Comprehensive analysis of 2097 Patients with Dystrophinopathy Based on a Database From 2011 to 2021

doi:10.21203/rs.3.rs-3146626/v1

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Research Article

Comprehensive analysis of 2097 Patients with Dystrophinopathy Based on a Database From 2011 to 2021

https://doi.org/10.21203/rs.3.rs-3146626/v1

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published 24 Aug, 2024

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Background

A growing number of clinical trials for new therapeutic strategies are being conducted or considered for dystrophinopathy. Detailed data on natural history will facilitate the evaluation of the effectiveness of new drugs for this rare disease. Nevertheless, there is a paucity of data regarding the long-term natural history and associated management in China. Here, we provide a comprehensive description of associated clinical and molecular findings and treatment outcomes in the Chinese population.

Methods

Institutional data on all patients with dystrophinopathy from August 2011 to August 2021 were reviewed retrospectively. The data included geographic distribution, age at diagnosis, genetic analysis, and treatment such as corticosteroids, cardiac interventions, and clinical outcomes.

Results

In total, 2097 patients with dystrophinopathy, including 1703 Duchenne muscular dystrophy (DMD), 311 Becker muscular dystrophy (BMD), 46 intermediate muscular dystrophy (IMD), and 37 “pending” (individuals with an undetermined phenotype) were registered in the Children’s Hospital of Fudan University database for dystrophinopathy from August 2011 to August 2021. The spectrum of identified mutations included exon deletions (66.7%), exon duplications (10.7%), nonsense mutations (10.3%), splice-site mutations (4.5%), small deletions (3.5%), small insertions (1.8%), and missense mutations (0.9%). Two deep intronic mutations were identified. Regarding treatment, 54.4% of DMD patients and 39.1% of IMD patients were treated with glucocorticoids. The median age at loss of ambulation was 2.5 years later in DMD patients with glucocorticoid treatment. Overall, 7.4% of DMD, 8.3% of IMD, and 2.6% of BMD patients were prescribed one cardiac medicine at least, and four DMD patients were under ventilator support. Those eligible for exon skipping therapy included 55.3% of DMD patients; among them, 12.9%, 10%, and 9.6% of these patients were eligible for skipping exons 51, 53, and 45.

Conclusions

This is one of the largest studies to have evaluated the natural history of dystrophinopathy in China, which is particularly conducive to recruiting eligible patients for clinical trials and providing real-world data to support drug development.

Dystrophinopathy

Duchenne and Becker muscular dystrophies

Patient management

Genetic diagnosis

Natural history

Dystrophinopathies cover a spectrum of X-linked muscle diseases caused by mutations in the dystrophin gene (OMIM 300377). Duchenne muscular dystrophy (DMD; OMIM 310200) is the most common form, which shows a severe phenotype clinically characterized by rapid progression in early childhood and loss of independent ambulation (LoA) by the age of 13 years; it affects 1 in 3600–6000 live male births[1]. The prevalence of DMD has been reported to be 1 in 4560 live male births in China[2]. Accordingly, about 1100 new cases are born every year in China. Milder allelic forms also exist, including intermediate muscular dystrophy (IMD) and Becker muscular dystrophy (BMD), which LoA typically at 13–16 years or over 16 years of age, respectively. Other types of dystrophinopathies occur at a lower incidence, including DMD and BMD manifesting in female carriers, isolated X-linked cardiomyopathy, and isolated quadriceps myopathy[3].

