genetics of neurodevelopmental disorders ap monaco

Genetics of Neurodevelopmental

Disorders

AP Monaco

Neurodevelopmental Disorders

• Autism

• Specific Language Impairment (SLI)

• Developmental Dyslexia

• Attention Deficit Hyperactivity Disorder

Summary of Talk

• Why we think autism has genetic origins.

• Strategies to identify genes involved in autism.

• What have we discovered thus far.

• The Autism Genome Project.

• The search for further autism susceptibility genes.

AUTISM: a severe neurodevelopmental disorder

‘…… an innate inability to form the usual biologically provided affective contact with people.’ Leo Kanner, 1943.Leo Kanner, 1943.

Characterised by impairments in three principal areas:• Verbal and non-verbal communication• Reciprocal social interaction• Repetitive and stereotyped patterns of interests and

behaviours

Onset apparent before 3 years of age and persists throughout life

Population prevalence of autism is ~17-61 per 10,000.Male to female ratio of ~4:1

Evidence for a genetic origin

• Large number of chromosome abnormalities associated with autism.

• Familial clustering of autism is well above the normal population prevalence.

• Twin based studies.

Twin Studies

• Identical twins share 100% of their genes.

• Non-identical twins share on average 50% of their genes.

mother father

child 1 child 2

Identical twins

child 3 child 4

mother father

Non-Identical twins

Twin StudiesIdentical twins Non-Identical twins

? ?

60% concordance 0-3% concordanceaffected

unaffected

GENETIC FACTORS IN AUTISM

The Wellcome Trust Centre for Human Genetics.

•Evidence from twin studies suggests a monozygotic (MZ) to dizygotic (DZ) concordance rate of 60%:0% (Bailey et al., 1995)

•Heritability estimates of >90%

•The rate among siblings of an autistic proband is ~3%

•Autism is one of the most strongly genetic of the childhood-onset psychiatric disorders but no known mode of inheritance

•Statistical modelling suggests the epistatic interaction of 3 genes, but possibly up to 10 loci

•Family and twin studies indicate evidence for a broader autistic phenotype including combinations of milder but related social and/or communicative abnormalities in people of normal intelligence

•MZ:DZ twin concordance rate for the broader phenotype ~92%:10%

Finding autism susceptibility genes:

I. Chromosome abnormalities

II. Linkage Studies

III. Association Studies

The Human Genome

a human

a human cell 23 pairs of chromosomes

23 from mother

23 from father

23 pairs of chromosomes

• Principle: To identify an individual (or several members of a family) with a chromosomal translocation and autism spectrum disorder.

Finding autism susceptibility genes:I. Translocations

Potential site for physical break in gene or regulatory element

• Principle: To identify an individual (or several members of a family) with a chromosomal translocation and reading impairment.

Finding dyslexia susceptibility genes:I. Translocations

23 pairs of chromosomes

Finding autism susceptibility genes:

• Principle: To identify regions of the genome inherited more often than by chance in individuals with autism.

child 1 child 2

mother father

Family 1

mother

child 1 child 2

father

Family 2

child 1 child 2

mother father

Family 3

Linked to autism

.... Family 300

II. Linkage Studies

Finding autism susceptibility genes:

• Principle: To identify a genetic variant that is disproportionately more frequent in individuals with autism.

III. Association Studies

Variant Freq. (%) in controls

Freq. (%) in autistic cases

1 25 24

2 11 13

3 28 27

4 17 34

5 11 12

Examples of genetic variants: - microsatellites

- single nucleotide polymorphisms (SNPs)

- insertions and deletions (INDELS)

- combinations of the above (haplotypes)

Finding dyslexia susceptibility genes:

• Principle: To identify a genetic variant that is disproportionately more frequent in individuals with autism.

Associated to autism

III. Association Studies

Variant Freq. (%) in controls

Freq. (%) in autistic cases

1 25 24

2 11 13

3 28 27

4 17 34

5 11 12

Examples of genetic variants: - microsatellites

- single nucleotide polymorphisms (SNPs)

- insertions and deletions (INDELS)

- combinations of the above (haplotypes)

The Wellcome Trust Centre for Human Genetics.

