Methylation is a term that is thrown around a lot in the functional medicine world. Heart disease, autism, Alzheimer’s and many other diseases have been linked to methylation problems. When most bloggers and functional doctors talk about methylation, they mean the process of the methylation cycle.
Methylation is so fundamental to every cell, that it is estimated to take place more than a billion times per second. Yep, it’s that important.
If you just want to know what to do, I highly recommend Chris Masterjohn's Start Here for MTHFR and Methylation post. It will give you some basic background and best strategies to address methylation problems.
Why methylation is important for ApoE4s
- Carrying both MTHFR and ApoE4 variants may increase your risk of AD. This presentation reported, "In our study we confirmed a high prevalence of ε4 alelle of ApoE among patients with AD and a higher prevalence of polymorphisms of MTHFR in homozygous state in AD patients in comparison with controls. The highest risk for developing AD had carriers of ApoE4 with TT genotype of MTHFR." (Sutovsky, S., et al., 2017) (Note: This link is to abstracts to conference presentations, so you have to hunt for the author.) Also see this studies for MTHFR and your risk. (Stoccoro, A., et al., 2017, Peng Q, et al., 2015)
- Methylation gene variants can lead to high homocysteine. When there are problems in the methylation cycle (from various genetic variants), homocysteine can run high. High homocysteine is correlated with risk of AD, and might be one of the factors in the link of MTHFR and APOE4 in increasing risk. High homocysteine is also found in other diseases besides AD, including coronary heart disease and osteoporosis.
- Get your B vitamin levels checked. Even if you are in the low end of the range for folate and B12, consider some low level of supplementation. Active or methylated forms are more suitable for those with MTHFR and other genetic variants (see also the section on folate and folic acid). For more details on how to supplement, check out the section below on Hacking Genetic Variants.
- Lower high homocysteine levels. This is critical because high homocysteine is often found in AD patients. Cause or effect? Still a bit unknown, but it can be driven down with supplementation. See Hacking Homocysteine for more details.
- If you have other chronic illnesses, consider working with a functional medicine doctor. Methylation is complicated but so important to so many processes in the body. If it isn't working right, it can cause a host of other health problems.
- Reduce your stress levels. Stress is just toxic on so many levels.
A deeper dive into the science: Methylation Cycle (aka One Carbon Cycle)
The methylation cycle’s main function is to convert methionine from the diet (eggs, meat, fish, brazil nuts and some seeds) to SAM (S-adenosylmethionine, also known as AdoMet, SAM-e). SAM is the primary active methyl donor in the body. If some process needs a methyl group, SAM is the man. Most SAM is produced and used in the liver.
This cycle supplies methyl groups, consisting of one carbon and three hydrogens (aka CH3), for a large number of methylation reactions, including those that methylate (and turn “off”) DNA, and processes involved in creating a wide variety of substances, including creatine, choline, carnitine, coenzyme Q-10, melatonin and myelin basic protein. Methylation is also used to metabolize dopamine, norepinephrine and epinephrine, to inactivate histamine, and to methylate phospholipids, promoting the integrity of and transmission of signals through cell membranes.
One study found 208 proteins that make up the human “methyltransferasome,” or those proteins that act as enzymes that transfer a methyl group from SAM. Those proteins represent only about 0.9% of all human gene products. However, 30% of those enzymes that transfer a methyl group, called methyltransferases, are associated with disease, most frequently cancer and mental disorders. These methytransferases include several well-known to the APOE4 forum including MTHFR, COMT, PEMT, HNMT and BHMT. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3013446/)
- S-Adenosyl-L-methionine (SAMe): from the bench to the bedside—molecular basis of a pleiotrophic molecule
- Nutrition and epigenetics: An interplay of dietary methyl donors, one-carbon metabolism, and DNA methylation
Once a methyl group transfer takes place, you are left with SAH (S-adenocyl homocysteine). The body is pretty efficient at recycling, so SAH gets recycled by an enzyme to create homocysteine and adenosine. From there, homocysteine is recycled back to methionine through transfer of a methyl group from the active form of folate, specifically 5-methyltetrahydrofolate (also known as 5-MTHF, L-methylfolate and other names). This is accomplished by one of the two paths.
- The first path uses the MTR gene and requires B12.
- The second path, found in the liver and kidney, is encoded by the BHMT gene, which transfers a methyl group to homocysteine from betaine (also known as trimethylglycine or TMG). Zinc is a cofactor.
In either path, methionine is then converted back to SAM, completing the cycle.
Now the body has other ways to keep this important cycle working well. When methylation is compromised, plasma homocysteine levels rise, increasing oxidative stress and the rate at which glutathione stores are depleted. If high homocysteine becomes chronic, BHMT and MTR enzymes are down-regulated and homocysteine is sent down the transsulfuration pathway. In this pathway, homocysteine is irreversibly converted to cystathionine by an enzyme created by the CBS gene, which requires the cofactor vitamin B6. The resulting byproduct, cysteine can be utilized in a number of cellular functions, including glutathione (GSH) production and protein synthesis. But in high homocysteine, with it’s increase in oxidative stress, the body focuses on increasing glutathione to battle the oxidative stress.
So the dance between these various pathways is a delicate one, where lots of things can go awry. Instead of typing a lot more on how methylation works, this picture is worth more than a thousand words.
PLEASE DO NOT SHARE THIS IMAGE OUTSIDE OF THE APOE.INFO SITE, AS IT IS COPYRIGHTED. I purchased the Seeking Health Planner kit, and provide this image as allowed under educational use.
Why do we have different pathways to the same end?
Some hints can be found in the following article.
“Dietary intakes of riboflavin, folate, choline, and betaine varied significantly by season; the most dramatic variation was seen for betaine. All metabolic biomarkers showed significant seasonality, and vitamin B-6 and folate had the highest fluctuations. Correlations between dietary intakes and blood biomarkers were found for riboflavin, vitamin B-6, active vitamin B-12 (holotranscobalamin), and betaine. We observed a seasonal switch between the betaine and folate pathways and a probable limiting role of riboflavin in these processes and a higher SAM/SAH ratio during the rainy season.” (Dominguez-Salas, et al., 2013)
Yes, methylation is a complicated system
So, it appears we evolved multiple methylation pathways in response to the variability of our food sources. Such a strategy ensures that we would have enough methyl groups to take care of business of everyday life. When you look at the diagram, you see that there are multiple feedback loops that help the body produce the right amounts of SAM. And yes, there are several co-factors needed by the enzymes to do their jobs, and some byproducts of every reaction, some good, some not so good.
