The functional genome Flashcards
What is the Whole Exome Sequencing (WES) and Whole Genome Sequencing (WGS)?
High throughput DNA next generation sequencing techniques which aim to identify potential disease causing variants
- WES captures sequence of coding region of genome
- WGS captures whole genome (not always necessary as most genetic diseases are in coding region)
Give the Steps of Candidate Gene Filtering using WES.
Filtering data with all of your variants:
1) ~3 million SNPs per individual in entire genome
2) Targeted Sequencing of Exons, resulting in 15,000-20,000 coding SNPs per individual
3) Synonymous variants removed (no amino acid change), filtering it down to 7,000-10,000 non-synonymous coding SNPs per individual
4) Remove previously identified variants that are common, resulting in 200-500 novel non-synonymous coding SNPs per individual
5) Restrict to variant fitting dominant/recessive model of inheritance, resulting in one or several putative genes
Describe Candidate Gene Filtering using WES.
15-20,000 coding SNPs reduced to one or several candidate genes
Checked for co-segregation in family members and validated by Sanger sequencing
How to prove that the candidate gene variant is pathogenic before giving a genetic diagnosis to a patient?
Detection of Protein
-if no protein present, it is a good indication that that variant has a detrimental effect on protein
Tissue/Cell Expression
-if examining a specific disorder of a tissue and gene is expressed in that tissue but not other tissue, then it is a good indication that the candidate gene is the correct one
Knockdown/Overexpression Effects
-examine how the knockdown or overexpression of gene of interest changes the phenotype of the cell line
Development of Cells/Tissues
-examine variants which might change the development of the tissue
Molecular Mechanism of Action
-does the gene of interest have common pathways which are already known to be involved in the disease?
How to detect if protein is affected from genetic variant?
IF gene of interest expressed in blood or tissue, take a BIOPSY and have a look to see if that protein is expressed in affected individual
What is the Disadvantage of biopsy technique?
Gene of interest will not always be expressed in the blood and might not be in an accessible affected tissue, making this technique very difficult
To counter this, we can start making in vitro cell culture models
Describe the Cell Culture Technique.
cells from animal removed and grown in favourable conditions in an artificial environment
primary cells (first taken out) have finite division but can be immortalised to provide continuous source
cheap, rapid and reproducible model for studying normal physiology and biochemistry of cells
reduces number of animals used in research; less restrictions
State the Cell Culture Techniques to Detect if protein is affected from genetic variant:
- Gene Knockdown
- Protein Localisation
- Induced Pluripotent Stem Cells (IPSCs)
What is Gene Knockdown?
Knocking down gene function by RNAi mediated gene silencing:
1) Gene of interest is packaged in DNA plasmid with RNA polymerase II promoter which controls its expression
2) When transfected into nucleus, short hairpin RNA (ShRNA) is transcribed and the protein Exportin-5 exports it through the nucleopore into the cytoplasm
3) Dicer protein complex cleaves RNA. Cleaved segments then bind to RNA induced silencing complex (RISC) and there is direct cleavage and degradation of complementary mRNA.
What is Short interfering RNA (siRNA)?
Similar to ShRNA, chemically synthesised, but not vector based
What is Protein Localisation?
We can look at where the gene of interest’s encoded protein is localised:
- transfect cells with green fluorescent protein tagged gene of interest (CMV promoter)
- transfect cells with GFP tagged mutated gene of interest (CMV promoter)
- antibody staining of protein interest and downstream target
What are Induced Pluripotent Stem Cells (IPSCs)?
Adult cells that have been artificially induced to dedifferentiate and revert to pluripotent stem cells capable of becoming many types of cells (e.g. taking dermal cells and de-differentiating them to become muscle fibre).
Useful in Duchenne Muscular Dystrophy, as patients have a mutation in gene dystrophin required for muscle fibre integrity (stop codon in exon 45 and missing exon 44).
TALEN or CRISPR genes can be used to cause changes in DNA which result in:
- exon skipping (translated DNA is in frame, but missing exon 44 and 45), causing a truncated but functional dystrophin protein
- frameshift mutations which produce a functioning dystrophin
- exon 44 knock in
Why are cell culture techniques not enough in determining whether a candidate gene variant is pathogenic?
because cells in the petri dish does not stimulate the actual conditions inside an organism
different signals come from a 3D environment than a dish
looking at cells in a dish doesn’t provide information about gene expression and function with regards to developmental phenotypes
This is why ANIMAL RESEARCH comes in
Describe how Animals are used in Research.
Most medicines come from animal research
Anything discovered using animals goes back to animal-ill health as well (veterinary science)
Research scientists seek to alleviate pain and suffering, and as a result there is a rigorous home office monitoring system in place that prevents any distress/suffering to occur in animals in research
What is the Animals (Scientific Procedures) Act 1986?
Act which makes sure animals are looked after correctly:
·Regulates the use of protected animals in any experimental or other scientific procedures which may cause pain, suffering, distress or lasting harm to the animal
· These protected animals are any living vertebrate animal (other than man) and any living cephalopod (squid, octopus)
· Animals are cared for with the best standards of modern animal husbandry
· Home office inspection system to ensure rules are not violated (can incur fines/imprisonment if rules not followed)
· Widely considered as the most stringent animal welfare act in the world
