Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status

Table 1 Frequency distributions of selected characteristics in CLC patients and control subjects

Variables	Cases			Control (n = 81)	P1
	Proximal	Distal	Total (n = 151)
Gender					0.8
Male (%)	28 (31.1)	62 (68.9)	90 (59.6)	50 (61.7)
Female (%)	32 (52.5)	29 (47.5)	61 (40.4)	31 (38.3)
Smoking status					0.1
Smokers2 (%)	22 (34.4)	42 (65.6)	64 (42.4)	25 (30.9)
Non-smokers	38 (43.7)	49 (56.3)	87 (57.6)	56 (69.1)
Age groups (yr)					0.4
< 60 (%)	24 (36.4)	42 (63.6)	66 (43.7)	40 (49.4)
≥60 (%)	36 (42.4)	49 (57.6)	85 (56.3)	41 (50.6)
Median (range)	61 (40-87)	60 (28-90)	60 (28-90)	60 (28-89)
Mean (SD)	62.22 (10.97)	59.24 (12.7)	60.42 (12)	58.98 (15.66)

¹Fisher’s exact test;

²Current and former smokers.

Table 2 Stratification analysis of tumors and genes promoter methylation frequencies

Variables	Methylation positive (%)						Methylated tumors2n (%)	OR (95% CI, P)3
	p16	P1	hMLH1	P1	hMSH2	P1
Sex
Male (90)	13 (14.4)	0.06	10 (11.1)	NS	4 (4.4)	NS	21 (23.3)	1
Female (61)	17 (27.9)		10 (16.4)		0 (0)		23 (37.7)	2.3 (1.1–5.04, 0.03)
Total (151)	30 (19.9)		20 (13.2)		4 (2.6)		44 (29.1)
Age (yr)
< 60 (66)	15 (22.7)	NS	7 (10.6)	NS	0 (0)	NS	19 (28.8)	1
≥ 60 (85)	15 (17.6)		13 (15.3)		4 (4.7)		25 (29.4)	1.1 (0.6–2.4, 0.7)
Tumor site
Proximal (60)	29 (48.3)	0.00	20 (33.3)	0.00	4 (6.7)	0.02	43 (71.7)	1
Distal (91)	1 (1.1)		0 (0)		0 (0)		1 (1.1)	0.002 (0.00–0.023, 0.00)
Serum folate (86)
Low (38)	2 (5.3)	0.04	4 (10.5)	NS	0 (0)	NS	4 (10.5)	1
High (48)	10 (20.8)		10 (20.8)		3 (6.3)		16 (33.3)	4.9 (1.4 -17.7, 0.01)
Serum vitamin B12 (86)
Low (42)	2 (4.8)	0.02	5 (11.9)	NS	0 (0)	NS	5 (11.9)	1
High (44)	10 (22.7)		9 (20.5)		3 (6.8)		15 (34.1)	3.9 (1.1-13.9, 0.03)
MTHFR 677 genotype4
CC (64)	13 (20.3)	NS	7 (10.9)	NS	0 (0)	NS	15 (23.4)	1
CT (80)	17 (21.3)	NS	12 (15)	NS	3 (3.8)	NS	28 (35)	1.92 (0.9-4.2, 0.09)
TT (7)	0 (0)	NS	1 (14.3)	NS	1 (14.3)	NS	1 (14.3)	0.7 (0.07-6.1, 0.7)
CT + TT (87)	17 (19.5)	NS	13 (14.9)	NS	4 (4.6)	NS	29 (33.3)	1.8 (0.8-3.9, 0.125)

¹Fisher's exact test;

²tumors with at least one methylated gene promoter;

³the first category was taken as reference. Odds ratio adjusted for age, sex, and smoking status;

⁴For genotype comparison CC used as the reference category. NS: Not significant.

Table 3 Association between MTHFR genotypes and tumor methylation in relation to clinical-biological features of CLC patients (case-control comparison, n)

