Introduction
Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited disorders characterized by progressive weakness, wasting of proximal muscles, and dystrophic features on muscle biopsy. LGMDs are subdivided into autosomal-dominant LGMD1 and autosomal-recessive LGMD2. A new classification of five autosomal-dominant LGMDs (D1–D5) and twenty-four autosomal-recessive LGMDs (R1–R24) was proposed at the 229th European Neuromuscular Center (ENMC) workshop and to date, at least 34 reported genes have been associated with LGMDs [
1].
The most common LGMDs are as follows: (a) Calpainopathy, also named LGMD-R1-calpain3-related, is one of the most prevalent forms of recessive LGMD, representing approximately 30–40% of LGMD cases with an estimated prevalence of 6.8–10.2 per million worldwide [
2]. Disease onset usually occurs between 6 and 18 years but can range from 2 to 49 years of age. Approximately 80% of patients usually become wheelchair-bound between the second and fourth decades of life [
3]. Calpainopathy is caused by a defect in the calpain-3 gene (
CAPN3) located in the chromosomal region 15q15.1-q21.1.
CAPN3 encodes the muscle-specific calpain-3 protein that plays an important role in promoting the release of Ca
2+ from skeletal muscle fibers, sarcomere remodeling, muscle contraction and NF-κB signaling [
4,
5]. To date, more than 580 different pathogenic mutations have been reported, and these mutations are distributed along the entire coding region of the
CAPN3 gene. Two hotspot mutations, c.2120A > G and c.550del, were identified in Chinese and some European populations, respectively [
6,
7]. Loss-of-function mutations of
CAPN3 inactivate its proteolytic function, playing an important role in the pathogenesis of calpainopathy, but the precise mechanism of calpainopathy has remained elusive. b) Autosomal recessive dysferlinopathy (R2) is one of the most commonly diagnosed LGMD subtypes in the world, with an estimated prevalence of 5.7–9.4 per million individuals. The clinical phenotypes of dysferlinopathy predominantly included Miyoshi myopathy, LGMD2B, and distal myopathy with anterior tibial onset (DMAT). The majority of patients with dysferlinopathy present progressive muscle weakness by the age of 30 years, but also later onset ranging to 60 years old has also been reported. Dysferlinopathy is caused by mutations in the
DYSF gene located on chromosome 2p13.2, which spans a genomic region of approximately 233 kb and comprises 55 exons. More than 900 potentially deleterious
DYSF variants have been reported, and hotspot mutations have been identified in some countries, such as c.2997G > T in Japanese patients and c.1375dup in Chinese patients [
8,
9].
DYSF encodes the 237 kDa transmembrane protein dysferlin with functions associated with Ca
2+ homeostasis, vesicle trafficking, sarcolemmal resealing and T‐tubule system shaping [
10,
11]. Patients with
DYSF mutations exhibit a deficiency of dysferlin protein in skeletal muscle membranes, and disruption of the aforementioned process contributes to the pathological mechanism.
