Polymorphisms Involved in Folate Metabolism Pathways and the Risk of the Development of Childhood Acute Leukemia

Abstract

Objective: Polymorphisms that reduce the activity of reduced folate carrier (RFC) and methylenetetrahydrofolate reductase (MTHFR) and double (2R2R) or triple (3R3R) 28-bp tandem repeats in the promoter region of thymidylate synthase (TS) have been associated with the risk of childhood acute leukemia (AL). A case-control genotyping study was conducted in Brazilian children with the aim of investigating RFC, MTHFR, and TS polymorphisms as risk factors. Methods: The polymerase chain reaction-restriction fragment length polymorphism method was employed in 177 AL cases and 390 controls. Results: The presence of the mutant 1298C, also RFC 80A, was linked to a decreased risk of developing acute lymphoid leukemia (ALL) (odds ratio (OR)=0.46, 95% confidence interval (CI)=0.30-071 and OR=0.51, 95% CI=0.28-0.0.93, respectively). Conclusions: The genotype 677 CT was associated with increased risk of developing ALL (OR=1.6, 95% CI=1.1-2.7). Further epidemiological study is needed to unravel the role of complex multiple gene-environment interactions in leukemogenesis.

Introduction

The biological mechanisms and etiology of malignant leukemia are not yet completely understood (Zeller et al., 2005). The known risk factors for childhood leukemia include ionizing radiation, Down's syndrome, and monogenic disorders, such as ataxia telangiectasia (Skibola et al., 1999; Gemmati et al., 2004); genetic disorders are unlikely to be caused by a single genetic defect because there is only a modest familial link (Gast et al., 2007). Therefore, unfavorable gene-environment interactions might be involved in the genesis of this childhood cancer, whose annual incidence rate is increasing (Vanesse et al., 1999).

The inherited susceptibility can potentially modulate acute leukemia (AL) risk from initiation through complete manifestation of the disease (Zingg and Jones, 1997). Several genetic alterations in multiple genes, due to incorrect DNA synthesis or altered methylation status of oncogenes and/or suppressor genes have been identified in the pathogenesis of malignant leukemia and lymphoma (Rosenblatt, 2001; Lucock, 2000).

Folate is a key element in the one-carbon group metabolism, and is essential in DNA synthesis as well as repair and methylation processes. One-carbon metabolism is divided into two main pathways leading to purine and methionine synthesis. Disruptions of the homeostasis of the one-carbon metabolism are attributed to folate deficiencies, leading to DNA damage (Homberger et al., 2000). Defects or polymorphisms in the genes of folate-dependent enzymes and deficiencies of micronutrients may influence cancer susceptibility (Weisberg et al., 1998; Homberger et al., 2000; Lucock, 2000).

Cellular uptake of the predominant plasma folate, 5-methyltetrahydrofolate, is primarily mediated by the reduced folate carrier (RFC-1). Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism and is essential for the regulation of methionine and homocysteine concentrations (Skibola et al., 2002). It catalyzes the reduction of 5, 10-methylenetetrahydrofolate (5,10-CH₂-THF) to 5-methyltetrahydrofolate (5-CH₂-THF), the carbon donor for the remethylation of homocysteine to methionine. Two common polymorphisms (C677T and A1298C) in the MTHFR gene have been described, which reduce the enzyme activity with altered distribution of intracellular folate (Weisberg et al., 1998).

The thymidylate synthase (TS) gene plays a critical role in maintaining a balanced supply of deoxynucleotides required for DNA synthesis. It catalyzes the synthesis of thymidylate, or deoxythymidine monophosphate (dTMP), from deoxyuridine monophosphate (dUMP) with 5,10-CH₂-THF as the methyl donor (Marsh et al., 2000, 2001).

Many studies have shown that a polymorphic tandem 28-bp repeat in the promoter region of the TS gene possesses a regulatory role and influences its expression (Kawakami et al., 1999; Marsh et al., 2001; Hishida et al., 2003). An allele of this TS enhancer region (TSER) polymorphism usually contains double or triple tandem repeats, leading to the genotypes 2R/2R, 2R/3R, and 3R/3R. Although rare, alleles with four and five repeats have also been described. The 2R allele has been shown to be associated with lower gene expression, when compared with the 3R allele. It has been proposed that the effect of this tandem repeat might be on the transcriptional and/or translational efficiency of the TS gene (Hishida et al., 2003).

