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University of Toronto. Etiology of Autism: A Role for Epigenetics?. Rosanna Weksberg NeuroDevNet September 21, 2012. Complex Etiology of Autism Spectrum Disorders. Genetics in ASD etiology. - PowerPoint PPT Presentation
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Etiology of Autism: A Role for Epigenetics?
University of Toronto
Rosanna WeksbergNeuroDevNet
September 21, 2012
Complex Etiology of Autism Spectrum Disorders
Genetics in ASD etiology• Until recently ASD was considered one of the most heritable of
neurodevelopmental disorders (~90% heritability)• Rare genetic variants :gene mutations/CNVs/chromosome
abnormalities or genetic syndromes account for ~10-20% of ASD cases
• Each individual rare variant is found not in more than ~1-2% of ASD cases– 15q11-13 maternal duplication, 16p11-12 deletion– SHANK3, NRXN1, NLGN3&4, PTCHD1
• Common variants: SNPs have been identified through several genome-wide association studies– MACROD2, CDH9, PITX1– Lack of replication among studies points to high genetic heterogeneity
and small effect size of the risk factor alleles
Environment in ASD etiology • Recent twin study - estimated that heritability of ASD is only 38%,
whereas shared environment contributes to 58% of the liability (Hallmayer et al. 2011)
• Environmental risk factors:• Sub-fertility/assisted reproduction?• In utero exposure to antiepileptic drugs
– Exposure to VPA increases ASD risk ~10 times • Pregnancy complications?• Viral infections?• Ecology?• Nutrition?
• Exposures can occur at different stages of development– Gametes– Prenatal development– Postnatal development
Epigenetics • Epigenetic modifications
can change gene expression patterns without changing primary nucleotide sequence (i.e. no mutation)
• Epigenetic Mechanisms include: DNA methylation, chromatin conformation, histone modifications, RNA silencing
Weksberg R, S.P., Smith AC, Tycko B. Epigenetics. in Emery and Rimoin's Principles and Practice of Medical Genetic
Epigenetic Modifications
• CAN BE STABLE: transmitted through mitotic cell division
• CAN BE DYNAMIC: sensitive to environmental stimuli (internal and external)
Cell differentiation
Memory
Circadian cycle
Nutrition (e.g. folic acid supplementation)
Medications (e.g. valproate)
• CAN BE CELL TYPE-SPECIFIC and DEVELOPMENTALLY-SPECIFIC
Site-specific H3K4H3K9H3K36……
Modification-specific AcetylationMethylationPhosphorylationUbiquitination ………
Complexity of Histone Modifications
Borrelli et al. Neuron 2008
Regulators of Epigenetic Modifications
Epigenetics in ASD etiology
• Evidence for role of epigenetics in ASD comes from both genetic and environmental risk factors
Genetic syndromes co-morbid with ASD and idiopathic ASD are caused by mutations in genes
involved in epigenetic regulation
Gene Function Locus Disorder OMIM Autism/autistic features
CHD7 ATPase/Helicase-
Chromatin remodeler 8q12
CHARGE syndrome
214800 15-50% risk of ASD (Hartshorne et al. 2005; Smith et al. 2005; Johansson et al.
2006)
CHD8 ATPase/Helicase-
Chromatin remodeler 14q11.2 ASD 610528
One of the most frequent recurrent de novo mutations found in idiopathic ASD by exome sequencing: 5 mutations/1144 cases (Neale et al. 2012; O'Roak et al.