Pharmacological and care-based interventions (corticosteroid, rehabilitative, cardiac, orthopedic, respiratory, gastrointestinal, speech/swallowing, nutrition, and psychosocial interventions) can improve the muscle function, quality of life, and longevity of DMD patients[1]. Moreover, some therapeutic medicines have been used in clinical settings. In August 2014, ataluren, used to treat people with DMD who have a nonsense mutation in the dystrophin gene in ambulatory boys aged ≥ 5 years, received market authorization from the European Commission; it is currently sold under the trade name Translarna in the European Union. The outcome of a phase III trial of Ataluren (ACT DMD; ClinicalTrials.gov identifiers: NCT01826487) showed that the benefits observed in patients with a baseline 6 min walk distance of 300 m or more to less than 400 m supported the clinical benefit of ataluren versus a placebo in patients with nonsense mutation DMD, particularly when considering the totality of supporting evidence. The data also confirmed the clinical benefit of ataluren in terms of preserving muscle function[4]. However, the United States Food and Drug Administration (FDA) filed a Refuse to File letter on February 22, 2016, claiming that the results of the phase IIb study[5] and ACT DMD phase III trial[4] did not demonstrate adequate evidence of effectiveness. Two international multicenter clinical trials of ataluren continued to examine the long-term efficacy and safety of ataluren (ClinicalTrials.gov identifiers: NCT03179631 and NCT01247207); five hospitals, including our hospital in China, participated in these trials. In September 2016, the FDA conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based antisense oligonucleotide drug, eteplirsen, developed for DMD exon 51 skipping, based on the outcomes of a clinical trial[6]. Charleston et al.[7] examined the quantification of novel dystrophin production in patients with DMD after long-term treatment with eteplirsen; their results showed a sustained increase in dystrophin protein production and the presence of a novel in-frame mRNA gene product. Taken together, these results serve as evidence for the successful mechanism of eteplirsen and support the use of dystrophin as a pharmacodynamic marker, thus suggesting that the therapeutic approach is working as intended. Subsequent clinical trials on eteplirsen are active or recruiting (ClinicalTrials.gov identifiers: NCT03992430, NCT04179409, NCT03985878, and NCT03218995). Vyondys 53 (golodirsen) to treat patients with DMD amenable to exon 53 skipping was approved in the United States in December 2019, based on positive results from a phase I/II clinical trial[8]. Frank et al. reported on the safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with DMD amenable to exon 53 skipping (ClinicalTrials.gov identifiers: NCT02310906.). They found that golodirsen was well-tolerated. Muscle biopsies from golodirsen-treated patients have shown increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization[9]. In March 2020, another intravenous medicine, Viltepso (viltolarsen), received its first global approval in Japan for treating DMD patients, with confirmed deletion of the dystrophin gene that is amenable to exon 53 skipping. Viltolarsen is under regulatory review in the U.S., and clinical trials continue globally (e.g., U.S. and Canada)[10]. In February 2021, Casimersen was approved for medical use in the U.S. It is the first FDA-approved targeted treatment for people with a confirmed mutation of the DMD gene that is amenable to skipping exon 45. In addition, many promising therapeutic strategies are currently underway, providing hope for identifying definitive treatments for this currently incurable disease, as summarized below[11–14].

(1) Gene replacement with adeno-associated virus. Currently, two phase-III independent clinical trials of slightly different microdystrophins, viral vectors, and gene promoters are ongoing in patients with DMD (ClinicalTrials.gov, Identifier: NCT05096221; ClinicalTrials.gov, Identifier: NCT04281485; ClinicalTrials.gov, Identifier: NCT05096221).

(2) Mutation-specific therapeutic approaches for repairing the genetic defects or the related transcripts, such as exon-skipping agents (e.g., exon 51, exon 45, and exon 53 skipping agents) and stop codon readthrough agents as mentioned above.

(3) Genome editing. CRISPR-Cas9 technology can be used to induce double-stranded DNA breaks at specific places in the genome. These breaks activate DNA repair systems, which can lead either to homologous recombination, which corrects mutations or to non-homologous end joining, which leads to random insertions and/or deletions (that is, additional mutations).

(4) Symptomatic agents that target the major pathologic changes for alleviating symptoms, including agents used to prevent muscle damage, reduce inflammation, accelerate muscle repair, increase blood flow to muscles, and stop muscle fibrosis. Several phase III clinical trials are recruiting (ClinicalTrials.gov, Identifier: NCT04371666 and NCT04632940; ClinicalTrials.gov, Identifier: NCT04587908).

(5) Dystrophin surrogates. Upregulation or replacement of utrophin, a developmental paralog of dystrophin was the first surrogate to show promise as a DMD treatment. Dystrophin surrogates may have the greatest potential in combination with dystrophin correction or replacement strategies.

(6) Cell therapy. Dystrophin expression derived from human induced pluripotent stem cells (iPSCs) showed modest gains but led to functional improvement in the mdx mouse. Patient-derived iPSCs have yielded a theoretically unlimited supply of self-renewing, fetal stage, therapeutic myogenic stem cells, allowing for gene replacement or correction before engraftment. Moreover, because skeletal muscle is relatively inhospitable to tumor formation, it represents an attractive target tissue for PSC therapy.

As for BMD, clinical trials that aim to improve motor function and heart problems are currently in progress (ClinicalTrials.gov, Identifier: NCT05166109; ClinicalTrials.gov, Identifier: NCT03238235; ClinicalTrials.gov, Identifier: NCT05160415; and ClinicalTrials.gov, Identifier: NCT04386304).

