Longitudinal Effects of Asymptomatic C9orf72 Carriership on Brain Morphology.

OBJECTIVE: We investigated effects of C9orf72 repeat expansion and gene expression on longitudinal cerebral changes before symptom onset. METHODS: We enrolled 79 asymptomatic family members (AFMs) from 9 families with C9orf72 repeat expansion. Twenty-eight AFMs carried the mutation (C9+). Participants had up to 3 magnetic resonance imaging (MRI) scans, after which we compared motor cortex and motor tracts between C9+ and C9- AFMs using mixed effects models, incorporating kinship to correct for familial relations and lessen effects of other genetic factors. We also compared cortical, subcortical, cerebellar, and connectome structural measurements in a hypothesis-free analysis. We correlated regional C9orf72 expression in donor brains with the pattern of cortical thinning in C9+ AFMs using meta-regression. For comparison, we included 42 C9+ and 439 C9- patients with amyotrophic lateral sclerosis (ALS) in this analysis. RESULTS: C9+ AFM motor cortex had less gyrification and was thinner than in C9- AFMs, without differences in motor tracts. Whole brain analysis revealed thinner cortex and less gyrification in parietal, occipital, and temporal regions, smaller thalami and right hippocampus, and affected frontotemporal connections. Thinning of bilateral precentral, precuneus, and left superior parietal cortex was faster in C9+ than in C9- AFMs. Higher C9orf72 expression correlated with thinner cortex in both C9+ AFMs and C9+ ALS patients. INTERPRETATION: In asymptomatic C9orf72 repeat expansion carriers, brain MRI reveals widespread features suggestive of impaired neurodevelopment, along with faster decline of motor and parietal cortex than found in normal aging. C9orf72 expression might play a role in cortical development, and consequently explain the specific brain abnormalities of mutation carriers. ANN NEUROL 2023;93:668-680.

MRI Clustering Reveals Three ALS Subtypes With Unique Neurodegeneration Patterns.

OBJECTIVE: The purpose of this study was to identify subtypes of amyotrophic lateral sclerosis (ALS) by comparing patterns of neurodegeneration using brain magnetic resonance imaging (MRI) and explore their phenotypes. METHODS: We performed T1-weighted and diffusion tensor imaging in 488 clinically well-characterized patients with ALS and 338 control subjects. Measurements of whole-brain cortical thickness and white matter connectome fractional anisotropy were adjusted for disease-unrelated variation. A probabilistic network-based clustering algorithm was used to divide patients into subgroups of similar neurodegeneration patterns. Clinical characteristics and cognitive profiles were assessed for each subgroup. In total, 512 follow-up scans were used to validate clustering results longitudinally. RESULTS: The clustering algorithm divided patients with ALS into 3 subgroups of 187, 163, and 138 patients. All subgroups displayed involvement of the precentral gyrus and are characterized, respectively, by (1) pure motor involvement (pure motor cluster [PM]), (2) orbitofrontal and temporal involvement (frontotemporal cluster [FT]), and (3) involvement of the posterior cingulate cortex, parietal white matter, temporal operculum, and cerebellum (cingulate-parietal-temporal cluster [CPT]). These subgroups had significantly distinct clinical profiles regarding male-to-female ratio, age at symptom onset, and frequency of bulbar symptom onset. FT and CPT revealed higher rates of cognitive impairment on the Edinburgh cognitive and behavioral ALS screen (ECAS). Longitudinally, clustering remained stable: at 90.4% of their follow-up visits, patients clustered in the same subgroup as their baseline visit. INTERPRETATION: ALS can manifest itself in 3 main patterns of cerebral neurodegeneration, each associated with distinct clinical characteristics and cognitive profiles. Besides the pure motor and frontotemporal dementia (FTD)-like variants of ALS, a new neuroimaging phenotype has emerged, characterized by posterior cingulate, parietal, temporal, and cerebellar involvement. ANN NEUROL 2022;92:1030-1045.

Sensitivity of brain MRI and neurological examination for detection of upper motor neurone degeneration in amyotrophic lateral sclerosis.

