A80G polymorphism of reduced folate carrier 1 (RFC1) and C776G polymorphism of transcobalamin 2 (TC2) genes in Down's syndrome etiology

ABSTRACT CONTEXT AND OBJECTIVE: There is evidence that polymorphisms of genes involved in folate metabolism may be associated with higher risk that mothers may bear a Down's syndrome (DS) child. This study therefore had the objective of investigating the A80G polymorphism of the reduced folate carrier 1 (RFC1) gene and the C776G polymorphism of the transcobalamin 2 (TC2) gene as maternal risk factors for DS among Brazilian women. DESIGN AND SETTING: Analytical cross-sectional study with control group, at Faculdade de Medicina de São José do Rio Preto (Famerp). METHODS: Sixty-seven mothers of DS individuals with free trisomy 21, and 113 control mothers, were studied. Molecular analysis of the polymorphisms was performed by means of the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP), followed by electrophoresis on 2% agarose gel. RESULTS: The frequencies of the polymorphic alleles were 0.51 and 0.52 for RFC1 80G, and 0.34 and 0.34 for TC2 776G, in the case and control groups, respectively. Thus, there were no differences between the groups in relation to either the allele or the genotype frequency, for both polymorphisms (P = 0.696 for RFC1 A80G; P = 0.166 for TC2 C776G; P = 0.268 for combined genotypes). CONCLUSION: There was no evidence of any association between the RFC1 A80G and TC2 C776G polymorphisms and the maternal risk of DS in the sample evaluated.


INTRODUCTION
Down's syndrome (DS) or trisomy 21 is a genetic disease associated with abnormal chromosomal segregation. 1Free trisomy 21 is found in 95% of DS cases and is due to chromosome 21 nondisjunction, which in most cases occurred during maternal meiosis. 2 The risk factors for meiotic nondisjunction are not very clear, except for advanced maternal age. 3,4Folic acid has an important role in the process of genetic material distribution during cell division, because of its importance to cellular methylation reactions. 5he folate metabolism is responsible for synthesizing S-adenosylmethionine (SAM), the main donor of methyl groups for cellular methylation reactions. 6It has been shown that DNA methylation is important for maintaining centromeric chromatin stability and plays an important role in chromosomal segregation. 7,8olymorphisms of genes that encode enzymes involved in folate metabolism have been associated with the etiology of DS. [9][10][11][12] A polymorphism of the reduced folate carrier 1 (RFC1) gene consisting of an adenine-to-guanine substitution at position 80 (A80G) has been associated with altered concentrations of products derived from the folate metabolic pathway. 13,14This has been indicated as a maternal risk factor for DS, in combination with other polymorphisms involved in this metabolism. 9,11The RFC1 gene encodes the reduced folate carrier 1 protein, which plays a role in folic acid absorption, thereby transporting 5-methyltetrahydrofolate, the metabolically active form of folate, into a variety of cells. 15olymorphisms of genes encoding cobalamin-transporting proteins such as transcobalamin 2 (TC2) may interfere with the availability of this vitamin in the organism. 16,17Co-balamin (vitamin B 12 ) plays an important role in folate metabolism because of its action as cofactor for methionine synthase (MTR) enzyme. 18Thus, genetic variants in the TC2 gene possibly have an infl uence on cellular methylation reactions and on the risk that the mother may bear a DS child.The continuing studies on the etiology of DS are motivated by the importance of this subject for families, given the repercussions from the birth of a child with DS.

OBJECTIVE
This study had the objective of investigating the RFC1 A80G and TC2 C776G polymorphisms as maternal risk factors for DS.

METHODS
This was an analytical cross-sectional study with a control group carried out at Faculdade de Medicina de São José do Rio Preto (Famerp).After informed consent had been obtained, peripheral blood samples were taken from 67 mothers of DS individuals with free trisomy 21 (case group) and 113 mothers of individuals without the syndrome (control group).Mothers of DS individuals with translocation or mosaicism were not included in the study.The average age among the mothers at the time of blood collection was 36.7 ± 10.4 years in the case group and 40.5 ± 8.2 years in the control group.
For molecular analysis, DNA was extracted from peripheral blood leukocytes, as described by Miller et al. 19 The RFC1 A80G polymorphism was investigated by means of the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP), using the primer sequences described by Födinger et al. 20 and the enzyme CfoI to recognize the polymorphic site.The evaluation of the TC2 C776G polymorphism was performed by means of PCR-RFLP using the forward primer 5'-CAT CAG AAC AGT GCG AGA GG -3' and the reverse primer 5'-GTG CCA GAC AGT CTG GGA AG -3', and the enzyme ScrF1 to recognize the polymorphic site. 21The resulting fragments from enzyme digestion were then subjected to electrophoresis on 2% agarose gel.
The chi-squared test was used for statistical analysis on genotype frequencies.Comparisons of maternal age between the groups was carried out by using Student's t test; P values ≤ 0.05 were taken to be statistically significant.

