POM MOCK 4 - Diagnostic virology, emerging treatments, prenatal testing, phenotypic variability, Flashcards
How is HTLV-1 transmitted?
Mother to infant (breastfeeding). Sexual contact. Blood transfusion.
What is HTLV-1 structure?
Enveloped virus. Single strand RNA virus. Contains reverse transcriptase.
HLTV-1 replication cycle?
HTLV-1 enters T-cell
ssRNA released into host cell cytosol
ssRNA reverse transcribed (RT-enzyme) to ssDNA
ssDNA converted to dsDNA
dsDNA enters nucleus and integrates into host genome
viral genome can replicate as part of the host chromosome
What triggers oncogenesis in T cell infected with HLTV?
The HTLV-1 Tax oncoprotein triggers oncogenesis and induces the production of its own RNAs.
How is a polymerase chain reaction done?
DNA sample is denatured by heating to 94 degrees celsius. Cooled down to 54 degrees to allow forward and reverse primers to anneal. Sample is heated to 72 degrees and new DNA chain is extended.
How many cycles is a typical PCR?
30-40.
What are the 5 key reagents of a PCR?
DNA template, Primers, DNA polymerase, deoxynucleotide triphosphates and reaction buffer.
How is DNA sample for HLTV taken?
Blood is taken from patient and mixed with seperation medium. It is then centrifuged. Peripheral blood mononuclear cell fraction is extracted and DNA is isolated.
How many PCR reactions would need to be setup if testing 4 patients for hltv?
- 4 patients PCR reactions. One positive control (HLTV). One negative control (no HLTV).
What electrode does DNA move towards in gel electrophoresis?
Positive electrode as DNA is negatively charged.
What is used to visualise the DNA in electrophoresis?
Intercalating DNA stain.
Why is a DNA loading dye used?
Increase weight of DNA sample. See which wells contain a sample. Indicate how far the DNA fragments have migrated during run.
Why is a DNA ladder used?
To estimate size of DNA fragment.
What is the point of a buffer in PCR?
Provides suitable pH for DNA polymerase.
How is the western blot method performed for HLTV?
Different HLTV viral proteins are separated based on their size on a protein gel using electrophoresis. The proteins will be transferred using an electro-transfer system onto a PVDF membrane that the viral proteins will stick to, and this will basically create an image of the gel. Incubate membrane with patient serum so antibodies can bind to viral proteins. Wash membrane to remove all unbound antibodies. Incubate membrane with secondary antibody that is conjugated with an enzyme. Secondary antibody binds to fc region of patient’s antibodies. Wash again to remove any unbound secondary antibodies. Substrate added, enzyme produces colour.
For a positive HLTV diagnosis what antibodies need to be present on western blot method?
MTA-1, p53, p24, p19 and gd21(recombinant glycoprotein).
What is the advantage of doing a quantitative real time PCR for diseases?
Provides information on amount of viral DNA present in sample. Can help predict severity of disease and how likely transmission is to occur.
What are the two ways in which quantitative real time PCR is done?
Fluorescence dye-based method and DNA probe-based method.
Describe the process of developing a treatment?
Preclinical.
Phase 1 - Health volunteers.
Phase 2 - Check therapeutic effect by testing on patients.
Phase 3 - Large scale therapeutic trials.
Approval.
How do pharmacological chaperones work?
Aggregation of misfolded protein. Drug folds protein correctly.
Disadvantage of pharmacological chaperones?
Mutation specific.
What organelle degrades misfolded proteins?
Endoplasmic reticulum
Example of a pharmacological modulator?
Receptor agonists/antagonist
Ion channel activators/blockers
Disadvantage of pharmacological modulators?
Mutation specific.
What is a non sense mutation? What does it result in?
Premature stop codon. Prevents protein production
What drugs are stop codon readthrough drugs based on?
Aminoglycoside antibiotics as they bind to ribosome and cause mistranslation.
How does mitochondrial disease therapy work?
IVF. Take DNA from fertilised patient egg and transfer to donor egg. Mutant mitochondrial DNA replaced with non mutant.
What are two ways in which antisense oligonucleotides can work?
Blocks translation by binding to mRNA’s causing them to be destroyed. Skipping of exons.
When does exon skipping work?
In large proteins.
How does RNAi work?
Cleaves mRNA.
How can virus gene therapy treat a deficiency of immune cells?
HSC’s harvested. Viruses infects cell. Cells are grown. Mutant HSC’s in bone marrow are killed and new HSC’S are implanted.
How is a nuchal scan done?
Ultrasound is used to see thickness of fluid at back of fetal neck. A thickness greater than 3mm in associated with chromosomal abnormalaties.
When is nuchal scan done?
Done at 12 weeks.
What non invasive prenatal testing can be done?
Cell free fetal DNA screening.
How is cell free fetal DNA screening done?
Analysis of fetal DNA in maternal bloodstream.
When is cffDNA screening done?
9 weeks.
In cffDNA screening what is looked at?
Sex determining region (if there is an x linked condition in family). Test for aneuploidy (invasive to confirm).
When is chorionic villus sampling done?
11-14 weeks.
Risks of chorionic villus sampling?
1-2% risk of miscarriage. Infection. Rhesus sensitisaton
When is amniocentesis done?
16 weeks.
Risks of amniocentesis?
Up to 1% chance of miscarriage. Infection. Rhesus sensitisation
How is CGH array done?
DNA sample compared to control using computer. Picks up microdeletions and duplications in DNA.
If structural anomalies are detected on prenatal ultrasound what test can be done?
Prenatal exome sequencing.
In preimplantation genetic diagnosis when are the cells removed for testing for the genetic condition in the family?
Day 5. Blastocyst stage.
What effects phenotypic variability?
Environmental impact. Sex. Effect of other genes. Different mutation in same gene. Unstable mutations such as tri-nucleotide repeat expansions.
If a disease is present in an extended family what is unlikely to occur that could affect phenotypic variability?
The family members have inherited different mutations.
If a disease is present in an extended family what is likely to occur that could affect phenotypic variability?
A second gene modifies the disease phenotype.
If a disease is caused by an unstable trinucleotide repeat and there is phenotypic variability across generations what could explain this?
Environmental factors can modify the disease phenotype.
If there is wide phenotypic variability between families with ‘same disease’ what is most likely to explain for this?
Different mutation in same gene.
If there is no phenotypic variability in a disease between monozygotic twins but there is genetic variability in non monozygotic twins what does this tell us?
Must be a genetic basis driving phenotypic variability.
If there is a wide variability of symptoms both within affected families and between affected families and no variability in monozygotic twins what is most likely to explain this?
Second gene modifying phenotype.
A disease is caused by mutation in mitochondrial DNA, the severity of the disease and age of onset is highly variable, what is the most likely explanation for this?
A variable number of mitochondria with the mutation are inherited.
Several members of an extended family have a disease. Age of onset and severity of the disease shows marked variability with no two members being affected in the same way.
Because no two members are affected in the same way environmental factors are the most likely explanation. A second gene affecting phenotype is possible but not the most likely given the marked variability.
Severity of trinucleotide repeat disorders down generations?
Increases as you have expansion of unstable trinucleotide repeat as you go down generations.