(c) Sarcoglycanopathies (R3-R6) are the most severe form of LGMD, accounting for 10–25% of LGMDs. The prevalence of sarcoglycanopathies is variable in different ethnicities and geographic regions, ranging from 0.31/100,000 to 0.58/100,000 [
2]. Most patients presented with progressive proximal muscle weakness with childhood onset, rapid progression and loss of ambulation in adolescence or early adulthood, although milder cases are still ambulant at age 60 years [
12,
13]. Sarcoglycanopathies include four subtypes of LGMD (LGMDR3-R6), caused by recessive mutations in the
SGCA,
SGCB,
SGCG and
SGCD genes. Their four encoded corresponding proteins, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan and δ-sarcoglycan, are components of the dystrophin-glycoprotein complex (DGC) in the muscle sarcolemma. Mutations in these four sarcoglycan-related genes result in deficiency of the corresponding protein subunits, perturbing the heterotetramer complex closely linked to the dystrophin-associated protein complex in the cell membrane, which plays a critical protective role in membrane integrity and provides a scaffold for transmitting important signals [
14]. (d) Several subtypes of LGMDs are dystroglycanopathies, primarily caused by mutations in eight genes, including
FKRP (R9),
POMT1 (R11),
FKTN (R13),
POMT2 (R14),
POMGnT1 (R15),
GMPPB (R19),
ISPD (R20), and
POMGNT2 (R24), involved in the glycosylation of the alpha-dystroglycan pathway [
15]. Dystroglycan is a highly glycosylated adhesion receptor complex that is composed of alpha-dystroglycan and beta-dystroglycan subunits. Alpha-dystroglycan acts as a receptor that interacts with extracellular matrix partners, whereas beta-dystroglycan is a transmembrane protein that binds to dystrophin intracellularly and interacts with alpha-dystroglycan extracellularly. Alpha-dystroglycan and beta-dystroglycan interact noncovalently and provide a vital molecular link connecting the extracellular matrix to the internal machinery [
16,
17]. Allelic mutations in dystroglycan-related genes encoding dystroglycan itself or glycosyltransferases and accessory proteins associated with the posttranslational modification of α-dystroglycan disrupt the O-glycosylation of α-DG and result in the loss of α-DG binding to its extracellular ligands, causing muscular dystrophy. Dystroglycanopathies have a wide spectrum of clinical phenotypes, ranging from severe congenital muscular dystrophy to a relatively milder form of LGMD [
18,
19]. Among LGMD-related dystroglycanopathies, LGMD-R9-
FKRP-related is the most frequent in North-European populations, with an estimated prevalence of 5.7–11.4 per million individuals [
20,
21]. Most LGMD-R9 patients harbor at least one copy of the common mutation c.826C > A in the
FKRP gene, and c.826C > A homozygotes manifest a milder phenotype than that of most other genotypes [
22].
The mutational spectrum and prevalence of LGMD subtypes vary greatly among different geographical regions and ethnic populations worldwide. The LGMD-R1-calpain3-related and dysferlinopathy subtypes are the most common in the United States, India and Brazil [
23‐
25], whereas LGMD-R1-calpain3-related and sarcoglycanopathies (LGMD-R3-5/LGMD2C-E) occur most frequently in the Netherlands. LGMD-R9-
FKRP-related exhibits the highest prevalence in some European regions [
26,
27]. To date, integrated data about LGMD epidemiology in the Chinese population are lacking, with only a few single-center studies on the LGMD subtypes in different geographic regions of China [
28‐
30]. Moreover, the frequencies and spectrum of LGMD subtypes in these Chinese studies differed substantially. A definitive molecular diagnosis is important for patient management and participation in clinical trials, prognosis-based counseling and therapy selection.
The precise diagnostic approaches for LGMD subtypes depend on targeted protein immunohistochemistry (IHC) and molecular testing results. Considering the increasing numbers of LGMD subtypes and their corresponding clinical phenotypes, the absence of specific tests to determine the defective proteins in the most recently described subtypes and the time-consuming nature of conventional sequencing technologies for complex LGMD subtypes present a challenge. High-throughput genetic diagnostic technologies, including whole-exome sequencing (WES) and next-generation sequencing (NGS), are widely used to screen mutations in complex genetic diseases and have facilitated high diagnostic efficiency. In this study, we validated the use of targeted NGS and WES for mutation detection in a cohort of Chinese patients clinically suspected of having LGMDs at single center in Southeast China. To better estimate the frequencies and spectrum of LGMD subtypes in Chinese patients, we analyzed detailed clinical phenotypes and variant data in 81 patients from 62 unrelated families. This study aimed to confirm the existence of recurrent mutations in different LGMD subtypes in the Chinese population and to describe the clinical features of these LGMD subtypes. To date, this study is the first of an LGMD cohort from a single center specializing in neuromuscular diseases (NMDs) in Southeast China, and the results expand the clinical phenotypes and genetic profiles of LGMD in Chinese patients.
