Hannah Hollinger

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.

Novel FUS/TLS Mutations and Pathology in Familial and Sporadic Amyotrophic Lateral Sclerosis

OBJECTIVE To determine the frequency of and clinicopathologic phenotypes associated with FUS/TLS mutations in a large cohort of amyotrophic lateral sclerosis (ALS) cases from the north of England. DESIGN Genetic screening project with neuropathologic examination of postmortem tissue in selected cases. The clinical details of selected cases are also presented. SETTING Neurology departments of 2 university teaching hospitals in the north of England. PARTICIPANTS The 15 exons of FUS/TLS were sequenced in an initial cohort of 42 familial ALS (FALS) and 117 sporadic ALS (SALS) cases. Exons 14 and 15 were subsequently screened in a larger cohort of 431 SALS cases. Regions mutated in ALS cases were also screened in 293 controls. MAIN OUTCOME MEASURE Evaluation of gene-sequencing chromatographs and detailed histopathologic analysis of the central nervous system. RESULTS Four heterozygous mutations, 1 of which is novel, were identified in 6 patients with ALS (4 with FALS and 2 with SALS). Two of the substitutions were not found to be present in controls, and neuropathology in these cases revealed neuronal and/or glial cytoplasmic inclusions positive for the FUS/TLS protein. One of these cases is also the first reported SALS case with an FUS/TLS mutation. The other 2 substitutions identified were also identified in control cases. Neuropathology in these cases revealed typical SALS pathology, suggesting that they are likely to represent benign polymorphisms. CONCLUSIONS FUS/TLS mutations represented approximately 5% of FALS cases screened. A FUS/TLS mutation was also identified in a single SALS case. Subsequent screening of this region in a larger cohort of SALS cases, however, did not reveal any additional mutations.

Broad clinical phenotypes associated with TAR-DNA binding protein ( TARDBP ) mutations in amyotrophic lateral sclerosis

The finding of TDP-43 as a major component of ubiquitinated protein inclusions in amyotrophic lateral sclerosis (ALS) has led to the identification of 30 mutations in the transactive response-DNA binding protein (TARDBP) gene, encoding TDP-43. All but one are in exon 6, which encodes the glycine-rich domain. The aim of this study was to determine the frequency of TARDBP mutations in a large cohort of motor neurone disease patients from Northern England (42 non-superoxide dismutase 1 (SOD1) familial ALS (FALS), nine ALS-frontotemporal dementia, 474 sporadic ALS (SALS), 45 progressive muscular atrophy cases). We identified four mutations, two of which were novel, in two familial (FALS) and two sporadic (SALS) cases, giving a frequency of TARDBP mutations in non-SOD1 FALS of 5% and SALS of 0.4%. Analysis of clinical data identified that patients had typical ALS, with limb or bulbar onset, and showed considerable variation in age of onset and rapidity of disease course. However, all cases had an absence of clinically overt cognitive dysfunction.

Lithium in patients with amyotrophic lateral sclerosis (LiCALS): a phase 3 multicentre, randomised, double-blind, placebo-controlled trial

Summary Background Lithium has neuroprotective effects in cell and animal models of amyotrophic lateral sclerosis (ALS), and a small pilot study in patients with ALS showed a significant effect of lithium on survival. We aimed to assess whether lithium improves survival in patients with ALS. Methods The lithium carbonate in amyotrophic lateral sclerosis (LiCALS) trial is a randomised, double-blind, placebo-controlled trial of oral lithium taken daily for 18 months in patients with ALS. Patients aged at least 18 years who had ALS according to the revised El Escorial criteria, had disease duration between 6 and 36 months, and were taking riluzole were recruited from ten centres in the UK. Patients were randomly assigned (1:1) to receive either lithium or matched placebo tablets. Randomisation was via an online system done at the level of the individual by block randomisation with randomly varying block sizes, stratified by study centre and site of disease onset (limb or bulbar). All patients and assessing study personnel were masked to treatment assignment. The primary endpoint was the rate of survival at 18 months and was analysed by intention to treat. This study is registered with Eudract, number 2008-006891-31. Findings Between May 26, 2009, and Nov 10, 2011, 243 patients were screened, 214 of whom were randomly assigned to receive lithium (107 patients) or placebo (107 patients). Two patients discontinued treatment and one died before the target therapeutic lithium concentration could be achieved. 63 (59%) of 107 patients in the placebo group and 54 (50%) of 107 patients in the lithium group were alive at 18 months. The survival functions did not differ significantly between groups (Mantel-Cox log-rank χ2 on 1 df=1·64; p=0·20). After adjusting for study centre and site of onset using logistic regression, the relative odds of survival at 18 months (lithium vs placebo) was 0·71 (95% CI 0·40–1·24). 56 patients in the placebo group and 61 in the lithium group had at least one serious adverse event. Interpretation We found no evidence of benefit of lithium on survival in patients with ALS, but nor were there safety concerns, which had been identified in previous studies with less conventional designs. This finding emphasises the importance of pursuing adequately powered trials with clear endpoints when testing new treatments. Funding The Motor Neurone Disease Association of Great Britain and Northern Ireland.