OBJECTIVES: To investigate sensitivity of brain MRI and neurological examination for detection of upper motor neuron (UMN) degeneration in patients with amyotrophic lateral sclerosis (ALS). METHODS: We studied 192 patients with ALS and 314 controls longitudinally. All patients visited our centre twice and underwent full neurological examination and brain MRI. At each visit, we assessed UMN degeneration by measuring motor cortex thickness (CT) and pyramidal tract fibre density (FD) corresponding to five body regions (bulbar region and limbs). For each body region, we measured degree of clinical UMN and lower motor neuron (LMN) symptom burden using a validated scoring system. RESULTS: We found deterioration over time of CT of motor regions (p≤0.0081) and progression of UMN signs of bulbar region and left arm (p≤0.04). FD was discriminative between controls and patients with moderate/severe UMN signs (all regions, p≤0.034), but did not change longitudinally. Higher clinical UMN burden correlated with reduced CT, but not lower FD, for the bulbar region (p=2.2×10-10) and legs (p≤0.025). In the arms, we found that severe LMN signs may reduce the detectability of UMN signs (p≤0.043). With MRI, UMN degeneration was detectable before UMN signs became clinically evident (CT: p=1.1×10-10, FD: p=6.3×10-4). Motor CT, but not FD, deteriorated more than UMN signs during the study period. CONCLUSIONS: Motor CT is a more sensitive measure of UMN degeneration than UMN signs. Motor CT and pyramidal tract FD are discriminative between patients and controls. Brain MRI can monitor UMN degeneration before signs become clinically evident. These findings promote MRI as a potential biomarker for UMN progression in clinical trials in ALS.

The Distinct Traits of the UNC13A Polymorphism in Amyotrophic Lateral Sclerosis.

OBJECTIVE: The rs12608932 single nucleotide polymorphism in UNC13A is associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) susceptibility, and may underlie differences in treatment response. We aimed to characterize the clinical, cognitive, behavioral, and neuroimaging phenotype of UNC13A in patients with ALS. METHODS: We included 2,216 patients with ALS without a C9orf72 mutation to identify clinical characteristics associated with the UNC13A polymorphism. A subcohort of 428 patients with ALS was used to study cognitive and behavioral profiles, and 375 patients to study neuroimaging characteristics. Associations were analyzed under an additive genetic model. RESULTS: Genotyping rs12608932 resulted in 854 A/A, 988 A/C, and 374 C/C genotypes. The C allele was associated with a higher age at symptom onset (median years A/A 63.5, A/C 65.6, and C/C 65.5; p < 0.001), more frequent bulbar onset (A/A 29.6%, A/C 31.8%, and C/C 43.1%; p < 0.001), higher incidences of ALS-FTD (A/A 4.3%, A/C 5.2%, and C/C 9.5%; p = 0.003), lower forced vital capacity at diagnosis (median percentage A/A 92.0, A/C 90.0, and C/C 86.5; p < 0.001), and a shorter survival (median in months A/A 33.3, A.C 30.7, and C/C 26.6; p < 0.001). UNC13A was associated with lower scores on ALS-specific cognition tests (means A/A 79.5, A/C 78.1, and C/C 76.6; p = 0.037), and more frequent behavioral disturbances (A/A 16.7%, A/C 24.4%, and C/C 27.7%; p = 0.045). Thinner left inferior temporal and right fusiform cortex were associated with the UNC13A single nucleotide polymorphism (SNP; p = 0.045 and p = 0.036). INTERPRETATION: Phenotypical distinctions associated with UNC13A make it an important factor to take into account in clinical trial design, studies on cognition and behavior, and prognostic counseling. ANN NEUROL 2020;88:796-806.

Pharmacogenetic interactions in amyotrophic lateral sclerosis: a step closer to a cure?

