Clinicopathological‐genetic features of congenital myasthenic syndrome from a Chinese neuromuscular centre

Abstract Congenital myasthenic syndrome (CMS) encompasses a heterogeneous group of inherited disorders affecting nerve transmission across the neuromuscular junction. The aim of this study was to characterize the clinical, physiological, pathohistological and genetic features of nine unrelated Chinese patients with CMS from a single neuromuscular centre. A total of nine patients aged from neonates to 34 years were enrolled who exhibited initial symptoms. Physical examinations revealed that all patients exhibited muscle weakness. Muscle biopsies demonstrated multiple myopathological changes, including increased fibre size variation, myofibrillar network disarray, necrosis, myofiber grouping, regeneration, fibre atrophy and angular fibres. Genetic testing revealed six different mutated genes, including AGRN (2/9), CHRNE (1/9), GFPT1 (1/9), GMPPB (1/9), PLEC (3/9) and SCN4A (1/9). In addition, patients exhibited differential responses to pharmacological treatment. Prompt utilization of genetic testing will identify novel variants and expand our understanding of the phenotype of this rare syndrome. Our findings contribute to the clinical, pathohistological and genetic spectrum of congenital myasthenic syndrome in China.

be the first-choice pharmacological strategy for treating CMS with DOK7 and could be a supplementary treatment for CMS with CHRNE mutations. 9,10 Specifically, AChE inhibitors should also be avoided in patients with CMS due to AChE deficiency, such as CMS with COLQ mutations or DOK7 mutations. [10][11][12] There is an ever-expanding panel of mutations associated with CMS. There are several case reports of Chinese CMS patients, and one study described 35 CMS patients from the northern part of China. 13 In this study, we describe nine patients from the southern part of China diagnosed with CMS to expand the clinical and pathological spectrum of CMS.

| Ethics approval and patients
With the approval of the Ethics Committee of Xiangya Hospital, Central South University, a total of nine Chinese patients in the Neuromuscular Center of Xiangya Hospital of Central South University from 2015 to 2020 were recruited, as mentioned in previous research. 14,15 The diagnosis of CMS was independently established by at least two neurologists according to clinical manifestations, pathological changes, EMG and genetic mutations.

| Serum antibody test
Serum samples were routinely tested by the DAAN Clinical Laboratory Central (Guangzhou, China) using an enzyme-linked immunosorbent assay (ELISA) method, and titres >0.45 nmol/L for anti-AChR antibody and >9.5 pmol/L for anti-MuSK antibody were defined as positive.

| Electromyography
All repetitive nerve stimulation (RNS) tests were performed at rest and with low-frequency (3 Hz) and high-frequency (20 Hz) stimulation in the following nerves: facial nerve, accessory nerve, peroneal nerve, spinal accessory nerve, nervus peroneus communis, median nerve, nervus tibialis, sural nerve and ulnar nerve. A decremental response in amplitude at the fourth potential that was higher than 10% compared with the first potential was considered abnormal. 16 Other EMG signals, such as spontaneous muscle activity or compound muscle action potential (CMAP), were also recorded.

| Biopsies and pathological examination
Open muscle biopsies were taken from the left biceps brachii muscles in all patients, except for patient 1, whose open muscle biopsy was taken from the left gastrocnemius muscles. The muscle tissue was immediately frozen in isopentane cooled with liquid nitrogen and stored at −80°C. Immunohistochemical staining was performed as described elsewhere with minor modifications. [17][18][19][20] Briefly, histological and immunohistochemical analyses were performed on 5 μm thick sections using a cryostat. Routine histological staining of muscle sections was performed using haematoxylin and eosin (HE), modified Gömöri trichrome, acid phosphatase, periodic acid-Schiff, oil red O (ORO), nicotinamide adenine dinucleotide dehydrogenasetetrazolium reductase (NADHTR), adenosine triphosphatase (ATPase) (pH 4.3, 4.6, 11.0), succinic dehydrogenase (SDH) and cytochrome C oxidase.

| Genetic testing
Genetic testing was performed as we previously reported with minor modifications. 21

| Demographic and clinical features
In our neuromuscular centre, we retrospectively screened patients diagnosed with CMS from 2015 to 2020 and found nine patients with CMS who were enrolled. All patients were Han Chinese and were from the southern part of China. All patients were male, and the median age of onset, disease duration and follow-up duration were 8 years (interquartile range, 5-18), 7 years (interquartile range, 3-10) and 12 months (interquartile range, [9][10][11][12][13], respectively ( Table 1).
All the patients denied having any family history of muscular disorders. All patients were ambulatory. All patients presented with varying degrees of limb weakness. Eight patients exhibited ptosis, five patients had facial muscle weakness, two patients presented with dysphagia and one patient had tachycardia. Two patients had an absent tendon reflex, and two had a decreased tendon reflex. Six patients were positive for the Gowers sign.
More details regarding the demographic and clinical features are shown in Table 1.
Except for patient 8, who lacked serum creatine kinase, the levels of creatine kinase were normal (20-170 U/L) in four patients, while the other four patients had low to moderately increased creatine kinase (Table 2). Specifically, one patient with GMPPB mutations had the highest creatine kinase at 2696 U/L ( Table 2). All patients were AChR antibody-negative. In addition, patients 1, 4 and 7 were also tested for MuSK antibody titres, and all three were seronegative for MuSK antibody.

