De Novo Pathogenic Variants in CACNA1E Cause Developmental and Epileptic Encephalopathy with Contractures, Macrocephaly, and Dyskinesias.

Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the α1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

A frameshift mutation in LRSAM1 is responsible for a dominant hereditary polyneuropathy.

Despite the high number of genes identified in hereditary polyneuropathies/Charcot-Marie-Tooth (CMT) disease, the genetic defect in many families is still unknown. Here we report the identification of a new gene for autosomal dominant axonal neuropathy in a large three-generation family. Linkage analysis identified a 5 Mb region on 9q33-34 with a LOD score of 5.12. Sequence capture and next-generation sequencing of the region of interest identified five previously unreported non-synonymous heterozygous single nucleotide changes or indels, four of which were confirmed by Sanger sequencing. Two sequence variants co-segregated with the disease, and one, a 2 bp insertion in the last exon of LRSAM1, was also absent in 676 ethnicity-matched control chromosomes. This frameshift mutation (p.Leu708Argfx28) is located in the C-terminal RING finger motif of the encoded protein. Ubiquitin ligase activity in transfected cells with constructs carrying the patient mutation was affected as measured by a higher level of abundance of TSG101, the only reported target of LRSAM1. Injections of morpholino oligonucleotides in zebrafish embryos directed against the ATG or last splice site of zebrafish Lrsam1 disturbed neurodevelopment, showing a less organized neural structure and, in addition, affected tail formation and movement. LRSAM1 is highly expressed in adult spinal cord motoneurons as well as in fetal spinal cord and muscle tissue. Recently, a homozygous mutation in LRSAM1 was proposed as a strong candidate for the disease in a family with recessive axonal polyneuropathy. Our data strongly support the hypothesis that LRSAM1 mutations can cause both dominant and recessive forms of CMT.

Cumulative effect of 5 daily sessions of θ burst stimulation on corticospinal excitability in amyotrophic lateral sclerosis.

INTRODUCTION: Excitotoxicity plays an important role in the pathogenesis of the preferential motor neuron death observed in amyotrophic lateral sclerosis (ALS). Continuous theta burst stimulation (cTBS) by transcranial magnetic stimulation has an inhibitory effect on corticospinal excitability (CSE). We characterized the neurophysiological changes induced by cTBS in ALS. METHODS: The patients received 5 daily sessions of cTBS. CSE was assessed at baseline and after each session of cTBS. RESULTS: The amplitude of a single pulse motor evoked potential was significantly decreased (34%) over the days. The amplitude returned to baseline a week after the last session. The resting motor threshold increased significantly, whereas intracortical inhibition and facilitation did not change over the sessions. CONCLUSIONS: Daily cTBS has a cumulative depressing effect on CSE in patients with ALS. These results suggest that modulation of CSE in ALS is possible, but repetitive sessions are needed to maintain the effect.

Structural MRI reveals cortical thinning in amyotrophic lateral sclerosis.

OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a fatal disease characterised by combined upper and lower motor neuron degeneration. An early and accurate diagnosis is important for patient care and might facilitate the search for a more effective therapy. MRI was used to study the whole cortical mantle, applying an unbiased surface based approach to identify a marker of upper motor neuron involvement in ALS. METHODS: Surface based cortical morphology analyses were performed on structural, 3T MRI data of 45 patients with ALS and 25 matched healthy controls in a case control study design. These analyses consisted of measuring cortical thickness, surface area and volume. The effects of disease progression were examined by correlating cortical measures with progression rate and by longitudinal measures in 20 patients. RESULTS: Cortical morphology analyses revealed specific thinning in the precentral gyrus, considered the primary motor cortex, in patients with ALS compared with controls (p=6.3×10(-8)). Surface area was reduced in the right inferior parietal region (p=0.049) and volume--the product of cortical thickness and surface area--was reduced in the right precentral gyrus (p=0.031). From these findings, it appears that cortical thickness is superior in detecting the degenerative effects of ALS. Relative cortical thinning in temporal regions was related to faster clinical progression (right inferior temporal gyrus: p=3.3×10(-4)). CONCLUSIONS: Cortical thinning of the primary motor cortex might be a diagnostic marker for upper motor neuron degeneration in ALS. Relative thinning in temporal regions was associated with a rapidly progressive disease course.

