Categorization of 77 dystrophin exons into 5 groups by a decision tree using indexes of splicing regulatory factors as decision markers

Abstract

Background: Duchenne muscular dystrophy, a fatal muscle-wasting disease, is characterized by dystrophin deficiency caused by mutations in the dystrophin gene. Skipping of a target dystrophin exon during splicing with antisense oligonucleotides is attracting much attention as the most plausible way to express dystrophin in DMD. Antisense oligonucleotides have been designed against splicing regulatory sequences such as splicing enhancer sequences of target exons. Recently, we reported that a chemical kinase inhibitor specifically enhances the skipping of mutated dystrophin exon 31, indicating the existence of exon-specific splicing regulatory systems. However, the basis for such individual regulatory systems is largely unknown. Here, we categorized the dystrophin exons in terms of their splicing regulatory factors.

Results: Using a computer-based machine learning system, we first constructed a decision tree separating 77 authentic from 14 known cryptic exons using 25 indexes of splicing regulatory factors as decision markers. We evaluated the classification accuracy of a novel cryptic exon (exon 11a) identified in this study. However, the tree mislabeled exon 11a as a true exon. Therefore, we re-constructed the decision tree to separate all 15 cryptic exons. The revised decision tree categorized the 77 authentic exons into five groups. Furthermore, all nine disease-associated novel exons were successfully categorized as exons, validating the decision tree. One group, consisting of 30 exons, was characterized by a high density of exonic splicing enhancer sequences. This suggests that AOs targeting splicing enhancer sequences would efficiently induce skipping of exons belonging to this group.

Conclusions: The decision tree categorized the 77 authentic exons into five groups. Our classification may help to establish the strategy for exon skipping therapy for Duchenne muscular dystrophy.

Figure 1

Preliminary decision tree to classify 77 authentic and 14 cryptic dystrophin exons. Exons are passed down the tree beginning at the top, where a "yes" result on any test means that it should be passed down to the left. The features tested in this tree are the maximum entropy at the 3' splice site (ss) (ME3'ss), the SF2/ASF density (SF2/ASF-D), the GC content at the 5'ss (GC5'ss), the maximum entropy at the 5'ss (ME5'ss), the free energy at the 5'ss U1 snRNP binding site (FE), the number of exonic splicing silencer (FESS), and the Shapiro score at the 3'ss (SH3'ss). The internal nodes of the tree represent index values that are tested for each exon as it is passed through the tree. Each successive node in the tree represents a decision that is based on those values, until a final classification is reached (the leaves). Authentic and cryptic exons were classified into four groups each.

Figure 2

Cloning of cryptic exon 11a. a. RT-nested PCR products. A fragment spanning exons 10 to 14 was amplified by RT-nested PCR. Two amplified products were obtained from peripheral lymphocytes of a DMD patient (P) but not a control (C). A schematic representation of the exon structure of the amplified fragments is shown on the right. b. Sequences at the exon junctions. Subcloning and sequencing of the amplified products revealed that the larger product contained a 157-bp insertion (exon 11a) between exons 11 and 12. c. Schematic description of the location of exon 11a. The 5' and 3' ends of exon 11a (hatched box) are 5.2 kb downstream of exon 11, and 24.3 kb upstream of exon 12, respectively. Both the AG and GT splicing consensus dinucleotides are present adjacent to exon 11a.

Figure 3

Final decision tree to classify 77 authentic and 15 cryptic dystrophin exons. The structure of the tree is as described for Figure 1. The features tested in this tree include seven indexes used in the preliminary tree (Figure 1) and one additional index, exon size (SIZE). This tree classified the authentic dystrophin exons into five groups (groups A to E), containing 30, 1, 42, 2, and 2 exons, respectively. The cryptic exons were classified into four groups (groups a to d).