Techniques of Cytogenetic Analysis

Question 1

Which out of the following tests would you use for whole genome and targeted testing?

G-Banding
Microarrays
FISH
MLPA
QF-PCR or qPCR

Answer 1

G-banding and Microarrays are used for whole genome analysis

FISH, MLPA and QF-PCR/ qPCR are used for targetted testing

Question 2

What is the difference between conventional cytogenetics and molecular cytogenetics?

Answer 2

Conventional cytogenetics involves the analysis of chromosomes at metaphase down a light microscope or image (g-banding)

Molecular cytogenetics involves chromosome analysis at the molecular resolution of all stages (FISH, array CGH)

Question 3

What three categories of samples can be taken for cytogenetics?

Answer 3

Prenatal- amniotic fluid, placenta and other foetal tissue
Postnatal- peripheral blood
Cancer- bone marrow, tumour

Question 4

How is blood in cytogenetics prepared and analysed?

Answer 4

5-10ml unclotted
culture t-lymphocytes 2-3 days using incubator to help division of cells
G-banding analysis can then be done

Question 5

How do you prepare and analyse amniotic fluid in cytogenetic ?

Answer 5

Portion for DNA extraction (QF-PCR)
Separate cells from remaining fluid
Culture cells as monolayer
7-14 days incubation
G-banded analysis

Question 6

Spontaneous abortions can provide tissue such as placenta, lungs, cartilage etc. What may be the problems with using this sort of tissue in cytogenetics and how can it be analysed?

Answer 6

It may be old and so macerated tissue (deteriorating)

Either way you can make it into a monolayer then G-banded analysis

Question 7

How does G-banding work? What preparation is needed?

Answer 7

Cell culture -> mitotic arrest -> hypotonic (swells and spreads out) -> fixation -> trypsin and leishman’s stain -> G-banding- AT (stain lighter) and GC (stain darker) rich regions

Question 8

What are four molecular cytogenetic techniques used for target areas?

Answer 8

Fluorescent in situ hybridisation (FISH)- used in conjunction with G-banding

Multiplex ligation dependent probe amplification (MLPA)

Microarray comparative genomic hybridisation (array CGH)

Quantitative Fluorescence-PCR (QF-PCR)

Question 9

What is Flourescent in situ hybridisation (FISH)? What are the steps and which colours mean what?

Answer 9

Detection of DNA material on slides using fluorescent
dyes & UV light. First step is to label them (flourochrome), denature them, hybridise, then after washing, you can visualise them. Blue is the chromosome and pink/green are the flourescent probes

Question 10

What types of probes are used in FISH?

What are each used for?

Answer 10

Unique sequence- locus specific + microdeletions/duplications & rearrangements in oncology

Centromeric- large regions of repetitive DNA + used much more in oncology

Chromosome paint- rearrangements/translocations and recombination

Question 11

What is MLPA used for?

Answer 11

DNA-based multiplex PCR test which identifies copy number changes in up to 50 different genomic locations simultaneously. It’s an alternative to FISH and detects deletions at ends of chromosomes which may be causing learning difficulties in children

Question 12

What is Microarray CGH?

Answer 12

A front line test for children with learning difficulties. A genome-wide screen in which hybridisation of a sample & control DNA to a microarray “chip” – BACs or SNPs or oligonucleotides (1000s of DNA spots) detects genomic imbalances (copy number variants) at high resolution (10-10000x conventional cytogenetics)

Question 13

How are the results of a microarray CGH read?

Answer 13

Using software which measures the ratio of red to green (correlating to levels of normal and test DNA that competed for a spot on the chip) which can then show any gains or losses of certain genes

Question 14

Which patients are often tested for with array CGH?

Answer 14

Those with learning difficulties and developmental disability (LDD). Many times the etiology is heterogeneous

Question 15

What are the disadvantages of array CGH?

