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S. Jill James, Ph.D.
Department of PediatricsArkansas Children’s Hospital Research InstituteUniversity of Arkansas for Medical Sciences
Little Rock, AR
Pathologic Implications of Low Glutathione LevelsAnd Oxidative Stress in Children with Autism: Metabolic Biomarkers and Genetic Polymorphisms
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Pathways of Folate/Methionine/Glutathione metabolism;Impact of Oxidative Stress
Mechanisms and Functions of Glutathione
Depletion of glutathione with Thimerosal in vitro
Abnormal Methylation and Oxidative Stress in Autistic Children: Treatment with Methyl B-12, Folinic Acid, and TMG
Selected Genetic Polymorphisms Associated with the Abnormal Metabolic Profile in Autism
Implications of Oxidative Stress in the Pathogenesis of Autism
Overview
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Homocysteine
B6
Methionine Transsulfuration to Cysteine and Glutathione
THF: tetrahydrofolate
Methionine
Enzymes
5-CH3THF
THF
B12MS5,10-CH2THF
MTHFR
1
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Homocysteine
B6
Methionine Transsulfuration to Cysteine and Glutathione
THF: tetrahydrofolate
Methionine
Enzymes
5-CH3THF
THF
B12MS5,10-CH2THF
MTHFR
1
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SAM
SAH
MTase
SAHH
Homocysteine
B6
BHMT
Choline
Betaine
Methionine Transsulfuration to Cysteine and Glutathione
THF: tetrahydrofolate
Methionine
Adenosine
Enzymes
5-CH3THF
THF
B12MS5,10-CH2THF
MTHFR
Cell Methylation1 2
MethylationPotential
(SAM/SAH)
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SAM
SAH
MTase
SAHH
Homocysteine
B6 CBS
BHMT
Choline
Betaine
Methionine Transsulfuration to Cysteine and Glutathione
THF: tetrahydrofolate
Cystathionine
Cysteine
GSH GSSG
Methionine
Adenosine
B6
Enzymes
B6
5-CH3THF
THF
B12MS5,10-CH2THF
MTHFR
MethylationPotential
(SAM/SAH)
Cell Methylation1 2
3 AntioxidantPotential
(GSH/GSSG)
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SAM
SAH
MTase
SAHH
Homocysteine
B6 CBS
BHMT
Choline
Betaine
Methionine Transsulfuration to Cysteine and Glutathione
Cystathionine
Cysteine
GSH GSSG
Methionine
Adenosine
B6
B6
5-CH3THF
THF
B12MS5,10-CH2THF
MTHFR
Cell Methylation1 2
3
1
2
3
Folate Cycle
Methionine Cycle
TranssulfurationPathway
MethylationPotential
(SAM/SAH)
AntioxidantPotential
(GSH/GSSG)
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Methionine is an essential AA: Methionine Cycle conserves methionine
Cysteine is a “conditionally” essential AA: >50% of cysteine is derived from methionine via the transsulfuration pathway; normal cysteine levels depend on normal methionine levels. Cysteine may be an essential amino acid in children with autism!
Cysteine is the rate-limiting amino acid for glutathione (Glu-Gly-Cys) synthesis: the thiol (-SH) group of cysteine is the active antioxidant component of glutathione.
RELEVANT BACKGROUND INFORMATION
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How Does Oxidative Stress InfluenceMethionine Metabolism?
