Estimating motor unit numbers from a CMAP scan: Repeatability study on three muscles at 15 centres.

OBJECTIVE: To assess the repeatability and suitability for multicentre studies of MScanFit motor unit number estimation (MUNE), which involves modelling compound muscle action potential (CMAP) scans. METHODS: Fifteen groups in 9 countries recorded CMAP scans twice, 1-2 weeks apart in healthy subjects from abductor pollicis brevis (APB), abductor digiti minimi (ADM) and tibialis anterior (TA) muscles. The original MScanFit program (MScanFit-1) was compared with a revised version (MScanFit-2), designed to accommodate different muscles and recording conditions by setting the minimal motor unit size as a function of maximum CMAP. RESULTS: Complete sets of 6 recordings were obtained from 148 subjects. CMAP amplitudes differed significantly between centres for all muscles, and the same was true for MScanFit-1 MUNE. With MScanFit-2, MUNE differed less between centres but remained significantly different for APB. Coefficients of variation between repeats were 18.0% for ADM, 16.8% for APB, and 12.1% for TA. CONCLUSIONS: It is recommended for multicentre studies to use MScanFit-2 for analysis. TA provided the least variable MUNE values between subjects and the most repeatable within subjects. SIGNIFICANCE: MScanFit was primarily devised to model the discontinuities in CMAP scans in patients and is less suitable for healthy subjects with smooth scans.

Inherited neuropathies with predominant upper limb involvement: genetic heterogeneity and overlapping pathologies.

BACKGROUND AND PURPOSE: In a subset of patients with inherited peripheral neuropathies the first symptom is atrophy and weakness of the intrinsic muscles of the hands, without involvement of lower limbs until later in the disease course. The exact pathomechanisms of this phenotype are currently unknown. The aim of this study was to characterize the clinical, neurophysiological and genetic features of a group of patients with a clinical diagnosis of upper limb predominant Charcot-Marie-Tooth disease (CMT). METHODS: The clinical, electrophysiology and genetic data of 11 patients with upper limb predominant peripheral neuropathy selected from a single-centre cohort of 461 patients diagnosed with inherited neuropathy were analysed and the clinical, electrophysiological and genetic characteristics of these patients reported. RESULTS: An overlapping phenotype of neuropathy and myopathy was detected in two patients. Four patients carry autosomal dominant mutations in GARS and a single patient had a homozygous mutation in SH3TC2. However, the underlying genetic diagnosis could not be confirmed in six patients by gene panel sequencing. CONCLUSIONS: Upper limb-onset inherited neuropathies are genetically heterogeneous and, in some cases, there is an overlapping myopathy. Autosomal dominant GARS mutations are the most common genetic cause; however, mutations in other CMT genes may also result in this phenotype in individual patients. The majority of these patients cannot be genetically diagnosed by gene panel testing of known CMT and myopathy genes, suggesting further genetic heterogeneity and highlighting the importance of further genetic investigations in these patients and families.

Correction: Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease.

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Video telemetry: current concepts and recent advances.

Epilepsy is a clinical diagnosis based on the history of the patient and of witnesses. Sometimes this is not available or is incomplete, thus making diagnosis uncertain. In other cases, specifically in patients with intractable epilepsy being considered for epilepsy surgery, the diagnosis of epilepsy is not in doubt but the precise localisation of the epileptogenic zone needs to be determined. In both these situations, video telemetry plays a key role and is now a routine in most neuroscience units. This review covers existing practice and the exciting recent development of home video telemetry.

Initial development and validation of a mitochondrial disease quality of life scale.

Mitochondrial diseases are a clinically diverse group of genetic disorders that often present to neurologists. Health related quality of life (HRQOL) is increasingly recognised as a fundamental patient based outcome measure in both clinical intervention and research. Generic outcome measures have been extensively validated to assess HRQOL across populations and different disease states. However, due to their inclusive construct, it is acknowledged that not all relevant aspects of a specific illness may be captured. Hence there is a need to develop disease specific HRQOL measures that centre on symptoms characteristic of a specific disease or condition and their impact. This study presents the initial conceptualisation, development and preliminary psychometric assessment (validity and reliability) of a mitochondrial disease specific HRQOL measure (Newcastle Mitochondrial Quality of life measure (NMQ)). NMQ is a valuable assessment tool and consists of 63 items within 16 unidimensional domains, each demonstrating good internal reliability (Cronbach's α≥0.83) and construct validity.

Mitochondrial disease in pregnancy: a systematic review.

