Integrative genetic analysis illuminates ALS heritability and identifies risk genes.

Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). We show that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk (P = 3.71.10-03; odds ratio = 3.29; 95%CI, 1.37 to 7.87) in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, our study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

Spatio-temporal clustering of amyotrophic lateral sclerosis in France: A population-based study.

OBJECTIVE: To assess spatial aggregates of amyotrophic lateral sclerosis (ALS) incident cases, using a solid geo-epidemiological statistical method, in France. METHODS: This population-based study (2003-2011) investigated 47.1 million person-years of follow-up (PYFU). Case ascertainment of incident ALS cases was based on multiple sources (ALS referral centers, hospital centres and health insurance data). Neurologists confirmed all ALS diagnoses. Exhaustiveness was estimated through capture-recapture. Aggregates were investigated in four steps: (a) geographical modelling (standardized incidence ratio (SIR) calculation), (b) analysis of the spatial distribution of incidence (Phothoff-Winttinghill's test, Global Moran's Index, Kulldorf's spatial scan statistic, Local Moran's Index), (c) classification of the level of certainty of spatial aggregates (i.e. definite cluster; probable over-incidence area; possible over-incidence area) and (d) evaluation of the robustness of the results. RESULTS: The standardized incidence of ALS was 2.46/100,000 PYFU (95% CI 2.31-2.63, European population as reference) based on 1199 incident cases. We identified 13 areas of spatial aggregates: one cluster (stable in robustness analysis), five probable over-incidence areas (2 stable in robustness analysis) and seven possible over-incidence areas (including 4 stable areas in robustness analysis). A cluster was identified in the Rhône-Alpes region: 100 observed vs 54.07 expected cases for 2,411,514 PYFU, SIR: 1.85 (95% CI 1.50-2.25). CONCLUSION: We report here one of the largest investigations of incidence and spatial aggregation of ALS ever performed in a western country. Using a solid methodology framework for case ascertainment and cluster analysis, we identified 13 areas that warrant further investigation.

Comparison of the ability of the King's and MiToS staging systems to predict disease progression and survival in amyotrophic lateral sclerosis.

Background: Assessing clinical progression in amyotrophic lateral sclerosis (ALS) remains a challenge. We evaluated the validity and predictive capabilities of the King's and Milano-Torino Staging (MiToS) systems in a cohort of patients with ALS to demonstrate their benefit in clinical practice.Methodology: A cohort study was performed by including ALS incident cases in a referral center from 2007 to 2016. The staging systems were determined at time of diagnosis and follow-up. The standardized median times to reach each stage were computed. A multi-state model in the framework of the Cox model evaluated the predictive value of measurements. The survival C-statistic was reported as a measure of prediction ability.Results: Overall, 298 incident cases were included. The King's and MiToS systems described a progressive increase in the risk of dying with each elapsed stage. However, a lower resolution for late disease description for the King's system was observed, and late stages overlapped for the MiToS system. Slight variations in the staging systems appeared to improve performance based on validity and prediction abilities: (i) in the King's (C-statistic = 0.783), by adding a new stage involving the need for both gastrostomy and NIV: (ii) in the MiToS (C-statistic = 0.792), by merging stage 3 and stage 4 into a single stage 3.Conclusion: Both King's and MiToS are valid systems but have certain limitations. Variations in the staging systems may provide a more suitable framework for describing progression and survival. Further research is needed to evaluate the variations in the staging systems.

Hypermetabolism is a reality in amyotrophic lateral sclerosis compared to healthy subjects.