Discussion
In this study, we comprehensively analyzed the clinical and molecular data of 41 unrelated families with LGMD originating from Southeast China. Our study revealed that LGMD-R2-dysferlin-related and LGMD-R1-calpain3-related were the most common subtypes and that dystroglycanopathies were the most common childhood-onset subtypes. We reported five novel variants in LGMD-related genes, a rare subtype of LGMD-R18-TRAPPC11-related, the frequency of cardiac dysfunction and respiratory involvement and the features of muscle MRI findings in relevant subtypes of LGMDs.
Consistent with previous reports from China, Korea and Japan, the most frequent LGMD subtype identified was dysferlinopathy (approximately 36.3% in our families). This subtype is also frequent in Brazil, with a relative frequency of 31.3% [
40,
41]. However, the frequency of dysferlinopathy is comparatively lower in some European populations, with a relative proportion of 10%–18.7% among LGMD patients [
42]. The second most frequent subtype in our study was LGMD-R1-calpain3-related, accounting for 29.3% of the families, which partially differed from previous reports that LGMD-R1-calpain3-related is the most prevalent form in Eastern, Southern Europe and the US. In Italy, approximately 28.4% of LGMD patients had LGMD-R1-calpain3-related, and the frequency was similar to that in the Netherlands. A previous large-sample study in the US revealed that LGMD-R1-calpain3-related was the most frequently diagnosed subtype (17% of LGMD phenotypes), followed by dysferlinopathy (16%) and LGMD-R9-
FKRP-related (9%). However, in contrast, the most frequent autosomal recessive LGMD was LGMD-R9-
FKRP-related in Denmark and the United Kingdom. The reported frequency of sarcoglycanopathies varied among each region in China, with the highest frequency of sarcoglycanopathies (29.2%) in the South region. In other regions of China, the frequencies of sarcoglycanopathies ranged from 7.7 to 15.4%; however, sarcoglycanopathies were not found in our cohort or in Korean studies. In Europe, sarcoglycanopathies are often considered the second most frequent form, with prevalence rates of 15–18.1% in Italy and 27% in the Netherlands. In the Brazilian population, sarcoglycanopathies are the most frequent cause of severe autosomal recessive LGMD (68%) and childhood-onset subtypes[
43].
In our cohort, dystroglycanopathies were the most common subtypes before 11 years of age. A single-center study from Taiwan revealed that dystroglycanopathies were identified among 23.1% of individuals with LGMD, the same percentage as that for dysferlinopathy, the most common diagnosis, accounting for 50% of subtypes with onset before 11 years of age [
44]. Overall, the most common subtypes in China were LGMD-R2-dysferlin-related and LGMD-R1-calpain3-related; however, the frequency of dystroglycanopathies and sarcoglycanopathies varied greatly among different regions. This phenomenon could be due to the relatively small sample size of previous LGMD cohorts and the different prevalences of some LGMD subtypes among different geographic regions. In addition, the diagnosis of LGMD subtypes in the Northeast region of China was made only according to testing for some mutant defective proteins, and the diagnosis was not further confirmed by genetic screening; therefore, some LGMD subtypes could not be identified.
All of our patients with LGMD-R7-telethonin-related carried the homozygous mutation c.26_33dupAGGTGTCG, except for one with homozygous c.110 + 5G > A mutations. Interestingly, 14% of our families had LGMD-R7-telethonin-related, which has rarely been reported in other LGMD subtype cohorts worldwide. To date, a total of 30 patients with LGMD-R7-telethonin-related have been diagnosed in the Chinese population, and five different pathogenic
TCAP mutations have been identified in these patients [
45,
46]. A previous study in Brazil reported that all of the families with LGMD-R7-telethonin-related were homozygous for a c.157C > T(p.Gln53*) mutation in
TCAP, which has also been detected in Caucasian and Portuguese populations but is not found in the Chinese population [
47,
48]. In addition, the hotspot variants in Europe and South Asia are c.75G > A and c.32C > A, respectively. In China, half of patients with LGMD-R7-telethonin-related carried at least one allele c.26_33dupAGGTGTCG mutation in
TCAP, and 36.7% of patients were homozygous for this mutation.
