Status epilepticus in Auckland, New Zealand: Treatment patterns and determinants of outcome in a prospective population-based cohort.

OBJECTIVE: Determination of the real-world performance of a health care system in the treatment of status epilepticus (SE). METHODS: Prospective, multicenter population-based study of SE in Auckland, New Zealand (NZ) over 1 year, with data recorded in the EpiNet database. Focus on treatment patterns and determinants of SE duration and 30-day mortality. The incidence, etiology, ethnic discrepancies, and seizure characteristics of this cohort have been published previously. RESULTS: A total of 365 patients were included in this treatment cohort; 326 patients (89.3%) were brought to hospital because of SE, whereas 39 patients (10.7%) developed SE during a hospital admission for another reason. Overall, 190 (52.1%) had a known history of epilepsy and 254 (70.0%) presented with SE with prominent motor activity. The mean Status Epilepticus Severity Score (STESS) was 2.15 and the mean SE duration of all patients was 44 min. SE self-terminated without any treatment in 84 patients (22.7%). Earlier administration of appropriately dosed benzodiazepine in the pre-hospital setting was a major determinant of SE duration. Univariate analysis demonstrated that mortality was significantly higher in older patients, patients with longer durations of SE, higher STESS, and patients who developed SE in hospital, but these did not maintain significance with multivariate analysis. There was no difference in the performance of the health care system in the treatment of SE across ethnic groups. SIGNIFICANCE: When SE was defined as 10 continuous minutes of seizure, overall mortality was lower than expected and many patients had self-limited presentations for which no treatment was required. Although there were disparities in the incidence of SE across ethnic groups there was no difference in treatment or outcome. The finding highlights the benefit of a health care system designed to deliver universal health care.

Characteristics and Neural Correlates of Emotional Behavior during Prefrontal Seizures.

OBJECTIVE: This study was undertaken to characterize clinical expression and intracerebral electroencephalographic (EEG) correlates of emotional expression during prefrontal epileptic seizures. METHODS: We performed a descriptive analysis of seizure semiology in patients explored with stereo-EEG (SEEG) for pharmacoresistant prefrontal epilepsy, using a semiquantitative score for seizure-related emotional behavior. Two independent observers scored occurrence and intensity of objective emotional features (face/body movements/vocalization/overall appearance), testing interobserver reliability. Intracerebral electrophysiological changes were documented. Cluster analysis and principal component analysis (PCA) compared behavioral signs with neural SEEG correlates. For each patient, the clinical and anatomoelectrophysiological scores were established, based on a prototypical emotional seizure. RESULTS: Forty-two patients (469 seizures) were included. Interobserver correlation for emotional signs was satisfactory (kappa = 0.6-0.8). Prevalence of any subjective and/or objective ictal emotional phenomena was 79% (33/42); objective emotional signs occurred in 27 of 42 subjects (64%). Negatively valenced emotional semiology (ictal feeling of fear, defensive and/or aggressive behaviors) was much more prevalent than positively valenced, prosocial behaviors. Cluster analysis and PCA identified 4 groups with different occurrence of emotional signs and cerebral correlates. Two main clusters of negatively valenced behavior were identified: "active threat response," associated with seizure organizations involving posterior orbitofrontal cortex, anterior cingulate, and dorsolateral and/or ventrolateral prefrontal cortex; and "passive fear," associated with amygdala, other mesial temporal structures, and posterior orbitofrontal cortex. INTERPRETATION: Emotional behaviors, especially fear/threat response, are common in prefrontal seizures, reflecting the role of the prefrontal cortex in emotional control. Different cortical seizure localizations were associated with "passive fear" and "active threat response" seizure behaviors at the group level. ANN NEUROL 2022;92:1052-1065.

Cognitive-behavioural therapy compared with standardised medical care for adults with dissociative non-epileptic seizures: the CODES RCT.