Answer 15

Only detects imbalanced rearrangements- not balanced
Limited for mosaicism
Non-pathogenic and uncertain pathogenic changes detected
Requires good quality DNA
Expensive initially (but long term cheaper than G-banding)
Doesn’t cover entire genome

Question 16

What is QF-PCR?

Answer 16

PCR amplification of short tandem repeats (STRs) using flourescent primers. Looks at lots of different areas simultaneously and the products are visualised and quantified as peak areas using an automated DNA sequencer

Question 17

How do prenatal aneuploidy detections happen? (testing for abnormalities in pregnancies)

Answer 17

DNA extraction from amniotic fluid
PCR amplification- primers from chromosome 13, 18 and 21
DNA dosage in 4-5 markers/chromosome
Aneuploidy= 2 markers with abnormal dosage
(Basically allows you to quantify chromosomes)

V.quick results= 24h

Question 18

What is shown from prenatal testing if there is trisomy 21 or 13?

Answer 18

Either 3 peaks or two peaks but one is double the size of the other

Question 19

What sort of patients are often send for blood chromosome studies?

Answer 19

Dysmorphic newborns
Gender assignment if not clear
Developmental and learning problems
Sexual development as some people do not reach puberty when they should
Heart defects
Reproductive problems
Family studies

Question 20

How many are affected by Digeorge syndrome? What are the traits?

Answer 20

1/5000
10-15% familial (got it from parents)
Low set ears
Sloping head
Facial abnormalities
Absent thyroid
Hypocalcaemia
Nasal speech
Short Stature
Learning difficulties
Mental health problems- schizophrenia/bipolar

Question 21

What is Williams syndrome? What gene does it affect? What traits are there with this condition?

Answer 21

7q11.23 deletion- critical gene involved in elastin and this affects the heart.
'cocktail-party' personality
Pixie-like face
Hypercalcaemia
Developmental delay
Infantile hypercalcaemia
Premature aging

Question 22

When are parents referred for prenatal tests?

Answer 22

When mother is older
Serum screen risk
Abnormal ultrasound
FH/previous chromosome abnormality

Question 23

What is the pattern/ correlation between down syndrome risk and maternal age?

Answer 23

From about 35 years old and older, the risk increases dramatically

Question 24

What are the two sampling procedures carried out to obtain material for prenatal diagnosis?

Answer 24

Amniocentesis at around 16 weeks (amniotic fluid)

Chorionic villus sampling at around 12 weeks (placental tissue)

Question 25

What happens to the tissue for a prenatal diagnosis?

Answer 25

Separate maternal from foetal tissue
Place in suspension culture for 24 hours or QF-PCR
Longer-term monolayer culture (8-20 days)
G-banded analysis

(Ideally want the QF-PCR and longer culture to be sure)

Question 26

What future tests could there be for prenatal diagnosis?

Answer 26

Array CGH or next gen. sequencing on NHS? Exomes or whole genome?
NIFTY (Non invasive fetal trisomy test)- an advanced test based on parallel sequencing- abnormal results would still need to be verified by other tests (used from 10 weeks onwards)

Question 27

How can cytogenetics help with cancer?

Answer 27

Can help detect chromosome changes and therefore diagnose it- leukaemia andsolid tumour tissue. Can also measure the progression or remission by measuring any more accumulating chromosome abnormalities and this may help with treatment decisions

Question 28

How can cytogenetic analysis help in the diagnosis of leukaemia cancer?

Answer 28

1 ml unclotted bone marrow
Suspension overnight
G-banded analysis/ FISH

Question 29

What is a philadelphia translocation? What are the symptoms of this?

Answer 29

It is an abnormality on chromosome 22 which makes it abnormally short due to a reciprocal translocation

Symptoms include tiredness and low white blood cell count

Question 30

How are solid tumours analysed?

Answer 30

Macerate+enzyme digest tumour tissue
short-term suspension and longer term monolayers
G-banded analysis/FISH analysis