Structural Chromosomal Abnormalaties Flashcards
What are some chromosomal structural abnormalities
Translocation:
- Reciprocal
- Robertsonian
Inversion
Deletion
Duplication
Rings
Isochromosomes
What is translocation
- Most common
- Exchange of two segments between non-homologous chromosomes
- Reciprocal or Robertsonian
How does translocation due to inappropriate NHEJ work
- Incorrectly sticks 2 ends of non-homologous chromosomes together.
- creates derivatives
how are unbalanced individuals produced?
- Chromosomes align normally as a bivalent structure but here it forms a tetravalent structure and separates either way.
What is the result 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
Explain what happens in robertsonian translocation
- The combination of Q arms of 2 chromosomes
What are the characteristics of Robertsonian translocations
- 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
What are some 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
List some other structural changes
- Terminal deletion- the end of a chromosome is deleted
- Interstitial deletion - the middle of a chromosome is taken out and resealed
- Inversion - The middle of a chromosome is inverted and resealed
- Duplication- a portion of the chromosome is duplicated and extends the chromosomes
- Ring Chromosome - The chromosomes sealed into a ring shape
What are some characteristics of deletion mutations?
- 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
What are some characteristics of microdeletion mutations?
- 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
Describe unequal crossing-over
- When homologous chromosomes don’t align equally
- Leads to genes not crossing over on the right locus
- Changes the length of the chromosomes
How and where do we acquire samples for testing
Prenatal:
- Amniocentesis, amniotic fluid
- Chorionic villus sampling, Placenta
- Cell-free fetal DNA, Maternal plasma
Postnatal:
- Blood
- Saliva
How does chromosome staining work
Most common = G-banding
G = Giemsa
Why bands?
- Chromatin
- 2 different sorts: euchromatin & heterochromatin
- Euchromatin = GC-rich; loosely packed; genes active
- Heterochromatin = AT-rich; tightly packed; genes inactive
- Stain differently
What are the steps in analysing a blood sample
- Draw 5ml of venous blood
- Add phytohemagglutinin and culture medium
- Culture at 37C for 3 days
- Add colchicine and hypotonic saline
- Cells fixed
- Spread cells onto the slide by dropping
- Digest with trypsin and stain with Giernsa
- Analyse metaphase spread
Summarise the G-banding technique
- How does karyotype of patient differ from expected?
- Uses a chemical stain
- Uses metaphase chromosomes
- Takes several days at least
- Looks for aneuploidies, translocations & very large deletions
What is FISH
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:
- Fluorescent probe
- Denature probe and target DNA
- Mix probe and target DNA
- Probe binds to target
What is a probe?
- 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
What is ArrayCGH
- Similar to microarray technologies
- Array comparative genomic hybridisation
- For detection of sub-microscopic chromosomal abnormalities
- Patient DNA labelled Green
- Control DNA labelled Red
What is QF-PCR
- Quantitative fluorescence polymerase chain reaction
- Trisomies 13, 18 and 21
- Uses microsatellites
What are micro satellites
- Short repeated sequences
- Number of repeats varies between individuals
- Total length of microsatellite sequence varies between individuals
How can we detect micro satellites
- Isolate DNA from individual
- Design primers specific to flanking sequences
- PCR amplification
- Gel electrophoresis
How does PCR work
Exponential amplification of a DNA fragment of known sequence
PCR consists of incubating at three different temperatures
This results in three different processes happening:
- Denaturation
- Annealing
- Extension
How is QF-PCR used to detect chromosomal abnormalities
- 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 is Non-invasive pre-natal testing and NGS
- Cell free fetal DNA
- Maternal blood sample
- Trisomy testing
- Next-generation sequencing
- “High chance” indicator for invasive test
