Structural chromosomal abnormalities Flashcards
What are the different types of structural abnormalities?
Translocations -reciprocal -robertsonian Inversion Deletion Duplication Rings Isochromosomes
What is a Translocation?
Translocation = the exchange of 2 segments between non-homologous chromosomes
- Reciprocal
- Robertsonian
How does Translocation occur?
Translocation is caused by inappropriate Non-Homologous End Joining (NHEJ).
NHEJ = A DNA repair mechanism which, repairs double strand breaks in DNA. double-strand DNA breaks separate the chromosome. NHEJ rejoins the broken off section to the same chromosome.
NHEJ incorrectly fixes chromosomes in translocation. 2 diff. chromosomes undergo a double-strand break. Instead of sticking broken chr back onto itself, genetic material is exchanged. Section of chr A is joined onto chr B. Section of chr B is joined onto chr A.
Produces derivative chromosomes (mixture of 2 chrs due to NHEJ).
Balanced translocation = no net gain/loss of genetic material = normal healthy individual
What is a Philadelphia chromosome ?
Philadelphia chromosome = Abnormal chr22 – Balanced translocation b/w chr9+chr22
- Leads to CML (Chronic Myeloid Leukaemia)
- Chr9 contains ABL gene = proto-oncogene (not cancer-causing when on chr9)
- Chr22 contains BCR gene = break point cluster region – prone to DNA double-strand break
- Chr9+Chr22 break + exchange genetic material to form derivative chrs
- Derivative Chr9 is fine but derivative chr22 = Philadelphia chr = creates a new fusion gene = activates oncogene ABL = leukaemia + myeloma
What is G-banding?
G-banding = Technique used in cytogenetics to produce a visible karyotype of stained condensed chromosomes.
What are the consequences of reciprocal translocations in meiosis ?
Reciprocal translocations in Meiosis form quadrivalents
In normal meiosis, chrs form bivalent structure.
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What are the results of unbalanced reciprocal translocation?
Many lead to miscarriage
Learning difficulties, Physical disabilities
What is Robertsonain translocation?
Robertsonain translocation involve acrocentric chromosomes.
The p arms (satellites) are lost and the q arms are joined together at the centromere.
Balanced carrier has 45 chromosomes
If 46 chromosomes are present including Roberstonian then must be unbalanced.
Satellites present on other chrs encode rRNA (multiple copies so not deleterious to lose some)
Roberstonian translocations 13,14 and 14,21 relatively common,
21;21 Translocations lead to 100% risk of Down syndrome in foetus
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What are acrocentric chromosomes ?
These are chromosomes in which the centromere is very close to one of the ends. The p arm is therefore very short.
13,14,15,21,22
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What are the outcomes of Translocations?
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
What are some of the other structural changes that can occour ?
-Terminal Deletions - Loss of a telomeric section
-Interstitial Deletions - Loss of a section in the middle of the chromosome
(Prader-willi, DiGeorge syndrome, Cri du chat).
-Inversion
-Duplication
-Ring Chromosome
Describe deletions and what they can result in
- Terminal deletion, Interstitial deletion
- Causes a region of monosomy in resulting zygote
- Haploinsufficiency of some genes
- Contiguous gene syndrome
- Phenotype is specific for size and place on deletion
- Gross deletions are seen on metaphase spread on G-banded karyotype.
Affect 1 in 7000 births
What are microdeletions/microduplications ?
Microdeletions/Microduplications = Few genes are lost/gained.
- No abnormality visible on metaphase spread. To view, use high resolution banding, FISH, array-CGH
- Caused by Unequal Crossing Over (Non-Allelic Homologous Recombination) - homologous chrs misalign + recombine in meiosis = simultaneous deletion+duplication on the individual chromatids that end up in the gametes = monosomic/trisomic for gene C when fertilised
Describe Array CGH
Array-CGH
Looks at dosage
i.e. 2 individuals should have the same number of copies of the autosome – no difference
- Take healthy individual (control) and affected individual DNA (patient)
- These anneal to probes on glass microarray
- Should be equal hybridisation if both patient and control have two copies of a region (disomic) – slide 39
- If there is a microdeletion/ microduplication in patient DNA i.e. 2 copies in healthy DNA and 1 copy in patient DNA, dosage will be different = Unequal Hybridisation
What are the different sources of samples which can be studied to detect abnormalities ?
Prenatal:
- Aminiocentesis (Amniotic fluid sample) - Invasive
- Chorionic villus sampling (Sample from placenta) - Invasive
- Cell-free foetal DNA - non-invasive, from maternal plasma
Post natal:
- Blood
- Saliva
Describe chromosome staining and what is it used for ?
E.g. G-banding (Giemsa) Chromosome Staining.
