Evaluation of genetic variants in the reduced folate carrier and in glutamate carboxypeptidase II for spina bifida risk

doi:10.1016/S1096-7192(03)00086-6

Molecular Genetics and Metabolism

Volume 79, Issue 3, July 2003, Pages 197-200

https://doi.org/10.1016/S1096-7192(03)00086-6 Get rights and content

Abstract

Genetic variants in folate metabolism have been reported to increase risk for neural tube defects (NTD). The first such sequence change was the 677C → T substitution in methylenetetrahydrofolate reductase (MTHFR), but additional sequence changes have been identified in enzymes or transporters for folates. Two recently identified variants are the 1561C → T (H475Y) mutation in glutamate carboxypeptidase II (GCPII) and the 80A → G (H27R) change in the reduced folate carrier RFC-1. We examined a group of mothers of spina bifida offspring, and a group of control women, for the above polymorphisms to assess their impact on NTD risk as well as on homocysteine and nutrient (RBC folate, serum folate, and serum cobalamin) levels. The GCPII variant (in the heterozygous state) did not influence NTD risk or metabolite levels; homozygous mutant (YY) women were not observed in our study group. The homozygous mutant (RR) genotype for the RFC-1 gene was not associated with a significant difference in NTD risk (OR=1.39, 95% CI=0.55–3.54), but there was a borderline significant (p=0.065) decrease in RBC folate levels, compared with the HH genotype. However, the combination of the RR genotype for RFC-1 and low RBC folate was associated with a significant 4.6-fold increase in NTD risk (OR=4.6, 95% CI=1.47–14.37). Since this small study is the first to demonstrate increased risk for women with the RFC-1 variant for having a child with a NTD, additional larger studies are required to confirm this change as another potential genetic modifier for spina bifida risk.

Introduction

Adequate folic acid intake in early pregnancy reduces the risk of neural tube defects (NTD) in the offspring. Although the mechanism by which this risk reduction occurs is unknown, research in recent years has focused on the identification of genetic variants in enzymes of folate metabolism that might modify risk for NTD. The first such polymorphism is the 677C → T substitution in 5,10-methylenetetrahydrofolate reductase (MTHFR) ([1], reviewed in [2]). In more recent studies, Devlin et al. [3] identified the 1561C → T (H475Y) variant in glutamate carboxypeptidase II (GCPII), which hydrolyzes the glutamate residue of folates at the intestinal brush border membrane; the presence of the mutant allele was associated with lower folate and higher homocysteine in an older English population. However, this variant was not preferentially transmitted to probands in an NTD study using TDT analysis [4].

A polymorphism in the gene for the reduced folate carrier (RFC-1), which transports 5-methyltetrahydrofolate across the plasma membrane, has been reported, but this variant (80A → G; H27R) was not associated with folate or homocysteine levels in a healthy French population [5]. Shaw et al. [6] found an increased risk for spina bifida in California newborns with the G80/G80 genotype whose mothers had not used vitamins. Although the spina bifida risk was not statistically significant, this study did reveal a modest gene-nutrient interaction between infant homozygosity for the RFC-1 G80/G80 genotype and maternal periconceptional intake of vitamins containing folic acid on the risk of spina bifida.

In this study, we assessed the potential impact of the RFC-1 and GCPII genotypes on nutrient and homocysteine levels, and on the risk for having an offspring with spina bifida.

Section snippets

Study population

We examined an extensively studied population reported in two other publications [7], [8]. Mothers of children with spina bifida were recruited from the Spina Bifida Clinic of The Montreal Children’s Hospital after approval of the protocol by the Institutional Review Board. The controls were mothers of other outpatients who were having a venipuncture at the Pediatric Test Center of The Montreal Children’s Hospital. A total of 19 mothers were excluded from the study population because of

Results

The distribution of RFC-1 genotypes is shown in Table 1. In the control population, 22% had the HH RFC1 genotype, while 54 and 24% had the HR and RR genotypes, respectively. For the GCPII genotype, 89% of control mothers had the HH genotype and 11% were heterozygous (HY). There were no homozygous YY mothers in our population. This distribution for GCPII was identical to that in the case mothers (data not shown).

There was no statistical association between spina bifida risk and RFC-1 or GCPII

Discussion

This is the first study to examine women for the H27R variant of the RFC-1 gene, to evaluate the risk of having an offspring with NTD. Women who were homozygous for the RR genotype had an increased risk for having a child with NTD, when compared with the HH genotype (OR=1.39), but this increase was not statistically significant. However, the RR genotype was associated with a significant 4.6-fold increase in risk when combined with low RBC levels. Our results are consistent with Shaw et al. [6]

Acknowledgements

This study was supported by a grant from the Canadian Institutes of Health Research to RR, who is also a Senior Scientist of the CIHR.