Genotype (%)	Controls	Cases	Methylated	OR (95% CI, P)1,2	Unmethylated	OR (95% CI, P)1,2
Total	81	151	44		107
CC	40 (49.4)	64 (42.4)	15 (34.1)	1	49 (45.8)	1
CT	31 (38.3)	80 (53)	28 (63.6)	2.5 (1.1-5.6, 0.02)	52 (48.6)	1.4 (0.7-2.6, 0.3)
CT or TT	41 (50.6)	87 (57.6)	29 (65.9)	2.1 (0.9-4.5, 0.07)	58 (54.2)	1.1 (0.7-2.1, 0.6)
Sex
Male	50	90	21		69
CC	28 (56)	33 (36.7)	5 (23.8)	1	28 (40.6)	1
CT	17 (34)	51 (56.7)	15 (71.4)	5.3 (1.6-18.2, 0.008)	36 (52.2)	1.9 (0.9-4.3, 0.09)
CT or TT	22 (44)	57 (63.3)	16 (76.2)	4.3 (1.3-14.3, 0.01)	41 (59.4)	1.7 (0.8-3.6, 0.1)
Female	31	61	23		38
CC	12 (38.7)	31 (50.8)	10 (43.5)	1	21 (55.3)	1
CT	14 (45.2)	29 (47.5)	13 (56.5)	1.1 (0.3-3.6, 0.9)	16 (42.1)	0.7 (0.2-1.9, 0.5)
CT or TT	19 (61.3)	30 (49.2)	13 (56.5)	0.9 (0.3-2.8, 0.8)	17 (44.7)	0.5 (0.2-1.4, 0.2)
Age
< 60	40	66	19		47
CC	18 (45)	31 (47)	6 (31.6)	1	25 (53.2)	1
CT	18 (45)	34 (51.5)	13 (68.4)	1.4 (0.4-5.1, 0.6)	21 (44.7)	0.8 (0.3-1.9, 0.6)
CT or TT	22 (55)	35 (53)	13 (68.4)	1.1 (0.3-3.8, 0.9)	22 (46.8)	0.7 (0.3-1.6, 0.3)
≥ 60	41	85	25		60
CC	22 (53.7)	33 (38.8)	9 (36)	1	24 (40)	1
CT	13 (31.7)	46 (54.1)	15 (60)	3.8 (1.2-12.0, 0.02)	31 (51.7)	2.6 (0.9-6.6, 0.09)
CT or TT	19 (46.3)	52 (61.2)	16 (64)	2.7 (0.9-8.2, 0.08)	36 (60)	1.9 (0.8-4.2, 0.1)
Site
Proximal		60	43		17
CC		22 (36.7)	15 (34.9)		7 (41.2)
CT		37 (61.7)	27 (62.8)	2.7 (1.2-6.2, 0.02)	10 (58.8)	1.4 (0.5-4.8, 0.4)
CT or TT		38 (63.3)	28 (65.1)	2.1 (1.03-4.99, 0.05)	10 (58.8)	1.2 (0.4-3.7, 0.7)
Distal		91	1		90
CC		42 (46.2)	0 (0)		42 (46.7)
CT		43 (47.3)	1 (100)	ND	42 (46.7)	1.3 (0.7-2.5, 0.4)
CT or TT		49 (53.8)	1 (100)	ND	48 (53.3)	0.9 (0.5-1.6, 0.7)

^1,2For odds ratio and 95% CI calculations, controls with CC genotype was used as reference category, odds ratio adjusted for age, sex and smoking status. ND: Not determined.

Table 4 Associations between serum folate/vitamin B₁₂ concentrations and tumor methylation in relation to the clinical-biological characteristics of patients

Variables	n(%)	Folate (ng/mL) mean (range)	P1	High folate/Low folate2n (%)	P3	Vit.B12 (pg/mL) mean (range)	P1	High B12/Low B122n (%)	P3
Controls (81)		6.3 (3.8-12)	0.14	49 (60.5)/32(39.5)	0.64	312.8 (108-995)	0.14	40 (49.4)/41 (50.6)	0.94
Total cases (86)		5.9 (2.1-12)		48 (55.8)/38(44.2)		269 (50-681)		44 (51.2)/42 (48.8)
Unmethylated	66 (76.7)	5.7 (2.4-12)		32 (48.5)/34 (51.5)		255.2 (79-681)		29 (43.9)/37 (56.1)
Methyalated	20 (23.3)	6.4 (2.1-10)	0.06	16 (80)/4 (20)	0.02	314.7 (50-571)	0.1	15 (75)/5 (25)	0.02
Sex
Male (55)		5.9 (2.4-12)		29 (52.7)/26 (47.3)		274.2 (67-681)		28 (50.9)/27(49.1)
Unmethylated	43 (78.2)	5.8 (2.4-12)		19 (44.2)/24 (55.8)		325.5 (67-571)		18 (41.9)/25 (58.1)
Methylated	12 (21.8)	6.5 (3.9-9.5)	0.1	10 (83.3)/2 (16.7)	0.02	254.4 (83-681)	0.04	10 (83.3)/2 (16.7)	0.02
Female (31)		5.7 (2.1-10)	0.65	19 (61.3)/12 (38.7)	0.55	259.7 (50-523)	0.75	16(51.6)/15 (48.4)	1.005
Unmethylated	23 (74.2)	5.6 (3-8)		13 (56.5)/10 (43.5)		260. 3 (79-521)		11 (47.8)/12 (52.2)
Methylated	8 (25.8)	6.2 (2.1-10)	0.4	6 (75)/2 (25)	0.4	257.8 (50-523)	0.9	5 (62.5)/3 (37.5)	0.7
Age
< 60 yr (36)		5.4 (2.1-8)		18 (50)/18 (50)		238.1(50-571)		14 (38.9)/22(61.1)
Unmethylated	30 (83.3)	5.4 (3-8)		14 (46.7)/16 (53.3)		229.8 (79-465)		11 (36.7)/19 (63.3)
Methylated	6 (16.7)	5.5 (2.1-7.9)	0.5	4 (66.6)/2 (33.3)	0.7	279.5 (50-571)	0.8	3 (50)/3 (50)	0.7
≥ 60 yr (50)		6.3 (2.4-12)	0.045	30 (60)/20 (40)	0.45	291.3 (67-681)	0.15	30 (60)/20(40)	0.15
Unmethylated	36 (72)	6.1 (2.4-12)		18 (50)/18 (50)		276.3 (83-681)		18 (50)/18 (50)
Methylated	14 (28)	6.8 (3.9-10)	0.1	12 (85.7)/2 (14.3)	0.03	329.7 (67-543)	0.1	12 (85.7)/2 (14.3)	0.03
Site
Distal (62)		5.8 (2.4-12)		32 (51.6)/30 (48.4)		260.6 (79-681)		28 (45.2)/34 (54.8)
Unmethylated	61 (98.4)	5.8 (2.4-12)		31 (50.8)/30 (49.2)		255.5 (79-681)		27 (44.3)/34 (55.7)
Methylated	1 (1.6)	7.9	0.3	1 (100)/0 (0)	ND	271 (271)	0.1	1 (100)/0 (0)	ND
Proximal (24)		6.1 (2.1-10)	0.45	16 (66.7)/8 (33.3)	0.25	290.7 (50-543)	0.55	16 (66.7)/8 (33.3)	0.15
Unmethylated	5 (20.8)	5.3 (4.5-6.4)		1 (20)/4 (80)		251 (109-483)		2 (40)/3 (60)
Methylated	19 (79.2)	6.3 (2.1-10)	0.06	15 (78.9)/4 (21.1)	0.03	301.2 (50-543)	0.5	14 (73.7)/5 (26.3)	0.3

