Study five well-documented diabetes risk scores (non-genomic) were tested, and all showed significant associations with development of incident diabetes. These five unique risk scores differed slightly by make-up of 5–10 traditional risk factors (e.g. hypertension, smoking, family history of diabetes, age, and waist circumference), but, in general, showed good specificity but poor sensitivity. Because of the low sensitivity, the authors concluded that an opportunity remains to develop a new, more sensitive diabetes prediction tools for black and white young adults [37]. The field is similar with respect to CVD risk prediction where an excess of models and different recommendations limit algorithm use [38, 39]. Given the importance of MetS to the cardiometabolic disease landscape, and that MRS may help refine risk metrics in diverse populations for important clinical sequelae, further evaluation of these scores should be considered for disease prediction.

While there are limitations to basing a MRS for MetS from blood-based DNA methylation (due to the proxy nature of blood as a surrogate tissue for organs involved in MetS) [40-42], the utility of blood-based DNA methylation has been proven to be highly feasible and replicable for population studies of glycemic, lipid, and other metabolic traits (Table 1). While we only had access to data derived from blood in GOLDN and HyperGEN, other studies have shown association whole blood and buffy coat are successful proxies for tissues in epigenetic studies [43-45]. Much of the success behind the use of blood/buffy coat as a proxy for tissue can be attributed to the development of new methods to account for things such as cell-type heterogeneity, which we can now accurately account and control for in statistical analyses [46]. To identify whether the MRS was capturing specific components of MetS, we also performed individual association analyses between the individual components of MetS and MRS. Our results suggest that relationship between DNA methylation biomarkers and the development of MetS may be driven by fasting glucose and fasting triglycerides, however, those associations were not significantly stronger than associations observed with other MetS components, suggesting relatively similar contributions from all components of MetS to the MRS in HyperGEN and GOLDN. Differential patterns of sex and MetS were also considered in this study. While both HyperGEN and GOLDN had higher proportions of females compared to males–particularly in HyperGEN–sensitivity analyses showed that the MetS score was fairly consistent across sex, with slightly higher associations noted in females compared to males. However, as noted previously, the interaction analyses between MRS and sex were not statistically significant, suggesting that the MRS is unlikely to have been impacted by sex in this study. The cross-sectional nature of this study, and lack of gold standard definition for MetS are potential limitations that should be considered in future MRS assessments. However, this study strengthened by the availability of CpGs paired metabolic data in two well characterized populations enabling both discovery and validation.

In summary, we developed a MRS for MetS using existing EWAS data from two population studies of different race groups. Addition of the calculated MRS variable to a basic model of MetS further improved model fit in the study used for score validation. Given the strength of association observed in the current study and the strong body of literature surrounding the CpG loci contributing to the methylation risk score, future studies may further development of this metric for evaluating risk of metabolic syndrome.

24 Jun 2021

PONE-D-21-04138

­A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies

PLOS ONE

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Reviewer #1: In this study Hidalgo, et al develop a risk score for metabolic syndrome, based on DNA methylation at 6 CpG sites identified through a prior literature review. Additionally, this study is novel for its focus on a population of primarily African ancestry in addition to one of European ancestry (in contrast to the many studies that primarily focus on the latter). The article is generally well-written but I believe that it does not go far enough into its analyses, and does not provide enough crucial details, to merit publication without substantial revisions.

My primary concern is the lack of detail regarding the selection process for the six CpGs. Based on my own (admittedly, cursory) literature search, it seems like a lot more has been published than that for MetS alone (not to mention its individual components). Why did these six CpGs make the cut, and others did not? MetS and/or related traits is a very, very broad category of the literature. Surely there are more than 6 CpGs that have been associated with them, so how did the authors narrow their list further? The reader needs more information on this selection process in order to evaluate its rigor. Where did they search? How many articles did they review? How many CpGs did they decide against including?

I find this particularly puzzling given the focus on African-ancestry populations in the introduction and discussion- four of the six CpGs used to construct the MRS were only validated in European- and Asian-ancestry individuals, and by the authors’ own arguments they shouldn’t be assumed to have a relationship in African-ancestry individuals. Additionally, a number of the single-CpG results are non-significant if you account for multiple testing. That isn’t necessarily a problem, but it does mean that the theoretical basis for selecting these six as candidates needs to be very well-justified.

