NCAM1 and GDF15 are biomarkers of Charcot-Marie-Tooth disease in patients and mice.

Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.

Results from a 3-year Non-interventional, Observational Disease Monitoring Program in Adults with GNE Myopathy.

BACKGROUND: GNE myopathy is a rare, autosomal recessive, muscle disease caused by mutations in GNE and is characterized by rimmed vacuoles on muscle biopsy and progressive distal to proximal muscle weakness. OBJECTIVE: Investigate the clinical presentation and progression of GNE myopathy. METHODS: The GNE Myopathy Disease Monitoring Program was an international, prospective, observational study in subjects with GNE myopathy. Muscle strength was assessed with hand-held dynamometry (HHD), with upper extremity (UE) and lower extremity (LE) composite scores reflecting upper and lower extremity muscle groups, respectively. The GNE myopathy-Functional Activity Scale (GNEM-FAS) was used to further assess impairment in mobility, upper extremity function, and self-care. RESULTS: Eighty-seven of 101 enrolled subjects completed the trial until study closure by the sponsor; 60 completed 36 months. Mean (SD) HHD UE composite score decreased from 34.3 kg (32.0) at baseline to 29.4 kg (32.6) kg at month 36 (LS mean change [95%CI]: -3.8 kg [-5.9, -1.7]; P = 0.0005). Mean (SD) HHD LE composite score decreased from 32.0 kg (34.1) at baseline to 25.5 kg (31.2) at month 36 (LS mean change [95%CI]: -4.9 [-7.7, -2.2]; P = 0.0005). GNEM-FAS scores were more severe at baseline in subjects who walked <200 meters versus ≥200 meters in 6 minutes; in both groups, GNEM-FAS total, mobility, UE, and self-care scores decreased from baseline through month 36. CONCLUSIONS: These findings demonstrate progressive decline in muscle strength in GNE myopathy and provide insight into the appropriate tools to detect clinically meaningful changes in future GNE myopathy interventional trials.

Economic Costs of Myasthenia Gravis: A Systematic Review.

OBJECTIVES: The objective of our study was to conduct a systematic literature review of economic costs (henceforth costs) associated with myasthenia gravis (MG). METHODS: We searched MEDLINE (through PubMed), CINAHL, Embase, PsycINFO, and Web of Science for studies reporting costs of MG published from inception up until March 18, 2020, without language restrictions. Two reviewers independently screened records for eligibility, extracted the data, and assessed included studies for risk of bias using the Newcastle-Ottawa Scale. Costs were inflated and converted to 2018 United States dollars ($). RESULTS: The search identified 16 articles for data extraction and synthesis. Estimates of costs of MG were found for samples from eight countries spanning four continents (Europe, North America, South America, and Asia). Across studies, the mean per-patient annual direct medical cost of illness was estimated at between $760 and $28,780, and cost per hospitalization between $2550 and $164,730. The indirect cost of illness was estimated at $80 and $3550. Costs varied considerably by patient characteristics, and drivers of the direct medical cost of illness included intravenous immunoglobulin and plasma exchange, myasthenic crisis, mechanical ventilatory support, and hospitalizations. CONCLUSIONS: We show that the current body of literature of costs of MG is sparse, limited to a few geographical settings and resource categories, mostly dated, and subject to non-trivial variability, both within and between countries. Our synthesis will help researchers and decision-makers identify gaps in the local health economic context of MG and inform future cost studies and economic evaluations in this patient population.

A phase 3 randomized study evaluating sialic acid extended-release for GNE myopathy.