Taking a systems view of the body, we see that the relationships between all of the parts of the body (e.g. liver, kidney and cells), and the processes (e.g. methylation) that take place within them are not isolated; they are very much inter-related. The methylation pathway is tightly regulated by intracellular levels of metabolites and cofactors. Push on one area of the methylation cycle, and some other effect takes place inside the cycle, both downstream and upstream of the action. The body tries it’s best to maintain some type of equilibrium. And sometimes that leads to side effects as various compensating pathways come into play.
So, for example, too much SAM puts the brakes on MTHFR (which converts folate to it’s active form) and it up-regulates CBS to flush homocysteine out the cycle, since the body has enough of what it needs. It’s just the body’s way to say, hey, we have enough so we need to open the floodgate to let some excess out.
But in low folate intake, the body aims to preserve the ability to make the DNA bases purines and pyrimidines (via the DNMT enzyme), and a side effect is a reduction in SAM production and the related process of recycling homocysteine. Call it greedy, but body has prioritized DNA work over other needs.
But it’s not just about diet. Downstream uses of methyl groups also come into play with regards to our health. Chronic viral loads can use up methyl groups, because methylation is used to suppress the transcription of some viral genomes, and is also needed for maintaining that suppression.
Side note for anyone wanting to know more about viral loads and methylation - ‘CpG’ is shorthand for the occurrence of a cytosine linked, through a phosphate bond, to a guanine. The immune system responds to un-methylated CpGs in DNA as a sign of foreign pathogen invasion and releases the hounds (dendritic cells, monocytes and B cells). Some viruses, like Epstein Barr, use a methylated form to escape detection by the immune system. For more detail on methylation and viruses and how some, like herpes simplex can reactivate, take a stab at the science dense, but useful http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2396429/.
Histamine production is an interesting one. Too little methylation flares histamine reactions, because the HNMT enzyme needs methyl groups to break down intracellular histamine. Too many methyl donors can increase methyl-histamine production in cells, which then requires downstream enzymes MAO, DAO, ADH to break it down. So, any genetic variants downstream can also cause an excess of histamine. It’s that whole intricate relationship of the balance of processes and responding to too little or too much of something.
And the last example is the effects on neurotransmitters. Too little SAM means less serotonin, epinephrine, and dopamine, and is often linked to depression. Too much SAM, and you get “wired” with side effects like headache, overstimulation, anxiety and insomnia.
With methylation there certainly is a Goldilocks effect.
A side note on Folates and Folic Acid
You’ll read on this site and other blogs about the importance of using “active” or methylated versions of folate. So what’s the difference between taking folic acid and methylfolate. We get folate from foods, and folic acid from enriched foods and supplements, while some brands of supplements provide an active methylfolate form. The term “folic acid” is used loosely, and when reading articles or research, know that the term can mean folic acid, folinic acid or methylfolate, which are all very different forms along the pathway to an activated form of folate.
Dietary folates are absorbed in the proximal jejunum and enter the enterohepatic (gut-liver) circulation bound to albumin. Because serum folate levels reflect very recent dietary ingestion but not body stores, a normal serum level may not reflect cellular deficiency. And the cells are where the action are. That’s why your doctor will typically order an RBC, or red blood cell, folate test. In the cells, dietary folates are in their inactive form until enzymes, with the help of vitamin B12, remove and keeps a methyl group to create the active form of folate. This step also activates vitamin B12 for its different uses in the body. Once folates and vitamin B12 are active, they are available for all things methylation.
What causes the methylation cycle to go awry?
Lack of folate and co-factors in the diet
As you can see from the above diagram, besides folate, you need a lot of co-factors like B12, zinc and choline to keep the methylation train on the tracks. Of course, a myriad of problems, including GI health, can be the source of low availability of co-factors. Certainly more than what I can cover in this primer. But here’s a quick introduction of micronutrient deficiency and it’s impact on disease.
And note, some studies suggest that the lack of B-vitamins is more important than the genetic contributions in hyperhomocysteinemia (high homocysteine).
“The genetic contribution to the variance in tHcy was estimated to be approximately 9%, compared with approximately 35% that could be attributed to low folate and vitamin B(12). Our study indicates that dietary factors are centrally important in the control of tHcy levels in young adults with additional, but somewhat weaker, genetic effects.” (Leo A. J. Kluijtmans, et al., 2003)
Other studies suggest that the lack of several nutrients combines with genes and environment to cause disease.
“Herein, we have highlighted data in which altered consumption of folate, choline, betaine, B vitamins, and methionine acts to modify methylation both globally and in the promoters of disease-related genes in animal and humans. Thus, nutri-epigenetics approaches provide a molecular foundation for understanding the role of diet throughout the life course and its prospective role in disease prevention and/or therapy.” (Olivia S. Anderson, et al., 2012)
This gene is very well studied regarding its effects on health. MTHFR can refer to either the gene or the enzyme (methylenetetrahydrofolate reductase) and it’s job is in the pathway to convert dietary or supplemented folic acid to a form that can be used in the methylation cycle to recycle homocysteine and create SAM.
Two of the most common genetic polymorphisms of MTHFR are C677T (rs1801133, T is the risk allele) and A1298C (rs1801131, C is the risk allele). In the 677C>T homozygous variant, there is an estimated 50-70% reduction in the conversion of folate to it’s active form. It’s quite frequent in the population - some 31-39% of the North American Caucasian population carries at least one T, and the homozygote (TT) frequency is 9-17%.
MTHFR C677T has been implicated in many diseases. Here’s a sampler. The MTHFR 677C>T variant leads to elevated serum tHcy and cholesterol levels; heart disease is more common; risks for diabetes, insulin resistance, inflammatory bowel disease and stroke increase; neural tube defects occur more often, especially in males; MTHFR levels are only 40-50% of normal in autistic children with the T allele; increased recurrent pregnancy loss and preeclampsia; spina bifida and cleft palate occur at higher rates; increased risk of certain cancers; and low folate levels common with the TT variant are associated higher allergy rates in children. With the MTHFR 1298A>C, the C allele is not relevant for heart disease risk but appears to be related to autism, pediatric stroke, and schizophrenia.
