Improving the measurement properties of the Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R): deriving a valid measurement total for the calculation of change.

BACKGROUND: The Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised (ALSFRS-R) total score is a widely used measure of functional status in Amyotrophic Lateral Sclerosis/Motor Neuron Disease (ALS), but recent evidence has raised doubts about its validity. The objective was to examine the measurement properties of the ALSFRS-R, aiming to produce valid measurement from all 12 scale items. METHOD: Longitudinal ALSFRS-R data were collected between 2013-2020 from 1120 people with ALS recruited from 35 centers, together with other scales in the Trajectories of Outcomes in Neurological Conditions-ALS (TONiC-ALS) study. The ALSFRS-R was analyzed by confirmatory factor analysis (CFA), Rasch Analysis (RA) and Mokken scaling. RESULTS: No definite factor structure of the ALSFRS-R was confirmed by CFA. RA revealed the raw score total to be invalid even at the ordinal level because of multidimensionality; valid interval level subscale measures could be found for the Bulbar, Fine-Motor and Gross-Motor domains but the Respiratory domain was only valid at an ordinal level. All four domains resolved into a single valid, interval level measure by using a bifactor RA. The smallest detectable difference was 10.4% of the range of the interval scale. CONCLUSION: A total ALSFRS-R ordinal raw score can lead to inferential bias in clinical trial results due to its non-linear nature. On the interval level transformation, more than 5 points difference is required before a statistically significant detectable difference can be observed. Transformation to interval level data should be mandatory in clinical trials.

Dyspnea (breathlessness) in amyotrophic lateral sclerosis/motor neuron disease: prevalence, progression, severity, and correlates.

Objective: Dyspnea, or breathlessness, is an important symptom in amyotrophic lateral sclerosis/motor neuron disease (ALS/MND). We examined the measurement properties of the Dyspnea-12. Methods: Rasch analysis enabled conversion of raw Dyspnea-12 scores to interval level metric equivalents. Converted data were used to perform trajectory modeling; those following different trajectories were compared for demographic, clinical, symptom, and functioning characteristics. Logistic regression examined differences between distinct trajectories. Results: In 1022 people, at baseline, mean metric Dyspnea-12 was 7.6 (SD 9.3). 49.8% had dyspnea, severe in 12.6%. Trajectory analysis over 28 months revealed three breathlessness trajectories: group 1 reported none at baseline/follow-up (42.7%); group 2 significantly increased over time (9.4%); group 3 had a much higher level at baseline which rose over follow-up (47.9%). Group 3 had worse outcomes on all symptoms, functioning and quality of life; compared to group 1, their odds of: respiratory onset sixfold greater; King's stage ≥3 2.9 greater; increased odds of being bothered by choking, head drop, fasciculations, and muscle cramps; fatigue and anxiety also elevated (p < .01). Conclusion: Dyspnea is a cardinal symptom in ALS/MND and can be quickly measured using the Dyspnea-12. Raw scores can easily be converted to interval level measurement, for valid change scores and trajectory modeling. Dyspnea trajectories reveal different patterns, showing that clinical services must provide monitoring which is customized to individual patient need. Almost half of this large population had worsening dyspnea, confirming the importance of respiratory monitoring and interventions being integrated into routine ALS care.

Delayed onset post-traumatic wound botulism.

A 41-year-old man developed rapidly progressive cranial neuropathies and muscle weakness followed by respiratory failure, requiring ventilation support. On examination, there was marked bilateral ptosis and ophthalmoplegia with bulbar, neck and proximal upper limb weakness. He had a recent open left humeral fracture that eventually required amputation. Despite immunoglobulin therapy, his progressive weakness continued. Multiple investigation results were inconclusive. Eventually, botulinum type A toxin was found positive, by which time the therapeutic window for antitoxin had passed. He continued on supportive management and was treated for concomitant infections and nosocomial illnesses. He was subsequently weaned from respiratory support and has made a good neurological recovery.

Phenotypical differences of C9ORF72 gene-positive and negative amyotrophic lateral sclerosis: a comparative case series.

