Methylation The Human Genome Project

• Completed in April 2003, the HGP gave us the ability to read nature's complete genetic blueprint for building a human being

• DNA contains all of our genes, and is made of 4 chemical bases, that pair up and make the “rungs” of the DNA molecule What is a SNP?

• Single Nucleotide Polymorphism • Variation in a single base pair in the genome • When the genome is copying to make a new cell, a single base pair can be SUBSTITUTED What is a SNP? • Low methylation = more RNA bases incorporated into DNA • This triggers repair mechanisms that increases frequency of DNA being read/repaired resulting in impaired function SNP’s

• Human genome = 10, 000 SNPS • Accounting for differences such as: – appearance, – response to drugs – pathology • Many show no change at all • SNPs are largely governed by epigenetics, toxic exposures and mental emotional health • Some KEY SNPs play a significant role in human health

Key SNPs

MTHFR MTR/MTRR MAT GSTM1 Cyto P450’s BHMT PEMT PON1 GAD GAMT MAO COMT TCN2 SUOX SULT IDO1 Methylation The Methyl Cycle

• Back bone of our physiology

• It’s functional status determines our resistance to environmental toxins and microbes

• We can’t CHANGE our DNA, but if we know your weak links, we can create “nutritional workarounds”

Glutathione

• Regulation of cell growth and division • DNA synthesis and repair • Protein synthesis • Amino acid transport • Enzyme catalysis and activation • Metabolism of toxins including carcinogens, heavy metals, xenobiotics, chemicals, endogenous by- products (HORMONES) • Regulation of homocysteine • Enhancement of systemic immune function including humoral immune function Methyl Cycle Mutation

• They are not disease specific or smoking gun genetic defects – PKU, Sickle Cell Anemia...but they do play a role in predisposing you to disease in general

• The more methylation defects, the greater is your susceptibility to toxicity and infection, and thus the greater will be your risk for disease (often times age related due to accumulation of toxins, or occur at vulnerable times such as brain growth) Epigenetic Modulation

• Impacts DNA methylation

• Modifies HISTONES

• Epigenetic control usually governs fn of SNP

• SNP mutations create gene instability (ex. MTHFR – Riboflavin/FAD) – Change in shape decreases co-factor binding Nutritional Workarounds

Bypassing the impact of SNPs by:

1. Increase concentration of co-factor 2. Supplement with end product

MTHFR – co-factor (active form of B2), end product methyltetrahydrafolate

Methylation

• 85% of the methylation cycle happens in the liver • Southern Ontario = very toxic • NFLD – 3rd most common liver dz • COMT and GSH rely heavily on MTHFR and CBS Methylation

• Turns on and off genes – usually off • Processes chemicals, endogenous toxins, xenobiotics • Builds and breaks down NEUROTRANSMITTERS • Builds immune cells (T-cell, NK cells) • Makes DNA and HISTONES • Produces energy (CoQ10, carnitine, creatine) • Produces myelin • Builds and maintains CELL MEMBRANES Methylation

• Essential to health in 2016 Ø 90, 000 TOXINS (land, air, water) • METHYLTRANSFERASES – mostly require SAMe to donate methyl. SAMe requires ATP.

• Mitochondrial function – need healthy MITO to make ATP – Mitochondrial DNA don’t have HISTONES – high rate of mt DNA mutagenesis = oxidative stress and lipid peroxidation of phospholipid bilayer – CELL DANGER RESPONSE slows methylation – Oxidative stress slows MTR enzyme down impairing methylation Methyl Donors and Essential nutrients

• B2/FAD – MTHFR • Methyl folate – donates methyl group to SAMe • B12 – needed for MS • SAMe – needed to support 200 different pathways – METHYLTRANSFERASES (MT) • Creatine* – uses up half the methyl in the body • Phosphatidylcholine* – consumes a large amounts of SAMe • Glycine - SHMT • Betaine (needs zinc) – needed for BHMT • B6 – needed for CBS, PST, SHMT and many more • Magnesium – needed for COMT

* Primary users of SAMe – creatine (GAMT) and phosphatidycholine (PEMT) Where do you start!?

