Genomics Flashcards
Epigenetics
As an organism grows and develops, carefully orchestrated chemical reactions activate and deactivate parts of the genome at strategic times and in specific locations. Epigenetics is the study of these chemical reactions and the factors that influence them.”
Video on epigenetics
https://www.youtube.com/watch?v=81rFpRsF80c&feature=youtu.be
Intro to Nutrigenomics
Health/Diet (Nutrition in between)
Diet/Genomics (Microarrays, proteomics, metabolomics, epigenomics between)
Genomics/Health (longevity, markers, behvaioral genomics in between)
within all three: (health, diet, genomics) Nutrigenomics
Nutrigenomics vs. Nutrigenetics
Nutritional genomics covers nutrigenomics, which explores the effects of nutrients on the genome, proteome and metabolome,
nutrigenetics, the major goal of which is to elucidate the effect of genetic variation on the interaction between diet and disease.”
If ApoE, may want to suggest dietary changes, however you cannot say it is because of higher risk of Alzheimers. Need to provide supportive literature for docs when sending referral. Need to have a kick-ass disclosure to protect yourself on every level.
Statutory Definition of Nutrigenomics
Nutritional genomics means the consideration of biochemical or genetic information to evaluate how genetics effect gene function and how genetic variation alters nutrient response, including the study of how dietary and other lifestyle choices influence the function of humans at the molecular, cellular, organismal and populational levels.”
Genetics 101/DNA Replication
https://www.facebook.com/AAMB.UK/videos/vb.305735409443436/304240952939950/?type=3
Methylation Basics
what is methylation?
The most common methylated base in humans is 5-methylcytosine.
1.A methyl group is 1 carbon and 3 hydrogens
2.Methylation is the process of adding or subtracting this group from different compounds.
What is the function of methylation?
1. Gene regulation
2. Biotransformation
3. Neurotransmitter synthesis
4. Hormone catabolism (estrogen)
5. DNA and histone synthesis
6. Energy production
7. Build and maintain cell membranes (phosphatidylcholine/serine)
To Add or not to add a methyl donor
-Adding a methyl group or hypermethylation silences gene expression
-Removing a methyl group or hypomethylation activates gene expression.
To silence gene – hyper methylation
To silence, adding in methyl donors.
If heathy, do not want to add methyl donors. Adding donors tells body to stop making DNA, stop transcription. Many supplements are full of methylated micronutrients.
Body should do a good job of methyl donors on it’s own.
Adding them in “shouts” at metabolic pathways. Do not want to add methyl donors in 99% of the time. Methylation only one way to turn on/off genes… many more: Acetylation, etc. Not always methyl donor turning things on/off.
What Negatively Impacts Methylation
- Insufficient cofactors or substrates
- Medications that block absorption (antacids, methotrexate, metformin, etc.)
- High dose niacin depletes methyl groups
- Environmental toxins, heavy metals, and chemical exposure
- Too much substrate causes feedback inhibition
- Genetic mutations
- Mental state
Blocked absorption creates insufficient cofactors and substrates. Too much Vitamin C causes diarrhea – lower doses to actually absorb. Transporters can only handle about 500 mg at a time. Absorption is 98% or so. One dose of 1000mg of Vit C, transporters only handle 50% and the rest is flushed out of the system. Vitamin C is not fat soluble. Vitamin D is, so want a liposomal delivery system. Not for Vitamin C.
What is a SNP?
https://www.youtube.com/watch?v=moQdU7nH-jg
Basics of homocystein metabolism
1.Methionine is methylated homocysteine
2. This is a breakdown product of SAM SAH via AHCY (adenosylhomocysteinase)
3. The only difference is the addition of a methyl group on homocysteine.
4. Methionine = homocysteine + Methyl group
homocysteine metabolism
Dietary Folate/Folic Acid
DHFR/NADPH (DHF > THF)
MTHFD/NADP > 10- Formyl THF
5, 10 Methylene THF>MTHFD1> 10-Formyl THF
5,10 Methylene THF > MTHFR (FAD, NADP),
MTHFR > 5-MTHF> MTR/MTRR (vitamin B12) > Methionine
Betaine > DMG
Methionine uses conversion of Betaine to DMG to make Homocysteine (CBS , B6)> Cystathionine >
Taurine, glutathione, sulfate
BHMT (Zn) is in the conversion with Homocysteine to Methionine
Methylation pathway also recycles B12. Recycles methyl donor to pass off to MTHFR to create methyl donor (methylate folate). Homocysteine can go back to methionine, but not as common as the Glutathione pathway.
Homocysteine catabolism
Three Possible Routes:
MTR/MTRR (50%) – Vitamin B12 and Folate
BHMT – TMG and Zinc
CBS (50%) – Vitamin B6 – Only eliminating route
May use choline/phosphatidylcholine in place of TMG for those with BHMT SNPs that are sensitive to methyl donors as choline is the precursor to betaine.
Betaine may be increased by consumption of beets and Swiss chard.
Not much zinc needed to get methlylation going. Folate interacts, but is not required (first route). CBS Cystationine beta synthase? When client says sensitive to active B vitamins… actually mean methylated.
MTR/MTRR
- Methionine synthase
- Methionine synthase reductase
- Function: Recycles homocysteine back into methionine in order to produce SAM (S-adenosylmethionine)
- SAM: the universal methyl donor
- MTR Cofactor: Zinc, blocked by lead, aluminum and mercury.
- MTRR Cofactors: SAM, FAD, NAD, blocked by lead
Methionine Synthase
(MTR/MTRR)
- Methionine Synthase
- Methionine Synthase Reductase
- Accounts for half of homocysteine catabolism.
- Recycles homocysteine back to methionine.
- IS the connection between one carbon metabolism and methylation.
Methionine>Homocysteine>MTR/MTRR (vitamin B12)> Methionine
BHMT
- Betaine-homocysteine S-methyltransferase
- Cofactor and enzymes: zinc, DMG->TMG via choline, blocked by glucocorticoids.
- Function: Supplies a methyl group to convert
homocysteine back to methionine and betaine to dimethylglycine (DMG) - Betaine + homocysteine → dimethylglycine +
methionine - Mutations may increase homocysteine levels
(theoretically)
How many patients do you have that are Zinc deficient.
Betaine Homocysteine Methyltransferase (BHMT)
- Betaine-Homocysteine S-Methyltransferase
- The short cut through the methylation cycle.
- Supplies a methyl group to convert homocysteine back to methionine and betaine to dimethylglycine (DMG).
CBS overview
- Cystathionine Beta Synthase
- Cofactor: Vitamin B6 (pyridoxal-5-phosphate)
- Function: Converts homocysteine to cystathionine
>Blocked by lead - Product: Hydrogen sulfide (H2S) and cystathionine
- CBS C699T, C1080T, and C1985T variants are fast forward enzymes
- CBS A360A and CBS C9150T variants are slow enzymes.
- Slow or fast CBS mutations may result in autism, down syndrome, connective tissue disease, cancer and homocystinuria.
- (much more on this later)
Cystathionine Beta Synthase
- Cystathionine Beta Synthase
- Accounts for half of homocysteine catabolism
- The only true disposal route
- Converts homocysteine to cystathionine
Case Study : Homocysteine and stress case study
John is a busy executive working 70 hours per week. His symptoms include insomnia, stress and has occasional diarrhea and anxiety.
>You ran an initial four-point adrenal stress index (ASI) which indicated that cortisol is elevated throughout the day.
>A serum homocysteine was also run and was lab high at 16 mmol/L (range is 4-16 mmol/L).
>An organic acids test was also run which includes methylmalonic acid (MMA), formiminoglutamate (FIGLU) and xanthurenate which assess vitamin B12, folate and vitamin B6 status respectively. When elevated these markers indicate a cellular insufficiency. While xanthurenate levels were normal, both MMA and FIGLU were elevated.
>How would you proceed? Are there any SNPs that you would expect to find? Do you think that there is a relationship between stress and homocysteine?
MTR/MTRR
Perhaps MTHFR
Probably block from Homocysteine to cystathionine – B6 deficiency, Probably blocked at Mtr/Mtrr – maybel MTHFR snp. Inflammatory markers not the whole picture.
Stress and methylation
> Talk to me about what you see here.
What is the chemical difference between Epinephrine and Norepinephrine?
What enzymatic process is being catalyzed by PNMT?
What do you think it means for those under chronic stress?
What do you think about their methylation status?
How about their Hcy Levels?
Difference between norepinephrine and Epinephrine is methyl donor – CH3. Stress uses up methyl donors = elevated levels of homocysteine whether system is broken or not.