BACKGROUND: Hexanucleotide repeat expansions of C9ORF72 account for a significant proportion of autosomal dominant neurodegenerative diseases in the amyotrophic lateral sclerosis (ALS)-frontotemporal dementia spectrum. In the absence of a family history, clinical identification of such patients remains difficult. We aimed to identify differences in demographics and clinical presentation between patients with C9ORF72 gene-positive ALS (C9pALS) versus C9ORF72 gene-negative ALS (C9nALS), to aid identification of these patients in the clinic and examine differences in outcomes including survival. METHODS: We retrospectively reviewed the clinical presentations of 32 patients with C9pALS and compared their characteristics with a cohort of 46 patients with C9nALS from the same tertiary neurosciences centre. RESULTS: Patients with C9pALS more commonly presented with mixed upper and lower motor signs (C9pALS 87.5%, C9nALS 65.2%; p=0.0352), but less frequently presented with purely upper motor neuron signs (C9pALS 3.1%, C9nALS 21.7%; p=0.0226). The C9pALS cohort had a higher frequency of cognitive impairment (C9pALS 31.3%, C9nALS 10.9%; p=0.0394) and bulbar disease (C9pALS 56.3%, C9nALS 28.3%; p=0.0186). There were no differences between cohorts in age at diagnosis, gender, limb weakness, respiratory symptoms, presentation with predominantly lower motor neuron signs or overall survival. DISCUSSION: Analysis of this ALS clinic cohort at a UK tertiary neurosciences centre adds to the small but growing understanding of the unique clinical features of patients with C9pALS. In the age of precision medicine with expanding opportunities to manage genetic diseases with disease-modifying therapies, clinical identification of such patients is increasingly important as focused therapeutic strategies become available.

Perry Syndrome with Intrafamilial Heterogeneity in Presentation and Survival Including Acute Respiratory Failure: Case Series.

Background: Perry syndrome is a rare autosomal dominant parkinsonian disorder characterized by respiratory failure. The variability in respiratory presentation in this condition is incompletely understood. Cases: We report 2 first-degree relatives with Perry syndrome attributed to the same mutation in the Dynactin 1 (DCTN1) gene. Their clinical presentations with respect to parkinsonism and respiratory failure were heterogeneous. The proband presented with acute respiratory failure requiring invasive ventilation on a background of parkinsonism and remains alive more than 3 years later with a good levodopa response. We contrast this with the published literature, in which acute respiratory presentations were associated with a poor outcome. The proband's brother presented with parkinsonism together with early falls and gait impairment and died following gradual hypoventilation despite noninvasive respiratory support. Conclusions: Perry syndrome can show intrafamily heterogeneity in both movement disorder and respiratory presentations. Acute respiratory failure is often but not always associated with a poor outcome.

Clinical trials in amyotrophic lateral sclerosis: a systematic review and perspective.

Amyotrophic lateral sclerosis is a progressive and devastating neurodegenerative disease. Despite decades of clinical trials, effective disease-modifying drugs remain scarce. To understand the challenges of trial design and delivery, we performed a systematic review of Phase II, Phase II/III and Phase III amyotrophic lateral sclerosis clinical drug trials on trial registries and PubMed between 2008 and 2019. We identified 125 trials, investigating 76 drugs and recruiting more than 15 000 people with amyotrophic lateral sclerosis. About 90% of trials used traditional fixed designs. The limitations in understanding of disease biology, outcome measures, resources and barriers to trial participation in a rapidly progressive, disabling and heterogenous disease hindered timely and definitive evaluation of drugs in two-arm trials. Innovative trial designs, especially adaptive platform trials may offer significant efficiency gains to this end. We propose a flexible and scalable multi-arm, multi-stage trial platform where opportunities to participate in a clinical trial can become the default for people with amyotrophic lateral sclerosis.

Cognition and behaviour in frontotemporal dementia with and without amyotrophic lateral sclerosis.

OBJECTIVE: The precise relationship between frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is incompletely understood. The association has been described as a continuum, yet data suggest that this may be an oversimplification. Direct comparisons between patients who have behavioural variant FTD (bvFTD) with and without ALS are rare. This prospective comparative study aimed to determine whether there are phenotypic differences in cognition and behaviour between patients with FTD-ALS and bvFTD alone. METHODS: Patients with bvFTD or FTD-ALS and healthy controls underwent neuropsychological testing, focusing on language, executive functions and social cognition. Behavioural change was measured through caregiver interview. Blood samples were screened for known FTD genes. RESULTS: 23 bvFTD, 20 FTD-ALS and 30 controls participated. On cognitive tests, highly significant differences were elicited between patients and controls, confirming the tests' sensitivities to FTD. bvFTD and FTD-ALS groups performed similarly, although with slightly greater difficulty in patients with ALS-FTD on category fluency and a sentence-ordering task that assesses grammar production. Patients with bvFTD demonstrated more widespread behavioural change, with more frequent disinhibition, impulsivity, loss of empathy and repetitive behaviours. Behaviour in FTD-ALS was dominated by apathy. The C9ORF72 repeat expansion was associated with poorer performance on language-related tasks. CONCLUSIONS: Differences were elicited in cognition and behaviour between bvFTD and FTD-ALS, and patients carrying the C9ORF72 repeat expansion. The findings, which raise the possibility of phenotypic variation between bvFTD and FTD-ALS, have clinical implications for early detection of FTD-ALS and theoretical implications for the nature of the relationship between FTD and ALS.

