Abstract
Background: Dihydrofolate reductase (DHFR) is essential for the conversion of folic acid to active folate needed for one-carbon metabolism. Common genetic variation within DHFR is restricted to the noncoding regions, and previous studies have focused on a 19 bp deletion/insertion polymorphism (rs70991108) within intron 1. Reports of an association between this polymorphism and blood folate biomarker concentrations are conflicting.
Objective: In this study, we evaluated whether the DHFR 19 bp deletion/insertion polymorphism affects circulating folate biomarkers in, to our knowledge, the largest cohort to address this question to date.
Methods: Healthy young Irish individuals (n = 2507) between 19 and 36 y of age were recruited between February 2003 and February 2004. Folic acid intake from supplements and fortified foods was assessed with the use of a customized food intake questionnaire. Concentrations of serum folate and vitamin B-12, red blood cell (RBC) folate, and plasma total homocysteine (tHcy) were measured. Data were analyzed with the use of linear regression models.
Results: Folic acid intake was positively associated with serum (P < 0.0001) and RBC (P = 0.0005) folate concentration and was inversely associated with plasma tHcy (P = 0.001) as expected. The DHFR 19 bp polymorphism was not significantly associated with either serum (P = 0.82) or RBC (P = 0.21) folate, or plasma tHcy (P = 0.20), even in those within the highest quintile of folic acid intake (>326 μg folic acid/d; P = 0.96). A nonsignificant trend toward lower RBC folate by genotype (P = 0.09) was observed in the lowest folic acid intake quintile (0–51 μg/d).
Conclusion: In this cohort of healthy young individuals, the DHFR 19 bp deletion allele did not significantly affect circulating folate status, irrespective of folic acid intake. Our data rule out a strong functional effect from this polymorphism on blood folate concentrations.
Keywords: DHFR, 19 bp polymorphism, rs70991108, red cell folate, homocysteine, plasma folate, folic acid
Introduction
Dihydrofolate reductase (DHFR)10 is an important enzyme that catalyzes the reduction of both dihydrofolate and folic acid to its biologically active metabolite, tetrahydrofolate, which accepts and donates one-carbon groups for reactions that are necessary for DNA synthesis and cellular remethylation (1). A number of polymorphisms within the DHFR gene have been investigated for their functional effects and/or association with disease and they include variants within the 3′ untranslated region (UTR), including 829 C > T (2), 35289 A > G (rs1232027) (3), and 721 A > T (rs7387) (4); the 5′ UTR and promoter region, including a 9 bp repeat polymorphism (5), 317A > G (rs408626), and 1610 C > G/T (rs1650694) (6); and intron 3, including 8890A > G (rs1643659) and 10372A > C (rs1677639) (7). However, a 19 bp deletion/insertion polymorphism within intron 1, rs70991108 (referred to as the 19 bp polymorphism from here), has received particular attention. The 19 bp polymorphism is located 60 bases from the splice donor site of the first intron, with the deletion (del) allele originally reported as being associated with maternal risk of neural tube defects (NTDs) in a sample size of 35 NTD families and 219 nonaffected controls (8). However, we showed that this allele is more likely to protect against maternal risk of NTDs in a larger cohort (n = 280 case mothers and n = 256 control mothers) and is in linkage disequilibrium with the 3′ UTR single-nucleotide polymorphism 721A > T (rs7387) (9). Another study found no effect from the 19 bp polymorphism (or the 9 bp repeat) on maternal (or case) risk (n = 101) (10). In terms of NTD risk, it is still unclear whether the DHFR 19 bp polymorphism is a true maternal risk factor for NTDs but it is unlikely to account for a large proportion of the risk based on the studies that have been published to date. What is probably more important to address is whether this polymorphism has a definitive functional effect that ultimately affects folate metabolism, leading to changes in an individual’s folate status—i.e., does it have relevance for personalized folate nutrition?
