Abstract
Background/Aim: 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) is responsible for folate metabolism, and we aimed to investigate its genetic role in colorectal cancer (CRC) among Taiwanese. Materials and Methods: A total of 362 cases and 362 controls were recruited and their MTRR rs1801394 (A66G) and rs1532268 (C524T) genotypes were examined. The behavioral factors and clinicalpathological factors were also analyzed. Results: MTRR rs1801394 genotypes were associated with CRC risk (p for trend=0.0087). In detail, G/G genotype was associated with lower risk (p=0.0049, OR=0.39, 95%CI=0.20-0.76). As for allelic frequency analysis, G allele was also associated with decreased CRC risk (p=0.0026, OR=0.68, 95%CI=0.53-0.88). There was no significant association as for MTRR rs1532268. Among non-smokers and non-alcohol drinkers, those with G/G genotype were at 0.38- and 0.46-fold odds of having CRC. There were no significant protective effects among smokers or alcohol drinkers. Conclusion: MTRR rs1801394 GG genotype can be a protective marker for CRC risk in Taiwan.
Colorectal cancer (CRC) is the third most common cancer among men and women worldwide (1-3). The incidence and mortality rates of CRC can change as high as ten folds among countries (1, 2, 4). Many factors may contribute to this variation, for instance, meat consumption, cigarette smoking, and exposure to carcinogens contribute to about 85% of CRC etiology (5, 6). In Taiwan, the problem of CRC is rather noticeable. The incidence rate of CRC is number one among all types of cancer, and the mortality rate of CRC is third, just behind lung and liver cancer. As 15-20% of CRC cases have a familial history of cancer (7, 8), genetic factors have been believed to play a very important part in the etiology of CRC. Although some genetic biomarkers for CRC have been revealed during recent years (9-14), however, the interactions between genomic and other risk factors are still of great interest among translational scientists. The understanding of genetic contribution to CRC can help translational scientists to achieve precise medication and therapy.
In the literature, it is demonstrated that folate metabolism has played a critical part in regulating DNA repair capacity and DNA methylation status (15, 16). There were a few famous genetic polymorphic sites of folate metabolism enzymes, including methionine synthase (MTR), methionine synthase reductase (MTRR), and 5,10-methylenetetrahydrofolate reductase (MTHFR). There were also studies reporting that the various genotypes could influence the levels of serum folate (17-19). Consequently, it is biologically plausible that these polymorphisms will affect DNA repair capacity or cause abnormal DNA methylation, which may subsequently lead to gene instability and give rise to development of various kinds of cancers. Among them, MTRR is a vital enzyme involved in folate metabolism. Folate, a water-soluble B-vitamin, is essential for the prevention of malignancy initiation (20). This vitamin functions as a coenzyme in the process of nucleotide synthesis as well as in the DNA and protein methylation (21). The enzymatic activity of MTRR can be affected by a missense MTRR A66G (rs1801394) polymorphisms. A substitution of A to G at the nucleotide 66 changes isoleucine to methionine at position 22 (Ile22Met) of MTRR (22, 23). The MTRR rs1801394 GG genotype is also associated with lower plasma homocysteine concentration compared to the AA genotype (24, 25). Mutation in the gene encoding this enzyme can cause hyper-homo-cysteinemia, which probably contributes to DNA hypomethylation and lowering DNA repair capacity, and leading to CRC initiation (26, 27). MTRR C524T (rs1532268) is considered to be a risk factor for the development of ventricular septal defects (VSD) (28, 29) and gastric cancer (30, 31), but not entirely concluded in CRC risk. Based on the above information, we hypothesized that the variant genotypes at MTRR rs801394 (A66G) and rs1532268 (C524T) may also be involved in determining the personal susceptibility for CRC among Taiwanese. In addition, we will check the genetic-behavioral and genetic-clinical interactions.
Materials and Methods
Collection of 362 CRC cases and 362 control subjects. The CRC cases were recruited as described in our previous studies (13, 14). Briefly and concisely, the CRC cases have been collected, and the pathological data were defined, graded and well recorded. Then, each of the case was well match by age, sex and behaviors including smoking and alcohol drinking. The collection protocols were approved by the Institutional Review Board of the China Medical University Hospital (coding number: DMR99-IRB-108). Specific characters which are analyzed in the study are presented in Table I.
Summary of selected data from 362 patients with colorectal cancer and 362 matched non-cancer healthy controls.
MTRR genotyping methodology. The genomic DNA from peripheral blood leukocytes of all participants were extracted and stored at –80°C as previously published (32, 33). The polymerase chain reaction (PCR) conditions set for MTRR genotyping were one cycle at 94°C for 5 min; 35 cycles at 94°C for 30 s, one cycle at annealing 55°C for 30 s and one cycle at elongating 72°C for 30 s and a final extension at 72°C for 10 min. The sequences of forward and reverse primers are designed by Terry Fox Cancer Research Lab and provided in Table II. In addition, the PCR products, corresponding restriction enzymes, and cutting adducts for MTRR rs1801394 and rs1532268 are also presented in Table II.
