methylation cycle and it\'s polymorphism

$: METHYLATION CYCLE AND IT\'S POLYMORPHISM$
Karl GoldkampKarl Goldkamp ND, ND, L.Ac., Dipl. Oriental MedicineL.Ac., Dipl. Oriental Medicine

The Center For Natural Medicine Family PracticeThe Center For Natural Medicine Family Practice81 Halls Road, Old Lyme CT. 81 Halls Road, Old Lyme CT.

The Methylation cycle The Methylation cycle

and and

its common polymorphismsits common polymorphisms

What is MethylationWhat is Methylation

It is the process by which a single carbon atom (a methyl group) is transferred from a methyl donor to another molecule.

Why is Methylation importantWhy is Methylation important

• The most important example is the epigenetic regulation of gene expression by DNA methylation.

– When DNA is methylated, it is incapable of being expressed, in other words it is turned off.

Why is methylation important con’tWhy is methylation important con’t

– Since all cells contain the same DNA, then the differences between cell types

• (heart muscle vs brain neurons vs liver cells) • are the specific pattern of DNA methylation for that

tissue.


• Development of tissues begins with the undifferentiated cells –stem cells

• Which are guided in changes by sequential shifts in their DNA methylations until they become the ultimate specific tissue (heart, brain, liver etc).


• Abnormal methylation– Then changes the development of the various

tissues.• This is process is thought to contribute to

neurodevelopment disorders such as Autism and ASD.

• Abnormal methylation has been considered a causative factor in Rett & fragile-X syndromes.

Deth, Richard. Molecular Aspects of Thmerosal-induced Autism

Homocysteine & MethylationHomocysteine & Methylation

• It was through the gradual acceptance of the

Homocysteine theory of arteriolsclerosis – that the components of the Methylation cycle

were identified

Homocysteine TheoryHomocysteine Theory of of ArteriosclerosisArteriosclerosis

• Attributes one of the underlying causes of vascular disease to elevation of blood homocysteine concentrations as the result of dietary, genetic, metabolic, hormonal, or toxic factors.

• Homocysteine is a key by-product of the methylation cycle.

McCully, Kilmer. Homocysteine, vitamins, and vascular disease prevention, 2007

The Homocysteine storyThe Homocysteine story

• Homocysteine in the body derives from methionine, – an essential amino acid present in large

amounts in protein from animal sources like meat, eggs and milk (but not in soy)

The Fall and Rise Of Kilmer McCully New York times Magazine, 8-7-1997

The Homocysteine storyThe Homocysteine story

• If there are adequate levels of vitamins B6, B12 and folic acid in the body, the homocysteine is broken down into harmless waste products or protein building blocks. But if there's a deficiency of those vitamins, the homocysteine begins its ravages on the blood vessels.

The Fall and Rise Of Kilmer McCully New York times Magazine, 8-7-1997

The road to discoveryThe road to discovery

• Dietary deficiency of vitamin B-6 and folic acid and absorptive deficiency of vitamin B-12, which result from traditional food processing or abnormal absorption of B vitamins, are important factors in causing elevations in blood homocysteine.



The homocysteine theory took nearly 30 years to be accepted in the U.S. medical community

• it has been one of the factors that resulted for the dramatic decline in cardiovascular mortality since the 50s

• And having B6 and folic acid added to the food supply

The Road to DiscoveryThe Road to Discovery

• At the time the proposal of the Homocysteine theory was in contrast to accepted theory of the day in the medical community: the cholesterol theory of arteriosclerosis

• Its’ chief proponent, Dr. Kilmer Mccully eventually lost his job at Harvard because of his alternative theory.

Kilmer McCully get his dueKilmer McCully get his due

19972000


• Dr. McCully was the first to identify the connection between elevated homocysteine and vascular disease

• Published a paper in 1969– Vascular disease which had reached it’s peak

in 1955 in the US– He made the connection between the case of

homocystinuria (from 1933) to a cobalamin C disease (1968)

Other cases Other cases

• In 1953 the amino acid methionine inhibited experimentally induced atherogenisis and eleveated cholesterol level monkeys

• suggesting an association between the sulfur amino acid vascular disease.

