Evaluation of Optical Genome Mapping in Clinical Genetic Testing of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy, caused by the contraction of the D4Z4 repeats on the permissive 4qA haplotype on chromosome 4, resulting in the faulty expression of the DUX4 gene. Traditional diagnostics are based on Southern blotting, a time- and effort-intensive method that can be affected by single nucleotide variants (SNV) and copy number variants (CNV), as well as by the similarity of the D4Z4 repeats located on chromosome 10. We aimed to evaluate optical genome mapping (OGM) as an alternative molecular diagnostic method for the detection of FSHD. We first performed optical genome mapping with EnFocus™ FSHD analysis using DLE-1 labeling and the Saphyr instrument in patients with inconclusive diagnostic Southern blot results, negative FSHD2 results, and clinically evident FSHD. Second, we performed OGM in parallel with the classical Southern blot analysis for our prospectively collected new FSHD cases. Finally, panel exome sequencing was performed to confirm the presence of FSHD2. In two patients with diagnostically inconclusive Southern blot results, OGM was able to identify shortened D4Z4 repeats on the permissive 4qA alleles, consistent with the clinical presentation. The results of the prospectively collected patients tested in parallel using Southern blotting and OGM showed full concordance, indicating that OGM is a useful alternative to the classical Southern blotting method for detecting FSHD1. In a patient showing clinical FSHD but no shortened D4Z4 repeats in the 4qA allele using OGM or Southern blotting, a likely pathogenic variant in SMCHD1 was detected using exome sequencing, confirming FSHD2. OGM and panel exome sequencing can be used consecutively to detect FSHD2.

Telemedicine in Neuromuscular Diseases During Covid-19 Pandemic: ERN-NMD European Survey.

BACKGROUND: Telemedicine (TM) contributes to bridge the gap between healthcare facilities and patients' homes with neuromuscular disease (NMD) because of mobility issues. However, its deployment is limited due to difficulties evaluating subtle neurological signs such as mild weakness or sensory deficits. The COVID-19 pandemic has disrupted healthcare delivery worldwide, necessitating rapid measures implementation by health care providers (HCPs) to protect patients from acquiring SARS-CoV-2 while maintaining the best care and treatment. OBJECTIVES: Given the challenges faced by remote healthcare assistance of NMD patients, we aim to evaluate the use of TM in NMD during the COVID-19 pandemic. METHODS: Based on the Model for Assessment-of-Telemedicine-Applications (MAST), we conducted a survey amongst clinicians of the ERN EURO NMD (European-Reference-Network-for-Rare-Neuromuscular-Diseases). RESULTS: Based on 42 responses over 76 expected ones, our results show that the COVID-19 pandemic significantly increased the number of HCPs using TM (from 60% to 100%). The TM types most used during the COVID-19 period are teleconsultation and consultation by phone, particularly in the context of symptoms worsening in NMD patients with COVID-19 infection. Most European HCPs were satisfied when using TM but as a complementary option to physical consultations. Many responses addressed the issue of technical aspects needing improvement, particularly for elderly patients who need caregivers' assistance for accessing the TM platform. CONCLUSIONS: TM has been essential during COVID-19, but its use still presents some limitations for NMD patients with cognitive deficits or for first-time diagnosis. Thus, TM should be used as complement to, rather than substitute, for face-to-face consultations.

Cone-beam computed tomography guided nusinersen administrations in adult spinal muscular atrophy patients with challenging access: a single- center experience.

BACKGROUND: The challenging anatomic predispositions in adult patients with spinal muscular atrophy (SMA) preclude the conventional lumbar punctures. Consequently, an introduction of alternative method for intrathecal delivery of nusinersen is required. Cone-beam CT (CBCT) allows volumetric display of the area of interest, pre-procedural planning and real time needle guidance which results in accurate anatomic navigation. The aim of the study was to evaluate technical success, safety, and feasibility of CBCT lumbar intrathecal delivery of nusinersen in the adult SMA patients with challenging anatomical access. PATIENTS AND METHODS: Thirty-eight adult SMA patients were treated in our institution. Patients with challenging access were selected by multidisciplinary board for image guided administration of nusinersen either due to implantation of the posterior fusion instrumentation, severe scoliosis defined as Cobb's angle > 40º or body mass index over 35. Technical success, radiation exposure and occurrence of adverse events were assessed. RESULTS: Twenty patients were selected, and 108 CBCT-guided procedures were performed. Each patient underwent at least 4 administrations. Transforaminal approach was performed in 82% of patients. The technical success was 100%, with primary success of 93.5%. The median radiation effective dose of the administrations was 5 mSv, the mean value equalled 10 mSv. Only mild adverse events were reported in the study. CONCLUSIONS: CBCT-guided lumbar intrathecal administrations of nusinersen in an adult SMA population with challenging access was feasible and safe image guided method.

