Altered PTPRD DNA Methylation Associates with Restricted Adipogenesis in Healthy First-Degree Relatives of Type 2 Diabetes Subjects

Figures & data

Table 1. Clinical characteristics of first-degree relative and CTRL subjects.

Measure	FDR	CTRL
n (females/males)	9 (5/4)	11 (6/5)
Age, years	42.3 ± 2.9	38.7 ± 2.3
BMI, kg/m^2	25.4 ± 0.5	24.5 ± 0.7
Fat percent, %	26.9 ± 2.4	24.0 ± 1.9
Waist to hip ratio (WHR)	0.90 ± 0.02^‡	0.81 ± 0.02
Total triglycerides, mmol/l	1.5 ± 0.6^‡	0.8 ± 0.2
HDL cholesterol, mmol/l	1.3 ± 0.2	1.6 ± 0.3
Cell size, μm	100.2 ± 1.7^§	89.4 ± 1.9
GIR/bw, mg/kg/min	7.9 ± 0.6^‡	11.5 ± 0.8
f-insulin, pmol/l	60.1 ± 8.0^‡	35.4 ± 3.9
fb-glucose, mmol/l	4.8 ± 0.1^‡	4.4 ± 0.1
OGTT p-glucose 2 h, mmol/l	6.6 ± 0.6	4.8 ± 0.4
HOMA-IR index	2.0 ± 0.8^§	1.1 ± 0.4

Clinical characteristics of FDR and CTRL subjects. All data are expressed as means ± SD. Statistical significance was tested by a two-tailed Mann“Whitney U-test.

  p<0.05.

^‡ p<0.01.

^§ p<0.001, FDR vs CTRL.

BMI: Body mass index; CTRL: Control; f-insulin: Fasting insulin; fb-glucose: Fasting blood-glucose; FDR: First-degree relatives; GIR/bw: Glucose infusion rate/body weight; HDL: High-density lipoproteins; HOMA-IR: Homeostatic model assessment of insulin resistance; OGTT: Oral glucose tolerance test; p-glucose: Plasma-glucose.

Table 2. DMR distribution in first-degree relative subjects.

Genomic features	Hypo-DMR in FDR	Hyper-DMR in FDR
Upstream	199	16
5²-UTR	25	-
Exon/CD	343	4
Intron	1955	188
3²-UTR	80	3
Downstream	26	2

DMR distribution in FDR subjects. The number and distribution of identified hypo- and hypermethylated DMR across different gene elements (e.g., 5²-UTR, Exons/CD, Intron, 3²-UTR) in the FDR samples.

CD: Coding sequence; DMR: Differentially methylated region; FDR: First-degree relatives; UTR: Untranslated region.

Figure 1. DNMT3A mRNA expression in subcutaneous pre-adipocytes from first-degree relative and CTRL subjects.

(A) DNMT3A mRNA expression was measured by qPCR in SVF from FDR (n = 9) and control (CTRL; n = 11) subjects. Data points represent AU from each individual subject. Mean values ± SD are also shown. *p<0.05 in a two-tailed Mann“Whitney U-test. (B) Representative immunoblot analysis of DNMT3A and β-ACTIN protein levels in SVF from FDR (n = 6) and control (CTRL; n = 6) subjects. Data are expressed as arbitrary units and shown as mean values ± SD. *p<0.05 in a two-tailed Mann-Whitney U-test. (C) Correlation between SVF DNMT3A mRNA levels and adipose cell size in FDR (n = 9) and CTRL (n = 11) subjects. r correlation coefficient and p-values are indicated on the graph.

AU: Absolute units; CTRL: Control; FDR: First-degree relatives; SD: Standard deviation; SVF: Stromal vascular fraction cells.

Table 3. List of the genes differentially methylated and expressed between first-degree relative and CTRL subjects.

Gene symbol	Gene name	log FC expression	log FC methylation	Relative location of DMR
PTPRD	Protein tyrosine phosphatase, receptor type D	1.43	-1.46	Inside intron
A2M	Alpha-2-macroglobulin	1.13	-0.62	Intron/exon
CADPS	Ca2+-dependent secretion activator	1.71	-0.87	Inside intron

List of the genes differentially methylated and expressed between FDR and CTRL subjects. Genes exhibiting significant expression changes associated to intragenic DMR in the FDR. Differences in mRNA expression and DNA methylation are given as logarithmic fold change (log FC), consistently standardized in relation to CTRL subjects.

  PTPRD-associated DMR exhibiting the higher significance as described further on.

CTRL: Control; DMR: Differentially methylated region; FC: Fold change; FDR: First-degree relatives.

Table 4. Characteristics of hypomethylated DMR associated with PTPRD in first-degree relative subjects.

GENE	DMR number	Chromosome and position	Relation to gene region	Relation to CpG island	q-value
PTPRD	Medip_406	chr9:10448607-10450000	Intron	Yes	3.62E-07
	Medip_3624	chr9:9617306-9618320	Intron	No	1.57E-02
	Medip_4422	chr9:10018162-10019196	Intron	Yes	3.17E-02
	Medip_1540	chr9:10171213-10172126	Intron	No	5.30E-04
	Medip_3179	chr9:8712562-8714121	Intron	Yes	9.89E-03
	Medip_4937	chr9:9133607-9134817	Intron	Yes	4.43E-02

Characteristics of hypomethylated DMR associated with PTPRD in FDR subjects. Summary of PTPRD-associated DMR according to chromosome position, gene element and CpG context. PTPRD-DMR validated by bisulphite sequencing is in bold. P-values have been adjusted for multiple testing using false discovery rate method.

