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Research paper
Clinical characterisation of Becker muscular dystrophy patients predicts favourable outcome in exon-skipping therapy
  1. J C van den Bergen1,
  2. S M Schade van Westrum2,3,
  3. L Dekker4,
  4. A J van der Kooi2,
  5. M de Visser2,
  6. B H A Wokke1,
  7. C S Straathof1,
  8. M A Hulsker5,
  9. A Aartsma-Rus5,
  10. J J Verschuuren1,
  11. H B Ginjaar6
  1. 1Department of Neurology, Leiden University Medical Center, Leiden, The Netherlands
  2. 2Department of Neurology, Academic Medical Center, Amsterdam, The Netherlands
  3. 3Department of Neurology, Martini Hospital, Groningen, The Netherlands
  4. 4Department of Cardiology, Catharina Medical Center, Eindhoven, The Netherlands
  5. 5Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
  6. 6Department of Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
  1. Correspondence to J C van den Bergen,  Department of Neurology, Leiden University Medical Center, Albinusdreef 2, K5-Q-110, Leiden 2333 ZA, The Netherlands; j.c.van_den_bergen{at}lumc.nl

Abstract

Objective Duchenne and Becker muscular dystrophy (DMD/BMD) are both caused by mutations in the DMD gene. Out-of-frame mutations in DMD lead to absence of the dystrophin protein, while in-frame BMD mutations cause production of internally deleted dystrophin. Clinically, patients with DMD loose ambulance around the age of 12, need ventilatory support at their late teens and die in their third or fourth decade due to pulmonary or cardiac failure. BMD has a more variable disease course. The disease course of patients with BMD with specific mutations could be very informative to predict the outcome of the exon-skipping therapy, aiming to restore the reading-frame in patients with DMD.

Methods Patients with BMD with a mutation equalling a DMD mutation after successful exon skipping were selected from the Dutch Dystrophinopathy Database. Information about disease course was gathered through a standardised questionnaire. Cardiac data were collected from medical correspondence and a previous study on cardiac function in BMD.

Results Forty-eight patients were included, representing 11 different mutations. Median age of patients was 43 years (range 6–67). Nine patients were wheelchair users (26–56 years). Dilated cardiomyopathy was present in 7/36 patients. Only one patient used ventilatory support. Three patients had died at the age of 45, 50 and 76 years, respectively.

Conclusions This study provides mutation specific data on the course of disease in patients with BMD. It shows that the disease course of patients with BMD, with a mutation equalling a ‘skipped’ DMD mutation is relatively mild. This finding strongly supports the potential benefit of exon skipping in patients with DMD.

  • MUSCULAR DYSTROPHY
  • MUSCLE DISEASE
  • DYSTROPHIN

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Introduction

Duchenne and Becker Muscular Dystrophy (DMD/BMD) are X-linked muscular dystrophies, both caused by mutations in the DMD gene.1 This gene encodes several isoforms of dystrophin, which are expressed primarily in muscle, brain and retina.2 In DMD, the absence of dystrophin in muscle leads to a quite uniform progressive disease course with patients loosing ambulance before the age of 12 years, needing mechanical ventilation around the age of 18 years and leading to death in the third or fourth decade due to respiratory or cardiac failure, as the heart muscle is also involved.3–5 In BMD, partially functional muscle dystrophin is present, leading to a highly variable disease course, ranging from a phenotype only slightly less severe than in patients with DMD to patients who remain ambulant throughout life.6 ,7 This variation is at least partly thought to be explained by the location of the mutation in the DMD gene.8–10

Currently, no long-term effective treatment for DMD and BMD exists. Several therapeutic interventions are being developed, of which exon skipping using antisense nucleotides is regarded as the most promising.11 ,12 Exon skipping restores the reading frame at the pre-mRNA level at the cost of an increased deletion size, resulting in the production of partially functional BMD-like dystrophin instead of a non-functional DMD-like dystrophin (figure 1).13 Thus, this therapy aims to alter a DMD phenotype into a BMD phenotype. Patients with BMD with a mutation equalling a DMD mutation after successful restoration of the reading frame represent a model of the potential beneficial effect of exon skipping in patients with DMD.

