1 INTRODUCTION

Congenital myasthenia syndrome (CMS) includes a large group of rare inherited endplate myopathies characterized by dysfunctions of neuromuscular junction transmission due to genetic defects (Engel et al., 2015; Finsterer, 2019). CMS shows great clinical and genetic heterogeneities characterized by abnormal fatigability, transient or permanent muscle weakness with varied age of onset. The main inheritance pattern of this disease is autosomal recessive, but a small part is inherited in autosomal dominant mode. There are at least 32 kinds of genes that have been identified in CMSs, while the number is still being updated (Iyadurai, 2020). Mutations in CHRNA1, CHRNB1, CHRND, or CHRNE are the most causative genes accounting for more than 30% of the cases, while mutations in RAPSN, COLQ, and DOK7 involve about 10% to 15% of the cases, and GFPT1 is accountable to approximately 3% of the cases (Engel et al., 2015; Finsterer, 2019).

Among the various types of CMS, the limb-girdle form is characterized by a muscle weakness and fatigability predominant in proximal muscles with minor or no involvement of ocular, facial, and bulbar muscles (Belaya et al., 2012). Mutations in the glutamine-fructose-6-phosphate transaminase 1 (GFPT1, Online Mendelian Inheritance in Man [OMIM]:138292) gene encoding a ubiquitous enzyme for biosynthesis pathway of protein glycosylation are responsible for a specific CMS subtype called limb-girdle myasthenia with tubular aggregates (OMIM:610542) (Huh et al., 2012; Senderek et al., 2011). Although genetic screening may be conveniently available for these patients through next-generation sequence (NGS), muscle biopsy is typically the first assessment conducted in these CMS patients who predominantly present with limb-girdle muscle weakness. Considering that some subtypes of CMS may be treatable genetic diseases, it is very important to make a timely diagnosis as early as possible (Farmakidis et al., 2018). Therefore, accurate identification of the various myopathological changes is very important to the diagnosis of CMS. However, not all patients show tubular aggregates in muscle biopsy (Guergueltcheva et al., 2012), suggesting a need to re-recognize and summarize the diversity of muscle pathology in patients with CMS associated with GFPT1 mutations.

In this study, we described two CMS patients with GFPT1 mutations: one presented with vacuolar myopathy with myofibrillar destruction, and the other showed typical myopathy with tubular aggregates. To further explore the pathological characteristics of CMS caused by GFPT1 mutation, we summarized the muscle pathological features in all reported GFPT1-related CMS cases with muscle biopsy.

2 MATERIALS AND METHODS 2.1 Subjects

Patients with GFPT1 mutations were recruited from our in-home database including 15 patients with CMS between January 2016 and June 2021. The inclusion criteria of CMS included fatigable muscle weakness presenting with ptosis, ophthalmoparesis, facial and bulbar, and generalized muscle involvement; positive changes of neuromuscular junction in electrophysiological assessments; and/or causative mutations in CMS-related genes. A battery of clinical and laboratory investigations were conducted to exclude the inflammatory, toxic, or metabolic origins. A detailed medical history was obtained from the subjects and their relatives. Information regarding age of onset, progression of disease, family history, and other clinical manifestations was collected. Electrophysiological study was performed in the nerves using a standard method with surface electrodes for stimulation and recording.

2.2 Ethical statement

All patients’ tissue samples were obtained after a written consent signed by each individual in compliance with the bioethics laws of China as well as the Declaration of Helsinki. The research was approved by ethics committee of the first affiliated hospital of Nanchang University.

