18.02.05 Copy number detection

Question 1

Give an overview of G-banding and it’s use.

Answer 1

Manipulation of cell cycle to obtain metaphase cells enabling comparison of banding pattern between homologues

1. To detect pre/postnatal aneuploidy
2. To detect unbalanced rearrangements in dysmorphic child
3. To detect balanced rearrangements in recurrent miscarriage couple
4. Detection of abnormal clones in cancer
5. Detect clinically significant structural rearrangementse.g. t(15;17) in APL

Question 2

What are the advantages of G-banding?

Answer 2

1. Whole genome screen
2. Detects balanced/unbalanced rearrangements
3. Provides positional information
4. Detects mosaicism
5. Relatively robust and inexpensive
6. Determine structural rearrangements e.g. ring chr 20

Question 3

What are the disadvantages of G-banding?

Answer 3

1. Low resolution (>5Mb)
2. Labour intensive
3. Slow turnaround time
4. Unable to detect UPD
5. Requires dividing cells and manipulation of the cell cycle
6. Risk of cultural artefacts e.g. prenatal cases
7. Some abnormalities (usually mosaic aneuploidies) not detected in cultured cells

Question 4

Give an overview of FISH.

Answer 4

Fluorescently labelled ssDNA probes are hybridised to specific denatured DNA sequences (metaphase spreads or interphase nuclei); centromere, telomere, locus-specific, whole chromosome paint or BACs

Question 5

What are the advantages of FISH?

Answer 5

1. Positional information if metaphases analysed
2. Detects mosaicism
3. Detects ploidy
4. Aids interpretation of G-banding
5. Fast turnaround time
6. Higher resolution than G-banding
7. Large number of individual cells can be examined
8. Can be used to analyse single cells
9. Probes available for almost any genomic region

Question 6

What are the disadvantages of FISH?

Answer 6

1. Targeted test
2. Probes can be expensive
3. Cannot detect MCC in certain cases
4. Cannot detect UPD
5. Limited number of probes can be used at one time, only 2 or 3 colours possible
6. Interphase FISH provides no positional info
7. May require metaphases
8. Atypical rearrangements may be normal by FISH e.g. some t(15;17) arrangements
9. Microdups may be undetected due to limited resolution on metaphase spread
10. Co-localisation can occur: two signals overlap and appear as one
11. Cross-hybridisation can occur: probe binds to regions with repetitive sequences

Question 7

What are the applications of FISH?

Answer 7

1. Aneuploidy screen
2. Microdeletion/duplication detection
3. Aid G-banding e.g.origin of marker chr
4. Detection of cryptic rearrangements e.g.t(12;21) in cancer
5. Used if G-banding fails e.g. AML screen using disease specific probes
6. Detection of mosaicism
7. Detection of abnormal clones/clonal evolution in cancer
8. Gene amplification e.g. HER2 in br. Ca. or N-MYC in neuroblastoma
9. Gene deletions e.g. TP53 or ATM
10. PGD
11. Variant gene fusionse.g. BCR-ABL1 in CML
12. Post-transplant chimaerism monitoring

Question 8

Give an overview of SKY/M-FISH/CGH and its potential applications.

Answer 8

Techniques enable the fluorescent labelling of the whole genome. Each chromosome is labelled with a different mix of fluorophores to give each chromosome a different colour

Can be used to aid interpretation of G-banding e.g. identification of marker chromosomes or interpretation of complex rearrangements

Question 9

What are the advantages and disadvantages of SKY/M-FISH/CGH?

Answer 9

Adv

1. Whole genome screen
2. Detects balanced/unbalanced rearrangements
3. Provides structural/positional information

Disadv

1. Low resolution (~5Mb)
2. Requires cell culture and manipulation to obtain good quality metaphases
3. Expensive (therefore not widely used)
4. Highly labour intensive

Question 10

Give an overview of QF-PCR and its potential applications.

Answer 10

Quantification of polymorphic repeat sequences to determine copy number. Uses fluorescently labelled primers

1. Prenatal aneuploidy screening
2. Detection of trisomy in pregnancy loss or PND
3. Post-transplant chimaerism monitoring

Question 11

What are the advantages of QF-PCR?

Answer 11

1. Detects mosaicism (above 15%), triploidy and aneuploidy
2. Requires little starting material
3. Fast turnaround times
4. Relatively inexpensive
5. High resolution/High through-put

Question 12

What are the disadvantages of QF-PCR?