Figure 1

Exon-skipping technology. A deletion of exon 45 results in a disruption of the reading frame of the dystrophin mRNA, causing a DMD phenotype. By ‘hiding’ exon 46 from the splicing machinery, this exon is excluded from the mRNA, resulting in a restoration of the reading frame. This leads to the production of partially functional dystrophin and a BMD phenotype. AON, antisense oligonucleotide.

We collected the clinical characteristics of patients with BMD with in-frame deletions corresponding to the predicted result after successful exon skipping of out-of-frame DMD mutations. We postulate that the clinical course in patients with BMD of our study with in-frame deletions reflects the result of a successful exon-skipping treatment of patients with DMD with the corresponding out-of-frame deletions.

Patients and methods

Patients

Patients were selected from the Dutch Dystrophinopathy Database (DDD), which represents over 50% of the Dutch DMD and BMD population, and contains information on all TREAT-NMD mandatory and recommended items. Patients were recruited for this registry through Dutch patient organisations, physicians and internet (http://www.lumc.nl/duchenne). Information was gathered about medical history, disease course, education and family through a written standardised questionnaire. All patients provided written informed consent. The educational data were compared with that of Duchenne patients registered in the DDD as well as to the general Dutch population using information from ‘Statistics Netherlands’ (statline.cbs.nl). Patients were included in the present study when their mutation equalled an out-of-frame DMD mutation that had been changed into an in-frame BMD mutation by skipping one exon. For clinical comparison, data were collected from the DDD of patients with DMD with corresponding out-of-frame mutations.

The study has been approved by the Local Medical Ethics Committee of Leiden University Medical Center as part of the study ‘Epidemiology, natural history and registration of dystrophinopathies in the Netherlands’.

Mutation analysis

DNA was extracted from whole blood taken from patients by a Gentra Puregene DNA purification Kit (Gentra Systems, Minneapolis, USA), following the manufacturer's instructions. Mutation analysis of the DMD gene was performed using multiplex ligation-dependent probe amplification (MLPA kit Salsa P034/P035 MRC-Holland, Amsterdam, The Netherlands).

Cardiac analysis

Data were retrieved from a previous study, which had collected information on a 12-lead ECG and echocardiography of dystrophinopathy patients and female carriers.14 The 12-lead ECG was screened for the following abnormalities: (1) increased R-wave in V1 (>4 mm), increased R-S ratio in V1 or V2 in the absence of a complete or incomplete right bundle branch block, or (2) pathological Q waves (>0.2 mV) in lateral (I, AVL, V6) or inferior leads (II, III, AVF), or (3) a complete or incomplete left bundle branch block or complete right bundle branch block.

Echocardiography was performed using a Vivid 5 GE echocardiograph equipped with a 5 MHz transducer, and measured Left Ventricle End Diastolic Diameter (LVEDD), and Left Ventricle End Systolic Diameter (LVESD), both in parasternal long axis projection. Global Left Ventricular Function (LVF) was judged as good, fair, or poor by an experienced cardiologist (LD). The Fractional Shortening Index (FSI) was calculated as follows: ((LVEDD-LVESD)/LVEDD)*100%. The LVEDD was corrected for weight and BSA.15 Dilated cardiomyopathy (DCM) was defined as an enlarged left ventricle with a global left ventricle dysfunction or fractional shortening of 28% or less.16

For patients not involved in the above mentioned study, cardiac data were collected from their treating cardiologist when possible. All ECGs were reassessed by an independent cardiologist (LD). For patients with DMD, available echocardiography reports were gathered from treating cardiologists.

Western blot analysis

A muscle biopsy from the anterior tibialis muscle was processed as previously described by van Deutekom et al.17 Protein lysates were generated from muscle biopsies and western blotting was performed according to previously described methods.17 ,18 Monoclonal NCL-DYS1 (dilution 1 : 100, Novacastra, UK) or polyclonal ab15277 (dilution 1 : 200 Abcam, UK) were used to detect dystrophin. Rabbit polyclonal antibody to sarcomeric α-actinin ab72592 (dilution 1/500 Abcam, UK) was used as a loading control. Blots were visualised and quantified with the Odyssey system and software (Li-Cor, USA) as described previously.17 ,18 Samples obtained from the tibialis anterior muscle and medial and vastus lateralis muscles of five healthy males were used as reference samples. For each patient sample at least two technical replicates were performed. The average level of both dystrophin antibodies was noted.