I also think that this discussion needs to be rewritten and expanded upon in more detail. There is no mention of the specific genes used to construct the MRS, their functions, their roles in MetS pathogenesis, or why there might be an association at all. Instead, the authors discuss other risk scores developed to measure related but different cardiovascular endpoints. There is very little presented here to relate the authors’ findings to the wider literature, beyond the fact that they all studied a methylation risk score for something.

On a related point, I think the authors could improve the impact of this study (and find more to talk about in the discussion) by running a set of secondary analyses. Specifically, similar models as for Table 4, but instead of MetS as the dichotomous outcome use each of the individual components of MetS and model them separately. By comparing these associations (even without assessing statistical significance) the authors can potentially identify which specific components the MRS is capturing, which in turn could provide some insight into the relationship between DNA methylation biomarkers and the development of MetS.

Some minor comments:

- Consider adding the MRS risk score to Table 1, as some descriptive analysis will be a good way to assess its performance.

- The methods section for HyperGEN should specify the source tissue for the DNA. It’s pretty clear elsewhere that this is blood, but it still should be specified in the methods.

- On page 8 the authors refer to Table 1 when talking about the consistency between their results and the prior literature, in terms of direction of association. However, this information is not presented in Table 1- only the citations are. The authors should either a) add this information to Table 1, or b) cite specific studies and not Table 1 when making this point in the discussion.

- Why was model fit in Table 5 compared in GOLDN but not HyperGEN?

- The authors may want to consider de-emphasizing the model fit results in their concluding paragraph. I’m not sure a ~70-point difference in AIC/BIC is worth making it a primary conclusion, even if does represent an improvement in fit.

Reviewer #2: The current study tries to create a methylation risk score (MRS) for metabolic syndrome (MetS) and performs an analysis to demonstrate the utility of the MRS across two different race groups. The study is well designed and executed and the topic is interesting for the scientific community and if validated in a longer cohort could have clinical utility. From my point of view there are no relevant concerns to be addressed, I have only minor comments:

-The main objective should be stated more clearly in both abstract and introduction.

-I suggest to include a paragraph in the introduction section regarding to the association between epigenetic mechanisms, mainly methylation marks, on the obesity and metabolic syndrome to highlight the rationale of this study more in depth. Some of these studies could be on help (PMID: 28211912, PMID: 27477082, PMID: 30887406).

Reviewer #3: It is a very well written article and authors should be commended for this. Few comments

1. Since MetS parameters are known to show differential patterns in sex, and owing to the fact both HYPER AND GENO studies have higher female participants-i would like authors to discuss more about how sexual dimorphism could have impacted the MRS score.

2. The authors should also comment why only one cell type was used in the validation but not all or the vice versa, because one would assume that the gene expression would alter between different cell types.

3. The authors tend to ignore referencing the facts throughout the study, I would like the authors to please go back to the introduction and discussion and use appropriate references. for example-in the first paragraph in introduction " To date, despite impressive effect sizes, strong statistical significance, and successful external replication in the EWAS literature

(even across race/ethnic groups), few studies have examined the polygenomic effects of CpG sites (e.g., methylation risk scores (MRS)) on complex diseases ". I don't see use of reference to literature

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Reviewer #2: No

Reviewer #3: Yes: Farha Ramzan

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18 Oct 2021

We thank the reviewers for their thorough review of our paper and the astute feedback provided. We have addressed the reviewers’ comments as follows:

Responses to Reviewer Comments

Reviewer #1:

Major comments

1. “…lack of detail regarding the selection process for the six CpGs.” We thank the reviewer for highlighting this. We have added additional details about our CpG selection process to the Methods section, specifically the subsection titled ‘CgG candidate selection’. (lines 170-188)

2. “…discussion needs to be rewritten and expanded upon in more detail.” We have added additional context to the discussion section accordingly. (lines 292-315 and 341-345)

3. “…running a set of secondary analyses. Specifically, similar models as for Table 4, but instead of MetS as the dichotomous outcome use each of the individual components of MetS and model them separately.” We performed additional analyses and have subsequently updated Table 2 and Table 4. In our prior work, we have shown the robustness of CpGs included in our score with each of the MetS components, as well as with MetS. We have included references to those studies in Table 1, including directions of effect and relevant references.

