1. Cytogenetics: Karyotypes and Chromosome Aberrations
Chapter 6

2. 6.2 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
Human chromosomes exist in pairs, with most cells having 23 homologous pairs, or 46 chromosomes.
This is the diploid, 2n, number
Eggs and sperm (gametes), contain only one copy of each chromosome, which is haploid, or n number.

3. Chromosome Number
Chromosome number in selected organisms

4. 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
Its location gives a chromosome its characteristic shape
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

5. Metaphase Chromosomes
Chromosomes are identified by size, centromere location, and banding pattern
Metacentric
Submetacentric
Acrocentric
Short arm (p)
Satellite p
Centromere p
Stalk
Long arm (q) q q 3 17 21

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

7. Types of Chromosomes Sex chromosomes Autosomes
In humans, the X and Y chromosomes 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
Human chromosomes are analyzed by construction of karyotypes
Karyotype - A complete set of chromosomes from a cell that has been photographed during cell division and arranged in a standard sequence

8. 6.3 Making a Karyotype
Allows any region to be identified by a descriptive address (chromosome number, arm, region, and band)
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

9. A Human Karyotype

10. Karyogram: Chromosome Banding Patterns3 6 1 4 5 2 11 7 8 9 10 12 16 17 18 13 14 15 19 20 21 22 Y X

11. System of Naming Chromosome Bands6
Region 5 3 4 3 2 1 2 2 1
Arm 3 1 p 2 1 q 1 1 2 1 2 3 2 4 5 1 3 2 1 2 4 3 4 1

12. 6.3 Making a Karyotype 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 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.

13. ANIMATION: Karyotype preparation
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14. Metaphase Chromosomes (a) Arranged Into a Karyotype (b)

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

16. Four Common Chromosome Staining Procedures
Banding technique
Appearance 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.
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.

17. Four Common Chromosome Staining Procedures
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 G-banding.
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.

18. Chromosomal Aberrations and Specific Syndromes

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

20. 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)

21. Amniocentesis
A method of sampling the fluid surrounding the developing fetus by inserting a hollow needle and withdrawing suspended fetal cells and fluid
A needle is inserted through the abdominal and uterine walls (avoiding placenta and fetus) into the amniotic sac surrounding the fetus.
10-30mL of fluid is withdrawn – contains cells shed from the skin, respiratory tract, and urinary tract of the fetus
Cell are collected by centrifugation and grown in a lab
Used in diagnosing fetal genetic and developmental disorders
Usually performed in the sixteenth week of pregnancy

22. Amniocentesis
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
Centrifugation
Quick determination of fetal sex and analysis of purified DNA
Fetal cells
Biochemical analysis for
the presence of alleles that
cause many different metabolic disorders
Growth for several days
in culture medium
(a)
Karyotype analysis

23. Amniocentesis Restricted to certain circumstances:
Mother is over age 35 – risk of chromosome abnormalities dramatically increases after 35
Mother has a child with a chromosomal aberration
Either parent carries one or more structurally abnormal chromosomes
Mother is a carrier of X-linked biochemical disorder that cannot otherwise be diagnosed prenatally and is willing to abort if the fetus is male
When couple has had a number of previous miscarriages or unexplained fertility problems

24. Chorionic Villus Sampling (CVS)
A method of sampling fetal chorionic cells (fetal tissue that forms part of the placenta) 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
Both amniocentesis and CVS can be coupled with genomic DNA testing for prenatal diagnosis of mutant alleles.

25. Chorionic Villus Sampling
Chorionic villi
Developing placenta
Ultrasound to monitor procedure
Developing fetus
Bladder
Uterus Chorion
Catheter
Amniotic cavity
Rectum
(a)

26. ANIMATION: Amniocentesis and CVS
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27. Exploring Genetics: Noninvasive Prenatal Diagnosis
Methods are being investigated to isolate fetal cells that can pass into the mother’s bloodstream, and cell-free fetal DNA (cff DNA) for genetic testing

28. 6.4 Variations in Chromosome Number
Changes in chromosome number or chromosome structure can cause genetic disorders
A karyotype can detect
A change in chromosomal number
A change in chromosomal arrangement

29. 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
Involves the gain or loss of a single chromosome
Monosomy – loss of a single chromosome (2n-1)
Trisomy – gain of a single chromosome (2n+1)

