Illustrating Exonic Splicing Enhancers

Exons may contain splicing regulatory elements (ESRs), such as exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs). ESEs and ESSs usually correspond to 6–8 nucleotide stretches that serve as binding sites for splicing activator or splicing repressor proteins. Variants that occur in ESEs or ESSs can cause splicing defects such as exon skipping (PLoS Genet 2016;12:e1005756).

Hexamer analysis

In 2011, Shengdong Ke and colleagues investigated a complete set of RNA 6-mer sequences (hexamers) by deep sequencing successfully spliced transcripts. All 4096 6-mers were substituted at five positions within two different internal exons in a 3-exon minigene, and millions of successfully spliced transcripts were sequenced after transfection of human cells and the splicing effect of each 6-mer could be quantified (Genome Res 2011;21:1360-74).

Daniela Di Giacomo and colleagues leveraged these hexamer scores to investigate candidate disease-associated variants by summing the the exonic splicing regulator sequence scores (ESRseq Scores) of the hexamers that overlap the variant position for the the reference sequence (ESRseq WT) and the alternate sequence (ESRseq VAR) and then calculating the change in total ESRseq score (i.e., ESRseq VAR minus ESRseq WT). The authors showed that experimental data had high sensitivity in detecting variants that increased exon skipping (Hum Mutat 2013;34:1547-57), a finding that was confirm by Omar Soukarieh and colleagues, who terms this score ΔtESRseq (PLoS Genet 2016;12:e1005756). For simplicity, we will refer to this score as ΔESR.

In the following example, we show a synonymous variant in exon 39 of FBN1 (NM_000138.4:c.4773A>C) that was shown to causes Exon Skipping and to be associated with Marfan syndrome (Li et al., 2020). The variant is associated with a ΔtESR of 0.22. The following graphic shows how the score was calculated from the ESRseq of the six hexamers that overlap with the variant.

Hexamer graphic for the variant NM_000138.4:c.4773A>C.

Creating Hexamer Plots with vmvt

The following code creates a hexamer plot. Note that the input Strings must be 11 nucleotides long for SNVs, whith the variant being at the middle position. Sequences can be provided in upper or lower case.

import org.monarchinitiative.vmvt.core;

String ref = "CCTGGAGGGGA";
String alt = "CCTGGCGGGGA";
VmvtGenerator vmvt = new VmvtGenerator();
String svg = vmvt.getHexamerSvg(ref, alt);

Heptamer analysis

In 2018, Shengdong Ke performed an analysis of 7-mer sequences (heptamers), whereby each 7-mer was assigned a normalized z-score as a measure of affinity relative to the mean of all 7-mers (Genome Res 2018;28:11-24). The following figure shows an analogous analysis of NM_000138.4:c.4773A>C.

Heptamer graphic for the variant NM_000138.4:c.4773A>C.