Consequences of dietary methyl donor supplements: Is more always better?

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JPBM1002_proof ■ 2 April 2015 ■ 1/7

Progress in Biophysics and Molecular Biology xxx (2015) 1e7

Contents lists available at ScienceDirect

Progress in Biophysics and Molecular Biology journal homepage: www.elsevier.com/locate/pbiomolbio

Review

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Consequences of dietary methyl donor supplements: Is more always better?

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Kimberly R. Shorter a, Michael R. Felder b, c, Paul B. Vrana b, c, * a

University of Florida School of Medicine, Department of Psychiatry at the McKnight Brain Institute, 1149 Newell Drive, Gainesville, FL 32611, USA University of South Carolina, Department of Biological Sciences, 715 Sumter Street, Columbia, SC 29208, USA c Peromyscus Genetic Stock Center, University of South Carolina, 715 Sumter Street, Columbia, SC 29208, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Available online xxx

Epigenetic mechanisms are now recognized to play roles in disease etiology. Several diseases increasing in frequency are associated with altered DNA methylation. DNA methylation is accomplished through metabolism of methyl donors such as folate, vitamin B12, methionine, betaine (trimethylglycine), and choline. Increased intake of these compounds correlates with decreased neural tube defects, although this mechanism is not well understood. Consumption of these methyl donor pathway components has increased in recent years due to fortification of grains and high supplemental levels of these compounds (e.g. vitamins, energy drinks). Additionally, people with mutations in one of the enzymes that assists in the methyl donor pathway (5-MTHFR) are directed to consume higher amounts of methyl donors to compensate. Recent evidence suggests that high levels of methyl donor intake may also have detrimental effects. Individualized medicine may be necessary to determine the appropriate amounts of methyl donors to be consumed, particularly in women of child bearing age. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Folic acid 5-MTHFR DNA methylation Epigenetics Dietary supplementation

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Key components of the methyl donor pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methyl donor components in supplements and fortified food/beverages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . FA/Methyl donor intake & ASD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Folic acid intake and cancer risk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal studies on effects of altering methyl donor intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Additional human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of variation in the MTHFR gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Epigenetic mechanisms have been shown to play roles in disease etiology. Thus, it is now widely believed that genetic and environmental factors act in tandem via epigenetic mechanisms to

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* Corresponding author. Peromyscus Genetic Stock Center, University of South Carolina, 715 Sumter Street, Columbia, SC 29208, USA. Tel.: þ1 949 400 3148. E-mail address: [email protected] (P.B. Vrana).

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underlie many diseases (Altobelli et al., 2013). Epigenetic modifications are now widely considered the missing heritability in diseases that do not follow traditional Mendelian inheritance patterns (Portela and Esteller, 2010; Robertson and Wolffe, 2000). For example, epigenetic effects such as genomic imprinting cause more complicated pedigrees and thus epidemiology (Handel et al., 2010). Environmental factors altering epigenetic patterns include maternal diet, stress, radiation exposure, infectious agents, and immunological factors (McGowan et al., 2008; Verma, 2003).

http://dx.doi.org/10.1016/j.pbiomolbio.2015.03.007 0079-6107/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Shorter, K.R., et al., Consequences of dietary methyl donor supplements: Is more always better?, Progress in Biophysics and Molecular Biology (2015), http://dx.doi.org/10.1016/j.pbiomolbio.2015.03.007

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K.R. Shorter et al. / Progress in Biophysics and Molecular Biology xxx (2015) 1e7