30. Polyploidy Triploidy Tetraploidy
A chromosomal number that is three times the haploid number, having three copies of all autosomes and three sex chromosomes
Found in 15-18% of all miscarriages
Approximately 75% of all cases of triploidy are 69,XYY and have two sets of paternal chromosomes
Triploid newborns have multiple abnormalities including enlarged head, fused fingers and toes, and malformations of the mouth, eyes, and genitals
Tetraploidy
A chromosomal number that is four times the haploid number, having four copies of all autosomes and four sex chromosomes
Found in 5% of all miscarriages but is extremely rare in live births

31. A Triploid Karyotype

32. A Triploid Infant

33. Causes of Aneuploidy Nondisjunction
The failure of homologous chromosomes to separate properly during anaphase in meiosis.
Two cell divisions in meiosis, and nondisjunction can occur in either the first or second, with different genetic consequences:
In meiosis I – all gametes will be abnormal and carry either both members of a chromosomal pair or neither member of the pair
In meiosis II – produces two normal haploid cells and two abnormal cells, one with an extra copy of a chromosome and one missing a chromosome.

34. ANIMATION: Nondisjunction
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35. Nondisjunction in Meiosis Leads to Aneuploidy
Extra chromosome (n + 1)
Nondisjunction
Extra chromosome (n + 1)
Missing chromosome (n − 1)
Missing chromosome (n − 1)
(a)
Meiosis I
Meiosis II
Gametes

36. Nondisjunction in Meiosis Leads to Aneuploidy
Extra chromosome (n + 1)
Normal division
Missing chromosome (n − 1)
Normal (n)
Normal (n)
(b)
Meiosis I
Meiosis II
Gametes

37. 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)

38. Trisomies in Spontaneous Abortions
7.5
Survey of 4,088 spontaneous abortions 5 4
Percentage of trisomies 3 2 1
Chromosome number

39. Trisomy 13: Patau Syndrome (47,+13)
A lethal condition - 1 in 10,000 births
Half of all affected individuals die in the first month.
Facial malformations, eye defects, extra fingers or toes, feet with large protruding heels
Internally – malformations of the brain and nervous system and congenital heart defects
Parents of children with trisomy 13 are older (average 32 years old) than parents who have normal children

40. Trisomy 18: Edwards Syndrome (47,+18)
A lethal condition - 1 in 11,000 births
Average survival – 2-4 months
80% are females
Small at birth, grow very slowly, mentally retarded, clenched fists (with second and fifth fingers overlapping the third and fourth fingers and malformed feet), heart malformations – heart failure or pneumonia are usual causes of death

41. Trisomy 21: Down Syndrome (47, +21)
First described by John Langdon Down in 1866
First chromosomal abnormality discovered in humans
1959 – Jerome Lejeune and his colleagues discovered that the presence of an extra copy of chromosome 21 is the underlying cause.
Trisomy 21 is the only autosomal trisomy that allows survival into adulthood – although few reach 50 years old
In US – occurs in 1 in 800 live births – leading cause of childhood mental retardation and heart defects
Wide, flat skull, folds in the corners of eyelids, spots on irises, large, furrowed tongues that cause mouth to remain open.
Physical growth, behavior, and mental development are retarded, approximately 40% have congenital heart defects

42. 6.6 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

43. Why is Maternal Age a Risk Factor?
Meiosis is not completed until ovulation
Primary oocytes are formed early in embryonic development and enter meiotic prophase I well before birth.
However, meiosis I stops and is not completed until ovulation, so oocytes produces at age 40 have been in meiosis I for over 40 years.
During this time, intracellular events or environmental agents may increase risk of nondisjunction, resulting in aneuploidy
Maternal selection – embryo-uterine interactions normally result in the miscarriage of chromosomally abnormal embryos.
Embryo-uterine interactions become less effective as women age

44. 6.7 Aneuploidy of the Sex Chromosomes
More common than autosomal aneuploidy
1 in 400 for males and 1 in 650 for females
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

45. Turner Syndrome (45,X)
Monosomy of the X chromosome that results in female sterility.
Short, wide-chested, with rudimentary ovaries and puffiness of the hands and feet
May have an aortic constriction, but no mental retardation
1 in 10,000 female births
Females require two X chromosomes for normal development and growth patterns

46. Klinefelter Syndrome (47, XXY)
Individuals (males) have some fertility problems but few additional symptoms
1 in 1,000 male births
60% of cases – maternal nondisjunction

