“OMG - I have the MTHFR gene!”
*This article is not medical advice. Before starting on any health related regimen, seek the advice of your Primary Care Physician or an M.D.
MTHFR: This article is a bit tongue in cheek
Every once in a while a potential client or somebody on social media will engage me with some sort of screaming emergency after they discover, “I have the MTHFR gene”. Yes, we all do, and some of us have variants on MTHFR 1298c and or c677t. In fact, most of us do. Yes, its an important gene, and in some cases, usually the homozygous variants need some strong consideration in terms of supports, but not without greater context. After all, we have ~20,000 genes, and millions of locations on those genes. Certainly, there are additional considerations for how much b12, folate, etc one should think about consuming beyond a single mutation on mthfr c677t. Rather than debate, lets get to some basic grounding facts around how common these variants/mutations are and what additional genes are involved in methylation…..
“OMG - I have mutations on MTHFR, quick somebody tell me how much methyl folate to take!”.
Basic Facts : Frequency of Variants From Two Data Sets:
Data Set #1 : Genome AD Browser : 807k people
MTHFR 1298c
Clean (no variants): 49%
Heterozygous or Compound / One Variant: 42%; 37% estimated at hetero alone
Homozygous / Two Variants: 9%
MTHFR C677T
Clean (no variants): 47%
Heterozygous or Compound/ One Variant: 42%; 37% estimated at hetero alone
Homozygous / Two Variants: 11%
Compound heterozygous - single variants on MTHFR 1298c and C677T;
Estimated:10%
Data Set #2 : Genomic Database across ~85,000 people, mostly in the USA
MTHFR 1298c
Clean (no variants): 48%
Heterozygous / One Variant or compound: 42%; 37% estimated hetero
Homozygous / Two Variants: 10%
MTHFR C677T
Clean (no variants): 42%
Heterozygous or compound / One Variant: 44%; 39% estimated as hetero
Homozygous / Two Variants: 13%
Compound heterozygous - single variants on MTHFR 1298c and C677T;
Estimated: 10%
Observationally, about 4-5% of my clients have had zero variants on both MTHFR 1298c and C677T.
What does all this tell us ?
MTHFR Variants are more common than being wild on either location
Homozygous is quite a bit more uncommon for either location than being heterozygous
One would expect to be heterozygous if anything, on at least one of the locations
In the sections below i will outline different aspects of the methylation cycle, although there may be slightly different nomenclature, it should help identify which aspects i am referring. Also, below i have identified the genes i am familiar with and often look at in clients for each part of the methylation cycle. As you can see, its not a short list, and also keep in mind, often there are many locations of interest on these genes of interest.
other aspects of “methylation/folate/b12 metabolism and transport/absorption”
Folate / Folic Acid Absorption, Transport, Delivery
Methylation “Long Cycle”
Methylation “Short Cut”
B12 Absorption, Transport, and Delivery
Production and Delivery Of Phosphatdyl Choline to Methylation Cycle
B6 Absorption, Metabolism, Delivery to Methylation Cycle
Metabolism of Homocysteine into Cysteine
Absorption, Metabolism, and Delivery of B2 to Methylation Cycle
Creatine production from methylation cycle
GENES INvolved in the Absorption, Transport, Delivery of Folate / Folic Acid to the methylation cycle
FOLH1 - Zn2+-dependent membrane glutamate carboxypeptidase; hydrolyzes poly-γ-glutamyl folates to monoglutamate; also NAAG peptidase in CNS. Dietary folates predominantly exist as polyglutamates and need to be deconjugated to monoglutamates prior to absorption using this gene. Uses cysteine residues structurally.
FOLR1 - High-affinity folate-binding, GPI-anchored receptor mediating endocytosis of 5‑MTHF/folates. Members of this gene family bind to folic acid and its reduced derivatives, and transport 5-methyltetrahydrofolate into cells. Uses cysteine residues structurally.
FOLR2 - High-affinity folate-binding GPI-anchored receptor; delivery of folates to cells (notably myeloid/placental). Members of this gene family bind to folic acid and its reduced derivatives, and transport 5-methyltetrahydrofolate into cells. Although this protein was originally thought to be specific to the placenta, it can also exist in other tissues. Uses cysteine residues structurally.
FOLR3 - Secreted folate receptor; binds folate and circulates; human‑specific expression pattern. The FOLR3 gene encodes a member of the folate receptor (FOLR) family of proteins, which have a high affinity for folic acid and for several reduced folic acid derivatives, and mediate delivery of 5-methyltetrahydrofolate to the interior of cells. Uses cysteine residues structurally.
SLCA19A1 - Reduced folate carrier; major facilitative importer of folates at neutral pH; folate : organic‑phosphate antiporter. The membrane protein encoded by the SLC19A1 gene is a transporter of folate and is involved in the regulation of intracellular concentrations of Folate. Uses non catalytic cysteine residues.
SLC46A1 - Proton‑coupled folate transporter; apical intestinal folate absorption; folate entry to CSF (choroid plexus). The SLC46A1 gene encodes a transmembrane proton-coupled folate transporter protein that facilitates the movement of folate and antifolate substrates across cell membranes. Uses non catalytic cysteine residues
DHFR - Dihydrofolate reductase; reduces DHF → THF for 1‑C metabolism (purines, thymidylate, Met). Dihydrofolate reductase catalyzes the reduction of Folic Acid (FA) to Dihydrofolate (DHF),. Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids. Dihydrofolate reductase deficiency has been linked to megaloblastic anemia. NADPH cofactor required. Uses non catalytic cysteine residues.