RATIONALE: Hypermetabolism (HM) in Amyotrophic lateral sclerosis (ALS) is the reflection of a high energy metabolic level, but this alteration seems controversial. The main objective of the study was to confirm the existence of HM during ALS compared to healthy subjects. METHODS: A cohort of ALS patients was compared to a control group without metabolic disorder. The assessment included anthropometric criteria measurements, body composition by bioelectric impedance analysis and resting energy expenditure (REE) by indirect calorimetry. HM was defined as a variation > +10% between measured and calculated REE. Statistical analysis used Mann-Withney and Chi2 tests. Multivariate analysis included logistic regression. RESULTS: 287 patients and 75 controls were included. The metabolic level was higher in ALS patients (1500 kcal/24 h [1290-1693] vs. 1230 kcal/24 h [1000-1455], p < 0.0001) as well as the REE/fat free mass ratio (33.5 kcal/kg/24 h [30.4-37.8] vs. 28.3 kcal/kg/24 h [26.1-33.6], p < 0.0001). 55.0% of ALS patients had HM vs. 13.3% of controls (p < 0.0001). HM was strongly and positively associated with ALS (OR = 9.50 [4.49-20.10], p < 0.0001). CONCLUSIONS: HM in ALS is a reality, which affects more than half of the patients and is associated with ALS. This work confirms a very frequent metabolic deterioration during ALS. The identification of HM can allow a better adaptation of the patients' nutritional intake.

Genetic screening of ANXA11 revealed novel mutations linked to amyotrophic lateral sclerosis.

ANXA11 mutations have previously been discovered in amyotrophic lateral sclerosis (ALS) motor neuron disease. To confirm the contribution of ANXA11 mutations to ALS, a large exome data set obtained from 330 French patients, including 150 familial ALS index cases and 180 sporadic ALS cases, was analyzed, leading to the identification of 3 rare ANXA11 variants in 5 patients. The novel p.L254V variant was associated with early onset sporadic ALS. The novel p.D40Y mutation and the p.G38R variant concerned patients with predominant pyramidal tract involvement and cognitive decline. Neuropathologic findings in a p.G38R carrier associated the presence of ALS typical inclusions within the spinal cord, massive degeneration of the lateral tracts, and type A frontotemporal lobar degeneration. This mutant form of annexin A11 accumulated in various brain regions and in spinal cord motor neurons, although its stability was decreased in patients' lymphoblasts. Because most ANXA11 inclusions were not colocalized with transactive response DNA-binding protein 43 or p62 deposits, ANXA11 aggregation does not seem mandatory to trigger neurodegeneration with additional participants/partner proteins that could intervene.

Increased resting energy expenditure compared with predictive theoretical equations in amyotrophic lateral sclerosis.

OBJECTIVES: Approximately 50% to 60% of amyotrophic lateral sclerosis (ALS) is characterized by an increase in metabolic rate. The Harris and Benedict (HB) 1919 formula is the equation mainly used to calculate resting energy expenditure (cREE) compared with measured REE (mREE) by indirect calorimetry (IC), but other formulas are also applied in current practice. The present study aimed to assess mREE in patients with ALS compared with 12 cREE formulas and study the relevant threshold of REE variation to screen patients with a higher evolving risk. METHODS: Nutritional assessments and body composition (by bioimpedance analysis) were performed in patients with ALS. mREE was measured by IC, and cREE was calculated using the HB 1919, HB 1984, World Schofield, De Lorenzo, Johnstone, Mifflin, World Health Organization/Food and Agriculture Organization, Owen, Fleisch, Wang, Rosenbaum, and Nelson formulas. Functional and respiratory evolution and survival by log-rank test according to two thresholds of REE variation (10% and 20%) were studied. RESULTS: A total of 315 patients with ALS were included in the study. The median mREE was 1503 kcal/24 h (range, 1290-1698 kcal/24 h), which was higher than all predictive equations (P < 0.0001). Depending on the predictive equation, REE variation >10% and 20% was found in 35.2% to 76.3% and 14.6% to 53.3% of patients with ALS, respectively. Patients with an REE variation >20% with HB 1919 and HB 1984 had a lower survival. Moreover, with this same threshold and the Mifflin formula, patients had higher functional and respiratory evolutions and lower survival. CONCLUSIONS: The increase in metabolic rate is present according to the different cREE formulas used compared with IC. In clinical practice, REE formulas (e.g., HB 1919, HB 1984, or Mifflin) can be used as a reference value compared with IC to screen patients with ALS with an REE variation >20% and a higher evolving risk.

