1. Karyotyping

2. What are Chromosomes
Chromosomes are structures found in the nucleus of cells
Chromosomes carry all of our genes, and therefore all of our genetic information
Humans have 46 chromosomes, or 23 pairs, to carry our approximately 25,000 genes
The first 22 pairs are called autosomes
The 23rd pair are the sex chromosomes or gonosomes this pair will either be XX or XY

3. Karyotyping
During mitosis, the 23 pairs of human chromosomes begin condensing during prophase, and reach their most condensed state during metaphase
A karyotype analysis usually involves blocking cells in mitosis and staining the condensed chromosomes with Giemsa dye.
The dye stains regions of chromosomes that are rich in the base pairs Adenine (A) and Thymine (T) producing a dark band

4. Karyotypes
A karyotype is a pattern or picture of chromosomes. The chromosomes are paired and arranged according to size
Each chromosome is paired with its homologous chromosome
its exact match in size and structure, though the homologous chromosomes may carry different alleles of the same gene
Then the chromosome pairs are labeled
The autosomes are numbered 1 through 22 according to size
The sex chromosomes are a different story
The X and X or the X and Y are paired, then placed at the end, even though they are not necessarily the smallest chromosomes
These chromosomes do not receive a number - just XX or XY

6. Human Karyotyping
A karyotype is an actual photograph of the chromosomes from one cell.
Karyotypes are usually done using blood cells, fetal skin cells (from amniotic fluid or the placenta) and occasionally bone marrow cells
It takes at least one week to complete a karyotype

7. Clinical Steps to Karyotyping
1. Sample Collection
2. Separating cells
3. Growing Cells
4. Synchronizing Cells
5. Releasing Chromosomes from their cells
6. Staining Chromosomes
7. Analysis
a. Counting & Sorting of chromosomes
b. Looking at Chromosomes Structure

8. Sample Collection
In newborns, a blood sample which contains red bloods cells, white blood cells, serum and other fluids is collected.
A karyotype will be done on the white blood cells which are actively dividing (a state known as mitosis).

9. How do we get blood samples from a fetus
During pregnancy, the sample can either be amniotic fluid collected during an amniocentesis
The amniotic fluid contains fetal skin cells which are used to generate a karyotype
or a piece of the placenta collected during a chorionic villi sampling test (CVS).
The important thing is to choose a cell type that is actively dividing, some cells, like nerve cells, divide infrequently or never!

10. Separating the Cells
In order to analyze chromosomes, the sample must contain cells that are actively dividing (or in mitosis).
In blood, the white blood cells are actively dividing cells.
Sometimes adult cells are treated with chemicals such as phytohemaglutenin (PHA) to induce mitotis
Most fetal cells are actively dividing.
Once the sample reaches the cytogenetics lab, the non-divided cells are separated from the dividing cells using special chemicals.

11. Growing Cells
In order to have enough cells to analyze, the dividing cells are grown in special media or a cell culture.
This media contains chemicals and hormones that enable the cells to divide and multiply.
This process of “culturing” the cells can take 3 to 4 days for blood cells, and up to a week for fetal cells

12. Synchronizing Cells
The best mitotic phase for chromosome analysis is prometaphase or metaphase
In order to get all the cells to this specific stage of cell division, the cells are treated with a chemical which stops cell division at the point where the chromosomes are the most compact.
cholicine

13. Releasing Chromosomes From Their Cells
In order to see these compact chromosomes under a microscope, the chromosomes have to be out of the white blood cells.
This is done by treating the white blood cells with a hypotonic solution that causes
Chromosomes to spread apart for easier viewing
And causes cells to burst
This is done while the cells are on a microscopic slide. The leftover debris from the white blood cells is washed away, and the chromosomes are fixed to the slide.

14. Staining the Chromosomes
Chromosomes are naturally colorless.
In order to be able to tell one chromosome from another, a special dye called Giemsa dye is applied to the chromosomes on the slide.
Giemsa dye stains regions of chromosomes that are rich in the bases adenine (A) and thymine (T).
When stained, the chromosomes look like strings with light and dark bands.
Each chromosome has a specific pattern of light and dark bands which enables cytogeneticist to tell one chromosome from another.

15. G Banding 1st use a protease to digest any protins
Giemsa reagent consists of a mixture od dyes including
Aminophenothiazine dyes(basic)
Eosin (acidic)
Produces light & dark bands
Patterns of banding change with the stage of mitosis
There may be as many as 1,000 bands in early prophase, and as few as 300 on a given chromosome in metaphase

16. G Banding GC rich Most genes are in light bands
Dark Bandss
GC rich
Most genes are in light bands
Light bands represent relatively open (uncoiled) regions of DNA making transcription easier
AT rich
Highly coiled regions of DNA
Contains few genes
Most genes in these regions are tissue specific, and so not transcribed in all cells

17. Analysis
Once chromosomes are stained, the slide is put under the microscope and the analysis of the chromosomes begins.
A picture is taken of the chromosomes and at the end of the analysis
This is known as a “metaphase spread”
Take a picture of the spread using a special microscope with attached camera

18. Typical Metaphase Spread

19. Critical Thinking!
When we draw chromosomes, we typically draw them in the shape of the letter X
What does this represent (ie, label the important parts of the X structure)
Why, in the above metaphase spread are the chromosomes NOT in the X shape, but rather linear?

