Transcript Slide 1

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
DNA SYNTHESIS
5-CH3THF
Methylation
Reactions
SAHH
Adenosine
Homocysteine
Folinic Acid
B6
Cystathionine
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
0.008
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)
aP
value refers to treatment effect
p valuea
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
Before
0
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
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
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
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
4
3.5
3
18
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
5,10-CH2-THF
B12
TC II
DMG
Methyl Acceptor
Methyltransferase COMT
MTHFR
Methylated Product
5-CH3-THF
RFC
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
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
1. Behavioral testing: ADOS; Vineland; PLS-2; SRS
Behavioral 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
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