Dietary effects on epigenetic status have been of particular interest since Barker and others have proposed that low protein levels during human development may predispose to specific diseases later in life (Barker, 1992, 1993; Wadhwa et al. 2009). Folate and related B vitamins contribute to the carbon/methyl donor pathway, critical for addition of epigenetic marks to DNA and proteins, among other functions. Maternal folic acid consumption has been strongly linked with reduction in the frequency of neural tube defects (Honeinm et al., 2001). This association led to the U.S. mandating fortification of grains and cereal in 1998 (Bailey and Gregory, 1999). Folate (as Folic acid, FA) and other methyl-donor pathway components are now added to a variety of consumables. For example, vitamin B12 is a common additive in energy drinks, and betaine/trimethylglycine (TMG) is used to reduce homocysteine levels (Wang et al., 2013). Products in this pathway are marketed as health products and for their ability to promote methylation (e.g. http://www.olaloa.com/ola-loa-and-the-healing-power-ofmethylation). Several diseases have increased in frequency within this postfortification time frame, leading to speculation that increased methylation may contribute to their etiology (Barua et al., 2014a; Hollingsworth et al., 2008; Kim, 2007; Van den Veyver, 2002). These diseases include cancers, neurological disorders, growth syndromes, respiratory disorders, and multiple sclerosis (Dominguez-Salas et al., 2012; Portela and Esteller, 2010; Schaevitz and Berger-Sweeney, 2012; Schanen, 2006; Sharp et al., 2013; Skinner, 2011). In particular, autism spectrum disorders (ASD) have increased from 1 in 250 in 2000 to 1 in 68 in 2013 (Schenkelberg et al., 2014). A possible connection between this increase and increased intake of methyl-donor pathway components is being hotly debated (Neggers, 2014; Schaevitz and Berger-Sweeney, 2012). Dietary methyl-donors were first shown to have epigenetic effects in classic animal studies which showed that high maternal intake can silence retroelement induced mutations at the agouti Avy allele and the similar AxinFu allele (Cooney et al., 2002; Waterland et al., 2006a; Waterland and Jirtle, 2003; Wolff et al., 1998). The diet used in these studies has also been shown to reduce the effects of de-methylating agents such as BPA (Dolinoy et al., 2007) and mitigate some effects of a high fat diet (Cordero et al., 2014). In addition, reduced amounts of dietary methyl donors have been shown to have negative effects (Altobelli et al., 2013). Several studies have suggested more broad effects of these supplements on the methylome. In addition, folic acid fortification has been associated with an increase in DNA methyltransferase (DNMT) activity (Ding et al., 2012). Consequently, a growing number of recent studies suggest deleterious effects of developmental exposure to high doses of methyl-donor pathway components (Barua et al., 2014a, 2014b; Mikael et al., 2013; Shorter et al., 2014; Vasquez et al., 2013). Alterations of methyl donors in the maternal diet have been associated with altered methylation at several genes and aberrant behavior phenotypes (Barua et al., 2014a). In this review, we focus on the broad potential effects of altered methyl donor levels in both maternal and post-weaning diets in animal and human studies, with specific discussion of some ASD relevant data. Notably, we spend little space on deficiencies of these molecules. We suggest genetic variation (both known and novel alleles) will ultimately be seen as a major factor in recommendations for dietary intake of these molecules. 2. Key components of the methyl donor pathway Dietary supplements that increase the level of methyl donor pathway components include FA, cobalamin (Vitamin B12), choline,