BACKGROUND: Dissociative (non-epileptic) seizures are potentially treatable by psychotherapeutic interventions; however, the evidence for this is limited. OBJECTIVES: To evaluate the clinical effectiveness and cost-effectiveness of dissociative seizure-specific cognitive-behavioural therapy for adults with dissociative seizures. DESIGN: This was a pragmatic, multicentre, parallel-arm, mixed-methods randomised controlled trial. SETTING: This took place in 27 UK-based neurology/epilepsy services, 17 liaison psychiatry/neuropsychiatry services and 18 cognitive-behavioural therapy services. PARTICIPANTS: Adults with dissociative seizures in the previous 8 weeks and no epileptic seizures in the previous year and meeting other eligibility criteria were recruited to a screening phase from neurology/epilepsy services between October 2014 and February 2017. After psychiatric assessment around 3 months later, eligible and interested participants were randomised between January 2015 and May 2017. INTERVENTIONS: Standardised medical care consisted of input from neurologists and psychiatrists who were given guidance regarding diagnosis delivery and management; they provided patients with information booklets. The intervention consisted of 12 dissociative seizure-specific cognitive-behavioural therapy 1-hour sessions (plus one booster session) that were delivered by trained therapists, in addition to standardised medical care. MAIN OUTCOME MEASURES: The primary outcome was monthly seizure frequency at 12 months post randomisation. The secondary outcomes were aspects of seizure occurrence, quality of life, mood, anxiety, distress, symptoms, psychosocial functioning, clinical global change, satisfaction with treatment, quality-adjusted life-years, costs and cost-effectiveness. RESULTS: In total, 698 patients were screened and 368 were randomised (standardised medical care alone, n = 182; and cognitive-behavioural therapy plus standardised medical care, n = 186). Primary outcome data were obtained for 85% of participants. An intention-to-treat analysis with multivariate imputation by chained equations revealed no significant between-group difference in dissociative seizure frequency at 12 months [standardised medical care: median of seven dissociative seizures (interquartile range 1-35 dissociative seizures); cognitive-behavioural therapy and standardised medical care: median of four dissociative seizures (interquartile range 0-20 dissociative seizures); incidence rate ratio 0.78, 95% confidence interval 0.56 to 1.09; p = 0.144]. Of the 16 secondary outcomes analysed, nine were significantly better in the arm receiving cognitive-behavioural therapy at a p-value < 0.05, including the following at a p-value ≤ 0.001: the longest dissociative seizure-free period in months 7-12 inclusive post randomisation (incidence rate ratio 1.64, 95% confidence interval 1.22 to 2.20; p = 0.001); better psychosocial functioning (Work and Social Adjustment Scale, standardised treatment effect -0.39, 95% confidence interval -0.61 to -0.18; p < 0.001); greater self-rated and clinician-rated clinical improvement (self-rated: standardised treatment effect 0.39, 95% confidence interval 0.16 to 0.62; p = 0.001; clinician rated: standardised treatment effect 0.37, 95% confidence interval 0.17 to 0.57; p < 0.001); and satisfaction with treatment (standardised treatment effect 0.50, 95% confidence interval 0.27 to 0.73; p < 0.001). Rates of adverse events were similar across arms. Cognitive-behavioural therapy plus standardised medical care produced 0.0152 more quality-adjusted life-years (95% confidence interval -0.0106 to 0.0392 quality-adjusted life-years) than standardised medical care alone. The incremental cost-effectiveness ratio (cost per quality-adjusted life-year) for cognitive-behavioural therapy plus standardised medical care versus standardised medical care alone based on the EuroQol-5 Dimensions, five-level version, and imputed data was £120,658. In sensitivity analyses, incremental cost-effectiveness ratios ranged between £85,724 and £206,067. Qualitative and quantitative process evaluations highlighted useful study components, the importance of clinical experience in treating patients with dissociative seizures and potential benefits of our multidisciplinary care pathway. LIMITATIONS: Unlike outcome assessors, participants and clinicians were not blinded to the interventions. CONCLUSIONS: There was no significant additional benefit of dissociative seizure-specific cognitive-behavioural therapy in reducing dissociative seizure frequency, and cost-effectiveness over standardised medical care was low. However, this large, adequately powered, multicentre randomised controlled trial highlights benefits of adjunctive dissociative seizure-specific cognitive-behavioural therapy for several clinical outcomes, with no evidence of greater harm from dissociative seizure-specific cognitive-behavioural therapy. FUTURE WORK: Examination of moderators and mediators of outcome. TRIAL REGISTRATION: Current Controlled Trials ISRCTN05681227 and ClinicalTrials.gov NCT02325544. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 43. See the NIHR Journals Library website for further project information.

