Sialyllactose ameliorates myopathic phenotypes in symptomatic GNE myopathy model mice.

Patients with GNE myopathy, a progressive and debilitating disease caused by a genetic defect in sialic acid biosynthesis, rely on supportive care and eventually become wheelchair-bound. To elucidate whether GNE myopathy is treatable at a progressive stage of the disease, we examined the efficacy of sialic acid supplementation on symptomatic old GNE myopathy mice that have ongoing, active muscle degeneration. We examined the therapeutic effect of a less metabolized sialic acid compound (6'-sialyllactose) or free sialic acid (N-acetylneuraminic acid) by oral, continuous administration to 50-week-old GNE myopathy mice for 30 weeks. To evaluate effects on their motor performance in living mice, spontaneous locomotion activity on a running wheel was measured chronologically at 50, 65, 72 and 80 weeks of age. The size, force production, and pathology of isolated gastrocnemius muscle were analysed at the end point. Sialic acid level in skeletal muscle was also measured. Spontaneous locomotion activity was recovered in 6'-sialyllactose-treated mice, while NeuAc-treated mice slowed the disease progression. Treatment with 6'-sialyllactose led to marked restoration of hyposialylation in muscle and consequently to robust improvement in the muscle size, contractile parameters, and pathology as compared to NeuAc. This is due to the fact that 6'-sialyllactose is longer working as it is further metabolized to free sialic acid after initial absorption. 6'-sialyllactose ameliorated muscle atrophy and degeneration in symptomatic GNE myopathy mice. Our results provide evidence that GNE myopathy can be treated even at a progressive stage and 6'-sialyllactose has more remarkable advantage than free sialic acid, providing a conceptual proof for clinical use in patients.

Mutation update for GNE gene variants associated with GNE myopathy.

The GNE gene encodes the rate-limiting, bifunctional enzyme of sialic acid biosynthesis, uridine diphosphate-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Biallelic GNE mutations underlie GNE myopathy, an adult-onset progressive myopathy. GNE myopathy-associated GNE mutations are predominantly missense, resulting in reduced, but not absent, GNE enzyme activities. The exact pathomechanism of GNE myopathy remains unknown, but likely involves aberrant (muscle) sialylation. Here, we summarize 154 reported and novel GNE variants associated with GNE myopathy, including 122 missense, 11 nonsense, 14 insertion/deletions, and seven intronic variants. All variants were deposited in the online GNE variation database (http://www.dmd.nl/nmdb2/home.php?select_db=GNE). We report the predicted effects on protein function of all variants well as the predicted effects on epimerase and/or kinase enzymatic activities of selected variants. By analyzing exome sequence databases, we identified three frequently occurring, unreported GNE missense variants/polymorphisms, important for future sequence interpretations. Based on allele frequencies, we estimate the world-wide prevalence of GNE myopathy to be ∼4-21/1,000,000. This previously unrecognized high prevalence confirms suspicions that many patients may escape diagnosis. Awareness among physicians for GNE myopathy is essential for the identification of new patients, which is required for better understanding of the disorder's pathomechanism and for the success of ongoing treatment trials.

Severe cardiomyopathy associated with the VCP p.R155C and c.177_187del MYBPC3 gene variants.

Inclusion Body Myopathy, Paget's Disease of Bone, with Frontotemporal Dementia is a progressive autosomal dominant disease that affects the ubiquitin-proteasome complex, that is caused by variants in the Valosin Containing Protein (VCP) gene. We report the first case of concurrent pathogenic variants in both MYBPC3 and VCP that led to earlier onset of congestive heart failure with features of dilated cardiomyopathy. Cardiomyopathy has previously been associated with VCP inclusion body myopathy mostly at an advanced stage of the disease. Due to acute onset of cardiomyopathy in a previous asymptomatic individual, a cardiomyopathy gene panel was obtained which revealed an additional c.177_187del variant of the MYBPC3 gene. We report a first case of concurrent pathogenic variants in both c.177_187del gene of MYBPC3 and p.R155C VCP that led to earlier onset and a more severe form of the cardiomyopathy.