Genetic mutations related to amyotrophic lateral sclerosis (ALS) act through distinct pathophysiological pathways, which may lead to varying treatment responses. Here we assess the genetic interaction between C9orf72, UNC13A, and MOBP with creatine and valproic acid treatment in two clinical trials. Genotypic data was available for 309 of the 338 participants (91.4%). The UNC13A genotype affected mortality (p = 0.012), whereas C9orf72 repeat-expansion carriers exhibited a faster rate of decline in overall (p = 0.051) and bulbar functioning (p = 0.005). A dose-response pharmacogenetic interaction was identified between creatine and the A allele of the MOBP genotype (p = 0.027), suggesting a qualitative interaction in a recessive model (HR 3.96, p = 0.015). Not taking genetic information into account may mask evidence of response to treatment or be an unrecognized source of bias. Incorporating genetic data could help investigators to identify critical treatment clues in patients with ALS.

Prognostic value of weight loss in patients with amyotrophic lateral sclerosis: a population-based study.

OBJECTIVE: To determine the prevalence and prognostic value of weight loss (WL) prior to diagnosis in patients with amyotrophic lateral sclerosis (ALS). METHODS: We enrolled patients diagnosed with ALS between 2010 and 2018 in a population-based setting. At diagnosis, detailed information was obtained regarding the patient's disease characteristics, anthropological changes, ALS-related genotypes and cognitive functioning. Complete survival data were obtained. Cox proportional hazard models were used to assess the association between WL and the risk of death during follow-up. RESULTS: The data set comprised 2420 patients of whom 67.5% reported WL at diagnosis. WL occurred in 71.8% of the bulbar-onset and in 64.2% of the spinal-onset patients; the mean loss of body weight was 6.9% (95% CI 6.8 to 6.9) and 5.5% (95% CI 5.5 to 5.6), respectively (p<0.001). WL occurred in 35.1% of the patients without any symptom of dysphagia. WL is a strong independent predictor of survival, with a dose response relationship between the amount of WL and the risk of death: the risk of death during follow-up increased by 23% for every 10% increase in WL relative to body weight (HR 1.23, 95% CI 1.13 to 1.51, p<0.001). CONCLUSIONS: This population-based study shows that two-thirds of the patients with ALS have WL at diagnosis, which also occurs independent of dysphagia, and is related to survival. Our results suggest that WL is a multifactorial process that may differ from patient to patient. Gaining further insight in its underlying factors could prove essential for future therapeutic measures.

Discussing personalized prognosis in amyotrophic lateral sclerosis: development of a communication guide.

BACKGROUND: Personalized ENCALS survival prediction model reliably estimates the personalized prognosis of patients with amyotrophic lateral sclerosis. Concerns were raised on discussing personalized prognosis without causing anxiety and destroying hope. Tailoring communication to patient readiness and patient needs mediates the impact of prognostic disclosure. We developed a communication guide to support physicians in discussing personalized prognosis tailored to individual needs and preferences of people with ALS and their families. METHODS: A multidisciplinary working group of neurologists, rehabilitation physicians, and healthcare researchers A) identified relevant topics for guidance, B) conducted a systematic review on needs of patients regarding prognostic discussion in life-limiting disease, C) drafted recommendations based on evidence and expert opinion, and refined and finalized these recommendations in consensus rounds, based on feedback of an expert advisory panel (patients, family member, ethicist, and spiritual counsellor). RESULTS: A) Topics identified for guidance were 1) filling in the ENCALS survival model, and interpreting outcomes and uncertainty, and 2) tailoring discussion to individual needs and preferences of patients (information needs, role and needs of family, severe cognitive impairment or frontotemporal dementia, and non-western patients). B) 17 studies were included in the systematic review. C) Consensus procedures on drafted recommendations focused on selection of outcomes, uncertainty about estimated survival, culturally sensitive communication, and lack of decisional capacity. Recommendations for discussing the prognosis include the following: discuss prognosis based on the prognostic groups and their median survival, or, if more precise information is desired, on the interquartile range of the survival probability. Investigate needs and preferences of the patients and their families for prognostic disclosure, regardless of cultural background. If the patient does not want to know their prognosis, with patient permission discuss the prognosis with their family. If the patient is judged to lack decisional capacity, ask the family if they want to discuss the prognosis. Tailor prognostic disclosure step by step, discuss it in terms of time range, and emphasize uncertainty of individual survival time. CONCLUSION: This communication guide supports physicians in tailoring discussion of personalized prognosis to the individual needs and preferences of people with ALS and their families.