The impact of this DHFR 19 bp polymorphism on circulating folate biomarkers has been investigated previously. One study examined 330 individuals ranging from 20 to 90 y of age, and reported the del allele to be associated with a decrease in plasma total homocysteine (tHcy) in comparison with the wild-type (wt) genotype (P = 0.006), but having no differences in serum or red blood cell (RBC) folate concentration, with adjustments for age and sex not changing the result (5). Another group found the del allele to be modestly associated with an elevated concentration of RBC and serum folate in women but not in men, with no differences seen in tHcy concentration, although the effects were annulled by smoking (11). This study was conducted on 430 young Northern Irish adults ranging from 20 to 26 y of age. However, because maternal low folate/high homocysteine concentrations have previously been shown to be associated with an increased risk of NTDs in offspring (12), these findings suggest that the del/del genotype may have a protective effect against NTDs, in agreement with our NTD study (9). In contrast, another group suggested that the potential functional effect of the DHFR 19 bp polymorphism depends on the amount of folic acid intake (13). This group examined 1215 individuals from the Framingham Offspring Study and reported that a folic acid intake of ≥500 μg/d increased the concentration of high circulating unmetabolized folic acid in individuals who had the del homozygous genotype compared with the other genotypes. An interaction between the DHFR 19 bp polymorphism and folic acid intake was also seen with respect to RBC folate. When folic acid intake was <250μg/d, the del/del genotype was associated with significantly lower RBC folate compared with the wt genotype. No interaction was found between the DHFR genotype and folic acid intake with tHcy and serum total folate (13).
The larger of the studies described above (13) suggests that the DHFR del allele may have an impact on circulating unmetabolized folic acid and RBC folate, but that folic acid intake and indeed sex may also need to be considered. We sought to decipher the definitive impact the DHFR 19 bp polymorphism may have on circulating folate biomarkers in what is, to our knowledge, the largest cohort (n = 2507) to address this issue to date, including detailed data on folic acid intake.
Methods
Study cohort.
The subjects for this study have been described previously (14–18) and consisted of healthy students with Irish grandparents who had attended the University of Dublin, Trinity College, during a 12 mo recruitment period between February 2003 and February 2004. Overall, 3569 students applied; of these, 2524 individuals who had Irish grandparents and had no major medical problems were invited to participate. Of these, blood samples and a completed lifestyle and nutrition questionnaire were obtained for 2507 students who were between 19 and 36 y of age. All samples were made anonymous before analysis and had appropriate ethical approval and written informed consent.
Supplement and fortified food intake.
A customized food intake questionnaire was used to assess folic acid intake as previously described (14, 18). Briefly, the design of the questionnaire allowed an assessment of vitamin supplements and fortified food intake over the previous week and the average over one month by asking participants to recall their fortified food and supplement intake over those 2 time periods. Of note, liberal voluntary folic acid fortification of foods was in place in Ireland during the collection period (19), and over 40 fortified products were available, including cereals, breads, milk, juices, and yogurts. The consumption of those fortified foods was captured in the questionnaire as follows. A list of fortified foods and serving size definitions were provided to each participant. Determination of nutrient intake from fortified food was based on a combination of fortified food consumed, the frequency of consumption, and the reported serving size. Supplement intake was recalled from within the previous week, and active nutrient information from supplements was converted to micrograms of nutrient per portion (tablet or liquid) or recorded in uniform (international unit) format. Data in international unit format were converted to micrograms of nutrient per portion, according to standard conversion rates. The average amount of individual nutrients consumed per day was calculated from the quantity and frequency of intake of both fortified foods and supplements in the previous week and month. Blood was collected from nonfasting participants on the day of the interview and processed within 3 h of collection.
Biochemical methods.