Dissociative seizures resemble epileptic seizures or faints, but can be distinguished from them by trained doctors. Dissociation is the medical word for a 'trance-like' or 'switching off' state. People with dissociative seizures commonly have other psychological or physical problems. Quality of life may be low. The condition accounts for about one in every six patients seen in hospitals because of seizures. We wanted to find out if people with dissociative seizures receiving standardised treatment would also benefit from a talking therapy, called cognitive­behavioural therapy, made specific to this disorder. We did a randomised controlled trial to find out if people with dissociative seizures given standardised treatment and cognitive­behavioural therapy (talking therapy) would do better than those given standardised treatment alone. Standardised treatment of dissociative seizures began with careful diagnosis from a neurologist and then further assessment and treatment from a psychiatrist. In total, 368 people with dissociative seizures participated, with half receiving standardised treatment alone and half having talking therapy plus standardised treatment. We measured seizures and psychological and physical health in both trial groups. We also investigated whether or not cognitive­behavioural therapy was good value for money. After 12 months, patients in both trial groups seemed to have fewer monthly seizures, but there was no advantage in the talking therapy group. Patients in the talking therapy group had more consecutive days without seizures, reporting less impact from them in everyday situations. Patients in the talking therapy group, and their doctors, considered improvements to be better, and patients in this group reported greater satisfaction with treatment. However, the talking therapy was expensive and not as cost-effective as hoped. Interviews with patients and study clinicians showed that they valued aspects of both treatments and of the care provided by the multidisciplinary teams. Overall, cognitive­behavioural therapy designed for dissociative seizures plus standardised treatment was not better at reducing the total numbers of seizures reported, but did produce several positive benefits for participants compared with standardised treatment alone.

The impact and challenges of the 2018 MHRA statement on the use of sodium valproate in women of childbearing age during the first year of implementation, in a UK epilepsy centre.

PURPOSE: On 24/04/2018, the United Kingdom (UK) Medicines and Healthcare Products Regulatory Agency (MHRA) clarified previous policies by issuing a statement, that the use of sodium valproate is contraindicated in women of childbearing potential unless the conditions of a pregnancy prevention programme are met, and only if other treatments are ineffective or not tolerated. We evaluated the impact of this over the first year of implementation in a tertiary epilepsy centre. METHODS: Cross-sectional study of all women under active follow up, or newly referred, of childbearing age (16-55 years), taking valproate for the treatment of epilepsy, over 12 months from 01/05/2018. RESULTS: We identified 125 cases, with 31 newly referred in response to MHRA regulations. 9.6% of patients did not attend their appointment, 35.2% had a learning disability (LD), which in 19.2% was sufficiently severe that they could not consent to a sexual relationship. Patients with LD prescribed valproate were significantly younger, and more likely to have a focal or uncharacterised epilepsy than patients without LD. In 46.4% of patients, MHRA regulations were followed: women were already using highly active contraception (HAC), HAC was started, or valproate withdrawn. In 24.8% of cases, women elected to continue valproate, and were not willing to use HAC. CONCLUSIONS: In 53.6% of cases, MHRA regulations contraindicating the use valproate in women of childbearing potential could not be followed fully, due to lack of patient attendance, lack of applicability in severe LD, or ethical concerns relating to patient choice.

Drug withdrawal in the epilepsy monitoring unit - The patsalos table.