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SAM
SAH
MTase
SAHH
Homocysteine
B6
THF
CBS
B12
Protein synthesis
BHMT
Choline
Betaine
Impact of Oxidative Stress on Methionine Transsulfuration
5-CH3THF
Cystathionine
Cysteine
Glutathione
Methionine
AdenosineAK
ADA
Inosine
AMP
B6
Methylation of DNA, RNA, Proteins, Catecholamines, Phospholipids, CreatineMS
Oxidative stress promotesa metabolic imbalance that further depletes glutathione
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SAM
SAH
MTase
SAHH
Homocysteine
B6 CBS
BHMT
ESTROGEN: THE FEMALE ADVANTAGE IN THIOL CHEMISTRY
Cystathionine
Cysteine
Glutathione
Methionine
Adenosine5-CH3THF
THF
B12MS5,10-CH2THF
MTHFR
PhosphatidylcholineMethylation
ESTROGEN
MAT
GCL
Betaine
Choline
Estrogen may protect little girlsfrom developing autism
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ANTIOXIDANT FUNCTION OF GLUTATHIONE
(active) (inactive)Major intracellular antioxidant: H2O2, superoxide, hydroxyl radical,
peroxynitrite, membrane lipid peroxidation
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Maternal/Fetal Drug/Carcinogen Exposures
Glutathione Conjugate
Mercapturic Acid(Cysteine conjugate)
Bile and Urine Excretion*
Glutamine
Glycine
GST
*Cysteine loss = increased requirement for de novo glutathione synthesis; sulfur loss in urine
Heavy Metals
DETOXIFICATION FUNCTIONS OF GLUTATHIONE
Detoxification: Hg, As, Pb, Cd bind to thiol (SH) group; Metal-cysteine conjugates excreted
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Functional Consequences of Low GSH/GSSG and Increased Oxidative Stress
Reduced ability to detoxify environmental toxicants and heavy metals:Neurotoxicity; Immunotoxicity
Oxidation of active site cysteine (-SH groups) in enzymes: altered structure/function:
Abnormal methionine metabolism; Altered membrane signaling
Decreased liver GSH synthesis: reduced export of cysteine to brain:Reduced astroglial/neuronal GSH synthesis: increased sensitivity to heavy metals; Neurotoxicity
Integrity of the gut epithelium compromised: Increased mucosal membrane permeability; malabsorption
Altered T-cell subpopulations:Decreased Th1:Increased Th2; autoimmunity; gut inflammation
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Neurotoxicity of Thimerosal in Human Brain Cellsis Associated with Glutathione Depletion:
Protective Effect of N-Acetyl Cysteine or Glutathione
S. Jill James, William Slikker, Elizabeth New, Stefanie Jernigan, Stepan Melnyk
Neurotoxicology, volume 26: pp1-8, 2005
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Methionine
S-adenosylmethionine
S-adenosylhomocysteine
HomocysteineB6
Methylated products:DNA, RNA, protein,phospholipids, neuro-transmittors
Methionine Synthase
Cystathionine
Cystathionine β Synthase
B12
GLUTATHIONE
Adenosine5-CH3THF
5,10-CH2THF
B6THF
Cysteine
Cystathionine Lyase
Cystathionine Lyase is not expressed in brain; therefore brain cells are dependent on plasma cysteine (derived primarily from the liver) for intracellular glutathione synthesis.
GLUTATHIONE SYNTHESIS IN THE BRAIN IS DEPENDENTON HEPATIC CYSTEINE SYNTHESIS AND EXPORT
B6
Lack of Cystathione Lyase renders developing brain highly sensitive to GSH depletion and oxidative stress and increases requirement for folate, methionine, and cysteine.
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WORKING HYPOTHESIS
The neurotoxicity of Thimerosal is associated with depletionof glutathione, the major intracellular antioxidant
Ethyl mercury in Thimerosal binds to cysteine thiol (–SH) groups on intracellular proteins and inactivates function.
The cysteine-SH group of glutathione, binds mercuryand protects essential proteins from functional inactivation.
Glutathione is the major mechanism of mercury detoxification
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Intr
a ce l
l ul a
r G
SH
(nm
ol/m
g p r
o te i
n)
0
10
20
30
40
50
0
10
20
30
40
50
Control +T T+NAC T+GSH Control +T T+NAC T+GSH
Intracellular glutathione levels in cells exposed to 15 µµµµM Thimerosal (T) in presence of 100 µµµµM N-acetylcysteine (NAC)
or glutathione ethyl ester (GSH)
GLIOBLASTOMA NEUROBLASTOMA
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A Project Funded by SafeMinds
Relative sensitivity to oxidative stress between Autistic and Control Lymphoblastoid cells
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Lymphoblastoid cell lines from autistic children withat least one affected sibling were obtained from AGRE.