Immunohistochemistry

Sections of 10 μm were cut from the anterior tibialis biopsies using a Shandon Cryotome (Thermo Fisher Scientific Co, Pittsburgh, Pennsylvania , USA). Sections were fixed for 1 min with ice-cold acetone. Goat polyclonal dystrophin diluted 1 : 50 (SC-7461, Santa Cruz Biotechnology, USA) and rabbit polyclonal β-dystroglycan diluted 1 : 50 (SC-28535, Santa Cruz Biotechnology, USA) and rabbit polyclonal γ-sarcoglycan diluted 1 : 50 (ab104478; Abcam, UK) were used to detect dystrophin, β-dystroglycan and γ-sarcoglycan. Alexa-fluor 488 donkey-antigoat IgG (A11055, Invitrogen, The Netherlands) diluted 1 : 1000 and Alexa-fluor 594 donkey-antirabbit IgG conjugate (A-21207, Invitrogen, The Netherlands) diluted 1 : 1000 were used as secondary antibodies. Slides were analysed using a fluorescence microscope (DM RA2; Leica Microsystems Wetzlar, Germany), and digital images were taken using a CCD camera (CTR MIC; Leica Microsystems). Staining of β-dystroglycan and γ-sarcoglycan was subjectively scored as absent, low (mainly cytoplasmatic staining), moderate (some (less than 50%) membrane-bound fibres) or good (the majority of fibres have membrane-bound staining).

Statistical analysis

Differences in disease course between our cohort and patients with BMD with other mutations were analysed using the Kaplan Meier Survival Analysis. Statistical analysis was performed on these data through a Log Rank Test. The possible effect of dystrophin quantity on age at first symptoms was analysed using Pearson's Correlation. Differences in average age at first symptoms between patients having absent/low β-dystroglycan or γ-sarcoglycan staining and those having moderate/good staining were assessed using an independent t test. All tests were performed at a significance level of 0.05.

Results

Participants

Out of the 120 patients with BMD known in our registry, 48 met the inclusion criteria of the present study, representing 43 different families. Of the excluded 72 patients with BMD, a mutation was known in 42. Their mutations were: point mutations8 duplications11 deletions not equalling a DMD mutation after skipping of one single exon19 and out-of-frame mutations.4 The median age of participants was 43 years (range 6–67 years). Three participants had died at the time of the study at an age of 45 years (stroke), 50 years (DCM) and 76 years (cause of death unknown), respectively. Information about these patients was gathered through their families. The 48 patients had eight different in-frame deletions that could theoretically mimic the rescue of 10 different out-of-frame deletions in patients with DMD by the skip of one exon (table 1). Thirty-two patients with DMD representing with six out-of-frame counterparts of these mutations were known in our national registry and analysed for comparison.

Table 1

Frequency of ‘skipped’ mutations per exon

Disease course

First symptoms occurred at a median age of 8 years (range 3–36). Most frequent first symptoms were sporting and walking difficulties, falling and myalgia (table 2). Age at diagnosis ranged from 4 years to 65 years (mean 19; median 16 years). Thirty-nine patients were ambulant, of whom four patients used a walking aid and 12 patients used a wheelchair intermittently. Nine patients were wheelchair users. The age at which these patients lost ambulation ranged from 26 years to 56 years of age. None of the patients had scoliosis surgery. Use of ventilatory support was rare, with only one patient using night-time non-invasive mechanical ventilation from the age of 48 years. All clinical data are summarised in the online supplementary data (see online supplementary table S1). The 48 patients with BMD with the ‘skipped’ DMD mutations showed a later onset of first symptoms (mean 12 vs 8.0 years; p=0.054) and a significantly milder course of disease as measured by age at wheelchair usage (estimated mean 53 vs 40 years; p=0.002) than the 42 patients with BMD with other DNA-proven mutations (figure 2). When comparing our patients with BMD with their 32 DMD ‘counterparts’, all disease milestones (wheelchair usage, ventilatory support and death) were reached at a later age in the patients with BMD, showing the theoretical benefit of (single) exon skipping (table 3). The groups were too small to perform statistical analysis.