• Mitochondrial support – CELL DANGER RESPONSE

• Oxidative stress SNPs – CBS, SHMT, BHMT

• Immune regulation / microbiome – IDO, FUT2 IDO1 (indoleamine 2, 3 dioxygenase)

• Catalyzes the degradation of L-tryptophan to N- formylkyrurenine using the superoxide anion as an oxygen donor

• 95% of tryptophan goes down the KYN pathway

• Stress, INF-gamma, and/or LPS shifts the pathway towards production of KYNURENIC ACID (neuroprotective) to help kill microbes

• INF-gamma pulls biopterin away to make neopterin = over time causes immune suppression IDO1 (indoleamine 2, 3 dioxygenase)

• B6/P5P needed to produce KA = deficiency will contribute to QUINOLINIC ACID production

• QUINOLINIC ACID - NMDA agonist = increased GLUTAMATE = excitotoxicity

• IDO SNPs or CDR can cause EXCITOTOXICITY via the KYN pathway

• IDO pathway when working well helps halt growth of microbes and alter T cells IDO1 Assessment

• High Kyurenic and high Quinolinic found in the organic acids test IDO Workarounds – decrease CDR

• Rosmarinic acid inhibits the expression of IDO via its cyclooxygenase inhibiting properties

• Alpha-methyl-tryptophan also inhibits IDO

• COX-2 inhibitors down-regulate IDO, leading to a reduction in kynurenine levels and reducing proinflammatory cytokine activity (CURCUMIN)

• 5-HTP, melatonin, GABA, B6/P5P, Magnesium, Kava, Taurine IDO Workarounds – decrease CDR

• 5-HTP, melatonin, GABA, B6/P5P, Magnesium, Kava, Taurine

• Removing grains from the diet – preferential craving to replenish TRYPTOPHAN

• The most important workaround is managing INFECTIONS by supporting MICROBIOME

• MANAGING STRESS!!!!!!!! MTHFR (methylenetetrahydrofolate reductase)

• #1 gene to look at in neural tube defects / birth defects • Supports 200 other genes

FUNCTIONS

1. Produce folate and SAMe to regulate methylation (supports 200 other enzymes)

2. Recylcles biopterin

Recycling BIOPTERIN

• Decreased by interferon gamma (which increases to fight pathogens) – sucks out oxidized biopterin to make neopterin (leaving less to be recylced to BH4)

• BH4 is required for 4 crucial enzymes that support mood regulation and cardiovascular health Recycling BIOPTERIN Phenylalanine

eNOS

MTHFR and Neurotransmitters How do we know when to treat? MTHFR

• MTHFR C677T Heterozygous – 40% loss of function • MTHFR C677T Homozygous – 75% loss of function

• MTHFR A1298C Heterozygous – 20% loss of function • MTHFR A1298C Homozygous – 40% loss of function

• MTHFR C677T & MTHFR A1298C Compound Heterozygous – 40% loss of function

• Approximately 45% of the population has 1 copy of the MTHFR C677T • 4.7 X increased risk of child with autism with MTHFR • 7X increased risk of child with autism with COMT

MTHFR C677T

• MTHFR C677T reduces the body’s ability to make 5- methyl folate

• MTHFR enzyme is shaped differently = co-factor has trouble binding

• But not just MTHFR C677T is associated with all these conditions 5-MTHF

• Supports production of neurotransmitters (with BH4)

• Helps regulate mood, behaviour and cognition

• Folate needed for methylation = supports 200 other pathways

ANTI-FOLATE DRUGS Analgesics Antibiotics BP Drugs HRT/BCP Diabetic medications Nitric Oxide Antacids Folic acid vs. Folate

