Exploring the molecular iceberg model for FASD: a …...Exploring the molecular iceberg model for...

Exploring the molecular iceberg model for FASD:a hope for the

futureShiva M. Singh & Bonnie Alberry

Research Lab

University of Western Ontario

Fetal Alcohol Spectrum Disorders

• Varied incidence rates depending on population being studied

• 4% in Canadian population

• Up to 52% of Swedish adoptees

• Annual cost in Canada: $7.6 billion

Cause: KNOWN (prenatal alcohol exposure)

Fetal Alcohol Syndrome

(FAS)

Alcohol Related Birth Defects

(ARBD)

Partial Fetal Alcohol Syndrome

(pFAS)

Alcohol Related Neurodevelopmental

Disorders(ARND)

(Flannigan et al., 2018)

Opinion Poll:

Based on latest evidence, would you recommend no alcohol consumption during pregnancy?

Opinion Poll:


2002

90%of Canadian family

physicians surveyed(Tough et al., 2005)

2015

90%of international

medical professionals

surveyed(BMJ Poll, October 2015)

2007

66%of Obstetricians &

Gynecologists

surveyed(Anderson et al., 2010)

2018

65%of online public

survey(Women’s Health Magazine, 2018)

Opinion Poll:


2002

90%of Canadian family

physicians surveyed(Tough et al., 2005)

2015

90%of international

medical professionals

surveyed(BMJ Poll, October 2015)

2007

66%of Obstetricians &

Gynecologists

surveyed(Anderson et al., 2010)

2018

65%of online public

survey(Women’s Health Magazine, 2018)

Alcohol Culture?

Need to find solutions…….

Fetal Alcohol Spectrum Disorders• Varied incidence rates depending on

population being studied

• 4% in Canadian population

• Up to 52% of Swedish adoptees

• Annual cost in Canada: $7.6 billion

Cause: KNOWN (prenatal alcohol exposure)

Mechanism: UNKNOWN

Treatment: non-existent

Fetal Alcohol Syndrome

(FAS)

Alcohol Related Birth Defects

(ARBD)

Partial Fetal Alcohol Syndrome

(pFAS)

Alcohol Related Neurodevelopmental

Disorders(ARND)

(Flannigan et al., 2018)

Working Hypothesis:

FASD molecular iceberg model

Only the tip is visible

Much of what goes on is below the surface

Underwater features are not visible nor easily

understood

Impossible to investigate directly in humans

How to look below

the surface?

Developed reproducible mouse model to investigate molecular events

• Gene expression and pathways disrupted by ethanol

• Mechanisms that may modulate gene expression

• Test reliability on humans

• Potential applications in diagnosis, prevention and amelioration

Our model: C57BL/6J mice

• Genome sequence 92% similar to humans

• Extensive genomic tools available

1. Alcohol treatment is easy to do – these mice prefer alcohol

2. Neurodevelopmental timeline correlated with humans

3. Similar symptoms to alcohol affect in humans

1. C57BL/6 mice prefer ethanol

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C57BL/6

DBA/2J

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1. C57BL/6 mice prefer ethanol

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C57BL/6

Pregnant C57BL/6

DBA/2J

3.77

3.52

2. Comparable neurodevelopmental timeline between humans & mice

Birth

Birth

3. Symptoms of prenatal alcohol exposure are similar between mice & humans

• Facial dysmorphologies

• Developmental delays

• Behavioural deficits• Learning and memory

• Hyperactivity

• Anxiety

How to assess learning & memory:Barnes Maze

Spatial Learning PhaseMemory

Recall Phase

Day 1 2 3 4 5 12

Singh lab FASD experimental protocol

• Mice mating overnight, confirmation of pregnancy

• Prenatal alcohol exposure via pregnant mother

• Birth of pups

• Assess development

• Assess FASD-related behaviours (day 50)

• Obtain adult brain – hippocampus (day 70)

• Isolate DNA and RNA

• Use molecular techniques to assess long-lasting changes:

• gene expression (RNA)

• DNA methylation

Alcohol-exposed mice are slower to learn the target

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Ethanol

**

*

(Kleiber et al., 2011)