Chr: Chromosome; DMR: Differentially methylated region; FDR: First-degree relatives.

Figure 2. PTPRD DNA methylation and mRNA expression in subcutaneous pre-adipocytes from first-degree relative and CTRL subjects.

(A) BS analysis of PTPRD DMR in SVF from FDR (n = 8) and CTRL (n = 9) subjects available from the study group. Data points represent DNA methylation percentage at the PTPRD-associated DMR. Mean values ± SD are also shown. ***p<0.001 in a two-tailed Mann“Whitney U-test. (B)PTPRD mRNA expression was measured by qPCR in SVF from FDR (n = 9) and control (CTRL; n = 11) subjects. Data points represent AU from each individual subject. Mean values ± SD are also shown. **p<0.01 in a two-tailed Mann“Whitney U-test. (C) Correlation between PTPRD DNA methylation and PTPRD mRNA levels in SVF from FDR (n = 8) and CTRL (n = 9) subjects. r correlation coefficient and p-values are indicated on the graph.

AU: Absolute units; BS: Bisulphite sequencing; CTRL: Control; DMR: Differentially methylated region; FDR: First-degree relatives; SD: Standard deviation; SVF: Stromal vascular fraction cells.

Figure 3. Correlation between subcutaneous adipose cell size, PTPRD DNA methylation and its mRNA levels.

(A) Correlation between SVF PTPRD DNA methylation and adipose cell size in FDR (n = 8) and CTRL (n = 9) subjects. (B) Correlation between SVF PTPRD mRNA levels and adipose cell size in FDR (n = 9) and CTRL (n = 11) subjects. r correlation coefficient and p-values are indicated on the graph.

AU: Absolute units; CTRL: Control; FDR: First-degree relatives; SVF: Stromal vascular fraction cells.

Figure 4. Effect of DNA methylation on PTPRD transcriptional activity.

The 590 bp sequence corresponding to the intronic PTPRD-associated DMR chr9:10448607-10450000, was cloned into a pCpGL-promoter vector (pCpGL-PTPRD DMR), upstream the luciferase gene. The construct was either methylated by M.SssI (methylated pCpGL-PTPRD DMR) or mock-treated (unmethylated pCpGL-PTPRD DMR) and then transfected in ChubS7 cells. The data were normalized using a co-transfected renilla luciferase vector and are presented as fold change relative to the mock-treated empty vector (pCpGL). Data are shown as means ± SD of n = three independent experiments. Statistical significance was tested by one-way ANOVA followed by Tukey’s multi-comparison test: ***p<0.001 for comparison versus pCpGL; ^§§§p<0.001 for comparison versus unmethylated pCpGL-PTPRD DMR.

ANOVA: Analysis of variance; DMR: Differentially methylated region; SD: Standard deviation.

Figure 5. Effect of PTPRD overexpression on differentiation of 3T3-L1 pre-adipocytes.

Un-transfected cells (Ctrl) and cells transfected with either the empty vector (pCMV) or the PTPRD expressing vector (pCMV-PTPRD), at differentiation day eight, were stained with Oil Red O and then subjected to phase contrast microscopy. Representative fields are shown in figure A. Lipid quantification (B) was assessed at 490 nm as described under Materials and methods. Expression levels of Pparγ2(C), Fabp4(D), and Glut4(E) were assessed by qPCR at the indicated time points. All data are presented as means ± SD of n = three independent experiments. Statistical significance was tested by one-way ANOVA followed by Tukey’s multi-comparison test at each indicated time point: *p<0.05; **p<0.01; ***p<0.001 for comparison versus Ctrl cells; ^§p<0.05, ^§§p<0.01, ^§§§p<0.001 for comparison versus pCMV transfected cells.

ANOVA: Analysis of variance; Ctrl: Control; OD: Optical density; pCMV: Cytomegalovirus promoter; qPCR: Quantitative PCR; REU: Relative expression units; SD: Standard deviation.

Figure 6. PTPRD DNA methylation in peripheral blood leukocytes from first-degree relative and CTRL subjects.

(A) BS analysis of PTPRD DMR in PBL from FDR (n = 8) and CTRL (n = 11) subjects available from the study group. Data points represent DNA methylation percentage at the PTPRD-associated DMR. Mean values ± SD are also shown. ***p<0.001 in a two-tailed Mann“Whitney U-test. Correlation between PBL PTPRD DNA methylation and adipose cell size in FDR (n = 8) and CTRL (n = 11) (B) or SVF PTPRD. DNA methylation in FDR (n = 8) and CTRL (n = 9) subjects (C)r correlation coefficient and p values are indicated on the graph.

BS: Bisulphite sequencing; CTRL: Control; DMR: Differentially methylated region; FDR: First-degree relatives; PBL: Peripheral blood leukocytes; SD: Standard deviation; SVF: Stromal vascular fraction cells.

Figure 7. PTPRD DNA methylation in peripheral blood leukocytes from obese and lean subjects.

(A) BS analysis of PTPRD DMR in PBL from obese (n = 10) and lean (n = 10) subjects from the replication group. Data points represent DNA methylation percentage at the PTPRD-associated DMR. Mean values ± SD are also shown. ***p < 0.001 in a two-tailed Mann“Whitney U-test. (B) Correlation between PBL PTPRD DNA methylation and BMI in obese (n = 10) and lean (n = 10) subjects. r correlation coefficient and p-values are indicated on the graph.

BS: Bisulphite sequencing; DMR: Differentially methylated region; PBL: Peripheral blood leukocytes; SD: Standard deviation.