• Folic acid does NOT equal folate • Folic acid is NOT found in nature (completely synthetic) – synthesized in 1947 and mandatory in wheat in 1998 • Folic acid must undergo transformations prior to utilization • Folate binding proteins show preferential binding to folic acid – high in breast milk, therefore decreased bioavailabilty of milk folate Mutagenesis. 2013 Nov;28(6):661-71. doi: 10.1093/mutage/get045. Epub 2013 Sep 25

Maternal gene polymorphisms involved in folate metabolism and the risk of having a Down syndrome offspring: a meta-analysis. Yang M1, Gong T, Lin X, Qi L, Guo Y, Cao Z, Shen M, Du Y. Author information: 1Department of Maternal and Child Health, Tongji Medical College, Huazhong University of Science and Technology, No 13 Hangkong Road, Wuhan, Hubei 430030, China.

Abstract Down syndrome (DS) is the most common chromosomal abnormality. Many studies have assessed the association between maternal gene polymorphisms involved in folate metabolism and the risk of having a DS offspring, but data are conflicting. Our study aimed to arrive at a more accurate estimation. Therefore, we carried out a meta-analysis of 26, 17, 9, 15, 9 and 6 case-control studies on the relationship between maternal methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MTR) A2756G, methionine synthase reductase (MTRR) A66G, reduced folate carrier 1 A80G and cystathionine β-synthase 844ins68 polymorphisms and the risk of having a DS offspring. The allele contrast and model- free approach were used. Results showed marginal significant associations for MTHFR C677T, overall [odds ratio (OR) = 1.28 (1.22, 1.46) and generalised odds ratio (ORG) = 1.35 (1.16, 1.57)] and in Caucasian [OR = 1.15 (1.03, 1.29) and ORG = 1.20 (1.04, 1.38)], Asian [OR = 1.68 (1.08, 2.63) and ORG = 1.74 (1.08, 2.80)] and Brazilian [OR = 1.22 (1.04, 1.43) and ORG = 1.28 (1.06, 1.55)] populations; for MTRR A66G, overall [OR = 1.22 (1.02, 1.46) and ORG = 1.31 (1.06, 1.62)]; and for RFC1 A80G, overall [OR = 1.16 (1.02, 1.31) and ORG = 1.18 (1.01, 1.37)]. MTHFR A1298C, MTR 12756G and CBS 844ins68 polymorphisms produced non-significant results. Since potential confounders could not be ruled out completely in this meta-analysis, further studies are needed to confirm these results. Dev Med Child Neurol. 2008 May;50(5):346-52. doi: 10.1111/j.1469-8749.2008.02053.x. Epub 2008 Mar 19.

A milk-free diet downregulates folate receptor autoimmunity in cerebral folate deficiency syndrome.

Ramaekers VT1, Sequeira JM, Blau N, Quadros EV. Author information: Eliminate Dairy Products 1Department of Paediatric Neurology, Centre Hospitalier Universitaire, Liège, Belgium.

Abstract In cerebral folate deficiency syndrome, the presence of autoantibodies against the folate receptor (FR) explains decreased folate transport to the central nervous system and the clinical response to folinic acid. Autoantibody crossreactivity with milk FR from different species prompted us to test the effect of a milk-free diet. Intervention with a milkfree diet in 12 children (nine males, three females; mean age 6y [SD 4y 11mo], range 1-19y), decreased autoantibody titer significantly from 2.08pmol of FR blocked per ml of serum (SD 2.1; range 0.24-8.35) to 0.35pmol (SD 0.49; range 0- 1.32; p=0.012) over 3 to 13 months, whereas FR autoantibody titer increased significantly to 6.53 (SD 6.08; range 0.54-14.07; p=0.013) in nine children who were reexposed to milk for 6 to 14 weeks. In 12 children on a normal diet (eight males, four females; mean age 5y 5mo [SD 4y 1mo], range 1y 6mo-16y 4mo), the antibody titer increased significantly from 0.84pmol of FR blocked per ml (SD 0.39; range 0.24-1.44) to 3.04pmol (SD 1.42; range 0.84-6.01; p=0.001) over 10 to 24 months. Decreasing the autoantibody titer with a milk-free diet in conjunction with folinic acid therapy may be advocated for these patients. BH4 - needed for serotonin, dopamine, NO - very sensitive to oxidative stress