Stare FJ. Experimental atherosclerosis in cebus monkeys. J Exp Med 1953; 98:195-218

Other cases con’tOther cases con’t

• In 1962 children with – Mental retardation, dislocated ocular lens,

accelerated growth, osteoporosis, and a tendency to thrombosis of arteries and veins and increased homocysteine in their urine

– Had an deficiency of cystathione synthase (an enzyme that links the methylation cycle to the transulfuration cycle requiring B-6).

Carson, NAJ. Metabolic abnormalities detected in a survey of mentally backward individuals in Northern Ireland. Arch Dis Child 1962


• 1968, two month old boy with growth failure and pneumonia

• Had homocysteine, cystathionine, and methylmalonic acid in the urine and advance atherioclerosis.

• Also a deficiency of Methionine synthase (MTR)

– An enzyme dependent on B12 and methylenetetrahydrofolate

• was later called cobalamin C disease



• 1976, a child with homocysteinuria, and atheriosclerotic plaques throughout the body was determined to have a deficiency of methylenetetrahydrofolate reductase – MTHFR

Putting the associations togetherPutting the associations together

• The previous cases suggested that somehow the three enzymes

• methionine synthase (MTR) • methylenetetrahydrofolate reductase (MTHFR) • and cystathione synthase (CBS)• And Methionine deficiency all had something to do with

elevated Homocysteine…….. What?

Copyright restrictions may apply.

Hubner, R. A. et al. Hum. Mol. Genet. 2007 16:1072-1077; doi:10.1093/hmg/ddm055

Schematic representation of folate metabolism

This diagram represents both the folate cycle and the methylation cycle (aka This diagram represents both the folate cycle and the methylation cycle (aka the SAM or Methionine cycle).the SAM or Methionine cycle).

Steps in the Methylation cycleSteps in the Methylation cycle

• Methionine is converted to SAMe – In the presents of magnesium, ATP (universal energy

donor) by the enzyme methionine- adenosyltransferase (MAT)

• SAMe is considered the universal methyl donor– It is the primary source of methyl groups for most

other biochemical reactions including methylation of DNA, RNA, protein, neurotransmitters, creatinine

Steps in the methylation cycleSteps in the methylation cycle

• Methionine is converted to SAMe (S-adenosylmethionine)

– In the presents of magnesium, ATP (universal energy donor) by the enzyme methionine- adenosyltransferase (MAT)

• SAMe is considered the universal methyl donoruniversal methyl donor– It is the primary source of methyl groups for most other

biochemical reactions including methylation of DNA, RNA, protein, neurotransmitters, creatinine

• Once same donates its methyl group it becomes SAH S-adenosylhomocysteine


• S-adenosylhomocysteine is then metabolized to homocysteine by SAHH

– If you need to know is called• S-adenosylhomocysteinehydrogenase.


• Homocysteine is removed as an intermediate in three ways

1. A reverible reaction that converts homocysteine back to methionine (folate cycle dependent).

2. An irreversible reaction through the trans-sulfuration to crystathione and on to glutathione

3. Converted back to methione independent of the folate cyle via betaine (aka trimethylglycine) BHMT (betainehomocysteinemethyltransferase)

The Methylation CycleThe Methylation Cycle

The Betainehomocysteine-The Betainehomocysteine-Methyltransferase (BHMT)Methyltransferase (BHMT)

conversion of Homocysteine conversion of Homocysteine

A Function of FolateA Function of Folate

• Folate is needed for the generation of SAM

(s-adenosylmethionine)

• A deficiency of SAM is associated with congenital abnormalities, cancers, cardiovascular disease, neural tube defect, and peripheral neuropathies.

Dietary Folate & the Folate cycleDietary Folate & the Folate cycle

The Folate cycle (simply put) :

Tetrahydrofolate (THF) is converted to

5,10 methyleneTHF which in turn gets converted to (with the help of B2B2)

5-methyl THF (aka MTHF and “Folapro”) is then converted back to THF.

Dietary Folate & the Folate cycleDietary Folate & the Folate cycle

• Dietary folate is converted into Dihydrofolate (DHFDHF) in the presence of B3.

• DHFDHF is then converted to THF with the help of B3.

• THFTHF is converted to 5,10 MTHF5,10 MTHF with the help of B6 and serine.