The Edinburgh Cognitive and Behavioral ALS Screen (ECAS) in frontotemporal dementia.

OBJECTIVES: To examine the usefulness of the Edinburgh Cognitive and Behavioral Amyotrophic Lateral Sclerosis (ALS) Screen (ECAS) as a cognitive screening tool for the detection of behavioral variant frontotemporal dementia (bvFTD). A secondary aim was to determine whether people with FTD combined with ALS (ALS-FTD) exhibit a similar ECAS profile to that of people with bvFTD alone. Methods: Patients with ALS-FTD and bvFTD and healthy controls were recruited. Participants were administered the ECAS, which comprises tests of language, verbal fluency, executive functions, memory, and visual-spatial functions. They also carried out analogous, full-length cognitive tests that examine naming, spelling, sentence completion, and social cognition skills. Results: The study cohort comprised 20 ALS-FTD patients, 23 with bvFTD, and 30 controls. Highly significant group differences were elicited for all cognitive domains, reflecting poorer performance in patients compared to controls. No significant differences in overall test scores were found between ALS-FTD and bvFTD patients, although ALS-FTD patients showed a higher frequency of impairment on verbal fluency. Correlative analyses revealed inter-relationships in patients (but not controls) between scores in different domains, most marked in bvFTD. There were strong correlations between performance on ECAS subtests and analogous cognitive tasks. Conclusion: The ECAS is a sensitive and valuable tool for the assessment of FTD. Executive, language and behavioral breakdown may, however, compromise performance in other cognitive domains, reducing the specificity of the 'frontotemporal' cognitive profile. Subtle differences observed between ALS-FTD and bvFTD raise questions regarding the precise relationship between bvFTD with and without ALS.

TCTEX1D1 is a genetic modifier of disease progression in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by pathogenic variants in the DMD gene leading to the lack of dystrophin. Variability in the disease course suggests that other factors influence disease progression. With this study we aimed to identify genetic factors that may account for some of the variability in the clinical presentation. We compared whole-exome sequencing (WES) data in 27 DMD patients with extreme phenotypes to identify candidate variants that could affect disease progression. Validation of the candidate SNPs was performed in two independent cohorts including 301 (BIO-NMD cohort) and 109 (CINRG cohort of European ancestry) DMD patients, respectively. Variants in the Tctex1 domain containing 1 (TCTEX1D1) gene on chromosome 1 were associated with age of ambulation loss. The minor alleles of two independent variants, known to affect TCTEX1D1 coding sequence and induce skipping of its exon 4, were associated with earlier loss of ambulation. Our data show that disease progression of DMD is affected by a new locus on chromosome 1 and demonstrate the possibility to identify genetic modifiers in rare diseases by studying WES data in patients with extreme phenotypes followed by multiple layers of validation.

Validation of genetic modifiers for Duchenne muscular dystrophy: a multicentre study assessing SPP1 and LTBP4 variants.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials. METHODS: Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates. RESULTS: We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss. CONCLUSIONS: This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials.

Agrin mutations lead to a congenital myasthenic syndrome with distal muscle weakness and atrophy.

Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of rare diseases resulting from impaired neuromuscular transmission. Their clinical hallmark is fatigable muscle weakness associated with a decremental muscle response to repetitive nerve stimulation and frequently related to postsynaptic defects. Distal myopathies form another clinically and genetically heterogeneous group of primary muscle disorders where weakness and atrophy are restricted to distal muscles, at least initially. In both congenital myasthenic syndromes and distal myopathies, a significant number of patients remain genetically undiagnosed. Here, we report five patients from three unrelated families with a strikingly homogenous clinical entity combining congenital myasthenia with distal muscle weakness and atrophy reminiscent of a distal myopathy. MRI and neurophysiological studies were compatible with mild myopathy restricted to distal limb muscles, but decrement (up to 72%) in response to 3 Hz repetitive nerve stimulation pointed towards a neuromuscular transmission defect. Post-exercise increment (up to 285%) was observed in the distal limb muscles in all cases suggesting presynaptic congenital myasthenic syndrome. Immunofluorescence and ultrastructural analyses of muscle end-plate regions showed synaptic remodelling with denervation-reinnervation events. We performed whole-exome sequencing in two kinships and Sanger sequencing in one isolated case and identified five new recessive mutations in the gene encoding agrin. This synaptic proteoglycan with critical function at the neuromuscular junction was previously found mutated in more typical forms of congenital myasthenic syndrome. In our patients, we found two missense mutations residing in the N-terminal agrin domain, which reduced acetylcholine receptors clustering activity of agrin in vitro. Our findings expand the spectrum of congenital myasthenic syndromes due to agrin mutations and show an unexpected correlation between the mutated gene and the associated phenotype. This provides a good rationale for examining patients with apparent distal myopathy for a neuromuscular transmission disorder and agrin mutations.

Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies.

Despite the recent progress in the broad-scaled analysis of proteins in body fluids, there is still a lack in protein profiling approaches for biomarkers of rare diseases. Scarcity of samples is the main obstacle hindering attempts to apply discovery driven protein profiling in rare diseases. We addressed this challenge by combining samples collected within the BIO-NMD consortium from four geographically dispersed clinical sites to identify protein markers associated with muscular dystrophy using an antibody bead array platform with 384 antibodies. Based on concordance in statistical significance and confirmatory results obtained from analysis of both serum and plasma, we identified eleven proteins associated with muscular dystrophy, among which four proteins were elevated in blood from muscular dystrophy patients: carbonic anhydrase III (CA3) and myosin light chain 3 (MYL3), both specifically expressed in slow-twitch muscle fibers and mitochondrial malate dehydrogenase 2 (MDH2) and electron transfer flavoprotein A (ETFA). Using age-matched sub-cohorts, 9 protein profiles correlating with disease progression and severity were identified, which hold promise for the development of new clinical tools for management of dystrophinopathies.

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.

Fibronectin is a serum biomarker for Duchenne muscular dystrophy.

PURPOSE: To identify and validate serum biomarkers for the progression of Duchenne muscular dystrophy (DMD) using a MS-based bottom-up pipeline. EXPERIMENTAL DESIGN: We used a bottom-up proteomics approach, including a protein concentration equalization step, different proteolytic digestions, and MS detection schemes, to identify candidate biomarkers in serum samples from control subjects and DMD patients. Fibronectin was chosen for follow-up based on the differences in peptide spectral counts and sequence coverage observed between the DMD and control groups. Subsequently, fibronectin levels were determined with ELISA in 68 DMD patients, 38 milder Becker muscular dystrophy patients, 33 patients with other neuromuscular disorders, and 15 age-matched adult and child controls. RESULTS: There was a significant increase in fibronectin levels in DMD patients compared to age-matched controls. Fibronectin levels in patients with Becker muscular dystrophy, Bethlem myopathy, or myasthenia gravis were comparable to control levels. Progressive elevation in fibronectin levels was observed in longitudinal samples from 22 DMD patients followed up for a period of 6 months up to 4 years. CONCLUSION AND CLINICAL RELEVANCE: This study suggests that serum fibronectin levels may constitute a promising biomarker to monitor disease progression in DMD patients.

Mutations in the Mitochondrial Citrate Carrier SLC25A1 are Associated with Impaired Neuromuscular Transmission.

BACKGROUND AND OBJECTIVE: Congenital myasthenic syndromes are rare inherited disorders characterized by fatigable weakness caused by malfunction of the neuromuscular junction. We performed whole exome sequencing to unravel the genetic aetiology in an English sib pair with clinical features suggestive of congenital myasthenia. METHODS: We used homozygosity mapping and whole exome sequencing to identify the candidate gene variants. Mutant protein expression and function were assessed in vitro and a knockdown zebrafish model was generated to assess neuromuscular junction development. RESULTS: We identified a novel homozygous missense mutation in the SLC25A1 gene, encoding the mitochondrial citrate carrier. Mutant SLC25A1 showed abnormal carrier function. SLC25A1 has recently been linked to a severe, often lethal clinical phenotype. Our patients had a milder phenotype presenting primarily as a neuromuscular (NMJ) junction defect. Of note, a previously reported patient with different compound heterozygous missense mutations of SLC25A1 has since been shown to suffer from a neuromuscular transmission defect. Using knockdown of SLC25A1 expression in zebrafish, we were able to mirror the human disease in terms of variable brain, eye and cardiac involvement. Importantly, we show clear abnormalities in the neuromuscular junction, regardless of the severity of the phenotype. CONCLUSIONS: Based on the axonal outgrowth defects seen in SLC25A1 knockdown zebrafish, we hypothesize that the neuromuscular junction impairment may be related to pre-synaptic nerve terminal abnormalities. Our findings highlight the complex machinery required to ensure efficient neuromuscular function, beyond the proteomes exclusive to the neuromuscular synapse.