CAPN3 c.1746-20C>G variant is hypomorphic for LGMD R1 calpain 3-related.

The investigated intronic CAPN3 variant NM_000070.3:c.1746-20C>G occurs in the Central and Eastern Europe with a frequency of >1% and there are conflicting interpretations on its pathogenicity. We collected data on 14 patients carrying the CAPN3 c.1746-20C>G variant in trans position with another CAPN3 pathogenic/likely pathogenic variant. The patients compound heterozygous for the CAPN3 c.1746-20C>G variant presented a phenotype consistent with calpainopathy of mild/medium severity. This variant is most frequent in the North/West regions of Russia and may originate from that area. Molecular studies revealed that different splicing isoforms are produced in the muscle. We hypothesize that c.1746-20C>G is a hypomorphic variant with a reduction of RNA and protein expression and only individuals having a higher ratio of abnormal isoforms are affected. Reclassification of the CAPN3 variant c.1746-20C>G from variant with a conflicting interpretation of pathogenicity to hypomorphic variant explains many unidentified cases of limb girdle muscular dystrophy R1 calpain 3-related in Eastern and Central Europe.

Shaky hands are a part of motor neuron disease phenotype: clinical and electrophysiological study of 77 patients.

BACKGROUND: In the sharp contrast with the existing literature, we frequently observe minipolymyoclonus, tremor and pseudodystonic thumb posturing in patients with motor neuron disease. We conducted a clinical and electrophysiological study to describe phenomenology, prevalence and pathophysiology of involuntary movements in motor neuron disease. METHODS: We included 77 consecutive patients. Involuntary movements were assessed at rest and on action. Patients were videotaped. Arm muscle tone, power and deep tendon reflexes were evaluated. Accelerometry with electromyography was recorded in a subset of patients. RESULTS: Involuntary movements were observed in 68.9% of patients and could be separated into rest minipolymyoclonus, thumb tremor, pseudodystonic thumb posture, action minipolymyoclonus, and action tremor. One-third of patients reported negative impact of involuntary movements on hand use. Logistic regression showed that rest minipolymyoclonus and thumb tremor were more likely to occur in patients with more prominent distal muscle weakness and less spasticity. Similarly, action involuntary movements were more likely to appear in weaker patients. Patients with brisk tendon reflexes were more likely to display action tremor than action minipolymyoclonus. Action tremor was characterized by accelerometer and corresponding electromyography peak frequency, which decreased with mass loading, suggesting a mechanical-reflex tremor. CONCLUSIONS: Involuntary movements are common, but poorly recognized feature of motor neuron disease that may add to functional impairment. Results of our study suggest that involuntary movements are likely of peripheral origin, with a non-fused contraction of enlarged motor units being a common driving mechanism. Minipolymyoclonus appears if no synchronization of motor units occurs. When synchronization occurs via stretch reflex, mechanical-reflex tremor is generated.

Axonal Polyneuropathy in 2 Brothers With a Homozygous Missense Variant in the First Catalytic Domain of PCYT2.

BACKGROUND AND OBJECTIVES: To expand the phenotype and genotype associated with PCYT2-related disorder. METHODS: Exome sequencing data from a patient with molecularly undiagnosed complex spastic paraplegia and axonal motor and sensory polyneuropathy were analyzed. Clinical data and nerve conduction studies of the patient and his affected brother were collected, and their phenotype and genotype were compared with previously reported patients with PCYT2-related disorder. RESULTS: A novel homozygous missense variant in PCYT2 (NM_001184917.2) c.88T>G; p.(Cys30Gly) was identified. This variant is located in a highly conserved tyrosine kinase site and is predicted damaging by several variant annotation tools. Both patients reported here and the previously published patients share several phenotypic features, including short stature, spastic tetraparesis, cerebellar ataxia, epilepsy, and cognitive decline. Axonal polyneuropathy, diagnosed in both brothers, was not previously reported. DISCUSSION: This family with a novel PCYT2 variant expands the clinical spectrum of PCYT2-related disorder to include axonal motor and sensory polyneuropathy and the genetic spectrum to include the variant located in the first catalytic domain, whereas all previously reported variants are located in the second catalytic domain. Further research is required to disentangle the underlying pathophysiologic mechanisms, leading to the complex phenotype of PCYT2-related disorder.

