Abstract
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway. We aimed to test the hypothesis that C677T and A1298C variants of MTHFR predispose to microsatellite instable (MSI) colorectal cancer. We determined MTHFR genotypes in 175 sporadic colorectal cancer patients and a total of 231 normal controls in Shiraz, Southern Iran. Among the genotypes found in our samples, MTHFR CT and CT + TT were associated with increased risk for CRC incidence [odds ratio (OR) = 2.4, 95% confidence interval (95%CI) = 1.8–4.4; OR = 2.4, 95%CI = 1.6–3.6, respectively]. Double heterozygotes 677CT/1298AC and double homozygote 677TT/1298AA and 677CC/1298CC genotypes also showed a significantly increased risk of developing CRC compared with the wild-type 677CC/1298AA genotypes of the controls. Among the 151 tumors tested, 36 (23.8%) were MSI+. MSI was more common in proximal tumors (OR = 10.4; 95%CI = 3.9–27.8) and in smokers (OR = 2.9; 95%CI = 1.3–6.7). In a case–control comparison, the MTHFR 677CT + TT genotype was strongly associated with MSI (OR = 2.6; 95%CI = 1.3–5.3). Hypermethylation of mismatch repair genes was positively related with MSI incidence in these tumor series (P = 0.00). Our data suggest that the MTHFR 677CT + TT variant genotype may be a risk factor for MSI+ cancer.
1. Introduction
The incidence of colorectal cancer (CRC) has rapidly increased in Iran during the last decade [1]. The increase has generally been ascribed to smoking and a Westernized diet, which is characterized by a high intake of fat and meat, but there have been a limited number of studies done on the molecular pathology of CRC in this region.
Sporadic CRC is a multifactorial disease caused by numerous interacting genetic, epigenetic, and environmental risk factors. There is increasing evidence suggesting that colorectal cancers develop through different molecular pathways [2]. Based on genetic and epigenetic profiles, Shen et al. [3] proposed three distinct subclasses of primary colon cancers, each with homogenous molecular features.
The enzyme methylene tetrahydrofolate reductase (MTHFR) functions at a critical juncture between DNA synthesis and methylation of DNA, proteins, and lipids [4]. Two common variant genotypes of the MTHFR gene, C677T in exon 4 (Ala222Val) and A1298C in exone 7 (Glu429Ala), are associated with reduced enzyme activity [5,6]. It has been suggested that mutations in MTHFR, which alter both DNA methylation and nucleotide synthesis, are related to CRC incidence [7,8].
The results of previous CRC studies with regard to its relationship with MTHFR polymorphism were controversial [9,10]. Several case–control studies have shown a reduction in CRC risk for individuals with the TT genotype compared with those with CC or CT genotypes, but the protective effect appeared to depend on an adequate dietary folate intake. Other studies did not find a reduced risk of CRC or rectal cancer among those with a variant genotype of the MTHFR polymorphism; rather, they observed an increased risk of colon cancer, suggesting that the effects of the MTHFR genotype may differ in populations with different levels of folate intake [11,12].
Microsatellites are short tandem sequence repeats in DNA, and their length changes are termed microsatellite instability (MSI), which have been found to be associated with about 15–20% of sporadic colorectal cancers [13]. MSI-positive colon cancers comprise a distinct group that also shows widespread gene promoter methylation, a feature referred to as the CpG island methylator phenotype. MSI occurs as a result of a deficiency in a group of nuclear proteins collectively known as the mismatch repair (MMR) system. Mutations in genes coding for enzymes involved in folate metabolism might play a role in altered gene promoter methylation and thus predispose to MSI CRC. On the basis of the functional effects of MTHFR genotypes and their role in methylation, variants may be expected to be associated with the risk of MSI in colorectal cancer. Little is known about the role of MTHFR variant genotypes in MSI-related colon carcinogenesis. A few studies have investigated the role of MTHFR variant genotypes in MSI-related colon carcinogenesis, but the findings were not consistent. While several investigators reported that the MTHFR homozygote TT genotype was associated with an increased risk of MSI-positive CRC [14–17], some studies found either no association or a reduced risk of MSI tumors associated with MTHFR variant genotypes [18,19].
As far as we know, there is currently no information in the available literature concerning the possible association of two MTHFR variants with CRC in Iranians. We recently examined the association between the MTHFR C677T genotype and hypermethylation of the promoter region of three tumor-associated genes (P16, hMLH1, and hMSH2) among 151 sporadic CRC patients in Iran [20]. We have now made MSI analysis and genetic typing studies of the MTHFR A1298C in these series of patients. Therefore, this should be considered as an extension of our previous study, which could be consulted for detailed information.
2. Materials and methods
2.1. Study population and DNA preparation
A total of 175 sporadic primary tumor samples (90 fresh and 85 paraffin-embedded samples) and their corresponding adjacent normal colon mucosa were obtained from surgical patients at university hospitals in Shiraz, Southern Iran. The Institutional Ethics Committee approved the study protocol. Fresh samples were snap-frozen immediately after surgery and stored at −70 °C. All samples were subjected to re-evaluation of the original histologic diagnosis made by an expert pathologist, who also selected representative tissue section areas for DNA extraction and further molecular analyses. Genomic DNA was extracted from tumor and normal tissues, as described previously [20].
2.2. MTHFR genotyping
DNA extracted from peripheral blood lymphocytes of healthy volunteers was used as normal controls for genotype analysis. Genotyping of MTHFR at codon 677 and 1298 of DNA from control and CRC cases was performed using the mutagenically separated polymerase chain reaction (MS-PCR) method, as described [20,21].