betaine/trimethylglycine (TMG), and L-methionine. Methionine metabolism is regulated by nutrient intake of B12, pyridoxine (Vitamin B6), and riboflavin (Vitamin B2) since these B vitamins are cofactors for hormones that regulate methionine metabolism (Kalhan and Marczewski, 2012). Methionine is the precursor molecule to S-adenosylmethionine (SAM) which actively donates a methyl group to more than 60 products including DNA, RNA, and histones (Hollenbeck, 2012). Choline is oxidized to betaine aldehyde by betaine aldehyde dehydrogenase, which is a niacin (Vitamin B3) dependent enzyme (Hollenbeck, 2012). Synthesis of betaine/trimethylglycine from choline is an irreversible process in humans. TMG, FA, and B12 are important coenzymes for conversion of homocysteine to methionine. The folate metabolism pathway leads to purine synthesis and synthesis of 5-methyltetrahydrofolate (5-MTHF) using the enzyme 5-methylenetetrahydrofolate reductase (5-MTHFR) and a cofactor Vitamin B2 (Fig. 1). Methionine is then produced with assistance from Vitamin B12 and methionine synthase (MS). Methionine is converted to S-adenosylmethionine (SAM), the methyl donor molecule. SAM donates a methyl group while methyltransferase enzymes add the methyl group to DNA, RNA, proteins, and lipids. 3. Methyl donor components in supplements and fortified food/beverages Fortification of grain products with FA began in the United States and other countries in the 1990s, as FA consumption was correlated with decreased neural tube defects (NTDs) (Berry et al., 1999; CDC, s, 1992; Honeinm et al., 2001; Godwin et al., Q2 1991; Czeizel and Duda 2008; Vanhees et al., 2014) and a decrease in low birthweights (Timmermans et al., 2009). While spina bifida and ostium secundum atrial septal defects were both decreased post fortification, there were increases in urinary tract obstructive defects and increased pyloric stenosis (Godwin et al., 2008). Folate/FA deficiency is linked to anemia, atherosclerosis, NTDs, adverse pregnancy outcomes, psychiatric disorders, and cancers (Bailey et al., 2003; Brito et al., 2012; Giovannucci, 2002; Reynolds, 2014), but FA intervention trials in humans are inconsistent and are not completely supportive of protective effects of FA supplementation except in the case of NTDs (Bønaa et al., 2006; Clarke et al., 2010; Lonn et al., 2006). Therefore, it has been questioned whether extra folic acid through food fortification is really beneficial to the majority of the population (Smith et al., 2008). Foods were fortified in the United States beginning in 1996 after the FDA approved fortification of grains at a dose of 140 ug FA/100 g of food to place approximately 100 ug FA more into the average adult diet (Table 1) (Hoyo et al., 2011a). Trials around the world have documented increased serum folate concentrations after foods were fortified with FA (Johansson et al., 2002; Neuhouser et al., 1998; O'Keefe et al., 1995; Tucker et al., 2004) or after supplementation with FA (Brouwer et al., 1999; Hao et al., 2008; Houghton et al., 2011; Hursthouse et al., 2011; Neuhouser et al., 1998; Venn et al., 2002). Notably, FA added to foods during fortification is 70e85% bioavailable (as folate) compared to only 50% bioavailability of FA/folate naturally occurring in foods (Hoyo et al., 2011a; Quinlivan and Gregory, 2007; Winkels et al., 2007). Note that it is commonly assumed that the maternal and fetal genotypes respond similarly to the recommended dosages. FA supplements and FA fortified foods are recommended to women who may become pregnant (Hoyo et al., 2011a). Therefore, if a mother consumes both supplements and fortified foods during pregnancy, it is possible both mother and fetus may be exposed to amounts of FA that exceed the recommended tolerable upper limit of 1000 ug/day for adult pregnant women (Hoyo et al., 2011a). Diets


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Fig. 1. Methyl Donor Metabolic Pathway. Folic acid (folate) is metabolized eventually to methionine, which is then converted to S-adenosylmethionine (SAM), the methyl donor molecule. SAM donates a methyl group to DNA, RNA, proteins, and lipids. Vitamins B2 and B12 are necessary in this process. Methylenetetrahydrofolate reductase (MTHFR) is largely responsible for this process as well, and mutations in the gene coding for this enzyme are linked to mild hyperhomocysteinemia as homocysteine can also be converted to methionine.

exceeding the recommended FA daily limit have been reported in women of child-bearing age (Lewis et al., 1999; Quinlivan and Gregory, 2003). Ten percent of women report daily FA supplementation that exceeds the tolerable upper limit before or during pregnancy, and 5 percent report exceeding the tolerable upper limit both before and during pregnancy (Hoyo et al., 2011a). 4. FA/Methyl donor intake & ASD An epidemiological study has correlated ingestion of prenatal vitamins with FA concentrations at or above the tolerable upper limit with increased autism/ASD risk (Beard et al., 2011). These findings are controversial, as another study has shown significantly lower plasma methionine and folate in mothers of children with ASDs when compared to control subjects (i.e. mothers of neurotypical children; James et al., 2010). Another recent study found no association between maternal whole blood folate levels and autistic behavior in their children (Braun et al., 2014). However, a number of links have been made between ASD and aberrant DNA methylation. Notably, Nguyen et al. (2010) found hypermethylation in CpG islands of 73 genes in autistic individuals which had monozygotic twins discordant for ASD. Other studies

have also found such associations in ASD related loci. These include FMR1 (associated with Fragile X syndrome) and MECP2 (associated with Rett Syndrome, psychiatric disorders) (Grafodatskaya et al., 2010; Nagarajan et al., 2006). Junaid et al. (2011) found that high FA supplementation led aberrant expression of FMR1 in an animal model. Several other regions in the human genome known to be regulated by DNA methylation harbor ASD susceptibility loci. These include the imprinted domains on 15q (PradereWilli, Angelman Syndromes) and 7q. RELN (associated with schizophrenia) is a nonimprinted ASD candidate gene regulated by DNA methylation (Chen et al., 2002; Grayson et al., 2006; Li et al., 2004). Interestingly, the MECP2 gene (aberrantly methylated in some ASD patients) is known to bind the 15q region on the maternally methylated allele to halt transcription (Yasui et al., 2011). GABRB1, whose disruption is implicated in ASD, is expressed in a concentration dependent manner in mouse neurons treated with FA (Vasquez et al., 2013). 5. Folic acid intake and cancer risk As noted, links between folic acid and cancer have largely been considered in terms of FA deficiency. However, a potential