Unaffected healthy control lymphoblastoid cell lines were obtained from ATCC
Pairs of autistic and control cells lines were cultured underidentical conditions. Rate of free radical generation, GSH/GSSG, , and mitochondrial caspase-3 were measured at baseline and after exposure to oxidative stress.
Experimental Procedures
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ROS generation with thimerosal dose
0
500
1000
1500
2000
0 0.625 1.25 2.5 5
Thimerosal Concentration (uM)
Vm
ax (m
un
its/m
in)
Control
Autistic
Cells from autistic children generate more free radicals than control cells
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fGSH:fGSSG
0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00
100.00
0 0.312 0.625 1.25 2.5
Thimerosal Concentration (uM)
Control
Autistic
Cells from autistic children have lower GSH/GSSG ratio than control cells
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uM Caspase-3/mg protein
00.05
0.10.150.2
0.250.3
0.350.4
0 0.3125 0.625 1.25 2.5
Thimerosal Concentration (uM)
Cas
pas
e-3
(uM
)
Control
Autistic
Cells from autistic children have higher cell death rate than control cells
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Since both cell lines were cultured at the same time under identical conditions with identical media, the differences at baseline and after exposure to oxidant stress must reflect inherent genetic differences.
These results provide experimental evidence that autistic children may be genetically more sensitive to pro-oxidant environmental exposures.
CONCLUSION
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Increased Oxidative Stress and Impaired Methylation Capacity in Children with Autism: Metabolic Biomarkers
and Genetic Predisposition
Results of Intervention Trial with Folinic Acid, TMG, and Methyl-B12
S. Jill James, Ph.D., Laurette Janak, M.O.M., Stepan Melnyk, Ph.D., Stefanie Jernigan, Paul Cutler, M.D.
American Journal of Clinical Nutrition volume 80: pp 611-17,2004
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Autistic Control p valuen=80 n=75
Methionine (µM/L) 21.2 28.0 <0.0001 Subset: 51% <20µM/L 16.8
SAM (nM/L) 81.5 94.2 <0.0001Subset: 28% <75µM/L 65.7
SAH (nM/L) 23.4 19.5 <0.0001Subset: 20% >28 µM/L 36.6
SAM/SAH Ratio 3.9 5.1 <0.0001Subset: 61% <4 3.0
Baseline Methionine Cycle Metabolites:Subset means within the overall means
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INTERPRETATION
1. The significant decrease in methionine, SAM and homocysteine levels in autistic children is consistent with reduced turnover of the methionine cycle.
2.The decrease in SAM and increase in SAH (decreased SAM/SAH ratio) provides metabolic evidence that methylation capacity may be reduced in autistic children.
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Baseline Transsulfuration Metabolites:Subsets within the overall means
Autistic Control p valuen=80 n=75
Cysteine (µM/L) 161 205 <0.0001 Subset: 71% <170µM/L 153
Total GSH (µM/L) 5.1 7.5 <0.0001Subset: 66% <5µM/L 4.4
Free GSH (µM/L) 1.4 2.1 <0.0001Subset: 86% >2µM/L 1.2
GSSG (µM/L) 0.4 0.24 <0.0001Sunset: 61% <0.3µM/L 0.5
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INTERPRETATION
1. The significant decreases in homocysteine, cystathionine, cysteine and glutathione suggests that the transsulfuration pathway is insufficient for adequate glutathione synthesis.
2. The increase in GSSG (oxidized inactive glutathione) and decrease in GSH (active antioxidant glutathione) is strong evidence that oxidative stress is increased in autistic children.
