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									Cytogenetics: Karyotypes and Chromosome
     The Human Chromosome Set
 The number and appearance of chromosomes in the
  nucleus of an organism is an important characteristic

 Chromosome analysis is a powerful and useful technique in
  human genetics
Chromosome Number

 Chromosome number in selected organisms
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

 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
Metaphase Chromosomes

 Chromosomes are identified by size, centromere
  location, banding pattern
        Metacentric   Submetacentric       Acrocentric

Short                                  Satellite
 (p)                  p                       Centromere

Long                  q                q

             3              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
A Human Karyotype
Chromosome Banding Patterns
System of Naming Chromosome Bands

 Allows any region to be
  identified by a descriptive
  address (chromosome
  number, arm, region, and
                                  Add a few           Add phytohemagglutinin
                                  drops of blood.     to stimulate mitosis.

                  Draw 10 to 20 ml
                  of blood.
                                                            Incubate at 37°C
                                                            for 2 to 3 days.

Transfer to tube                     Transfer            Add Colcemid to
containing fixative.                 cells to tube.      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          Digitized
                                     microscope.           chromosome
    Stain slide                                            images processed
    with Giemsa.                                           to make karyotype.
Metaphase Chromosomes (a)
Arranged Into a Karyotype (b)
ANIMATION: Preparation of a karyotype

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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
  Banding technique                    Appearance of chromosomes

G-banding — Treat metaphase
                                             Darkly stained G bands.
spreads with trypsin, an enzyme that
digests part of chromosomal protein.
Stain with Giemsa stain. Observe
banding pattern with light
    Banding technique                   Appearance of chromosomes

Q-banding — Treat metaphase                   Bright fluorescent bands
spreads with the chemical                     upon exposure to
quinacrine mustard. Observe                   ultraviolet light; same as
fluorescent banding pattern with a             darkly stained G bands.
special ultraviolet light microscope.
   Banding technique                   Appearance of chromosomes

R-banding — Heat metaphase                   Darkly stained R bands
spreads at high temperatures to              correspond to light bands
achieve partial denaturation of DNA.         in G-banded chromosomes.
Stain with Giemsa stain. Observe             Pattern is the reverse of G-
with light microscope.                       banding.
  Banding technique                Appearance of chromosomes

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

 Sampling cells before birth
  • Amniocentesis
  • Chorionic villus sampling (CVS)

 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
  • Usually performed in the sixteenth week of
      Removal of about 20 ml of amniotic fluid 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                     Fetal cells
of purified DNA
                                          Growth for
Biochemical analysis for                  several days
the presence of alleles                   in culture
that cause many different                 medium
metabolic disorders
                         Karyotype analysis
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
Chorionic Villus Sampling
      Developing                 Ultrasound to monitor
      placenta                   procedure

      Developing                              Bladder



Exploring Genetics: Noninvasive Prenatal
 Methods are being investigated to isolate fetal cells
  that can pass into the mother’s bloodstream
  (placental cells, white blood cells, immature red
  blood cells) for genetic testing
ANIMATION: Amniocentesis

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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
A Triploid Infant
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
      Nondisjunction                          (n + 1)

                                              (n + 1)

                                              (n − 1)

                                              (n − 1)

         Meiosis I     Meiosis II   Gametes
      Normal division                              (n + 1)

                                                   (n − 1)



         Meiosis I       Meiosis II      Gametes
Effects of Monosomy and Trisomy

 Autosomal monosomy is a lethal condition
  • Eliminated early in development (spontaneous

 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
                                           Survey of 4,088
                                        spontaneous abortions

Percentage of trisomies





                                1   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
die within 1st month       system & heart defects
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           • High risk of leukemia &
  tongues                    Alzheimer’s disease
 40% have congenital      • Few reach the age of 50
  heart defects
Trisomy 21: Down Syndrome (47,+21)
                  Monosomy and trisomy involve the
                  loss and gain of a single chromosome
                  to a diploid genome
ANIMATION: Nondisjunction

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ANIMATION: Duplicating chromosomes

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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


                                         Risk for
Trisomy 21/1,000 births               Down syndrome






                                 15   20 25 30 35      40 >44
                           (a)          Maternal age

Percentage of clinically recognized pregnancies
                                                                         Maternal age and
                                                                      trisomic conceptions



                                                         15 16 18 20 22 24 26 28 30 32 34 36 38 40   ≥42
                                                                           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
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
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
Structural Alterations
Within Chromosomes

 Changes in the structure of chromosomes
  •   Deletion
  •   Duplication
  •   Translocation
  •   Inversion
Structural Changes in Chromosomes

 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
  • 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
  • By comparing the region
    deleted with its associated
    phenotype, investigators
    have identified regions of
    the chromosome that
    carry genes involved in
    developing the larynx.

 Translocation involves exchange of chromosome
  • Often produces no overt phenotypic effects
  • Can result in genetically imbalanced and aneuploid
 Chromosomes can lose, gain, or rearrange
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
ANIMATION: Inversion

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ANIMATION: Translocation

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ANIMATION: Duplication

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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
   The frequency of aneuploidy changes dramatically
   over developmental time. Between 6 to 8 weeks and 20
   weeks, about 35% of spontaneous abortions are
   aneuploid. Around 20 weeks, the frequency falls by an
   order of magnitude to about 4% stillbirths.

                Gametes       Gestation (weeks)
               Sperm   Eggs   0                6–8                 20                   40
                                  Preimplantation    Spontaneous         Stillbirths    Live births
                                  embryos            abortions
Incidence of
aneuploidy 1–2%        ~20%       ~20%               35%                 4%            0.3%

Common             Various                           45, X, + 16        + 13, + 18,    + 13, + 18, + 21
aneuploidies                                         +21, +22           + 21           XXX, XXY, XYY

                The frequency decreases again by an
                order of magnitude, with about 0.3% of
                newborns being aneuploid.
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
Gene Imprinting

 A small proportion (<1%) of genes are imprinted
  where gene expression occurs from only one allele
 Ie: only the maternal or paternal chromosome allele
  is expressed
Gene Imprinting, ie: Uniparental Disomy
 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)
Two Mechanisms of Generating Uniparental
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
Sites B, C, and D                • Some fragile
are common                         sites are
sites and are
found on almost                    associated
all copies of the
X chromosome.
                                   with mental
A, E, and F are                    retardation
rare sites;
expression of A
is associated
with fragile-X
                    FRAX D   FRAX A
                             FRAX E
                    FRAX F

The relationship between copy number variation (CNV) in the
PMP22 gene and the symptoms of Charcot-Marie-Tooth (CMT)
syndrome. Having more than two copies of this gene causes
(CMT). As the copy number increases, the symptoms become
more severe.

PMP22 Genotypes

Gene copies       2          3            4             1
Phenotype     Normal      CMT1A       Severe CMT1A    HNPP
Human Diseases Associated with Copy
Number Variants
ANIMATION: Chromosome abnormalities exercise

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