2.3. MSI analysis
DNA from 151 tumor samples were tested for MSI at the Bethesda Consensus recommended loci (BAT25, BAT26, D2S123, D5S346, and D17S250). Target DNA sequences were amplified by PCR in a 50-μL reaction volume containing 100 ng genomic DNA. The PCR products were analyzed under denaturing conditions on a 6.7% polyacrylamide gel containing 50% urea. After electrophoresis, the polyacrylamide gel was fixed and the PCR products were visualized by silver staining. MSI analysis was based on the interpretation of the mobility shift of tumor DNA in comparison to normal DNA. MSI was defined by the presence of a ladder-like expansion or contraction of the microsatellite repeat unit. MSI analysis results for 53 samples and their normal matched tissues were also verified by an automatic DNA sequencer (ABI Prism 3130 Genetic Analyzer; Applied Biosystems, Foster City, CA). Tumors were classified as having microsatellite instability (MSI) if two or more microsatellite markers showed changes compared with matched normal tissue. Tumors with only one MSI-positive marker defined as MSI-low (MSI-L) and were grouped together with no MSI+ specimens as microsatellite stable (MSS).
2.4. Statistical analysis
Depending upon the sample size, associations between clinical, biologic, and genotypic features were evaluated using either the chi-square test or the Fisher’s exact test. The level of significance was assessed at P < 0.05. Logistic regression was used to calculate odds ratio (OR) and 95% confidence intervals (95%CI). We also adjusted for potential confounding variables such as age, gender, tumor site, and cigarette smoking. All statistical computations were performed by SPSS software (version 11.5; SPSS Inc., Chicago, IL).
3. Results
In this study, we investigated the association of two common functional polymorphisms of MTHFR with reduced enzyme activity (C677T and A1298C ), as well as CRC incidence among Iranian sporadic colon cancer patients. Sixty percent of patients had distal and 40% had proximal tumors. Cases were more likely to be males older than 60 years.
3.1. Distribution of genotypes
Genotype frequencies and odds ratios for MTHFR genotypes and colon cancer are presented in Table 1. Prevalence of the MTHFR 677T allele did not differ significantly between controls (20.3%) and cases (31.1%). The distribution of MTHFR 677 genotypes among controls (CC, 64.9%; CT, 29.4%; TT, 5.6%) agreed with that expected from the Hardy-Weinberg equilibrium (χ2 = 1.22, P = 0.3). However, significant departures from Hardy-Weinberg equilibrium were observed for MTHFR 677 genotypes among cases (P = 0.004). The genotype distributions of CRC patients (CC, 42.4%; CT, 53%; TT, 4.6%) were significantly different from controls (P = 0.00). To increase precision, we combined heterozygous and homozygous variant genotypes. The MTHFR 677CT + TT genotype in controls was less frequent than in CRC cases (P = 0.00;Table 1). Similar frequencies of the A1298C genotypes were seen in patients and controls. Both patients and controls were in Hardy-Weinberg equilibrium for the distribution of the A1298C polymorphism (χ2 = 0.77, P = 0.4; χ2 = 0.01, P = 9, respectively).
Table 1.
The frequencies of the C677T and A1298C genotypes of the MTHFR gene in sporadic CRC patients and controls
| MTHFR genotypes | Cases n (%) | Controls n (%) | P * | OR (95%CI)** | P ** |
|---|---|---|---|---|---|
| C677T | |||||
| CC ref | 64 (42.4) | 150 (64.9) | 1 | ||
| CT | 80 (53) | 68 (29.4) | 2.8 (1.8–4.4) | 0.000 | |
| TT | 7 (4.6) | 13 (5.6) | 0.000 | 0.7 (0.3–1.9) | 0.6 |
| CT + TT | 87 (57.6) | 81 (35.1) | 0.000 | 2.4 (1.6–3.6) | 0.000 |
| Total | 151 | 231 | |||
| A1298C | |||||
| AA ref | 38 (37.3) | 79 (42.5) | 1 | ||
| AC | 52 (51) | 85 (45.7) | 1.3 (0.8–2.2) | 0.3 | |
| CC | 12 (11.8) | 22 (11.8) | 0.6 | 1.2 (0.5–2.7) | 0.7 |
| AC + CC | 64 (62.7) | 107 (57.5) | 0.5 | 1.2 (0.8–2.1) | 0.4 |
| Total | 102 | 186 | |||
| Combined genotype | |||||
| CC/AA ref | 12 (14) | 46 (24.7) | 0.03 | 1 | |
| CC/AC | 20 (23.3) | 57 (30.6) | 1.4 (0.6–3.1) | 0.5 | |
| CC/CC | 10 (11.6) | 14 (7.5) | 2.6 (0.9–7.5) | 0.07 | |
| CT/AA | 14 (16.3) | 27 (14.5) | 2.0 (0.8–4.9) | 0.1 | |
| CT/AC | 23 (26.7) | 25 (13.4) | 3.6 (1.5–8.6) | 0.004 | |
| CT/CC | 1 (1.2) | 5 (2.7) | ND | ||
| TT/AA | 6(7) | 6 (3.2) | 3.9 (1.0–14.4) | 0.04 | |
| TT/AC | 0 (0) | 3 (1.6) | ND | ||
| TT/CC | 0 (0) | 3 (1.6) | ND |
OR was adjusted for age, sex, and smoking status.
