Abstract
The aim of the present study was to evaluate the effects of the genotypic polymorphisms in methylenetetrahydrofolate reductase (MTHFR) and its interaction with early-onset breast cancer risk in Taiwan. Two well-known polymorphic variants of MTHFR, C677T (rs1801133) and A1298C (rs1801131), were analyzed and their joint effects with individual age- and estrogen-related factors on breast cancer risk were discussed. In total, 1,232 patients with breast cancer and 1,232 healthy controls were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The MTHFR C677T genotype, but not the A1298C, was differently distributed between cancer and control groups. The T allele of MTHFR C677T was significantly more frequently found in controls than in patients with cancer. In addition, females carrying MTHFR C677T CT or TT genotypes had a higher odds ratio of 1.21 (95% confidence interval=1.03-1.42, p=1.85E-5) for breast cancer, especially before the age of 45.4 years (odds ratio=1.51 and 95% confidence interval=1.20-1.90). Our results indicate that MTHFR C677T T allele was associated with increased risk of breast cancer in Taiwan, especially in cases who were 45.4 old or younger and with earlier menarche age (<12.2 years).
Breast cancer is now ranked first among cancer affecting women throughout the world and the latest estimates suggest that more than 1,050,000 new breast cancer cases occur worldwide annually (1, 2). Breast cancer in Asia is characterized by a lower incidence than in Western populations, but is still the leading type of cancer in Asian women, and a significantly increasing tread indicates that it is an issue of particular public health importance. In Taiwan, breast cancer is the second leading type of cancer, important for its high incidence, high mortality, and early onset (3, 4). Previous studies revealed that oriental women affected by breast cancer, such as those in Taiwan, were significantly younger than white women and had racial/ethnic difference in their survival patterns (5, 6). Recently, scientists began to explore the mechanisms underlying breast cancer formation at the molecular level. Further investigation into these racial/ethnic differences may help unravel the genomic and environmental etiology of breast cancer, and aid cancer detection, therapy and prevention.
Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism and catalyzes 5,10-methylene-tetrahydrofolate to 5-methyltetrahydrofolate. The importance of MTHFR in cancer susceptibility arises from its involvement in two pathways of folate metabolism: one leads to numerous methylation processes that are dependent on S-adenosyl-methionine (SAM), while the other, via thymidylate synthesis, contributes to DNA replication and cell division. Reduced activity of MTHFR may decrease the methylation of homocysteine to methionine and in turn the level of SAM, resulting in DNA hypomethylation. On the other hand, a reduced level of MTHFR substrate, required for thymidylate synthesis, could lead to uracil mis-incorporation into DNA, diminished DNA repair and increased frequency of chromosomal breaks and damage. Malignancies that are derived from rapidly proliferating tissues, which have a higher requirement for DNA synthesis, should be more susceptible to folate deficiency and resultant DNA damage. The DNA variants causing reduced MTHFR activity were found to be associated with a reduced risk of leukemia, lymphoma and colorectal carcinoma. The mechanism proposed to explain these associations was the shunt of folate metabolism versus thymidine and purine synthesis, which would slow the incorporation of uracil into DNA and protect cells against carcinogenesis (7).
The primer sequences, polymerase chain reaction and restriction fragment length polymorphism conditions for methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms.
Previous investigations of MTHFR genetic variations focused on the catalytic domain and the two polymorphisms C677T and A1298C, which slightly change enzymatic activity. In the case of C677T polymorphism, the cytosine base at position number 677 changes to a thymidine base, which in turn affects the amino acid sequence at position number 222 (alanine→valine). The MTHFR enzyme resulting from the C677T polymorphism becomes thermolabile, causing a loss of its activity with increasing temperature. The modified protein also loses its co-factor flavin adenine dinucleotide (FAD) more quickly and has a lower stability. The mutation effect can be suppressed by addition of folate, which causes a higher FAD affinity and an increase in MTHFR stability. The MTHFR A1298C polymorphism is localized in the coding regulatory region domain (8). Studies investigating the MTHFR A1298C variant have found positive associations with colorectal cancer (9), breast cancer (10), acute lymphocytic leukemia (11), childhood leukemia (12).
