Numerical Chromosomal Abnormalities

Question 1

What are the stages of the cell cycle?

Answer 1

G1 = Cell makes a variety of proteins needed for DNA replication and grow
S = synthesis; chromosomes are replicated so that each chromosome now consists of two sister, identical chromatids
G2 – synthesis of proteins especially microtubules and finish growing in size
Some cells don’t replicate; some are senescent.

Question 2

What happens during metaphase?

Answer 2

Metaphase is a stage during the process of cell division (mitosis or meiosis).
Usually, individual chromosomes cannot be observed in the cell nucleus.
However, during metaphase of mitosis or meiosis the chromosomes condense and become distinguishable as they align in the centre of the dividing cell.
Metaphase chromosomes are used during the karyotyping procedure that is used to look for chromosomal abnormalities.

Question 3

What two purposes does meiosis serve?

Answer 3

It produces (haploid) gametes with 23 chromosomes and introduces variation in the resulting cells through independent assortment (which chromosome of which pair ends up in each gamete is down to chance) and recombination, the exchange of genetic material between paired (homologous) chromosomes.
Therefore through meiosis, genetic variation is introduced into the next generation.
In males each cell in the testis which undergoes meiosis results in the production of four haploid sperm cells.
In females, only one of the four haploid cells produced forms the egg, the other three products of meiosis form the polar bodies attached to the egg, but which are then discarded.

Question 4

How does chromosomes recombination occur?

Answer 4

Homologous chromosomes align
Form a bivalent structure
Exchange genetic material (recombine)

The synaptonemal complex, a lattice of proteins between the homologous chromosomes, first forms at specific locations and then spreads to cover the entire length of the chromosomes.
The tight pairing of the homologous chromosomes is called synapsis.
In synapsis, the genes on the chromatids of the homologous chromosomes are aligned precisely with each other.
The synaptonemal complex supports the exchange of chromosomal segments between non-sister homologous chromatids, a process called crossing over.
Crossing over can be observed visually after the exchange as chiasmata (singular = chiasma) (Figure).
In species such as humans, even though the X and Y sex chromosomes are not homologous (most of their genes differ), they have a small region of homology that allows the X and Y chromosomes to pair up during prophase I.
A partial synaptonemal complex develops only between the regions of homology.)

Question 5

What happens during each phase of meiosis?

Answer 5

Meiosis I
- Homologous chromosomes align as 23 bivalents
- Allows for chiasma formation (i.e. recombination)
- Pulls apart homologues from one another
- Daughter cells have 23 chromosomes (each with 2 chromatids)
Meiosis II
- Align as independent chromosomes
- Sister chromatids pulled apart
- Daughter cells have 23 chromosomes (each of 1 chromatid)
Very important to introduce natural variation via independent assortment of chromosomes and recombination
Daughter cells genetically unique

Question 6

What are the different types of chromosomes?

Answer 6

Humans have 23 pairs of chromosomes
	- 22 pairs autosomes, 1 pair sex chromosomes XX or XY
Different types of chromosomes:
Metacentric
	- p & q arms even length
	- 1-3, 16-18
Submetacentric
	- p arm shorter than q
	- 4-12, 19-20, X
Acrocentric
	- Long q, small p 
	- p contains no unique DNA
	- 13-15, 21-22, Y

Question 7

What are the different numerical terms for chromosomes?

Answer 7

HAPLOID:
- one set of chromosomes (n=23) as in a normal gamete.
DIPLOID:
- cell contains two sets of chromosomes (2n=46; normal in human)
POLYPLOID:
- multiple of the haploid number (e.g. 4n=92), inconsistent with life
ANEUPLOID:
- chromosome number which is not an exact multiple of haploid number - due to extra or missing chromosome(s) (e.g. 2n+1=47) (trisomy, monosomy)

Question 8

How does aneuploidy arise?

Answer 8

When there has been an error in the segregation of the chromosomes
Either an error in the segregating of the pair of homologous chromosomes in meiosis I or non-disjunction in meiosis II which is where both chromatids go into a single cell
Causes an imbalance of chromosomes
So either zygotes are trisomic for that chromosome or monosomic for that chromosome

Question 9

What are the autosomal and sex aneuploidies that carry to term?

Answer 9

Trisomy 13 (Patau’s, 2 in 10,000 births)
Trisomy 18 (Edward’s, 3 in 10,000)
Trisomy 21 (Down’s, 15 in 10,000)
Other aneuploidies happen but do not carry to term
Turner’s (45,X) (1 in 5000 female births)
Triple X syndrome (47,XXX) (1 in 1000 female births)
Klinefelter’s (47,XXY) (1 in 1000 male births)
47,XYY (1 in 1000 male births)

Question 10

Can nondisjunction occur in mitosis?

Answer 10

Nondisjunction can also occur in mitosis e.g. at day 16 of blastocyst development
It will only affect a proportion of the cells
This is called mosaicism
Mosaicism is the presence of two or more genetically different cell lines derived from a single zygote
The monosomic cells will die out, but the trisomic ones will continue

Question 11

What is trisomic rescue?

