Chapter 13 Methylenetetrahydrofolate Reductase, Common Polymorphisms, and Relation to Disease

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Methylenetetrahydrofolate Reductase, Common Polymorphisms, and Relation to Disease Philip Thomas* and Michael Fenech* Contents 376 377 381 383 384 385 386 386

I. Introduction A. Methylenetetrahydrofolate reductase B. MTHFR and disease C. MTHFR and pregnancy outcomes D. MTHFR–environment interactions E. MTHFR–other gene interactions II. Conclusions References

Abstract Folate plays a key role in maintaining genomic stability and providing methyl groups for the formation of dTMP from dUMP which is required for DNA synthesis and repair and for the maintenance of methylation patterns involving cytosine or specific sites such as CpG islands. Under conditions of low folate, dUMP accumulates producing DNA strand breaks and micronucleus formation as a result of excessive uracil incorporation into DNA in place of thymine. Methylenetetrahydrofolate reductase (MTHFR) is an important folate metabolizing enzyme that catalyzes the irreversible conversion of 5,10-methylenetretrahydrofolate, which is the methyl donor for the conversion of dUMP to dTMP, into 5-methyltetrahydrofolate, which is the methyl donor for remethylation of homocysteine to methionine. Certain common polymorphisms within the MTHFR gene (C677T, A1298C) result in reduced enzymatic activity and have been associated with reduced risk for a variety of cancers such as acute lymphocytic leukemia, lung and colorectal cancer. These common polymorphisms are also associated with hyperhomocysteinemia that has been reported to be an increased risk factor for neural tube defects and cardiovascular disease.

*

CSIRO Human Nutrition, Adelaide BC, Adelaide, South Australia 5000

Vitamins and Hormones, Volume 79 ISSN 0083-6729, DOI: 10.1016/S0083-6729(08)00413-5

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2008 Elsevier Inc. All rights reserved.

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In this chapter, we consider the role that MTHFR plays in relation to folate metabolism and the possible contribution made in relation to certain important clinical outcomes. ß 2008 Elsevier Inc.

I. Introduction Folate (vitamin B9) is an essential B vitamin that is crucial to the prevention of genomic instability and hypomethylation of DNA (Choi and Mason, 2000, 2002). Folate is required for the synthesis of deoxythymidine monophosphate (dTMP) from deoxyuridine monophosphate (dUMP), which is essential for DNA synthesis and repair (Fig. 13.1). Under conditions of folate deficiency, dUMP accumulates resulting in excessive uracil incorporation into DNA leading to single- and doublestrand DNA breaks, chromosome breakage, and ultimately micronucleus (MN) formation (Blount and Ames, 1995; Blount et al., 1997; Fenech, 2001). Folate and vitamin B12 are required in the synthesis of methionine through the remethylation of homocysteine (Hcy) that ultimately leads to the synthesis of S-adenosylmethionine (SAM). SAM plays an important role as a methyl donor required for the maintenance of genomic methylation patterns that determine gene expression, DNA conformation, and is required for the synthesis of myelin, neurotransmitters, and membrane phospholipids (Calvaresi and Bryan, 2001; Zingg and Jones, 1997). Folate deficiency reduces SAM levels resulting in lower DNA cytosine methylation and elevated levels of Hcy. Additionally, folate deficiency may lead to demethylation of centromeric DNA repeat sequences and centromere dysfunction leading to abnormal chromosome distribution during nuclear Folic acid Cell proliferation and protein synthesis DHF B12 Cob (III)

Methionine

MTRR

SAM MTR CH3 Homocysteine DNA methylation

B6 Cystathione

THF B6 5,10-methyl THF 5,10-methylene THF

B12 Cob (II) B12 Cob (I)

5-methyl THF

Dietary folate

MTHFR (B2)

dUMP dTMP

DNA synthesis and repair

Gene expression

Figure 13.1 Metabolism of folic acid. Adapted from Wagner (1995). SAM: S-adenosyl methionine, MTRR: methionine synthase reductase, MTR, methionine synthase; THF, tetrahyrdofolate; DHF, dihydrofolate; MTHFR, methylene tetrahydrofolate reductase; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; Cob(I), reduced form of vitamin B12; Cob(III), oxidized form of vitamin B12 .