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Methionine: Low methionine is correlated with low SAM
Total Homocysteine: Elevated homocysteine is correlated with elevated adenosine and SAH
Cysteine: Low cysteine is correlated with low GSH and taurine
GSH: Low GSH indicates low detox and antioxidant capacity
MINIMUM ESSENTIAL THIOL PROFILE AVAILABLE COMMERCIALLY
Note: AA profiles measure “free” homocysteine which is usually negligible: Ask for “total” homocysteine (protein bound plus free)
(FASTING)
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Pharmacologic doses of nutrient cofactors can break the vicious cycles, restore normal flux, and release metabolic blocks by mass action
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Folinic Acid, Betaine, and Methyl B-12 Supplementation in Children with Autism
Eight of the children participated in an interventiontrial and were given 800 µg folinic acid and 1000 mg betaine b.i.d. for 3 months and the plasma metabolites were re-measured.
The children were then given injectible methyl-B12 (75 µg/Kg 2x/week) and the plasma profile was repeated after 4 weeks of combined folinic acid, betaine, and methyl B12
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Methionine
SAM
SAH
MTase
SAHH
Homocysteine
B6
Methylation of DNA,RNA, histones,creatinephospholipids
CBS
Protein synthesis
TMG
DMG
Folinic Acid
dNTPs
DNA synthesis
TranssulfurationPathway
Glutathione
Cysteine
Cystathionine
Adenosine
5,10 CH2 THF
Supplementation:800 µg folinic acid, b.i.d.1000 mg betaine, b.i.d.75 µg/Kg methyl-B12
BHMT
Me-B12
5CH3THF
THF
MS
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Proportion of Autistic Children within Normal RangeBefore and After Supplementation
Folinic+BetaineMetabolite Normal Range a Baseline Folinic+Betaine +methylB12Methionine (µmol/L) > 24 1/8 5/8 7/8SAM (nmol/L) > 80 2/8 8/8 8/8SAH (nmol/L) < 23 2/8 7/8 7/8SAM/SAH > 4 1/8 7/8 7/8Adenosine (µmol/L) < 0.3 4/8 8/8 8/8Homocysteine (µmol/L) < 5.5 3/8 8/8 8/8 Cysteine (µmol/L) <180 0/8 2/8 7/8GSH (µmol/L) > 5.4 0/8 2/8 7/8GSSG (µmol/L) < 0.33 0/8 2/8 8/8GSH/GSSG > 16 0/8 3/8 8/8
____________________________________________________________________________a Range estimated to include 90% of control children
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INTERPRETATION
Folinic Acid and Betaine brought all the methionine cycle metabolites into normal range.
The combined regimen of Folinic Acid, Betaine, and Methyl B12 brought all the transsulfuration metabolites into the normal range.
Best predictors of impaired methylation are low methionineand SAM/SAH ratio OR elevated adenosine.
Best predictors of impaired antioxidant defense are low cysteine, and low glutathione (low GSH/GSSG ratio).
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Expanded Baseline Data and Lessons LearnedWe now know after analyzing baseline metabolites from over 100 autistic children:
1. Each child is unique – one size (dose) does not fit all
2. Low cysteine, low free glutathione, low GSH/GSSG Ratio (reduced antioxidant capacity) are present in over 80% of autistic children.
3. Increased SAH and elevated adenosine (reduced methylation capacity) only occurs in a subset of about 21% of autistic children.
4. About 20% of children with autism do not tolerateTMG (become hyperactive) and about 5% do not tolerate folinic acid or methylB12.
