1. Loss of Function variants and Transcription
Manuel Rivas, University of Oxford
Geuvadis RNA Sequencing Analysis Meeting
15/04/2012

2. Loss of Function variants
(Putative) LoF variants:
Splice site variants
Nonsense and Readthrough mutations
Frameshift indels.
Exon Deletion and Duplication
Gene Deletion and Duplication
Gene Fusion* 5’ AG
GT[AG]AG AG G 3’

3. What can we learn about LoF using RNA sequencing data
Effect of splice variants on transcripts
Exon Skipping
Intron Retention
mRNA Surveillance Pathways
Escape NMD
Trigger NMD
What are the sequence properties that trigger NMD pathway or dictate escaping NMD pathway.
3. NMD efficiency in individuals. Do rates vary between individuals?
CNVs
Gene Fusion – impact of gene fusions in transcription? Do they properly get transcribed?
Exon/Gene Deletions and Duplications – impact of exon/gene deletions and duplications in transcription?

4. Splice Site Variants ` ` ` `
Initial analysis of Exon Quantification Data using splice variant annotation successfully recapitulates splicing event.
(IFIH1 Exon Skipping c.IVS14+1G>A) ` G A ` ` `
Annotation -> Evaluate Empirical Effect on Transcript

5. mRNA surveillance – Nonsense Mediated Decay (NMD)
IFIH1 Transcript Escapes Nonsense Mediated Decay.
Model 1: NMD is not triggered
Model 2: NMD is triggered ` G A ` ` `
Propose Statistic that detects Nonsense Mediated Decay
EXAMPLE 1: ESCAPE NMD

6. mRNA surveillance – Nonsense Mediated Decay (NMD)
CIAO1 premature stop variant triggers Nonsense Mediated Decay.
Model 1: NMD is not triggered
Model 2: NMD is triggered
Propose Statistic that detects Nonsense Mediated Decay
EXAMPLE 2: TRIGGER NMD
REDUCTION OBSERVED THROUGHOUT TRANSCRIPT

7. Loss of Function variants and ASE
Added Annotation Layer to VAT output from annotation program co-developed at Oxford.
Fraction of LoF observations with allele specific expression detected. Crude
measure at the moment based on Tuuli’s first run (n = 180 samples).
Determines a two-sided confidence interval from the beta posterior.

8. Loss of Function variants and eQTL Analysis
Dependent on frequency range.
ASE most powerful for rare variants.
eQTL suitable for common variants.

9. Methods and Analytical Strategies for LoF variants
Splicing Effects based on Annotation
Exon Skipping
Intron Retention
mRNA Surveillance Pathways
Escape NMD
Trigger NMD
CNVs
Gene Fusion
Exon/Gene Deletions and Duplications

10. Splice Site Variants (Putative) LoF variants: Splice site variants
Nonsense and Readthrough mutations
Frameshift indels.
Exon Deletion and Duplication
Gene Deletion and Duplication
Gene Fusions*
Identify additional splice variants based on transcript assembly
M. Sammeth and other groups.