Serum and RBC folate were measured with the use of a validated microbiological assay based on chloramphenicol-resistant Lactobacillus casei as previously described (20, 21). Serum vitamin B-12 was measured with the use of a validated microbiological assay based on colistin-resistant Lactobacillus delbreuckii (22). Plasma tHcy was measured by gas chromatography–mass spectrometry (23). Serum ferritin assays were performed with the use of an Abbott AxSYM analyzer (Abbott Laboratories Ireland) (24). The interassay CVs were as follows: serum folate, <11%; RBC folate, <10.5%; serum vitamin B-12, <10.6%; serum ferritin, <6.0%; and plasma tHcy, <2.2%.
Genotyping methods.
The DHFR 19 bp polymorphism (rs70991108) genotypes were determined with the use of melting curve analysis with HybProbes on a LightCycler 480 Real Time PCR machine (Roche Diagnostics). The following reagents were used: forward primer (5′-TGGGCATCGGCAAGAAC-3′) (0.75 μM), reverse primer (5′-TCTGGCCCCATCCTCTC-3′) (0.25 μM), sensor probe (5′-CCAGGTACCCCGACCGTG-Fluorescein-3′) (0.25 μM), anchor probe (5′-BODIPY 630/650-CAGCCTGCGCCCGTTTGGG-Phosphate-3′) (0.25 μM), and DMSO. The conditions used were as follows: preincubation for 10 min at 95°C, followed by 45 amplification cycles of 95°C for 10 s, 59°C for 15 s, and 72°C for 15 s, followed by melting curve analysis, 95°C for 1 min, 30°C for 1 min, and acquisition ramp-up to 75°C (ramp rate 0.1°C/s), followed by 40°C for 30 s. Genotyping quality was verified by repeat genotyping of 10% of the samples with a concurrence of >99%.
Statistical methods.
Biomarker association analysis was performed for 3 folate metabolites, RBC and serum folate, and tHcy with the DHFR 19 bp deletion polymorphism. Univariate analysis was used to check the normality of the 3 biomarkers by Kolmogorov-Smirnov test. Because the biomarker measurements are not normally distributed (P < 0.01), rank-based inverse normal transformation was applied. Linear regression was used to examine the association between genotype and each folate biomarker with the use of an additive effect model. A second analysis was performed, adjusting for vitamin B-12 concentration, sex of the participants, and folic acid intake in micrograms per day over the previous week. To explore the effect of high vs. lower folic acid intake, a further analysis consisted of testing for DHFR 19 bp genotype effects by dividing into quintiles of folic acid intake.
Results
Characteristics of the cohort.
The characteristics of the study cohort participants are shown in Table 1. Because men and women differed with respect to some folate biomarker concentrations and supplement intake, we adjusted for sex in our analyses.
TABLE 1.
Characteristics of the healthy young Irish adults from the study1
| Total (n = 2503) | Men (n = 1036) | Women (n = 1467) | P2 | |
| Age, y | 22 [21, 24] | 23 [21, 24] | 22 [21, 23] | <0.001 |
| BMI, kg/m2 | 22.6 [21.0, 24.4] | 23.1 [21.4, 24.9] | 22.2 [20.7, 24.0] | <0.001 |
| Serum creatinine, μmol/L | 64.7 [56.6, 74.3] | 74.2 [67.0, 82.1] | 58.8 [52.9, 65.7] | <0.001 |
| Hemoglobin, g/dL | 14.1 [13.1, 15.2] | 15.3 [14.6, 16.0] | 13.3 [12.7, 14.0] | <0.001 |
| Serum ferritin, μg/L | 32.2 [17.5, 56.6] | 57.5 [38.6, 82.9] | 21.5 [13.1, 34.0] | <0.001 |
| Serum vitamin B-12, pmol/L | 307 [231, 410] | 326 [253, 426] | 291 [214, 398] | <0.001 |
| Serum total folate, nmol/L | 29.9 [20.9, 43.4] | 28.5 [20.6, 40.8] | 30.7 [21.2, 45.7] | 0.002 |
| Red blood cell folate, nmol/L | 1000 [767, 1310] | 1030 [799, 1310] | 994 [740, 1300] | 0.10 |
| Plasma homocysteine, μmol/L | 8.13 [7.00, 9.64] | 8.92 [7.66, 10.3] | 7.65 [6.59, 8.96] | <0.001 |
| Total folic acid intake,3 μg/d | 140 [65.5, 287] | 137 [68.3, 293] | 141 [64.3, 284] | 0.73 |
| Supplement use | ||||
| Yes | 576 (23.0) | 202 (19.5) | 374 (25.5) | |
| No | 1903 (76.0) | 833 (80.4) | 1070 (72.9) | |
| No information given | 24 (1.0) | 1 (0.0) | 23 (1.6) |
Values are medians [IQRs] or n (%).