MTHFR ***Aluminum/bacteria, CBS, A1298C

OAT

Epi &NE MTHFR Workarounds – The trouble with PALEO • C667T – increases risk of some cancers • Best approach – no grains (fortified with 3-5 mg daily of synthetic folic acid • No grains = no dihydrafolate } UNCOOKED GREEN LEAFY VEGETABLES a must • No grains = reduced B6 levels (impacts CBS, SHMT, PST and many more) • Yeast depletes B6 • Dairy – creates inflammation, decreases absorption of cysteine Diet and methylation

• Animal protein provides creatine, B12, carnitine, choline • Vegans – higher risk for weak methylation • Paleo – removing grains decreases dietary B6 (supplement or assure adequate nut intake • Athletes – create more free radicals, monitor methylation • Type A / high stress – requires more methyl donors MTHFR Workarounds

• B2, B6, B12, choline, zinc • Add activated folate (~ 1 mg) – consider COMT B2, B6, B12 is needed • Choline • Zinc DO NOT ADD FOLATE FIRST! ADD B12… then MTHF MTR/MTRR (methionine synthase/methionine synthase reductase)

• Unregulated therefore uses up B12 at a faster rate • Methyl or Hydroxy B12 injections based on COMT enzyme • Workarounds – B12, Mg, SAMe

Support MTR/MTRR by supporting BHMT which is active in liver and kidney cells • BHMT – zinc, TMG MTR/MTRR

Slowed by OXIDATIVE STRESS, lead, mercury, nitric oxide, microbes elevated TNF, acetylaldehydes CBS (cystathionine beta synthase)

• Transsulfuration pathway major route of cysteine biosynthesis

• Only pathway capable of removing sulfur-containing amino acids under conditions of excess

• CBS can catalyze alternative reactions that produce hydrogen sulfide, a novel neuromodulator in the brain

• Glutathione is made from cysteine

• Sensitive to oxidative stress

CBS – teeter-totter

• Environmental toxins are more difficult to detoxify • Heavy metal provocation test will often show high lead and mercury • Yeast by products deplete B6

Too much sulfite impairs detoxification ***key function CBS pathway indicators

● Sulfation symptoms such as itchy, hives, rashes, headaches, headaches with red wine. ● Or anxiety and agitation when add in B vitamins ● Most clinically important CBS’s slow down the pathway ● CBS upregulation – oxidative stress, BHMT, CBS C699T CBS Workarounds Dr. Anderson, N.D.

1. Support SUOX with 500 mcg Mo BID for 4-8 weeks

2. Support CBS with P5P and Magnesium

3. Continue those two and try GSH (this can be one week from starting CBS support in most folks - but start low

4. Slowly add methyl support CBS Workarounds – Dr. Anderson, N.D.

• If you get sulfite or methyl Sx then back off on the latter steps and reinforce (or do for a longer time) the former steps for a few weeks then add Tx on again.

• Sulfite excess you see itchiness, headaches, hives or hive like reactions like skin irritations and joint pain.

• Methyl reaction are agitation and catecholamine reactions CBS - NCC “hacks” It’s all about the fats

• NO BEEF – 25, 000 vs. hundreds • B6 P5P, B12, folate, molybdenum, taurine, and magnesium glycinate, carnitine, vitamin E, creatine, PC/PS, SAMe. • Test and treat metals and dysbiosis • PALEO/egg free • Curcumin if can’t do NAC or GSH or DMSA • Worse carnitine? More MITO support • B6 not P5P GAD (glutamic acid decarboxylase)

• CBS produces alpha-ketoglutarate from homocysteine; is converted to glutamate, an EXCITOTOXIN

