To our knowledge, this is the first study performed in China that investigated the associations between genetic variants in one-carbon metabolism genes and BC risk as well as their interactions with high adherence to MDP on BC risk. Among 15 SNPs involved in the present study, none of them were statistically significantly associated with the risk of BC despite the menopausal status. However, some promising positive results were obtained when analyzing the associations between high adherence to MDP and BC risk based on the stratification of genotypes of the SNPs studied. We found that the associations between high adherence to MDP and risk reduction of BC were affected by the SNPs genotype associated with the one-carbon metabolism pathway. Furthermore, we observed a significant overall interaction between MTHFD1 G > A (rs8003567) genotype and high adherence to MDP on BC risk. Our study suggests that the one carbon-metabolism genes may act as modifiers between diet and BC risk.
The numerous health benefits of the Mediterranean dietary pattern are widely recognized. Since this dietary pattern is composed of many foods rich in fiber and antioxidants, such as flavonoids, vitamins and carotenoids, it has been linked to the prevention of various diseases, including several common cancers. The underlying mechanisms by which the Mediterranean dietary pattern modulated BC risk were previously identified as the decrease of endogenous estrogens,(Carruba, et al. 2006) neutralization of free radicals to prevent DNA damage(Visioli, et al. 2004) as well as reduction of oxidative stress(Mitjavila, et al. 2013). Although an inverse association of adherence Mediterranean dietary pattern with BC risk has been unveiled by many studies,(van den Brandt and Schulpen 2017) such observation failed to be confirmed by others,(Couto, et al. 2013, Demetriou, et al. 2012) or was confined to specific subject subgroups defined by menopausal status(Buckland, et al. 2013, Trichopoulou, et al. 2010). The inconsistent results across different studies may be attributed to differences in study design, racial background, sample size and dietary assessment method. Meanwhile, the influences of unknown genetic variations on BC risk as potential effect modifiers cannot be ignored. Our findings demonstrated that compliance with the Mediterranean diet pattern was associated with an inverse association of breast cancer risk among postmenopausal but not premenopausal women among the Chinese population. Besides, SNPs of one carbon-metabolism could modulate the association between MDP and BC risk.
The nutrients associated with one-carbon metabolism could not naturally be manufactured by the human body, which means they need to be achieved from foods. Any disruption on the one-carbon metabolism pathway could interfere with DNA-repair, DNA-replication, and gene expression regulation, which could be highly carcinogenetic(Kim 2004, Locasale 2013). This has motivated lots of studies focusing on the potential associations between the nutrients associated with one-carbon metabolism pathway and carcinogenesis(Milne, et al. 2017, Weinstein, et al. 2006). As previous studies showed, some SNPs of one-carbon metabolism gene have interactions with folate intake to affect the BC risk(Kakkoura, et al. 2017). However, the majority of the previous studies focus on the effect of individual nutrients rather than a combination of foods(Lissowska, et al. 2007), which have been limited in interpreting the high degree of intercorrelation among various nutrients. Most of these associations were weak evidence because it is hard to attribute effects to single independent component foods(Kim 2004). In this context, we choose a ‘posterior’ dietary pattern instead of individual nutrients, which could better capture specific diet characteristics and cumulative effects of nutrients.
The Mediterranean dietary pattern is characterized by high consumption of vegetables, fruit, legumes, and fish, rich in folate, choline, vitamins and methionine, sufficient levels of micronutrients play an important role in the one-carbon metabolism(Park, et al. 2012, Woodside, et al. 2005). Because specific enzymes and co-enzymes in one-carbon metabolism require ample quantities of dietary micronutrients (e.g., folate, methionine and other specific amino acids and B2, B6 and B12 and other vitamins) as substrates to achieve their biological functions(Lucock 2000, Stevens, et al. 2007).
