Cytogenetics - 1

Question 1

Which phase are chromosomes viewed for in cytogenetic studies?

Answer 1

Metaphase

Question 2

Describe the process for chromosome preparation from blood cultures.

Answer 2

Phytohaemagglutinin stimulation in a suspension culture.
Arrest culturing through colchicine (stops formation of the mitotic spindle)
Cell is swelled with hypotonic solution (KCl) thus bursting the nuclear membrane.
Fixation then proceeds with methanol-acetic acid mixture
Drop on slides as single cell suspension
Band and stain
Analyse under a microscope

Question 3

Describe the tissue culture preparations for karyotyping.

Answer 3

From amniotic fluid, chorionic villus sampling and other solid tissues. No stimulation required as cells adherent on substrate.
Culturing is arrested using colchicine and cells are removed from substrate with trypsin-versene if cultured on tube.
Swell cells with hypotonic solution (KCl)
Fix cells with methanol-acetic acid mixture.
Drop solution on slide.
Band and stain, analyse under microscope.

Question 4

What is a karyotype?

Answer 4

An orderly arrangement of all the chromosomes in a metaphase state. Chromosomes are arranged in descending order of size and grouped by banding pattern and shape to allow consistent description of chromosome abnormalities.

Question 5

Describe high resolution G-banding.

Answer 5

Trypsin reagent is used to denature chromosomes, lichens stain used for banding patterns.

Question 6

What is the main problem with karyotyping?

Answer 6

Metaphase cytogenetics is crude; smallest abnormality detectable by routine banded analysis is 3-5 Mb.

Question 7

Describe FISH methodology.

Answer 7

A probe (small DNA fragment derived from human genome) is labelled with a reporter molecule (biotin = green, digoxin = red)
The probe and target DNA are denatured. The probe is then hybridised to the target region in metaphase.
Indirect labelling does not require a fluorescent tag, antibody detection stain is performed after hybridisation.
Then visualised under fluorescence microscope.

Question 8

What are the different types of probes/ labelling used for chromosome identification?

Answer 8

Centromeric probes: identification of individual chromosomes. These bind highly repetitive DNA sequences in the pericentromeric region of chromosomes, suitable for interphase and metaphase analysis.
Unique sequence probes: identification of particular genes or regions. Hard to visualise because they bind to relatively small regions. Used to detect microdeletions (and microduplications) and number of chromosome rearrangements found in cancer.
Chromosome paints: identification of whole or large segment of chromosomes. Probes are made from whole chromosomes collected by flow sorting or microdissection. Used to identify chromosomes or chromosome fragments and some chromosome rearrangements. Suitable for metaphase BUT NOT INTERPHASE.

Question 9

What is the difference between balanced and unbalanced chromosomes?

Answer 9

Balanced = no loss or gain of genetic material
Unbalanced = loss/gain of genetic material

Question 10

What are constitutional abnormalities?

Answer 10

Those which are seen in all or most of the cells of the body.

Question 11

What are acquired abnormalities?

Answer 11

Abnormalities limited to cells that result in cancer leukaemia (malignancy) and are a form of mosaicism.

Question 12

Why are constituional unbalanced chromosome abnormalities so important?

Answer 12

They almost always result in a phenotypic effect in the carrier, which is usually significant because the chromosome abnormalities are relatively large.

Question 13

When are unbalanced abnormalities searched for?

Answer 13

Developmental delay/ mental retardation
Dysmorphic features
Behavioural problems (autism)
Where there are feature of a known chromosome abnormalities.
Also looked for in multiple miscarriages.

Question 14

Describe the clinical value of constitutional balanced chromosome abnormalities.

Answer 14

Balanced rearrangements rarely result in a phenotypic effect in the carrier: only where the breakpoint disrupts the normal function of important genes.
Can be a problem in meiosis as it will produce an offspring with unbalanced chromosome abnormalities (abnormal phenotype) or an increase in the rate of miscarriage.

Question 15

When are balanced chromosomal abnormalities looked for?

Answer 15

Couples with a history of recurrent foetal loss

Parent of children with unbalanced abnormalities (inherited from a parent carrying a balanced rearrangement)

Question 16

What are the two main classification of chromosome abnormalities?

Answer 16

Numerical: changes in the ploidy, loss or gain in whole chromosome set(s). Loss or gain of whole chromosomes.

Structural: interchromosomal/ intrachromosomal.

Question 17

What are the ploidy classifications?

Answer 17

Haploidy = only seen in gametes (23 chromosomes)
Diploidy = normal (46 chromosomes)
Triploidy: not compatible with life but seen in miscarriages (69 chromosomes)
Tetraploidy: not compatible with life but seen in some cells in the body (92 cells).

Question 18

What are the classifications of chromosome losses/gains?

Answer 18

Monosomy: loss of a single chromosome. Autosomal monosomy is generally not compatible with life. Gonosomal: 45,X0 = Turner syndrome, may survive but have abnormal phenotype.

Trisomy: gain of a single chromosome. Autosomal: some may be compatible with life but have abnormal phenotype (Trisomy 21/18/13: Down’s/Edwards/ Patau). Gonosomal: majority is compatible with life but may appear to be mildly affected or unaffected. (Trisomy X, XXY: Klinefelter syndrome: extra breast tissues and infertility issues) and XYY (asymptomatic).

Question 19

What are the intrachromosomal structural abnormalities?

