Clinical Cytogenetics

19.2.07

What is cytogenetics?
• Study of chromosomal pathology.
• Chromosomes are protein/DNA macromolecules.
• They carry genes.
• When they mutate, they cause phenotypic changes - some are recognised as syndromes.
• Constitutional changes.
• Acquired changes.

Cell and tissue culture
• There are a variety of tissues received in the Cytogenic laboratory, which include:
-Blood - lymphocytes.
-Amniotic fluid - foetal cells.
-Chorionic villus.
-Bone marrow.
-Products of conception, skin and tumour tissue.
• Most cells and tissues need to be cultured - exception to this:
-Bone marrow cells.
-Trophoblasts seen in chorionic villus tissue.

Investigations on blood samples
• Neonates - know syndromes and unexplained dismorphism and/or congenital abnormalities.
• Childhood developmental/intellectual delay.
• Pubertal failure.
• Infertility.

Prenatal diagnosis
• Indications for prenatal diagnosis include Downs risk from sera screening.
• Abnormalities seen on ultrasound scan.
• Known carriers of single gene/chromosomal disorder.
• Parents who have had previously chromosome-affected child.

Methodologies
1. Cell culture: mitotic cell division stopped at metaphase.
2. Stain by G banding: molecular Cytogenic - special identity by FISH.
3. Microscopy: molecular genetic methods e.g. MLPA, RTFCR.
4. Microarrays.
5. Write detailed interpretative reports.

Cell and tissue culture
• Aspects to be considered in cell culture include:
-Sterility - aseptic techniques, biological safety cabinets, antibiotics.
-Culture vessels - plastic/glass.
-Culture media - provides nutrients, maintains pH.
-Temperature - 370C maintained using incubator.
-Culture environment - carbon dioxide and oxygen levels can be altered.
-Mitogens.
-Harvesting procedure.
-Stop cells growing at metaphase using colcemid.
-Hypotonic solution swells cells.
-Fixative (methanol/acetic acid).

Molecular cytogenics - fluorescence in-situ hybridisation (FISH)
• Hybridises probes with complementary sequences to target DNA.
• Procedure consists of:
-Denaturation.
-Hybridisation.
-Stringency washing.
-Visualisation.
• Extend cytogenetic analysis to non-dividing cells.

New technologies - arrays
• Array CGH, BAC arrays and oligonucleotide arrays.
• Arrays consist of specific DNA sequences, which may be spotted onto a glass slide/some other substrate.
• Hybridisation with target DNA then performed.

What might cause non-disjunction?
• Failure of chromosomes to separate at meiosis I/chromatids at meiosis II.
• Asynapsis = Failure of chromosomes to pair.
• Asynapsis can be deduced by observing that number of chiasma reduced/non-existent.
• Can be estimated by recombination frequency - known that older women (>35 years) have half number recombinations compared to younger women.

Viable trisomy conditions
• Trisomy 21 = Down's.
• Trisomy 13 = Patau.
• Trisomy 18 = Edwards.

Sex chromosome aneuploidy
• 45, X = Turner.
• 47, XXX = Triple X.
• 47, XXY = Klinefelter.
• 47, XYY.

X chromosome inactivation
• In normal female with 2Xs, one X inactivated.
• In male with Klinefelter, one X inactivated.

Chromosomal abnormalities (structural)
• Deletion.
• Duplication.
• Isochromosome.
• Inversion.
• Translocation.

Deletions
• Deletion with >2% of total haploid genome will result in lethal outcome.
• Smallest viable loss from chromosome ~4Mb.
• Therefore, large number of contiguous genes lost, resulting in intellectual retardation and congenital malformations.

Microdeletions
• Describes small chromosomal loss only detectable by FISH.
• Well-described syndromes include:
-Prader-Willi.
-Angelman.
-William's.
-Wolf Hirschorn.
-Cri-du-chat.
-DiGeorge.
-Miller Dicker.

Reciprocal translocations
• Associated with:
-Impaired spermatogenesis.
-Miscarriages.
-Congenital abnormalities due to segregation at meiosis, resulting in unbalanced gametes.
• Identified as exchange of chromosomal material between non-homologous chromosomes.
• Break points can be in short/long arm - generally in non-transcribing DNA.
• Therefore little, if any, phenotypic effect.

Robertsonian translocations
• Affects acrocentric chromosomes 13, 14, 15, 21 and 22.
• Associated with recurring miscarriage and impaired spermatogenesis.
• Mal-segregation can give rise to Down's if 21 involved or Patau's if 13 involved.

Inversions
• Paracentric gametes - normal/inversion/dicentric/acentric.
• Pericentric gametes normal, inversion, duplication/deletion.
• In large pericentric inversions, duplicated and deleted segments very small therefore, newborns may survive.

Haematological malignancies - acquired disorders
• Clonal expansion of neoplastic cells derived from (or resembling) normal haematological cells.
• In general, leukaemias and lymphomas classified according to stage of normal haematopoiesis at which cells appear to be blocked.

Bone marrow samples
• Bone marrow obtained via iliac crest puncture by haematologist.
• Reasons for referral include:
-Confirmation of diagnosis for chronic myeloid leukaemia.
-Acute myeloid leukaemia (M2, M3, M4).
• Indication of prognosis, particularly in childhood ALL.