welcome to today’s air-p/atn advances in autism research ... · aim 1 – explore relationship...

This research activity was supported by a cooperative agreement UA3 MC11054 through the U.S. Department of Health and Human Services, Health Resources and Services Administration, Maternal and Child Health Research Program to the Massachusetts General Hospital. This work was conducted through the Autism Speaks

Autism Treatment Network serving as the Autism Intervention Research Network on Physical Health.

Welcome to today’s AIR-P/ATN Advances in Autism

Research & Care Webinar Series!

RELATIONSHIP BETWEEN GASTROINTESTINAL DISORDERS AND STRESS REACTIVITY, IMMUNITY, AND BLOOD SEROTONIN IN AUTISM SPECTRUM DISORDERS

David Beversdorf, M.D. Principal Investigator AIR-P/ATN Webinar Monday, November 24, 2014

Relationship Between Gastrointestinal Disorders and Stress

Reactivity, Immunity, and Blood Serotonin in Autism Spectrum Disorder

Investigators / Collaborating Sites:

David Beversdorf, M.D. Missouri

Jeremy Veenstra-VanderWeele, M.D. VUMC

Relationship Between Gastrointestinal Disorders and Stress Reactivity, Immunity, and Blood Serotonin in Autism Spectrum Disorder

BACKGROUND

• High rate of GI disorders in ASD, but biology and etiology is unknown

• In general population, strong relationship between stress and GI

• There is an augmented stress response in ASD • Therefore, it is possible for GI symptoms in ASD to vary as a

function of stress reactivity


Pilot Study

• Conducted at the University of Missouri Thompson Center for Autism and Neurodevelopmental Disorders

• 16 children(mean age 12.3±3.6 years(±sdev), range 5-19)

• 8 children with ASD with GI problems, 8 with ASD without GI

• GI symptomatology verified by parent report on the Questionnaire on Pediatric Gastrointestinal Symptoms – Rome III Version (QPGS Rome-III)

• Skin Conductance data collected during rest and in response to vibrotactile and cold pressor stimulation (stressors)

Figure 1. Mean skin conductance (microsiemens) for ASD GI

and ASD NO GI groups during baseline. *p < .05.


Pilot Data *


Figure 2. Mean skin conductance (microsiemens) for ASD GI

and ASD NO GI groups during vibrotactile stimulation (p = 0.083)

Pilot Data


• Next Steps

• Explore in a more definitive manner in a larger sample

• Examine other systems that may be affected by GI disorders in ASD

• A three-aim study was proposed…

80 Children

from ATN

Registry from

Missouri

Aim 1 – Explore Relationship Between GI Problems and Stress

40 Children

from ATN

Registry from

Vanderbilt

ECG and GSR

Data

Recordings

During Stress

Stimuli

HRV and Mean

Skin

Conductance

Is Stress

Implicated in GI

Problems in

ASD?


• Hypothesis • Heart Rate Variability as GI symptoms increase • Skin Conductance as GI symptoms increase

• Skin Conductance Level (SCL)

• Average conductance over time

• Heart Rate Variability (HRV)

• pNN50 – percentage of normal R-R

interval pairs that differ by 50ms or more.


80 Children

from ATN

Registry from

Missouri

Aim 2 – Examine Immune and Endocrine Markers in GI Disorders in ASD

40 Children

from ATN

Registry from

Vanderbilt

Collect Blood

and Saliva

Samples

Examine IL-6

and TNF-alpha

and salivary

cortisol

Are there immune

and endocrine

differences in

those with GI and

ASD?

Hypothesis • Immunological Functioning

• Altered IL-6 and TNF-alpha cytokine levels in those with greater stress reactivity

• Cortisol

• Salivary cortisol as GI symptoms


Atypical Immune Response in ASD • Immune markers affected in ASD: IL-12, IFN-γ, IL-2, IL-

6, IL-10, and TNF-alpha (Ashwood et al., 2006) • TNF-α, IL-12, IL-10, and IL-6 are associated with GI

symptomatology (Ashwood et al, 2006; Jyonouchi et al., 2011)

• Significant overlap between what is observed in ASD and immune markers associated with the stress response (IL-6 and TNF-α)

Cortisol Levels Increased in ASD and in GI Alone •Increased stress response in ASD (Corbett et al., 2008) •In general population, increased stress response in those with GI disorders (Lyte, et al., 2011)


80 Children from ATN Registry

from Missouri

40 Children from ATN Registry

from Vanderbilt

Collect Whole Blood Samples

Examine Whole Blood Serotonin

Levels

Are there differences in

serotonin levels in ASD and GI?