Statistical comparisons between sexes were carried out with the use of the nonparametric Mann-Whitney test for independent samples.
Calculated as the average micrograms per day based on reported intake in the previous week from combined supplement use and folic acid from fortified foods.
DHFR 19 bp polymorphism genotype and allele frequencies.
The genotype and allele frequencies of the DHFR 19 bp polymorphism are shown in Table 2. Analyses were carried out in both men and women together and separately. The genotype and allele frequencies seen were similar to those of a control Irish population previously genotyped (9) and did not differ significantly from the expected frequencies based on the Hardy-Weinberg equilibrium.
TABLE 2.
Genotypes and allele frequencies of the DHFR 19 bp (rs70991108) polymorphism in healthy young Irish adults1
| DHFR 19 bp polymorphism | Total | Men | Women |
| wt/wt | 829 (33.1) | 340 (32.8) | 489 (33.3) |
| wt/del | 1204 (48.1) | 507 (48.9) | 697 (47.5) |
| del/del | 470 (18.9) | 189 (18.2) | 281 (19.2) |
| wt | 2862 (57.2) | 1187 (57.3) | 1675 (57.1) |
| del | 2144 (42.8) | 885 (42.7) | 1259 (42.9) |
Values are n (%). del, deletion; DHFR, dihydrofolate reductase; wt, wild-type.
The DHFR 19 bp polymorphism and folate biomarker concentrations.
The mean folate biomarker values within each DHFR 19 bp genotype group are shown in Table 3. The unadjusted linear regression analysis showed that the DHFR 19 bp deletion had no statistically significant association with RBC, plasma folate, or tHcy (Table 3 and Supplemental Table 1). Adjusting for vitamin B-12 concentrations, sex, or folic acid intake did not significantly alter the results (Supplemental Table 2).
TABLE 3.
Folate metabolite concentrations by DHFR 19 bp genotype group in healthy young Irish adults1
| DHFR 19 bp genotype | Red blood cell folate, nmol/L | Serum folate, nmol/L | Plasma tHcy, μmol/L |
| wt/wt (n = 828) | 1010 (786, 1320) | 30.0 (21.0, 42.6) | 8.2 (7.0, 9.7) |
| wt/del (n = 1204) | 1010 (755, 1320) | 29.3 (20.7, 43.6) | 8.2 (7.1, 9.7) |
| del/del (n = 470) | 990 (757, 1270) | 30.9 (21.2, 44.5) | 8.0 (6.9, 9.4) |
| P | 0.20 | 0.77 | 0.21 |
Values are medians (IQRs). P values are from linear regression analysis after application of rank-based inverse normal transformation. Full regression analysis values are in Supplemental Table 1. del, deletion; DHFR, dihydrofolate reductase; tHcy, total homocysteine; wt, wild-type.
Folic acid intake and the DHFR 19 bp polymorphism.
In order to determine whether total folic acid intake has an influence on genotype association with folate biomarkers as previously reported (13), the population was divided into quintiles by total folic acid intake. This analysis showed that RBC folate and serum folate concentrations increased as folic acid intake increased across all genotypes going from the lowest intake (quintile 1) to the highest intake (quintile 5) (Table 4). Similarly to Kalmbach et al. (13), RBC folate concentrations did decrease in individuals carrying 1 or 2 DHFR del alleles (wt/del or del/del vs. wt/wt) in the lowest folic acid intake quintile (quintile 1), but this was not significant (P = 0.09). There was no association between DHFR 19 bp genotype, folic acid intake, and serum folate or RBC folate in the remaining quintiles. In addition, plasma tHcy concentrations were inversely associated with folic acid intake as expected. However, no association was seen between DHFR 19 bp genotype and either folic acid intake or plasma tHcy.