Given these promising results, it becomes clear that the clinical trials for dystrophinopathy require international collaboration to establish sufficient patient enrollment for therapeutic significance to be demonstrated statistically. Based on this, we created a database for patients with dystrophinopathy at Children’s Hospital of Fudan University (referred to as the CHFU Database for Dystrophinopathy) in 2011. Over the past 10 years, 2097 patients with dystrophinopathy have been registered in our database. Thus, this database provides a general picture of the natural history and the current status of diagnosis and treatment of patients with dystrophinopathy in East China. Clearly, a database with a large number of patients with dystrophinopathy will be critical for international efforts to develop experimental therapies, facilitate trial design for new treatment strategies, and provide real-world data as external controls. Here, we describe the genetic diagnosis, clinical outcomes, and treatment status of the patients registered in our database.

2.1 Institution and organization of the project

The CHFU database for dystrophinopathy is supported by Children’s Hospital of Fudan University. The development and management of the registry is led by the Neuromuscular research unit, department of neurology.

2.2 Patients

The CHFU database includes patients with dystrophinopathy (all male Chinese) whose diagnosis has been confirmed by genetic analysis or muscle biopsy (see below section Phenotypic classification).

2.3 Method of registration and data collection

Information about the registry was provided to the patients with dystrophinopathy through DMD Outpatient Clinic in our hospital. Patient records were entered into the database on the basis of informed consent, which explained the content and aims of the database with voluntary registration. No identifiable personal data will be shared with any third party without the patient’s permission and the subject will be removed from the database immediately on request. This study was approved by the Institutional Review Board at Children’s Hospital of Fudan University.

2.4 Structure of the registry form and Patients’ follow-up

The registry form was designed as reported previously[15].The database was semi-annually or annually updated by outpatient visits for ambulant patients and through telephone for non-ambulant patients if they are not able to get to the outpatient clinic or patients aged 18 years over.

2.5 Diagnosis of dystrophinopathy

The diagnosis of a dystrophinopathy is established in a proband with the characteristic clinical findings and elevated CK concentration and/or by identification of a hemizygous pathogenic variant in DMD gene on molecular genetic testing [16]. The patients who diagnosed by the results of muscle biopsy with dystrophin immunostaining are also included in our database. Multiplex ligation-dependent probe amplification (MLPA), which allows detection of deletions and /or duplications in all 79 exons has been developed and widely used[17]. For single exon deletion, further verification will be carried out by PCR and sequencing. When no mutations are detected by MLPA, genetic sequencing will be done to screen for the remaining types of mutations that are attributed to dystrophinopathy or a muscle biopsy sample will be tested for the presence of dystrophin protein by immunohistochemistry of tissue cryosections.

2.6 Phenotypic Classification

Phenotypic classification is determined by neurologists and genetic curators together and based on genetic analysis (reading frame rule) with consideration of clinical information and pathological data (including dystrophin immunostaining, if available). Four phenotypic subgroups have been defined including the “DMD”, “BMD”, and “IMD” (Intermediate Muscular Dystrophy); and “pending” (for patients where the phenotype cannot be ascertained because of the age of the patient or incomplete clinical data) [18].

2.7 Loss of ambulation (LoA)

We defined LoA as participant-caregiver-reported age at continuous wheelchair use, approximated to the nearest month, and verified by a trained clinical evaluator with the inability to perform the 10 m run/walk assessment[19].

2.8 Glucocorticoid (GC) treatment.

At baseline and follow-up visits, we recorded time of beginning/discontinuation, drug, dose, and pattern of administration of previous and current GC regimens. Because of a low number of participants subject to intermittent regimens (10-days-on/10-days-off, 10 days/month, and every other day), we grouped these regimens together. The patients with GC regimens < 1 month were grouped into untreated registrants. For comparisons of each milestone between GC-treated and untreated registrants, we considered “GC-treated” only those patients who had been administered GCs for ≥ 1 year, starting 1 year before LoA, because a long-term effect cannot be attained with a short-term treatment[20].

2.9 Statistical analysis.

We performed a time-to-event analysis of each milestone (with median [IQR]) with age (years) as time variable and patients reaching milestone as event. Median age at each milestone was estimated by Kaplan Meier curves for participant groups defined by GC treatment administered while ambulatory, with the following grouping: untreated (< 1 month’s use or never treated) and treated (≥ 1 year’ use, and starting ≥ 1 year before LoA). Median age at event was compared on the basis of GC treatment with a log-rank test. Statistical significance was set at p < 0.05. All analyses were performed with the SPSS software, version 25.

