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Copy Number Variants in Autism …what are we missing?
Lauren A. Weiss
Dept. PsychiatryInstitute for Human Genetics
Center for Neurobiology and Psychiatry
Autism
• Specific impairment in:– Language and communication– Social skills– Restricted and repetitive behavior
• Clinically very heterogeneous– MR, epilepsy, hyperserotonemia, GI
problems, regression, savant skills, sensory hypersensitivity, head growth
What we know
• A Pervasive Developmental Disorder– Prevalence of ASDs ~0.6%– 4:1 male to female ratio
• Estimates of >90% heritability – Siblings at increased risk– Large role for inherited genetics vs. sporadic
events and environmental factors– no genes identified in majority
Known Genetic Causes of Autism
• Chromosomal/Structural variants associated with autism
• Rare mutations associated with autism
• Account for ~10%
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
• FMR1 (Fragile X)
• 15q11-q13 duplication (PWS/AS)
• 7q11.23 (Williams Syndrome)
• 22q11.2 (diGeorge/VCFS)
• 22q13 (Phelan-McDermid syndrome)
• 17p11.2 (Smith-Magenis/Potocki-Lupski)
Single Base Mutations in Autism
• NLGN3, NLGN4X
• MECP2 (Rett Syndrome)
• TSC1/2 (Tuberous Sclerosis)
• PTEN (Cowden Disease)
• SHANK3
• CACNA1C (Timothy Syndrome)
• DCHR7 (Smith-Lemli-Opitz Syndrome)
Complex Genetics
disease
genes QTs environment
Complex Genetics
Common
polymorphism
Rare
variant
Study Design
>1000 multiplex families
Linkage analysis SNP/CNV association
IdentificationOf risk genes
In linkage/CNVregions
Replication studies
Follow up of autismrisk factors
CNV analysis
Validation andTyping of
CNVs
Genome-Wide Association
• Common polymorphism
• Usually case-control– TDT for family-based association
T U
A 5 0
B 0 5
case control
A 0.61 0.28
B 0.39 0.72
Genome-Wide Association
• Common polymorphism
• Usually case-control– TDT for family-based association
• LARGE sample size required– 1,000s to 10,000s
• MANY markers across the genome– 500,000 to 1,000,000
Copy Number Variation
• Greater than 1 kb in/del– Normal copy number 2– Rare, common?
• Detection methods– Karyotype, CGH, FISH, MLPA, qPCR
• SNP arrays– Whole genome, small events, boundary
estimates
Datasets
Broad Affy 5.0 AGRE 850 mpx
JHUAffy
5.0/500KNIMH 240 mpx
CHOP(linkage only)
Illumina HH550
AGRE 120 mpx
Many!(replication only)
Affy 5.0/500K, Sequenom
Varied 2400 trios
Site Platform Source N families
Analysis Methods
• Genotype calling by Birdseed– Careful QC pipeline
• Linkage analysis in MERLIN
• Association analysis by TDT in PLINK
Linkage Results
NP
L L
OD
Sco
re
3
2
1
0
•16,150 high quality markers in 878 multiplex families
Top Linkage
Chr6 Chr15
Chr17 Chr20
Chr6 Chr15
Chr17 Chr20
SNP Association Results
11 22 33 44 55 66 77 88 99 1010 1111 1212 1313 1414 15151616 18 18 20 20 2222
17 17 1919 2121 XX
6
5
4
3
2
1
-log 1
0P
chr
Replication
• Top results (P < 10-4) into replication– Additional Affy microarray data– Sequenom genotyping (TaqMan)– extra cases vs. controls
• rs10513025 on 5p15 replicates P < 0.006– MAF 0.04, OR 0.5
• Combined P = 2 x 10-7
Top Association
Chr5, kb
SEMA5A TAS2R1
Top Association
Chr5, kb
SEMA5A TAS2R1
rs10513025 meta-P = 2 x 10-7
SEMA5A Brain Expression
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Control Autism
Fol
d E
xpre
ssio
n
P = 0.039
Control Autism
SNP Results Summary
• Genome-wide significant linkage on 20p
• Association on 5p15 near SEMA5A– Replicated independently
• Very low/high frequency variation implicated
• More to find!