Table 1 Amounts of folic acid and/or B12 in supplements, energy drinks, and foods. High amounts of folic acid are available in supplement form equaling the upper tolerable limit of 1000ug per day. Amounts of B12 are available in very high quantities in supplements, some reaching over 40,000% of the recommended daily intake. Energy drinks and foods supplemented with folic acid and/or B12 provide additional amounts of these nutrients in a more bioavailable form.


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connection has recently been suggested between excess FA consumption and increases in the rate of colon cancer (Mason et al., 2007; Cole et al. 2007). However, direct evidence of causality is still lacking (Mehta and Shike, 2014). To date, variation at loci involved in folate uptake and distribution does not appear linked with colon cancer risk (Figueiredo et al., 2010). Evidence for similar FA-tumor links has been found in prostate cancer (Figueiredo et al., 2009; Tomaszewski et al., 2011). A recent study examined the potential effects of excess (~2.5) FA on toxin-inducted mammary tumor growth in a rat model of breast cancer (Deghan Manshadi et al., 2014). These data indicated that increased FA both promoted greater tumor weight and progression. Thus, an emerging consensus appears to be that FA likely aids in cancer prevention in normal tissue, but promotes progression of pre-cancerous cells/tumors (Ulrich and Potter, 2007). 6. Animal studies on effects of altering methyl donor intake There is now no question varying intake of 1-carbon metabolism nutrients affects epigenetic status of genes in the offspring (Burdge et al., 2012). FA supplementation and grain fortification have resulted in decreased NTDs, but the impact on non-NTD changes are not thoroughly studied (Godwin et al., 2008). There has been an implicit hypothesis that the epigenetic changes induced by greater amounts of these nutrients are uniformly beneficial across genotypes. We present here methyl donor supplementation models that suggest there may be deleterious effects associated with high intake of methyl donor pathway components. Perhaps the best known studies are those that examined the effects of a methyl donor diet on Agouti expression. Viable yellow agouti (Avy) mice have an IAP endogenous retrovirus-like transposable element in the agouti gene (Waterland and Jirtle, 2003; Wolff et al., 1998). DNA methylation and IAP activation regulate IAP expression. Dietary supplementation of a/a dams with FA, B12, choline and betaine altered phenotype of Avy offspring by increased methylation of CpGs at the Avy locus (Cooney et al., 2002; Waterland and Jirtle, 2003). The murine metastable epiallele known as AxinFu has been found to exhibit a similar epigenetic plasticity to the methyl donor diet. Tail kinking was decreased in offspring of mothers given the same periconceptional methyl donor diet as in the Avy studies (Waterland et al., 2006a). A similar study was done in Peromyscus maniculatus (deer mice, native North American rodents). Wide-band agouti (ANb) Peromyscus naturally overexpress agouti, with no evidence of an IAP insertion as in Mus Avy. Using the same periconceptional and postweaning methyl donor diet as in Mus agouti studies (MS diet e moderately supplemented), coat color changes were seen (Shorter et al., 2014, 2012). Repetitive behavior significantly increased in methyl diet female offspring while weight was significantly higher in methyl diet female offspring as well (Shorter et al., 2014). Defects of cataracts, ovarian cysts, for example, were also seen (Shorter et al., 2014). In C57BL/6 mice, a 10 FA diet was given as a periconceptional diet and as a maintenance diet post-weaning. Newborn pup cerebral hemispheres had down-regulation of 4 genes and upregulation of 5 genes, one of which was the X chromosome inactivation locus Xist. Pups had increased ultrasonic vocalizations, higher anxiety-like behaviors, and hyperactivity (Barua et al., 2014a). In an additional C57BL/6 study using the same diet, cerebral hemispheres of offspring had widespread changes in methylation patterns of genes involved in development. Some of these genes are imprinted while others are autism candidate susceptibility genes (Barua et al., 2014b). Offspring of mothers given a synthetic methyl donor deficient diet had loss of imprinting at the Igf2 locus (Waterland et al., 2006b). Meanwhile, a separate study in

