Recent Advances in the Genetics
of Autism Spectrum Disorder
27th ECNP Congress
Session S11: October 19th 2014
Christian R. Marshall, PhD
Genome Diagnostics,
Department of Paediatric Laboratory Medicine
The Hospital for Sick Children
University of Toronto, Canada
No conflict of interest or financial
relationships to disclose
Autism Spectrum Disorder (ASD)
1. Enormous variation in clinical presentation in autism
• collection of rare disorders/conditions that share features
2. Enormous variation in the genetic factors involved in autism
• dozens of (known) genes are involved in autism
• understanding the genes and the factors that influence them
may lead to personalized treatment
• Our lab is focused on delineating the role of genomic variation in
ASD and how it relates to variable clinical presentation by:
- Identifying new genes/genetic loci associated with Autism
- Studying the variable expressivity in families
- Looking at the overlap of genetic risk variants with other
neuropsychiatric disorders
Autism Spectrum Disorder Genetics
• 50-90% MZ twin concordance for broader traits vs. 10%
for DZ twin
• ~18% risk to siblings (> the ~1% general risk)
• 4:1 male female ratio
• ~20% of parent(s) have ASD or BAP
• Variability of clinical presentation within the same
family
• Common genetic variants have a role, but many rare
genetic variants are known to be causative
Genomic Variation in 2014
STRUCTURAL VARIATION
TCATGCCAGGCTA
AGTACTCGTCAAT
CGCTTGCATATGT
CTAGTCGCTAGCT
GTACTGAAATCGC
INDELS & SNPs
Sequencing, PCR
Copy Number Variant (CNV)
Chromosomal
rearrangements
Frequency
CGH arrays
SNP arrays
Karyotyping; FISH
>5Mb
3Mb
10kb
1kb
1bp
Total size of genomic change
2003
10K probes
2005
500K probes
2008
2M probes
2011
5M probes
Whole
genome
• Increasing resolution means increased findings and need for interpretation of rare
genomic variation
Genetic Variation associated with ASD
Effect Size
(Penetrance)
High
Highly Penetrant
Common Variants
are rare
Rare Variants
that are Highly
Penetrant
Intermediate
Uncommon
Variants of
moderate effect
Moderate
Rare Variants of
low effect are
hard to find
Common Variants
of low effect in
common disorders
Low
0.001
Very Rare
0.005
Rare
Low Frequency
0.05
Common
Allele Frequency
• Searching for Rare Variants has led to discovery of many genes associated
with Autism (often de novo/spontaneous).
adapted from McCarthy et al . Nat Rev Gen
NRXN1 deletion: a highly penetrant variant in autism
Autism
300kb de novo deletion
1/1000
(0.1%)
• Using low resolution SNP array, we detected a 300kb de novo deletion in NRXN1 in one out of
~1000 unrelated probands not present in controls
• Good candidate for ASD – does it contribute to the phenotype?
• Challenge is that highly penetrant CNVs are inherently rare – need more cases and controls
for interpretation
NRXN1 deletion: a highly penetrant variant in autism
Autism
42/16,618
(0.25%)
Controls
9/52,690
(0.017%)
•
NRXN1 exonic deletions significantly associated with ASD p=4.9X10-19 (OR 15.6)
•
Exonic deletions also associated with other neuropsychiatric disorders
•
NRXN1 sequence level loss of function variants have also been identified in ASD
Daisuke Sato and data from Szatmari Nature Genetics 2007; Marshall et al AJHG
2008; Pinto et al Nature 2010; Bucan et al Plos Genetics 2009
Overview of Autism Project Design
• Canadian Autism Genome project High resolution SNP microarray and Whole
Exome sequencing (WES) and genome (WGS) workflow (mainly trio design)
Genomic DNA of
families
N= >2000
Illumina 2.5M
Affymetrix cytoHD
High throughput
Sequencing
High resolution
SNP microarray
N=400
CGI
Illumina
Copy number
Variation
Whole genome
Sequencing
Copy Number
Variation
N=600
OR
Whole Exome
Sequencing
Solid 5500xL
Ion Proton
SNVs and Indels
Combined High Resolution Genome Analysis
• Microarray analysis lead to the discovery of many loci/genes
GenotypePhenotype
Summary of CNV discoveries in ASD genetics
• Rate and size of CNVs across genome equal in cases and controls
• Increased burden in # of genes affected by rare CNV in ASD cases
compared to controls (1.41 increase, p= 1.0x10-5)
• ~5% de novo CNVs in ASD families (~1% in controls); many are highly
penetrant:
Identified from
ASD research
P-value;
Odds Ratio (95% CI)
(del)16p11.2
(del)NRXN1
(del)PTCHD1 (chrX)
(dup)7q11.23
(dup)1q21.1
(del) SHANK1-SHANK3
2.0x10-10; 9.5 (5.2-17.4)
4.9x10-19; 15.6 (7.6-32.1)
1.2x10-5; ∞ (5.89-∞)
8.0x10-4; 30.7 (3.4-275.1)
3.6x10-5; 8.0 (3.5-18.4)
9.0x10-3; ∞ (1.76-∞)
Nature Genetics 2007 ; AJHG
2008; Nature 2010; Nature
Genetics 2010; Science
Transl. Med. 2011; AJHG
2014.
