Cytogenetics: Karyotypes and Chromosome Aberrations

Cytogenetics = The study of chromosome number, structure, function, and behavior in relation to gene inheritance, organization and expression Chromosome Chromo = colored in response to dye Some = body Chromosome of Eukaryotes have been the traditional subject for cytogenetic analysis because they are large enough to be examined with light microscope

Chromosome Number
Chromosome number in selected organisms

Cytogenetic methods to detect chromosomal abnormalities underlying human birth defects usually involve analysis of mitotic chromosomes

Why Analyse Chromosomes and Genes?

Genetic errors arise from deletions or insertions of genetic material, abnormal numbers of whole chromosomes or genes, and even from misplacement of a single base in the DNA sequence. Genetic abnormalities can range from relatively harmless to severe: from vitamin deficiencies and food allergies to cancer, birth defects and infant mortality.

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

Centromere Location
Metacentric
A chromosome that has a centrally placed centromere

Submetacentric
A chromosome whose centromere is placed closer to one end than the other

Acrocentric
A chromosome whose centromere is placed very close to, but not at, one end

Human Chromosomes
Replicated chromosomes at metaphase consist of sister chromatids joined by a single centromere

Fig. 6-1, p. 122

Chromosome

Sister Chromatids

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

Metacentric

Submetacentric

Acrocentric

Short arm (p)

Satellite p p Centromere Stalk

Long arm (q)

17

21

Types of Chromosomes
Sex chromosomes
In humans, the X and Y chromosomes that are involved in sex determination. These have different sizes and shapes

Autosomes
Chromosomes other than the sex chromosomes In humans, chromosomes 1 to 22 are autosomes

A Set of Human Chromosomes

Human chromosomes are analyzed by construction of karyotypes Karyotype
A complete set of chromosomes from a cell that has been photographed during cell division at the metaphase stage and arranged in a standard sequence

A Human Karyotype

Karyogram: Chromosome Banding Patterns

System of Naming Chromosome Bands

Allows any region to be identified by a descriptive address (chromosome number, arm, region, and band)

Add a few drops of blood. Draw 10 to 20 ml of blood.

Add phytohemagglutinin to stimulate mitosis.

Incubate at 37C for 2 to 3 days.

Transfer to tube containing fixative.

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.

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

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 technique 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.

Appearance of chromosomes Darkly stained G bands.

Banding technique

Appearance of 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 technique

Appearance of 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 Gbanding.

Banding technique 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.

Appearance of chromosomes

Darkly stained C band centromeric region of the chromosome corresponds to region of constitutive heterochromatin.

Chromosomal Aberrations and Specific Syndromes

Chromosome Painting
New techniques using fluorescent dyes generate unique patterns for each chromosome

Obtaining Cells for Chromosome Studies

Any nucleus can be used to make karyotype
Lymphocytes, skin cells, cells from biopsies, tumor cells

Sampling cells before birth

Amniocentesis Chorionic villus sampling (CVS) Cord Blood

Amniocentesis
A method of sampling the fluid surrounding the developing fetus by inserting a hollow needle and withdrawing suspended fetal cells and fluid
Used in diagnosing fetal genetic and developmental disorders Usually performed in the sixteenth week of pregnancy

Amniocentesis

Removal of about 20 ml of amniotic uid containing suspended cells that were sloughed off from the fetus

A few biochemical analyses with some of the amniotic fluid Quick determination of fetal sex and analysis of purified DNA

Centrifugation

Fetal cells Growth for several days in culture medium

Biochemical analysis for the presence of alleles that cause many different metabolic disorders Karyotype analysis (a)

Chorionic Villus Sampling (CVS)

A method of sampling fetal chorionic cells by inserting a catheter through the vagina or abdominal wall into the uterus
Used in diagnosing biochemical and cytogenetic defects in the embryo Usually performed in the eighth or ninth week of pregnancy