RESULTS
Table 1 presents the distribution of the groups according to maternal age.The mean maternal age in the case group was 32 ± 8.6 years, and in the control group, it was 27.4 ± 5.5 years (P < 0.0001).
The frequencies of the polymorphic alleles RFC1 80G and TC2 776G were 0.51 and 0.34 in the case group and 0.52 and 0.34 in the control group, respectively.The genotype distribution of the polymorphisms is presented in Table 2.There were no significant differences in genotype distribution between the groups (P = 0.696 for RFC1 A80G; P = 0.166 for TC2 C776G).
Analysis of the combined genotypes of the two polymorphisms did not show any statistically significant differences between the groups (P = 0.268).Nor did the genotypes differentiate between the groups when only women with maternal age under 35 years were considered (P = 0.714 for RFC1 A80G; P = 0.166 for TC2 C776G; P = 0.759 for combined genotypes).

DISCUSSION
Several technological advances in molecular cytogenetics, making it possible to identify most chromosomal aberrations (both structural and numerical) and the genes responsible for some diseases, have been achieved. 22evertheless, although the chromosomal basis of DS has been well characterized, the etiology of chromosome 21 nondisjunction remains unclear.
Among the risk factors associated with DS occurrence, advanced maternal age is the best established factor. 3,4In fact, it has been shown that the frequency of chromosomal aberrations becomes greater with advancing maternal age, 3,24,25 especially with regard to trisomy of chromosomes 13, 18 and 21 in women who give birth when over 35 years old. 26According to data from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), 40% of live births with DS were born from mothers who were between 40 and 44 years old, although women of this age account for only 2% of all births. 27In our study, the mean maternal age was significantly higher in the case group than in the control group.This corroborates with data in the literature regarding the association between maternal age and the risk of DS.
Occurrences of births of children with DS among young mothers suggests that advanced maternal age is not the only risk factor involved.Investigation of genetic polymorphisms that lead to abnormalities in folate metabolism products is the approach furthest investigated at the present time. 28ames et al. 29 reported that mothers with DS children have abnormal folate metabolism.They suggested that a variant of the methylenetetrahydrofolate reductase gene (MTHFR C677T), which regulates cellular methylation reactions, could lead to DNA hypomethylation and consequently to chromosomal segregation errors.Other genes involved in the folate metabolic pathway have been investigated as maternal risk factors for DS, such as RFC1, MTR, methionine synthase reductase (MTRR), cystathionine B-synthase (CBS).][11][12] However, the few studies that have evaluated the influence of RFC1 A80G polymorphism, including a previous study by our group, 9 did not observe any association between this variant per se and the maternal risk of bearing a DS child, 9,10,30 thereby corroborating the present study.Higher maternal risk of DS has been observed in the presence of this polymorphism, in combination with other genetic polymorphisms relating to folate metabolism, such as MTHFR C677T, MTHFR A1298C and MTR A2756G. 9,10This may be due to the small impact of RFC1 A80G polymorphism on the affinity and transport efficiency of the variant enzyme in relation to the wild-type enzyme. 31ith regard to TC2 C776G polymorphism, the present study is, to our knowledge, the first to investigate the contribution of polymorphisms of the cobalamin-transporting gene (an important cofactor for folate metabolism), in relation to the maternal risk of bearing a DS child.There is evidence for an association between this genetic variant and the maternal risk of bearing a child with neural tube defects, 32 which are influenced by genetic determinants involved in folate metabolism.
Evidence showing a higher frequency of DS cases in families with a risk of neural tube defects, and vice versa, 33 strengthens the notion that the same genetic determinants of folate metabolism influence both disorders. 34owever, in the present study, no association was observed between the TC2 C776G polymorphism and the maternal risk of bearing a DS child.It is widely accepted that supplementation or fortification with folic acid reduces the risk of neural tube defects, 35,36 and it is believed that supplementation with cobalamin in combination with folate could contribute towards reducing this risk. 18,32NCLUSION No evidence for an association between RFC1 A80G and TC2 C776G polymorphisms and the maternal risk of bearing a DS child was observed in this study.Thus, further stud-ies including these and other polymorphisms involved in folate metabolism could provide a better understanding of the role of genetic variants in the etiology of the chromosomal nondisjunction that results in DS.

Table 2 .
Genotype distribution of the RFC1 A80G and TC2 C776G polymorphisms between the case (n = 67) and control (n = 113) groups

Table 1 .
Distribution of the case (n = 67) and control (n = 113) groups according to maternal age