Table 1
The primers used for multiplex PCR.
Loci
|
Primers
|
PCR
Product (bp)
|
Restriction Enzyme
|
Bands (bp)
|
MTHFR C677T
|
5′-TGAAGGAGAAGGTGTCTGCGGGA-3′
5′-AGGACGGTGCGGTGAGAGTG-3
|
198-bp
|
HinfI
TT
CT
CC
|
175
198 − 175
198
|
MTHFR A1298C
|
5′-CAAGGAGGAGCTGCTGAAGA-3′
5′-CCACTCCAGCATCACTCACT-3′
|
128-bp
|
MobII
AA
AC
CC
|
72
72–100
100
|
bp: base pair.
Table 2
Demographic and clinical data of the studied cases.
|
No= (50)
|
%
|
Gender
Male
Female
|
36.0
14.0
|
72.0
28.0
|
Age (mean ± SD)
|
55.42 ± 6.57
|
Child-Pugh score
Class A
Class B
Class C
|
1
20
29
|
2.0
40.0
58.0
|
Size
|
4.86 ± 1.71
|
Number of mass
1
2
|
36.0
14.0
|
72.0
28.0
|
Grade
1
2
3
4
|
11
21
15
3
|
22.0
42.0
30.0
6.0
|
Barcelona classification
Stage 0
Stage A
Stage B
Stage C
|
15
21
12
2
|
30.0
42.0
24.0
4.0
|
Site
Right
Left
|
30
20
|
60.0
40.0
|
Data are expressed as Mean ± SD for continuous data and or no and % for discrete data.
Table 2 shows the distribution of age, gender, and tumor characters among cases. More than two-thirds of the cases were male with a mean age of 55 years old. More than half of the cases were a class c Child-Pugh score (58%). The mean size of the mass was 4.8 cm. Two-thirds of the cases had one mass. Most of them were grade 3 and stage A Barcelona classification (42%). Two-thirds of them (60%) were right-sided tumors.
Table 3
Distributions of laboratory findings in HCC patients and control subjects.
|
Case
|
Control
|
t-test
|
P value
|
WBCs (mean ± SD)
*10³/ml
|
7.22 ± 3.96
|
7.55 ± 2.22
|
t-test= -0.52
|
0.603
|
Hb (mean ± SD) gm/dl
|
13.122 ± 2.48
|
13.11 ± 1.79
|
t-test = 0.03
|
0.97
|
Platelets (mean ± SD) *10³/ml
|
156.98 ± 83.61
|
221.44 ± 61.04
|
t-test = -4.4
|
< 0.001*
|
INR (mean ± SD)
|
1.31 ± 0.49
|
1.19 ± 0.15
|
t-test = 1.6
|
0.09
|
Albumin(mean ± SD) gm/dl
|
3.72 ± 0.53
|
3.99 ± 0.53
|
t-test = -2.5
|
0.01*
|
ALT (IU/L)
Median (min-max)
|
45 (20.06-85.0)
|
28 (20.0–37.0)
|
Mann Whitney
Z= -5.9
|
< 0.001*
|
AST (IU/L)
Median (min-max)
|
56 (20.0–96.0)
|
27 (21–34)
|
Mann Whitney
Z= -6.9
|
< 0.001*
|
AFP (IU/L) (mean ± SD)
|
137.71 ± 110.3
|
5.94 ± 2.21
|
t-test = 8.4
|
< 0.001*
|
WBCs: White blood cells, Hb: hemoglobin, INR: international normalized ratio, ALT: alanine aminotransferase, AST: aspartate aminotransferase, AFP: alpha-fetoprotein.* Significant p ≤ 0.05 |
As in Table 3, platelets, albumin, ALT, AST and AFP were higher in cases than in control with a significant difference (P 0.01 − 0.001). No significant difference was found regarding WBCs, Hb, and INR levels between the two groups.
Table 4
The joint genotypes of the MTHFR677 and MTHFR1298 polymorphisms among cases and controls and risk of HCC.