• GOAL – convert glutamate to GABA – GABA is calming • GAD enzyme mutation reduces GABA • Heavy metals, environmental toxins, viral exposure all increase GLUTAMATE GAD Workarounds

• Glutamine converts to GABA via GAD • Kava Kava stimulates GABA • Diet (grain free!!!), GABA, B6/P5P, NAC • Support glutathione production – mops up glutathione • Remove food additives BHMT (betaine homocysteine methyl transferase)

• Betaine-homocysteine methyltransferase (BHMT) is a zinc metallo-enzyme that catalyzes the transfer of a methyl group from betaine to homocysteine to produce dimethylglycine and methionine respectively

• This enzyme is central to the methylation short cut – active in liver and kidneys only

• It is recommended to address BHMT before addressing MTHFR

• Some BHMT SNPS (2 & 4) will result in more pressure in the CBS pathway requiring more support of managing ammonia BHMT (betaine homocysteine methyl transferase)

• BHMT mutations require support from phosphatidylserine and phosphatidylcholine

• BHMT mutations require support from SAMe and TMG

• Eventually, DMG can be used to regulate this pathway but early use decreases enzyme activity

• Zinc is needed to support this pathway BHMT Treatment

• “BACK DOOR” pathway that pulls homocysteine away from CBS

• If you have CBS or BHMT mutations, you need to support its function...TMG

• PC and PS stimulates BHMT to increase SAMe (the bodies largest methyl donour)

• Enzyme activity influenced by stress; can affect attention (ADHD) BHMT Workarounds

• BHMT requires zinc • Betaine reduces lipid levels in the liver • PC/PS, SAMe + TMG, betaine (beets have high betaine) • Diet: No Dairy • Dimethlyglycine (DMG) inhibits BHMT • BHMT backs up CBS • High betaine and choline protect BC - Reduce BC mortality GAMT (guanidinoacetate methyltransferase)

1/3 of SAMe is spent making CREATINE

- involved in ATP energy recycling and fatty acid oxidation GAMT Related Conditions

Intellectual Disability Limited speech development Autism (social interaction and communication) Self-injurious behaviours Tremors and tics

Creatine is an essential pregnancy support Anyone with methylation impairment can benefit GAMT Workarounds

• Creatine

• Omega 3 and 6

• Vitamin E

• Carnitine

• Phosphatidylcholine COMT (catechol-O-methyl transferase)

• Controls breakdown of epinephrine, dopamine and norepinephrine • Introduces a methyl group to catecholamine by SAM • Determines the amount of methyl donors a person can tolerate (can use hydroxyB12) • SNPS – advantage is more DOPAMINE • Very sensitive to LEAD STRESS • Controls how brain responds to

COMT Workarounds

• Adequate SAM (S-adenosyl methionine) – not too much • Inositol, B6, B12 (HYDROXY) • Magnesium Bisglycinate • DIM • Lithium orotate • Folate, methyl folate – not too much • Curcumin • Melatonin • L-theanine • Mitochondrial support – vitamin E/K, carnitine MAO (monoamine oxidase)

• A family of enzymes that catalyze the oxidation of monamines

• Monoamines are neurotransmittors such as phenylalaine, tyrosine, tryptophan, serotonin, melatonin, dopamine, noradrenaline and adrenaline and the thyroid hormones

• Involved in the regulation of cognitive processes such as emotion, arousal, and certain types of memory

• Monoamine neurotransmitters play an important role in the secretion and production of serotonin breakdown

• “Warrior Gene” – on the X chromosome so males have a greater tendency towards aggression MAO

Anything that injure mitochondria such as oxidative stress, toxin exposure, and the aging process in general will cause an increase in MAO activity regardless of genetics MAO Workarounds?

