Expanding the clinicopathological‐genetic spectrum of GNE myopathy by a Chinese neuromuscular centre

Abstract GNE myopathy is a heterogeneous group of ultrarare neuromuscular disorders caused by mutations in the GNE gene. An estimated prevalence of 1~21/1,000,000 leads to a deficiency of data and a lack of availability of samples to conduct clinical research on this neuromuscular disorder. Although GNE, which is the mutated gene responsible for the disease, is well known as the key enzyme in the biosynthesis pathway of sialic acid, the clinicopathological‐genetic spectrum of GNE mutant patients is still unclear and expanding. This study presents ten unrelated patients with GNE myopathy, discovering five novel missense mutations. Clinical, electrophysiological, imaging, pathological and genetic data are presented in a retrospective manner. Interestingly, several patients in the cohort were found to have peripheral neuropathy and inflammatory cell infiltration in muscle biopsies, which have seldom been reported. This study, conducted by a neuromuscular centre in China, is the first attempt to highlight these abnormal clinicopathological features and associate them with genetic mutations in GNE myopathy.


| INTRODUC TI ON
GNE myopathy, also known as distal myopathy with rimmed vacuoles (DMRV), hereditary inclusion body myopathy (HIBM) or inclusion body myopathy 2 (IBM2), was first reported in Japanese patients by Nonaka in 1981. 1 It is a rare, recessively inherited muscle disease caused by mutations in the GNE gene (9p13.3) encoding the bifunctional enzyme UDP-N-acetylglucosamine (GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase, a significant rate-limiting enzyme of the sialic acid biosynthesis pathway. 2 GNE myopathy is clinically characterized by progressive weakness and atrophy of distal lower-limb muscles that preferentially involve the tibialis anterior muscles and spare the quadriceps, 3 with normal or mildly increased serum creatine kinase (CK) levels. 4 Peripheral neuropathy is not a typical presentation but can be seen in several cases.
Pathological features of GNE myopathy include specific rimmed vacuoles, muscle fibre atrophy and a muscle volume decrease. Notably, inflammatory infiltrations are rarely seen in GNE myopathy, different from sporadic inclusion body myositis (sIBM), but without any satisfactory explanation for the clinical presentation.
To date, more than 201 GNE mutations associated with GNE myopathy have been reported, 5  In this study, we described the clinicopathological and genetic profiles of ten Chinese patients with GNE myopathy, among which five novel mutations were found, broadening the mutation spectrum of the GNE gene. In addition, we analysed the presence of two relatively rare clinicopathological manifestations in GNE myopathy, peripheral neuropathy and muscle inflammation, and summarized the genotype-phenotype correlations of the GNE mutations.

| Ethics approval
Ethics approval was granted by the Ethics Committee of Xiangya Hospital, Central South University. Informed consent for participation in our research was obtained from all of the patients, as previously reported in our centre. 9

| Patients and clinical evaluation
From 2014 to 2021, ten patients were diagnosed with GNE myopathy based on clinical manifestations, pathological findings and genetic testing in the neuromuscular centre of Xiangya Hospital, Central South University. Clinical assessment of the patients consisted of a physical examination and laboratory investigations, such as serum creatine kinase (CK), electromyography (EMG), muscle biopsy, magnetic resonance imaging (MRI) of the thigh and leg muscles, and genetic testing, as previously used in our centre. 10

| Biopsies and pathological examination
Muscle biopsies were obtained from the tibialis anterior or biceps brachii muscles. Nerve biopsies were performed on the sural nerves.
Pathological examination was performed as described elsewhere with minor modifications. 11,12 First, the samples were frozen in isopentane cooled with liquid nitrogen and cut into 5 μm thick sections using a cryostat. The sections were stained with haematoxylin and eosin (HE), modified Gömöri trichrome, acid phosphatase, nicotina-

| Genetic analysis
Genomic DNA (gDNA) was extracted from peripheral blood (MyGenostics) using a DNeasy Blood and Tissue Kit (Qiagen, Venlo) as previously mentioned 13,14 according to the manufacturer's instructions.
Next-generation sequencing (NGS) analysis covering 2082 genes known to be associated with neuromuscular disorders was performed. The sequences obtained were compared with those in the human genome database. Functional prediction software, polymorphism Phenotyping version 2 (PolyPhen-2, http://genet ics.bwh.harva rd.edu/pph2/) and Mutation Taster (http://www.mutat ionta ster.org/) were used to predict the possible impact of the identified substitution on protein structure and function.