2.3 Genetic screening

Genomic DNA was extracted from peripheral blood samples. The NGS was commercially supported by Running Gene Inc. (Beijing, China). In brief, targeted exon enrichment was performed using SureSelect Human All Exon V5 (Agilent Technologies). The exon-enriched DNA libraries were subjected to paired-end sequencing with the Hiseq 2000 platform (Illumina, Inc.). Sequence data were mapped with BWA (Li & Durbin, 2009) and SAMTOOLS (Li et al., 2009) onto the hg19 human genome as a reference. Calls with variant quality less than 20 were filtered out, and 95% of the targeted bases were covered sufficiently to pass our thresholds for calling single nucleotide polymorphisms (SNP), nonsynonymous/splice acceptor and donor site, insertions or deletions (NS/SS/InDel) variants in the dbSNP v137, ESP6500, and 1000 Genome were removed. Synonymous changes were filtered using SIFT software (http://sift.jcvi.org). Sanger sequencing with specific primers was conducted to confirm the GFPT1 mutation in the patients and their available family members.

2.4 Muscle pathological examination

Muscle biopsies were performed from the right bicep or left gastrocnemius of the two cases, respectively. The muscle tissue was frozen and then cut at 8 μm sections. These sections were stained according to standard histological and enzyme histochemical procedures with hematoxylin and eosin (H&E), modified Gomori trichrome (MGT), periodic acidic Schiff (PAS), oil red O (ORO), nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), succinate dehydrogenase (SDH), cytochrome c oxidase (COX), nonspecific esterase (NSE), and ATPase stain. Antibodies of desmin (Abcam, ab6322, 1:100), dystrophin (Leica Biosystems, NLC-DYS2, 1:20), dysferlin (Leica Biosystems, Ham1/7B6, 1:40) and MHC-I (Dako, R7000, 1:200) were used to detect the distribution of protein in the muscle specimens by immunohistochemical stain.

For electron microscopy, muscle specimens were fixed in 2.5% glutaraldehyde in phosphate buffer and post-fixed in 1% osmium tetroxide in the same buffer. Specimens were then dehydrated and embedded in Epon 812. The ultrathin sections of muscle tissue were double stained with uranyl acetate and lead citrate, and then examined with an electron microscope (JEM-1230 JEOL Inc. Tokyo, Japan).

2.5 Literature review

We searched the literature in multiple databases including PubMed, EMBASE, Scopus, Web of Science, EBSCO, and Google Scholar database using the keywords “congenital myasthenia syndrome” and “GFPT1 gene.” All included cases were required to have muscle biopsy, then the clinical characteristics, laboratory results, treatments, complications and outcomes of all patients were summarized and reanalyzed.

3 RESULTS 3.1 Clinical features 3.1.1 Patient one

The patient was a 37-year-old man who had limb weakness for more than 30 years. At age 5, the patient has noticed poor ability in walking and running compared to his peers. At age 15, he had difficulty in standing up after squatting, and frequently falling down. The symptom of muscle weakness was better in the morning, but worse in the evening. He was diagnosed with myasthenia gravis. Corticosteroid was initially administered, but no efficacy was observed. Afterward, he showed some responses to pyridostigmine, while the muscle weakness gradually progressed to walking difficulty and bath inability. His family history was unremarkable.

Physical examination on admission revealed symmetrical limb weakness without facial, bulbar, neck, and respiratory muscle involvement. Muscle strength (Medical Research Council, MRC) was 4 grade in the proximal upper limbs, 5- grade in the distal upper limbs, 3 grade in the proximal lower limbs, and 4 grade in the distal lower limbs. Deep tendon reflexes were decreased. Pathological reflexes were negative. No muscle atrophy or fasciculation could be observed. There was no evidence of sensory disturbance, ataxia, or autonomic dysfunction.

The blood count, blood biochemistry, thyroid function, parathyroid hormone, blood acylcarnitines and urine organic acid profiles, paraneoplastic antibody spectrum, and antibodies of myasthenia gravis were all negative. Muscle MRI of thigh revealed a slightly diffused hyperintensity on T1WI except adductor magnus and semimembranosus muscles; and muscle MRI of leg also showed a slightly diffused hyperintensity except medial gastrocnemius (Figure 1a). Nerve conduction velocity (NCV) and electromyography (EMG) were not obvious abnormal. Nevertheless, repetitive nerve stimulation (RNS) at 3 Hz revealed positive decrements of compound muscular action potential (CMAP) in the deltoid.