Simplified Focus on MTHFR- The celebrity SNP
1.URea cycle
2. BH4 Cycle (Tryptophan to Serotonin, Tyrosine> Dopamine)
3. MTHFR
4. Folate Cycle with 5-MethylTHF+ > Methyl B12
5. Mehtylation Cycle (Mehionine) , SAMe, SAH,
6. Homocysteine> B6 input
7. Cysteine>
8. Glutathione
MTHFR
- Methylene Tetrahydrofolate Reductase
- Key Variants: C677T and A1298C
a. C677T heterozygous=40% loss of function
b. C677T homozygous=75% loss of function
c. A1298C heterozygous=20% loss of function
d. A1298C homozygous=40% loss of function
e. Compound heterozygous=40%loss of function - Cofactor(s): Riboflavin, NADH, and ATP
- Approximately 45% of the population has 1 copy of MTHFR C677T (Esp. Mexican, Italian, and Chinese).
MTHFR pathway
Draw and study
Consequences of MTHFR
- C677T variant will increase homocysteine, A1298C will not.
- Increases risk for neural tube defects, miscarriage, dementia, mood disorders, peripheral artery disease, colon cancer, and acute leukemia.
- Common in those with ADHD, autism, depression, Alzheimer’s disease, Parkinson’s disease, coronary artery disease and those with detoxification challenges.
MTHFR Case Study
A 25 year old woman who is 5’4” tall and weighs 178 lbs has been trying to get pregnant for the last year. She has conceived three times only to miscarry early in the first trimester. She has come to you for help in figuring out the problem.
You start by looking at a recent CBC that her gynecologist ran and notice that her MCV is functionally high at 98 (80.0-98.0).
As the next step you decide to run homocysteine, MMA and FIGLU.
Homocysteine: 18
MMA: elevated
FIGLU: elevated
Which SNPs would you expect to find and why?
What role do you think obesity plays in fertility?
MTHFR, MTR/MTRR
PCOS/estrogen dominance.
Macrocytic anemia
3 -6 minutes in
DHF dietary folate
Dihydrofolate Reductase (DHFR)
rs7387, rs19972026, rs1643649, rs1643659, rs1677693, rs1650697
Cofactor NADPH
Codes the enzyme dihydrofolate reductase used in conversion of dihydrofolate into tetrahydrofolate
Dihydrofolate is reduced to tetrahydrofolate and NADPH is oxidized to NADP+
Is the precursor to both one carbon metabolism AND neurotransmitter synthesis.
Causes difficulty in conversion of synthetic folic acid into the active form of folate
Fortification with folic acid is a problem as it requires two DHFR steps for conversion as compared to only one required with natural folate
DHFR is the target for anticancer and antibiotic therapies such as methotrexate and trimethoprim (folate is required by rapidly dividing cancer cells to make thymine therefore inhibition of DHFR can limit growth and proliferation of cancer cells)
“Leucovorin does not require reduction by DHFR to participate in reactions in which folates are used as a source of 1-carbon moieties. Moreover, leucovorin is rapidly converted to other reduced folates, thereby restoring the pool of reduced folates.” (Chuang V.T.G. and Suno, M., 2012 p. 1350)
Consider dosing folinic acid (calcium folinate) rather than L-methylfolate
DHFR and Folic Acid Case Study
A middle-aged, depressed man eating a SAD diet comes to you after visiting a naturopathic physician. The ND prescribed Deplin (prescription L-methylfolate) after running the following labs:
Homocysteine: 10
Serum folate: elevated
FIGLU: Elevated
MMA: Normal
Xanthurenate: Normal
What SNPs would you expect to find and why? Do you think that Deplin is the correct intervention?
Answer:Absence of B12 def., folic acid and methyl folate is not working because of DHFR
Probably has MTHFR
Probably getting too much folic acid through SAD inducing the condition without the SNP?
catecholamine production
Neurotransmitters and hormones from phenylalanine and Tyrosine
The essential amino acid phenylalinine breaks down to tyrosine.
Tyrosine is used in the production of Thyroid hormones, melatonin in skin, and the neurotransmitters, dopamine, norepinephrine, and epinephrine are excreted jointly as vanilmandelate.
High levels of these breaksdown products in the urine identify a high turnover, low levels may indicate inadequate production.
Tetrahydrobiopterin (BH4)
Cofactor required for hydroxylation of aromatic amino acids (i.e. phenylalanine to tyrosine, tyrosine to DOPA and tryptophan to serotonin)
Cofactor in nitric oxide production from arginine
Guanosine triphosphate (GTP) is converted to dihydroneopterin triphosphate catalyzed by guanosine triphosphate cyclohydrolase (GTPCH)
6-Pyruvoyl tetrahydropterin synthase (PTPS) (cofactor magnesium) converts dihydroneopterin triphosphate into 6-pyruvoyl-tetrahydropterin
A final conversion to BH4 is made via the enzyme sepiapterin reductase (SR) using NADPH and NADP as cofactors
BH4 is subsequently used to produce the monoamines dopamine, norepinephrine, epinephrine and serotonin as well as nitric oxide.
BH4 Status
“Restricted BH4 cofactor availability has also been suggested as an etiologic factor in neurological diseases, including DOPA-responsive dystonia, Alzheimer disease, Parkinson disease, autism and depression; as well as in other conditions such as insulin sensitivity and vascular disease. BH4 is obligatory for the activity of all nitric oxide (NO) synthase isoforms and glycerl-ether mono-oxygenase. By its regulation of neuronal NO synthase, BH4 is a neuroprotective factor. Low BH4 levels are also associated with impaired eNOS activity, leading to endothelial dysfunction.”
May be measured via BH4 blood levels, 5-HIAA, 5-MTHF, HVA, Phe/Tyr ratio and Phe blood levels.
Lower BH4 status is often found in depressed individuals.
Depressed individuals often low in BH4
Depression, not get out of bed, not eat, not going places – serotonin
Dopamine wants to go out and do things, don’t enjoy them. – binge eating, sex addicts, etc may be dopamine deficient.
Anxious often excess of neurotransmitter, epinephrine, norepinephrine. Anything pushing dopamine also pushes epinephrine, norepinephrine. Work on anxiety first then address dopamine.
Without proper methyl donors, BH4 status is low
BH4 and Neurotransmitter Synthesis
Notice the connection between DHFR and BH4…what do you think occurs when you have a functional block in DHFR? What causes a functional block in DHFR again? Folic acid
Can look at neurotransmitters through OATs example, elevated HVA and low VMA then dopamine not moving to norepinephrine then cofactor deficiency or snp.
Walsh theory re: depression – copper, vitamin C affects conversion of dopamine to epinepherine
Elevated dopamine – halucinations, mood changes, bipoloar, schizophrenia – induced by supplements may not be able to correct and must be careful.
BH4 and Folate
BH2 is important for the regeneration of BH4 (susceptible to oxidation).
Dihydrofolate reductase (DHFR) is thought to be involved in BH4 regeneration.
“Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 toBH4, leading to eNOS recoupling.”
Folate (5-MTHF) is responsible for regenerating oxidized BH4.
“In the absence of an adequate amount of BH4, 5-MTHF “stands in” for BH4 at the enzyme level. The chemical structures of BH4 and 5-MTHF are similar enough that eNOS will accept 5-MTHF as a substitute cofactor.” (Miller 2008 p.220)
May account for why folate may have an antidepressant effect.
BH4, Phenylanine, and Tyrosine
BH4 Rx ( sapropterin) and discuss supplementation for Bh4 ( niacin, Vit C, folate, b6, mg)
Dopamine depression may also have hypothyroid
Iron overload may lead to hyperthyroid
BH4 is required for the conversion of phenylalanine to tyrosine as a cofactor along with iron for phenylalanine 4-hydroxylase (PAH).
It is also required for the subsequent conversion of tyrosine to DOPA as the cofactor along with iron for tyrosine hydroxylase (TH).
Therefore without proper production of BH4 you will have low dopamine levels which ultimately will also cause decreased production of norepinephrine and epinephrine.
Methylation and Neurotransmitters **DHFR
PAH requires BH4 and iron to catalyze Phenylalanine to Tyrosine
BH4 is also required for the conversion of tyrosine to DOPA
Remember that BH4 is formed via methylation.
VMA is the metabolic breakdown product of norepinephrine and epinephrine
HVA is the metabolic breakdown product of dopamine
Decreased levels of these neurotransmitters can cause depression, sleep disturbances, anxiety and fatigue.
This is the link between methylation and neurotransmitters.
Which SNP/Gene is it that again directly links methylation and neurotransmitters?
