DNA methylation, colon cancer and Mediterranean diet: results from the EPIC-Italy cohort

Figures & data

Figure 1. Diagram illustrating CpG selection.

Illumina = Infinium Human Methylation450 BeadChips IMI = Italian Mediterranean Index

Figure 2. Diagram illustrating the data sets used in the different stages of the analysis.

ca = casesco = controls Illumina = Infinium HumanMethylation450 BeadChips pyro = pyrosequencing.

Table 1. Descriptive statistics of the case-control study nested in the EPIC-Italy cohort.

Variables	Controls	Cases	all	P-value*
N	161	161	322
Median of (IQR)
age, years	55 (9)	55 (9)	55 (9)
BMI, kg/m^2	25.01 (5)	26.42 (5)	25.85 (5)	0.0019
Counts of
Gender
Men	88 (55%)	88 (55%)	176 (55%)
Women	73 (45%)	73 (45%)	146 (45%)
Centre
Varese	64 (40%)	64 (40%)	128 (40%)
Ragusa	10 (6%)	10 (6%)	20 (6%)
Turin	87 (54%)	87 (54%)	174 (54%)
Educational level				0.078
1°tertile RII	51 (34%)	53 (36%)	104 (35%)
2°tertile RII	54 (36%)	37 (25%)	91 (30%)
3°tertile RII	45 (30%)	59 (39%)	104 (35%)
Total physical activity				0.801
Inactive	34 (22%)	37 (24%)	71 (23%)
moderately inactive	65 (42%)	69 (44%)	134 (43%)
moderately active	36 (23%)	29 (19%)	75 (21%)
Active	20 (13%)	20 (13%)	40 (13%)
Smoking status				0.828
never smokers	71 (46%)	67 (43%)	138 (44%)
former smokers	48 (31%)	53 (34%)	101 (33%)
current smokers	36 (23%)	35 (23%)	71 (23%)
Fasting status				0.360
Yes	119 (74%)	126 (78%)	245 (76%)
No	42 (26%)	35 (22%)	77 (24%)
Cell Types
CD8T	8.1%	7.1%	7.5%	0.129
CD4T	13%	14%	14%	0.528
NK	7.4%	7.5%	7.5%	0.851
Bcell	5.2%	5.4%	5.3%	0.777
Mono	7.4%	7.1%	7.2%	0.543
Gran	63.0%	63.9%	63.2%	0.791

BMI = Body Mass Index; RII = Relative Index of Inequality; NK = Natural Killer; Bcell = B lymphocytes; Mono = Monocytes; Gran = Granulocytes.

*differences between cases and controls are assessed using Mann Whitney Test for continuous variables and Chi-Square Test for categorical variables.

Table 2. CpG sites selected by EN with 10-fold CV with an estimated coefficient higher than 0.15 in absolute value in the case-control study nested in the EPIC-Italy cohort.

Probe Name	Estimated coefficient	Gene	Chromosome	Location	MAPINFO	Delta	Frequency
cg13104385	0.56	IL6	7	Body	22767384	0.020	62.4%
cg18773937	−0.37	IL1B	2	TSS1500	113594611	0.016	72.1%
cg12195446	0.37	IRS2	13	Body	110424497	0.076	64.6%
cg05265849	−0.34	IL6	7	Body	22767390	0.010	9.3%
cg02749784	−0.25	MAL2	8	TSS1500	120219927	0.018	72.4%
cg17968347	−0.23	SERPINE1	7	Body	100777740	0.012	55.7%
cg12252547	0.23	MAL2	8	TSS1500	120220032	0.051	57.3%
cg01265860	−0.23	RUNX1	21	Body	36256316	0.016	56.7%
cg24312520	−0.22	STAT3	17	Body	40489584	0.017	57.9%
cg16308790	−0.22	NFATC1	18	Body	77225973	0.011	51.2%
cg08053846	0.21	SERPINE1	7	TSS1500	100769605	0.018	64.4%
cg15363134	−0.20	NFATC1	18	Body	77161214	0.015	62.7%
cg20674490	−0.17	RUNX3	1	Body	25240932	0.019	50.9%
cg27026615	−0.17	PTX3	3	Body	157156326	0.021	43.6%
cg08510264	0.16	IRS2	13	First Exon	110438288	0.012	32.9%
cg06493806	0.15	NFATC1	18	Body	77278806	0.031	44.9%

Delta = mean absolute difference in methylation percentage between cases and controls.

