copy number variants in autism …what are we missing? lauren a. weiss dept. psychiatry institute...

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