Autism Research in Arkansas

Autism Research in Arkansas:

On-going clinical trials and the Arkansas Autism Alliance
S. Jill James, PhD

Professor, Department of Pediatrics Director, Autism Metabolic Genomics Laboratory Arkansas Children’s Hospital Research Institute University of Arkansas for Medical Sciences Little Rock, AR

OVERVIEW
Review of metabolic pathways: folate/methionine/glutathione Efficacy of methylB12 and folinic acid treatment on glutathione redox status and core behaviors in autism Parent Metabolic Profiles Specific Aims of our 5 year NIH-funded study Placebo-controlled double-blind cross-over study of broad spectrum nutritional supplementation AAA and ATN in Arkansas

Methionine Transsulfuration to Cysteine and Glutathione
Methionine THF 5,10-CH2THF
MTHFR

1

B12

MS

5-CH3THF Homocysteine
B6

THF: tetrahydrofolate
Enzymes

Methionine Transsulfuration to Cysteine and Glutathione
Methionine THF 5,10-CH2THF
MTHFR

SAM
MS

Methylation Potential
(SAM/SAH)

1

2

B12

SAH
SAHH

MTase Cell Methylation

5-CH3THF

Adenosine

Homocysteine
B6

THF: tetrahydrofolate
Enzymes

Methionine Transsulfuration to Cysteine and Glutathione
Methionine THF 5,10-CH2THF
MTHFR

SAM
MS

Methylation Potential
(SAM/SAH)

1

2

B12

SAH
SAHH

MTase Cell Methylation

5-CH3THF

Adenosine CBS

Homocysteine
B6 B6

THF: tetrahydrofolate
Enzymes

3

Cystathionine
B6

Cysteine GSH

GSSG

Antioxidant Redox Potential (GSH/GSSG)

Methionine Transsulfuration to Cysteine and Glutathione
Methionine THF 5,10-CH2THF
MTHFR

SAM
MS

Methylation Potential
(SAM/SAH)

1

2

B12

SAH
SAHH

MTase Cell Methylation

5-CH3THF

Adenosine

1 2 3

Folate Cycle Methionine Cycle Transsulfuration Pathway 3

Homocysteine
B6 B6 B6

CBS

Cystathionine Cysteine GSH

GSSG

Antioxidant Redox Potential (GSH/GSSG)

Vital Importance of these Interdependent Metabolic Pathways
Methionine THF 5,10-CH2THF

SAM
MS

METHYLATION
Methylation Reactions

1

2

MTase Cellular

B12

SAH

Purines and Thymidylate
DNA SYNTHESIS

5-CH3THF

SAHH

Adenosine

Homocysteine
B6 Cystathionine

3 PROLIFERATION

Cysteine GSH GSSG

REDOX HOMEOSTASIS

AN OPEN LABEL TRIAL OF METHYLCOBALAMIN AND FOLINIC ACID IN AUTISTIC CHILDREN

Can supplementation with methyl-B12 and folinic Acid improve glutathione levels and core behaviors in autistic children?
Intervention: (3 months) Inclusion Criteria: MethylB12 (75µg/Kg every 3 days) Folinic Acid (400 µg bid) Autistic Disorder (DSM-IV; CARS) Age 3-7 No previous supplements GSH < 6.0 Methylation and glutathione metabolites Vineland Adaptive Behavioral Scales

Endpoints:

STUDY DESIGN
Each child served as their own control in the open label trial in which both parents and investigators were aware that the child was receiving supplements ofmethyl-B12 and folinic acid for a period of three months. Plasma metabolites in the transmethylation and transsulfuration pathways were measured at baseline and again after the 3 month intervention period. The study nurse administered and scored the Vineland Adaptive Behavior Scales parent questionnaire before and after the 3 month intervention.