GENES INvolved in THE Methylation “Long Cycle”
DHFR - Dihydrofolate reductase; reduces DHF → THF for 1‑C metabolism (purines, thymidylate, Met). Dihydrofolate reductase catalyzes the reduction of Folic Acid (FA) to Dihydrofolate (DHF),. Dihydrofolate reductase converts dihydrofolate into tetrahydrofolate, a methyl group shuttle required for the de novo synthesis of purines, thymidylic acid, and certain amino acids. Dihydrofolate reductase deficiency has been linked to megaloblastic anemia. NADPH cofactor required. Uses non catalytic cysteine residues.
ALDH1L1 - Oxidizes 10-formyl-THF → THF + CO₂, generating NADPH; key cytosolic sink for 1C units and redox support. NADP cofactor required. The protein encoded by the ALDH1L1 gene catalyzes the conversion of 10-formyltetrahydrofolate, NADP+, and H2O to Tetrahydrofolate, NADPH, and Carbon Dioxide. Cysteine residues required for catalytic activity.
MTHFD1 - Trifunctional enzyme: 5,10‑methylene‑THF dehydrogenase + 5,10‑methenyl‑THF cyclohydrolase + 10‑formyl‑THF synthetase (purine synthesis). Trifunctional enzyme: 5,10‑methylene‑THF dehydrogenase + 5,10‑methenyl‑THF cyclohydrolase + 10‑formyl‑THF synthetase (purine synthesis). The MTHFD1 gene encodes a protein that has three distinct enzymatic activities: 1) 5,10-methylenetetrahydrofolate dehydrogenase ; 2) 5,10-methenyltetrahydrofolate cyclohydrolase; 3) 10-formyltetrahydrofolate synthetase. Each of these enzymatic activities catalyzes one of three sequential reactions in the interconversion of 1-carbon derivatives of tetrahydrofolate, which are substrates for methionine, thymidylate, and de novo purine syntheses. NADP, Mg, and ATP are required cofactors.
MTHFR C677 -Reduces 5,10‑methylene‑THF → 5‑methyl‑THF for methionine synthase/Re-methylation; C677T variants yield thermolabile enzyme with lower activity/stability. NADPH, and FAD required cofactors.
SHMT1 - Transfers C1 unit from serine to THF → 5,10‑methylene‑THF + glycine; central to thymidylate/purine synthesis. Transfers C1 unit from serine to THF → 5,10‑methylene‑THF + glycine; central to thymidylate/purine synthesis. The SHMT1 gene encodes the cytosolic form of serine hydroxy methyltransferase, a pyridoxal phosphate-containing enzyme that catalyzes the reversible conversion of Serine and Tetrahydrofolate to Glycine and 5,10-Methylenetetrahydrofolate. PLP (B6) is a required cofactor.
GENES IN METHIONE METABOLISM / “SHORT Cycle” for the methylation cycle
MTR - Remethylates homocysteine → methionine using 5‑methyl‑THF; central to methionine/folate cycles. Methionine synthase uses Vitamin B12 in the form of Methylcobalamin as a cofactor to convert Homocysteine back to Methionine . B12 and zinc are required cofactors along with Cysteine for catalytic activity. MTRR activity required for MTR, which requires FAD, and NADPH.
MTRR - Reactivates oxidized cobalamin in MTR using electrons from NADPH (restores active cob(I)alamin state). This gene attaches a methyl group onto B12. Variants here, will lower the B12 available for the MTR enzyme to process Homocysteine into Methionine. The situation gets more complex if there is an MTR variant, which is an upregulation, trying to go faster. There is a need for Methyl B12 with this variant, unless the individual has excess methyl groups, and then Hydroxocobalamin may be more helpful. FAD and NADPH are required cofactors.
MAT1A -Catalyzes methionine + ATP → S‑adenosyl‑methionine (SAM); predominant hepatic isoform. The MAT gene supports the Methionine to SAMe conversion. SAMe is critical for many functions. In addition to mutations on MAT, Hydroxyl Radicals from the Fenton reaction will suppress MAT. The MAT1A enzyme converts methionine into SAMe. Variants in this gene may cause high methionine. If you suspect an issue, may want to do the Doctor's Data Plasma test. Mg required for cofactor, K, will stimulate activity.
MAT2A - SAM synthesis in extra‑hepatic tissues; regulates cellular methylation capacity. Mg required cofactor, K will stimulate activity.
PEMT - ER‑membrane enzyme: PE → PC via three SAM‑dependent methylations (endogenous PC biosynthesis). PEMT uses SAMe to methylate Phosphatidylethanolamine to form Phosphatidylcholine. This reaction generates SAH as a byproduct. In humans, phosphatidylcholine is only made by the PEMT enzyme. Estrogen is required for the PEMT enzyme to activate and function normally. Men and postmenopausal women have an elevated risk of choline deficiency due to low estrogen levels. The PEMT enzyme is commonly slowed down by polymorphisms, making it unresponsive to estrogen levels. 74% of women have at least one copy of a slowed PEMT. Homozygous carriers of PEMT have much higher risk of choline deficiency. Men, postmenopausal women, and premenopausal women with PEMT SNPs need to increase choline intake in the diet to offset elevated risk of liver dysfunction. SAM is cofactor.
AHYC - Reversible SAH ⇌ adenosine + homocysteine; clears SAH to permit SAM‑dependent methylation reactions. AHCY is the only enzyme that mediates the reversible catalysis of S-adenosylhomocysteine (SAH) to adenosine and Homocysteine. AHCY converts SAH into homocysteine. Variants here can be quite tricky. Be extra cautious when there are GAMT and MAT variants. If there are many AHCY, MAT, and GAMT variants, there is the possibility of high methionine. In this case, supporting the BHMT enzyme or even B12 can have adverse reactions. It may be best to do the Doctor's Data Blood Plasma test to see the status of methionine, SAMe, SAH, homocysteine, and cysteine. NAD+ is the required cofactor, and K may help modulate.