Fast-channel congenital myasthenic syndrome with a novel acetylcholine receptor mutation at the α-ε subunit interface.

Congenital myasthenic syndromes (CMS) result from the failure to achieve muscle depolarisation due to disorders in the structure and/or function of the neuromuscular synapse. Mutations of the nicotinic acetylcholine receptor (nAChR) form a major subset of CMS. We describe a patient who presented with recurrent apnoeic crises in the neonatal period requiring ventilator support. Electromyography revealed compound muscle action potential decrement upon repetitive stimulation. Sequencing of nAChR subunit genes revealed two missense mutations. One previously reported null mutation p.εTyr15His, and a second novel missense mutation, p.εThr38Lys, that is well expressed in mammalian cell culture and thus likely to exert its effect via alteration of ion channel kinetics. Functional analysis revealed abbreviated ion channel bursts characteristic of a fast channel CMS. The mutation p.εThr38Lys occurs at the interface between the α and ε subunits of the nAChR pentamer and leads to instability of the open channel. The effects of this mutation on channel function were investigated in relation to other fast channel mutants at an analogous subunit interface within the nAChR pentamer. Fast channel syndromes are frequently characterised by severe myasthenic weakness with apnoeic crises; knowledge of the underlying mutation and its functional consequences can be vital for appropriate therapy and patient management.

Salbutamol therapy in congenital myasthenic syndrome due to DOK7 mutation.

INTRODUCTION: Salbutamol is a selective B2-adrenergic agonist, which has previously been described to be associated with partial improvement of myasthenia gravis and congenital myasthenic syndromes (CMS). In this study, we analyzed the effect of salbutamol in five patients with Dok-7 CMS. METHODS: We studied 5 patients (2 male and 3 female), with a mean age of 27±11.06 years, who harbored c.1124_1127dupTGCC, p.G64R and/or p.S45L mutations in DOK7 gene. Salbutamol was given at a dose of 2mg three times daily (6 mg/day) to all patients. The response was assessed by QMG score at baseline, 3, 6, 9 and 12 months; ADL-MG score and 6 minute walk test at baseline and after 12 months during follow-up clinic visits. Side effect profile of salbutamol was also evaluated. RESULTS: We noted an increasingly positive response as measured by the QMG score after 3 months of salbutamol treatment. Improvement in specific subcomponents of the QMG score such as leg outstretched in 45° supine was most marked. In ADL-MG scores and 6 minute walk test, comparison between baseline and after 12 months revealed a clear beneficial response. Salbutamol was well tolerated in all patients. CONCLUSIONS: Salbutamol is an effective treatment in Dok-7 CMS. This study provides class IV evidence that salbutamol given at a dose 6 mg/day improves function as measured by the QMG score, ADL-MG score and 6 minute walk test.

Presymptomatic late-onset Pompe disease identified by the dried blood spot test.

Pompe disease or glycogen storage disease type II is an autosomal recessive disorder caused by mutations in the GAA gene leading to muscle weakness. Here we describe a 15 years old presymptomatic patient with normal muscle MRI, unspecific muscle biopsy findings but abnormal acid maltase activity in a dried blood spot test. Sequencing the GAA-gene identified a heterozygous novel splice-site and a heterozygous previously described mutation. The case highlights the variability in clinical phenotype and difficulties to diagnose late-onset Pompe disease. Dried Blood Spot (DBS) might be the most sensitive tool to pick up mildly symptomatic patients.

Congenital myasthenic syndromes: achievements and limitations of phenotype-guided gene-after-gene sequencing in diagnostic practice: a study of 680 patients.

Congenital myasthenic syndromes (CMSs) are clinically and genetically heterogeneous disorders characterized by a neuromuscular transmission defect. Even though CMSs are genetic disorders, they are highly treatable, and the appropriate drug treatment depends on the underlying genetic defect. This highlights the importance of genetic testing in CMS. In recent years, the molecular basis of CMS has constantly broadened and disease-associated mutations have been identified in 14 genes encoding proteins of the neuromuscular junction. In the dawn of novel sequencing strategies, we report on our 14-year experience in traditional Sanger-based mutation screening of a large cohort of 680 independent patients with suspected CMS. In total, we identified disease-causing mutations in 299 patients (44%) of patients in various known CMS genes, confirming the high degree of genetic heterogeneity associated with the disease. Apart from four known founder mutations, and a few additional recurrent mutations, the majority of variants are private, found in single families. The impact of previously reported genotype-phenotype correlations on efficiency of genetic testing was analyzed in our population. Taking our experiment into account, we present our algorithm for genetic testing in CMS.