20. Analysis: Step 1
The first step of the analysis is counting the chromosomes.
Most humans have 46 chromosomes.
People with Down syndrome have 47 chromosomes.
It is also possible for people to have missing chromosomes or more than one extra chromosome.
By looking at just the number of chromosomes, it is possible to diagnose different conditions including Down syndrome, Turners, Kleinfelters

21. Analysis: Step 2 Sort the chromosomes by
comparing chromosome length,
the placement of centromeres (the areas where the two chromatids are joined),
the location and sizes of G-bands.
The chromosomes pairs are numbered from largest (number 1) to smallest (number 22).
There are 22 pairs of chromosomes, called autosomes, which match up exactly.
2 sex chromosomes
two X‘s is a female
X and a Y is a male.

22. Analysis: Step 3
In addition to looking at the total number of chromosomes and the sex chromosomes, the cytogeneticist will also look at the structure of the specific chromosomes to make sure that there is no missing or additional material, no structural abnormalities like
Translocations
Deletions
Duplications

23. What we will do
Unfortunately in high school we aren’t allowed to stick needles in our students and/or work with human bodily fluids
Possible spread of infectious diseas
Parents complain
We will practice karyotyping from the analysis standpoint

24. 3 Criteria used to Identify a Chromosome
The analysis involves comparing chromosomes for their
Length
placement of centromeres (areas where the two chromatids are joined)
location and sizes of G-bands.

25. Typical Chromosome Structure
Each chromosome has 2 arms separated by a constriction point called the centromere
Metacentric: term used to describe chromosomes with centromere at or near the center of the chromosome
Submetacentric: term used to describe chromosomes with centromere slightly displaced from the center

26. Typical Chromosome Structure
Very submetacentric: term used to describe chromosomes with centromere halfway between the middle and the tip of the chromosome
Acrocentric: term used to describe chromosomes with centromere very near one end

27. Typica Chromosome Structure

28. Typical Chromosome Structure
Telocentric: term used to describe chromosomes with centromere at the very tip of one end
These are absent in human chromosomes

29. Typical Chromosome Structure
Notice, the centromere divides the chromosome into 2 separate regions
In general
The shorter chromosome arm is designated p for petite
The longer arm is designated the q arm, because the letter q comes after the letter p in the alphabet

32. How To Start Open your worksheet titled Key to Human Chromosomes
It is a dichotomous key
Start by cutting chromosomes out of your metaphase spread
Divide all chromosomes into one of the 2 class sizes
Class 1 = 1st 12 chromosome pairs (24), and X chromosomes
So class 1 will contain 25 chromosomes for a boy and 26 chromosomes for a girl
Class 2 = the rest of the smaller chromosomes

33. Nomenclature Normal female  46, XX Normal Male  46, XY
Telomeres, centromeres, and a number of prominent bands areused as landmarks on a chromosome

34. Nomenclature
A section of a chromosome between 2 landmarks is called a region
Regions are numbered 1,2,3 in both directions starting with the centromere
Bands within a region are numbered according to the same rule
Example: the 1st band in the 2nd region of the p arm of chromosome #1 is indicated as follows
1p21

35. So what does this mean: 14q32
14 refers to chromosome # 14
q refers to the q arm (the longer arm) of chromosome 14
3 refers to region 3 of the q arm of chromosome 14
And 2 refers to the 2nd band in the 3rd region of the q arm in chromosome 14

36. Nomenclature There may also be sub-bands
Decimal points are used to indicate what sub-band
Example: 14q32.3
Refers to the 3rd (last) sub-band within the 2nd band of region 3 in the q arm of chromosome 14

37. Numerical Chromosome Abberations for Sex Chromosomes
45, X  45 chromosomes, 1 X chromosome
47, XXY  45 Chromosomes, two X chromsomes one Y chromosome

38. Numerical Chromosome Abberations
Use a + or – sign
before the symbol to indicate additional or missing whole chromosomes
After a symbol to indicate additional or missing segments of a chromosome
47, XY, +21  an additional 21st chromosome
46,XX, 1q+  female karyotype with 46 chromosomes showing an increase in the length of the long arm of chromosome 1

39. Nomenclature Question marks are used to indicate uncertainty
45, XY, -?8
45 chromosomes
XY is male
Missing a chromosome, probably #8
There is special nomenclature for inversions, deletions, translocations, insertions, etc.; but its is beyond the scope of this class

40. Other Types of Staining
C-Banding: stains constitutive heterochromatin
Regions of chromosomes that are highly compacted containing highly repetitive DNA
Results from selective removal of the chromosomes except from regions of the C-Bands
Used for determining presence or absence of a centromere, as well as for studying abnormalities of the centromeric region

41. Other Types of Staining
Silver Staining for NOR
Ribosomal DNA, which codes for ribosomal RNA, occurs in multiple copies on the short arms of all acrocentric chromosomes
Silver stain results in the deposition of silver grains on the active nucleolar organizer regions (NORs)
In other words, NORs which were actively transcribing their rRNA’s in the preceding interphase, contain the proteins to which thre silver grains bind