DHFR…not MTHFR
Neurotransmitter case study
A 35 year old woman who has been complaining of fatigue, binge eating and depression. Her internist ran some lab work with the following results:
MCV: 80 (80.0-98.0)
Serum iron: 39 mcg/dL (40-155 mcg/dL)
Ferritin: 20 mcg/L (12-150 mcg/L)
TSH: 5.0 (0.35-5.5)
Free T4: 1.0 ng/dL (0.8-2.8 ng/dL)
Functional range is 1.0-2.0 ng/dL
Free T3: 2.7 nmol/L (1.23-3.53 nmol/L)
Functional range is 3.0-3.5 nmol/L)
After looking at these labs you decide to run a NutrEval to assess her neurotransmitter and amino acid levels. Her results are as follows:
Phenylalanine: elevated
Tyrosine: low
HVA: low
VMA: low
What SNPs would you expect to find and why? Do you see any clinical diagnoses? What recommendations would you make (remember cofactors)?
BH4 deficiency, possible PAH
Not converting phe to tyr
Fe is cofactor
Microcytic presentation of red blood cell – Iron or B6 deficiency
Serine Hydroxymethyltransferase (SHMT)
rs9909104, rs1979299
Cofactor—vitamin B6 as the active P5P
Encodes the enzyme serine hydroxymethyltransfase which is involved in cellular one-carbon pathways
We are going to go around the Wheels, so follow along! SHMT is not on the “big” picture, but it is important. It is known as the “gut dysbiosis” SNP.
Requires Vitamin B6 for one carbon metabolism
Catalyzes the reversible, simultaneous conversions of L-serine to glycine and tetrahydrofolate to 5,10-methylenetetrahydrofolate (requires SAM)
Also catalyzes conversion of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate
“The SHMT1 1420C>T SNP (rs1979277; exon 12) has been associated with protection from rectal cancer and with CVD risk in a gene-gene interaction with MTHFR rs1801133 (C677T). SHMT1 rs1979277 reduces the sumoylation of SHMT1, impairing its translocation to the nucleus, suggesting that it reduces nuclear folate metabolism and thymidylate synthesis. The SHMT1 TA haplotype, which includes rs1979277, was associated with higher RBC folate and the CG haplotype was lower RBC folate.”(Zick, Groh and MacFarlane, 2015 pp. 1301-2)
On its own SHMT is the “gut dysbiosis gene” resulting in an increased risk of CVD and CR cancer risk- however in association with MTHFR C671, the risk is remediated. Thus not all SNPs are bad.
CBS overview
- Cystathionine Beta Synthase
- Cofactor: Vitamin B6 (pyridoxal-5-phosphate)
- Function: Converts homocysteine to cystathionine
>Blocked by lead - Product: Hydrogen sulfide (H2S) and cystathionine
- CBS C699T, C1080T, and C1985T variants are fast forward enzymes
- CBS A360A and CBS C9150T variants are slow enzymes.
- Slow or fast CBS mutations may result in autism, down syndrome, connective tissue disease, cancer and homocystinuria.
CBS pathway overview
- Cystathionine Beta Synthase
- Accounts for half of homocysteine catabolism
- The only true disposal route
- Converts homocysteine
CBS Upregulation
- Decreased levels of vitamin B6 and magnesium
- Decreased levels of homocysteine, GSH
- Compromised ability to recycle homocysteine back the universal methyl donor SAM)
- Increased levels of ammonia drain methyl donors and increases levels of nitric oxide to counteract increased ammonia.
- Hydrogen Sulfide increases cortisol and glutamate (excitotoxicity)
Hypomethylating changing balance between SA and SO. When not methylating… creating ammonia and increases cortisol levels.
Glutamate, inflammatory
Excess ammonia – relieve strain on kidney, lessen protein – presentation is brain fog
CBS Upregulation and SUOX
1.Increased flux of sulfur through CBS forces sulfur too quickly downstream
2.This will put undo stress on the enzyme 3. SUOX (Sulfite Oxidase) and depletes molybdenum, SUOX’s cofactor
SUOX has a low capacity
4. A reduced sulfur diet for a few days (never long term) and molybdenum supplementation can help with sulfur intolerance
CBS Upregulation Clinical/Lab Findings
- Elevated urinary sulfates: typically greater than 800. If consistently greater than 1000 then high CBS activity is likely; Sulfur flatulence, sulfur food intolerance, Sulfa drug reactions and halitosis. (Brain Fog!)
- Elevated ammonia and/or sulfate levels along with elevated Orotate, Citrate and Isocitrate on an Organic Acids Test (OAT) indicate elevated ammonia. (Ammonia depletes BH4, leading to insufficient dopamine and serotonin production)
- Typically has an elevated xanthurenate on an OAT test
- Also will have high Taurine - Taurine is synthesized via Cysteine – found on amino acids tests.
- To combat sulfur intolerance: Try a reduced sulfur diet (cruciferous vegetables, eggs) and reduced protein diet for a few days along with molybdenum, the cofactor for SUOX.
CBS Downregulation
- Hypertension/Cardiovascular disorders
- Potentially elevates homocysteine levels and moderately elevates xanthurenate—check MTHFR C677T status.
- Consider N-acetyl cysteine (NAC) to help increase GSH levels, magnesium and P5P.
- Decreased risk of breast cancer with CBS A360A
- Elevates nitrous oxide
- Breathing difficulty/asthma
- Often found in those with autism spectrum disorders
Nutritional deficiencies and Enzyme Function of CBS (down-regulation)
- P5P
- Ammonia
- Homocysteine
- NADH
- Taurine
- Sulfate
- Glutathione
CBS- Transulfuration Pathway
Functional Blocks in the Pathway
So, if you have too much Hcy, do you think you will make the DNA, RNA, protein and lipids that you are supposed to? Methyl donors (SAM) are needed to make creatine. Some suggest that this is why supplementation is helpful in relieving the methylation burden. Most studies suggest that the majority of the methylation drain comes from BHMT while serine, which feeds one carbon unit into the folate pool, contributed 100% of the methyl groups required for remethylation.
Stead, L.M., Brosnan, J.T., Brosnan, M.E., Vance, D.E., and Jacobs, R.L. (2006) Is it time to reevaluate methyl balance in humans?
CBS Case Study
A mother brings her 6 year old autistic son in to see you as she has noticed that when he defecates it smells like rotten eggs. He has currently been on the GAPS diet to help decrease his autistic symptoms, though so far there has been no change.
What labs would you run and why?
What SNPs would you expect to find?
CBS upregulation ( Fast forward)
Can anyone tell us what the GAPS diet is?
OAT/xan
Sulfate should be high
CBS Case Study
You have a 23andme report for a 20 year old female who complains about chemical sensitivities. You notice that she has a homozygous SNP to CBS A360A. Additional testing reveals the following:
Xanthurenate: elevated
Glutathione: low
RBC Magnesium: low
Glutamine: Low
Glycine: Low
Cystine: High
Cysteine: Low
Is this an upregulation or downregulation CBS case? How would you intervene?
CBS down regulation expect high homocysteine
Increase B6, supplement with NAC (N acytle cistine) 100 to 150 mg of p5p
Looking for impairment in methyl donor creation cycle and homocysteine cycle
Glycine and Cysteine required for glutathione
Cobalamin Mechanism
Normal B12 Deficiency
- TCN1 (aka haptocorrin) is secreted by the salivary glands and is a B12 binding protein. TCN1 helps B12 survive the HCL in the stomach and the complex travels to the intestine.
- Gastric intrinsic factor (GIF) is produced by the parietal cells and is also a B12 binding protein.
- Once in the less acidic environment of the intestine, the TCN1/B12 complex can then bind to intrinsic factor and be absorbed in the ileum.
- Once inside the enterocytes of the ileum, B12 breaks apart from TCN1 to bind to TCN2, which then carries the B12 to the liver.
Lots of transporters for B12 because so important
TCN1 problem – hydroxocobolamine
TCN2 problem – andenosine cobolamine
Cyanocobalomine – most widely used in market, kicking out a cyano group, increases cyanide in the body increasing stress
Cobalamin Mechanisms
- It can take up to 3-5 years for a vitamin B12 deficiency to develop.
- About 2-4 mg of vitamin B12 is stored in the body, of which 50% is found in the liver.
- Adenosylcobalamin represents 70% of the vitamin B12 stored in the body, mostly found in the liver.
- Methylcobalamin is the main form of vitamin B12 found in the blood.
- Hydroxocobalamin and methylcobalamin are also stored but to a lesser extent in the muscles, bone, kidney, heart, brain and spleen.
- Methylcobalamin comprises 60-80% of vitamin B12 found in the blood and adenosylcobalamin comprises up to 20% of total plasma cobalamin (Gropper, 2013).
