Upload
marlow
View
48
Download
0
Embed Size (px)
DESCRIPTION
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
Citation preview
Searching for autism susceptibility genes
Elena MaestriniUniversity of Bologna
International Molecular Genetic Study of Autism Consortium
Helsinki June 2 2006
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:
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
Approaches to identify susceptibility genes
• LINKAGE studies using non-parametric methods (allele sharing methods)
• ASSOCIATION studies
•Chromosomal abnormalitiesCopy number variation
candidate genes
whole genome
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
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
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)
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*
Further investigations of the linkage loci
1) Detailed linkage analysis to improve mapping
2) Candidate gene analysis
3) Association studies using high density SNPs
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
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
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)
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
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)
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.
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
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
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
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)
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
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
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
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).
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
Chromosome 7Probands v/s internal controls (family-based analysis)Experiment-wise posterior probability of association
IMMP2L
WNT16
CUTL1
MUC3A/B
LHFPL3
FBXL13
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).
Chromosome 2Probands v/s unrelated controls Experiment-wise posterior probability of association
NOSTRIN
TAI2HUMAN NM018981ZNF533
OSBPL6
Chromosome 2Probands v/s internal controls (family-based analysis)Experiment-wise posterior probability of association
UPP2NOSTRIN NM024770 ZNF533
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
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.
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)
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
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