Non-coding variation: determining function Flashcards
What are the gold standard tests for regulatory function?
- reporter gene assays: test variants in functional assays in vitro
- Electrophoretic mobility shift assay (EMSA): test for differentially interacting regulatory proteins
What are the problems with gold standard tests?
- not in vivo (context?)
- type I errors (false-positives)
- type II errors (false negatives)
What are the problems with gene reporter assays?
- cell-specific effects (might see different effect in different cells) e.g. differences in T cells but not B cells
- inducible effects - stimulation etc (e.g. transfection method so lipid-mediated compared to electroporation have an effect)
- also cell culture conditions
- experimental effects/errors (dirty DNA, normalisation etc)
What are the problems with EMSAs?
- probe limiting effects (higher order complexes)
- cell type issues
- in vitro only (no chromatin context)
What are the solutions to the problems of gold standard tests?
- develop a credible model of how the genetic variation affects function
- develop a vivo assays that assess allele-specific chromatin arrangement
- develop in vivo reporter gene assays in different cellular contexts
- genome editing
What is the Vanin-1 San Antonio Family Heart study?
- Major project at TBRI to find genes involved in heart disease
- focusing on 1400 individuals of 40 large Mexican American families with high incidence of heart disease and diabetes
What cardiovascular gene was identified as a disease gene?
Vanin 1
What was found from eQTL analysis for blood HDL-cholesterol?
- BQTN analysis on 113 Vanin1 SNVs found SNVs in LD with one another (haplotype) - makes analysis harder
- then performed gold standard tests
What is a chromatin accessibility assay - CHART-PCR?
- Assigns relative value of chromatin accessibility
- maps DNA binding proteins to a specific region
- value indicates localised chromatin arrangement (transcriptional activity)
What is the workflow of a high throughout functional SNP screen?
- harvest nuclei
- fragment genome with nuclease
- prepare DNA library for NGS (illumina etc)
- selection
- amplify
- deep-sequencing (100-fold depth)
- analysis
Assays should identify SNPs which influence?
- TF binding
- Transcriptional potential
- methylation
- miRNA binding?
What does a reporter gene assay entail?
Cloning a regulatory region/promoter upstream of a reporter gene (e.g. GFP, luciferase). Easy to measure
What is an example of a reporter gene assay experiment?
- Studying Tumour Necrosis Factor (TNF) -308 SNP
- cloned a region of the -308 TNF promoter upstream of luciferase reporter
- can measure the reporter in cell lines
- can make a single base change within our promoter sequence (e.g. -308A or -308G) and determine this influence of transcriptional activity
What is an EMSA?
An electrophoretic mobility shift assay, looks at the ability of sequences to bind TFs.
- can assess whether single nucleotide changes within a sequence can affect the binding of transcription
What is the process of an EMSA experiment?
- take a cell line, isolate nuclear protein, design synthetic oligonucleotides, then take that nuclear extract and bind it to the oligonucleotide
- then run the bound protein complexes on a gel