GNMT - Transfers methyl from SAM to glycine → sarcosine; buffers hepatic SAM; allosterically regulated by 5‑MTHF. The protein encoded by the GNMT gene is an enzyme that catalyzes the conversion of S-adenosyl-L-methionine (along with glycine) to S-adenosyl-L-homocysteine and sarcosine. This protein is found in the cytoplasm and acts as a homo tetramer. Defects in this gene are a cause of GNMT deficiency (hypermethioninemia). SAM is the substrate.
BHMT - Transfers methyl from betaine to homocysteine → methionine; hepatic/kidney re-methylation route. Betaine-Homocysteine S-Methyltransferase- BHMT is the shortcut secondary pathway through the middle of the methionine cycle. The BHMT enzyme uses TMG (made by the PEMT genes) to convert homocysteine into methionine, so more SAMe can be made. The BHMT gene encodes a cytosolic enzyme that catalyzes the conversion of Betaine and Homocysteine to Dimethylglycine and Methionine. Variants in BHMT impede the conversion, while it is believed that the BHMT-08 variants cause excess activity in the transulfuration pathway, creating more glutamate and ammonia, and often leading to anxiety and adrenal fatigue. BHMT is involved in the recycling of homocysteine to methionine. Zinc is used as a cofactor. Polymorphisms can contribute to high homocysteine levels. Zn is required cofactor, Betaine is the substrate, cysteine residues required for catalytic activity.
BHMT2 - Homolog of BHMT using S‑methylmethionine (SMM) as methyl donor to remethylate homocysteine. The BHMT2 gene encodes a cytosolic enzyme that catalyzes the conversion of Betaine and Homocysteine to Dimethylglycine and Methionine . Homocysteine is a sulfur-containing amino acid that plays a crucial role in methylation reactions. Transfer of the methyl group from betaine to homocysteine creates methionine, which donates the methyl group to methylate DNA, proteins, lipids, and other intracellular metabolites. The protein encoded by the BHMT2 gene is one of two methyl transferases that can catalyze the transfer of the methyl group from betaine to homocysteine. Zn is required cofactor, and cysteine residues are required for catalytic activity.
GENES INVOLVED IN the Absorption, Transport, and Delivery of b12 to the methylation cycle
TCN1 - Haptocorrin; salivary/serum B12-binding glycoprotein that protects cobalamin in the stomach. Haptocorrin is encoded by the TNC1 gene. When vitamin B12 binds to haptocorrin, haptocorrin carries vitamin B12 along the gastrointestinal tract. The TCN1 gene makes a protein that binds and carries Vitamin B12 through the body. It is found in various tissues and in neutrophils, helping transport B12 into cells. If this protein does not work properly, B12 transport may be reduced. Cysteine residues required for catalytic activity.
TCN2 - Transcobalamin II; plasma transporter delivering B12 to cells via CD320-mediated endocytosis. The absorbed Vitamin B12 then binds to Transcobalamin II within the enterocyte and is then released into the blood stream. Transcobalamin II is encoded by the TCN2 gene. The TCN2 gene makes a protein called Transcobalamin II that binds Vitamin B12 in the blood and helps transport it into cells. If this protein does not work properly, B12 delivery to cells may be reduced. Mutations in this gene can lead to Transcobalamin deficiency → infantile megaloblastic anemia, infections; ↑MMA + ↑homocysteine; responds to parenteral hydroxocobalamin. Cysteine residues required for catalytic activity.
MMUT - Methylmalonyl‑CoA mutase (mitochondrial); converts methylmalonyl‑CoA → succinyl‑CoA. The MMUT gene encodes the mitochondrial enzyme methylmalonyl Coenzyme A mutase. In humans, the product of this gene is a vitamin B12-dependent enzyme which catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. On organic acids tests, elevations in methylmalonic acid is used as a B12 deficiency marker, but its specific to adeno b12, not other forms. However, methylmalonic acid can also buildup due to too much input from propionic acid through the PCCA gene. Adenocobalamin B12 is the cofactor.
CD320 - Transcobalamin receptor; mediates cellular uptake of holo‑TCN2. The Transcobalamin II/Vitamin B12 complex then binds to the transcobalamin receptor (encoded by the CD320 gene) and is taken up by cells via endocytosis. The CD320 gene makes a cell surface receptor that allows cells to take in Vitamin B12 bound to Transcobalamin II. It also helps support B-cell growth and immune function. If this receptor does not work properly, B12 uptake into cells may be reduced, which can contribute to methylmalonic aciduria. CD320 deficiency → cellular B12 uptake defect; megaloblastic anemia ± neurologic findings; ↑MMA + ↑homocysteine. No cofactors, but cysteine residues are required for catalytic activity.
FUT2 - Golgi α1,2‑fucosyltransferase; creates H antigen on mucosal/secreted glycans (secretor status). FUT2 is involved in the synthesis of antigens of the Lewis blood group. These antigens mediate the attachment of gastric pathogens to the gastric mucosa. This can then affect the absorption of Vitamin B12. The FUT2 gene makes an enzyme that helps add sugar molecules to proteins and cells, which affects blood group markers and how cells interact with each other. If this enzyme does not work properly, it can change blood group expression and cell surface markers. GDP fucose required as cofactor.
FUT6 - Golgi α1,3‑fucosyltransferase; fucosylates N‑glycans (e.g., sLe^x synthesis). FUT6 is involved in forming Lewis associated antigens. These antigens attach gastric pathogens to the gastric mucosa. Research has found that these gastric pathogens can then reduce the absorption of vitamin B12 in the gut. The FUT6 gene makes an enzyme in the Golgi that adds sugar molecules to proteins, helping form markers like sialyl-Lewis X on cell surfaces. If this enzyme does not work properly, it can cause fucosyltransferase-6 deficiency . GDP fucose required as cofactor.