One-carbon metabolism is interconnected to the biological processes of DNA methylation and DNA synthesis(Xu and Chen 2009). Both processes are thought to play key roles in carcinogenesis(Lewis, et al. 2006, Maruti, et al. 2009). DNA methylation is an epigenetic mechanism by which cells regulate gene expression, which involves the addition of a methyl (-CH3) to the 5-carbocytosine residue, predominantly in the cytosine guanine dinucleotide (CPGs). Dietary micronutrients involved in one-carbon metabolism play an essential role in DNA methylation, such as folic acid, choline, betaine, riboflavin, vitamins B6 and B12, and the amino acid methionine (Fig. 3)(Crider, et al. 2012, Locasale 2013). Especially folic acid (or called vitamin B9), whose role is crucial in the DNA methylation process, producing the methyl group donor, S-adenosylmethionine.
The significant interaction results of MTHFD1 G > A (rs8003567) also imply that a one-carbon metabolism gene may modify the association between diet and BC risk, which may be associated with DNA methylation. The MTHFD1 gene product is a multifunctional enzyme possessing the activities of methylene-THF dehydrogenase, methenyl-THF cyclohydrolase and formyl-THF synthetase in one-carbon metabolism pathway(MacFarlane, et al. 2011). It usually catalyzes sequential and reversible reactions in multiple conversion of tetrahydrofolate (THF), the active form of folate, into 5,10 methylene-THF which is essential for the de novo purine and thymidylate synthesis as well as the supply of one-carbon units for subsequent DNA methylation. The deficiency or dysregulation of MTHFD1 enzyme may influence cell division and global methylation pattern, eventually contributing to tumorigenesis.(Ding, et al. 2018, MacFarlane, et al. 2011) Since rs8003567 located in the intronic region of the MTHFD1 gene and no disease-related studies on SNPs have been reported before, another possible explanation cannot be excluded that there are additional functional genetic variants in linkage disequilibrium with these two SNPs that modify BC risk in Chinese female population. Furthermore, the interpretation of a nominally significant interaction between MTHFD1 G > A (rs8003567) polymorphism and adherence to Mediterranean dietary pattern should be cautious, we have not corrected the P-values for multiple comparisons, the gene-diet interaction obtained may be a false positive result. Thus, replication of the findings in other independent studies is needed before the firm conclusions can be drawn.
Recently, there is evidence that one-carbon nutrients may affect methylation levels, dietary intake of one-carbon metabolites and cofactors in rural African women's diet fluctuates with seasons. The concentration of biomarkers of maternal carbon metabolism nutrients during pregnancy was associated with the methylation of metastable epi-alleles in DNA from birth infant’s lymphocytes and hair follicles. Specifically, plasma concentrations of riboflavin and vitamin B6 indicate this association,(Dominguez-Salas, et al. 2014) and previous studies(Dominguez-Salas, et al. 2013) had shown a positive correlation between the two biomarkers and carefully measured dietary intake. However, a recent big cross-sectional study included 5186 adults does not found any log-linear association between the intake of one-carbon metabolic nutrients and individual CpG methylation. Overall, the relationship between nutrient intake, blood nutrient concentration and DNA methylation is complicated, and there is no unified conclusion now.
The strength of our study is that we focus on the genetic variants in the one-carbon metabolism pathway associated with BC risk linking a specific dietary pattern. Most of the previous studies historically focused on individual nutrients(Kim, et al. 2016, Matejcic, et al. 2017) could not capture the complicated interrelationships among nutrients and their cumulative effects(Turati, et al. 2018). The dietary pattern is a combination of food groups rather than isolated nutrients. A ‘posterior’ Mediterranean dietary pattern contains adequate levels of micronutrients associated with one-carbon metabolisms, such as Vitamins B2, B6, B12, folate and choline, which are all involved in DNA methylation and synthesis(Mas, et al. 2007). Thus, it may be more predictive for vivo situation(Brennan, et al. 2010, Gerber 2003) and interpretative for disease risk and biological mechanism.
Several limitations should also be taken into account in our study. First, we did not obtain adequate information on various subtypes of breast cancer, such as hormone receptor status. The BC risk based on the stratification analysis of breast cancer subtypes is not able to be further evaluated. Second, although we examined a number of SNPs in multiple key genes thought to be important in the one-carbon metabolism pathway, some other potential polymorphic sites may not be involved in the present study. Finally, since the number of cases and controls enrolled in this project is relatively small, the associations we founded requires replication in other larger sample independent studies. Further work should assess associations of BC risk and the concentrations of these nutrients in plasma associated with one-carbon metabolism and DNA methylation.