Cytogenetics B.Sc. MLT fourth semester

Chromosome Abnormalities A chromosome abnormality reflects an abnormality of chromosome number or structure. There are many types of chromosome abnormalities. They can be organized into two basic groups: Numerical Abnormalities: – When an individual is missing either a chromosome from a pair (monosomy) or has more than two chromosomes of a pair (trisomy) or have abnormal sets of genome Aneuploidy Polyploidy

Structural Abnormalities: When the chromosome's structure is altered. This can take several forms: – Deletions: A portion of the chromosome is missing or deleted. – Duplications: A portion of the chromosome is duplicated, resulting in extra genetic material. – Translocations: When a portion of one chromosome is transferred to another chromosome. In a reciprocal translocation, segments from two different chromosomes have been exchanged. In a Robertsonian translocation, an entire chromosome has attached to another at the centromere. – Inversions: A portion of the chromosome has broken off, turned upside down and reattached, therefore the genetic material is inverted. – Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material.

Chromosomal abnormalities that lead to disease in humans include – Turner syndrome results from a single X chromosome (45, X or 45, X0). – Klinefelter syndrome, the most common male chromosomal disease, otherwise known as 47, XXY is caused by an extra X chromosome. – Edwards syndrome is caused by trisomy (three copies) of chromosome 18. – Down syndrome, a common chromosomal disease, is caused by trisomy of chromosome 21. – Patau syndrome is caused by trisomy of chromosome 13.

Cri du chat (cry of the cat), from a truncated short arm on chromosome 5. The name comes from the babies' distinctive cry, caused by abnormal formation of the larynx. 1p36 Deletion syndrome, from the loss of part of the short arm of chromosome 1. Angelman syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the maternal genes, example of imprinting disorder. Prader-Willi syndrome – 50% of cases have a segment of the long arm of chromosome 15 missing; a deletion of the paternal genes, example of imprinting disorder.

How do chromosome abnormalities happen? Chromosome abnormalities usually occur when there is an error in cell division.. In mitosis and meiosis processes, errors in cell division can result in cells with too few or too many copies of a chromosome. Errors can also occur when the chromosomes are being duplicated. Other factors that can increase the risk of chromosome abnormalities are: – Maternal Age: – Environment:

The number size and shape of the chromosomes of a somatic cell arranged in a standard manner. – position of centromere - arm length ratio – secondary constrictions (nucleolar organisers) The normal human karyotype has 46 chromosomes – 23 derived from each parent – sex is determined by X and y chromosomes – Males are XY – Females are XX – The sex of an offspring is determined by the sex chromosome carried in the sperm

A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. The karyotype is also used for the complete set of chromosomes in a species, or an individual organism. Karyograph is a diagram or photograph of the chromosomes of a cell, arranged in homologous pairs and in a numbered sequence also called idiogram

Chromosome Shape As chromosomes condense and become visible during cell division, certain structural features can be recognized Centromere – A region of a chromosome to which microtubule fibers attach during cell division – The location of a centromere gives a chromosome its characteristic shape

The position of the centromere in the chromosome (which is constant to a given chromosome) varies i.e., it may occupy different positions. Based on this, four morphological shapes have been identified in chromosomes. – Metacentric: – The Centromere occupies a middle position with reference to the length of the chromosoem. – Sub metacentric: – When the centromere is located some distance away from the middle region of the chromosome, the position is said to be median and the chromosome will be shorter than the other – Acrocentric: – In this case, the centromere is situated almost near one end of the chromo­some. – Telocentric: – When the centromere is situated exactly at one end, the chromosome will be having only one long arm.

Human chromosomes are divided into 7 groups & sex chromosomes – A 1-3 Large metacentric 1,2 or submetacentric – B 4,5 Large submetacentric, all similar – C 6-12, X Medium sized, submetacentric - difficult – D medium-sized acrocentric plus satellites – E short metacentric 16 or submetacentric 17,18 – F Short metacentrics – G 21,22,Y Short acrocentrics with satellites. Y no satellites.

