MTHFR Mutations, Cardiovascular Disease, and Riboflavin (B2): Scientists Zone in on a Neglected Ménage à Trois

You can test your MTHFR gene either directly or by plugging your raw data from 23andme, or another provider into evaluation tools such as Genetic Genie.

If you have no idea what #MTHFR means, here's the Reader's Digest version: MTHFR is an enzyme that is affected by a mutation in the MTHFR gene. The latter encodes the enzyme methylene-tetrahydrofolate reductase aka MTHFR effectively unless there's a single-nucleotide polymorphism (SNP) affecting the 677th base pair of the MTHFR gene... not helping?

Well, let's just say if you have a certain variation of this gene, you're having a hard time processing B-vitamins; and it's not totally unlikely that you're affected: According to Marini et al. 2008, this mutation affects 29% of the global population.

Previous studies, however, report much lower estimates for prevalence of the MTHFR 677TT genotype, i.e. 10% worldwide, with values ranging from 4 to 18% in the United States, over 20% in Northern China to up to 32% in Mexico (Wilcken 2003 | see Figure 1).

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Based on observational studies, we've known for quite some time that there is a link between having the TT-allele of MTHFR 677 (henceforth only "MTHFR") and cardiovascular disease. Studies quantify the risk increase for hypertension, a major driver of cardiovascular disease, at 24–87% and report CVD risk increases of up to 40%. In view of the large geographical variation in the extent of excess disease risk, scientists have speculated that there may be gene-environment interaction way before the MTHFR mutation was identified. As soon as its effect on the metabolism of folic acid and the importance of the latter in the methylation of homocysteine (Hcy) was clear, it appeared obvious that the previously specified risk increase was a result of accumulating Hcy levels in the in the blood of people who have TT allele of the MTHFR gene. This theory, however, may now have to be revised or at least expanded in view of recent research.

Figure 1: Prevalence of homozygous TT genotype (two 677C>T alleles) among 7130 newborns of different ethnicities from 16 areas in Europe, Asia, the Americas, the Middle East, and Australia (Wilcken 2003) - Note: Others estimate the global prevalence at almost 30% (Marini 2008).

As McNulty et al. report in their 2017 paper in "Molecular Aspects of Medicine", there's "[e]merging evidence [...] that the relevant environmental factor may be riboflavin". The "yellow" vitamin (B2) you pee out in large quantities if you're still willing to pay for crazily high dosed super-vitamins, is an MTHFR co-factor with a previously largely ignored "genotype-specific effect on blood pressure" (McNulty 2017). And in fact, randomized trials in hypertensive patients (with and without overt CVD) "show that targeted riboflavin supplementation in homozygous individuals (MTHFR 677TT genotype) lowers systolic blood pressure by 6 to 13 mmHg, independently of the effect of antihypertensive drugs" (McNulty 2017) - an effect that could reduce the risk of stroke, infarction and co.

Subgroup analysis for the association between MTHFR C677T genotype frequencies and the risks of NAFLD.

The TT-allele is not the only problematic mutation of the MTHFR gene: Studies like Sun et al. 2016 show that the having the CC-allele (albeit at a different position of the MTHFR gene) is similarly problematic - not in terms of CVD, though, but with respect to your risk of developing non-alcoholic fatty liver disease. In their meta-analysis, the researchers from the Tianjin Union Medicine Center & Tianjin People’s Hospital in Tianjin, China, were able to show that both the TT (~50% increased risk) and the CC genotype (50-180% increased risk depending on the model, the scientists used) of the MTHFR C677T and MTHFR A1298C, respectively, "are more likely to be associated with the susceptibility to NAFLD" (Sun 2016).