Mus examined the effects of FA supplementation on five NTD knockouts. FA supplementation resulted in exacerbated NTDs in some knockouts while it improved in others (Marean et al., 2011). Sperm of male mice fed a low folate diet had differential methylation of genes associated with development, cancer, diabetes, autism and schizophrenia (Lambrot et al., 2013). Global hypomethylation occurs in brains of offspring of mothers with micronutrient imbalances in FA and B12 (Sable et al., 2013). Low maternal choline also leads to DNA hypomethylation and an increase in expression of genes involved in cell cycle progression and early cell differentiation (Niculescu et al., 2006). Maternal supplementation with FA induced prolonged colitis in offspring associated with mucosal epigenetic changes as methylation of some genes was decreased in offspring of FA supplemented mothers (Johnson and Belshaw, 2014; Schaible et al., 2011). A 10 FA supplementation in Wistar rats induced a reduction in body weight and altered glucose response (Cho et al., 2013). The same 10 FA supplementation in rats before and during gestation led to offspring with a 42% decrease in seizure threshold while in vitro, acute FA treatment in neurons induced hyper-excitability and cell bursting (Girotto et al., 2013). Rats fed a maternal high FA and low B12 diet had pups with lower mRNA levels of two key enzymes in the methyl donor metabolic pathway, MTHFR and methionine synthase (Khot et al., 2014). Mother rats with an imbalance in B12 have pups with reduced antioxidant enzymes at birth, while brain oxidative stress was higher in pups of mothers given a high FA and low B12 diet (Roy et al., 2014). Pups of rat mothers given a high FA diet were at increased risk for mammary adenocarcinomas if kept on the FA diet postweaning, while maternal FA supplementation was linked to accelerated development of the same mammary tumors in offspring (Ly et al., 2011). Maternal, but not postweaning FA supplementation significantly decreased global DNA methylation while postweaning, but not maternal FA, significantly decreased DNA methyltransferase activity in non-neoplastic mammary glands of the offspring (Ly et al., 2011). 7. Additional human studies Maternal FA supplement use after 12 weeks of gestation in humans is associated with higher methylation of the imprinted IGF2 locus and reduced methylation of PEG3 and LINE-1 (Haggarty et al., 2013). IGF2 had increased methylation at its own DMRs with maternal FA consumption of around 400ug per day, which is associated with lower birthweight (lower birthweight is associated with increased chronic diseases later in life) (Steegers-Theunissen et al., 2009; Vanhees et al., 2014). Methylation at the H19 imprinting control region (ICR) decreased in umbilical cord blood leukocytes with increased FA consumption, especially in male infants (Hoyo et al., 2011b). Increased B12 serum during pregnancy was associated with a decrease in global DNA methylation in newborns (McKay et al., 2012). Offspring Low B12 and high folate levels are more prone to insulin resistance, higher body fat percentages, and higher abdominal fat, which puts offspring at risk for type 2 diabetes (Yajnik et al., 2008). High FA supplementation during early pregnancy has been correlated with enhanced vocabulary development, communication skills, and verbal comprehension (Chatzi et al., 2012). However, FA supplementation later in pregnancy has been correlated with childhood asthma (Hollingsworth et al., 2008; Sharland et al., 2011). 8. Effects of variation in the MTHFR gene Apart from a polymorphism in the reduced folate carrier (RFC1) gene (James et al. 2010), studies of human genetic variation