Multicentre, cross-cultural, population-based, case-control study of physical activity as risk factor for amyotrophic lateral sclerosis.

OBJECTIVE: To investigate the association between physical activity (PA) and amyotrophic lateral sclerosis (ALS) in population-based case-control studies in three European countries using a validated and harmonised questionnaire. METHODS: Patients with incident ALS and controls were recruited from five population-based registers in The Netherlands, Ireland and Italy. Demographic and data regarding educational level, smoking, alcohol habits and lifetime PA levels in both leisure and work time were gathered by questionnaire, and quantified using metabolic equivalent of task scores. Logistic regression models adjusting for PA-related factors were used to determine the association between PA and ALS risk, and forest plots were used to visualise heterogeneity between regions. RESULTS: 1557 patients and 2922 controls were included. We found a linear association between ALS and PA in leisure time (OR 1.07, P=0.01) and occupational activities (OR 1.06, P<0.001), and all activities combined (OR 1.06, P<0.001), with some heterogeneity between regions: the most evident association was seen in the Irish and Italian cohorts. After adjustment for other occupational exposures or exclusion of patients with a C9orf72 mutation, the ORs remained similar. CONCLUSION: We provide new class I evidence for a positive association between PA and risk of ALS in a large multicentre study using harmonised methodology to objectively quantify PA levels, with some suggestions for population differences.

C9orf72 expansion differentially affects males with spinal onset amyotrophic lateral sclerosis.

INTRODUCTION: The C9orf72 repeat expansion has been reported as a negative prognostic factor in amyotrophic lateral sclerosis (ALS). We have examined the prognostic impact of the C9orf72 repeat expansion in European subgroups based on gender and site of onset. METHODS: C9orf72 status and demographic/clinical data from 4925 patients with ALS drawn from 3 prospective ALS registers (Ireland, Italy and the Netherlands), and clinical data sets in the UK and Belgium. Flexible parametric survival models were built including known prognostic factors (age, diagnostic delay and site of onset), gender and the presence of an expanded repeat in C9orf72. These were used to explore the effects of C9orf72 on survival by gender and site of onset. Individual patient data (IPD) meta-analysis was used to estimate HRs for results of particular importance. RESULTS: 457 (8.95%) of 4925 ALS cases carried the C9orf72 repeat expansion. A meta-analysis of C9orf72 estimated a survival HR of 1.36 (1.18 to 1.57) for those carrying the expansion. Models evaluating interaction between gender and C9orf72 repeat expansions demonstrated that the reduced survival due to C9orf72 expansion was being driven by spinal onset males (HR 1.56 (95% CI 1.25 to 1.96). CONCLUSIONS: This study represents the largest combined analysis of the prognostic characteristics of the C9orf72 expansion. We have shown for the first time that the negative prognostic implication of this variant is driven by males with spinal onset disease, indicating a hitherto unrecognised gender-mediated effect of the variant that requires further exploration.

C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits.

OBJECTIVE: How hexanucleotide (GGGGCC) repeat expansions in C9ORF72 cause amyotrophic lateral sclerosis (ALS) remains poorly understood. Both gain- and loss-of-function mechanisms have been proposed. Evidence supporting these mechanisms in vivo is, however, incomplete. Here we determined the effect of C9orf72 loss-of-function in mice. METHODS: We generated and analyzed a conditional C9orf72 knockout mouse model. C9orf72(fl/fl) mice were crossed with Nestin-Cre mice to selectively remove C9orf72 from neurons and glial cells. Immunohistochemistry was performed to study motor neurons and neuromuscular integrity, as well as several pathological hallmarks of ALS, such as gliosis and TDP-43 mislocalization. In addition, motor function and survival were assessed. RESULTS: Neural-specific ablation of C9orf72 in conditional C9orf72 knockout mice resulted in significantly reduced body weight but did not induce motor neuron degeneration, defects in motor function, or altered survival. INTERPRETATION: Our data suggest that C9orf72 loss-of-function, by itself, is insufficient to cause motor neuron disease. These results may have important implications for the development of therapeutic strategies for C9orf72-associated ALS.