OBJECTIVE: To investigate the efficacy and safety of aceneuramic acid extended-release (Ace-ER), a treatment intended to replace deficient sialic acid, in patients with GNE myopathy. METHODS: UX001-CL301 was a phase 3, double-blind, placebo-controlled, randomized, international study evaluating the efficacy and safety of Ace-ER in patients with GNE myopathy. Participants who could walk ≥200 meters in a 6-minute walk test at screening were randomized 1:1, and stratified by sex, to receive Ace-ER 6 g/d or placebo for 48 weeks and assessed every 8 weeks. The primary endpoint was change in muscle strength over 48 weeks measured by upper extremity composite (UEC) score. Key secondary endpoints included change in lower extremity composite (LEC) score, knee extensor strength, and GNE myopathy-Functional Activity Scale (GNEM-FAS) mobility domain score. Safety assessments included adverse events (AEs), vital signs, and clinical laboratory results. RESULTS: Eighty-nine patients were randomized (Ace-ER n = 45; placebo n = 44). Change from baseline to week 48 for UEC score between treatments did not differ (least square mean [LSM] Ace-ER -2.25 kg vs placebo -2.99 kg; LSM difference confidence interval [CI] 0.74 [-1.61 to 3.09]; p = 0.5387). At week 48, there was no significant difference between treatments for the change in key secondary endpoints: LEC LSM difference (CI) -1.49 (-5.83 to 2.86); knee extension strength -0.40 (-2.38 to 1.58); and GNEM-FAS mobility domain score -0.72 (-2.01 to 0.57). Gastrointestinal events were the most common AEs. CONCLUSIONS: Ace-ER was not superior to placebo in improving muscle strength and function in patients with GNE myopathy. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that for patients with GNE myopathy, Ace-ER does not improve muscle strength compared to placebo.

GNE genotype explains 20% of phenotypic variability in GNE myopathy.

OBJECTIVE: To test the hypothesis that common GNE mutations influence disease severity; using statistical analysis of patient cohorts from different countries. METHODS: Systematic literature review identified 11 articles reporting 759 patients. GNE registry data were used as a second data set. The relative contributions of the GNE mutations, homozygosity, and country to the age at onset were explored using linear modeling, and relative importance measures were calculated. The rate of ambulation loss for GNE mutations, homozygosity, country, and age at onset was analyzed using Cox proportional hazards models. RESULTS: A spectrum of symptoms and large variability of age at onset and nonambulatory status was observed within families and cohorts. We estimated that 20% of variability is explained by GNE mutations. Individuals harboring p.Asp207Val have an expected age at onset 8.0 (s.e1.0) years later than those without and probability of continued ambulation at age 40 of 0.98 (95% confidence interval [CI] 0.96-1). In contrast, p.Leu539Ser results in onset on average 7.2 (s.e.2.7) years earlier than those without this mutation, and p.Val603Leu has a probability of continued ambulance of 0.61 (95% CI 0.50-0.74) at age 40, but has a nonsignificant effect on age at onset. CONCLUSIONS: GNE myopathy severity significantly varies in all cohorts, with 20% of variability explained by the GNE mutation. Atypical symptoms and clinical presentation suggest that physical and instrumental examination should include additional clinical tests. Proven and measurable effect of GNE mutations on the disease severity should be factored in patient management and clinical research study for a better data interpretation.

GNE myopathy in the bedouin population of Kuwait: Genetics, prevalence, and clinical description.

INTRODUCTION: GNE myopathy is a rare recessive myopathy caused by mutations in the GNE gene. It is mainly a distal myopathy with relative sparing of the quadriceps muscle. METHODS: Patients with distal myopathies from Kuwait were examined and tested for the Middle Eastern GNE gene founder mutation, p.M743T. Patients were further studied for disease-associated features. RESULTS: GNE myopathy was confirmed in 14 of the 37 patients (37.8%) screened. All cases were caused by the p.M743T mutation. Age of onset and time from disease onset to loss of ambulation were variable. Both wasted and hypertrophied calf muscles were noted. Severely affected quadriceps were present in 1 patient, and ptosis, ophthalmoplegia, and tongue wasting in another. DISCUSSION: The scope of the p.M743T mutation now includes the Arabian Peninsula. Variations in age of onset, disease progression, and distribution in patients harboring the same mutation suggest the role of other genetic- and environment-modifying factors. Muscle Nerve 58: 700-707, 2018.

GNE myopathy: from clinics and genetics to pathology and research strategies.

GNE myopathy is an ultra-rare autosomal recessive disease, which starts as a distal muscle weakness and ultimately leads to a wheelchair bound state. Molecular research and animal modelling significantly moved forward understanding of GNE myopathy mechanisms and suggested therapeutic interventions to alleviate the symptoms. Multiple therapeutic attempts are being made to supplement sialic acid depleted in GNE myopathy muscle cells. Translational research field provided valuable knowledge through natural history studies, patient registries and clinical trial, which significantly contributed to bringing forward an era of GNE myopathy treatment. In this review, we are summarising current GNE myopathy, scientific trends and open questions, which would be of significant interest for a wide neuromuscular diseases community.