Muscle MRI changes of lower limb in patient one (a) and patient two (b) with GFPT1-related CMS. Thigh level showed a slightly diffused hyperintensity on T1WI except adductor magnus and semimembranosus muscles; leg level showed a slightly diffused hyperintensity except medial gastrocnemius that simultaneously had mild high-signal on STIR

3.1.2 Patient two

The patient was a 21-year-old man who had limb weakness for 14 years. At age 7, he showed a poor performance in physical education class, and had a little difficulty in running and stairs climbing. Since then, he had complained of muscle fatigue and fluctuating weakness. He was diagnosed with lipid storage disease at age 12, and was given riboflavin and coenzyme Q10, but no benefits were observed. On this admission, he showed permanent weakness characterized by difficulties in climbing stairs, standing up after squatting, and combing hair.

Physical examination showed waddling gait and symmetrical proximal limb weakness without facial, bulbar, neck, and respiratory muscle involvement. Muscle strength was 3+ grade in the proximal upper limbs, 5- grade in the distal upper limbs, 3 grade in the proximal lower limbs, and 4+ grade in the distal lower limbs. Deep tendon reflexes could be induced. Pathological reflexes were negative. No muscle atrophy or fasciculation could be observed. No evidence of sensory disturbance, ataxia, or autonomic dysfunction was noticed.

The blood count, blood biochemistry, thyroid function, parathyroid hormone, blood acylcarnitines and urine organic acid profiles, and antibodies of myasthenia gravis were all negative. Muscle MRI of thigh revealed a mildly diffused hyperintensity on T1WI; and muscle MRI of leg also showed a slightly diffused hyperintensity except medial gastrocnemius that simultaneously had mild hyperintensity on STIR (short tau inversion recovery) (Figure 1b). NCV had no abnormality. EMG showed rapid recruitment of motor units suggestive of a myopathic pattern. In addition, RNS at 3 Hz revealed positive decrements in the deltoid and abductor digiti minimi muscle.

3.2 Genetic findings

Genetic sequencing disclosed compound heterozygous mutations in GFPT1: c.331C > T (p.R111C) and c.332G > A (p.R111H) in the patient one (Figure 2a); c.331C > T (p.R111C) and c.1534C > T (p.R512W) in the patient two (Figure 2b). The variants co-segregated with their parents: c.332G > A was from the mother and c.331C > T was from the father; c.331C > T was from the mother and c.1534C > T was from the father. All variants have been previously reported in other patients, and had a very low allele frequency in gnomAD database (http://gnomad.broadinstitute.org, v2.1.1, Table S1). A homology search in different species demonstrated that the amino acids at residues 111 and 512 were evolutionally highly conserved, respectively (Figure 2c). The variants were predicted to be damaging by several in silico tools. The significance of variants was evaluated as pathogenic according to the American College Medical Genetics and Genomics (ACMG) criteria (Li et al., 2017). No causative mutations associated with other CMS or myopathies were found in the genetic screening.

Genetic mutations in the GFPT1 gene. Genetic sequencing disclosed compound heterozygous mutations c.331C > T and c.332G > A in patient one (a); c.331C > T and c.1534C > T in patient two (b). The variants co-segregated with their parents. Residues arginine 111 and 512 have high evolutionary conservations (c)

3.3 Muscle pathological changes

The myopathological changes of patient one showed an appearance of multiple small vacuoles (Figure 3a) and a few rimmed vacuoles (Figure 3b) in some fibers, accompanied with variation of fiber size, central nuclei, fiber splitting and mild interstitial proliferation. Some fibers with small vacuoles had dark aggregations on MGT stain (Figure 3c). The small vacuoles were negative to ORO, PAS, and NADH stain (Figure 3d,e), but some affected fibers were positive to NSE (Figure 3f), dystrophin (Figure 3g), desmin (Figure 3h), dysferlin, and MHC-I (Figure 3i). On the other side, the muscle pathological features of patient two revealed tubular aggregates myopathy characterized by multiple basophilic materials deposition, variation of fiber size, central nuclei, and mild interstitial proliferation (Figure 3j). The affected fibers with tubular aggregates also showed abnormal depositions on MGT (Figure 3k), NADH (Figure 3l,m), and NSE (Figure 3n) stain. Some fibers had an immuno-reactivity to MHC-I (Figure 3o), but absence of desmin (Figure 3p) or other proteins aggregation.