There is banding structure in chromosomes due to :
Chromatin
-Euchromatin - GC rich, loosely packed active genes
-Heterochromatin - AT rich, tightly packed inactive genes
Staining is used because euchromatin + heterochromatin stain differently.
Karyotype is visible under microscope = detect large scale abnormalities.
What is the process of chromosome staining?
- Extract 5mL venous blood is extracted
- Culture blood sample for 3 days at 37oC to get to metaphase
- Fix Cells
- Spread cells onto slide by dropping
- Digest with trypsin + Stain with Giemsa
- Analyse Metaphase spread
- Karyotype to detect aneuploidies, translocations and very large deletions
What is the FISH method and what is it used for ?
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.Dentaure probe and target DNA 3.Mix probe and target DNA 4.Probe binds to target The part annealed to the probe will be fluorescent
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What is a Probe?
- Probe = A single stranded DNA/ RNA molecule.
- Typically 20-1000 bases in length
- Labelled with a fluorescent/luminescent/radioactiveIsotope molecule
- Probe is mixed with target DNA.
- Some techniques use thousands/millions of probes simultaneously
- FISH uses 1 probe which is specific to a region of the genome that we want to detect
What is Cri-du-chat and how can it be studied?
Cri-du-chat = Deletion of part of 5p chromosome.
- Telomeric deletion
- Use G-banding/FISH
- Probe anneals to a specific region of chromosome 5p.
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What is Spectral Karyotyping ?
Karyotype in which the homologous chr pairs are manipulated to have distinctive colours.
SKY technique = easier to detect chromosomal abnormalities
Describe Array CGH and what it is used for
Array CGH is used to detect sub-microscopic chromosomal abnormalities.
(Microdeletions/Microduplications)
Patient DNA is labelled green
Control DNA is labelled red.
1.Patient DNA and control DNA are labelled with fluorescent dyes (green, red) and applied to microarray.
2.Patient and control DNA compete to attach/hybridise to the microarray.
(Equal hybridisation=Yellow fluorescence)
- Microarray scanner measures the fluorescent signals
- Computer software analyses the data and generates a plot
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What is QF-PCR?
- Quantitative fluorescence polymerase chain reaction.
- Diagnoses different trisomies (13,18,21)
- Uses microsatellites
What does the QF-PCR output look like ?
It shows a different number of peaks.
Disomic- 2 peaks
Trisomic: Either 1 peak bigger than the other OR 3 peaks
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What are microsatellites?
- STR Short Tandem Repeats
- Short repeated sequences
- Number of repeats varies between individuals = length polymorphism
- Total length of microsatellite sequence varies b/w individuals
- Distributed across entire genome but most are not within genes
What are the different types of microsatellite length polymorphism ?
Dinucleotide Trinucleotide Tetranucleotide Pentanucleotide Hexanucleotide
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What is a unique flanking sequence ?
A DNA sequence which is located adjacent to a microsatellite, either upstream/downstream
Flanking seq. is unique + specific to each microsatellite – used to detect microsatellite
slide 46
How can we detect microsatellites?
- Isolate DNA from the individual
- Design primers specific to unique flanking sequence
- PCR amplification to amplify specific region
- Gel electrophoresis to determine which alleles are present
What are the types of products from PCR?
- PCR amplification of the microsatellite region
- Run PCR fragments on gel electrophoresis = genotyping, determining fragments size
- Homozygotes = single product of specific size
- Heterozygotes = 2 different sized products
What are the components of PCR?
PCR = Exponential amplification of a DNA fragment of known sequence
Components of the PCR reaction:
- Template -DNA to amplify
- Primers-Short pieces of ssDNA
- Polymerase-Thermostable enzyme (Taq)
- Nucleotides-Single base mixture (dNTPS)
- Buffer-To maintain pH
- MgCl2-Essential for polymerase activity
What are the processes within PCR?
PCR involves incubating at 3 different temperatures
3 Processes:
- Denature -Double to single stranded (94-95oC)
- Anneal-Primers to anneal (50-65oC)
- Extend (72oC)
Cyclical process
How is QF-PCR used to diagnose aneuploidies?
Perform PCR using primers for microsatellite known to be on chromosome 21 (Specifically for Down’s syndrome )
Should be 2 copies of microsatellite
If homozygous there will be a single peak of high signal
If heterozygous, there will be two peaks of similar lower signal
Describe the difference between the two trisomy peaks
One trisomy will show 1 high peak + 1 low peak.
The second trisomy shows 3 same-level peaks
Describe the Non Invasive Pre-Natal Testing (NIPT) and NGS
- Obtain cell-free foetal DNA from maternal blood sample
- Trisomy testing
- Next generation sequencing
- High chance indicator for invasive test (High risk mothers).
- Must then follow up with invasive test - amniocentesis/chorionic villus sampling
-There will be greater representation of the affected chromosome
