HUMAN KARYOTYPING

Sarwat Maroof
Senior. Technologist Cytogenetics section Histopathology

AGA KHAN UNIVERSITY HOSPITAL

What is cytogenetics?
The study of chromosomes and the related

disease states caused by abnormal chromosome number and/or structure.

Macro-visualization of the DNA in

terms of chromosomes

MAIN PURPOSE
Is the visualization of changes, which

affect the normal structure and number of chromosomes of an individual. Visualization of evolution

Setting the number of chromosomes

1891 - 1956 (between 44 & 48) 1956 set as 2n = 46 (Tjio & Levan).

How is the sex of the offspring determined?

In mammals, determined by chromosome

composition Humans: 46 chromosomes total 23 chromosome pairs, 22 pairs are autosomes 1 pair of sex chromosomes Y chromosome carries genes for male development XX = female XY = male

KARYOTYPING
Karyotyping is the arrangement of chromosomes

according to their size.

Banding pattern Centomeric position

Why do we do karyotyping? Post-natal Samples

Pre-natal Samples
Amniotic Fluid Product of Conception

Pre-natal Diagnosis
Autosomal Anomalies Trisomy 13 Trisomy 18 Trisomy 21

Blood Bone Marrow Post-natal Diagnosis Autosomal Anomalies Trisomy 13 Trisomy 18 Trisomy 21 Turners syndrome Klinefelters syndrome Trisomy X Double Y syndrome Proliferated Syndrome

CHROMOSOME STRUCTURE

Human Karyotype

46,XY

...and centromere position.

HOW TO GET CHROMOSOMES

A variety of tissue types can be used to obtain chromosome preparations. Blood Bone marrow Amniotic fluid Product of conception.

Human Karyotype Procedure: blood sample centrifugation isolation of lymphocytes (phytohemaglutinin) incubation in 0.075M KCl (Hypotonic bath) 2 fixation (methanol/ethanol:acetic acid; 3:1) drop on frozen slide from 15 - 25 cm. Stain (Giemsa 4%) visualization of well spread samples. Photo karyotyping.

Banding

G: G banding pattern. Staining with Giemsa. Q: G banding pattern. staining with quinacrin orange. R: Reverse G banding pattern. Staining with Giemsa). C: Centromere and telomere banding pattern (constitutive heterochromatin). Staining with Giemsa )

Smear

Human Karyotype

46,XY

Chromosomes can be identified by their size and banding pattern...

Karyotype

What can we learn from a karyotype? Sex of individual Chromosome number Euploid = 46 chromosomes Aneuploid = extra or missing chromosome(s) Trisomy: three homologous chromosomes Monosomy: one chromosome Polyploid = extra chromosome sets Large changes in chromosome structure Sex determination

Chromosome Abnormalities SEX CHROMOSOMAL DISORDER

Turners Syndrome (XO) Klinefelters Syndrome XYY Syndrome XXX Syndrome

AUTOSOMAL DISORDER

Downs Syndrome (3 of type 21) Edwards Syndrome (3 of type 18) Pataus Syndrome (3 of type 13) Translocations: Chronic myelogenous leukemia (type 9 and type 22)

Changes
Structural
translocations inversions insertions

Numerical euploidy aneuploidy

ALL autosomal monosomics die, BUT XO individuals survive and are relatively normal!!! There is something different about the autosomes and sex chromosomes.

XXX Females are normal

XXXX and XXXXX females also exist

Another difference between sex chromosomes and autosomes. Autosomal trisomies die, but XXY, XYY, XXX, XXXX and XXXXX survive.

Abnormalities
15% of pregnancies end up in spontaneous abortions. 50% are chromosomal abnormalities. 25% are aneuploidies

Nomenclature
For a normal human being: 46, XY/46, XX For an abnormal number: 47, XX +21.(Downs

Syndrome)

Downs Syndrome
Studied in the 1860s - Landon Down.
Frequency: 1/800 - 1/700 In every cell: 95%

Mosaics: 2%
Robertsonian translocation: 3% Cause: Trisomy of chromosome 21

(47, XY +21/47, XX +21) (Lejeun -1959)

Symptoms

Trisomy 21; Down Syndrome

Can survive to age 50 Mental retardation

Patau Syndrome
Trisomy of the 13
47, XY +13/47, XY+13 Frequency = 1/12000 Life term = up to 4 years (10%)

Symptoms
Forebrain folds. cleft lip proximal heel low set ears

Trisomy 13; Patau Syndrome

Most fetuses spontaneously abort

Edwards Syndrome
Trisomy of the 18 47, XY +18/47, XX +18 Frequency = 1/7000 - 1/3500 80% straight trisomy 10% mosaics Life term = up to one year (10%)

Symptoms

Presents most of the Patau syndrome

symptoms, but in a less

severe manner.

Overlapping fingers

Trisomy 18; Edwards Syndrome

Human Aneuploidy in Sex Chromosomes

Turner Syndrome

Frequency = 1/2000 45, X apparently independent of the mother's age. It seems to be the fathers chromosome that is missing.

Symptoms

Turner syndrome, XO
Female Short, wide-chested Underdeveloped breasts Rudimentary ovaries Sterile Normal intelligence

Klinefelter Syndrome

Frequency = 1/500 47, XXY (47, XYY tall & violent) Symptoms: small testis gynecomastia (male breast) mild mental retardation psychotic tendency

Klinefelter syndrome, XXY

Male Phenotype of syndrome not apparent until puberty Breast development Low fertility Subnormal intelligence

Modified Klinefelter
49, XXXXY: very retarded, skeletal anomalies, hypogenitalism, strabismus, wide eye set. 48, XXYY: violent behavior, taller than normal men. Others: XXXYY, XYYY, XYYYY. 47, XXX: normal women, but sometimes with abnormal sex development.

Modified Klinefelter
49, XXXXY: very retarded, skeletal anomalies, hypogenitalism, strabismus, wide eye set. 48, XXYY: violent behavior, taller than normal men. Others: XXXYY, XYYY, XYYYY. 47, XXX: normal women, but sometimes with abnormal sex development.

XYY Individuals
~5% criminals with violent and antisocial behavior are XYY, but only 0.1% of males in population XYY

XYY Syndrome

THANX

THE END