Genetic Screening - Part 2

Question 1

What techniques are may be used in prenatal diagnosis?

Answer 1

1) . Invasive - high risk
- Amniocentesis
- Chorionic villus sampling
- Foetal blood sampling

2) . Semi invasive - intermediate risk
- Coelocentesis
- Uterine lavage

3) . Non invasive - low risk
- Foetal cells in maternal circulation
- Foetal DNA in maternal circulation
- Preimplantation Diagnosis

The problem at the moment is that definitive diagnostic tests are the invasive tests.

Question 2

Describe current techniques used for prenatal diagnosis.

Answer 2

1) . Amniocentesis - rapid result by FISH / QF-PCR - final result in 2-3 weeks - chromosome quality is good.
2) . Chorionic Villus - rapid result from direct preparations or FISH / QF-PCR - final result in 2-3 weeks - chromosome quality is intermediate and poor from the direct test.
3) . Fetal blood sampling - FISH or 24-48 hour rapid result - final result in 3-4 days - chromosome quality is very good.

Question 3

Outline the principle of amniocentesis.

Answer 3

• Amniocentesis - traditional test used for many years.
• 10-20ml amniotic fluid withdrawn trans abdominally using a needle under ultrasound guidance.
• Relies on cells from foetal skin, kidney, bladder, gut, GI tract and extra embryonic membranes present that may be grown for karyotyping - multiple tissue sampling.
• Miscarriage 0.5-1% - only suitable for high prior risk cases - need for screening.
• Quality chromosome preparations within 14-16 days.
• Considered a reliable indicator of foetal karyotype.
• Approximately 0.3% of cases may be mosaic; a false positive or true findings.
• Drawback 16-18 weeks diagnosis; late medical termination of pregnancy (legal limit 24 weeks).
• It has to be a late test due to the amount of amniotic fluid present in the sac. Need to be able to safely draw off material.

Question 4

Outline the differences between open and closed culturing systems.

Answer 4

1) . Open system - 37degrees + CO2 + H20
- The principle of an open system is free exchange of gas between culture medium/cells and incubator chamber.
- pH buffer system (pH 7.2 - 7.4) = bicarbonate (base) / 5% CO2 (acid).
- Humidification via incubator - vessels / petri dishes ventilated.
- Typical application is for in situ cultures (cover slip or slide) or loosely capped flasks / tubes.
2) . Closed system - 37degrees and dry
- The principle of a closed system is no exchange of air between medium / cells and incubator chamber.
- pH buffer system (pH 7.2 - 7.4) = Buffer (e.g. Hepes) / bicarbonate / internal CO2.
- Humidification - internal - air tight vessels.
- Typical application - flask or leighton tube (tightly sealed) culture vessels.

Question 5

What are the advantages and disadvantages of open systems?

Answer 5

ADVANTAGES OF OPEN SYSTEMS:

• Cultures reported to grow more rapidly.
• Final results available in a shorter time.

DISADVANTAGES OF OPEN SYSTEMS:

• Expensive incubators with CO2 regulator.
• External supply of CO2 and back up change over unit.
• Increased risk of infection / cross contamination in humidified atmosphere of incubator.
• Maintenance of incubators - corrosion.

Question 6

What are the advantages and disadvantages of closed systems?

Answer 6

ADVANTAGES OF CLOSED SYSTEMS:

• Less risk of infection or air born (cross) contamination - no incubator humidification.
• Simple (less expensive) incubators.

DISADVANTAGES OF CLOSED SYSTEMS:

• Cultures reported to grow less rapidly.
• Longer time to report.

Question 7

In what ways can cells be grown?

Answer 7

• Can grow cells in suspension but amniotic fluid cells and long term CVS will not grow like this as they need a matrix to grow. Cells are grown as monolayer long term culture (ltc) (e.g. amniotic fluid, chorionic villus and solid tissues from foetal products or tumours).
• CVS long term cultures ad amniotic fluid cells are grown in a medium on a coated matrix - coated plastic vessels.
• You need to get them to divide as if they are in an in utero situation. Grown with medium containing growth factors and carbon source and is pH controlled.
• Can grow to colony harvests, we can synchronise cultures and we can disperse cells to get optimal cell growth.

Question 8

Outline QA control for prenatal cultures.

Answer 8

• Set up multiple cultures - at least 3 for each sample.
• Use at least 2 or 3 different sources of medium.
• All work done in class II cabinet under aseptic conditions / procedure - mainly to protect the sample.

Question 9

Outline the process of harvesting prenatal cultures.

Answer 9

• Cells need to be taken off the monolayer. Cells are trypsinised and put into a suspension.
• Cells then exposed to a hypotonic solution, fixative and then slide preparation takes place.
• Slides will be G-banded or stained.
• Cell prep environment crucial - humidity and temp very important.
• Long term tissue culture of amniotic fluid cells take 7-10 days in culture to be sufficient for analysis.