The myopathological changes in the two patients. Muscle biopsy in patient one showed multiple small vacuoles on HE stain (a), a few rimmed vacuoles (b) on MGT stain. Some fibers with small vacuoles had dark aggregations on MGT stain (c), negative to NADH stain (d,e), but positive to NSE (f), dystrophin (g), desmin (h), and MHC-I (i). The muscle biopsy in patient two revealed tubular aggregates (j), which were dark on MGT (k), NADH (l,m), and NSE (n) stain. Some fibers had an immuno-reactivity to MHC-I (o), but not to desmin (p)

Ultrastructural examination of patient one revealed that numerous fibers harbored dilated and degenerating vesicular profiles (Figure 4a) in which were filled with autophagic vacuoles (Figure 4b), pleomorphic myeloid bodies, vacuolated mitochondria, lipofuscin granules, and bizarre debris (Figure 4c). Some fibers showed disorganization of myofibrillar structure with Z line disturbance, and some electronic dense granulofilamentous deposits under the sarcolemma and between the myofibrils (Figure 4d). In addition, some endplates appeared reduced and poorly developed junctional folds with electronic dense materials (Figure 4e). Ultrastructural examination of patient two showed local destructions of myofibrillar structure with multiple tubular aggregates (Figure 4f).

Muscle ultrastructural changes in the two patients. The muscle fibers harbored many degenerating vacuoles (a, arrow) and autophagic vacuoles (b, arrow) with myeloid bodies, vacuolated mitochondria (c, arrow), and bizarre debris (c, arrow head). Some fibers showed Z line disturbance and electronic dense granulofilamentous deposits between the myofibrils (d, arrow). Some endplates appeared reduced junctional folds (e, arrow) with ring-like electronic dense materials (e, arrow head). Ultrastructural examination of patient two showed multiple tubular aggregates (f, arrow)

3.4 Response to therapy

The patient one has been taking pyridostigmine (180 mg/day) since the age of 15. The medicine worked well at first 15 years while the response became less pronounced gradually. After a definite diagnosis, he was prescribed salbutamol (6 mg/day) and fluoxetine (20 mg/day), but his symptoms showed no significant alleviation. After joint prescription to patient two of pyridostigmine (180 mg/day) and albuterol (6 mg/day), his symptoms of muscular weakness improved considerably.

3.5 Muscle pathological review

We summarized all reported cases of GFPT1-related CMS in the past 10 years from 2011 to the present. A total of 77 patients with clinical details were reviewed (Table S2), of which 51 patients with muscle biopsy were summarized (Aharoni et al., 2017; Bauché et al., 2017; Guergueltcheva et al., 2012; Helman et al., 2019; Huh et al., 2012; Luo et al., 2019; Ma et al., 2021; Maselli et al., 2014; Matsumoto et al., 2019; Natera-De Benito et al., 2017; O'grady et al., 2016; Prior & Ghosh, 2021; Selcen et al., 2013; Selvam et al., 2018; Senderek et al., 2011; Szelinger et al., 2020; Yiş et al., 2017; Zhao et al., 2021). The first symptoms were noted in the first decade of life in 42 of 51 patients (range from 0 to 19, median 6 years old). Besides apneic spells and survival crisis in a few patients at birth, most of them started with muscle weakness, fatigue or frequent falls due to the involvement of proximal limbs. All 51 patients showed limb-girdle weakness, 26 (51.0%) had distal muscle weakness, 6 (11.8%) had neck weakness, 6 (11.8%) had respiratory muscle involvement, 5 (9.8%) had bulbar paralysis, and only 2 (3.9%) patients had slight ptosis.