As of August 2021, 2097 patients had been registered in our database consisting of DMD (1703), BMD (311), IMD (46), and individuals with an undetermined phenotype (“pending”) (37). Most registrants were under 18 years of age. 139 (6.6%) patients were lost to follow-up, and 20 (1%) died during the follow-up period. Genetic testing identified a molecular diagnosis in 1949 boys. Twenty-seven patients whose genetic tests did not confirm dystrophinopathy were diagnosed by immunohistochemical staining of muscle biopsy specimens. Another 5.8% of patients without genetic testing were diagnosed via muscle biopsy. As of August 2021, the top three registrants at follow-up by age group were 9–10, 8–9, and 6–7 years old (Fig. 1). A diagnosis was made with the highest proportion at 3–4 years, 16.3% for DMD, and 23.8% for BMD because of the medical examination of transaminases for kindergarten enrollment, which is a widespread practice in many provinces in China (Fig. 2).

Figure 1 Ages of registered individuals. Most registrants were under 18 years of age.

Figure 2 Age at the diagnosis. Most patients were diagnosed at 3–4 years old.

Patients with dystrophinopathy registered in our database were from 27 provinces in China; most (80%) were from East China, including Shanghai (163) and the provinces surrounding Shanghai, namely, Jiangsu (461), Anhui (386), Zhejiang (303), Jiangxi (201), Fujian (73), and Shandong (94). A small proportion of registrants resided in South Central China (199), Southwest China (106), Northwest China (61), Northeast China (19), South China (19), and North China (12) (Fig. 3).

Figure 3 Geographical distribution of 2097 registrants.

In total, 1949 male patients in our database were subjected to molecular diagnosis of the disease. The spectrum of identified mutations contained exon deletions (66.6%), exon duplications (10.7%), nonsense mutations (10.3%), splice-site mutations (4.5%), small deletions (3.5%), small insertions (1.8%), and missense mutations (0.9%). In the 418 patients harboring a small mutation, 293 mutations were reported and 125 mutations were unreported (Table S1). However, the remaining 27 genetically undiagnosed patients were diagnosed based on muscle biopsy, and two deep intronic mutations were identified. Exon deletion was the most frequent (65.7% and 78.5%), followed by point mutations (15.7% and 11.6%), duplications (11.1% and 7.5%), and small insertion/deletion (6.3% and 1%) for DMD and BMD, respectively (Table 1). Some DMD patients (55.3%) were eligible for exon-skipping therapy, among whom 12.9%, 10%, and 9.6% were eligible for skipping exon 51, 53, and 45, respectively (Table 2).

Table 1

Distribution of mutations in DMD/BMD patients
	DMD patients BMD patients		BMD patients
Distribution of mutation	No. of case	% of cases	No. of case	% of cases
Deletion	1052	65.7%	230	78.5%
Duplication	177	11.1%	22	7.5%
Deletion and duplication	2	0.1%	0	0
Nonsense mutation	186	11.6%	7	2.4%
Small insertion	35	2.2%	0	0
Small deletion mutations	65	4.1%	3	1%
Splice site mutations	58	3.6%	20	6.8%
Missense mutation	6	0.4%	7	2.4%
Synonymous mutation	1	0.1%	0	0
No mutation found*	19	1.2%	4	1.4%
	1601	100%	293	100%

* Diagnosis was made on the basis of absence of dystrophin expression and clinical manifestation.

Table 2

**Candidate for to 51, 53 and 45 skipping for restoration of dystrophin expression**

(source: www.clinicaltrials.gov)
51 exon skipping			53 exon skipping			45 exon skipping
Deleted exon(s)	No. of individuals	% of registrants	Deleted exon(s)	No. of individuals	% of registrants	Deleted exon(s)	No. of individuals	% of registrants
19–50	1	0.1%	45–52	51	3.2%	12–44	1	0.1%
45–50	61	3.8%	47–52	3	0.2%	44	38	2.4%
47–50	4	0.2%	48–52	32	2%	46	2	0.1%
48–50	53	3.3%	49–52	35	2.2%	46–47	38	2.4%
49–50	41	2.6%	50–52	12	0.7%	46–48	29	1.8%
50	19	1.2%	52	28	1.7%	46–49	9	0.5%
52	28	1.7%				46–51	25	1.5%
						46–53	1	0.1%
						46–55	8	0.5%
						46–57	1	0.1%
						46–59	1	0.1%
						46–60	1	0.1%
total	207	12.9%		161	10%		154	9.6%

Some DMD (54.4%), IMD (39.1%) and BMD (1%) boys received glucocorticoid (GC) treatment, and 67.7% of DMD boys and 78.6% of IMD boys were ambulant, whereas all BMD boys were ambulant. Cardiac function was examined in 49.8% of DMD boys, 63% of IMD, and 38.4% of BMD boys. Respiratory functions were examined for only 26.6% of DMD boys, 45.7% of IMD, and 11.5% of BMD boys. Low cardiac function (EF < 55%) was found in 0.6% of DMD, 4.3% of IMD, and 1% of BMD. 7.4% of DMD, 8.3% of IMD, and 2.6% of BMD registrants were prescribed one cardiac medication at least. Four patients in our database used ventilator support (Table 3). None of the patients received scoliosis surgery.