Methyl B12 Folinic Acid

Methionine THF

SAM
MS

5,10-CH2THF

1

2

MTase Cellular

B12

SAH

Methylation Reactions

Purines and Thymidylate
DNA SYNTHESIS

5-CH3THF

SAHH

Adenosine

Homocysteine
Folinic Acid
B6 Cystathionine

3

Cysteine GSH GSSG

METABOLIC DATA
Plasma Metabolite Concentration Methionine SAM (nmol/L) SAH (nmol/L) SAM/SAH (µmol/L) Homocysteine (µmol/L) Cysteine (µmol/L) Total Glutathione (µmol/L) Free Glutathione (µmol/L) GSSG (µmol/L) tGSH/GSSG fGSH/GSSG
a

Control Children (n = 42) 24 ± 3 78 ± 22 14.3 ± 4.3 5.6 ± 2.0 5.0 ± 1.2 210 ± 18 7.5 ± 1.8 2.8 ± 0.8 0.18 ± 0.07 47 ± 18 17 ± 6.8

Autism Pre-treatmentb (n = 40) 21 ± 4 66 ± 13 15.2 ± 5 4.7 ± 1.5 4.8 ± 1.8 191 ± 24 5.4 ± 1.3 1.5 ± 0.4 0.28 ± 0.08 21 ± 6 6±2

Autism Post-treatment (n = 40) 22 ± 3 69 ± 12 14.8 ± 4 5.0 ± 2.0 5.3 ± 1.1 215 ± 19 6.2 ± 1.2 1.8 ± 0.4 0.22 ± 0.06 30 ± 9 9±3

p valuea ns ns ns ns 0.04 0.001 0.001 0.008 0.001 0.001 0.001

P value refers to treatment effect

Cysteine
300 250
µmol/L

200 150 100 50 0

Before

After

Total Glutathione
10 9 8 7
µmol/L
x

6 5 4 3 2 1 0
Before After

GSSG
0.6 0.5

µmol/L

0.4 0.3 0.2 0.1 0

Before

After

Total GSH/GSSG
60 50 40 30 20 10 0

Before

After

SUMMARY OF METABOLIC RESULTS
1. All baseline metabolites were significantly different from age-matched controls (except for SAH) 2. The treatment did not significantly improve levels of methionine, SAM or SAM/SAH • The treatment did significantly improve cysteine, glutathione, and GSH/GSSG 10. Although significantly improved, glutathione and GSH/GSSG did not reach levels in control children

Behavioral Evaluation
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.

BEHAVIOR SCORES
Vineland Category Baseline Score (mean ± SD) 65.3 ± 12.9 67.0 ± 76 68.2 ± 9.3 75.6 ± 9.7 66.5 ± 9.2 Post-Treatment Score (mean ± SD) 72.0 ± 15.5 76.0 ± 17.7 75.7 ± 14.7 79.0 ± 14.7 73.9 ± 17.0 Change in Score (mean; 95% C I) 6.7 (3.5, 10) 9.0 (4.0, 14) 7.5 (3.5, 11) 3.3 (0, 8) 6.6 (2.3, 11) p value <0.001 <0.007 <0.005 0.12 <0.003

Communication Daily Living Skills Socialization Motor Skills Composite Score

SUMMARY OF BEHAVIOR RESULTS
Although treatment with methylB12 and folinic acid significantly improved core behaviors, they did not reach standard scores for unaffected children (100 ± 15)

CONCLUSIONS
Improvement in measures of both metabolic and behavioral endpoints converge to suggest that some children may benefit from targeted nutritional intervention

What about the parents?

Maternal Methionine Cycle Metabolites:
Autism Moms
(n = 46)

Control Moms
(n= 200)

Methionine (µM/L) SAM (nM/L) SAH (nM/L) SAM/SAH Ratio Homocysteine (µM/L)

24 ± 5 80 ± 19 33 ± 14* 3.1 ± 1.7* 11 ± 3.9*

26 ± 6 83 ± 13 23 ± 8.4 4.0 ± 1.4
7.6 ± 1.6
*statistically significant

It would be a very good idea to ask your physician to check your “total” homocysteine

