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Searching for autism susceptibility genes Elena Maestrini University of Bologna International Molecular Genetic Study of Autism Consortium Helsinki June 2 2006

Searching for autism susceptibility genes

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Searching for autism susceptibility genes. Elena Maestrini University of Bologna I nternational M olecular G enetic S tudy of A utism C onsortium. Helsinki June 2 2006. Autism. Neurodevelopmental disorder characterised by impairments in 3 domains:. Verbal and non-verbal communication - PowerPoint PPT Presentation

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Page 1: Searching for autism susceptibility genes

Searching for autism susceptibility genes

Elena MaestriniUniversity of Bologna

International Molecular Genetic Study of Autism Consortium

Helsinki June 2 2006

Page 2: Searching for autism susceptibility genes

Autism

Verbal and non-verbal communication Reciprocal social interaction Repetitive and stereotyped patterns of behaviours and interests

Onset before 3 years of agePopulation prevalence of autism is ~10-20 per 10,000.Male to female ratio of ~3:1.

75% of autistic people have mental retardation~30% of cases have epilepsy

Belongs to the spectrum of Pervasive Developmental Disorders (PDDs) which include Asperger syndrome, Atypical autism, Childhood disintegrative disorder, PDD NOS. Prevalence of PDDs ~ 60/10,000

Neurodevelopmental disorder characterised by impairments in 3 domains:

Page 3: Searching for autism susceptibility genes

Autism is a complex disorder

No disorder

Cognitive disorder

4%Social

disorder 12%

Social and

cognitive

Autism60%

No disorder

Cognitive disorder

10%

1) TWIN studies

(Bailey et al, Psychol Med 25:63-67, 1995)

Monozygotic Twins (MZ) Dizygotic Twins (DZ)

92%92% 10%10%

Sibling recurrence risk (~ 3%) for autism at least 30 times higher than general population risk (~ 10/10,000). A significant proportion of relatives are affected by the milder phenotypes.

2) FAMILY studies

Page 4: Searching for autism susceptibility genes

Approaches to identify susceptibility genes

• LINKAGE studies using non-parametric methods (allele sharing methods)

• ASSOCIATION studies

•Chromosomal abnormalitiesCopy number variation

candidate genes

whole genome

Page 5: Searching for autism susceptibility genes

Non-parametric linkage studies of complex disorders

• They are insensitivelarge numbers of sibpairs are required to detect a significant

increase in allele sharing • They are imprecise

no assumption on number of genes and inheritance model can not rely on recombination events for fine mapping

More robust than model based approaches but ….

• Significance thresholdMLS > 3.6 genome-wide significanceoften use simulations to estimate significance

Replication in an independent data-set

Page 6: Searching for autism susceptibility genes

International Molecular Genetic Study of Autism Consortium ( IMGSAC )

Team of clinicians and researchers from 9 countries, coordinated by Univ of Oxford (Prof A Monaco, Prof A Bailey)

Collection of > 290 multiplex families

Inclusion criteria• Autism Diagnostic Interview (ADI-R)• Autism Diagnostic Observation Schedule (ADOS-G) • IQ > 30 Exclusion of other medical disorders

Fragile X, tuberous sclerosis Cytogenetic abnormalities

Page 7: Searching for autism susceptibility genes

Chromosome

1 2 4 6 8 10 12 15 18 223 5 7 9 1113141617192021X00.511.522.533.5

ASPEX MLS

D2S2314 (2.54)

D7S530 (2.31)

D9S161 (2.12)

D16S497 (1.73 )

152 ASP

219 ASP

(IMGSAC, 2001)

(Lamb et al, 2005)

IMGSAC GENOME SCREENInitial genome screen - 354 microsatellite markers in 83 sib-pairs, 11 extended families.Typed additional markers under peaks of linkage in up to 268 ASP (307 ARP total)

Page 8: Searching for autism susceptibility genes

Summary of genome scans

** MLS > 3.6* MLS > 2.2^ MLS > 1

I: IMGSAC (Lamb et al, 2005)