Diagnostic yield of exome sequencing in myopathies: Experience of a Slovenian tertiary centre.

BACKGROUND: Our aim was to present the experience of systematic, routine use of next generation sequencing (NGS) in clinical diagnostics of myopathies. METHODS: Exome sequencing was performed on patients with high risk for inherited myopathy, which were selected based on the history of the disease, family history, clinical presentation, and diagnostic workup. Exome target capture was performed, followed by sequencing on HiSeq 2500 or MiSeq platforms. Data analysis was performed using internally developed bioinformatic pipeline. RESULTS: The study comprised 86 patients, including 22 paediatric cases (26%). The largest group were patients referred with an unspecified myopathy (47%), due to non-specific or incomplete clinical and laboratory findings, followed by congenital myopathies (22%) and muscular dystrophies (22%), congenital myotonias (6%), and mitochondrial myopathies (3%). Altogether, a diagnostic yield was 52%; a high diagnostic rate was present in paediatric patients (64%), while in patients with unspecified myopathies the rate was 35%. We found 51 pathogenic/likely pathogenic variants in 23 genes and two pathogenic copy number variations. CONCLUSION: Our results provide evidence that phenotype driven exome analysis diagnostic approach facilitates the diagnostic rate of complex, heterogeneous disorders, such as myopathies, particularly in paediatric patients and patients with unspecified myopathies.

Biallelic loss-of-function variants in NEMF cause central nervous system impairment and axonal polyneuropathy.

We aimed to detect the causative gene in five unrelated families with recessive inheritance pattern neurological disorders involving the central nervous system, and the potential function of the NEMF gene in the central nervous system. Exome sequencing (ES) was applied to all families and linkage analysis was performed on family 1. A minigene assay was used to validate the splicing effect of the relevant discovered variants. Immunofluorescence (IF) experiment was performed to investigate the role of the causative gene in neuron development. The large consanguineous family confirms the phenotype-causative relationship with homozygous frameshift variant (NM_004713.6:c.2618del) as revealed by ES. Linkage analysis of the family showed a significant single-point LOD of 4.5 locus. Through collaboration in GeneMatcher, four additional unrelated families' likely pathogenic NEMF variants for a spectrum of central neurological disorders, two homozygous splice-site variants (NM_004713.6:c.574+1G>T and NM_004713.6:c.807-2A>C) and a homozygous frameshift variant (NM_004713.6: c.1234_1235insC) were subsequently identified and segregated with all affected individuals. We further revealed that knockdown (KD) of Nemf leads to impairment of axonal outgrowth and synapse development in cultured mouse primary cortical neurons. Our study demonstrates that disease-causing biallelic NEMF variants result in central nervous system impairment and other variable features. NEMF is an important player in mammalian neuron development.

Improvements in the multidisciplinary care are beneficial for survival in amyotrophic lateral sclerosis (ALS): experience from a tertiary ALS center.

Objective: The Ljubljana ALS Centre, established in 2002, is the only tertiary center for amyotrophic lateral sclerosis (ALS) in Slovenia. The aim of our study was to evaluate the impact of therapeutic interventions and improvements in the multidisciplinary care on the survival of our patients.Methods: All patients diagnosed with ALS at our center during years 2003-2005 (early group) and 2011-2012 (late group) were included in this retrospective cohort study (n = 124). Kaplan-Meier survival analysis and multiple regression analysis with Cox proportional hazards model were performed to compare survival and to evaluate the differences between the two cohorts.Results: Median survival from the time of diagnosis was 13.0 (95% CI 10.2-15.8) months in the early group and 21.8 (95% CI 17.2-26.4) months in the late group (p = 0.005). In the Cox proportional hazards analysis, the late group of patients was associated with better survival independently of all other prognostic factors (hazard ratio (HR)=0.51, 95% CI = 0.32-0.81, p = 0.004). Survival was also associated with patients' age, use of noninvasive ventilation (NIV) and gastrostomy. The model fit significantly improved when the interaction between the NIV use and the observed time period was added to the model (HR = 0.34, 95% CI = 0.12-0.96, p = 0.041).Conclusions: Our findings suggest that improvements in the multidisciplinary care were beneficial for survival of our patients with ALS. The survival benefit in the late group of our patients could be partially explained by the improvements in the NIV use at our center.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome due to RFC1 repeat expansion.