• St. John’s Wort

• 5-HTP

• Lavender

• Vitamin C

• B6 PEMT (phosphatidyethanolamine N- methyltransferase)

• A family of enzymes that catalyze the oxidation of monamines. • Monoamines = neurotransmitters • Phenylalaine, tyrosine, tryptophan, serotonin, melatonin, dopamine, noradrenaline and adrenaline and the thyroid hormones • Regulates cognitive processes such as emotion, and certain types of memory • Secretion and production of serotonin as well as breakdown • “Warrior Gene” – on the X chromosome so males have a greater tendency towards aggression • PEMT is induced by estrogen PC functions

• Methyl donation, neurotransmission, cell membranes, bioactive phospholipids and cell signalling

• PC makes the neurotransmitter acetylcholine

• Choline is critical during fetal development, when it alters DNA methylation and thereby influences neural precursor cell proliferation and apoptosis.

• There is not enough prenatal PC supplementation or dietary needs met PEMT Workarounds

• Provides PHOSPHATIDYLCHOLINE and potentially, DHA, phosphatidylserine and carnitine

• MTHFR requires more phosphatidylcholine

• Betaine may also be needed

• Phosphatidylcholine vs choline: phosphatidylcholine enhances immune function over choline and is better utilized. Other NCC “Hacks”

• Corn and gluten } HASHIMOTO’s • Vegans needs creatine • Paleo peeps needs B6 • Creatine and PC essential for prenatal care • SOD better with PQQ • SOD better w gingko • SUOX - molybdenum 500mcg • PST – no P5P • CBS – needs carnitines and PC, PS • CBS – increased LYME risk??? References for MTHFR

1. Bae J, et al. Fertil Steril. 2007.Prevalent genotypes of methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) in spontaneously aborted embryos. 2. Brandalize AP, et al. Am J Med Genet A. 2009. Evaluation of C677T and A1298C polymorphisms of the MTHFR gene as maternal risk factors for Down syndrome and congenital heart defects. 3. D'Elia PQ, et al. Reprod Biomed Online. 2014.MTHFR polymorphisms C677T and A1298C and associations with IVF outcomes in Brazilian women. 4. Hecht S, et al. Fertil Steril. 2009.Common 677C-->T mutation of the 5,10-methylenetetrahydrofolate reductase gene affects follicular estradiol synthesis. 5. Holwerda KM, et al. Pregnancy Hypertens. 2016. The association of single nucleotide polymorphisms of the maternal cystathionine-β-synthase gene with early-onset preeclampsia. 6. La Marca A, et al. Fertil Steril. 2013. Polymorphisms in gonadotropin and gonadotropin receptor genes as markers of ovarian reserve and response in in vitro fertilization. 7. Mohammad NS, et al. Psychiatr Genet. 2009.Aberrations in folate metabolic pathway and altered susceptibility to autism. 8. Nordqvist S, et al. Reprod Biomed Online. 2015. Ovarian response is affected by a specific histidine-rich glycoprotein polymorphism: a preliminary study. 9. Rosen MP, et al. Fertil Steril. 2007. Methylenetetrahydrofolate reductase (MTHFR) is associated with ovarian follicular activity. 10. Schalin-Karrila M, et al. Br J Dermatol. 1987.Evening primrose oil in the treatment of atopic eczema: effect on clinical status, plasma phospholipid fatty acids and circulating blood prostaglandins. 11. Song W, et al. Zhonghua Yu Fang Yi Xue Za Zhi. 2014. [An association study between gene polymorphism of the key enzyme's folacin metabolism pathway and plasmatic homocysteine levels in fertile woman]. 12. Wang BJ, et al. Genet Mol Res. 2015.Association between SNPs in genes involved in folate metabolism and preterm birth risk. 13. Weiner AS, et al. Fertil Steril. 2014. Polymorphisms in folate-metabolizing genes and risk of idiopathic male infertility: a study on a Russian population and a meta-analysis. References for MTR/MTRR

