What Can We Learn From Clinical Trials of Exon Skipping for DMD?

OPEN

Qi-long Lu1, Sebahattin Cirak2 and Terence Partridge2

1. 1
Department of Neurology, McColl Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, North Carolina,
USA
2. 2
Center for Genetic Medicine, Children’s National Medical Center, NW, Washington, DC, USA

Correspondence: Qi-long Lu, McColl Lockwood Laboratory for Muscular Dystrophy Research, Department of Neurology, Carolinas Medical Center, 1000 Blythe
Blvd., Charlotte, North Carolina 28203, USA. E-mail:Qi.lu@Carolinashealthcare.org; Terence Partridge, Children’s National Medical Center, 111 Michigan Ave,
NW, Washington, DC 20010, USA. E-mail: TPartridge@childrensnational.org

On 20 September 2013, GlaxoSmithKline (GSK) and Prosensa announced that GSK’s Phase III clinical trial
(NCT01254019) of Drisapersen, an exon skipping drug for Duchenne muscular dystrophy (DMD), failed to meet the
primary endpoint of a statistically significant improvement in the 6 Minute Walking Distance Test (6MWT) compared to
placebo.1 On 12 November 2013, Sarepta Therapeutics announced that the US Food and Drug Administration (FDA)
has considered its application for accelerated approval of Eteplirsen as a DMD drug to be premature. 2 The news came
as a great disappointment to the scientific community and more specifically to the families and foundations that had
followed the trail of research articles and press announcements. Moreover, the clinical trial results and the FDA’s view
of the relationship between the dystrophin biomarker and functional endpoint of 6MWT in clinic will have a far reaching
impact beyond exon skipping therapy in DMD.

Currently, the most promising therapies for DMD are arguably gene therapy and exon skipping, both aiming to restore
the expression of dystrophin. For exon skipping, the general strategy of restoring expression of the mutated
dystrophin gene by excluding frame-disrupting mutations was vindicated by early experiments in dystrophic animal
models.3,4,5,6 This principle has been substantiated for DMD by clinical trials over the last 7 years with two chemistries,
the 2’O-methyl phosphorothioate backbone (2OMePS, named PRO051/Drisapersen initiated by Prosensa/GSK) and the
morpholino backbone (PMO, named Eteplirsen initiated by AVI Biopharma, now Sarepta Therapeutics). 7,8,9,10 Both drugs
target dystrophin exon 51 and both elicited the expected skipping of exon 51 and production of dystrophin protein
following intramuscular injections.

In a subsequent open-label, dose-escalation systemic study, five weekly subcutaneous injections of Prosensa/GSK’s
PRO051 at 0.5, 2, 4, or 6 mg/kg induced skipping of exon 51 accompanied by low levels of dystrophin in 12 DMD boys.
But, importantly, this data lacked pretreatment controls. 10 This lack of pretreatment controls in combination with the
use of the highly sensitive dystrophin antibody MANDYS106 led to reports of expansion of dystrophin positive fibre
counts to up to 100%. The follow-up extension for 12-weeks at 6 mg/kg of the PRO051 reported stabilisation of motor
function in the boys.10However, the study included several boys below 7 years of age where natural history would
predict improved motor function within the experimental period. A subsequent Phase IIb placebo-controlled 6 
mg/kg/week study (NCT01153932) of Drisapersen on 53 DMD patients again reported a significant benefit in 6MWT in
the treatment over the placebo group. Clinical benefits were maintained, but with reduced significance, after 48 weeks
of treatment. However, no muscle biopsy results have yet been reported. 11 This was followed by the phase III trial,
with 186 patients, that failed to show statistically significant improvements in the primary outcome measure of the
6MWT. The difference in conclusion between the Phase III and earlier studies with the exact same treatment regime is
therefore attributed almost solely to the high variability of the 6MWT endpoint measurement within the time-frame of
the subject population and difference in sample sizes.