Genetic and Clinical Spectrum of GNE Myopathy in Russia.

GNE myopathy (GNEM) is a rare hereditary disease, but at the same time, it is the most common distal myopathy in several countries due to a founder effect of some pathogenic variants in the GNE gene. We collected the largest cohort of patients with GNEM from Russia and analyzed their mutational spectrum and clinical data. In our cohort, 10 novel variants were found, including 2 frameshift variants and 2 large deletions. One novel missense variant c.169_170delGCinsTT (p.(Ala57Phe)) was detected in 4 families in a homozygous state and in 3 unrelated patients in a compound heterozygous state. It was the second most frequent variant in our cohort. All families with this novel frequent variant were non-consanguineous and originated from the 3 neighboring areas in the European part of Russia. The clinical picture of the patients carrying this novel variant was typical, but the severity of clinical manifestation differed significantly. In our study, we reported two atypical cases expanding the phenotypic spectrum of GNEM. One female patient had severe quadriceps atrophy, hand joint contractures, keloid scars, and non-classical pattern on leg muscle magnetic resonance imaging, which was more similar to atypical collagenopathy rather than GNEM. Another patient initially had been observed with spinal muscular atrophy due to asymmetric atrophy of hand muscles and results of electromyography. The peculiar pattern of muscle involvement on magnetic resonance imaging consisted of pronounced changes in the posterior thigh muscle group with relatively spared muscles of the lower legs, apart from the soleus muscles. Different variants in the GNE gene were found in both atypical cases. Thus, our data expand the mutational and clinical spectrum of GNEM.

Visualizing Muscle Sialic Acid Expression in the GNED207VTgGne-/- Cmah-/- Model of GNE Myopathy: A Comparison of Dietary and Gene Therapy Approaches.

BACKGROUND: GNE myopathy (GNEM) is a rare, adult-onset, inclusion body myopathy that results from mutations in the GNE gene. GNE encodes UDP-GlcNAc epimerase/ManNAc-6 kinase, a protein with two enzymatic activities that comprise the committed step in biosynthesis of sialic acid (SA), an essential glycan that appears on the terminal positions of many extracellular oligosaccharide chains. These GNE mutations can cause a reduction of SA in many tissues, although pathology is restricted to skeletal muscles through a poorly understood mechanism. OBJECTIVE: Despite recent advances in the field, it remains unclear which therapeutic avenue is most promising for the restoration of SA level in skeletal muscle affected by GNEM. Our objective was to assess dietary and gene therapy strategies for GNEM in Cmah-deficient GNED207VTgGne-/- mice, a model that allows for the visualization of orally delivered N-glycolylneuraminic acid (Neu5Gc), one of the two predominant SA forms in muscle. METHODS: Methods included in situ physiology studies of the tibialis anterior muscle, studies of ambulation and limb grip strength, and muscle staining using MAA, SNA, and anti-Neu5Gc antibody, along with qPCR, qRT-PCR, western blot, and HPLC studies to assess virally introduced DNA, GNE gene expression, GNE protein expression, and SA expression. RESULTS: We found that a diet enriched in Neu5Gc-containing glycoproteins had no impact on Neu5Gc immunostaining in muscles of GNEM model mice. Delivery of a single high dose oral Neu5Gc therapy, however, did increase Neu5Gc immunostaining, though to levels below those found in wild type mice. Delivery of a single dose of GNE gene therapy using a recombinant Adeno Associated Virus (rAAV) vector with a liver-specific or a muscle-specific promoter both caused increased muscle Neu5Gc immunostaining that exceeded that seen with single dose monosaccharide therapy. CONCLUSIONS: Our findings indicate that dietary loading of Neu5Gc-containing glycoproteins is not effective in increasing muscle Neu5Gc expression, while single dose oral Neu5Gc monosaccharide or GNE gene therapy are. Neu5Gc immunostaining, however, showed greater changes than did lectin staining or HPLC analysis. Taken together, these results suggest that Neu5Gc immunostaining may be more sensitive technique to follow SA expression than other more commonly used methods and that liver expression of GNE may contribute overall muscle SA content.