CLIBF (GIF/TCN3) - Intrinsic factor; gastric secreted glycoprotein that binds B12 for intestinal absorption via CUBN/AMN. In the duodenum (the first part of the small intestine), pancreatic proteases break the Haptocorrin/vitamin B12 complex. Vitamin B12 then binds to Intrinsic factor to form an Intrinsic factor/vitamin B12 complex. The Intrisic Factor is encoded by the CBLIF gene. The GIF (TCN3) gene makes a protein called Intrinsic Factor that is secreted by the stomach and is needed for Vitamin B12 absorption. If this protein does not work properly, it can lead to congenital pernicious anemia. Hereditary intrinsic factor deficiency (HIFD) → megaloblastic anemia from B12 malabsorption; treat with parenteral B12. Cysteine required for catalytic activity.
CUBN - Cubilin; endocytic receptor for IF‑B12 complex in ileum; also in renal proximal tubule (with AMN). Cubilin (encoded by the CUBN gene) binds the Intrinsic factor/vitamin B12 complex to another protein called Amnionless (encoded by the AMN gene) to facilitate the entry of vitamin B12 into the intestinal cells. The CUBN gene makes a protein called cubilin that acts as a receptor in the intestines and kidneys. It helps cells take in certain nutrients, including Vitamin B12. If this protein does not work properly, it can contribute to autosomal recessive megaloblastic anemia. Imerslund–Gräsbeck syndrome (IGS) leads to selective B12 malabsorption and proteinuria. Ca required as cofactor. Cysteine residues required for catalytic activity.
ANM - Amnionless; transmembrane adaptor that partners with cubilin (CUBAM complex) for IF‑B12 endocytosis. The AMN gene makes a transmembrane protein that helps cells interact with growth signals and other proteins. If this protein does not work properly, it can affect nutrient uptake and other cellular functions. Imerslund–Gräsbeck syndrome (IGS) due to AMN mutations → B12 malabsorption. Cysteine residues required for catalytic activity.
ABCD4 - Lysosomal/cytosolic ABC transporter (with LMBD1) exporting cobalamin to cytosol. The protein encoded by the ABCD4 gene is a member of the superfamily of ATP-binding cassette (ABC) transporters. Research suggests that ABCD4 is involved in intracellular Vitamin B12 processing and is involved in transporting Vitamin B12 from lysosomes to the Cytosol. The ABCD4 gene makes a transporter protein that moves molecules across membranes inside cells. It is part of a larger family of transporters that help move fatty acids and other substances. If this protein does not work properly, certain molecules may not reach where they are needed in the cell. ABCD4 deficiency can lead to elevations in MMA + homocystinuria. ATP and Mg are required cofactors.
LMBRD1 - Lysosomal membrane protein partnering with ABCD4 for cobalamin egress. Previous research has shown that the protein encoded by the LMBD1 gene (which is responsible for the lysosomal export of Vitamin B12) can interact with the ABCD4 protein. However, the mechanisms of interaction between LMBD1 and ABCD4 remains unclear, but, it is believed that variations in the LMBRD1 gene and ABCD4 may prevent translocation of the Vitamin B12 from the Lysosome to the Cytoplasm. The LMBRD1 gene makes a lysosomal membrane protein that helps move certain molecules, including Vitamin B12, out of lysosomes and into the cell. If this protein does not work properly, B12 may not reach where it is needed, which can contribute to metabolic disorders like homocystinuria-megaloblastic anemia.
MMACHC - Cobalamin processing (dealkylation/decyanation) and trafficking to cytosol/mitochondria. The MMACHC gene makes a protein that helps cells handle Vitamin B12. It may help bind and move B12 inside the cell. If this protein does not work properly, B12 processing can be disrupted, which can contribute to methylmalonic aciduria and homocystinuria type cblC. Required cofactors are Glutathione, FAD, and NADPH. Mutations in this gene can lead to combined MMA + homocystinuria; neuro‑ophthalmologic involvement common.
MMADHC - Adaptor/targeting factor directing cobalamin to cytosolic (MTR) or mitochondrial (MMUT) branches. The MMADHC gene makes a mitochondrial protein that helps with an early step in processing Vitamin B12 inside cells. If this protein does not work properly, active forms of B12 may be low, which can contribute to methylmalonic aciduria and homocystinuria type cblD. cblD (variable) → isolated MMA, isolated homocystinuria, or combined, depending on domain/location affected.
MMAA - GTPase/chaperone assisting MMUT cofactor insertion and repair. The protein encoded by the MMAA gene is involved in the translocation of cobalamin into the mitochondrion, where it is used in the final steps of adenosylcobalamin synthesis. Adenosylcobalamin is a coenzyme required for the activity of methylmalonyl-CoA mutase. cblA → isolated methylmalonic acidemia (variable B12 responsiveness). GTP and Mg are the required cofactors.
MMAB - Adenosyltransferase forming adenosylcobalamin from cobalamin + ATP. The MMAB gene encodes a protein that catalyzes the final step in the conversion of vitamin B12 into adenosylcobalamin (AdoCbl), a vitamin B12-containing coenzyme for methylmalonyl-CoA mutase. ATP and Mg are the required cofactors, cobalamin is the substrate.
GENES INVOLVED IN the Absorption, Metabolism, Delivery OF B6 to THE Methylation Cycle
ALPL - Tissue‑nonspecific alkaline phosphatase; dephosphorylates PPi and other substrates to enable bone mineralization. Alkaline phosphatase, encoded by ALPL, has been hypothesized to have an important role in the clearance of vitamin B6. Zn and Mg are the required cofactors.