We analyzed genotype–phenotype correlations with age at onset and clinical severity of disease in those with LGMD-R1-calpain3-related and dysferlinopathy. Our study showed that compared with two missense mutations, one missense/null mutation in
CAPN3 resulted in relatively late onset, but only 4 patients had one missense/null mutation in our cohort, and this association should be investigated in larger patient populations in the future. Previous studies have revealed genotype–phenotype correlations with age at onset in those with LGMD-R1-calpain3-related and dysferlinopathy, with earlier symptom onset in patients with two null mutations than in patients with at least one missense mutation [
49]. Differences in age at onset according to dysferlinopathy genotype were similar to the current results. Our data also revealed that patients with two null mutations tended to have earlier symptom onset than those with at least one missense mutation, although the difference was not statistically significant. Previous Japanese studies have found that the c.2997G > T mutation in the
DYSF gene is associated with relatively late onset [
50,
51]; however, this mutation was not found in our study. Our study also evaluated the effects of null mutations on the clinical severity of dysferlinopathy, but we did not find a significant difference between patients with two null mutations and those with at least one missense mutation. This finding might be because of different disease durations between the two groups. Additionally, intrafamilial variability was observed in dysferlinopathy, suggesting that other additional factors, such as environmental factors and modifier gene(s), also play important roles in the clinical phenotype of the disease. These data suggest that the clinical severity of dysferlinopathy is related to the type(s) of mutation(s), the presence of mutations in residual inner domains, and other unknown modifying factors.
Cardiac involvement is common in many subtypes of LGMDs, which could lead to an increased risk of sudden death [
52]. A total of 22.0% (9/41) of all patients identified in our study had cardiac dysfunction. The ECG findings of our patients included conduction defects (right bundle branch block and intraventricular block), arrhythmias (atrial flutter and sinus arrhythmia), and early repolarization syndrome. Cardiac complications are the most common causes of death in
LMNA-related muscular dystrophies and were found in only one patient with
LMNA-related muscular dystrophy in this study [
53]. This event corroborated previous reports that cardiac involvement in patients with
LMNA mutations is more frequent and severe than in patients with other types of muscular dystrophies. Another patient with LGMD-R9-
FKRP-related showed a right bundle branch block, which is not an uncommon finding in the population and is not directly related to the diagnosis of cardiomyopathy. However, her CMR indicated cardiomyopathy involving fatty infiltration of the subepicardial layer of the LV. Therefore, CMR can be used to detect early cardiac abnormalities in patients with LGMDs, monitor disease progression and assess treatment effects during follow-up. Furthermore, early diagnosis is necessary for the timely commencement of cardiac management, and therapeutic interventions are appropriate for patients with severe cardiac arrhythmias.
A total of 15.4% of all patients experienced restrictive respiratory insufficiency, but none required nocturnal noninvasive ventilation, which warrants longer follow-up. Previous studies revealed that 80% of patients with LGMD-R9-
FKRP-related experienced respiratory impairment, and 3%–45% required assisted ventilation [
54,
55]. One of two patients with LGMD-R9-
FKRP-related in our study had relatively severe respiratory insufficiency and cardiac involvement. Our findings support that cardiorespiratory dysfunction is a frequent complication of LGMD-R9-
FKRP-related. Respiratory impairment was not observed in our patients with other subtypes of dystroglycanopathies, suggesting that respiratory involvement could be more frequent in LGMD-R9-
FKRP-related than in other dystroglycanopathy subtypes. In addition, we reported that a rare subtype, LGMD-R18-
TRAPPC11-related, presented as severe respiratory impairment. Although no correlation between muscle weakness and respiratory insufficiency was observed in previous reports, patients with LGMD-R9-
FKRP-related and LGMD-R18-
TRAPPC11-related with respiratory involvement showed a relatively severe phenotype with walking aid dependency. This finding requires further investigation in larger numbers of patients with LGMD subtypes at greater risk for developing respiratory complications. It is important for clinicians to be aware of symptoms of ventilatory insufficiency in patients with LGMDs and to monitor pulmonary function during follow-up.