Upper body involvement in GNE myopathy assessed by muscle imaging.

Upper body muscle involvement has never been systematically investigated in GNE myopathy (GNEM). Aims of our study were to explore upper body involvement in GNEM patients by means of muscle MRI, to compare the degree of pathology with that of lower body and to validate the MRI pattern of the lower limbs in novel patients. MRI scans of 9 GNEM patients were retrospectively evaluated. T1-weighted and short-tau inversion recovery images were scored. As a result, serratus anterior was involved in all patients, followed by subscapularis and trapezius muscles. The majority of scans consistently showed hypotrophy of pectoralis minor. Conversely, cranial muscles including the tongue were always spared while pectoralis major and latissimus dorsi were relatively spared. We confirmed the known pattern of involvement in the pelvic girdle and limbs, that were more significantly affected than the upper girdle in all disease stages. Paraspinal muscles were also frequently affected displaying both a cranio-caudal and latero-medial gradient of severity along the body axis. Upper girdle MRI highlights a selective muscle involvement in GNEM, offering an added value in patients' diagnostic workup and deep stratification.

Corrigendum: Identification of the CFTR c.1666A>G Mutation in Hereditary Inclusion Body Myopathy Using Next-Generation Sequencing Analysis.

[This corrects the article DOI: 10.3389/fnins.2018.00329.].

Identification of the CFTR c.1666A>G Mutation in Hereditary Inclusion Body Myopathy Using Next-Generation Sequencing Analysis.

Hereditary Inclusion Body Myopathy (HIBM) is a rare autosomal dominant or recessive adult onset muscle disease which affects one to three individuals per million worldwide. This disease is autosomal dominant or recessive [corrected] and occurs in adulthood. Our previous study reported a new subtype of HIBM linked to the susceptibility locus at 7q22.1-31.1. The present study is aimed to identify the candidate gene responsible for the phenotype in HIBM pedigree. After multipoint linkage analysis, we performed targeted capture sequencing on 16 members and whole-exome sequencing (WES) on 5 members. Bioinformatics filtering was performed to prioritize the candidate pathogenic gene variants, which were further genotyped by Sanger sequencing. Our results showed that the highest peak of LOD score (4.70) was on chromosome 7q22.1-31.1.We identified 2 and 22 candidates using targeted capture sequencing and WES respectively, only one of which as CFTRc.1666A>G mutation was well cosegregated with the HIBM phenotype. Using transcriptome analysis, we did not detect the differences of CFTR's mRNA expression in the proband compared with healthy members. Due to low incidence of HIBM and there is no other pedigree to assess, mutation was detected in three patients with duchenne muscular dystrophyn (DMD) and five patients with limb-girdle muscular dystrophy (LGMD). And we found that the frequency of mutation detected in DMD and LGMD patients was higher than that of being expected in normal population. We suggested that the CFTRc.1666A>G may be a candidate marker which has strong genetic linkage with the causative gene in the HIBM family.

GNE myopathy caused by a synonymous mutation leading to aberrant mRNA splicing.

GNE myopathy is a rare autosomal recessive myopathy caused by bi-allelic mutations in GNE. We report the case of a 36-year-old man who presented with typical clinical and pathological features of GNE myopathy including distal dominant muscle weakness from the age of 29 and numerous rimmed vacuoles on muscle biopsy. Targeted next-generation sequencing revealed a novel synonymous mutation, c.1500A>G (p.G500=), together with a common Japanese mutation c.620A>T (p.D207V). The cDNA analysis of the biopsied muscle revealed that this synonymous mutation creates a cryptic splice donor site that causes aberrant splicing. This report will expand our understanding of the genetic heterogeneity of GNE myopathy emphasizing the importance of interpreting synonymous variants in genetic testing.

Patient reported outcomes in GNE myopathy: incorporating a valid assessment of physical function in a rare disease.

