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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 Autism Neurodevelopmental disorder characterised by impairments in 3 domains: 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 Autism is a complex disorder 1) TWIN studies Monozygotic Twins (MZ) Social disorder 12% Cognitive disorder 4% Social and cognitiv e No disorder 92% Autism 60% Dizygotic Twins (DZ) No disorder 10% Cognitive disorder 10% (Bailey et al, Psychol Med 25:63-67, 1995) 2) FAMILY studies 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. Approaches to identify susceptibility genes • LINKAGE studies using nonparametric methods (allele sharing methods) • ASSOCIATION studies candidate genes whole genome • Chromosomal abnormalities Copy number variation Non-parametric linkage studies of complex disorders More robust than model based approaches but …. • 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 • Significance threshold MLS > 3.6 genome-wide significance often 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 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) 3.5 3 152 ASP (IMGSAC, 2001) 2.5 ASPEX MLS D9S161 (2.12) D16S497 (1.73 ) 2 219 ASP (Lamb et al, 2005) 1.5 1 0.5 0 1 2 3 4 5 6 7 8 9 10 Chromosome 11 12 13 14 15 16 17 18 19 20 21 22 X Summary of genome scans D^ M* S^ A* S^ C* F* I* B^ D^ I* D^ F** F** 7 6 5 4 3 A^ I^ 9 8 11 10 2 C* S^ I^M^ P^ A* a* M^ C* 12 A* A^ P* 1 I^ I^ 13 14 15 16 17 P^M^ D^ S^ 18 19 F* D* 20 21 22 X ** MLS > 3.6 * MLS > 2.2 ^ MLS > 1 I: IMGSAC (Lamb et al, 2005) Further investigations of the linkage loci 1) Detailed linkage analysis to improve mapping 2) Candidate gene analysis 3) Association studies using high density SNPs Reduce heterogeneity by sample stratification based on different component traits of the autism phenotype Phenotype Language Repetitive behaviours and sterotyped patterns Developmental regression Sex of probands NЎ of ARPs Chr region Markers Highest LOD scores Reference 49 (PSD) 2q D2S364/D2S335 NPL 3.32 Buxbaum et al (2001) 45 (PSD) 2q D2S116 M MLS 2.86 Shao et al. (2002) 50 (PSD) 7q 13q D7S1813 D13S800 M-HLOD 2.17 M-HLOD 2.54 Bradford et al (2001) 152 (QTL) 7q D7S1799/D7S3058 QTL-Z 2.85 (WORD) Alarcon et al (2002) 291 (QTL) 3q 17q D3S3045/D3S1763 QTL-Z 3.10 (WORD) D17S1290/D17S1301 QTL-Z 2.84 (WORD) Alarcon et al (2005) 23 (IS) 15q GABRB3 OSA-LOD 3.19 Shao et al (2003) D1S1656 62 (OC) 1q 6q 19p Buxbaum et al (2004) D19S714 NPL 3.06 NPL 2.61 NPL 2.31 34 7q 21q D7S483 D21S1437 NPL 3.7 NPL 3.0 Molloy et al (2005) 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) 219 (145 MO, 74 FC) 7q 16p 15q D7S480/D7S530 M MLS 2.55 (MO) M MLS 2.48 (MO) M MLS 2.62 (FC) Lamb et al. (2005) D6S1270 D16S407/D16S497 D16S407/D16S497 IMGSAC: effect of affected sibling sex on linkage All (219 ASPs) Lamb et al (2005) J Med Genet 42:132 3 3 chromosome 7 chromosome 16 2.5 2.5 Males p=0.075 MLS Male pairs (145 ASP) Female containing pairs (74 ASP) Males p=0.026 2 2 1.5 1.5 1 1 0.5 0.5 0 0 0 50 100 150 200 250 0 20 40 60 80 100 120 140 160 3 chromosome 15 Non-males p=0.0011 3 chromosome 2 3 2.5 chromosome 17 2.5 2.5 2 2 MLS 2 1.5 1.5 1.5 1 1 1 0.5 0.5 0.5 0 0 0 0.5 1 1.5 2 2.5 3 0 20 0 0 20 40 60 80 100 120 140 • Multipoint MLS calculated using ASPEX sib_phase under additive model • Significance assessed by permuting sibling sex 10,000x. 40 60 80 100 120 140 160 IMGSAC: Parent of origin linkage modelling Chromosome 7, all ASP Chromosome 9, all ASP 2.5 2.5 2 2 All (219 ASPs) Paternal linkage Maternal linkage 1.5 1.5 1 1 Lamb et al (2005) 0.5 0.5 0 50 100 150 200 250 0 40 80 120 160 • Possible parent of origin specific effects • Involvement of an imprinted gene(s), & 2 loci underlying linkage to chr 7q ? Sex-specific linkage in chromosome 17q Sex-stratified genome scan in 257 AGRE families All 267 AGRE + 73 Vanderbilt families MO FC Sutcliffe et al. Am J Hum Genet 77:265 (2005) Stone et al. Am J Hum Genet 75:1117 (2005) Cantor et al. Am J Hum Genet 76:1050 (2005) 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) IMGSAC Candidate gene studies 2q24.2-q32.2 GENE FUNCTION RPRM KCNJ3 NR4A2 TBR1 GAD1 DLX1 DLX2 RAPGEF4 CHN1 ATF2 HOXD1 KIAA1604 UBE2E3 NEUROD1 FRZB NCKAP1 GULP INPP-1 NAB1 apoptosis ~40 Mb neuronal cells excitability transcriptional regulation brain development neurotransmitter metabolism brain development brain development neuronal signal transduction neuronal signal transduction transcriptional regulation brain development possible role in brain development and function ~ 190 genes Resequencing or DHPLC screening of coding and regulatory regions in 32 - 48 affected individuals from autism families that are contributing to the linkage peak ubiquitination brain development