1 23 4 5 6 7 8 9 10 11

1213 14 15 16 17 18 19 20 21 22

X

I*B^I*

P^C*

I^

I^

I^

I^

C*

C*P*

P^

S^

S^

S^

S^A*

A*A*

A^

A^D^

D^

D^

F**D^

D*

F*

F*

F**

M^

M^

M*

M^a*

Page 9: Searching for autism susceptibility genes

Further investigations of the linkage loci

1) Detailed linkage analysis to improve mapping

2) Candidate gene analysis

3) Association studies using high density SNPs

Page 10: Searching for autism susceptibility genes

49 (PSD) 2q D2S364/D2S335 NPL 3.32 Buxbaum et al (2001)

45 (PSD) 2q D2S116 M MLS 2.86 Shao et al. (2002)

7q D7S1813 M-HLOD 2.1713q D13S800 M-HLOD 2.54

152 (QTL) 7q D7S1799/D7S3058 QTL-Z 2.85 (WORD) Alarcon et al (2002)

3q D3S3045/D3S1763 QTL-Z 3.10 (WORD)17q D17S1290/D17S1301 QTL-Z 2.84 (WORD)

23 (IS) 15q GABRB3 OSA-LOD 3.19 Shao et al (2003)

1q D1S1656 NPL 3.06

6q D6S1270 NPL 2.61

19p D19S714 NPL 2.31

7q D7S483 NPL 3.721q D21S1437 NPL 3.0

257 (170 MO, 145 FC)

17q D17S1294/D17S798 M MLS 4.3 (MO) Stone et al (2004)

109 (56 MO) 17q D17S2180 M MLS 4.1 (MO) Cantor et al (2005)

7q D7S480/D7S530 M MLS 2.55 (MO)

16p D16S407/D16S497 M MLS 2.48 (MO)

15q D16S407/D16S497 M MLS 2.62 (FC)

Alarcon et al (2005)

62 (OC) Buxbaum et al (2004)

Developmental regression

34

Chr region

291 (QTL)

Language

Sex of probands

219 (145 MO, 74 FC)

Molloy et al (2005)

Lamb et al. (2005)

Repetitive behaviours and

sterotyped patterns

ReferenceN° of ARPs

Bradford et al (2001)50 (PSD)

Highest LOD scoresPhenotype Markers

Reduce heterogeneity by sample stratification based on different component traits of the autism phenotype

Page 11: Searching for autism susceptibility genes

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100 120 140 160

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200 250

MLS

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100 120 140 160

All (219 ASPs)

Male pairs(145 ASP)Female containing pairs (74 ASP)

chromosome 7 chromosome 16

0

0.5

1

1.5

2

2.5

3

0 0.5 1 1.5 2 2.5 3

MLS

chromosome 2

IMGSAC: effect of affected sibling sex on linkage

Lamb et al (2005) J Med Genet 42:132

Males p=0.075 Males p=0.026

• Multipoint MLS calculated using ASPEX sib_phase under additive model• Significance assessed by permuting sibling sex 10,000x.

0

0.5

1

1.5

2

2.5

3

0 20 40 60 80 100 120 140

chromosome 15

Non-males p=0.0011chromosome 17

Page 12: Searching for autism susceptibility genes

IMGSAC: Parent of origin linkage modelling

0.5

1

1.5

2

2.5

0 40 80 120 160

0.5

1

1.5

2

2.5

0 50 100 150 200 250

Chromosome 9, all ASPChromosome 7, all ASP

• Possible parent of origin specific effects

• Involvement of an imprinted gene(s), & 2 loci underlying linkage to chr 7q ?

All (219 ASPs)

Paternal linkage

Maternal linkage

Lamb et al (2005)

Page 13: Searching for autism susceptibility genes

Sex-specific linkage in chromosome 17q

Sutcliffe et al. Am J Hum Genet 77:265 (2005)

267 AGRE + 73 Vanderbilt families

Stone et al. Am J Hum Genet 75:1117 (2005)Cantor et al. Am J Hum Genet 76:1050 (2005)

All

MO

FC

Sex-stratified genome scan in 257 AGRE families

Page 14: Searching for autism susceptibility genes

Serotonin transporter locus (SLC6A4)SLC6A4/ 5HTT - 17q11.2High blood platelet serotonin levels consistently detected in subgroups of

autistic individuals and their relativesSerotonin-reuptake inhibitors ameliorate some symptoms

Several association studies focused on two functional polymorphisms insertion/deletion polymorphism in the promoter (HTTLPR) HTT-VNTR in intron 2

Inconsistent results or modest association with S alleleRare non-synonymous variants (Sutcliffe et al 2005)Variants in SLC6A4 may have a small effect on serotonin blood levels

•ITGB3 identified as a QTL locus for blood serotonin levels in the Hutterites population (Weiss et al. 2004, 2005). This effect is seen primarily in males

•ITGB3 is localized on chrom 17 ~ 20 cM distal to 5HTT.