What have we learnt from the Prosensa/GSK trials? According to the comment from the FDA in response to Sarepta’s
application for Eteplirsen targeting the same dystrophin exon 51, the failure of the Drisapersen trial indicates a
“disconnect between increased expression of dystrophin and clinical efficacy.” This criticism has serious implications far
beyond exon skipping in DMD raising questions about our assessment of all experimental therapies that aim to restore
or produce functional dystrophins in DMD, including gene replacement therapies that deliver a known functional gene
product as a drug.

So, is there clear evidence indicating a disconnect between the levels of dystrophin expression and clinical efficacy?
Fortunately, the answer is no. Results, both from animal models 6 and clinical studies on Becker muscular dystrophy
(BMD) patients, all point to a positive connection. Much of the confusion about levels of dystrophin expression come
from over emphasizing assessment of dystrophin expression solely by immunohistochemistry (IHC) without
pretreatment control biopsies for each patient and the use of highly sensitive antibody MANDYS106. Prosensa reported
that up to 100% of fibers were dystrophin positive in the muscle samples of some Drisapersen-treated patients in its
phase II study.10 IHC is critical to provide us with a rough estimate of the levels of protein expression, the proportion
of cells expressing the protein, and especially patterns of distribution. However, one must be aware that judgment of
positivity of dystrophin staining with IHC is highly subjective and especially troublesome in DMD samples due to many
factors, including the degeneration-related background staining, and highly variable distribution of both revertant
fibers (spontaneously expressing dystrophin) and antisense oligomer-induced dystrophin expression, all of which call
for comparison with a pretreatment biopsy. Small areas of clustered revertant fibers, reaching a few dozen or more,
could easily be misinterpreted to result from treatment. For these reasons, IHC, even though very valuable for
assessing distribution and localization, should never be the sole source of evidence for levels of dystrophin expression
and require confirmatory backup.

Currently, the most generally available and reliable assessment of dystrophin levels in muscles is by western blots
although the data are still, arguably semiquantitative, and it is generally difficult to convincingly demonstrate levels of
dystrophin below 10% of normal levels. However, dystrophin levels higher than 10% of normal level can be
demonstrated without much difficulty in most laboratories as observed from western blots using tenfold dilutions of
positive control protein.7,8,10 Unfortunately, data from western blots in the Drisapersen systemic trials showed only
trace amounts of dystrophin in the treated muscle biopsy samples. 10 In fact, the image presented in the publication
shows no clear difference in signal intensity between the baseline samples and those from 2/7 weeks after
treatment.10 The FDA also cited the failure of PTC Therapeutics’s Ataluren trial as another example of the disconnect
between levels of dystrophin and clinic outcomes. Ataluren has been selected to induce significant read-through of
nonsense mutations of DMD gene, resulting in restoration of dystrophin expression in patients carrying such
mutations. However, the screening assay method initially used to identify the specificity of PTC124 has been
contradicted by more recent study. 12 Furthermore, studies to validate the effect of PTC124 in vitro did not demonstrate
any significant read-through of nonsense mutations.13 More importantly, clinical trials of Ataluren have shown only the
phase IIa results with little evidence of dystrophin restoration by IHC with a very subjective scoring
method.14 Unfortunately, the phase IIb muscle biopsy results have not been presented and reported to the public.
Therefore, the disappointing functional data from the Ataluren and Drisapersen trials are, in fact, entirely consistent
with the failure of either agent to induce production of significant amounts of dystrophin protein and do not support
the notion of the “disconnect” proclaimed in the FDA report. The rationale of both exon-skipping and stop-codon read-
through is that any benefit derives from production of significant amounts of dystrophin and the degree of efficacy
should be related to the level of dystrophin. This has been clearly demonstrated in animal models of DMD. 6,15 This
conclusion is also consistent with the data from Sarepta’s clinical trials of the exon skipping drug Eteplirsen (formerly
AVI-4658). The first open label, dose escalation (cohorts 1–6: 0·5, 1, 2, 4, 10, and 20 mg/kg bodyweight
respectively), repeated intravenous administration study showed that Eteplirsen was well tolerated. There was a
statistically significant dose-response as well as remarkable variability in dystrophin production in patient muscles. In
the low dose cohorts 1–4, there was no increase in dystrophin expression, with the exception of one subject in cohort
3. However, 6 of 8 subjects in the two highest dose cohorts 5 and 6 showed an increase in dystrophin expression.
Three patients, one in each of cohorts 3, 5, and 6, showed a substantial number of dystrophin-positive fibers, 21, 15,
and 55%, respectively. Western blot analysis of these patients also showed an increase following treatment of protein
levels from 2 to 18%, from 0.9 to 17% and from 0 to 7.7% of normal muscle values, respectively. 8 This was followed
by a placebo-controlled phase IIb trial using higher doses (30 and 50 mg/kg/week, each cohort had n = 4 subjects) of
Eteplirsen.16 Ambulant boys were treated for 24 weeks and primary outcome was the percentage of increase in the
number of dystrophin positive fibres in comparison to baseline biopsies. The secondary outcome was safety and the
6MWT. After another 24 weeks of open label extension, a final muscle biopsy was taken at 48 weeks. The results
showed the number of dystrophin positive fibres up to 52 and 43% in the 30 and 50 mg/kg cohorts
respectively.16 Overall, the Eteplirsen trials showed a clear trend of dose-dependent increase in dystrophin expression.