PDXK - Pyridoxal kinase; phosphorylates B6 vitamers (PL/PN/PM) → PLP/PNP/PMP. Pyridoxal kinase, encoded by PDXK, has an important role in the phosphorylation of vitamin B6 vitamers, which is required for PLP synthesis: PLP is the active form of vitamin B6. ATP and Mg are the required cofactors.
PDXP - Pyridoxal phosphate phosphatase (haloacid dehalogenase family); hydrolyzes PLP → PL for salvage; also known as chronophin. PDXP has an important role in the degradation of the active form of vitamin B6, pyridoxal 5'-phosphate (PLP), into pyridoxine (PN) and pyridoxamine (PM). Mg is the required cofactor.
NBPF3 - Neuroblastoma breakpoint family member; DUF1220‑rich protein; function not well defined in metabolism. NBPF3 is part of the neuroblastoma breakpoint family (NBPF) and may have a role in clearing vitamin B6.
PROSC - PLP‑binding protein that buffers cellular PLP and supports B6 cofactor homeostasis. PROSC, also known as PLPBP, encodes for pyridoxal 5′-phosphate (PLP)-binding protein: PLP is the active form of vitamin B6. Research suggests PLPBP has a role in intracellular PLP regulation. Escorts B6 to genes like CBS.
PNPO - Pyridox(am)ine‑5′‑phosphate oxidase; converts PNP/PMP → PLP. PNPO, encodes for pyridoxamine 5'-phosphate oxidase, catalyzes the rate-limiting step in the synthesis of the active form of vitamin B6, pyridoxal 5'-phosphate (P5P). B2 (FMN) required as a cofactor.
GENES INVOLVED IN THE Metabolism of Homocysteine into Cysteine
CTH - Cystathionine γ-lyase; PLP-dependent enzyme in transsulfuration producing cysteine, α‑ketobutyrate, NH3, and H2S. The CTH gene encodes a cytoplasmic enzyme in the trans-sulfuration pathway that converts cystathione derived from methionine into cysteine. Glutathione synthesis in the liver is dependent upon the availability of cysteine. Mutations in this gene cause cystathioninuria. LPL (B6) is the cofactor. CTH deficiency → cystathioninuria (often benign; may have subtle neurodevelopmental features).
CBS - Cystathionine β-synthase; PLP enzyme condensing homocysteine + serine → cystathionine; produces H2S. Cystathionine Beta Synthase (CBS) is the enzyme responsible for converting homocysteine into cystathionine, the first step in the transsulfuration pathway that ultimately produces glutathione (the master antioxidant). With the help of vitamin B6, the CBS enzyme is responsible for removing excess sulfur containing amino acids from the pathway. The CBS polymorphism creates an upregulation, potentially causing homocysteine to rush down the transsulfuration pathway at up to 10 times faster than without the variant (Mullan, 2011). Conversion impairments prevent homocysteine from being properly used, creating low homocysteine, diminished glutathione levels, and excesses in ammonia (a by-product of amino acid metabolism), sulfites and sulfates. Classic homocystinuria due to CBS deficiency (↑homocysteine, lens dislocation, thrombosis); some B6‑responsive. PLP (B6) and Zn are the required cofactors; cysteine residues are required for catalytic activity.
GENES INVOLVED IN THE ABSORPTION, METABOLISM, AND DELIVERY OF B2 TO THE METHYLATION CYCLE
SLC52A1 - Riboflavin transporter 1 (RFVT1); intestinal/placental riboflavin transport. Biological redox reactions require electron donors and acceptor. Vitamin B2 is the source for the flavin in flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) which are common redox reagents. The SLC52A1 gene encodes a member of the riboflavin (vitamin B2) transporter family. Highly expressed in placenta (and intestine). Human cases show that impaired RFVT1 causes transient neonatal riboflavin deficiency when mom carries the variant—infants present with MADD-like labs and improve with riboflavin. In polarized gut cells, RFVT1 has been observed predominantly at the basolateral membrane (export side into blood). Riboflavin transporter deficiency (RTD1/Brown‑Vialetto–Van Laere type 1); riboflavin‑responsive neuropathy/cranial neuropathies.
SLC52A2 - Riboflavin transporter 2 (RFVT2). SLC52A2 encodes for the riboflavin (vitamin B2) transporter known as RFVT2 and has an important role in riboflavin absorption. Broadly expressed and especially important for neural tissue uptake. Biallelic SLC52A2 variants cause the treatable neuronopathy historically called Brown-Vialetto–Van Laere (BVVL) syndrome (now “riboflavin transporter deficiency type 2”). In intestine, RFVT2 is enriched on the basolateral side (serosal), complementing RFVT3’s apical uptake to move riboflavin into the bloodstream. Riboflavin transporter deficiency (RTD2/Brown‑Vialetto–Van Laere type 2): pontobulbar palsy, sensorimotor neuropathy; riboflavin‑responsive.
SLC52A3 - Riboflavin transporter 3 (RFVT3). The main function of SLC52A3 is to absorb Riboflavin from the diet. Once Riboflavin is absorbed, it can transported by SLC52A1 or SLC52A2 to the bloodstream and distributed to tissues where it is needed. SLC52A3 is a riboflavin (vitamin B2) transporter, and is highly expressed in the intestines; thus, this gene likely has a role in the intestinal absorption of riboflavin. The major apical transporter on intestinal enterocytes that takes dietary riboflavin from the lumen into the cell. Intestinal knockout in mice collapses gut B2 uptake and causes systemic riboflavin deficiency—rescued by high-dose riboflavin. Also expressed in placenta, kidney, testis. FMN/FAD are not transported efficiently (the transporters move riboflavin itself). Sublingual FMN may work around SLC52A3 compromise - Predominantly intestinal SLC52A3 impairment (or severe GI malabsorption/surgeries): bypassing the intestinal lumen/brush border may help you get riboflavin into circulation, as long as SLC52A2/1 are functional for basolateral/tissue uptake. (RFVT3 is the apical gut transporter.). Rare riboflavin transporter deficiency (RTD3); neonatal or maternal‑fetal riboflavin deficiency states; riboflavin‑responsive.