•Possible association of a Leu/Pro variant in ITGB3 with autism susceptibility, with different effects in males and females (Weiss et al. 2006)

Page 15: Searching for autism susceptibility genes

2q24.2-q32.2 ~40 Mb ~ 190 genes

IMGSAC Candidate gene studiesIMGSAC Candidate gene studies

GENE FUNCTION

RPRM apoptosis

KCNJ3 neuronal cells excitability

NR4A2 transcriptional regulation

TBR1 brain development

GAD1 neurotransmitter metabolism

DLX1 brain development

DLX2 brain development

RAPGEF4 neuronal signal transduction

CHN1 neuronal signal transduction

ATF2 transcriptional regulation

HOXD1 brain development

KIAA1604 possible role in brain development and function

UBE2E3 ubiquitination

NEUROD1 brain development

FRZB brain development

NCKAP1 apoptosis

GULP apoptosis

INPP-1 phosphoinositides metabolism

NAB1 transcriptional regulation

Resequencing or DHPLC screening of coding and regulatory regions in 32 - 48 affected individuals from autism families that are contributing to the linkage peak

Test common variants for association with autism by case-control and/or TDT studies in the whole IMGSAC family sample.

Page 16: Searching for autism susceptibility genes

Chromosome 7NCAM Neuronal cell adhesion moleculeRELN ReelinLAMB1 Laminin beta-1 chain precursor LRNN1 Leucine-rich repeat protein, neuronal 1PTPRZ1 Protein tyrosine phosphatase receptor-

type Z, polypeptide 1CUTL1 Cut-like 1 DLX5 & DLX6 Distal-less homeobox genes 5 & 6FOXP2 Forkhead box P2CHRM2 Cholinergic receptor, muscarinic 2COPG2 Coatamer protein complex, subunit

gamma 2SRPK2 Serine/Arginine rich protein kinase 2SYPL Synaptophysin-like proteinGRM8 Glutamate receptor, metabotropic 8CPA1 & CPA5 Carboxypeptidase isoform 1 & 5MEST Mesoderm specific transcript homologEN2 Engrailed 2

Chromosome 16TBX6 T-box 6UBN1 Ubinuclein 1A2BP1 Ataxin 2-binding proteinABAT 4-Aminobutyrate aminotransferaseCREBBP CREB binding protein GRIN2A Glutamate receptor, ionotropicKIAA1243 KIAA1243 proteinBFAR Bifunctional apoptosis regulatorEMP2 Epithelial membrane protein 2SSTR5 Somatostatin receptor 5

• Rare missense variants found in RAPGEF4 (chr2) and RELN (chr7)• Some evidence of association with common variants in ABAT and

GRIN2 (chr16)

IMGSAC Candidate gene studiesIMGSAC Candidate gene studies

Page 17: Searching for autism susceptibility genes

Autism candidate gene studies Over 150 candidate genes studied in the last 10 years

No clear association with autism

Heterogeneity and clinical complexity of autism Different diagnostic and inclusion criteria used in different studies

Too small sample sizes No comprehensive analysis of variation (only 1- few SNPs/gene)

Position of genes and frequency of publications in candidate gene/association studies over the last decade

1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 1815 17 20 2321220

2

4

6

8

10

12

14

Chromosome

Number of studies

HOXA1

FOXP2

RELN GABRB3

SLC6A4

MECP2

FMR1UBE3A HOXB1

EN2SLC25A12

Page 18: Searching for autism susceptibility genes

HapMap

Public database of common human variation: > 3 millions SNPs genotyped in 269 DNA samples from 4 populations