Abbreviation: ND, not determined.
Statistical test for case–control comparisons, chi-square test.
For OR and 95%CI calculations, controls with the wild-type CC and AA MTHFR genotype were used as reference category.
We examined the relationship between two MTHFR polymorphisms and the risk of CRC. According to the logistic regression model, in the entire group of patients, MTHFR CT and CT + TT genotypes were strongly associated with a higher risk of cancer (Table 1). The adjusted OR for CT and CT + TT genotypes were 2.8 (95%CI = 1.8 – 4.4) and 2.4 (95%CI = 1.6 – 3.6), respectively. No CRC risk associated with MTHFR A1298C genotypes was found when we compared patients with controls and considered the AA genotype as the referent (Table 1). For MTHFR 677 and MTHFR 1298 haplotypes, double heterozygotes (677CT/1298AC ) had 3.6-fold (95%CI 1.5 – 8.6) increased risk compared with the wild-type (677CC/1298AA) genotypes of controls. The homozygous 677TT/1298AA genotype was also associated with an increased risk of developing CRC in comparison to controls (OR = 3.9, 95%CI = 1.0 – 14.4; Table 1). Due to the small number of cases in the current study, it was not possible to perform analyses of CRC risk associated with other combined MTHFR genotypes.
3.2. Correlation between MTHFR genotypes and patients’ characteristics
Patients’ clinicopathologic characteristics in relation to MTHFR genotypes are summarized in Tables 2 and 3. No differences in frequencies of the MTHFR A1298C genotypes were found in patients stratified by age, sex, smoking status, and tumor differentiation stage (Table 2). Grouping the patients according to their tumor localization, significant differences in MTHFR 1298 genotype distribution were observed between patients with proximal colon tumor (AA, 20.7%; AC, 55.2%; CC, 1.7%) and patients with distal CRC (AA, 43.8%; AC, 49.3%; CC, 6.8%), as verified by the chi-square test (P = 0.02). The frequency of the MTHFR 1298AC + CC genotype in patients with proximal cancer was significantly higher than distal CRC cases (P = 0.02). We did not also find a CRC risk associated with the AC + CC genotype for any of the sex and age groups, when cases were compared with controls and the AA genotype was used as a reference category (Table 2). However, the AC + CC genotype was associated with a threefold-increased CRC risk in proximal patients compared with controls (OR = 3; 95%CI = 1.1–7.6).
Table 2.
Characteristics of patients and controls according to MTHFR 1298A/C genotypes
| Cases |
Controls |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Number | AA n (%) | AC n (%) | AC + CC n (%) | OR (95%CI)* | Number | AA n (%) | AC n (%) | AC + CC n (%) | OR (95%CI)** | |
| Total | 102 | 38 (37.3) | 52 (51) | 64 (62.7) | 186 | 79 (42.5) | 85 (45.7) | 107 (57.5) | 1.3 (0.8–2.1) | |
| Age | ||||||||||
| < 60 | 40 | 12 (30) | 24 (60) | 28 (70) | 1 | 86 | 36 (41.9) | 45 (52.3) | 50 (58.1) | 1.7 (0.7–3.9) |
| ≥ 60 | 62 | 26 (41.9) | 28 (45.2) | 36 (58.1) | 0.6 (0.3–1.4) | 100 | 43 (43) | 40 (40) | 57 (57) | 1.01 (0.5–1.9) |
| Sex | 186 | |||||||||
| Male | 68 | 26 (38.2) | 41 (60.3) | 42 (61.8) | 1 | 117 | 48 (41) | 60 (51.3) | 69 (59) | 1.2 (0.6–2.2) |
| Female | 34 | 12 (35.3) | 11 (32.4) | 22 (64.7) | 1.1 (0.4–2.6) | 69 | 31 (44.9) | 25 (36.2) | 38 (55.1) | 1.4 (0.6–3.4) |
| Smoking | 74 | |||||||||
| Never | 51 | 17 (33.3) | 29 (56.9) | 34 (66.7) | 1 | 50 | 17 (34) | 23 (46) | 33 (66) | 0.9 (0.4–2.3) |
| Current/past | 51 | 21 (41.2) | 23 (45.1) | 30 (58.8) | 0.8 (0.3–1.8) | 24 | 9 (37.5) | 12 (50) | 15 (62.5) | 0.8 (0.3–2.4) |
| Location | ||||||||||
| Proximal | 29 | 6 (20.7) | 16 (55.2) | 23 (79.3) | 1 | – | – | – | – | 3 (1.1–7.6) |
| Distal | 73 | 32 (43.8) | 36 (49.3) | 41 (56.2) | 0.3 (0.1–0.8) | – | – | – | – | 0.9 (0.6–1.7) |
| Differentiation | 94 | |||||||||
| Well/moderate | 89 | 34 (38.2) | 43 (48.2) | 55 (61.8) | 1 | – | – | – | – | 1.2 (0.8–2.0) |
| Poor | 5 | 0(0) | 5 (100) | 5 (100) | ND | N.D | ||||
OR was adjusted for age, sex, and smoking status.
Abbreviation: ND, not determined.
OR and 95%CI for frequency of the AC or CC genotype in CRC patients; the first category was taken as reference.
OR and 95%CI for frequency of the AC or CC genotype in CRC patients compared with the control group; controls with the wild-type AA genotype were used as reference category.
Table 3.
