Exon Skipping Applicability

Antisense-mediated exon skipping is a mutation specific approach. As the incidence of the disease is quite high (1 in 3,500 newborn boys) and one in three mutations are de novo, the mutation spectrum is vast. So far an estimated 6,000 different mutations have been identified in DMD and BMD patients worldwide, close to 5,000 of which are reported in the Leiden DMD mutation database (Aartsma-Rus et al. 2009a). The majority of patients (65%) has a deletion of one or more exons, while 27 % has a small mutation, 8% has a duplication of one or more exon and less than 1% has a translocation, inversion or intronic mutation (Aartsma-Rus et al. 2009a).

The deletions tend to cluster around the major hotspot (exons 45-53, 75% of all deletions) and the minor hotspot (exons 2-20, 20% of all deletions) and thus the skipping of exons in these regions is applicable to larger patient groups (Aartsma-Rus et al. 2006b) (Table 5.1). Due to the high occurrence of deletion breakpoints in intron 50 and intron 51, exon 51 skipping is applicable to the largest number of

Table 5.1 Overview of exons applicable to the largest number of patients (Aartsma-Rus et al. 2009a)

All mutations

Deletions

Small

Single exon

Ranking

Exon(s)

(%)

(%)

mutations (%)

duplications (%)

1

51

13

19.14

0.31

2.98

2

45

8.05

11.79

0.24

2.18

3

53

7.67

11.37

0.08

1.45

4

44

6.17

8.83

0.39

2.72

5

46

4.28

6.15

0.23

1.61

6

52

4.11

5.70

0.46

2.31

7

50

3.96

5.64

0.23

1.92

8

43

3.81

5.32

0.16

2.63

9

6 and 7

2.98

3.56

0.08

6.31

10

8

2.27

2.32

7.97

patients (13% of all patients), while exon 45 and 53 skipping would both individually be applicable to 8% of the patients (Aartsma-Rus et al. 2009a). AONs to skip each individual exon have been identified and theoretically exon skipping would be applicable to 80% of deletions, 91% of small mutations and 73% of duplications, or 83% of all patients (Table 5.2).

However, mutations in the essential cysteine-rich domain invariably cause DMD, regardless of whether deletions are in-frame or out-of-frame (Aartsma-Rus et al. 2006b). Thus, for patients with mutations in the part of the transcripts that encodes the cysteine-rich domain (exons 64-70, Fig. 5.1) exon skipping will in all likelihood not be beneficial. Since there are two N-terminal actin-binding domains, and a third domain located in the central rod domain (Fig. 5.1), there is more flexibility for deletions affecting one or two actin-binding domains, as the additional domains retain some of the functionality (Aartsma-Rus et al. 2006b). Nevertheless, BMD patients with mutations in the minor mutation hotspot generally suffer from a more severe phenotype and in-frame deletions affecting all actin-binding domains result in DMD (Aartsma-Rus et al. 2006b) indicating that for out-of-frame mutations affecting all actin-binding domains exon skipping is not beneficial. Finally, exon skipping is not possible for patients with mutations that affect the first or the last exon, or large rearrangements (e.g., translocation). Fortunately, these mutations are very rare and together make up less than 5% of all mutations (Aartsma-Rus et al. 2009a).

However, even in the parts encoding the nonessential rod domain, skipping different exons for (different) mutations will lead to qualitatively different dystrophins for which the functionality will likely vary, as, e.g., exemplified by the finding that in-frame deletions in the proximal part of the rod domain are generally associated with very mild phenotypes and deletions in the distal part of the rod domain are found in typical Becker patients (Aartsma-Rus et al. 2006b). As the phenotype of Becker patients varies a lot, even within families carrying identical genetic mutations (Dastur et al. 2008), it is impossible to exactly predict the extent of the effect of reading frame restoration for DMD patients. There are some mutations that are

Table 5.2 Applicability of single and double exon skipping for different mutation types (Aartsma-Rus et al. 2009a)

Mutation type

Single skipping Double skipping

Total

All mutations

Small mutations (%) 44

Duplications (%) 61

All mutations (%) 64

47 12 19

79 91 73

54 23 6 83

generally found in mildly affected BMD patients, such as the exon 45-55 deletion (Beroud et al. 2007). This suggests that the resulting dystrophin is likely quite functional. As exons 45-55 include a significant amount of DMD mutations (30% of the Leiden DMD mutation database and 63% of the French DMD UMD database) we and others envisaged an exon 45-55 multiexon skipping approach (Beroud et al. 2007; van Vliet et al. 2008; Aartsma-Rus et al. 2006a). This would allow treating a large group of patients with a single treatment and likely conversion into a mild BMD phenotype. Unfortunately, the simultaneous skipping of a large number of exons at a stretch is very challenging and thus far multiexon skipping levels are too low to result in detectable levels of dystrophin and are thus too low to be beneficial (van Vliet et al. 2008).

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