Techniques of cytogenetic analysis

Question 1

What techniques are involved in conventional cytogenetics?

Answer 1

G banding

Question 2

How is G-banding performed?

Answer 2

Cell culture -> mitotic arrest -> hypotonic -> fixation -> trypsin & Leishman’s stain -> banding - AT and GC rich regions

Question 3

What techniques are used in molecular cytogenetics?

Answer 3

• Fluorescent in situ hybridisation (FISH)
• Multiplex ligation dependent probe amplification (MLPA)
• Microarray comparative genomic hybridisation (array CGH)
• Next generation sequencing
• Quantitative fluorescent PCR (QF-PCR)
• qPCR

Question 4

What is fluorescent in situ hybridisation?

Answer 4

Detection of DNA material on slides using fluorescent dyes & UV light

Question 5

What is the FISH process?

Answer 5

Labelling -> denaturation -> target -> hybridisation -> post-hybridisation washing -> visualisation (24 hours)

Question 6

What types of probes may be used in FISH?

Answer 6

Sequence specific, centromeric or ‘paint’ (ie stain the whole chromosome)

Question 7

What might unique sequence probes be used for?

Answer 7

Detection of deletions (ie DiGeorge).

Question 8

What might centrometic probes be used for?

Answer 8

Detection of aneuploidy.

Question 9

What might chromosome paints be used for?

Answer 9

Translocations

Question 10

What are the applications of FISH?

Answer 10

Copy number imbalance

Aneuploidy

Confirmation/ clarification of G-banding

Confirmation of array CGH

Identifying specific abnormalities in cancer

Question 11

What is copy number variation?

Answer 11

• “A DNA segment with a variable copy no. compared with a reference genome”
• Range 1Kb – several Mb
• 12% of human genome
• One gene or contiguous genes
• Pathogenic or benign
• Familial or de novo

Question 12

What are the potential consequences of copy number variation?

Answer 12

• Disease
  
  • Autism
  • Cancer (acquired)
• Resistance & susceptibility
  
  • High copy no. of CCL3L1 = susceptibility to HIV
  • Low copy no. of FCGR3B = susceptibility to inflammatory autoimmune disorders

Question 13

What techniques might be used to assess copy number variation?

Answer 13

• FISH
• MLPA
• Microarray CGH
• Next generation sequencing
• QF-PCR
• qPCR

Question 14

What is Multiplex Ligation-dependent Probe Amplification?

Answer 14

A variation of the multiplex polymerase chain reaction that permits multiple targets to be amplified with only a single primer pair.

Each probe consists of two oligonucleotides which recognize adjacent target sites on the DNA.

One probe oligonucleotide contains the sequence recognised by the forward primer, the other contains the sequence recognised by the reverse primer.

Only when both probe oligonucleotides are hybridised to their respective targets, can they be ligated into a complete probe.

The advantage of splitting the probe into two parts is that only the ligated oligonucleotides, but not the unbound probe oligonucleotides, are amplified. If the probes were not split in this way, the primer sequences at either end would cause the probes to be amplified regardless of their hybridization to the template DNA, and the amplification product would not be dependent on the number of target sites present in the sample DNA.

Each complete probe has a unique length, so that its resulting amplicons can be separated and identified by (capillary) electrophoresis.

Question 15

What is a Microarray (CGH array)?

Answer 15

• Hybridise sample & control DNA to a microarray “chip” 1000s of DNA spots (oligonucleotides)
• Genomic imbalances (copy number variants) at high resolution (10-10000x conventional cytogenetics)
• detection rates
• Replacing karyotyping as 1st line test

Question 16

What are the requirements for microarray?

Answer 16

• 3ml blood in EDTA (also 1-2ml lithium heparin blood for cell culture if follow up studies needed)
• Control DNA from same sex

Question 17

For which patients would you use Microarray?

Answer 17

Moderate to severe learning & developmental disability (LDD) = 1-3% (IQ<70)

Question 18

What are the advatages of using microarray CGH?

Answer 18

Early diagnosis -1st line test, reduces need for other tests and avoids the “diagnostic odyssey”

High resolution = increased diagnostic hit rate

Greater accuracy of location/size of imbalances

Information on relevant genes

Question 19

What are the disadvantages of using microarray CGH?

Answer 19

• Dosage changes only – not balanced rearrangements or mutations
• Low level mosaics not detected
• Non-pathogenic & uncertain pathogenic changes detected
• Needs good quality DNA

Question 20

What is Quantitative fluorescent PCR (QF-PCR)?

