Structural chromosomal abnormalities Flashcards
What are the different types of structural abnormalities?
Translocations -reciprocal -robertsonian Inversion Deletion Duplication Rings Isochromosomes
What is a Translocation?
This is the exchange of two segments between non-homologous chromosomes
-reciprocal or robertsonian
How can Translocation occur?
It can occur dur to inappropriate Non-homologous end joining (NHEJ).
Its a DNA repair mechanism which can cause double strand breaks in DNA which separates the chromosome.
Normally it would re-join the broken off section to the same chromosome.
However it can incorrectly fix the chromosome.
It creates a derivative chromosome.
Balanced translocation =no net gain or loss of genetic material
What is a Philadelphia chromosome ?
An abnormality of chromosome 22 in which part of chromosome 9 is transferred to it.
This can lead to leukaemia and myeloma.
What is G-banding?
This is a technique used in cytogenetics to produce a visible karyotype of stained condensed chromosomes.
What are the consequences of reciprocal translocations in meiosis ?
These form quadrivalents during meiosis
What are the results of unbalanced reciprocal translocation?
Many lead to miscarriage
Learning difficulties,physical disabilities
What is robertsonain translocation?
These involve acrocentric chromosomes which contain genes which code for RNA molecules.
The p arms (sattelites) 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.
P arms 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
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
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 -The 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
Affect 1 in 7000 births
May be terminal or interstitial
Causes a region of monosomy
Haploinsufficiency of some genes
Contiguous gene syndrome
phenotype is specific for size and place on deletion
Gross deletions seen on metaphase spread on G-banded karyotype.
What are microdeletions/microduplications ?
These are when few genes are lost or gained.
Many patients had no abnormality visible on metaphase spread.
Can be caused by unequal crossing over.
Describe Array CGH
ARRAY-CGH
Looks at dosage
i.e. two individuals should have the same number of copies of the autosome – no difference
Take healthy individual and affected individual DNA
These anneal to probes on glass array
Should be equal hybridisation If both patient and control have two copies of a region (disomic) – 5 probes will have patient DNA
and 5 will have healthy DNA = DOSAGE WILL BE EQUAL
If there is a microdeletion/microduplication in patient DNA i..e two copies in healthy DNA and one
in patient DNA :
Dosage will be different
Hybridisation will be unequal
What are the different sources of samples which can be studied to detect abnormalities ?
Prenatal -Aminiocentesis (Amniotic fluid sample )-Invasive -Chroionic villus sampling (Sample from placenta )-Invasive -Cell-free foetal DNA
Post natal
Blood
Saliva
Describe chromosome staining and what is it used for ?
Most common form is G-banding using Giemsa.
There is banding structure in chromosomes due to :
Chromatin
Euchromatin- GC rich ,loosely packed and genes active
Heterochromatin-AT rich , tightly packed genes are inactive.
Staining is used because they both stain differently.
Can be viewed under the microscope therefore will detect large scale abnormalities.
What is the process of chromosome staining?
-5mL venous blood is extracted
(A few days required because a metaphase spread is used as they are most condensed to ensure the cells have divided)
- phytohemagglutinin is added and medium is cultured.
- Culture at 37oC for 3 days
- Add colchicine and hypotonic saline
- Cells are fixed
- Spread cells onto slide by dropping
- Digest with trypsin and stain with Giemsa
- Analyse Metaphase spread
-Karyotype
(Look for 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
What is a Probe?
A single stranded DNA/ RNA molecule.
Typically around 20-1000 bases in length
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope.
The probe is mixed with target DNA.
In some techniques thousands or millions of probes may be used simultaneously.
The FISH technique uses one probe which is specific to a region of the genome which we want to detect
What is Cri-du-chat and how can it be studied?
It is a condition where there is a deletion of part of the 5p chromosome.
It can be studied using G-banding and FISH.
The probe has been designed to anneal to a specific section of chromosome 5p.
What is spectral karyotyping ?
Karyotype in which the homologous pairs of chromosomes are manipulated in such a way that they have distinctive colours. The SKY technique makes it easier for scientists 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.Pateint and control DNA are labelled with fluorescent dyes and applied to microarray.
2.Pateint and control DNA compete to attach/hybridise to the microarray.
(Equal hybridisation)-Yellow
- The microarray scanner measure the fluorescent signals
- Computer software analyses the data and generates a plot.
What is QF-PCR?
This is quantitative fluorescence polymerase chain reaction.
It was developed to specifically diagnose different trisomies (13,18,21)
It uses microsatellites
What does the QF-PCR output look like ?
It shows a different number of peaks.
Disomic- Two peaks
Trisomic:
Either one peak bigger than the other or three peaks
What are microsatellites?
STR (Short tandem repeats )/SSR(Simple sequence repeat)
These are short repeated sequences
The number of repeats varies between individuals
Total length of microsatellite sequence varies between individuals
They are also distributed across the whole genome but most are not within genes
What are the different types of microsatellite length polymorphism ?
Dinucleotide Trinucleotide Tetranucleotide Pentanucleotide Hexanucleotide
What is a unique flanking sequence ?
A DNA sequence which is located adjacent to a gene either upstream or downstream
How can we detect microsatellites?
Isolate DNA from the individual
Design primers specific to flanking sequences
PCR amplification
Gel electrophoresis to determine alleles which are present
What are the types of products from PCR?
PCR amplification of the microsatellite region
Genotype size of fragments on gel based system
Homozygotes=single product of specific size
Heterozygotes=two different sized products
What are the components of 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 consists of incubating at three different temperatures
Processes as a result:
Denaturing -Double to single stranded (94-95oC)
Annealing -Primers to anneal (50-65oC)
Extension (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 syndrome )
Should be two 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 one high peak and one low peak.
The second trisomy is when there are three same level peaks
Describe the 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 (High risk mothers)
-There will be greater representation of the affected chromosome