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division, resulting in elevated rates of aneuploidy, altered gene dosage and increased cancer risk (Beetstra et al., 2005; Duesberg et al., 1999; Rasnick and Duesberg, 1999; Wang et al., 2004).

A. Methylenetetrahydrofolate reductase One of the intriguing aspects of the relationship between folate status and cancer risk is the potential modifying effect of polymorphisms in key folate metabolizing enzymes. Methylenetetrahydrofolate reductase (MTHFR) is a pivitol enzyme within the folate methionine pathway which can influence both the bioavailability of folate for dTMP synthesis and maintain methylation patterns at CpG islands known to regulate gene expression. MTHFR catalyzes the reduction of 5,10-methylenetretrahydrofolate into 5-methyltetrahydrofolate, which is the major circulating form of folate, and acts as a methyl donor in the remethylation of Hcy to methionine (Crott et al., 2001; Goyette et al., 1998, 1994; Rozen, 1997; Sibani et al., 2003). The MTHFR gene was cloned in 1998 and found to be 20.3 kb long, consisting of 11 exons ranging in size from 102 to 432 bp (Frosst et al., 1995; Goyette et al., 1998). The major gene product is a catalytically active 77 kDa protein consisting of 656 amino acids, which has been shown to map to the short arm of chromosome 1 at 1p36.3 (Goyette et al., 1994). MTHFR enzymatic activity can be affected in a number of ways. First, polymorphisms within the gene sequence could alter the affinity of the enzyme for either substrate or its cofactor flavin adenine dinucleotide (FAD or vitamin B2). Second, high concentrations of methionine or SAM are inhibitory to MTHFR activity, and finally, insufficient levels of FAD cofactor may lead to reduced enzymatic activity (Hustad et al., 2000; Kimura et al., 2004; Rivlin, 1996). Under conditions of reduced MTHFR activity, 5,10methylenetetrahydrofolate concentration increases with a resultant subsequent lowering of 5-methyltetrahydrofolate concentration. This shift in balance favors dTTP synthesis over CpG island methylation, a reduction in the number of chromosome breaks by minimizing uracil incorporation, an increase in DNA hypomethylation that could favor chromosome loss, and a resultant increase in the concentration of plasma Hcy (Blount and Ames, 1995; Blount et al., 1997; Castro et al., 2004; Kimura et al., 2004; Stern et al., 2000). Many polymorphisms within the MTHFR gene have been reported within the literature; however, very few have been conclusively studied in relation to disease and population dynamics. The two most widely studied of these are the common C677T and A1298C polymorphisms. 1. MTHFR C677T polymorphism A common genetic variant of MTHFR involves a cytosine (C) to thymine (T) transition at position 677 within exon 4 of the gene, resulting in an alanine to valine substitution and reduced enzyme activity. This C677T