42. FISH Fluorescence In Situ Hybridization
Involves precise annealing of ssDNA probes to complementary target sequences
Can be visualized with a fluorescent microscope by using a probe directly labeled with a fluorophore
Can be used to find
If a specific gene is present (and or duplicated)
And if it is in the correct region of the correct chromosome

45. WCP Whole Chromosome Painting
Same idea as FISH, but you use a mixture of many fluorophore labeled probes
Must also use unlabeled blocking DNA to supress highly repetitive regions common to many different chromosomes
Allows for
Chromosome enumeration
Identification of chromosomal structural rearrangements

46. Whole Chromosome Walking

47. WCP
Particularly useful in solid tumor and linical genetic research because it has the ability to aid in the analysis of complex and cryptic translocations
Also helpful in research that is evaluating the extent of chromosomal breakage and translocations following exposure to mutagenic chemicals or radiation

49. Using A Karyotype
A karyotype allows a cyto-geneticist or lab technician to examine the chromosomes and see if there is anything extra or missing, or if the structure of the chromosomes is grossly different than usual

50. Trisomy 21 Trisomy 21 is the presence of 3 chromosome 21’s.
Trisomy 21 causes the condition commonly known as Down syndrome
The extra chromosome leads to the specific characteristics of Down syndrome, some of which are very familiar
not all individuals with Down syndrome will show the exact same characteristics there is a great deal of variability

51. Trisomy 21/Down syndrome
Individuals with Down syndrome have a typical facial appearance
All have some degree of mental retardation, but for most it is mild or moderate
Can learn to read, write, do some math, and live day-to to-day with minimal assistance from others
Others require a lot of attention and care
There are several possible health concerns, including
heart problems, hearing loss, feeding concerns, and others
Individuals with Down syndrome are now living longer than in the past – into their 50’s and 60’s
We are now discovering that those individuals who survive to this age are at very high risk of developing Alzheimer Alzheimer’s disease

52. Trisomy 13
Trisomy 13 is the presence of 3 chromosome 13’s, and causes the condition sometimes known as Patau syndrome
Trisomy 13 is a very serious condition with only about 5% of babies with the disorder survive past their first year
Most pregnancies involving Trisomy 13 end in miscarriage
All individuals with Trisomy 13 have severe mental retardation
Other characteristics commonly seen in people with Trisomy 13 include
a small head
extra fingers and/or toes
and a cleft lip or cleft palate
Trouble breathing, especially in their sleep
seizures

53. Trisomy 18
Trisomy 18 is the presence of 3 chromosome 18’s, & causes the condition sometimes known as Edwards syndrome
Only about 10% of babies with the disorder survive past their first year.
A majority of babies who survive are female.
Most pregnancies involving Trisomy 18 end in miscarriage

54. Trisomy 18
Children with Trisomy 18 usually have breathing problems, difficulty eating, and many have seizures. Some have serious heart conditions.
All individuals with Trisomy 18 have severe mental retardation.Most babies with Trisomy 18 are very small and have certain recognizable facial features. They also tend to overlap their fingers in a very distinct pattern

55. 47, XXY
An individual with the genotype 47, XXY is male 􀁺 The person has 47 chromosomes just like someone with Trisomy 21, 13, or 18, but does not have a typical “trisomy,” as he has two of one chromosome and one of another
One could say, though, that such an individual has a sex chromosome trisomy
The extra X chromosome leads to features of the condition commonly known as Klinefelter syndrome

56. 47, XXY

57. Klienfelters Syndrome
Affects about 1 in 1000 males
Most males are taller than average and may have a different distribution of body fat (e.g. more than usual in the hips or chest)
Also tend to have sparse facial and body hair
Some have a degree of mental retardation, but many have normal intelligence
The most common feature is infertility
It is estimated about 2% of infertile men have Klinefelter Syndrome
Since the characteristics of the syndrome are not always obvious, many males with Klinefelter will never be diagnosed

58. 45, X An individual with the genotype 45, X is phenotypically female
The person has 45 chromosomes instead of the usual 46 Instead of a trisomy, this would be called a monosomy X
One copy of an X chromosome = monosomy
Monosomy X is the only monosomyknown to be compatible with life
Having only one copy of an X chromosome leads to the features of the condition known as Turner syndrome

59. 45, X

60. Turner Syndrome Affects about 1 in 5000 newborn females
Physical features
Swelling of the hands and feet
webbing of the neck
broad chest
May also have features which affect their health, including
heart disease
a“horseshoe-shaped”kidney
Individuals with Turner syndrome will not typically have mental retardation, but may have specific learning disabilities

61. Discussion Questions
Why would the presence (or absence) of a chromosome lead to certain characteristics in a person?
Why wouldn’t the physical, mental, and behavioral features be exactly the same for two people with the same extra (or missing) chromosome?
Why are babies with trisomy 21 more likely to survive than babies with trisomy 13 or 18?
Why do you think monosomy X (Turner syndrome) is the only monosomy in which the individual survives?