TABLE 4.
Folate metabolites by DHFR 19 bp (rs70991108) genotype group in quintiles of folic acid intake in healthy young Irish adults1
| Folic acid intake |
|||||
| Q1 (n = 500) | Q2 (n = 501) | Q3 (n = 501) | Q4 (n = 501) | Q5 (n = 500) | |
| RBC folate, nmol/L | |||||
| wt/wt | 850 (661, 1120) | 949 (727, 1250) | 982 (796, 1300) | 1100 (881, 1370) | 1239 (954, 1620) |
| wt/del | 841 (659, 1060) | 917 (696, 1160) | 1020 (775, 1310) | 1110 (872, 1490) | 1220 (924, 1610) |
| del/del | 782 (634, 979) | 944 (692, 1100) | 1010 (810, 1220) | 1060 (826, 1450) | 1260 (962, 1580) |
| P | 0.09 | 0.85 | 0.81 | 0.34 | 0.96 |
| Serum folate, nmol/L | |||||
| wt/wt | 22.7 (15.9, 29.6) | 26.6 (19.6, 37.7) | 29.6 (23.1, 40.1) | 34.9 (24.3, 47.8) | 40.6 (27.8, 58.1) |
| wt/del | 23.0 (16.4, 30.7) | 26.3 (17.9, 37.4) | 29.4 (21.2, 41.9) | 36.1 (24.8, 50.7) | 43.0 (27.1, 59.2) |
| del/del | 21.2 (13.7, 30.3) | 26.6 (18.2, 37.7) | 31.7 (24.3, 41.3) | 36.0 (26.5, 53.7) | 45.3 (27.3, 60.5) |
| P | 0.48 | 0.43 | 0.82 | 0.23 | 0.56 |
| Plasma tHcy, μmol/L | |||||
| wt/wt | 9.4 (7.8, 11.2) | 8.3 (7.2, 9.8) | 8.2 (7.1, 9.4) | 7.6 (6.7, 9.1) | 7.7 (6.6, 8.6) |
| wt/del | 9.2 (7.8, 10.5) | 8.4 (7.1, 10.0) | 8.2 (7.0, 9.6) | 7.8 (6.8, 8.9) | 7.6 (6.6, 9.1) |
| del/del | 8.7 (7.4, 11.0) | 8.6 (7.3, 9.8) | 8.0 (6.9, 9.2) | 7.3 (6.5, 8.3) | 7.6 (6.7, 9.0) |
| P | 0.43 | 0.55 | 0.31 | 0.14 | 0.71 |
Values are medians (IQRs). Total folic acid intake was calculated as the average micrograms per day based on reported intake in the previous week from combined supplement use and folic acid from fortified foods. Range of folic intake: Q1, 0–51 μg/d; Q2, 52–104 μg/d; Q3, 105–192 μg/d; Q4, 193–327 μg/d; and Q5, 328–14,920 μg/d. P values were generated with the use of an F test by ANOVA on rank-based inverse normal transformations of the data. del, deletion; DHFR, dihydrofolate reductase; Q, quintile; tHcy, total homocysteine; wt, wild-type.