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transition is a mutation that causes thermolability as the activity of the encoded enzyme is reduced at 37
C (Botto, 2000; Kang et al., 1991). Enzyme activity of the CT heterozygote and the TT homozygote is reduced by 35% and 70%, respectively, when compared to the normal CC genotype ( James et al., 1999). Homozygosity for the T allele is associated with reduced enzyme activity resulting in mild to moderately elevated Hcy levels (Frosst et al., 1995). The population frequency of the C677T allele shows significant differences based upon geographical location and ethnic background. Wilcken et al. (2003) studied the C677T polymorphism in more than 7000 newborns from 16 areas within Europe, Asia, the Americas, the Middle East, and Australia. The TT homozygosity was particularly common in northern China (20%), southern Italy (26%), and Mexico (32%). There was also some evidence for changes in geographic gradients in Europe (north to south increase) and China (north to south decrease). The TT genotype frequency was low among new born individuals of African ancestry, intermediate among newborns of European origin, and high among newborns of American Hispanic ancestry. Areas at the extremes of the frequency distribution showed deviations from Hardy-Weinberg expectations (Helsinki, southern Italy, and southern China). The findings suggested the existence of selective pressures leading to the marked variation. It has been shown from other studies that C677T homozygosity in white Europeans ranged from 7.2% in Germany to 22% in northern Italy (Cattaneo et al., 1997; Koch et al., 1998). In Japanese populations, the homozygous frequency was shown to range between 10.2% and 12.2% whereas in black Africans, the homozygosity was not reported but the allelic frequency was 7% (Arinami et al., 1997; Morita et al., 1998; Schneider et al., 1998). This suggests that the allele is less common within the black African population. It is plausible that dietary folate may exert a selective pressure if high maternal folate determines the survival of C677T TT homozygotes in utero; preliminary data suggests that this may be possible but evidence is as yet inconclusive (Lucock et al., 2003; Munoz-Moran et al., 1998). 2. MTHFR A1298C polymorphism A second polymorphism in the MTHFR gene involves an A to C transition at position 1298 within exon 7 that results in a change from a glutamate to an alanine residue. This mutation alters an mboII recognition site and has an allelic frequency of 0.33 (van der Put et al., 1998). The activity of the enzyme is decreased but not to the same extent as the C677T allele (Weisberg et al., 1998a). It has been shown that neither the homozygous nor the heterozygous state of A1298C leads to an elevation in plasma Hcy or lower plasma folate concentration, which is evident for the homozygous state for C677T (Lievers et al., 2001). However, compound heterozygosity for both the C677T and the A1298C is associated with reduced MTHFR

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enzyme activity, higher plasma Hcy, and lower plasma folate concentrations (Botto et al., 2003; Friedman et al., 1999; Weisberg et al., 1998b). The combined association of these two alleles produces a similar biochemical profile to those individuals who are homozygote for the T allele of C677T. The population frequency for A1298C homozygosity is not as well documented as for the C677T allele and is thought to have a prevalence of about 10% (van der Put et al., 1998; Weisberg et al., 1998a). To date, over 30 other mutations have been identified as being associated with severe MTHFR deficiency (Sibani et al., 2000, 2003). The relationship between these rare polymorphisms and population dynamics and disease outcomes has yet to be fully determined. 3. MTHFR and genomic instability Damage to the genome could lead to altered gene dosage and gene expression as well as contribute to the risk of accelerated cellular death. Certain genomic instability biomarkers have been found to be altered in individuals possessing polymorphisms within the MTHFR gene. MN formation is a biomarker of chromosome malsegregation and fragility and has been found to be elevated in individuals carrying the variant C677T, TT genotype compared with the wild-type or heterozygous genotype (Andreassi et al., 2003; Botto et al., 2003; Kimura et al., 2004). Nucleoplasmic bridges (a biomarker for chromosomal rearrangement) were also found to be significantly increased in C677T, TT cells grown under low folate conditions compared with C677C or C677T cells (Leopardi et al., 2006). Kimura et al. also showed that nuclear buds (a biomarker for gene amplification) were markedly higher under conditions of low folate and high riboflavin compared with low folate and low riboflavin conditions, indicating a potential genotoxic effect of elevated riboflavin concentrations under conditions of low folate. Note that the level of nuclear buds was lower in C677T, TT cells compared with C677C, or C677T cells. This suggests that nuclear bud formation under low folate conditions may be exacerbated when riboflavin concentration increases or in the presence of the MTHFR wild-type genotype, both of which increase MTHFR activity (Kimura et al., 2004). Abnormal folate and methyl metabolism have been shown to lead to chromosome malsegregation and DNA hypomethylation (Fenech, 2001). Hypomethylation of repeat sequences within the centromeric regions of chromosomes may lead to faulty kinetochore assembly or despiralization leading to the loss of chromosomes as micronuclei (Botto et al., 2003). It has been shown that individuals who have the C677T, TT polymorphism are associated with genomic DNA hypomethylation and have an increased risk for having children with three copies of chromosome 21 (Down syndrome) (Friso et al., 2002; Stern et al., 2000). James et al. (1999) found a 2.6-fold higher risk in individuals with the C677T polymorphism of having a Down syndrome child compared with wild-type individuals. It has also been