2012)
NSD1 H3K36 methyltransferase 5q35 Sotos syndrome 117550
Autistic features (Rutter and Cole 1991; Mouridsen and Hansen 2002; Sarimski 2003; Ball et al. 2005) +Case reports of
ASD (Morrow et al. 1990; Zapella 1990; Trad et al. 1991; Mouridsen and Hansen
2002)
CREBBP, EP300 Histone acetyltransferase 16p13 Rubinstein-Taybi
syndrome 180849 Autistic features (Schorry et al. 2008)
MECP2 Methyl binding protein Xq28 Rett syndrome 300672 Overlap in phenotype between Rett and ASD (White et al. ; Weaving et al. 2004;
Russo et al. 2009)
MLL2 H3K4 methyltransferase 12q13.12 Kabuki syndrome 147920 Autistic features (Ho and Eaves 1997; Oksanen et al. 2004) and case report of
ASD (Akin Sari et al. 2008)
EHMT1 H3K9 methyltransferase 9q34 9q subtelomeric
deletion syndrome 610253 Case report of ASD (Kleefstra et al. 2009)
KDM5C H4K4 demethylase Xp11 Intellectual disability
300534 Case report of ASD (Adegbola et al. 2008)
Environmental Factors Associated with Epigenetic Regulation: Valproate and Autism
• The risk of ASD in children exposed in utero to valporate during pregnancy is estimated to be ~9% vs 0.9% in general population
(Moore et al. 2000, J Med Genet ; Rasalam et al. 2005 Dev Med Child Neurol)
• Possible mechanisms of valproate teratogenic action: o oxidative stress and cell toxicity caused by metabolites of
valproate o interference with folate metabolismo inhibition of histone deacetylases
Folate Metabolism: Combination of genetics, environment and epigenetics
• Dietary folate and vitamin B12 are important components of the S-adenosylmethionine (SAM) synthesis pathway, which is the main donor of methyl group for DNA and histone methylation
• Multiple studies have reported association of functional polymorphisms in the SAM synthesis pathway with ASD (Boris et al 2004; Pasca et al. 2008; Adams et al. 2007; Goin-Kochel et al. 2009; Mohammad et al. 2009)
• Increased risk of ASD in children– If mothers did not take periconceptional vitamin supplementation– If mothers were carriers of a functional polymorphism in one of the
SAM pathway genes (Schmidt et al. 2011)
Epigenetic alterations in ASD• Epigenetic alterations in ASD could occur due to:
– Genetic alterations in genes involved in epigenetic regulation
– Environmental exposures causing epigenetic alterations– Unknown/stochastic factors
• Challenges of identifying epigenetic alterations in ASD:– Etiological heterogeneity– Tissue specificity of epigenetic marks
Interplay of genetic and epigenetic factors: lessons from
KDM5C mutation
KDM5C• Mutations in X-linked gene KDM5C cause intellectual
disability (mild to severe)• More than 20 mutations are identified to date
(Rujirabanjerd et al. 2010)• KDM5C encodes H3 lysine4 (K4) demethylase specific for
demethylating H3K4me3/2 (Iwase et al. 2007)• KDM5C escapes X-inactivation, and has a Y-linked
functional homologue KDM5D• All forms of H3K4 methylation protect DNA from de novo
methylation in embryonic development by blocking DNMT3A/L binding (Ooi et al. 2007)
Hypothesis • KDM5C loss of function
mutations result in loss of DNA methylation at specific genomic targets– Advantage of studying DNA
methylation – accessibility in clinical samples
• Identification of dysregulated epigenetic targets of KDM5C will elucidate the molecular pathophysiology of intellectual disability
Study Design • Illumina Methylation27 array containing 27,578 CpG sites
covering >14,000 genes was used for genome-wide comparison of CpG methylation patterns in blood samples of 10 patients with KDM5C mutations vs 19 male controls
• Mann-Whitney U test with permutation analysis was used for group comparisons
• Targeted validation by Sodium bisulfite pyrosequencing• DNA methylation analysis of top candidate CpG sites using
publically available control datasets• Comparison of DNA methylation in KDM5C targets between
normal XY males (KDM5C/KDM5D) and XX females (KDM5C/KDM5C) in blood and brain using published dataset
DNA methylation
•DNA methylation level = C/C+T
•DNAm at CpG site is a quantitative variable ranging from fully methylated (100%) to completely unmethylated (0%), representing the mixture of methylated and unmethylated cells and alleles
Number of CpG sites detected by multivariate permutation analysis for different levels of
confidence (1-α) and false discovery proportion limit (γ)
1-α γ = 0 γ = 0.005 γ = 0.01 γ = 0.050.995 53 loss/0 gain 53 loss/0 gain 125 loss/11 gain 362 loss/44 gain
0.99 98 loss/5 gain 98 loss/5 gain 207 loss/23 gain 625 loss/103 gain
0.95 207 loss/23 gain 362 loss/44 gain 568 loss/89 gain 1098 loss/207 gain
Targeted Validation: Loss of DNA methylation observed in several
CpGs in cis
Array results for 3 top candidate genes implicated in ubiquitin-mediated protein
degradation
C- controlsK- individuals with ID and KDM5C mutations
Loss of DNA methylation in top three candidate genes was not found in >900 population
controls
GEO datasets AF (Aging in females, GSE20236), AP1 (aging pediatric 1,GSE27097), AP2 (aging pediatric 2, GSE36064), CO (cancer ovarian, GSE19711), DB (diabetes, GSE20067), DS (Down syndrome, GSE25395).K-C are controls from our study (), K-M are KDM5C mutation cases. For CO and DS only control samples were included.