Table 2

First symptoms for patients in our cohort as indicated by patients themselves

Table 3

Individual clinical comparison of patients with BMD and their DMD mutation counterparts

Figure 2

(A) Survival analysis of age at first symptoms. The dotted line represents the survival curve for participants of the current study, in black all other DNA-proven patients with BMD in the DDD. (B) Survival analysis of ambulatory status. The dotted line represents the survival curve for participants of the current study; in black all other DNA-proven patients with BMD in the DDD.

Education

Educational levels were similar to those of the general Dutch population, and higher than those of Dutch Duchenne patients (table 4). Thirty-eight out of 45 patients (84%) attended a regular primary school. Two patients attended a school for chronically disabled children, while five patients went to a school for children with behavioural difficulties. Six patients (15%) had to attend a secondary school for chronically disabled children.

Table 4

Highest level of education for patients aged 15 years and older compared to patients with DMD and the general Dutch population

Cardiac features

Cardiac data were available for 36 patients. Twenty-nine patients had participated in the previous cardiac study. Cardiac analysis in these patients was performed at a median age of 31 years (range 8–55 years). For seven other patients (median age 40; range 7–70 years), recent cardiac data were retrieved from regular cardiac follow-up. Of 12 patients, no cardiac data were included; four patients did not undergo any cardiac screening, while we were unable to retrieve cardiac data from the other eight.

ECG results were present for 32 patients (26 study participants/six others); echocardiography results for all 36 patients, and Holter ECG were performed in 25 patients (21 study participants/four others). Eight patients showed no abnormalities in all three examinations (median age at examination 31; range 8–55 years). None of the 25 patients who underwent Holter ECG showed ventricular tachycardia (VT), none-sustained VT, atrial-ventricular conduction defects or atrial fibrillation. Ten patients (28%) met the criteria for DCM. There was no correlation between the occurrence of cardiomyopathy and ambulation (see online supplementary table). Only three of the 13 patients with DMD for whom we were able to retrieve cardiac data met the criteria for a DCM (table 3).

Muscle biopsy

Muscle biopsy data were present for 13 patients with BMD with five different mutations. The average dystrophin level was 38% (range 7–71%) (see online supplementary table S1). β-Dystroglycan staining revealed that in six patients, β-dystroglycan was primarily cytoplasmatic, while in another six patients it was primarily membrane bound in either some (3/13) or the majority (3/13) of muscle fibres. For one biopsy, β-dystroglycan staining was uninformative, as the material was too fibrotic. For γ-sarcoglycan, staining was absent for one patient (exon 45–47 deletion), primarily cytoplasmatic for 2/13, and primarily membrane bound for 8/13 patients (in some fibres for 6/13 patients and in the majority of fibres for 2/13). γ-Sarcoglycan staining could not be assessed for two biopsies due to the poor quality of the muscle tissue (fibrotic).

No correlation was found between dystrophin levels and age at first symptoms (R −0.037 p 0.90). Furthermore, no difference was observed between the average age of first disease symptoms for patients showing absent or low β-dystroglycan or γ-sarcoglycan staining compared with patients with moderate or good staining (β-dystroglycan 18 vs 13% p 0.21; γ-sarcoglycan 10 vs 16% p 0.38). The occurrence of other disease milestones was too low to perform a meaningful statistical analysis.

Discussion

Our study suggests that successful skipping of exons 22, 44, 45, 51, 53 or 55 in patients with DMD would significantly ameliorate the course of the disease. All 48 patients with BMD with various in-frame deletions showed a disease course that was milder than that of patients with DMD, in terms of cognitive function, mobility, cardiac and respiratory function, as shown by comparison with patients with DMD from our national registry as well as with the DMD disease course as known from literature. Remarkably, the milder disease course was further underlined by the percentage of patients who were wheelchair users in this subset of patients when compared with the 42 patients with BMD with other mutations (ie, a deletion other than of exons 10–33, 30–44, 45–47, 45–48, 45–49, 45–53, 45–55 or 50–51). This might be explained by the localisation of the mutations within the DMD gene, and highlights the importance of investigating each subgroup of patients with BMD with a mutation equalling a DMD mutation after successful exon skipping separately.