In 2003, the association between single nucleotide polymorphisms (SNPs) of MTHFR and breast cancer susceptibility was firstly examined in a Taiwanese population, indicating the C677T SNP is not associated with breast cancer risk (13). However, the sample size was rather small (controls/cases=232/59), and only one SNP was investigated in that study. In 2006, the same group performed a genotype–phenotype correlation study showing that the combined genotype (677CT+TT with 1298AC+CC) conferred greater reduction of breast cancer risk among Taiwanese females with lower plasma folate levels (14), with a sample size of controls/cases=295/146. In the present work, we analyzed both MTHFR C677T and A1298C in a more representative population (controls/cases=1232/1232), and investigated the correlation between MTHFR genotypes and early onset of breast cancer in Taiwanese women.
Materials and Methods
Study population and sample collection. A total of 1232 patients diagnosed with breast cancer were recruited at our hospital. All patients voluntarily participated, completed a self-administered questionnaire and provided peripheral blood samples. An equal number of age-matched non-breast cancer healthy volunteers as controls were selected from the Health Examination Cohort of the hospital. Our study was approved by the Institutional Review Board of the China Medical University Hospital (DMR96-IRB-240).
Genotyping assays. Genomic DNA was prepared using a QIAamp Blood Mini Kit (Blossom, Taipei, Taiwan) as described in previous studies (15-20). The polymerase chain reaction (PCR) cycling conditions were: one cycle at 94°C for 5 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 min. Pairs of PCR primer sequences and restriction enzyme for each DNA product are listed in Table I.
Statistical analyses. Only those with both genotypic and clinical data (controls/cases=1232/1232) were selected for final analysis. Pearson's Chi-square test or Fisher's exact test was used to compare the distribution of the genotypes. The data were recognized as significant when the statistical p-value was less than 0.05.
Results
The frequency distributions of selected characteristics of 1232 breast cancer patients and 1232 non-cancer controls are shown in Table II. These characteristics of patients and controls are all well matched (p>0.05) (Table II). As for the individual behaviors, cigarette smoking and alcoholism were both risk factors for breast cancer in this population (p<0.05) (Table II).
The frequencies of the genotypes for the MTHFR C677T and A1298C in controls and patients with breast cancer are shown in Table III. The genotype distribution of the MTHFR C677T was significantly different between breast cancer and control groups (p=1.85×10−5), while that for A1298C polymorphisms was not (p>0.05) (Table III). Those who carried the TT genotype were found to have a 1.88-fold odds of breast cancer compared with those with CC genotype (95% CI=1.44-2.47).
Distributions of demographic and life-style of breast cancer patients and the matched controls.
Distribution of methylenetetrahydrofolate reductase (MTHFR) genotypes among breast cancer and control groups.
Since age and estrogen exposure are the predominant risk factors for breast cancer, and Taiwan is well-known for early-onset breast cancer, the interactions between MTHFR genotype and age- and estrogen-related factors were also analyzed and presented in Tables IV, V, VI and VII. The average age of all participants for age at onset, age at menarche, age at first birth of child, and age at menopause were 45.4, 12.2, 29.6 and 49.0 years, respectively, and were set as the cutting point for stratification. We noticed that those with CT or TT genotypes for MTHFR C677T had higher risk of breast cancer than those with CC genotype in the younger group (<45 years), but not in the case of the elder group (≥45 years) (Table IV). Those with CT or TT genotypes for MTHFR C677T also had higher risk of breast cancer than those with CC genotype in the group with earlier menarche (<12.2 years) but not in the case of later menarche (Table V). To sum up, there was an interaction between age at early menarche and onset with MTHFR C677T genotype for breast cancer susceptibility.
