SNPs

1. Introduction
Cynthia Smith
Cynthia Smith has completed a conflict of interest disclosure form and does not have
any conflicts of interest to disclose.
Saturday
January 17, 2015
1

2. Methylation
• Methylation is required for:
– Energy (CoQ10, carnitine, creatine, ATP via Krebs cycle)
– Bile production https://www.ncbi.nlm.nih.gov/pubmed/25309573
– Nerves (myelination) https://www.ncbi.nlm.nih.gov/pubmed/24652042
– Building and maintaining cell membranes
– Generating & degrading neurotransmitters
– https://www.ncbi.nlm.nih.gov/pubmed/25084335
– Generating purines for new DNA and RNA
– Generating SAMe http://www.ncbi.nlm.nih.gov/pubmed/18950248
– Performing Phase II liver conjugation of heavy metals, hydroxyestradiols,
nor-epi, epi, histamine, etc.
– http://modernherbalmedicine.com/articles/the-low-down-on-liver-detoxification-2.html?page=3
2

3. What is a SNP?
• S = Single
• N = Nucleotide
• P = Polymorphism
• SNPs occur in:
– Coding regions
– Non-coding regions
– Between genes
• Aka Intergenic
3
http://blog.23andme.com/wp-content/uploads/2010/11/snp-snip.png

4. General Info About SNPs
• SNPs vary in terms of
severity and benefit due
to location and
redundancy.
• Our bodies have back-up
pathways for
redundancy, but they are
typically not as efficient
as the primary pathways.
• http://ghr.nlm.nih.gov/handbook/genomicresearch/snp
4

5. RNA and DNA strand
(we are focusing on nuclear DNA)
5

6. What is a SNP? Cont’d
• A SNP is a nucleotide swap at a “rung”
location of the DNA “ladder”.
– Wildtype
– Heterozygous SNP
– Homozygous SNP
• The rung location is referred to as an “rs
number” (e.g., rs 1801133)
• http://learn.genetics.utah.edu/content/pharma/snips/
6

7. De Novo Synthesis of Nucleotides (in
Liver)
Purines
(A) Adenine -----------
(G) Guanine -----------
Synthesis requires
- DHFR
- 5, 10 methylene THF (plus
glutamine, glycine, aspartate and
formate)
Disorders in Purine Metabolism:
- Gout
- Lesch-Nyhan syndrome (severe
gout and nervous system disorder)
Pyrimidines
(T) Thymine (aka 5-methyl uracil)
(C) Cytosine
Synthesis requires
- DHFR
- 5, 10 methylene THF (plus
glutamine and aspartate)
NOTE: DHFR inhibitors:
- methotrexate
- aminopterin
- trimethoprim
7

8. Nuts and bolts of SNPs
• Sequential DNA ladder
rungs provide “blueprints”
for protein synthesis
(polypeptide synthesis).
• The polypeptide may be
a receptor, an enzyme, a
carrier protein, etc.
• Pearl necklace metaphor
8

9. OK, not so much a pearl necklace; more ribbon
like, with nutrient cofactor docking stations and
critical bends.
9

10. Example
• At location rs1801133 (rung) of the DNA genome, or
position 677 of the MTHFR-encoding portion of DNA, one
expects to find two cytosine (C) nucleotides.
10

11. Example Cont’d
Heterozygous SNP
•If one of the cytosine nucleotides has been replaced by a
thymine (T), the SNP (“swap”) is referred to as an MTHFR
C677T heterozygous SNP
– The resulting MTHFR enzymatic activity may be reduced by
20-30%.
11

12. Example Cont’d
Homozygous SNP
•If both cytosine nucleotides have been replaced by thymine,
the SNP is referred to as an MTHFR C677T homozygous
SNP.
– The resulting MTHFR enzymatic activity may be reduced by as
much as 60%.
12

13. How do SNPs play a role?
A SNP:
• Will result in the selection of an alternate amino
acid, which may:
• Change the shape of the resulting polypeptide,
which may:
• Impact the function of the polypeptide enzyme,
receptor, carrier protein.
•https://www.ncbi.nlm.nih.gov/pubmed/25429430
13

14. Why Do We Care?
• SNPs may translate into (mostly) down-
regulated, or slower, enzymatic activity, receptor
function and carrier protein function.
– COMT V158M ++ -> Slow adrenaline clearing
• A few SNPs may translate into up-regulated, or
faster, enzymatic activity, receptor function and
carrier protein function.
– FCER1A +- -> Hyper coupling allergen to MAST cell
14