THE INTERNATIONAL MOLECULAR GENETIC STUDY OF AUTISM CONSORTIUM (IMGSAC)

Initial aims:

Identify 200 multiplex families with two or more individuals with autism

Clear inclusion (ADI and ADOS) and exclusion criteria

Carry out a genome screen for autism susceptibility loci.

340 families now identified through clinics internationally U.K, Italy, Denmark, France, Greece, Germany, The Netherlands, U.S.A.

Autism Pathway

A

B

C

D

E

F

Patient1

A*

C*

D*

Patient2

B*

C*

E*

F*

Patient3

A*

C*

E*

Patient4

B*

C*

E*

Normal

B*

The Wellcome Trust Centre for Human Genetics.

GENOME SCREEN RESULTS

Chromosome

1 2 4 6 8 10 12 15 18 223 5 7 9 11 13 14 16 17 19 20 21 X

0

0.5

1

1.5

2

2.5

3

3.5

AS

PEX

MLS

152 ASP

219 ASP

MLS > 3.6MLS > 2.2MLS > 1

The Wellcome Trust Centre for Human Genetics.

AUTISM GENOME SCREEN RESULTS8 genome screens for autism susceptibility loci carried out to date

Genetic heterogeneity between studies?Some encouraging convergence of linkage findings eg chr 2, 7

The Wellcome Trust Centre for Human Genetics.

POSITIONAL CANDIDATE GENE SCREENING

•Common Disease, Common Variant model-High Resolution SNP/haplotype association mapping-Regional or Whole Genome Association studies

Strategy depends on the model for autism susceptibility

•Multiple rare variants model -Exhaustive Candidate gene screening-No clear aetiological mutations involved in the majority of families -Rare mutations identified: NLGN, NRXN, SHANK3

Chromosome

1 2 4 6 8 10 12 15 18 223 5 7 9 11 13 14 16 17 19 20 21 X

0

0.5

1

1.5

2

2.5

3

3.5

AS

PEX

MLS

Chromosome 2, 219 ASP

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200 250 300

Position (cM)

AS

PE

X M

LS

The Wellcome Trust Centre for Human Genetics.

REGIONS FOR ILLUMINA SNP GENOTYPING

Chromosome 7, 219 ASP

0

0.5

1

1.5

2

2.5

3

0 50 100 150 200 250

Position (cM)A

SP

EX

MLS

International HapMap Project• Genotyped individuals from four populations:

– Yoruba (Nigerian)– Japanese– Han Chinese– Utah (US) = CEPH samples:

• Northern and western European ancestry.

• PHASE I to genotype 1,000,000 SNPs

• PHASE II to genotype 5,000,000 more SNPs

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

• Create haplotype blocks:– Regions of LOW recombinations.

Haploview

Daly Lab at the Whitehead Institute

LOW

recombination

HIGH

recombination

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

• Create haplotype blocks.

• Identify haplotype-tagging SNPs (htSNPs).

1 2 3 4 5 6 7 8 9 Freq

A T A A G C T A G 0.75C C A G A T C A G 0.10C C C A A T T G A 0.08C C A A A T T A G 0.06

1 2 3 4 5 6 7 8 9^ ^ ^A T A A G C T A GC C A G A T C A GC C C A A T T G AC C A A A T T A G

SNP number

1234

Haplotype number

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

• Create haplotype blocks.

• Identify haplotype-tagging SNPs (htSNPs).

• Align haplotype blocks with genes.

Important Points: Gene not covered by haplotype block, so can not be tested.

Haplotype block not covering any gene, therefore less need

to genotype htSNPs.

Segments of genes not covered by

haplotype block, so can not be tested.

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

• Create haplotype blocks.

• Identify haplotype-tagging SNPs (htSNPs).

• Align haplotype blocks with genes.