Loci
|
Genotypes and alleles
|
Case
(n = 50)
|
Control
(n = 50)
|
OR
|
95% CI
|
P
|
N
|
%
|
N
|
%
|
MTHFR C677T
|
Genotypes
|
CC
|
30
|
60
|
37
|
74
|
r
|
|
|
CT
|
4
|
8
|
10
|
20
|
2.02
|
0.58–7.11
|
0.27
|
TT
|
16
|
32
|
3
|
6
|
0.15
|
0.04–0.57
|
< 0.005*
|
Alleles
|
C
|
64
|
64
|
84
|
84
|
0.34
|
0.17–0.66
|
0.001*
|
T
|
36
|
36
|
16
|
16
|
Hardy-Weinberg
|
x2
P
|
34.1
< 0.001*
|
3.27
0.07
|
|
MTHFR A1298C
|
Genotypes
|
AA
|
21
|
42
|
26
|
52
|
r
|
|
|
AC
|
15
|
30
|
20
|
40
|
1.08
|
0.45–2.6
|
0.8
|
CC
|
14
|
28
|
4
|
8
|
0.23
|
0.07–0.81
|
0.02*
|
Alleles
|
A
|
55
|
55
|
52
|
52
|
1.09
|
0.62–1.9
|
0.67
|
C
|
45
|
45
|
48
|
48
|
Hardy-Weinberg
|
x2
P
|
7.01
0.008*
|
0.003
0.9
|
|
|
C677T A1298C
|
Case
|
Control
|
|
|
Combined analysis
|
CC
|
AA
|
11
|
52.4
|
19
|
73.1
|
0.58
|
0.23–1.44
|
0.19
|
CC
|
AC
|
11
|
78.6
|
15
|
75
|
0.73
|
0.28–1.9
|
0.48
|
CC
|
CC
|
8
|
53.3
|
3
|
75
|
2.6
|
0.59–13.56
|
0.15
|
CT
|
AA
|
3
|
14.3
|
7
|
26.9
|
0.43
|
0.08–1.99
|
0.22
|
CT
|
AC
|
1
|
7.1
|
3
|
15
|
0.33
|
0.01–3.8
|
0.32
|
CT
|
CC
|
0
|
0
|
0
|
0
|
-
|
-
|
-
|
TT
|
AA
|
7
|
33.3
|
0
|
0
|
-
|
-
|
0.01*
|
TT
|
AC
|
2
|
14.3
|
2
|
10
|
1
|
0.1-10.44
|
1
|
TT
|
CC
|
7
|
46.7
|
1
|
25
|
7
|
0.81–156.8
|
0.04*
|
OR: odds ratio, CI: confidence interval, r: reference category (OR = 1.0) |
* Significant p ≤0.05.
|
Three genetic forms for each polymorphic locus were identified by RFLP. In cases, wild-type MTHFR 677CC was found in 30 (60%), the MTHFR 677CT heterozygous variant was present in 4 (8%), and the MTHFR 677 TT homozygous variant was found in 16 (32%). The MTHFR allele C was 64%, and T was 36%. While, in controls, wild-type MTHFR 677CC was found in 37 (74%), the MTHFR 677CT heterozygous variant was present in 10 (20%), and the MTHFR 677 TT homozygous variant was found in 3 (6%). The MTHFR allele C was 84% and T was 16%. For the allelic model C vs. T, the OR was 0.34 (95% CI: 0.17–0.66, p 0.001) (Table 4). In cases, wild-type MTHFR 1298AA was observed in 21 (42%) while MTHFR 1298AC heterozygous variant was noted in 15 (30%) and the MTHFR 1298CC homozygous variant was seen in 14 (28%) OR (0.23) (95% CI: 0.07–0.81, p 0.02). While, in controls, wild-type MTHFR 1298AA was observed in 26 (52%) while MTHFR 1298AC heterozygous variant was noted in 20 (40%) and the MTHFR 1298CC homozygous variant was seen in 4 (8%). For the allelic model A vs. C, the OR was 1.09 (95% CI: 0.62–1.9, p 0.67).