The debate about MTHFR on the internet has, just as the discourse in the scientific community, focused almost exclusively on the effects of the genetic mutation on folate/folic acid metabolism. I guess that's because the impaired folic acid metabolism in people who carry the TT-allele of the MTHFR gene increases the risk of neural tube and other birth defects in unborn babies - this and the association of having the TT allele with increased homocysteine (Hcy) levels (0.4-8.8µmol/L) and significantly reduced effects of folic acid supplementation on Hcy levels (Lewis 2005; Fezeu 2018). As previously hinted at, there's yet "no strong evidence [...] to support an association of the MTHFR 677 C→T polymorphism and coronary heart disease in Europe, North America, or Australia" - in other words, the potential increase in homocysteine in TT-allele carriers is not just smaller than previously thought it can, in contrast to what the interwebs may say,  also be ameliorated w/ folic acid supplements | Lewis 2005). Contemporary research does yet suggest that "lowering homocysteine concentrations, through supplementation with folic acid" cannot as of now be linked to significantly reduced risks of "heart disease" (Lewis 2005).

Let's briefly forget the folic acid (B9) <> homocysteine theory of heart disease and turn to the more recent revelations about the modulatory effect riboflavin (B2) seem to play:

With the technological advances in gene-testing and interest in gene-environment interactions, there's an increasing number of studies investigating the role of the genetically determined MTHFR activity in cardiovascular disease - and, as of late, its interaction with riboflavin intake and status. The latest set of studies has been presented at the conference of the Nutrition Society in June 2018 and here are a few sneak peaks on what the scientists found.

• MTHFR & endothelial function: Rooney, et al. (2018) show for the first time that individuals with the MTHFR TT genotype have poorer endothelial function compared to their age-matched CC genotype counterparts.
  

  

  Table 1: Differences between groups were assessed by ANOVA; values within a row with different superscript letters are significantly different, by Tuckey post-hoc test. Abbreviations: AIx, augmentation index; DBP, diastolic blood pressure; PWV, pulse wave velocity; SBP. Systolic BP (Rooney 2018).

  More specifically, they observed in healthy individuals, aged 18–60 years, from Northern Ireland, who were screened for MTHFR genotype, that systolic BP is markedly higher(~9%) in participants with the TT genotype, compared to CC and CT genotypes, with a similar, albeit non-significant, trend for diastolic BP. In conjunction with the significantly elevated pulse wave velocity in individuals with the TT compared to CC genotype, this indicates "poorer endothelial compliance in this genetically at-risk group" (Rooney 2018).
  
  What the scientists could not tell at the conference (yet) is whether the increase in blood pressure is modified by the subjects riboflavin intake as it was observed in the studies summarized in the previously cited paper by McNulty et al. (2017).
• MTHFR & gestational hypertension: O'Sullivan et al. (2018) conducted the subset analysis that's (as of yet) lacking for the data from the previously discussed study by Rooney et al. (2018). They found based on data from the ongoing Optimal Nutrition for the Prevention of Hypertension (OptiPREG) project that "women with the TT genotype and low riboflavin status undergo a markedly greater increase in systolic BP from the 14th to the 36th GW". A similar trend was also seen in diastolic BP, but did not reach statistical significance.
  

  

  Figure 2: Changes in systolic and diastolic blood pressure from gestational week 14 to 36 according to riboflavin status of the pregnant subjects | plotted based on data in O'Sullivan 2018.

  If you look at the actual values I've plotted for you in Figure 2 the effect is not as large as one may expect based on the phrase "markedly greater increases", but the important mediating effect of riboflavin intake (high vs. low) is obvious... and, even though this may not be significant - it looks as if it would help everyone, i.e. independent of the MTHFR gene variant.
  
  At this point, it is obviously important to conduct a randomized controlled trial to investigate the effect of riboflavin supplementation on pregnant women's blood pressure levels - needless to say that this RCT is already "underway at [the researchers'] centre with results from the trial expected in early 2019" (O'Sullivan 2018).
• MTHFR & blood pressure in normal people: With Hughes' recent study, the follow-up to the previously discussed study does already exist. However, it was conducted in 18-65-year-old non-pregnant adults, and not mothers-to-be O'Sullivan et al. are interested in. In their study, Hughes et al. (2018) assigned n = 81 adult subjects with the TT-allele of the MTHFR gene randomly (but stratified by baseline systolic BP) to two groups: the B2 group in which subjects received 10 mg/day riboflavin (that's "only" 8x the RDA and much less than I've seen in many multivitamin products) and the placebo (PLA) group which supplemented with indistinguishable placebo pills, both for 16 weeks.
  
