Clinical and genetic spectrum of sarcoglycanopathies in a large cohort of Chinese patients

Of 3638 patients who underwent muscle biopsy for a suspected neuromuscular disorder (1733 with inherited myopathies, 1557 with acquired myopathies, and 348 unknown) at Peking University First Hospital from January 2013 to August 2018, 756 patients highly suspected of inherited myopathies had next-generation sequencing (NGS) diagnostic panel covering all exons and flanking sequences of genes known to be associated with inherited neuromuscular diseases (Additional file 1: Table S1) according to the following inclusion and exclusion criteria. Inclusion criteria: 1) clinically presented with muscle weakness verified by muscle-strength examination, delayed motor milestones, muscle pain, or exercise intolerance; 2) muscle biopsy showing (1) dystrophic or myopathic changes, i.e., the presence of degenerated and regenerated muscle fibers, with or without variation in fiber size, proliferation of connective tissue, and/or (2) immunohistochemical staining or western blot results showing either decreased expression or accumulation of muscle related proteins; 3) agreed to provide DNA samples for NGS. Exclusion criteria: 1) clinical, histopathologic, and/or genetic diagnosis of facioscapulohumeral muscular dystrophy or myotonic muscular dystrophy; 2) deletion/duplication of exons detected in the DMD gene using multiplex ligation-dependent probe amplification (MLPA) assay; 3) muscle biopsy and genetic confirmation of mitochondrial myopathy, glycogen storage myopathy, or lipid storage myopathy; 4) muscle biopsy confirmation of normal histological appearance without any specific pathological findings [13]. Of the 441 patients showing varying reduction of sarcoglycans with or without reduction of dystrophin on muscle biopsy, 25 were confirmed to have the primary genetic defect in SGCA, SGCB, and SGCG, 2 were confirmed to have the primary genetic defect in FKRP, and 392 were confirmed to have the primary genetic defect in DMD. The primary genetic defect in the remaining 22 patients remained unclear. A total of 218 patients were diagnosed with LGMD based on their clinical manifestations, muscle biopsy results, and genetic analysis, 25 of which were diagnosed with sarcoglycanopathies. Eighteen of these 25 patients were confirmed to have LGMD2D, 6 to have LGMD2E, and one to have LGMD2C, with these patients originating from 12 separate provinces in China (Additional file 2: Figure S1). The proportion of different LGMD subtypes was shown in Additional file 3: Figure S2. Clinical characteristics at the time of diagnosis were evaluated by review of medical records and a detailed physical examination. Walking ability was graded from 1 to 5 according to the scoring system devised by Tasca et al. [4]. Muscle strength was evaluated by manual muscle testing and graded according to Medical Research Council.

Genomic DNA was extracted using standard procedures from peripheral blood samples or muscle tissues taken from all patients. Sequence variants were detected by NGS diagnostic panel (Additional file 1: Table S1). Sanger sequencing with specific primers was performed to confirm the variants detected by NGS. In patients who had large deletion or large duplication variants detected by NGS, we further performed MLPA assay (patients 10, 11, and 15) or fluorescence quantitative polymerase chain reaction (patient 19) to confirm these variants. MLPA was also performed in four patients with only one mutation identified in SGCA or SGCB to rule out deletions/duplications on the other allele. Variants were described according to the Human Genome Variation Society (HGVS) nomenclature using nucleotide and amino acid numbering based on published coding DNA reference sequences (SGCA, NM_000023.2; SGCB, NM_000232.4; SGCG, NM_000231.2; and PMP22, NM_000304.2) and protein reference sequences (SGCA, NP_000014.1; SGCB, NP_000223.1; SGCG, NP_000222.1; and PMP22, NP_000295.1).

When interpreting and classifying a sequence variant in our study population, we checked to see if it has been previously reported as a pathogenic variant in the Human Gene Mutation Database [16], ClinVar [17], and Google Scholar [18]. Each novel sequence variant was classified as pathogenic, likely pathogenic, uncertain significance, likely benign, or benign according to the rules specified in the 2015 American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG-AMP) guidelines [19].

When assessing the frequencies of variants in large populations, 100 healthy control participants (100HC) of Chinese origin were screened, and we also checked for allele frequencies in the Genome Aggregation Database (gnomAD) [20], NHLBI Exome Sequencing Project (ESP6500) Exome Variant Server [21], 1000 Genomes Project (TGP) [22], and Exome Aggregation Consortium (ExAC) [23]. The evidence for pathogenicity was deemed to be moderate (PM2) for variants that were absent or present at extremely low frequencies with alternative allele frequency < 0.5% [24] in population databases. Multiple pieces of computational evidence were derived from various in silico analyses where the FATHMM [25], Mutation Taster [26], PolyPhen-2 [27], and SIFT [28] were used to predict deleteriousness and GERP [29] was used to assess evolutionary conservation. The splicing impact of a variant spanning the exon and intron region was inferred by the Human Splicing Finder (HSF) [30]. Segregation analysis of the variants was performed in available family members. We used the wInterVar tool [24] to automatically generate predictions on 6 (PS1, PM1, PM5, PP2, BP1, BP7) of 28 criteria specified in the 2015 ACMG-AMP guidelines; the rest were interpreted by manual review and adjustment on the basis of variants’ detailed information (such as a variant’s de novo status) and our own domain knowledge. These criteria were then combined to arrive at a final interpretation.