Lithium lacks effect on survival in amyotrophic lateral sclerosis: a phase IIb randomised sequential trial.

OBJECTIVES: To determine the safety and efficacy of lithium for the treatment of amyotrophic lateral sclerosis (ALS) in a randomised, placebo controlled, double blind, sequential trial. METHODS: Between November 2008 and June 2011, 133 patients were randomised to receive lithium carbonate (target blood level 0.4-0.8 mEq/l) or placebo as add-on treatment with riluzole. The primary endpoint was survival, defined as death, tracheostomal ventilation or non-invasive ventilation for more than 16 h/day. Secondary outcome measures consisted of the revised ALS Functional Rating Scale and forced vital capacity. Analysis was by intention to treat and according to a sequential trial design. RESULTS: 61 patients reached a primary endpoint, 33 of 66 in the lithium group and 28 of 67 patients in the placebo group. Lithium did not significantly affect survival (cumulative survival probability of 0.73 in the lithium group (95% CI 0.63 to 0.86) vs 0.75 in the placebo group (95% CI 0.65 to 0.87) at 12 months and 0.62 in the lithium group (95% CI 0.50 to 0.76) vs 0.67 in the placebo group (95% CI 0.56 to 0.81) at 16 months). Secondary outcome measures did not differ between treatment groups. No major safety concerns were encountered. CONCLUSIONS: This trial, designed to detect a modest effect of lithium, did not demonstrate any beneficial effect on either survival or functional decline in patients with ALS. TRIAL REGISTRATION NUMBER: NTR1448. Name of trial registry: Lithium trial in ALS.

TDP-43 plasma levels are higher in amyotrophic lateral sclerosis.

Our objective was to investigate TDP-43 plasma levels in patients with amyotrophic lateral sclerosis (ALS). TDP-43 has been identified as a major component of protein inclusions in the brain of patients with ALS; mutations in the corresponding gene (TARDBP) have also been identified. Although increased TDP-43 levels have been reported in the cerebrospinal fluid, plasma levels have not yet been assessed in patients with ALS. TDP-43 levels were quantified by sandwich ELISA in plasma of 219 patients and 100 controls. In addition, we sequenced exon 6 of TARDBP, and performed longitudinal TDP-43 plasma measurements in a subset of patients. Results showed that TDP-43 plasma levels were significantly increased in patients with ALS (p=0.023) and we found a positive correlation with age in patients and controls. Longitudinal measurements of TDP-43 plasma levels showed an increase in only one patient, with stable levels in five others. Three TARDBP variations were identified in the ALS group (1.7%), but the association with TDP-43 plasma levels was ambiguous. In conclusion, our data indicate that TDP-43 plasma levels may have potential as a marker for ALS. A genotype-phenotype relationship could not, however, be established in this cohort.

Population based epidemiology of amyotrophic lateral sclerosis using capture-recapture methodology.

