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Genomic and Functional Profiling of Duplicated Chromosome Human Molecular Genetics, 2006, Vol. 15, No. 6 853–869 doi:10.1093/hmg/ddl004 Advance Access published on January 30, 2006 Genomic and functional profiling of duplicated chromosome 15 cell lines reveal regulatory alterations in UBE3A-associated ubiquitin–proteasome pathway processes Colin A. Baron1, Clifford G. Tepper2, Stephenie Y. Liu1, Ryan R. Davis1, Nicholas J. Wang3, N. Carolyn Schanen4 and Jeffrey P. Gregg1,* 1Department of Pathology and 2Department of Biochemistry and Molecular Medicine, University of California, Davis School of Medicine, Sacramento, CA 95817, USA, 3Department of Human Genetics, University of 4 California–Los Angeles, Los Angeles, CA, USA and Center for Pediatric Research, Nemours Biomedical Research, Downloaded from Alfred I. duPont Hospital for Children, Wilmington, DE, USA Received November 21, 2005; Revised and Accepted January 25, 2006 Autism is a complex neurodevelopmental disorder having both genetic and epigenetic etiological elements. hmg.oxfordjournals.org Isodicentric chromosome 15 (Idic15), characterized by duplications of the multi-disorder critical region of 15q11–q14, is a relatively common cytogenetic event. When the duplication involves maternally derived con- tent, this abnormality is strongly correlated with autism disorder. However, the mechanistic links between Idic15 and autism are ill-defined. To gain insight into the potential role of these duplications, we performed a comprehensive, genomics-based characterization of an in vitro model system consisting of lymphoblast cell lines derived from individuals with both autism and Idic15. Array-based comparative genomic hybridiz- ation using commercial single nucleotide polymorphism arrays was conducted and found to be capable of by guest on December 17, 2010 sub-classifying Idic15 samples by virtue of the lengths of the duplicated chromosomal region. In further analysis, whole-genome expression profiling revealed that 112 transcripts were significantly dysregulated in samples harboring duplications. Paramount among changing genes was ubiquitin protein ligase E3A (UBE3A; 15q11–q13), which was found to be nearly 1.5–2.0-fold up-regulated in duplicated samples at both the RNA and protein levels. Other key findings from gene expression analysis included two down- regulated genes, APP and SUMO1, with well-characterized roles in the process of apoptosis. We further demonstrate in this lymphoblast model that the gene-dosage directed increases in UBE3A levels can lead to dysregulation of the process of ubiquitination in response to genotoxic insult. This study provides insight into the direct and indirect effects of copy number gains in chromosome 15 and provides a framework for the study of these effects in neuronal systems. INTRODUCTION factors for autism has been well established in family and twin studies. Additionally, there is an emerging evidence Childhood autism (MIM 209850) is characterized as a that the presence of chromosomal abnormalities may play a pervasive neurodevelopmental disorder with onset before significant role in the etiology of autism. This is based on 30–36 months of age, resulting in impairment in social inter- the high frequency of large chromosomal anomalies identified action and communication and manifestation of repetitive in patients with autism spectrum disorders (ASD), which stereotypic behavior. The estimated prevalence of this disorder range from 1.7 to 4.8% depending on the population screened is one to two per 1000 births (1). The involvement of genetic and the method of detection applied (2,3). These have *To whom correspondence should be addressed at: MIND Institute Wet Lab, 2805 50th St., No. 2420, Sacramento, CA 95817, USA. Tel: +1 9167030362; Fax: +1 9167030367; Email: [email protected] # The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 854 Human Molecular Genetics, 2006, Vol. 15, No. 6 included unbalanced translocations, inversions, rings and isodicentric chromosomes. Analysis of differentially expressed interstitial deletions and duplications. Although most of these genes demonstrated an overabundance of genes in pathways rearrangements are unique to a single patient or family, a associated with ubiquitination and apoptosis, suggesting that number of recurrent chromosome abnormalities have been these pathways may be altered in Idic15 subjects. Of reported in unrelated patients, including duplication of 15q, prominence was UBE3A, located within the 15q11–q13 deletions of 18q, Xp, 2q and sex chromosome aneuploidies: contig area, which was found to be overexpressed at both the 47,XYY and 45,X (4,5). transcriptional and protein levels. Furthermore, functional In these studies, chromosomal abnormalities have been data