| Clinical characteristics
In our current research, ten patients diagnosed with GNE myopathy

| Muscle and nerve pathological features
Muscle biopsies were performed for all the patients, and nerve biopsies were conducted in one patient. Increased fibre-size variation, rimmed vacuoles and internal nuclei were the most common pathologic changes in nearly all of the muscle samples ( Figure 2).   Table 3. diseases, and we defined these variants are benign (Table S1-S5).

| Genetic analysis
To our knowledge, this is the first report of two novel heterozygous missense mutations, c.2099G>A (p.G700E) and c.539C>T  (Table 4). Subsequently, we screened hundreds of alleles from normal Chinese individuals, and we did not identify any of these genetic changes. We think that these five novel mutations are likely to be pathogenic based on the predictions of PolyPhen-2 and MutationTaster. The predicted scores and results of the functional prediction software programs are shown in Table 5.   Table 6. 19,20 In our cohort, three patients were found to carry c.830G>A

| DISCUSS ION
(p.R277Q) heterozygous mutation. They all presented in their early twenties and had relatively severe lower-limb weakness, especially of the distal muscles. In comparison with patients with c.620A>T (p.D207V) mutation, the muscles of the shoulder, neck and waist were mildly or not involved; however, nerve impairment tended to be much more common, accounting for 2/3 patients in our cohort.
Another four cases with c.830G>A (p.R277Q) homozygous mutation and five cases with heterozygous variants reported in previous studies are listed in Table 6. 15,21,22 Notably, the median onset age of the patients who were homozygous (median, interquartile range:   presented with simple muscle atrophy in appearance of the lower distal limbs, without functional weakness or numbness. It is still uncertain whether the difference in primary symptoms is associated with c.124C>T (p.R42W) mutation.
In recent years, due to the increased availability of genetic testing, a growing number of cases with GNE mutations have been reported. Surprisingly, some patients also showed a notable association with peripheral neuropathy. 26 In our study, nearly half of the patients presented with myopathic lesions accompanied by neuropathic changes during the progression of the disease, suggesting potential nerve involvement in the pathogenesis of GNE myopathy.
The specific aetiology of neuropathy complications remains unknown. In a previous study, 27  The spectrum of diseases caused by GNE mutations is constantly growing. Interestingly, no patient has been identified carrying biallelic nonsense mutations or frameshifting mutations thus far, 15 suggesting that some basic activity of GNE is required during embryonic or early development. In mice, the GNE protein is expressed and plays an important role in an early embryonic stage, and Gne −/− is lethal to mice, which is consistent with the clinical lack of biallelic null mutations and only 'mildly deleterious' mutations reported in GNE myopathy patients. 28 Recently, NGS has become more widely available, leading to an increasing understanding not only of GNE myopathy-related mutations, but also of other myopathies. 29 At present, there is no effective therapy available for GNE myopathy. 30,31 Preclinical studies have identified that the use of oral monosaccharides reversed muscle hyposialylation in a GNE myopathy mouse model. 32 However, phase II and III randomized studies evaluating sialic acid extended-release for GNE myopathy showed two distinct results; the phase II study was positive for the curative effect of N-acetylneuraminic acid (Ace-ER), while the latter study showed no improvement of muscle strength after Ace-ER intake compared with placebo. 33 Additional studies are urgently needed to identify an effective treatment for GNE myopathy.

This work was supported by the Science and Technology Innovation
Program of Hunan Province, China (Grant No. 2021RC2023, KH).

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.