Aim 3 – Examine Whole Blood Serotonin and GI in ASD

Gastrointestinal Disorder Assessment

• Questionnaire on Pediatric Gastrointestinal Symptoms, Rome III Version (QPGS-RIII)

• Measures frequency, severity, and duration of functional GI disorders

• Can be used to assess whether a person meets criteria for a GI disorder

• We created a scoring algorithm to assess a range of GI symptoms

• Higher scores = greater GI impairment

Selection of Participants at Each Site

Screened ATN

Registry

Patients

Contacted

QPGS Rome-III

Questionnaire

Administered

Inclusion Criteria • ASD Diagnosis

• Age 6-18

Exclusion Criteria •Mitochondrial/genetic disorder • Sensitivity to adhesives •Inability to remain still •Bleeding disorder •Medical Condition •Lack of reliable informant •Inability/unwilling to provide consent

Invited to

Participate in

Study

Study Demographics

Sex Number Percentage of Sample

Male 108 90

Female 12 10

Mean Age SD Min Max N

11.89 3.75 6 18 120

Aim 1 - Results

Draft analysis -- not audited

MGH Biostatistics Center

Data assembled on 20141110

ATN/AIR-P -- GI/Stress

Root-Arcsine Transformed pNN50 Baseline, Cold Pressor-Baseline, Vibro-Baseline on GI

Sum Scores

Scatterplot With Regression Line

0 10 20 30 40 50

Lower GI Sum R3

-0.4

0.0

0.4

Vib

ro-B

ase

0 10 20 30 40 50

Upper GI Sum R3

-0.4

0.0

0.4

Vib

ro-B

ase

0 10 20 30 40 50

Lower GI Sum R3

-0.4

-0.2

0.0

0.2

0.4

Cold

-Base

0 10 20 30 40 50

Upper GI Sum R3

-0.4

-0.2

0.0

0.2

0.4

Cold

-Base

0 10 20 30 40 50

Lower GI Sum R3

0.0

0.2

0.4

0.6

0.8

Base

line

0 10 20 30 40 50

Upper GI Sum R3

0.0

0.2

0.4

0.6

0.8

Base

line





Root-Arcsine Transformed pNN50 Baseline, Cold Pressor-Baseline, Vibro-Baseline on GI

Sum Scores


0 10 20 30 40 50

Lower GI Sum R3

-0.4

0.0

0.4

Vib

ro-B

ase

0 10 20 30 40 50

Upper GI Sum R3

-0.4

0.0

0.4

Vib

ro-B

ase

0 10 20 30 40 50

Lower GI Sum R3

-0.4

-0.2

0.0

0.2

0.4

Cold

-Base

0 10 20 30 40 50

Upper GI Sum R3

-0.4

-0.2

0.0

0.2

0.4

Cold

-Base

0 10 20 30 40 50

Lower GI Sum R3

0.0

0.2

0.4

0.6

0.8

Base

line

0 10 20 30 40 50

Upper GI Sum R3

0.0

0.2

0.4

0.6

0.8

Base

line





GSR Baseline and Cold Pressor on GI Sum Scores


0 10 20 30 40 50

Lower GI Sum R3

-6

-2

2

6

Cold

-Base

0 10 20 30 40 50

Upper GI Sum R3

-6

-2

2

6

Cold

-Base

0 10 20 30 40 50

Lower GI Sum R3

2.5

5.0

7.5

10.0

12.5

Cold

Pre

sor

0 10 20 30 40 50

Upper GI Sum R3

2.5

5.0

7.5

10.0

12.5

Cold

Pre

sor

0 10 20 30 40 50

Lower GI Sum R3

0

5

10

15

Base

line

0 10 20 30 40 50

Upper GI Sum R3

0

5

10

15B

ase

line

Aim 1 - Results

Summary of findings Spearman Correlation (95% CI)

P-value

pNN50 baseline Upper GI

0.18 (-.02, 0.36) †0.076

pNN50 baseline Lower GI

0.21 (.01, 0.39) *0.034

pNN50 vibration-base Lower GI

-0.18 (-.37, 0.02) †0.072

pNN50 cold pressor-base Lower GI

-0.24 (-.42, -.04) *0.018

GSR: cold pressor- base Upper GI

-0.21 (-.42, 0.02) †0.070

Aim 1 - Results

SUMMARY OF REGRESSION ANALYSIS -UPPER GI predominantly related to pNN50 baseline (small contribution from heart rate) -LOWER GI predominantly driven by pNN50 cold pressor minus baseline (but very closely interrelated with baseline