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reported that mothers of Down syndrome children have a higher frequency of joint heterozygotic MTHFR polymorphisms (677 and 1298) compared to those with chromosomally normal offspring (OR: 5.7) (Grego´rio Lorenzo Aca´cio, 2005). The interactive relationships between folate, MTHFR genotype, and its cofactor riboflavin are complex and have been shown to affect a number of biomarkers of genomic instability. A flow diagram of these relationships and potential outcomes are highlighted in Fig. 13.2. The framework predicts that (1) as folate concentration increases, genomic instability events arising from aneuploidy and breakage-fusion-bridge (BFB) cycles are minimized, (2) genome hypomethylation and aneuploidy are minimized during high MTHFR activity, (3) the risk of BFB cycles is increased under high riboflavin and low folate conditions, (4) the risk of genome hypomethylation and aneuploidy are maximized under both low riboflavin and low folate conditions, and (5) reduced MTHFR activity may decrease MN frequency caused by uracil incorporation and subsequent chromosome breakage but may increase MN originating from chromosome loss or gain resulting from changes in methylation patterns. These complex

Low R

Low F

Low CpG

Low MTHFR activity G E N O T Y P E

INCREASED CANCER RISK?

methylation * Low uracil in DNA

High

High CpG High MTHFR activity

methylation * High uracil in DNA

High R

High MNi (Chromosome Loss/Gain) frequency increased ANEUPLOIDY

Low F

Low MNi (Chromosome breaks) Low NPB (Chromosome rearrangement) Low NBUDs (Gene amplification)

REDUCED CANCER RISK?

Low MNi (Chromosome loss/gain) frequency decreased ANEUPLOIDY High MNi (Chromosome breaks) High NPB (Chromosome rearrangement) High NBUDs (Gene amplification)

INCREASED CANCER RISK?

Figure 13.2 Mechanistic framework explaining the interrelationship between MTHFR genotype, riboflavin (R), and folic acid (F) with respect to (1) CpG methylation and uracil in DNA, (2) aneuploidy and micronuclei (MNi) originating from chromosome loss events, (3) MNi (originating from acentric chromosome fragments), nuclear buds (NBUDs), nucleoplasmic bridges (NPBs), and breakage-fusion-bridge (BFB) cycles, (4) initiation of cancer caused by CpG hypomethylation and aneuploidy, and (5) initiation of cancer caused by increased BFB cycles, MNi (originating from acentric chromosome fragments), NBUDs, and NPBs. *For brevity, other carcinogenic mechanisms induced by altered genome methylation, such as silencing of tumor suppressor genes and/or activation of oncogenes, are not included in the diagram.

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relationships may go some way to explain how the MTHFR C677T polymorphism may influence the relative risk for certain important clinical outcomes. The prevention of chromosome breakage and BFB cycles caused by uracil incorporation into the DNA may be more relevant to conditions such as leukemia or lymphoma, whereas prevention of CpG hypomethylation may be more relevant to cancers caused by DNA hypomethylation or aneuploidy-driven developmental defects such as Down syndrome.