• Genotype 1536 htSNPs on the Illumina platform, utilising the GoldenGate assay, in the selected samples (cases and controls).

G/G A/C A/T G/C T/T A/CT/CG/CT/T

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

• Create haplotype blocks.

• Identify haplotype-tagging SNPs (htSNPs).

• Align haplotype blocks with genes.

• Genotype 1536 htSNPs on the Illumina platform, utilising the GoldenGate assay, in the selected samples (cases and controls).

• Reconstruct haplotypes based on the htSNPs’ genotypes.

G/G A/C A/T G/C T/T A/CT/CG/CT/T

TGC 0.80

TCG 0.15

AGC 0.05

AT 0.60

AA 0.25

CT 0.15

AT 0.60

AA 0.25

CT 0.15

AT 0.60

AA 0.25

CT 0.15

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

• Create haplotype blocks.

• Identify haplotype-tagging SNPs (htSNPs).

• Align haplotype blocks with genes.

• Genotype 1536 htSNPs on the Illumina platform, utilising the GoldenGate assay, in the selected samples (cases and controls).

• Reconstruct haplotypes based on the htSNPs’ genotypes.

• Test haplotypes for association to autism.

G/G A/C A/T G/C T/T A/CT/CG/CT/T

association p-values0.021 0.7480.451 0.001

TGC 0.80

TCG 0.15

AGC 0.05

AT 0.60

AA 0.25

CT 0.15

AT 0.60

AA 0.25

CT 0.15

AT 0.60

AA 0.25

CT 0.15

Summary Strategy Overview• Download CEPH HapMap genotypes.

• Process genotypes (filter & clean).

• Create haplotype blocks.

• Identify haplotype-tagging SNPs (htSNPs).

• Align haplotype blocks with genes.

• Genotype 1536 htSNPs on the Illumina platform, utilising the GoldenGate assay, in the selected samples (cases and controls).

• Reconstruct haplotypes based on the htSNPs’ genotypes.

• Test haplotypes for association to autism.

• Attempt to replicate any significant resultsin INDEPENDENT samples.

G/G A/C A/T G/C T/T A/CT/CG/CT/T

TGC 0.80

TCG 0.15

AGC 0.05

AT 0.60

AA 0.25

CT 0.15

AT 0.60

AA 0.25

CT 0.15

AT 0.60

AA 0.25

CT 0.15

association p-values0.021 0.7480.451 0.001

Autism Candidate Gene

0.847 0.002

IMMP2L

PTPRZ1/2 NM015328

Chromosome 7

Strong evidence for association (90%)

Positive evidence for association (75%)

Probands v/s unrelated controls (population-based analysis)Experiment-wise posterior probability of association

Family Based Analysis

Probands v/s internal controlsExperiment wise posterior probability of association

Strong evidence for association 90%Positive evidence for association 75%

CUTL1

MUC3A/B

LHFPL3 IMMP2L

WNT16

Summary of molecular genetics research

The Wellcome Trust Centre for Human Genetics.

• Encouraging convergent evidence for linkage in some regions.

• Many candidate gene screening and association studies have been carried out, but no etiological variants have been so far conclusively identified in the majority of families with autism.

• High density SNP genotyping for association to autism on chromosome 7q and 2q have identified several positive risk haplotypes that need to be replicated.

• Several groups have carried out genome screens for linkage to autistic disorder.

Autism Genome Project

• Genome-wide SNP genotypes (10k, 1M)

• Copy Number Variation (CNV)

• Linkage analyses

• Association analyses

• Trait subsets, endophenotypes, QTLs

Autism Genome Project• Consortium of consortia

• Autism Genetics Cooperative (AGC)• Autism Genetics Research Exchange (AGRE)• Collaborative Programs of Excellence in Autism (CPEA)• International Molecular Genetic Studies of Autism

Consortium (IMGSAC)

• Pool autism family samples, phenotype data and expertise

• Phase I• Genome-wide genotyping (10k)• Initial linkage analysis

• Phase II• Association and Copy Number Variants

Autism Genome Project: Phase I

• ~1400 multiplex families• Affymetrix 10k SNP genotypes• Basic linkage analysis

• “autism” = affected (categorical Dx)• Narrow and broad autism and “heterogeneous