Aim 1 - Results

• Aim 2 results are forthcoming… • IL-6 and TNF-alpha ELISAs are being analyzed at the moment

Aim 2 - Results

Discussion

• Psychophysical factors

– Parasympathetic markers (pNN50) appear to be related to Lower GI and to some extent Upper GI

– GSR limited findings- technically challenging

– Baseline and stimulated HRV closely related

– Post-hoc frequency analysis underway to more closely examine sympathetic and parasympathetic components from HRV

Discussion

– Psychophysical factors

• GI symptoms also related to some behavioral aspects (Vineland, ABC)

• Cytokines completed, being analyzed

Discussion

• Would be of interest in future to look at other factors, how stress genetics relate, microbiome

• Future implications may also include impact on treatment

• May serve as a biomarker that impacts treatment plan

Missouri • David Beversdorf, M.D. • Brad Ferguson, M.A. • Jill Akers, Sr. LPN • Micah Mazurek, Ph.D. • Kaitlyn Hartnett • Briana Kille • Univ. of Missouri – Thompson Center for

Autism & Neurodevelopmental Disorders

Vanderbilt University Medical Center • Jeremy Veenstra-VanderWeele, M.D./

Columbia University • Evon Lee, Ph.D. • Sarah Marler, M.A. • Vanderbilt Kennedy Center for Research on

Human Development

MGH Biostats • Eric Macklin, Ph.D. • Lily Alstein, Ph.D. • Erin McDonnell, M.S.

Thank you!

Jeremy Veenstra-VanderWeele, M.D.

Associate Professor of Psychiatry, Pediatrics, and Pharmacology

Medical Director, Treatment and Research Institute for Autism Spectrum Disorder

Kennedy Center for Research on Human Development

Vanderbilt Brain Institute

Hyperserotonemia in man and mouse: leveraging a biomarker to

gain insights in autism

Funding / Disclosures

• National Institute of Mental Health

• National Institute of Child Health and Development

• Autism Speaks

• American Academy of Child and Adolescent Psychiatry

• Brain and Behavior Research Foundation (NARSAD)

• Agency for Health Care Research and Quality

• Health Resources and Services Administration

• Seaside Therapeutics

• Roche Pharmaceuticals

• Novartis

• Forest

• Sunovion

• SynapDx

Outline

• Hyperserotonemia as Endophenotype

• SERT Ala56 mouse – Brain

– Behavior

– Gut

• Serotonin and GI symptoms in ASD

Outline


• SERT Ala56 mouse

HYPERSEROTONEMIA IN ~25% OF CHILDREN WITH AUTISM

SCHAIN AND FREEDMAN, 1961

Hyperserotonemia in Autism: Half a Century of Mystery

Whole blood serotonin

• Serotonin = 5-HT

• whole blood 5-HT – Schain and Freedman, 1961

• Platelets =>99% of blood 5-HT – 5-HT actually produced in intestinal

enterochromaffin cells

– Taken up into platelets via SERT

• Whole blood 5-HT is highly heritable – Broad heritability ~ 0.99

– Associated with serotonin transporter gene SLC6A4 in males

Weiss et al., 2005

Genetic Linkage of Autism on Chromosome 17

Barondes,1994

Families with only affected males (189 families)

All families (327 families)

Families with affected females (138 families)

Sutcliffe et al AJHG, 2005

hSERT

Multiple Rare SERT Variants Identified in Autism Probands

• Rare amino acid variants – Conserved amino acids

– Collectively associated with autism in males • Rigid-compulsive behavior

• Variants increased 5-HT uptake in lymphoblastoid cell lines and transiently transfected HeLa cells – Ala56 ~30%

Prasad et al, 2009

SERT Gly56Ala

• 3:1 transmission rate to

affected males

– Rigid-compulsive symptoms

(P = 0.0085)

– Sensory aversion (P = 0.0005)

Caveats

• Gly56Ala found in some healthy subjects

• Amino acid variants add to complexity of multiple functional SERT variants

– Enable studies in mouse models

Outline


• SERT Ala56 mouse

Normal growth.

No health problems.