Predictive factors for gastrostomy at time of diagnosis and impact on survival in patients with amyotrophic lateral sclerosis.

BACKGROUND: Gastrostomy is recommended in patients with Amyotrophic Lateral Sclerosis (ALS) in the presence of weight loss over 10% as compared to usual weight, repeated aspirations or meal time duration longer than 45 min. Currently, the impact of gastrostomy on survival of ALS patients is not clear. AIMS: i) to describe diagnosis factors associated with the indication for gastrostomy ii) to evaluate survival of ALS patients with gastrostomy indication according to their acceptance of feeding tube placement. METHODS: Patients with ALS were included and followed in the ALS referral centre of Limoges's teaching hospital between 2006 and 2017. Neurological, nutritional and respiratory status was assessed prospectively from diagnosis to death. Statistical analysis was performed using Mann-Whitney test, Chi2 tests, Cox model and multivariate logistic regression. RESULTS: Two hundred and eighty-five patients were included. Among the 182 for whom gastrostomy was indicated, 63.7% accepted the placement. The median time was 7.3 months [IQR: 3.2-15.0] and 2.7 months [IQR: 0.9-5.8] respectively from diagnosis to indication and from indication to placement. Weight loss >5% significantly increased the risk of death by 17% (p < 0.0001). At time of diagnosis, bulbar onset, a loss of one point in the body mass index or on the bulbar functional scale were all positively associated with indication for gastrostomy (aOR = 10.0 [95%CI: 1.96-25.0]; p = 0.002, aOR = 1.17 [95%CI: 1.02-1.36]; p = 0.025 and aOR = 1.19 [95%CI: 1.06-1.32]; p = 0.002, respectively). However, gastrostomy placement did not have any impact on survival (aHR = 1.25 [95%CI: 0.88-1.79]; p = 0.22). CONCLUSION: Both neurological and nutritional criteria were associated with an indication for gastrostomy at diagnosis. Gastrostomy placement had no impact on survival. The study of earlier gastrostomy placement might be of interest in further prospective studies.

Resting energy expenditure equations in amyotrophic lateral sclerosis, creation of an ALS-specific equation.

INTRODUCTION: Resting energy expenditure (REE) formulas for healthy people (HP) are used to calculate REE (cREE) in amyotrophic lateral sclerosis (ALS) patients. In 50-60% of ALS cases an increase of measured REE (mREE) in indirect calometry (IC) compared to cREE is found. The aims here were (i) to assess the accuracy of cREE assessed using 11 formulas as compared to mREE and (ii) to create (if necessary) a specific cREE formula for ALS patients. METHOD: 315 Patients followed in the ALS expert center of Limoges between 1996 and 2014 were included. mREE assessed with IC and cREE calculated with 11 predictive formulas (Harris Benedict (HB) 1919, HB 1984, WSchofield, De Lorenzo, Johnstone, Mifflin, WHO/FAO, Owen, Fleisch, Wang and Rosenbaum) were determined at the time of diagnosis. Fat free mass (FFM) and fat mass (FM) were measured with impedancemetry. A Bland and Altman analysis was carried out. The percentage of accurate prediction ±10% of mREE, and intraclass correlation coefficients (ICC) were calculated. Using a derivation sample, a new REE formula was created using multiple linear regression according to sex, age, FFM and FM. Accuracy of this formula was assessed in a validation sample. RESULTS: ICC ranged between 0.60 and 0.71 (moderate agreement), and percentage of accurate prediction between 27.3% and 57.5%. Underestimation was found from 31.7% to 71.4% of cases. According to these unsatisfactory results we created an ALS-specific formula in a derivation sample (130 patients). ICC and percentage of accurate prediction increased in a validation sample (143 patients) to 0.85 (very good agreement) and 65.0% respectively, with 17.5% underestimation. CONCLUSION: REE formulas for HP underestimate REE in ALS patients compared to mREE. Our new ALS-specific formula produced better results than formulas for HP. This formula can be used to estimate REE in ALS patients if IC is not accessible.