Theoretically, this enhanced expression may increase the conversion of dUMP to dTMP, limit DNA damage in rapidly dividing tissues that have the greatest requirement for DNA, such as those involved in hematopoeisis, and afford some protection from leukemia risk (Marsh et al., 2001). Recently, an epidemiological study showed that some differences in the incidence ratio of childhood cancer occur in some regions of Brazil, particularly in some Northeast regions (De Souza Reis et al., 2011). Therefore, we analyzed the effect of genetic polymorphism of MTHFR C677T and A1298C, RFC1 G80A, and the double or triple tandem repeats in the TS gene to evaluate the risk of development of AL in Brazilian children.

Materials and Methods

Study population

The study genotyped biological samples obtained from children diagnosed with AL from January 2003 to December 2009 at the hospital in the city of Recife, Brazil. The hospital is a regional referral center for childhood cancer treatment in the Northeast region of Brazil. There were 144 ALL and 33 AML children cases and young adults aged ≤19 years. The ascertained patients were diagnosed by morphology and/or immunophenotyping methods before any treatment. Peripheral blood (PB) samples were taken at the time of initial diagnosis, and the control group consisted of PB samples obtained from unselected individuals with the same age and from the same Brazilian regions as those of the cases (n=390). In the control group, the diagnosis of malignant disease was excluded before the subject was included in the study.

Informed consent was obtained from all parents or guardians of the subject, and the Research and Ethics Institutional Committee approved the study.

MTHFR 1298 genotyping

The MTHFR A1298C polymorphism was determined by allele-specific polymerase chain reaction (PCR), using primers to separately amplify wild-type and mutated alleles, according to the protocol described by Ranjith et al. (2003).

MTHFR 677 genotyping

The genotyping protocol for the detection of MTHFR 677 C→T polymorphism was adapted from the study by Frosst et al. (1995). The amplified and digested products were visualized in a 3% agarose gel with ethidium bromide. Fragments obtained after HinfI enzyme digestion were considered as Wild types (677CC) that produced a singlet band at 198 bp. The heterozygotes (677CT) produced 198-, 175-, and 23-bp fragments, while the homozygous mutants (677TT) produced 175- and 23-bp fragments.

TS 2R33R genotyping

The presence of tandem repeat sequences in the 5′-terminal of the regulatory region of the TS gene was detected using a protocol adapted from the study by Hishida et al. (2003). The amplified PCR products were visualized on a 3% agarose gel with ethidium bromide. Homozygotes for the double repeat (2R2R) produced a single 220-bp band, while the heterozygotes (2R3R) produced 220-bp and 250-bp fragments. Furthermore, homozygotes for the triple repeat (3R3R) produced a 250-bp fragment.

RFC1 G80A genotyping

The RFC G80A genotyping was carried out using a PCR-restriction fragment length polymorphism method adapted from a previous publication by Chango et al. (2000). The amplified and digested fragments were analyzed in a 3% agarose gel. Fragments obtained after Hha1 enzyme digestion were 125, 62, and 37 pb for allele G, and 162 and 62 pb for allele A. Heterozygotes produced bands for each allele.

Statistical analysis

Maximum likelihood method was used to estimate the allelic frequencies, and the goodness of fit of phenotype distribution with respect to Hardy-Weinberg equilibrium was tested by chi-square method. The analysis was carried out using the Biostat (Version 5.0) to estimate odds ratios (OR) and confidence intervals at 95% (95% CI) significance level for the presence of leukemia with each polymorphism using univariate logistic regressor models.

Results

The genotypic distribution of MTHFR C677T and A1298C, RFC G80A, and TS variants were found to be in Hardy-Weinberg equilibrium in both the case and control groups. The frequencies obtained for the mutant alleles in the ALL, AML, and control groups were 0.78 and 0.64 for RFC G80A, 0.48 and 0.40 for the MTHFR 1298C allele, 0.43, 0.57, and 0.33 for the MTHFR 677T allele, and 0.68 and 0.66 for the TS tandem repeats, respectively. The analysis of heterogeneity between the cases and controls showed no significant differences for the TS tandem repeats, ALL, and AML; however, statistical significance was found for the RFC1 G80A, MTHFR C677T MTHFR A1298c genotypes distribution for ALL (χ²=7.52; p=0.023 and χ²=18.78, p=0.0001, respectively), and for the MTHFR C677T and A1298C genotypes distribution for ALL (χ²=6.141, p=0.0464 and χ²=16.048, p=0.0003, respectively).

