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

MS

B12

5-CH3THF Homocysteine B6

THF: tetrahydrofolate Enzymes

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

1

SAM MS

B12

5-CH3THF

2

SAH SAHH

B6

Enzymes

(SAM/SAH)

MTase Cell Methylation

Homocysteine

THF: tetrahydrofolate

Methylation Potential

Adenosine

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

1

Methylation Potential

SAM MS

2

B12

5-CH3THF

(SAM/SAH)

MTase Cell Methylation

SAH SAHH

Adenosine

Homocysteine B6 B6

THF: tetrahydrofolate Enzymes

3

CBS

Cystathionine B6

Cysteine GSH

Antioxidant Redox Potential (GSH/GSSG)

GSSG

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

1

SAM MS

2

B12

5-CH3THF

1

Folate Cycle

2

Methionine Cycle

3

Methylation Potential

Transsulfuration Pathway

(SAM/SAH)

MTase Cell Methylation

SAH SAHH

Adenosine

Homocysteine B6 B6

3

CBS

Cystathionine B6

Cysteine GSH

Antioxidant Redox Potential (GSH/GSSG)

GSSG

Vital Importance of these Interdependent Metabolic Pathways Methionine THF

1

5,10-CH2THF

SAM

MTase Cellular

2

MS

5-CH3THF

SAHH

Adenosine

Homocysteine B6 Cystathionine

DNA SYNTHESIS

3 PROLIFERATION

Methylation Reactions

SAH

B12

Purines and Thymidylate

METHYLATION

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)

MethylB12 (75µg/Kg every 3 days) 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 metabolites Vineland Adaptive Behavioral Scales

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

Methionine

Folinic Acid

THF 5,10-CH2THF

1

SAM MTase Cellular

2

MS

SAH

B12

Purines and Thymidylate

5-CH3THF

Methylation Reactions

SAHH

Adenosine

Homocysteine Folinic Acid

B6 Cystathionine

DNA SYNTHESIS

3

Cysteine GSH

GSSG

METABOLIC DATA Plasma Metabolite Concentration

Control Children (n = 42)

Autism Pre-treatmentb (n = 40)

Autism Post-treatment (n = 40)

Methionine

24 ± 3

21 ± 4

22 ± 3

ns

SAM (nmol/L)

78 ± 22

66 ± 13

69 ± 12

ns

SAH (nmol/L)

14.3 ± 4.3

15.2 ± 5

14.8 ± 4

ns

SAM/SAH (µmol/L)

5.6 ± 2.0

4.7 ± 1.5

5.0 ± 2.0

ns

Homocysteine (µmol/L)

5.0 ± 1.2

4.8 ± 1.8

5.3 ± 1.1

0.04

Cysteine (µmol/L)

210 ± 18

191 ± 24

215 ± 19

0.001

Total Glutathione (µmol/L)

7.5 ± 1.8

5.4 ± 1.3

6.2 ± 1.2

0.001

Free Glutathione (µmol/L)

2.8 ± 0.8

1.5 ± 0.4

1.8 ± 0.4

0.18 ± 0.07

0.28 ± 0.08

0.22 ± 0.06

0.001

tGSH/GSSG

47 ± 18

21 ± 6

30 ± 9

0.001

fGSH/GSSG

17 ± 6.8

6±2

9±3

0.001

GSSG (µmol/L)

a

P value refers to treatment effect

p valuea

0.008

Cysteine 300

µmol/L

250 200 150 100 50 0

Before

After

Total Glutathione 10 9

x

8 µmol/L

7 6 5 4 3 2 1 0

Before

After

GSSG 0.6

µmol/L

0.5 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)

Post-Treatment Score (mean ± SD)

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)

Composite Score

66.5 ± 9.2

73.9 ± 17.0

6.6 (2.3, 11)

Change in Score (mean; 95% C I)

p value

0.12 <0.003

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)

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

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

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

0.30 ± 0.08*

0.24 ± 0.04

GSSG (µM/L) Total GSH/GSSG

17 ± 8

31 ± 10*

*statistically significant

Metabolite imbalance and the risk of being a mother of a child with autism Control Mothers

Case Mothers (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

Stratified Group

(N=200)

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

METABOLIC BIOMARKERS OF AUTISM:

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

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:

FAIL = High Risk

M-CHAT (18-30 months)

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

Metabolic Profile

(1-6 months) Visit 2:

(6 months)

M-CHAT Repeat

M-CHAT Repeat

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

PASS Not Autism Control

AUTISM PROSPECTIVE STUDY DESIGN Visit 1:

M-CHAT (18-30 months)

FAIL = High Risk

PASS = Developmental Delay and Normal CONTROLS

Metabolic Profile

Metabolic Profile

(1-6 months) Visit 2:

(6 months)

M-CHAT Repeat FAIL

FAIL = High risk Regression

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

M-CHAT Repeat PASS

Not Autism Control

AUTISM PROSPECTIVE STUDY DESIGN Visit 1:

M-CHAT (18-30 months)

FAIL = High Risk

PASS = Developmental Delay and Normal CONTROLS

Metabolic Profile

Baseline

(1-6 months) Visit 2:

(6 months)

M-CHAT Repeat FAIL

Autism Diagnosis

M-CHAT Repeat

FAIL = High risk Regression

Visit 3: DSM-IV; CARS; ADOS

Metabolic Profile

PASS

Not Autism Final 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)

Vmax ROS Rate

900 800

Control Autistic

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

Control Autistic

140 120 100 80 60 40 20 0 0

0.16

0.32

0.62

1.25

2.5

Thimerosal Concentration (uMol/L)

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

MITOCHONDRIAL REDOX IMBALANCE IN LYMPHOBLASTOID CELL LINES 18

4 3.5 3

Autistic

16

Control

14

GSH/GSSG RATIO

12

2.5

10

2

8

1.5

6

1

4

0.5

2

0

0

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 B12

5,10-CH2-THF

TC II

DMG

Methyl Acceptor Methyltransferase COMT Methylated Product

MTHFR

5-CH3-THF

SAH

RFC 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

WASHOUT

B

Thiols, Complete Lab, Behavioral Testing

B A

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

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

Necessary but Not Sufficient

ENVIRONMENT

(Vulnerability/Resistance) Multiple, Additive Variable Factors

FROM EPIDEMIOLOGY TO MECHANISM BEHAVIOR

Necessary but Not Sufficient

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

Mechanism

(Redox Imbalance; Methylation)

Necessary but Not Sufficient

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