N= 93 398 9 257 99 21 19 10 N= 93 398 9 257 99 21 19 10 N= 93 398 9 257 99 21 19 10
FBXL5 DNA methylation depends on KDM5C/D dosage in brain and blood
Direction of differences is consistent with KDM5C/D dosage:2 copies of KDM5C (females) have higher H3K demethylating activity than KDM5C/KDM5D (males) resulting in higher DNA methylation
Frontal and Temporal Cortex data is a published dataset of 150 neurologically normal individuals (Gibbs et al. 2010)Blood - pyrosequencing 13 males and 13 females
p=0.029 p=0.00026 p=0.01
Conclusions: KDM5C • Loss of DNA methylation at specific genes was found to be
associated with KDM5C mutation:– Large degree of change 20-50% similar to changes seen in imprinting
disorders– Supports interplay between H3K4 methylation and DNA methylation
in humans– Supports the feasibility of studying DNA methylation in
neurodevelopmental disorders, including ASD
• Dependence of FBXL5 and CACYBP DNA methylation on KDM5C/D dosage in normal males and females– Suggest that loss of DNA methylation at FBXL5 and CACYBP promoters
in blood of patients with KDM5C mutations could be a biomarker of similar changes occurring in brain
Interplay of environmental and epigenetic factors in ASD etiology
DOES ART/SUBFERTILITY INCREASE RISK OF ASD?
• Cohort study: – California, University of California, San Francisco: 4 fold increase of ASD in
children born following assisted reproduction (Croughan et al. American Society for Reproductive Medicine conference 2006, 1699 naturally conceived/1008 conceived using ART or FT)
• Case control studies: – Denmark: 2.3 decrease of ART in ASD(N=461) compared to controls (n-461)
(Maimburg and Vaeth, Human Reproduction, 2007)– California: 2.2 increase of history of infertility in ASD twins (N=21) compared
to twin controls (N=54), no association in singletons (349 cases vs 1.847 controls) (Grether et al. 2012, J Autism Dev Disord)
– Boston: 1.7 increased rated of fertility treatments in ASD born to advanced age mothers>35 (N=164) compared to controls born to advanced age mothers (N=857) )(Lyall, et al., Paediatric and Perinatal Epidemiology
• Comparison to general population: – Israel: 10.7% rate of ART in 507 ASD cases vs 3% rate of ART in Israeli
population (Zachor and Itzchak, Research in Developmental Disabilities, 2011)– Japan: 4.5% rate of ART in in 466 ASD cases vs 2,5% of ART in Japan population
(Shimada et al. Research in Autism Spectrum Disorders, 2012)
Sources of inconsistency
• Parental age• Twinning (number of transferred embryos)• Variability in ART/FT procedures: type/dose of
medications and embryo culture• ASD diagnosis
General Population ART
BWSBWS
19/2019/20
5/195/19
ASAS
Enrichment of epigenetic defects in Beckwith-Wiedemann (BWS) and Angelman (AS) Syndromes ART populations
DeBaun et al., (2003)Sutcliffe et al. (2006)Ludwig et al. (2005)
EPIGENETIC DYSREGULATION and ASSISTED REPRODUCTION IN IMPRINTING DISORDERS
Fertility Treatments Can Change DNA Methylation Patterns
• Ovulation stimulation (FSH/clomid):
– Maturation and ovulation of oocytes with incomplete/aberrant DNA methylation
• In vitro fertilization (IVF):
– In vitro embryo culture disrupts proper imprint maintenance during global genome
demethylation