The block-like structure of LDHOTSPOTS

•A large part of the genome falls into segments of strong LD, known

as “haplotype blocks”, separated by segments of low LD

•Within a block, variants are strongly correlated to each other and a

small number of distinct allele combinations (haplotypes) account

for most of the genetic variation in a population

Page 19: Searching for autism susceptibility genes

Tagging SNPs

Select subset of SNPs which adequately summarises genetic variability within the gene

TAG SNPsMay not be common to different populationsMay depend on definition method employed

•Gabriel et al. (block based selection of tag SNPs)•r2 (htSNPs selected so that all SNPs are highly correlated (r2>0.8) to at least 1 SNP in the tag set)

Page 20: Searching for autism susceptibility genes

High density SNP association study for the investigation of autism loci on chrom 7q and 2q

0

0.5

1

1.5

2

2.5

0 0.5 1 1.5 2 2.5 3

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200 250

Position (cM)

ASPEX MLS

2q24.2-q32.2~ 40 Mb

7q21.3q3340 Mb

Power calculations carried out to determine the optimal selection of SNPs and samples.

126 parent-child trios from autistic multiplex families selected for IBD sharing 200 gender-matched controlsTest statistics: HHRR (family based approach); Case-Control

Prof Anthony Monaco

Page 21: Searching for autism susceptibility genes

Define blocks of strong LD

891 LD BlocksAverage SNPs/block = 8

Identify htSNPs

Average N of htSNPs / Block = 3.3

G/G A/C A/T G/C T/T A/C T/C

Genotype intragenic block htSNPs in selected families

Test htSNPs for association to autism

Number of Haplotype Blocks Required = 419

Number of htSNPs to Genotype = ~1480

Genotyped 1536 SNPs on both chr 2 & chr 7 in 576

samples.

18,389 HapMap SNPs1 SNP/3.4kb

Download all HapMap SNP genotypes in chromosome 2 candidate regionHapMap release 13

(phase 1) CEU

Strategy overview

Align blocks with genesNumber of Genes = 183

Page 22: Searching for autism susceptibility genes

ResultsGenotyped 1536 SNPs on both chr 2 & chr 7 in 576 samples on Illumina Platform

•97.7% SNPs successfully typed (35/ 1536 excluded)

•Sample genotyping success ~ 99%

•99.79% genotyping efficiency after removing failed SNPs/samples

0 0,20 0,40 0,60 0,80 1

Normalized Theta

Genotype calls for rs2368352

0

0,20

0,40

0,60

0,80

1

1,20

1,40

1,60

N

o

r

m

a

li

z

e

d

R

247 171 56

AA

AB

BB

NA11881

NA12006

Page 23: Searching for autism susceptibility genes

Statistical analysis

Case-control analyses.• Single-locus logistic regression allowing for additive and dominance effects,

adjusting for gender main effect.• Block-based haplotype analysis using GENEBPM algorithm (Morris A, 2005)

with dominance, adjusting for (i) gender main effect and (ii) gender main effect and interaction.

Family-based analyses.• Single locus TDT.• Block-based haplotype analysis using TRANSMIT.• Block-based haplotype analysis using GENEBPM algorithm, allowing for dominance and parent of origin effects, comparing probands with internal controls.

Stratification analysis (STRUCTURE)No evidence for population substructure on chromosome 2 or 7 between autism, control and HapMap CEPH samples.

Assign prior probability of 0.01 to each block to overcome multiple testing issue. Takes account of linkage signal and allows for underlying LD and heterogeneity (equivalent to expectation that each region will contain at least 2 associated blocks).

Page 24: Searching for autism susceptibility genes

Chromosome 7

Probands v/s unrelated controls Experiment-wise posterior probability of association

Strong evidence for association (90%)

Positive evidence for association (75%)

IMMP2L

PTPRZ1/2

NM015328

Page 25: Searching for autism susceptibility genes

Chromosome 7Probands v/s internal controls (family-based analysis)Experiment-wise posterior probability of association

IMMP2L

WNT16

CUTL1

MUC3A/B

LHFPL3

FBXL13

Page 26: Searching for autism susceptibility genes

Parent of origin effects

FBXL13 (Block 188)Individuals at greatest risk when inheriting causal variant from mother alone.Posterior mean (SD) of the parent of origin effect of the causal variant: -3.02 (0.50).

