Post on 27-Dec-2015
Autism Research and On-Going Clinical Trials in Arkansas
S. Jill James, PhD
Professor, Department of PediatricsDirector, Autism Metabolic Genomics LaboratoryArkansas Children’s Hospital Research Institute
University of Arkansas for Medical SciencesLittle Rock, AR
OVERVIEW
A little basic biochemistry: folate/methionine/glutathione
Abnormal metabolic profile in children with autism Efficacy of methylB12 and folinic acid treatment
Parent Metabolic profiles
Specific Aims of our 5 year NIH-funded study
Placebo-controlled double-blind cross-over study of broad spectrum nutritional supplementation
Autism Treatment Network, Department of Defense Grant
Autism: Beyond the Brain
SAM
SAH
MTase
SAHH
Homocysteine B6 CBS
Methionine Transsulfuration to Cysteine and Glutathione
Cystathionine
Cysteine GSH GSSG
Methionine
Adenosine
B6
B6
5-CH3THF
THF
B12MS5,10-CH2THF Cell Methylation1
1
2
3
Folate Cycle
Methionine Cycle
Transsulfuration Pathway
Methylation Potential(SAM/SAH)
2
3 Antioxidant Redox Potential (GSH/GSSG)
SAM
SAH
MTase
SAHH
Homocysteine B6Cystathionine
Cysteine GSH GSSG
Methionine
Adenosine 5-CH3THF
THF
B12MS5,10-CH2THF
Cellular MethylationReactions
Purines and Thymidylate
DNA SYNTHESIS
PROLIFERATION
METHYLATION
REDOX HOMEOSTASIS
Vital Importance of these Interdependent Metabolic Pathways
1 2
3
Evidence for increased oxidative stress and impaired
methylation capacity in children with autism
Results of intervention trial with methylB12and folinic acid treatment
American Journal of Clinical Nutrition 2004American Journal of Medical Genetics 2006 American Journal of Clinical Nutrition 2008
Autistic Control p value
n=80 n=75
Methionine (µM/L) 20.6 ± 5.2 28.0 ± 6.5 <0.0001
SAM (nM/L) 84.3 ± 11 93.8 ± 18 <0.0001 SAH (nM/L) 23.3 ± 7.9 18.8 ± 4.5 <0.0001 SAM/SAH Ratio 4.0 ± 1.7 5.5 ± 2.8 <0.0001 Homocysteine 5.7 ± 1.2 6.0 ± 1.3 0.03
Fasting Plasma Transmethylation Metabolites
Means ±SD
INTERPRETATION
The significant decrease in methionine and SAM and increase in SAH levels in autistic children
provides metabolic evidence that methylation capacity may be reduced in autistic children.
Fasting Plasma Transsulfuration Metabolites
Autistic Control p value n=80 n=75
Cysteine (µM/L) 165 ± 14 207 ± 22 <0.0001
Total GSH (µM/L) 5.1± 1.2 7.5 ± 1.7 <0.0001
Free GSH (µM/L) 1.4 ± 0.5 2.2 ± 0.9 <0.0001
GSSG (µM/L) 0.4 ± 0.2 0.24 ± 0.1 <0.0001
Free GSH/GSSG 4.9 ± 2.2 7.9 ± 2.5 <0.0001
Means ±SD
INTERPRETATION
1.The significant decreases in homocysteine, cysteine and glutathione suggest 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.
Intervention: MethylB12 (75µg/Kg every 3 days) (3 months) Folinic Acid (400 µg bid)
Inclusion Criteria: Autistic Disorder (DSM-IV; CARS) Age 3-7
No previous supplements GSH < 6.0
Endpoints: Methylation and glutathione metabolitesVineland Adaptive Behavioral Scales
Can supplementation with methyl-B12 and folinic Acid improve glutathione levels and core behaviors
in autistic children?