Serum B12 gives proxy form of methylation
Cobolamin Transporter SNPs
- Transcobalamin I (TCN1) helps B12 survive digestion. TCN1 CG rs526934
>Transports up to 80% of vitamin B12
>Thought to function as a circulating storage form and may prevent bacterial use of the vitamin - Transcobalamin II (TCN2) is a major part of the secondary granules found in neutrophils and facilitates the transport of cobalamin to the liver. rs1801198 risk allele G
>20-30% of cobalamin is transported on the TCN2
>Has a half-life of less than two hours
TCN1/TCN2 Consequences
1.It can take up to 3-5 years for a vitamin B12 deficiency to develop.
2.About 2-4 mg of vitamin B12 is stored in the body, of which 50% is found in the liver.
3.Adenosylcobalamin represents 70% of the vitamin B12 stored in the body, mostly found in the liver.
4.Methylcobalamin is the main form of vitamin B12 found in the blood.
5.Hydroxycobalamin and methylcobalamin are also stored but to a lesser extent in the muscles, bone, kidney, heart, brain and spleen.
6.Methylcobalamin comprises 60-80% of vitamin B12 found in the blood and adenosylcobalamin comprises up to 20% of total plasma cobalamin (Gropper, 2013).
May cause an increase in serum vitamin B12 (often even without supplementation) with an increase in MMA
This means that there will be high serum levels and low tissue concentration
Sometimes, if only TCN1 is present, serum levels may be low
Use of Hydroxocobalamin can bypass this transport issue
TCN1/TCN2 Intervention
- Hydroxocobalamin is helpful for those with SNPs to TCN1 and TCN2 as it bypasses the transport mechanism.
- Adenosylcobalamin and hydroxocobalamin is helpful for those with mitochondrial issues. This is because hydroxocobalamin must undergo cytosolic methylation to generate methylcobalamin which can then be reduced with ATP in the mitochondria to yield adenosylcobalamin (Gropper, 2013).
- Those with malabsorption issues require more vitamin B12 supplementation do to enterohepatic circulation (Gropper, 2013).
»Vegans and those with pernicious anemia will also require more vitamin B12 supplementation.
Pure Encapsulations Adenosyl/Hydroxy B12
Overall with snps supplement with nutrient cofactors
B complexes (no more than 400 mcg of folate) many full of junk vitamins, too much niacin
Want to get pregnant? OATs and genomic testing is good to use for these
Correct for B12 and test for B12 deficiency before dosing with folate
TCN3 (AKA GIF)
- Gastric Intrinsic Factor – B12 binding protein that allows absorption into the enterocytes.
- rs558660 – risk allele G
- Associated with:
a.Familial Pernicious Anemia
b.Acute Lymphoblastic Leukemia
c.High-altitude polycythemia
4.In gastritis ( non-H. pylori related), those with the FUT2 secretor variant had decreased amounts of GIF secretion. This was true even in heterozygous FUT2 mutations.
Vitamin B12 Case Study
MG is a 72-year-old female complaining of fatigue and neuropathy in her hands. Patient also has fasciculations in her legs. She has been gluten free for eight years. Her diet is healthy and consists of quinoa with blueberries and sunflower seed butter for breakfast; salmon or sardines with a salad for lunch; and chicken or turkey, sweet potato or squash and green vegetables for dinner. She is currently on no medication. She takes a one-a-day multivitamin.
Homocysteine high – 17 (high)
MMA – 739 (87-318)
FIGLU – 2 (<2.2)
Xanthurenate – 0.99 (<0.96)
Serum vitamin B12 – 1338 (180-914) NOTE: Patient was not on vitamin B12 supplements at the time
RBC – 3.9 (functional range is 3.9-4.5)
Hemoglobin – 13.4 (functional range is 13.5-13.5)
Hematocrit – 42.7 (functional range is 37-44)
Mean Corpuscular Volume – 97 (functional range is 85-92 cu microns)
What abnormalities can you find in her lab work and what SNPs would you expect to find? Are there any diagnosable diseases that you think are likely? What types of recommendations would you make for this woman?
What’s the Issue? Megaloblastic anemia
Macrocytic anemia
Folic acid blocking DHFR from MV
Folate trapping ( Hcy, mthfr, MTR, MTRR, TCN2, MMA, FIGLU)
HCy catabolism issues/detoxification issues – BHMT, CBS, AHCY, Xanthurate
Zinc
Mag
Vitamin D Receptor (VDR) Polymorphins
vitamin D is both a nutrient and a hormone. The gene regulatory effects interact with retinoic receptors and wide classes of immuno-modulatory mediators.
VDR BSM
VDR-BSM vitamin D3 receptor (1, 25-dihydroxyvitamin D3 receptor, nuclear receptor subfamily 1 group I member 1)
Provides instructions for making nuclear vitamin D receptors
rs1544410
Involved in the binding of calcitriol and calcitriol receptor activity
This is the active form of vitamin D
Polymorphism often causes rapid conversion of vitamin D2 to the active vitamin D3
Results in elevated calcitriol/1,25 dihydroxy vitamin D with normal to low 25(OH)D levels
Also look at 1, 25 ohd as well as looking at calcitriol levels
2000 is a safe dose
Over supplementation of Vitamin D can create autoimmune with these people
If autoimmune, don’t stimulate the immune system.
Capsule form in lipid form for Vitamin D supplementation; Liquid form has less contaminants
VDR TAQ
- VDR-TAQ SNPs are often linked to better tolerance of methyl donors (circumstantial evidence only)
- Will potentially cause a decrease in 25(OH)D levels
- “There was limited overlap between genetic determinants of vitamin D status and those associated with non-skeletal health outcomes: polymorphisms in DBP, CYP2R1 and DHCR7 were the most frequent to be reported to associated with circulating concentrations of 25(OH)D, while polymorphisms in VDR were most commonly reported to associate with non-skeletal health outcomes, among which infectious and autoimmune diseases were the most represented.” (Jolliffe, Walton, Griffiths and Martineau, 2015 p. 33153)
Vitamin D Case Study
- You have a new client who comes to you regarding a vitamin D deficiency that their internist has diagnosed. While their 25(OH)D level is lab low they feel awful when taking 50,000 IUs of prescription vitamin D.
>Are there any additional labs that you think would be helpful for this case?
>What genetic SNP do you think is present and why
Discuss Toxicity of Vitamin D, Cacitriol levels need to be checked.
PEMT
rs12325817, rs7946, rs46464006, rs4244593
Cofactor–SAMe
Phosphatidylethanolamine N-methyltransferase (PEMT)—converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver.
Requires three sequential methylations steps using three molecules of SAM
Choline may be obtained from diet (i.e. eggs) or by conversion of choline-containing lipids to PC using PEMT (the only source of choline other than diet)
Required in choline synthesis, hepatocyte membrane structure, bile secretion and VLDL secretion
“Though more than one-half of premenopausal women were resistant to choline deficiency-induced organ dysfunction, those premenopausal women that did require a dietary source of choline had a SNP in PEMT (rs21325817; 74% of women in North Carolina had 1 or more variant alleles), making them unresponsive to estrogen induction of PEMT. This common SNP greatly increased the risk for developing organ dysfunction when participants were fed a low-choline diet (OR=2; P<0.00005, based on 64 women studied).” (Zeusekm S.H. 2011 p.533)
PEMT is also used in the estrogen pathway for the degradation of estrogen.
Estrogen is a positive regulator of hepatocyte PEMT production
Choline (and folate) are needed to form methionine and consequently SAMe
Very important during pregnancy for proper fetal brain development
COMT
Catechol-O-Methyltransferase
Important variants: rs4680, rs769224, rs4633
Cofactors: SAM and magnesium
Functions:
Transfers methyl group from SAM to the catecholamines
Breaks down dopamine, epinephrine, and norepinephrine
Transfers methyl group from SAM to catechols from foods, (green tea, potatoes), antioxidants (quercetin), and hormone catechols (estrogen)
Perfectionism Case Study
You have two workaholic nutritional genomics professors who have tendencies towards perfectionism, occasional anxiety, insomnia and suffer from estrogen dominance. They complain that their brains are always spinning. One has chronically depleted vitamin D. The other has normal vitamin D levels.
Which SNPs would you expect to find in the chronically depleted vitamin D levels professor and why?
Which SNPs would you expect to find in the professor with normal vitamin D levels and why?
Chronically depleted-VDRbsm/ homozygous COMT
Normal Vitamin D- VDR taq- heterozygous COMT
MAO-A
- Monoamine Oxidase A
- Cofactor: Riboflavin (FAD)
- Function: Catalyzes the deamination of amines
>Dopamine, serotonin, epinephrine, norepinephrine - Metabolizes xenobiotic amines
>Heterocyclic amines (meat), tyramines, histamine
>Connected to tryptophan via methylation; look at UAA - Important variant: rs6323 (G variant encodes for the higher activity)
>Mutations increased enzyme activity leading to decreased amounts of amines
>Fast forward=Decreased amines
>Note: there are also slow variants (rs5906883, rs5906957, rs909525, rs9593210 and rs2072743) which will cause increased amines
If upregulated, low levels of catecholines. Upregulation of MAO-A often found in depression, typically serotonin, but can be dopamine also.