Table 3

Walking capability, medications and intervention in the registrants with DMD and BMD
	DMD patients		BMD patients
	No.of case	% of cases	No.of case	% of cases
Walking capability
Not acquiring the ability to walk independently	7	0.5	0	0
Normal walking	1046	67.5%	305	100%
Not able to walk	497	32.1%	0	0
	1550	100%	305	100%
Cardiac function
Normal	763	49.2%	114	37.4%
Dysfunction	9	0.6%	3	1%
Not performed	778	50.2%	188	61.6%
	1550	100%	305	100%
Respiratory function
Normal	366	23.6%	35	11.5%
Dysfunction	47	3%	0	0
Not performed	1137	73.4%	270	88.5%
	1550	100%	305	100%
Steriod use
Current	777	50.1%	2	0.7%
Used to	67	4.3%	1	0.3%
Never	706	45.6%	302	99%
	1550	100%	305	100%
Cardiac medication
Prescribed	114	7.4%	8	2.6%
Not prescribed	1436	92.6%	297	97.4%
	1550	100%	305	100%
Drug
ACE-inhibitor	93	81.6%	6	75%
β-blocker	27	23.7%	5	62.5%
Diuretics	4	3.5%	3	37.5%
Other	6	5.3%	3	37.5%
	114*1		8*1
*1. The number of registrants who were prescribed with cardiovascular medicines.

Table 3Walking capability, medications and intervention in the registrants with DMD and BMD

Since the establishment of our database, nearly 250 patients with dystrophinopathy have been registered annually. Based on this large patient population, we feel that our database provides a relatively good representation of the mutation spectrum, genotype/phenotype correlation, and treatment efficacy of patients with dystrophinopathy.

4.1 Mutation distribution and genotype/phenotype correlation

From August 2011 to August 2021, 2097 patients with dystrohinopathy were registered in the CHFU database. Among them, 1976 patients had genetic testing, and pathologic variants were found in 1949 patients. Exon deletion was the most frequent, followed by duplications, nonsense point mutations, splice site mutations, and small insertion/deletion. This is similar to the pattern described in other databases[18, 21] and those reported previously in the Chinese population[22–24], with some differences. In DMD patients, exons 45–52 were most frequently deleted. The deletion of exons 45–50 (61 times) was the most common deletion, and exon 2 duplication (17 times) was the most common duplication in DMD patients, accounting for 5.8% (61/1052) of deletions and 9.6% (17/177) of duplications. The deletions of exons 45–47 (48 times) and 45–48 (32 times) represent 34.8% (80/230) of the BMD deletions. Table 4 showed the most commonly reported large deletions and large duplications in our database. Monaco et al. postulated an explanation for the phenotypic difference between Duchenne and Becker patients in the reading-frame rule[25]. In our database, 95.3% of patients with DMD/BMD were consistent with this rule; exon 3–7 deletions are well-documented exceptions to the rule[26]. In our series, this deletion was found in eight DMD, one BMD, and six IMD patients, as well as in two patients with a pending phenotype. We also found that exon 3–13 deletions (seven times), exon 3–44 deletions/duplications (five/two times), exon 3–30 deletions (five times), and exon 3–18 deletions (three times) are primarily common exceptions to the reading frame rule. Annemieke Aartsma-Rusin et al. [26] indicated that deletions removing both the actin-binding domain and part of the central rod domain usually cause DMD, which may be explained by the fact that an additional actin-binding site is present in the central rod domain.

Table 4

The Most Frequent Patterns of Deletions and Duplications in DMD and BMD Patients
DMD
5’ hot spot	3’ hot spot	5’ hot spot	3’ hot spot
dup 2 (17)	del 45–50 (61)	del 3–4 (4)	del 45–47 (48)
dup 3–7 (12)	del 48–50 (53)	dup 2–7 (4)	del 45–48 (32)
dup 8–9 (11)	del 45–52 (51)		del 45–53 (20)
del 3–7 (8)	del 51 (48)		del 45–55 (19)
	del 45 (44)		del 48 (15)
	del 49–50 (41)		del 48–49 (14)
	del 46–47 (38)
	del 44 (38)
	del 49–52 (35)
	del 48–52 (32)
	del 46–48 (29)
	del 52 (28)
	del 46–52 (27)
	del 46–51 (25)
	del 45–54 (24)
	del 46–50 (21)
	del 50 (19)
	del 48–54 (17)
	del 51–53 (14)
	del 50–52 (12)
	del 51–55 (11)
	del53-55 (10)