Answer 20

• PCR amplification of short tandem repeats (STRs) [chromosome-specific, repeated DNA sequences] using fluorescent primers
• Products visualised & quantified as peak areas using an automated DNA sequencer

Question 21

What might PCR-QF be used for?

Answer 21

• Prenatal aneuploidy detection.
  
  • DNA extraction from amniotic fluid or chorionc villi
  • PCR amplification – primers from chromosomes 13, 18, 21, X and Y
  • DNA dosage in up to 4-5 markers/chromosome
  • aneuploidy =>2 markers with abnormal dosage

Question 22

What is qPCR quantification?

Answer 22

• Relative Quantitation (RQ) - compares difference in concentration between patient sample & normal control assessed by 2 different primer sets
• RQ value is expressed as a ratio relative to 1 - a deletion has an expected value of 0.5 & a duplication an expected value of 1.5

Basically I have no idea.

Question 23

How long does each method of analysis take?

Answer 23

G-banding = 30 mins - 4 hours/case

FISH = 10 mins -1 hour/case

Microarrays = 10 mins -2 hours/case

NGS/qPCR = 30 mins – 2 hours per case

MLPA/QF-PCR = 10 mins/30 mins/case

Question 24

What referrals might be recieved for cytogenetics?

Answer 24

• Dysmorphic newborns
• Gender assignment
• Developmental problems – intellectual, physical, sexual
• Heart defect
• Reproductive problems
• Family studies

Question 25

What is a Robertsonian translocation?

Answer 25

A Robertsonian translocation is a type of nonreciprocal translocation involving two homologous (paired) or non-homologous chromosomes (i.e. two different chromosomes, not belonging to a homologous pair). A feature of chromosomes that are commonly found to undergo such translocations is that they possess an acrocentric centromere, partitioning the chromosome into a large arm containing the vast majority of genes, and a short arm with a much smaller proportion of genetic content. During a Robertsonian translocation, the participating chromosomes break at their centromeres and the long arms fuse to form a single, large chromosome with a single centromere. The short arms also join to form a smaller reciprocal product, which typically contains nonessential genes and is usually lost within a few cell divisions. This type of translocation is cytologically visible, and can reduce chromosome number if the smaller chromosome that results from a translocation is lost over cellular divisions. However, the smaller chromosome lost may carry so few genes that it can be lost without an ill effect to the individual

Question 26

What are the potential consequences of a Robertsonian translocation?

Answer 26

In humans, when a Robertsonian translocation joins the long arm of chromosome 21 with the long arm of chromosome 14 (or 15), the heterozygous carrier is phenotypically normal because there are two copies of all major chromosome arms and hence two copies of all essential genes. However, the progeny of this carrier may inherit an unbalanced trisomy 21, causing Down Syndrome.

Question 27

What are the indicators for prenatal diagnosis?

Answer 27

* increasing maternal age * serum screen risk * abnormal ultrasound scan (USS) * FH/previous chromosome abnormality

Question 28

Which two invasive prenatal tests may be used in Down syndrome screening?

Answer 28

Amniocentesis and CVS.

Question 29

What analysis would be performed on aminiotic fluid?

Answer 29

1. Separate maternal from foetal tissue 2. QF-PCR 3. Culture cells (7-14 days) if QFPCR result abnormal 4. G-banded analysis

Question 30

What kind of analysis would be performed for CVS?

Answer 30

1. Separate maternal from foetal tissue 2. QF-PCR 3. Culture cells (7-14 days) if QFPCR result abnormal 4. G-banded analysis

Question 31

What analyses are preformed in the case of a spontaneous abortion?

Answer 31

1. Macerate tissue 2. Either cell culture (7-28 days) & G-banding, or QF-PCR/MLPA to assess aneuploidy, or array CGH (extracted DNA) to assess dosage

Question 32

How is cytogenetics used in cancer?

Answer 32

•Disease specific acquired chromosome changes –Mostly translocations –Several different acquired abnormal clones –Diagnosis –Prognosis –Treatment (stratified medicine) * Leukaemia - bone marrow * Solid tumour - tumour tissue

Question 33

In what kind of cancers might cytogenetics be used?

Answer 33

CML (Philadelphia chromosome, Bcr-Abl fusion) MYCN gene amplification in neuroblastoma