Down regulated can cause histamine issues, further depressions, psychosis especially with COMT issues as well.
MAO-B
Monoamine Oxidase B
rs1799836, rs10521432 (C112982T), rs6651806 (T57758G)
Catalyzes the oxidative deamination of biogenic and xenobiotic amines (i.e. histamine and tyramine)
Part of the metabolism of neuroactive and vasoactive amines in the CNS and peripheral tissues
Preferentially oxidizes dopamine whereas MAO-A preferentially oxidizes serotonin and noradrenaline
Associated with depression
MAO-B inhibitors common treatment for those with Parkinson’s disease
Glutathione
Glutamate > Peptide Bonds> Cysteine> Glycine
Dao, apb1, MAU snps, on pubmed for research
Histamine and probiotics in pubmed
Just published series of articles on role of probiotics in disease
Histamine issues generally mast cell
Histamine intolerance buy wild-caught frozen
Glutathione Synthase
Glutathione: major anti-oxidant in detoxification
GSS/GSH
GSS (glutathione synthase) is an enzyme within the gamma-glutamyl cycle.
Aids in the production of glutathione, our major antioxidant used in detoxification
Glutathione:
Protects cells from damaging effects of unstable molecules
Helps process medications and carcinogens,
Helps build DNA, proteins and other important cellular components
Cofactor is magnesium.
Decreased GSS levels are associated with hemolytic anemia
Gamma-Glutamyl Cycle
Pyroglutamate – urinary organic acid – elevated means strong need for glutathione, may be low or on the way to becoming low
GSS mutations may lead to increased levels of pyroglutamate (5-Oxoproline) in the urine (OAT test).
Foods high in glutamic acid such as those containing MSG and high protein foods can be a dietary source of increased pyroglutamate when GSS SNPs are not present.
Supplementation with N-acetyl-cysteine (NAC) may be helpful in increasing glutathione levels.
Taurine may also be used to help spare sulfur based amino acids and is also a good antioxidant.
Other helpful supplements include curcumin, selenium, milk thistle, vitamin C, Vitamin E, MSM, B vitamins.
Increased intake of cruciferous vegetables, onion, garlic and eggs are also helpful in increasing glutathione.
If CBS C699T SNP is present consider using liposomal glutathione instead of NAC.
Other snp to deplete mg = COMT which almost everyone has
MSG can create this issue – eliminate before diagnosing?
Taurine is inhibitory neurotransmitter and is not well tolerated. Can induce histamine response.
Supplement with amino acid only if clearly indicated.
Reduced glutathione if liposomal too expenseive or can’t handle taste.
“Glutathione synthase deficiency is the most common genetic defect seen in the GGC pathway. The autosomal recessive form of this defect in the ɣ-glutamyl cycle causes recurrent kidney stones, whereas the homozygous expression results in vomiting, diarrhea, and abdominal pain.”
Detoxification: Cytochrome P450 Enzymes
Cytochrome P450 enzymes are a superfamily of enzymes used in detoxification divided into families.
They are all found in phase I detoxification which converts a lipid-soluble molecule entering the liver into a more water soluble intermediary metabolite.
This metabolite is often more toxic, not less.
Phase 1 takes from lipid soluble to water soluble. Remember to get phase 2 moving as well as phase 1
CYP 450
- Phase II detoxification includes:
Acetylation (NAT1, NAT2)
Glucaronidation (UGT1A1, UGT2A1)
Glutathione Conjugation (GGT1, GPX3, GSR, GSTM, GSTP)
Peptide Conjugation (glycine, taurine)
Methylation (MTHFR, MTR, MTRR, COMT),
Sulfation (SULT1A1, SULT1A2)
All are named using “CYP” for cytochrome P450, followed by an Arabic numeral.
I.E. CYP1, CYP2, CYP3, etc.
Families are further divided into subfamilies with the addition of a capital letter.
I.E. CYP1A, CYP1B, CYP1C, etc.
Individual members of each subfamily are then numbered in the order in which they were identified.
I.E. CYP1A1, CYP1A2, CYP1A3, etc.
Substance That Can Induce P450 Enzymes
Drugs: alcohol, barbiturates, sulfonamides
Dietary: high protein or brassica diets, saturated fats
Hormones: steroid hormones
Foods: charcoal-broiled meats, oranges, sassafras, tangerines
Vitamins: niacin, riboflavin
Xenobiotics: carbon tetrachloride, dioxin, exhaust fumes, organophosphorus pesticides, paint fumes
Detoxification Case Study
You have an 18 year old female college student studying for her first semester’s final exams. She has been pulling lots of all night study sessions and has been drinking at least a pot of coffee per day. She notices that she is more jittery than can be explained by anxiety. She is even experiencing bouts of tachycardia.
What SNPs do you think she might have and why?
CYP1A2: caffeine detoxification SNPProblems with porferin pathway, anemias, phase 1 is tanked; heme is used in these pathways
Heme is cofactor for every cytochrome 450 pathway
These pathways important for breaking down botanicals as well, can have as many adverse reactions with botanicals
Acetylation
N-acetyltransferase 1 protein (NAT1) is involved in phase II xenobiotic metabolism and helps with the biotransformation of aromatic amines and heterocyclic amines.
rs4986782 increases the risk for smoking-induced lung cancer, head and neck cancers
Considered the most common “slow acetylator” variant
N-acetyltransfease 2 protein (NAT2) is also used in phase II detoxification for the biotransformation of aromatic and heterocyclic amines.
Detoxifies hydrazine and acrylamine drugs.
SNPs to NAT1 and NAT2 are associated with rapid, intermediate or slow metabolizers.
It takes two slow metabolizer alleles to give risk to a slow metabolizer phenotype. This means that the rapid metabolizer allele is dominant, and the slow metabolizer allele is recessive.
Sulfation
Sulfotransferase 2A1 (SULT2A1) is an enzyme within the sulfation pathway.
Sulfotransferases are important for the metabolism of drugs and endogenous compounds and convert them into more hydrophilic water-soluble sulfate conjugates that then may be excreted.
SULT2A1 catalyzes sulfation of steroids and bile acids in the liver and adrenals.
May have a role in adrenal androgen excess in women with polycystic ovary syndrome (PCOS)
IR/PCOS
Associated with androgen excess in women with PCOS, because regulates DHEA levels.
Acytlators – foods with dark blue colors
GAD
Glutamic Acid Decarboxylase
Converts the excitatory neurotransmitter glutamate into the inhibitory neurotransmitter GABA
Requires vitamin B6 as the active pyridoxal-5’-phosphate (P5P)
Associated with anxiety (panic attacks)
Often will lead to functional insufficiency of vitamin B6 as evidenced by presence of elevated xanthurenate (organic acid)
In the pancreas the GAD enzyme is used to regulate insulin release
Type 1 diabetes common in Celiac and other autoimmune
GAD and Anxiety significant lower GAD1 methylation
GAD vs COMT Case Study
You have a 35 year old client who has had a recent panic attack while shopping at the local mall. She has also noticed generally that she has been feeling more and more anxious for no apparent reason. Her naturopathic physician has run the following tests:
Xanthurenate: Elevated
RBC Magnesium: Normal
Insulin: Functionally Low
Fasting Blood Glucose: 115 (normal is less than 99)
Would you expect this to be a GAD or COMT case and why?
GAD runs on B6
++ GAD
Xanthurenate low = low b6
Mg needed for COMT, so if COMT, Mg would be low
ACE Angiotensin Converting Enzyme
Angiotensin converting enzyme (deletion, GG)
rs4343 is a proxy for ACE Del16 - GG
Deletion results in up-regulation of activity
Results in higher rate of conversion of angiotensin 1 to angiotensin 2
Result of high levels of angiotensin 2 are an increased level of aldosterone which can result in anxiety and learning/memory problems
Electrolyte balance issues, specifically, decreased sodium excretion (so sodium retention/ swelling) and increased excretion of potassium excretion (low potassium); cardiovascular complications.
Salt Sensitive Hypertension
“Almost 50% of hypertensive individuals manifest blood pressure changes in response to salt depletion or repletion and are termed “salt sensitive”
angiotensinogen gene AGT M235T and angiotensin-converting enzyme insertion/deletion polymorphism ACE I/D
AGT
- Angiotensin gene(CC)- 20% CEU
rs699, M235T
Specifically increases angiotensin levels, exacerbating the ACE deletion
Associated with insulin resistance, especially when combined with ACE
Associated with increased hypertension and its associated diseases like pre-eclampsia and heart disease
Maintaining adequate water intake and balance is imperative as well as adrenal support
FTO
Fat mass and obesity associated gene
Requires molecular oxygen, alpha-ketoglutarate and iron.