1. MTR/MTRR 2. 3. O'Leary, V.B., Mills, J.L., Pangilinan, F., Kirke, P.N., Cox, C., Conley, M., Weiler, A., Peng, K., Shane, B., Scott, J.M., Parle-McDermott, A., Molloy, A.M., Brody, L.C. Mol. Genet. Metab. (2005). Analysis of methionine synthase reductase polymorphisms for neural tube defects risk association. 4. Shi, Q., Zhang, Z., Li, G., Pillow, P.C., Hernandez, L.M., Spitz, M.R., Wei, Q. Pharmacogenet. Genomics (2005) . Polymorphisms of methionine synthase and methionine synthase reductase and risk of lung cancer: a case-control analysis. 5. Yamada, K., Gravel, R.A., Toraya, T., Matthews, R.G. Proc. Natl. Acad. Sci. U.S.A. (2006).Human methionine synthase reductase is a molecular chaperone for human methionine synthase. 6. Lincz, L.F., Scorgie, F.E., Kerridge, I., Potts, R., Spencer, A., Enno, A. Br. J. Haematol. (2003). Methionine synthase genetic polymorphism MS A2756G alters susceptibility to follicular but not diffuse large B-cell non-Hodgkin's lymphoma or multiple myeloma. 7. Lee, H.C., Jeong, Y.M., Lee, S.H., Cha, K.Y., Song, S.H., Kim, N.K., Lee, K.W., Lee, S. Hum. Reprod. (2006) . Association study of four polymorphisms in three folate-related enzyme genes with non-obstructive male infertility. 8. Gemmati, D., Ongaro, A., Scapoli, G.L., Della Porta, M., Tognazzo, S., Serino, M.L., Di Bona, E., Rodeghiero, F., Gilli, G., Reverberi, R., Caruso, A., Pasello, M., Pellati, A., De Mattei, M. Cancer Epidemiol. Biomarkers Prev. (2004). Common gene polymorphisms in the metabolic folate and methylation pathway and the risk of acute lymphoblastic leukemia and non-Hodgkin's lymphoma in adults. 9. Bosco, P., Guéant-Rodríguez, R.M., Anello, G., Romano, A., Namour, B., Spada, R.S., Caraci, F., Tringali, G., Ferri, R., Guéant, J.L. J. Neurol. Neurosurg. Psychiatr. (2004)Association of IL- 1 RN*2 allele and methionine synthase 2756 AA genotype with dementia severity of sporadic Alzheimer's disease. 10. Ma, J., Stampfer, M.J., Christensen, B., Giovannucci, E., Hunter, D.J., Chen, J., Willett, W.C., Selhub, J., Hennekens, C.H., Gravel, R., Rozen, R. Cancer Epidemiol. Biomarkers Prev. (1999). A polymorphism of the methionine synthase gene: association with plasma folate, vitamin B12, homocyst(e)ine, and colorectal cancer risk. 11. Homocysteine concentrations and molecular analysis in patients with congenital heart defects. Galdieri, L.C., Arrieta, S.R., Silva, C.M., Pedra, C.A., D'Almeida, V. Arch. Med. Res. (2007) 12. Niclot, S., Pruvot, Q., Besson, C., Savoy, D., Macintyre, E., Salles, G., Brousse, N., Varet, B., Landais, P., Taupin, P., Junien, C., Baudry-Bluteau, D. Blood (2006) . Implication of the folate- methionine metabolism pathways in susceptibility to follicular lymphomas. 13. Bosco, P., Guéant-Rodriguez, R.M., Anello, G., Barone, C., Namour, F., Caraci, F., Romano, A., Romano, C., Guéant, J.L. Am. J. Med. Genet. A (2003). Methionine synthase (MTR) 2756 (A --> G) polymorphism, double heterozygosity methionine synthase 2756 AG/methionine synthase reductase (MTRR) 66 AG, and elevated homocysteinemia are three risk factors for having a child with Down syndrome. 14. Morita, H., Kurihara, H., Sugiyama, T., Hamada, C., Kurihara, Y., Shindo, T., Oh-hashi, Y., Yazaki, Y. Arterioscler. Thromb. Vasc. Biol. (1999). Polymorphism of the methionine synthase gene : association with homocysteine metabolism and late-onset vascular diseases in the Japanese population. 15. Sarbia, M., Stahl, M., von Weyhern, C., Weirich, G., Pühringer-Oppermann, F. Br. J. Cancer (2006) . The prognostic significance of genetic polymorphisms (Methylenetetrahydrofolate Reductase C677T, Methionine Synthase A2756G, Thymidilate Synthase tandem repeat polymorphism) in multimodally treated oesophageal squamous cell carcinoma. 16. Jacques, P.F., Bostom, A.G., Selhub, J., Rich, S., Ellison, R.C., Eckfeldt, J.H., Gravel, R.A., Rozen, R. Atherosclerosis (2003). Effects of polymorphisms of methionine synthase and methionine synthase reductase on total plasma homocysteine in the NHLBI Family Heart Study. 17. Mosharov, E., Cranford, M.R., Banerjee, R. Biochemistry (2000). The quantitatively important relationship between homocysteine metabolism and glutathione synthesis by the transsulfuration pathway and its regulation by redox changes. 18. Skibola, C.F., Smith, M.T., Hubbard, A., Shane, B., Roberts, A.C., Law, G.R., Rollinson, S., Roman, E., Cartwright, R.A., Morgan, G.J. Blood (2002). Polymorphisms in the thymidylate synthase and serine hydroxymethyltransferase genes and risk of adult acute lymphocytic leukemia. 19. Ulrich, C.M., Curtin, K., Potter, J.D., Bigler, J., Caan, B., Slattery, M.L. Cancer Epidemiol. Biomarkers Prev. (2005). Polymorphisms in the reduced folate carrier, thymidylate synthase, or methionine synthase and risk of colon cancer. References for CBS

