Molecular Psychiatry (2010) 15, 996–1005 & 2010 Macmillan Publishers Limited All rights reserved 1359-4184/10 www.nature.com/mp ORIGINAL ARTICLE High-density SNP association study of the 17q21 chromosomal region linked to autism identifies CACNA1G as a novel candidate gene SP Strom1, JL Stone1, JR ten Bosch1, B Merriman1, RM Cantor1,2,3, DH Geschwind1,2,3,4 and SF Nelson1,2,3 1Department of Human Genetics, University of California, Los Angeles, CA, USA; 2Center for Neurobehavioral Genetics, University of California, Los Angeles, CA, USA; 3Department of Psychiatry and Biobehavioral Sciences, University of California, Los Angeles, CA, USA and 4Department of Neurology, University of California, Los Angeles, CA, USA Chromosome 17q11-q21 is a region of the genome likely to harbor susceptibility to autism (MIM(209850)) based on earlier evidence of linkage to the disorder. This linkage is specific to multiplex pedigrees containing only male probands (MO) within the Autism Genetic Resource Exchange (AGRE). Earlier, Stone et al.1 completed a high-density single nucleotide polymorphism association study of 13.7 Mb within this interval, but common variant association was not sufficient to account for the linkage signal. Here, we extend this single nucleotide polymorphism-based association study to complete the coverage of the two-LOD support interval around the chromosome 17q linkage peak by testing the majority of common alleles in 284 MO trios. Markers within an interval containing the gene, CACNA1G, were found to be associated with Autism Spectrum Disorder at a locally significant level (P = 1.9 Â 10À5). While establishing CACNA1G as a novel candidate gene for autism, these alleles do not contribute a sufficient genetic effect to explain the observed linkage, indicating that there is substantial genetic heterogeneity despite the clear linkage signal. The region thus likely harbors a combination of multiple common and rare alleles contributing to the genetic risk. These data, along with earlier studies of chromosomes 5 and 7q3, suggest few if any major common risk alleles account for Autism Spectrum Disorder risk under major linkage peaks in the AGRE sample. This provides important evidence for strategies to identify Autism Spectrum Disorder genes, suggesting that they should focus on identifying rare variants and common variants of small effect. Molecular Psychiatry (2010) 15, 996–1005; doi:10.1038/mp.2009.41; published online 19 May 2009 Keywords: autism; Autism Spectrum Disorder; association; chromosome 17q; CACNA1G Introduction not otherwise specified under the umbrella of Autism Spectrum Disorders (ASD). Autism (MIM(209850)) is a pervasive developmental Twin and family studies have provided strong disorder defined by impairment along three dimen- evidence of heritability and suggest a high likelihood sions: language development, development of social of genetic contribution for the susceptibility to behaviors and the presence of stereotypic or rigid autism. The monozygotic twin concordance rate is behavior. The diagnosis of ‘autistic disorder’ encom- reported to be as high as 90% for ASD, whereas passes a broad range of phenotypically diverse sibling concordance rates are B10%.2,3 This indicates conditions with wide variation along the three a strongly heritable yet genetically complex disorder.4 dimensions of impairment, making autism a particu- The sibling relative risk is B15–20-fold higher than larly heterogeneous disorder. ‘Autistic disorder’ is the population frequency. The inheritance pattern of commonly grouped with Asperger’s syndrome ASD is not consistent with a Mendelian disease (MIM(608638)) and pervasive developmental disorder model and can likely be better explained by the involvement of multiple interacting loci and environ- Correspondence: Dr SF Nelson, Department of Human Genetics, mental factors.5 However, the degree of genetic University of California, 695 Charles E. Young Drive South, Gonda complexity has not yet been established. 5554, Los Angeles, CA, 90095-7088, USA. The high prevalence6,7 and strong heritability of E-mail: [email protected] Received 22 September 2008; revised 31 March 2009; accepted 8 ASD has encouraged multiple groups to complete April 2009; published online 19 May 2009 whole-genome linkage studies8 searching for genomic CACNA1G as a novel candidate gene for Autism Spectrum Disorder SP Strom et al 997 regions likely harboring autism susceptibility alleles. materials available through AGRE at the time of assay One of the few genomic regions identified in initial design were included in this study. linkage studies and replicated at a genome-wide Ethnicity was recorded through self-report, with significance is 17q11-q21.9,10 The linkage signal was 79% of those reporting identified