Clearly, one major difference between the two chemistries of exon skipping clinic trials is that Eteplirsen has been
administrated systemically at up to 50 mg/kg, more than eight times higher than Drisapersen. The limited dose of 6 
mg/kg for Drisapersen was largely the result of kidney toxicity whereas no clear toxicity has so far been identified for
Eteplirsen. Most preclinical studies have reported higher efficiency of exon skipping with the PMO chemistry than with
equivalent amounts of reagent based on the 2OMePS chemistry. 4,5,6 It is therefore not surprising that the Eteplirsen
trial reported detectable dystrophin by western blot. The Eteplirsen treated patients also showed a benefit of 67 m less
decline in 6MWT over the placebo group.16 Stabilization of the motor function in DMD boys participating in Eteplirsen
extension study has now being observed over 2 years. However one needs to be aware of the small sample sizes (only
12 subjects) and the bias associated with the open label nature of the extension study. Nevertheless, the levels of
dystrophin were limited, clearly below 10% by western blot, and from only one sample (judged from the image
presented in the publication) despite the high percentage of dystrophin positive fibers reported. 16This again illustrates
the inflation of using IHC and number of dystrophin positive fibers as a marker of overall levels of dystrophin
expression. One also needs to be aware that similar results of 6MWT stabilization have been reported from the
Drisapersen open label extension studies and recently published natural history studies, although the interpretation is
complex because of different age ranges and subpopulations. 17,18 It therefore remains to be seen whether the reported
levels of dystrophin under the current Eteplirsen treatment regime can be maintained. Upcoming placebo controlled
phase III studies will be able to show if Eteplirsen can significantly delay the long-term disease progression.

There should be no doubt that dystrophin is critical for normal muscle functions, for its lack is the cause of DMD and
BMD.19 One could argue that increase of any amount of dystrophin might benefit the dystrophic muscle. However, the
presence of low amounts of dystrophin in these trials is associated with limited distribution of the dystrophin positive
fibers. The small number of these fibers in dystrophic muscles is unlikely to provide widespread and measurable
functional improvement to the general mass of muscle, a view supported by studies in animal models. 4,5,6 Therefore,
therapy based on dystrophin restoration must ask the question: can the treatment produce sufficient amounts of
dystrophin, measureable by both IHC and western blot, and sufficiently widely distributed, to effectively delay the
disease progression? To answer this question, one would first ask how much dystrophin is required to have a
significant impact on the disease outcome. Analyses of biopsies from BMD patients provide some insights. BMD is
associated with natural occurring in-frame deletions from which reduced amounts of slightly shortened dystrophins are
produced. The aim of exon skipping therapy in DMD patients is to mimic BMD by causing production of in-frame
transcripts from the out-of-frame dystrophin gene. Published work on dystophin levels in near-asymptomatic Becker
patients suggests that this degree of amelioration can result from around 30% of normal dystrophin levels in western
blots.20 Dystrophinopathy patients of intermediate clinical severity have been associated with dystrophin levels of
between 10 and 25% of normal levels21 while in-frame deletions in BMD patients with severe DMD phenotype have
been associated with less than 10% dystrophin. From this data, it is reasonable to postulate that significant
improvement in the disease phenotype by exon skipping will require expression of truncated dystrophin to at least
10% of normal levels with wide distribution. Such levels of dystrophin have not so far been convincingly demonstrated
in any of the systemic trials of exon skipping and read-through treatments. Significantly higher levels of dystrophin are
likely required to improve disease phenotype of individuals with diminished remaining muscle mass and higher
variation in dystrophin distribution. This assumption does not exclude possible limited benefit to diseased muscle of
lower levels of dystrophin, but the incidence of severe BMD patients with low levels of dystrophin indicates that this is
unlikely.

