FISH and Karyotype analysis

Question 1

What causes reduced fertility in humans?

Answer 1

Inheritance of structurally abnormal chromosomes.
Abnormal complement of chromosomes

Question 2

How do the two causes of reduced fertility result in reduced fertility?

Answer 2

By disruption of gametogenesis
By spontaneous loss of pregnancy

Question 3

What are the main two technologies used to visualise human chromosomes?

Answer 3

G-banding and associated karyotype analysis
Fluorescence in-situ hybridisation

Question 4

What is aneuploidy? What is this a major cause of?

Answer 4

Gain or loss of one or few chromosomes.
Major cause of pregnancy loss.

Question 5

Describe Edwards syndrome

Answer 5

Trisomy of chromosome 18.
Likely that foetus will spnteneously abort.
Under 5% chance of pregnancies coming to term.

Question 6

What stage of cell cycle should cells be in for Karyotype analysis?

Answer 6

Metaphase

Question 7

Describe interphase

Answer 7

The genetic content of a cell is contained within the nucleus, surrounded by the nuclear lamina and nuclear envelope.

Question 8

Descrobe prometaphase

Answer 8

In prometaphase membrane structures begin to breakdown. The nuclear lamina dissociates, and the nuclear envelope breaks down into vesicles.

Question 9

Describe metaphase

Answer 9

By metaphase the genetic content of the cell exists as condensed chromosomes that are free in the cytoplasm.

Question 10

What must be done to provide patients with appropriate diagnosis

Answer 10

investigate relevant issue.

Question 11

Are blood samples sufficient to provide a diagnosis?

Answer 11

Blood sample normally sufficient (as all cells expected to have same genotype, any nucleated cells in body are assumed to have same genotype – so any nucleated cells in body are assumed to be representative of all others). However exception in mosaic individuals.

Question 12

What happens during prenatal investigation?

Answer 12

DNA from foetus harvested from amniotic fluid. Epithelial cells from foetus fall off during development and suspended in liquid.

Question 13

Placental tissue

Answer 13

Leads to higher risk of spontaneous miscarriage.
Placental tissue also diverged from foetal tissues early on during pregnancy – therefore it is possible for genotype of placenta to differ from that of the foetus.

Question 14

WHat is foetal blood used and why?

Answer 14

Associated with high risk of miscarriage, last resort

Question 15

How is chromosome morphology described?

Answer 15

P arm = Short arm
q arm = Long arm
Metacentric
Submetacentric
Acrocentric

Question 16

Descrobe chromosome 21 P arm

Answer 16

P arm satellited and rich with repetitive sequences such as rDNA genes. Possible for individuals to lose whole satellited p arm from the genome and be normal.

Question 17

What is FISH? and what can it be used for?

Answer 17

Fundamental tool for rapid confirmation of genetic abnormalities for prenatal referrals.

Detection of chromosomal rearrangements which can result in reduced fertility.

Question 18

WHat does FISH use?

Answer 18

Uses labelled oligonucleotide probes, complementary to particular regions of genome to report on the copy number of these genetic loci and their approx. position in the genome.
Able to provide information at level of the individual cell.

Question 19

Explain colocalisation

Answer 19

Look at slides

Question 20

When is FISH used?

Answer 20

Childhood leukaemia and new acute leukaemia – in particular.

Prenatal Diagnosis (PND).

Question 21

Why is FISH a useful technique? however whats starting to happen?

Answer 21

Generated quickly.

Shift in clinical pathology towards the analysis of free foetal DNA by qPCR, (qPCR wont be covered in lecture but there is further reading).

Question 22

What is reduced fertility, infertility and pregnancy loss caused by?

Answer 22

By gross structural rearrangements to the genome and genomic imbalance (gain or loss of genetic information).

Question 23

Exaplain Gene-specific probes and what is it used to report on?

Answer 23

Reports on copy number of disease critical regions – ie Down syndrome critical region.

Down syndrome – trisomy of chromosome 21. Disease caused by hemizygous gain of down syndrome critical region located on the long arm of chromosome 21.

This abnormality accounts for significant proportion of spontaneous abortions.

Vast majority of pregnancies affected by this genomic imbalance do not come to term.

Question 24

Centromeric probe

Answer 24

Complementary to alpha satellite sequences that are located in sub-centromeric regions of chromosomes.

Target regions very large – probes very bright.

Probes often used to report copy number of chromosomes including 13, 18, 21, X and Y.

Question 25

Telomeric probes and whole chromosome paints.

Answer 25

Help geneticist characterise chromosomes that have been observed to be abnormal by G-banding. Help to characterise gross structural rearrangements in a foetus, and whether the abnormality was inherited from one of the parents.

Question 26

Where is TBX1 gene located? which disorder is it deleted in?

Answer 26

On the long arm of chromosome 22 within a 2Mpb region that is commonly deleted in patients with DiGeorge Syndrome (approx. 90% of cases).

Question 27

WHat sequences are important in FISH and why?

Answer 27

Control sequences are essential component of FISH experiments – used to report on the efficiency of probe hybridisation and to help lower rates of false positive and false negative results in clinical experiments.

Question 28

WHat is DiGeorge syndrome

Answer 28

DiGeorge Syndrome is one of the human microdeletion syndromes, that are often detected in newborns and infants following observation of developmental delay, intellectual disability, characteristic morphological abnormalities, heart defects, and many other health complications. Deletion of the region is in fact associated with a spectrum of disorders that affect the copy number of other genes in this chromosomal region.

Question 29

Why is TBX1 investigation important?

Answer 29

The TBX1 investigation is important as it can provide additional prognostic information to the consultant with additional problems – maybe no treatment. Detection of a genetic abnormality will inform the healthcare of a newborn.