Contributes to the regulation of the global metabolic rate, energy expenditure, energy homeostasis, body size and body fat accumulation.
Molecular oxygen from oxygen. Biochemically get it from mitochondrial function. Mitochondrial function, B1 is gatekeeper,
Alpha-ketoglutarate – Krebs cycle
Iron – deficiency increases risk of obesity
Apolipoprotein E (APOE)
Three variations, ε2, ε3 and ε4
Associations with cardiovascular disease and neurovascular diseases like dementia and Alzheimer’s disease.
Determined by two SNPs, rs429358 and rs7412
Carrying a single copy of the ε4 variant is associated with about 1.5 to two times increased odds of developing Alzheimer’s disease.
Two copies of the ε4 variant is associated with approximately 9 times increased odds risk of Alzheimer’s disease populations of European ancestry.
ApoE is a lipoprotein
Type 3 Diabetes because on the same pathways as diabetes
KEto Diet contraindicated.
ε2/ε2 is protective of Alzheimer’s disease but associated with familial hypercholesterolemia.
Dysbetalipoproteinemia is a rare familial dyslipidemia characterized by approximately equally elevated serum cholesterol and triglyceride levels due to accumulated remnant lipoproteins in apolipoprotein ε2/ε2 homozygotes. Those with this genotype should limit the saturated fat intake to 12g/day.
APOE Case Study: Cardiovascular vs Alzheimer’s
A new client who is a 65 year old male and has a history of cardiovascular disease visits you after his physician ran a LabCorp APOE genetic test. The results are as follows:
APOE E4/E3
Total cholesterol: 225 mg/dL (Below 200 mg/dL)
Triglycerides: 75 mg/dL (30-150 mg/dL)
Functional range: 70-100 mg/dL
HDL: 40 mg/dL (40-90 mg/dL)
Optimal range is above 55 mg/dL
LDL: 185 mg/dL (60-130 mg/dL)
Optimal is less than 120 mg/dL
What questions should you ask about his family history? Are there any tests that you should perform to assess cognitive ability?
Mediteranean diet is implicated here
BCMO1
Beta-carotene 15,15’-monooxygenase 1 (BCMO1)
Converts beta-carotene into the active form of retinol, vitamin A (beta-carotene -> trans-retinol)
Cofactor: iron
Highly polymorphic
rs12934922, rs7501331, rs11645428, rs6564851, rs6420424 may cause between a 32-59% decrease in conversion of beta-carotene to trans-retinol
Overall will increase plasma beta-carotene and decrease plasma retinol
Vitamin A is involved in T regulatory cells, immune function, embryonic development, reproduction, cell growth and differentiation, and eye health
Vegan is contraindicated with this polymorphism
Autoimmune, deperession, anxiety all corelated
HLA Genes and Gluten Sensitivity/Celiac Disease
1.Major risk variants are HLA-DQ2 and HLA-DQ8
2. Relevant SNPs include rs2395182, rs7775228, rs2187668, rs4639334, rs7454108, rs4713586
3. Presence identifies risk for celiac disease and not the disease itself
4. May also present as non-celiac gluten sensitivity (NCGS)—a milder form of gluten intolerance that doesn’t cause small intestine damage or nutrient malabsorption
>Symptoms include diarrhea, abdominal pain, fatigue, and headaches after exposure to gluten
Guidelines for CD diagnosis:
Signs and symptoms suggestive of CD
Anti-transglutaminase type 2 antibody (anti-TG2) levels more than 10 times the upper limit of normal
Positive confirmation tests of anti-endomysium-IgA antibodies (EMA)
At-risk HLA-DQ2 or HLA-DQ8
40% of the genetic susceptibility to CD. In fact, approximately 90%-95% of celiac patients are HLA-DQ2 positive, while half of the remaining patients are HLA-DQ8-positive
Gluten Case Study
A new client had seen a chiropractor who ran a Cyrex Array 3 looking at celiac disease and gluten sensitivity. The results revealed several elevated antibody levels to gluten. As a result you decided to run a Nutrigenomix NGx Gluten test which reveal the following:
HLA-DQ2: homozygous
HLA-DQ8: homozygous
What is the likelihood of a celiac diagnosis from a gastroenterologist?
With the information presented would you recommend a gluten free diet and why?
SOD2/SOD3
SOD2 and SOD3
Superoxide dismutase is an antioxidant enzyme required to catalyze the dismutation of two superoxide radicals into hydrogen peroxide and oxygen.
Protects tissues from oxidative stress
Sprouts and algae are rich in superoxide dismutase
SOD2 cofactor is manganese
SOD3 cofactors are copper and zinc
SOD SNPs and Cardiopulmonary Disease. This shift explains the paradox of how a single polymorphism can simultaneously reduce the risk for lung disease while increasing the risk of vascular disease.”
raw data
Genetic Genie
Promethease
Livewello
MTHFRsupport
Nutrigenomix
Genetic Genie: Pros and Cons
Genetic Genie is free!
It does ask for a small donation (though not required)
It is a data collection service, so not completely free.
Recently, they have corrected the errors in VDR Taq, and formerly MAO-A.
Processors are great, but you get what you pay for. Use 23andme to check behind your work and make sure that you deliver a good product. This is a good first pass and teaching tool for yourself. Your clients will bring you these reports, so know how to use them and interpret these SNPs. They are important.
Promethease
Pros
$5.00
Electronic access
Downloadable immediately
Professional service available
Nutrigenetic functions (disease processor)
Links directly with genomic data processors (23and me or Ancestry.com)
Cons
Only available for 45-days electronically (though may be downloaded for later use)
Can take up to 20 minutes to process
Can be confusing or overwhelming at first, especially for clients
Loading function does not work well with mobile devices
Is constantly updated with new disease information (pro/con)
Functional Testing
Organic Acids Testing (OAT)
Urinary Amino Acids (UAA)
Erythrocyte Minerals
Key Markers
-Xanthurenate
- A Marker for Vitamin B6 insufficiency
-Result of lack of pyridoxal -5-phosphate (P5P) in kynurenine pathway
-May also cause elevation of Kynurenate
-Will cause oxidative stress
-Estrogen may inhibit the initial conversion of tryptophan to kynurenine and may cause losses of vitamin B6
-Will cause oxidative stress
-Estrogen may inhibit the intitial conversion of tryptophan to kynurenine and may cause losses of Vitmain B6
>Possible connection to COMT and PEMT mutations resulting in estrogen dominance
-Xanthurenic acid forms complexes with insulin decreasing circulating concentrations and decreasing insulin’s activity.
>Xanthurenate is often elevated in diabetics
>likely connection to the GAD enzyme which requires P5P as its cofactor
CBS snps, GAD snps, SHMT (precursor to MTHFR)
Look at if high marker, means diffifiency in vitamin
Some organic acids are problems when low
CBS or GAD
If you have a client with an elevated xanthurenate in a urinary organic acids test how would you differentiate whether the cause is CBS (upregulation) or GAD?
How would these SNPs effect the following tests?
Amino acids
Organic acids
Oxidative Stress
Homocysteine
Both will be B6 de;
Increased Gly and decreased GABA ( GAD)
Decrease HCY, Check CTH, decrease in serine ( UAA)
OX stress for CBS will be abnormally high
CBS upregulation – feels awful with sulfer foods, increased sulfate levels
GAD – anxiety/panic attack; insulin levels low; glucose, slightly high, A1c normal to slightly high
Look at ammonia metabolism
Key Markers (MMA)
Methylmalonic Acid (MMA)
Functional marker for vitamin B12 insufficiency
MMA is converted into succinic acid by methylmalonyl-CoA mutase which requires vitamin B12 as its cofactor
Generally homocysteine will also be elevated as vitamin B12 is required in the conversion of homocysteine to methionine via MTR/MTRR
-SNPs in MTR and MTRR can contribute to MMA elevation in this way
Polymorphism of methylmalonyl-CoA mutase can prevent cofactor or substrate binding that could cause MMA accumulation that does not respond to Adenosylcobalamin
-Mild polymorphisms are common
MMA Continued:
Polymorphism in the transcobalamin transporters (TCN1 and TCN2) may cause transport issues making it difficult to move vitamin B12 into the cell.