15. As a Result
• A person with an MTHFR C677T hetero- or
homozygous SNP, may have a decreased ability
to convert (substrate A) to (product B).
A B
100%
A B
60%
W/O SNP
W/ SNP
5,10-methylenetetrahydrofolate
5-methyltetrahydrofolate
15

16. So….
• An MTHFR C677T SNP indicates a potential poor
conversion of dietary folic acid and folate, into its
usable form, 5-methyltetrahydrofolate.
– NOTE: Supplements that contain synthetic folic acid
are not advised and can block folate receptors.
http://ajcn.nutrition.org/content/87/3/517.full
– Also, look upstream at DHFR (2 enzymatic steps) and
MTHFD1 (3 enzymatic steps)
16

17. Nutrient Cofactors
• Enzymatic function can be compromised as a result of
lacking cofactor(s) and NOT SNPs.
– Replete enzymatic cofactors as an initial step
• Minerals
• B vitamins
• The cofactor may be a vitamin, mineral or other byproduct
of biochemical pathway processing (e.g., NAPD)
17

18. Cofactors
• COMT V158M - -
– “Normal” catecholamine
clearing
– Plus depleted
Magnesium
• COMT V158M ++
– “Slow” catecholamine
clearing
– Plus repleted
Magnesium
https://www.ncbi.nlm.nih.gov/pubmed/18804702
http://www.ncbi.nlm.nih.gov/pubmed/3607001
C D
60%
=
http://www.ncbi.nlm.nih.gov/pubmed/7836621
18

19. Do Not Treat SNPs!
• SNPs are Guideposts
• SNPs are not a
diagnosis
19

20. SNPs are Guideposts
• Example: 5 MTHF
– If a client has MTHFR and/or MTHFD1 and/or DHFR
SNPs, she/he MAY need to supplement with 5MTHF
AT SOME POINT IN TIME.
– There are initial steps that should be addressed first.
• pushing on the “accelerator” biochemical pathway (e.g.,
administering 5MTHF and methyl B12), before ensuring that
the “braking system” (e.g., neurotransmitter balance, GI,
Adrenals) is in place, will yield poor results. More on this
later….
20

21. A Bit About Epigenetics
• Most SNPs do NOT govern genetic function and
expression…
• Rather it is our and (our parents and grandparents)
– Diet
– Lifestyle
– Attitude
– Nutrients
– Environment / Exposures
• that manipulates expression of our genes (hardware) via
their interface with our Epigenome (software)
21

22. If genes are the hardware, diet and lifestyle and exposures are the
software, via the Epigenome.
“….a folic acid–rich diet can influence DNA and histone methylation, which
leads to phenotypic changes in subsequent generations”
22

23. Epigenetics
• Epigenetic changes to parent’s DNA
(toggled on or off) are passed via gametes
to the next generation
– Excellent documentary by NOVA entitled
“Ghost in Your Genes”
• http://topdocumentaryfilms.com/the-ghost-in-our-
genes/
23

24. Bottom Line
• SNPs should be considered as another
tool in the toolbox of Practitioners
24

25. What is a Variant Report?
• A listing of selected SNPs derived from raw
data, generated from results of a saliva or blood
DNA test
»PDF format
»Information about methylation and
much more
»The list can vary, depending on
which rs-numbers are plucked from
the raw data by the App
25

26. An Example of a Variant Report
26

27. A Variant Report; Now What?
• Address the basics…first,
the Braking System
• Consider:
–
•
– (b) Health history and
IFM principles
– (c) GI
– (d) Neurotransmitter
– (e) HPATG Axis
– (f) Replete Enzyme
Cofactors
27

28. Potential First Steps
(a) ID SNP Patterns
28

29. Example, CYP1B1 & COMT SNP Combo;
Phase 1 & II Estrogen Metabolism
29

30. Potential First Steps, con’t
• (b) A comprehensive health history
– Principles of Functional Medicine
• Time line
• Antecedents
• Triggers
• Exposures
• Listening skills!!
30

31. Potential First Steps, con’t
Braking System
• (c) GI Health
– 5 R’s
• Remove
• Replace
• Repair
• Reinoculate
• Rebalance
• More on this later
• GI Testing
– Stool
• DNA vs Plated
– Food intolerance
– Autoimmune markers
• Cyrex
– Zonulin, Occludin, LPA
31

32. Potential First Steps, con’t
Braking System
– (d) Balancing calming
with excitatory
neurotransmitters
• Serotonin and GABA
• Dopamine, Nor-epi, Epi,
Glutamate
– Consider
• Neurotransmitter testing
– Adrenal testing
http://www.nimh.nih.gov/health/educational-
resources/brain-basics/brain-basics.shtml
32