• Some risk CNVs are intergenic (***PTCHD1 locus)
• Many of these CNVs/genes are found in across other disorders
• No single gene/locus accounts for >1% of Autism
ASD genes/proteins at the Synapse
• Although individually rare, several of the‘ASD genes’code for proteins
involved in regulating and maintaining neuronal synapse development
and plasticity
Adapted from State, Nat Gen 2010
Gene-Set Enrichment for rare exonic deletions
Gene-set enrichment for rare exonic
deletions (de novo and inherited) in
ASD vs. control subjects
D. Merico and AGP, 2014
Neuron synaptic spine volume is reduced in
ASD cases haploinsufficient for SHANK2
Control
SHANK2 mut
• Over expression of SHANK2 nonsense mutation in neurons
With Gudrun Rappold,
Nature Genetics 2010;
Human Molecular Genetics, 2011
Interpretation depends on technology
Microarray CNV analysis:
15q11.2 535kb loss
15q11.2 loss
NRXN1 G989*
(HERC2P2, CYFIP1, NIPA2, NIPA1, TUBGCP5,
WHDC1L1, GOLGA9P)
Known ASD association with variable expressivity
Whole Exome Sequencing:
NRXN1 exon 15 Gly989stop
NRXN1 G989*
15q11.2 loss
(chr2: 50,724,505 C>A)
Neurexin 1 – nervous system cell adhesion
molecule and ASD candidate gene
INTERPRETATION: NRXN1 G989* may be pathogenic variant with
15q11.2 CNV also contributing to phenotype
Highly-penetrance risk genes from array and WES
Locus
identified by
CNV studies1
Cytoband
Combined
#of events
in cases/
controls
P-value
(cases vs.
controls)2
Freq.
in ASD
Cases
Gene
identified by
Exome
Sequence3
CNV16p11.2
16p11.2
23/3,271;
2/3,512
<0.0001
0.7%
PTCHD1/
PTCHD1AS
Xp22.11
11/2,821;
1/1,1644
(males)
0.067
NRXN1
2p16.3
CNV7q11.23
CNV22q11.2
CNV1q21.1
7q11.23
22q11.2
1q21.1
CNV15q13.3
15q13.3
CNV15q11-q13
15q11q13
SHANK2
11q13.3
SHANK3
22q13.33
10/3,271;
2/3,512
6/3,271;
0/3,512
8/3,271;
1/3,512
7/3,271;
1/3,512
7/3,271;
1/3,512
6/3,271;
0/3,512
3/3,271;
0/3,512
3/3,271;
0/3,512
Cytoband
Combined
#of events
in cases/
controls
P-value
(cases vs.
