Can symptomatic nmDuchenne carriers benefit from treatment with ataluren? Results of 193-month follow-up.

Duchenne's muscular dystrophy (DMD) is an X-linked neuromuscular disorder caused by deletions (75%), duplications (15-20%) and point mutations (5-10%) in the dystrophin gene. Among the latter, stop-codon point mutations are rare. Female carriers of dystrophin gene mutations are usually asymptomatic as they are "protected" by the second X-chromosome, which produces a normal dystrophin protein. However, about 8-10% of them can present symptoms that set the clinical picture of the manifesting or symptomatic carrier. Although no causative cure there is for DMD, therapies are available to slow the decline of muscle weakness and delay the onset of heart and respiratory involvement. However, there is limited data in the literature documenting the treatment of symptomatic carriers, often entrusted to the sensitivity of individual doctors. In this paper, we report the follow-up outcomes of four European symptomatic nmDMD carriers treated with ataluren, overall followed for 193 months. Annual assessment of muscle strength, pulmonary lung function tests, and echocardiography, indicate a mild attenuation of disease progression under treatment.. There were no adverse clinical effects or relevant abnormalities in routine laboratory tests. We can conclude that ataluren appears to stabilize, if not slightly improve, the clinical course of patients with a good safety profile, especially if we consider that the treatment was late for 3/4 patients, at a mean age of 36.6 ± 10.6 years. ©2021 Gaetano Conte Academy - Mediterranean Society of Myology, Naples, Italy.

Introduction

Duchenne’s muscular dystrophy (DMD) is an X-linked neuromuscular disorder affecting muscles and heart in young boys [1,2], caused by deletions (75%), duplications (15-20%) and point mutations (5-10%) in the dystrophin gene. Among the latter, the stop codon point mutations are rare [3,4].

Females carrying a dystrophin gene mutation on one of the two X-chromosomes, are usually asymptomatic as they are “protected” by the second X-chromosome which products a normal dystrophin protein. However, about 8-10% of these females can present symptoms, which causes the clinical picture of the manifesting or symptomatic carrier. Both terms have been widely used since the 1970s [5-12] to define females with a history of Duchenne muscular dystrophy in their pedigree who have symptomatic weakness. These females can also develop myalgia, cramps, fatigue, and show enlarged calf muscles (pseudo-hypertrophy). The severity of symptoms may range from a Duchenne-like progression to a very mild Becker-like phenotype. A considerable percentage of carriers may develop cardiomyopathy, at an advanced stage [13-15]. Cognitive impairment was also reported, mainly associated with mutations in the distal part of the DMD gene [16,17]. An increase in serum creatine kinase (CK) levels up to ten times the upper normal limit was reported in approximately 40-50% of carriers, especially in childhood [18,19]. Several mechanisms leading to reduced dystrophin production, were hypothesized to explain the onset of clinical manifestations and in particular the role played by the skewed X-chromosome inactivation (XCI). Though this role is still questioned, several papers [20-22] showed that DMD-manifesting carriers have a preferential inactivation of the X-chromosome carrying the normal allele, while non-manifesting carriers and healthy females showed a random (50:50) XCI pattern.

From a clinical point of view, symptomatic carriers should be considered as affected as males with disease are, and be able to benefit from the same therapeutic opportunities.

There is no causative cure for DMD, but therapies are available to slow the decline of muscle weakness and delay the onset of heart and respiratory involvement. Among others, steroids, ACE-inhibitors and beta-blockers, are the gold standard of the treatment [23,24]. However, in the literature there is limited data documenting treatment of symptomatic carriers [25,26], often entrusted to the sensitivity of individual doctors.

In the last decade, different therapeutic approaches have been tested with encouraging results in patients with dystrophin gene deletions or duplications. Among them we mention gene therapy (which consists of introducing a transgene coding for full-length or a truncated version of dystrophin complementary DNA (cDNA) in muscles) [27], and exon-skipping techniques with antisense oligonucleotides which convert an out-of-frame mutation into an in-frame mutation [28]. For DMD patients having stop codon mutations in the DMD gene [4], potential drugs such as gentamicin[29] and ataluren (PTC124) [30] were explored as an alternative approach. These drugs allow ribosomal readthrough of premature stop codons, enabling the production of a functional dystrophin that might ameliorate the disease progression [30,31]. About 10-15% of DMD patients could potentially benefit from treatment with ataluren [31]. This drug has been available in Europe since 2014 [32] under the name (Translarna®).