Just go to PubMed and put in MTHFR and your favorite disease! In reality, most of these diseases relate directly to low RBC folate levels, so, even with MTHFR variants, adequate folate intake through diet and supplements can help level the playing field.
The 1298A>C mutation is less well studied, but we do know that the combined heterozygosity for the 1298(one C) and 677(one T) mutations is associated with reduced MTHFR enzyme activity, higher homocysteine, and decreased plasma folate levels. The combined heterozygosity for both MTHFR mutations results in similar features as observed in homozygotes for the 677C>T mutation. In addition, studies do show that wild type AA Caucasian carriers have significantly lower plasma folate. A study of Chinese hypertensive patients showed patients with the MTHFR 1298AC or CC genotypes had a higher serum folate level than those with the wild-type genotype. (For more info see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555142/, http://www.ncbi.nlm.nih.gov/pubmed/19283448)
- Snpedia C677T
- Snpedia A1298C
- NIH MTHFR Gene
- A 2018 meta-analysis that demonstrates that the MTHFR rs1801133 polymorphism is associated with increased risk of CAD and elevated levels of TC and LDL-C.
Related to MTHFR: MTHFD1
The MTHFD1 gene encodes methylenetetrahydrofolate dehydrogenase 1 and converts dietary folates into the form needed by MTHFR. With MTHFD1 1958G>A (or rs2236225), the A variant reduces the production of the precursors for MTHFR to create active folate. Deficiencies in MTHFD1 may also reduce the synthesis of phosphatidylcholine and/or increase the demand for choline as a source of methyl donors, causing overall choline deficiency.
- Genetic variants in phosphatidylethanolamine N-methyltransferase (PEMT) and methylenetetrahydrofolate dehydrogenase (MTHFD1) influence biomarkers of choline metabolism when folate intake is restricted
- Genetic variation of folate-mediated one-carbon transfer pathway predicts susceptibility to choline deficiency in humans
The MTR gene encodes methionine synthase, which is an important enzyme for one pathway that converts homocysteine back to methionine. MTR A2756G (or rs1805087) transfers a methyl group from methyl folate to B12 (cobalamin) to activate the enzyme and the methyl-B12 is used to transfer a methyl group to homocysteine to create methionine. There are mixed results in studies on which variant is bad. GeneticGenie says G is the risk allele, but A is listed as bad for homocysteine in the study below. I’ve added the researchers’ comments on why there might be conflict between studies.
“For HCY, adjusted mean concentrations differed among the MTR (p < 0.01) and the MTHFR genotypes (p = 0.04) with higher levels of HCY in those with the MTR ‘AA’ or the MTHFR ‘TT’ genotypes.
Our results and those of previous investigations of the MTR–HCY relationship suggest the possibility that the MTR A2756G polymorphism may actually increase enzyme activity which would enhance the conversion of HCY to methionine, leading to lower levels of HCY. However, the biochemical effects of MTR A2756G polymorphism have not been examined in vitro due to the inability to express human MTR at sufficient levels in an active form (Harmon et al. 1999). Twenty-five studies have investigated the MTR–HCY association in healthy non-pregnant populations…However, of the 25 studies, only 10 were conducted among a study population of greater than 100 participants with similar age distribution and ethnic background (Ma et al. 1999; Klerk et al. 2003; Chen et al. 2001; Jacques et al. 2003; Tsai et al. 2009; Harmon et al. 1999; D’Angelo et al. 2000; Fillon-Emery et al. 2004; Geisel et al. 2001; Summers et al. 2008); two of the 10 comparable studies support the association found in this research (Chen et al. 2001; Harmon et al. 1999). In particular, sample size is of concern due to the low prevalence of the MTR polymorphism, which would limit the power of a study to detect an association if one truly exists; as well, it is unknown whether the association between MTR and HCY concentrations differs by age. In the literature, it is postulated that genetic factors may have more impact on HCY concentration among youths and that cumulative environmental factors may be more important in modifying HCY as individuals reach middle age (Kluijtmans et al. 2003). Finally, important lifestyle, environmental or additional genetic factors that may interact with MTR polymorphism could differ in their distributions between ethnic groups and thus limit comparison of results obtained across different ethnic populations.” (Vikki Ho, et al., 2013)
When looking at folate status, studies show that AG or GG variants can cause lower folate levels. And the risk is compounded with each addition of MTHFR C677T, MTHFR A1298C, and MTRR A66G variants. (Wen-Xing Li, et al., 2015)
The MTRR gene encodes methyltransferase reductase. This enzyme helps recycle the B12 leftover from the MTR conversion to return it to a methylated form using SAM. So the B12 is ready for another call of duty when MTR converts homocysteine to methionine. This is another gene where the risk allele seems to depend on the disease.
There are several MTRR snps, but MTRR A66G (or rs1801394) is associated with late onset Alzheimer’s Disease (LOAD), where G is the risk allele. (See Fabio Coppedè, et al., 2011)
As far as homocysteine levels and heart disease, MTRR A66G was found to be the culprit in this study. But in this instance, A is the risk allele. “The frequency of MTRR A allele was higher in CAD patients than in controls (0.48 [95% CI: 0.44-0.52] vs 0.38 [95% CI: 0.32-0.44], P = 0.0081) while no difference was observed for MTHFR 677T frequency. In multivariate analysis, t-Hcys > median and MTRRAA genotype were two significant independent predictors of CAD with respective odds ratios of 3.1 (95 % CI: 1.8-5.1, P < 0.0001) and 4.5 (95% CI: 1.5-13.1, P = 0.0051). In conclusion, in contrast to North Europe studies, MTRRAA genotype is a genetic determinant of moderate hyperhomocysteinemia associated with CAD in a French population without vitamin fortification.” (Rosa-Maria Guéant-Rodriguez, et al., 2005)
The fucosyltransferase 2 (FUT2 gene) encodes a membrane protein that is involved in vitamin B12 absorption. For the common snp rs601338, the G allele is associated with lower B12 levels. The A allele of the SNP rs492602 is associated with lower vitamin B12 concentrations (− 0.06 pg/ml) among Caucasians from the US. The A allele of the SNP rs492602 is associated with lower vitamin B12 concentrations (− 0.06 pg/ml) among Caucasians from the US. The most commonly studied variant of the FUT2 gene is the SNP rs602662. The A allele carriers were at a lower risk of vitamin B12 deficiency in a population of Canadian adults.