47. XYY Syndrome (47,XYY)
1965 – cytogenetic survey of 197 males imprisoned for violent and dangerous antisocial behavior – 9 were XYY
7 of the 9 were of subnormal intelligence
Affected individuals are usually taller than normal and some, but not all, have personality disorders
Frequency of XYY in penal and mental institutions is significantly higher than in the population at large
Some defendants have attempted to use their XYY karyotype as legal defense in criminal trials
Vast majority of XYY males lead socially normal lives

48. 6.8 Structural Changes in Chromosomes
Changes in the structure of chromosomes
Deletion – loss of a chromosome part
Duplication – extra copies of chromosome parts
Translocation – transfer of a chromosome part to another, nonhomologous chromosome
Inversion – order of chromosome segments is reversed

49. ANIMATION: Translocation
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50. ANIMATION: Deletion To play movie you must be in Slide Show Mode
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51. Structural Changes in Chromosomes
Deletion
Deletion of segment F

52. Structural Changes in Chromosomes
(b)
Duplication

53. Deletions Involve loss of chromosomal material
Associated with several genetic disorders
Cri du chat syndrome
Prader-Willi syndrome

54. Cri du chat syndrome
Caused by a deletion in the short arm of chromosome 5 and occurs in 1 in 20,000 to 1 in 50,000 births
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

55. Translocations Two major types:
Reciprocal translocation – two nonhomologous chromosomes exchange genetic parts
No genetic information gained or lost from the cell, but genes are moved to new chromosomal locations.
In some cases, there are no phenotypic effects, and the translocation is passed through a family for generations.
Robertsonian translocations – can produce genetically unbalanced gametes with duplicated or deleted chromosomal segments that can result in embryonic death or abnormal offspring
About 5% of Down Syndrome cases involve this

56. Robertsonian Translocation14 21
Robertsonian translocation
Normal cell
14/21 Translocation carrier
Meiosis and gamete formation
Normal gamete
Fertilization
Translocation carrier
Normal
Translocation Down syndrome
Monosomy 21 lethal
Trisomy
14q lethal
Monosomy 14 lethal
Phenotype
Chromosome number 45 46 46 45 46 45

57. 6.8 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
Especially leukemia

58. 6.10 Other Forms of Chromosome Changes
Uniparental disomy (UPD) - A condition in which both copies of a chromosome are inherited from a single parent
Can occur in meiosis or in mitotic divisions after fertilization.
Associated with several genetic diseases
Females affected with rare X-linked disorders, such as hemophilia
Father-to-son transmission of rare, X-linked disorders where the mother is homozygous for the normal allele
Children affected with rare autosomal recessively inherited disorders where only one parent is heterozygous
Autosomal recessive disorders (Prader-Willi syndrome, Angelman syndrome) – deletions in the long arm of chromosome 15 or by UPD
If both copies of chromosome 15 are inherited from the mother, the child will have Prader-Willi syndrome
If both copies of chromosome 15 are inherited from the father, the child will have Angelman syndrome

59. Mechanisms of Generating UPD
Nondisjunction in one parent, followed by duplication in embryo
Nondisjunction
in both parents
Normal
Gamete
Zygote
Duplication
Embryo
(a)
(b)
(c)

60. 6.10 Other Forms of Chromosome Changes (contd.)
Copy number variation
A particular gene or chromosomal region is present in multiple copies
The relationship between copy number variation in the PMP22 gene and the symptoms of Charcot-Marie-Tooth Syndrome
A duplication encodes a protein involved in making a sheath surrounding nerve cells.
Duplication disrupts the production of this protective sheath and causes an autosomal dominant disease
Normal individuals – two copies of region – one on each homologue
CMT – three copies of this chromosome region, two on one homologue, and one on the other

61. Human Diseases Associated with Copy Number Variants

62. 6.10 Other Forms of Chromosome Changes (contd.)
Fragile sites
Appear as gaps or breaks in chromosomes
Over 100 fragile sites have been identified in the human genome.
Regions susceptible to breakage
One fragile site on the X chromosome is associated with a common form of mental retardation in males know as Fragile X Syndrome (Martin-Bell Syndrome)

63. Fragile Sites on the X Chromosome
FRAX B
FRAX C
FRAX D
FRAX A
FRAX E
FRAX F
(a)