SLC52A Family Of Transporters- Intestinal absorption: luminal riboflavin enters via SLC52A3 (apical) → exits the enterocyte to blood via SLC52A1/SLC52A2 (basolateral). Circulation and tissue delivery: tissues (esp. nervous system) rely heavily on SLC52A2; placenta relies on SLC52A1, and SLC52A3 also contributes to placental transport in mice. Intracellular conversion: riboflavin → FMN via RFK, then FMN → FAD via FLAD1; SLC25A32 moves FAD across the mitochondrial inner membrane to supply flavoenzymes. (The SLC52A transporters do not transport FMN/FAD.). SLC52A3 loss: global KO mice die neonatally with riboflavin deficiency; intestinal-specific KO causes growth failure and low systemic riboflavin—both rescued by high-dose riboflavin. SLC52A2 or SLC52A3 variants (RTD2/RTD3): progressive cranial and peripheral neuropathy; often riboflavin-responsive if treated early/high-dose. SLC52A1 variants: placental transport failure → transient neonatal riboflavin deficiency (MADD-like acylcarnitines/organic acids) that resolves with riboflavin therapy. Polarity in gut: RFVT3 = apical, RFVT2 (and evidence for RFVT1) = basolateral. This apical→basolateral handoff is how dietary riboflavin reaches blood. Not FMN/FAD: SLC52A transporters prefer riboflavin; FMN and FAD are poor substrates—cells make FMN/FAD internally.
RFK - Riboflavin kinase; phosphorylates riboflavin → FMN. The Riboflavin kinase enzymed encoded by the RFK gene catalyzes the phosphorylation of Riboflavin to form Flavin Mononucleotide. RFK encodes for riboflavin kinase which catalyzes the critical phosphorylation of riboflavin (vitamin B2) to flavin mononucleotide (FMN), an essential step in the utilization of the vitamin. FMN is a precursor to FAD and is involved in a myriad of metabolic reactions. RFK deficiency: neurometabolic disorder with seizures/ataxia; responsive to high‑dose riboflavin/FMNs (case reports). ATP and Mg are required cofactors.
FLAD1 - FAD synthase; converts FMN → FAD (adenylyltransferase). The FLAD1 gene encodes the enzyme that catalyzes adenylation of Flavin Mononucleotide to form Flavin Adenine Dinucleotide (FAD). FLAD1 catalyzes the conversion of flavin mononucleotide (FMN) to form flavin adenine dinucleotide (FAD) coenzyme: an important riboflavin-dependent cofactor for a myriad of reactions, including the electron transport chain. FAD synthase deficiency → lipid storage myopathy / multiple acyl‑CoA dehydrogenase‑like; riboflavin‑responsive in many. ATP and Mg are required cofactors.
GENES INVOLVED IN THE PRODUCTION OF CREATINE FROM Methylation cycle / SAMe
AGAT - Arginine:glycine amidinotransferase; synthesizes guanidinoacetate from Arg + Gly (first step of creatine biosynthesis). Reaction (rate-limiting, “first step”): transfers the amidino group from L-arginine to glycine → guanidinoacetate (GAA) + ornithine. GAA is then methylated by GAMT to make creatine. Creatine buffers ATP in high-demand tissues (brain, muscle). Where it lives: predominantly mitochondrial (intermembrane space/inner-membrane–associated), highly expressed in kidney (and also pancreas); the second step (GAMT) is mainly hepatic—together they create a kidney→liver axis for creatine supply. Regulation: product feedback—creatine (and ornithine) suppress AGAT activity/expression to balance synthesis with need. AGAT (GATM) deficiency is one of the three cerebral creatine deficiency syndromes (CCDS), along with GAMT deficiency and SLC6A8 (creatine transporter) deficiency. Shared features: global developmental delay, language impairment, hypotonia; AGAT cases skew toward myopathy/weakness, while seizures are more prominent in GAMT/CRTR. Biomarkers: low GAA with low creatine (plasma/urine/CSF) and low brain creatine on MRS—a distinct pattern from GAMT (high GAA) and SLC6A8 (high urine creatine/creatinine ratio). Treatment: typically oral creatine monohydrate (early initiation improves outcomes); dietary restriction is not usually required for AGAT deficiency. Cysteine residues are required catalytically.
GAMT - Guanidinoacetate methyltransferase; methylates GAA → creatine (SAM donor). The GAMT gene provides instructions for making the enzyme guanidinoacetate methyltransferase. This enzyme participates in the two-step production of the compound creatine from the amino acids glycine, arginine, and methionine. In the first step, glycine and arginine combine via the AGAT enzyme (Arginine:Glycine amidinotransferase) to form ornithine and guanidinoacetate. Guanidinoacetate then receives a methyl donation from SAMe via the enzyme guanidinoacetate methyltransferase (GAMT), which produces S-adenosylhomocysteine and creatine. Creatine is needed for the body to store and use energy properly. Deficiencies in GAMT are a bit more severe (although equally rare) relative to AGAT, resulting in severe mental retardation and autism-like symptoms. GAMT gene mutations impair the ability of the guanidinoacetate methyltransferase enzyme to participate in creatine synthesis, resulting in a shortage of creatine. The effects of GAMT deficiency are most severe in organs and tissues that require large amounts of energy, especially the brain. Creatine is also a neurological nutrient. Individuals who cannot produce endogenous creatine suffer from a form of mental retardation with autistic-like symptoms due to deficiencies in the enzymes of creatine synthesis (AGAT or GAMT). http://ghr.nlm.nih.gov/condition/guanidinoacetate-methyltransferase-deficiency. SAME is the required cofactor.