  Primary outcomes of the study were the biomarker status of riboflavin, which was measured using the erythrocyte glutathione reductase activation coefficient (EGRac) assay (Graham 2005), and, obviously the subjects' blood pressure (BP), which was measured in form of both clinic BP and ambulatory BP (the latter gives a much better picture of the 'real' blood pressure of an individual and this is the first study to evaluate in the B2 <> blood pressure context)- in accordance with NICE guidelines.
  
  The results are interesting and cannot be summarized solely as "B2 worked" or "B2 failed". That's mostly because the blood pressure response to riboflavin (i.e. whether there was a reduction or not) was "found to be strongly dependent on baseline BP" (Hughes 2018).

  • 

    Table 2: Response to intervention analyzed by repeated measures ANCOVA, adjusting for sex. † Higher EGRac values are indicative of lower riboflavin status. ≠ mean of participant daytime/awake hrs which was personalized for each participant (Hughes 2018).

    Participants with a baseline systolic BP of less than 125 mmHg showed no response to riboflavin supplementation (data not shown).
  • In participants with a baseline systolic BP ≥125 mmHg, B2 supplementation resulted in a significant BP lowering of daytime systolic BP by 3·8 mmHg vs. 0·2 mmHg in the placebo group (see Table 2).
  Is that bad news? No, quite the opposite is the case. A systolic blood pressure of <125mm/Hg is absolutely within the no-danger zone (learn more about optimal blood pressure levels in "Pre-hypertension ain't benign").
  
  But is the effect even relevant? With only -3.8 mmHg, the effect may seem to be irrelevant. You have to keep in mind, however, that it was observed in a healthy study population, not in patients with hypertension, and lowering an already optimal systolic BP at e.g. 110 mmHg by another 10-15 mmHg could actually make the subjects pass out. Accordingly, it seems to be more worthwhile to ignore the effect size for the time being and start speculating about the reasons why the provision of 8-9 times (for men and women respectively) the RDA of B2 failed to increase the riboflavin status of the subjects (see Table 2). I mean, the subjects in the supplement group did, in fact, end up with a lower B2 status than the placebo group.
  
  Before the scientists hope comes true and "these findings [...] offer a personalised approach for BP management in [...] at risk sub-populations", there's thus IMHO still a lot of work to do.
• MTHFR & Messed Up Methylation: The additional studies I asked for towards the end of the discussion of the Hughes study could also deal with the epigenetic effects of riboflavin, as studies like Amenyah et al. 2018 which examine the global and MTHFR gene DNA methylation response to riboflavin supplementation may explain many of the questions we haven't answered yet.
  
  In the corresponding paper, Amenyah and colleagues from the Ulster University point out that, while the "mechanism linking this gene-nutrient [MTHFR <> B2] interaction is currently unknown", it is possible if not likely that it "involve[s] aberrant DNA methylation, which has been implicated in hypertension" (Amenyah 2018) before. To confirm the importance of this interaction and investigate the methylation response, the scientists analyzed blood samples of 120 subjects who had previously participated in a 16-week B2 supplementation trial (1.6mg/d ~1xRDA).  While the scientists found changes in gene-methylation, most importantly a reduction in MTHFR north shore methylation in TT genotype participants, it's not clear how this or the concomitant reduction in LINE-1 methylation are related to the blood pressure lowering effects of riboflavin in genetically at-risk adults. It is possible, though, that the slight changes in MTHFR methylation partly restore its function in TT-allele carriers and thus contribute to significant improvements in their ability to effectively process folate and other B-vitamins - an improvement that could have significant downstream effects on CVD.