The genotyping distribution of polymorphism for AML and controls are shown in Table 1. The frequency of the MTHFR 1298AC heterozygous genotype was higher among all the patients, when compared with controls, and the difference was statistically significant (p=0.028, OR=0.58, 95% CI=0.36-0.92), imposing a 1.7-fold protection risk for AL. The frequency of the MTHFR 1298CC homozygous genotype was even higher among the ALL patients (p=0.000, OR=0.25, 95% CI=0.13-0.49), when compared with the controls, imposing a 3.9-fold protection risk for ALL. Furthermore, the frequency of the MTHFR 1298AC+CC genotype was also significantly higher among the ALL patients, when compared with the controls, showing a 2.1-fold higher risk for ALL. Regarding RFC1, the frequency of the RFC1 80AA homozygous genotype was significantly higher among the patients, when compared with the controls (p=0.0104, OR=0.38, 95% CI=0.19-0.76), indicating a 2.6-fold risk. The genotyping distribution of gene polymorphism for ALL and controls is shown in Table 2.

Table 1.

Genotyping Frequencies of RFC-1 G80A, MTHFR C677T, MTHFR A1298C Polymorphisms, and Thymidylate Synthase Repeat of Acute Myeloid Leukemia and Controls

	Cases n (%)	Controls n (%)	p	OR	CI 95%	p (OR)
RFC1 G80A	21 (100%)	137 (100%)
GG	4 (19.05%)	49 (35.77%)	0.2938	1
GA	10 (47.62%)	56 (40.87%)		0.4571	0.1348-1.5504	0.3205
AA	7 (33.33%)	32 (23.36%)		0.3732	0.1010-1.3786	0.2323
GA+AA	17 (80.95%)	88 (64.23%)		0.4226	0.1346-1.3264	0.2066
MTHFR C677T	33 (100%)	224 (100%)
CC	19 (57.57%)	95 (42.41%)	0.2580	1
CT	12 (36.37%)	108 (48.21%)		1.8000	0.8305-3.9013	0.1900
TT	2 (6.06%)	21 (9.38%)		2.1000	0.4539-9.7155	0.5152
CT+TT	15 (45.45%)	129 (57.59%)		1.7200	0.8314-3.5584	0.1976
MTHFR A1298C	31 (100%)	248 (100%)
AA	13 (41.94%)	147 (59.27%)	0.112	1
AC	13 (41.94%)	82 (33.06%)		0.5578	0.2469-1.2600	0.2284
CC	5 (16.12%)	19 (7.67%)		0.3361	0.1078-1.0473	0.1128
AC+CC	18 (58.06%)	101 (40.73%)		0.4962	0.2328-1.0579	0.0994
TS (2R/3R)	31 (100%)	390 (100%)
3R3R	10 (32.26%)	130 (33.33%)	0.9418	1
2R3R	15 (48.39%)	194 (49.74%)		0.9949	0.4336-2.2825	0.8418
2R2R	6 (19.35%)	66 (16.93%)		0.8462	0.2947-2.4292	0.9711
2R3R+2R2R	21 (67.74%)	260 (66.66%)		0.9524	0.4357-2.0816	0.9396

p by χ² test OR, odds ratio; CI 95%, confidence interval 95%, significance level (p<0.05).

Table 2.

Genotyping Frequencies of RFC-1 G80A, MTHFR C677T, MTHFR A1298C Polymorphisms, and Thymidylate Synthase Repeat of Acute Lymphoblast Leukemia and Controls

