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CytogeneticsB.Sc. MLT fourth semesterChromosome 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 genomeAneuploidyPolyploidy

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 syndromeresults 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 extraXchromosome.Edwards syndromeis caused bytrisomy(three copies) of chromosome 18.Down syndrome, a common chromosomal disease, is caused by trisomy of chromosome 21.Patau syndromeis 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 ofimprintingdisorder.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 ratiosecondary constrictions (nucleolar organisers)The normal human karyotype has 46 chromosomes23 derived from each parentsex is determined by X and y chromosomesMales are XYFemales are XXThe sex of an offspring is determined by the sex chromosome carried in the sperm

Akaryotype is the number andappearanceofchromosomesin thenucleusof aeukaryoticcell. The karyotype is also used for the complete set of chromosomes in aspecies, 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 calledidiogram

Chromosome ShapeAs 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 divisionThe location of a centromere gives a chromosome its characteristic shape15

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 otherAcrocentric: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 chromosomesA 1-3 Large metacentric 1,2 or submetacentricB 4,5 Large submetacentric, all similarC 6-12, X Medium sized, submetacentric - difficultD 13-15 medium-sized acrocentric plus satellitesE 16-18 short metacentric 16 or submetacentric 17,18F 19-20 Short metacentricsG 21,22,Y Short acrocentrics with satellites. Y no satellites.

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

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MetacentricSubmetacentricAcrocentricShort arm (p)SatellitepCentromerepStalkLong arm (q)qq3172122Figure 6.2Human mitotic metaphase chromosomes are identified by size, centromere location, and banding pattern. The relative size, centromere locations, and banding patterns for three representative human chromosomes are shown. Chromosome 3 is one of the largest human chromosomes and, because the centromere is centrally located, is a metacentric chromosome. Chromosome 17 is a submetacentric chromosome because the centromere divides the chromosome into two arms of unequal size. Chromosome 21 has a centromere placed very close to one end and is called an acrocentric chromosome. In humans, the short arm of each chromosome is called the p arm, and the long arm is called the q arm. The symbol p was chosen for the short arm because it stands for the word petit, which in French means small. The symbol q was chosen for the long arm because it is the next letter in the alphabet.Sample collection for cytogenetic analysisPeripheral bloodBone marrow Spinal FluidAmniotic fluidChorionic villi samplingSkin biopsyStorage of the cellsCollected in anticoagulant containing vialsTemporarily(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 37C 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 preparation25The steps in the process of creating a karyotype for chromosome analysis.PREPARATION OF CHROMOSOMES

26METHODOLOGYAseptic precautionsPreparation of RPMI 1640 mediumCollection of 10ml of blood with heparinSetting of culture8 ml of medium0.1 ml of PHA-M0.5 ml of blood/plasma2 ml of FCS/bovineIncubate at 37C for 72 hours

27METHODOLOGYHarvesting of cultureSpindle inhibitors Colchicine/colcemed (0.1g/ml)Hypotonic treatment 0.075M KClFixation (3:1 methanol : acetic acid)Preparation of slidesSlides stained with 4% Giemsa for 20-25minScreening of slides to study the morphology of chromosomeConstruction of karyotype28Metaphase Chromosomes (a) Arranged Into a Karyotype (b)

29(a) A metaphase array of chromosomes as viewed in the microscope. (b) The computer-derived karyotype. Note that the computer has straightened the chromosomes as they were processed and arranged into a karyotype.

Chromosome BandingDeveloped 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 KaryotypesKaryotype construction and analysis are used to identify chromosome abnormalitiesDifferent stains and dyes produce banding patterns specific to each chromosomeKaryotypes reveal variations in chromosomal structure and number1959: Discovery that Down syndrome is caused by an extra copy of chromosome 21 Chromosome banding and other techniques can identify small changes in chromosomal structure31Information Obtained from a KaryotypeNumber of chromosomes

Sex chromosome content

Presence or absence of individual chromosomes

Nature and extent of large structural abnormalities32Four Common Chromosome Staining Procedures

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Banding techniqueAppearance of chromosomesG-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 .34Four common staining procedures used in chromosomal analysis. Most karyotypes are prepared using G-banding. R-banding produces a pattern of bands that is the reverse of those in G-banded chromosomes.G-BandingG-banding of human female metaphase chromosomes

Q-banding Treat metaphase spreads with the chemical quinacrine mustard. Observe uorescent banding pattern with a special ultraviolet light microscope.Bright uorescent 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. 36Four common staining procedures used in chromosomal analysis. Most karyotypes are prepared using G-banding. R-banding produces a pattern of bands that is the reverse of those in G-banded chromosomes.Q-BandingQ-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 chromosomesReverse 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.38Four common staining procedures used in chromosomal analysis. Most karyotypes are prepared using G-banding. R-banding produces a pattern of bands that is the reverse of those in G-banded chromosomes.R-BandingR-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 chromosomes40Four common staining procedures used in chromosomal analysis. Most karyotypes are prepared using G-banding. R-banding produces a pattern of bands that is the reverse of those in G-banded chromosomes.C-Banding C-banding stains the constitutive heterochromatin, which usually lies near the centromere.