Structural chromosomal abnormalities

Question 1

Name some examples of structural chromosomal abnormalities (and the ones you need for know in CAPITALS)

Answer 1

TRANSLOCATIONS (2 types:)
RECIPROCAL
ROBERTSONIAN
Inversion
DELETION
DUPLICATION
Rings
Isochromosomes

Question 2

What is translocation?

Answer 2

→ Exchange of two segments between non-homologous chromosomes
→ Reciprocal or Robertsonian
→ DUE TO INAPPROPRIATE NON-HOMOLOGOUS END JOINING (NHEJ)

Question 3

What are the consequences of reciprocal translocations in meiosis?

Answer 3

→ You have a tetravalent formed. then the impact/effects depend on where the chromosomes split. eg can lead to trisomy/monosomy (not sure of the word lol) but if they are a balanced carrier in the structure of the chromosomes then all fine!

Question 4

What is reciprocal translocation?

Answer 4

→ Reciprocal translocation is a chromosome abnormality caused by exchange of parts between non-homologous chromosomes.
→ Two detached fragments of two different chromosomes are switched.

Question 5

What is robertsonian translocation?

Answer 5

→ When two acrocentric chromosomes break at or near their centromeres, when the fragments are joined together again it’s possible for just the two sets of long arms to be brought together and there’s loss of the satellites.
→ This is called a Robertsonian translocation.
If this happens in a cell, how many chromosomes will there be? 45
→ The only genetic material we’ve lost are these satellites and the cell can do without those and so this isn’t a problem for the cell.
→ For now, let’s move on to another type of translocation – the Robertsonian translocation.
→ This is named after American cytogeneticist who first described them
→ Only affect acrocentric chromosomes – ie. Those which have the centromere near the chromosome tip.
→ These are chromosomes 13, 14, 15, 21 & 22
→ Most common Robertsonian translocation involves chromosomes 13 and 14, which accounts for approximately 1/3 of all Robertsonian translocations
→ Results in loss of two short arms and fusion of the two long arms, with either one or two centromeres
→ The resultant chromosome usually contains the long arms of different chromosomes (unusual to see, for example, the maternal and paternal long arms of chromosome 13 fused together)
→ Robertsonian translocation: A common and significant type of chromosome rearrangement that is formed by fusion of the whole long arms of two acrocentric chromosomes (chromosomes with the centromere near the very end).

Question 6

How many chromosomes does a balanced carrier have as a result of robertsonian translocations?

Answer 6

→ Two acrocentric chromosomes join near centromere with the loss of p arms
→ Balanced carrier has 45 chromosomes
→ If 46 chromosomes present including Robertsonian then must be unbalanced
p arms encode rRNA (multiple copies so not deleterious to lose some)
→ Robertsonian translocations 13;14 and 14;21 relatively common. 21;21 translocation leads to 100% risk of Down syndrome in fetus

Question 7

What are the general outcomes/ features of outcomes of translocations?

Answer 7

→ Very difficult to predict
→ Only have approximate probability of producing possible gametes
→ Some unbalanced outcomes may lead to spontaneous abortion of conceptus so early that not seen as problem
→ Some unbalanced outcomes may lead to miscarriage later on and present clinically
→ Some may result in live-born baby with various problems

Question 8

What are deletions?

Answer 8

→ 1:7000 live births
→ Deletion may be terminal or interstitial
→ Causes a region of monosomy
→ Haploinsufficiency of some genes
→ Contiguous gene syndrome (multiple, unrelated clinical features)
→ Phenotype is specific for size and place on deletion
→ Gross deletions seen on metaphase spread on G-banded karyotype

Question 9

What are microdeletions?

Answer 9

→ Many patients had no abnormality visible on metaphase spread
→ High resolution banding, FISH and now CGH showed ‘micro’ deletions
→ Only a few genes may be lost or gained
→ Velocardiofacial (DiGeorge), 22q11
→ Wolf-Hirschhorn, 4p16
→ Williams, 7q11
→ Smith-Magenis, 17p11

Question 10

What do deletions and microdeletions mostly occur due to?

Answer 10

Deletions and microdeletions occur mostly due to UNEQUAL CROSSING OVER

Question 11

What do deletions and microdeletions mostly occur due to?

Answer 11

Deletions and microdeletions occur mostly due to UNEQUAL CROSSING OVER (you get exchange ‘ve between homologous chromosomes but they’ve not aligned properly)

Question 12

On which chromosomes can robertsonian translocation only occur?

Answer 12

→ Robertsonian translocation can only occur on chromosomes 13,14,15, 21 and 22
→ 2 acrocentric chromosomes get stuck together, making one large chromosome- the most common chromosomes coming together are chromosomes 13 and 14. (also common on chromosome 14 and 21 coming together)
→ Then when having a child will pass on chromosome and depending on which combination of chromosomes are handed over depends on the child’s health/complications etc, eg trisomy 14 leads to a miscarriage and trisomy 13 (known as patau syndrome) also miscarry but some may survive til birth but not much longer!