Discussion
In order to determine the role of MTHFR and to find potential biomarkers of breast cancer, in the present study, we selected two SNPs of MTHFR and investigated their associations with breast cancer risk in Taiwan. We found CT and TT genotypes of MTHFR C677T were significantly associated with a lower susceptibility for breast cancer (Tables III). These data are consistent with those finding the T allele to confer a higher risk (21-23), but not those reporting no association (13, 24-28). This may be caused by differences in ethnicity; moreover, our sample size was much larger than that of Jeng et al. and more representative of Taiwanese patients (13). Thus, the effects of MTHFR C677T polymorphism on carcinogenesis are complex, exerting either an adverse effect on DNA methylation or an advantageous influence on nucleotide synthesis in determining cancer risk.
Distribution of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genotypes and age-related demographic characteristics (onset age).
Distribution of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genotypes and age-related demographic characteristics (age at menarche).
We further analyzed the association between C677T genotype and breast cancer risk in those aged younger to investigate the role of MTHFR in early onset of breast cancer in Taiwan. Interestingly, the interaction between MTHFR C677T and age is clear, younger females (diagnosed with breast cancer at an age earlier than 45.4 years) with the CT or TT genotype have a 1.51-fold greater odds of breast cancer than those younger females with the CC genotype, which is not the case in the elder group (Table IV). This trend also fits well with the history of earlier menarche in the same population, those with the TT and CT+TT genotypes have a 2.54- and 1.38-fold greater odds for breast cancer than those with younger age at first menarche with CC genotype, which is not the case in the elder group (Table V). Since early first full-term pregnancy and late menopause were considered to be protective (29-31) and risky (32-34) factors of breast cancer in Taiwan, respectively, we also analyzed the interactions of MTHFR genotype with these two age- and estrogen-related factors. The data showed that TT genotype of MTHFR C677T has a 1.59 -fold greater odds for breast cancer than those with CC at early (<29.6 years) birth of first child, while CT, TT and CT+TT genotypes have a 1.26-, 2.22- and 1.36 -fold greater odds for breast cancer respectively than those with CC at late (>29.6 years) birth of first child (Table VI). As for age of menopause, TT genotype of MTHFR C677T has a 1.75-fold greater odds for breast cancer than those with CC at early (<49 years) menopause, while TT and CT+TT genotypes have a 2.03- and 1.31-fold greater odds for breast cancer respectively than those with CC at late (>49 years) menopause (Table VII).
Distribution of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genotypes and age-related demographic characteristics (age at birth of first child).
Distribution of methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C genotypes and age-related demographic characteristics (age at menopause).
We propose that T allele at C677T may affect MTHFR activity, influencing the normal function of MTHFR. In literature, it has been shown that MTHFR 677T variants result in 70% lower functional activity (35). Those with T allele(s) not only have an imbalance in their folate pool available for DNA synthesis and cell proliferation, but may not be able to remove the DNA adducts caused by estrogen-induced insults (36) as soon as those with C allele, or cannot regulate the methylation status of other genes normally. All the above mechanisms may lead to earlier onset of breast cancer. To sum up, the alterations toward early breast carcinogenesis may be caused by decreased functions of MTHFR and cascading effects, which may finally lead to early onset of breast cancer.
In conclusion, as far as we are aware of, this is the first study focusing on MTHFR genotype and joint effects with age- and estrogen-related risk factors for early-onset breast cancer. The presence of T allele at C677T was not only associated with a higher cancer risk, but involved in early breast carcinogenesis. The T allele of MTHFR C677T may be a useful marker in breast oncology and early cancer detection.
Acknowledgements
We thank Tzu-Chia Wang, Yun-Ru Syu, Lin-Lin Hou and Chia-En Miao for their technical assistance. This study was supported by research grants from Terry Fox Cancer Research Foundation, and Taichung Armed Forces General Hospital (grant number: 103A24 and 103A25).
Footnotes
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↵* These Authors contributed equally to this work.
- Received May 7, 2014.
- Revision received June 19, 2014.
- Accepted June 20, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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