Widespread structural brain involvement in ALS is not limited to the C9orf72 repeat expansion.

BACKGROUND: In patients with a C9orf72 repeat expansion (C9+), a neuroimaging phenotype with widespread structural cerebral changes has been found. We aimed to investigate the specificity of this neuroimaging phenotype in patients with amyotrophic lateral sclerosis (ALS). METHODS: 156 C9- and 14 C9+ patients with ALS underwent high-resolution T1-weighted MRI; a subset (n=126) underwent diffusion-weighted imaging. Cortical thickness, subcortical volumes and white matter integrity were compared between C9+ and C9- patients. Using elastic net logistic regression, a model defining the neuroimaging phenotype of C9+ was determined and applied to C9- patients with ALS. RESULTS: C9+ patients showed cortical thinning outside the precentral gyrus, extending to the bilateral pars opercularis, fusiform, lingual, isthmus-cingulate and superior parietal cortex, and smaller volumes of the right hippocampus and bilateral thalamus, and reduced white matter integrity of the inferior and superior longitudinal fasciculus compared with C9- patients (p<0.05). Among 128 C9- patients, we detected a subgroup of 27 (21%) with a neuroimaging phenotype congruent to C9+ patients, while 101 (79%) C9- patients showed cortical thinning restricted to the primary motor cortex. C9- patients with a 'C9+' neuroimaging phenotype had lower performance on the frontal assessment battery, compared with other C9- patients with ALS (p=0.004). CONCLUSIONS: This study shows that widespread structural brain involvement is not limited to C9+ patients, but also presents in a subgroup of C9- patients with ALS and relates to cognitive deficits. Our neuroimaging findings reveal an intermediate phenotype that may provide insight into the complex relationship between genetic factors and clinical characteristics.

Cortical thickness in ALS: towards a marker for upper motor neuron involvement.

OBJECTIVE: Examine whether cortical thinning is a disease-specific phenomenon across the spectrum of motor neuron diseases in relation to upper motor neuron (UMN) involvement. METHODS: 153 patients (112 amyotrophic lateral sclerosis (ALS), 19 patients with a clinical UMN phenotype, 22 with a lower motor neuron (LMN) phenotype), 60 healthy controls and 43 patients with an ALS mimic disorder were included for a cross-sectional cortical thickness analysis. Thirty-nine patients with ALS underwent a follow-up scan. T1-weighted images of the brain were acquired using a 3 T scanner. The relation between cortical thickness and clinical measures, and the longitudinal changes were examined. RESULTS: Cortical thickness of the precentral gyrus (PCG) was significantly reduced in ALS (p=1.71×10(-13)) but not in mimic disorders (p=0.37) or patients with an LMN phenotype (p=0.37), as compared to the group of healthy controls. Compared to patients with ALS, patients with a UMN phenotype showed an even lower PCG cortical thickness (p=1.97×10(-3)). Bulbar scores and arm functional scores showed a significant association with cortical thickness of corresponding body regions of the motor homunculus. Longitudinal analysis revealed a decrease of cortical thickness in the left temporal lobe of patients with ALS (parahippocampal region p=0.007 and fusiform cortex p=0.001). CONCLUSIONS: PCG cortical thinning was found to be specific for motor neuron disease with clinical UMN involvement. Normal levels of cortical thickness in mimic disorders or LMN phenotypes suggest that cortical thinning reflects pathological changes related to UMN involvement. Progressive cortical thinning in the temporal lobe suggests recruitment of non-motor areas, over time.