SAM
SAH
MTase
SAHH
Homocysteine B6Cystathionine
Cysteine GSH GSSG
Methionine
Adenosine 5-CH3THF
THF
B12MS5,10-CH2THF
Cellular MethylationReactions
Purines and Thymidylate
DNA SYNTHESIS
1 2
3
Methyl B12
Folinic Acid
Folinic Acid
Intervention Trial with MethylB12 and Folinic Acid
Plasma Metabolite Concentration
Autism Pre-treatment
(n = 40)
Autism Post-treatment
(n = 40) p valuea
Methionine 21 ± 4b 22 ± 3 ns
SAM (nmol/L) 66 ± 13b 69 ± 12 ns
SAH (nmol/L) 15.2 ± 5 14.8 ± 4 ns
SAM/SAH (µmol/L) 4.7 ± 1.5b 5.0 ± 2.0 ns
Homocysteine (µmol/L) 4.8 ± 1.8 5.3 ± 1.1 0.04
Cysteine (µmol/L) 191 ± 24b 215 ± 19 0.001
Total Glutathione (µmol/L) 5.4 ± 1.3b 6.2 ± 1.2 0.001
Free Glutathione (µmol/L) 1.5 ± 0.4b 1.8 ± 0.4 0.008
GSSG (µmol/L) 0.28 ± 0.08b 0.22 ± 0.06 0.001
tGSH/GSSG 21 ± 6b 30 ± 9 0.001
fGSH/GSSG 6 ± 2b 9 ± 3 0.001
b Signficantly different from age-
matched control children
a Treatment effect
SUMMARY OF METABOLIC RESULTS
1. Baseline metabolites were significantly different from age-matched controls
2. The treatment did not significantly improve levels of methionine, SAM or SAM/SAH
3. The treatment did significantly improve cysteine, glutathione, and GSH/GSSG
4. Although significantly improved, glutathione and GSH/GSSG did not reach levels in control children
The Vineland Adaptive Behavior Scales (VABS) provides a numerical score for adaptive functioning in the areas of communication, socialization, daily living skills, motor skills, and an adaptive behavior composite (ABC) score.
The data are presented as the mean score for each category before and after intervention.
Behavioral Evaluation
Vineland Category
Baseline Score
(mean ± SD)
Post-Treatment
Score (mean ± SD)
Change in Score
(mean; 95% C I)
p value
Communication 65.3 ± 12.9
72.0 ± 15.5 6.7 (3.5, 10) <0.001
Daily Living Skills
67.0 ± 76 76.0 ± 17.7 9.0 (4.0, 14) <0.007
Socialization 68.2 ± 9.3 75.7 ± 14.7 7.5 (3.5, 11) <0.005
Motor Skills 75.6 ± 9.7 79.0 ± 14.7 3.3 (0, 8) 0.12
Composite Score
66.5 ± 9.2 73.9 ± 17.0 6.6 (2.3, 11) <0.003
BEHAVIOR SCORES
SUMMARY OF BEHAVIOR RESULTSAlthough treatment with methylB12 and folinic acid significantly improved core behaviors, they did not reach standard scores for unaffected children (100 ± 15)
Improvement in measures of both metabolic and behavioral endpoints converge to suggest that some children may benefit from targeted nutritional intervention.
This open label trial provides strong preliminary evidence for a double-blind placebo-controlled clinical trial.