Table 4. The Most Frequent Patterns of Deletions and Duplications in DMD and BMD Patients

Whole-gene deletions were identified in three patients. Furthermore, a definite rare causative mutation in intron 48, c.7098 + 1813G > T, was identified via skeletal muscle RNA analysis as being hemizygous in two affected siblings, and the mother was found to be a carrier. The novel deep intronic mutation resulted in the truncation of dystrophin protein by pseudoexon inclusion and a frameshift of coding sequences. The proband presented with progressive muscle weakness since the age of 3 years. Clinical examination of the proband revealed calf muscle pseudohypertrophy, reduced tendon reflexes, winged scapula, lordosis, and Gower’s maneuver when the boy came to the outpatient clinic at 3.5 years of age. Multiplex ligation probe amplification analysis of the DMD gene and next-generation sequencing were applied to identify the mutations, and the results were negative. A muscle biopsy was subsequently performed, and the results were consistent with the diagnosis of DMD. The younger sibling of the proband presenting with a similar phenotype was later diagnosed with DMD. A patient with c.9225–647A > G, as described in several studies [27–31], exhibited an IMD phenotype. He showed a positive Gower sign and a waddling gait, but he could still jog when he was 8 years and 7 months. Therefore, as to the patients with mutations which are difficult to determine the pathogenicity and show negative genomic testing but abnormal dystrophin, an analysis of muscle-derived RNA is an important diagnostic step. A definitive molecular diagnosis facilitates accurate genetic counseling and diagnostic testing of family members.

Our data represented the largest cohort of proband male patients with independent mutations reported in East China to date, and such a genetic DMD mapping allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, with effects on population genetic characteristics and new personalized therapies of dystrophinopathy. Furthermore, some patients with certain mutations would potentially benefit from novel genetic therapies for DMD, including stop codon readthrough therapies (11.6% of total mutations) and exon skipping therapy (84% of deletion and 55.3% of total mutations) within our database. The top 10 exon skips that would be applicable to the largest group of patients were skipping of exon 51 (12.9% of total mutations), 53 (10%), 45 (9.6%), 44 (5.7%), 50 (4.8%), 52 (4.4%), 55 (3.9%), 43 (3.2%), and 8 (0.7%).

4.2 Family history and carrier screening

In our study, 13% of the probands had a positive family history of the disease. The carrier status of 1242 probands’ mothers have been established, and 799 mothers carried the same mutation (64.3%), similar to previous European and Chinese reports of a large population with dystrophinopathy[18, 22, 23]. Moreover, five cases with mosaicism in the mothers were found through the prenatal diagnosis of the fetus or the probands’ siblings.

4.3 Glucocorticoid treatment

Of the 1550 DMD patients, 777 (50.1%) follow-up patients were treated with GC; 67 (4.3%) of the 1550 DMD patients were past GC users treated within the prior ≥ 1 month with GC but were not currently receiving GC therapy, and 706 of the 1550 (45.6%) were GC-naïve. The rate of GC therapy has increased significantly compared to that cited in our previous report[15]. A steady increase in the use of GC treatment is expected in China[24]. In our database, 99.3% of DMD patients receiving GC therapy chose daily PRED, the most frequently prescribed regimen; 11 DMD patients chose intermittent use (10-days-on/10-days-off, 10 days/month, and every other day). Nineteen DMD patients switched from PRED to DFZ; three later switched back to PRED.

The average age of the start of GC treatment (excluding treatment started after LoA) was 6.3 ± 1.6 years (range, 1.8–12 years). Most DMD patients (26.8%) started GC treatment at 4–5 years of age. The mean duration of treatment while ambulatory was 3 ± 1.7 years (range, 0.1–15.4 years), as shown in Table 5. Kaplan–Meier analysis showed that median age at LoA was 2.5 years later in participants treated ≥ 1 year while ambulatory vs. untreated or treated < 1 month (12.6 vs. 10.1 years, n = 665 vs. 688, log-rank p < 0.0001) (Fig. 4). Several DMD studies have reported time to LoA as the primary outcome. King et al. [32] reported that DMD males treated with corticosteroids for at least 1 year (n = 75) ambulated 3.3 years longer than untreated males (n = 68). Kim et al. [20] found that long-term treatment was positively associated with time to LoA at an earlier stage of treatment. Cases in the long-term treatment group stopped walking 2 years later (at age 12.3 years). The CINRG Duchenne Natural History Study[33] found that GC treatment for 1 year or longer was associated with increased median age at LoA by 3.4 years, compared to treatment for less than 1 month or having never been treated (13.4 vs. 10 years, n = 330 vs. 73, log-rank p < 0.0001). Recently, a study with 1163 Chinese DMD patients indicated significant beneficial responses to GC treatment. The median ages at LoA for GC-naïve, prednisone/prednisolone-treated, and deflazacort-treated groups were 10.23, 12.02, and 13.95 years, respectively[34]. We also compared patients treated with GC versus GC-naïve patients for age at transition to ≥ 5 s and ≥ 10 s stand from supine which tended to be more delayed in patients treated with GC than in GC-naïve patients. In addition, age at transition to ≥ 6 s and ≥ 12 s walk/run (10 MWT) also tended to be more delayed in patients treated with GC than in those that were GC-naïve (Fig. 4). Obviously, GC treatment had a more substantial protective effect on DMD patients’ decline in motor function. However, we found that due to rapid weight gain, a proportion of patients may not benefit from GC treatment in terms of delaying the age of LoA, and some chose to halt the treatment. Nevertheless, the situation may improve in the near future. A New Drug Application filing for vamorolone, a first-in-class steroid medication that targets the same receptors as corticosteroids but with fewer side effects, has been submitted to the FDA[35].

