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.

AN OPEN LABEL TRIAL OF METHYLCOBALAMINAND FOLINIC ACID 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

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300

CysteineM

icro

mol

/L

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GSSGM

icro

mol

/L

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ol/L

GSH/GSSG REDOX RATIO

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

What about the parents?

Journal Of Autism and Developmental Disabilities 2008

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

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fGSH GSSG (x 10)

nm

ol/m

g p

rote

in Control

Autistic

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20

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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)

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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)

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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

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fGSH GSSG

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Control Autistic

GSH/GSSG RATIOAutistic Control

(X 10)

PERCENT DECREASE IN MITOCHONDRIAL MEMBRANE POTENTIAL WITH NITRIC OXIDE EXPOSURE

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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

AUTISM TREATMENT NETWORK (ATN) IN ARKANSAS

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

DEPARTMENT OF DEFENSE AUTISM IDEA DEVELOPMENT AWARD

2008-2011

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

AUTISM: BEYOND THE BRAIN

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

You Are HereCellular Metabolic Pathways

Putting it all into Perspective……we see what we know

AcknowledgementsAutism Metabolic Genomics Laboratory

Stepan Melnyk, PhDStefanie JerniganAlena SavenkaShannon PalmerSarah Blossom, PhDLesya Pavliv

Study Nurses Rachael Seach, Nancy Chambers, Amanda Hubanks, Nancy Lowery