Answer 19

Intrachromosomal (within chromosomes) 
Deletions (terminal or interstitial) - unbalanced
Duplications -U
Isochromosome - U 
Rings - U
Breaks - U 
Inversions - B 
Some insertions - B

Question 20

What are the interchromosomal abnormalities?

Answer 20

Interchromosomal (between chromosomes)
Reciprocal translocations - B
Robertsonian translocations - B
Some insertions - B

Question 21

Describe Deletions:

Answer 21

Can occur on any chromosome, almost always interstitial as chromosomes need stable telomeres. Reduction in genetic material = series phenotypic effects, dependent on size of deletion. Cri-du-Chat syndrome (5p:13.2)
DiGeorge/ velocardiofacial syndrome (22q11.2)

Question 22

Describe microdeletions and microduplications.

Answer 22

More than 100 syndromes associated with very small deletions or duplications. Difficult to detect by light microscopy. Readily detected by FISH, MLPA or microarray.
Many occur in the exact same region as a result of the DNA architecture: during meiosis and mitosis, small tandem repeat sequences can misalign to result in deletions or their reciprocal duplication.

Question 23

Describe duplications.

Answer 23

Can occur anywhere in the genome, however are rare. Almost inevitably associated with phenotypic abnormality: dosage sensitivity.

Question 24

Describe ring chromosomes.

Answer 24

A form of deletion; formed when genetic material is lost from both ends of the chromosome. Deleted chromosome stabilised by the 2 deleted ends fusing to form a ring.
Severity associated depends on the chromosome involved and size of deletion. However even if very small deletion occurs, carrier may still have ring syndrome (developmental delay, short stature, failure to thrive)

Question 25

Describe isochromosomes.

Answer 25

Formed by centromere misdivision: horizontal division rather than vertical.
Results in duplication and deletion, can occur in any chromosome.
Gonosomal isochromosomes are usually viable h/ever with abnormal phenotype.
Autosomal isochromosomes are not usually viable but syndromes exist that are associated with liveborn but with abnormal phenotype (mosaic form). Pallister-Killian syndrome = an extra isochromosome of the short arm of chromosome 12.

Question 26

Describe insertions.

Answer 26

Insertions are very rare and can occur within the same chromosome or between chromosomes. They involve 3 or 4 breakpoints and the majority of insertions are balanced.

Question 27

What are the two types of inversions?

Answer 27

Paracentric: one arm only.
Pericentric: involving both arms.
They are balanced however there are problems for reproduction.

Question 28

What is the effect of maternal age and trisomy?

Answer 28

Increasing maternal age increases the risk of common trisomies; most children with an autosomal trisomy have inherited their additional chromosomal as a result of non-disjunction occurring during one of the maternal meiotic divisions. The Down syndrome risk is 1/880 at age 32 and 1/400 at 35 years.

Question 29

What are the sample collecting strategies for prenatal diagnosis?

Answer 29

Amniocentesis; collecting a sample of fluid from around the foetus; contains viable foetal cells.
Chorionic villus sampling; taking a sample of chorionic villi which are part of the placenta but foetal in origin.
Foetal blood sampling; taking a sample of foetal blood from the umbilical cord.

Question 30

When can amniocentesis be performed?

Answer 30

Reliably after approx. 13th weeks of pregnancy however most performed between 16th and 18th week. Full analysis cultures 8-14 days. The results more reliable than CVS in addition to an easier procedure adn lower risk than either CVS or foetal blood sampling. 10-20ml of amniotic fluid is collected using a fine needle transabdominally or transvaginally under ultrasound guidance.

Question 31

When can chorionic villus sampling (CVS)?

Answer 31

Performed in the first trimester at about 10 weeks of gestation. Direct preparation can give results in 4-24 hours however pure quality. Final long-term culture result takes 8-14 days. Greater risk of miscarriage than amniocentesis and less accurate because:
Greater chance of sampling maternal cells
placental tissue does not always reflect foetal karyotype
Occasional discrepancies between direct and culture results.
A small sample of chorionic villi is collected using a fine needle transabdominally or transvaginally under ultrasound guidance.

Question 32

Describe foetal blood sampling.

Answer 32

Cannot be performed until after 18th week of gestation. Quicker result compared with amniocentesis or CVS: 48 hours compared to 8days/more. Permits high quality chromosome preparation. Mostly used when abnormalities are found on ultrasounds. FBs carries higher risk of procedure related abortion than either CVS or amniocentesis so should only be used for high risk pregnancies.

Question 33

After amniocentesis, which procedures can be performed?

Answer 33

FISH or QF-PCR: give result in 6-24 hours, only detect common trisomies: 13/18/21 and numerical sex chromosome abnormalities. These abnormalities make up about 80% of all significant chromosome abnormalities.

Question 34

Describe the process of maternal serum screening for Down Syndrome

Answer 34

1st trimester: nuchal thickness (the elevation of the skin above the neck) measured by ultrasound at 10-11 weeks gestation
materal serum PAPP-A (pregnancy associated plasma protein-A)
Free beta hCG: human chorionic gonadotrophin.
In the second trimester: uses maternal serum conjugated oestriol
maternal serum alpha-fetoprotein
maternal serum human chorionic gonadotrophin.

Question 35

What are the new molecular genetic methods for detecting chromosomal abnormalities?

Answer 35

QF-PCR,
MLPA
SNP array
NIPT
cffDNA analysis