With all the data available from animal model studies of exon skipping and adeno-associated virus gene therapy and
clinic trials of exon skipping, we may conclude that dystrophin protein level, far from being dubious, is a fundamental
biomarker to be taken into account when assessing experimental therapies that aim to produce and restore the
expression of dystrophin in DMD. This principle is applicable to any therapy that aims to produce therapeutic protein.

We are still in the early stages of clinical trials for the exon skipping therapy. It is therefore critical to collect such data
as accurately as possible, so that real guidance may be provided for later clinical trials targeting other exons or for
gene replacement therapy. The efforts made by several large collaborative groups of physicians and scientists in the
design and execution of these large clinical trials can’t be appreciated enough as we have learnt invaluable lessons
about the natural history of DMD, the practicability and utility of clinical trial procedures and outcome measures, and
have created a sustainable clinical infrastructure for future clinical trials. The most important lesson learnt is that,
clinical endpoints such as 6MWT are important for assessing the disease progression, but total dominance of this
principle appears to have led to the misconception that clinical benefit can be achieved without the production of a
therapeutic amount of relevant protein product.

Another valuable and important lesson from the two exon skipping trials is that the behaviour of a particular antisense
chemistry in the mouse and dog models appears to provide a good guide to what it will do in human DMD. The
relationship of dose per body weight to exon-skipping efficiency and dystrophin production with the PMO chemistry in
animal models has been strongly paralleled in man. It reinforces the rationale for conducting initial investigations of
the efficacy/toxicity window for a given chemistry in animals with a reasonable expectation that the general principles
of dose and regime may be transferable to man.

In summary, detection of dystrophin is fundamental to exon-skipping therapy in DMD and it is important to gather
reliable data relating levels of dystrophin expression to clinic outcome. This will provide guidance for later trials testing
other antisense chemistries and targeting other exons as well as for gene replacement therapy. Preclinical studies in
the dystrophic mouse and dog have shown a dose dependent dystrophin induction and therapeutic effect on dystrophic
muscles. In fact, clinical and biochemical outcome from the treatment regimes of the two different chemistries are
strongly predicted from animal model studies. Data from the pivotal Ataluren and Drisapersen clinical trials of exon
skipping have, so far demonstrated to our best knowledge too little dystrophin to justify the notion of a disconnection
between the levels of dystrophin expression and clinical outcome measurement. The newly started Ataluren phase III
trial (NCT01826487) might be able to determine clinical benefit to DMD patients, but unfortunately no muscle biopsies
have been planned in this phase III study. Determination of dystrophin levels should rely more heavily on western
blot, currently the most available method, with supporting data from IHC, although more reliable and accurate
detection methods are currently being developed.22 Lastly, we would remind the FDA and ourselves that dystrophin is
not a purely phenomenological biomarker for DMD; lack of dystrophin is the primary biochemical defect in DMD and
denial of the importance of the principle of substantial dystrophin restoration in DMD would have future impact on all
therapeutic investigations of inherited disorders.