In 2017, McDonald and al. [33] presented the results of a phase 3, multicentre, randomised, double-blind, placebo-controlled trial (ACT DMD) that assessed the ability of ataluren to stabilise ambulation, with a focus on a pre-specified subgroup of patients with ambulatory decline. The primary endpoint of change in 6-min walk distance (6MWD) from baseline to week 48, with a hypothesis of a difference of at least 30m between ataluren-treated and placebo-treated patients, was not reached (difference 13.0 m [SE 10.4], 95% CI -7.4 to 33.4; p = 0.213). However, a benefit of ataluren was observed in the subgroup of patients with a baseline 6MWD between 300 and 400 m (difference vs placebo 42.9m [SE 15.9], 95% CI 11.8-74.0; p = 0.007) and confirmed in papers that appeared in subsequent years [34-36].

Articles recently published on the long-term ataluren treatment indicated a delay in loss of ambulation, as well effects on cardiac and respiratory parameters and upper limb motor function, even after loss of ambulation [37,38]. An early treatment with ataluren has also been suggested [39]. The response to the treatment with ataluren was investigated by D’Ambrosio et al. [40] in a 26-year-old symptomatic nmDMD female carrier who reported an improvement in motor skills after 9 months of treatment.

In this paper we report the follow-up outcomes of the patient described by D’Ambrosio et al., still on treatment with ataluren, together with those of three further European symptomatic nmDMD carriers overall followed for 193 months (average 48.25).

Patients and methods

Clinical data of the four European DMD carriers so far treated with ataluren were retrospectively collected and included country’s origin of female patients, age at first symptoms, age at muscle biopsy, time between first symptoms and muscle biopsy, age at genetic confirmation, time between first symptoms and laboratory abnormality or genetic confirmation. Age at first and last visit for ataluren, age at informed consent, prior and concomitant medications, age at start- and end-date of ataluren, duration of treatment, age at loss of ambulation were also collected. Motor function outcomes such as six minute walking test (6MWT), North Star Ambulatory Assessment (NSAA) total score, dynamic tests (Gowers time, time to climb 4 steps) were evaluated at the start and at the last visit; data on forced vital capacity (FVC) and left ventricular ejection fraction (LVEF) were also evaluated when available.

The drug was administered orally, at a dosage according to the weight of the patients.

Data are shown as mean, range and standard deviation when applicable.

Results

Baseline patient demographics & characteristics

Demographics and characteristics of symptomatic nmDMD carriers treated with ataluren are shown in Table I. Two patients are from Italy, one from Israel and one from UK. The age of onset of the first symptoms was before 2 years in two, and at 17 and 30 years in the other 2 carriers. Muscle biopsy was performed in three carriers, immediately after the onset of symptoms in two, 4 years after the onset of symptoms in the third carrier. The mean time between the onset of symptoms and muscle biopsy was 1.3 years, ranging from 0 to 4 years. The mean age, at the molecular confirmation of the diagnosis, was 21, ranging from 3 to 38 years. The two carriers with onset of symptoms in childhood were on deflazacort, antioxidants, calcium and vitamin D3 treatment, which they continued to take concomitantly with ataluren.

Table I.

Demographics of symptomatic nmDMD carriers treated with ataluren.

Reference Center	Current age (years)	Age at first symptoms (years)	Age at muscle biopsy (years)	Time between first symptoms and muscle biopsy (years)	Age at genetic confirmation of nmDMD (years)	Time between MB and genetic confirmation(years)	Time between first symptoms and genetic confirmation(years)
IT001	30	1.6	1.6	0	24	22	25
UK001	13	< 2	2.6	0.3	3	0.5	1
IL001	49	30	n.p.	n.ap	38	n.ap.	8
IT002	31	17	21	4	21	0.5	0,5
Mn	30.75	16.2	8.4	1.43	21.5	7.67	8.63
Range	13-49	1.6-30	1.6-21	0-4	3-38	0.5-22	0.5-25

Abbreviations: MB: Muscle Biopsy; n.p.: not performed; n.a.: not applicable

The mean age at the first visit for ataluren was 26.9 (range 9.6-43 years); the start date of ataluren was between May 2015 and November 2017. All patients were under treatment at the time of last visit. The mean age at last visit was 30.7, ranging from 13 to 49 years. The average follow-up period was 48.25 months, ranging from 23 to 77, for an overall period of 193 months. During the follow-up, one patient stopped to walk after 6 years of starting treatment, at the age of 49. Another carrier is still able to walk with a waddling gait and lumbar hyperlordosis, but with a search for support. The other two carriers are still able to walk independently.

Motor function tests

6MWT was performed at the first visit in 3/4 patients, showing a mean value of 262 ± 10.47 m, but it was available for only one patient (217 m) at last visit. In the older carrier, an initial improvement under treatment was observed in 6MWT, passing from 270 up to 315m and followed by a gradual decline. NSAA total score was available at the first visit in two patients, showing a mean score of 22/34 which passed to 23.5/34 at the last visit. The time to get up from the floor, an ability present in 2/4 patients, changed from an average value of 8.95 sec at the first visit to 11.5sec at the last visit.