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.

Clinical Outcomes in Duchenne Muscular Dystrophy: A Study of 5345 Patients from the TREAT-NMD DMD Global Database.

BACKGROUND: Recent short-term clinical trials in patients with Duchenne Muscular Dystrophy (DMD) have indicated greater disease variability in terms of progression than expected. In addition, as average life-expectancy increases, reliable data is required on clinical progression in the older DMD population. OBJECTIVE: To determine the effects of corticosteroids on major clinical outcomes of DMD in a large multinational cohort of genetically confirmed DMD patients. METHODS: In this cross-sectional study we analysed clinical data from 5345 genetically confirmed DMD patients from 31 countries held within the TREAT-NMD global DMD database. For analysis patients were categorised by corticosteroid background and further stratified by age. RESULTS: Loss of ambulation in non-steroid treated patients was 10 years and in corticosteroid treated patients 13 years old (p = 0.0001). Corticosteroid treated patients were less likely to need scoliosis surgery (p < 0.001) or ventilatory support (p < 0.001) and there was a mild cardioprotective effect of corticosteroids in the patient population aged 20 years and older (p = 0.0035). Patients with a single deletion of exon 45 showed an increased survival in contrast to other single exon deletions. CONCLUSIONS: This study provides data on clinical outcomes of DMD across many healthcare settings and including a sizeable cohort of older patients. Our data confirm the benefits of corticosteroid treatment on ambulation, need for scoliosis surgery, ventilation and, to a lesser extent, cardiomyopathy. This study underlines the importance of data collection via patient registries and the critical role of multi-centre collaboration in the rare disease field.

Reduced serum myostatin concentrations associated with genetic muscle disease progression.

Myostatin is a highly conserved protein secreted primarily from skeletal muscle that can potently suppress muscle growth. This ability to regulate skeletal muscle mass has sparked intense interest in the development of anti-myostatin therapies for a wide array of muscle disorders including sarcopenia, cachexia and genetic neuromuscular diseases. While a number of studies have examined the circulating myostatin concentrations in healthy and sarcopenic populations, very little data are available from inherited muscle disease patients. Here, we have measured the myostatin concentration in serum from seven genetic neuromuscular disorder patient populations using immunoaffinity LC-MS/MS. Average serum concentrations of myostatin in all seven muscle disease patient groups were significantly less than those measured in healthy controls. Furthermore, circulating myostatin concentrations correlated with clinical measures of disease progression for five of the muscle disease patient populations. These findings greatly expand the understanding of myostatin in neuromuscular disease and suggest its potential utility as a biomarker of disease progression.

GNE myopathy in Roma patients homozygous for the p.I618T founder mutation.

GNE myopathy is an autosomal-recessive disorder caused by mutations in the GNE gene, encoding the key enzyme in the sialic acid biosynthetic pathway, UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase. We studied 50 Bulgarian Roma patients homozygous for p.I618T, an ancient founder mutation in the kinase domain of the GNE gene, dating before the Gypsy exodus from North West India. The clinical features in the Bulgarian GNE group can be described with disease onset mostly in the third decade, but in individual cases, onset was as early as 10 years of age. The majority of patients had foot drop as the first symptom, but three patients developed hand weakness first. Muscle weakness was early and severe for the tibialis anterior, and minimal or late for quadriceps femoris, and respiratory muscles were only subclinically affected even in the advanced stages of the disease. During a 15-year follow-up period, 32 patients became non-ambulant. The average period between disease onset and loss of ambulation was 10.34 ± 4.31 years, ranging from 3 to 20 years. Our analysis of affected sib pairs suggested a possible role of genetic modifying factors, accounting for significant variation in disease severity.

Muscle-Derived Proteins as Serum Biomarkers for Monitoring Disease Progression in Three Forms of Muscular Dystrophy.