• Intracytoplasmic sperm injection (ICSI):
– Sperm with incomplete/aberrant methylation bypass natural selection
Epigenetic alterations, specifically DNA methylation, play an important role in ASD etiology
Subfertility/fertility treatments are associated with an increased rate of epigenetic errors that contribute to the ASD phenotype
HYPOTHESIS
GroupAge
(Mean ±SD)Female/Male
Ethnicity: Caucasian/Asian/Africa
n
Subfertility/Fertility treatments
ASD-FT 7±4 3/9 7/3/1 3 Subf, 7 OI, 2 IVF
ASD 7±4 3/9 8/2/2 None
Controls 11±4 5/7 8/1/3 None
Research Subjects
Subf. is sub-fertility, defined as time to conception >2 years, OI is ovulation induction, and IVF is in vitro fertilizationDNA was extracted from white blood cells
Experimental Outline
Illumina HumanMethylation27 dataset of frontal and temporal cortex of 150 neurologically normal individualsGEO Accession Number: GSE15745 (Gibbs et al., 2010)
DNA methylation
•DNA methylation level = C/C+T
•DNAm at CpG site is a quantitative variable ranging from fully methylated (100%) to completely unmethylated (0%), representing the mixture of methylated and unmethylated cells and alleles
Global Methylation Analysis
A: Small, but significant reduction in average DNA methylation of 26, 486 autosomal CpGs in ASD-FT group compared to naturally conceived ASD and controls B: Loss results from differences between CpG sites with 10-20% and 0-10% DNA methylation levels
ASD –FT
ASD
Controls
Microarray Analysis• Common variant analysis:
– Mann-Whitney test with correction for multiple testing and difference in DNA methylation ≥ 10% did not reveal any significant changes
• Individual Analysis: – Selection of CpG sites with at least one sample with methylation level
10% lower or higher than the minimum and maximum values in controls: • ASD-FT Group: 13 CpG sites with gain and 36 CpG sites with loss of
DNA methylation • ASD Group: 2 CpG sites with gain and 6 CpG sites with loss of DNA
methylation
ASD ASD-FT C FC TC
ASD: ASD blood samples (N=12)ASD-FT: ASD-FT blood samples (N=12)C: control blood samples (N=12)FC: frontal cortex control samples (N=150)TC: temporal cortex control samples (N=150)
DIRAS family, GTP-binding RAS-like 3
Loss of DNA methylation at imprinted gene DIRAS3 in ASD-FT cases
ConclusionsStudying cases with 1. neurodevelopmental phenotypes and genetic alterations in epigenetic regulators or 2. certain environmental exposures can identify epigenetic dysregulation
– Likely to play important role in molecular pathophysiology of disorder
– Could be further studied in idiopathic cases
Epigenetics plays at least as important a role in ASD etiology as genetics
AcknowledgmentsWeksberg LabAutism project:Daria GrafodatskayaDarci Butcher Brian ChungRageen RajendramSarah Goodman
Sanaa ChoufaniChunhua ZhaoYouliang LouJonathan ShapiroYi-an ChenTanya GuhaHailey Jin Liis UuskulaMichal FeigenbergKhadine Wiltshire
Cinical GeneticsCheryl Cytrynbaum Cheryl Shuman
Steve Scherer , The Centre for Applied Genomics
Wendy Roberts, Autism Research Unit
Evdokia Anagnostou , Bloorview
Andrei Turinsky, Centre for Computational Biology
KDM5C project• C.E. Schwartz• F.E. Abidi• C. Skinn
– Greenwood Genetic Center, South Carolina, USA
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