BACKGROUND: Variation in the incidence rate in epidemiological studies on amyotrophic lateral sclerosis (ALS) may be due to a small population size and under ascertainment of patients. The previously reported incidence decline in the elderly and a decrease in the male:female ratio in postmenopausal age groups have yet to be confirmed. METHODS: ALS epidemiology in a large population based register in The Netherlands was studied between 1 January 2006 and 31 December 2009, and applied capture-recapture methodology in separate age and gender groups to adjust for the number of unobserved patients. RESULTS: 1217 incident patients were observed, and a capture-recapture incidence of 2.77 per 100 000 person-years (95% CI 2.63 to 2.91). Prevalence on 31 December 2008 was 10.32 per 100 000 individuals (95% CI 9.78 to 10.86). The incident cohort had a higher median age at onset (63.0 vs 58.1 years) and more bulbar onset patients (30.0% vs 19.1%) compared with the prevalent cohort. Incidence and prevalence peaked in the 70-74 year age group followed by a rapid decline in older age. The male:female ratio in the premenopausal age group (1.91, 95% CI 1.32 to 2.79) was not significantly higher than that in the postmenopausal age group (1.50, 95% CI 1.34 to 1.67). CONCLUSION: The marked difference in patient characteristics between incident and prevalent cohorts underscores the importance of including incident patients when studying susceptibility or disease modifying factors in ALS. The incidence decline in the elderly may suggest that ALS is not merely the result of ageing. Absence of a significant postmenopausal drop in the male:female ratio suggests that the protective role of female sex hormones in ALS is limited.

Transcranial direct current stimulation does not modulate motor cortex excitability in patients with amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive disease caused by the degeneration of upper and lower motor neurons. The etiology of ALS is unclear, but there is evidence that loss of cortical inhibition could be related to motor neuron degeneration. We sought to determine whether cathodal transcranial direct current stimulation (tDCS) can reduce cortical excitability in patients with ALS. METHODS: Three sessions of cathodal tDCS, lasting 7, 11, or 15 minutes, were performed in 10 patients and 10 healthy controls. Corticospinal excitability was measured before and after the tDCS. RESULTS: Cathodal tDCS induced a consistent decrease in corticospinal excitability in healthy controls, but not in ALS patients. CONCLUSIONS: The failure of tDCS to produce an excitability shift in the patients supports the potential diagnostic value of tDCS as a marker of upper motor neuron involvement. However, variation in corticospinal excitability measurements both inter- and intraindividually will limit its usefulness.

Randomized sequential trial of valproic acid in amyotrophic lateral sclerosis.

OBJECTIVE: To determine whether valproic acid (VPA), a histone deacetylase inhibitor that showed antioxidative and antiapoptotic properties and reduced glutamate toxicity in preclinical studies, is safe and effective in amyotrophic lateral sclerosis (ALS) using a sequential trial design. METHODS: Between April 2005 and January 2007, 163 ALS patients received VPA 1,500mg or placebo daily. Primary end point was survival. Secondary outcome measure was decline of functional status measured by the revised ALS Functional Rating Scale. Analysis was by intention to treat and according to a sequential trial design. This trial was registered with ClinicalTrials.gov (number NCT00136110). RESULTS: VPA did not affect survival (cumulative survival probability of 0.72 in the VPA group [standard error (SE), 0.06] vs 0.88 in the placebo group [SE, 0.04] at 12 months, and 0.59 in the VPA group [SE, 0.07] vs 0.68 in the placebo group [SE, 0.08] at 16 months) or the rate of decline of functional status. VPA intake did not cause serious adverse reactions. INTERPRETATION: Our finding that VPA, at a dose used in epilepsy, does not show a beneficial effect on survival or disease progression in patients with ALS has implications for future trials with histone deacetylase inhibitors in ALS and other neurodegenerative diseases. The use of a sequential trial design allowed inclusion of only half the number of patients required for a classic trial design and prevented patients from unnecessarily continuing potentially harmful study medication.

Normal values for quantitative muscle ultrasonography in adults.

Ultrasonography can detect structural muscle changes caused by neuromuscular disease. Quantitative analysis is the preferred method to determine if ultrasound findings are within normal limits, but normative data are incomplete. The purpose of this study was to provide normative muscle ultrasonography data for muscle thickness and echo intensity for five different muscle groups in adults. Bilateral scans of the sternocleidomastoid, biceps brachii/brachialis, forearm flexor group, quadriceps femoris, and tibialis anterior were made in 95 volunteers, aged 17-90 years. Both muscle thickness and echo intensity showed gender differences and a muscle-specific non-linear correlation with age. The muscles of the upper extremities showed right-left differences. These data demonstrate the effect of age on muscle characteristics and provide normative values that can be used in clinical practice.