CONCLUSIONS
Autism Moms Control Moms (n = 46) (n= 200)
Methionine (µM/L) 24 ± 5 26 ± 6
SAM (nM/L) 80 ± 19 83 ± 13
SAH (nM/L) 33 ± 14* 23 ± 8.4
SAM/SAH Ratio 3.1 ± 1.7* 4.0 ± 1.4
Homocysteine (µM/L) 11 ± 3.9* 7.6 ± 1.6
*statistically significant
Maternal Methionine Cycle Metabolites:
Maternal Transsulfuration Metabolites
Autism Moms Control Moms
Cysteine (µM/L) 232 ± 40 231 ± 20
Total GSH (µM/L) 5.1 ± 1.7* 7.3 ± 1.5
Free GSH (µM/L) 1.5 ± 0.5* 2.6 ± 0.6
GSSG (µM/L) 0.30 ± 0.08* 0.24 ± 0.04
Total GSH/GSSG 17 ± 8 31 ± 10*
*statistically significant
Metabolite imbalance and the risk of being a mother of a child with
autism
Stratified GroupControlMothers
(N=200)
CaseMothers
(N=46)
Odds Ratio (Risk)
SAH >30µMol/L) 14% 54% 6.9
SAM/SAH <2.5 10% 54% 10.7
tGSH/GSSG <20 11% 65% 15.2
SAM/SAH <2.5 and tGSH/GSSG <20
3% 41% 46
It is not possible to determine from this data whether the abnormal metabolic profile in parents is genetically determined or whether it simply reflects the stress of living with an autistic child
IMPORTANT CAVEAT
METABOLIC BIOMARKERS OF AUTISM:PREDICTIVE POTENTIAL AND GENETIC SUSCEPTIBILITY
A 5 YEAR NIH-FUNDED STUDY (2006-2011)
Specific Aim 1: To determine whether the observed metabolite imbalance is associated with quantitative measures of autistic behavior
An expanded database of metabolic profiles will allow us to determine whether the metabolite imbalance is associated with abnormal behaviors.
SPECIFIC AIM 1: METABOLITES AND BEHAVIOR
SPECIFIC AIM 2: PROSPECTIVE STUDY
Specific Aim 2: To investigate whether the abnormal metabolic profile is present before the diagnosis of autism among toddlers 18-30 months of age who are identified in developmental delay clinics to be at increased risk of developing autism.
An autism screening test (MCHAT) and plasma metabolic biomarkers will be measured at Visit 1 and children will be followed for subsequent diagnosis of autism (case) or developmental delay (control).
Metabolic data will be analyzed statistically to determine whether metabolic abnormalities precede the behavioral diagnosis of autism and could serve as predictive biomarkers for risk of autism.
SPECIFIC AIM 2: PROSPECTIVE STUDY
Autism Diagnosis
Visit 1: M-CHAT (18-30 months)
FAIL = High Risk PASS = Developmental Delay and Normal
CONTROLS
Visit 2: M-CHAT Repeat M-CHAT Repeat
(1-6 months) (6 months)
Metabolic Profile Metabolic Profile
PASS
Visit 3: DSM-IV; CARS; ADOS
Control
Not Autism
AUTISM PROSPECTIVE STUDY DESIGN
FAIL
If the metabolic profile is found to precede the behavioral diagnosis of autism, subsequent studies would determine whether early intervention to normalize the metabolic profile can reduce or prevent the development of autism.
IMPLICATIONS OF AIM 2 AUTISM PROSPECTIVE STUDY
Specific Aim 3: To establish whether cells from children with autism exhibit evidence of increased oxidative stress and oxidative damage.
This mechanistic aim will determine whether lymphocytes from autistic children are inherently more vulnerable to oxidative stress than control cells
SPECIFIC AIM 3: CELLULAR CONSEQUENCES
Lymphoblastoid cell lines from autistic individuals with at least one affected sibling were compared with lymphoblastoid cell lines from unaffected controls*
Pairs of autistic and control cells lines were cultured under identical conditions. Rate of free radical generation, GSH/GSSG were measured at baseline and after exposure to thimerosal as oxidative stress.