IMMP2L (Block 376)Individuals at greater risk when inheriting causal variant from father.Posterior mean (SD) of the parent of origin effect of the causal variant: 1.25 (0.80).

LHFPL3 (Block 220)Individuals at greatest risk when inheriting causal variant from father alone.Posterior mean (SD) of the parent of origin effect of the causal variant: 0.72 (1.38).

Page 27: Searching for autism susceptibility genes

Chromosome 2Probands v/s unrelated controls Experiment-wise posterior probability of association

NOSTRIN

TAI2HUMAN NM018981ZNF533

OSBPL6

Page 28: Searching for autism susceptibility genes

Chromosome 2Probands v/s internal controls (family-based analysis)Experiment-wise posterior probability of association

UPP2NOSTRIN NM024770 ZNF533

Page 29: Searching for autism susceptibility genes

NOSTRIN (nitric oxide synthase trafficker) and ZNF533 (zinc finger protein 533) genes on chromosome 2, and IMMP2L (IMP2 inner mitochondrial membrane protease-like) gene on chromosome 7 give positive results in both case-control and family based analysis.

Strong evidence of differential risk according to the parental origin of the causative variant for IMMP2L, FBLX13 (F-box and leucine-rich repeat protein 13) and LHFPL3 (lipoma HMGIC fusion partner-like 3 ) genes on chromosome 7.

This effect is not seen on chromosome 2.

Results summary

Page 30: Searching for autism susceptibility genes

What is known about these genes?

NOSTRIN: encoding nitric oxide synthase trafficker.Nitric oxide (NO) is a potent mediator in biologic processes such as neurotransmission, inflammatory response, and vascular homeostasis. Binds eNOS and triggers translocation of eNOS to vescicle like subcellular structures, leading to inhibition of NO release

IMMP2L: inner mitochondrial membrane peptidase-like.Implicated in Gilles de la Tourette Syndrome, a complex neuropsychiatric disorder showing phenotypic overlap with autism spectrum disorder.LRRN3 (leucine rich repeat neuronal 3) gene is transcribed in the opposite orientation within an intron of IMMP2L

FBXL13: F-box and leucine-rich repeat protein 13.Substrate-recognition component of the SCF (SKP1-CUL1-F- box protein)-type E3 ubiquitin ligase complex. LRRC17 leucine rich repeat containing 17 gene is transcribed in the opposite orientation within an intron of FBXL13

Sequenced the entire coding sequence and putative regulatory regions of NOSTRIN, ZNF533, IMMP2L and LRRN3 in individuals with the most significant risk haplotype.

No novel coding variants identified.

Page 31: Searching for autism susceptibility genes

Replication!

•Test top 5% of associated haplotype blocks from each chromosomal region in a new independent sample (~200 trios + 200 controls) using the same SNPs (420 htSNPs)

•Use higher density Phase II HapMap data to refine the haplotypic structure in the 4 top genes (NOSTRIN, ZNF533, IMMP2L and FBXL13)

Page 32: Searching for autism susceptibility genes

Large scale, high throughput analysis of genome variation

Better characterization of the phenotype

• component traits• study of milder phenotypes in relatives International collaborations

NAAR AUTISM GENOME PROJECT: Analysis of >1000 multiplex autism families (Europe, USA, Canada)

Perspectives

Page 33: Searching for autism susceptibility genes

Acknowledgements

University of BolognaInternational

Molecular

Genetic

Study of

Autism

Consortium

Funding

Elena BacchelliFrancesca BlasiClaudio TomaSimona CaroneProf Giovanni Romeo

Department of Biology

Medical Genetics LaboratoryS.Orsola-Malpighi Hospital

Wellcome Trust Centre for Human GeneticsJanine LambGabrielle BarnbyNuala SykesAndrew MorrisProf Anthony Monaco

Department of PsychiatryProf Anthony Bailey

University of Oxford• The Wellcome Trust

• The Nancy Lurie Marks

Family Foundation

• UK Medical Research

Council

• NAAR

•Telethon Italy

•European Commission