Table 5

Glucocorticoid (GC) treatment of DMD patients while ambulatory
	GC treatment	GC previously	GC naïve	Total
	n = 741	n = 67	n = 698	n = 1506
Age(years)	9.2 ± 2.2	12.5 ± 2.3	8.1 ± 3.4	8.8 ± 2.9
Age at diagnosis	4.9 ± 2.1	7.4 ± 1.6	4.6 ± 2.5	4.9 ± 2.4
Age at loss of ambulation	10.7 ± 1.6	10.5 ± 1.8	9.9 ± 1.3	10.2 ± 1.5
GC initiated	6.2 ± 1.5	7.7 ± 1.5	N/A	6.3 ± 1.6
GC duration	3.1 ± 1.7	2.7 ± 2	N/A	3 ± 1.7
GC stopped	N/A	10.1 ± 1.9	N/A	10.1 ± 1.9

Age and timing of milestones in years ± SD. N/A: not applicable.

Figure 4: Time to ambulatory milestones. Kaplan-Meier analyses comparing cumulative glucocorticoid use (< 1 month or never treated vs ≥ 1 year) for (A) age at transition to stand from supine of 5s or greater; (B) age at transition to stand from supine of 10s or greater; (C) age at transition to walk/run 10m of 6s or greater; (D) age at transition to walk/run 10m of 12s or greater; and (E) age at loss of ambulation (unable to ambulate 10 m).

4.4 Cardiac and pulmonary management

Cardiac care is vitally important to improve the quality of life of DMD patients and is focused on surveillance and management, with the goal of slowing the onset and progression of heart failure complications. Due to a growing recognition that adverse myocardial changes occur before overt cardiac dysfunction and at a younger age than previously thought, as specified in the 2018 DMD Care Considerations, it is now recommended to begin yearly cardiac screening at diagnosis[36]. The initial cardiac visit should include a past and present cardiac medical and family history. In addition, the clinician should perform a physical examination, electrocardiogram, and noninvasive imaging with either an echocardiogram or cardiac MRI (CMRI) based on the child’s age and ability to cooperate[37]. For patients with established cardiac abnormalities, more frequent visits may be needed.

Based on our database, we found that electrocardiography can detect abnormalities at a very early age prior to other cardiac function tests in DMD patients. Characteristic electrocardiographic changes included left ventricular high voltage, sinus arrhythmia, short PR interval, T wave change, left ventricular hypertrophy, right ventricular hypertrophy, prolonged corrected QT interval, and Q waves. Some DMD patients (53%) who accepted electrocardiographic examination before 6 years of age showed electrocardiographic abnormalities well before the onset of clinical symptoms. Similarly, electrocardiographic abnormalities were frequently noted in patients with BMD, but were generally nonspecific, as mentioned in previous reports[38]. Given the absence of dystrophin-specific, targeted cardiac therapies, traditional treatment strategies for heart failure should be used. Traditionally, angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers have been used as first-line therapies for the treatment of heart disease associated with DMD[37]. Our cardiologists usually initiated the use of ACE inhibitors in asymptomatic boys approaching 10 years of age with normal left ventricular systolic function, after discussing the potential benefits and risks with the family. Irrespective of age, pharmacological therapy should be initiated with the onset of heart failure symptoms or when abnormalities are noted in imaging studies (electrocardiogram, CMR, or echocardiogram). In our database, 7.4% of DMD, 8.3% of IMD, and 2.6% of BMD registrants were prescribed at least one cardiac medication, and an ACE-inhibitor was the most commonly used, as shown in Table 3. The average age at the start of such treatment was 10 ± 2 years (range, 3–17.6 years) for DMD patients, 13.2 ± 2.7 years (range, 9.3–17 years) for IMD patients and 9.9 ± 3.4 years (range, 3.9–14.3 years) for BMD patients.