– THF get a CH2- (methylene group) from serine to become 5-10 5-10 MTHFMTHF

– Folinic acid (5-formyl THF5-formyl THF) is also converted to 5,10 MTHF5,10 MTHF simultaneously

The Methylation CycleThe Methylation Cycle

The Methylation cycle and the Folate cycle

Another way to view itAnother way to view it

MS / MSR

MTHFR

CBS

5,10 methyleneTHF

Think of this as a coupling of Think of this as a coupling of different cyclesdifferent cycles

Redox Rep 2003, 8(1): 57-63

MethylationMethylation -> -> Trans-sulfurationTrans-sulfuration –> –> glutathionine synthesisglutathionine synthesis

Under conditions where homocysteine conversion to methionine is dominant then folate and B12 are critical micronutrients.

During oxidative stressDuring oxidative stress

With oxydative stress the conversion of homocysteine to cysteine and glutathione,is the dominant flow and B6 is the critical micronutrient. While the transmethylation of homocysteine to methionine is inhibited.

Homocysteine regulationHomocysteine regulation

Homocysteine is removed either

1. by its irreversible conversion to cysteine

(trans-sulfuration) or

2. to methionine (remethylation).

Think of this as a coupling of Think of this as a coupling of different cyclesdifferent cycles

Redox Rep 2003, 8(1): 57-63

Homocysteine regulation con’tHomocysteine regulation con’t

There are two separate remethylation reactions:

1. Catalyzed by betaine (aka tri-methylglycine): homocysteine methyltransferase and

2. methionine synthase, respectively.

The reactions that remove homocysteine are very sensitive to B vitamin

Trans-sulfuration enzymes require pyridoxal phosphate (B6), methionine synthase contains cobalamin (B12) and receives its

methyl group from folic acid.

MTHFRMTHFR((methylenetratrahydrofolate reductasemethylenetratrahydrofolate reductase))

The MTHFR MTHFR enzyme is an essential enzyme in the folate cycle.

It catalyzes

5,10-methylenetetrahydrofolate5,10-methylenetetrahydrofolate 5-methyltetrahydrofolate5-methyltetrahydrofolate. .

The later serves as a methyl donor for the remethylation of homocysteine to methionine through B12 to methyl-B12.

MTHFR is the most common SNP in the pathway.

MTHFR con’tMTHFR con’t• Methylenetetrahydrofolate exists in the cytoplasm of cells,

except vascular tissue.

• It directs folate species either to DNA synthesis or to homocysteine remethylation to methionine.

• MTHFR irreversibly reduces 5,10-methylenetetrahydrofolate5,10-methylenetetrahydrofolate (substrate) to 5-methyltetrahydrofolate.5-methyltetrahydrofolate.

• 5-Methyltetrahydrofolate is used to convert homocysteine (potentially toxic amino acid) to methionine by the enzyme methionine synthase.

The Methylation cycle and the Folate cycle

The polmorphismsThe polmorphisms

• A gene mutation is a permanent change in the DNA sequence that makes up a gene.

• Mutations range in size from a single DNA base to a large segment of a chromosome.

• A Single Nucleotide polymorphism is a small genetic change, or variation. – The genetic code is specified by four nucleotide “letters” AA

(adenine), CC (cytosine), TT (thymine), GG (guanine). – SNP occurs when a single variation occurs when a single

nucleotide is replaced by another one, eg C -> T or A -> G.

• It is a difference in the DNA sequence from that of the most common type in the population (the wild type).

Single nuclear polymorphismSingle nuclear polymorphism

SNPs of MTHFRSNPs of MTHFR

• There are approximately 24 known snps for this enzyme, however only two have primarily been studied

• C677T (the most studied) and

• A1298G

MTHFR C677T Polymorphism MTHFR C677T Polymorphism and its role in diseaseand its role in disease

• A common SNP in MTHFR is at basepair 677 (a change from a CytosineCytosine

to ThymineThymine, C T) that results in a enzyme with decreased enzymatic activity.

• 10% of North American population are homozygous for this polymorphism, estimated reduction in function is approx. 70% less then the wild type MTHFR.

• Individuals of 677TT are predisposed to mild hyperhomocysteinemia because they have less – or slower functioning MTHFR -available to produce 5-methyltetrahydrofolate (which is used to decrease homocysteine).

• Low dietary intake of the folic acid can also cause mild hyperhomocysteinemia. This polymorphism and mild hyperhomocysteinemia are associated with neural tube defects in offspring, arterial and venous thrombosis, and cardiovascular disease.