BACKGROUND: The aim of this analysis was to evaluate the psychometric properties of three patient reported outcome (PRO) measures characterizing physical function in GNE myopathy: the Human Activity Profile, the Inclusion Body Myositis Functional Rating Scale, and the Activities-specific Balance Confidence scale. METHODS: This analysis used data from 35 GNE myopathy subjects participating in a natural history study. For construct validity, correlational and known-group analyses were between the PROs and physical assessments. Reliability of the PROs between baseline and 6 months was evaluated using the intra-class correlation coefficient model; internal consistency was tested with Cronbach's alpha. RESULTS: The hypothesized moderate positive correlations for construct validity were supported; the strongest correlation was between the human activity profile adjusted activity score and the adult myopathy assessment endurance subscale score (r = 0.81; p < 0.0001). The PROs were able to discriminate between known high and low functioning groups for the adult myopathy assessment tool. Internal consistency of the PROs was high (α > 0.8) and there was strong reliability (ICC >0.62). CONCLUSION: The PROs are valid and reliable measures of physical function in GNE myopathy and should be incorporated in investigations to better understand the impact of progressive muscle weakness on physical function in this rare disease population. Implications for Rehabilitation GNE myopathy is a rare muscle disease that results in slow progressive muscle atrophy and weakness, ultimately leading to wheelchair use and dependence on a caregiver. There is limited knowledge on the impact of this disease on the health-related quality of life, specifically physical function, of this rare disease population. Three patient reported outcomes have been shown to be valid and reliable in GNE myopathy subjects and should be incorporated in future investigations to better understand how progressive muscle weakness impacts physical functions in this rare disease population. The patient reported outcome scores of GNE myopathy patients indicate a high risk for falls and impaired physical functioning, so it is important clinicians assess and provide interventions for these subjects to maintain their functional capacity.

GNE Myopathy With Novel Mutations and Pronounced Paraspinal Muscle Atrophy.

GNE myopathy is characterized by distal muscle weakness, and caused by recessive mutations in GNE. Its onset is characteristically in young adulthood, although a broad spectrum of onset age is known to exist. A large number of mutations in GNE are pathogenic and this clinical phenotype can be difficult to differentiate clinically from other late-onset myopathies. We describe two families with novel mutations in GNE, and describe their clinical and MRI features. We also describe the presence of striking paraspinal muscle involvement on MRI of the lumbar spine, which is an under-recognized feature of GNE myopathy.

Phenotypic stratification and genotype-phenotype correlation in a heterogeneous, international cohort of GNE myopathy patients: First report from the GNE myopathy Disease Monitoring Program, registry portion.

GNE myopathy is a rare distal myopathy, caused by mutations in the GNE gene, affecting sialic acid synthesis. Clinical presentation varies from asymptomatic early stage patients to severely debilitating forms. This first report describes clinical presentations and severity of the disease, using data of 150 patients collected via the on-line, patient-reported registry component of the GNE Myopathy Disease Monitoring Program (GNEM-DMP). Disease progression was prospectively analysed, over a 2-year period, using the GNE myopathy functional activity scale (GNEM-FAS). The average annual rates of decline in function were estimated at -9.6% and -3.2% in ambulant and non-ambulant patients respectively. 4.3% of participants became non-ambulant within one year. The mean time from onset to required use of a wheelchair was 11.9 years. Mean delay of genetic diagnosis from symptom onset was 5.2 years. Mutation specific analysis demonstrated genotype-phenotype relationships; i.e. p.Ala662Val may be associated with a more severe phenotype, compared to p.Val727Met. Patients with compound heterozygous mutation in epimerase and kinase domain appeared to have a more severe phenotype compared to patients with both mutations located within one domain. Acknowledging the limitations of the study, these findings suggest that the severity of the GNE mutations affects disease severity. The GNEM-DMP is a useful data collection tool, prospectively measuring the progression of GNE myopathy, which could play an important role in translational and clinical research and further understanding of genotype-phenotype correlations.

Hereditary inclusion body myopathy: A myopathy with unique topography of weakness, yet frequently misdiagnosed: Case series and review of literature.

