Autism Research in Arkansas: On-going clinical trials and the Arkansas Autism Alliance

Autism Research in Arkansas: On-going clinical trials

and the Arkansas Autism Alliance

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

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

Homocysteine B6

Methionine Transsulfuration to Cysteine and Glutathione

THF: tetrahydrofolate

Methionine

Enzymes

5-CH3THF

THF

B12MS5,10-CH2THF

MTHFR

1

SAM

SAH

MTase

SAHH

Homocysteine B6



Methionine

Adenosine

Enzymes

5-CH3THF

THF

B12MS5,10-CH2THF

MTHFR

Cell Methylation1

Methylation Potential(SAM/SAH)

2

SAM

SAH

MTase

SAHH

Homocysteine B6 CBS



Cystathionine

Cysteine

GSH GSSG

Methionine

Adenosine

B6

Enzymes

B6

5-CH3THF

THF

B12MS5,10-CH2THF

MTHFR


Cell Methylation1 2

3 Antioxidant Redox Potential (GSH/GSSG)

SAM

SAH

MTase

SAHH

Homocysteine B6 CBS


Cystathionine

Cysteine GSH GSSG

Methionine

Adenosine

B6

B6

5-CH3THF

THF

B12MS5,10-CH2THF

MTHFR

Cell Methylation1

1

2

3

Folate Cycle

Methionine Cycle

Transsulfuration Pathway


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

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?

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.

STUDY DESIGN

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

METABOLIC DATA

Plasma Metabolite Concentration

ControlChildren(n = 42)

Autism Pre-treatmentb

(n = 40)

Autism Post-treatment

(n = 40)p valuea

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 0.008

GSSG (µmol/L) 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 0.001a P value refers to treatment effect

Cysteine

0

50

100

150

200

250

300

Before After

µm

ol/L

0

1

2

3

4

5

6

7

8

9

10

Total Glutathione

Before After

µm

ol/L

x

0

0.1

0.2

0.3

0.4

0.5

0.6

GSSG

µm

ol/L

Before After

Total GSH/GSSG

0

10

20

30

40

50

60

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

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 RESULTS

Although 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

CONCLUSIONS

What about the parents?

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:

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

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 severity and specificity of the metabolite imbalance is associated with the severity and specificity of behavioral abnormalities.

SPECIFIC AIM 1: METABOLITES AND BEHAVIOR

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.

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.

SPECIFIC AIM 2: PROSPECTIVE STUDY

Visit 1: M-CHAT (18-30 months)

FAIL = High Risk PASS = Developmental Delay and Normal

CONTROLS

Metabolic Profile Metabolic Profile

AUTISM PROSPECTIVE STUDY DESIGN



CONTROLS

Visit 2: M-CHAT Repeat M-CHAT Repeat

(1-6 months) (6 months)



Autism Diagnosis



CONTROLS




PASS

Visit 3: DSM-IV; CARS; ADOS

Control

Not Autism


FAIL

Autism Diagnosis



CONTROLS




FAIL = High risk Regression


Control

Not Autism


PASS FAIL

Autism Diagnosis



CONTROLS




FAIL = High risk Regression


Control

Not Autism


PASS FAIL

Baseline

Final diagnosis

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.

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 children withat 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.

EXPERIMENTAL PROCEDURES

*Preliminary data supported by SafeMinds

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)

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.

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 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.

2. 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

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

Our Dream for Autism in Arkansas

Arkansas Autism Alliance (AAA)

UAMS/ACH/ACHRI

http://www.masternewmedia.org/images/puzzle_pieces_id150248_size500o.jpg

Clinical Evaluation & Treatment Center

Resource and Outreach Center Translational Research Center

UAMS/ACH/ACHRI Arkansas Autism Alliance

BEHAVIOR

(Genetic/Epigenetic) (Vulnerability/Resistance)

Multiple, AdditiveVariable Genes

Multiple, AdditiveVariable Factors

Necessary but Not Sufficient


FROM EPIDEMIOLOGY TO MECHANISM

GENE EXPRESSION ENVIRONMENT

GENE EXPRESSION ENVIRONMENT

BEHAVIOR

(Genetic/Epigenetic) (Vulnerability/Resistance)



Metabolic Endophenotype (GSH/GSSG) (SAM/SAH)

Mechanism(Redox Imbalance; Methylation)

TREATMENTMultiple, AdditiveVariable Factors

Multiple, AdditiveVariable Genes

FROM EPIDEMIOLOGY TO MECHANISM

AcknowledgementsAutism Metabolic Genomics Laboratory

Stepan Melnyk, PhDStefanie JerniganAlena SavenkaShannon PalmerSarah Blossom, PhDLesya Pavliv

Study Nurses Nancy Chambers, Dana Schmidt, Amanda Hubanks, Nancy Lowery

Autism Research in Arkansas: On-going clinical trials and the Arkansas Autism Alliance

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