controls)2
Freq. in
ASD
cases
CHD8
14q11.2
2/965;
0/3,416
0.048
0.2%
0.3%
(0.4%)
KATNAL2
18q21.1
2/965;
0/3,416
0.048
0.2%
0.019
0.3%
POGZ
1q21.3
0.048
0.2%
0.013
0.2%
DYRK1A
21q22.13
0.048
0.2%
0.018
0.2%
SCN2A
2q24.3
0.048
0.2%
0.033
0.2%
GRIN2B
12p13.1
0.220
0.1%
0.033
0.2%
CTTNBP2
7q31.2
0.220
0.1%
0.013
0.2%
RIMS1
6q13
0.220
0.1%
0.112
0.1%
NRXN1
2p16.3
0.220
0.1%
0.112
0.1%
SHANK2
11q13.3
0.220
0.1%
2/965;
0/3,416
2/965;
0/3,416
2/965;
0/3,416
1/965;
0/3,416
1/965;
0/3,416
1/965;
0/3,416
1/965;
0/3,416
1/965;
0/3,416
• All <1% frequency….100s of genes involved
• Only 4 genes found by CNV (113) or LoF SNV (122) overlap between
datasets (different technologies)
AGP, AJHG 2014
Estimated diagnostic yield of WES and CMA
9%
Resolved
Potential
19%
66%
10%
Unknown
Essential (n=185)
(Idiopathic Autism)
81%
15%
38%
42%
Complex (n=65)
(Autism + syndromic
features)
20%
• Cohort of 250 probands from Newfoundland
• All assessed for Autism and examined by a clinical geneticist
• Combined genetic diagnostic yield from whole exome and chromosome
microarray is roughly 20% in the ASD population
Bridget Fernandez and Kristiina Tammimies
What about the rest of the genetic risk?
Considerations:
1. Estimated to be >100 risk genes involved
2. CNV studies and exome studies yield different
genes/loci
3. Resolution issues with both CNVs (probe coverage)
and WES (sequence capture)
4. ~5% of Canadian cohort have presumed
intronic/intergenic (eg. PTCHD1, NRXN1) or noncoding RNA mutations (PTCHD1AS)
Whole Genome Sequencing in Autism
• 85 quartet family sequenced (170 autistic children)
• Advantage: higher resolution + capture all types of variants
• Question: what are all the risk variants in two affected?
Father
Mother
Proband
Affected sibling
Rare 1.7 kb exonic deletion in SCN2A
c
SCN2A
1.7kb del
SCN2A
1.7kb del
SCN2A
1.7kb del
• Through WGS, detected a rare 1.7kb loss in an ASD proband at SCN2A
• SCN2A is a voltage gated sodium channel function of gene linked to seizure
disorders
• Both affected females carry the ‘de novo’ deletion in exon 18, which causes a
frameshift of the rest of the protein
• Germline mosaic or more complex rearrangement?
• Sanger Sequencing showed variant is inherited from father (balanced)
Jiang/Yuen et al 2013
Summary of Genetic findings in WGS
• In 42% (36 out 85) of families, potentially relevant
mutations (CNV + SNV) were identified using WGS
• But presumed penetrant de novo/inherited ASD risk
variants identified in the index case were also found
in the other sibling who developed ASD only ~1/3 of
the time (13/36)
• Siblings with ASD often carry different pathogenic
mutations
• Genome wide screening also uncovers other
medically relevant information
WGS and pharmacogenetic variants
• WGS gives you ‘other’ information (secondary)
• Currently testing the utility of genotyping relevant
pharmacogenetic variants from WGS
Ron Ammar, Iris Cohn, Tara Paton
Summary and Observations
• Autism spectrum disorder is a heterogeneous spectrum
disorder with varied clinical presentation
• Underlying genetic risk is complex – 100s of genes are likely
involved (rare penetrant versus common variation) and many
of these overlap with other neuropsychiatric disorders
• Gene-sets and pathways emerge - some genes are targets for
drugs/development -> personalized treatment
• Genetic risk factors detected by microarray technology and
sequencing uncover different genes (overall ~20% cases will
have a genetic variants that is likely causative).
• Technology moving into WGS (detects all types of variation)
Acknowledgments
Toronto TCAG team
in Autism Speaks Blue on Autism Awareness Day
Rosanna Weksberg, Wendy Roberts, Peter Szatmari, John Vincent, Bridget
Fernandez, Eric Fombonne, Evdokia Anagnostou, Jim Stavropoulos, Peter Ray,
Lonnie Zwaigenbaum, Michael Brudno, and teams.
Funders: Autism Speaks, Canadian Institutes of Heath Research, Canada Foundation for Innovation,
Genome Canada/Ontario Genomics Institute, Canadian Institutes for Advanced Research, NeuroDevNet,
The Centre for Applied Genomics, Ontario Ministry of Research and Innovation, Ontario Brain Institute,
University of Toronto McLaughlin Centre, +generous philanthropic donors