Investigation of possible candidates for epilepsy surgery will usually require inpatient EEG to capture seizures and allow full operative planning. Withdrawal of antiepileptic drugs increases the yield of this valuable diagnostic information and the benefits of this should justify any increase in the risk of harm associated with these seizures This paper outlines our opinion on what would constitute proposed best practice for management of antiepileptic drug (AED) dosing when patients are admitted for monitoring of seizures to an epilepsy monitoring unit (EMU). In the vast majority of cases EMU admissions are safe and, even if seizures occur, will pass off without complication. Previous guidance has concentrated on ensuring practice around technical aspects of EEG monitoring itself and staffing within the unit. In this guidance we aim to outline optimally safe ways of ensuring that EMUs ensure the minimisation of risk to the patients admitted under their care. We propose an algorithm for enhancing the safety of AED withdrawal in VT admissions while ensuring adequate seizure yields. Risk minimisation requires planned management of drug dosing (with reduction if appropriate), provision of adequate rescue medication, and adequate supervision to allow rapid response to generalised seizures. This algorithm is accompanied by a table which uses knowledge of the clinical and pharmacological properties of each AED to ensure dose withdrawal and reduction is timely and safe taking into account the severity and frequency of the individual's seizures.

Status epilepticus in Auckland, New Zealand: Incidence, etiology, and outcomes.

OBJECTIVE: To determine the incidence, etiology, and outcome of status epilepticus (SE) in Auckland, New Zealand, using the latest International League Against Epilepsy (ILAE) SE semiological classification. METHODS: We prospectively identified patients presenting to the public or major private hospitals in Auckland (population = 1.61 million) between April 6, 2015 and April 5, 2016 with a seizure lasting 10 minutes or longer, with retrospective review to confirm completeness of data capture. Information was recorded in the EpiNet database. RESULTS: A total of 477 episodes of SE occurred in 367 patients. Fifty-one percent of patients were aged <15 years. SE with prominent motor symptoms comprised 81% of episodes (387/477). Eighty-four episodes (18%) were nonconvulsive SE. Four hundred fifty episodes occurred in 345 patients who were resident in Auckland. The age-adjusted incidence of 10-minute SE episodes and patients was 29.25 (95% confidence interval [CI] = 27.34-31.27) and 22.22 (95% CI = 20.57-23.99)/100 000/year, respectively. SE lasted 30 minutes or longer in 250 (56%) episodes; age-adjusted incidence was 15.95 (95% CI = 14.56-17.45) SE episodes/100 000/year and 12.92 (95% CI = 11.67-14.27) patients/100 000/year. Age-adjusted incidence (10-minute SE) was 25.54 (95% CI = 23.06-28.24) patients/100 000/year for males and 19.07 (95% CI = 16.91-21.46) patients/100 000/year for females. The age-adjusted incidence of 10-minute SE was higher in Maori (29.31 [95% CI = 23.52-37.14]/100 000/year) and Pacific Islanders (26.55 [95% CI = 22.05-31.99]/100 000/year) than in patients of European (19.13 [95% CI = 17.09-21.37]/100 000/year) or Asian/other descent (17.76 [95% CI = 14.73-21.38]/100 000/year). Seventeen of 367 patients in the study died within 30 days of the episode of SE; 30-day mortality was 4.6%. SIGNIFICANCE: In this population-based study, incidence and mortality of SE in Auckland lie in the lower range when compared to North America and Europe. For pragmatic reasons, we only included convulsive SE if episodes lasted 10 minutes or longer, although the 2015 ILAE SE classification was otherwise practical and easy to use.

EpiNet study of incidence of status epilepticus in Auckland, New Zealand: Methods and preliminary results.

The EpiNet project has been commenced to facilitate investigator-led collaborative research in epilepsy. A new Web-based data collection tool has been developed within EpiNet to record comprehensive data regarding status epilepticus and has been used for a study of status epilepticus in Auckland, New Zealand. All patients aged >4 weeks who presented to any of the five public hospitals and the major private hospital within Auckland city (population = 1.61 million) with an episode of status epilepticus between April 6, 2015 and April 5, 2016 were identified using multiple overlapping sources of information. For this study, status epilepticus was defined as any seizure exceeding 10 minutes in duration, or repeated seizures lasting >10 minutes without recovery between seizures. Patients who had either convulsive or nonconvulsive status epilepticus were included. Episodes of status epilepticus were classified according to the 2015 International League Against Epilepsy ILAE status epilepticus classification. A total of 477 episodes in 367 patients were considered as definite or probable status epilepticus; 285 episodes (62%) lasted >30 minutes, which is the duration that has previously been used for epidemiological studies of status epilepticus.