Minor comments:

1. “Consider adding the MRS risk score to Table 1, as some descriptive analysis will be a good way to assess its performance.” We have included the MRS risk score to table 2, and have included all references of published methylation risk scores (MRS) to Table 1.

2. “The methods section for HyperGEN should specify the source tissue for the DNA. It’s pretty clear elsewhere that this is blood, but it still should be specified in the methods.” We have updated the methods section to make this detail clearer. (line 158)

3. “On page 8 the authors refer to Table 1 when talking about the consistency between their results and the prior literature, in terms of direction of association. However, this information is not presented in Table 1- only the citations are. The authors should either a) add this information to Table 1, or b) cite specific studies and not Table 1 when making this point in the discussion. We have added further details about each of the components and relevant literature to the text (lines 92-96), added results from analyses performed in each of the components, as well as results from our sex-stratified analyses. (Table 4)

4. “Why was model fit in Table 5 compared in GOLDN but not HyperGEN?” We have removed this information to avoid confusion about model fits, and have added results for additional analyses performed in tables 2 and 4.

5. “The authors may want to consider de-emphasizing the model fit results in their concluding paragraph. I’m not sure a ~70-point difference in AIC/BIC is worth making it a primary conclusion, even if does represent an improvement in fit.” We have made changes to the conclusion paragraph accordingly. (line 356)

Reviewer #2: “…there are no relevant concerns to be addressed, I have only minor comments”:

1. “The main objective should be stated more clearly in both abstract and introduction.” We thank the reviewer for this recommendation. We have updated the text in the introduction and abstract to make this point clearer. (lines 35-37 and lines 74-75)

2. “I suggest to include a paragraph in the introduction section regarding to the association between epigenetic mechanisms, mainly methylation marks, on the obesity and metabolic syndrome to highlight the rationale of this study more in depth. Some of these studies could be on help (PMID: 28211912, PMID: 27477082, PMID: 30887406).” We thank the reviewer for the suggested references. We have integrated them into both the introduction section and Table 1 of our paper. (line 82 and Table 1)

Reviewer #3: It is a very well written article and authors should be commended for this. Few comments

1. “Since MetS parameters are known to show differential patterns in sex, and owing to the fact both HYPER AND GENO studies have higher female participants-i would like authors to discuss more about how sexual dimorphism could have impacted the MRS score.” We performed these additional analyses and have added the results to the paper. (lines 218-219, 255-260, and 305-315)

2. “The authors should also comment why only one cell type was used in the validation but not all or the vice versa, because one would assume that the gene expression would alter between different cell types.” We agree with the reviewer that additional cell types would have been important to study, however, for this study we only had one cell type available in both HyperGEN and GOLDN. We have expanded on this point in the discussion section of the paper. (lines 341-345)

3. “The authors tend to ignore referencing the facts throughout the study, I would like the authors to please go back to the introduction and discussion and use appropriate references.” We regret that we did not include this information previously, and have added this literature and relevant references throughout, including Table 1 of the paper.

Submitted filename: Reviews for MetS paper_clean.docx

Click here for additional data file.

28 Oct 2021

­A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies

PONE-D-21-04138R1

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Kyle J Burghardt

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PLOS ONE

Additional Editor Comments (optional):

Dr. Hidalgo, Thank you for your revisions to your manuscript. You have answered all reviewer critiques in a clear and complete manner. Thank you for your patience as well. I did receive your earlier query regarding manuscript status but the manuscript was not in my control at that time. I received the revision this morning and processed immediately. Best. Kyle

4 Nov 2021

PONE-D-21-04138R1

­­A 6-CpG Validated Methylation Risk Score Model for Metabolic Syndrome: The HyperGEN and GOLDN Studies

Dear Dr. Hidalgo:

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