Mitochondrial diseases are heterogeneous in clinical presentation and genotype. The incidence of known pathogenic mitochondrial DNA mutations in the general population is 1 in 500. Little is known about the implications of pregnancy for women with mitochondrial disease. We undertook a systematic review of the literature on mitochondrial disease in pregnancy. Ten case reports were identified. The most common complications were threatened preterm labour (5 women) and preeclampsia (4 women). Two women experienced magnesium sulphate toxicity. Pregnancy had a varied effect on mitochondrial disease with some women being asymptomatic; others developing mild symptoms such as exercise intolerance or muscle weakness which resolved postnatally; and others developed more serious, persistent symptoms such as symptomatic Wolff-Parkinson-White syndrome, persistent paraesthesia and focal segmental glomerulosclerosis. Women with mitochondrial disease appear to be at increased risk of complications during pregnancy and labour but further prospective cohort studies are needed.

Multi-system neurological disease is common in patients with OPA1 mutations.

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal 'dominant optic atrophy plus' variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44-6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08-4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

A diagnostic tattoo.

The hallmarks of the myoclonic epilepsy with ragged red fibers (MERRF) syndrome are myoclonic epilepsy, ataxia and ragged red fibres detected on muscle biopsy. We present a case of a 25-year-old male who first presented to his general practitioner at the age of 22 years with myoclonic jerks affecting the arms and legs, fatigue and mild ataxia. He was found to carry an A>G transition at nucleotide 8344 in mitochondrial DNA. This mutation is the most common cause of the MERRF syndrome, found in more than 80% of affected patients. Our patient had the diagnosis tattooed on his arm, both out of frustration at how few people had heard of it, and as a way of accepting that his condition was a part of who he was. Although the MERRF syndrome is one of the more common forms of mitochondrial encephalomyopathy, with a prevalence estimated at between 0.25 and 0.39 per 100,000, it is still a rare disorder. We are always striving to increase the public's understanding of these important conditions. Our patient has perhaps helped more than most towards this aspiration.

Detecting seizure origin using basic, multiscale population dynamic measures: preliminary findings.

Many types of electrographic seizures are readily identifiable by direct visual examination of electroencephalographic or electrocorticographic recordings. This process can, however, be painstakingly slow, and much effort has been expended to automate the process using various dynamic properties of epileptiform waveforms. As methods have become more subtle and powerful they have been used for seizure subclassification, seizure prediction, and seizure onset identification and localization. Here we concentrate on the last, with reference to seizures of neocortical origin. We briefly review some of the methods used and introduce preliminary results from a very simple dynamic model based on key electrophysiological properties found in some seizure types: occurrence of very fast oscillations (sometimes called ripples), excess gamma frequency oscillations, electroencephalographic/electrocorticographic flattening, and changes in global synchrony. We show how this multiscale analysis may reveal features unique to seizure onset and speculate on the underlying cellular and network phenomena responsible.

Prevalence and progression of diabetes in mitochondrial disease.

AIMS/HYPOTHESIS: The aims of this study were (1) to determine the prevalence and rate of progression in diabetes secondary to mitochondrial DNA (mtDNA) mutations; and (2) to determine whether percentage heteroplasmy predicts clinical outcome in patients carrying the m.3243A>G mutation. METHODS: We prospectively assessed 242 patients attending a specialist neuromuscular clinic using a validated mitochondrial disease rating scale. Retrospective clinical data on these patients from up to 25 years of follow-up were also included. Percentage heteroplasmy in blood, urine and muscle was determined for the m.3243A>G group and correlated against clinical features. RESULTS: Patients carrying the m.3243A>G mutation formed the largest group of patients with diabetes (31/81 patients). The highest prevalence of diabetes was in the m.12258C>A group (2/2 patients), the lowest in the multiple mtDNA deletions group (3/43 patients). The earliest age of onset was in the m.3243A>G group (37.9 years) with the highest age of presentation in the multiple deletion group (56.3 years). Of patients presenting with m.3243A>G, 12.9% required insulin; an additional 32.3% progressed to insulin requirement over a mean of 4.2 years after presentation. Percentage heteroplasmy in blood, urine or muscle did not predict progression of diabetes or risk of developing complications. Early age of presentation with diabetes did predict poor clinical outcome. CONCLUSIONS/INTERPRETATION: Although patients carrying the m.3243A>G mutation account for the majority of cases of diabetes secondary to mtDNA mutations, several other genotypes are also associated with the development of diabetes, some with high penetrance. All show a gradual progression to insulin requirement. Percentage heteroplasmy is a poor predictor of severity of diabetes in the m.3243A>G group.

Myotonic dystrophy: practical issues relating to assessment of strength.

BACKGROUND: Myotonic dystrophy type 1 is a slowly progressive multisystem disease in which skeletal muscle involvement is prominent. As novel physical and pharmacological treatments become available, it is crucial to be able to measure their efficacy accurately. METHODS: 158 consecutive patients with myotonic dystrophy were assessed annually in a specialist muscle clinic. Strength was measured using both the Medical Research Council (MRC) scale and a hand-held dynamometer. Dynamometer readings were obtained from 108 normal subjects (controls). RESULTS: The movements showing the greatest rate of change in strength were ankle dorsiflexion and pinch grip. Both of these showed a decline of only 0.06 points/year on the MRC scale. Using a hand-held dynamometer, a change in strength of 1.18 kgN/year for women and 1.61 kgN/year for men was detected. CONCLUSIONS: The MRC scale is unsuitable for detecting the small changes in strength seen in a slowly progressive disease such as myotonic dystrophy. Dynamometry provides a simple alternative that can give meaningful data over the duration of a typical clinical trial.