Minimal assessment of pulmonary function (such as the measurement of forced vital capacity (at least annually) allows the child to become familiar with the equipment and the team to assess the maximum respiratory function achieved while a boy with DMD is still walking. The main emphasis of pulmonary assessment is after the loss of independent walking[36]. However, pulmonary management is still far from ideal in our patient cohort. Only 26.6% of DMD patients received pulmonary function testing, mainly because some patients are cognitively impaired and unable to perform the test reliably. In our database, abnormalities were found in only 3% of DMD patients, primarily due to poor compliance with follow-up of most patients who were wheelchair users. Ventilatory assistance significantly improves the survival of DMD patients, and the median survival age in these patients now reaches 40, with no impairment in their quality of life[39]. However, only four patients used ventilator support in our database. After all of these years, little has changed in the care of these patients, due to the lack of awareness by physicians and families of the significance of these measures to the patient’s quality of life and the lack of medical insurance to cover treatment expenses. As GC treatment have become more widely used and the approval of new drugs has markedly increased in recent years, it reinforces the urgency to raise awareness of the disease to the public, physicians, and government organizations.

4.5 Cause of death in DMD children

Overall, 20 patients died during the follow-up, 17 of which were attributable to DMD complications. The mean age at death due to DMD was 14.4 ± 2.2 (10.8–20.9) years, except for two patients who did not die of DMD complications. One patient died of pneumonia when he was 3 years old, and the other died of acute leukemia at 7 years. The exact cause of death in one patient was unclear.

The CHFU database for dystrophinopathy has been instrumental in providing clinicians with a essential tool for the selection of patients based on predetermined genotypic and phenotypic criteria for clinical trials of new drugs for dystrophinopathy. The registration of 2097 patients in the database is significant, as it allows for gaining accurate knowledge of the natural history of patients with dystrophinopathy in China, which provides clinicians with insights into the effectiveness of therapeutic interventions and is conducive to developing practical and cost-effective strategies for the management of patients with dystrophinopathy.

DMD

Duchenne muscular dystrophy

IMD

Intermediate muscular dystrophy

BMD

Becker muscular dystrophy

MLPA

Multiplex Ligation-dependent Probe Amplification

LoA

Loss of ambulation

glucocorticoid

DFZ

deflazacort

PRED

prednisone or prednisolone.

Ethic approval and consent to participate

All clinical information and materials used in the present study were obtained with written informed consent from patients or their legal guardians. The study was approved by the Research Ethics Board of Children’s Hospital of Fudan University([2022]300A).

Consent for publication

Not applicable.

Availability of data and materials

The data that support the findings of this study are not openly available due to reasons of sensitivity and are available from the corresponding author upon reasonable request. Data are located in controlled access data storage at Children’s Hospital of Fudan University.

Competing interests

The authors declare that they have no competing interests.

Funding

This study was funded by the National Key Research & Development Plan of China (2022YFC2703600)

Author’s contributions

XL participated in planning and leading this study, curating the molecular and pathological data and guiding the writing of the manuscript. Lei Zhao participated in data entry, analysis and interpretation, generation of the tables and figures, in writing of the manuscript. SZ supervised in planning this study and curating clinical data. YS and CH contributed to data collection, data analysis and manuscript preparation. HL, Lifeng Zhang, CQ and YZ participated in curating clinical data and data analysis. All authors read and approved of the final manuscript.

Acknowledgements

We express our heartfelt gratitude for the patients, families, physicians, and The Special Fund For Rare Disease to support the dystrophinopathy registry.

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Comprehensive analysis of 2097 Patients with Dystrophinopathy Based on a Database From 2011 to 2021

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Abstract

Background

Methods

Results

Conclusions

Figures

1. Background

2. Materials and methods

2.1 Institution and organization of the project

2.2 Patients

2.3 Method of registration and data collection

2.4 Structure of the registry form and Patients’ follow-up

2.5 Diagnosis of dystrophinopathy

2.6 Phenotypic Classification

2.7 Loss of ambulation (LoA)

2.8 Glucocorticoid (GC) treatment.

2.9 Statistical analysis.

3. Results

4. Discussion

4.1 Mutation distribution and genotype/phenotype correlation

4.2 Family history and carrier screening

4.3 Glucocorticoid treatment

4.4 Cardiac and pulmonary management

4.5 Cause of death in DMD children

5. Conclusions

Abbreviations

Declarations

References

Supplementary Files

Status:

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