BACKGROUND: Identifying translatable, non-invasive biomarkers of muscular dystrophy that better reflect the disease pathology than those currently available would aid the development of new therapies, the monitoring of disease progression and the response to therapy. OBJECTIVE: The goal of this study was to evaluate a panel of serum protein biomarkers with the potential to specifically detect skeletal muscle injury. METHOD: Serum concentrations of skeletal troponin I (sTnI), myosin light chain 3 (Myl3), fatty acid binding protein 3 (FABP3) and muscle-type creatine kinase (CKM) proteins were measured in 74 Duchenne muscular dystrophy (DMD), 38 Becker muscular dystrophy (BMD) and 49 Limb-girdle muscular dystrophy type 2B (LGMD2B) patients and 32 healthy controls. RESULTS: All four proteins were significantly elevated in the serum of these three muscular dystrophy patient populations when compared to healthy controls, but, interestingly, displayed different profiles depending on the type of muscular dystrophy. Additionally, the effects of patient age, ambulatory status, cardiac function and treatment status on the serum concentrations of the proteins were investigated. Statistical analysis revealed correlations between the serum concentrations and certain clinical endpoints including forced vital capacity in DMD patients and the time to walk ten meters in LGMD2B patients. Serum concentrations of these proteins were also elevated in two preclinical models of muscular dystrophy, the mdx mouse and the golden-retriever muscular dystrophy dog. CONCLUSIONS: These proteins, therefore, are potential muscular dystrophy biomarkers for monitoring disease progression and therapeutic response in both preclinical and clinical studies.

Atrial fibrillation associated with ivabradine treatment: meta-analysis of randomised controlled trials.

OBJECTIVE: To quantify any risk of atrial fibrillation (AF) associated with ivabradine treatment by meta-analysis of clinical trial data. METHODS: Medline, Embase, Web of Knowledge and the Cochrane central register of controlled trials were searched for double-blinded randomised controlled trials of ivabradine with a minimum follow-up period of 4 weeks. For studies where AF data were unpublished, safety data were obtained from the European Medicines Agency (EMeA) website and personal communications. Studies were appraised for risk of bias using components recommended by the Cochrane Collaboration. Meta-analyses were performed of relative risk of AF and absolute risk difference of AF per year of treatment. The main outcome measure was incident AF during the follow-up period. RESULTS: AF data were available from 11 studies: one from the published report, six from the EMeA and four from personal communications. Ivabradine treatment was associated with a relative risk of AF of 1.15 (95% CI 1.07 to 1.24, p=0.0027) among 21 571 patients in the meta-analysis. From this we estimated that the number needed to harm for ivabradine would be 208 (95% CI 122 to 667) per year of treatment. CONCLUSIONS: AF is a substantially more common side effect of ivabradine treatment than one patient in 10 000, the risk presently reported in the product literature. The incidence of AF has not routinely been reported in clinical trials of ivabradine.

Two recurrent mutations are associated with GNE myopathy in the North of Britain.

OBJECTIVE: GNE myopathy is a rare recessive myopathy associated with inclusion bodies on muscle biopsy. The clinical phenotype is associated with distal muscle weakness with quadriceps sparing. Most of the current information on GNE myopathy has been obtained through studies of Jewish and Japanese patient cohorts carrying founder mutations in the GNE gene. However, little is known about GNE myopathy in Europe where the prevalence is thought to be very low. METHODS: Patients were referred through the National Specialist Commissioning Team service for limb-girdle muscular dystrophies at Newcastle (UK). All patients harbouring mutations in the GNE gene were recruited for our study. Detailed clinical and genetic data as well as muscle MRIs and muscle biopsies were reviewed. RESULTS: We identified 26 patients harbouring mutations in the GNE gene. Two previously reported mutations (c.1985C>T, p.Ala662Val and c.1225G>T, p.Asp409Tyr) were prevalent in the Scottish, Northern Irish and Northern English populations; with 90% of these patients carrying at least one of the two mutations. Clinically, we confirmed the homogenous pattern of selective quadriceps sparing but noted additional features like asymmetry of weakness at disease onset. CONCLUSIONS: GNE myopathy is an important diagnosis to consider in patients presenting with distal leg muscle weakness. We report, for the first time, two common mutations in the north of Britain and highlight the broader spectrum of clinical phenotypes. We also propose that the prevalence of GNE myopathy may be underestimated due to the frequent absence of rimmed vacuoles in the muscle biopsy.