The functional genome Flashcards
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)
Steps of Candidate Gene Filtering using WES
Filtering data with all of your variants to find the specific causal variant:
1) Millions of SNPs per person 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 rare non-synonymous coding SNPs per individual
5) Restrict to variant fitting dominant/recessive model of inheritance, resulting in one or several putative genes
= one or several putative genes
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? Filters WES variants does not prove causality - need further functional evidence
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
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
Cell Culture Technique (in vitro)
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
Cell Culture Techniques to Detect if protein is affected from the putative genetic variant that was narrowed down:
- Gene Knockdown
- Protein Localisation
- Induced Pluripotent Stem Cells (IPSCs)
Gene Knockdown
Knocking down gene function by RNAi mediated gene silencing: (ShRNA - short hairpin)
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.
Difference between ShRNA and Short interfering RNA (siRNA)
Similar to ShRNA, chemically synthesised, but not vector based
Protein Localisation
We can look at where the gene of interest’s encoded protein is localised:
- transfect (infect) 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
Induced Pluripotent Stem Cells (IPSCs)
Take a skin biopsy, and fibroblast from this can be grown in a dish, and through gene expression - the fibroblast can be caused to de differentiate and become pluripotent stem cells - can be reprogrammed to become a cell type of your choice
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).
- convert fibroblast into muscle cells
Genome editing by TALEN or CRISPR 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
Animals (Scientific Procedures) Act 1986
Act which makes sure animals are looked after correctly
Licenses for Animal Research
3-tier licensing system authorised by home office:
> Establishment License- certificate of designation (makes sure party has correct facilities in place to look after animals)
> Project License- specific research/testing programme
> Personal License- specific individual/competency
Licenses to commence animal research only approved if:
- benefits outweighs cost
- prove there is no non-animal alternative
- show that minimum number of possible animals will be used
- prove that animals with the lowest sensitivity to pain are being used
- pain is minimised
- research premises have necessary facilities to care for animals
Advantage of using mouse models
- closely related to humans
- accelerated lifespan (1 year is 30 human years)
- small, reproduce quickly, relatively easy to handle and transport
- lots of mouse strains and models already exist
- more ethical than using larger animals/non-human primate/humans
- modern genome engineering has allowed us to make precise mutations to recreate disease
Making a Conditional Mutant Mouse
1) Take out embryonic stem cells
2) Introduce changes in DNA through homologous recombination and select for cells expressing desired gene
3) Embryonic stem cells injected into inner cell mass of blastocyst in mouse
4) Implanted into pseudopregnant female mouse
5) Test offspring for presence of gene
Advantages of using zebrafish models for human genetic disease
- develop really quickly
- produce lots of eggs
- easy to genetically manipulate
- cheap
Gene knockdown in zebrafish
Morpholino = synthetic oligonucleotides (MOs) used to knock down gene function because zebrafish have a morpholino backbone instead of a ribose backbone
MOs block gene specific translation or splicing (blocks movement of initiation complex
Disadvantage of genetic knockdown in zebradish
MOs can have off target genetic effects (toxic effects)
difference between in vitro and in vivo
in vitro study occurs in a controlled environment, such as a test tube or petri dish. In vivo is Latin for “within the living.” It refers to tests, experiments, and procedures that researchers perform in or on a whole living organism
Making a Zebrafish Mutant
Methods:
1) Bathing zebrafish in ENU (N-Ethyl-Nitrosourea)
2) CRISPR/Cas9 System
Bathing Zebrafish in ENU
> Potent mutagen targeting spermatogonial stem cells
> Causes point mutations at a rate of 1 per 700 gametes
Then you can cross the mutagenized males with females and look at their offspring. You can do:
- Phenotype based ENU screening (forward genetics): look for obvious changes (e.g. lack of eyes)
- Genotype based ENU screening (reverse genetics): taking DNA out of the fish and WES and finding mutations and looking for a mutation in phenotype after that
CRISPR-Cas9 system
a technique for editing genes in living cells, involving a bacterial protein called Cas9 associated with a guide RNA complementary to a gene sequence of interest
Cas9 causes double stranded DNA breaks, and cell repairs this by non-homologous end joining or homologous directed repair, adding or deleting nucleotides, causing mutations
RNA Rescue experiments
the real proof of whether a variant is causing the disease is if you can rescue this genetic knockdown