Maternal Transsulfuration Metabolites
Autism Moms Cysteine (µM/L) Total GSH (µM/L) Free GSH (µM/L) GSSG (µM/L) Total GSH/GSSG 232 ± 40 5.1 ± 1.7* 1.5 ± 0.5* 0.30 ± 0.08* 17 ± 8 Control Moms 231 ± 20 7.3 ± 1.5 2.6 ± 0.6 0.24 ± 0.04 31 ± 10*

*statistically significant

Metabolite imbalance and the risk of being a mother of a child with autism
Stratified Group SAH >30µMol/L) SAM/SAH <2.5 tGSH/GSSG <20 SAM/SAH <2.5 and tGSH/GSSG <20 Control Mothers
(N=200)

Case Mothers
(N=46)

Odds Ratio (Risk) 6.9 10.7 15.2 46

14% 10% 11% 3%

54% 54% 65% 41%

IMPORTANT CAVEAT
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

PREDICTIVE POTENTIAL AND GENETIC SUSCEPTIBILITY A 5 YEAR NIH-FUNDED STUDY (2006-2011)

METABOLIC BIOMARKERS OF AUTISM:

SPECIFIC AIM 1: METABOLITES AND BEHAVIOR
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 severity and specificity of the metabolite imbalance is associated with the severity and specificity of behavioral abnormalities.

SPECIFIC AIM 2: PROSPECTIVE STUDY
Specific Aim 2: To investigate whether the abnormal metabolic
profile precedes 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.

SPECIFIC AIM 2: PROSPECTIVE STUDY
M-CHAT autism screening test 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.

AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk

PASS = Developmental Delay and Normal CONTROLS

Metabolic Profile

Metabolic Profile

AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk

PASS = Developmental Delay and Normal CONTROLS

Metabolic Profile (1-6 months) Visit 2: M-CHAT Repeat

Metabolic Profile (6 months) M-CHAT Repeat

AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk

PASS = Developmental Delay and Normal CONTROLS

Metabolic Profile (1-6 months) Visit 2: M-CHAT Repeat FAIL Visit 3: DSM-IV; CARS; ADOS Autism Diagnosis Not Autism

Metabolic Profile (6 months) M-CHAT Repeat PASS

Control

AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk

PASS = Developmental Delay and Normal CONTROLS

Metabolic Profile (1-6 months) Visit 2: M-CHAT Repeat FAIL FAIL = High risk Regression Not Autism

Metabolic Profile (6 months) M-CHAT Repeat PASS

Visit 3: DSM-IV; CARS; ADOS Autism Diagnosis

Control

AUTISM PROSPECTIVE STUDY DESIGN
Visit 1: M-CHAT (18-30 months)

FAIL = High Risk

PASS = Developmental Delay and Normal CONTROLS

Metabolic Profile (1-6 months) Visit 2: M-CHAT Repeat FAIL

Baseline

Metabolic Profile (6 months) M-CHAT Repeat PASS

FAIL = High risk Regression Not Autism Final diagnosis

Visit 3: DSM-IV; CARS; ADOS Autism Diagnosis

Control

IMPLICATIONS OF AIM 2 AUTISM PROSPECTIVE STUDY If the metabolic profile is found to precede the behavioral diagnosis, subsequent studies would determine whether early intervention to normalize the metabolic profile can reduce or prevent the development of autism.

SPECIFIC AIM 3: CELLULAR CONSEQUENCES

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

EXPERIMENTAL PROCEDURES
Lymphoblastoid cell lines from autistic children with at least one affected sibling were compared with unaffected control lymphoblastoid cell lines* 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.
*Preliminary data supported by SafeMinds

Relative Free Radical Generation (DCF)
900 800

Control Autistic

Vmax ROS Rate

700 600 500 400 300 200 100 0 0 0.3125 0.625 1.25 2.5

Thimerosal Concentration (uMol/L)
Cells from autistic children generate more free radicals than control cells

Glutathione Redox Ratio (GSH/GSSG)
160 140 120 100 80 60 40 20 0 0 0.16 0.32 0.62 1.25 2.5 Thimerosal Concentration (uMol/L)

Control Autistic

Cells from autistic children have lower GSH/GSSG ratio than control cells

MITOCHONDRIAL REDOX IMBALANCE IN LYMPHOBLASTOID CELL LINES
4 3.5 3 2.5 2 1.5 1 0.5 0

18

Autistic Control

16 14 12 10 8 6 4 2 0

GSH/GSSG RATIO

fGSH

GSSG (X 10)

Control

Autistic

CONCLUSION
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 sensitive to pro-oxidant environmental exposures.

