Karola Rehnström Karola Karola Rehnström Karola Rehnström Genetic Heterogeneity in RESEARCH RESEARCH Genetic Heterogeneity in Autism Spectrum Autism Spectrum Disorders Disorders in a Population Isolate in a Population Isolate in a Population Isolate Population a in Disorders Spectrum Autism in Heterogeneity Genetic Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization, despite active research for decades. In this study, we have utilized modern molecular genetics methods combined with the special characteristics of the isolated Finnish population to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within the Finnish population. The lack of founder mutations in a subset of distantly related families from an internal subisolate suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. The results of this study are consistent with other recent studies of genetic risk factors for ASDs. The underlying genetic architecture for ASDs has emerged to be highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and family specific linkage, homozygosity mapping and resequencing efforts are needed to identify this type of genetic risk factors. .!7BC5<2"HIEGFG! National Institute for Health and Welfare P.O. Box 30 (Mannerheimintie 166) FI-00271 Helsinki, Finland 21 ISBN 978-952-245-132-3 Telephone +358 20 610 6000 21 2009 21 www.thl.fi Karola Rehnström GENETIC HETEROGENEITY IN AUTISM SPECTRUM DISORDERS IN A POPULATION ISOLATE ACADEMIC DISSERTATION To be presented with the permission of the Medical Faculty, University of Helsinki, for public examination in The Small Lecture Hall, Haartman Institute, on October 30th 2009, at 12 noon. National Institute for Health and Welfare and Institute for Molecular Medicine Finland and Department of Medical Genetics, University of Helsinki Helsinki 2009 Helsinki University Biomedical Dissertations No. 126 ISSN 1457-8433 © National Institute for Health and Welfare ISBN 978-952-245-132-3 (print) ISSN 1798-0054 (print) ISBN 978-952-245-133-0 (pdf) ISSN 1798-0062 (pdf) Cover art: Anne Pernaa, ’Lost Chromosomes’. Pastel. www.annepernaa.fi Helsinki University Print Helsinki, Finland 2009 Supervised by Professor Leena Peltonen-Palotie Institute for Molecular Medicine Finland FIMM Helsinki, Finland Wellcome Trust Sanger Institute Hinxton, UK and Tero Ylisaukko-oja, Ph. D. National Public Health Institute Department of Molecular Medicine Helsinki, Finland Reviewed by Professor Jim Schröder University of Helsinki Department of Biological and Environmental Sciences Helsinki, Finland and Adjunct Professor Tarja Laitinen Helsinki University Central Hospital Clinical Research Unit of Pulmonary Diseases Helsinki, Finland O p p o n e n t Professor Kerstin Lindblad-Toh Vertebrate Genome Biology The Broad Institute Cambridge MA, USA Department of Medical Biochemistry and Microbiology Uppsala University Uppsala, Sweden “What does it matter to Science if her passionate servants are rich or poor, happy or unhappy, healthy or ill? She knows that they have been created to seek and to discover, and that they will seek and find until their strength dries up at its source. It is not in a scientist‟s power to struggle against his vocation: even on his days of disgust or rebellion his steps lead him inevitably back to his laboratory apparatus.” -From Madame Curie – A biography by Eve Curie Karola Rehnström, Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate. National Institute for Health and Welfare, Research 21|2009. 192 pages. Helsinki, Finland 2009. ISBN 978-952-245-132-3 (print); 978-952-245-133-0 (pdf) ABSTRACT Positional cloning has made it possible to perform hypothesis-free, genome-wide scans for genetic factors affecting a disorder or trait. Traditionally linkage analysis using microsatellite markers has been used as a first step in this process to identify regions of interest, followed by meticulous fine mapping and candidate gene screening using association methods and subsequent sequence analysis. More recently, genome-wide association analysis has enabled a more direct approach to identify specific genetic variants explaining a part of the variance of the phenotype of interest. In addition, data produced for genome-wide association analysis has also made it possible to assay small, submicroscopic variation on the chromosomes, referred to as copy number variants, which have been shown to confer susceptibility to some complex disorders. Isolates have proven useful in the identification of genes causing Mendelian, or monogenic, disorders. The Finnish population is genetically homogenous, and has been molded by founder effect, multiple consecutive bottlenecks and genetic drift. These features can be utilized in identification of genetic risk factors for complex disorders, although population isolates have not been shown to be as useful for the genetic mapping of complex traits as for Mendelian disorders. The genetic risk factors for complex disorders in Finland could, however, prove to be less heterogeneous due to the limited range of susceptibility alleles carried into the gene pool by the original settlers. Autism spectrum disorders (ASDs) are a group of childhood onset neuropsychiatric disorders with shared core symptoms but varying severity. Although a strong genetic component has been established in ASDs, genetic susceptibility factors have largely eluded characterization, despite active research for decades. In this study, we have utilized modern molecular genetics methods combined with the special characteristics of the Finnish population to identify genetic risk factors for ASDs. The results of this study show that numerous genetic risk factors exist for ASDs even within a population isolate. Stratification based on clinical phenotype resulted in encouraging results, as linkage to 3p14-p24 identified in the Finnish genome-wide linkage scan for Asperger Syndrome (AS) was replicated in an independent family set. The success of linkage mapping of susceptibility regions for AS has interesting THL Research 21|2009 Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate implications for the underlying genetic architecture, suggesting that genetic risk factors for AS could possibly be less heterogeneous than for the wider spectrum of ASDs. Fine-mapping of the previously identified linkage peak for ASDs at 3q25-q27 revealed association between ASDs and a subunit of the 5-hydroxytryptamine receptor 3C (HTR3C). The 5-hydroxytryptamine pathway has previously been robustly implicated in the etiology of ASDs but this is the first time the 5- hydroxytryptamine receptors on 3q have been evaluated as risk factors for ASDs. However, association to HTR3C only accounted for a part of the observed linkage signal, suggesting that other predisposing factors exist at this locus. As a part of this study, we used dense, genome-wide single nucleotide polymorphism (SNP) data to characterize the population structure. We observed significant population substructure caused by the multiple consecutive bottle-necks experienced by the Finnish population during the population history. We used this information to ascertain a genetically homogenous subset of autism families from Central Finland to identify possible rare, enriched risk variants from dense, genome- wide SNP data. However, no rare enriched genetic risk factors were identified in this dataset, although a subset of families could be genealogically linked to form two extended pedigrees which would suggest shared susceptibility factors. The lack of founder mutations in this isolated population suggests that the majority of genetic risk factors are rare, de novo mutations unique to individual nuclear families. We also attempted to use gene ontology (GO)-categories to characterize the biological pathways involved in ASDs, but found significant heterogeneity in identified GO- categories among different genome wide SNP and gene expression datasets. The results of this study are consistent with other recent studies of genetic risk factors for ASDs. The underlying genetic architecture for this group of complex disorders seems to be highly heterogeneous, with common variants accounting for only a subset of genetic risk. The majority of identified risk factors have turned out to be exceedingly rare, and only explain a subset of the genetic risk in the general population in spite of their high penetrance within individual families. The results of this study, together with other results obtained in this field, indicate that family specific linkage, homozygosity mapping and resequencing efforts are needed to identify these rare genetic risk factors. Keywords: Autism spectrum disorder, Asperger syndrome, linkage analysis, expression analysis, genome-wide association analysis, isolated population, population genetics, serotonin receptor THL Research 21|2009 Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate Karola Rehnström, Genetic Heterogeneity in Autism Spectrum Disorders in a Population Isolate. Terveyden ja hyvinvoinnin laitos, Tutkimus 21|2009. 192 sivua. Helsinki 2009.