Two previous studies reported a mild disease course in 19 patients with BMD with in-frame deletions ending with exon 51, 53 and (multi-exon skip) 55.19 ,20 Our data support these results and, in addition, provide information about the phenotypes of patients with BMD with deletions ending with four other exons (22, 44, 45 and 46). These results are relevant for a large proportion of patients with DMD (an estimated 13, 8, 2, 0.6, 6 and 8% for exon 51, 53, 55, 22, 44 and 45, respectively).11

In our BMD cohort, all clinical disease parameters were delayed compared with patients with DMD. In patients with DMD, first symptoms are clearly present at the age of four, while first symptoms in our patients with BMD were only noticed at a median age of eight years.21 The progression of ambulatory difficulties is significantly slower, with the youngest BMD patient becoming a wheelchair user at 26 years, while almost all patients with DMD have lost ambulation when reaching puberty.3

Another milestone in disease progression for DMD is mechanical ventilation, with pulmonary failure being a major cause of death. However, the introduction of respiratory support has significantly improved survival, from an average survival in the teenage years to the twenties and thirties.3–5 While the average DMD patient becomes ventilator dependent before the age of 20 years, only one of the patients with BMD in our study used non-invasive night-time ventilator support, starting at the age of 48 years.3 ,5

Mortality in DMD is also often caused by cardiac failure.4 We found DCM in 28% of the patients with BMD, which is consistent with previous studies, in which DCM was observed in 17% and 32%, respectively.6 ,22 Our study showed ECG changes in 13 out of 32 patients (41%). Previous studies found ECG abnormalities in 45–89% of patients with BMD,23–25 but the small size of the populations in all studies (<30 patients) and the different ages at cardiac examination, could have biased the results. A study in 328 patients with DMD by Nigro et al showed echocardiographic signs of a DCM in 45% of patients with DMD between 14 and 18 years.26 In the adult DMD population, they found signs of cardiomyopathy or conduction defects in all patients, with 72% meeting the criteria for DCM. The present study showed a DCM in only 7/29 (24%) adult patients with BMD. Unfortunately, no data about age at presentation of DCM were available for these patients.

Although the delivery of antisense oligonucleotides (AON) to the heart for exon-skipping technology is more difficult than to skeletal muscle, current developments show expression of the exon-skipping therapeutics in murine heart muscle.27–29 Though part of the difference in prevalence of DCM between our BMD study and that of patients with DMD by Nigro et al could be explained by a difference in definition of DCM, our data feature an important possible benefit of exon skipping in patients with DMD.

AON delivery to brain is another concern as systemically delivered AONs do not cross the blood brain barrier.30 Since DMD can be accompanied by cognitive impairment, brain expression of dystrophin is important.31 Notably, it has been shown that upon intraventricular treatment of adult mice, exon skipping-mediated dystrophin restoration resulted in correction of neuronal plasticity and behaviour, suggesting that part of the cognitive impairment could be reversible.32 ,33 The patients presented in the current study provide a distribution of educational level similar to the general Dutch population, showing no signs of intellectual deficits. However, in previous studies, intellectual deficits in patients with BMD have been described.7 ,34 This difference could signify clinical differences between BMD mutations, but could also be caused by our relatively small sample size.

Ten percent of our patients attended a school for children with behavioural difficulties. This percentage is higher than the prevalence of the general population, but lower than the prevalence of neuropsychiatric disorders (ADHD, autism and obsessive-compulsive disorders) in patients with DMD (14%), although a strict comparison is difficult, since the level of impairments caused by these neuropsychiatric disorders in patients with DMD is unknown.35 If the concerns of AON delivery to the brain could be overcome, the differences in cognitive functioning and occurrence of neuropsychiatric disorders could constitute another possible improvement in the disease course of patients with DMD.