Assessment of risk of ALS conferred by the GGGGCC hexanucleotide repeat expansion in C9orf72 among first-degree relatives of patients with ALS carrying the repeat expansion.

OBJECTIVES: We aimed to estimate the age-related risk of ALS in first-degree relatives of patients with ALS carrying the C9orf72 repeat expansion. METHODS: We included all patients with ALS carrying a C9orf72 repeat expansion in The Netherlands. Using structured questionnaires, we determined the number of first-degree relatives, their age at death due to ALS or another cause, or age at time of questionnaire. The cumulative incidence of ALS among first-degree relatives was estimated, while accounting for death from other causes. Variability in ALS risk between families was evaluated using a random effects hazards model. We used a second, distinct approach to estimate the risk of ALS and FTD in the general population, using previously published data. RESULTS: In total, 214 of the 2,486 (9.2%) patients with ALS carried the C9orf72 repeat expansion. The mean risk of ALS at age 80 for first-degree relatives carrying the repeat expansion was 24.1%, but ranged between individual families from 16.0 to 60.6%. Using the second approach, we found the risk of ALS and FTD combined was 28.7% (95% CI 17.8%-54.3%) for carriers in the general population. CONCLUSIONS: On average, our estimated risk of ALS in the C9orf72 repeat expansion was lower compared to historical estimates. We showed, however, that the risk of ALS likely varies between families and one overall penetrance estimate may not be sufficient to describe ALS risk. This warrants a tailor-made, patient-specific approach in testing. Further studies are needed to assess the risk of FTD in the C9orf72 repeat expansion.

Association Between Hypothalamic Volume and Metabolism, Cognition, and Behavior in Patients With Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: Dysfunction of energy metabolism, cognition, and behavior are important nonmotor symptoms of amyotrophic lateral sclerosis (ALS), negatively affecting survival and quality of life, but poorly understood. Neuroimaging is ideally suited to studying nonmotor neurodegeneration in ALS, but few studies have focused on the hypothalamus, a key region for regulating energy homeostasis, cognition, and behavior. We evaluated, therefore, hypothalamic neurodegeneration in ALS and explored the relationship between hypothalamic volumes and dysregulation of energy metabolism, cognitive and behavioral changes, disease progression, and survival. METHODS: Patients with ALS and population-based controls were included for this cross-sectional and longitudinal MRI study. The hypothalamus was segmented into 5 subregions and their volumes were calculated. Linear (mixed) models, adjusted for age, sex, and total intracranial volume, were used to compare hypothalamic volumes between groups and to analyze associations with metabolism, cognition, behavior, and disease progression. Cox proportional hazard models were used to investigate the relationship of hypothalamic volumes with survival. Permutation-based corrections for multiple hypothesis testing were applied to all analyses to control the family-wise error rate. RESULTS: Data were available for 564 patients with ALS and 356 controls. The volume of the anterior superior subregion of the hypothalamus was smaller in patients with ALS than in controls (ß = -0.70 [-1.15 to -0.25], p = 0.013). Weight loss, memory impairments, and behavioral disinhibition were associated with a smaller posterior hypothalamus (ß = -4.79 [-8.39 to -2.49], p = 0.001, ß = -10.14 [-15.88 to -4.39], p = 0.004, and ß = -12.09 [-18.83 to -5.35], p = 0.003, respectively). Furthermore, the volume of this subregion decreased faster over time in patients than in controls (ß = -0.25 [0.42 to -0.09], p = 0.013), and a smaller volume of this structure was correlated with shorter survival (hazard ratio = 0.36 [0.21-0.61], p = 0.029). DISCUSSION: We obtained evidence for hypothalamic involvement in ALS, specifically marked by atrophy of the anterior superior subregion. Moreover, we found that atrophy of the posterior hypothalamus was associated with weight loss, memory dysfunction, behavioral disinhibition, and survival, and that this subregion deteriorated faster in patients with ALS than in controls. These findings improve our understanding of nonmotor involvement in ALS and could contribute to the identification of new treatment targets for this devastating disease.

Determining the role of sarcomeric proteins in facioscapulohumeral muscular dystrophy: a study protocol.

