Differentially methylated regions (DMRs) in PON3 gene between responders and non-responders to a weight loss dietary intervention: a new tool for precision management of obesity

Figures & data

Table 1. Anthropometric and metabolic parameters before and after the intervention period in non-responders and responders

	Non-responders (NR) N = 63			Responders (R) N = 64			NR vs R Baseline ^#P-value
	Baseline	After intervention	*P-value	Baseline	After intervention	*P-value
Body weight (kg)	88.4 ± 1.8	83.0 ± 1.7	<0.001	87.8 ± 1.4	75.2 ± 1.2	<0.001	NS
Body mass index (kg/m^2)	31.9 ± 0.5	29.89 ± 0.45	0.003	31.8 ± 0.39	27.2 ± 0.36	<0.001	NS
Fat mass (kg)	33.8 ± 1.1	29.1 ± 1.0	<0.001	33.2 ± 0.9	23.7 ± 0.9	<0.001	NS
Waist circumference (cm)	103.0 ± 1.4	96.7 ± 1.4	<0.001	102.3 ± 1.3	89.7 ± 1.3	<0.001	NS
Diastolic pressure (mm Hg)	77 ± 2	78 ± 1	0.654	80 ± 1	73 ± 1	<0.001	NS
Systolic pressure (mm Hg)	126 ± 3	119 ± 2	0.017	127 ± 2	116 ± 2	<0.001	NS
Total cholesterol (mg/dL)	216.5 ± 5.0	210.6 ± 5.1	0.078	221.3 ± 4.9	189.0 ± 4.0	<0.001	NS
HDL cholesterol (mg/dL)	53.2 ± 1.4	51.3 ± 1.4	0.008	58.0 ± 1.9	52.2 ± 1.2	<0.001	0.048
LDL cholesterol – Friedelwald (mg/dL)	143.6 ± 4.8	137.7 ± 5.3	0.108	143.1 ± 4.1	122.2 ± 3.5	<0.001	NS
Triglycerides (mg/dL)	98.3 ± 7.4	96.8 ± 7.4	0.671	101.3 ± 5.8	72.4 ± 3.4	<0.001	NS
Glucose (mg/dL)	96.1 ± 1.3	93.1 ± 1.2	0.008	96.9 ± 1.5	90.3 ± 1.1	<0.001	NS
Insulin (mU/L)	8.20 ± 0.63	6.07 ± 0.46	0.001	7.86 ± 0.62	4.17 ± 0.27	<0.001	NS
ALT (U/L)	22.50 ± 1.46	22.23 ± 1.94	0.809	23.30 ± 1.52	19.12 ± 1.52	<0.001	NS
AST (U/L)	21.30 ± 0.76	22.78 ± 1.69	0.383	21.23 ± 0.84	20.44 ± 0.75	0.353	NS
Uric acid (mg/dL)	5.00 ± 1.33	5.07 ± 0.15	0.732	5.19 ± 0.15	4.85 ± 0.12	<0.001	NS
Leptin (ng/dL)	38.9 ± 3.6	25.8 ± 2.4	<0.001	39.6 ± 3.6	12.4 ± 1.1	<0.001	NS
Adiponectin (ng/mL)	11.5 ± 0.6	11.5 ± 0.6	0.904	11.3 ± 0.7	12.1 ± 0.6	0.04	NS
TNF-alpha (pg/mL)	0.89 ± 0.40	0.89 ± 0.39	0.840	0.86 ± 0.48	0.85 ± 0.41	0.865	NS
C- Reactive protein (mg/L)	3.27 ± 0.40	2.31 ± 0.31	<0.001	2.69 ± 0.30	1.93 ± 0.33	0.001	NS
HOMA-IR	1.99 ± 0.17	1.41 ± 0.11	0.001	1.94 ± 0.17	0.95 ± 0.07	<0.001	NS
Leptin/Adiponectin ratio	3.75 ± 0.39	2.59 ± 0.27	<0.001	3.79 ± 0.36	1.15 ± 0.12	<0.001	NS
Atherogenic Index (AIP)	−0.135 ± 0.03	−0.123 ± 0.03	0.520	−0.143 ± 0.030	−0.237 ± 0.020	<0.001	NS

Data is represented as mean ± SE. *P-value was calculated by paired t-test and ^#P-value was calculated by unpaired t-test, p < 0.05 was considered statistically significant.

