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

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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 Slide 2 Autism Verbal and non-verbal communication Reciprocal social interaction Repetitive and stereotyped patterns of behaviours and interests Onset before 3 years of age Population 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: Slide 3 Autism is a complex disorder 1) TWIN studies (Bailey et al, Psychol Med 25:63-67, 1995) Monozygotic Twins (MZ)Dizygotic Twins (DZ) 92% 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 Slide 4 Approaches to identify susceptibility genes LINKAGE studies using non- parametric methods (allele sharing methods) ASSOCIATION studies Chromosomal abnormalities Copy number variation candidate genes whole genome Slide 5 Non-parametric linkage studies of complex disorders They are insensitive large 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 threshold MLS > 3.6 genome-wide significance often use simulations to estimate significance Replication in an independent data-set Slide 6 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 Slide 7 D2S2314 (2.54) D7S530 (2.31) D9S161 (2.12) D16S497 (1.73 ) 152 ASP 219 ASP (IMGSAC, 2001) (Lamb et al, 2005) IMGSAC GENOME SCREEN Initial 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) Slide 8 Summary of genome scans ** MLS > 3.6 * MLS > 2.2 ^ MLS > 1 I: IMGSAC (Lamb et al, 2005) 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 19 20 21 22 X I* B^B^ I*I* P^P^ C*C* I^I^ I^I^ I^I^ I^I^ C*C* C*C* P*P* P^P^ S^S^ S^S^ S^S^ S^S^ A*A* A*A* A*A* A^A^ A^A^ D^D^ D^D^ D^D^ F** D^D^ D*D* F*F* F*F* M^M^ M^M^ M*M* M^M^ a*a* Slide 9 Further investigations of the linkage loci 1)Detailed linkage analysis to improve mapping 2)Candidate gene analysis 3)Association studies using high density SNPs Slide 10 Reduce heterogeneity by sample stratification based on different component traits of the autism phenotype Slide 11 All (219 ASPs) Male pairs (145 ASP) Female containing pairs (74 ASP) chromosome 7chromosome 16 chromosome 2 IMGSAC: effect of affected sibling sex on linkage Lamb et al (2005) J Med Genet 42:132 Males p=0.075Males p=0.026 Multipoint MLS calculated using ASPEX sib_phase under additive model Significance assessed by permuting sibling sex 10,000x. chromosome 15 Non-males p=0.0011 chromosome 17 Slide 12 IMGSAC: Parent of origin linkage modelling 0.5 1 1.5 2 2.5 04080120160 0.5 1 1.5 2 2.5 050100150200250 Chromosome 9, all ASP Chromosome 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) Slide 13 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 Slide 14 Serotonin transporter locus (SLC6A4) SLC6A4/ 5HTT - 17q11.2 High blood platelet serotonin levels consistently detected in subgroups of autistic individuals and their relatives Serotonin-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 allele Rare 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) Slide 15 2q24.2-q32.2 ~40 Mb ~ 190 genes IMGSAC Candidate gene studies 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. Slide 16 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 studies Slide 17 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) HOXA1 FOXP2 RELN GABRB3 SLC6A4 MECP2 FMR1 UBE3A HOXB1 EN2 SLC25A12 Slide 18 HapMap Public database of common human variation: > 3 millions SNPs genotyped in 269 DNA samples from 4 populations The block-like structure of LD HOTSPOTS 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 Slide 19 Tagging SNPs Select subset of SNPs which adequately summarises genetic variability within the gene TAG SNPs May not be common to different populations May depend on definition method employed Gabriel et al. (block based selection of tag SNPs) r 2 ( htSNPs selected so that all SNPs are highly correlated (r 2 >0.8) to at least 1 SNP in the tag set) Slide 20 High density SNP association study for the investigation of autism loci on chrom 7q and 2q 2q24.2-q32.2 ~ 40 Mb 7q21.3q33 40 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 controls Test statistics: HHRR (family based approach); Case-Control Prof Anthony Monaco Slide 21 Define blocks of strong LD 891 LD Blocks Average SNPs/block = 8 Identify htSNPs Average N of htSNPs / Block = 3.3 G/GA/CA/TG/C T/TA/CT/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 SNPs 1 SNP/3.4kb Download all HapMap SNP genotypes in chromosome 2 candidate region HapMap release 13 (phase 1) CEU Strategy overview Align blocks with genes Number of Genes = 183 Slide 22 Results Genotyped 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 Slide 23 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). Slide 24 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 Slide 25 Chromosome 7 Probands v/s internal controls (family-based analysis) Experiment-wise posterior probability of association IMMP2L WNT16 CUTL1 MUC3A/B LHFPL3 FBXL13 Slide 26 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). Slide 27 Chromosome 2 Probands v/s unrelated controls Experiment-wise posterior probability of association NOSTRIN TAI2HUMAN NM018981 ZNF533 OSBPL6 Slide 28 Chromosome 2 Probands v/s internal controls (family-based analysis) Experiment-wise posterior probability of association UPP2 NOSTRIN NM024770 ZNF533 Slide 29 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 Slide 30 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. Slide 31 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) Slide 32 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 Slide 33 Acknowledgements University of Bologna International Molecular Genetic Study of Autism Consortium Funding Elena Bacchelli Francesca Blasi Claudio Toma Simona Carone Prof Giovanni Romeo Department of Biology Medical Genetics Laboratory S.Orsola-Malpighi Hospital Wellcome Trust Centre for Human Genetics Janine Lamb Gabrielle Barnby Nuala Sykes Andrew Morris Prof Anthony Monaco Department of Psychiatry Prof Anthony Bailey University of Oxford The Wellcome Trust The Nancy Lurie Marks Family Foundation UK Medical Research Council NAAR Telethon Italy European Commission