To our knowledge this is only the second study to assess SNPs in the folate pathway enzymes, FPGS and GGH, in relation to plasma homocysteine levels, and the first in a population without universal folate fortification and limited alcohol consumption. In this study, FPGS
rs10106 and rs10987742 do not appear to influence plasma homocysteine levels. For rs10106, our findings are consistent with those of DeVos et al
rs11545076, rs1800909, and rs3758149 did appear to slightly alter plasma homocysteine levels, but only rs11545076 remained statistically significant after adjusting for multiple comparisons. DeVos et al
did not find rs11545076 to be associated with homocysteine concentrations, but they did find that the variant G allele was associated with increased DNA uracil content [7
rs11545076 and rs3758149 are both located upstream (−5′) of the GGH
gene. An in vitro
study found that variant alleles for both SNPs increase promoter activity [17
], suggesting the potential to influence enzyme levels, and ultimately, intracellular folate concentrations. The third SNP, rs1800909, is located in exon 1 and results in a missense mutation [18
], however, bioinformatics prediction suggests that this is a benign amino acid change (Cys>Arg) [19
]. Since these three SNPs do appear to be in linkage disequilibrium, it is possible that the observed variations in homocysteine levels could be due to genetic polymorphisms of one of the related SNPs alone or in combination, such as the haplotype. In this study, individuals carrying at least one variant allele had lower levels of plasma homocysteine than those homozygous for the wild-type, suggesting the variant allele results in decreased enzyme activity. Findings from the cell culture study by Chave et al
indicate that variant alleles for rs11545076 and rs3758149 increase GGH expression. If the Chave et al
findings are true, our observations of lower plasma homocysteine concentrations with the variant GGH
alleles are not consistent with our hypothesis that higher GGH activity would lead to higher homocysteine levels. Additional research is needed to clarify this discrepancy.
There is some evidence from pharmacologic studies to indicate that alterations in FPGS
function may alter cellular retention of folate. A study by Sadahiro et al
suggests variation in FPGS
can influence tissue folate levels after administration of 5-formyl THF (leucovorin) treatment for colorectal cancer [20
]. Additionally, studies examining FPGS and GGH activity in relation to methodrexate (MTX), which is chemically similar to folate and taken into cells by the same mechanism as folate, suggest that decreased FPGS activity and increased GGH activity are associated with MTX resistance by limiting the amount of MTX retained in the cells [21
]. MTX retention in a cell uses the same polyglutamation mechanism as folate. MTX that is highly polyglumated is retained in the cell, while short polyglutamate chains are associated with reduced cellular retention. Some studies have assessed the effects of SNPs in FPGS and GGH on MTX levels, efficacy and toxicity in rheumatoid (RA) and juvenile idiopathic arthritis patients [23
]. Van der Straaten et al
found no association with FPGS SNP rs10106 and MTX response in RA patients[25
]. Consistent with our results, at three months this study suggests that the variant allele (CC) for rs1800909 may decrease enzyme activity, however, after 6 months no associations were observed. A second study examined associations between the SNP, rs1800909 and toxicity and efficacy of MTX in juvenile idiopathic arthritis patients [24
]. This study found that liver dysfunction was associated with the heterozygous and variant homozygous (CC) rs1800909 genotypes, suggesting this polymorphism may reduce enzyme activity. This is consistent with our study results that found lower homocysteine levels (lower enzyme activity) among those heterozygous or homozygous (CC) variant for rs1800909. Dervieux et al
, found that genotype for rs3758149 altered red blood cell levels of long chain polyglutamated MTX levels in RA patients. Participants homozygous for the variant TT genotype had lower levels of long chain polyglutamated MTX (increased enzyme activity) in red blood cells (OR: 4.8, 95% CI: 1.8 – 13.0) than homozygotes for the wild-type or heterozygotes [23
]. This is consistent with our results that suggest the wild-type (CC) for rs3758149 has lower activity than the homozygous variant (TT) genotypes with the highest homocysteine levels observed in those with the variant genotype. While these studies are assessing different questions, and assess glutamation of MTX instead of folate, the results of these studies are generally consistent with the results from this study.
There were some limitations in the current study. This study evaluated circulating plasma homocysteine levels as a surrogate for intracellular folate. Erythrocyte folate concentration data were not available, but would likely have been a better measure of intracellular folate levels, and more indicative of relative FPGS and GGH activity. Erythrocyte folate concentration would also allow for a more complete picture of long-term status than a single, cross-sectional measurement of plasma folate.
The observed differences in mean homocysteine among genotypes for the significant SNPs are small and would not be considered clinically meaningful. This could be related to the relatively similar homocysteine levels among participants, but it could also be due to the many other enzymes and transport proteins that could influence the levels of plasma homocysteine but were not evaluated in the present study. It is possible that alternative, non-folate dependent, homocysteine detoxification pathways may be more influential, or that additional genetic variants in other pathways interact with FPGS
SNPs in relation to plasma homocysteine levels. Additionally, this study did not have sufficient numbers of participants to assess combinations of SNPs or to stratify the analyses by higher vs lower folate intake levels. Previous research has shown folate intake and genotype can interact, with genotype having different effects as high versus low folate levels. For example, studies of MTHFR
C677T have shown that the variant T-allele is associated with increased risk of colon cancer when folate intake is low, but may be protective against colon cancer with sufficient folate intake [26
This study also had several strengths. This was only the second study to assess the potential impact of genetic variation in FPGS and GGH on plasma homocysteine. This study differed from the previous study by DeVos et al
in that our study population, Singapore Chinese, live in a country without mandatory folate fortification and tend not to drink alcohol or smoke cigarettes. The participants in the DeVos et al
study were from the Puerto Rican community of Boston, and had higher prevalence of alcohol consumption and smoking [27
]. Unlike DeVos et al
, instead of evaluating many genes, we assessed multiple SNPs within only two genes, which allowed us to assess the potential influence of multiple SNPs for each gene.
More research is needed to understand the functional relevance of the FPGS and GGH SNPs. Future studies should aim to explore the impact of these genetic variants on long term folate status by using erythrocyte folate and multiple homocysteine measurements, since it is likely long term status that influences disease risk. It has been reported that expression of GGH is tissue specific [28
], so when examining GGH and FPGS polymorphisms it may be important to take this into consideration.