MTHFR (C677T and MTHFR A1298C) polymorphisms and the risk of HCC.
Using the C/C genotype as the reference for MTHFR C677T, ORs were 2.02 (95% CI: 0.58–7.11) and 0.15 (95% CI: 0.04–0.57 P < 0.005) for the C/T and T/T genotypes, respectively with significant difference. OR for the T allele was 0.34 (95% CI: 0.17–0.66 P 0.001) when compared with the C alleles. Using the A/A genotype as the reference group for MTHFR A1298C, ORs were 1.08 (95% CI: 0.45–2.6) for the A/C without significant difference and 0.23 (95% CI: 0.62–1.9 P 0.02) for the C/C genotypes, with significant difference. Applying Hardy Weinberg equation (HWE), revealed that MTHFR C677T and in MTHFR A1298C in control subjects was in HWE while cases of both genotypes weren't in HWE. We evaluated the combined effect of different exposures on the risk of HCC. MTHFR 677 CC or TT genotype combined with MTHFR 1298 CC genotype were associated with an increased risk of HCC, with the OR, 2.6 and 7 respectively.
Table 5
Genotypes and alleles distribution in different HCC presentation.
Child-Pugh score
|
(677 C-T)
|
|
Class A
|
Class B
|
Class C
|
P value
|
CC
|
1(3.3%)
|
14(46.7%
|
15(50.0%)
|
0.312
|
CT
|
0
|
1(25.0%)
|
3(75.0%)
|
0.76
|
TT
|
0
|
5(31.2%)
|
11(68.8%)
|
0.49
|
C
|
2(3.1%)
|
29(45.3%)
|
33(51.6%)
|
0.16
|
T
|
0
|
11(30.6%)
|
25(69.4%)
|
(1298 A-C)
|
AA
|
1(4.8%)
|
6(28.6%)
|
14(66.7%)
|
0.2
|
AC
|
0
|
7(46.7%)
|
8(53.3%)
|
0.68
|
CC
|
0
|
7(50.0%)
|
7(50.0%)
|
0.58
|
A
|
2(3.6%)
|
19(34.5%)
|
34(61.8%)
|
0.24
|
C
|
0
|
21(46.7%)
|
24(53.3%)
|
No of mass
|
|
|
One mass
|
Two masses
|
|
(677 C-T)
|
CC
|
20(66.7%)
|
10(33.3%)
|
0.24
|
CT
|
4(100.0%)
|
0
|
0.25
|
TT
|
12(75.0%)
|
4(25.0%)
|
0.5
|
C
|
44(68.8%)
|
20(31.2%)
|
0.23
|
T
|
28(77.8%)
|
8(22.2%)
|
|
(1298 A-C)
|
AA
|
16(76.2%)
|
5(23.8%)
|
0. 4
|
AC
|
9(60.0%)
|
6(40.0%)
|
0.16
|
CC
|
11(78.6%)
|
3(21.4%)
|
0.39
|
A
|
39(70.9%)
|
16(29.1%)
|
0.48
|
C
|
33(73.3%)
|
12(26.7%)
|
Grade
|
|
|
1
|
2
|
3
|
4
|
|
(677 C-T)
|
CC
|
8(26.7%
|
12(40.0%
|
10(33.3%
|
0
|
0.13
|
CT
|
0
|
1(25.0%)
|
1(25%)
|
2(50%)
|
0.002*
|
TT
|
3(18.8%)
|
8(50.0%)
|
4(25.0%)
|
1(6.2%)
|
0.88
|
C
|
16(25.0%)
|
25(39.1%)
|
21(32.8%)
|
2(3.1%)
|
0.26
|
T
|
6(16.7%)
|
17(47.2%)
|
9(25.0%)
|
4(11.1%)
|
(1298 A-C)
|
AA
|
4(19.0%)
|
9(42.9%)
|
5(23.8%)
|
3(14.3%)
|
0.19
|
AC
|
4(26.7%)
