Splicing Characteristics of Dystrophin Pseudoexons and Identification of a Novel Pathogenic Intronic Variant in the DMD Gene

Abstract

Pseudoexon (PE) inclusion has been implicated in various dystrophinopathies; however, its splicing characteristics have not been fully investigated. This study aims to analyze the splicing characteristics of dystrophin PEs and compare them with those of dystrophin canonical exons (CEs). Forty-two reported dystrophin PEs were divided into a splice site (ss) group and a splicing regulatory element (SRE) group. Five dystrophin PEs with characteristics of poison exons were identified and categorized as the possible poison exon group. The comparative analysis of each essential splicing signal among different groups of dystrophin PEs and dystrophin CEs revealed that the possible poison exon group had a stronger 3' ss compared to any other group. As for auxiliary SREs, different groups of dystrophin PEs were found to have a smaller density of diverse types of exonic splicing enhancers and a higher density of several types of exonic splicing silencers compared to dystrophin CEs. In addition, the possible poison exon group had a smaller density of 3' ss intronic splicing silencers compared to dystrophin CEs. To our knowledge, our findings indicate for the first time that poison exons might exist in DMD (the dystrophin gene) and present with different splicing characteristics than other dystrophin PEs and CEs.

Keywords: DMD; canonical exon; intronic variants; pseudoexon; splicing characteristics.

Figure 1

Graphic representation of the non-canonical splicing event caused by a de novo and novel genomic intronic variant in the dystrophin (DMD) gene. The intronic variant c.7310-19A>G in intron 50 created a new 3′ splice site that was stronger than the natural acceptor site of exon 51. This caused the inclusion of an 18 bp sequences into the mature transcript, which was predicted to create a premature termination codon. (A) Patient genome (NC_000023.10); (B) dystrophin pre-mRNA; (C) dystrophin mRNA (NM_004006.2).

Figure 2

Graphic representation of the forty-two dystrophin pseudoexons. (A) Graphic representation of PEs activated by deep intronic single nucleotide variants in DMD. (B) Graphic representation of PEs introduced by small and large rearrangements in DMD. Each PE is shown as a light blue box, and its genomic coordinate is indicated under the light blue box. The PE-activating genomic variant of each PE is shown above an arrow. A dark blue box indicates a canonical exon. The more detailed genetic information about the PEs is provided in Table S2. PE, pseudoexon; ins, insertion; inv, inversion; dup, duplication; del, deletion; delins, deletion-insertion.

Figure 3

Comparative analyses of different splicing signals among different groups of dystrophin pseudoexons and the group of dystrophin canonical exons. Comparative analysis of the 3′ ss strength (A) revealed that the possible poison exon group had a significantly stronger 3′ ss compared to other groups. Comparative analyses of the SREs revealed that different groups of dystrophin PEs had a smaller density of diverse types of ESEs (B), a higher density of several types of ESSs (D), and a smaller ratio of total ESEs to total ESSs (C) compared to the CEs group. Some groups of dystrophin PEs had a smaller density of 3′ ss ISSs compared to the CEs group (D). Only splicing signals with significant difference among different groups were included in these figures. Statistics data of other splicing signals are presented in Table S3. Density was calculated as numbers per base pair. Descriptive statistics are presented as box plots, displaying the minimum, first quartile, median, third quartile, and maximum. PE, pseudoexon; SRE, splicing regulatory element; HSF, Human Splicing Finder; MaxEnt, maximum entropy; MM, first order Markov model; ss, splice site; ESE, exonic splicing enhancers; ESS, exonic splicing silencers; ISS, intronic splicing silencers; EIE, exon-identity element; IIE, intron-identity element; NI, neighborhood inference. * p < 0.05; ** p < 0.001.

Figure 4

Position weight matrix-based splice site consensus motifs of dystrophin pseudoexons and canonical exons. Sequence logos for donor (A) and acceptor (B) splice site consensus motifs of dystrophin canonical exons and the consensus sequences are MAG|GTAAGW and TTTWTTTTTTTTTTTTTTWYAG|G, respectively. Sequence logos for donor (C) and acceptor (D) splice site consensus motifs of dystrophin pseudoexons and the consensus sequences are MAG|GTAAGT and TTTTTHTTTYTTTTYYTTNCAG|R, respectively. The height of each letter reflects the relative frequency of that nucleotide in the respective position. “|” indicates the exon–intron boundary in the consensus sequence. H stands for any nucleotide except G, Y for C or T, N for any nucleotide, R for A or G, M for A or C, and W for A or T.