33. 33
e.g. , Address glutamate/GABA ratio
and lower serotonin
Potential First Steps’ con’t; Neurotransmitter Biomarkers
Braking System

34. Potential First Steps’ con’t; Neurotransmitter
Biomarkers Braking System
34

35. Potential First Steps, con’t
Braking System
• (e) HPATG Axis
– Pituitary
• ACTH level
– Adrenal
• Free Cortisol, vs
• Metabolized Cortisol
• Cortisone
• DHEAs
– Thyroid
• TSH, fT3, fT4, rT3,
TPOAb, TgAb, TRAb
35

36. HPATG Axis
Braking System
36

37. Potential First Steps, con’t
Braking System
- Gonads
• Estrogen and its
metabolites,
• Testosterone and its
metabolites,
• Progesterone and its
metabolites
- Hormone liver Detox
SNPs
• CYPs, COMT, MTHFR,
SULT, etc.
37

38. Potential First Steps, con’t
Braking System
• (f) Ensure enzymatic
Nutrient Cofactors
– B1 Thiamin
– B2 Riboflavin
– B3 Niacin
– B5 Pantothenic Acid
– B6 Pyridoxine and/or
Pyridoxal-5-Phosphate
– B7 Biotin
– Minerals including
lithium
– Specific form of B12,
maybe…depending on
robustness of “braking
system”
– B1, B2, B3 needed for
Krebs and generating
cofactors
38

39. Let’s Talk SNPs …
39
http://www.dramyyasko.com/wp-content/files_flutter/1327512160_9_1_1_8_pdf_02_file.pdf

40. Overview of well-known Methylation
SNPs
Reprinted from Holistic Heal
Pathway A
Pathway B
Pathway C
40
Pathway D

41. Pathway A
41
®

42. Pathway B
42
®

43. 43
Pathway C
®

44. Pathway D
44
®

45. Pathway B1
45
®

46. Phase I & II Liver Detox Pathways
46
®

47. YEAST & ALCOHOL METABOLISM
METHYLATION INHIBITION & LEAKY GUT & LOW ENERGY PRODUCTION (decreased Kreb’s)
47

48. Page 1
48

49. Page 2
49

50. Page 3
50

51. Page 4
51

52. Page 5
52

53. Page 6
53

54. Page 7
54

55. Page 8
55

56. Page 9
56

57. Page 10
57

58. Page 11
58

59. Page 12
59

60. Page 13
60

61. Page 14
61

62. Page 15
62

63. Page 16
63

64. Page 17
64

65. Page 18
65

66. Page 19
66

67. Coming up...
• Sterling will discuss SNPs and clinical pearls
• We will address SNP interpretation via case
studies
– Example1: GI and autoimmune
– Example2: Leaky gut, inflammatory skin issues
– Example3: Estrogen dominance, poor bile
production, high excitatory neurotransmitters, low
GABA, potential addiction
67

68. SNP Insights And Clinical Pearls
Sterling Hill Erdei
Sterling Hill Erdei has completed a conflict of interest disclosure form and does not
have any conflicts of interest to disclose.
Saturday
January 17, 2014
68

69. Vitamin B6 Dependent Enzymes
• Pyridoxal Phosphate (PLP, pyridoxal 5’phosphate, P5P),
the active form of vitamin B6, is a co-enzyme (nutrient
cofactor) in a variety of enzymatic reactions.
– http://scholarbank.nus.sg/bitstream/handle/10635/16089/Thesis%2020080612.pdf?sequence=1
• A B6-dependent pathway can become dysfunctional if B6
is depleted
– Regardless of whether the pathway includes SNPs
– Pathway dysfunction can “mimic” SNPs, or can
exacerbate SNPs*
* Note: We are using the word “SNP” in this context to refer to the SNP-
affected enzyme
69

70. CBS – P5P Dependent
cystathionine β-synthase
/cys·ta·thi·o·nine β-syn·thase/
(sin´thās) a pyridoxal
phosphate–containing lyase
that catalyzes a step in the
catabolism of methionine.
CBS http://medical-
dictionary.thefreedictionary.com/cystathionine+beta-
synthase
70

71. CBS
71

72. CBS Clinical Pearls:
• Suspect CBS
dysregulation and/or
depleted B6 in those with:
– Sulfur sensitive
– Sulfa drug sensitive
– High homocysteine
– Low glutathione
– High ammonia
– High oxalates (OATs)
– “Fibro”
– http://www.mdpi.com/1099-4300/15/1/372/pdf
Suggestions:
– Replete B6
• Pyridoxal Phosphate
• P5P
– Remove GMO Foods
• Thought to deplete
molybdenum, also a
CBS cofactor
http://www.drperlmutter.com/another-important-threat-
posed-gmo-food/
– Reduce high oxalate
foods (biggie!!)
• More about oxalates later
72