Exploring the diagnosis delay and ALS functional impairment at diagnosis as relevant criteria for clinical trial enrolment.

Objectives were: i) to describe the phenotypic heterogeneity of incident amyotrophic lateral sclerosis (ALS) patients diagnosed in 2012 in French ALS centres; ii) to look at the associations between ALSFRS-R score and ALSFRS-R slope (ΔFS) at time of diagnosis with diagnosis delay, ALS phenotypes and Airlie House diagnosis criteria (AHDC); iii) to describe the rate of progression on ΔFS, according to diagnosis delay. METHODS: Incident ALS cases diagnosed in French ALS centres were included. The rate of progression was evaluated as follows: ΔFS = (48 - ALSFRS-R at time of diagnosis)/duration from onset to diagnosis (months). Fast and slow progressors were defined by ΔFS >1 and <0.5, respectively. RESULTS: At time of diagnosis, 476 patients were classified into eight phenotypes: bulbar (33.0%), spinal lumbar (28.2%), spinal cervical (23.1%), flail leg (4.4%), ALS/FTD (4.2%), possible flail arm (4.0%), respiratory (2.1%), dropped-head (1.0%). Median ΔFS (n = 358/476) was 1.0 [0.5-2.0]. ΔFS was associated with AHDC (p = 0.009), but not with clinical phenotype (p = 0.902). Stratification on diagnosis delay (<12 months or ≥18 months) allowed to differentiate fast progressors from slow progressors. CONCLUSION: At time of inclusion in therapeutic trial closed to diagnosis, ΔFS or diagnosis delay may discriminate the rate of progression.

Novel UBQLN2 mutations linked to amyotrophic lateral sclerosis and atypical hereditary spastic paraplegia phenotype through defective HSP70-mediated proteolysis.

Mutations in UBQLN2 have been associated with rare cases of X-linked juvenile and adult forms of amyotrophic lateral sclerosis (ALS) and ALS linked to frontotemporal dementia (FTD). Here, we report 1 known (c.1489C>T, p.Pro497Ser, P497S) and 3 novel (c.1481C>T, p.Pro494Leu, P494L; c.1498C>T, p.Pro500Ser, P500S; and c.1516C>G, p.Pro506Ala, P506A) missense mutations in the PXX domain of UBQLN2 in familial motor neuron diseases including ALS and spastic paraplegia (SP). A novel missense mutation (c.1462G>A, p.Ala488Thr, A488T) adjacent to this hotspot UBQLN2 domain was identified in a sporadic case of ALS. These mutations are conserved in mammals, are absent from ExAC and gnomAD browsers, and are predicted to be deleterious by SIFT in silico analysis. Patient lymphoblasts carrying a UBQLN2 mutation showed absence of ubiquilin-2 accumulation, disrupted binding with HSP70, and impaired autophagic pathway. Our results confirm the role of PXX repeat in ALS pathogenesis, show that UBQLN2-linked disease can manifest like a SP phenotype, evidence a highly reduced disease penetrance in females carrying UBQLN2 mutations, which is important information for genetic counseling, and underline the pivotal role of ubiquilin-2 in proteolysis regulation pathways.

[Epidemiology of amyotrophic lateral sclerosis]. / Épidémiologie de la sclérose latérale amyotrophique. Près de 6 000 cas en France, majoritairement sporadiques et sans facteurs de risque identifiés.