BACKGROUND: Hereditary inclusion body myopathy (HIBM) continues to be underrecognized clinically despite a characteristic topography of weakness with total sparing of quadriceps muscles and patient being wheelchair bound. We report seven patients of HIBM from four families in North India. METHODS AND RESULTS: Seven patients from four different families were diagnosed to have HIBM. There was no consanguinity in any of the families. While one patient had two affected siblings, another had one affected siblings and the family history was noncontributory in two patients. Two of the siblings were available for examination and confirmed clinically to be suffering from HIBM. Among the seven patients, only one was still ambulatory at the time of diagnosis. DISCUSSION: This is the first case report of occurrence of HIBM in North Indian population. Despite its unique clinical presentation, HIBM is frequently misdiagnosed resulting in unnecessary diagnostic and therapeutic interventions. A high index of suspicion of this rare myopathy along with proper clinical examination may go a long way in accurate prognostication and management of these patients.

Aceneuramic Acid Extended Release Administration Maintains Upper Limb Muscle Strength in a 48-week Study of Subjects with GNE Myopathy: Results from a Phase 2, Randomized, Controlled Study.

BACKGROUND: GNE Myopathy (GNEM) is a progressive adult-onset myopathy likely caused by deficiency of sialic acid (SA) biosynthesis. OBJECTIVE: Evaluate the safety and efficacy of SA (delivered by aceneuramic acid extended-release [Ace-ER]) as treatment for GNEM. METHODS: A Phase 2, randomized, double-blind, placebo-controlled study evaluating Ace-ER 3 g/day or 6 g/day versus placebo was conducted in GNEM subjects (n = 47). After the first 24 weeks, placebo subjects crossed over to 3 g/day or 6 g/day for 24 additional weeks (dose pre-assigned during initial randomization). Assessments included serum SA, muscle strength by dynamometry, functional assessments, clinician- and patient-reported outcomes, and safety. RESULTS: Dose-dependent increases in serum SA levels were observed. Supplementation with Ace-ER resulted in maintenance of muscle strength in an upper extremity composite (UEC) score at 6 g/day compared with placebo at Week 24 (LS mean difference +2.33 kg, p = 0.040), and larger in a pre-specified subgroup able to walk ≥200 m at Screening (+3.10 kg, p = 0.040). After cross-over, a combined 6 g/day group showed significantly better UEC strength than a combined 3 g/day group (+3.46 kg, p = 0.0031). A similar dose-dependent response was demonstrated within the lower extremity composite score, but was not significant (+1.06 kg, p = 0.61). The GNEM-Functional Activity Scale demonstrated a trend improvement in UE function and mobility in a combined 6 g/day group compared with a combined 3 g/day group. Patients receiving Ace-ER tablets had predominantly mild-to-moderate AEs and no serious adverse events. CONCLUSIONS: This is the first clinical study to provide evidence that supplementation with SA delivered by Ace-ER may stabilize muscle strength in individuals with GNEM and initiating treatment earlier in the disease course may lead to better outcomes.

GNE Myopathy and Cell Apoptosis: A Comparative Mutation Analysis.

In a number of genetic disorders such as GNE myopathy, it is not clear how mutations in target genes result in disease phenotype. GNE myopathy is a progressive neuro-degenerative disorder associated with homozygous or compound heterozygous missense mutations in either epimerase or kinase domain of UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). This bifunctional enzyme catalyses the rate limiting step in sialic acid biosynthesis. Many mechanisms have been suggested as possible cause of muscle degeneration. These include hyposialylation of critical proteins, defects in cytoskeletal network, sarcomere organization and apoptosis. In order to elucidate the role of GNE in cell apoptosis, we have used HEK cell-based model system overexpressing pathologically relevant GNE mutations. These cells display a reduction in the levels of sialic acid-bound glycoconjugates. These mutants GNE overexpressing cells have defect in cell proliferation as compared to vector or wild-type GNE (wtGNE) controls. Moreover, effect of different GNE mutations on cell apoptosis was also observed using staining with annexin V-FITC and TUNEL assay. The downstream apoptosis signalling pathway involving activation of caspases and increased PARP cleavage were observed in all GNE mutant cell lines. In addition, morpho-structural changes in mitochondria in cells overexpressing different GNE mutants were noticed by transmission electron microscopy, and mitochondrial transmembrane potential was found to be altered in absence of functional GNE. Our results clearly indicate role of GNE in mitochondria-dependent cell apoptosis and provide insights into the pathomechanism of GNE myopathy.

