DHFR 19-bp Deletion and SHMT C1420T Polymorphisms and Metabolite Concentrations of the Folate Pathway in Individuals with Down Syndrome

Introduction

Down syndrome (DS) results from the presence and expression of three copies of the genes located on chromosome 21 (Shin et al., 2004; Ishinohe et al., 2005). The Cystathionine β-synthase (CBS) gene, located on chromosome 21, is responsible for the condensation of homocysteine (Hcy) and serine to cystathionine and is overexpressed in individuals with DS (Ishinohe et al., 2005). Increased concentrations of the CBS enzyme results in lower concentrations of Hcy, methionine, S-adenosylhomocysteine, and S-adenosylmethionine (Pogribna et al., 2001; Coppus et al., 2007; Meguid et al., 2010), substrates of folate metabolism.

Studies have shown that, in addition to overexpression of the CBS gene, polymorphisms in genes involved in folate/Hcy metabolism may also influence metabolite concentrations of this pathway (Fillon-Emery et al., 2004; Guéant et al., 2005; Licastro et al., 2006; Biselli et al., 2008; Biselli et al., 2012). A 19-base pair (bp) deletion polymorphism in intron-1 of the Dihydrofolate reductase (DHFR) gene, located on chromosome 5q11.2, has been identified (Johnson et al., 2004) and Kalmbach et al. (2008) demonstrated that this is a functional polymorphism. Study shows that the 19-bp deletion polymorphism is associated with increased expression of the DHFR gene, responsible for the conversion of dihydrofolate in tetrahydrofolate (THF) (Xu et al., 2007), and changes of folate/Hcy metabolism (Gellekink et al., 2007; Kalmbach et al., 2008; Stanisiawska-Sachadyn et al., 2008; Mendes et al., 2010).

Another polymorphism, C1420T, which results in substitution of leucine by phenylalanine, was identified in the Serine hydroxymethyltransferase (SHMT) gene, located on chromosome 17p11.2 (Heil et al., 2001). This gene encodes the enzyme that catalyzes the reversible conversion of serine and THF to glycine and 5,10-methylene THF (Fowler, 2001) and Fu et al. (2005) showed that the SHMT C1420T polymorphism may compromise the formation of the SHMT enzyme.

Both the DHFR 19-bp deletion and SHMT C1420T polymorphisms involved in folate/Hcy metabolism have been associated with variations in the concentrations of Hcy and folate in several populations (Heil et al., 2001; Chen et al., 2004; Lim et al., 2005; Gellekink et al., 2007; Kalmbach et al., 2008; Stanisiawska-Sachadyn et al., 2008; Mendes et al., 2010; Marucci et al., 2012). Thus, the aim of the present study was to investigate the association between the DHFR 19-bp deletion and SHMT C1420T polymorphisms and the serum folate and plasma Hcy and methylmalonic acid (MMA) concentrations in individuals with DS.

Materials and Methods

This study was composed by eighty-five individuals with full trisomy 21 confirmed by karyotype (median age 1.36, range 0.07-30.35 years old; 47 male and 38 female) recruited at the General Genetics Outpatient Service of Hospital de Base, São José do Rio Preto, SP, Brazil. The study protocol was approved by the Research Ethics Committee of São José do Rio Preto Medical School (CEP-FAMERP, 165/2004), in São Paulo state, and informed consent was obtained for all families.

Fasting blood samples were collected for molecular and biochemical analysis (serum folate and plasma Hcy and MMA). DNA extraction was performed as previously described by Miller et al. (1988) and polymorphisms in DHFR and SHMT genes were analyzed by polymerase chain reaction (PCR) using difference in the size of fragments and real-time PCR allelic discrimination, respectively. The 19-bp deletion polymorphism in the DHFR gene was detected using primer sequences described by Dulucq et al. (2008) and SHMT C1420T was detected using TaqMan^® probes and primer sequences described by Skibola et al. (2004). Serum folate was quantified by chemiluminescence (Immulite Kit, DPC Medlab, Brazil) and liquid chromatography-tandem mass spectrometry was used to determine concentrations of plasma Hcy and MMA, as previously described (Haddad et al., 2001; de Andrade et al., 2006; Carvalho and Kok, 2008).

The Hardy-Weinberg equilibrium was tested by the chi-square test, using the BioEstat program (version 5.0). The folate, Hcy, and MMA concentrations among different genotypes were compared, after adjustment for age, using multiple linear regression. The computer-assisted statistical analyses were carried out using the Minitab for Windows program (Release 14). Values of p≤0.05 were considered significant.

In this study, the allele with a 19-bp deletion in the DHFR gene was denominated D and the allele without the deletion was named I.

Results

Table 1 presents genotype frequencies of DHFR 19-bp deletion and SHMT C1420T polymorphisms in individuals with DS and both the genotype distributions were in the Hardy-Weinberg equilibrium (χ²=2.079; p=0.15; χ²=0.004; p=0.95, respectively). The mean concentrations of folate, Hcy, and MMA observed in the individuals were 19.69±11.87 ng/mL, 5.83±3.30 μM, and 0.54±0.84 μM, respectively. The distribution of the Hcy, folate, and MMA concentrations and age according to the combined genotypes are presented in Table 2.