Frequency = percentage of times the CpGs are selected when EN is applied on a random subset of the initial dataset.

The CpG sites with a frequency higher than 50% are indicated in red.

The CpG sites analysed in Table 3 (those with a frequency higher than 50% and an association with MD in the control group in line with its protective effect) are indicated in grey.

Table 3. Detailed analysis of the CpG sites Selected for Validation (those reported in Table 2 with a frequency higher than 50% and an association with MD in the control group in line with its protective effect).

	Association with colon cancer*						Association with MD (only control group)**
IL1B	ca	co	OR	95% CI	p-value		IMI category	coef	95% CI	P-value
cg18773937
model A	157	157	0.69	(0.53,0.89)	0.005	linear model	0–2 (20)	reference
model B	134	134	0.61	(0.44,0.84)	0.003		3–4 (71)	0.25	(−0.13,0.63)	0.200
model C	134	134	0.59	(0.42,0.84)	0.003		5–11 (51)	0.46	(0.05,0.87)	0.029
SERPINE1									p-trend	0.025
cg17968347
model A	161	161	0.78	(0.62,0.98)	0.030	linear model	0–2 (22)	reference
model B	138	138	0.64	(0.47,0.86)	0.003		3–4 (71)	0.04	(−0.09,0.18)	0.552
model C	138	138	0.58	(0.42,0.81)	0.001		5–11 (52)	0.12	(−0.03,0.28)	0.122
RUNX1									p-trend	0.089
cg01265860
model A	161	161	0.77	(0.61,0.98)	0.036	linear model	0–2 (22)	reference
model B	138	138	0.72	(0.54,0.97)	0.030		3–4 (71)	0.11	(−0.06,0.27)	0.197
model C	138	138	0.57	(0.37,0.84)	0.006		5–11 (52)	0.13	(−0.05,0.31)	0.154
STAT3									p-trend	0.203
cg24312520
model A	161	161	0.78	(0.62,0.98)	0.037	linear model	0–2 (22)	reference
model B	138	138	0.81	(0.62,1.07)	0.142		3–4 (71)	−0.02	(−0.21,0.15)	0.762
model C	138	138	0.82	(0.61,1.12)	0.209		5–11 (52)	0.10	(−0.09,0.29)	0.323
NFATC1									p-trend	0.171
cg15363134
model A	161	161	0.82	(0.67,0.99)	0.043	linear model	0–2 (22)	reference
model B	138	138	0.76	(0.59,0.98)	0.032		3–4 (71)	0.22	(0.04,0.41)	0.018
model C	138	138	0.74	(0.56,0.98)	0.038		5–11 (52)	0.05	(−0.15,0.25)	0.617
RUNX3									p-trend	0.738
cg20674490
model A	160	160	0.70	(0.55,0.91)	0.008	linear model	0–2 (22)	reference
model B	137	137	0.66	(0.48,0.91)	0.012		3–4 (71)	0.15	(−0.10,0.20)	0.246
model C	137	137	0.64	(0.46,0.90)	0.009		5–11 (51)	0.09	(−0.19,0.38)	0.511
SERPINE1									p-trend	0.719
cg08053846
model A	134	134	1.28	(1.02,1.60)	0.037	linear model	0–2 (22)	reference
model B	114	114	1.27	(0.95,1.70)	0.103		3–4 (71)	−0.10	(−0.43,0.23)	0.548
model C	114	114	1.30	(0.96,1.74)	0.084		5–11 (51)	−0.20	(−0.56,0.15)	0.262
									p-trend	0.242

ca = cases; co = controls.