References (10)

N.M.J van der Put et al.
Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida
Lancet
(1995)
A Chango et al.
A Polymorphism (80G → A) in the reduced folate carrier gene and its associations with folate status and homocysteinemia
Molec. Genet. Metab.
(2000)
L.D Botto et al.
5,10-methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGe review
Am. J. Epidemiol.
(2000)
A.M Devlin et al.
Glutamate carboxypeptidase II: a polymorphism associated with lower levels of serum folate and hyperhomocysteinemia
Hum. Mol. Genet.
(2000)
A.R Vieira et al.
Studies with His475Tyr glutamate carboxypeptidase II polymorphism and neural tube defects
Am. J. Med. Gen.
(2002)

There are more references available in the full text version of this article.

Cited by (66)

Trophic Factor, Nutritional, and Hormonal Regulation of Brain Development
2017, Fetal and Neonatal Physiology, 2-Volume Set
Molecular basis for pharmacokinetics and pharmacodynamics of methotrexate in rheumatoid arthritis therapy
2014, Drug Metabolism and Pharmacokinetics
Methotrexate (MTX) is a derivative of folic acid (folate) and commonly used as an anchor drug for the treatment of rheumatoid arthritis (RA). The pharmacokinetics (PK) and pharmacodynamics (PD) of MTX entirely depends on the function of specific transporters that belong to the two major superfamilies, solute carrier transporters and ATP-binding cassette transporters. Several transporters have been identified as being able to mediate the transport of MTX, and suggested to be involved in the disposition in the body and in the regulation of intracellular metabolism in target cells, together with several enzymes involved in folate metabolism. Thus, drug–drug interactions through the transporters and their genetic polymorphisms may alter the PK and PD of MTX, resulting in an interpatient variability of efficacy. This review summarizes the PK and PD of MTX, particularly in relation to RA therapy and focuses on the roles of transporters involved in PK and PD with the aim of facilitating an understanding of the molecular basis of the mechanism of MTX action to achieve its effective use in RA therapy.
Association of glutamate carboxypeptidase II (GCPII) haplotypes with breast and prostate cancer risk
2013, Gene
Citation Excerpt :
The positive association between folate and prostate cancer risk observed in the current study is consistent with earlier studies (Collin et al., 2010a, 2010b). Out of the 52 SNPs reported in GCPII, only one SNP i.e. H475Y has been investigated extensively for possible association with different diseases (Afman et al., 2003; Chen et al., 2004; Födinger et al., 2003; Lopreato et al., 2008; Morin et al., 2003; Roffman et al., 2011; Winkelmayer et al., 2003). Study by Chen et al. showed a null association of this variant in colorectal cancer (Chen et al., 2004).
In view of the pivotal role of glutamate carboxypeptidase II (GCPII) in carcinogenesis, its expression as prostate specific membrane antigen (PSMA) and folate hydrolase (FOLH1) may be influenced by its haplotypes, contributing to the etiology of prostate and breast cancer. To test this hypothesis, breast and prostate cancer cases and controls were subjected to whole gene screening of GCPII and correlated with plasma folate levels and PSMA expression. The impact of variants on a 3-dimensional structure of GCPII was explored by in silico studies. Six novel variations i.e. V108A, P160S, Y176H, D191V, G206R and G245S; and two known variations i.e. R190W and H475Y were identified in GCPII. All-wild haplotype and a haplotype harbouring D191V showed association with breast cancer risk while haplotypes harbouring V108A and P160S reduced the risk. Haplotypes with V108A and G245S variants showed increased risk for prostate cancer due to high PSMA expression while P160S conferred protection against prostate cancer. In silico studies suggests that P160S and R190W variants result in relaxed substrate binding facilitating either rapid catalysis or exchange of substrates and products in the active site which was substantiated by high plasma folate levels associated with these variants. On the contrary, D191V was associated with very low plasma folate levels despite having a high PSMA expression. This is the first comprehensive study examining variations in GCPII in relation to breast and prostate cancer risk. Changes in the plasma folate levels and changes in PSMA expression are associated with breast and prostate cancer risk respectively.
A single nucleotide polymorphism of reduced folate carrier 1 predicts methotrexate efficacy in Japanese patients with rheumatoid arthritis
2013, Drug Metabolism and Pharmacokinetics
Methotrexate (MTX) exhibits large inter-individual and inter-ethnic differences in the dose required for its anti-rheumatic effect. To maintain low disease activity, patients may require increased MTX doses or co-administration of biologic disease-modifying anti-rheumatic drugs (bDMARDs). The availability of a marker predicting the effect of MTX will make it possible to increase the MTX dose and prescribe bDMARDs to patients at an early stage. To establish individualized medication for rheumatoid arthritis (RA), we investigated genetic polymorphisms of the folate pathway in Japanese RA patients. Eighty-nine patients were treated with MTX alone (MTX group). MTX and bDMARDs were co-administered to 81 patients because of insufficient MTX efficacy (MTX + bDMARDs group); an equally stable therapeutic effect was achieved in both groups. Polymorphism analyses using bDMARD co-treatment as the objective variable revealed a significant association between age and the G80A polymorphism of the reduced folate carrier 1 gene (RFC1) as an explanatory variable. Compared to patients with the A allele, patients with the G allele may have less intracellular MTX uptake and, therefore, poor efficacy; a greater number of them were found to be bDMARD concomitant cases. The results of this study suggest that the RFC1 G80A polymorphism may be a useful marker for predicting MTX efficacy in Japanese patients with RA.