GENES INVOLVED IN THE Production and Delivery Of Phosphatdyl Choline to Methylation Cycle
CERS1 - Ceramide synthase 1; acylates sphinganine/sphingosine with C18:0-acyl-CoA → (dihydro)ceramide (brain-enriched). The CERS1 gene encodes a ceramide synthase enzyme, which catalyzes the synthesis of ceramide, the hydrophobic moiety of sphingolipids. The encoded enzyme synthesizes 18-carbon (C18) ceramide in brain neurons. Elevated expression of this gene may be associated with increased longevity, while decreased expression of this gene may be associated with myoclonus epilepsy with dementia in human patients. This protein is transcribed from a monocistronic mRNA as well as a bicistronic mRNA, which also encodes growth differentiation factor 1. Acetyl-CoA is substrate. Biallelic variants reported with progressive neurodegeneration/epilepsy; rare human data.
CERS2 - Ceramide synthase 2; uses very‑long‑chain acyl‑CoAs (C22–C24) for ceramide (myelin/liver). The CERS2 gene encodes a protein that may play a role in the regulation of cell growth. Acetyl-CoA is substrate. Human LOF linked to hypomyelinating leukodystrophy–like features (emerging); KO mice: severe myelin defects.
CERS3 - Ceramide synthase 3; ultra‑long‑chain acyl‑CoAs (C24–C36) for epidermal ceramides. The CERS3 gene is a member of the ceramide synthase family of genes. The ceramide synthase enzymes regulate sphingolipid synthesis by catalyzing the formation of ceramides from sphingoid base and acyl-coA substrates. This family member is involved in the synthesis of ceramides with ultra-long-chain acyl moieties (ULC-Cers), important to the epidermis in its role in creating a protective barrier from the environment. The protein encoded by this gene has also been implicated in modification of the lipid structures required for spermatogenesis. Acetyl-CoA substrate. Autosomal recessive congenital ichthyosis with alopecia and skin barrier defects.
CERS4 - Ceramide synthase 4; prefers C18–C20 acyl‑CoAs (skin, liver, nervous system). Enables sphingosine N-acyltransferase activity. Involved in ceramide biosynthetic process. Acetyl-CoA substrate.
CERS5 - Ceramide synthase 5; favors C16:0 acyl‑CoA (ubiquitous). The CERS5 gene encodes a protein that belongs to the TLC (TRAM, LAG1 and CLN8 homology domains) family of proteins. The encoded protein functions in the synthesis of ceramide, a lipid molecule that is involved in a several cellular signaling pathways. Acetyl-CoA substrate. No established Mendelian phenotype; metabolic/cardiovascular associations reported.
CERS6 - Ceramide synthase 6; favors C14–C16 acyl‑CoA (kidney, adipose, brain). Enables sphingosine N-acyltransferase activity. Involved in ceramide biosynthetic process. Located in membrane. Acetyl-CoA Substrate. No established Mendelian phenotype; variants linked to metabolic traits (emerging).
SPHK1 - Sphingosine kinase 1; phosphorylates sphingosine → S1P (cytosol → secretion). The protein encoded by the SPHK1 gene catalyzes the phosphorylation of sphingosine to form sphingosine-1-phosphate (S1P), a lipid mediator with both intra- and extracellular functions. Intracellularly, S1P regulates proliferation and survival, and extracellularly, it is a ligand for cell surface G protein-coupled receptors. This protein, and its product S1P, play a key role in TNF-alpha signaling and the NF-kappa-B activation pathway important in inflammatory, antiapoptotic, and immune processes. Phosphorylation of this protein alters its catalytic activity and promotes its translocation to the plasma membrane.
SPHK2 - Sphingosine kinase 2; nuclear/ER/mito pools of S1P; gene regulation & mito function. The SPHK2 gene encodes one of two sphingosine kinase isozymes that catalyze the phosphorylation of sphingosine into sphingosine 1-phosphate. Sphingosine 1-phosphate mediates many cellular processes including migration, proliferation and apoptosis, and also plays a role in several types of cancer by promoting angiogenesis and tumorigenesis. ATP and Mg cofactors. No established Mendelian disease; rare variants described in research settings.
SGPP1 - Sphingosine‑1‑phosphate phosphatase 1 (ER); dephosphorylates S1P → sphingosine + Pi. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biologic processes. SGPP1 catalyzes the degradation of S1P via salvage and recycling of sphingosine into long-chain ceramides.
ETNK1 - Ethanolamine kinase 1; phosphorylates ethanolamine → P‑Etn (first step to PE). The ETNK1 gene encodes an ethanolamine kinase, which functions in the first committed step of the phosphatidylethanolamine synthesis pathway. This cytosolic enzyme is specific for ethanolamine and exhibits negligible kinase activity on choline. ATP and Mg are cofactors.
ETNK2 - Ethanolamine kinase 2; overlaps with ETNK1 (tissue‑biased expression). The protein encoded by the ETNK2 gene is a member of choline/ethanolamine kinase family which catalyzes the first step of phosphatidylethanolamine (PtdEtn) biosynthesis via the cytidine diphosphate (CDP) ethanolamine pathway. ATP and Mg are cofactors.
PYCT1A - CTP:phosphocholine cytidylyltransferase α; makes CDP‑choline (rate‑limiting for PC synthesis). The PCYT1A gene belongs to the cytidylyltransferase family and is involved in the regulation of phosphatidylcholine biosynthesis. CTP and Mg are cofactors. Spondylometaphyseal dysplasia with cone‑rod dystrophy; lipodystrophy/steatosis spectrum.
