BACKGROUND

DNA Copy Number

• It was previously thought that genes are present in two copies in the genome

• Large segments of DNA can vary in copy number

– Thousands to millions of base pairs

• Lead to dosage imbalances

Copy Number and Diseases

• Predisposition to diseases

• Biomarkers

– Molecular alterations specific to cancer

• Gene amplifications

• Gene deletions

• Genomic rearrangement leading to gene fusion

• Target of drugs and therapies

CNVs and CNAs

Copy Number Variation

• Germline cells

• Genetic marker

• Associations in diseases – Autism,

Glomerulonephritis, CNS disorders

Copy Number Alteration

• Somatic cells

• Biomarker

• Cancer studies

Obstacles and Difficulties

• Expensive

• Extensive bioinformatics support

• Requires considerable quantities of genomic DNA (>5 μg)

• Not easily accessible

Objectives

• How small are micro-CNAs that can be detected?

• Can they reliable detect small CNA using small quantities of DNA from biopsy samples?

• Does the amplification process introduce artifacts that can confound analysis of data?

• What are the limitations of array CGH?• 244K

• 1 M array

METHODOLOGY

Cultured MCF-7 cell line

Harvest of cells in exponential phase

Extraction of genomic DNA using QIAmp kit

60 ng for whole genome amplification

Amplification using φ29 DNA pol and random

primers

250 μg for analysis w/o amplification

Digestion with Alu I and Rsa I

Integrity check using NanoDrop and AGE

Genomic DNA Preparations

Array CGH and Data analysis

Labeled by random priming by Cy5-dUTP

and Cy3-dUTP

Purification by Microcon

Centrifgation Filters, UltracelYM-30

Hybridization by probes at 65°C for 40

hrs

WashingScan by DNA

Microarray ScannerFeature Extraction Software 10.7.3.1

Analysis by Genome Workbench 5.0.14

Identification of DNA copy-number

aberration using ADM-2 algorithm

CNV identification using Agilent

Genomic Workbench Database

Comparative Genome Hybridization

• Developed to survey CNVs in the genome

• Labeled sample and reference genomic DNA are co-hybridized to normal metaphase chromosomes

CGH analysis of a tumor cell

Array CGH

• BAC, P1, cosmid or cDNA clones are used for hybridization

• Microarray technology

• Increased resolution

RESULTS AND DISCUSSION

The detection of micro-CNAs using array-CGH

Detected micro-CNA

Amplification Deletion

With genes involved 22 15

Single genes 9 5

Highest # of genes 14 7

Total 24 15

Summary of Affected Genes

• Total of 84 genes with discovered CNA

• Common biological process affected:– Cell Cycle

• 9 genes

– Estrogen Receptor signaling• 5 genes

• FOXA1: candidate biomarker of poor prognosis in breast tumors

• BMP7: biomarker of bone metastasis in breast cancer

• VAV3: oncogene known to be overexpressed in MCF-7 cell lines

RESULTS AND DISCUSSION

Detection of Breakpoints of Chromosomal Rearrangements

Breakpoints in Chromosomal Rearrangement

• Double-stranded DNA breaks• Translocation

• Amplification

• Deletion

• Intragenic alterations in DNA copy number– Gene fusion events are more common than

previously believed

• Complex sequence rearrangement– Chromosomal segments break down to smaller

regions which differ in copy number values

– Detection due to dense spacing of probes in array

RESULTS AND DISCUSSION

Ultradense array CGH reveals micro-amplifications and micro-deletions which are artifacts inherent to whole genome amplification

Genome and Chromosomal Level

• Reproducible results

– Four separate experiments

• Three WGA

• One without amplification

• No apparent change whether the DNA was amplified or not

Zoom in of Chromosome 1

Zoom in of chromosome 2

Subchromosomal level

• Repetitive, periodic aartifacts in amplified samples

• Wave effects– Discreet decreases in DNA copy number values (10-

100 kb)– Intervals of 50 – 500 kb– Hardly visible with 244 K array

• Considerably confounded the analysis of ADM-2 algorithm

• Does not obscure detection of true CNAs

CONCLUSION

Summary of Findings

• Limit of sensitivity for 244 K array:• ~100 kb

• Two novel amplicons• USP6 and PECAM1

• Micro-CNAs that cut through exonic sequences may indicate potential sites of chromosomal rearrangements and translocation

• Complex intragenic DNA copy number changes caused gene fusion events

Comments on the Technology Used

• Cluttered 1 M microarray profile• Wave artifacts from whole genome amplification

• Large number of artifacts limits analysis of data at subchromosomal level

• Limitations on the use of ADM-2 algorithm

• Requires excellent quality of genomic DNA

• Highly sensitive to confounding effects of whole genome amplifications

Issues and Concerns

• Privacy and confidentiality

• Psychological impact

• Clinical issues• Highly sensitive technology requires high quality

data gathering procedures

• Uncertainties associated with gene tests for susceptibilities and complex conditions

• Genes versus the environment

End of Presentation

Gomez Marineil C.