Age at onset was predominantly in childhood in patients with dystroglycanopathies, LGMD-R10-
TTN-related and
LMNA-related muscular dystrophy and in childhood or adolescence in those with LGMD-R1-calpain3-related, dysferlinopathy, LGMD-R7-telethonin-related and LGMD-R18-
TRAPPC11-related. A loss of ambulation was observed in one patient with
IPSD-related dystroglycanopathy at the early age of 21, suggesting that
IPSD-related dystroglycanopathy phenotypes are relatively severe. Previous studies revealed that patients with LGMD-R18-
TRAPPC11-related had infantile-onset or early childhood onset and presented with progressive muscle weakness and extramuscular manifestations, including motor delay, ataxia, cataracts, liver disease, and intellectual disability [
56,
57]. Our patient with LGMD-R18-
TRAPPC11-related had a relatively late onset of 19 years and presented with a myopathic syndrome and signs of foot drop associated with fatty liver and diabetes.
We performed muscle MRI imaging on 28 patients with different subtypes of LGMD. LGMD-R1-calpain3-related and LGMD-R2-dysferlin-related exhibited clinical similarity in some aspects; however, some features of muscle MRI patterns can help distinguish these two subtypes. The patients with LGMD-R1-calpain3-related showed more severe fatty infiltration of the posterior thigh muscles than those with LGMD-R2-dysferlin-related, consistent with the findings of a previous report. However, there were no differences in the fatty infiltration of the anterior thigh muscles between patients with LGMD-R1-calpain3-related and those with LGMD-R2-dysferlin-related, although fatty infiltration of these muscles was previously reported to be less severe in patients with LGMD-R1-calpain3-related [
58]. Furthermore, muscle edema was more frequent and severe in the lower legs of patients with LGMD-R2-dysferlin-related than in patients with LGMD-R1-calpain3-related. The edema pattern in LGMD-R2-dysferlin-related was rather heterogeneous and prominent and involved different compartments of the lower legs. In contrast to patients with other subtypes, muscle imaging in our patient with LGMD-R9-
FKRP-related indicated more severe fatty infiltration of the peroneus longus muscle and extensor digitorum longus, while the tibialis anterior muscle was relatively spared. According to previous literature, a specific pattern of muscle imaging in LGMD-R9-
FKRP-related could facilitate differential diagnosis from other LGMDs [
59]. In addition, muscle imaging in patients with LGMD-R18-
TRAPPC11-related revealed an advanced stage pattern that has not been previously reported, and more patients must be enrolled for further analysis.
In conclusion, we described the detailed clinical phenotypes, muscle imaging findings, and genetic spectrum of an LGMD cohort of 50 patients in 41 families. We comprehensively analyzed the frequency of LGMD subtypes in different regions in China, which revealed that LGMD-R2-dysferlin-related and LGMD-R1-calpain3-related were the most common subtypes. We described the distinct characteristic patterns of muscle imaging in different subtypes of LGMDs, which may improve the differential diagnoses of the diseases. We reported a rare subtype of LGMD, recurrent mutations and novel pathogenic mutations, which contributed to expanding the clinical phenotype and genetic spectrum of LGMD. Cardiac involvement and respiratory insufficiency are common complications in many subtypes of LGMDs, and it is essential to regularly screen cardiac and pulmonary function in the future management of LGMDs. These results improve our understanding of the epidemiology of different LGMD subtypes in China, facilitating diagnostic processes, allowing for the timely provision of cardiorespiratory treatment and benefitting future clinical trials for LGMDs.
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