This may cause an increase in serum vitamin B12 (often even without supplementation) with an increase in MMA
Use of Hydroxocobalamin can bypass this transport issue
SNPs to gastric intrinsic factor (GIF) may also contribute to MMA elevation
Intrinsic factor is secreted by the parietal cells and is required for proper absorption of vitamin B12
Use of sublingual lozenge or drops may be indicated for mild cases
Prescription vitamin B12 shots may be needed if frank macrocytic anemia is present
MCV is above 100
Hemoglobin below 12.5 for men or below 11.5 for women
Hematocrit is below 36% for men or below 34% for women
RBCs below 4.2 for men or 3.8 for women
Formiminoglutamate (FIGLU)
-FIGLU is an intermediate in the deamination of the amino acid histidine and requires folate as a cofactor for formiminotransferase, the enzyme that converts FIGLU to glutamic acid.
-Homocysteine may elevate along with FIGLU
> Methyl donors from L-methylfolate are required to support the methylation of vitamin B12
> The addition of methyl groups in the homocysteine pathway requires methylfolate, while folate is only required for the FIGLU pathway
> Unmethylated folate is sufficient for normalizing FIGLU levels
>May use folinic acid (calcium folinate)
THFR-MTHFR may be assocaite
Hydrxy or adnysil B12, then 6 to 12 weeks, retest
Folate, use folinic acid (calcium colinate)
Levicoran (when taking methyltrxate)
FIGLU Continued:
Mutations to MTHFR C677T and MTHFR A1298C decrease conversion of folate to L-methylfolate and may contribute to decreased levels of L-methylfolate. MTHFR C677T may cause increased homocysteine, but will not necessarily cause increased FIGLU.
DHFR (NADP cofactor) converts dihydrofolate into tetrahydrofolate and may increase FIGLU levels.
MTHFD1 (NADP cofactor) is a protein that allows for 3 different enzymatic reactions in the folate pathway and allow conversion of THF to 10-Formyl THF, conversion of 10-Formyl THF to 5,10-Methenyl THF, and finally 5,10-Methenyl THF to 5,10-Methylene THF and can therefore increase FIGLU levels.
FIGLU Case Study
A mother brings in her four month old infant boy with a history of irritability, sleep disturbances and cerebellar ataxia (spastic paraplegia and diskinesia). The child recently had a seizure that was not related to febrile illness. You run the following tests:
FIGLU: Elevated
MCV: 90 (80-100)
Hemoglobin: 14.0 g/dL (12.5-17.0 g/dL)
DHFR: Homozygous
FR1: Homozygous
Folate receptor protein 1 (FR1): this protein facilitates active transport of folate into the central nervous system
What do you think the diagnosis is?
Cerebral Folate Deficiency: DHFR/Leucovorin low dose of 0.5-1 mg/kg/day; higher doses are sometimes needed 2-3 mg/kg/day to normalize the CSF/5MTHF values
Elevated Figlu= low folate
DHFR homozygous affects neurotransmitters and conversion to THF
Most of these kids get misdiagnosed as autism
Key Markers (VMA) and Homovanillate (HVA)
- Vanilmandelate (VMA) and Homovanillate (HVA)
-VMA is the metabolic breakdown product of norepinephrine and epinephrine
-HVA is the metabolic breakdown product of dopamine
-Decreased level sof these neurotransmitters can cause depression, sleep disturbance, anxiety, and fatigue
-Supplementation with tyrosine which is the precursor to these neurotransmitters may also help.
-Elevated levels signal increased neurotransmitters synthesis and degradation.
>May occur from increased pituitary adrenocorticotropic hormone (ACTH) and cortisol from sympathetic activation and stress.
Mucan pruriens is also an excellent way to support dopamine production; Consider supporting protein digestion with digestive enzymes and HCL
VMA and HVA Continued:
Drugs like risperidone, a dopamine reuptake inhibitor, will cause a pattern of low HVA with elevated VMA showing (nor)epinephrine overload.
Copper deficiency may lead to increased dopamine and lower norepinephrine levels from decreased activity of dopamine-beta-monooxygenase resulting in increased HVA and suppressed levels of VMA
VMA and HVA Continued:
The COMT (catechol-O-methyltransferase) enzyme is important for the breakdown of dopamine, norepinephrine and epinephrine. SNPs may slow this process down causing increased anxiety, stress, and OCD-like behaviors.
May result in decreased levels of HVA and VMA with supplementation with tyrosine and methyl donors leading to increased anxiety and irritability.
MAOA (monoamine oxidase A) and MAOB (monoamine oxidase B) breakdown dopamine, norepinephrine, epinephrine, and serotonin with the aid of FAD as a cofactor
Will cause decreased levels of HVA and VMA with elevated levels of dopamine, norepinephrine, epinephrine and serotonin
5-HIAA
5-Hydroxyindoleacetate (5-HIAA)
5-HIAA is the catabolic breakdown product of serotonin
SSRIs, 5-HTP and St. John’s wort generally cause increased 5-HIAA levels
Increased 5-HIAA levels cause depletion of L-tryptophan, the amino acid that is the precursor for both serotonin and niacin synthesis
MAOA (monoamine oxidase A) preferentially oxidizes serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (NE), whereas MAO B preferentially oxidizes phenylethylamine (PEA). Both forms can oxidize dopamine (DA). Cofactor is FAD.
Will cause decreased levels of 5-HIAA with elevated levels of dopamine, norepinephrine, epinephrine and serotonin
Slow variant – depression
Low or high on 5-hiaa, will have issues that need to be addressed
Depression: Serotonin vs Dopamine Case Study
You have a 30 year old female who comes to see you complaining that she is depressed. When asked to elaborate she explains that while she thinks she will enjoy going out with friends and doing things she is always disappointed in the end. She also has been experiencing some binge eating. Even though she knows that she should eat healthier foods in more reasonable portions she just cannot stop herself.
Do you think that the following markers will be high or low? Why?
HVA
VMA
5-HIAA
Tyrosine
Tryptophan
RBC Copper
Are these symptoms indicative of dopamine or serotonin depression?
HVA and VMA could be high or low
5-HIAA will likely be normal
Tyrosine: low
Tryptophan: normal
RBC Copper: low
Dopamine
D-Lactate (Marker)
D-Lactate
Marker of Lactobacillus overgrowth
May occur with short-bowel syndrome resulting from poor dietary carbohydrate absorption
FUT2 SNPs may result in D-lactate elevation by causing decreased levels of Bifidobacteria and the decreased competition causes the Lactobacillus overgrowth (may be confirmed with a stool test showing decreased Bifidobacteria levels)
Symptoms of elevated D-Lactate:
Altered mental status (drowsiness to coma)
Slurred speech
Disorientation
Impaired motor coordination
Hostile, aggressive, abusive behavior
Inability to concentrate
Nystagmus
Delirium
Hallucinations
Irritability
Excessive hunger
Headache
Weak, droopy eyelid
Hand tremor when the wrist is extended
Blurred vision
Urinary Amino Acids
Amino acids are important for:
Neurotransmitter function
Pain control
pH regulation
Detoxification
Cholesterol metabolism
Control of inflammation
Clinically they are useful for looking into:
Protein inadequacy
Gastrointestinal imbalances
Inflammatory responses
Detoxification impairments
Chronic fatigue
Cardiovascular disease
Ammonia toxicity
Food and chemical sensitivities
Depression or behavioral disorders, including autism and ADHD
Neurological dysfunction
Inborn errors of metabolism
Key marker (Histidine)
I’m starting with the one you can’t see, but it is the most popular because of MTHFR
Histidine:
Low histidine is associated with rheumatoid arthritis, folate deficiency, and/or salicylate/steroid use.
Check 3-Methylhistidine, if both are high, then muscle catabolism is present.
Key Marker (Methionine and Serine)
Methionine and serine:
Low Methionine: possible poor-quality protein diet. Adverse effects on sulfur metabolism.
High Methionine: excessive intake of methionine rich protein or inefficient metabolism. If other sulfur-containing AAs are low, then enhance methionine utilization by adding the necessary cofactors, magnesium and vitamin B6.
Serine - low levels can lead to disordered methionine metabolism and deficits in acetylcholine synthesis. If simultaneous high threonine or phosphoserine, then need for vitamin B6, folate, and manganese is indicated.
High Serine - when accompanied by low threonine, indicates glucogenic compensation and catabolism. Supplement threonine and BCAAs.
Tyrosine, Phenylalanine and Tryptophan:
Tyrosine, phenylalanine and tryptophan:
Low Tyrosine - implicated in depression, hypothyroidism, and blood pressure disorders. If phenylalanine is normal or high (barring PKU), iron, vitamin C, and niacin supplementation might be indicated to help convert phenylalanine to tyrosine.
High Tyrosine - inadequate utilization of tyrosine. Supplement the cofactors needed here including iron, copper, vitamin B6, and ascorbate.
Low Phenylalanine - can result in altered thyroid function and catecholamine deficits including symptoms of depression, cognitive disorders, memory loss, fatigue, and autonomic dysfunction.