Chorionic Villus Sampling

Chorionic villi Developing placenta Ultrasound to monitor procedure

Developing fetus Uterus Chorion Amniotic cavity

Bladder

Catheter

Rectum

(a)

Variations in Chromosome Number

Changes in chromosome number or chromosome structure can cause genetic disorders Two major types of chromosomal changes can be detected in a karyotype
A change in chromosomal number A change in chromosomal arrangement

Changes in Chromosome Number

Polyploidy
A chromosomal number that is a multiple (3n or 4n) of the normal haploid chromosomal number

Aneuploidy
A chromosomal number that is not an exact multiple of the haploid number

Polyploidy Changes the Number of Chromosome Sets

Triploidy
A chromosomal number that is three times the haploid number, having three copies of all autosomes and three sex chromosomes

Tetraploidy
A chromosomal number that is four times the haploid number, having four copies of all autosomes and four sex chromosomes

A Triploid Karyotype

Keep In Mind

Polyploidy results when there are more than two complete sets of chromosomes

Aneuploidy Involves the Gain or Loss of Individual Chromosomes

Monosomy
A condition in which one member of a chromosomal pair is missing; one less than the diploid number (2n 1)

Trisomy
A condition in which one chromosome is present in three copies, and all others are diploid; one more than the diploid number (2n + 1)

Causes of Aneuploidy

Nondisjunction
The failure of homologous chromosomes to separate properly during meiosis

Nondisjunction in Meiosis I Leads to Aneuploidy

Nondisjunction

Extra chromosome (n + 1)

Extra chromosome (n + 1)

Missing chromosome (n 1)

Missing chromosome (n 1) Meiosis I (a) Meiosis II Gametes

Nondisjunction Extra chromosome (n + 1)

Normal division

Missing chromosome (n 1)

Normal (n)

Normal (n) Meiosis I (b) Meiosis II Gametes

Effects of Monosomy and Trisomy

Autosomal monosomy is a lethal condition
Eliminated early in development (spontaneous abortion)

Some autosomal trisomies are relatively common

Most result in spontaneous abortion Three types can result in live births (13, 18, 21)

Trisomies in Spontaneous Abortions

7.5

Survey of 4,088 spontaneous abortions

5
Percentage of trisomies

3 2

2 3 4 5 6

7 8

9 10 11 12 13 14 15 16 17 18 19 20 21 22

Chromosome number

Trisomy 13: Patau Syndrome (47,+13)

A lethal condition Usually have polydactyly, eye 1 in 10,000 births, most defects, severe brain, nervous system & heart defects die within 1st month

Trisomy 18: Edwards Syndrome (47,+18)

A lethal condition Survival only 2-4 mths 1 in 11,000 births 80% are females Very slow growth, Mental retardation, Heart malformations

Trisomy 21: Down Syndrome (47, +21)

Occurs in 1/800 births Trisomy 21 is the only autosomal trisomy that allows survival into adulthood Mental retardation Characterized by epicanthic fold (corner of eye) large furrowed tongues 40% have congenital heart defects

High risk of leukemia & Alzheimers disease Few reach the age of 50

Trisomy 21: Down Syndrome (47,+21)

Monosomy and trisomy involve the loss and gain of a single chromosome to a diploid genome

ANIMATION: Nondisjunction

To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

What Are the Risks for Autosomal Trisomy?

The causes of autosomal trisomy are unknown Factors that have been proposed include:
Genetic predisposition Exposure to radiation Viral infection Abnormal hormone levels

Maternal age is the leading risk factor for trisomy

94% of nondisjunctions occur in the mother

18 16 14
Trisomy 21/1,000 births

Risk for Down syndrome

12 10 8 6 4 2 15 (a) 20 25 30 35 Maternal age 40 >44

35
Percentage of clinically recognized pregnancies

30 Maternal age and trisomic conceptions 25 15

10

15 16 18 20 22 24 26 28 30 32 34 36 38 40

42

(b)

Maternal age

Why is Maternal Age a Risk Factor?