Ataxia, causing imbalance, dizziness and falls, is a leading cause of neurological disability. We have recently identified a biallelic intronic AAGGG repeat expansion in replication factor complex subunit 1 (RFC1) as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and a major cause of late onset ataxia. Here we describe the full spectrum of the disease phenotype in our first 100 genetically confirmed carriers of biallelic repeat expansions in RFC1 and identify the sensory neuropathy as a common feature in all cases to date. All patients were Caucasian and half were sporadic. Patients typically reported progressive unsteadiness starting in the sixth decade. A dry spasmodic cough was also frequently associated and often preceded by decades the onset of walking difficulty. Sensory symptoms, oscillopsia, dysautonomia and dysarthria were also variably associated. The disease seems to follow a pattern of spatial progression from the early involvement of sensory neurons, to the later appearance of vestibular and cerebellar dysfunction. Half of the patients needed walking aids after 10 years of disease duration and a quarter were wheelchair dependent after 15 years. Overall, two-thirds of cases had full CANVAS. Sensory neuropathy was the only manifestation in 15 patients. Sixteen patients additionally showed cerebellar involvement, and six showed vestibular involvement. The disease is very likely to be underdiagnosed. Repeat expansion in RFC1 should be considered in all cases of sensory ataxic neuropathy, particularly, but not only, if cerebellar dysfunction, vestibular involvement and cough coexist.

IMG-01 The spectrum of involuntary movements in patients with motor neuron disease: a cross-sectional study.

Background: We have commonly observed involuntary jerks and tremor in patients with motor neuron disease (MND), even though these features are not considered typical for the disease.Objectives: We conducted prospective clinical and electrophysiological study to explore the prevalence, phenomenology and pathophysiology of involuntary movements in MND.Methods: Seventy-four consecutive patients were clinically examined and video-recorded. Based on regularity and distribution, movements observed at rest position were classified as minipolymyoclonus (MPMC) or rest thumb tremor (RTT) and movements present during action as action MPMC or action tremor. In 11 patients with tremor, accelerometry was recorded at (a) rest position, (b) with arms outstretched (postural condition) and (c) at postural condition with 500 g mass attached to the hand.Results: Involuntary movements were present in 54 patients (73%). Rest MPMC was present in 26 patients (35%), RTT in 22 patients (31%), action MPMC in 22 patients (30%) and action tremor in 20 patients (27%), with some overlap. Sixteen patients (22%) reported negative impact of involuntary movements on their ability to use hands. Regression model showed that lower distal muscle power and less prominent upper motor neuron involvement significantly increased the odds of MND patient having involuntary movements. Sex, age and disease duration did not significantly predict the occurrence of involuntary movements. At rest, tremor frequency ranged from 5.2 to 8.2 Hz, at postural position from 4.9 Hz to 7.6 Hz and during postural position with mass attached from 3.6 Hz to 7.6 Hz. On the group level, tremor peak frequency statistically significantly decreased from 6.1 Hz to 5 Hz without versus with loading.Discussion and conclusions: Involuntary movements are very common yet largely overlooked feature of MND that may also have negative impact on patient's functional abilities. Lower distal muscle power increases and the presence of upper motor neuron signs decreases the probability of involuntary movements. Together with finding of decrease in tremor frequency with mass loading, these results suggest that generation of involuntary movements is of peripheral origin.

Selective screening of late-onset Pompe disease (LOPD) in patients with non-diagnostic muscle biopsies.