Prevalence and distribution of fasciculations in healthy adults: Effect of age, caffeine consumption and exercise.

Our objective was to determine the prevalence and distribution of fasciculations in healthy adults and to assess the effect of age, caffeine and exercise. Fasciculations were studied with ultrasonography in 58 healthy adults in various age categories. Questionnaires were used to determine effect of caffeine and regular exercise on the presence of fasciculations. Finally, we tested the effect of strenuous exercise on fasciculations in 10 healthy adults. Twenty-five subjects (43%) showed fasciculations on ultrasonography, mostly in the abductor hallucis longus muscle. Fasciculations were only sporadically encountered in muscle groups above the knee. Subjects with fasciculations were significantly older than those without. Caffeine and regular physical exercise did not influence the prevalence of fasciculations. However, strenuous physical exercise caused a temporary increase in fasciculations, but only in lower leg muscles. Fasciculations above the knee should raise suspicion and may warrant further investigation.

Breath analysis in detecting epilepsy.

The aim of this proof of concept study is to investigate if an electronic nose (eNose) is able to make a distinction between breath profiles of diagnosed epilepsy patients and epilepsy-free control subjects. An eNose is a non-invasive device, with a working mechanism that is based on the presence of volatile organic compounds (VOCs) in exhaled breath. These VOCs interact with the sensors of the eNose, and the eNose has to be trained to distinguish between breath patterns from patients with a specific disease and control subjects without that disease. During the measurement participants were asked to breathe through the eNose for five minutes via a disposable mouthpiece. Seventy-four epilepsy patients and 110 control subjects were measured to train the eNose and create a classification model. To assess the effects of anti-epileptic drugs (AEDs) usage on the classification, additional test groups were measured: seven patients who (temporarily) did not use AEDs and 11 patients without epilepsy who used AEDs. The results show that an eNose is able to make a distinction between epilepsy and control subjects with a sensitivity of 76%, a specificity of 67%, and an accuracy of 71%. The results of the two additional groups of subjects show that the created model classifies one out of seven epilepsy patients without AEDs and six out of 13 patients without epilepsy but with AEDs correctly. In this proof of concept study, the AeonoseTM is able to differentiate between epilepsy patients and control subjects. However, the number of false positives and false negatives is still high, which suggests that this first model is still mainly based on the usage of various AEDs.

CSF hypocretin-1 levels are normal in patients with amyotrophic lateral sclerosis.

Hypocretin (orexin) neurotransmission is not only crucially involved in the regulation of sleep and wake, but serves in multiple autonomic and cognitive functions as well. This is reflected in the widespread connections between the hypothalamic hypocretin neurons and the rest of the brain, such as dense projections to the frontal cortex. Both frontal cognitive impairment and autonomic disturbances have been described in ALS. Furthermore, in some ALS patients there may be sleep disturbances other than sleep related breathing disorders, including REM sleep behaviour disorder. In addition, a role for the hypocretin system in the regulation of motor functions has been suggested. Hypocretin defects have been described in several neurodegenerative disorders. We therefore speculated that the hypocretin system is also involved in ALS and measured hypocretin-1 levels in cerebrospinal fluid samples from 20 patients. All results were well within the normal range (>200 pg/ml) and individual values showed no correlation with age, gender and disease duration. We conclude that it is unlikely that the hypocretin system is involved in the degenerative process of ALS.

TDP-43 pathology in familial frontotemporal dementia and motor neuron disease without Progranulin mutations.

