Structural chromosomal abnormalities Flashcards
Describe the normal human karyotype
- 22 pairs of autosomal chromosomes
- 1 pair of sex chromosomes
Name some structural abnormalities and briefly describe what they are
- Terminal deletion - From end of chromosome
- Interstitial deletion - From within chromosome
- Inversion - 2 breakpoints within same chromosome and when these are repaired the middle section is ‘upside down’
- Duplication - Region of chromosome repeated
- Ring chromosome - 2 breaks in same chromosome and NHEJ mechanism joins 2 ends together resulting in ring
What is translocation?
Chromosome breaks, and 2 fragmented pieces re-attach to different chromosomes
What is reciprocal translocation?
Part of one chromosome is exchanged with another
No loss or gain of material, so little consquence to cell carrying reciprocal translocation. However, during meiosis this can have negative consquences.
The chromosomes can separate so that there’s ultimately an unbalanced rearrangement
Forms a quadravalent structure
What are the consequences of unbalanced reciprocal translocation?
- Many lead to miscarriage (women with high number of unexplained miscarriages should be screened for balanced translocation)
- Learning difficulties, physical disabilities
- Can be more specific and individualiased though, so risks and features vary
What is robertsonian translocation?
When 2 acroentric chromosomes break at or near their centromeres, these fragments join together again and it’s possible for just the 2 sets of long arms to be brought together, and there’s loss of the satellites - This is robertsonian translocation
If this happens in a cell, there will be 45 chromosomes, this is because the short arm join and are often excluded by the cell, meaning 1 chromosme pair is missing
If 46 chromosomes are present including robertsonian then it must be unbalanced
This only affects acrocentric chromosomes, which are chromosomes 13,14,15,21 and 22
Most common robertsonian translocation contains chromosomes 13 and 14 and involves the loss of 2 short arms so 2 long arms fuse, with either 1 or 2 centromeres
The resultant chromosome usually contains the long arms of different chromosomes. It forms a trivalent structure.
Robertsoninan translocation of chromosomes 21;21 results in 100% down’s syndrome development
What do P arms encode?
rRNA
Explain the process of translocation due to inappropriate non-homologous end joining
- DNA repair mechanism pairs chromosome fragments incorrectly
- This is non-homologous end joing because it’s joining together 2 ends that’s irrespective of the DNA sequence joined together
- This is also called balanced translocation
- These can have no negative effects, but in some cases these can be oncogenic
Explain the process of non-homologous end joining as a DNA repair mechanism
If part of a chromosome breaks off, NHEJ will just reattach that part to its parent chromosome
What is a balanced translocation?
When the translocation doesn’t result in any loss or gain of genetic material
What happens to homologous chromosomes during metaphase 1 of meiosis?
They align and form a bivalent structure.
This is also when independent assortment occurs - the random orientation of homologous chromosome pairs during metaphase I allows for the production of gametes with many different assortments of homologous chromosomes
How does unbalanced translocation occur?
One normal chromosme could end up with the short arm and the other with a long arm. The long arm carries most of the genetic material, so cells with the long arm have one extra chromosome in essence
What is trisomy?
Presence of 3 chromosomes instead of a pair of choromsomes
What is monosomy?
Condition of having a diploid chromosome in which one chromosome lacks its homologous partner, so there are 45 chromosomes within the body
What are the consequences of deletions?
Deletions cause a region of monosomy, haploinsufficiency on some genes (if the gene leads to a clinical phenotype), contiguous gene syndrome. The phenotype is specific for size and place on deletion. gross deletions seen on metaphase spread on G-branded karytotype
The situation that occurs when one copy of a gene is inactivated or deleted and the remaining functional copy of the gene is not adequate to produce the needed gene product to preserve normal function.
Explain how unequal crossing over can lead to deletions and duplications
- This is also called non-allelic homologous recombination
- Exchange of genetic material b/w homologous chromosomes that incorrectly align
- At meiosis the genes should align perfectly, but if misaligned, there can be simultaneous deletions and duplications on the invidual chromatids in the gametes.
- This means that during crossing over, the incorrect parts of the chromosome will be exchanged. 1 chromosome will experience deletion and the other duplication, as can be seen below
How can microdeletions be detected?
- Many patients had no abnormality visible on metaphase spread
- FISH and CGH show microdeletions
What are sources that can be sampled to detect sturctural abnormalities?