Answer 12

1. No positional information obtained
2. Targeted test
3. MCC may prevent interpretation of results
4. May not detect low-level mosaicism (<15%)
5. Limited ability to detect multiple targets in a single assay due to spectral overlap of dyes

Question 13

Give an overview of real-time PCR.

Answer 13

PCR amplification in which the amount of productis measured during each PCR cycle. Uses fluorescent dyes (non-specific e.g. SYBR green) or probes (sequence specific e.g. Taqman). During the exponential phase the amount of amplified product is proportional to the amount of starting material.

Question 14

What are the advantages of real-time PCR?

Answer 14

1. Quantitative
2. Very high resolution
3. Rapid and easy to perform
4. Fast turnaround time
5. Requires little starting material
6. Single cell analysis possible
7. Detects UPD if methylation specific Res are used
8. Post-PCR processing is eliminated, reducing labour, cost and possibility of cross-contamination

Question 15

What are the disadvantages of real-time PCR?

Answer 15

1. No positional information obtained
2. Targeted test
3. Specialist equipment required – thermal cycler and optical instrument to measure fluorescence
4. Unlikely to detect low level mosaicism
5. Multiple reactions required to examine multiple loci
6. Sequence-specific probes expensive
7. Non-specific fluorescent dyes such as a SYBR green intercalate with any dsDNA which may lead to false positive signals

Question 16

What are the potential applications of real-time PCR?

Answer 16

1. Quantify gene expression (mRNA)
2. Minimal residual disease monitoring of gene fusion products e.g. PML-RARA in AML, BCR-ABL1 in CML
3. Detection of mutations e.g. JAK2 V617F, NPM1/FLT3-ITD
4. Detection of microdels/dups e.g. 22q11
5. Detection of subtle del/dup

Question 17

Give an overview of MLPA.

Answer 17

PCR-based multiplex reaction allowing amplification of ~40-45 targets in a single reaction.
Detects copy number imbalances in gDNA and RNA sequences. Probes anneal to target and are then amplified and quantified

Question 18

What are the advantages of MLPA?

Answer 18

1. Can test for ~40-50 different imbalances in one reaction
2. High throughput technique
3. High resolution– detects sequences of only ~60nt-can therefore detect dups/dels of a single exon
4. Fast turnaround time
5. Inexpensive
6. Requires little starting material
7. Detects methylation

Question 19

What are the disadvantages of MLPA?

Answer 19

1. Targeted test
2. More labour-intensive than QF-PCR
3. Can’t be used for single cell screening i.e. PGD
4. No positional information obtained
5. Doesn’t detect balanced rearrangements
6. Cannot detect low-level mosaicism (<20-30%)&unreliable detection at higher levels
7. Unreliable for detection of maternal cell contamination
8. Cannot detect triploidy (69,XXX) and unreliable for other triploidy detection
9. Analysis gives average copy number per cell. Tumour analysis difficult if sample contains <50% cancer cells
10. SNPs under probes can prevent binding (false positive result)
11. Sensitive to contaminants
12. Not easily scaled up

Question 20

What are the potential applications of MLPA?

Answer 20

1. Aneuploidy screen
2. Sub-telomeric imbalance screen
3. Microdeletion screen
4. MS-MLPA – PWS/AS testing
5. Commercial gene dosage kits available for BRCA1 and BRCA2 screening, Duchenne/Becker Muscular Dystrophy, Fanconi Anaemia A and many more
6. Bespoke MLPA
7. Methylation and cancer screening

Question 21

Give an overview of MAPH.

Answer 21

Multiplex Amplifiable Probe Hybridisation - gDNA is fixed to membrane and hybridised with target probes. Unbound probe washed off, amount of bound probe proportional to copy no of target. Probes stripped from membrane and amplified, products separated by electrophoresis.

Question 22

What are the advantages of MAPH?

Answer 22

1. Can multiplex 40 probes
2. High resolution
3. High throughput
4. Requires small amount of starting material
5. Probes are very long (100-200bp) so SNPs under probes less likely to prevent binding than MLPA

Question 23

What are the disadvantages of MAPH?

Answer 23

1. Targeted test
2. Can’t detect triploidy
3. Time consuming
4. More labour intensive than QF-PCR
5. Does not give structural/positional information

Question 24

What are the potential applications of MAPH?

Answer 24

1. DMD gene deletions, subtelomeric deletions, CML tumour typing.
2. Can be used for similar testing to MLPA.
3. Not widely used due to requirement of membrane bound probes (MLPA is more commonly used).

Question 25

Give an overview of BAC arrays.

Answer 25

Patient and reference DNA labelled with different fluorescent dyes and then hybridised to different BAC clones spread throughout the genome

Question 26

What are the advantages of BAC arrays?