EpiNet as a way of involving more physicians and patients in epilepsy research: Validation study and accreditation process.

OBJECTIVE: EpiNet was established to encourage epilepsy research. EpiNet is used for multicenter cohort studies and investigator-led trials. Physicians must be accredited to recruit patients into trials. Here, we describe the accreditation process for the EpiNet-First trials. METHODS: Physicians with an interest in epilepsy were invited to assess 30 case scenarios to determine the following: whether patients have epilepsy; the nature of the seizures (generalized, focal); and the etiology. Information was presented in two steps for 23 cases. The EpiNet steering committee determined that 21 cases had epilepsy. The steering committee determined by consensus which responses were acceptable for each case. We chose a subset of 18 cases to accredit investigators for the EpiNet-First trials. We initially focused on 12 cases; to be accredited, investigators could not diagnose epilepsy in any case that the steering committee determined did not have epilepsy. If investigators were not accredited after assessing 12 cases, 6 further cases were considered. When assessing the 18 cases, investigators could be accredited if they diagnosed one of six nonepilepsy patients as having possible epilepsy but could make no other false-positive errors and could make only one error regarding seizure classification. RESULTS: Between December 2013 and December 2014, 189 physicians assessed the 30 cases. Agreement with the steering committee regarding the diagnosis at step 1 ranged from 47% to 100%, and improved when information regarding tests was provided at step 2. One hundred five of the 189 physicians (55%) were accredited for the EpiNet-First trials. The kappa value for diagnosis of epilepsy across all 30 cases for accredited physicians was 0.70. SIGNIFICANCE: We have established criteria for accrediting physicians using EpiNet. New investigators can be accredited by assessing 18 case scenarios. We encourage physicians with an interest in epilepsy to become EpiNet-accredited and to participate in these investigator-led clinical trials.

Unravelling the enigma of selective vulnerability in neurodegeneration: motor neurons resistant to degeneration in ALS show distinct gene expression characteristics and decreased susceptibility to excitotoxicity.

A consistent clinical feature of amyotrophic lateral sclerosis (ALS) is the sparing of eye movements and the function of external sphincters, with corresponding preservation of motor neurons in the brainstem oculomotor nuclei, and of Onuf's nucleus in the sacral spinal cord. Studying the differences in properties of neurons that are vulnerable and resistant to the disease process in ALS may provide insights into the mechanisms of neuronal degeneration, and identify targets for therapeutic manipulation. We used microarray analysis to determine the differences in gene expression between oculomotor and spinal motor neurons, isolated by laser capture microdissection from the midbrain and spinal cord of neurologically normal human controls. We compared these to transcriptional profiles of oculomotor nuclei and spinal cord from rat and mouse, obtained from the GEO omnibus database. We show that oculomotor neurons have a distinct transcriptional profile, with significant differential expression of 1,757 named genes (q < 0.001). Differentially expressed genes are enriched for the functional categories of synaptic transmission, ubiquitin-dependent proteolysis, mitochondrial function, transcriptional regulation, immune system functions, and the extracellular matrix. Marked differences are seen, across the three species, in genes with a function in synaptic transmission, including several glutamate and GABA receptor subunits. Using patch clamp recording in acute spinal and brainstem slices, we show that resistant oculomotor neurons show a reduced AMPA-mediated inward calcium current, and a higher GABA-mediated chloride current, than vulnerable spinal motor neurons. The findings suggest that reduced susceptibility to excitotoxicity, mediated in part through enhanced GABAergic transmission, is an important determinant of the relative resistance of oculomotor neurons to degeneration in ALS.