Characteristics of patients and controls according to MTHFR 677CIT genotypes
| Cases |
Controls | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Number | CC n (%) | CT n (%) | CT + TT n (%) | 1OR (95% CI) | Number | CC n (%) | CT n (%) | CT + TT n (%) | 2OR (95% CI) | |
| Total | 151 | 64 (42.4) | 80 (53) | 87 (57.6) | 231 | 150 (64.9) | 68 (29.4) | 81 (35.1) | 2.4 (1.6–3.6) | |
| Age | ||||||||||
| < 60 | 66 | 31 (47) | 34 (51.5) | 35 (53) | 1 | 100 | 60 (60) | 35 (35) | 40 (40) | 1.3 (0.7–2.7) |
| ≥ 60 | 85 | 33 (38.8) | 46 (54.1) | 52 (61.2) | 1.3 (0.7–2.5) | 131 | 90 (68.7) | 33 (25.2) | 41 (31.3) | 2.7 (1.5–4.8) |
| Sex | ||||||||||
| Male | 90 | 33 (36.7) | 51 (56.7) | 57 (63.3) | 1 | 140 | 87 (62.1) | 46 (32.9) | 53 (37.9) | 1.6 (0.8–3.2) |
| Female | 61 | 31 (50.8) | 29 (47.5) | 30 (49.2) | 0.6 (0.3–1.2) | 91 | 63 (69.2) | 22 (24.2) | 28 (30.8) | 2.5 (1.3–4.6) |
| Smoking | *81 | |||||||||
| Never | 87 | 40 (46) | 44 (50.6) | 47 (54) | 1 | 56 | 23 (41.1) | 24 (42.9) | 33 (58.9) | 0.8 (0.4–1.6) |
| Current/Past | 64 | 24 (37.5) | 36 (56.3) | 40 (62.5) | 1.2 (0.6–2.4) | 25 | 17 (68) | 7 (28) | 8 (32) | 4.4 (1.6–12.5) |
| Location | ||||||||||
| Proximal | 60 | 22 (36.7) | 37 (61.7) | 38 (63.3) | 1 | – | – | – | – | 3.4 (1.8–5.9) |
| Distal | 91 | 42 (46.2) | 43 (47.3) | 49 (53.8) | 0.6 (0.3–1.2) | – | – | – | – | 1.8 (1.1–3.2) |
| Differentiation | ||||||||||
| Well/Moderate | 131 | 58 (44.3) | 66 (50.4) | 73 (55.7) | 1 | – | – | – | – | 2.3 (1.5–3.6) |
| Poor | 20 | 6 (30) | 14 (70) | 14 (70) | 2.1 (0.7–6.1) | – | – | – | – | 4.5 (1.7–12.3) |
Odds ratio and 95% confidence interval for frequency of the CT or TT genotype in CRC patients taking the first category as reference;
Odds ratio and 95% confidence interval for frequency of the CT or TT genotype in CRC patients compared with the control group; controls with the wild type CC genotype were used as reference category.
Adjusted for age, sex, and smoking status.
ND: Not Determined.
Missing information on variable of smoking status explains why n for controls does not equal 231.
In case–case comparisons, we observed no differences in frequencies of MTHFR C677T genotypes in patients stratified by the clinicopathologic variables (Table 3). In case–control comparisons, however, we found an increased cancer risk associated with the CT + TT genotypes in females (OR = 2.5; 95%CI 1.3 – 4.6), in older patients (OR = 2.7; 95%CI 1.5 – 4.8), and in both proximal (OR = 3.4; 95%CI 1.8 – 5.9) and distal (OR = 1.8; 95% CI 1.1 – 3.2) tumors (Table 3). Compared with controls, the CT + TT genotype was strongly associated with CRC in smokers (OR = 4.4; 95%CI 1.6 – 12.5), but not in nonsmokers. This genotype also showed a significant association with both well/moderately differentiated and poorly differentiated CRC (Table 3).
3.3. Relationship between MTHFR genotypes and MSI
We examined the relationship between MTHFR polymorphisms and MSI in CRC from 151patients. PCR amplification of the extracted DNA was performed for the five microsatellite markers recommended by the National Cancer Institute (i.e. BAT25, BAT26, D17S250, D2S123, and D5S346 [22]. Illustrative examples of MSI analysis of the five microsatellite markers in CRC tumors are shown in Fig. 1.
Figure 1.
MSI analysis in colorectal cancer using five microsatellite markers (Bat-25, Bat-26, D2S123, D17S250, and D5S346) by PCR-SSCP. The gels were stained by the silver nitrate method. N, normal DNA pattern; T, tumor specimens.
Table 4 provides the OR with 95%CI for the association between MSI and clinicopathologic characteristics among colon cancer cases. Among the 151 tumors, 36 (23.8%) exhibited a high degree of MSI (MSI+). Proximal location of the tumor was significantly associated with the likelihood of having MSI (P = 0.00). A total of 26/60 (43.3%) patients with proximal CRC showed MSI tumors, and only 10/91 (11%) patients with distal CRC showed MSI. Patients with MSI CRC were smokers and more likely to have poorly differentiated tumors. A small trend for a higher risk of MSI was observed in younger patients compared with the old ones (P = 0.06; Table 4).
Table 4.