PCYT1B - CTP:phosphocholine cytidylyltransferase β; neuronal/retinal isoform. The protein encoded by the PCYT1B gene belongs to the cytidylyltransferase family. It is involved in the regulation of phosphatidylcholine biosynthesis. CTP and Mg are cofactors. Emerging/rare neurodevelopmental presentations reported (limited cases).
PCYT2 - CTP:phosphoethanolamine cytidylyltransferase; makes CDP‑ethanolamine (PE synthesis). The PCYT2 gene encodes an enzyme that catalyzes the formation of CDP-ethanolamine from CTP and phosphoethanolamine in the Kennedy pathway of phospholipid synthesis. CTP and Mg are cofactors. Recessive PCYT2 deficiency → neuromuscular disease/spasticity, failure to thrive.
CEPT1/SEPT1- Choline/ethanolamine phosphotransferase 1 (ER): CDP‑choline or CDP‑ethanolamine + DAG → PC or PE. The CEPT1 gene codes for a choline/ethanolaminephosphotransferase, which functions in the synthesis of choline- or ethanolamine- containing phospholipids. Mg/Mn cofactors.
SELENOI - Ethanolamine phosphotransferase 1 (Golgi/ER): CDP‑ethanolamine + DAG → PE (selenoprotein). The multi-pass transmembrane protein encoded by the SELENOI gene belongs to the CDP-alcohol phosphatidyltransferase class-I family. It catalyzes the transfer of phosphoethanolamine from CDP-ethanolamine to diacylglycerol to produce phosphatidylethanolamine, which is involved in the formation and maintenance of vesicular membranes, regulation of lipid metabolism, and protein folding. This protein is a selenoprotein, containing the rare selenocysteine (Sec) amino acid at its active site. Sec is encoded by the UGA codon, which normally signals translation termination. The 3' UTRs of selenoprotein mRNAs contain a conserved stem-loop structure, designated the Sec insertion sequence (SECIS) element, that is necessary for the recognition of UGA as a Sec codon rather than as a stop signal. Mg/Mn cofactors along with selenocysteine (cysteine required). SELENOI deficiency → neurodevelopmental disorder with epilepsy/spasticity; PE deficiency in cells.
CHPT1 - Choline phosphotransferase 1 (Golgi): CDP‑choline + DAG → PC. Enables diacylglycerol cholinephosphotransferase activity. Involved in phosphatidylcholine biosynthetic process and platelet activating factor biosynthetic process. Mg/Mn cofactors. Biallelic CHPT1 variants → neurodevelopmental disorder (microcephaly, hypomyelination, optic atrophy).
CHKA - Choline kinase α; phosphorylates choline → phosphocholine (first step to PC). CHKA encodes for choline kinase alpha and catalyzes a major step in CDP-choline synthesis, and thus phosphatidylcholine. Research suggests CHKA mutations may contribute to shifts in the metabolic fate of choline. ATP and Mg are cofactors. CHKA deficiency → neurodevelopmental disorder with epilepsy and hypotonia.
CHKB - Choline kinase β; muscle‑biased isozyme. CHKB encodes for choline kinase beta, which catalyzes the first step of phosphatidylcholine/ phosphatidylethanolamine synthesis. ATP and Mg are cofactors. Recessive CHKB deficiency → congenital muscular dystrophy with megaconial myopathy.
PEMT - ER‑membrane enzyme: PE → PC via three SAM‑dependent methylations (endogenous PC biosynthesis). PEMT uses SAMe to methylate Phosphatidylethanolamine to form Phosphatidylcholine. This reaction generates SAH as a byproduct. In humans, phosphatidylcholine is only made by the PEMT enzyme. Estrogen is required for the PEMT enzyme to activate and function normally. Men and postmenopausal women have an elevated risk of choline deficiency due to low estrogen levels. The PEMT enzyme is commonly slowed down by polymorphisms, making it unresponsive to estrogen levels. 74% of women have at least one copy of a slowed PEMT. Homozygous carriers of PEMT have much higher risk of choline deficiency. Men, postmenopausal women, and premenopausal women with PEMT SNPs need to increase choline intake in the diet to offset elevated risk of liver dysfunction. SAM is cofactor.
SLC44A - Choline transporter‑like proteins (plasma membrane/mitochondria); supply choline for PC/Bet pathway. SLC44A1 encodes for a ubiquitous choline transporter, which has an important role in the regulation and transport of choline across the cellular and mitochondrial membrane. SLC44A1 may also have a role in membrane, and myelin synthesis. Cysteine required for catalytic activity. No single established Mendelian disorder for SLC44A family; SLC44A2 risk allele in thrombosis; antibody target in immune hearing loss.
CHDH - Choline dehydrogenase (mitochondrial); oxidizes choline → betaine aldehyde (→ betaine for methylation). CHDH is the first step in oxidation of Choline to Betaine. Choline dehydrogenase, a flavin-protein encoded by CHDH, is primarily found in mitochondria and catalyzes the production of betaine (TMG) from choline. Mutations in this gene may alter susceptibility to choline deficiency, choline metabolism, and thus PEMT activity. FAD is required cofactor. No classic Mendelian deficiency; variants associated with male infertility and choline oxidation capacity.
SUMMARY
As one can see from the above, determining supports for the methylation cycle, involves more than just methyl folate and b12. It involves many nutrients and cofactors for the above genes. The order in which you support this process is also highly individual - everybody is a bit different both in genetics and in nutritional status. Before you jump into taking methyl folate or b12 - pausing and having somebody support you in your exploration who is well versed in the various aspects is likely a prudent choice.