B. MTHFR and disease 1. Cancer Low folate intake has been shown to be associated with increased cancer risk (Giovannucci et al., 1993, 1995; Sellers et al., 2001; Zhang et al., 1999), raising the possibility of a potential role for the C677T mutation in carcinogenesis or cancer progression. The association between MTHFR and cancer susceptibility has been examined in a number of different cancers. Individuals with C677T homozygosity have been shown to have a 2.8-fold increased risk for endometrial cancer, whereas patients with ovarian tumors have been shown to have allelic deletions in the MTHFR gene (Esteller et al., 1997; Viel et al., 1997). In contrast, various studies have shown that TT homozygotes have a 1.2- to 3.0-fold reduced risk for colorectal cancer and a 4.3-fold reduced risk for acute lymphocytic leukemia, but the protective effect may be lost in individuals who are folate deficient (Chen et al., 1996; Ma et al., 1997; Skibola et al., 1999). Reduced MTHFR enzymatic activity may prove to be protective by inhibiting hypermethylation that could lead to CpG island silencing of certain tumor suppressor genes. For example, it has been shown in patients with lung cancer that the TT allele is associated with the increased expression of the tumor suppressor gene p16 (Kamiya et al., 1998). It is also thought that the protective effect afforded by the polymorphism toward conditions such as lung and colorectal cancers may be due to the diversion of folate to purine and thymidine synthesis. This leads to the increased availability of 5,10-methylenetetrahydrofolate and subsequent methyl groups for the conversion of dUMP to dTMP, thereby reducing uracil incorporation and subsequent DNA instability (Crott et al., 2001; Kimura et al., 2004; Ueland et al., 2001). A small number of studies have investigated the association between the A1298C polymorphism and colorectal cancer risk (Chen et al., 2002; Keku et al., 2002; Le Marchand, 2002). In all studies, a reduced risk was found to be evident in CC individuals compared to AA subjects. Relative risks were in the range of 0.6–0.8 but were not found to be statistically significant. It has also been reported that the A1298C result was not due to a confounding C677T effect (Chen et al., 2002) and that a greater protective effect occurred in individuals who carried both 677T and 1298C alleles compared to wild-type subjects (Le Marchand, 2002).

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2. Cardiovascular disease Homozygosity for the C677T allele leads to a relative deficiency in the remethylation process of Hcy into methionine resulting in mild-to-moderate hyperhomocysteinemia, a condition recognized as an independent risk factor for atherosclerosis (Clarke et al., 1991; Danesh and Lewington, 1998). A number of studies have investigated whether an association between MTHFR polymorphisms and cardiovascular disease exists. A meta-analysis was performed in 1996 combining all studies where MTHFR genotyping data was available for patients with coronary heart disease. It was shown that individuals who were 677T homozygotes had a significant 19% higher risk for coronary heart disease compared with the other MTHFR genotypes (Blom, 1998). A more recent meta-analysis of over 72 studies in which MTHFR genotypes were available in patients that had been diagnosed with ischemic heart disease, deep vein thrombosis, or pulmonary embolism showed a significantly higher risk in people with the MTHFR mutation (Wald et al., 2002). Further meta-analysis investigation involving over 80 studies examining increased risk for coronary disease showed an odds ratio of 1.14 (95% confidence intervals (CI): 1.05–1.24) for TT versus CC genotype (Lewis et al., 2005). Although there is not a strong allelic association in most studies, it may be that the resulting elevated Hcy may also interact with other risk factors that may induce vascular events in those individuals who have underlying conditions such as thrombophilia that may predispose to cardiovascular disease (Refsum and Ueland, 1998; Ueland et al., 2001). 3. Alzheimer’s disease Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized clinically by cognitive decline, memory loss, visuospatial and language impairment, and is the commonest form of dementia (Burns et al., 2002; Kawas, 2003; Mattson, 2004; St George-Hyslop, 2000). Regions of the brain that are involved in short-term memory and learning such as the temporal and frontal lobes are impaired as a result of neuronal loss and the breakdown of the neuronal synaptic connections (Mattson et al., 1998). Many case control studies have shown that Alzheimer’s patients have been found to be deficient in certain micronutrients such as folate, vitamin B12, and have elevated levels of the sulfur-based amino acid Hcy (Aisen et al., 2003; Shea and Rogers, 2002). These factors are associated with increased MN formation and the alteration of methylation patterns that could modify gene expression (Fenech and Crott, 2002; Scarpa et al., 2003; Suzuki et al., 2002). It has been shown that adequate folate intake improves global cognitive function (de Lau et al., 2007) and that increased Hcy levels have a significant effect in the reduction of effective cognitive function (Kim et al., 2007).