ASDs”

• Copy Number Variation• Use signal intensity data from adjacent/contiguous

SNPs to infer copy number gains or losses

• Linkage analyses in data subsets• Sex: Male-only, Female-containing• Ancestry: Western European• CNV: Removing CNV by different algorithms

Autism Genome ProjectCopy Number Variation and

Genetic Linkage

Copy Number Variants

1 2

2p16 deletion:2 affected siblings

NRX1

1q21 dups/dels:three families

Previously implicated in MR

17

17p12 dups:three families SMS, CMT

22

22q11.2 deletions:two families

Interpretation complicated

17 de novo CNVs (10 found in both ASP) 18 CNVs overlap ASD-related

rearrangements Numerous overlapping/recurrent CNVs Families with transmission of maternal 15q

gains

Copy Number Variation and Autism Risk:De Novo Mutation vs. Inherited Risk

CNV in Simplex and Multiplex Autism Families

Sebat et al, Science 2007

Are CNVs more frequent in simplex families?

Sebat et al, Science 2007

Are autism associated CNVs more likely to be sporadic “mutations” or inherited risk factors?

AGP Phase IIDesign

Summary

• Autism has a complex genetic etiology• CNV is a major class of autism risk and causation• Need to clarify what is disease-related vs rare polymorphism• New information from genetic linkage studies• 11p and other regions targeted for candidate gene analysis• Whole genome association (WGA) studies will (hopefully)

identify common liability alleles– Common = common in the general population

• WGA does not identify genes with rare or infrequent risk or causative alleles– Sequence (autism genomes?)

• Higher resolution CNV analysis• Collaboration is important to make progress in such complex

disorders

AcknowledgmentsNuala SykesInes SousaAlistair PagnamentaRichard HoltKirsty Wing Gaby BarnbyPenny Farrar Elena BonoraTom Scerri Elena MaestriniAndrew MorrisJanine LambTony Monaco Tony Bailey

autism@well.ox.ac.uk

FundingMRC, Wellcome Trust, NLM Family Foundation, Simons Foundation, EU

The Wellcome Trust Centre for Human Genetics.

International Molecular Genetic Study of Autism Consortium (IMGSAC)

http://www.well.ox.ac.uk/~maestrin/iat.html

The Wellcome Trust Centre for Human Genetics.

• U.K.• Dept of Psychiatry, University of Oxford• Wellcome Trust Centre, Univ of Oxford• Institute of Psychiatry, London• University of Cambridge, Dept of

Psychiatry • Guy’s Hospital, London• University of Newcastle, School of

Clinical Medical Sciences• University of Manchester, School of

Epidemiology & Health Science• ECACC, Porton Down

• Denmark, Centre for Autisme, Bagsvaerd• France, Hôpital La Grave, Toulouse• Germany, Molecular Genome Analysis,

HeidelbergDept of Psychiatry, Frankfurt

• Greece, Agia Sophia Children’s Hospital, Athens

• Italy, University of Bologna• The Netherlands, Dept of Psychiatry,

Utrecht• U.S.A

University of Chicago, Dept of PsychiatryUCLA Center for Neurobehavioural Genetics, Psychiatric InstituteYale UniversityUniversity of Pittsburgh, Dept of Human Genetics and BiostatisticsUniversity of Michigan, Autism & Communicative Disorders Center

Genome-Wide Analysis of Linkage and Copy Number Variation

Autism Genome Project Consortium:Autism Genetics Collaborative (AGC)

Autism Genetics Resource Exchange (AGRE)Collaborative Programs of Excellence in Autism (CPEA)

International Molecular Genetics Study of Autism Consortium (IMGSAC)

Funding:UK: Medical Research Council

USA: Autism SpeaksIreland:HRB

Canada: Genome Canada

Autism Genome ProjectAutism Genome Project