as Caucasian. These strengthened based on the stratification of linkage reports are consistent with population structure data conditioned on the sex of affected siblings, analyses of the genotype data using the structure which resulted in genome-wide significant linkage software package16 (data not shown). Owing to the centered at 25–28 Mb in multiplex families with family-based design and the outbred nature of the exclusively male probands (male-only, or MO fa- population sampled (the United States), ethnicity is milies) within the Autism Genetic Resource Exchange not used as a factor in this study. This distribution (AGRE).11 A replication study in a set of 109 will lead to an overrepresentation of the Caucasian additional MO families from AGRE showed an alleles overall, which will in turn increase the evidence of sex specific linkage extending over the probability that discovered associated alleles will be same region, and fine mapping identified a region of specific to or enriched in the United States’ Caucasian linkage extending an additional 18 Mb from the end of population. The follow-up sample has a similar the initial linkage peak9 centered at 61 cM. ethnicity distribution. Earlier, Stone et al.1 tested approximately half of the All individuals were diagnosed using the Autism linkage interval on 17q11-q21 for association to Diagnosis Interview Revised (ADI-R).17 A subset of common variants through high-density single nucleo- individuals were also diagnosed using the Autism tide polymorphism (SNP) genotyping. Although the Diagnostic Observation Schedule (ADOS).18 In total, results showed suggestive evidence of the association 12 out of 296 individuals diagnosed as either autistic of ASD to several interesting and novel candidates, or broad spectrum on the ADI-R scored as ‘Not the association signals were not sufficiently strong to Spectrum or Autism’ on the ADOS (Table 1). These account for the observed linkage signal within the individuals were excluded from study because of AGRE MO families. Thus, we sought to cover the their ambiguous phenotype. The 284 individuals remaining likely linkage interval by testing common surviving genotype cleaning and diagnostic criteria DNA variants comprehensively within the remaining were included for all subsequent analysis. 17q linkage region defined by fine mapping,9 testing Genomic DNA samples were obtained from the 1975 SNPs at an average marker density of 6.3 kb. NIMH cell repository (Rutgers, Piscataway, NJ, USA). This provided strong coverage of the majority of Concentrations were determined using the Nanodrop common haplotypes over the extended region of (Wilmington, DE, USA) instrument. The UCLA Inter- linkage, testing for ASD association within 295 genes nal Review Board has approved all aspects of this in 284 independent trios from MO families in AGRE. study. A complete list of samples used in this study, We report the overall association analyses, which along with gender, demographic and diagnostic data highlight CACNA1G as a novel candidate gene. is found in Supplementary Table 1. SNP selection Materials and methods Single nucleotide polymorphisms were selected Genetic material and preparation using several criteria, including the linkage disequili- The AGRE, an organization facilitating the collection brium (LD) data from the Hapmap project and ability of biomaterials and phenotypic information of fa- to develop a working genotyping assay. To perform milies with autistic individuals, provided DNA the SNP selection, we first requested all possible samples for this study. AGRE has a standardized set dbSNP genotypes that will perform well in the of criteria for inclusion, which have been published Golden Gate assay as per the manufacturer’s guide- earlier12 and are available at www.agre.org. AGRE lines (Illumina Inc., San Diego, CA, USA). To focuses on collecting genetic material from families maximize the amount of common variation tested with more than one individual diagnosed with ASD while minimizing the number of markers typed, the (as defined by Liu et al.13). software package Tagger19 was used to select a subset For this study, both parents and one affected son of these markers for genotyping such that the subset were typed in 302 MO trios. After data cleaning, 296 would cover the region of interest in the Hapmap remained (Supplementary Materials 1). Families (Build #21 July 2006) European Ancestry population containing individuals flagged for non-idiopathic with a minimum r2 value of 0.9. In total, 2042 SNPs autism because of other medical conditions, such as were selected for genotyping in the region. Markers Fragile-X syndrome, birth trauma and dysmorphic span 34.3–47 Mb on chromosome 17 (17q12-q21.33). features, were not included in this study.