Choline, which has become a deficiency nutrient since choline-rich foods like eggs have been demonized is necessary another co-factor in homocysteine metabolism. But that's by no means the only reason why you should keep an eye on your choline intake | learn more in my 2014 article "Choline Deficiency, Its Consequences"

MTHFR is a factor to consider... even beyond heart disease: Heart disease is the #1 killer in the US. According to the CDC, 610,000 people die of heart disease in the United States every year–that's 1 in every 4 deaths. It is, however, not yet clear how many of the 610,000 victims of this serial killer carry the TT-allele of the MTHFR gene. What is clear is that those who belong to the (as of now) elusive group of "MTHFR mutants" are at an increased risk of heart disease - an increase of which the studies discussed in this article suggest that it may be modified by one's riboflavin intake.

The emerging theory, which could provide an alternative to the old "homocysteine-folate"- theory scientists used to refer to when trying to explain increased CVD risks in carriers of MTHFR TT-allele. As of late, this link has yet been increasingly scrutinized.

In fact, many scientists are convinced "that HCY is a marker, rather than a cause, of CVD" (Wierzbicki 2007, my emphasis). Hence, its elevation in MTHFR-mutants would not be causally, but corollary related to heart disease. That's in contrast to hypertension, which is so intricately linked to major cardiovascular disease that an MTHFR TT-allele carrier who reduces his systolic blood pressure by 13 mmHg (the upper range of improvements in the meta-analysis by McNulty et al. (2017)) would experience a significant 20-30% decrease in overall cardiovascular disease risk and a 10-25% decrease in his risk of dying from any form of cardiovascular disease (Bundy 2017).

These figures make the previously discussed ménage à trois (MTHFR mutation <> CVD <> riboflavin) all the more interesting. What we should not forget, though, is that increases in CVD risk are by no means the only side effects of MTHFR mutations: Migraines (Scher 2006; Liu 2014), autism (Rai 2016), colorectal cancer (Chen 1999; Shiao 2017), breast cancer (Sharp 2002; Shrubsole 2004; Zhang 2017), lung cancer (Chen 2015) and a plethora of other, non-communicable diseases have at least initially evidence suggesting that the MTFHR mutants among us are at significantly increased risk of contracting them | Comment!

References:

• Amenyah, S.D., et al. “Epigenetic Effects of Riboflavin Supplementation on Hypertension in Adults Screened for the MTHFR C677 T Polymorphism.” Proceedings of the Nutrition Society, vol. 77, no. OCE3, 2018, p. E62., doi:10.1017/S0029665118000666.
• Bundy, Joshua D., et al. "Systolic blood pressure reduction and risk of cardiovascular disease and mortality: a systematic review and network meta-analysis." JAMA cardiology 2.7 (2017): 775-781.
• Chen, Jia, Edward L. Giovannucci, and David J. Hunter. "MTHFR polymorphism, methyl-replete diets and the risk of colorectal carcinoma and adenoma among US men and women: an example of gene-environment interactions in colorectal tumorigenesis." The Journal of nutrition 129.2 (1999): 560S-564S.
• Chen, Hsiao-Ling, et al. "Abstract A47: Meta-analysis of MTHFR polymorphisms in lung cancer: Population health and mutations in the world." (2015): A47-A47.
• Fezeu, Leopold K., et al. "MTHFR 677C→ T genotype modulates the effect of a 5-year supplementation with B-vitamins on homocysteine concentration: The SU. FOL. OM3 randomized controlled trial." PloS one 13.5 (2018): e0193352.
• Graham, Joanne M., et al. "Erythrocyte riboflavin for the detection of riboflavin deficiency in pregnant Nepali women." Clinical chemistry 51.11 (2005): 2162-2165.
• Haerian, Monir Sadat, et al. "MTRR rs1801394 and its interaction with MTHFR rs1801133 in colorectal cancer: a case–control study and meta-analysis." Pharmacogenomics 18.11 (2017): 1075-1084.
• Hughes, C.F., et al. “A Randomised Controlled Trial to Investigate Ambulatory Blood Pressure Response to Riboflavin Supplementation in Adults with the MTHFR 677TT Genotype.” Proceedings of the Nutrition Society, vol. 77, no. OCE3, 2018, p. E56., doi:10.1017/S0029665118000605.
• Lewis, Sarah J., Shah Ebrahim, and George Davey Smith. "Meta-analysis of MTHFR 677C→ T polymorphism and coronary heart disease: does totality of evidence support causal role for homocysteine and preventive potential of folate?." Bmj 331.7524 (2005): 1053.
• Liu, Ruozhuo, et al. "MTHFR C677T polymorphism and migraine risk: a meta-analysis." Journal of the neurological sciences 336.1-2 (2014): 68-73.
• Marini, Nicholas J., et al. "The prevalence of folate-remedial MTHFR enzyme variants in humans." Proceedings of the National Academy of Sciences (2008).
• Mason, Joel B. "Folate status and colorectal cancer risk: a 2016 update." Molecular aspects of medicine 53 (2017): 73-79.
• McNulty, Helene, et al. "Riboflavin, MTHFR genotype and blood pressure: a personalized approach to prevention and treatment of hypertension." Molecular aspects of medicine 53 (2017): 2-9.
• National Institute for Health and Care Excellence (2011) Hypertension in adults: diagnosis and management nice.org.uk/guidance/cg127.
• O'Sullivan, E., et al. “MTHFR Genotype and It's Interaction with Riboflavin in Relation to Blood Pressure Increase during Normal Pregnancy; Preliminary Findings from the OptiPREG Project.” Proceedings of the Nutrition Society, vol. 77, no. OCE3, 2018, p. E58., doi:10.1017/S0029665118000629.
• Rai, Vandana. "Association of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with autism: evidence of genetic susceptibility." Metabolic brain disease 31.4 (2016): 727-735.
• Rooney, M., et al. “B-Vitamins, Blood Pressure and Endothelial Compliance in Healthy Adults Stratified by MTHFR Genotype.” Proceedings of the Nutrition Society, vol. 77, no. OCE3, 2018, p. E61., doi:10.1017/S0029665118000654.
• Scher, Ann I., et al. "Migraine and MTHFR C677T genotype in a population‐based sample." Annals of Neurology: Official Journal of the American Neurological Association and the Child Neurology Society 59.2 (2006): 372-375.
• Sharp, L., et al. "Folate and breast cancer: the role of polymorphisms in methylenetetrahydrofolate reductase (MTHFR)." Cancer letters 181.1 (2002): 65-71.
• Shiao, S. Pamela K., Amanda Lie, and Chong Ho Yu. "Meta-analysis of homocysteine-related factors on the risk of colorectal cancer." Oncotarget 9.39 (2018): 25681.
• Shrubsole, Martha J., et al. "MTHFR polymorphisms, dietary folate intake, and breast cancer risk: results from the Shanghai Breast Cancer Study." Cancer Epidemiology and Prevention Biomarkers 13.2 (2004): 190-196.
• Sun, Man-Yi, et al. "Associations between methylenetetrahydrofolate reductase (MTHFR) polymorphisms and non-alcoholic fatty liver disease (NAFLD) risk: a meta-analysis." PloS one 11.4 (2016): e0154337.
• Wilcken, B., et al. "Geographical and ethnic variation of the 677C> T allele of 5, 10 methylenetetrahydrofolate reductase (MTHFR): findings from over 7000 newborns from 16 areas world wide." Journal of medical genetics 40.8 (2003): 619-625.
• Wierzbicki, Anthony S. "Homocysteine and cardiovascular disease: a review of the evidence." Diabetes and Vascular Disease Research 4.2 (2007): 143-149.
• Zhang, Yun, et al. "Cumulative review and meta-analyses on the association between MTHFR rs1801133 polymorphism and breast cancer risk: a pooled analysis of 83 studies with 74,019 participants." (2017): 57-73.