AIMS: As of 2016, there were five patients with Pompe in Slovenia (two infantile, one childhood and two adult onset) with a prevalence of 1:400 000; however, the prevalence of late-onset Pompe disease (LOPD) in some other countries means this ratio could be an underestimate. Since an LOPD muscle biopsy could be unspecific or even normal, the purpose of this study is to assess the prevalence of LOPD in patients with non-diagnostic muscle biopsies. METHODS: Six hundred biopsies were recorded at the Neuromuscular Tissue Bank of the University of Ljubljana for the period 2004-2014. All adult patients with non-diagnostic muscle biopsies were invited to the National Slovenian Neuromuscular Centre for dried blood spot testing for LOPD. RESULTS: A total of 90 patients (56% of those invited) responded. No patient with LOPD was found. A total of 49 patients (54%) had fixed muscle weakness, 31 (34%) had mild symptoms and no weakness and 10 (11%) had asymptomatic hyperCKemia. Ventilatory insufficiency associated with proximal muscle weakness was found in two patients (2%). No patients exhibited vacuolar myopathy, globular accumulations of glycogen or regions of increased acid phosphatase activity within the sarcoplasm. CONCLUSIONS: The study results do not support the hypothesis that LOPD is underestimated in Slovenian patients with non-diagnostic muscle biopsies; this could be consistent with the fact that LOPD is of low prevalence in Slovenia, as is the case in the populations of Finland, French-speaking Belgium, west Sweden and west Denmark.

Differential expression of microRNAs and other small RNAs in muscle tissue of patients with ALS and healthy age-matched controls.

Amyotrophic lateral sclerosis is a late-onset disorder primarily affecting motor neurons and leading to progressive and lethal skeletal muscle atrophy. Small RNAs, including microRNAs (miRNAs), can serve as important regulators of gene expression and can act both globally and in a tissue-/cell-type-specific manner. In muscle, miRNAs called myomiRs govern important processes and are deregulated in various disorders. Several myomiRs have shown promise for therapeutic use in cellular and animal models of ALS; however, the exact miRNA species differentially expressed in muscle tissue of ALS patients remain unknown. Following small RNA-Seq, we compared the expression of small RNAs in muscle tissue of ALS patients and healthy age-matched controls. The identified snoRNAs, mtRNAs and other small RNAs provide possible molecular links between insulin signaling and ALS. Furthermore, the identified miRNAs are predicted to target proteins that are involved in both normal processes and various muscle disorders and indicate muscle tissue is undergoing active reinnervation/compensatory attempts thus providing targets for further research and therapy development in ALS.

Differential Expression of Several miRNAs and the Host Genes AATK and DNM2 in Leukocytes of Sporadic ALS Patients.

Genetic studies have managed to explain many cases of familial amyotrophic lateral sclerosis (ALS) through mutations in several genes. However, the cause of a majority of sporadic cases remains unknown. Recently, epigenetics, especially miRNA studies, show some promising aspects. We aimed to evaluate the differential expression of 10 miRNAs, including miR-9, miR-338, miR-638, miR-663a, miR-124a, miR-143, miR-451a, miR-132, miR-206, and let-7b, for which some connection to ALS was shown previously in ALS culture cells, animal models or patients, and in three miRNA host genes, including C1orf61 (miR-9), AATK (miR-338), and DNM2 (miR-638), in leukocyte samples of 84 patients with sporadic ALS. We observed significant aberrant dysregulation across our patient cohort for miR-124a, miR-206, miR-9, let-7b, and miR-638. Since we did not use neurological controls we cannot rule out that the revealed differences in expression of investigated miRNAs are specific for ALS. Nevertheless, the group of these five miRNAs is worth of additional research in leukocytes of larger cohorts from different populations in order to verify their potential association to ALS disease. We also detected a significant up-regulation of the AAKT gene and down-regulation of the DNM2 gene, and thus, for the first time, we connected these with sporadic ALS cases. These findings open up new research toward miRNAs as diagnostic biomarkers and epigenetic processes involved in ALS. The detected significant deregulation of AAKT and DNM2 in sporadic ALS also represents an interesting finding. The DNM2 gene was previously found to be mutated in Charcot-Marie-Tooth neuropathy-type CMT2M and centronuclear myopathy (CNM). In addition, as recent studies connected AATK and frontotemporal dementia (FTD) and DNM2 and hereditary spastic paraplegia (HSP), these two genes together with our results genetically connect, at least in part, five diseases, including FTD, HSP, Charcot-Marie-Tooth (type CMT2M), CNM, and ALS, thus opening future research toward a better understanding of the cell biology involved in these partly overlapping pathologies.

Template-operated MUP analysis is not accurate in the diagnosis of myopathic or neuropathic changes in the diaphragm.

