91
Journal of Neuromuscular Diseases 1 (2014) 91–94
DOI 10.3233/JND-140002
IOS Press
Research Report
Prolonged Ambulation in Duchenne Patients
with a Mutation Amenable to Exon 44
Skipping
J.C. van den Bergena,∗ , H.B. Ginjaarb , E.H. Niksa , A. Aartsma-Rusc and J.J.G.M. Verschuurena
a Department
of Neurology, Leiden University Medical Center, Leiden, The Netherlands
of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
c Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
b Department
Abstract. Duchenne muscular dystrophy has a severe disease course, though variability exists. Case reports suggest a milder
disease course of patients amenable to exon 44 skipping. In this study, we analyzed this and show that age at wheelchair
dependence in patients with a dystrophin deletion requiring exon 44 skipping is postponed compared to patients with a deletion
skippable by exon 45, 51 and 53 (10.8 versus 9.8 years; P 0.020). This may be explained by more frequent spontaneous exon
44 skipping in patients with a deletion flanking exon 44. This finding has important implications for the development of future
Duchenne trials.
Keywords: Duchenne muscular dystrophy, exon skipping, disease progression
INTRODUCTION
Duchenne and Becker Muscular Dystrophy
(DMD/BMD) are X-linked muscle diseases, caused
by mutations in the DMD gene, coding for the muscle
membrane-stabilizing dystrophin protein. DMD is
generally caused by out-of-frame mutations in the
DMD gene, leading to the absence of dystrophin,
while in-frame mutations allow production of internally deleted or duplicated but functional dystrophin
proteins and are associated with the less severe BMD.
However, there are some exceptions. First, mutations
at the 5 end of the DMD gene often violate the
reading-frame rule, as there are many examples of
BMD patients with out-of-frame mutations. This
∗ Correspondence to: J.C. van den Bergen, Department of Neurology, Leiden University Medical Center, Albinusdreef 2, 2333
ZA Leiden, The Netherlands. Tel.: +31 71 5262197; Fax: +31 71
5248221; E-mail: j.c.van den [email protected].
inconsistency is generally thought to be caused by
the activation of alternative translation initiation sites
and alternative splicing [1–4]. Secondly, there are
several reports describing patients with a deletion of
exon 45 (out-of-frame) and a BMD or intermediate
phenotype [5, 6]. This could result from low level
spontaneous exon 44 skipping, which would restore
the reading frame. Most reports of exon 45 deleted
patients doing better than expected are anecdotal. A
recent study by Pane et al showed a trend towards
a slower decline in the 6MWT over a period of one
year in exon 44 skippable patients compared to other
skippable mutations [7]. To study this in more detail
we have compared the age at wheelchair dependence
of DMD patients with out-of-frame mutations who
would benefit from skipping of exon 44 to patients
who would benefit from skipping of the three other
most prevalent skippable exons (45, 51 and 53) in a
large cohort of 107 DMD patients [8].
ISSN 2214-3599/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved
This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License.
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J.C. van den Bergen et al. / Mild Disease in Exon 44 Skippable DMD Patients
PATIENTS AND METHODS
Patients were recruited from the Dutch Dystrophinopathy Database, which contains clinical and
DNA information about 363 DNA-confirmed DMD
patients. Patients were included if they had an out-offrame mutation for which the reading frame would be
restored by skipping of exons 44, 45, 51 or 53 respectively. Age at wheelchair dependence and steroid use
were recorded. Steroid treatment was defined as “using
steroids for one year or more prior to loss of ambulation”. This limit was chosen to ensure there was
sufficient time for the steroid treatment to influence
the age at wheelchair dependence. Patients who started
less than one year prior to loss of ambulation or who
started less than one year ago and were still ambulant
were excluded.
A survival analysis for loss of ambulation was performed using Cox Regression Analysis, with exon 44
skippable or 45, 51 and 53 skippable and steroid use as
covariates. As the patients with a mutation skippable by
exon 45, 51 and 53 show similar results, these data were
pooled. Our hypothesis included a loss of ambulation
at a later age for the exon 44 skippable patients, therefore tests were performed using a one-sided T-test, at
a significance level of 0.05. Differences in frequency
of steroid users and steroid regimen between the two
different groups were analysed using the Chi Square
test.
