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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:
Based on latest evidence, would you recommend no alcohol consumption during pregnancy?
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:
Based on latest evidence, would you recommend no alcohol consumption during pregnancy?
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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rget (s
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Control
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?
(Kleiber et al., 2012)
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
(Kleiber et al., 2013)
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
(Kleiber et al., 2014)
FASD as a continuum of genetic and epigenetic events
(Kleiber et al., 2014)
FASD as a continuum of genetic and epigenetic events
(Kleiber et al., 2014)
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
(Laufer et al., 2015)
Alterations in DNA methylation are associated with FASD diagnosis
Children with known prenatal alcohol exposure cluster clearly together separately from controls, without any overlap
(Laufer et al., 2015)
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
(Laufer et al., 2015)
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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et
(s)
Day of Testing
Control
Ethanol
• Prenatal ethanol exposed mice are
slower to reach the target
(Alberry et al., 2016)
Maternal separation stress results in learning deficits
Maternal
separation stress
• Prenatal ethanol exposed mice are
slower to reach the target
• Postnatal stress mice perform more
like ethanol mice
0
50
100
150
0 1 2 3 4
Late
ncy
to T
arg
et
(s)
Day of Testing
Control
Stress
Ethanol
(Alberry et al., 2016)
Maternal separation stress results in learning deficits
Maternal
separation stress
• Prenatal ethanol exposed mice are
slower to reach the target
• Postnatal stress mice perform more
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
et
(s)
Day of Testing
Control
Stress
Ethanol
Ethanol-Stress
(Alberry et al., 2016)
Maternal separation stress results in learning deficits
Maternal
separation stress
• Prenatal ethanol exposed mice are
slower to reach the target
• Postnatal stress mice perform more
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
et
(s)
Day of Testing
Control
Stress
Ethanol
Ethanol-Stress
(Alberry et al., 2016)
Can we alter the environment to
improve outcomes?
Prenatal ethanol & postnatal stress also
implicate genes with FASD-
relevant functions
Ontologies and pathways p-value
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
Canonical pathways (number of genes affected)
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
• Prenatal ethanol exposed mice are
slower to reach the target
Environmental
enrichment
0
50
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150
0 1 2 3 4
Late
ncy t
o T
arg
et
(s)
Day of Testing
Control
T3 Ethanol
Environmental enrichment improves learning
• Prenatal ethanol exposed mice are
slower to reach the target
• Postnatal environmental enrichment
improves learning in control mice
Environmental
enrichment
0
50
100
150
0 1 2 3 4
Late
ncy t
o T
arg
et
(s)
Day of Testing
Enriched
Control
T3 Ethanol
Environmental enrichment improves learning
• Prenatal ethanol exposed mice are
slower to reach the target
• Postnatal environmental enrichment
improves learning in control mice
• Postnatal enrichment improves
learning in prenatal ethanol exposed
mice
Environmental
enrichment
0
50
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150
0 1 2 3 4
Late
ncy t
o T
arg
et
(s)
Day of Testing
Enriched
Control
Ethanol-Enriched
T3 Ethanol
Environmental enrichment after prenatal ethanol also implicates
genes with FASD-relevant functions
Ontologies and pathways p-value
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
Canonical pathways (number of genes affected)
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
(Kleiber et al., 2014)
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:
Based on latest evidence, would you recommend no alcohol consumption during pregnancy?
This work was made possible by…