Muscle biopsies revealed tubular aggregates in most patients, while some showed multiple pathological features (Table 1): 36 (70.6%) patients showed pure tubular aggregates; 10 (19.6%) patients presented with unspecific or mild myopathy changes but tubular aggregates accompanied in 6 patients; rimmed vacuoles occurred in 4 (7.8%) cases but simultaneously with tubular aggregates; ragged red fibers were found in 4 (7.8%) cases; neurogenic features were presented in 3 (5.9%) cases; 2 (3.9%) patients showed mild necrotizing myopathy with extensive autophagic vacuolar pathology; and 2 (3.9%) patients showed a dystrophic pattern.

TABLE 1. The clinical and pathological summarization of GFPT1-related CMS patients with muscle biopsy Muscle biopsy findings References Patient Sex/AAO/AAD/ethnic Clinical features GFPT1 mutations Light microscope Electron microscope Guergueltcheva 2012 1 M/6/31/Iranian Fatigue, fluctuating LGM, distal involvement p.D348Y (homo) TAs, type 1 fibre predominance, atrophy fibers ND Guergueltcheva 2012 2 F6/26/Turk Fluctuating LGM, fatigue, pain p.W240X (homo) TAs, type 2 fibers predominance, chronic myopathy ND Guergueltcheva 2012 3 NA/6/23-35/Libyan LGM, fatigue p.R111C (homo) Small TAs TAs Guergueltcheva 2012 4 M/14/55/Spanish LGM p.M492T; c.*22C > A TAs, RRF, mild myopathic changes, type 1 fibre predominance TAs Guergueltcheva 2012 5 M/10/50/Spanish Fluctuating LGM, falls p.M492T; c.*22C > A TAs, RRF, unspecific myopathic changes, type 1 fibre predominance TAs Guergueltcheva 2012 6 M/5/16/German Fluctuating LGM p.D43V; p.I121T TAs, unspecific myopathic changes ND Guergueltcheva 2012 7 M/8/23/British LGM, facial and distal muscle involvement p.R385H; p.R434H TAs; vacuoles, denervation changes ND Guergueltcheva 2012 8 M/6/37/British Fluctuating LGM, distal limb involvement p.T15M; p.R496W TAs ND Guergueltcheva 2012 9 F/13/26/German Fluctuating LGM, fatigue p.V199F; c.*22 > A TAs ND Guergueltcheva 2012 10 F/1/7/Senegalese Fluctuating LGM p.R512W (homo) TAs, uneven oxidative staining, mitochondria accumulation ND Guergueltcheva 2012 11 M/7/19/Spanish LGM p.M491T (homo) Unspecific myopathic changes ND Guergueltcheva 2012 12 M/1/37/Spanish Fluctuating LGM c.1278_1281dup; c.*22C > A Unspecific myopathic changes ND Guergueltcheva 2012 13 M/10′s/39/Spanish Fluctuating LGM c.1278_1281dup; c.*22C > A TAs, unspecific myopathic changes TAs Guergueltcheva 2012 14 M/10/55/Italian LGM p.T15A; c.621-622del TAs TAs Guergueltcheva 2012 15 M/7/36/Italian LGM UD TAs ND Guergueltcheva 2012 16 M/10′s/40/Swedish Fluctuating LGM p.222-223insA; p.R111C TAs TAs, PMS Guergueltcheva 2012 17 F/8/9/Maltese Fluctuating LGM, fatigability, learning difficulty p.M491T; c.714_715insA Size variability, uneven enzyme stain, type 2 fiber predominance ND Guergueltcheva 2012 18 M/7/13/Maltese Fluctuating LGM, fatigability, learning difficulty p.M491T; c.714_715insA Size variability, uneven enzyme stain ND Huh 2012 19 M/13/15/Korean LGM p.E256Q; p.M499T TAs ND Selcen 2013 20 M/0/16/NA Poor cry, apneic spells, LGM, distal limb involvement c.1700-17 16dup17; c.