Binge-like trimester exposure also results in learning deficits

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Trimester 1 Trimester 2 Trimester 3

(Mantha et al., 2013; 2014)

Mouse model is comparable to human FASD features

• Learning deficits

• Memory deficits

• Growth deficits

• Developmental delay

• Hyperactivity

• Anxiety-like behaviours

• Lasts for life

The model is suitable to assess

underlying mechanism of prenatal

alcohol exposure

Mouse model is comparable to human FASD features

• Learning deficits

• Memory deficits

• Growth deficits

• Developmental delay

• Hyperactivity

• Anxiety-like behaviours

• Lasts for life

The model is suitable to assess

underlying mechanism of prenatal

alcohol exposure

What may be the underlying

mechanism?

1) Gene expression

2) DNA methylation?

DNA – a gene

mRNA

Protein

Central Dogma: this is how genes work

Gene

expression

DNA – a gene

mRNA

Protein

Measuring gene expression, mRNA

DNA – a gene

mRNA

Protein

DNA

methylation

DNA may changeDNA methylation: cytosine to methyl-cytosine

DNA – a gene

DNA

methylation

Methylation change may stop gene expression

No

transcript

DNA – a gene

DNA

methylation

Measure DNA methylation by methyl-Cytosine

No

transcript

Methylation changes can be brought

about by environment, including alcohol

Pups exposed to prenatal alcohol show many changes in

adult brain gene expression

• Pups exposed to ethanol or saline prenatally

• Assessed as adults at postnatal day 70

• Hundreds of genes differentially expressed between groups

What are genes affected responsible for?


Gene expression:

Genes affected by prenatal alcohol exposure

Timing of exposure results in different combinations of genes affected

• Trimester 1:

• Htt, App, Fos, Bdnf, Tp53

• Trimester 2:

• Htt, App, Fos, Jun, Notch

• Trimester 3:

• Htt, Apoe, Grin, Ntf3

Genes are compatible with FASD-related functions in the brain

• Htt – prenatal neuron development

• App – neurite growth, axonogenesis

• Fos – proliferation, transformation, apoptosis

• Ntf3 – survival, neuron differentiation

• Bdnf, Grin – psychiatric disorders


Gene expression:

Gene expression conclusions

Pups exposed to alcohol develop FASD-related

defects

Alcohol exposed animals show changes in adult brain

gene expression

Affected genes are involved in FASD-related behaviours

and brain functions

Gene expression conclusions

Pups exposed to alcohol develop FASD-related

defects

Alcohol exposed animals show changes in adult brain

gene expression

Affected genes are involved in FASD-related behaviours

and brain functions

What causes alcohol to initiate and maintain gene

expression changes for life?

Gene

expression

DNA – a gene

mRNA

Protein

DNA

methylation

Alcohol may alter DNA methylation that regulates gene expression

Prenatal alcohol exposure leads to changes in DNA methylation in mice

• Pups exposed to ethanol or saline prenatally

• Assessed as adults at postnatal day 70

• Hundreds of genomic regions differentially methylated between groups

Are these regions related to changes in gene expression?

(Laufer et al., 2013)

Alcohol causes changes in DNA methylation that is related to brain gene expression

• More methylation (less expression):• H19, Gtl2, Npy, Akt1, Ghr, Ntrk1, Apoe, Grin2c

• Less methylation (more expression):• App, Mbp, Atp1a2, Grin1

DNA

methylation

Gene

expression

Model: FASD as a continuum of genetic and epigenetic events


FASD as a continuum of genetic and epigenetic events


Summary

Prenatal alcohol exposure causes:

• Lifelong changes in gene-specific DNA methylation

• Corresponding long-term alterations in brain gene expression

• Genes are associated with FASD

EthanolDNA

methylationGene

expressionFASD

FASD iceberg

Gene expression

• During development

• Throughout life

Epigenetics

• DNA methylation

• miRNA

• Histone modifications

What is the future potential of these results?

Early and reliable

diagnosis

Can we use DNA methylation as a marker for

diagnosis?