“For the FUT2 gene, seven SNPs including rs281379, rs492602, rs516316, rs601338, rs602662, rs838133 and rs1047781 were previously reported to be associated with vitamin B12 levels [12, 18–22, 27–29]. To identify loci associated with plasma vitamin B12, a meta-analysis of three genome-wide association scans (n = 4763) was carried out in a Caucasian population residing in the USA . The SNP rs601338, also known as 428 G/A nonsecretor variant allele (W143X variant), was significantly associated with plasma vitamin B12 levels (P = 6.92 × 10−15), with the allele ‘A’ being positively associated with plasma vitamin B12 levels (β = 0.06 pg/ml) . This finding was further confirmed in another study looking at 37,283 Icelandic adults (P = 2.40 × 10−95, β = 0.162 pmol/l) , as well as in two Indian populations of children (β = 0.18–0.25 pmol/l) . Notably, the minor allele frequency (MAF) of rs601338 varies widely between ethnicities, contributing to genetic heteroegeneity in FUT2-B12 associations.” (S. Surendran, et al., 2018)
NBPF3 and ALPL Genes
The snp rs4654748 on NBPF3 influences the clearance of Vitamin B6. Research has found approximately 2.9 ng/ml lower plasma concentrations of the active form pyridoxal 5′-phosphate, (PLP), in CC carriers and 1.45 ng/ml less in CT carriers. The lower vitamin B6 in C allele carriers most likely results from more efficient clearance of the vitamin. The G allele of snp rs1256335 on ALPL also produces lower plasma PLP levels as compared with the homozygous A allele, possibly due to increased hydrolysis of PLP. If you carry either snp and show lower levels of B6 (PLP), you may require higher intakes of B6.
- Genome-wide Association Study of Vitamin B6, Vitamin B12, Folate, and Homocysteine Blood Concentrations (2009)
Choline is an essential micronutrient. As a source of methyl groups, choline supports cellular methylation reactions, including genomic methylation, which influences gene expression and DNA stability. Choline is also used to create the neurotransmitter acetylcholine. But the primary use for choline is in the synthesis of phosphatidylcholine (PC), the most abundant phospholipid in cell membranes.
Choline contributes to PC synthesis through two distinct pathways. Either choline can be phosphorylated, entering the cytidine diphosphate (CDP)-choline pathway where it is converted directly to PC, or, its methyl groups can be used in the triple-methylation of phosphatidylethanolamine (PE) to PC, which occurs by the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. This pathway is also responsible for endogenous choline production as PC can be produced through the PEMT pathway using PE and folate or methionine-derived methyl groups in the absence of sufficient dietary choline.
Part of the second homocysteine recycling path, BHMT found in the liver and kidney, and the enzyme transfers a methyl group to homocysteine from betaine (also known as trimethylglycine or TMG). It is responsible for about 50% of homocysteine remethylation. Zinc is a cofactor.
“Betaine homocysteine methyltransferase (BHMT) is a cytosolic zinc metalloenzyme that is highly expressed in the liver, kidney and lens of the eye [1, 2]. BHMT catalyzes the transfer of a methyl group from betaine to homocysteine, resulting in the generation of dimethylglycine and methionine. Homocyteine remethylation is a critical step in the synthesis of S-adenosylmethionine (AdoMet), the methyl donor for most methylation reactions . A methionine-deficient diet induces BHMT expression and increases its activity in mice , while excess AdoMet can down regulate BHMT expression through an NFkB-dependent mechanism . By competing with transsulfuration, BHMT-catalyzed remethylation of homocysteine helps to maintain homocysteine homeostasis.” (Qiping Feng, et al., 2011)
GeneticGenie says the ones to check are BHMT-02 (rs567754, T is the risk allele), BHMT-04 (rs617219, C is the risk allele) and BHMT-08 (rs651852, T is the risk allele). I could not find references for rs617219.
Here’s what we know about the other two, along with some snps not mentioned in GeneticGenie. Please note, with the exception of rs651852, there is currently not a lot of research on these other BHMT variants and how to treat them.
- rs6860725 is directly associated with homocysteine levels (T is the risk allele)
- rs651852 is associated with folate and B12 levels (A is the risk allele)
- rs567754 is associated with B6 status (T is the risk allele)
But we also have this set of SNPs with reduced activity, from a study that used adult hepatic biopsy samples to measure protein and enzyme levels: rs41272270, rs16876512, rs6875201, rs7700790. (Qiping Feng, et al., 2012)
And also the following studies:
- About rs3733890, they note “One unit increase in this SNP would increase the value of log(nHcy) by 0.084 units in these participants (both men and women) with hetero type or variant genotypes.” A is the risk allele. (Andrew J. Clifford, et al., 2012)
- With regards to BHMT rs3733890 (c.716 G > A, also Known as c.742 G > A; p.Arg239Gln), "these results indicate that the variant favors the use of dietary choline for CDP-PC synthesis at the expense of betaine synthesis." (Ariel B. Ganz, et al., 2017)
Phosphatidylethanolamine N-methyltransferase (PEMT) is responsible for the synthesis of phosphatidylcholine (PC) which supplies structural integrity to cell membranes, used to create acetylcholine in the brain and used by the liver to secrete triglycerides into very low density lipoproteins (VLDL). The T allele of rs7946 is associated with low PC production.
The CBS enzyme, cystathionine β-synthase, represents the start of the third homocysteine pathway, called trassulfuration, for homocysteine. Once homocysteine goes down this pathway, it is not reversible - no recycling of homocysteine occurs. CBS variants cause the enzyme to not be as effective in eliminating homocysteine via the transsulfuration pathway. CBS variants are certainly something to consider where homocysteine stays high after other interventions, or you have thrombosis or a family history of thrombosis.
