Role of Chemical lexposure in Generating Spontaneous Copy Number Variants (CNVs) Jennifer Freeman Assistant Professor of Toxicology School of Health Sciences Purdue University
CNV Discovery Reference Genetic variation A B C Single nucleotide polymorphisms (SNPs) Deletion Variable tandem repeats (e.g., mini- and A C micro-satellites) Duplication Presence/absence of transposable elements A B C C 2004: Discovery of wide-spread copy number variation Gain or loss of intermediate-sized DNA sequence (50 bp to a few Mb) Refined human CNV maps 2007 www.sciencemag.org
CNVs and Disease Classical cytogenetics: copy number alterations ti and disease Neutral function if not result in earlyonset genomic disorders = No Spontaneous CNVs and complex disease association Autism Schizophrenia Others 22q11 deletion Di George Syndrome k www.sanger.ac.uk
CNVs in Other Species Characterized in a variety of vertebrate species Non-human primates, cattle,,pg, pigs, dogs, g, rodents, zebrafish
Does Chemical Exposure Induce Spontaneous CNVs? Why important? What we know CNVs play a role in Complex diseases are complex diseases influenced by environmental factors What we don t know Are environmental stressors generating spontaneous CNVs? Which environmental stressors?
Does Chemical Exposure Induce Spontaneous CNVs? Why not thoroughly investigated before?
Does Chemical Exposure Induce Spontaneous CNVs? How can we assess? Assays: efficient i genome-wide analysis Array Comparative Genomic Hybridization (CGH) Whole Genome Sequencing
Principles of Array CGH Similar concept as gene expression microarrays Test Ref Cy3 Cy5 DNA fragments mapped to a specific genomic locations on a glass slide Detects gain or loss of DNA sequence through competitive hybridization Deviation from the 1:1 expected signal fluorescence ratio (or log 2 value of 0) indicates difference in copy number of DNA sequence Resolution is dependent on size and density of genomic elements Green/Red Ratio Copy number loss Copy number gain
Principles of Whole Genome Sequencing Genomic DNA Isolation High-throughput ihh h genome sequencing Template Preparation Specific methods based on sequencing approach Sequencing Genome Alignment and Assembly Comparative Genome Analysis
Does Chemical Exposure Induce Spontaneous CNVs? How can we assess? Assays: efficient i genome-wide analysis Array Comparative Genomic Hybridization (CGH) Whole Genome Sequencing Zebrafish model system In vitro and in vivo Spontaneous CNV identification and functional impacts
Zebrafish Model System Small tropical fish native to eastern India 1930s: Classical developmental biology research 1970s: Inexpensive vertebrate model system for forward genetic analysis of complex biological processes 1990s through today: expansion to all areas of biological research Haffter et al. 1996. Development
Strengths of Zebrafish Model System Hundreds of eggs weekly Ex-utero embryonic development Singular, transparent t embryos Short developmental periods Embryonic 3-4 days Sexually mature ~3 months Short life span (~1 year) Small-sized adult organism Amenable to genetic manipulations
Finished Genome Sequence Zebrafish Sequencing Project: http://www.sanger.ac.uk/projects/ ac D_rerio/ 15 1.5 x 10 9 bp of DNA (~1/2 size of human genome) 25 pairs of chromosomes Cytogenetic ti characterization ti and mapping of genome Freeman et al. 2007. BMC Genomics
Genetic Conservation among Vertebrates Gene function is conserved among vertebrates ~80% of zebrafish genes are homologous to human genes Mutations in zebrafish genes display phenotypes similar to human diseases
CNVs in Zebrafish Effective resolution of ~4 kb CNVEs Brown et al. 2012 Proc. Natl. Acad. Sci. USA
CNVs in Zebrafish Plasticity of zebrafish genome permits CNV formation Suitable model for assessing role of chemical exposure in generation of spontaneous CNVs Copy Number Loss Copy Number Gain Line Length = Frequency Brown et al. 2012 Proc. Natl. Acad. Sci. USA
Proof of Principle Experiment Does Chemical Exposure Induce Spontaneous CNVs? Using known genotoxic chemical Zebrafish cell line (Freeman et al. 2007. BMC Genomics) Multiple chemical concentrations
Control Chemical Treatment DNA isolation Cy3 Fluorescently Label Combine and Hybridize Cy5 Array CGH Analysis Cy3/C Cy5 Log 2 Ratio Loss Gain Chromosome Position Peterson and Freeman. 2009. Zebrafish.
Detecting Multiple Spontaneous CNVs at All Exposure Concentrations ti
Spontaneous CNVs in Similar Genomic Regions at Multiple l Concentrations ti
Comparative Gene Expression Analysis Control Chemical Treatment RNA isolated and converted into cdna cdna labeled with Cy3 Labeled cdna hybridized onto microarray Gene expression increased relative to control t Treatment Control Multiple replicates are compared to determine relative level of gene expression Gene expression decreased relative to control Peterson and Freeman. 2009
Comparative Gene Expression Analysis Spontaneous CNVs correlate to altered gene expression Example
Does Chemical Exposure Induce Spontaneous CNVs? In vivo model Developmental exposure Spontaneous CNV identification Biological and functional significance
Summary: Does Chemical Exposure Induce Spontaneous CNVs? Proof of principle p experiment confirms chemical exposure can result in spontaneous CNVs and impact gene expression Need? Assess environmental chemical contaminants Characterization i of genomic regions and functional significance of alterations Factors influencing spontaneous CNVs? Environmental chemical exposure? Why important? New mechanisms influencing disease onset and pathogenesis Disease prevention and treatment Biological and functional impact?