Pattern of spread and prognosis in lower limb-onset ALS

Abstract 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.

Gene expression signatures in motor neurone disease fibroblasts reveal dysregulation of metabolism, hypoxia-response and RNA processing functions

Amyotrophic lateral sclerosis (ALS) and primary lateral sclerosis (PLS) are two syndromic variants within the motor neurone disease spectrum. As PLS and most ALS cases are sporadic (SALS), this limits the availability of cellular models for investigating pathogenic mechanisms and therapeutic targets. The aim of this study was to use gene expression profiling to evaluate fibroblasts as cellular models for SALS and PLS, to establish whether dysregulated biological processes recapitulate those seen in the central nervous system and to elucidate pathways that distinguish the clinically defined variants of SALS and PLS.

Evaluation of two different methods for per-oral gastrostomy tube placement in patients with motor neuron disease (MND): PIG versus PEG procedures

Abstract Placement of a gastrostomy tube remains the gold standard procedure to maintain nutrition in patients with motor neuron disease (MND) and bulbar muscle weakness. Percutaneous endoscopic gastrostomy (PEG) is the most commonly used procedure in this context. Per-oral image guided gastrostomy (PIG) is a new hybrid technique used successfully in non-MND patients. We have modified the PIG technique to improve patient tolerability and have undertaken a pilot evaluation of PIG compared to PEG in MND patients. Nineteen PIG and 16 PEG procedures performed over a period of four years were evaluated. Pre-procedural forced vital capacity (FVC), procedural oxygen saturation, post-procedural complications and survival duration were recorded. Results showed that a gastrostomy tube was successfully placed in 95% of the PIG group and 80% of the PEG group. Rates of minor complications were comparable in both groups (21% in PIG, 23% in PEG). No life-threatening complications occurred in either group. Procedural mean oxygen saturations were higher in the PIG group compared to the PEG group (p < 0.001). No significant survival differences were observed. This study provides evidence for the use of the PIG procedure as a safe and well tolerated alternative to PEG in MND patients.

Lack of unique neuropathology in amyotrophic lateral sclerosis associated with p.K54E angiogenin (ANG) mutation

Five to 10% of cases of amyotrophic lateral sclerosis are familial, with the most common genetic causes being mutations in the C9ORF72, SOD1, TARDBP and FUS genes. Mutations in the angiogenin gene, ANG, have been identified in both familial and sporadic patients in several populations within Europe and North America. The aim of this study was to establish the incidence of ANG mutations in a large cohort of 517 patients from Northern England and establish the neuropathology associated with these cases.

Prognosis for patients with amyotrophic lateral sclerosis: development and validation of a personalised prediction model