The results did not show any individual association between these polymorphisms and folate and Hcy and MMA concentrations. However, the DHFR DD/SHMT TT combined genotypes were associated with elevated folate concentrations (Coefficient=25.69; p=0.004) and the DHFR II/SHMT TT combined genotypes were associated with increased MMA concentrations (Coefficient=1.78; p=0.008). In addition, the MMA concentrations were associated with age (Coefficient=−0.04; p=0.04).

Discussion

The overexpression of genes results in biochemical alterations that affect the multiple interacting metabolic pathways culminating in cellular dysfunction and contributing to the pathogenesis of DS (Pogribna et al., 2001). The presence of three copies of the CBS gene, located on chromosome 21, and Methylenetetrahydrofolate reductase (MTHFR) C677T, Methionine synthase (MTR) A2756G, Transcobalamin 2 (TC2) C776G, and Betaine homocysteine methyltransferase (BHMT) G742A polymorphisms, involved in folate/Hcy metabolism, have been associated with variations on the concentrations of metabolites of this pathway (Pogribna et al., 2001; Fillon-Emery et al., 2004; Guéant et al., 2005; Licastro et al., 2006; Coppus et al., 2007; Biselli et al., 2008; Meguid et al., 2010; Biselli et al., 2012).

Guéant et al. (2005) observed that individuals with DS who present the MTHFR 677T allele and an elevated Hcy concentration had a low intelligence quotient and Licastro et al. (2006) found that the MTHFR 677TT genotype increases the concentrations of Hcy in these individuals. However, Fillon-Emery et al. (2004) found no difference in Hcy concentrations according to the MTHFR C677T genotype in adults with DS. In another study, the heterozygous genotype MTR 2756AG was associated with increased plasma Hcy concentrations in individuals with DS (Biselli et al., 2008). Recent study that evaluated the association between twelve polymorphisms in genes involved in the folate/Hcy metabolism and folate, Hcy and MMA concentrations indicated that the MTHFR C677T, MTR A2756G, TC2 C776G, and BHMT G742A polymorphisms are predictors of the Hcy concentration. In this study, individuals with DS and MTR 2756 AG or GG genotype presented an increased Hcy concentration and MTHFR 677 TT, TC2 776 GG, and BHMT 742 AA genotypes were associated with a decreased Hcy concentration. (Biselli et al., 2012).

In the present study, we observed that the DHFR DD/SHMT TT combined genotypes were associated with increased folate concentrations and DHFR II/SHMT TT genotypes were associated with elevated MMA concentrations. The finding concerning the association between genotypes and the MMA concentrations must be interpreted with some caution considering that we observed a negative association between MMA concentrations and age and that the individuals with DHFR II/SHMT TT genotypes presented the lower mean age than the individuals with other combined genotypes. Other special attention should be paid to the sample size.

DHFR is an important folate-metabolizing enzyme responsible for reduction of folic acid into THF (Stanisiawska-Sachadyn et al., 2008). A common polymorphism in this gene, a 19-bp deletion polymorphism in intron-1, was associated with alterations on the concentration of metabolites involved in the folate/Hcy pathway (Gellekink et al., 2007; Kalmbach et al., 2008; Stanisiawska-Sachadyn et al., 2008; Mendes et al., 2010). Gellekink et al. (2007) reported that the DHFR DD genotype is associated with a lower concentration of plasma Hcy in Caucasian individuals, but no association between this genotype and concentrations of serum and erythrocyte folate was observed. Another study found no effect of this polymorphism on Hcy concentration in healthy adults, but the DD genotype was associated with increased concentrations of serum and erythrocyte folate relative to the II genotype in women (Stanisiawska-Sachadyn et al., 2008). Kalmbach et al. (2008) also observed no association between genotypes and plasma Hcy or plasma total folate in young adults; however, the DHFR DD genotype was associated with a lower concentration of erythrocyte folate compared to DHFR ID and II genotypes. A recent study with women also demonstrated that folate, Hcy, and MMA concentrations did not differ between the genotypes (Mendes et al., 2010).

The SHMT enzyme plays a pivotal role in the folate/Hcy metabolism by carrying out the reversible conversion of serine and glycine with THF and 5, 10-methyleneTHF (Fowler, 2001). Heil et al. (2001) identified the SHMT C1420T polymorphism and reported that individuals with neural tube defects and the SHMT CC genotype had decreased concentrations of erythrocyte and plasma folate and an increased Hcy concentration. In a study involving men with cardiovascular disease, the SHMT TT genotype was associated with the lower Hcy concentration (Lim et al., 2005); yet, Chen et al. (2004) found no significant association between SHMT C1420T and plasma folate and Hcy concentrations in colorectal cancer. In addition, another study observed that the SHMT C1420T polymorphism does not affect the folate, Hcy, and MMA concentrations in women (Marucci et al., 2012).

This is the first study that evaluated the association between the DHFR 19-bp deletion and SHMT C1420T polymorphisms and metabolite concentrations of the folate pathway. Despite the small sample size, our results suggest that there is an association between DHFR DD/SHMT TT and DHFR II/SHMT TT combined genotypes and the folate and MMA concentrations, respectively, in individuals with DS. However, further studies are needed to confirm these associations.