|
5(33.3%)
|
6(40.0%)
|
0
|
0.45
|
CC
|
3(21.4%)
|
7(50.0%)
|
4(28.6%)
|
0
|
0.86
|
A
|
11(29.1%)
|
22(40.0%)
|
16(20.0%)
|
6(10.9%)
|
0.15
|
C
|
11(24.4%)
|
20(44.4%)
|
14(31.1%)
|
0
|
BCLC
|
|
|
Zero
|
1
|
2
|
3
|
|
(677 C-T)
|
CC
|
11(36.7%)
|
14(46.7%)
|
5(16.7%)
|
0
|
0.1
|
CT
|
0
|
1(25.0%)
|
3(75.0%)
|
0
|
0.09
|
TT
|
4(25.0%)
|
6(37.5%)
|
4(0.25%)
|
2(12.5%)
|
0.2
|
C
|
22(34.4%)
|
29(45.3%)
|
13(20.3%)
|
0
|
0.02*
|
T
|
8(22.2%)
|
13(36.1%)
|
11(30.6%)
|
4(11.1%)
|
(1298 A-C)
|
AA
|
6(28.6%)
|
10(47.6%)
|
4(19.0%)
|
1(4.8%)
|
0.88
|
AC
|
3(20.0%)
|
8(53.3%)
|
4(26.7%)
|
0
|
0.51
|
CC
|
6(42.9%)
|
3(21.4%)
|
4(28.6%)
|
1(7.1%)
|
0.29
|
A
|
15(27.3%)
|
26(47.3%)
|
12(21.8%)
|
2(3.6%)
|
0.7
|
C
|
15(33.3%)
|
16(35.6%)
|
12(26.7%)
|
2 (4.4%)
|
|
Site
|
|
|
|
Right
|
Left
|
|
(677 C-T)
|
CC
|
17(56.7%)
|
13(43.3%)
|
0.38
|
CT
|
2(50.0%)
|
2(50.0%)
|
0.52
|
TT
|
11(68.8%)
|
5(31.2%)
|
0.29
|
C
|
36(56.2%)
|
28(43.8%)
|
0.21
|
T
|
24(66.7%)
|
12(33.3%)
|
|
(1298 A-C)
|
AA
|
13(61.9%)
|
8(38.1%)
|
0.52
|
AC
|
8(53.3%)
|
7(46.7%)
|
0.37
|
CC
|
9(64.3%)
|
5(35.7%)
|
0.47
|
A
|
32(58.2%)
|
23(41.8%)
|
0.41
|
C
|
28(62.2%)
|
17(37.8%)
|
0.17
|
BCLC: Barcelona clinic liver cancer
* Significant p ≤ 0.05
CT genotype of MTHFR variant C677T showed significant difference in different grade of HCC. The C allele of variant C677T showed significant difference BCLC stages. No significant difference in genotypes and alleles distribution exists between classes of Child-Pugh score or according to No or site of tumor mass (Table 5).
Table 6
Multivariate analysis for HCC predictors.
|
B
|
P value
|
Exp(B)
|
95.0% C.I.
for EXP(B)
|
Lower
|
Upper
|
Platelets
|
0.01
|
0.4
|
1.01
|
0.99
|
1.02
|
Albumin
|
0.62
|
0.49
|
0.54
|
0.09
|
3.11
|
ALT
|
0.17
|
0.00
|
0.84
|
0.77
|
0.92
|
AST
|
0.004
|
0.58
|
1.0
|
0.99
|
1.02
|
AFP
|
0.34
|
0.00
|
0.71
|
0.59
|
0.86
|
TT (C677T)
|
3.68
|
0.1
|
3.28
|
0.48
|
3.28
|
CC (A1298C)
|
1.09
|
0.2
|
2.99
|
0.57
|
15.68
|
CI: confidence interval, ALT: alanine aminotransferase, AST: aspartate aminotransferase, AFP: alpha-fetoprotein.
Regarding Multivariate analysis for HCC risk predictors, the most important predictors for HCC are T/T genotype of variant (C677T) and C/C genotype of variant (A1298C) with odds ratio 39.76, 2.01 and 2.99 respectively (Table 6).