EXPERIMENTAL PROCEDURES
0
5
10
15
20
25
30
35
fGSH GSSG (x 10)
nm
ol/m
g p
rote
in Control
Autistic
0
20
40
60
80
100
120
140
Control AutisticfG
SH
/GS
SG
Baseline intracellular glutathione status in autistic and control lymphoblastoid cell lines
*
*
*
* p < 0.05
Cells from autistic children generate more free radicals than control cells
Relative Free Radical Generation (DCF)
0
100
200
300
400
500
600
700
800
900
0 0.3125 0.625 1.25 2.5
Thimerosal Concentration (uMol/L)
Vm
ax
RO
S R
ate
Control
Autistic
Cells from autistic children have lower GSH/GSSG ratio than control cells
Glutathione Redox Ratio (GSH/GSSG)
0
20
40
60
80
100
120
140
160
0 0.16 0.32 0.62 1.25 2.5
Thimerosal Concentration (uMol/L)
Control
Autistic
MITOCHONDRIAL REDOX IMBALANCE INLYMPHOBLASTOID CELL LINES
0
0.5
1
1.5
2
2.5
3
3.5
4
fGSH GSSG
0
2
4
6
8
10
12
14
16
18
Control Autistic
GSH/GSSG RATIOAutistic Control
(X 10)
PERCENT DECREASE IN MITOCHONDRIAL MEMBRANE POTENTIAL WITH NITRIC OXIDE EXPOSURE
0
2
4
6
8
10
12
14
16
18
20
Control Autistic
% D
ecre
ase
in J
C-1
Flu
ore
scen
ce In
ten
sity
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 or epigenetic differences.
These results provide experimental evidence that cells from autistic children may be more vulnerable to pro-oxidant environmental exposures.
CONCLUSION
SPECIFIC AIM 4: METABOLIC GENETICS
Specific Aim 4: Using a case-control design, we will determine whether the frequency of relevant genetic polymorphisms is increased among autistic children and whether specific genotypes are associated with the abnormal metabolic phenotype.
We have access to 500 trios (child, mother, father) from NIH genetic repository to look at relevant SNP frequencies and transmission
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
GST
COMT
RFC
A Targeted Approach to Autism Genetics:Using the Metabolic Endophenotype as a
Guide to Candidate Genes
CBS
GCL
A RANDOMIZED DOUBLE-BLIND PLACEBO-CONTROLLED CROSS-OVER STUDY
Treating Oxidative Stress and the Metabolic Pathology of Autism
A significant proportion of autistic children have impaired methylation and antioxidant/detoxification capacity that results in chronic oxidative stress.
Targeted nutritional intervention that is designed to correct the metabolic imbalance will significantly improve their metabolic profile and improve measures of autistic behavior.
HYPOTHESIS
Specific Aim 1. We will screen children with a diagnosis of autism for evidence of impaired methylation (↓SAM/SAH) and impaired antioxidant capacity (↓GSH/GSSG)
Specific Aim 2. Children who exhibit evidence of impaired methylation and antioxidant capacity will be randomized into a double blind placebo-controlled cross-over trial of targeted nutritional intervention designed to correct metabolic deficiencies and to improve scores on standardized behavioral evaluation tests.
SPECIFIC AIMS
Thiols, Complete Lab, Thiols, Complete Lab, Thiols, Complete Lab, Behavioral Testing Behavioral Testing Behavioral Testing
B A
A BWASHOUT
A is supplement first, placebo secondB is placebo first, supplement second
RANDOMIZED DOUBLE-BLIND PLACEBO-CONTROLLED CROSS-OVER DESIGN
Children are randomly assigned to either the placebo first or the treatment firstfor 3 months before 1 month wash out period and cross-over
The supplements have been selected to impact three core cellular functions that are altered with chronic oxidative stress (www.clinicaltrials.gov)
1) Decreased SAM/SAH ratio and cellular methylation capacity
2) Antioxidant and detoxification support (mitochondrial and cytosolic)
3) Cell membrane integrity
1. Behavioral testing: ADOS; Vineland; PLS-2; SRSBehavioral testing will be videotaped and administered by PhD psychologists
2. Metabolic evaluation:Plasma: Thiol metabolic profile; CBC; amino
acid profile, P5P, B12; sulfate; nitrotyrosine; vitamin D;
uric acid Urine: Sulfate, organic acids; creatinine; FIGlu,
MMA Cellular: RBC membrane phospholipids;
GSH/GSSG
3. Immunologic evaluation: Flow cytometry for intracellular cytokine expression
OUTCOME MEASURES
The ATN is a consortium of 15 national sites composed of experts in developmental pediatrics, neurology, genetics, metabolism, sleep, and gastroenterology who are dedicated to improving the standard of care of children with autism.