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relevant to the methyl donor cycle has largely focused on the methylenetetrahydrofolate reductase (MTHFR) locus. Low blood FA concentrations are associated with a low intake of FA and in persons homozygous for the C667T variant of MTHFR (Anderson et al., 2013). MTHFR catalyzes the reduction of 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, a process which transfers a methyl group to cobalamin which then transfers the methyl group to methionine (Anderson et al., 2013). Therefore, people with a C to T mutation in MTHFR can have mild hyper€m et al., 1998). Blood FA concentrations homocysteinemia (Brattstro improve with modest doses of FA supplements; larger responses to 400ug FA supplementation were seen in people homozygous for the T variant of MTHFR (Anderson et al., 2013). The MTHFR C667T and A1298C alleles are both associated with increased risk of congenital heart disease (Zidan et al., 2013). Men homozygous for the MTHFR C667T allele were given 300 mg choline/day in addition to a supplement of choline (total of 550 mg/day or 1100 mg/day) for 12 weeks, and men with the mutant genotype had higher TMG:choline ratios in their urine and plasma than control subjects, which favors choline as a methyl donor for persons with the mutant MTHFR genotype (Yan et al., 2011). An important question is whether these MTHFR mutations affect DNA methylation upon supplementation with FA. Pregnant female mice with a mild MTHFR deficiency were fed a 10 FA diet. At E14.5, offspring of FA supplemented MTHFR þ/þ and MTHFR þ/ mothers had embryonic loss, embryonic delay, higher incidence of ventricular septal defects, and thinner left and right ventricular heart walls (Mikael et al., 2013). There was no difference in effects of FA supplementation between offspring of wild type and heterozygous MTHFR mothers (Mikael et al., 2013). Persons homozygous for the T polymorphism had lower DNA methylation and higher homocysteine in peripheral lymphocytes while there was an inverse correlation between DNA methylation and RBC FA levels (Stern et al., 2000). Additionally, leukocytes are lower in DNA methylation in persons with the MTHFR TT genotype; DNA hypomethylation in leukocytes is a characteristic of some cancers (Stern et al., 2000). Early studies of human MTHFR variants in relation to cancer are inconclusive (Nazki et al., 2014). Results to date have suggested that higher FA is associated with lower survival rates in women with ovarian cancer while the MTHFR SNP is associated with a better survival rate (Dixon et al., 2014). This oddly suggests that higher levels of homocysteine may help survival in ovarian cancer, although other studies indicate there is no link between MTHFR SNPs, FA consumption, and ovarian cancer (Li et al., 2013). Gastric cancer risk in patients homozygous for the MTHFR mutation is twofold higher, and patients who consumed 310ug FA per day or more had more protective effects against the cancer while the risk for getting gastric cancer was modified as well (Gao et al., 2013). Finally, the MTHFR C677T allele is associated with increased risk of ASD in countries without FA grain fortification (Pu et al., 2013).

9. Future directions The Western diet, which often includes high sugars and fats, can also interact with FA dosage as high-fat fed offspring of high FA dams were more susceptible to obesity, glucose intolerance, and insulin resistance (Huang et al., 2014). Our own unpublished data indicates body fat effects in Peromyscus raised on the methylsupplemented diet. Therefore, fortification and supplementation may lead to unintentional changes in DNA (and histone) methylation which could lead to disease in offspring. Environmental factors such as diet even have the potential to change the germline to

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induce transgenerational effects (Skinner, 2011; Skinner et al., 2013). Individualized medicine may be an appropriate approach to determining appropriate dosages of FA and other methyl donors. We suggest other variation at many other loci besides those already considered (e.g. MTHFR, RFC1) will also be shown to have effects. As individualized genotyping programs (e.g. as offered by companies such as “23 and Me”) become more readily available to patients, such an approach seems inevitable). Determining the genotype of in utero offspring will be beneficial as well, as fetal genotype may have significantly different sensitivities than the maternal genotype. Timing of FA supplementation (both in utero and postpartum) should be taken into account as well, as studies show the window of exposure affects the effects that FA supplementation can have on offspring, with negative effects being more prominent in later-in-pregnancy supplementation.

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Enhancing informed decision making: is more information always better?

Why more is not always better: new Pap smear guidelines.

Is easier always better?

Is thinner always better?

fMRI exploration of pedagogical benefits of repeated testing: when more is not always better.

Asthma, allergy, and responses to methyl donor supplements and nutrients.

Is More Always Better for Verbs? Semantic Richness Effects and Verb Meaning.

pelvic pain: Is more always better?

When more is better.

Biventricular pacing: more is better!

Once you're choosing, nobody's perfect: is more information necessarily better in oocyte donor selection?

Change isn't always better.

Bigger isn't always better.

Dietary Methyl Donor Depletion Suppresses Intestinal Adenoma Development.

Large diameter femoral heads: is bigger always better?

If one is good, are two always better?

Hazards of dietary supplements.

More is not always better: modeling the effects of elastic exoskeleton compliance on underlying ankle muscle-tendon dynamics.

New renal guidelines; is more better?

Akt as a target for cancer therapy: more is not always better (lessons from studies in mice).

Cancer Immunotherapy: More Is (Much) Better.

Recurrent Cervical Cancer: Can We Agree That More Is Not Always Better?

Paediatric palliative care: There is always more we can do.

Less is not always more: embracing (appropriate) medical intensity.