Large Scale Variation Among Human and Great Ape Genomes Determined by Array Comparative Genomic Hybridization Devin P. Locke, Richard Segraves, Lucia Carbone, Nicoletta Archidiacono, Donna G. Albertson, Daniel Pinkel, and Evan E. Eichler (2003) Genome Research. Presented by Nicholas Dubé

B.Sc. University of Saskatchewan, MB PhD Baylor College, Houston, TX Currently, Assistant Prof. & Director of the Bioinformatics Core Facility at Case Western Reserve School of Medicine, Ohio Research Interest: T o understand the evolution, pathology and mechanisms of gene duplication and DNA transposition within the human genome The Authors Dr. Evan Eichler

The Authors Dr. Donna Albertson Associate Professor, Cancer Research Institute at UCSF Dr. Dan Pinkel Professor, Lab Medicine at UCSF Dr. N. Archidiacono Staff Member, Dept. of anatomy and genetics, University of Bari, Italy

The Authors Lucia Carbone Student, Dept. of anatomy and genetics, Bari, Italy Richard Segraves Student, Pinkel lab, UCSF Devin Locke Student, Eichler lab, Case Western

Previous Findings Evolutionary change of human & nonhuman primates studied at 2 levels: 1. Karyotypically (i.e FISH) 2. At the single base-pair level (i.e. SNPs) Array CGH used to detect within-species DNA copy- number variation associated with tumor progression

Previous Findings Previous studies implicated gene- deficient heterochomatic regions as the location of greatest variation between genomes of the great apes & humans But this would all change…

Aim of the Paper 1. To further the understanding of our genetic history and evolution 2. To identify sites of large-scale rearrangement, gain or loss of DNA sequence, among humans and great apes 3.To pioneer the use of array CGH for interspecies genomic comparison

Major Findings of The Paper Identified 63 sites of DNA copy-number variation between Humans and apes Confirmed rearrangements ranging from kb Localized the majority of variant sites to interstitial euchromatin Presented the first genome-wide comparison of the great apes

Array CGH To measure DNA sequence copy number gains and losses Allows for mapping of gains & losses Fig.1 Array CGH. To determine DNA copy number. Lung tumor (Cy3)/Reference (Cy5)

Array CGH: How it works Genomic DNA isolated from blood Label DNAs with fluorochromes Hybridize human & primate DNA probes to Arrays that contain 2460 different Human BACs Primate-human intensity ratio at each locus assessed for variation

Methods: Array CGH Compared relative hybridization intensities among 4 great apes (chimp, bonobo, gorilla, and orangutan) pairwise with humans Array loci designated as “variant” IFF a consistent increase/decrease in intensity ratio was observed in each species in all trials

Results 63 variant sites detected (38 reductions & 25 increases) Quantity of variant sites in each species consistent with amount of evolutionary time diverged

Results Fig. 1. Graphs depicting 2460 loci. Variant sites circled in red. PPA=bonobo PTR=chimpanzee PPY=orangutan GGO=Gorilla

Results: Validation Examined a subset of 63 variant sites (7 putative gains and 7 potential losses) using FISH 7 gains: Observed both intra/inter- chromosomal duplication (gains) 7 losses: Found 1 instance of complete loss of signal (loss of ~175kb or 1 BAC) Partial signal detected for other 6 sites- FISH not suitable

Results: Validation Developed BAC end Sequence-based approach to tackle deletion validation problem Compared insert size of primate BAC to estimated human BAC size Discrepancy in sizes indicates a partial deletion

Verified Variant Sites

Rearrangements occur at euchromatic regions 86% of variant sites mapped to euchromatic regions Suggests large-scale rearrangements not confined to pericentromeric or subtelomeric regions Question: Does rearrangement occur exclusively in heterochromatic regions as previously reported?

Chromosomal Bias Appears to be a chromosomal bias for the number of variant sites (Table 2) Correlation between enrichment for variant sites and segment duplications Found 5/9 experimentally validated rearrangements were proximate to a segmental duplication

Sites of ape/human variation

Summary Results illustrate the power & feasibility of CGH for comparing large-scale differences between species Large-scale rearrangements do occur in gene-rich regions as well as gene-poor regions Large-scale changes are not randomly distributed across the genome

Discussion Questions What other applications might Array CGH have? Can the authors conclude there is a chromosomal bias for variant site location given that they examined only 12% of the genome? Is this paper significant for the greater research community?