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Hyperhomocysteinemia has been shown to be a strong independent risk factor for AD in a number of epidemiological studies (Clarke et al., 1998; McCaddon et al., 1998; Morris, 2003; Seshadri et al., 2002; Wang et al., 2001). It appears that nervous tissue may be extremely sensitive to excessive Hcy as it promotes excitotoxicity and damages neuronal DNA giving rise to apoptosis (Kruman et al., 2002). Studies have also shown a strong correlation between a reduction in hippocampal width, which is associated with shortterm memory loss and concentrations of plasma Hcy (Williams et al., 2002). Recently MRI measurements have shown that an inverse relationship exists between plasma Hcy and cortical and hippocampal volume (den Heijer et al., 2003). The above findings have been interpreted as involving neuronal damage within the hippocampal regions leading to memory loss, which is characteristic of AD. Elevated Hcy has also been implicated as playing a role in an iron dysregulation/oxidative stress cycle that is thought to be central to the pathogenesis of the disease (Dwyer et al., 2004). As AD is related to low folate, vitamin B12, and elevated Hcy levels, investigators have looked toward genetic polymorphisms within the folate methionine pathway (Fig. 13.1) to explain the effect of micronutrient differences. In determining the risk factor of C677T in relation to AD, no association between C677T and increased susceptibility to Alzheimer’s risk was found (Brunelli et al., 2001). However, another study found that female TT homozygotes have significant cognitive decline compared to wild type and heterozygotes (Elkins et al., 2007). However, if combinations of polymorphisms within a gene are considered together, then sometimes effects become apparent that are not always evident if those same polymorphisms are considered in isolation. Wakutani et al. (2004) found that the MTHFR 677C-1298C-1793G haplotype to be protective in Japanese populations against late onset AD.

C. MTHFR and pregnancy outcomes 1. Neural tube defects Under low folate conditions, individuals possessing C677T polymorphisms are predisposed to hyperhomocysteinemia. It has been shown that hyperhomocysteinemia is an increased risk factor for neural tube defects (NTD) (Finnell et al., 1998; Steegers-Theunissen et al., 1994, 1995). Initial studies reported an increased frequency of the C677T allele in both affected mothers and children inferring an increased risk for NTD. Data generated from metaanalysis studies indicate that TT individuals have a doubling in risk of having an affected child (Botto, 2000). Interestingly, it has been observed that low maternal blood folate and no periconceptional folate supplementation elevate the risk associated with the T allele (Christensen et al., 1999; Shaw et al., 1998). The combination of a TT genotype and low folate concentration increases Hcy concentration, while impairing the formation of embryonic

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methionine which may result in abnormalities in myelin synthesis that could contribute to NTD formation (Aubard et al., 2000). Limited data is available regarding the association between A1298C and the incidence of NTD. In one study, a significant association was found in a subset of cases between the allele and an increased spina bifida risk but this result could not be replicated (Botto, 2000; Trembath et al., 1999). Compound heterozygosity for both the C677T and A1298C alleles may increase spina bifida risk compared to the wild-type combinations. Two separate studies have calculated odds ratios of 2.0 (95% CI: 0.9–5.1) and 2.8 (95% CI: 1.1–7.6) for allelic compound heterozygosity in relation to increased risk for spina bifida (Botto, 2000; Trembath et al., 1999; van der Put et al., 1998). 2. Preeclampsia Hyperhomocysteinemia in conjunction with a TT genotype has been associated with an increased risk for spontaneous abortion, placental abruption, and preeclampsia (Ray and Laskin, 1999). It is thought that the elevated levels of Hcy produce placental endovascular damage as a result of oxidative stress. It has been shown that 17% of women suffering with severe preeclampsia were found to have hyperhomocysteinemia compared with a 2% prevalence found in the general population (Dekker et al., 1995; Leeda et al., 1998). Recent studies have not been able to associate a clear relationship between MTHFR polymorphisms and placenta-mediated diseases such as preeclampsia (Els et al., 2000; Laivuori et al., 2000; Thomas Kaiser and Moses, 2000). However, an increased incidence of the TT allele has been reported in women who have experienced abruption placentae, interuterine fetal growth retardation, and still births compared to women who have had normal pregnancies (Kupferminc et al., 1999). These pregnancy outcomes are associated with elevated Hcy and emphasizes the importance of maintaining adequate folate levels periconceptionally, especially in individuals who possess MTHFR polymorphisms that may contribute to increased risk for abnormal pregnancies.