- Prenatal
- Amniocentesis
- Chorionic villus sampling
- Cell-free fetal DNA
- Postnatal
- Blood
- Saliva
Describe G banding
G = Giemsa
This is metaphase chromosome staining
Giemsa highlights heterchromatic regions which are less likely to contain genes. Banding can be used to differentiate b/w chromosomes and to compare them. It’s generally done at metaphase when the chromosomes are highly condensed.
Looks for aneuploidies, translocations and large deletions
Take several days at least
Bands because:
- 2 types of chromatin - Euchromatin and heterochromatin
- Euchromatin - GC rich, loosely packed, activate genes
- Heterochromatin - AT-rich, tightly packed, genes inactive
- Stain differently
What is FISH?
- Fluorescent in situ hybridisation
- Hydbridisation - Single stranded nucleic acid strand binds to a new single stranded nucleic acid strand (DNA/DNA or DNA/RNA)
- Uses metaphase chromosomes
- Looks for aneuploidies, translocations and large deletions
- Take several days at least
- Cultured cells, metapase spread:
- Fluorescent probe
- Denature probe and target DNA
- Mix probe and target DNA
- Probe binds to target
What is a probe?
- Single stranded DNA or RNA molecule
- Typically 20-1000 bases in length
- Labelled with fluorescent or luminescent molecule (less commonly a radioactive isotope)
Probes vary in size from oglionucleotides to genomic clones.
Describe array-CGH
- Patient and control DNA labelled with fluorescent dyes, applied to microarray
- Patient and control DNA compete to attach or hybridise to microarray
- Microarray scanner measures fluorescent signals
- Computer software analyses data and generates plot
Used for detection of sub-microscopic chromosomal abnormalities. Patient DNA is labelled green and controlled DNA is labelled red. Looks for microdeletions and microduplications
Microarrays are created by deposit and immobilisation of small amounts of DNA (probes) on solid support e.g. glass slide.
As probes are much, much smaller than metaphase chromosomes, the theoretical resolution of aCGH is proportionally higher than that of traditional CGH.
Lvl of resolution is determined by considering both probe size and genomic distance b/w DNA probes.
Main question being asked using this technique is how many copies of a particular genomic region does the patient have?
Describe QF-PCR
- Quantitative fluorescence polymerase chain reaction
- Uses microsatellites
- Trisomies 13 (Patau syndrome), 18 (Edwards syndrome) and 21 (Down’s syndrome)
- This method of analysis looks for how many copies of a chromosome patients have
What are microsatellites?
- Short repeated sequences
- Number of repeats varies b/w individuals
- Total length of microsatellite sequence varies b/w individuals
- They’re distributed across the whole genome, most are not within genes
How can microsatellites be detected?
- Isolate DNA from individual
- Design primers specific to flanking sequences
- PCR amplification of microsatellite region
- Gel electrophoresis
Homozygotes - Single product of specific size, produces single peak of high signal on PCR
Heterozygotes - 2 different sized products, produces two peaks of similar, lower signal on PCR
Describe the process of PCR and its components
- Exponentional amplification of DNA fragment of known sequence
- Componets of PCR reaction:
- Template- DNA to amplify
- Primers - Short pirces of ssDNA
- Polymerase - Thermostable enzyme (Taq)
- Nucleotides - Single base mixture (dNTPs)
- Buffer - To maintain pH
- MgCl2 - Essential for polyemrase activity
- Incubating at 3 diff temperatures (thermal cycling), results in 3 different processes happening:
- Denaturation (94)
- Annealing (60)
- Extension (72)
- PCR cycle 1 - Starting point
- Template DNA - 1 molecule
- dsDNA template needs amplifying
- DNA denatured at 94 degrees to separate the DNA strands
- Primers anneal with template (50-65 degrees). When the temparature drops down from 94 degrees in the absence of any other DNA, the large strands re-anneal BUT in the presence of primers, these bind quicker to the DNA template, and once the primer is bound the template strands cannot bind to this region
- DNA polymerase extends strand from primer, this is at 72 degrees. The primers extended in the 5’ to 3’ direction by Taq.
- After 1 cycle, we have 2 identical regions of interest, and this PCR process repeats to keep amplifying the amount of DNA.
- This process is exponential, and so will be ongoing until the enzyme activity stops.
Describe non-invasive pre-natal testing (NIPT) and NGS
- Cell free fetal DNA
- Maternal blood sample
- Trisomy testing
- Next-genration sequencing
- “High chance” idicator for invasive test
- Looks for aneuploidies
- Screening not diagnostic
- Parallel sequencing of total DNA present in maternal plasma, alignment of sequencing reads to human genome sequence and deterination of relative chromosome representation