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References

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Deletion mutations in Duchenne muscular dystrophy (DMD) in Western Saudi

children
 Mohammed T. Tayeb
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doi:10.1016/j.sjbs.2010.04.008
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Open Access funded by King Saud University

Under a Creative Commons license

Abstract
Duchenne and Becker muscular dystrophy (DMD and BMD) are caused, in the majority of cases, by deletions in the dystrophin gene
(DMD). The disease is an X-linked neuromuscular diseases typically caused by disrupting (DMD) or non-disrupting (BMD) the reading
frame in the dystrophin (DMD) gene. In the present study, amplifications of the genomic DNAs of unrelated 15 Saudi DMD males were
carried out using multiplex polymerase chain reaction (PCR) for nine-hotspot regions of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51. We
detected six Saudi patients having deletions in a frequency of 40%. The frequency of deletions in exon 51 (20%) was the most common
deletion frequently associated with our Saudi sample males. Exons 19, 45, and 48 were present in a frequency of 6.7% each. All
deletions were recognized as an individual exonic deletions, while no gross deletion where detected. Finally, the molecular deletions in
the Saudi males was expected to be characterized by a moderate frequency among different populations due to the geographical KSA
region, which it is in the crossroad of intense migrations and admixture of people coming from continental Asia, Africa, and even Europe.
In conclusion, attempts to include an extra DNA samples might reflect a valid vision of the deletions within the high frequency deletion
regions (HFDR’s) in the DMDgene mutations in KSA.

Keywords
 Dystrophin gene;
 Duchenne muscular dystrophy;
 Deletion mutation;
 Saudi patients
1. Introduction
Duchenne muscular dystrophy (DMD, MIM #310200) and its milder allelic variant, Becker muscular dystrophy (BMD), are both caused by
mutations in the dystrophin gene (DMD, MIM #300377) located on Xp21. DMD represents the most common genetic neuromuscular
disease of childhood. It is relatively frequent, with an incidence of 1 in 3500 male livebirth ( Worton and Thompson, 1988).
Symptoms in DMD patients start to appear at the age of 3 years with progressive muscle weakness. The patient is non ambulatory by
the age of 9 or 10 years and usually by 20 years old; death will occur after cardiac or respiratory complications (Sbiti et al., 2002). DMD
is the more severe phenotype of muscular dystrophies (Lo et al., 2006). BMD is the allelic and the milder form of DMD, but it is less
frequent in which the birth prevalence is 1 in 18,500 live born male ( Zhou et al., 2006 and Ramellia et al., 2006). It characterized by
slower rate of progression in which the mean age of the onset of symptoms such as muscle weakness and poor walking, arise around
12 years of age. Loss of ambulation varies from adolescence to adulthood (Ramellia et al., 2006), while death generally occurs in the
third or fourth decade (Sbiti et al., 2002).
The DMD gene consists of 79 exons and encodes a 14-kb mRNA ( Koenig et al., 1988, Ahn and Kunkel, 1993 and Freund et al., 2007).
The protein product of the gene, with a molecular weight of 427 kDa, is a sarcolemma-associated protein, which binds cytoskeletal actin
through its N-terminal domain, and a complex of dystrophin associated proteins (DAP) through its C-terminal domain ( Ahn and Kunkel,
1993). Intragenic deletions and duplications together account for over two thirds of the mutations leading to DMD and BMD ( Koenig et
al., 1989 and den Dunnen et al., 1989).
Intragenic deletions and duplications together account for over two thirds of mutations leading to DMD and BMD (den Dunnen et al.,
1989 and Koenig et al., 1989). Most can be detected by multiplex PCR (Beggs and Kunkel, 1990 and Chamberlain et al., 1988) and are
clustered in two high frequency deletion regions (HFDRs), one in the 5′ (centromeric) portion of the gene, the other in the 3′ half of the
gene (Baumbach et al., 1989, Kim et al., 2002 and Koenig et al., 1989). A small proportion ranging from 0% to 6% of the mutations
within the dystrophin gene involves duplications (Hu et al., 1990 and Mendell et al., 2001). More than 200 dystrophin point mutations
are known (http://www.dmd.nl/). Previous study of dystrophin deletion mutations in Saudi males (Al-Jumah et al., 2002) has not dealt
with the frameshifting hypothesis.
In the present paper, we presented an analysis of exceptions to the frameshift rule and their implications for dystrophin in males.