Question 10

How does mosaicism occur?

Answer 10

Post-zygotic nondisjunction events leading to mixed populations of cells.

Question 11

Outline the different classifications of mosaicism.

Answer 11

Mosaicism is the post zygotic loss or gain of chromosomes in two or more cells.

1) . Genuine Mosaicism - affecting somatic (and germ) cells of individual.
2) . Pseudo mosaic - false mosaic diagnosis arising as a tissue culture artefact.
3) . Inter tissue mosaicism - mosaicism restricted to some tissues.
4) . Confined placental mosaicism - restricted to placenta tissue and not in the foetus (and associated UPD risks) (chorionic villus analysis).

Question 12

What is the % mosaicism in trisomy 21?

Answer 12

3%

Question 13

What is the % mosaicism in trisomy 18?

Answer 13

10%

Question 14

What is the % mosaicism in trisomy 13?

Answer 14

5%

Question 15

What is the % mosaicism in 45, X?

Answer 15

35%

Question 16

What is the % mosaicism in trisomy 8?

Answer 16

100%

Question 17

What is the % mosaicism in XXY?

Answer 17

20%

Question 18

What is the % mosaicism in i(12)(p10)?

Answer 18

100%

Question 19

What is the % mosaicism for markers?

Answer 19

20-30%

Question 20

List % mosaicism for different conditions.

Answer 20

Trisomy 21 = 3%
Trisomy 18 = 10%
Trisomy 13 = 5%
Trisomy 8 = 100%
45, X = 35% 
XXY = 20%
i(12)(p10) = 100%
markers = 20-30%

Question 21

Describe i(12)(p10)?

Answer 21

• i(12)(p10) is Pallister Killian - extra chromosome 12p - inter tissue mosaicism could confound the diagnosis.
• Coarse features, diaphragmatic hernia, pigmented skin abnormalities, profound MR and seizures, supernumerary nipples.
• Supernumerary isochromosome for 12p - tetrasomic for 12p.
• If you choose the wrong tissue type to test you may not diagnose an individual with Pallister Killian.

Inter tissue mosaicism:

• Peripheral blood cells - 0-2% cells abnormal
• Skin fibroblast cultures - 50-100% cells abnormal
• Amniocytes/bone marrow cultures - 100% cells abnormal
• Can lead to problems with diagnosis of isochromosme 12p if wrong tissue analysed.

Question 22

Outline some of the physical features of Pallister Killian.

Answer 22

• Coarse facial features, frontal bossing.
• Localised alopecia and sparse eyebrows.
• Low set dysplastic ears, hypertelorism, long philtrum.
• Small nose, upturned nares, wide flat nasal bridge.
• Prominent upper lip.

Question 23

What do you do if you find a single cell that is abnormal against a background of normal cells? How do you assess likelihood of mosaicism in prenatal cultures?

Answer 23

• Usually would just look at more cells - use Hook’s tables to determine how many cells you need to analyse to get a certain degree of confidence.
• Assumes independently derived cells and is not applicable to long term cultures. This is a problem in prenatal diagnosis as we use long term cultures.
• In prenatal samples we assess mosaicism by looking at various cultures - three levels of mosaicism - multiple culture or cover slip / slide systems.
• Have defined three levels of abnormality that works on culture vessels and in situ systems also.
  1) . LEVEL 1 = when a single cell is abnormal in a single flask or cover slop prep.
• 2.5-7% of amniocentesis
• Unlikely to represent a genuine mosaicism in the individual = pseudomosaic.
  2) . LEVEL 2 = when multiple cells are abnormal but only in one slide or one tube.
• 0.7%-1.1% of amniocentesis samples
• In long term culture likely to represent a tissue culture artifact = mainly a pseudomosaic

BUT 1% of level 2 mosaics present a true foetal chromosome abnormality!

• Contingent to chromosomes involved - see BPGs for mosaicism evaluation.
  3) . LEVEL 3 = when multiple cells in more than one culture or slide are abnormal.
• approximately 0.2% of amniocentesis - likely to be a genuine mosaicism.

Question 24

Outline the principles of chorionic villus sampling.

Answer 24

• CVS is the only first trimester diagnosis sample that we have got at the moment.
• Like any villus it is composed of 2 layers, an outer cytotrophoblast layer and an inner mesenchymal core layer.
• The outer cytotrophoblast layer is spontaneously dividing so we can perform on this material a suspension harvest - it doesn’t need to grow in a monolayer.
• Also have the mesenchymal core layer which requires a layer to attach to and grow on.
• Direct (cytotrophoblast) same day / 24 hour result - aneuploidy / large chromosome abnormality.
• Culture (mesenchyme core) - 2-3 weeks - small chromosome abnormalities.
• Direct CVS are of poor quality and can be hard to interpret with certainty. May see something else on higher re mesenchyme culture karyotype.