Information about primers, PCR products, restriction enzymes, and cutting adducts for MTRR rs1801394 and rs1532268.
Statistical analysis. To compare the distribution of MTRR genotypic and allelic distributions between stratified sub-groups, the Pearson’s Chi-square test without Yates’ correction was applied. To check the associations between MTRR genotypes and CRC risk, odds ratios (ORs) together with 95% confidence intervals (CIs) was applied. Adaptation of confounding factors as indicated was applied when examining the interactions of behavioral factors with MTRR genotypes.
Results
Basic indexes between the CRC patient and control groups. The distribution of age, sex and other indexes for the 362 CRC patients and 362 non-cancer healthy controls are shown in Table I. There were 203 (56.1%) males and 159 (43.6%) females both in the CRC case and control groups. Ninety-one (25.1%) of the CRC group had the smoking habits, while 44 (12.2%) had alcohol drinking habits, and the percentages were not significant different from those of the control group (both p>0.05, Table I). The BMI was not differentially distributed between case and control groups (Table I).
Association of MTRR genotypes with CRC risk. The genotypes of MTRR rs1801394 (A66G) and rs1532268 (C524T) among the 362 CRC patients and 362 controls are shown in Table III. First, the various genotypic frequencies of MTRR rs1801394 were differentially distributed between CRC and control groups (p for trend=0.0087). In detail, the MTRR rs1801394 heterozygous A/G and homozygous G/G genotypes were associated with lower risk for CRC than the wild-type A/A genotype (p=0.0825 and 0.0049, OR=0.76 and 0.39, 95%CI=0.55-1.03 and 0.20-0.76). In the recessive model, the G/G genotype conferred a decreased risk for CRC compared to combination of A/A+A/G genotypes (p=0.0110, OR=0.43, 95%CI=0.22-0.84). In the dominant model, those who carry A/G+G/G conferred a decreased susceptibility of CRC compared to the A/A genotype carriers (p=0.0151, OR=0.69, 95%CI=0.51-0.93). On the contrary, as for rs1532268, there was no difference in genotype distribution in any models analyzed (Table III, lower panel). To sum up, the MTRR rs1801394 genotypes play a critical role in determining personal susceptibility to CRC in Taiwan.
Distributions of MTRR rs1801394 and rs1532268 genotypic frequencies among colorectal cancer patients and healthy controls.
The allelic frequency analysis of MTRR with CRC risk. Further analysis of allelic frequency was performed and is presented in Table IV. There is an obvious difference in the distribution of allelic frequencies among the CRC patients and healthy controls regarding MTRR rs1801394 (OR=0.68, 95%CI=0.53-0.88, p=0.0026). On the contrary, there is no difference found as for rs1532268 of MTRR (OR=0.94, 95%CI=0.70-1.27, p=0.7027). This is consistent with the finding in Table III.
Allelic frequencies for MTRR rs1801394 and rs1532268 polymorphisms among the colorectal cancer patients and healthy controls.
Influence of smoking habit and MTRR rs1801394 genotype on CRC risk. Cigarette smoking is a risk factor for Taiwan CRC, we intended to examine the influence of cigarette smoking and MTRR rs1801394 genotypes on CRC risk. As for non-smokers, those with MTRR rs1801394 G/G genotype were at 0.38-fold odds of CRC risk (95%CI=0.18-0.83, p=0.0123). There was no significant association between MTRR rs1801394 A/G genotypes and CRC risk (OR=0.74, 95%CI=0.52-1.07, p=0.1150). After adjusting for age, sex, alcohol drinking and BMI status, the trends are similar for both MTRR rs1801394 G/G and A/G (OR=0.36 and 0.70, 95%CI=0.15-0.80 and 0.48-1.12, respectively). There was no any significant association found among the smokers (Table V).
Odds ratio for MTRR rs1801394 genotype and colorectal cancer after stratification by smoking status.
Influence of alcohol drinking habit and MTRR rs1801394 genotype on CRC risk. Then we aimed to investigate the influence of alcohol drinking and MTRR rs1801394 genotypes on CRC risk, another risk factor for Taiwan CRC. As for those non-drinkers, those with MTRR rs1801394 G/G genotype were at 0.46-fold odds of CRC risk (95%CI=0.22-0.95, p=0.0336). There was no significant association between MTRR rs1801394 A/G genotypes and CRC risk (OR=0.74, 95%CI=0.53-1.03, p=0.0702). After adjusting for age, sex, smoking and BMI status, the trends are similar for both MTRR rs1801394 G/G and A/G (OR=0.42 and 0.71, 95%CI=0.18-0.83 and 0.47-1.02, respectively). There was no any significant association found among the alcohol drinkers (Table VI).
