Cytogenetics

Definition

Cytogenetics is the study of chromosomes. It includes the study of normal and abnormal chromosomes, and investigation of the causes of chromosomal abnormalities. It is actually a visual study of chromosomes at microscopic level.

Chromosomes

  • Centromere-movement during cell division divides the chromosomes into short (p) Long (q) arms
  • Telomere-tip of each chromosome
    • seal chromosomes and retain integrity
    • telomere consists of tandem repeats TTAAGGG
    • maintained by enzyme – telomerase
    • reduction in telomerase and decrease in number
    • repeats important in ageing and cell death

Chromosomes are dense bodies found in the nucleus of cells. They contain all the genetic information necessary to create a living being.

Chromosome pairs look a bit like stick figures of people-each with two arms and two legs-the longarms and the short arms joined in the middle-centromere.

CHROMOSOMES DIVIDED INTO GROUPS LABELLED A_G

  • A - 1-3
  • B - 4-5
  • C - 6-12+X
  • D - 13-15
  • E - 16-18
  • F - 19-20
  • G - 21-22+Y

Chromosomes are classified according to:

  • Length
  • Position of centromere
  • Presence or absence of sattelites

KARYOTYPE

  • Staining methods to identify chromosomes:
  • G Banding - Giesma
  • Q Banding - Quinacrine
  • R Banding - Reverse
  • C Banding - Centromeric (heterochromatin)
  • Ag Banding - Nucleolar Organizing Regions (active)

Karyotyping – cell preparation

  • Need metaphases
  • Culture cells until sufficient mitotic activity
  • Add colchicines (or colcemid) to arrest in metaphase(prevents mitotic spindle fibres forming
  • Add hypotonic salt solution to swell cells
  • Fix with mix of methanol;acetic acid
  • Want long chromosomes with none overlapping

G banding

  • Most common method used
  • Chromosomes treated with trypsin (denatures protein)
  • Geisma stain;
  • each chromosome characteristic light and dark bands
  • 400 bands per haploid genome
  • each band corresponds to 5-10 megabases
  • high resolution (800 bands ; prometaphase chromosome)
  • use methotrexate and colchicines

G banding

  • Metaphase spreads
  • Count chromosomes in 10-15 metaphases
  • If mosaicism suspected, count 30
  • Detailed analysis of 3-5 metaphases
  • Used to photograph and cut out
  • Now Computer programme

NORMAL MALE KARYOTYPE

  • 13,18,21 gene poor(very dark chromosomes)
  • 21 smaller than 22 (wrong way around)
  • 22 twice as many genes as 21 (200 on 21) (400 on 22)

Q Banding

  • Used for Y chromosome abnormalities/mosaicism
  • Similar pattern to G banding(can detect Polymorphisms)
  • Needs fluorescent microscopes

R banding

  • Used to identify X chromosome abnormalities
  • Heat chromosomes before staining with Giesma
  • Light and dark bands are reversed

C banding

  • Used to identify
  • 1 centromeres
  • 2 heterochromatin
  • Heterochromatic regions
  • contain repetitive sequences
  • highly condensed chromatin fibres
  • Treat with chromosomes with
    • Acid
    • Alkali
    • then G band

ISCN

  • International System for Human Cytogenetic Nomenclature
  • Each area of chromosome given number
  • Lowest number closest (proximal) to centrome
  • Highest number at the tips (distal) to centromere

ISCN

  • del – deletion
  • dic – dicentric
  • fra – frafile site
  • i - isichromosome
  • inv – inversion
  • p - short arm
  • r - ring
  • der – derivative
  • dup – duplication
  • h - heterochromatin
  • ins - insertion
  • mat – material origin
  • q - long arm
  • t - translocation

ISCN

  • 46,XX,del(5p) • separates chrosome numbers sex chromosomes chromosome abnormalities
  • 46,XX,t(2;4)(q21;q21) • separates altered chromosomes break points in structural rearrangements involving more than 1 chromosome NORMAL MALE -- 46,XY NORMAL FEMALE -- 46,XX