Cytogenetic Techniques and their Application

Question 1

What are the pros and cons of G-band analysis?

Answer 1

Pros:

• G-band analysis is a genome wide screen
• Un-targeted analysis
• Provides positional information

Cons:

• Resolution is relatively low - within the range of 3-5mb
• Requires viable tissue

Question 2

What is C-banding?

Answer 2

• C-banding is another chromosome staining technique. Constitutive heterochromatin (non-coding genetic material) stains dark, euchromatin regions remain pale.
• Useful in excluding variants or establishing significance of markers (do they contain coding euchromatin regions?)
• Heterochromatin is present in the peri-centromeric regions.
• The C-band positive regions can vary in size - most notably on chromosomes 1, 9, and 16.

Question 3

How will coding material stain on C-banding?

Answer 3

Constitutive heterochromatin (non-coding genetic material) stains dark, euchromatin regions remain pale.

Question 4

How will non-coding constitutive heterochromatin material stain on C-banding?

Answer 4

Constitutive heterochromatin (non-coding genetic material) stains dark, euchromatin regions remain pale.

Question 5

What is the silver staining technique?

Answer 5

• Silver staining utilises silver nitrate.
• It results in a dark deposit corresponding to the ‘satellite stalks’ of the acrocentric chromosomes.
• Not all acrocentric chromosomes will be positive for silver nitrate. This is a heritable variation.
• If a marker chromosome includes a silver nitrate positive region then it is derived from an acrocentric chromosome and that part of the marker is non-coding.

Question 6

What can be deduced if a marker chromosome includes a silver nitrate positive region?

Answer 6

If a marker chromosome includes a silver nitrate positive region then it is derived from an acrocentric chromosome and that part of the marker is non-coding.

Question 7

What parts of an acrocentric chromosome does silver nitrate staining stain?

Answer 7

Silver nitrate staining results in a dark deposit corresponding to the ‘satellite stalks’ of the acrocentric chromosomes.

Question 8

What are the pros and cons of FISH?

Answer 8

Pros:

• Very high resolution - in the hundreds of kilobase range
• When used on chromosome preps it provides positional information
• Can also be used on uncultured material thus are not always required to have viable fresh tissue

Cons:
- Targeted - specific question that gives a specific answer

Question 9

Briefly describe how aCGH works.

Answer 9

Patient genomic DNA is labelled with a red fluorochrome and mixed in equal quantity with a normal ref DNA labelled with a green fluorochrome. It then competitively hybridises to a DNA microarray containing probes representing genomic regions of interest. DNA then competes for same probe site on microarray. The spot colour ratio analysis shows which DNA regions are under or over represented.

Question 10

What considerations should be taken into account when analysing array results? What might you see in a patient that has a large ring chromosome?

Answer 10

• Consider a large ring chromosome - the array may detect small deletions at each end of the chromosome.
• If there is dynamic mosaicism as a result of misdivision at mitosis there may be a range of different cell lines with loss of the ring, doubling of the ring. The DNA will contain a mixture of all different cells.
• The patient’s phenotype is related more to generalised ring syndrome than to deletions at the telomeres.

Question 11

Consider a case in which an amplification is detected on array in a proband - will array results for the parents of the proband be sufficient to determine the recurrence risk?

Answer 11

No. The parents may be balanced. Array analysis might look normal but the parents may carry a balanced insertion of the amplified sequence. Positional information is essential in establishing recurrence risks.

Question 12

What are the pros and cons of aCGH?

Answer 12

Pros:

• High resolution
• Genome wide
• Un-targeted
• Does not require viable tissue or cell culture

Cons:

• Requires good quality DNA
• Only detects genetic imbalances and will not detect balanced rearrangements - will not detect imprinting defects - UPD may be detected id snp based array is used
• Provides no positional information
• Mosaics not detected

Question 13

Summarise genetic techniques and learning disorders.

Answer 13

• Because of the variability of phenotype and the vast array of viable genetic imbalance it is important to use an un-targeted/genome wide and high resolution technique - such as aCGH.
• In order to understand the nature of the imbalances detected by aCGH and the recurrence risks for families positional information is required - chromosome preparations and FISH are still important.

Question 14

What genetic techniques might you want to use to investigate patients with learning difficulties?

Answer 14

• Investigating proband by aCGH will provide best detection rate
• Array result will not provide positional information and may fail to detect mosaicism
• Gold standard for parental follow up is to use a technique which provides positional information - FISH on to chromosome preparations or G-banded analysis. Will not always be possible.

Question 15

What is the gold standard for parental follow up to aCGH?

Answer 15

Gold standard for parental follow up is to use a technique which provides positional information - FISH on to chromosome preparations or G-banded analysis. Will not always be possible.