OBJECTIVES: The aim of this study was to evaluate the quantitative motor unit potential (MUP) analysis in the diagnosis of myopathy and neuropathy of the diaphragm. METHODS: Diaphragm template-operated quantitative EMG were performed in 30 patients with myotonic dystrophy type 1 (DM1), 17 with myotonic dystrophy type 2 (DM2) and 40 with amyotrophic lateral sclerosis (ALS). RESULTS: Low MUP amplitude precluded MUP analysis in 21% of DM1 patients. Only a single DM1 patient had EMG findings consistent with myopathy. In this patient, and another 4 DM1 and 3 DM2 patients, findings were consistent with neuropathy. Neuropathic MUP changes were found in 92% of ALS patients, but due to motor neuron cell loss in only 60% MUPs analyses could be done. Phrenic nerve conduction studies correlated with respiratory function tests, whereas MUP parameters did not. CONCLUSION: Quantitative MUP analysis was not able to adequately sample diaphragm MUPs in patients with chronic myopathy or motor neuronopathy.

Genome-wide association analyses identify new risk variants and the genetic architecture of amyotrophic lateral sclerosis.

To elucidate the genetic architecture of amyotrophic lateral sclerosis (ALS) and find associated loci, we assembled a custom imputation reference panel from whole-genome-sequenced patients with ALS and matched controls (n = 1,861). Through imputation and mixed-model association analysis in 12,577 cases and 23,475 controls, combined with 2,579 cases and 2,767 controls in an independent replication cohort, we fine-mapped a new risk locus on chromosome 21 and identified C21orf2 as a gene associated with ALS risk. In addition, we identified MOBP and SCFD1 as new associated risk loci. We established evidence of ALS being a complex genetic trait with a polygenic architecture. Furthermore, we estimated the SNP-based heritability at 8.5%, with a distinct and important role for low-frequency variants (frequency 1-10%). This study motivates the interrogation of larger samples with full genome coverage to identify rare causal variants that underpin ALS risk.

Single fiber EMG as a prognostic tool in myasthenia gravis.

INTRODUCTION: Single fiber electromyography (SFEMG) is the most sensitive diagnostic tool for diagnosis of myasthenia gravis (MG). Its prognostic value is not known. METHODS: We retrospectively analyzed the clinical course of 232 MG patients who presented with only mild symptoms and had SFEMG of the orbicularis oculi muscle. We correlated their SFEMG results with the severity of their later clinical course. RESULTS: During the observation period 39 patients (17%) developed severe disease exacerbations, and 193 (83%) remained stable. Patients with severe disease exacerbation had a significantly higher mean jitter value (P < 0.0001), a greater percentage of fibers with increased jitter (P < 0.0001), and/or impulse blocking (P < 0.0001) on SFEMG. CONCLUSIONS: The extent of the SFEMG abnormalities in this study correlated with the later clinical course of MG. Muscle Nerve 54: 1034-1040, 2016.

Fatal recurrent dermatoneuro syndrome associated with systemic AL amyloidosis.

A male patient is presented with long-lasting paraproteinemia of monoclonal IgG λ, who suffered from recurrent, and until the last one, mostly reversible episodes of dermatoneuro syndrome, described exclusively in scleromyxedema. The skin biopsy revealed λ-light chain amyloid deposition instead of changes typical for scleromyxedema. Systemic AL amyloidosis was diagnosed post mortem since the patient had no clinical signs of any other organ impairment except skin and brain. Neuropathology is described and possible etiopathogenesis of brain involvement is considered.

Reference values for jitter recorded by concentric needle electrodes in healthy controls: A multicenter study.

INTRODUCTION: The aim of this study was to create reference values for jitter measured with concentric needle electrodes. METHODS: Operators worldwide contributed recordings from orbicularis oculi (OO), frontalis (FR), and extensor digitorum (ED) muscles in healthy controls. Criteria for acceptable signal quality were agreed upon in advance. Fifteen or 20 recordings of acceptable quality from each muscle were required for voluntary and electrical stimulation recordings, respectively. RESULTS: Recordings from 59 to 92 subjects were obtained for each muscle and activation type. Outlier limits for mean consecutive difference and individual jitter data for voluntary activation were: OO, 31 and 45 µs; FR, 28 and 38 µs; ED, 30 and 43 µs; and for electrical stimulation they were: OO, 27 and 36 µs; FR, 21 and 28 µs; ED, 24 and 35 µs. CONCLUSION: Reference jitter values from concentric needle electrode recordings were developed from signals of defined quality while seeking to avoid creating supernormal values.