1. Bhattacharyya S, et al. PLoS One. 2013 Cystathionine beta-synthase (CBS) contributes to advanced ovarian cancer progression and drug resistance. 2. Sarov M, et al. J Neurol Sci. 2014. A case of homocystinuria due to CBS gene mutations revealed by cerebral venous thrombosis. 3. Boas WV, et al. Rev Bras Ginecol Obstet. 2015. Metabolism and gene polymorphisms of the folate pathway in Brazilian women with history of recurrent abortion. 4. Wu X, et al. Hered Cancer Clin Pract. 2014. Plasma homocysteine levels and genetic polymorphisms in folate metablism are associated with breast cancer risk in chinese women. 5. Fiorito G, et al. Nutr Metab Cardiovasc Dis. 2014. B-vitamins intake, DNA-methylation of One Carbon Metabolism and homocysteine pathway genes and myocardial infarction risk: the EPICOR study. 6. Nuño-Ayala M, et al. Physiol Genomics. 2012. Cystathionine β-synthase deficiency causes infertility by impairing decidualization and gene expression networks in uterus implantation sites. 7. Masud R, et al. Mol Cell Biochem. 2011. Tetra primer ARMS-PCR relates folate/homocysteine pathway genes and ACE gene polymorphism with coronary artery disease. 8. Szczepańska M, et al. Eur J Obstet Gynecol Reprod Biol. 2011. Polymorphic variants of folate and choline metabolism genes and the risk of endometriosis-associated infertility. 9. Holwerda KM, et al. Pregnancy Hypertens. 2016.The association of single nucleotide polymorphisms of the maternal cystathionine-β-synthase gene with early-onset preeclampsia. 10. Suri F, et al. J Neurol Sci. 2014.Diagnosis of cystathionine beta-synthase deficiency by genetic analysis. 11. P.J. Kelly, MB MSc, MRCPI, K.L. Furie, MD MPH, J.P. Kistler, MD, M. Barron, BS, E.H. Picard, MD, R. Mandell, BA and V.E. Shih, MD. N eurology January 28, 2003 vol. 60 no. 2 275-279. Stroke in young patients with hyperhomocysteinemia due to cystathionine beta-synthase deficiency References for GAD

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