Clinico-pathological features in amyotrophic lateral sclerosis with expansions in C9ORF72.

Intronic expansion of the GGGGCC hexanucleotide repeat within the C9ORF72 gene causes frontotemporal dementia and amyotrophic lateral sclerosis/motor neuron disease in both familial and sporadic cases. Initial reports indicate that this variant within the frontotemporal dementia/amyotrophic lateral sclerosis spectrum is associated with transactive response DNA binding protein (TDP-43) proteinopathy. The amyotrophic lateral sclerosis/motor neuron disease phenotype is not yet well characterized. We report the clinical and pathological phenotypes associated with pathogenic C9ORF72 mutations in a cohort of 563 cases from Northern England, including 63 with a family history of amyotrophic lateral sclerosis. One hundred and fifty-eight cases from the cohort (21 familial, 137 sporadic) were post-mortem brain and spinal cord donors. We screened DNA for the C9ORF72 mutation, reviewed clinical case histories and undertook pathological evaluation of brain and spinal cord. Control DNA samples (n = 361) from the same population were also screened. The C9ORF72 intronic expansion was present in 62 cases [11% of the cohort; 27/63 (43%) familial, 35/500 (7%) cases with sporadic amyotrophic lateral sclerosis/motor neuron disease]. Disease duration was significantly shorter in cases with C9ORF72-related amyotrophic lateral sclerosis (30.5 months) compared with non-C9ORF72 amyotrophic lateral sclerosis/motor neuron disease (36.3 months, P < 0.05). C9ORF72 cases included both limb and bulbar onset disease and all cases showed combined upper and lower motor neuron degeneration (amyotrophic lateral sclerosis). Thus, clinically, C9ORF72 cases show the features of a relatively rapidly progressive, but otherwise typical, variant of amyotrophic lateral sclerosis associated with both familial and sporadic presentations. Dementia was present in the patient or a close family member in 22/62 cases with C9ORF72 mutation (35%) based on diagnoses established from retrospective clinical case note review that may underestimate significant cognitive changes in late disease. All the C9ORF72 mutation cases showed classical amyotrophic lateral sclerosis pathology with TDP-43 inclusions in spinal motor neurons. Neuronal cytoplasmic inclusions and glial inclusions positive for p62 immunostaining in non-motor regions were strongly over-represented in the C9ORF72 cases. Extra-motor pathology in the frontal cortex (P < 0.0005) and the hippocampal CA4 subfield neurons (P < 0.0005) discriminated C9ORF72 cases strongly from the rest of the cohort. Inclusions in CA4 neurons were not present in non-C9ORF72 cases, indicating that this pathology predicts mutation status.

Downregulation of genes with a function in axon outgrowth and synapse formation in motor neurones of the VEGFdelta/delta mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that stimulates vasculogenesis. It has also been shown to act as a neurotrophic factor in vitro and in vivo. Deletion of the hypoxia response element of the promoter region of the gene encoding VEGF in mice causes a reduction in neural VEGF expression, and results in adult-onset motor neurone degeneration that resembles amyotrophic lateral sclerosis (ALS). Investigating the molecular pathways to neurodegeneration in the VEGFdelta/delta mouse model of ALS may improve understanding of the mechanisms of motor neurone death in the human disease. RESULTS: Microarray analysis was used to determine the transcriptional profile of laser captured spinal motor neurones of transgenic and wild-type littermates at 3 time points of disease. 324 genes were significantly differentially expressed in motor neurones of presymptomatic VEGFdelta/delta mice, 382 at disease onset, and 689 at late stage disease. Massive transcriptional downregulation occurred with disease progression, associated with downregulation of genes involved in RNA processing at late stage disease. VEGFdelta/delta mice showed reduction in expression, from symptom onset, of the cholesterol synthesis pathway, and genes involved in nervous system development, including axonogenesis, synapse formation, growth factor signalling pathways, cell adhesion and microtubule-based processes. These changes may reflect a reduced capacity of VEGFdelta/delta mice for maintenance and remodelling of neuronal processes in the face of demands of neural plasticity. The findings are supported by the demonstration that in primary motor neurone cultures from VEGFdelta/delta mice, axon outgrowth is significantly reduced compared to wild-type littermates. CONCLUSIONS: Downregulation of these genes involved in axon outgrowth and synapse formation in adult mice suggests a hitherto unrecognized role of VEGF in the maintenance of neuronal circuitry. Dysregulation of VEGF may lead to neurodegeneration through synaptic regression and dying-back axonopathy.