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

A Targeted Approach to Autism Genetics: Using the Metabolic Endophenotype as a Guide to Candidate Genes
Methionine THF SAM 5,10-CH2-THF
MTHFR

Methyl Acceptor Methyltransferase COMT Methylated Product

B12 5-CH3-THF RFC

TC II

DMG

SAH Homocysteine Cystathionine CBS Cysteine GCL Glutathione GST Adenosine

Treating Oxidative Stress and the Metabolic Pathology of Autism

A RANDOMIZED DOUBLE-BLIND PLACEBOCONTROLLED CROSS-OVER STUDY

HYPOTHESIS
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.

SPECIFIC AIMS
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.

RANDOMIZED DOUBLE-BLIND PLACEBOCONTROLLED CROSS-OVER DESIGN
A is supplement first, placebo second B is placebo first, supplement second
A B
WASHOUT

B A

Thiols, Complete Lab, Behavioral Testing

Thiols, Complete Lab, Behavioral Testing

Thiols, Complete Lab, Behavioral Testing

Children are randomly assigned to either the placebo first or the treatment first for 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

OUTCOME MEASURES
• Behavioral testing: ADOS; Vineland; PLS-2; SRS Behavioral testing will be videotaped and administered by PhD psychologists 3. Metabolic evaluation: Plasma: Thiol profile; CBC; amino acid profile, P5P, HoloTCII; sulfate; nitrotyrosine; lactate/pyruvate; 25-hydroxy vitamin D; uric acid; Urine: Sulfate, organic acids; creatinine; FIGlu, MMA Cellular: RBC membrane phospholipids; leukocyte GSH/GSSG. • Immunologic evaluation: Flow cytometry for CRP, cytokine mRNA expression and protein levels for TNFα; g-IFN, IL-1; IL-4, IL-6; IL-10; IL-13; T-regs

AUTISM TREATMENT NETWORK (ATN) IN ARKANSAS

The ATN
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 autism and their parents.

Our Dream for Autism in Arkansas

UAMS/ACH/ACHRI

Arkansas Autism Alliance (AAA)

Clinical Evaluation & Treatment Center

UAMS/ACH/ACHRI Arkansas Autism Alliance
Resource and Outreach Center Translational Research Center

FROM EPIDEMIOLOGY TO MECHANISM
BEHAVIOR

Necessary but Not Sufficient

Necessary but Not Sufficient

GENE EXPRESSION
(Genetic/Epigenetic) Multiple, Additive Variable Genes

(Vulnerability/Resistance) Multiple, Additive Variable Factors

ENVIRONMENT

FROM EPIDEMIOLOGY TO MECHANISM
BEHAVIOR

Necessary but Not Sufficient

(Redox Imbalance; Methylation)

Mechanism

Necessary but Not Sufficient

GENE EXPRESSION
(Genetic/Epigenetic) Multiple, Additive Variable Genes

Metabolic Endophenotype ENVIRONMENT (GSH/GSSG) (SAM/SAH) (Vulnerability/Resistance)

TREATMENT

Multiple, Additive Variable Factors

Acknowledgements
Autism Metabolic Genomics Laboratory Stepan Melnyk, PhD Stefanie Jernigan Alena Savenka Shannon Palmer Sarah Blossom, PhD Lesya Pavliv Study Nurses Nancy Chambers, Dana Schmidt, Amanda Hubanks, Nancy Lowery