Cytogenetics

What is cyto-genetics?

• Chromosome Analysis
• FISH test e.g. TBX1 gene in Chromosome 22 for Di George syndrome (most common microdeletion syndrome in the world)
• MLPA - Multiplexing PCR so mutliple tests can be ran on the same sampel
• Quatitative Fluorescent-PCR
• array CGH/SNP array

Cyto-genetics resolution is 5Mb and usually used cell culture and microscopy where as molecular genetics is 1bp and used DNA extraction and PCR.

Primary tissues for testing: blood, bone marrow, amniotic fluid, chorionic villus biopsies, skin biopsies.

37'C incubator,  media that includes nutirents required and growth factors. For blood PHA which stimulates T-cells. Then add  Colcemid (derived from kidney beans) to stop cell division at metaphase. The cells are then processed and the suspension is put onto a slide to view under the microscope or use a slide scanner which finds 120 metaphases per patient which reduces time.

Chromosome Classification

Short arm = p (petit) and long arm = q

1.Metacentric - p arm and q arm are approx the same length e.g chromosomes 1,3,16,19 & 20

2. Sub-metacentric - p arm shorter than q arm e.g 4,5,17,18, most others

3. Acrocentric - satellite structure present on p arm, p arm very small e.g 13,14,15,21,22

X and Y are sex chromosomes, all others are autosomes

Giemsa Banding

• Pre-treatment with proteolytic enzyme trypsin
• Dark bands - tightly packed, enzyme cannot degrade chromatin = gene poor
• Light bands - open structure, enzyme can degrade the chromatin = gene rich
• G-banding is very reproducible

Can change the amount of time cells have in colcemid in order to change resolution of the banding pattern.

Karyotyping has a resolution of 4-5Mb

Constitutional Cytogenetics

Prenatal - 600 samples (400 CVS, 200 Amniotic fluid) and Postnatal case load - 1500 samples (venous blood, buccal swab)

Main reasons for referral:

Prenatal: pregnant women at risk of having a chromosomally abnormal baby.

• Two tests available: Chorionic villus biopsy where tissue is taken from the placenta and amniocentesis where fluid is taken from the sac surrounding the baby
• Need to have a risk of over 1/200 to have these invasive tests because there is 0.5% chance of miscarriage caused by the test

Prenatal case study

• 25ml of amniotic fluid. 0.5ml of amniotic fluid QF-PCR, rapid result (<48hours). 20+mls culture in flasks (>10 days)
• An abnormal QF-PCR result -> Karyotype, if normal QF-PCR results -> SNP array (better resolution)
• Look for the most common autosomal trisomy's: 13,18 and 21

QF-PCR gives you a graph with peaks to show where the alleles are located. 2 peaks = one maternal marker and one paternal marker, both are markers are slightly different. Can be the same marker which resutls in only one peak would show which is uninformative. 3 peaks shows that there are 2 alleles and therefore a trisomy.

Robertsonian Translocation - 2 chromosomes joined together.

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