Is it true in humans?

DNA methylation is

dynamic

Does it work in humans?

Assessing DNA methylation in human FASD

• Children with clinically diagnosed FASD compared to age-matched controls

• DNA from a cheek swab

• Infinium Human Methylation 450K BeadChip array was used

Pateint # (Age) 1 (6) 2 (6) 3 (5) 4 (4) 5 (3) 6 (3)

Clinical Features

Developmental delay and/or mental

retardation x x x x x x

Learning disorders x x x

Hyperactivity, poor impulse control x x x x x x

Short attention span and inattention x x x

Conduct disorder x x x x

Oppositional defiant disorder x x x

Social difficulties x x x

Nervousness and anxiety x x x

Mood disorder x x

Macrocephaly/macrotia x x

Microcephaly/microtia x x

Distinctive facial features x x x x


Alterations in DNA methylation are associated with FASD diagnosis

Children with known prenatal alcohol exposure cluster clearly together separately from controls, without any overlap


Implicated genes have

FASD-relevant functions

Ontologies and pathways p-value

Biological processes

Nervous system development (GO:0007399) 1.0 x 10-8

Anatomical structure development (GO:0048856) 8.0 x 10-8

Cell adhesion (GO:0007155) 2.2 x 10-5

Calcium-dependent cell–cell adhesion (GO:0016339) 7.4 x 10-4

Skeletal system development (GO:0001501) 0.006

Cell projection assembly (GO:0030031) 0.015

Cell–cell adhesion (GO:0016337) 0.014

Molecular functions

Neuropeptide binding (GO:0042923) 0.024

Neuropeptide receptor activity (GO:0008188) 0.022

Sodium channel activity (GO:0005272) 0.016

Protein binding (GO:0005515) 0.007

Voltage-gated sodium channel activity (GO:0005248) 0.009

RNA polymerase II transcription factor activity (GO:0003702) 0.026

Cation channel activity (GO:0005261) 0.043

Canonical pathways (number of genes affected)

Hippo signaling pathway (25 genes) 2.0 x 10-4

Glutamatergic synapse (18 genes) 0.001

Calcium signaling pathway (23 genes) 0.005

Retrograde endocannabinoid signaling (16 genes) 0.006

Serotonergic synapse (16 genes) 0.009

Axon guidance (17 genes) 0.012

Cell adhesion molecules (18 genes) 0.013

Long-term depression (8 genes) 0.035

Focal adhesion (21 genes) 0.038

Dorso–ventral axis formation (5 genes) 0.047

Cholinergic synapse (14 genes) 0.055

Apoptosis (10 genes) 0.061

Brain

development

Brain

function


Manhattan plot for human chromosome 5

• Genomic location for differentially methylated sites in children with known prenatal alcohol exposure

• Arrow shows clustered protocadherin genes

Protocadherins

• Important for connecting cells in the brain

• Other research groups have also found DNA methylation differences at protocadherin genes in children with FASD

Can we find FASD biomarkers?

DNA

methylation

patterns?

Something

we haven’t

understood

yet?

Underlying defects highly variable…..

DNA methylation conclusions

• DNA methylation changes are apparent in children diagnosed with FASD

• Every patient is unique

• Diagnosis is challenging

What is the future potential of these results?

Early and reliable diagnosis using DNA methylation

DNA methylation is dynamic

Can we change it using the postnatal

environment?

Bonnie Alberry : Ph.D. candidate

Long-lasting timeline: comparable postnatal development

Each stage is influenced by environment

Birth

Birth

How can we alter postnatal environment to affect FASD outcomes?