Answer 11

Sometimes a process called trisomic rescue occurs
Also known as anaphase lag
The cell recognises that it has the wrong number of chromosomes in it and it will throw out one of those copies
Which one is thrown out is random
This only happens in a proportion of the cells so you end up with mosaicism

Question 12

What are the sources of sample for detecting chromosomal disorders?

Answer 12

Prenatal:
	- Amniocentesis
	- Chorionic villus sampling
	- Cell-free fetal DNA
Postnatal
	- Blood
	- Saliva

Question 13

Why is G-banding and why do chromosomes have bands?

Answer 13

G = Giemsa (chromosome stain)
Why bands?
- Chromatin
- 2 different sorts: euchromatin & heterochromatin
- Euchromatin = GC-rich; loosely packed; genes active
- Heterochromatin = AT-rich; tightly packed; genes inactive
- Stain differently
Giemsa highlights heterochromatic regions which are less likely to contain genes.
But the crucial thing is that the banding can be used to differentiate between chromosomes and to compare chromosomes.
Generally done at metaphase when chromosomes are highly condensed.
Looks for aneuploidies, translocations & very large deletions

Question 14

What is FISH?

Answer 14

Fluorescent in situ hybridisation
Hybridisation = single stranded nucleic acid strand binds to a new single stranded nucleic acid strand (DNA/DNA or DNA/RNA)
Cultured cells, metaphase spread
	1. Fluorescent probe
	2. Denature probe and target DNA
	3. Mix probe and target DNA
	4. Probe binds to target

Question 15

What is a probe?

Answer 15

A single stranded DNA (or RNA) molecule
Typically 20 – 1000 bases in length
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
In some techniques thousands or millions of probes are used simultaneously

Question 16

What is an Array CGH, how does it work and how can it differ?

Answer 16

• Array comparative genomic hybridisation
  
  • For detection of sub-microscopic chromosomal abnormalities
  • Patient DNA labelled Green
  • Control DNA labelled Red
    These microarrays are created by the deposit and immobilization of small amounts of DNA (known as probes) on a solid support, such as a glass slide, in an ordered fashion.
    Because probes are several orders of magnitude smaller than metaphase chromosomes, the theoretical resolution of aCGH is proportionally higher than that of traditional CGH.
    The level of resolution is determined by considering both probe size and the genomic distance between DNA probes.
    For example, a microarray with probes selected from regions across the genome that are 1 Mb apart will be unable to detect copy number changes of the intervening sequence.

Question 17

What is QF-PCR?

Answer 17

Quantitative fluorescence polymerase chain reaction
Trisomies 13, 18 and 21
Uses microsatellites
Perform QF-PCR ONLY based on national screening programmes.
Takes only about 48 hours

Question 18

How do you detect microsatellites?

Answer 18

Isolate DNA from individual
Design primers specific to flanking sequences
PCR amplification
Gel electrophoresis
The way that we detect microsatellites in the lab is by using PCR and gel electrophoresis
The first step is to isolate DNA from the person or people you want to study.
Then you design primers specific to the unique flanking sequences
Then perform the PCR amplification and run the products out on a gel by electrophoresis
By looking at how far the fragment migrates in the gel we can work out how big the fragment is and from there we can determine the number of repeats in the microsatellite.

Question 19

What are the different components of PCR?

Answer 19

Components of the PCR reaction:
- Template – DNA to amplify
- Primers – Short pieces of ssDNA (15-30bp)
- Polymerase – thermostable enzyme (Taq)
- Nucleotides – single base mixture (dNTPs)
- Buffer – To maintain pH
- MgCl2 – Essential for polymerase activity
In the simplest PCR there are two primers but there are variations of PCR where there are more.
ssDNA is single-stranded DNA

Question 20

What happens during PCR thermal cycling?

Answer 20

PCR, Cycle 1, Step 1 (Denaturation)
Heat and Separate DNA strands at 94oC

PCR, Cycle 1, Step 2 (Annealing)
Primers anneal with template (50-65oC)
When the temperature drops down from 94oC in the absence of any other DNA the large strands of template DNA re-anneal.
However, with large amounts of short single-stranded DNA in the reaction (the primers), these bind more quickly to the DNA template, before the large strands can do so.
Once then primer is bound the large template strands cannot bind to this region.

PCR, Cycle 1, Step 3 (Extension)
DNA polymerase extends strand from primer, 72oC
The primers are extended in the 5’ to 3’ direction by the thermostable DNA polymerase
This happens very quickly. Taq polymerase adds 35-100 nucleotides per second.
Note that we don’t have the PCR fragment of interest at this stage as Taq will just extend until it reaches the end of the template strand or it falls off naturally.

Question 21

What is NIPT?

Answer 21

Non-invasive pre-natal testing (NIPT) and NGS
Cell free fetal DNA
Maternal blood sample
Trisomy testing
Next-generation sequencing
“High chance” indicator for invasive test