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A fragile homeostatic balance underliesincreased sensitivity to oxidative stress
in autistic children
SAM
SAH
Homocysteine
B6 CBS
Methionine Transsulfuration to Cysteine and Glutathione
Cystathionine
Cysteine
Methionine
Adenosine
B6
B6
5-CH3THF
THF
B12MS5,10-CH2THF
MethylationPotential
(SAM/SAH)
Cell Methylation
AntioxidantPotential
(GSH/GSSG)Glutathione
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Chronic oxidative stress decreases bothmethionine cycle flux and glutathione synthesis
creating a self-perpetuating vicious cyclein autistic children
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Adenosine
SAM
SAH
MTase
SAHH
Homocysteine
B6
THF
CBS
5-CH3THF
Cystathionine
Cysteine
Glutathione
Methionine
Methylation of DNA, RNA, Proteins, Catecholamines, Phospholipids, CreatineMS
MAT
Oxidized B12B12
Chronic oxidative stress decreases bothmethionine cycle flux and glutathione synthesis
SELF-PERPETUATINGCYCLE LEADING TO LOWER SAM AND
LOWER GSH
Increased susceptibilty to infection anddecreased ability to resolve inflammation
ROS
B6
B6
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Viral infection depletes GSH and increases oxidative stress
Influenza, herpes, measles virus infection induce GSH export
GSH blocks cytokine induction required for viral replication, reduces viral reverse transcriptase activity and blocks expression of viral proteins
Maintains cell-mediated immune response (Th1/Th2 ratio)
GLUTATHIONE AND VIRAL INFECTION
GSH is a potent anti-viral agent!
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Treatment strategies that increase SAM and GSH are protective against viral infection
Low SAM and low GSH self-amplify and increase susceptibility to viral infection, increase virulence, and impair timely resolution of the viral infection
Many autistic children have low SAM and low GSH
AUTISM, GSH, AND VIRAL INFECTION
High levels of SAM and GSH are anti-viral
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Is there a genetic basis for increased vulnerability to oxidative stress?
e.g.,
Mercury and other heavy metal toxicity; cell death
Autoimmunity: increased T helper2 cells
Gut Inflammation: increased inflammatory cytokines
Redox imbalance in the brain: inflammation; cell death
Redox enzyme inhibition: excess dopamine, glutamate
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THF
5,10-CH2-THF
5-CH3-THF
B12
Cystathionine
DMG
Methionine
Homocysteine
SAM Methyl Acceptor
Methyltransferase
Methylated ProductMTHFR
TC II
SAH
Cysteine
Glutathione
Adenosine
Metabolic Response to Genetic Polymorphisms in the Methionine Cycle
GST
COMT
RFC
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MTHFR: Methylenetetrahydrofolate reductaseTransfers methyl groups for methionine synthesis
MTRR: Methionine synthase reductaseMay reduce methionine synthase activity
RFC: Reduced Folate Carrier;Transports folate into the cell
GSTs: Glutathione-S- TransferaseImportant for glutathione detoxification capacity
TCII: Transcobalamin II; Transports methylB12 into the cell
COMT: Catechol-O-methyltransferase; Methylates dopamine and prevents dopamine-induced oxidative stress in the brain
APO E4: May help detoxify mercury
Functions of Polymorphic Genes Analyzed
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1. MTHFR 677 TT Frequency Odds Ratio p valueControl Individuals (203): 10.7% Autistic Children (360): 13.1% 1.47 NS**
2. MTHFR 677 CTControl Individuals (205): 43.9% Autistic Children (360): 49.2% 1.35 NS
3. MTHFR 677CT/1298ACControl Individuals (205): 19.1%Autistic Children (360): 23.7% 1.7 NS; 0.07
Polymorphisms in the Methionine Cycle Pathway
**NS: Not statistically significant
Methylenetetrahydrofolate Reductase (677C→T;1298A→C)
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Polymorphisms in the Methionine Cycle Pathway
Transcobalamin II (TCII 776 66C→G)
1. TCII 776 GG Frequency Odds Ratio p valueControl Individuals (203): 16.0% Autistic Children (360): 25.8% 1.8 0.05**
2. TCII 776 CC+CGControl Individuals (205): 84.0% Autistic Children (360): 74.2% 0.55 0.01**
**Statistically significant
TCII G allele is significantly more frequent in autistic children
Theoretically, this could reduce B12 transport inside the celland could decrease methionine synthase activity
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Polymorphisms in the Methionine Cycle Pathway
Reduced Folate Carrier RFC1 80A→G
RFC1 80 GG Frequency Odds Ratio 95% C.I.