BACKGROUND: Although muscle weakness is a hallmark of facioscapulohumeral muscular dystrophy (FSHD), the molecular mechanisms that lead to weakness in FSHD remain largely unknown. Recent studies suggest aberrant expression of genes involved in skeletal muscle development and sarcomere contractility, and activation of pathways involved in sarcomeric protein degradation. This study will investigate the contribution of sarcomeric protein dysfunction to the pathogenesis of muscle weakness in FSHD. METHODS/DESIGN: Evaluation of sarcomeric function using skinned single muscle fiber contractile studies and protein analysis in muscle biopsies (quadriceps femoris and tibialis anterior) from patients with FSHD and age- and gender-matched healthy controls. Patients with other forms of muscular dystrophy and inflammatory myopathy will be included as disease controls to assess whether results are due to changes specific for FSHD, or a consequence of muscle disease in general. A total of 56 participants will be included. Extensive clinical parameters will be measured using MRI, quantitative muscle studies and physical activity assessments. DISCUSSION: This study is the first to extensively investigate muscle fiber physiology in FSHD following an earlier pilot study suggesting sarcomeric dysfunction in FSHD. The results obtained in this study will increase the understanding of the pathophysiology of muscle weakness in FSHD, and possibly identify novel targets for therapeutic intervention.

Genome-wide study of DNA methylation shows alterations in metabolic, inflammatory, and cholesterol pathways in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an estimated heritability between 40 and 50%. DNA methylation patterns can serve as proxies of (past) exposures and disease progression, as well as providing a potential mechanism that mediates genetic or environmental risk. Here, we present a blood-based epigenome-wide association study meta-analysis in 9706 samples passing stringent quality control (6763 patients, 2943 controls). We identified a total of 45 differentially methylated positions (DMPs) annotated to 42 genes, which are enriched for pathways and traits related to metabolism, cholesterol biosynthesis, and immunity. We then tested 39 DNA methylation-based proxies of putative ALS risk factors and found that high-density lipoprotein cholesterol, body mass index, white blood cell proportions, and alcohol intake were independently associated with ALS. Integration of these results with our latest genome-wide association study showed that cholesterol biosynthesis was potentially causally related to ALS. Last, DNA methylation at several DMPs and blood cell proportion estimates derived from DNA methylation data were associated with survival rate in patients, suggesting that they might represent indicators of underlying disease processes potentially amenable to therapeutic interventions.

