Comparative Genome Hybridization

Rob Holt, PhDHead of Sequencing, GSCSr. Scientist, BCCA

Medical Genetics 505University of British ColumbiaJanuary 29, 2004

Karyotype• Chromosome constitution (46,XX) plus any abnormalities (46,XX del(4) (p16.3))

Chromosomal abnormalities•Polymorphic. Prevalent in population at large•Can be benign, disabling, or lethal•Important in cancer, mental retardation, various mendelian disorders (Velo Cardio Facial Syndrome ,Angelmans Syndrome, Rett Syndrome), and maybe other complex diseases

Chromosome Abnormalities

Euploidy – complete chromosome set

Polyploidy – extra set of chromosomes (lethal). Failure to complete first zygotic division (tetraploidy)

Aneuploidy – one or more complete chromosomes extra or missing. Due to failure of paired chromosomes to separate and be incorporated into a daughter nuclei.

Duplication/Deletion – portion of chromosome extra/missing. Often due to repeat mismatch and unequal crossing over.

Translocation – Ends of broken chromosomes are mispaired then rejoined

G banding (1970s)

• Chromosomes subjected to controlled digestion with trypsin and staining with DNA binding chemical dye. Dark bands (G bands) have more condensed chromatin. Resolution ~5-10 Mbp

FISH (1990s) Fluorescent In Situ Hybridization

•Air dried microscope slide preparation of denatured metaphase chromosomes hybridized with fluorescently labeled DNA probe (cosmid or BAC clone).

•Interrogate one locus at a time. Resolution = probe size.

Chromosome paintingMultiplex FISH (M-FISH)Spectral Karyotyping (SKY)

•DNA probes composed of large number of fluorescently labeled fragments from a single chromosome. Hybridization causes whole chromosome to fluoresce.•Combinatorial labeling of chromosome specific probes with more than one fluorophore allows entire karyotype to be defined (each chromosome assigned a pseudocolour)

Loss of Heterozygosity (LOH)• screening paired samples of constitutional DNA (ex. from blood) and somatic DNA (ex. from tumor tissue) with polymorphic markers spaced across the genome

•Detects chromosomal loss or loss followed by re-duplication

Comparative Genome Hybridization (CGH)• Test DNA and reference DNA are labeled with different fluorophors and co-hybridized to a representation of the genome (metaphase spread, genomic array) to detect DNA copy number variations

DNA probe 1 DNA probe 2

Assay signal ratio on of each genomic DNA fragments (BACs)

Normal Cells Abnormal Cells

Array CGH

Chromosomal Aberrations

Albertson DG et al.,2003 Nat Genentics, 34;369

BAC array CGH Protocol

1. Label ~50ng test and reference DNA with dCTP-Cy5 and dCTP-Cy3, respectively, by nick translation.

BAC array CGH Protocol

2. Incubate with excess (~35ug) Cot1 DNA to block repetitive sequence

Cot 1 DNA

Cot-1 DNA

• DNA randomly sheared to ~500bp, heated to separate strands, cooled to renature

• Rate of renaturation depends on concentration of sequence (Co) and time (t). Measured in M/s.

• Reassociation kinetics first suggested that complex genomes comprise 3 different sequence classes based on copy number

Cot-1 derived by re-association of 3mM DNA for 5.5 min

(330s)

1. Highly repetitive (10%). High chance of quickly finding partner. Instantaneous re-association

2. Moderately repetitive (30%)

3. Single or low copy (60%). Re-association takes several decades

BAC array CGH Protocol

3. Add to array, cover with lifter slip, seal, incubate 48h

4. Wash (SSC) and scan (Cy3 and Cy5)

SPOT

Array location map

Clone position map

Results

Genomic copy number profile

BAC Array CGH

Humarray 2.0

•developed by Albertson Lab (UCSF)

•2460 BAC clones in triplicate on 12x12mm array

•average resolution of 1.4 Mbp

•clones confirmed by FISH to be single copy

•successfully used for studies in cancer, neurofibromatosis, mental retardation and primate phylogenetics

Advantages and Limitations of Array CGH

•Does not provide information on location of sequences that have altered copy number

•Can’t detect chromosomal aberrations that do not result in copy number differences (balanced translocations)

• heterogeneous cell populations (ex. mixed tumor and normal stromal cells) can result in appearance of partial copy number differences.

Applications

Cancer

Cancer

Hanahan and Weinberg,2000, Cell 100;57

Hanahan and Weinberg,2000, Cell 100;57

Tumor progression

•Generally few chromosomal aberrations in early stages

•Sharp increase in genome complexity which then levels off

•Sheer number of aberrations suggests noise, but more likely represent selection during tumor evolution

•Identifying important cancer related genes difficult because losses or gains may be several Mbp, and contain hundreds of genes

Schizophrenia

A severe mental illness characterized by:

• Reality distortion (delusions, hallucinations)

• Thought disorganization

• Psychomotor abnormalities (poverty to excitement)

• Blunted affect

Onset: late teens, early 20’s

Affects: 1/100 people (lifetime prevalence). Incidence 1/10,000

Life expectancy: decreased 20%. 10% suicide rate.

Cost to Canadian health care: $2.5B/year

There are no robust neurological, behavioral or genetic markers…

Schizophrenia

Risk Factors for Schizophrenia

Genetic: 80-85% of liability

Non-Genetic: 15-20% of liability• Developmental abnormalities• Birth complications• Place (urban) and time (winter) of birth• Heavy cannabis intake• Viral exposure or under-nutrition during gestation• Socio-economic status (10x more likely to be in lowest group)

(Cannon and Jones, J.Neurol. Neurosurg. Psychiatry, 60,604)

Average risk of developing schizophrenia

Bray NJ and Owen MJ (2001) Trends in Molecular Medicine 7;169.

Candidate susceptibility regions for schizophrenia

Evidence from morethan 1 data set

Genome wide significance

Bray NJ and Owen MJ (2001) Trends in Molecular Medicine 7;169.

Insights from Cytogenetics

• Velo-Cardio-Facial Syndrome: • 3 Mbp hemizygous microdeletion at 22q11 • congenital heart defects, cleft palate, facial dysmorphology, learning disability/mental retardation• highest known risk factor for schizophrenia (25%) aside from having either both parents or a monozygotic twin with the disease

• DISC-1, DISC-2:•Scottish family with balanced translocation t(1:11) that segregates with SCZ, BP and MDD. The breakpoint at 1q42.1 is gene dense and two novel and uncharacterized genes, DISC-1 and DISC-2 disrupted

• Anecdotal case reports of chromosomal abnormalities for almost all other chromosomes, esp. 5,9,18,X

•Full scope of chromosomal aberrations in SCZ and BP unknown. Findings complimentary to linkage analysis for identifying candidate genes

Bipolardisorder

Unaffectedcontrol