Reduced folate carrier A80G polymorphism and susceptibility to neural tube defects: A meta-analysis
2012, Gene
Citation Excerpt :
Fig. 1 presents a flowchart for the process of articles inclusion/exclusion. Among these articles, 6 studies investigated the influence of RFC1 A80G polymorphism in mothers of NTDs offspring on NTDs risk (De Marco et al., 2001; Morin et al., 2003; O'leary et al., 2006; Pei et al., 2009; Relton et al., 2004b; Shang et al., 2008), 5 studies investigated the association of RFC1 A80G polymorphism in infants with NTDs (De Marco et al., 2001; O'leary et al., 2006; Pei et al., 2009; Relton et al., 2004b; Shaw et al., 2002). Table 1 summarizes the general characteristics of the studies included in the meta-analysis.
The reduced folate carrier (RFC1) plays a crucial role in mediating folate delivery into a variety of cells. RFC1 polymorphism (A80G) has been reported to be associated with increased risk of neural tube defects (NTDs). However, results derived from individually underpowered studies are conflicting. We performed a systematic search of MEDLINE and EMBASE databases and carried out a meta-analysis on the association between RFC1 polymorphism (A80G) and NTDs risk. Overall, a significant correlation between RFC1 A80G polymorphism and NTDs risk was found neither in infants nor in maternal (allele contrast in infants: OR_RE = 1.15, 95% CI: 0.92–1.45; allele contrast in mothers: OR_RE = 1.24, 95% CI: 0.98–1.56). The present meta-analysis failed to support a positive association between RFC1 A80G polymorphism and susceptibility to NTDs. It is important to realize, however, that socio-economic factors, and gene–environment and gene–gene interactions, could have influenced the outcome of our meta-analysis. For this reason, a relationship between the A80G polymorphism and NTD risk cannot be entirely discounted.
Paradoxical role of C1561T glutamate carboxypeptidase II (GCPII) genetic polymorphism in altering disease susceptibility
2012, Gene
Citation Excerpt :
This was consistent with Relton et al., study showing the increased risk of anencephalic pregnancy in GCPII H475Y variants (Relton et al., 2003). However, Morin et al., have observed that the GCPII variant (in the heterozygous state) did not influence NTD risk or metabolite levels with no homozygous mutant women observed in their study group (Morin et al., 2003). The association of GCPII H475Y variant with CAD risk was probably mediated through homocysteine.
Glutamate carboxypeptidase II (GCPII) is predominantly expressed in brain, intestinal mucosa and prostate cancer in the form of three splice variants i.e. N-acetylated-α-linked acidic dipeptidase (NAALADase), folyl poly-γ-glutamate carboxypeptidase (FGCP) and prostate specific membrane antigen (PSMA) respectively. Its inhibition was found to confer protection against certain neurological disorders and cancer. Despite the pivotal role of this enzyme, the most common polymorphism i.e. H475Y has not been explored comprehensively in all its splice variants. In this study, we have determined the role of this variant in different disease conditions such as breast and prostate cancers, autism, coronary artery disease (CAD) and miscarriages (N = 1561). Genotyping was done by PCR-RFLP and dideoxy sequencing. Plasma folate levels were estimated by Axysm folate kit. GCPII expression was studied by semi-quantitative RT-PCR. In silico model was developed using PYMOL. We observed the protective role of H475Y variant in cancers [breast cancer; OR (95% CI): 0.81 (0.55–1.19), prostate cancer: OR (95% CI): 0.00 (0.00–0.66)], and in autism (OR (95% CI): 0.47 (0.21–1.03), whereas inflated risk was observed in CAD (OR (95% CI): 1.69 (1.20–2.37) and miscarriages [Maternal OR (95% CI): 3.26 (2.11–5.04); Paternal OR(95% CI): 1.99 (1.23–3.21)]. Further, this variant was found to impair the intestinal folate absorption in subjects with dietary folate intake in the lowest tertile (CC vs. CT in lowest tertile; 7.56 ± 0.85 ng/ml vs. 2.73 ± 045 ng/ml, p = 0.005). In silico model of GCPII showed steric hindrance with H475Y resulting in stereochemical alteration of catalytic site, thus interfering with ligand binding. Statistically significant association was not observed between dietary folate levels and GCPII expression. However, a positive correlation was seen between plasma folate levels and GCPII expression (r = 0.70, p < 0.05). To conclude, our data suggests that GCPII H475Y variant shows inverse association with autism and cancer while showing positive association with CAD and miscarriages.

View all citing articles on Scopus

View full text

Evaluation of genetic variants in the reduced folate carrier and in glutamate carboxypeptidase II for spina bifida risk

Abstract

Introduction

Section snippets

Study population

Results

Discussion

Acknowledgements

Lancet

Molec. Genet. Metab.

5,10-methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGe review

Am. J. Epidemiol.

Glutamate carboxypeptidase II: a polymorphism associated with lower levels of serum folate and hyperhomocysteinemia

Hum. Mol. Genet.

Studies with His475Tyr glutamate carboxypeptidase II polymorphism and neural tube defects

Am. J. Med. Gen.