Substrate preference profiles of proteases released by allergenic pollens.

BACKGROUND: Pollens are important triggers for allergic asthma and seasonal rhinitis. We have recently reported that proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of epithelial integrity by proteases released following deposition of pollens on mucosal surfaces could promote sensitization and induce inflammation. OBJECTIVE: To compare protease activities released by allergenic pollens of various genera. METHODS: We used a rapid microassay which quantifies cleavage of dipeptide ester substrates to characterize the substrate preference profiles of serine proteases in diffusates of the pollens of perennial ryegrass (Lolium perenne), Kentucky blue grass (Poa pratensis), Bermuda grass (Cynodon dactylon), Western ragweed (Ambrosia spp.), white birch (Betula spp.) and Sydney golden wattle (Acacia longifolia). RESULTS: Comparison of the profiles revealed notable differences as well as similarities between serine protease activities released by these pollens. Diffusates of Kentucky blue grass pollen exhibited very high substrate preference for arginine and lysine. For other pollens, cleavage of the cysteine substrate was usually the most rapid and was associated with marked preference for leucine and methionine. There was considerable variation between these pollens in the rates of cleavage of the histidine substrate. In addition, we observed high rates of cleavage of arginine and lysine substrates by Acacia pollen diffusate. CONCLUSION: At least two dominant patterns of substrate preference are identifiable in the mixtures of proteases released by hydrated pollens. Purification of the proteases responsible for these patterns of activity will facilitate investigation of their role in airway epithelial injury and allergic disease.

A transfection compound series based on a versatile Tris linkage.

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Efficacy of fusion peptide homologs in blocking cell lysis and HIV-induced fusion.

Contrary to earlier reports, we have found that tri- and hexapeptides analogous or homologous with segments of the 23-residue N-terminal fusion sequence (FS) of the viral transmembrane glycoprotein gp41 (residues 517-539) did not significantly inhibit HIV-1-induced syncytium formation, using an uninfected cell-infected cell fusion assay. In contrast, we found that the high molecular weight apolipoprotein A-1 and a 23-residue analog of the FS, with the phenylalanine residues at positions 524 and 527 replaced with alanine residues, were effective inhibitors. Although the tripeptides were ineffective as inhibitors of syncytium formation, we found a number of them inhibited red cell lysis induced by the synthetic peptide AVGIGALFLGFLGAAGSTMGARS (based on the HIV-1 gp41 FS). This effect was also seen with apolipoprotein A-1. The Ala524,527 analog of the fusion sequence could not be tested in this system because it was hemolytic. We concluded that the smaller peptides were effective inhibitors of hemolysis because they interfered with pore formation by the fusion sequence peptide, either by disrupting the pores or by preventing the peptide from adopting the alpha-helical conformation found in the pores. On the other hand, membrane fusion, which is a prelude to syncytium formation, has been shown to require the fusion sequence in the beta-strand conformation. We argue that small peptides would be unable to block interaction between such strands, although larger molecules, such as apolipoprotein A-1 and the Ala524,527 analog, would be able to do so and thus inhibit fusion. It seems, therefore, that a successful drug directed against the FS-cell membrane interaction stage of syncytium formation would need to be of relatively high molecular weight and complexity.

Purification and characterization of S1 mutants of alpha-lytic protease having altered catalytic properties.

A procedure is described for purifying alpha-lytic protease and its mutants from culture supernatants of recombinant Escherichia coli. The method affords substantial amounts (approx. 80 mg) of homogeneous enzyme. We compared the cleavage preferences of wild-type alpha-lytic protease and of mutants containing the substitutions Ala190 ("parent"), Ala190/Val192/His213/Met218 (mutant 1), Ala190/His213/Leu218 (mutant 9), and Ala190/Thr213/Leu218 (mutant 55), and for each enzyme we found broad agreement between the results obtained with synthetic ester and amide substrates. Kinetic constants were determined for the purified enzymes using selected tetrapeptide p-nitroanilide substrates. Mutant 55 had broad specificity and high activity. In terms of kcat/Km it cleaved at Met and Phe residues two to three times as effectively as the Ala190 enzyme and cleaved at Ala 7 times more effectively than the wild-type protease. The Ala190/His213 enzymes showed a preference for cleavage at His and Met residues. Not only were their kcat values for cleavage at His increased (in relation to the Ala190 enzyme) by an order of magnitude, but they also exhibited large decreases in kcat/Km for cleavage at other residues; for example, the value for cleavage at Phe was 400- to 600-fold lower. Mutant 9 cleaved a recombinant IGF-II fusion protein at a unique His residue and also at a nearby Asn residue.