Non-coding variation: determining function

Question 1

What are the gold standard tests for regulatory function?

Answer 1

• reporter gene assays: test variants in functional assays in vitro
• Electrophoretic mobility shift assay (EMSA): test for differentially interacting regulatory proteins

Question 2

What are the problems with gold standard tests?

Answer 2

• not in vivo (context?)
• type I errors (false-positives)
• type II errors (false negatives)

Question 3

What are the problems with gene reporter assays?

Answer 3

• 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)

Question 4

What are the problems with EMSAs?

Answer 4

• probe limiting effects (higher order complexes)
• cell type issues
• in vitro only (no chromatin context)

Question 5

What are the solutions to the problems of gold standard tests?

Answer 5

• 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

Question 6

What is the Vanin-1 San Antonio Family Heart study?

Answer 6

• 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

Question 7

What cardiovascular gene was identified as a disease gene?

Answer 7

Vanin 1

Question 8

What was found from eQTL analysis for blood HDL-cholesterol?

Answer 8

• BQTN analysis on 113 Vanin1 SNVs found SNVs in LD with one another (haplotype) - makes analysis harder
• then performed gold standard tests

Question 9

What is a chromatin accessibility assay - CHART-PCR?

Answer 9

• Assigns relative value of chromatin accessibility
• maps DNA binding proteins to a specific region
• value indicates localised chromatin arrangement (transcriptional activity)

Question 10

What is the workflow of a high throughout functional SNP screen?

Answer 10

• harvest nuclei
• fragment genome with nuclease
• prepare DNA library for NGS (illumina etc)
• selection
• amplify
• deep-sequencing (100-fold depth)
• analysis

Question 11

Assays should identify SNPs which influence?

Answer 11

• TF binding
• Transcriptional potential
• methylation
• miRNA binding?

Question 12

What does a reporter gene assay entail?

Answer 12

Cloning a regulatory region/promoter upstream of a reporter gene (e.g. GFP, luciferase). Easy to measure

Question 13

What is an example of a reporter gene assay experiment?

Answer 13

• 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

Question 14

What is an EMSA?

Answer 14

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

Question 15

What is the process of an EMSA experiment?

Answer 15

• 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

Question 16

What are some in vivo functional tests?

Answer 16

HaploChIP (knight): tests for haplotype effects, to test for allele specific mRNA expression differences

Develop novel chromatin accessibility assay: SNP centric tests for differences in Chromatin arrangement

Question 17

How does CHART-PCR work?

Answer 17

If open chromatin –> incubate nuclei with nuclease (get cleavage of accessible DNA) –> real time PCR (less PCR product)

If condensed chromatin –> incubate nuclei with nuclease (no cleavage of DNA) –> Real time PCR (more PCR product)

Question 18

Can we use what we have learned from CHART-PCR to identify functional variants across the genome?

Answer 18

YES.