Metaphase Chromosomes Chromosomes are identified by size, centromere location, banding pattern

MetacentricSubmetacentricAcrocentric Short arm (p) Satellite p Centromere p Stalk Long arm (q) q q 31721

Sample collection for cytogenetic analysis Peripheral blood Bone marrow Spinal Fluid Amniotic fluid Chorionic villi sampling Skin biopsy

Storage of the cells Collected in anticoagulant containing vials Temporarily(3-4 hrs)- room temperature > 4 hrs(~5 days)- in refrigerator > 5 days-Cryopreservation

Add a few drops of blood. Add phytohemagglutinin to stimulate mitosis. Draw 10 to 20 ml of blood. Incubate at 37°C for 2 to 3 days. Transfer cells to tube. Add Colcemid to culture for 1 to 2 hours to stop mitosis in metaphase. Centrifuge to concentrate cells. Add low-salt solution to eliminate red blood cells and swell lymphocytes. Drop cells onto microscope slide. Examine with microscope. Digitized chromosome images processed to make karyotype. Stain slide with Giemsa. Cell culture and metaphase chromosome slide preparation

PREPARATION OF CHROMOSOMES

METHODOLOGY Aseptic precautions Preparation of RPMI 1640 medium Collection of 10ml of blood with heparin Setting of culture 8 ml of medium 0.1 ml of PHA-M 0.5 ml of blood/plasma 2 ml of FCS/bovine Incubate at 37  C for 72 hours

METHODOLOGY… Harvesting of culture Spindle inhibitors – Colchicine/colcemed (0.1  g/ml) Hypotonic treatment – 0.075M KCl Fixation (3:1 methanol : acetic acid) Preparation of slides Slides stained with 4% Giemsa for 20-25min Screening of slides to study the morphology of chromosome Construction of karyotype

Metaphase Chromosomes (a) Arranged Into a Karyotype (b)

Chromosome Banding -Developed based on the presence of heterochromatin and euchromatin. - Heterochromatin is darkly stained where as euchromatin is lightly stained during chromosome staining. - A band is defined as that part of a chromosome which is clearly distinguishable from its adjacent segments by appearing darker or brighter with one or more banding techniques. -There are a few types of chromosome banding: G-banding, C-banding, Q-banding, R-banding etc.

Constructing and Analyzing Karyotypes Karyotype construction and analysis are used to identify chromosome abnormalities Different stains and dyes produce banding patterns specific to each chromosome Karyotypes reveal variations in chromosomal structure and number – 1959: Discovery that Down syndrome is caused by an extra copy of chromosome 21 Chromosome banding and other techniques can identify small changes in chromosomal structure

Information Obtained from a Karyotype Number of chromosomes Sex chromosome content Presence or absence of individual chromosomes Nature and extent of large structural abnormalities

Four Common Chromosome Staining Procedures

Banding techniqueAppearance of chromosomes G-banding — Treat metaphase spreads with trypsin, an enzyme that digests part of chromosomal protein. Stain with Giemsa stain. Observe banding pattern with light microscope. Darkly stained G bands. It yields a series of lightly and darkly stained bands - the dark regions tend to be heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic, early-replicating and GC rich.

G-Banding G-banding of human female metaphase chromosomes

Q-banding — Treat metaphase spreads with the chemical quinacrine mustard. Observe fluorescent banding pattern with a special ultraviolet light microscope. Bright fluorescent bands upon exposure to ultraviolet light; same as darkly stained G bands. Banding techniqueAppearance of chromosomes - This method requires a fluorescence microscope (quinacrine fluoresces strongly in the ultraviolet) and is no longer as widely used as G-banding.

Q-Banding Q-banding of human male metaphase chromosomes

R-banding — Heat metaphase spreads at high temperatures to achieve partial denaturation of DNA. Stain with Giemsa stain. Observe with light microscope. Darkly stained R bands correspond to light bands in G- banded chromosomes. Pattern is the reverse of G-banding. Banding techniqueAppearance of chromosomes -Reverse banding (R-banding) requires heat treatment and reverses the usual white and black pattern that is seen in G-bands and Q- bands. -R-banding is the reverse of G-banding (the R stands for "reverse"). - the dark regions are euchromatic (guanine-cytosine rich regions) and the bright regions are heterochromatic (thymine-adenine rich regions). - telomeres are stained well by this procedure.

R-Banding R-banding of human female metaphase chromosomes

C-banding — Chemically treat metaphase spreads to extract DNA from the arms but not the centromeric regions of chromosomes. Stain with Giemsa stain and observe with light microscope. Darkly stained C band centromeric region of the chromosome corresponds to region of constitutive heterochromatin. Banding techniqueAppearance of chromosomes

C-Banding - C-banding stains the constitutive heterochromatin, which usually lies near the centromere.