Table 1
Estimated age at wheelchair dependence for patients with different
skippable mutations
Exon to be
skipped
Exon 44
- del 19–43
- del 23–43
- del 35–43
- del 43
- del 45
- del 45–54
Exon 45
- del 12–44
- del 44
- del 46
- del 46–47
- del 46–49
- del 46–51
Exon 51
- del 45–50
- del 48–50
- del 49–50
- del 50
- del 52
Exon 53
- del 45–52
- del 48–52
- del 49–52
- del 50–52
Overall
Steroid treated
patients
Non-steroid treated
patients
n
Age (SE)
n
Age (SE)
11
1
1
1
13.0 (0.97)
22
10.2 (0.40)
11.4 (0.91)
4
16
2
10
8.6 (0.56)
6
2
9
2
1
4
2
15
2
3
1
8
1
13
4
4
4
1
48
11.0 (0.65)
10.2 (0.52)
11.3 (0.44)
2
1
2
1
4
17
6
4
3
2
2
17
5
3
6
3
66
9.4 (0.52)
8.7 (0.53)
9.4 (0.26)
Data are presented as mean (standard error) for patients amenable for
exon 44, 45, 51 and 53 skip respectively. The numbers of patients are
subdivided into the different presented mutations for each skippable
mutation individually.
RESULTS
One-hundred-and-twenty-seven patients were registered with a mutation skippable by either exon 44,
45, 51 or 53. Thirteen patients were excluded due to
unknown steroid status. This led to the inclusion of
33 patients with an exon 44 skippable mutation in
the study and 81 controls (19 skippable by exon 45,
32 by exon 51 and 30 by exon 53; Table 1). Thirtythree patients were still ambulant at the time of the
study (10, 7, 11 and 5 for the exon 44, 45, 51 and 53
skippable mutations respectively). Forty-eight patients
(42%) used steroids, of whom 40 (83%) followed a 10
days on/10 days off or 10 days on/20 days off regimen. There was no significant difference in frequency
of steroid users or steroid regimen between the exon
44 skippables versus the others (P 0.23 and P 0.72).
The age at loss of ambulation was significantly higher
for the 33 patients with an exon 44 skippable mutation
than for the 81 patients with other skippable mutations
(10.8 versus 9.8 years; P 0.020) (Table 1 and Fig. 1).
Fig. 1. Survival analysis of age at wheelchair dependence for
patients with a mutation eligible to skipping of exon 44 (dotted line),
compared to the exons 45, 51 and 53 (black line).
J.C. van den Bergen et al. / Mild Disease in Exon 44 Skippable DMD Patients
93
There was also a significant effect of steroid use on age
at wheelchair dependence (P 0.001).
more detailed studies are necessary to investigate this
relation in more detail and to further look into the
physiological mechanism of this difference.
DISCUSSION
ACKNOWLEDGEMENTS
Our results confirm that patients with a deletion
amenable to exon 44 skipping have a less severe disease course than patients with 45, 51 or 53 skippable
mutations. For patients with an exon 45 deletion on
DNA level, but a disease course more consistent with
BMD, low level of spontaneous exon 44 skipping has
been reported previously [5, 6, 9, 10]. This restores the
reading frame, allowing the production of some dystrophin. The finding that exon 44 skippable patients
have a milder disease course suggests that spontaneous
skipping of exon 44 occurs frequently, although the
present study lacks data from dystrophin RNA and
dystrophin expression studies.
Interestingly, a positive effect of corticosteroid use
on ambulation was seen for all four different groups of
patients. The potential low expression of dystrophin in
the exon 44 skippable patient group does not clearly
change the beneficial effect of corticosteroids.
The effect of mutation on disease course could have
implications for future trials. As patients with an exon
44 skippable mutation show a slower pace of disease
progression, stratification for mutation should be considered when planning trials for drugs that are not
mutation specific. Secondly, caution should be used
when ‘sharing’ placebo groups for different exon skipping trials. As patients with an exon 44 skippable
mutation show a less severe disease course than other
patients, they cannot be used as placebo group for e.g.
exon 53 skipping trials, nor the other way around.
This study has some limitations. Firstly, we here
suggest that the possible differences in disease course
are caused by spontaneous skipping of exon 44. However, we did not perform a muscle biopsy to investigate
dystrophin RNA and dystrophin expression to validate
this hypothesis. Material from previous biopsies was
not available, and performing a new muscle biopsy in
the boys for this study was impractical for obvious
medical and ethical reasons. A second limitation is the
retrospective nature of our study. However, recent studies by Pane et al and Servais et al (unpublished data)
including prospective data also showed differences in
disease severity between patients with different skippable mutations.[7]
In conclusion, in this study, we present data that
suggest a less severe disease course for DMD patients
with a deletion eligible for exon 44 skipping. Larger,
We thank dr. A van der Kooi, dr. C Faber, dr. R.
de Coo, dr A. Fock, dr. I. de Groot, dr. M. van Tol,
dr. A. Verrips, dr. L. Smit, dr. R. Pangalila, E. Vroom
and A. Horemans, and the Dutch patient organisations
“Duchenne Parent Project” and “Spierziekten Nederland” for their help in recruiting patients for the DDD.
We thank Mrs. R. Wilson for her statistical analyses. The study was supported by grants from ZonMw,
AFM, and the Prinses Beatrix Spierfonds.
CONFLICTS OF INTEREST
The authors report no conflicts of interest.
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