Read also: http://www.omim.org/entry/613381 for list of diseases and background
The CBS variant listed in this next study is c.833T>C (aka I278T, rs5742905, C is the risk allele). Their recommendation is to test CBS if homocysteine levels are very high. SNPedia says this variant is vitamin B6 responsive (https://www.snpedia.com/index.php/Rs5742905)
"Among the 413 eligible patients, 184 (45%) patients agreed to participate in the present follow-up study. A MTHFR(3)c.677TT genotype was found in 49% of the patients. Eight patients were found to have mutations in CBS(2). Of those, two were homozygous for c.833T>C (p.I278T), and four were compound heterozygous for c.833T>C. One c.833T>C (p.I278T) compound heterozygote was identified by lowering the threshold for sequencing from tHcy at 100μM to 50μM. The most prominent clinical presentation among patients with a CBS(2) mutation was thrombosis presenting at a median age of 25years. In case of arterial or venous thrombosis without any explanation in individuals below 40years, tHcy should be part of the thrombophilia screening. When tHcy is between 50 and 100μM genotyping for the MTHFR(3) c.677TT is relevant, and when tHcy >100μM CBS should be genotyped." (Jannie Tanderup Sørensen, et al., 2016)
Two additional variants have been uncovered that also impact homocysteine: rs234709 and rs2851391. (Joyce BJ van Meurs, et al., 2013)
Also note that higher selenium levels are seen with CBS variants, because selenium metabolism happens as a result of processes downstream of CBS. If CBS is not doing its job, the selenium pathway will not have what it needs. (Marilyn C. Cornelis, et al., 2015)
We all know that chronic stress is not good. Stress leads to epigenetic changes that alter gene expression by affecting DNA methylation patterns.
- Genome-wide DNA methylation levels and altered cortisol stress reactivity following childhood trauma in humans
And here’s one last sobering thought - stress not only affects our DNA methylation, but impacts future offspring.
- DNA methylation mediates the effect of exposure to prenatal maternal stress on cytokine production in children at age 13½ years: Project Ice Storm.
- DNA methylation mediates the impact of exposure to prenatal maternal stress on BMI and central adiposity in children at age 13½ years: Project Ice Storm.
A Special Note about Alzheimer’s Disease
Some studies show that DNA methylation levels are lower (called hypomethylation), than in comparison with normal individuals. They see aberrant methylation in neurons, reactive astrocytes, and microglia, and in neurons from control entorhinal cortex layer II, and hippocampus. With hypomethylation, genes like PSEN1 overexpress (i.e. turn on) and that leads to increased Aβ production. There are also studies of cell and mouse models that show withholding B vitamins increased PSEN1 hypomethylation (the lack of methyl groups which essentially turns PSEN1 on) and adding SAM reversed it (turning it off). (http://www.ncbi.nlm.nih.gov/pubmed/19329227/, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2766770/)
The jury is out on this because other studies, using different assay methods found mixed or opposite results, that methylation was not involved in PSEN1 expression. (http://www.ncbi.nlm.nih.gov/pubmed/16040194)
But researchers do know that higher plasma homocysteine is an independent AD risk factor and occurs prior to the pathogenesis of AD. Going back to the diagram, we see that homocysteine accumulation leads to increases to SAH, which is a strong DNMT inhibitor leading to DNA hypomethylation.
Since researchers see an elevation in the homocysteine level preceding the onset of dementia, studies imply that global DNA hypomethylation could be a cause of AD.
“Several hypotheses have been formulated to explain the increased AD risk associated with high serum Hcy levels and low serum folate. For istance, folate deficiency fosters a decline in SAM, decreasing DNA methylation during aging and AD [14,120]. Folate deficiency and resultant SAM depletion lead to increased levels of Hcy, which in turn potentiate Aβ peptide toxicity . Hcy is a critical branch point metabolite that can influence cellular levels of SAM and SAH, which regulate the activity of methyltransferases during DNA methylation and posttranslational modification of proteins . Studies in rodents showed that Hcy accumulation reduces cellular levels of SAM, stimulates glutamate excitotoxicity and increases oxidative damage . Hcy has been also associated to vascular disease in AD, with attention focused on vascular changes related to AD as a consequence of Aβ peptide toxicity and its deposition . Several studies suggest a correlation between plasma Hcy concentrations and plasma Aβ levels [129,130]. Moreover, there is indication that elevated Hcy causes tau hyperphosphorilation, NFT formation and SP formation via inhibition of methyltransferases and reduced methylation of protein phosphatase 2A [131,132]. However, one of the most exciting hypothesis linking one-carbon metabolism to AD risk suggests that impaired folate/Hcy metabolism and subsequent reduction of SAM levels might result in epigenetic modifications of the promoters of AD-related genes leading to increased Aβ peptide production [133,134].” (Coppedè, 2010)
Besides homocysteine, aging is generally considered to be one of the highest risk factors for AD. Studies have shown a strong correlation between DNA methylation dysregulation and aging in AD patients and LOAD brains. LOAD may represent an extreme form of normal aging where methylation changes effect the epigenetic variability of genes, leading to some tipping point that results in brain malfunctions and symptoms of AD.
For more on this topic of methylation and AD, see:
- Oxidative DNA damage and level of thiols as related to polymorphisms of MTHFR, MTR, MTHFD1 in Alzheimer's and Parkinson's diseases
Hacking Nutritional Support for Methylation
Here are some of the important ones that you can get through diet and, for some, you might need add some supplementation.
- For folate, eat uncooked green vegetables, and for you beef liver lovers, it’s the top source. Folate requires active transport across the intestinal mucosa, so if you have IBS or other gut problems, you might not be absorbing enough.
- Vitamin B12 (aka cobalamin) is the yin to folate’s yang - both are required at a critical point in the methylation cycle. If you’re a vegetarian, you will need to supplement regardless of genetic variants because it is found in highest quantities in animal products. Some supplements use cyanocobalamin, but if you have the MTRR genetic variant, you might want to consider methylcobalamin if your blood levels stay low after supplementing.