The muscle biopsies were evaluated and rated by two independent evaluators (WZ and YY), both of whom were experienced in interpretation of muscle biopsies and muscle immunoanalysis and blinded to the underlying genotypes of the patients. Muscle biopsies were obtained from quadriceps femoris (patients 6 and 8), gastrocnemius (patients 4 and 11), tibialis anterior (patients 10, 16 and 20), or biceps brachii (patients 1–3, 5, 7, 9, 12–15, 17–19, and 21–25, and the normal control subjects). The muscle specimens were frozen in isopentane, cooled in liquid nitrogen, and then stored at − 80 °C. Routine histological and histochemical staining was performed [31] and standard techniques were used for immunohistochemical staining [32]. Primary antibodies against the following proteins were used: α-SG, β-SG, and γ-SG (all from Leica Biosystems Newcastle Ltd., Newcastle upon Tyne, UK). Protein expression on sections was scored according to the intensity of staining of the sarcolemma as follows [12]: score 1, normal (complete staining of all fibers); score 2, slight reduction (partial or incomplete staining of a few fibers); score 3, reduction (between severe reduction and slight reduction); score 4, severe reduction (partial or incomplete staining of most fibers); score 5, absence (absence of staining of cell membrane). The genotype was predicted on the basis of the rule that the SG (α, β, or γ) with the most severely reduced expression was the one primarily affected; if there was a similar reduction in two or three of the SGs, prediction was considered impossible.

The Shapiro-Wilk test was used to confirm that the measured variables were not normally distributed. The median patient age, age at onset, disease duration, and muscle strength were treated as descriptive statistics. Hierarchical analysis and graphical representation of the muscle strength values in the form of a heatmap were performed using R software version 3.1.3 (The R Foundation for Statistical Computing, Vienna, Austria; http://​www.​r-project.​org). The software established the order of the patients and muscle strength in the heatmap automatically and generated dendrograms that linked patients or muscles with similar involvement. Mann-Whitney U tests were used to compare the main clinical characteristics (age at onset, disease duration, CK value, and disease severity) between patients with LGMD2D and those with LGMD2E. A two-tailed Pearson correlation coefficient (r) was used to analyze the relationship between the main clinical characteristics and the degree of SG protein deficiency. Positive and negative Pearson’s correlations were considered statistically significant if the P-value was < 0.01. The statistical analyses were performed using SPSS for Windows version 22.0 (IBM Corp., Armonk, NY, USA).

The clinical details of the patients with sarcoglycanopathies were listed in Table 1. Muscle involvement and disease severity determined by hierarchical analysis were shown in Fig. 1. Patients with LGMD2D or LGMD2E did not cluster according to their molecular diagnosis but rather to the severity of muscle involvement. The patients were divided into four subgroups according to the results of the hierarchical analysis, i.e., hyperCKemia without muscle weakness (n = 7) and hyperCKemia with muscle weakness that was mild (n = 5), intermediate (n = 7), or severe (n = 6). There was no significant difference in age at onset, disease duration, CK value, or disease severity between the patients with LGMD2D and those with LGMD2E (P = 0.545, 0.739, 0.386, and 0.836, respectively). Therefore, the clinical characteristics of the patients with LGMD2D and LGMD2E were summarized together.

The median patient age was 10.1 (3.2–27.4) years, the median age at onset was 4.5 (0.8–11) years, and the median duration of disease at the time of diagnosis was 4.6 (0.7–16.4) years. In 16 patients (66.7%), the symptoms at the time of disease onset were associated with proximal lower limb weakness and included early fatigue, frequent falls, gait abnormalities, delayed motor milestones, exercise intolerance, and difficulty in running, climbing and jumping; in 6 patients (25.0%), the symptom at onset was post-exercise muscle pain without muscle weakness. Two patients (8.3%) were diagnosed to have sarcoglycanopathy after an incidental finding of hyperCKemia. Four patients were no longer able to ambulate independently at a median age of 18.2 (range 12–26.4) years. Motor signs included calf hypertrophy (in 54.2% of patients), tendon contractures (in 33.3%), and scapular winging (in 12.5%). Muscle pain was reported by 29.2% of patients. Physical examination revealed that 17 patients (70.8%) had proximal weakness involving the axial, pelvic, and shoulder girdle muscles, and that 7 patients (29.2%) had asymptomatic hyperCKemia or exercise-induced myalgia without muscle weakness. The distal muscles were affected in 5 patients (20.8%), all of whom had a severe disease severity. The hip and neck flexors and hip adductors were the muscle groups most often involved and the plantar flexors were the least often involved.