presented here suggest that this overexpression of identified by using traditional cytogenetics and fluorescence UBE3A is associated with dysregulated ubiquitination triggered in situ hybridization (FISH), which allow detection of whole- by genotoxic stress and altered apoptosis in patients with chromosome losses or gains or large chromosomal deletions Idic15. Therefore, we demonstrate a systems biology approach and duplications. Over the last several years, however, to analyze copy number changes, demonstrating a link between smaller chromosomal aberrations in the range of 5 kb to DNA copy, transcriptional activity and protein production, as 10 Mb, which have traditionally been undetectable with well as identifying functional repercussions. classic cytogenetics, have been detected in a number of dis- eases. New genomic technologies have been developed, which can scan the genome at a very high resolution (6) in RESULTS Downloaded from order to analyze DNA copy number. Large insert clones that Affymetrix mapping arrays identify Idic15 amplifications contain mapped genomic segments have been utilized for in samples previously characterized by CGH copy number alteration (CNA) detection either by directly spotting onto glass slides or by amplifying these regions and To identify chromosomal amplifications using commercially then spotting them (6). Similarly, oligonucleotides represent- available SNP arrays and a nascent array-based CGH ing specific spacing and regions in the genome have been approach, we profiled 10 samples with known chromosome developed and utilized for copy number analysis (7). Recently, 15 duplications and nine control cell lines with Affymetrix hmg.oxfordjournals.org single nucleotide polymorphism (SNP) arrays, initially uti- arrays. The samples with duplications have been previously lized for loss of heterozygosity in cancer (8–10), have been found to have variable duplications of chromosome 15q11– applied for use in copy number analysis (11). q13, a region spanning 10 Mb. The Affymetrix mapping In this study, we applied the SNP array-based comparative 10K array includes 32 SNPs in this region separated by a genomic hybridization (CGH) approach to a known dupli- median distance of 191 kb. cation of the chromosome 15q11–q13 region that is associated To evaluate whether the SNP arrays would be able to with autism. Duplications of chromosome 15q11–q13 account differentiate between control and duplicated samples, we by guest on December 17, 2010 for 1–3% of autism and are the second most prevalent hybridized genomic DNA from nine samples from typically genomic aberration associated with autism next to fragile X developing children to the arrays and analyzed using the syndrome. These duplications range from 8 to 12 Mb and, Chromosomal Copy Number Tool (CCNT) for regions of sig- when associated with autism, are derived from the maternal nificant consecutive copy change, which we defined to be chromosome (12,13). These duplications can occur either above a 2log10(meta P-value) of 5. None of the samples through generation of a supernumerary isodicentric chromo- from the typically developing children showed meta P-value some 15 (idic15) or as an interstitial duplication. With proof scores above 5 for the target region (Fig. 1A). Conversely, of principle that the SNP array approach is robust in accurately when the same region was examined in the samples with identifying and determining the breakpoints of this region, this known duplications, chromosomal copy number increase for technology could be extended to analyze a large cohort of the 15q11–q13 region was identified in all samples. The children with autism. meta P-values for these samples were found to be at the theor- 220 The importance of this genomic approach in autism is that etical maximum of 20 (P-value ¼ 1 Â 10 ) for the entire there is a significant co-morbidity with mental retardation length of the duplication, a region containing 22–31 SNPs (MR). Recently, Lugtenberg et al. (14) applied array-based (Fig. 1B). Additionally, the data from the CCNT discriminated CGH to a cohort of mentally retarded patients who were con- the characteristic breakpoints of each sample, making it poss- sidered to be cytogenetically normal based on classic cyto- ible to classify them by the length of their duplications genetics and FISH. These studies demonstrated that 10% of (Fig. 2). In summary, these data support the utilization of these MR cases carried small chromosomal abnormalities the SNP arrays and CCNT as a valid approach for the identi- ranging from 100 kb to 12 Mb. This suggests that in autism fication of copy number changes in chromosomal regions. there may be a large number of undetected chromosomal aberrations. Beyond identifying these aberrant
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