73. GAD
• Glutamate decarboxylase 2
• Glutamate decarboxylase or
glutamic acid decarboxylase
(GAD) is an enzyme that
catalyzes the decarboxylation of
glutamate to GABA and CO2.
GAD uses P5P as a nutrient
cofactor.
• Glutamate is excitatory
• GABA is calming
• http://www.uniprot.org/uniprot/Q05329
73

74. GAD
74

75. GAD Clinical Pearls:
• Suspect GAD dysregulation
and/or depleted B6 in those
with:
– Seizures
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4204538/
• http://adc.bmj.com/content/90/5/512.full.pdf
– Anxiety
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2959081/
– High glutamate
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3679127/
– Low GABA
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2077924/
– “Brain fog”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3611040/
• Microglial activation
Suggestions:
– Replete B6
• Pyridoxal Phosphate
• P5P
– Reduce high oxalate
foods
– NOTE: A P5P deficiency, alone,
may cause production of
endogenous oxalates
75

76. P5P Deficiency and SNPs --- The Domino Effect pt.1
DHFR: Key enzyme in folate metabolism. Contributes to the de novo mitochondrial thymidylate
biosynthesis pathway. Catalyzes an essential reaction for de novo glycine and purine synthesis,
and for DNA precursor synthesis.[1]
FOLR: Binds to folate and reduced folic acid derivatives and mediates delivery of 5-
methyltetrahydrofolate and folate analogs into the interior of cells.
MTHFR: Catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a
co-substrate for homocysteine remethylation to methionine.[2]
MTHFD1: This gene encodes a protein that possesses three distinct enzymatic activities; 5,10-
methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-
formyltetrahydrofolate synthetase. Each of these activities catalyzes one of three sequential
reactions in the interconversion of 1-carbon derivatives of tetrahydrofolate, which are substrates for
methionine, thymidylate, and de novo purine syntheses. (Source: http://www.genecards.org/)
[1] http://www.uniprot.org/uniprot/P00374 [2] http://www.uniprot.org/uniprot/P15328
[3] http://www.genecards.org/cgi-bin/carddisp.pl?gene=SUOX&search=1cf0da205eef5cd2999879fe29ef19e3
76

77. 77

78. DHFR Clinical Pearls
• Fortified foods with
synthetic folic acid are not
recommended
– Unmetabolized folic acid ->
colon cancer
– http://www.greenmedinfo.com/blog/synthetic-folic-acid-
may-contribute-colorectal-cancer2
-> GI inflammation
– https://www.drfuhrman.com/faq/question.aspx?sid=21&qi
ndex=0
-> low serum folate
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317000/
• A B6 deficiency and
subsequent
endogenous
production of oxalates
will adversely impact
the folate cycle.
78

79. FOLR Clinical Pearls
• Folate receptors:
– Cerebral folate transport deficiency
• Sometimes seen in children on the Spectrum
• http://www.genecards.org/cgi-bin/carddisp.pl?gene=FOLR1
– Synthetic folic acid can down-regulate cerebral
folate receptors in those with FOLR SNPs
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759072/
79

80. MTHFR Clinical Pearls
Huge impact from MTHFR
SNPs, but a few things
• Catalytic activity requires
5MTHF, FAD and NAD
(Krebs…B1, B2, B3)
• http://www.uniprot.org/uniprot/P42898
• Enzymatic regulation via SAMe
• Low folate linked to:
– Low T
cellshttp://www.ncbi.nlm.nih.gov/pubmed/15322179
– Low NK cells
– http://www.ncbi.nlm.nih.gov/pubmed/16365081
Anecdotal
• Of eleven people with nine
or more homozygous
SNPs on their Variant
Report; Two had dystonia,
one had Parkinsons, nine
had low dopamine and six
had low B6. Several had
been told that they had
tetrahydrobiopterin
deficiency
80

81. MTHFD1 Clinical Pearls
• Choline dependent
– Impacted by PEMT
SNPs
– Impacted by low
estrogen as PEMT PE
to PC (choline)
conversion requires
estrogen (e.g.,
menopause)
– http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1574369/
– http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2655101/
• MTHFD1 SNPs:
-> low folate
-> low choline
• http://www.pnas.org/content/102/44/16025.short
– Associated with neural
tube defects
– http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2732995/
– If low cholesterol,
suspect low choline and
MTHFD1 impact
– http://www.sciencedirect.com/science/article/pii/S0002822
308020397
81