¹P-value from Mann-Whitney and t-test where appropriate;

²Median was taken as cut off point for low and high categorical value for serum folate/vitamin B₁₂ levels (5.5 ng/mL, and 240 pg/mL, respectively);

³P value from Fisher's exact test;

⁴Case/control comparison;

⁵Subgroups comparison, cases only. ND:Not determined.

Table 5 Combined effects of MTHFR 677 genotype and serum folate/vitamin B₁₂ concentrations on risk of tumor methylation (case-case comparison)

MTHFR 677genotype	n(%)	Folate (ng/mL) mean (range)	P1	High folate/Low folate2n (%)	P3	Vit. B12 (pg/mL) mean (range)	P1	High B12/Low B122n (%)	P3
Cases (86)
CC (42)		5.8 (2.1-8.8)		25 (59.5)/17 (40.5)		254.6 ( 50-673)		20 (47.6)/22 (52.4)
Unmethylated	34 (81)	5.9 (3.4-8.8)		20 (58.8)/14 (41.2)		267.3 (107-673)		16 (47.5)/18 (53.9)
Methylated	8 (19)	5.4 (2.1-7.8)	0.6	5 (62.5)/3 (37.5)	1.00	200.9 (50-429)	0.2	4 (50)/4 (50)	1.00
CT (38)		6.3 (3-12)	0.55	22 (57.9)/16 (42.1)	1.00	296 (79-681)	0.45	21 (55.3)/17 (44.7)	0.5
Unmethylated	27 (71.1)	5.9 (3-12)		12 (44.4)/15 (55.6)		254.1 (79-681)		11 (40.7)/16 (53.9)
Methylated	11 (28.9)	7.2 (5.3-10)	0.06	10 (90.9)/1 (9.1)	0.01	398.7 (150-571)	0.007	10 (90.9)/1 (9.1)	0.01
TT (6)4		4.2 (2.4-5.9)	0.035	1 (16.7)/5 (83.3)	0.08	198.5 (83-300)	0.45	3 (50)/3(50)	1.00
Unmethylated	5 (83.3)	3.8 (2.4-5.1)		0/5 (100)		178.2 (83-258)		2 (40)/3 (60)
Methylated	1 (16.7)	5.9		1 (100)/0		300		1 (100)/0
CT + TT (44)		5.9 (2.4-12)	0.95	23 (52.3)/21 (47.7)	0.5	282.7 (79-681)	0.55	24 (54.5)/20 (45.5)	0.7
Unmethylated	32 (72.7)	5.6 (2.4-12)		12 (37.5)/20 (62.5)		242.3 (79-681)		13 (40.6)/19 (59.4)
Methylated	12 (27.3)	7.1 (5.3-10)	0.002	11(91.7)/1 (8.3)	0.002	390.5 (150-571)	0.002	11 (91.7)/1 (8.3)	0.003

¹P-values for methylated and unmethylated cases, and genotype comparison, Mann-Whitney, t-test and Kruskal Wallis where appropriate;

²Median was taken as cut off point for low and high categorical value for serum folate/vitamin B₁₂ levels (5.5 ng/mL, and 240 pg/mL, respectively);

³P-value from Fisher's exact test;

⁴Since we did not have enough TT cases, the association of serum methyl donors and tumor methylation was not determined in these individuals;

⁵Comparison with the CC genotype, cases only.