Question 25

What are the advantages and disadvantages of CVS?

Answer 25

ADVANTAGES:

• true first trimester diagnosis
• prompt molecular and cytogenetic studies possible
• two stage results system including direct (cytotrophoblast) 24 hr result and culture (mesenchyme core) 2-3 week result.

DISADVANTAGES:

• skill to perform the technique safely (approximately 1% foetal loss)
• false positive (1-2%) / false negative issues (0.01%-0.6%) -follow up test
• mosaicism / CPM vs TFM - requiring follow up invasive testing (1-2%)
• trisomic rescue and UPD issues
• The real problems with CVS are the way it differentiates. After fertilisation the embryo goes through cleavage forming the morula surrounded by the zona pelucida differentiates up to day 5 or 6 where it then forms a blastocyst forming the trophoblast and the ICM. From one cell only from the ICM will the embryo truly form. The cytotrophoblast differentiates out early in the developmental tree so if you get a mitotic error after this point forming a trisomic cell you will find that you have abnormal cells in the trophoblast but the embryo is normal. You have what is called a type I confined placental mosaic where the direct is abnormal but the culture and embryo are potentially normal. Might get the inverse situation for cells in the mesenchymal core where you get an abnormal culture result but a normal direct test, this is called type II confined placental mosaicism. It is all about what point the cells differentiate and how divergent they are from the cells that go on to form the embryo.
• Can also get trisomic rescue scenarios. Firstly may actually have an abnormal baby but we pick up normal cells in the placenta. Alternatively we may pick up abnormal cells in the placenta but the baby may be fully normal - this may indicate an inherent risk of UPD leading to imprinting defects.

Question 26

What is Type I confined placental mosaicism?

Answer 26

Type I confined placental mosaicism = CVS direct is abnormal but the culture is normal - foetus normal.

Question 27

What is Type II confined placental mosaicism?

Answer 27

Type II confined placental mosaicism = CVS culture is normal but the direct is abnormal - foetus normal.

Question 28

Outline the different types of confined placental mosaicism.

Answer 28

CMP Type I - CPM - Direct Abnormal - Culture Normal - Foetus Normal - Relative Frequency 48%

CMP Type II - CPM - Direct Normal - Culture Abnormal - Foetus Normal - Relative Frequency 25%

CMP Type III - CPM - Direct Abnormal - Culture Abnormal - Foetus Normal - Relative Frequency 16%

CMP Type IV - TFM - Direct Abnormal - Culture Normal - Foetus Abnormal - Relative Frequency 0%

CMP Type V - TFM - Direct Normal - Culture Abnormal - Foetus Abnormal - Relative Frequency 4%

CMP Type VI - TFM - Direct Abnormal - Culture Abnormal - Foetus Abnormal - Relative Frequency 7%

Question 29

What conditions can arise as a result of UPD?

Answer 29

• Maternal UPD of chromosome 2 = IUGR - normal postnatal development - CPM involvement.
• Paternal UPD of chr 6 = neonatal diabetes or autosomal recessive gene.
• Maternal UPD of chr 7 = Silver Russell syndrome (10%). CPM involvement.
• Paternal UPD of chr 11 = BWS 10-20% of cases segmental UPD.
• Maternal UPD of chr 14 = short stature, precocious puberty.
• Paternal UPD of chr 14 = MR, skeletal abnormalities.
• Mat UPD 15 = 15% PWS cases.
• Pat UPD 15 = 5% AS cases.
• Mat UPD 16 = IUGR (dev delay). CPM involvement.

Question 30

Concluding remarks about CVS testing:

Answer 30

• Very useful (rapid) method of prenatal diagnosis.
• Only first trimester diagnostic test available currently - first trimester screening programme compliant.
• Issues of accuracy need to be handled carefully in the absence of USS markers.
• Prenatal diagnosis strategies (including counselling) are well documented.
• Concerning mosaicism issues - more data emerging with time.

(EUCHROMIC = European chromosome mosaicism in CVS study, Association of Clinical Cytogeneticists (UK)).

Question 31

Describe fetal blood sampling.

Answer 31

• 2% miscarriage risk.
• Technically more difficult.
• Risk of maternal sampling if cord insertion used.
• Better chromosome preps than AF or CVS.
• Preliminary results in 48 hrs.
• Full results within 3-4 days.
• 18+ weeks gestation.

Question 32

From what gestational age can CVS be used?

Answer 32

10 weeks onwards.

Question 33

From what gestational age can Early Amniocentesis be used?

Answer 33

14 weeks onwards.

Question 34

From what gestational age can Foetal Blood be used?

Answer 34

18 weeks onwards.