- Vitamin B6 in it’s coenzyme form is involved in more than 100 enzyme reactions, mostly concerned with protein metabolism. Pyridoxal 5' phosphate (PLP) and pyridoxamine 5' phosphate (PMP) are the active coenzyme forms of vitamin B6. PLP is involved in the metabolism of one-carbon units, carbohydrates, and lipids. Tuna is the best source, followed by spinach, cabbage and bok choy. Go easy on the cooking since prolonged heat can degrade B6. Inflammation can lead to low B6 levels.
- Mind your zinc levels. I could do a whole separate post on the role of zinc in immune health! Oysters, red meat, sesame and pumpkin seeds and lentils rank highest as food sources, but phytates bind zinc and will limit it’s absorption from grains, legumes, nuts and seeds. Another reason to soak them before using. GI disease, diabetes, and liver disease can decrease zinc absorption.
- Choline is not a vitamin, but considered an essential nutrient. Humans only synthesize a small amount, so we all need to bump our dietary or supplement intake. If you don't consume or supplement enough folate, the body draws on choline to recycle homocysteine, reducing the amount that can be used to produce phosphatidylcholine, and essential to keep your cell membranes fluid and functional. Shrimp, eggs and scallops top the list of food sources. If supplementing, the best type of choline is up for debate. You can search APOE4.info for those discussions. Citicoline is recommended by Dr. Bredesen for the ReCODE protocol.
- Consider using a nutrition tracker, like Cron-o-meter, for a short while to see if you are getting enough micronutrients. They can be tedious to use, but eye-opening. And make sure to test your blood levels and see if you are within range before deciding to supplement. In addition, prescription drugs, other supplements and aging can impact your ability to absorb and utilize vitamins. If you’re looking for specific foods that can boost specific nutrients, I like to use http://www.whfoods.com/.
- And lastly, if you go back to the large Seeking Health diagram, you can look for other co-factors that could potentially be tweaked.
Hacking Genetic Variants
Supplementation might be needed with some of these genetic variants. Work with your doctor to get the appropriate labs done, and always start with low doses and work up to a level that works for your target range. For that reason, I do not recommend amounts because they should be based on your individual needs, except for a comment on where to start with methylfolate, mostly driven by personal experience. Some doctors may suggest higher doses, so I'm suggesting a starting point.
- Start any B supplementation regime with methyl-B12 first, especially if your labs show low B12 status. This avoids a potential “methylfolate trap” where methylfolate can’t get converted due to lack of B12. For more on that, see Y. M. Smulders et al., 2006.
- Use a methylated version of folate. This allows you to bypass the less efficient process of converting dietary folates to their active form. [Golja, et al]
Too much methylfolate is a problem, too, so start low (400 mcg/day or less), and go up slowly. Side effects of too much methylfolate include irritability, insomnia, anxiety, headaches, migraines, palpitations, sore muscles and achy joints. If you feel any of these symptoms after taking folate, try taking 50-100 mg of niacin as nicotinic acid. Niacin is broken down by SAM, so it’s a good way to use up excess SAM (from excess folate).
It is also important to use methylfolate with MTHFR variants because there is competition between “active” folate and “inactive” folic acid. So, it’s best to avoid vitamins or processed food products with folic acid.
- Avoid Nitrous Oxide gas at the dentist. This study shows that patients with a homozygous MTHFR 677C>T or 1298A>C mutation are at a higher risk of developing abnormal plasma homocysteine concentrations after nitrous oxide anesthesia. (Peter Nagele, et al., 2008)
- Avoid Methotrexate. The analysis highlighted a significant association of 677C>T polymorphism with overall MTX toxicity, hepatotoxicity, hematological toxicity, and neurotoxicity. It also revealed an association with MTX toxicity in patients with rheumatoid arthritis. (Kalliopi P Spyridopoulou, et al., 2012)
- Avoid Lamisil. "It was later noted that Lamisil's mechanism of action interferes with cells' methylation cycle, which we suspect compromises cellular function in people with the methylenetetrahydrofolate reductase genetic mutation." (Trachtman and Pagano, 2015)
- Check for hypothyroidism. In hypothyroid conditions, the hepatic activity of MTHFR is decreased. (Friemann and S Schmitz, 1992)
Other Genetic Hacks
With the remaining genetic variants, I have not found many direct studies testing specific treatments. So, if you want to treat any of the following, here’s a starting point and feel free to check the Seeking Health diagram for other substrates and co-factors and go from there. N=1!
If you also carry PEMT variants along with MTHFD1, supplementing folate will help control homocysteine and ensure sufficient creation of PC. (Alexandre Ivanov, et al., 2009)
MTR or FUT2
Test your folate, B12 and zinc levels. Supplement as needed to get within range.
Additionally, if you have FUT2 variants, they can inhibit uptake of vitamin B12 from the intestine. With these variants, sublingual B12 might work better to bypass intestinal absorption.
Test your folate levels, because this one needs SAM, a product of folate metabolism. You can also look at riboflavin, because it is a precursor to FAD (flavin adenine dinucleotide). See the riboflavin entry below. NAD (nicotinamide adenine dinucleotide) is synthesized from niacin and tryptophan.
NBPF3 and ALPL
If you carry either snp listed above and show lower levels of B6 (PLP), you may require higher intakes of B6.
Adequate choline is important, but if it’s not enough, try adding trimethylglycine (TMG, also known as betaine, not to be confused with betaine HCL). TMG is known to cause upset stomaches, so take it with food. Start low, as low as 250mg/day and titer up.
This might also be affected by choline gene variants. For more information, see Common genetic polymorphisms affect the human requirement for the nutrient choline. (Kerry-Ann da Costa, et al, 2006).
Increase dietary choline intake. See Masterjohn’s choline article for a choline calculator to determine your needs, and a choline database for dietary sources and amounts to eat. PC can also be supplemented directly, which will spare methyl groups (and therefore folate), for other uses.