Finally, the milder disease progression in our BMD cohort compared with patients with DMD concerns survival. In our cohort, the median age is 43 years, with 11 patients being older than 50 years. Two patients had died at the relatively young age of 45 and 50 years, respectively. In DMD, however, the average survival is in the 30s age range.3 ,4

In our study, we did not find a significant correlation between dystrophin levels or β-dystroglycan/γ-sarcoglycan expression and disease severity as measured by age at first symptoms. However, our data concerning muscle biopsies was limited to a group of only 13 patients with five different mutations.

Our data show the possible result of exon skipping in DMD when achieved from very young or even conceptual age, and therefore represents an optimistic viewpoint. The outcome might be less favourable if exon skipping becomes a clinical reality. Since the average age of diagnosis is 4.5 years, the potentially positive effects of exon-skipping treatment may be less than described here.36 A systematic neonatal CK screening for DMD might be warranted, to enable starting therapy as early as possible. Aside from the diagnostic delay, unforeseen technical problems might impair an optimal treatment with AON, possibly due to the less efficient exon skipping in some tissues like the heart or the brain.

One could argue that our BMD cohort has been biased towards the selection of less affected patients. However, as our database covers over 50% of all Dutch patients with BMD, and patients were recruited using multiple sources, like patient organisations, neurologists, rehabilitation specialists, centres for ventilatory support, genetic diagnostic databases and the internet, a selection bias is unlikely. This is supported by the fact that the clinical characteristics of our BMD cohort in the DDD were similar to another study on a large number of patients with BMD, with a similar frequency and age of wheelchair usage.7 Within our study cohort, patients with cardiac data did not differ from the other patients, when compared on age, age at first symptoms, use of a walking aid and wheelchair usage.

In conclusion, our report strongly supports the potential benefit of exon-skipping treatment for DMD. Compared with patients with DMD, patients with BMD with an in-frame deletion equalling the mutation achieved after successful exon skipping of an out-of-frame DMD deletion have a milder disease course, as can be concluded from data concerning ambulation, education, pulmonary and cardiac function and survival. Hopefully, the outcome of exon-skipping trials will support these results and indeed show clinically significant improvements in the health and physical function of patients with DMD.

References

Supplementary materials

  • Supplementary Data

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Footnotes

  • Contributors JC vdB was involved in the setup of the study, gathering of data, interpretation of data and writing of the manuscript. SMSvW was involved in gathering the cardiac data and editing of the manuscript. LD was involved in the cardiac analysis and editing of the manuscript. AJvdK was involved in patient selection, gathering of cardiac data and editing of the manuscript. MdV, CSS and AA-R were involved in editing of the manuscript. BHAW and MAH were involved in gathering and analysing of the muscle biopsy data and editing of the manuscript.

    JJV and HBG were involved in the setup of the study, interpretation of data and writing of the manuscript.

  • Competing interests xxxx

  • LD reports to receive speakers’ fees from St Jude Medical, Medtronic and MSD and research fees from Medtronic and St Jude Medical. AJvdK and MdV report being an investigator in the EMPOWER trial in ALS conducted by Biogen and receiving research support from Biogen.

  • MdV participated in the Myositis Advisory Board Meeting and received funding for the trip to the meeting (Atlanta, Georgia, USA) from MedImmune, Gaithersburg, Maryland, USA.

  • AA-R reports being employed by LUMC and receiving salary from LUMC. LUMC has patents on exon skipping of some of which AA-R is coinventor. Upon sublicensing some of these patents to Prosensa Therapeutics and GSK, AA-R has received a share of royalty payments from LUMC. JJV reports to be involved in clinical trials for Duchenne muscular dystrophy for GSK, Prosensa and Santhera. JJV is consultant for Prosensa on MRI studies, without receiving personal fees. All payments are made to the LUMC.

  • Ethics  approval The study has been approved by the Local Medical Ethics Committee of Leiden University Medical Center as part of the study ‘Epidemiology, natural history and registration of dystrophinopathies in the Netherlands’.

  • Provenance and peer review Not commissioned; internally peer reviewed.

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