Frontotemporal dementia is accompanied by motor neuron disease (FTD + MND) in approximately 10% of cases. There is accumulating evidence for a clinicopathological overlap between FTD and MND based on observations of familial aggregation and neuropathological findings of ubiquitin-positive neuronal cytoplasmatic inclusions (NCI) in lower motor neurons, hippocampus and neocortex in both conditions. Several familial forms exist with different genetic loci and defects. We investigated the familial aggregation and clinical presentation of FTD + MND cases in a large cohort of 368 FTD patients in The Netherlands. Immunohistochemistry of available brain tissue of deceased patients was investigated using a panel of antibodies including ubiquitin, p62 and TAR DNA-binding protein of 43 kDa antibodies. A total of eight patients coming from six families had a family history positive for FTD + MND (mean age at onset 53.2 +/- 8.4 years). Five patients presented with behavioural changes and cognitive changes followed by motor neuron disease, whereas symptoms of motor neuron disease were the presenting features in the remaining three patients. Other affected relatives in these families showed dementia/FTD, MND or FTD + MND reflecting the clinical interfamilial variation. No mutations were identified in any of the candidate genes, including Superoxide Dismutase 1, dynactin, angiogenin, Microtubule-Associated Protein Tau, valosin-containing protein and progranulin. Available brain tissue of five patients with familial FTD + MND showed NCI in hippocampus, neocortex and spinal cord in all, and neuronal intranuclear inclusions (NII) in two brains. TDP-43 antibody showed robust staining of neuronal inclusions similar in distribution and morphology to NCI and NII. Additionally, TDP-43 antibody also stained ubiquitin-negative glial inclusions in the basal striatum of one case. In conclusion, there exists considerable clinical variation within families with FTD + MND, which may be determined by other genetic or environmental factors. NII are also found in some cases of familial FTD + MND without Progranulin mutations. The observation of glial TDP-43 positive inclusions in one brain is very interesting, although their pathophysiological significance is yet unknown.

Quantitative muscle ultrasonography in amyotrophic lateral sclerosis.

In this study, we examined whether quantitative muscle ultrasonography can detect structural muscle changes in early-stage amyotrophic lateral sclerosis (ALS). Bilateral transverse scans were made of five muscles or muscle groups (sternocleidomastoid, biceps brachii/brachialis, forearm flexor group, quadriceps femoris and anterior tibialis muscles) in 48 patients with ALS. Twenty-five patients were also screened for fasciculations. Quantitative analysis revealed a significant increase in echo intensity in all muscles and a decrease in muscle thickness of the biceps brachii, forearm flexors and quadriceps femoris on both sides. Fasciculations were easy to detect in multiple muscles of all screened patients except one. We conclude that quantitative ultrasonography can be used to detect muscle changes caused by ALS in an early phase of the disease. (E-mail: m.zwarts@neuro.umcn.nl).

Psychogenic nonepileptic seizures in adults with epilepsy and intellectual disability: A neglected area.

PURPOSE: To describe the main characteristics of psychogenic nonepileptic seizures (PNES) in adults with epilepsy and intellectual disability (ID), and to analyse the differences regarding psychosocial functioning, epilepsy severity and ID between patients with PNES and a control group without PNES. METHODS: Medical records of adults with ID and epilepsy living at an epilepsy care facility (N = 240) were screened for PNES and evaluated by a neurologist. A control group consisting of patients with epilepsy and ID, without PNES, was matched according to age, sex and level of ID. Characteristics of PNES and epilepsy were provided by the subject's nursing staff or retrieved from patient charts, psychosocial data were collected by standardised questionnaires and level of ID was individually assessed using psychometric instruments. RESULTS: The point prevalence of PNES was 7.1%. The patients with PNES (n = 15) were most often female and had a mild or moderate level of ID. Compared to controls, they showed more depressive symptoms, experienced more negative life events and had more often an ID discrepancy (ID profile with one domain particularly more impaired than another). Stress-related triggers were recognised in a large majority by the nursing staff. CONCLUSION: PNES appears to be a relatively rare diagnostic entity among inpatients with both epilepsy and ID. However, the complexity of diagnosing PNES in this population, and the similarities in stress-related triggers for PNES in patients with and without ID, suggest that PNES may be underdiagnosed in the ID population. Diagnostic challenges of PNES and, as subcategory, reinforced behavioural patterns are discussed.