CHDH - a deeper dive into choline variants
So, your BHMT might be just fine, but you have to make sure you are converting choline to TMG. So, also take a look at CHDH, which converts choline to TMG. https://commons.wikimedia.org/w/index.php?curid=20265140
- First up is rs9001, where A is the risk allele for higher homocysteine. "We have also found that individuals with CC genotype (homozygous for minor allele) have lower homocysteine levels than the AA genotype [of CHDC A119C]." (Jitender Kumar, et al., 2009)
- A variant of rs12676 is another risk. T is the risk allele (NA on my 23andme, but 45% of the population has one copy and 9% have two copies of the minor T allele. (Zeisel, 2012)
If you have either of these variants, you might need to supplement with TMG.
B6 and zinc are the two important ones here.
And lastly, you can look beyond these genes for problem areas to investigate further:
Homocysteine can usually be reduced by skipping or pushing the faulty parts of the recycling pathways by supplying active forms of precursors and co-factors.
WRT heart disease
Do note, there are unsettled questions about homocysteine's role with regards to cardiovascular disease:
Serum homocysteine is not independently associated with an atherogenic lipid profile: The Very Large Database of Lipids (VLDL-21) study.'
“Although high levels of tHcy were associated with 2-6% higher TG-rich lipoproteins in unadjusted analysis, after adjustment for confounders our findings do not support the hypothesis that hyperhomocysteinemia is associated with an atherogenic lipid profile.”
The nutritional burden of methylation reactions.
“Recent evidence has clearly demonstrated that transmethylation reactions can consume a significant proportion of the flux of methionine. In particular, synthesis of creatine and phosphatidylcholine consume most methyl groups and their dietary provision could spare methionine. Importantly, methionine can become limiting for protein and phosphatidylcholine synthesis when creatine synthesis is upregulated. Other research has shown that betaine and choline seem to be more effective than folate at reducing hyperhomocysteinemia and impacting cardiovascular outcomes suggesting they may be limiting.”
In regards to cognition, evidence is accumulating that lowering homocysteine has a positive effect on cognitive function. There have been two trials showing positive results with reducing homocysteine (FACIT trial of folic acid in the Netherlands & VITACOG trial of folic acid, B12 and B6 in people with Mild Cognitive Impairment in Oxford).
The current experts in the field are Drs. Smith and Refsum. See these links to recent talks below where they carefully outline the homocysteine connection to cognitive decline. (You need to register with Nestle Nutrition Institute for free access.)
Dr. Smith: How Valid is the Homocysteine Hypothesis of Brain Disease? Dr. Refsum: Vitamin B12 and the Brain
The quick hack guide to homocysteine
Supplements per Dr Bredesen's protocol - B12: 1mg methylcobalamin (1mg "milligram"=1000mcg "microgram"), folate: 0.8mg methyl-tetrahydrofolate, B6: 20mg pyridoxal-5-phosphate aka P5P. These are the active forms. Those people with what we call methylation defects do not efficiently convert the usual forms of cyanocobalamin and folic acid to the active forms the body needs.
If you are feeling unhealthy, have a history of reacting to supplements or know your methylation genetics contain variants from GeneticGenie or Strategene, my caution is to start supplementation slowly to avoid side effects. Start with a week on B12 at 500 mcg/day, to avoid something called "methyl trapping" which happens if your B12 levels are low (more on the wiki about this.) Then add folate at 400 mcg/day for a week. Last, add the B6. If your homocysteine does not go down enough, then increase to the amounts listed above. If it still doesn't go down, try adding 500-1000 mg/day of TMG, which is commonly used to effectively decrease levels in people with familial hyperhomocysteinemia.
More about Methylfolate and Methyl-B12
Studies generally show homocysteine levels will be normal when folate and B12 status are good even in those with genetic variants. So, this is the place to start, especially if you have MTHFR or MTRR variants. The efficiency of MTR to recycle homocysteine depends on both.
More about B6
Used by the CBS enzyme to flush homocysteine down the transsulfuration pathway. Pyridoxal-5-phosphate (P5P) is the active form. Start with 20 mg/day and work up to 50mg/ day if needed.
More about Trimethylglycine (aka TMG, Betaine)
Helps overcome problems in the BHMT portion of the methylation cycle. TMG is created from choline, so deficiencies in choline are also a factor.
- “When looking at the human evidence at this moment in time, it appears that betaine is effective and reliable for reducing homocysteine concentrations when taken daily at 3g or more. A single dose of betaine reduces homocysteine levels, which remain suppressed as long as supplementation is continues. Betaine has been found to reduce homocysteine by 10% in persons with normal levels or by 20-40% in persons with elevated homocysteine levels.” http://www.ncbi.nlm.nih.gov/pubmed/24022817
Notice the high dose of TMG mentioned, up to 3 g/day. As above, I recommend starting much lower, perhaps as low as 250mg/day and increasing from there to avoid upset an stomach.
More information about TMG can be found at https://examine.com/supplements/Trimethylglycine/
I rarely recommend one supplement company over another, but several of us on the forum have found that Life Extension's TMG is more effective. I suspect it is because it uses a type of water-soluble form of TMG, which may be absorbed better by the liver. This study shows how food sources of choline (a precursor to TMG) are absorbed, which suggests water-soluble forms might work better:
- “Excellent sources of dietary choline include liver, eggs and wheat germ (32, 33). In foods, choline is found free and as choline esters. Though it is likely that these forms are fungible, there is some evidence that they may have different bioavailability (34) because the lipid-soluble forms bypass the liver when absorbed from the diet while the water soluble forms enter the portal circulation and are mostly absorbed by the liver.“ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430110/
And finally, this mouse model suggests restricting methionine works better than TMG - that means cutting down on animal protein. http://www.ncbi.nlm.nih.gov/pubmed/26231230
Adequate choline is also necessary to support the BHMT portion of the cycle.
- “Other research has shown that betaine and choline seem to be more effective than folate at reducing hyperhomocysteinemia and impacting cardiovascular outcomes suggesting they may be limiting.“ http://www.ncbi.nlm.nih.gov/pubmed/23196816
- And menopausal women have a higher requirement for choline (the precursor to betaine). There is a PEMT variant (rs12325817, C is the risk allele) that is induced by estrogen and affects choline levels. Dietary choline requirements of women: effects of estrogen and genetic variation. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954445/
However, the effects of choline on homocysteine may depend on your folate status. Choline can be used as a methyl donor in instances of low folate and not be available to be converted into TMG to push the BHMT pathway.