brain development apoptosis apoptosis phosphoinositides metabolism transcriptional regulation Test common variants for association with autism by case-control and/or TDT studies in the whole IMGSAC family sample. IMGSAC Candidate gene studies Chromosome 7 NCAM RELN LAMB1 LRNN1 PTPRZ1 CUTL1 DLX5 & DLX6 FOXP2 CHRM2 COPG2 SRPK2 SYPL GRM8 CPA1 & CPA5 MEST EN2 Neuronal cell adhesion molecule Reelin Laminin beta-1 chain precursor Leucine-rich repeat protein, neuronal 1 Protein tyrosine phosphatase receptortype Z, polypeptide 1 Cut-like 1 Distal-less homeobox genes 5 & 6 Forkhead box P2 Cholinergic receptor, muscarinic 2 Coatamer protein complex, subunit gamma 2 Serine/Arginine rich protein kinase 2 Synaptophysin-like protein Glutamate receptor, metabotropic 8 Carboxypeptidase isoform 1 & 5 Mesoderm specific transcript homolog Engrailed 2 Chromosome 16 TBX6 UBN1 A2BP1 ABAT CREBBP GRIN2A KIAA1243 BFAR EMP2 SSTR5 T-box 6 Ubinuclein 1 Ataxin 2-binding protein 4-Aminobutyrate aminotransferase CREB binding protein Glutamate receptor, ionotropic KIAA1243 protein Bifunctional apoptosis regulator Epithelial membrane protein 2 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) Autism candidate gene studies Over 150 candidate genes studied in the last 10 years No clear association with autism Position of genes and frequency of publications in candidate gene/association studies over the last decade 14 12 SLC6A4 Number of studies 10 HOXA1 8 GABRB3 RELN FOXP2 6 HOXB1 UBE3A FMR1 EN2 SLC25A12 4 MECP2 2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 21 22 23 Chromosome 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) 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 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) • 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 2.5 3 2 2.5 2 ASPEX MLS 1.5 1 1.5 1 0.5 0.5 0 0 0 0.5 1 1.5 2 2.5 2q24.2-q32.2 ~ 40 Mb 3 0 50 100 150 200 250 Position (cM) 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 Strategy overview Download all HapMap SNP genotypes in chromosome 2 candidate region HapMap release 13 (phase 1) CEU 18,389 HapMap SNPs 1 SNP/3.4kb Define blocks of strong LD 891 LD Blocks Average SNPs/block = 8 Identify htSNPs Average N of htSNPs / Block = 3.3 Align blocks with genes Number of Genes = 183 Genotype intragenic block htSNPs in selected families Number of Haplotype Blocks Required = 419 Number of htSNPs to Genotype = ~1480 G/G A/C A/T G/C T/T A/C T/C Test htSNPs for association to autism Genotyped 1536 SNPs on both chr 2 & chr 7 in 576 samples. 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 Genotype calls for rs2368352 1,60 1,40 No rma li zed R 1,20 AA 1 AB BB 0,80 NA11881 0,60 NA12006 0,40 0,20 0 247 0 171 0,20 0,40 56 0,60 Normalized Theta 0,80 1 Statistical analysis Stratification analysis (STRUCTURE) No evidence for population substructure on chromosome 2 or 7 between autism, control and HapMap CEPH samples. 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. 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 1.0 Experiment-wise posterior probability of association IMMP2L 0.8 Strong evidence for association (90%) NM015328 0.2 0.4 0.6 PTPRZ1/2 0.0 Posterior probability Positive evidence for association (75%) 0 200 400 600 Block 800 Chromosome 7 Probands v/s internal controls (family-based analysis) Experiment-wise posterior probability of association 1.0 FBXL13 LHFPL3 0.6 MUC3A/B WNT16 0.2 0.4 CUTL1 0.0 Posterior probability 0.8 IMMP2L 0 200 400 600 Block 800 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 2 Probands v/s unrelated controls 1.0 Experiment-wise posterior probability of association TAI2HUMAN 0.6 ZNF533 NM018981 0.2 0.4 OSBPL6 0.0 Posterior probability 0.8 NOSTRIN 0 200 400 Block 600 800 Chromosome 2 Probands v/s internal controls (family-based analysis) 0.8 1.0 Experiment-wise posterior probability of association UPP2 ZNF533 0.2 0.4 0.6 NM024770 0.0 Posterior probability NOSTRIN 0 200 400 Block 600 800 Results summary 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. 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) Perspectives 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) Acknowledgements University of Bologna Elena Bacchelli Francesca Blasi Claudio Toma Simona Carone Prof Giovanni Romeo Department of Biology Medical Genetics Laboratory S.Orsola-Malpighi Hospital University of Oxford Wellcome Trust Centre for Human Genetics Janine Lamb Gabrielle Barnby Nuala Sykes Andrew Morris Prof Anthony Monaco Department of Psychiatry Prof Anthony Bailey International Molecular Genetic Study of Autism Consortium Funding • The Wellcome Trust • The Nancy Lurie Marks Family Foundation • UK Medical Research Council • NAAR • Telethon Italy • European Commission