Applicability of exon skipping for DMD mutations

 

The majority of patients (~70%) has a deletion of one or more exons, while duplications are found in ~7% and small mutations in almost 25% of patients [1]. Exon skipping has been confirmed to restore the open reading frame  patients with deletions, point mutations and small duplications [2-5].

Tables indicating which exon(s) needs to be skipped to restore the reading frame for common deletions, duplications and point mutations can be accessed through the respective links. Please note that these tables are based on a 2004 version of the Leiden DMD mutation database and that new mutations have been added since. If a certain mutation is not present in the tables, exon skipping could still be possible. You can email us your mutation and we will be able to determine whether exon skipping is theoretically feasible and if so which exon(s) needs to be skipped.

Exon skipping for deletions

Most patients carry a mutation of one or more exons, e.g. exon 45 (Figure 1). As exon 45 contains 176 nucleotides (not divisible by 3) the deletion disrupts the open reading frame, resulting in the incorporation of aberrant amino acids and a premature truncation codon in exon 46. Since a deletion of both exons 45 and 46 covers 324 nucleotides (divisible by 3), this deletion maintains the open reading frame. To restore the open reading frame for patients with a deletion of exon 45, exon 46 is therefore hidden from the splicing machinery by a specific AON. This leads to the skipping of the targeted exon and an mRNA transcript for which the reading frame is restored. In addition to an exon 45 deletion (exon 46 skip), we have confirmed the therapeutic applicability of single exon skipping in cultured cells from patients with a deletion of exon 45-50 (exon 51 skip), exon 45-54 (exon 44 skip), exon 48-50 (exon 51 skip), exon 49-50 (exon 51 skip), exon 50 (exon 51 skip), exon 51-55 (exon 50 skip) and exon 52 (exon 51 or exon 53 skip).

Figure 1. Single exon skipping for deletions (back to text)

 

For a small number of deletions, skipping a single exon is not sufficient to restore the open reading frame, e.g. a deletion of exon 46-50 (Figure 2). The total number of deleted nucleotides is 695 (not divisible by 3) and neither a deletion of 45-50 (871 nucleotides) or a deletion of 46-51 (928 nucleotides) is divisible by 3 and thus exon 45 skipping or exon 51 skipping will not restore the open reading frame. However, a deletion of exon 45-51 involves 1106 nucleotides, which is divisible by 3. Therefore, to restore the open reading frame for this patient both exon 45 and exon 51 have to be skipped. This can be achieved by using a combination of AONs targeting exon 45 and AONs targeting exon 51. We have indeed confirmed the applicability of double exon skipping in cultured cells from a patient with a deletion of exon 46-50.

Figure 2. Double exon skipping for deletions (back to text)

 

Exon skipping for duplications

For patients with a duplication of a single exon e.g. exon 45 (176 nucleotides) (Figure 3), skipping either the original or the duplicated exon will restore the wild type transcript, resulting in normal dystrophin. This has indeed been achieved in cultured cells from a patient with a duplication of exon 45.

Figure 3. Single exon skipping for duplications (back to text)

Unfortunately, skipping only of the duplicated exons appeared less straightforward for a duplication of exon 44 (Figure 4), where skipping was so efficient that both exons 44 were deleted, resulting in a deletion of exon 44, which contains 148 nucleotides - not divisible by 3 - and thus disrupts the open reading frame. However, using a combination of AONs targeting exon 43 and AONs targeting exon 44, multiexon skipping of exon 43-44-44 could be achieved, thus restoring the open reading frame.

Figure 4. Triple exon skipping for duplications (back to text)

For patients with larger duplications, the situation is more complex as the AON will target both the original exons (the skipping of which is detrimental for the reading frame even when the duplicated exons are skipped) and the duplicated exons (the skipping of which is beneficial for the reading frame). So far, we attempted to restore the reading frame for only a single patient with a large, atypical duplication (exon 52-62 duplicated between original exons 63 and 64). This was unsuccessful, but it is possible that reading frame restoration is feasible for less complex multiple exon duplications.

Exon skipping for point mutations

Small mutations can directly lead to premature stop codons (nonsense mutation) or disrupt the reading frame (intra exonic deletion or duplication of a number of nucleotides that is not divisible by 3). Either way, if the mutation is present in an in-frame exon, such as exon 49 (Figure 5) that contains 102 nucleotides (divisible by 3), the mutation can be bypassed by skipping said exon. This has been confirmed in cultured cells from a patient with a nonsense mutation in exon 49.

Figure 5. Single exon skipping for point mutations (back to text)

If the small mutation is present in an exon that contains a number of nucleotides not divisible by 3 (e.g. exon 43 in Figure 6 cotains 173 nucleotides), skipping of the mutated exon will bypass mutation, but cause a disruption of the reading frame at the same time (i.e. exon 42 and exon 44 do not fit in Figure 6). However, a deletion of exon 43 and exon 44 is in-frame (321 nucleotides, divisible by 3). Thus by inducing the combined skipping of both exon 43 and exon 44 the mutation can be bypassed, while the reading frame is maintained. Treating cultured cells from a patient with the deletion of a single nucleotide in exon 43 with AONs targeting exon 43 and AONs targeting exon 44 indeed resulted in the generation of dystrophin.

Figure 6. Double exon skipping for point mutations (back to text)

 

Mutation specificity and applicability

The exon skipping approach is mutation specific, i.e. for different mutations, the skipping of different exons is required to restore the open reading frame. However, deletions (present in 65% of patients) and duplications (present in 7% of patients) do not occur random throughout the DMD gene, but are located in deletion hotspots. Of all deletions, 70% is located between exon 45 and exon 55 (major deletion hotspot), while 23% lies between exon 2 and exon 20 (minor deletion hot spot). Therefore, the skipping of some exons can restore the reading frame for different deletions, e.g. skipping exon 51 is  theoretically therapeutic for the following deletions: exon 13-50,  29-50, 43-50, 45-50, 47-50, 48-50, 49-50 and 52, which totals on 25% of all deletions (or 16% of all mutations). By choosing exons strategically, skipping of only 10 different exons can be therapeutic for over 50% of patients in the Leiden DMD mutation database. These exons are: 2, 8, 43, 44, 45, 46, 50, 51, 52 and 53.

There are some mutations for which exon skipping is not applicable. These include deletions containing the first or the last exon, mutations involving exon 64-70, which code for the essential cysteine-rich domain, large deletions that involve both the actin-binding domain and a major part of the central rod domain and large rearrangements such as inversions and translocations. However, these mutations are rare and together make up less than 10% of all mutations.

References

1        Aartsma-Rus A, van Deutekom JC, Fokkema IF,et al: Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve, 2006; 34: 135-144.
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2        van Deutekom JC, Bremmer-Bout M, Janson AA et al: Antisense-induced exon skipping restores dystrophin expression in DMD patient derived muscle cells. Hum Mol Genet 2001; 10: 1547-1554.
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3        Aartsma-Rus A, Janson AA, Kaman WE et al: Therapeutic antisense-induced exon skipping in cultured muscle cells from six different DMD patients. Hum Mol Genet 2003; 12: 907-914.
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4        Aartsma-Rus A, Janson AA, Kaman WE et al: Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet 2004; 74: 83-92.
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5      Aartsma-Rus A, Janson AM, van Ommen G-J et al.: Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy. BMC Med Genet 2007; 8: 43
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