Associations between MSI status and clinicopathological features of CRC
| Number | MSI n (%) | MSS n (%) | OR (95% CI) | p Value | |
|---|---|---|---|---|---|
| Total | 151 | 36 (23.8) | 115 (76.2) | ||
| Age | |||||
| < 60 | 66 | 20 (30.3) | 46 (69.7) | 1 | |
| ≥ 60 | 85 | 16 (18.8) | 69 (81.2) | 0.5 (0.5–1.0) | 0.06 |
| Sex | |||||
| Male | 90 | 23 (25.6) | 67 (74.4) | 1 | |
| Female | 61 | 13 (21.3) | 48 (78.7) | 0.97 (0.4–2.3) | 0.9 |
| Smoking | |||||
| Never | 87 | 14 (16.1) | 73 (83.9) | 1 | |
| Current/past | 64 | 22 (34.4) | 42 (65.6) | 2.9 (1.3–6.7) | 0.01 |
| Location | |||||
| Distal | 91 | 10 (11.0) | 81 (89) | 1 | |
| Proximal | 60 | 26 (43.3) | 34 (56.7) | 10.4 (3.9–27.8) | 0.00 |
| Differentiation | |||||
| Well/Moderate | 131 | 27 (20.6) | 104 (79.4) | 1 | |
| Poor | 20 | 9(45) | 11 (55) | 3.5 (1.2–9.9) | 0.02 |
The associations between MSI and the genetic features of tumors are shown in Table 5. The frequency of MTHFR 677 and MTHFR 1298 genotypes were not statistically different in MSI tumors compared with MSS tumors. In case–control comparisons, however, a significant positive association was observed between the MTHFR CT + TT genotype and MSI status (OR = 2.6, 95%CI 1.3 – 5.3; P = 0.01).
Table 5.
Association between MTHFR genotypes and MSI and MSS CRC
| Genotype | MSI cancers n (%) | MSS cancers n (%) | 1OR (95% CI) | 2OR (95% CI) |
|---|---|---|---|---|
| MTHFR C677T | ||||
| CC | 15 (41.7) | 49 (42.6) | Ref | 2.3 (0.5–9) |
| CT | 18 (50) | 62 (53.9) | 0.9 (0.4–2.1) | 2.6 (1.3–5.6) |
| CT + TT | 21 (58.3) | 66 (57.4) | 1.1 (0.4–2.2) | 2.6 (1.3–5.3) |
| MTHFR A1298C | ||||
| AA | 11 (64.7) | 21 (30.4) | Ref | N.D |
| AC | 6 (35.3) | 37 (53.6) | 2.7 (0.8–9.3) | N.D |
| AC + CC | 6 (35.3) | 48 (69.6) | 2.9 (0.8–9.7) | 0.4 (0.14–1.1) |
OR and 95% CI for MSI-cases versus MSS-cases, OR adjusted for age, sex, and smoking status
OR and 95% CI for MSI-cases versus controls, controls with the wild type genotype were used as reference category
OR adjusted for age, sex, and smoking status
ND: Not determined
The majority of CRC exhibiting the MSI phenotype result from hypermethylation of the hMLH1 gene promoter [23]. The CRC series investigated in this study for MSI analysis (151 tumors) had been characterized previously for hypermethyaltion of the promoter region of hMLH1 and hMHSH2 genes [20]. Therefore, this allowed us to examine the association between MSI status and hypermethylation of the two MMR genes in the present study. Ninety-five percent of the patients with methylated hMLH1 (19/20) and 75% of those with methylated hMSH2 (3/4) exhibited a high degree of MSI, indicating a strong association between hypermethylation of MMR genes and MSI incidence in CRC tumors (P = 0.00; data not shown).
4. Discussion
The current case–control study aimed to explore the role of two common functional polymorphisms of MTHFR gene, C677T and A1298C, in altering the susceptibility to cancer and MSI in sporadic CRC in a Southern Iranian population. These variants may be involved in the development of CRC by aberrant methylation of genes promoter and thus predispose to microsatellite instability.
The frequencies of MTHFR 1298 genotypes in patients corresponded to those of the controls (Table 1). The distribution of MTHFR 677 genotypes among patients, however, was significantly different from that of the controls (P = 0.00). The TT genotype frequency observed in our control group (5.6%) was lower than the 12% generally reported for Caucasian and Asian populations [24].
Previous studies on the association between the C677T polymorphism and susceptibility to CRC showed no consistent results. Some studies have reported a significantly reduced risk of developing CRC in TT individuals with an adequate dietary folate intake [25,26], which suggests a protective effect of this genotype. Several other studies, however, have failed to show a protective effect of the MTHFR 677TT genotype against CRC, even when folate intake was high [11,27,28].
Because of the relatively small numbers, we combined individuals with MTHFR 677TT and CT genotypes. In agreement with the previous findings of an increased CRC risk associated with the MTHFR-TT genotype, we found more than a twofold-increased risk of CRC incidence associated with the CT + TT genotype in the entire group of patients compared with age-matched controls (Table 3). The proportion of CT + TT individuals was not significantly elevated in male patients (P = 0.06). Compared with controls, however, the MTHFR CT + TT genotype showed a higher risk of cancer in females (OR = 2.5; 95%CI = 1.3 – 4.6), but not in males (Table 3). We observed no significant difference in the distribution of the CT + TT genotype between patients with proximal and distal tumors. In case–control comparisons, the CT + TT genotype was positively associated with cancer incidence in older cases. Shannnon et al. [14] suggested that the increased risk of CRC associated with the MTHFR TT genotype in older populations is possibly due to age-related disturbances in folate metabolism. In our previous study, we examined the association among the MTHFR C677T genotype, serum folate status, and hypermethyaltion of the promoter region of three tumor-related genes (p16, hMLH1, and hMHSH2) in CRC patients [20]. In that study, we found that levels of serum folate and vitamin B12 were positively associated with tumor methylation, especially for those with the MTHFR CT + TT genotype.