Control Individuals (203): 30% Autistic Children (360): 39% 2.0 1.15,3.33*
A decrease in the transport of folate into the cells would producea functional folate deficiency
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1. GST M1 Null Frequency Odds Ratio p valueControl Individuals (205): 57.2%Autistic Children (360): 49.2% 1.4 0.07, NS
2. GST T1 NullControl Individuals (183): 25.8%Autistic Children (233): 22.7% 0.86 NS
Polymorphisms Glutathione-S-Transferase (GST) Affecting Antioxidant Capacity
GST M1 and GST T1
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COMT 472GG:(high activity variant) Frequency Odds Ratio 95% C.I.
Control Individuals (205): 16.3%Autistic Children (360): 26% 2.34 1.06,2.85*
Polymorphisms Affecting Methylation andIncreased Oxidative Stress
Catechol-O-Methyltransferase (COMT 1947A→G)
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Gene-Gene Interactions Affecting Methylation and Increased Oxidative Stress
TCII GG/COMT GG Frequency Odds Ratio 95% C.I.Control Individuals (203): 2.5% Autistic Children (360): 9.7% 7.0 2.32,21.2*
Combined TCII GG plus COMT GG in the same individual
The TCII and COMT gene polymorphisms may interact to synergistically increase the risk of autism
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Gene-Gene Interactions Affecting Methylation and Increased Oxidative Stress
TCII GG/GSTM1 null Frequency Odds Ratio 95% C.I.Control Individuals (203): 7.6% Autistic Children (360): 13.4% 2.2 1.05,4.4*
Combined TCII GG plus GSTM1 null in the same individual
The TC II and GSTM1 null gene polymorphisms interact to increase the risk of autism
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RFC1 GG/COMT GG Frequency Odds Ratio 95% C.I.Control Individuals (203): 6.7% Autistic Children (360): 13.8% 2.5 1.05,5.75*
Combined RFC1 GG plus COMT AG in same individual
Polymorphisms in the RFC1 and COMT genes interact to increase risk of autism
Gene-Gene Interactions Affecting Methylation and Increased Oxidative Stress
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No single polymorphism alone can predict increased risk of autism because, by definition, polymorphisms are highly prevalent in normal people as well. It is possible, however, that specific combinations of these polymorphisms interact to shift specific metabolic pathways that are important in the pathogenesis of autism.
Important Caveat
To determine if a relationship exists between metabolic profile and genetic profile will require statistical analysis of ~1500 cases and ~1500 control children.
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Environment
Hormones
Genes
Glutathione depletionOxidative stress
Gut inflammation: Dysbiosis; leaky gut;
Immune Dysfunction:Frequent infections, Chronic inflammation
Emotional stress
Timing, duration, developmental stage, severity
FINAL COMMON PATHWAY
Dietary deficienciesToxinsViral/Bacterial infections
EpigeneticsSNPsMutations
EstrogenTestosteroneThyroxin
Neuropathology
↑ cytokines; inflammation
Mitochondrial Dysfunction
↑ ROS; ↓ ATP production
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Reduced Methionine(Oxidative inhibition of MS)
Reduced SAM (Low methionine)
Reduced Cysteine(Low methionine, Homocysteine)
Reduced Glutathione(Low Cysteine)
Reduced Cellular Antioxidant Capacity in Autismand Increased Vulnerability to Oxidative Stress
Metabolic Indicators
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You Are HereCellular Metabolic Pathways
Putting it all into Perspective……we see what we know
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The abnormal metabolic profile in children with autism strengthens the hypothesis that a genetic inability to control oxidative stress may be central to the development of neurologic, immunologic, and gastrointestinal dysfunction that occurs with autism.
OVERALL CONCLUSION MAY 2006
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Dr. Moms
ResearchersAnd Physicians
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EnvironmentGenes
InflammationInfection
Hormones
Autism
Timing
Factors Contributing to Oxidative Stressin Autistic Children
Gut Inflammation Brain InflammationImmune dysfunction