2. Subjects and methods

Dystrophin patients were selected from Saudi dystrophin families registered in the database records in neurologic out-patient clinics of
governmental and military hospitals, and Handicapped Children Societies in Western Saudi regions. Informed consents were obtained
from all patients’ family. Clinical data sheet of a patient was registered on the database records of Molecular Genetics laboratory in the
Department of Medical Genetics, Faculty of Medicine, Umm Al-Qura University. Clinical information was independent of any molecular
data of DMD gene or its protein. For the sake of accuracy, we excluded the patients who lacked clinical evidence of the disease.
We categorized patients according to the severity of the phenotype of the disease, as ‘DMD’ if they were confined to a wheelchair at or
before the age of 12 years and as ‘BMD’ if they were still ambulant at age 16 years. Patients were classified as intermediate (B/DMD) if
they became wheelchair bound (WCB) between the ages of 12 and 16 years ( Hodgson et al., 1989). They defined as ‘ND – not
determined’ phenotype if the patient was not wheelchair bound or too young to be diagnosed as BMD. Our sample contained 15
unrelated proband males. The age of the DMD patients ranged from 5.0 to 19.0 years of a mean age ± standard deviation (SD) was
11.8 ± 3.4 years. Diagnosis of DMD/BMD probands included elevated serum creatine phosphokinase (CPK), age of onset, calf
pseudohypertrophy, age of wheelchair confinement, presence of cardiomyopathy, and myopathic changes to EMG pattern
(http://www.dmd.nl/).

2.1. DNA analysis

Genomic DNA was isolated from EDTA-peripheral leukocytes using Mini Spin-Column protocol (QIAGEN, USA). Multiplex PCR of the high
frequency deletion regions in the DMD gene was performed ( Fig. 1). The amplification was carried out using multiple 18 primers
flanking exons 4, 8, 12, 17, 19, 44, 45, 48, and 51 (Chamberlain et al., 1988). PCR cycling was programmed as: initial denaturing at
95 °C for 6 min (1 round), then 95 °C for 30 s; 53 °C for 30 s, 65 °C for 4 min (repeated for 23 rounds), and an initially extension 65 °C
for 7 min. PCR products were separated on 3% NuSieve agarose. The gel was photographed using Gel Documentation and Analysis
System (UVitec, Cambridge, UK).
 Figure 1.
Multiplex PCR of the human DMD gene electrophoresed on a 3% NuSieve gel-ethidium dye. ‘C’ represented ‘normal control male’ with no deletions, MW is size
marker (100 bp ladder). Lanes 1, 2, 3, and 4 showed missing of exon 19, 51, 45 and 48, respectively.
Figure options

3. Results and discussion

3.1. Deletion analysis

To detect DMD gene deletions in Chamberlain’s set, genomic DNA was successfully amplified. The DMDdeletion frequency was 40% (6 of
15 patients). The missing exons in the central region represented the majority of deletions (83%) confined to exons 51, 48 and 45, while
the remaining deletions (27%) were represented only in exon 19 in the proximal region. These results might be in agreement with the
Egyptian studies ( Elhawary et al., 2004) and other comparative studies in Asian populations ( Lu et al., 2006, Hwa et al.,
2007, Hallwirth-Pillay et al., 2007, Hassan et al., 2008 and Marini et al., 2008). In the present study, the deletion frequency of exon 51
was the most common deletion (20%), while exons 19, 45 and 48 showed a frequency of 6.7% each ( Table 1). All deletions recognized
in this study were detected as an individual exonic deletions, while no gross deletion where detected.
Table 1.

Exon deletion frequencies in 15 unrelated DMD Saudi patients.