Copy Number Analysis
Physical position (kb)
Chr 15 Duplications
Indi
vidu
al
mom
dad
autism
autism0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
16p11.2 microdeletion/duplication
SPNQPRT
c16orf54
MAZPRRT2
MVPCDIPT
c16orf53
KIF22SEZ6L2
ASPHD1
KCTD13LOC124446
TAOK2HIRIP3
CCDC95DOC2A
FAM57BALDOA
PPP4C
TBX6YPEL3
GDPD3
MAPK3 CORO1ABOLA2GIYD1/2SULT1A3/4
BOLA2GIYD1/2SULT1A3/4
29.5 29.6 29.7 29.8 29.9 30.0 30.1
29 29.5 30 30.5 31
16p11.2
16p11.2 microdeletion/duplication
Sample Deletion Duplication
freqcase vs freqcontrol
P-valuedel+dup
AGRE
751 multiplex autism families
2814 controls
5 cases
3 controls
7 cases
0 controls
0.8% vs 0.1%
1.1 x 10-4
16p11.2 microdeletion/duplication
Sample Deletion Duplication
freqcase vs freqcontrol
P-valuedel+dup
Children’s Hospital
512 DD/MR/ASD
434 congenital anomalies
(not including DD/MR/ASD)
5* cases
0 controls
4 cases
0 controls
1.6% vs. 0
7.1 x 10-3
16p11.2 microdeletion/duplication
Sample Deletion Duplication
freqcase vs freqcontrol
P-valuedel+dup
deCODE
299 ASD cases
18,834 unscreened controls
5,019 psych/lang disorders
3 cases
2 controls
5 psych/
lang
0 cases
5 controls
1% vs. 0.01%
4.2 x 10-4
(0.1% in psych/lang)
Copy Number Summary
• 16p11.2 microdeletion/duplication found in 1-2% of autism cases, <0.1% controls– Three independent samples– Large Icelandic population sample
• 0.1% in psych/lang disorder, 0.01% population
• 15q11-q13 duplications, including partial
• Follow-up and additional controls required for more common and inherited events
• 16p11.2– Deletion, high penetrance, variable expression– Duplication, reduced penetrance, variable expression
• 15q13.3– Deletion, high penetrance, variable expression– Duplication, reduced penetrance, variable expression
• 1q21.1– Deletion, high penetrance, variable expression– Duplication, reduced penetrance, variable expression
Novel CNVs associated with autism
• 16p11.2– Autism, PDD/Asperger’s, ADHD, MR/DD, epilepsy,
schizophrenia, bipolar disorder, dyslexia
• 15q13.3– Epilepsy, MR/DD, schizophrenia, autism, language
delay, dysmorphism
• 1q21.1– Micro/macrocephaly, MR/LD, autism, ADHD,
schizophrenia, epilepsy, dysmorphism, congenital anomalies, short stature
Novel CNVs associated with autism
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
• FMR1 (Fragile X)
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
• FMR1 (Fragile X)
• 15q11-q13 duplication (PWS/AS)
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
• FMR1 (Fragile X)
• 15q11-q13 duplication (PWS/AS)
• 7q11.23 (Williams Syndrome)
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
• FMR1 (Fragile X)
• 15q11-q13 duplication (PWS/AS)
• 7q11.23 (Williams Syndrome)
• 22q11.2 (diGeorge/VCFS)
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
• FMR1 (Fragile X)
• 15q11-q13 duplication (PWS/AS)
• 7q11.23 (Williams Syndrome)
• 22q11.2 (diGeorge/VCFS)
• 22q13 (Phelan-McDermid syndrome)
Structural Variants in Autism
• X chromosome (Turner, Klinefelter)
• FMR1 (Fragile X)
• 15q11-q13 duplication (PWS/AS)
• 7q11.23 (Williams Syndrome)
• 22q11.2 (diGeorge/VCFS)
• 22q13 (Phelan-McDermid syndrome)
• 17p11.2 (Smith-Magenis/Potocki-Lupski)
• Reverse genetic screen
• Common, small CNV associations?
Implications for CNV studies
Common
polymorphism
Single Base Mutations in Autism
• NLGN3, NLGN4X
• MECP2 (Rett Syndrome)
• TSC1/2 (Tuberous Sclerosis)
• PTEN (Cowden Disease)
• SHANK3
• CACNA1C (Timothy Syndrome)
• DCHR7 (Smith-Lemli-Opitz Syndrome)
• Are narrow phenotype definitions useful?
• Population re-sequencing?
• Genetic overlap (e.g. autoimmune syndromes)
• Meta-analysis?
Implications for SNP studies
Human Molecular Genetics, 2008, Vol. 17, Review Issue 2
Guillaume Lettre and John D. Rioux
• Underlying QT– E.g. Diabetic retinopathy, neuropathy,
periodontal disease, nephropathy, cardiovascular disease, reproductive disorders
• Heritable modifiers?
Implications for Neurobiology
Acknowledgements
• Mark Daly
• Pamela Sklar
• Aravinda Chakravarti
• Dan Arking
• AGP, CHOP, and replication groups
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