→ Also there is trisomy 21- downs syndrome

Question 13

What are some sources of samples you can obtain- pre-nataly and post-nataly, for detecting chromosomal abnormalities?

Answer 13

Prenatal:
→ Amniocentesis
→ Chorionic villus sampling
→ Cell-free fetal DNA

Postnatal:
→ Blood
→ Saliva

Question 14

What is chromosome staining as a method of detecting chromosomal abnormalities?

Answer 14

Most common = G-banding
G = Giemsa

Why bands? 2 different types of chromatin: euchromatin & heterochromatin which stain differently

Euchromatin = GC-rich; loosely packed; genes active
Heterochromatin = AT-rich; tightly packed; genes inactive

→ 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 ant to compare chromosomes
→ Generally done at metaphase when chromosomes are highly condensed method usually takes a few days

Question 15

Summarise G banding as a method for detecting chromosomal abnormalities

Answer 15

→ Essentially looking at how the karyotype of patient differs from expected
→ Uses a chemical stain
→ Uses metaphase chromosomes
→ Takes several days at least
→ Looks for aneuploidies, translocations & very large deletions - not small so couldn’t detect micro deletions e.g.

Question 16

Summarise the method- FISH

Answer 16

→ Fluorescent in situ hybridisation
→ Hybridisation = when a single stranded nucleic acid strand binds to a new single stranded nucleic acid strand (DNA binding to DNA or DNA binding to RNA)
→ Cultured cells, metaphase spread
→ Fluorescent probe
→ Denature probe and target DNA
→ Mix probe and target DNA
→ Probe binds to target takes several days as you need to generate metaphase chromosome

Question 17

What is a probe?

Answer 17

→ 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 18

What happens in array CGH?

Answer 18

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.
→ Probes vary in size from oligonucleotides manufactured to represent areas of interest (25–85 base pairs) to genomic clones such as bacterial artificial chromosomes (80,000–200,000 base pairs).
→ 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

Question 19

Summarise what aCGH does (and the benefit it has as a method for detecting chromosomal abnormalities in comparison to eg using G banding)

Answer 19

→ How many copies of a particular genomic region does the patient have?
→ Uses fluorescent probes to differentiate between patient and control
→ Uses extracted DNA
→ Looks for microdeletions and microduplications (whereas G banding couldn’t look for microdeletions etc)

Question 20

Describe QF-PCR as another method

Answer 20

→ Quantitative fluorescence polymerase chain reaction
used to specifically detect Trisomies 13, 18 and 21
→ Uses microsatellites:
Trisomy 13 = Patau syndrome
Trisomy 18 = Edwards syndrome
Trisomy 21 = Down’s syndrome

Perform QF-PCR ONLY based on national screening programmes. Takes only about 48 hours

Question 21

What are microsatellites?

Answer 21

→ Short repeated sequences
→ Number of repeats varies between individuals
→ Total length of microsatellite sequence varies between individuals

Question 22

What is PCR and what components are required?

Answer 22

→ Exponential amplification of a DNA fragment of known sequence
→ 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
→ Primers are often called oligonucleotides – oligo means “a few” – in this case 10-30.
→ Primers are also sometimes named on the basis of the number of nucleotides – e.g. a hexamer has six bases, a nonamer would have 9, above 10 they are usually oligomers, or the number plus –mer, e.g. 15-mer, 20-mer.
→ PCR consists of incubating at three different temperatures

This results in three different processes happening:
→ Denaturation
→ Annealing
→ Extension

Question 23

Using QF-PCR for testing for Downs syndrome as an example, what would you see in regards to peaks?

Answer 23

→ Perform PCR using primers for microsatellite known to be on chromosome 21 (if testing for Down’s)
→ Should be two copies of microsatellite (one from mother, one from father, like any other autosomal locus, gene, whatever)
→ If homozygous, there will be a single peak of high signal
→ If heterozygous, there will be two peaks of similar, lower signal

Question 24

Summary of QF-PCR

Answer 24

→ Uses fluorescent probes for SPECIFIC microsatellite markers on SPECIFIC chromosomes
→ Uses extracted DNA
→ Quick (~48hrs)
→ Looks for aneuploidies
→ Need to know what you’re looking for

Question 25

What does Non-invasive pre-natal testing (NIPT) and NGS (next generation sequencing) involve?

Answer 25

→ Cell free fetal DNA
→ Maternal blood sample
→ Trisomy testing
→ Next-generation sequencing
→“High chance” indicator for invasive test
→ Essentially (a summary of NIPT:)
→ Uses extracted DNA from mother
→ Looks for aneuploidies
→ Utilises next generation sequencing
→ Screening not diagnostic