What are the methods of chromosome testing?
- Karyotyping
- Array comparative genomic hybridisation
- FISH
- QF-PCR
What stage of the cell cycle is looked at in karyotyping?
Cell division
Why is cell division looked at in karyotyping?
Because chromosomes are only visible microscopically in dividing cells
How is cell division visualised in karyotyping?
Non-dividing cells are taken and cultured to encourage cell division. They are then stained and examined
What cells are taken for karyotyping?
Usually peripheral blood lymphocytes
What is looked for in karyotyping?
Any extra or missing chromosomes, or large scale chromosomal abnormalities
What will karyotyping miss?
Any changes smaller than 5-10 mega bases (5-10 million bases)
What is the result of karyotyping missing any changes smaller than 5-10 mega bases?
It is unsuitable for detection of most micro-deletions or duplications
What might karyotyping be helpful in identifying?
The structural basis for abnormalities detected by other methods
What is array comparative genomic hybridisation (aCGH) also known as?
Microarray
What is microarray?
A relatively new technique which looks for chromosomal copy number variations (CNVs) by comparing a patient’s DNA to normal control DNA
How has the use of microarray in practice changed?
It has taken over from karyotyping as the first line test for most chromosomal abnormalities in paediatric practice
How does microarray work?
- Patient DNA is labelled green, and normal control DNA is labelled red.
- 1000’s of probes (fragments of DNA) which bind specifically to regions spanning the whole genome are immobilised on a slide (the array).
- The array is immersed in a solution containing equal proportions of the flourescently-labelled test and control DNA, allowing green ‘patient’ DNA to compete with red ‘control’ DNA to hybridise with each of the probes.
- At probes where there is a deletion (i.e. lower patient copy number than control) the array appears red. When there is a duplication, the array appears green
- The array is scanned with a high resolution camera, and data interpreted using computer software
What size deletions and duplications can microarray deter?
Typically deletions/duplications larger than 50,000 bases
How does the resolution of microarray compare to karyotyping?
100 times higher
What does microarray allow for the detection of?
- Micro-deletions or duplications
- Larger chromosomal copy number abnormalities, including aneuploidies
What is the limitation of microarray?
It cannot detect balance chromosomal rearrangements, and does not distinguish unbalanced chromosome rearrangement caused by different mechanisms
Give an example of an unbalanced chromosome rearrangement that microarray cannot distinguish between?
A conventional trisomy 21, and a trisomy 21 caused by Robertsonian translocation
What is FISH?
A modification of conventional chromosome analysis using flourescently labelled probes
What does FISH allow?
Targeted testing for copy number variation and structural rearrangement
How does FISH work?
- Chromosomes are immobilised and denatured on a microscope slide and exposed to a solution containing a flourescently labelled prove specific to a specific chromosomal region.
- After hybridization, the slide is washed and examined microscopically.
- Where the probe has hybridized, fluorescent spots are seen over the relevant chromosome.
What is meant by hybridisation in FISH?
The formation of double stranded DNA from complementary single strands
Using 22q11 deletion syndrome as an example, how are the results of FISH interpreted?
If a child had a 22q11 deletion syndrome, FISH using a 22q11-specific probe should show 1 pair of fluorescent spots, rather than 2
What does FISH require to be a useful investigation?
Clinical recognition of the likely causative mechanism
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Chromosomal Disorders ✅31
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Genetic Testing ✅28
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Imprinting Disorders ✅87
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Chromosome Testing ✅28
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Mechanisms of Chromosomal Abnormality ✅42
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Autosomal Aneuplodies ✅39
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Autosomal Dominant Genetic Disorders ✅56
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Sex Chromosome Aneuploidies ✅20
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Partial Chromosomal Deletions And Duplications ✅31
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Single Gene Disorders ✅39
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Autosomal Recessive Genetic Disorders ✅14
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X-Linked Inheritance ✅39
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Mitochondrial Disorders ✅38