Odds ratios for MTRR rs1801394 genotype and colorectal cancer after stratification by alcohol drinking status.
Correlations among genotypes of MTRR rs1801394 and clinical indexes. The correlations among genotypes of MTRR rs1801394 and clinical features were analyzed (Table VII). No statistically significant correlation was observed between MTRR rs1801394 genotypic distributions and age, sex, BMI, tumor size or location, or lymph node metastasis status (all p>0.05) (Table VII).
Correlation between MTRR rs1801394 genotypes and clinicopathological properties of 362 colorectal cancer patients.
Discussion
CRC has been the third most common cause of cancer-related mortality worldwide (1-3), and in Taiwan (35). During the last years, the role of folate and its related genetic variations on CRC have attracted great interest of translational scientists. However, several studies have been conducted, with conflicting findings from various populations with different genetic backgrounds (36-43). Taiwan is genetically and geographically conserved and our collection of CRC cases are representative for Eastern Asia. Our results provided evidence for positive association of MTRR rs1801394 genotypes with CRC risk (Table III and Table IV). In a meta-analysis consisting of 17 studies investigating 8,371 cases and 12,574 control subjects, the overall results indicated that the genotypes of MTRR rs1801394 may be ethnically, associated with CRC risk, which means that Asian populations are more likely to be influenced by MTRR rs1801394 while Caucasian populations are not (44).
MTRR is one of the major players in folate metabolism, and plays essential roles in nucleotide neo-synthesis and methylation status of DNA, histones and proteins. The MTRR rs1801394 G/G genotype has been reported to have lower affinity with MTR (45). High folate intake may be a protective diet for CRC risk (46-48). However, all of these studies neither linked with personal MTRR genotype, nor assessed the dietary folate intake with the real serum folate status to reveal how folate influences CRC risk. The regulation of folate in serum is complicated, and lots of enzymes may be involved, for instance, MTHFR, MTRR and MTR. In 2018, we provided evidence for the significant association of MTHFR rs1801133 T allele serves as a predictive marker for CRC risk (49). It seems that those MTHFR rs1801394 G allele carriers can have a lower level of serum homo-cysteine, which by some unknown reason causes a higher efficiency in DNA repair activity, and keeps the human genome from instability and CRC carcinogenesis. The detailed mechanisms of how MTRR interact with other molecules leading to CRC needs further investigation in the future.
There have been various clinical or behavioral indexes reported to play a role in determining CRC risk, such as age, sex, familial cancer history, diet, alcohol consumption, obesity, tumor site, size, grade, histologic type, TNM stage, and carcinoembryonic antigen (CEA) level, and so on (50-53). But these kinds of epidemiological studies always lack genetic data for any genetic-linked analysis. In the present study, we combined numerous clinical indexes with genotyping data for analysis, and come up with the findings that MTRR rs1801394 G/G genotype can interact with non-smoking (Table V) and non-alcohol drinking habits (Table VI) to affect CRC risk. It is interesting, but complicated to figure out how MTRR rs1801394 G/G genotype can affect non-smokers and non-alcohol drinkers with their CRC risk, but not affect smokers and alcohol drinkers. We temporarily found no significant interactions between MTRR rs1801394 genotype and those clinical indexes (Table VII). However, some indexes such as tumor size (p=0.0664), may have significant results when larger sample size could be collected for bringing up a conclusive finding.
In conclusion, we provided evidence for the significant association of MTRR rs1801394 with Taiwan CRC risk. Our results also suggest that the G/G genotype of MTRR rs1801394 may have its protective effects specifically among non-smokers and non-alcohol drinkers. It can serve as a useful genomic marker for Taiwan CRC risk.
Acknowledgements
The Authors are grateful to Yu-Chen Hsiau and Yu-Ting Chin for their excellent technical assistance. All the participants including those who were not selected into the control group of the study are appreciated. This study was supported mainly by Taichung Armed Forces General Hospital (grant number: TCAFGH-D-110016), China Medical University and Hospital (grant number CMU110-ASIA-03) and Taichung Veterans General Hospital (grant number TCVGH-1115302B). The funders had no role in study design, data collection, statistical analysis, or decision to publish or preparation of the manuscript.
Footnotes
Authors’ Contributions
Research design: Wu MH, Chen CH, Bau DT, Pei JS and Chang WS; patient and questionnaire summaries: Wu MH, Chen CP and Yueh TC; experimental work: Huang TH, Chang WS and Tsai CW; statistical analysis: Wang ZH, Mong MC and Yang YC; article writing: Chang WS and Bau DT; review and revision: Bau DT and Chang WS.
Conflicts of Interest
The Authors declare no conflicts of interest regarding this study.
- Received March 10, 2022.
- Revision received March 31, 2022.
- Accepted April 7, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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