Negative

environment:

stress

Regular

environment

Positive

environment:

enrichment

Postnatal day 70

Hippocampus

The double whammy: postnatal stress after prenatal alcohol exposure

Ethanol+Stress

Stress

Ethanol

Control

Gestation

EthanolWater

Water

Postnatal Days

2-14

Separation

Maternal separation stress results in learning deficits

Maternal

separation stress

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• Prenatal ethanol exposed mice are

slower to reach the target

(Alberry et al., 2016)


Maternal

separation stress



• Postnatal stress mice perform more

like ethanol mice

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Stress

Ethanol



Maternal

separation stress




like ethanol mice

• Ethanol+stress mice perform worse

• Negative postnatal environment is

detrimental for FASD development

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(s)

Day of Testing

Control

Stress

Ethanol

Ethanol-Stress



Maternal

separation stress




like ethanol mice

• Ethanol+stress mice perform worse

• Negative postnatal environment is

detrimental for FASD development

0

50

100

150

0 1 2 3 4

Late

ncy

to T

arg

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(s)

Day of Testing

Control

Stress

Ethanol

Ethanol-Stress


Can we alter the environment to

improve outcomes?

Prenatal ethanol & postnatal stress also

implicate genes with FASD-

relevant functions


Biological processes

negative regulation of protein binding 1.0 x 10-4

regulation of GTP binding 1.5 x 10-4

organic substance metabolic process 1.7 x 10-4

activation of membrane attack complex 2.0 x 10-4

gene expression 3.1 x 10-4

regulation of dendrite development 3.6 x 10-4

regulation of Schwann cell migration 4.8 x 10-4

Molecular functions

nucleic acid binding 8.1 x 10-4

granulocyte colony-stimulating factor binding 1.4 x 10-3

phosphomannomutase activity 1.4 x 10-3

heterocyclic compound binding 2.6 x 10-3

G-quadruplex RNA binding 3.6 x 10-3


RNA degradation (8 genes) 0.005

Rap1 signaling pathway (15 genes) 0.008

Arginine and proline metabolism (6 genes) 0.010

Adherens junction (7 genes) 0.011

Glycerolipid metabolism (6 genes) 0.015

Longevity regulating pathway (8 genes) 0.018

Sphingolipid metabolism (5 genes) 0.022

Circadian entrainment (8 genes) 0.024

Hippo signaling pathway (11 genes) 0.027

Brain

development

Brain

function

(Alberry et al., unpublished)

Gene

expression

Environmental Enrichment

• Groups in large cages

• Running wheels

• Swings

• Ropes

• Tunnels

• Toys with varied texture shape, and colours

• New objects every week

Environmental enrichment improves learning



Environmental

enrichment

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• Postnatal environmental enrichment

improves learning in control mice

Environmental

enrichment

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T3 Ethanol




• Postnatal environmental enrichment

improves learning in control mice

• Postnatal enrichment improves

learning in prenatal ethanol exposed

mice

Environmental

enrichment

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Control

Ethanol-Enriched

T3 Ethanol

Environmental enrichment after prenatal ethanol also implicates

genes with FASD-relevant functions


Gene ontologies

Heterocyclic compound binding 2.9 x 10-6

Macromolecule metabolic process 3.0 x 10-4

RNA metabolic process 0.002

Lymphocyte activation 0.023

Response to stress 0.029


Destabilization of mRNA by AUF1 (2 genes) 0.015

Serotonin receptors (2 genes) 0.017

TNF signaling pathway (3 genes) 0.018

Gene

expression

Brain

function

(Chokroborty-Hoque et al., unpublished)

FASD as a continuum of genetic and epigenetic events


Summary

• Even a single dose of alcohol during pregnancy can cause changes in the brain

How?

• Alcohol leads to FASD outcomes via changes in DNA methylation and related gene expression

• Brain development is a long-lasting continuum

• It is responsive to prenatal and postnatal environment

• Changes during prenatal development can be modified by postnatal environment

Current Approach: early diagnosis, improved care through enrichment

Future Approach: medication to alter methylation status

How? This is the challenge in FASD research

Environmental Enrichment for FASD

• Early intervention programs

• Adopted, maltreated children diagnosed with FASD

• Marked improvements in child developmental functioning

• Improvements for parents:

• Caregiving skills

• less stress

Can we change DNA methylation?

• Drugs – different compounds are known to alter DNA methylation

• Molecular techniques –targeted control over DNA methylation at specific sites

Opinion Poll:


This work was made possible by…

Exploring the molecular iceberg model for FASD: a …...Exploring the molecular iceberg model for...

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