Mutations in CHMP2B in lower motor neuron predominant amyotrophic lateral sclerosis (ALS).

BACKGROUND: Amyotrophic lateral sclerosis (ALS), a common late-onset neurodegenerative disease, is associated with fronto-temporal dementia (FTD) in 3-10% of patients. A mutation in CHMP2B was recently identified in a Danish pedigree with autosomal dominant FTD. Subsequently, two unrelated patients with familial ALS, one of whom also showed features of FTD, were shown to carry missense mutations in CHMP2B. The initial aim of this study was to determine whether mutations in CHMP2B contribute more broadly to ALS pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Sequencing of CHMP2B in 433 ALS cases from the North of England identified 4 cases carrying 3 missense mutations, including one novel mutation, p.Thr104Asn, none of which were present in 500 neurologically normal controls. Analysis of clinical and neuropathological data of these 4 cases showed a phenotype consistent with the lower motor neuron predominant (progressive muscular atrophy (PMA)) variant of ALS. Only one had a recognised family history of ALS and none had clinically apparent dementia. Microarray analysis of motor neurons from CHMP2B cases, compared to controls, showed a distinct gene expression signature with significant differential expression predicting disassembly of cell structure; increased calcium concentration in the ER lumen; decrease in the availability of ATP; down-regulation of the classical and p38 MAPK signalling pathways, reduction in autophagy initiation and a global repression of translation. Transfection of mutant CHMP2B into HEK-293 and COS-7 cells resulted in the formation of large cytoplasmic vacuoles, aberrant lysosomal localisation demonstrated by CD63 staining and impairment of autophagy indicated by increased levels of LC3-II protein. These changes were absent in control cells transfected with wild-type CHMP2B. CONCLUSIONS/SIGNIFICANCE: We conclude that in a population drawn from North of England pathogenic CHMP2B mutations are found in approximately 1% of cases of ALS and 10% of those with lower motor neuron predominant ALS. We provide a body of evidence indicating the likely pathogenicity of the reported gene alterations. However, absolute confirmation of pathogenicity requires further evidence, including documentation of familial transmission in ALS pedigrees which might be most fruitfully explored in cases with a LMN predominant phenotype.

Pattern of spread and prognosis in lower limb-onset ALS.

Our objective was to establish the pattern of spread in lower limb-onset ALS (contra- versus ipsi-lateral) and its contribution to prognosis within a multivariate model. Pattern of spread was established in 109 sporadic ALS patients with lower limb-onset, prospectively recorded in Oxford and Sheffield tertiary clinics from 2001 to 2008. Survival analysis was by univariate Kaplan-Meier log-rank and multivariate Cox proportional hazards. Variables studied were time to next limb progression, site of next progression, age at symptom onset, gender, diagnostic latency and use of riluzole. Initial progression was either to the contralateral leg (76%) or ipsilateral arm (24%). Factors independently affecting survival were time to next limb progression, age at symptom onset, and diagnostic latency. Time to progression as a prognostic factor was independent of initial direction of spread. In a regression analysis of the deceased, overall survival from symptom onset approximated to two years plus the time interval for initial spread. In conclusion, rate of progression in lower limb-onset ALS is not influenced by whether initial spread is to the contralateral limb or ipsilateral arm. The time interval to this initial spread is a powerful factor in predicting overall survival, and could be used to facilitate decision-making and effective care planning.

Large-scale pathways-based association study in amyotrophic lateral sclerosis.