	Cases n (%)	Controls n (%)	p	OR	CI 95%	p^a
RFC1 G80A	95 (100%)	137 (100%)
GG	21 (22.11%)	49 (35.77%)	0.0232^a	1
GA	38 (40%)	56 (40.87%)		0.6316	0.3276-1.2177	0.2257
AA	36 (37.89%)	32 (23.36%)		0.3810	0.1894-0.7661	0.0104^a
GA+AA	74 (77.89%)	88 (64.23%)		0.5097	0.2804-0.9262	0.0372^a
MTHFR C677T	144 (100%)	224 (100%)
CC	82 (56.94%)	95 (42.41%)	0.0234^a	1
CT	53 (36.81%)	108 (48.21%)		1.7589	1.1306-2.7364	0.0163^a
TT	9 (6.25%)	21 (9.38%)		2.0140	0.8739-4.6415	0.1423
CT+TT	62 (43.06%)	129 (57.59%)		1.7959	1.1763-2.7419	0.0089^a
MTHFR A1298C	136 (100%)	248 (100%)
AA	55 (40.44%)	147 (59.27%)	0.0001^a	1
AC	53 (38.97%)	82 (33.06%)		0.5789	0.364-0.9207	0.0278^a
CC	28 (20.59%)	19 (7.67%)		0.2539	0.1312-0.4911	0.0000^a
AC+CC	81 (59.56%)	101 (40.73%)		0.4665	0.3047-0.7143	0.0006^a
TS (2R/3R)	140 (100%)	390 (100%)
3R3R	45 (32.14%)	130 (33.33%)	0.9522	1
2R3R	70 (50%)	194 (49.74%)		0.9593	0.6207-1.4828	0.9394
2R2R	25 (17.86%)	66 (16.93%)		0.9138	0.5159-1.6186	0.8712
2R3R+2R2R	95 (67.86%)	260 (66.66%)		0.9474	0.6272-1.4310	0.8790

p^a by χ² test OR, odds ratio; CI 95%, confidence interval 95%, significance level (p<0.05).

In the combined analysis, the frequency of the MTHFR 677CT heterozygous genotype was lower among the patients (65/177, 36.72%), when compared with the controls (108/224, 48.21%), and the difference was statistically significant (p=0.0016, OR=1.75, 95% CI=1.13-2.73), imposing a 1.6-fold risk. Also, the frequency of the MTHFR 677CT heterozygous genotype plus 677TT homozygous genotype was significantly lower among the patients (p=0.0172, OR=1.6179, 95% CI=1.1050-2.3689), when compared with the controls, imposing a 1.6-fold risk (Table 2).

However, when analyzed separately, only ALL value showed statistical significance. The frequency of the MTHFR 677CT heterozygous genotype was lower among patients (53/144, 36.8%), when compared with the controls (108/224, 48.21%), and the difference was statistically significant (p=0.016, OR=1.75, 95% CI=1.13-2.73), imposing a 1.6-fold risk. Also, the frequency of the MTHFR 677CT heterozygous genotype plus 677TT homozygous genotype was significantly lower among the patients (p=0.089, OR=1.79, 95% CI=1.17-2.74), when compared with controls, imposing a 1.7-fold risk (Table 2).

Discussion

Previous studies have demonstrated that folate levels and genetic regulation of folate metabolism are involved in the risk factor of childhood leukemia (Skibola et al., 1999, 2002; Gemmati et al., 2004; de Jonge et al., 2009), predicating the creation or expansion of chromosomal aberrations as well as the critical component on the risk of relapse and level of toxicity of ALL treatment (Assaraf, 2007). However, ambiguous results have been described regarding the polymorphisms of folate genes (Pereira et al., 2006). The present investigation demonstrated significant decreased risk for MTHFR 1298AC (1.7-folds) and MTHFR 1298CC genotypes (3.9-folds). This lower risk is thought to be caused by the increased fidelity of DNA synthesis afforded by the greater availability of the MTHFR substrate, 5,10-CH₂-THF, for DNA synthesis, and, in particular, the increased availability of methyl groups for the conversion of uracil to thymidine. These findings corroborate the recent data reported by Kamel et al. (2007), who described a protective effect for this polymorphism (p=0.001, OR=0.382, 95% CI=0.222-0.658), imposing a 2.6-fold protection.

Similar results were also noted in RFC1 80AA (2.0-folds), an important modulator of leukemia protection. However, in contrast to our results, De Jonge et al. (2009) observed 1.5- and 2.1-fold increased risk in A-allelic carriers and 80AA homozygotes, respectively. Interestingly, patients carrying the polymorphic RFC1 80A allele showed an increased percentage of extracellular folate relative to intracellular folate. These data suggest that the RFC1 80GA polymorphism renders a reduced efficiency in the cellular uptake of methyl-THF (Yates and Lucock, 2005). Recent studies did not show a relation between RFC1 G80A polymorphism and the risk of non-Hodgkin lymphoma (Lightfoot et al., 2005), neural tube defects (Relton et al., 2004), and colon cancer (Ulrich et al., 2005).