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive, fatal motor neuron disease with a variable natural history. There are no accurate models that predict the disease course and outcomes, which complicates risk assessment and counselling for individual patients, stratification of patients for trials, and timing of interventions. We therefore aimed to develop and validate a model for predicting a composite survival endpoint for individual patients with ALS. METHODS We obtained data for patients from 14 specialised ALS centres (each one designated as a cohort) in Belgium, France, the Netherlands, Germany, Ireland, Italy, Portugal, Switzerland, and the UK. All patients were diagnosed in the centres after excluding other diagnoses and classified according to revised El Escorial criteria. We assessed 16 patient characteristics as potential predictors of a composite survival outcome (time between onset of symptoms and non-invasive ventilation for more than 23 h per day, tracheostomy, or death) and applied backward elimination with bootstrapping in the largest population-based dataset for predictor selection. Data were gathered on the day of diagnosis or as soon as possible thereafter. Predictors that were selected in more than 70% of the bootstrap resamples were used to develop a multivariable Royston-Parmar model for predicting the composite survival outcome in individual patients. We assessed the generalisability of the model by estimating heterogeneity of predictive accuracy across external populations (ie, populations not used to develop the model) using internal-external cross-validation, and quantified the discrimination using the concordance (c) statistic (area under the receiver operator characteristic curve) and calibration using a calibration slope. FINDINGS Data were collected between Jan 1, 1992, and Sept 22, 2016 (the largest data-set included data from 1936 patients). The median follow-up time was 97·5 months (IQR 52·9-168·5). Eight candidate predictors entered the prediction model: bulbar versus non-bulbar onset (univariable hazard ratio [HR] 1·71, 95% CI 1·63-1·79), age at onset (1·03, 1·03-1·03), definite versus probable or possible ALS (1·47, 1·39-1·55), diagnostic delay (0·52, 0·51-0·53), forced vital capacity (HR 0·99, 0·99-0·99), progression rate (6·33, 5·92-6·76), frontotemporal dementia (1·34, 1·20-1·50), and presence of a C9orf72 repeat expansion (1·45, 1·31-1·61), all p<0·0001. The c statistic for external predictive accuracy of the model was 0·78 (95% CI 0·77-0·80; 95% prediction interval [PI] 0·74-0·82) and the calibration slope was 1·01 (95% CI 0·95-1·07; 95% PI 0·83-1·18). The model was used to define five groups with distinct median predicted (SE) and observed (SE) times in months from symptom onset to the composite survival outcome: very short 17·7 (0·20), 16·5 (0·23); short 25·3 (0·06), 25·2 (0·35); intermediate 32·2 (0·09), 32·8 (0·46); long 43·7 (0·21), 44·6 (0·74); and very long 91·0 (1·84), 85·6 (1·96). INTERPRETATION We have developed an externally validated model to predict survival without tracheostomy and non-invasive ventilation for more than 23 h per day in European patients with ALS. This model could be applied to individualised patient management, counselling, and future trial design, but to maximise the benefit and prevent harm it is intended to be used by medical doctors only. FUNDING Netherlands ALS Foundation.

Complementary therapies for people with motor neurone disease: extending a cancer care service

Background Cavendish Cancer Care (CCC) is a charitable organisation in Sheffield providing assessment, counselling and complementary therapies for people with cancer and their families. Motor Neurone Disease (MND), is characterised by progressive deterioration: muscle wasting, speech, swallowing, and breathing difficulties. A year-long pilot study for patients and carers, funded by South Yorkshire MND Association was set up using the CCC model of care, in conjunction with the MND Team at the Royal Hallamshire Hospital. We report here on patient experiences, using our standard evaluation procedures. Aim To assess the value and outcomes of a supportive care service for people diagnosed with MND. Method Data was collected over 1 year. Clients were seen at home or at the Centre, and completed the Measure Yourself Concerns and Wellbeing (MYCAW) scale, in which clients score their nominated main concerns on a 7-point scale at assessment, repeated at post-therapy review, and give comments on the service received. Results Twenty-seven people, with ages ranging from 50 to 83, of whom 14 were men, used the service during the year. Fifteen were seen in their own homes, and twelve at the Centre. Physical symptoms were prominent among the concerns, along with emotional issues, the need for relaxation, for information, and for general wellbeing. Twenty-four patients received therapy: massage/aromatherapy massage, shiatsu and reflexology were chosen most often. Follow-up scores were available for 16 patients, with improvements in first nominated concerns for eleven, static scores for three and deterioration for two. Comments from those reporting no improvement suggest that there were benefits, but not specific to the concern, or transient. Conclusion Compared to our usual cancer populations, patients with MND showed a higher proportion of symptom-based concerns. Improvement was reported by a smaller majority with MND compared to cancer patients. General benefits of the therapies were highlighted.