Sporadic amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, most likely results from complex genetic and environmental interactions. Although a number of association studies have been performed in an effort to find genetic components of sporadic ALS, most of them resulted in inconsistent findings due to a small number of genes investigated in relatively small sample sizes, while the replication of results was rarely attempted. Defects in retrograde axonal transport, vesicle trafficking and xenobiotic metabolism have been implicated in neurodegeneration and motor neuron death both in human disease and animal models. To assess the role of common genetic variation in these pathways in susceptibility to sporadic ALS, we performed a pathway-based candidate gene case-control association study with replication. Furthermore, we determined reliability of whole genome amplified DNA in a large-scale association study. In the first stage of the study, 1277 putative functional and tagging SNPs in 134 genes spanning 8.7 Mb were genotyped in 822 British sporadic ALS patients and 872 controls using whole genome amplified DNA. To detect variants with modest effect size and discriminate among false positive findings 19 SNPs showing a trend of association in the initial screen were genotyped in a replication sample of 580 German sporadic ALS patients and 361 controls. We did not detect strong evidence of association with any of the genes investigated in the discovery sample (lowest uncorrected P-value 0.00037, lowest permutation corrected P-value 0.353). None of the suggestive associations was replicated in a second sample, further excluding variants with moderate effect size. We conclude that common variation in the investigated pathways is unlikely to have a major effect on susceptibility to sporadic ALS. The genotyping efficiency was only slightly decreased ( approximately 1%) and genotyping quality was not affected using whole genome amplified DNA. It is reliable for large scale genotyping studies of diseases such as ALS, where DNA sample collections are limited because of low disease prevalence and short survival time.

Screening of the transcriptional regulatory regions of vascular endothelial growth factor receptor 2 (VEGFR2) in amyotrophic lateral sclerosis.

BACKGROUND: Vascular endothelial growth factor (VEGF) has neurotrophic activity which is mediated by its main agonist receptor, VEGFR2. Dysregulation of VEGF causes motor neurone degeneration in a mouse model of amyotrophic lateral sclerosis (ALS), and expression of VEGFR2 is reduced in motor neurones and spinal cord of patients with ALS. METHODS: We have screened the promoter region and 4 exonic regions of functional significance of the VEGFR2 gene in a UK population of patients with ALS, for mutations and polymorphisms that may affect expression or function of this VEGF receptor. RESULTS: No mutations were identified in the VEGFR2 gene. We found no association between polymorphisms in the regulatory regions of the VEGFR2 gene and ALS. CONCLUSION: Mechanisms other than genetic variation may downregulate expression or function of the VEGFR2 receptor in patients with ALS.

Expression of vascular endothelial growth factor and its receptors in the central nervous system in amyotrophic lateral sclerosis.

Vascular endothelial growth factor (VEGF) prolongs survival in the mutant SOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS), whereas dysregulation of VEGF through deletion of its hypoxia-regulatory element causes motor neuron degeneration in mice. We investigated the expression of VEGF and its major agonist receptors in the normal central nervous system and in patients with ALS. Immunohistochemistry demonstrated similar expression patterns of VEGF and VEGF receptor 2 (VEGFR2) in the spinal cord with finely punctate staining of the neuropil and strong expression in anterior horn cells (AHCs). Granular staining on the surface of some AHCs, similar to that seen with synaptic markers, suggested synaptic labeling. VEGFR2 staining was reduced in the neuropil of ALS cases (p=0.018) associated with a reduction of synaptophysin but not SNAP25 expression. A greater proportion of AHCs in ALS cases showed low expression of VEGF (p=0.006) and VEGFR2 (p=0.009) compared with controls. Expression of VEGF and VEGFR2 was confirmed by Western blotting and quantitative reverse transcriptase-polymerase chain reaction (QPCR). The similar expression patterns of VEGF and VEGFR2 suggests autocrine/paracrine effects on spinal motor neurons, and the reduction in their expression seen in ALS cases would support the hypothesis that, as in mouse models of the disease, reduced VEGF signaling may play a role in the pathogenesis of ALS.