A few recent studies have suggested the protective role of MTHFR polymorphism in leukemogenesis (Franco et al., 2001; De Jonge et al., 2009; Krajinovic et al., 2009). However, the present study provides contradictory evidence on the association between MTHFR 677 polymorphism and the risk of AL.

This investigation demonstrated that the MTHFR 677 CT genotype is linked to a 1.6-fold increased risk of developing ALL and that the MTHFR 677CT heterozygous genotype plus 677TT homozygous genotype is linked to 1.7-fold increased risk. However, the homozygous MTHFR 677TT genotype did not show this risk effect, which may be due to the smaller number of homozygous cases. This finding does not support the idea that increased availability of 5,10-MTHF, which is a feature of MTHFR 677T, influences the leukemogenesis process by conferring protection against the occurrence of ALL. Similarly, Kim et al. (2009) showed that MTHFR 677TT increased the risk of developing ALL in childhood cancer.

Different from our results, De Jonge et al. (2009) found that MTHFR 677CT and T-allelic carriers exhibited decreased risk of pediatric ALL (OR=0.7, 95% CI=0.5-1.0, p=0.03) in 245 West European pediatric ALL patients (<18 years of age) and 500 healthy controls. In a recent meta-analysis, Pereira et al. (2004) concluded that MTHFR 677TT reduces the risk of adult ALL, but not childhood ALL.

In contrast to the risk of MTHFR 677T variant allele, a significantly decreased risk was observed for MTHFR 1298 AC genotype (1.7-folds; 95% CI=1.1167-2.7007, p=0.0189), MTHFR 1298 CC genotype (3.4-folds, 95% CI=1.7629-6.4990, p=0.0003), and MTHFR 1298AC heterozygous genotype plus 1298CC homozygous genotype (two-folds, 95% CI=1.3603-3.0794, p=0.0008). This finding corroborates the recent data reported by Kamel et al. (2009), who found a protective effect for this polymorphism (p=0.001, OR=0.382, 95% CI=0.222-0.658), imposing a 2.6-fold protection.

In general, the ALL incidence ratio has been found to be correlated to the levels of socioeconomic development, suggesting that environmental factors linked to affluence of opportunities are important in ALL pathogenesis (Stiller and Parkin, 1996).

In our study, TS polymorphism showed no impact. Several case-control studies investigated the impact of TS polymorphism in leukemia, including both AML and ALL. However, in contrast to our results, Skibola et al. (2002) found a 2.8-fold reduction in ALL risk (OR=0.36, 95% CI=0.16-0.83, p=0.01) in 2R3R individuals. This protective effect was even greater when TS 3R3R individuals were compared with those with 2R2R, presenting a 4.0-fold reduction in ALL risk among TS 3R3R variants (OR=0.25, 95% CI=0.08-0.78, p=0.017). Likewise, Hishida et al. (2003) found that subjects with at least one TS 2R allele had an approximately 1.6-fold higher susceptibility to lymphoma than those without the TS 2R allele. On the other hand, De Jonge et al. (2009) observed a protective effect of the TS 2R variant in ALL patients.

The limitations of the present study are related to the sample ethnic characteristics as well as the lack of information on environmental exposures with biological effects, depending on the nature of potential agents that cause chromosomal alterations. Despite careful selection of the controls at the same socioeconomic levels, skin color and/or self-declared ethnicity are poor ethnicity indicators due to the high-grade miscegenation of Brazilian populations (Parra et al., 2003).

On the other hand, the strength of this study is that the polymorphisms, according to age and type of leukemia, demonstrated regional differences in geographical settings, where the lower incidence could be associated with polymorphisms in the folate genes. However, more ecological studies analyzing other variables are necessary to confirm our preliminary results.

Footnotes

Acknowledgments

This work was partly supported by Fundação de Amparo a Ciência e Tecnologia de Parnambuco (FACEPE) and Swissbridge Foundation (Switzerland). The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico and Freitas, EM from Universidade de Pernambuco for statistic review.

Author Disclosure Statement

No competing financial interests exist.

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