Construction of mSERT Gly56Ala Knock-In Mouse

loxP

X 129S4/Jae Protamine-Cre Transgenic Mouse

129S6/SvEvTac

Tammy Jessen, Brent

Thompson

Brain

• Increased 5-HT clearance

– Increased SERT phosphorylation

• Increased receptor sensitivity

• Increased 5-HT neuron firing

Biomarker

P < 0.05

Behavior

• Decreased sociability

– Complicated by genetic background

• Back out in tube test

• Decreased ultrasonic vocalizations

• Increased climbing/hanging behavior in home cage

Gastrointestinal System

Gastrointestinal System in ASD

• Increase in constipation in autism • Pat Levitt and others

• Constipation + (Diarrhea or Soiling) • ~ Overflow fecal incontinence / encopresis

• Associated with parent report of repetitive behavior and compulsivity (P’s < 0.001)

• OCD diagnosis (P < 0.001)

• Observed rituals on ADOS (P = 0.009)

• DSM-IV rigid-inflexible behavior (P = 0.014)

Peters et al., JADD, 2014

Gastrointestinal System in SERT Mutant Mice

• Mice lacking SERT have decreased transit time

– Intermittent watery stool

• SERT Ala56 mice:

– Rectal prolapse in mice > 1 year

– Increased intestinal transit time

Unpublished:

Kara Gross

Mike Gershon

Total and late-born

submucosal neurons are deficient in

SERT Ala56 mice

• Total neurons

– HuC/D

• Late born neurons

– TH and CGRP

WT Ala56 WT Ala56 WT Ala56

WT Ala56

Unpublished:

Kara Gross

Mike Gershon

So does hyperserotonemia predict constipation in ASD?

• “GI Stress” population, as described by Dr. Beversdorf

– 82 subjects not taking 5-HT-related medications

• Age 11.3 +/- 4.0

• 74 males, 8 females

• 75 Caucasian

• IQ 81.5 +/- 24.5 (N = 51)

Characterizing GI Symptoms

• 46 have no Rome III GI disorders

• 36 have a Rome III GI disorder

– 32 meet criteria for Functional Constipation

• Some of these also meet broader criteria

• Next most common = Irritable Bowel Syndrome

– Can also score quantitatively lower GI symptom score

Characterizing whole blood serotonin levels

• Quantitative trait

– Log transformation to normalize

– Age effect (-0.02 +/- 0.01, P = 0.05)

• Hyperserotonemia compared to historical samples (McBride et al., 1998)

– 20 hyperserotonemic (24%)

– 62 normoserotonemic (76%)

Primary analyses

• Categorical functional constipation v. Ø

– Hyperserotonemia vs. historical control • 31% vs. 22% hyperserotonemia

• OR 1.6 [0.52-5.3], P = 0.43

– Log 5-HT corrected for age and sex • Effect 0.14 [-0.03-0.31], P = 0.11

• Continous lower GI score

– Hyperserotonemia vs. historical control • 14.5±10.5 vs. 18.9±13.4, P = 0.13

– Log 5-HT corrected for age and sex • Pearson r = 0.24 [0.01-0.45], P = 0.037, Spearman <0.05

Secondary analyses

• Hyperserotonemia not associated with any behavioral variables

– Self-injury

– Stereotypies

– Compulsivity (P = 0.08)

– Tactile sensitivity

– IQ

Next questions

• Is this a real effect?

• What specific lower GI symptoms drive correlation?

• Are lower GI symptoms independently associated with behavior?

• What comes first, the lower GI symptoms or altered 5-HT system function?

– Treat and repeat

• What accounts for hyperserotonemia?

Tryptophan hydroxylase activity could explain altered blood 5-HT levels

• Tph1 expression is elevated in SERT Ala56

mice

– 5-HT synthesis increases to compensate for

increased SERT-mediated re-uptake?

• TPH1 genotype is associated with whole

blood 5-HT in relatives of children with

ASD (Cross et al., 2007)

Unpublished: Kara Gross, Mike

Gershon

WT Ala56

Acknowledgements • Sarah Marler

• Evon Lee

• Brittany Peters

• Chris Muller

• Jenny Sauer

• Travis Kerr

• Ran Ye

• Tammy Jessen

• Brent Thompson

• Ray Johnson

• David Beversdorf

• Brad Ferguson

• Erin McDonnell

• Lily Altstein

• Eric Macklin

• Randy Blakely

• Jim Sutcliffe

• Ana Carneiro

• Jacki Crawley

• Randy Blakely

• Ed Cook

• Bennett Leventhal

Questions?

• From the audience for the presenters

• From the presenters for the audience

• What other factors might serve as biomarkers for gastrointestinal problems in ASD for future studies?

• What might the implications for treatment be?

Thank you for attending today’s ATN / AIR-P Advances in Autism Research & Care Webinar Series!

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welcome to today’s air-p/atn advances in autism research ... · aim 1 – explore relationship...

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