*22C > A Small TAs, RV PMS, PPM, multiple myeloid structures Selcen 2013 21 F/8/12/NA LGM, distal limb involvement p.R545P; c.*22C > A Small TAs, type 1 fiber predominance PMS, PPM Selcen 2013 22 F/12/20/NA LGM, distal limb involvement c.606-8A > G and c.*22C > A Neurogenic features PMS, PPM Selcen 2013 23 M/19/56/NA NA p.D113G; p.M492T Large TAs, small vacuoles PMS, PPM Selcen 2013 24 M/12/12/NA LGM, distal limb involvement p.R17X; c*22C > A Neurogenic features Normal EPs, myeloid structures Selcen 2013 25 F/0/1 m/NA Hypotonia, arthrogryposis, all weakness except ocular muscles c.686-2A > G; p.R304X Small TAs, RV, AV, regenerating fibers, type 1 fiber preponderance PMS, PPM, multiple autophagic vacuoles Selcen 2013 26 M/10/18/NA LGM, distal limb involvement p.R111C (homo) TAs ND Selcen 2013 27 F/9/64/NA LGM, distal limb involvement p.T350I; c.1337delA Small TAs, RV, neurogenic features ND Selcen et al., 2013 28 M/4/9/NA LGM, distal limb involvement p.M1fsX2; p.T15M TAs ND Maselli 2014 29 F/13/68/American LGM, neck and distal limb involvement c IVS7-8A > G; c.*22C > A Type I fiber predominance and type II fiber atrophy PMS O'Grady 2016 30 F/0/13/Australian Congenital hypotonia, contractures, scoliosis c.686-2A > G; p.M358V Dystrophic pattern ND Yis 2017 31 M/1/17/Turk LGM, axial weakness c.686-2A > G (homo) Dystrophic pattern ND Bauche 2017 32 NA/10′s/68/French LGM, distal limb involvement p.G39_ K75delinsE; p.R111H TAs TAs, PMS, PPM Bauche 2017 33 NA/1/49/French LGM, distal limb involvement p.R111C (homo) TAs ND Bauche 2017 34 NA/6/18/French LGM, distal limb involvement p.T392P; p.M499R TAs TAs, PMS, PPM Bauche 2017 35 f/6/16/French LGM, distal limb involvement, transient ptosis p.R111C (homo) TAs ND Bauche 2017 36 f/2.5/15/French LGM, distal limb involvement p.R111C (homo) TAs PMS, PPM Bauche 2017 37 NA/15/21/French LGM, distal limb involvement p.R111H; p.M317L TAs ND Helman 2019 38 M/5/7/Nepalese LGM, bilateral retinoschisis p.R14L (homo) RRF, fiber degeneration ND Helman 2019 39 M/0/5/Afghans Hypotonia, LGM p.T151K (homo) RRF, fiber degeneration ND Matsumoto 2019 40 F/1.5/38/Japanese LGM, axial muscle atrophy c.722_723 insG (homo) TAs, mild myopathic changes ND Luo 2019 41 M/5/23/Chinese Transient LGM, fatigue p.K154D; p.D363S TAs ND Szelinger 2020 42 M/0/8/Mexican Congenital hypotonia, low muscle bulk, dysphagia p.R230X (homo) Unspecific myopathic changes ND Szelinger 2020 43 M/0/2/Mexican Intubated and resuscitation p.R230X (homo) Necrotizing myopathy, AV PMS Ma 2021 44 F/4/15/Chinese LGM, mild ptosis p.F5Y; p.F194S TAs, RV TAs Zhao 2021 45 M/0/4/Chinese Lower limbs weakness p.R111C; p.A550T Nonspecific myopathies ND Zhao 2021 46 M/6/14/Chinese LGM p.V650A (homo) TAs ND Zhao 2021 47 M/5/18/Chinese LGM p.T15M TAs ND Zhao 2021 48 M/0/17/Chinese