Figure 1. Volcano plot showing the differential methylation probes (DMP) between responders and non-responders. Volcano plot for DMP with raw p-value < 0.05, deltaBeta ≥ |5|% and B-Limma ≥ 0. Effect size is displayed along X-axis, highlighting deltaBeta ≥ |5|%. On the Y-axis is displayed the negative logarithm of the association (adjusted p-value) for each CpG site. Points below the horizontal grey line are not statistically significant according to B-Limma and FDR 0.05. The red dots represent the 63 CpGs significant after filtering by B-Limma ≥ 0, and deltaBeta ≥ |5|% (adjustment 1).

Table 2. Genomic data of CpG sites in a DMR located at PON3 gene at baseline. Methylation levels (beta values) of CpG sites located at PON3 gene in non-responders and responders

Illumina_ID	Chromosomal position^1	Strand^2	Gene	Region^3	CGI^4	Non-responders	Responders	P-value	*Methylation status
cg15927196	7:95,025,905	R	PON3	TSS1500	Island	0.152 ± 0.033	0.153 ± 0.038	0.926	-
cg15500865	7:95,025,924	F	PON3	TSS1500	Island	0.068 ± 0.012	0.072 ± 0.012	< 0.0001	Hypermethylated
cg25572105	7:95,025,955	R	PON3	TSS1500	Island	0.058 ± 0.010	0.062 ± 0.012	0.096	-
cg25161512	7:95,026,066	R	PON3	TSS1500	Island	0.097 ± 0.024	0.115 ± 0.030	0.001	Hypermethylated
cg11435506	7:95,026,073	R	PON3	TSS1500	Island	0.138 ± 0.028	0.165 ± 0.036	< 0.0001	Hypermethylated
cg03301582	7:95,026,095	R	PON3	TSS1500	Island	0.103 ± 0.025	0.120 ± 0.032	0.002	Hypermethylated
cg08898155	7:95,026,097	R	PON3	TSS1500	Island	0.145 ± 0.026	0.163 ± 0.035	0.002	Hypermethylated
cg04080282	7:95,026,106	R	PON3	TSS1500	Island	0.302 ± 0.036	0.324 ± 0.038	0.001	Hypermethylated
cg26457160	7:95,026,121	F	PON3	TSS1500	Island	0.469 ± 0.028	0.490 ± 0.036	< 0.0001	Hypermethylated
cg10329418	7:95,026,181	R	PON3	TSS1500	Shore	0.230 ± 0.035	0.252 ± 0.042	0.031	Hypermethylated
cg27166921	7:95,026,193	F	PON3	TSS1500	Shore	0.239 ± 0.027	0.253 ± 0.037	0.015	Hypermethylated
cg24750391	7:95,026,211	F	PON3	TSS1500	Shore	0.333 ± 0.033	0.355 ± 0.038	0.001	Hypermethylated
cg08461772	7:95,026,248	R	PON3	TSS1500	Shore	0.393 ± 0.043	0.418 ± 0.045	0.001	Hypermethylated

¹CpG locations were mapped using GRCh37 version of the genome from Ensembl platform. FDR < 0.05. ² Strand: Reverse (R), Forward (F) designation of the design strand; ³TSS1500: Transcription Start Site that covers 200 to 1500 nucleotides upstream of TSS. ⁴CGI: location of the CpG relative to the CpG island. Data are expressed as beta values mean ± SEM. P-value was calculated by paired t-test, p < 0.05 was considered significant. *Methylation status in responders in comparison to non-responders.

Figure 2. Differentially methylated regions (DMRs) identification. Plot representing the comparison (between responders and non-responders) of the methylation levels of the individual CpGs included in the DMR identified in PON3 gene. Solid lines represent mean values and dashed lines represent the Loess line, where a hypermethylation is observed in the responders group (pink solid line). TSS1500: Transcription Start Site that covers 200 to 1500 nucleotides upstream of TSS.

Figure 3. BMI loss and DNA methylation. (a) Correlation between DNA methylation levels (z-score) in PON3 DMR and BMI loss and (b) correlation between DNA methylation levels (z-score) in PON3 DMR and PON3 protein levels in serum.

Figure 4. Oxidized LDL and paraoxonase activity. (a) Oxidized LDL levels (oxLDL) in Non-Responders and Responders at baseline. (b) Correlation between oxLDL levels and paraoxonase (PON) activity in serum. ns: non-significant.

Figure 5. Z-score and glucose metabolism. Relation between z-scores of PON3 DMR methylation and glucose metabolism parameters. Values reflect the significant change in z-scores from DMR methylation (11 CpG sites) according to insulin (a) or HOMA-IR at baseline (b).