| Electromyography
Electromyography revealed myopathic changes in seven patients (7/9) and a decremental response to RNS (7/9) ( Table 2). Except for patient 9 (an SCN4A-CMS patient), who showed a decremental response to high-frequency (20 Hz) RNS, all the other patients showed a decremental response to low-frequency (3 Hz) RNS. One patient (patient 8) with a PLEC mutation exhibited normal EMG. Due to technical limitations, single fibre electromyography (SFEMG) was not available at our centre.

| Genetic testing and mutations
Mutation analysis of 1546 genes known to be associated with hereditary neuropathies and myopathies was performed for all patients.

| Response to treatment
Pyridostigmine was the most commonly used medication. All patients were prescribed pyridostigmine, and two patients (2/9), including patient 1 and patient 9, showed a slight benefit. Two patients (patients 6 and 8) had moderate improvement in response to pyridostigmine, and three patients (patients 3, 4 and 5) displayed strong improvement with pyridostigmine. Patient 7, with PLEC mutations, did not respond to pyridostigmine but showed moderate TA B L E 1 Clinical features and physical examination of the nine patients with CMS

| DISCUSS ION
In the current study, we report the clinical and genetic findings of   34 Patients with CMS who have AGRN mutations respond quite differently to albuterol and pyridostigmine. CMS with AGRN mutations usually respond to albuterol but not to pyridostigmine. 35 In our two patients, one responded to pyridostigmine, and one worsened, suggesting that pyridostigmine should be avoided in CMS patients with AGRN mutations.
The CHRNE gene encodes the epsilon subunit of AChR.
Mutations in CHRNE fall into two groups: kinetic mutations with or without minor AChR deficiency and low-expressor mutations with or without minor kinetic effects, which are also called primary AChR deficiency. Kinetic mutations consist of two classes: slow-channel syndromes and fast-channel syndromes. 36 Patient 3 had been diagnosed with myasthenia gravis (MG) for several years and was prescribed other immunosuppressors, such as prednisolone and azathioprine. However, the treatment effect was not satisfactory, and genetic testing was conducted. After the genetic diagnosis was established, we prescribed pyridostigmine, and the patient had a good response.
GFPT1 is the rate-limiting enzyme in the hexosamine biosynthetic pathway, which is indispensable for protein and lipid glycosylation. Mutations in GFPT1 cause CMS characterized by fatigable muscle weakness owing to impaired neurotransmission. The precise pathomechanisms at the NMJ due to GFPT1 deficiency have yet to be discovered. 37 In our study, the two missense mutations in the GFPT1 mutant patient were previously reported. [22][23][24] Previously reported patients with the GFPT1 mutation p. R111C began having difficulty walking and climbing stairs in the second decade of life. 24 The p. R111C mutation has no effect on the enzymatic activity of GFPT1. 22 The reported patient with the p. R212Q mutation had an onset at 14 years old and presented with tubular aggregates under a light microscope. 23 These patients with previously reported mutations all benefited from pyridostigmine and derived additional benefit from 3,4-DAP and salbutamol. Our patient also exhibited a good response to pyridostigmine, suggesting that pyridostigmine may be the first choice for CMS patients with these specific mutations.
GMPPB encodes the enzyme GDP-mannose pyrophosphorylase B, which catalyses the conversion of mannose-1-phosphate and GTP to GDP-mannose, 38  Treatment with pyridostigmine has been reported to be effective in these patients. 40 Skeletal muscle sodium channelopathies due to SCN4A gene mutations have a broad clinical spectrum. 47 CMS is a rare phenotype of SCN4A mutations. 48 Several patients with recessive mutations in SCN4A were diagnosed with CMS/congenital myopathy. [48][49][50] Further investigations are required to understand the mechanism of fatigue in CMS/congenital myopathy caused by mutations in SCN4A.
Since no symptoms or signs of myotonia or paralysis were found,  10 and COLQ 54 , and should be carefully prescribed for AGRN-CMS. 13 The most common misdiagnosis in our cohort of CMS was MG. Seronegative MG patients unresponsive to immunosuppressive treatment should always be re-evaluated for CMS. Another common misdiagnosis for CMS is myopathy, especially limb girdle muscular dystrophy (LGMD) since they also have elevated serum creatine kinase levels and myopathic changes in the EMG.
LGMD-CMSs are usually caused by DOK7, GFPT1, ALG2 and ALG14 mutations. The presence of fluctuations, daily or over longer time periods, should raise a suspicion of CMS, even in patients previously diagnosed with myopathy, including LGMD, and careful electrophysiological studies are needed, including a search for repetitive CMAP and single fibre EMG if the RNS studies are negative. 55 Electrophysiological tests are very helpful for the differential diagnosis of CMS. Muscle biopsy usually discloses generally nonspecific findings, which play a limited role in differential diagnosis. Genetic testing should be considered for differential diagnosis. In our cohort, RNS revealed a ≥ 10% decrease in most patients (7/9). The differentiation of CMS subtypes, which is usually very difficult clinically, can be achieved using molecular genetic tests. Whole-exome sequencing should be considered in patients suspected of having CMS.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original data that described in this study are available from the corresponding author upon reasonable request.