ORIGINAL RESEARCH ARTICLE Analysis of reelin as a candidate gene for autism E Bonora 1 , KS Beyer 2 , JA Lamb 1 , JR Parr 4 , SM Klauck 2 , A Benner 3 , M Paolucci 1 , A Abbott 1 , I Ragoussis 1 , A Poustka 2 , AJ Bailey* , 4 , AP Monaco* , 1 and the International Molecular Genetic Study of Autism Consortium (IMGSAC) 5 1 The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK; 2 Department of Molecular Genome Analysis, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 3 Department of Biostatistics, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 4 Centre for Social, Genetic and Developmental Psychiatry and Department of Child and Adolescent Psychiatry, Institute of Psychiatry, London, UK Keywords: autism; candidate genes; brain development; reelin; mutation screening Genetic studies indicate that chromosome 7q is likely to contain an autism susceptibility locus (AUTS1). We have followed a positional candidate gene approach to identify relevant gene(s) and report here the analysis of reelin (RELN), a gene located under our peak of linkage. Screening RELN for DNA changes identified novel missense variants absent in a large control group; however, the low frequency of these mutations does not explain the relatively strong linkage results on 7q. Furthermore, analysis of a previously reported triplet repeat polymorphism and intragenic single nucleotide polymorphisms, using the transmission disequilibrium test, provided no evidence for association with autism in IMGSAC and German singleton families. The analysis of RELN suggests that it probably does not play a major role in autism aetiology, although further analysis of several missense mutations is warranted in additional affected individuals. Molecular Psychiatry (2003) 8, 885–892. doi:10.1038/ sj.mp.4001310 Introduction Autism (MIM209850) is a neurodevelopmental dis- order characterised by impairments in communica- tion and social interactions, with restricted and stereotyped patterns of behaviours and interests. The population prevalence of autism is 12–17 in 10 000, 1 with a male to female ratio of 4 : 1 2 and onset in the first 3 years of life. Twin and family studies point to a strong role for genetic factors in autism, 3–5 with a complex mode of inheritance. Statistical models suggest that between 2 and 10 loci are probably implicated 6 (for a review see Lamb et al 7 ). The first genome screen reported a multipoint max- imum LOD score (MLS) of 2.53 on the long arm of human chromosome 7 (AUTS1). 8 Further analysis of the AUTS1 locus has been presented by IMGSAC, 9 with a multipoint MLS of 3.37 on chromosome 7q22. Several independent genome screens support the presence of an autism locus on chromosome 7q; also cytogenetic studies strengthen the role of chromo- some 7q in autism (reviewed in Lamb et al 7 ). The human reelin gene maps to chromosome 7q22, close to the peak of linkage reported by IMGSAC. Reelin protein plays an important role in neuronal migration during brain development. 10 Reeler mice, natural null mutants, carry large deletions of the reelin gene and show pronounced neurological symptoms (eg dysto- nia, ataxia and tremors) and neuroanatomical abnormalities in the positioning of neurons through- out the cerebral cortex, cerebellum and hippocam- pus. 11 The male heterozygous mouse (rln/) does not show the same phenotypic abnormalities, but a postnatal decrease in the number of Purkinje cells. 12 Mutations in the human gene have been identified in two cases of autosomal recessive lissencephaly. 13 Several lines of evidence suggest a role for reelin in autism: the gene maps to one of the regions of interest and there is significant overlap between the areas of developmental alterations in reeler mice and regions where abnormalities in autistic brains have been found, for example, the cerebral cortex, brainstem and cerebellum. 14,15 Persico et al 16 reported prefer- ential transmission of long alleles of a triplet repeat at the 5 0 UTR in autism. However, further independent studies reported conflicting findings (reviewed in Fatemi 17 ). We report here the molecular screening of the gene and an association study of the triplet repeat and intragenic polymorphisms to identify possible variants leading to autism susceptibility. Results In total, 55 independent autistic patients (49 males, six females) were selected from those IMGSAC multiplex families showing increased marker allele sharing identical by descent (IBD) at the peak of linkage on chromosome 7q22, 9 to enrich for indivi- duals likely to carry aetiological variants. The promoter region was defined using the program PromoterInspector. The exon–intron organisation was obtained by BLAST search using the human cDNA sequence (GenBank Accession no. U79716); alignment of the mRNA with the genomic sequence demonstrates that the gene covers a genomic region of at least 340 kb. Ten missense variants were identified in the 55 autistic individuals. Exons harbouring Molecular Psychiatry (2003) 8, 885–892 & 2003 Nature Publishing Group All rights reserved 1359-4184/03 $25.00 www.nature.com/mp