The percentage mean values of FVC passed from 89.7 ± 24.3 to 76.3 ± 20.4, with an annual average decline of 3.3%. The EF values, available in 2/4 carriers, varied on average from 65.5 to 61%, with an annual decline of 1.1% (Tab. II).

Table II.

Clinical data of symptomatic nmDMD carriers treated with ataluren.

Reference center	Age at first visit (years) for ataluren	Ataluren start date	Duration of treatment (years)	Loss of ambulation (years)	6MWD at ataluren start date (meters)	6MWD at ataluren end date (or last visit)	NSAA total score at ataluren start date	NSAA total score at ataluren end date (or last visit)	Gowers time at ataluren start date	Gowers time at ataluren end date (or last visit)	Percentage FVC at ataluren start date	Percentage FVC at ataluren end date (or last visit)	Percentage ejection fraction at ataluren start Date	Percentage ejection fraction at ataluren end date (or last visit)
IT001	26	01/10/2017	48	30	100,00	u	n.p.	n.p.	u.t.p.	u.t.p.	68	53	65	58
UK001	9.6	01/12/2017	45		n.a.	n.p.	31/34	32/34	2.5	2.7	116	91	n.a.	n.a.
IL001	43	01/05/2015	21	49	270.00	u.t.p.	n.p.	n.p.	u.t.p.	u.t.p.	n.p.	n.p.	n.p.	n.p.
IT002	29	23/11/2019	23		255.20	217	13/34	15/34	15.4	20.6	85	85	66	64
Mn	26.90		34.25	39.5	262.60		22/34	23.5/34	8.95	11.65	89.67	76.33	65.50	61.00
Range	9.6-43		21-48	30-49	100-270		13-31/34	15-32/34	2.5-15.0	2.7-20.6	68-116	53-91	65-66	58-64

Abbreviations: n.e. not evaluable; n. p.: not performed; n.a.: not available; u.t.p.: unable to perform

Discussion

By definition, the term ‘carrier’ refers to someone who has a heterozygous mutation in his/her DNA, without presenting the symptoms related to the disease.

The prevalence of skeletal muscle damage among Duchenne female carriers, including asymptomatic carriers is estimated to be between 2.5-19%, and the incidence of dilated cardiomyopathy between 7.3-16.7% [13,14,16,41]. Viggiano et al. [21] observed that DMD carriers with moderate/severe muscle involvement exhibit a moderate or extremely skewed XCI, in particular if presenting with an early onset of symptoms, while carriers with mild muscle involvement present a random XCI. Moreover, when comparing muscle with heart manifesting carriers, the former group showed a higher degree of skewing [21,22].

The frequency of manifesting carriers complicated by cardiomyopathy increases with age [13,16,41] and studies begin to appear on how and when to best treat these patients [26,42]. However, there is limited high-quality evidence to guide the treatment of female carriers of Duchenne /Becker muscular dystrophy. The available evidence is mainly based on expert opinions and clinical experience.

Here, we report our experience in four symptomatic nmDMD female carriers treated with ataluren for 193 months overall. Routine investigations included muscle strength, dynamic tests, cardiac function and pulmonary function tests. We compared changes in 6MWT, Gower’s time, FVC and LVEF at baseline and at the last visit from the start of ataluren. All patients were ambulant at the start of treatment, and two remained so at the last follow-up visit, after 48 and 45 months of treatment, respectively. Under ataluren, the annual assessment of muscle strength, pulmonary lung function tests, and echocardiography indicated a mild attenuation of the disease progression. No adverse clinical effects were reported by the patients nor relevant abnormalities observed in routine laboratory values.

We are aware that the study has the limitations of a retrospective study, which put together data collected spontaneously by researchers who wanted to test the efficacy of treatment with ataluren in nmDMD symptomatic carriers they had in care. The number of carriers treated may also seem too small, but we must remember that the estimated number of nmDMD patients is about 10-15% of the entire Duchenne population and that symptomatic carriers are an even smaller percentage.

Despite these limitations, we believe that ataluren has a good safety profile and stabilizes, if not slightly improves the clinical course of nmDMD female patients, in whom the treatment started much later than in affected males. However, larger clinical trials, and possibly on younger subjects are required to assess the role of ataluren and its long-term impact on disease progression in symptomatic nmDMD carriers.

Ethical consideration

The project was approved by the Ethical Committee of the University of Campania (Protocol number 769 of 23/11/2018).

Acknowledgement

We thank the patients and their families for collaboration.

The unconditional support for medical writer received by the Medical Affairs PTC Italia has been greatly appreciated.

Funding

None.

Conflict of interest

The Authors have no conflicts of interest to declare that are relevant to the content of this article.

Author contributions

LP: conceptualization, methodology, writing original and draft preparation, writing review and editing, and supervision; AD, MG, MS, LPa, AT: investigation and data collection.

All authors have read and agreed to the published version of the manuscript.