We found no significant difference in the risk associated with the MTHFR AC + CC genotype when all the CRC patients were compared with controls and the wild-type AA genotype was used as a reference category (Table 2). A significant risk difference was observed between homozygous AA and the AC + CC genotypes when proximal cases were compared with controls and the wild-type AA was used as the reference category (OR = 3, 95% = CI 1.1–7.6). Such a relationship was not observed when cases with distal tumors or any sex and age group of CRC patients were compared with controls.
While the genotype of MTHFR 1298 was not associated with the overall risk of CRC in our study, a significantly increased cancer risk was observed in individuals with double heterozygotes (677CT/1298AC ), as well as homozygous 677TT/1298AA and 677CC/1298CC genotypes (Table 1). The lack of risk associated with the MTHFR A1298C alone could be due to no significant alteration in the activity of the enzyme caused by this polymorphism [5,29]. However, the coincidence of the 1298C polymorphism with the 677T allele, which adversely affects the MTHFR activity [6], could increase the risk of cancer development.
We had previously reported that the MTHFR TT genotype, combined with a high serum folate status, may be a risk factor for tumor-specific gene promoter hypermethylation among Iranian CRC patients [20]. We have now conducted MSI analysis in these patients. We aimed to test the hypothesis that polymorphisms in the MTHFR enzyme that are involved in folate metabolism may play a role in altered promoter-specific hypermethylation and, thus, predispose to MSI CRC.
We scored MSI status by examining a combination of three dinucleotide markers (D2S123, D5S346, and D17S250) and two mononucleotide markers (BAT25 and BAT26) in sporadic CRC based on National Cancer Institute (NCI) workshop criteria [22]. MSI was scored if two or more markers showed changes. Some investigators have defined tumors as microsatellite instable if only mononucleotide markers show instability [30]. In our study, all tumors showing instability in dinucleotide makers were also MSI-positive at least in one mononucleotide maker.
In agreement with previous reports [15,31], MSI CRC cases in our study group were more likely to be proximally located and poorly differentiated (Table 4). Our result is consistent with the findings of other studies showing a positive association between cigarette smoking and microsatellite instability in CRC [32,33].
Several studies have addressed the relationship among MTHFR polymorphisms and MSI in colon cancer. The findings of some previously published studies suggest that individuals with one or two MTHFR 677T alleles were at increased MSI CRC risk [15,17]. Compared with age-matched controls, we observed that the MTHFR 677CT + TT genotype is also associated with MSI cancer (OR = 2.6, 95%CI = 1.3 – 5.3; Table 5). Such a relationship was not observed in case–case comparisons. Clarizia et al. [16] observed no association between the C677T polymorphism and microsatellite instability in the Brazilian population. Our results suggest that the MTHFR C677T variant genotype might be associated with an increased risk of MSI tumors. In case–case comparisons, we found a small trend of higher MSI risk in cases with the MTHFR 1298AC + CC variant genotype (OR = 2.9, 95%CI = 0.8 – 9.7; P = 0.07; Table 5).
MSI occurs as a result of a deficiency of the MMR genes [34]. This deficiency is identified in 15 – 20% of all CRC [35]. Our finding is in general agreement with previous studies showing that silencing of MMR gene expression by hypermethylation is associated with MSI-CRC [36]. A strong association was observed between genes promoter methylation and MSI incidence in these tumors (data not shown). Ninety-one percent of 22 tumors, which had at least one methylated MMR gene, were MSI+. The balance of DNA synthesis and DNA methylation determined by MTHFR genotypes may play an important role in the regulation of gene expression influencing cancer risk.
In conclusion, we found the MTHFR 677T allele to be strongly associated with CRC and MSI. Our data suggest that this risk may be mediated, at least in part, by inactivation of MMR genes caused by their promoter hypermethylation. The major limitation of this study is its sample size, and it is also necessary to investigate gene–nutrient interactions to better predict risk factors.
Acknowledgments
We thank Dr. Mahmood Vessal of Shiraz University of Medical Sciences for carefully reading the manuscript. This work has been supported by the office of the Vice Chancellor for Research, Shiraz University of Medical Sciences.