The patient with LGMD2C in this study (patient 25) had a severe disease severity and was no longer able to ambulate independently by the age of 18 years, and was diagnosed to have sarcoglycanopathy because of frequent falls. In this patient, physical examination revealed proximal and distal muscle weakness as well as tendon contractures and scapular winging.

CK levels were elevated in all patients (345–35,120 IU/L, normal range 25–195 IU/L). Nerve conduction study of patient 1 revealed that the motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) reduced severely in all nerves examined, and the compound muscle action potential (CMAP) amplitude and sensory nerve action potential (SNAP) amplitude decreased in some of the nerves examined (Additional file 4: Table S2). Concomitant mutations in SGCA and PMP22 were confirmed by genetic analysis in patient 1; in this patient, the diagnosis was coexistence of LGMD2D and Charcot-Marie-Tooth 1A (CMT1A).

In total, 35 mutations were identified in SGCA (n = 26), SGCB (n = 7), SGCG (n = 1) and PMP22 (n = 1), 19 of which have been previously reported as pathogenic [2, 13, 33‐42] and the remaining 16 were novel (Table 3). Twenty-one patients (16 with LGMD2D, 4 with LGMD2E, and one with LGMD2C) had a complete molecular diagnosis and were found to have two mutations in SGCA, SGCB or SGCG and 4 (2 with LGMD2D and 2 with LGMD2E) were found to have only one mutation in SGCA or SGCB. In patient 1, we also identified a previously reported mutation in PMP22 (duplication of exons 1–5) [41, 42] in addition to the mutations identified in SGCA. The allele frequencies in various population databases, results of in silico analysis and clinical interpretation of novel candidate variants detected in SGCA, SGCB and SGCG according to the 2015 ACMG-AMP guidelines were summarized in Tables 2 and 3. Except for the missense variant c.218C > T in SGCA (0.00000815 in gnomAD) and the missense variant c.320C > T in SGCG (0.00000813 in gnomAD, 0.000008 in ExAC), none of the novel candidate variants were detected in the various population databases or in the 100 healthy control subjects. Not all of the in silico programs tested agreed on the prediction of the missense variant c.956G > A in SGCA, so PP3 evidence was not used when classifying this variant. When interpreting and classifying other novel candidate variants that could be predicted by the in silico programs, PP3 evidence was counted as supporting because all of the in silico programs tested agreed on the prediction. After combining the criteria specified in the 2015 ACMG-AMP guidelines [19], all 14 novel candidate variants were classified as pathogenic or likely pathogenic. Spectrum and location of mutations in SGCA, SGCB and SGCG were shown in Fig. 2.

The results of muscle biopsy and immunohistochemistry analysis were summarized in Fig. 3 and Additional file 5: Table S3. The majority of muscle biopsy specimens (76.0%) showed a dystrophic pattern, i.e., increased variation in fiber size, proliferation of connective tissue, and necrotic and regenerated fibers. The muscle biopsies in 6 patients with LGMD2D who had a mild form of disease severity showed mild myopathic changes, including a few hypertrophic, atrophic, hypercontracted and whorled fibers, as well as fiber splitting and a small number of internal nuclei. We were able to correctly predict the genotype in 36.0% of the patients according to α-, β-, or γ-SG that was most reduced on the sections. In 52.0% patients, it was impossible to predict the genotype because there was a similar reduction in expression of two or three of α-, β-, and γ-SG. Furthermore, the prediction was incorrect in 12.0% of patients.

In the patients with LGMD2D, there was variable reduction in expression of α-SG, ranging from a slight decrease to absence, except in patient 17, in whom expression of α-SG was positive and that of β-SG was slightly reduced. Expression of β-SG and γ-SG was also affected, with varying degrees of deficiency in patients with LGMD2D, except for 3 patients. In 14 patients, expression of α-SG was found to be similarly or more severely reduced than the expression levels of β-SG and/or γ-SG; in 3 patients, the most pronounced reduction was in β-SG. In the patients with LGMD2E, β-SG was absent in all but one case (patient 19), and expression levels of α-SG and γ-SG were reduced to varying degrees. The expression of β-SG was found to be more decreased than that of α-SG and γ-SG in 4 patients and reduced to a similar extent of α- and/or γ-SG in 2 patients. There was a concomitant absence of α-SG, β-SG, and γ-SG in one patient with LGMD2D (patient 1) and in one with LGMD2E (patient 22). In the patient with LGMD2C, the expression levels of all three SGs were decreased, particularly for γ-SG.

No statistically significant correlations were found between age at onset, duration of disease, CK value, and disease severity in the patients with LGMD2D or LGMD2E. There was a statistically significant positive correlation of reduction of the α-SG level with disease severity in the patients with LGMD2D (r = 0.689, P = 0.002), indicating that the greater the amount of residual protein, the milder the disease. This correlation was not found in patients with LGMD2E.