D. MTHFR–environment interactions A number of studies have examined the interaction between nutritional status and the C677T polymorphism in relation to clinical outcomes. Two studies suggest that neural tube risk associated with C677T, TT, homozygosity may be dependent on nutritional status. The first study showed a 13-fold increased risk for spina bifida in C677T, TT, homozygotes with a red blood cell folate value in the lowest study quartile (Christensen et al., 1999). The second found that maternal multivitamin use (containing folic acid) reduced the risk for spina bifida in individuals with wild-type or heterozygous alleles (OR ¼ 0.3) and in those with the homozygous TT

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genotype (OR ¼ 0.2) (Shaw et al., 1998). Studies investigating colorectal cancer showed that the inverse association with TT homozygosity was greatest in those individuals with high folate or methionine intake (Chen et al., 1996; Ma et al., 1997). These results suggest that dietary methyl supply is particularly important among TT individuals. When dietary methyl supply is high, TT individuals may be at reduced risk of colorectal cancer as the higher levels of 5,10-methylenetetrahydrofolate make purines and thymidines available for the nucleotide pool during DNA synthesis and repair. Alcohol consumption was found to remove the reduced risk associated with TT individuals for colorectal cancer (Ma et al., 1997). In fact TT individuals who consumed large volumes of alcohol were at even greater risk than those without the T allele who consumed similar volumes of alcohol (Chen et al., 1996). This would suggest that when 5-methyltetrahydrofolate is depleted by alcohol consumption, TT individuals may be less able to compensate, leading to alterations in methylation patterns that may lead to altered expression of oncogenes.

E. MTHFR–other gene interactions Other genes whose alleles have been studied in combination with MTHFR alleles include transcobalamin, cystathionine-b-synthase, methionine synthase reductase, and methionine synthase. Polymorphisms in the MTHFR and transcobalamin genes (C776G) have been shown to influence Hcy metabolism that may in turn increase the risk for spontaneous abortion. It has been shown that embryos with a combined T677T and transcobalamin C776G or G776G genotype have an odds ratio of 3.8 for spontaneous abortion compared with embryos with only one of these genotypes (Zetterberg et al., 2003). Cystathionine-b-synthase catalyzes the conversion of Hcy to cystathionine, thus providing an alternative route for Hcy metabolism. It has been shown that individuals who have polymorphisms for both the C677T allele and the cystathionine-b-synthase 844ins68 allele are at an increased risk for spina bifida (OR ¼ 5.2; 95% CI: 1.4–21.2). The risk is higher than would be expected if each of the polymorphisms for C677T (OR ¼ 2.1; 95% CI: 1.1–3.9) or 844ins68 (OR ¼ 0.8; 95% CI: 0.4–1.4) were considered individually suggesting a significant gene–gene interaction (Ramsbottom et al., 1997). It has been shown that individuals who are homozygous for polymorphisms within the methionine synthase reductase gene (A66G) and MTHFR C677T appear to have an increased risk for spina bifida (OR ¼ 4.1; 95% CI: 1.0–16.4) (Morrison et al., 1998). Further evidence for potential gene–gene interactions involving MTHFR was shown in individuals that possess both the C677T, T allele, together with the methionine synthase A2756G, G allele. This combination was found to reduce the risk of colorectal cancer and may afford a certain degree of protection (Le Marchand, 2002).

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II. Conclusions The MTHFR C677T polymorphism has been implicated in a number of clinical diseases. Under conditions of low folate, the MTHFR C677T, TT genotype is associated with increased risk for NTDs. Conversely when folate levels are adequate, the TT genotype may reflect a selective advantage for the reduction in risk for both lung and colorectal cancer. Individuals who are TT homozygotes or compound heterozygotes for both the A1298C and C677T alleles may have to pay more attention to adjusting their folate and riboflavin intake in order to reduce genomic instability and the effect of potential clinical outcomes associated with hyperhomocysteinemia.

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