References
- [1].Yazdizadeh B, Jarrahi AM, Mortazavi H, Mohagheghi MA, Tahmasebi S, Nahvijo A. Time trends in the occurrence of major GI cancers in Iran. Asian Pac J Cancer Prev. 2005;6:130–4. [PubMed] [Google Scholar]
- [2].Jass JR. Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology. 2007;50:113–30. doi: 10.1111/j.1365-2559.2006.02549.x. [DOI] [PubMed] [Google Scholar]
- [3].Shen L, Toyota M, Kondo Y, Lin E, Zhang L, Guo Y, Hernandez NS, Chen X, Ahmed S, Konishi K, Hamilton SR, Issa JP. Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer. Proc Natl Acad Sci USA. 2007;104:18654–9. doi: 10.1073/pnas.0704652104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [4].Botto LD, Yang Q. 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review. Am J Epidemiol. 2000;151:862–77. doi: 10.1093/oxfordjournals.aje.a010290. [DOI] [PubMed] [Google Scholar]
- [5].Van der Put NM, Gabreëls F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, van den Heuvel LP, Blom HJ. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Gene. 1998;62:1044–1051. doi: 10.1086/301825. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [6].Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJH, denHeijer M, Kluijtmans LAJ, vandenHeuve LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10:111–3. doi: 10.1038/ng0595-111. [DOI] [PubMed] [Google Scholar]
- [7].Murtaugh MA, Curtin K, Sweeney C, Wolff RK, Holubkov R, Caan BJ, Slattery ML. Dietary intake of folate and co-factors in folate metabolism, MTHFR polymorphisms, and reduced rectal cancer. Cancer Causes Control. 2007;18:153–63. doi: 10.1007/s10552-006-0099-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [8].Yin G, Kono S, Toyomura K, Hagiwara T, Nagano J, Mizoue T, Mibu R, Tanaka M, Kakeji Y, Maehara Y, Okamura T, Ikejiri K, Futami K, Yasunami Y, Maekawa T, Takenaka K, Ichimiya H, Imaizumi N. Methylenetetrahydrofolate reductase C677 T and A1298C polymorphisms and colorectal cancer: the Fukuoka Colorectal Cancer Study. Cancer Sci. 2004;95:908–13. doi: 10.1111/j.1349-7006.2004.tb02201.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [9].Hubner RA, Houlston RS. MTHFR C677 T and colorectal cancer risk: a meta-analysis of 25 populations. Int J Cancer. 2007;120:1027–35. doi: 10.1002/ijc.22440. [DOI] [PubMed] [Google Scholar]
- [10].Kim DH. The interactive effect of methyl-group diet and polymorphism of methylenetetrahydrofolate reductase on the risk of colorectal cancer. Mutat Res. 2007;622:14–8. doi: 10.1016/j.mrfmmm.2007.04.012. [DOI] [PubMed] [Google Scholar]
- [11].Guerreiro CS, Carmona B, Gonçalves S, Carolino E, Fidalgo P, Brito M, Leitão CN, Cravo M. Risk of colorectal cancer associated with the C677 T polymorphism in 5,10-methylenetetrahydrofolate reductase in Portuguese patients depends on the intake of methyl-donor nutrients. Am J Clin Nutr. 2008;88:1413–8. doi: 10.3945/ajcn.2008.25877. [DOI] [PubMed] [Google Scholar]
- [12].Kim DH, Ahn YO, Lee BH, Tsuji E, Kiyohara C, Kono S. Methylenetetrahydrofolate reductase polymorphism, alcohol intake, and risks of colon and rectal cancers in Korea. Cancer Lett. 2004;216:199–205. doi: 10.1016/j.canlet.2004.08.014. [DOI] [PubMed] [Google Scholar]
- [13].Chapusot C, Martin L, Bouvier AM, Bonithon-Kopp C, Ecarnot-Laubriet A, Rageot D, Ponnelle T, Laurent Puig P, Faivre J, Piard F. Microsatellite instability and intratumoural heterogeneity in 100 right-sided sporadic colon carcinomas. Br J Cancer. 2002;87:400–4. doi: 10.1038/sj.bjc.6600474. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [14].Shannon B, Gnanasampanthan S, Beilby J, Iacopetta B. A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability. Gut. 2002;50:520–4. doi: 10.1136/gut.50.4.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [15].Hubner RA, Lubbe S, Chandler I, Houlston RS. MTHFR C677 T has differential influence on risk of MSI and MSS colorectal cancer. Hum Mol Genet. 2007;16:1072–7. doi: 10.1093/hmg/ddm055. [DOI] [PubMed] [Google Scholar]
- [16].Clarizia AD, Bastos-Rodrigues L, Pena HB, Anacleto C, Rossi B, Soares FA, Lopes A, Rocha JC, Caballero O, Camargo A, Simpson AJ, Pena SD. Relationship of the methylenetetrahydrofolate reductase C677 T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer. Genet Mol Res. 2006;5:315–22. [PubMed] [Google Scholar]
- [17].Chang SC, Lin PC, Lin JK, Yang SH, Wang HS, Li AF. Role of MTHFR polymorphisms and folate levels in different phenotypes of sporadic colorectal cancers. Int J Colorectal Dis. 2007;22:483–9. doi: 10.1007/s00384-006-0190-x. [DOI] [PubMed] [Google Scholar]
- [18].Eaton AM, Sandler R, Carethers JM, Millikan RC, Galanko J, Keku TO. 5,10-methylenetetrahydrofolate reductase 677 and 1298 polymorphisms, folate intake, and microsatellite instability in colon cancer. Cancer Epidemiol Biomarkers Prev. 2005;14:2023–9. doi: 10.1158/1055-9965.EPI-05-0131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [19].Toffoli G, Gafà R, Russo A, Lanza G, Dolcetti R, Sartor F, Libra M, Viel A, Boiocchi M. Methylenetetrahydrofolate reductase 677 C → T polymorphism and risk of proximal colon cancer in north Italy. Clin Cancer Res. 2003;9:43–8. [PubMed] [Google Scholar]