The ATN believes that treatment of medical issues can improve core behaviors and improve quality of life for children and adults with autismand their parents.
The ATN
Specific Aim 1: We will determine whether glutathione potential in primary lymphocytes can be used as a biomarker for regressive autism and whether it is predictive of the subsequent diagnosis of autism.
Specific Aim 2: We will determine whether targeted treatment to increase normal glutathione potential in autistic children will improve immune function and reverse DNA methylation alterations associated with low redox status in lymphocytes.
DEPARTMENT OF DEFENSE AUTISM IDEA DEVELOPMENT AWARD
2008-2011
Difficulties with purely genetic approach to autism
Estimated between 10 and 100 different small effect genes are required for the autistic phenotype
Different combination of genes in different autistic individuals
If genetic susceptibility requires an environmental trigger, same genetic risk factors will be present in people without autism Genome-wide array studies have been disappointing
The Autism Triad: Brain-Gut-Immune Axis
Brain/Nervous System
Gut Immune
System
GUT BRAIN: Vagus afferents; Gut neuropeptides
BRAIN GUT: Endorphins; Neuropeptides
IMMUNE BRAIN: Cytokines; Microglia activation
BRAIN IMMUNE: Endorphins; Neuropeptides; Cortisol
GUT IMMUNE: Gut neuropeptides; microbial products
IMMUNE GUT: Cytokines; GALT
Beyond the Brain
The Autism Triad: Brain-Gut-Immune Axis
All 3 systems highly sensitive to oxidative stressespecially during critical developmental windows
Brain/Nervous System
Gut Immune
System
Intracelluar Redox
GSH/GSSG
Developmental trajectories of all three systems depend on appropriate environmental signals
Gene-environment interactions affect intracellular redox and maturation of all 3 systems
Environmental Influences
Brain/Nervous System
Gut Immune
System
Intracelluar Redox
GSH/GSSG
Genetic Influences
Brain/Nervous System
Gut Immune
System
Intracelluar Redox
GSH/GSSG
Toxic insult to one will indirectly affect the development and function of the others
Brain/Nervous System
Gut Immune
System
Intracelluar Redox
GSH/GSSG
Toxic insult to one will indirectly affect the development and function of the others
METALS Mercury Cadmium Aluminum Lead Nickel Arsenic Cobalt Manganese
SOLVENTS Alcohol
Chlorinated Solvents Benzene
INDUSTRIAL CHEMICALS PCBs Pesticides Herbicides
Glutathione depletion/oxidative stress may be a final common pathway of toxicity for many structurally diverse environmental exposures
Simultaneous sub-toxic doses can reach a toxic threshold
Induce oxidative stress and GSH depletion
GSH/GSSG
GSH/GSSG
TOXICITY
TOXIC THRESHOLD
Toxic InsultsNormal Homeostasis
Fragile Homeostasis (limited reserve)
Toxic Insults
Do we need a broader paradigm for autism pathogenesis?
A more systemic approach beyond brain/behavior?
Could there be a component of metabolic encephalopathy?
The oxidative stress hypothesis encompasses the possibility of a gut-brain-immune interaction and
gene-environment interactions
New Questions
GENE EXPRESSION ENVIRONMENT
BEHAVIOR
(Genetic/Epigenetic) (Vulnerability/Resistance)
Necessary but Not Sufficient
Necessary but Not Sufficient
Metabolic Endophenotype (GSH/GSSG) (SAM/SAH)
Mechanism(Redox Imbalance; Methylation)
TREATMENTMultiple, AdditiveVariable Factors
Multiple, AdditiveVariable Genes
FROM EPIDEMIOLOGY TO MECHANISM
EnvironmentGenes
Inflammation Infection
Hormones
Autism
Timing
Factors Contributing to Oxidative Stress in Autistic Children
Gut Inflammation Brain InflammationImmune dysfunction