Answer 26

1. Whole genome screen 2. Can detect unbalanced copy no changes to ~1Mb resolution 3. Can concentrate probes to particular regions of interest e.g. gene rich (can design custom arrays to target specific areas/genes as well as providing a lower resolution coverage over the whole genome) 4. Simultaneously process large numbers of samples

Question 27

What are the disadvantages of BAC arrays?

Answer 27

1. No positional/structural information 2. Doesn’t detect balanced rearrangements/ ploidy 3. Lower resolution than other platforms (0.5-1Mb detection levels) 4. Expensive 5. Labour intensive 6. Difficulty with interpreting some results 7. Resolution not even across the genome 8. Requires relatively large amounts of good quality DNA

Question 28

What are the potential applications of BAC arrays?

Answer 28

1. Genome screen of individuals with idiopathic mental retardation, dev. del, dysmorphism 2. Prenatal samples with abnormal scans/solid tissue samples 3. Interpretation of abnormal karyotypes

Question 29

Give an overview of oligo arrays and there potential application.

Answer 29

Synthetic oligos used in hybridisation stage which are smaller than BAC clones therefore allowing greater coverage 1. Genome screen of individuals with idiopathic mental retardation, dev. del, dysmorphism 2. Interpretation of abnormal karyotypes

Question 30

What are the advantages of oligo arrays?

Answer 30

1. High resolution imbalance detection (~50-200Kb depending on coverage) 2. Can concentrate probes to particular regions e.g. gene rich (can design custom arrays to target specific areas/genes as well as providing a lower resolution coverage over the whole genome) 3. Simultaneously process large numbers of samples 4. Analysis of single cells possible (with whole genome amplification) 5. Uses DNA from uncultured or cultured material

Question 31

What are the disadvantages of oligo arrays?

Answer 31

1. No positional information obtained 2. Doesn’t detect balanced rearrangements/ploidy 3. Difficulty with interpreting some results-CNVs of unclear significance 4. Expensive 5. Labour intensive 6. Requires relatively large amounts of good quality DNA

Question 32

Give an overview and potential application of SNP arrays.

Answer 32

Array-based technology as above but platform also has additional SNP specific oligos present throughout the genome 1. Screen for LOH in cancers 2. Possible application to detect UPD 3. Could elucidate non-paternity or incidental findings

Question 33

What are the advantages of SNP arrays?

Answer 33

1. High resolution | 2. Can detect copy number changes and also genotype to reveal LOH and UPD

Question 34

What are the disadvantages of SNP arrays?

Answer 34

1. Doesn’t detect balanced rearrangements 2. No positional/structural information 3. Coverage dependent on SNP locations 4. Difficulty with interpreting some results 5. Expensive 6. Labour intensive

Question 35

Give an overview of NGS.

Answer 35

A number of different methods are employed, but all involve step wise addition of nucleotides to fragmented DNA. WGS + WES have become primary strategies for NGS in CNV detection

Question 36

What are the advantages of NGS?

Answer 36

1. Very high resolution (single base change detection) 2. Genome wide or targeted 3. Provides positional information 4. Detects UPD and LOH 5. High throughput 6. Detects balanced/unbalanced rearrangements (paired-end/mate-pair seq) 7. More tolerant of poor quality DNA 8. Detection of both single base variants and CNVs in a single assay 9. WES is lower cost, higher coverage + less complex data analysis than WGS 10. Exome represents a highly function enriched subset of human genome + CNV in exome are more likely to be disease causing aberrations than in nongenic regions

Question 37

What are the disadvantages of NGS?

Answer 37

1. Difficulty with interpreting results-CNVs of unclear significance 2. Vast amount of data obtained 3. Requires a lot of starting material 4. Expensive 5. Labour intensive 6. Not suitable for small genes 7. Need to confirm abnormal results 8. WES data introduces biases due to hybridisation which doesn’t exist in WGS data

Question 38

What are the potential applications of NGS?

Answer 38

1. Targeted genome resequencing to study a particular gene(s) or disease 2. Reduced representation sequencing to e.g. generate a SNP map of the human genome 3. Transcriptome sequencing to study gene expression profiling 4. Gene panels e.g.TruSight Cancer Panel (96 genes) 5. Whole exome sequencing – assessment of all coding regions (DDD project) 6. WES targeted to protein coding regions (less than 2% of the genome) 7. WES used for identifying clinically relevant aberrations in cancer – challenges due to: 1. Sequence data 2. WES technical problems 3. Tumour complexity 8. Copy number detection is a relatively new use of this technology