GENOMICS Structural chromosomal abnormalities Flashcards
What is translocational structural abnormality?
Exchange of two segments between non-homologous chromosomes
What are the 2 types of translocation
-Reciprocal
-Robertsonian
What is reciprocal translocation?
In a reciprocal translocation, two different chromosomes have exchanged segments with each other.
What is robertsonian translocation?
In a Robertsonian translocation, an entire chromosome attaches to another at the centromere.
What is translocation due to inappropriate non homologous end joining?
Before:
-No net gain or loss of genetic material
-Involves any chromosome and any size fragment
After:
-Plus copy of normal 1 and normal 22
-Carrier of balanced translocation
Why is carriers of balanced translocation not always lucky? Give an example
Philadelphia chromosome
-Translocation involving chromosome 9 ABL and 22 BCR
-This encodes BCR-ABL protein which can lead to the development of chronic myelogenous leukemia
What is reciprocal translocation in meisois?
A reciprocal translocation usually involves breakage of two non-homologous chromosomes with exchange of the fragments.
What are the results of unbalanced reciprocal translocation?
-Many lead to miscarriage (hence why a woman with a high number of unexplained
miscarriages should be screened for a balanced translocation)
-Learning difficulties, physical disabilities
-Tend to be specific to each individual so exact risks and clinical features vary
What is robertsonian translocation?
Two acrocentric chromosomes join near centromere with the loss of p arms
What chromosomes does robertsonian translocation involve and why?
This type of translocation involves only chromosomes 13, 14, 15, 21 and 22, because the ends of their short arms have similar repetitive DNA sequences that predispose to their fusion.
How many chromosomes does an individual who is a balanced carrier of robertsonian translocation have?
Balanced carrier has 45 chromosomes
If there are 46 chromosomes present with robertsonian translocation then what does this mean?
If 46 chromosomes present including Robertsonian then must be unbalanced
What do the p arms of chromosomes encode?
p arms encode rRNA
What does a robertsonian translocation of 21;21 lead to?
21;21 translocation leads to 100% risk of Down syndrome in fetus
What does a terminal deletion result in?
A new telomere
What are examples of structural changes that represent interstitial deletion?
Examples:
-Prader-Will
-DiGeorge syndrome
-Cri du chat
What may a deletion structural chane to chromosomes be?
Deletion may be terminal or interstitial
What do deletions cause a region of?
Causes a region of monosomy
What can deletions cause insufficiency of in some genes?
Haploinsufficiency of some genes
What is phenotype specific for in deletion?
Phenotype is specific for size and place on deletion
What do gross deletions spread on?
Gross deletions seen on metaphase spread on G-banded karyotype
What techniques show microdeletions in chromosomes?
High resolution banding, FISH and now CGH showed ‘micro’ deletions
What microdeletion causes DiGeorge syndrome?
22q11
What microdeletion causes Wolf-Hirschhorn?
4p16
What microdeletion causes Williams syndrome?
7q11
What microdeletion causes Smith-Magenis syndrome?
17p11
What does unequal crossing over result in?
Explain how unequal crossing over can result in (micro)deletions and (micro)duplications
What are prenatal sources of sample in detecting chromosomal abnormalities?
-Amniocentesis
-Choronic villus sampling
-Cell-free fetal DNA
What are postnatal sources of sample in detecting chromosomal abnormalities?
-Blood
-Saliva
What is the most common form of chromosome staining?
G-banding
Why do we use bands in chromosome staining?
Because of chromatin
-2 different sorts: Euchromatin and heterochromatin
What is Euchromatin rich in, how is it packed and what does this mean for gene activity?
-GC rich
-Loosely packed
-Gene active
What is heterochromatin rich in, how is it packed and what does this mean for gene activity?
-AT rich
-Tightly packed
-Genes inactive
What are the steps involved in the production of a karyotype?
- Extract 5mL of venous blood
- Add phytohemagglutinin and culture medium
- Culture at 37 degrees celcius for 3 days
- Add colchicine and hypotonic saline
- Cells fixed
- Spread cells onto slide by dropping
- Digest with trypsin and stain with Giemsa
- analyse ‘metaphase spread’
- Have final karyotype
What does G-banding look for?
Looks for aneuploidies, translocations & very large deletions
What does FISH stand for?
Fluorescent in situ hybridisation
What is hybridisation?
single stranded nucleic acid strand binds to a new single stranded nucleic acid
strand (DNA/DNA or DNA/RNA)
What are the steps involved in FISH?
Cultured cells, metaphase spread
1. Fluorescent probe
2. Denature probe and target DNA
3. Mix probe and target DNA
4. Probe binds to target
What is a probe?
A single stranded DNA (or RNA) molecule
How long is a probe?
A single stranded DNA (or RNA) molecule
What is a probe labelled with?
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive
isotope)
What type of chromosomes does FISH use?
Uses metaphase chromosomes
What is the complete process of array CGH?
- Patient and control DNA are labelled with fluorescent dyes and applied to the microarray
- Patient and control DNA compete to attach, or hybridise, to the microarray
- the microarray scanner measures the fluorescent signals
- Computer software analyses the data and generates a plot
What does array CGH look for?
Looks for microdeletions and microduplications
What does QF-PCR stand for?
Quantitative fluorescence polymerase chain reaction
What does QF-PCR detect?
Trisomies 13, 18 and 21
What does QF-PCR use?
Uses microsatellites
What are microsatellites?
Short repeated sequences with variable numbers of repeats
What is the variability of microsatellites like between individuals?
Number of repeats varies
between individuals
Total length of microsatellite
sequence varies between
individuals
What are short tandem repeats also known as?
Short tandem repeats are also known as microsatellites and simple sequence repeat(SSR)
What are the steps involved in detecting microsatellites?
Isolate DNA from individual
Design primers specific to flanking sequences
PCR amplification
Gel electrophoresis
What is PCR?
Exponential amplification of a DNA fragment of known sequence
What are the steps involved in PCR?
- Heat and Separate DNA strands at 94oC
- Primers anneal with template (50-65oC)
- DNA polymerase extends strand from primer, 72oC
-2 identical copies of the region of interest are formed - After 2 cycles we have 4 copies of the region of interest
What are the steps involved in QF=PCR when testing for downs?
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
What does Non-invasive pre-natal testing (NIPT) and NGS use?
Cell free fetal DNA
What type of testing is NIPT and NGS?
Trisomy testing
What type of sequencing is NIPT and NGS?
Next-generation sequencing
What are the steps involved in NIPT and NGS?
- Cell free DNA in placenta is extracted from maternal plasma
- Massively parallel sequencing of total DNA present in maternal plasma
- Alignment of sequencing reads to human genome sequence an determination of relative chromosome representation
- Detection of aneuploidy like trisomy 21