Associations between lifestyle and amyotrophic lateral sclerosis stratified by C9orf72 genotype: a longitudinal, population-based, case-control study.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is considered to be caused by both genetic and environmental factors. The causal cascade is, however, not known. We aimed to assess lifestyle during the presymptomatic phase of ALS, stratified by C9orf72 mutation, and examine evidence supporting causality of lifestyle factors. METHODS: This study was a longitudinal, population-based, case-control study that used data from the Prospective ALS study the Netherlands. We included patients with a C9orf72 mutation (C9+ group), patients without a C9orf72 mutation (C9- group), and controls. Patients fulfilled the revised El Escorial criteria and were recruited through neurologists and rehabilitation physicians in the Netherlands as well as the Dutch Neuromuscular Patient Association and ALS Centrum website. 1322 population-based controls, matched for age and sex, were enrolled via the patients' general practitioners. Blood relatives or spouses of patients were not eligible as controls. We studied the relationship between ALS risk and smoking, alcohol, physical activity, body-mass index (BMI), and energy intake by the use of structured questionnaires. Smoking, physical activity, and BMI were longitudinally assessed up to 50 years before onset (defined as the period before onset of muscle weakness or bulbar symptoms for cases, or age at completing the questionnaire for controls). We calculated posterior probabilities (P(θ|x)) for causal effects of smoking, alcohol, and BMI, using Bayesian instrumental variable analyses. FINDINGS: Between Jan 1, 2006 and Jan 27, 2016, we included 143 patients in the C9+ group, 1322 patients in the C9- group, and 1322 controls. Compared with controls, cigarette pack-years (C9+ group mean difference from control 3·15, 95% CI 0·36 to 5·93, p=0·027; C9- group 3·20, 2·02 to 4·39, p<0·0001) and daily energy intake at symptom onset (C9+ group 712 kJ, 95% CI 212 to 1213, p=0·0053; C9- group 497, 295 to 700, p<0·0001) were higher in the C9+ and C9- groups, whereas current BMI (C9+ group -2·01 kg/m2, 95% CI -2·73 to -1·29, p<0·0001; C9- group -1·35, -1·64 to -1·06, p<0·0001) and lifetime alcohol consumption (C9+ group -5388 units, 95% CI -9113 to -1663, p=0·0046; C9- group -2185, -3748 to -622, p=0·0062) were lower in the C9+ and C9- groups. Median BMI during the presymptomatic phase for the C9+ group was lower (-0·69 kg/m2, 95% CI -1·24 to -0·13, p=0·015) and physical activity was similar (-348 metabolic equivalent of task [MET], 95% CI -966 to 270, p=0·27) to controls, whereas both the median BMI during the presymptomatic phase (0·27 kg/m2, 95% CI 0·04 to 0·50, p=0·022) and physical activity (585 MET, 291 to 878, p=0·0001) were higher in the C9- group than controls. Longitudinal analyses showed more cigarette pack-years in the C9- (starting 47 years pre-onset) and C9+ (starting 24 years pre-onset) groups, and higher physical activity over time in the C9- group (starting >30 years pre-onset). BMI of the C9+ group increased more slowly and was significantly lower (starting at 36 years pre-onset) than in controls, whereas the BMI of the C9- group was higher than controls (23-49 years pre-onset, becoming lower 10 years pre-onset). Instrumental variable analyses supported causal effects of alcohol consumption (P(θ|x)=0·9347) and smoking (P(θ|x)=0·9859) on ALS in the C9- group. We found evidence supporting a causal effect of increased BMI at younger age (mean 33·8 years, SD 11·7) in the C9- group (P[θ|x]=0·9272), but not at older ages. INTERPRETATION: Lifestyle during the presymptomatic phase differs between patients with ALS and controls decades before onset, depends on C9- status, and is probably part of the presymptomatic causal cascade. Identification of modifiable disease-causing lifestyle factors offers opportunities to lower risk of developing neurodegenerative disease. FUNDING: Netherlands ALS Foundation.

Innovating Clinical Trials for Amyotrophic Lateral Sclerosis: Challenging the Established Order.

Development of effective treatments for amyotrophic lateral sclerosis (ALS) has been hampered by disease heterogeneity, a limited understanding of underlying pathophysiology, and methodologic design challenges. We have evaluated 2 major themes in the design of pivotal, phase 3 clinical trials for ALS-(1) patient selection and (2) analytical strategy-and discussed potential solutions with the European Medicines Agency. Several design considerations were assessed using data from 5 placebo-controlled clinical trials (n = 988), 4 population-based cohorts (n = 5,100), and 2,436 placebo-allocated patients from the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) database. The validity of each proposed design modification was confirmed by means of simulation and illustrated for a hypothetical setting. Compared to classical trial design, the proposed design modifications reduce the sample size by 30.5% and placebo exposure time by 35.4%. By making use of prognostic survival models, one creates a potential to include a larger proportion of the population and maximize generalizability. We propose a flexible design framework that naturally adapts the trial duration when inaccurate assumptions are made at the design stage, such as enrollment or survival rate. In case of futility, the follow-up time is shortened and patient exposure to ineffective treatments or placebo is minimized. For diseases such as ALS, optimizing the use of resources, widening eligibility criteria, and minimizing exposure to futile treatments and placebo is critical to the development of effective treatments. Our proposed design modifications could circumvent important pitfalls and may serve as a blueprint for future clinical trials in this population.

Common and rare variant association analyses in amyotrophic lateral sclerosis identify 15 risk loci with distinct genetic architectures and neuron-specific biology.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.