82. P5P Deficiency and SNPs --- The Domino Effect pt.2
SULT1a1: Sulfotransferase that utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as sulfonate donor
to catalyze the sulfate conjugation of catecholamines, phenolic drugs and neurotransmitters. Has
also estrogen sulfotransferase activity. Responsible for the sulfonation and activation of minoxidil. It
Mediates the metabolic activation of carcinogenic N-hydroxyarylamines to DNA binding products
and could so participate as modulating factor of cancer risk. [1]
SULT2a1: Sulfotransferase that utilizes 3'-phospho-5'-adenylyl sulfate (PAPS) as sulfonate donor
to catalyze the sulfonation of steroids and bile acids in the liver and adrenal glands. [2]
SUOX: Sulfite oxidase is a homodimeric protein localized to the intermembrane space of
mitochondria. Each subunit contains a heme domain and a molybdopterin-binding domain. The
enzyme catalyzes the oxidation of sulfite to sulfate, the final reaction in the oxidative degradation of
the sulfur amino acids cysteine and methionine. (Source: http://www.genecards.org/)
[1] http://www.uniprot.org/uniprot/P50225 [2] http://www.uniprot.org/uniprot/Q06520
[3] http://www.genecards.org/cgi-bin/carddisp.pl?gene=SUOX&search=1cf0da205eef5cd2999879fe29ef19e3
82

83. SULT
• SULT encodes Phase II
liver detox enzymes
– The “Sulfation Pathway”
– http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3426368/
– Utilizes 3'-phospho-5'-adenylyl
sulfate (PAPS) as a sulfur
donor to conjugate some
hormones, neurotransmitters,
phenolytic drugs, some
catecholamines, and
xenobiotics.
– http://www.uniprot.org/uniprot/P50225
– Low Vit B6 will impact SULT
function!!!
• Oxalate connection
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1847
840/
83

84. SULT1A1 Clinical Pearls
– Plays a role in estrogen
(estradiol) breakdown
• SNPs can negatively impact
estrogen breakdown in Phase II
liver detox step
• Strongly suspect “estrogen
dominance” if SULT SNPs in
conjunction with COMT and
CYP1B1 SNPs
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1410
736/?report=reader
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2767
256/
Susan Costen Owens MD:
When Vit B6 is depleted, endogenous
oxalate production may ensue.
The endogenous oxalates may negatively
impact oxidative phosphorylation
(Krebs).
This negative impact on phosphorylation is
decreased energy production at the
cellular level (ATP).
That can cause sulfur wasting into urine
since the sulfate cannot then be used
for sulfation. Sulfate has to be
converted into PAPS (3′-
phosphoadenosine-5′-phosphosulfate) to be
used to sulfate anything, and that
requires ATP.
84

85. SULT2A1 Clinical Pearls
• Utilizes PAPS as sulfonate
donor to catalyze the
sulfonation of:
– Steroids
– Bile acids
• in the liver
• In the adrenal glands
• Estrogen induced cholestasis
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2767
256/
• http://www.uniprot.org/uniprot/Q06520
• Anecdotal
– Individuals who have run
Sterling’s app and have had
more than five homozygous
SULT2a1 SNPs have
experienced gallbladder
issues. Several have had
their gallbladders removed
– COMT SNPs further
exacerbate
• COMT H62H
• COMT V158M
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436139/
85

86. SUOX
• SUOX encodes an
enzyme in the “Trans-
sulfuration pathway”
• http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3590070/
– Downstream from
homocysteine and CBS
enzyme
• Vit B6 dependant
– Converts sulfites to sulfates,
the final reaction in the
oxidative degradation of the
sulfur amino acids cysteine
and methionine
http://www.sciencedirect.com/science/article/pii/S03877604090
02484
Pathway:
86

87. SUOX Clinical pearls
• SOUX SNPs
– Suspect expressing if
low molybdenum, low
B6 and/or high oxalates
• Oxalates and sulfates
are regulated by a
shared
transporter/exchanger
• http://www.uniprot.org/uniprot/A3M0G5
• Glycophosphates
– PON1
– Round-up; think GMO
grains:
• Are known to chelate/deplete
molybdenum
• Eventual impact to the trans-
sulfuration pathway
• High ammonia may result
• http://www.glyphosate.eu/response-glyphosate-
task-force-study-published-journal-entropy
• Appears as a CBS up-
regulation
• http://www.degruyter.com/view/j/intox.2013.6.issue
-4/intox-2013-0026/intox-2013-0026.xml
87