- “Choline deprivation did not increase plasma homocysteine concentration in rats fed 20C, but it markedly enhanced plasma homocysteine concentration when rats were fed folate-deprived 20C. This indicates that choline deprivation reinforced folate deprivation-induced hyperhomocysteinemia. Increased hepatic DMG concentration was also associated with such an effect. These results support the concept that folate deficiency impairs homocysteine metabolism not only by the MS pathway but also by the BHMT pathway.“ https://www.jstage.jst.go.jp/article/jnsv/58/2/58_69/_article
The most direct way to supplement choline for the purpose of reducing homocysteine would be to take phosphatidylcholine, 200-400 mg/day. This is the active form needed to support cell walls, bile flow and brain function. Because it's already active, your body doesn't need extra methyl groups from folate to convert choline to this active form.
There have been many discussions about choline, betaine and the dreaded metabolite, TMAO. It’s more than I want to go into, but feel free to search the APOE4.info site for those discussions. I will note that there might be a link to diabetes - this study is suggesting that diabetes accentuates the relationship of elevated TMAO and increased cardiovascular risk. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114969. And there is also a link to the composition of bacteria in your gut. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453578/
Creatine is another supplement that can help spare your methyl groups because a lot of the methylfolate you use is actually used to make creatine. Both Drs Ben Lynch and Chris Masterjohn suggest supplementing creatine. Masterjohn suggests 3-5 grams per day of "creapure" powdered creatine and to expect 4-6 weeks to see any effects. I would suggest starting much lower than that and work up because it can cause insomnia and diarrhea for some people.
The RDA upper limit for daily zinc is 40 mg of elemental zinc. Take care to watch your zinc/copper ratio. It should be close to 1. You can also search APOE4.info for those discussions.
“Animal in vivo and in vitro studies have shown that zinc deficiency decreases the absorption and metabolism of dietary folate (Ghishan et al., 1986; Quinn et al., 1990; Favier et al., 1993) because of its function as a cofactor for the folate-metabolizing enzymes dihydrofolate reductase and γ-glutamyl hydrolase. Zinc itself, however, is not a cofactor of the MTHFR enzyme; however, it is a cofactor for methionine synthetase and for betaine-homocysteine methyltransferase. ” http://humupd.oxfordjournals.org/content/13/2/163.full
One last thing to push is riboflavin if you have the MTHFR C677T polymorphism. Riboflavin, or vitamin B2, is a precursor for flavin adenine dinucleotides (FAD), which is a cofactor to MTHFR.
“Supplementation of riboflavin (1.6mg) for 12 weeks in subjects positive for the MTHFR 677C->T polymorphism reduced homocysteine levels in only in subjects who were MTHFR 677TT homozygous, where homocysteine levels decreased by 22% in those with normal riboflavin status. Larger decreases (in upwards of 40%) were noted in homozygous individuals with lower riboflavin status, suggesting that riboflavin intake affects homocysteine levels in these individuals.
In a large assessment of MTHFR mutations, it was found that overall subjects who had the MTHFR 677T allele had higher homocysteine concentrations, but this increase was not found in individuals whose riboflavin status was considered optimal (only in those with lower riboflavin status).” http://examine.com/supplements/vitamin-b2/
There seems to be a multiplier effect when omega-3s are added to B vitamins. Homocysteine was found to be significantly associated with cortical Aβ in people with low levels of omega-3s, even after controlling for E4 status in this study. But more importantly, not associated in those who had a high baseline index..
- "The average homocysteine-lowering effect was greater when omega-3 supplementation was combined with folic acid and B-group vitamins (-1.37μmol/L, 95%CI: (-2.38, -0.36), P<.01) compared to omega-3 supplementation alone (-1.09μmol/L 95%CI: (-2.04, -0.13), P=.03). Omega-3 polyunsaturated fatty acid supplementation was associated with a modest reduction in homocysteine. For the purposes of reducing homocysteine, a combination of omega-3s (0.2-6g/day), folic acid (150 - 2500μg/day) and vitamins B6 and B12 may be more effective than omega-3 supplementation alone." http://www.ncbi.nlm.nih.gov/pubmed/27188895
- "Exploratory analysis showed that homocysteine was however significantly associated with cortical Aβ in subjects with low baseline omega-3 index (< 4.72 %) after adjustment for Apolipoprotein E ε4 status (B-coefficient 0.041, 95 % CI: 0.017,0.066, p = 0.005, n = 10), but not in subjects with a high baseline omega-3 index (B-coefficient -0.010, 95 % CI: -0.023,0.003, p = 0.132, n = 66). CONCLUSIONS: The role of n-3 PUFAs on the relationship between homocysteine and cerebral Aβ warrants further investigation." https://www.ncbi.nlm.nih.gov/pubmed/29188863
- "For all three outcome measures, higher concentrations of docosahexaenoic acid alone significantly enhanced the cognitive effects of B vitamins, while eicosapentaenoic acid appeared less effective. When omega-3 fatty acid concentrations are low, B vitamin treatment has no effect on cognitive decline in MCI, but when omega-3 levels are in the upper normal range, B vitamins interact to slow cognitive decline. A clinical trial of B vitamins combined with omega-3 fatty acids is needed to see whether it is possible to slow the conversion from MCI to AD." http://content.iospress.com/download/journal-of-alzheimers-disease/jad150777?id=journal-of-alzheimers-disease%2Fjad150777
Other genetic variants
If you want to dig further, other genetic variants effecting homocysteine can be found at:
- The Effect of Multiple Single Nucleotide Polymorphisms in the Folic Acid Pathway Genes on Homocysteine Metabolism http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3913508/
- Common genetic loci influencing plasma homocysteine concentrations and their effect on risk of coronary artery disease http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321227/
- Novel Associations of CPS1, MUT, NOX4 and DPEP1 with Plasma Homocysteine in a Healthy Population: A Genome Wide Evaluation of 13,974 Participants in the Women’s Genome Health Study http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2745176/
A Gentle Reminder About ANY Hacks
Remember: you can’t supplement just on the results of your genetic variants - you have to fix your lifestyle, diet, and stress levels, too. But the bottom line is that properly functioning methylation is a basic foundation for anyone’s good health.