- [20].Mokarram P, Naghibalhossaini F, SaberiFiroozi M, Hosseini SV, Izadpanah A, Salahi H, Malek-Hosseini SA, Talei A, Mojallal M. Methylenetetrahydrofolate reductase C677 T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status. World J Gastroenterol. 2008;14:3662–71. doi: 10.3748/wjg.14.3662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [21].Naghibalhossaini F, Mokarram P, Khalili I. Easy detection of 5, 10-methylenetetrahydrofolate reductase 1298A/C genotype by mutagenically separated PCR assay. Clin Chem Lab Med. 2008;46:987–9. doi: 10.1515/CCLM.2008.184. [DOI] [PubMed] [Google Scholar]
- [22].Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, Meltzer SJ, Rodriguez-Bigas MA, Fodde R, Ranzani GN, Srivastava S. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res. 1998;58:5248–57. [PubMed] [Google Scholar]
- [23].Watanabe T, Kobunai T, Toda E, Yamamoto Y, Kanazawa T, Kazama Y, Tanaka J, Tanaka T, Konishi T, Okayama Y, Sugimoto Y, Oka T, Sasaki S, Muto T, Nagawa H. Distal colorectal cancers with microsatellite instability (MSI) display distinct gene expression profiles that are different from proximal MSI cancers. Cancer Res. 2006;66:9804–8. doi: 10.1158/0008-5472.CAN-06-1163. [DOI] [PubMed] [Google Scholar]
- [24].Ueland PM, Hustad S, Schneede J, Refsum H, Vollset SE. Biological and clinical implications of the MTHFR C677 T polymorphism. Trends Pharmacol Sci. 2001;22:195–201. doi: 10.1016/s0165-6147(00)01675-8. [DOI] [PubMed] [Google Scholar]
- [25].Slattery ML, Potter JD, Samowitz W, Schaffer D, Leppert M. Methylenetetrahydrofolate reductase, diet, and risk of colon cancer. Cancer Epidemiol Biomarkers Prev. 1999;8:513–8. [PubMed] [Google Scholar]
- [26].Ma J, Stampfer MJ, Giovannucci E, Artigas C, Hunter DJ, Fuchs C, Willett WC, Selhub J, Hennekens CH, Rozen R. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res. 1997;57:1098–102. [PubMed] [Google Scholar]
- [27].Keku T, Millikan R, Worley K, Winkel S, Eaton A, Biscocho L, Martin C, Sandler R. 5,10-Methylenetetrahydrofolate reductase codon 677 and 1298 polymorphisms and colon cancer in African Americans and whites. Cancer Epidemiol Biomarkers Prev. 2002;11:1611–21. [PubMed] [Google Scholar]
- [28].Heijmans BT, Boer JM, Suchiman HE, Cornelisse CJ, Westendorp RG, Kromhout D, Feskens EJ, Slagboom PE. A common variant of the methylenetetrahydrofolate reductase gene (1p36) is associated with an increased risk of cancer. Cancer Res. 2003;63:1249–53. [PubMed] [Google Scholar]
- [29].Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab. 1998;64:169–72. doi: 10.1006/mgme.1998.2714. [DOI] [PubMed] [Google Scholar]
- [30].Lipton LR, Johnson V, Cummings C, Fisher S, Risby P, EftekharSadat AT, Cranston T, Izatt L, Sasieni P, Hodgson SV, Thomas HJ, Tomlinson IP. Refining the Amsterdam Criteria and Bethesda Guidelines: testing algorithms for the prediction of mismatch repair mutation status in the familial cancer clinic. J Clin Oncol. 2004;22:4934–43. doi: 10.1200/JCO.2004.11.084. [DOI] [PubMed] [Google Scholar]
- [31].Sugai T, Habano W, Jiao YF, Tsukahara M, Takeda Y, Otsuka K, Nakamura S. Analysis of molecular alterations in left- and right-sided colorectal carcinomas reveals distinct path ways of carcinogenesis: proposal for new molecular profile of colorectal carcinomas. J Mol Diagn. 2006;8:193–201. doi: 10.2353/jmoldx.2006.050052. [DOI] [PMC free article] [PubMed] [Google Scholar]
- [32].Slattery ML, Curtin K, Anderson K, Ma KN, Ballard L, Edwards S, Schaffer D, Potter J, Leppert M, Samowitz WS. Associations between cigarette smoking, lifestyle factors, and microsatellite instability in colon tumors. J Natl Cancer Inst. 2000;92:1831–6. doi: 10.1093/jnci/92.22.1831. [DOI] [PubMed] [Google Scholar]
- [33].Yang P, Cunningham JM, Halling KC, Lesnick TG, Burgart LJ, Wiegert EM, Christensen ER, Lindor NM, Katzmann JA, Thibodeau SN. Higher risk of mismatch repair-deficient colorectal cancer in alpha(1)-antitrypsin deficiency carriers and cigarette smokers. Mol Genet Metab. 2000;71:639–45. doi: 10.1006/mgme.2000.3089. [DOI] [PubMed] [Google Scholar]
- [34].Noda H, Kato Y, Yoshikawa H, Arai M, Togashi K, Nagai H, Konishi F, Miki Y. Microsatellite instability caused by hMLH1 promoter methylation increases with tumor progression in right-sided sporadic colorectal cancer. Oncology. 2005;69:354–62. doi: 10.1159/000089768. [DOI] [PubMed] [Google Scholar]
- [35].Giardiello FM, Brensinger JD, Petersen GM. AGA technical review on hereditary colorectal cancer and genetic testing. Gastroenterology. 2001;121:198–213. doi: 10.1053/gast.2001.25581. [DOI] [PubMed] [Google Scholar]
- [36].Grady WM, Rajput A, Lutterbaugh JD, Markowitz SD. Detection of aberrantly methylated hMLH1 promoter DNA in the serum of patients with microsatellite unstable colon cancer. Cancer Res. 2001;61:900–2. [PubMed] [Google Scholar]