Epidemiology of amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is the most common motorneuron disease. The incidence of ALS in France is 2.5/100 000 persons-years of follow-up, or 1,500 cases per year. The peak incidence is between 65 and 75 years. The prevalence of ALS is 8/100 000, or 6 000 cases in France with a sex ratio close to 1. The average survival time is close to 36 months after the onset of symptoms, with a large variation between patients. The main prognostic factors are the age of onset, initial site of involvement, the time to diagnosis, respiratory status and nutritional status. 5 to 10% of cases are family related to a mutation of the four major genes SOD1, FUS, TARDP and C9ORF72. In sporadic forms an interaction between a genetic susceptibility factor and an environmental factor is suspected. There is to date no association between exogenous risk factor for sporadic ALS occurrence of which could be demonstrated reproducibly with the notable exception of smoking.

Épidémiologie de la sclérose latérale amyotrophique. La sclérose latérale amyotrophique est la plus fréquente des maladies du motoneurone. Son incidence en France est de 2,5/100 000 personnes-années, soit 1 500 cas par an. Le pic d'incidence est compris entre 65 et 75 ans. Sa prévalence est de 8/100 000, soit 6 000 cas en France, avec un sex-ratio proche de 1. La durée moyenne de survie est proche de 36 mois après le début des premiers symptômes avec une importante variation selon les patients. Les principaux facteurs pronostiques sont l'âge de début, le siège initial de l'atteinte, le délai diagnostique, le statut respiratoire et l'état nutritionnel. Cinq à 10 % des cas sont familiaux, liés à une mutation des quatre principaux gènes SOD1, FUS, TARDP et C9ORF72. Dans les formes sporadiques, une interaction entre un facteur de susceptibilité génétique et un facteur environnemental est suspectée. Il n'existe à ce jour aucune association entre un facteur de risque exogène et la survenue d'une sclérose latérale amyotrophique sporadique qui ait pu être démontrée de manière reproductible, à l'exception notable du tabagisme qui favoriserait la survenue de la maladie.

[Epidemiology, clinical spectrum of ALS and differential diagnoses]. / Épidémiologie, spectre clinique de la SLA et diagnostics différentiels.

Amyotrophic Lateral Sclerosis (ALS) is the most common motor neuron disease in adults. Its incidence in France is estimated at 2.5 per 100,000 population and its prevalence between 5 and 8 per 100,000 inhabitants. Good prognostic factors are age of early onset, a longer time to diagnosis, initial damage to the spinal onset, early management of undernutrition and restrictive respiratory failure. The diagnosis of ALS is primarily clinical and is based on the evidence of involvement of the central motor neuron and peripheral neuron (NMP) in different territories or spinal or bulbar. The EMG confirms the achievement of NMP, shows the extension to clinically preserved areas and allows to exclude some differential diagnoses. The clinical spectrum of ALS is broad: conventional forms beginning brachial, lower limb or bulbar onsets, rarer forms to start breathing, pyramidal forms, forms with cognitive and behavioural impairment. In 5-10% of cases, ALS is familial. In 15% of cases, it is associated with frontotemporal degeneration rather than orbito-frontal type. The main differential diagnoses are guided by the clinic: combining pure motor neuropathy with or without conduction block, post-polio syndrome, cramp-fasciculation syndrome, myasthenia gravis, paraneoplastic syndromes, Sjögren syndrome, retroviral infections, some endocrine disorders, some metabolic diseases, genetic diseases (Kennedy and SMA) and inclusion body myositis.

Early onset Parkinsonism associated with an intronic SOD1 mutation.

We report on a patient belonging to a large family with autosomal-dominant amyotrophic lateral sclerosis, who developed asymmetrical akineto-rigid symptoms at 33 years of age. He had no signs of lower motor neuron disease after four years of follow-up. All seven ALS patients from this family harboured a mutation located in the fourth intron of the SOD1 gene. The proband also harboured the same mutation, associated with a 40% decrease in SOD1 erythrocyte activity. This case report suggests that SOD1 mutations might be associated with marked phenotypic variability (ALS or early onset Parkinsonism in this family).