*Associations between DNA methylation and colon cancer are assessed using conditional logistic regression models with DNA methylation levels included as an independent variable and standardized to 1 standard deviation.

model A = crude model (adjusted for study centre, sex, age and seasonality by design)

model B = model A adjusted also for BMI, smoking status, physical activity, level of education and IMI.

model C = model B adjusted also for differential cell types.

** Associations between the M-values of DNA methylation sites and IMI are assessed using a multivariate linear-mixed effect model with chip as random effect and IMI, sex, age, centre, BMI, smoking status, total physical activity, level of education and the differential cell types as fixed effects.

Table 4. Validation analysis: associations between colon cancer/MD and DNA methylation levels at SERPINE1 and RUNX3 CpG sites obtained by pyrosequencing in the validation set.

	Association with colon cancer*						Association with MD (only control group)**
SERPINE1	ca	co	OR	95% CI	P-value		IMI category	coef	95% CI	P-value
cg17968347
model A	47	47	0.82	(0.59,1.15)	0.257	linear model	0–2 (6)	reference
model B	42	42	0.69	(0.45,1.05)	0.080		3–4 (23)	0.30	(−0.08,0.69)	0.117
model C	42	42	0.80	(0.37–1.76)	0.588		5–11 (15)	0.39	(−0.02,0.80)	0.061
RUNX3									p-trend	0.091
cg20674490
model A	47	47	0.74	(0.47,1.16)	0.196	linear model	0–2 (6)	reference
model B	42	42	0.59	(0.29,1.19)	0.144		3–4 (23)	0.39	(−0.20,0.98)	0.188
model C	42	42	0.40	(0.14–1.08)	0.071		5–11 (15)	0.09	(−0.52,0.72)	0.756
									p-trend	0.627

ca = cases; co = controls.

*Associations between DNA methylation and colon cancer are assessed using conditional logistic regression models with DNA methylation levels included as an independent variable and standardized to 1 standard deviation.

model A = crude model (adjusted for study centre, sex, age and seasonality by design).

model B = model A adjusted also for BMI, smoking status, physical activity, level of education and IMI.

model C = model B adjusted for differential cell types.

** Associations between M-values of DNA methylation sites and IMI are assessed using linear models adjusting for age, sex, centre, BMI, smoking status, physical activity, level of education and differential cell types.

Table 5. Replication analysis: Associations between colon cancer/MD and DNA methylation levels at SERPINE1 and RUNX3 CpG sites obtained by pyrosequencing in the replication set.

	Association with colon cancer*						Association with MD (only control group)**
SERPINE1	ca	Co	OR	95% CI	P-value		IMI category	coef	95% CI	P-value
cg17968347
model A	93	93	1.08	(0.82,1.42)	0.573	linear model	0–2 (17)	reference
model B	87	87	1.12	(0.79,1.56)	0.522		3–4 (44)	0.005	(−0.22,0.23)	0.961
							5–11 (27)	0.10	(−0.17,0.36)	0.431
RUNX3									p-trend	0.377
cg20674490
model A	92	92	0.80	(0.60,1.07)	0.132	linear model	0–2 (17)	reference
model B	86	86	0.80	(0.57,1.13)	0.210		3–4 (44)	0.29	(−0.12,0.70)	0.162
							5–11 (27)	0.38	(−0.09,0.85)	0.114
									p-trend	0.133

ca = cases; co = controls.

*Associations between DNA methylation and colon cancer are assessed using conditional logistic regression models with DNA methylation levels included as an independent variable and standardized to 1 standard deviation.

model A = crude model (adjusted for study centre, sex, age, year of recruitment and seasonality by design).

model B = model A also for BMI, smoking status, physical activity, level of education and IMI.

** Associations between M-values of DNA methylation sites and IMI are assessed using linear models adjusting for age, sex, centre, BMI, smoking status, physical activity and level of education.

Figure 3. Results of the mixed effects meta-analysis of the discovery and replication studies on the validated signals.