RFC-1 80G>A Polymorphism in Case-Mother/Control-Mother Dyads Is Associated with Risk of Nephroblastoma and Neuroblastoma

Introduction

Genetic susceptibility, prenatal factors, and neonatal exposure are probably involved in the development of embryonic tumors, which develop in very young children. Microscopically, they resemble structures seen in developing tissues of the embryo and fetus (Dehner, 1998). The risk of developing these cancers may be modified by polymorphic variants in genes (of the children or parents) involved in metabolism, growth, and development, as well as in DNA repair and the cell cycle (Blount et al., 1997; Gemmati et al., 2004).

Folate plays an important role in embryogenesis and early fetal development through its effects on DNA methylation and synthesis (Blount et al., 1997). Folate insufficiency, due to low folate intake or defects in folate metabolism, may lead to DNA hypomethylation and genomic instability, which may play a role in carcinogenesis by altering gene expression or increasing DNA damage. Prenatal folic acid supplementation has been shown to decrease several congenital anomalies and is recommended for women of childbearing potential and during early pregnancy to decrease the risk of neural tube defects (NTD) in several countries (Berry et al., 1999). Multivitamin supplementation is thought to be associated with a reduced incidence of neuroblastoma, brain tumors, retinoblastoma, and nephroblastoma (Wilms' tumor) (Preston-Martin et al., 1998; Goh et al., 2007; Schuz et al., 2007). Folic acid food fortification was associated with a decline in neuroblastoma in Canada and nephroblastoma incidence in the United States (French et al., 2003; Grupp et al., 2011; Linabery et al., 2012).

Methylenetetrahydrofolate reductase (MTHFR) is one of the main regulatory enzymes of the folate metabolic pathway since it controls the balance between DNA methylation and synthesis through the irreversible conversion of 5,10-methylenetetrahydrofolate required for DNA synthesis to 5-methyltetrahydrofolate, a methyl donor for conversion of homocysteine to S-adenosylmethionine. Reduced folate carrier-1 (RFC-1) is a well-characterized folate transporter in the cell membrane with a high affinity for physiological folate. Genes encoding these proteins harbor polymorphisms that may decrease the enzymatic activity and impair folate entrance into the cell and are associated with the development of several malignancies (Yin et al., 2004; Kennedy et al., 2012; Yu and Chen, 2012).

Recently, Miranda et al. (2014) demonstrated that the RFC A80A genotype was significantly associated with the risk of developing neuroblastoma. MTHFR variants play a controversial role in the development of childhood acute leukemia (da Costa Ramos et al., 2006; Zanrosso et al., 2006; Metayer et al., 2011). In a previous Brazilian series, MTHFR A1298C polymorphism was associated with an increased risk of acute lymphoblastic leukemia and acute myelogenous leukemia in nonwhite children (da Costa Ramos et al., 2006; Zanrosso et al., 2006). In embryonic solid childhood tumors, several data have suggested an association of MTHFR C677T with nephroblastoma, MTHFR A1298C with embryonic central nervous system tumors, and MTR (methionine synthase) A2756G with retinoblastoma (Sirachainan et al., 2008; Ferrara et al., 2009; De Lima et al., 2010).

The present study aimed to evaluate the association between nephroblastoma and neuroblastoma with the polymorphisms of folate metabolism genes through case-mother/control-mother design.

Materials and Methods

Results

Discussion

Embryogenesis and carcinogenesis are complex multifactorial events arising from an array of genetic and environmental interactions. To the best of our knowledge, this is the first report evaluating the association of case-mother/control-mother dyads MTHFR rs1801133 (C677T) and RFC-1 rs1051266 (G80A) polymorphisms with neuroblastoma and nephroblastoma.

In the present report, first, we analyzed the influence of each child and mother polymorphism separately on the risk of each tumor and found significant increased risk association conferred by child RFC-1 A80G genotype. Next, we investigated the combination of child and mother genotype of MTHFR rs1801133 (C677T) and RFC-1 rs1051266 (G80A) on the risk of developing tumors. The combination of mother and child genotype of RCF-1 G80A showed an even higher risk of developing nephroblastoma and neuroblastoma. Our findings demonstrate a role for both maternal and child folate polymorphism genotypes on the risk of nephroblastoma and neuroblastoma and highlight the importance of the combined genotype analysis. We believe that the mother has a crucial role in the early-onset disease predisposition as she provides the prenatal environment and can influence her offspring's risk of disease not only as a genetic donor but also through the effects of her genes acting directly on the intrauterine milieu or indirectly through child maternal gene-gene interactions.

Zhao et al. (2001) shed light on the critical role that RFC-1 plays in embryonic development. RFC-1-null mice embryos (genetically engineered by gene knockout) died in utero due to failure of hematopoietic organs. The embryonic lethality could be rescued by maternal folate supplementation. Folic acid is a very poor substrate for RFC-1, which has a much higher affinity for reduced folate that comes from natural resources. It is likely that appreciable quantities of folic acid may be delivered into cells by passive diffusion as well as other secondary transport routes. As RFC-1 rs1051266 (G80A) polymorphism may reduce the intracellular folate concentration, based on the above data on RFC-1-null mice embryos, we can speculate that higher doses of folate might overcome this deficit.

The complex interaction between genetic variants in the folate pathway and nutrient intake during pregnancy warrants further exploration. The preventive effect of folic acid supplementation is not questionable, but its protective mechanism is still poorly understood. The demand for folate during pregnancy has increased because it is required for growth and development of the fetus. It is well known that folic acid supplementation or fortification has reduced NTD and other congenital malformations (Berry et al., 1999; Mezzomo et al., 2007; Orioli et al., 2011). Introduction of folic acid fortification of flour and grains in Canada produced a 30% reduction in the risk of nephroblastoma (Grupp et al., 2011). Regarding neuroblastoma, the number of cases decreased from 1.57 cases per 10,000 births to 0.62 cases per 10,000 births (p<0.0001) (French et al., 2003). Rates of nephroblastoma were significantly lower after mandatory US folic acid fortification, with stronger effects detected in infants (Linabery et al., 2012). Schuz et al. (2007) observed similar results with the use of vitamins, folate, and/or iron supplements during pregnancy in Germany. In Brazil, fortification of foods has been mandatory since 2004 (www.anvisa.gov.br/alimentos/farinha.htm). Supplementation with folic acid is also recommended during pregnancy in Brazil, but data on compliance are scarce. On the other hand, some countries do not mandate folic acid fortification, in part, because of concerns about the potential for enhancing cancer development (Wright et al., 2007). The main strength of this study was our ability to investigate maternal genotypes together with children's genotypes in case and control samples. Maternal effects can arise because the genetic variants carried by the mother influence the prenatal environment in which the fetus develops, thereby increasing disease risk (Vermeulen et al., 2009). The major limitation is the lack of information about folate levels and supplementation. Based on literature data, we assume that polymorphisms in these genes influence enzyme activity and receptor function (Kim, 1999; Kim et al., 2011).

Further studies assessing folate levels and gene-environment interactions in childhood embryonic tumor development are still necessary.

In summary, our results suggest that individual carriers of RFC variant genotype present a higher risk for the development of nephroblastoma and neuroblastoma. It is likely they have an impaired absorption of folic acid and this may affect the folate metabolism pathway causing genomic instability and promoting cancer development.