Novel Mutation of the GNE Gene Presenting Atypical Mild Clinical Feature: A Korean Case Report.

Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is caused by mutations in GNE, a key enzyme in sialic acid biosynthesis. Here, we reported a case of GNE that presented with atypical mild clinical feature and slow progression. A 48-year-old female had a complaint of left foot drop since the age of 46 years. Electromyography (EMG) and muscle biopsy from left tibialis anterior muscle were compatible with myopathy. Genetic analysis led to the identification of c.1714G>C/c.527A>T compound heterozygous mutation, which is the second most frequent mutation in Japan as far as we know. Previous research has revealed that c.1714G>C/c.527A>T compound heterozygous mutation is a mild mutation as the onset of the disease is much later than the usual age of onset of GNE myopathy and the clinical course is slowly progressive. This was the first case report in Korea of the clinicopathological characteristics of GNE myopathy with GNE (c.1714G>C/c.527A>T compound heterozygous) mutation.

Targeted sequencing and identification of genetic variants in sporadic inclusion body myositis.

Sporadic inclusion body myositis (sIBM) has clinical, pathologic and pathomechanistic overlap with some inherited muscle and neurodegenerative disorders. In this study, DNA from 79 patients with sIBM was collected and the sequencing of 38 genes associated with hereditary inclusion body myopathy (IBM), myofibrillar myopathy, Emery-Dreifuss muscular dystrophy, distal myopathy, amyotrophic lateral sclerosis and dementia along with C9orf72 hexanucleotide repeat analysis was performed. No C9orf72 repeat expansions were identified, but; 27 rare (minor allele frequency <1%) missense coding variants in several other genes were identified. One patient carried a p.R95C missense mutation in VCP and another carried a previously reported p.I27V missense mutation in VCP. Mutations in VCP cause IBM associated with Paget's disease of the bone (PDB) and fronto-temporal dementia (IBMPFD). Neither patient had a family history of weakness or manifested other symptoms reported with VCP mutations such as PDB or dementia. In vitro analysis of these VCP variants found that they both disrupted autophagy similar to other pathogenic mutations. Although no clear genetic etiology has been implicated in sIBM pathogenesis, our study suggests that genetic evaluation in sIBM may be clinically meaningful and lend insight into its pathomechanism.

GNE Myopathy: Two Clusters with History and Several Founder Mutations.

GNE myopathy (previous names: HIBM, DMRV, IBM2) is a unique distal myopathy with quadriceps sparing. This recessively inherited myopathy has been diagnosed in various regions of the world with more than 150 disease-causing mutations already identified. Several of those are proven or suspected to be founder mutations in certain regional clusters and are described in this review. The review also discusses some historical aspects that might be relevant to the mutational distribution.

A Novel Mutation of the GNE Gene in Distal Myopathy with Rimmed Vacuoles: A Case with Inflammation.

Distal myopathy with rimmed vacuoles (DMRV) is an autosomal recessive or sporadic early adult-onset myopathy caused by mutations in the UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase (GNE) gene. Characteristic pathologic features of DMRV are rimmed vacuoles on muscle biopsy and tubulofilamentous inclusion in ultrastructural study. Presence of inflammation in DMRV is unusual. We report a sporadic case of DMRV in a 40-year-old Thai man who presented with slowly progressive distal muscle weakness. Gene analysis revealed a compound heterozygous mutation of the GNE gene including a novel mutation c.1057A>G (p.K353E) and a known mutation c.2086G>A (p.V696M). The latter is the most common mutation in Thai DMRV patients. The muscle pathology was compatible with DMRV except for focal inflammation.