Schwahn B, Rozen R (2002). "Polymorphisms in the methylenetetrahydrofolate reductase gene: clinical consequences.". American journal of pharmacogenomics : genomics-related research in drug development and clinical practice

MTHFR C677T Polymorphism MTHFR C677T Polymorphism and its role in diseaseand its role in disease

• It is interesting to note that 677TT individuals are at a decreased risk for certain leukemias and colon cancer, but only when their dietary intake of folate is high.

• The MTHFR gene could be one of the factors of overall schizophrenia risk. Schizophrenic patients having the risk allele (T\T) show more deficiencies in executive function tasks.

Schwahn B, Rozen R (2002). "Polymorphisms in the methylenetetrahydrofolate reductase gene: clinical consequences.". American journal of pharmacogenomics : genomics-related research in drug development and clinical practice

C677T MTHFR & DepressionC677T MTHFR & Depression

• In a large population study from Norway increased plasma homocysteine was associated with increased risk of depression but not anxiety.

• There is now substantial evidence of a common decrease in serum/red blood cell folate, serum vitamin B12 and an increase in plasma homocysteine in depression.

Coppen, A. J Psychopharmacol. 2005 Jan;19(1):59-65. Treatment of depression: time to consider folic acid and vitamin B12.

MTHFR & depressionMTHFR & depression

• Furthermore, the MTHFR C677T polymorphism that impairs the homocysteine metabolism is shown to be overrepresented among depressive patients…

• On the basis of current data, we suggest that oral doses of both folic acid (800 microg daily) and vitamin B12 (1 mg daily) should be tried to improve treatment outcome in depression.

•Coppen, A. J Psychopharmacol. 2005 Jan;19(1):59-65. Treatment of depression: time to consider folic acid and vitamin B12.

Neural Tube DefectNeural Tube Defect

• “It is proven that folic acid supplied in the periconceptional period can lower the recurrence and occurrence rate of neural tube defects (NTDs).

• Preventable NTDs are partly based on hyperhomocystinemia and a genetic predisposition mutation of the methylenetetrahydrofolate-reductase gene (MTHFR) …….

The bottom line:• This genetic metabolic defect can be overcome by This genetic metabolic defect can be overcome by

treatment with folic acid and/or vitamin B12.treatment with folic acid and/or vitamin B12.

Folates and the fetus, European Journal of Obstetrics & Gynecology and Reproductive Biology,

Volume 71, Issue 2, February 1997, Pages 105-111

Where can we supplementWhere can we supplement

TestingTesting5 year old ASD (Austistic spectrum disorder) child

Metametrix labs www.metametrix.com

Oxidative Stress IndicatorsOxidative Stress Indicators

Lipid Peroxides - SerumDNA/Oxidative Stress Marker - Urine

Standard testing offered by a number of labs

Intracellular testing Intracellular testing of antioxidant functionof antioxidant function

Spectracell Total Antioxidant Function

isolated peripheral blood mononuclear cells (lymphocytes, WBCs) that arestimulated to grow in a chemically-defined, serum and protein-free media. Cumene hydroperoxide is added to the media to provide an oxidative stress,and after several days of incubation, lymphocyte growth is measured bytritiated thymidine incorporation – this measures total cell proliforation

All factors that influence antioxidant status are taken into account since live, metabolically active cells are used.

Since the function of antioxidants is to protect biomolecules from oxidativedamage, this test measures the net ability of antioxidants and repairmechanisms of each individual's own cells, reporting a total assessment ofantioxidant function.

http://www.spectracell.comhttp://www.spectracell.com

Female 60 years old, Hx stroke, Female 60 years old, Hx stroke, meds lipitor with CoQ10, fish oil, vitamin Dmeds lipitor with CoQ10, fish oil, vitamin D

Female 60 years old, hx stroke, Female 60 years old, hx stroke, meds lipitor with CoQ10, fish oil, vitamin Dmeds lipitor with CoQ10, fish oil, vitamin D

Female 56, Hx ulcerative colitis, Female 56, Hx ulcerative colitis, meds: sulfalazine, lipitor,meds: sulfalazine, lipitor,Supplements: folate, fish oil, antioxidant drink, coQ10Supplements: folate, fish oil, antioxidant drink, coQ10

See any patterns?See any patterns?

Female 68, recent dx of sarcoidosisFemale 68, recent dx of sarcoidosis

See any Patterns ?

methylation cycle and it\'s polymorphism

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