Meiosis is not completed until ovulation
Intracellular events may increase risk of nondisjunction, resulting in aneuploidy

Maternal selection
Embryo-uterine interactions that normally abort abnormal embryos become less effective Age of the mother is the best known risk factor for trisomy

Aneuploidy of the Sex Chromosomes

More common than autosomal aneuploidy Can involve both X and Y chromosomes A balance is needed for normal development
At least one copy of the X chromosome is required for development Increasing numbers of X or Y chromosomes causes progressively greater disturbances in phenotype and behavior

Turner Syndrome (45,X)

Monosomy of the X chromosome that results in female sterility. Other phenotypic characteristics but otherwise normal.

Fig 6.20

Klinefelter Syndrome (47, XXY)

Individuals (males) have some fertility problems but few additional symptoms

Fig 6.22

XYY Syndrome (47,XYY)

Affected individuals are usually taller than normal and some, but not all, have personality disorders
Changes in the number of sex chromosomes have less impact than changes in autosomes

Structural Alterations Within Chromosomes

Changes in the structure of chromosomes

Deletion Duplication Translocation Inversion

Structural Changes in Chromosomes

Deletions
Deletions involve loss of chromosomal material Deletions of chromosomal segments are associated with several genetic disorders
Cri du chat syndrome Prader-Willi syndrome

Deletion in Chromosome 5 and cri du chat syndrome

Associated with an array of malformations, the most characteristic of which is an infant cry that resembles a meowing cat due to defects in the larynx By comparing the region deleted with its associated phenotype, investigators have identified regions of the chromosome that carry genes involved in developing the larynx.

Translocations

Translocation involves exchange of chromosome parts

Often produces no overt phenotypic effects Can result in genetically imbalanced and aneuploid gametes

Chromosomes can lose, gain, or rearrange segments

Robertsonian Translocation

A translocation resulting in Down syndrome

Robertsonian translocation makes Down syndrome a heritable genetic disease Potentially present in one in three offspring

Robertsonian Translocation and Down Syndrome

ANIMATION: Inversion

To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

ANIMATION: Translocation

To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

ANIMATION: Duplication

To play movie you must be in Slide Show Mode PC Users: Please wait for content to load, then click to play Mac Users: CLICK HERE

What Are Some Consequences of Aneuploidy?

Aneuploidy is the leading cause of reproductive failure in humans

Results in miscarriages and birth defects

Aneuploidy also is associated with many cancers

Chromosomal Abnormalities in Miscarriages

Other Forms of Chromosome Changes

Uniparental disomy
A condition in which both copies of a chromosome are inherited from a single parent

Copy number variation

A situation in which a particular gene or chromosomal region is present in multiple copies

Fragile sites
Appear as gaps or breaks in chromosome-specific locations

Gene Imprinting, ie: Uniparental Disomy (UPD)

UPD is associated with several genetic diseases, also called gene imprinting
X-linked disorders Autosomal recessive disorders (Prader-Willi syndrome, Angelman syndrome) Prader-Willi syndrome: missing part of paternal chromosome 15, obesity, reduced mental ability & muscle tone, little sex hormone production Angelman syndrome: missing part of maternal chromosome 15, jerky movements, laughter (happy puppet syndrome)

Fragile Sites
Appear as gaps or breaks in chromosomes One fragile site on the X chromosome is associated with a common form of mental retardation in males know as Fragile X Syndrome

Fragile Sites on the X Chromosome

FRAX B

FRAX C

The fragile sites on the human X chromosome. Sites B, C, and D are common sites and are found on almost all copies of the X chromosome. A, E, and F are rare sites; expression of A is associated with fragile-X syndrome.

Some fragile sites are associated with mental retardation

FRAX D FRAX F (a)

FRAX A FRAX E