Exon(s)
deleted No. of deletions Deletion frequency (%)
Exon 19 1 6.7
Exon 45 1 6.7
Exon 48 1 6.7
Exon 51 3 20.0

Total 6 40
deletions

Table options
Again, the deletion frequency in the Saudi DMD patients (40%) is relatively lower than other Arabs or neighboring populations, but
comparable with most of the Asian data (Hassan et al., 2008, Marini et al., 2008 and Hwa et al., 2007). In the North countries of Africa,
Egypt, for example, represented a relatively high frequency of DMD gene deletion frequency (51%; 78/152) ( Elhawary et al., 2004),
which is the case in Moroccan population (51%; 37/72) ( Sbiti et al., 2002).
Our data results might lie between that of the highest frequency in Turk (60%) (Onengut et al., 2000) and the lowest deletion frequency
in Philippines (33%) (Cutiongeo et al., 1995). Some Asian populations, for example, Pakistani (40.75%) (Hassan et al., 2008), Malaysian
(42%) (Marini et al., 2008), Chinese (49%) (Lu et al., 2006) and Taiwanese (35.3%) (Hwa et al., 2007) have nearly the same magnitudes
and patterns of deletion frequencies to this Saudi study. This might be rationalized as strong genetic linkage or genetic drifts.

3.1.1. In-frame and out-frameshifting and DMD phenotypes

A hypothesis known as the reading-frame hypothesis proposes that deletions that alter the reading frame of dystrophin mRNA produce
no functional dystrophin and cause severe DMD, while in-frame deletions may produce partly-functional internally deleted dystrophin
leading to the milder Becker disease (Monaco et al., 1988).
 Deletion of exon 19 resulted in a disruption of the reading frame, resulting in the severe DMD phenotypes. Deletion of exon 45 resulted
in the intermediate D/BMD phenotype (Table 2). On the other hand, deletion of exon 51 gave rise to severe DMD phenotypes as shown in
two cases (#11 and #15) and in ‘not determined phenotypes’ phenotype in (#7) that might be suspected to have DMD phenotype.
Deletions due to exons (4, 8, 12, 17 and 44) have never been shown deletions. Our results agreed the frameshift rule in a frequency of
(66.7%) of patients. This result showed a high concordance with Monaco et al. (1988) besides other relevant studies (Elhawary et al.,
2004, Lu et al., 2006, Hassan et al., 2008, Marini et al., 2008 and Hwa et al., 2007). The deviation from the frameshift hypothesis was
not clearly shown because of the presence of ND phenotypic cases.
Table 2.

Clinical data and molecular finding of dystrophin Saudi patients within the high frequency
deletion regions (HFDR’s).
Fam.
no.a Ageb (y) Sex Diagnosisc WCBd (y) F.H.e Exon deletedf Frame-shift g
1 15 M DMD 4 + 19 +
5 7 M ND − − 51 +
8 14 M DMD/BMD 12 − 45 +
9 11 M DMD 8 + 51 +
12 15 M DMD 9 − 51 +
20 9 M ND − + 48 −

a
These numbers only refer to one affected proband.

b
The age of initial examination.

c
Patients were classified as intermediate (B/DMD) if they became wheelchair bound (WCB) between the ages of 12 and 16 y (Dubowitz, 1990). Patients labeled as ‘not determined’ “ND” were too

young to permit a definitive diagnosis, and are grouped separately (Table 1).

d
Wheelchair bound.

e
Family history (F.H.) considered (+) if there are more than one affected individuals in the family and (−) if the affected male was a sporadic case.

f
Deletion detection were focused to the high frequency deletion regions including exons 4, 8, 12, 17, 19, 44, 45, 48, and 51.

g
In-frame (−) and outframeshift (+) were assigned according to Monaco et al. (1988).

Table options
In conclusion, the molecular deletions in the Saudi males should be expected to characterize a moderate frequency. This might be due
to the geographical KSA region, where it is in the crossroad of intense migrations and admixture of people coming from continental Asia,
Africa, and even Europe. Attempts to involve an extra DNA samples might reflect the real map of the deletions within the high frequency
deletion regions (HFDR’s) in the DMD gene mutations in KSA.

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Tapping Therapy for Muscular Dystrophy

Tapping Therapy, sometimes also identified as EFT, uses the old principles of chinese acupuncture to address your emotions (especially the
negative feelings) about issues you experience on a standard basis. For example: fears and phobias, sadness, anger, disappointment, frustration,
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