High Phe - high protein intake or a block in the conversion of phenylalanine to tyrosine. Iron, vitamin C, and niacin are necessary for this enzymatic step. Check tyrosine level and, if low, supplement tyrosine and iron.
Low Tryptophan - commonly correlated with depression, insomnia, and schizophrenia. Supplementation with 5-Hydroxytryptophan (5-HTP) may help. 5-HTP is one enzymatic step away from serotonin.
High Trp - possibly inadequate metabolism of tryptophan. Required nutrients for this process include niacin and vitamin B6.
Vegan Case study
A 17-year-old girl has been vegan for the past few years and currently consumes high carbohydrate, high fat processed foods. She complains about fatigue, dry skin and hair and slight weight gain. You run the following tests and find the following:
TSH: 6.0 (0.35-5.5 uIU/ml)
Free T4: 0.5 ng/dL (0.8-2.8 ng/dL)
Free T3: 1.0 nmol/L (1.23-3.53 nmol/L)
Tyrosine: low
Phenylalanine: elevated
MMA: Elevated
What clinical diagnosis does she have and how does it relate to the markers listed above?
Copper, Vitamin C, B6, iron and BH4 will all be low
BH4 and PAH
Cystathionine, cystine and Homocystine (Key Markers)
Cystathionine, cystine and Homocystine :
High cystathionine: possible B6 functional deficit as P5P is required for the conversion of cystathionine to cysteine. Low cysteine can result.
Low cystine: possible dietary deficiency of methionine and/or cystine. Low cystine can impair taurine synthesis. High cysteine is resultant of excessive dietary intake or impaired cystine metabolism. Cystine is converted to cysteine (reduced cystine) via a B2 and copper.
Homocystine: high levels indicate an increased risk for atherosclerosis and abnormalities in the ocular, neurological and musculo-skeletal systems. The enzyme that converts homocysteine (reduced homocystine) to cystathionine is B6 dependent; remethylation of homocysteine to methionine requires B12, folate and betaine. Supplementation of these nutrients plus magnesium is effective for the proper metabolism of homocystine.
RBC Mineral Testing
Mineral and toxic element (heavy metal) levels may be assessed from erythrocyte (RBC) testing.
For magnesium in particular this is the preferred way to assess usable levels as serum levels are under tight regulation. Changes in RBC magnesium levels have been linked to hypertension, premenstrual syndrome and chronic fatigue syndrome.
Low RBC levels of magnesium may be a sign of an active COMT SNP
While difficult to assess zinc status, RBC zinc may be helpful because of the high concentration of zinc enzymes such as carbonic anhydrase.
High cadmium may decrease zinc levels
Excessive zinc intake may cause decreased copper levels which may then lead to iron-iron based anemia via the impaired mobilization and use of iron due to decreased ferroxidase activity of hephaestin in enterocytes and of ceruloplasmin required for oxidation of iron.
Decreased zinc levels may be found in SOD1RBC
copper is a good marker for copper deficiency because it is linked with copper-superoxide dismutase (SOD1)
Most of the copper in erythrocytes is associated with SOD
RBC manganese is associated with long-term levels and are a good assessment of manganese status.
Deficiency may be associated with SOD2 SNPs (CVD-associated/Detoxification SNP)
RBC case study
You have a new client that is a 55 year old postmenopausal female with exercise intolerance. Her naturopathic physician had run a RBC mineral test that showed the following:
Zinc: Low
Copper: Low
Manganese: Low
Which SNPs do you think might be present and why?
SOD3 and SOD2 (mito) Manganese shows 2
SOD3 cytosollular base
Neurotransmitter Case Study
DD, a 46 year old female diagnosed with histamine intolerance, Ehlers Danlos Syndrome (hypermobility type), anxiety and depression. Reports difficulty turning off mind, difficulty sleeping (cannot stay asleep), occasional paranoia, intermittent depression and periodic anxiety. No history of antidepressant use but does take Ativan occasionally. Feels best when eating a low histamine diet with lots of vegetables. Avoids refined sugar and fried foods. Does not drink any alcohol because both parents were alcoholics. Anxiety first started when working in a prison for 20 years which has caused paranoia and hyper vigilance. Anxiety worsens just before period.
Salient SNPs
COMT rs6269 AG heterozygous
COMT H62H rs4633 CT heterozygous
COMT V158M rs4680 AG heterozygous
DAO rs3741775 CC homozygous (associated with histamine intolerance)
GAD1 seven heterozygous SNPs
MAOA rs6323 GG homozygous
MTHFR A1298C rs1801131 GT heterozygous
MTHFR C677T rs1801133 AG heterozygous
PEMT rs4244593 TT homozygous
VDR BSM rs1544410 CT heterozygous
VDR TAQ rs731236 AG heterozygous
Organic Acids Test
Formiminoglutamate (FIGLU): 2.0 (upper fourth quintile—functionally high)
Vanilmandelate: 5.3 (high)
Homovanillate: 7.1 (high)
5-Hydroxyindoleacetate: 6.1 (high)
Other Laboratory Markers
RBC magnesium: 27 (low—range is 30.1-56.5 mcg/g)
Need B6 for GAD to function
COMT drains Magnesium
High cortisol – lots of stress, maybe PTSD
Histidine/FIGLUHistidine – amino acid; THF related to histadine breakdown; forms glutamate, excitatory
Histidine binds to same receptor as GABA
What does it mean?Three heterozygous SNPs to COMT have resulted in magnesium insufficiency and increased anxiety (mind cannot stop). This combined with 7 heterozygous SNPs to GAD have caused anxiety issues.
COMT SNPs will cause increased breakdown of dopamine, norepinephrine and epinephrine.
GAD SNPs will cause decreased conversion of glutamate to GABA.
Increased anxiety just before period is because the COMT and PEMT enzymes are also responsible for estrogen breakdown.
Dose 300-500 mg magnesium glycinate at bedtime to help improve COMT function.
Vitamin B6 as the active P5P may be helpful for helping the GAD enzyme function better. Dose 50 mg BID.
If anxiety continues around period consider phosphatidylcholine to help with PEMT SNP.Compound heterozygous MTHFR status has caused increased FIGLU and decreased cellular folate levels.
Consider dosing 400-800 mcg of folate as calcium folinate (folinic acid).
L-methylfolate may not be well tolerated give the COMT SNPs
Folate deficiency may contribute to depression
Homozygous SNP to MAO A causes increased breakdown of serotonin, dopamine, norepinephrine and epinephrine.
This has caused the increased levels of HVA, VMA and 5-HIAA which are related to the depression and anxiety.
Consider dosing 5-HTP 50-100 mg TID and tyrosine 1000 mg BID-TID between meals. Mucana pruriens may also be dosed instead to increase dopamine levels.
Cardiovascular Case Study
Your new client is a 40 year old male who is 5’6” and 220 lbs. He has been diagnosed with hypercholesterolemia, hypertension and pre-diabetes. He eats a SAD diet that his high in sodium. He complains of edema and swelling in his lower body. He comes to you with the following labs:
APOE: E2/E2
Homocysteine: 18
Fasting Blood Glucose: 125
Hemoglobin A1C: 5.9
Blood Pressure: 145/90
Methionine: Low
Cystathionine: Low
Potassium: Low
What additional SNPs might he have that may be contributing to his condition?
Are there any additional diagnoses that are likely in this case?
ACE deletion, AGT, CBS, BHMT, MTR/MTRR: homocysteine routes of disposal
SNPs that we are likely to find include:
ACE deletion
ACE deletion is associated with salt-sensitive hypertension
Causes electrolyte imbalance and cardiovascular complications
AGT
AGT exacerbates the ACE deletion and is associated with insulin resistance
CBS
If cystathionine is low then a downregulated CBS SNP is present. This will cause decreased conversion of homocysteine to cystathionine.
MTR/MTRR
MTR/MTRR are important for conversion of homocysteine back to methionine and are the most likely cause of the decreased methionine.
BHMT
This is the short cut route through the methylation cycle. While this SNP may be present and could possibly contribute to decreased methionine levels (it converts homocysteine to methionine), it is not likely to be the primary cause.
FTO
rs1421085 (T-C alteration) is the causal SNP for fat mass and obesity
Almost 50% of individuals with hypertension have the ACE and AGT alterations.
For CBS downregulation we would still expect to find low levels of vitamin B6, increased homocysteine and decreased cystathionine.
For MTR/MTRR supplementation with the correct form of cobalamin is indicated (not enough information to determine proper form—look at COMT, TCN1/TCN2, VDR-TAQ).
For BHMT supplementation with TMG maybe helpful if the methyl donors are tolerated. Zinc, as the cofactor for BHMT could also help upregulate BHMT.