Discussion
Human DHFR activity is thought to be quite variable between individuals (25), and although rare mutations with dramatic effects on enzyme activity have been reported in metabolic disorders (26, 27), common nonsynonymous variants within this enzyme have not been found. Recently, a second DHFR-like enzyme, DHFRL1, has been identified (28, 29), but it has much lower enzyme activity than DHFR (28) and is unlikely to account for the reported differences in enzyme activity between individuals (25). This points toward common polymorphisms that control the regulation and expression of the DHFR gene. The promoter region, which often extends to include the first intron (30), is the most likely area of a given gene to harbor functional polymorphisms that affect its control. Sequence analysis of the 19 bp polymorphism within intron 1 of DHFR suggest that some individuals may have alleles with an Sp2 transcription factor binding site, whereas others lack this consensus control sequence. This could have major implications for DHFR expression and could in part explain the variation in reductase activity that has been reported (25). However, this has not been proven to date, but clarification is required, given the widespread mandatory and voluntary fortification of foods with folic acid (31) that require DHFR activity to become bioavailable.
Any potential functional effect of the DHFR 19 bp polymorphism is likely to relate to the regulation of expression of the DHFR gene. Johnson et al. (8) originally proposed that the del allele might lead to loss of an Sp1 transcription factor–binding site, affecting the control of the expression of the DHFR gene. Our more recent sequence analysis (www.genomatix.com) showed that the del allele actually spans a consensus Sp2 sequence that has been shown to function as a negative regulator of gene expression (32). We and others (9, 33) suggested that the del allele causes an increase in mRNA levels in a cell line model and lymphocytes of healthy blood donors (n = 36), respectively, suggesting the loss of an inhibitory effect, but van der Linden et al. (10) observed no such association in the mRNA of spina bifida patients (n = 66).
A number of previous studies have attempted to resolve whether the DHFR 19 bp polymorphism has a direct impact on circulating folate biomarkers, with conflicting conclusions (5, 11, 13). In this study, we examined the relation and the effect of the DHFR 19 bp polymorphism genotype on serum folate, RBC folate, and plasma tHcy in the largest population studied to date in order to resolve the conflict. We found that the DHFR 19 bp del allele is not associated with any of the 3 circulating folate biomarkers examined, regardless of whether the data from men and women were analyzed in combination or separately. Previous studies that have showed an association had a smaller number of participants and/or had an older age profile. One of the studies also found that the functional effect of the DHFR polymorphism depends on the amount of folic acid intake (13). Although DHFR enzyme activity is required to reduce folic acid to its active form, the human enzyme was reported to have a slow rate of conversion of folic acid compared with the rat version of the enzyme, suggesting that high folic acid intake could saturate it (25). This would be particularly problematic in individuals with lower than average DHFR activity (25), with DHFR genetic polymorphisms being one obvious source of this variation. However, our study found that folic acid intake does not significantly interact with the DHFR 19 bp polymorphism in our population with detailed data on folic acid intake. This indicates that the source of DHFR variation between individuals previously reported (25) is unlikely to be due to the 19 bp polymorphism, because intake of its synthetic substrate, folic acid, has no effect on folate status. This data also implies that the DHFR 19 bp polymorphism will have no influence on the concentrations of circulating unmetabolized folic acid in an individual as reported previously (13), although we did not measure unmetabolized folic acid in our samples. We cannot, however, completely rule out a more subtle functional effect that simply does not reflect at the level of the circulation. Also, the finding that RBC folate was nonsignificantly lower in those carrying at least one del allele compared with wt/wt genotype (P = 0.09) within the lowest folic acid intake group (Table 4, quintile 1), leaves open the possibility that the DHFR del allele might affect folate status in a folate-deficient population. In conclusion, our results indicate that the DHFR 19 bp polymorphism does not have a significant impact on circulating folate biomarkers irrespective of sex or folic acid intake.
Acknowledgments
AMM, JLM, LCB, and AP-M designed the research; MO, AMM, and ERG collected the data and conducted the research; MO, AMM, and AP-M analyzed the data; RF and YW performed the statistical analysis; JLM, BS, and LCB contributed to data interpretation; and MO and AP-M wrote the manuscript. All authors read and approved the final manuscript.
Footnotes
Abbreviations used: del, deletion; DHFR, dihydrofolate reductase; NTD, neural tube defect; RBC, red blood cell; tHcy, total homocysteine; UTR, untranslated region; wt, wild-type.
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