Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the gradual death of motor neurons in the brain and spinal cord, leading to fatal paralysis. Socioeconomic status (SES) is a measure of an individual's shared economic and social status, which has been shown to have an association with health outcomes. Understanding the impact of SES on health conditions is crucial, as it can influence and be influenced by health-related variables. The role of socioeconomic status in influencing the risk and progression of ALS has not been established, and understanding the various factors that impact ALS is important in developing strategies for treatment and prevention. To investigate this relationship, we recruited 413 participants with definite, probable, or possible ALS according to the El Escorial criteria, from three tertiary centers in London, Sheffield, and Birmingham. Logistic regression was used to examine the association between case-control status, socioeconomic criteria, and ALS risk. Linear regression was used to examine the association between age of onset and socioeconomic variables. Two sensitivity analyses were performed, one using an alternative occupational classifier, and the other using Mendelian Randomization analysis to examine association. There was no significant relationship between any variables and ALS risk. We found an inverse relationship between mean lifetime salary and age of ALS onset (Beta = -0.157, p = 0.011), but no effect of education or occupation on the age of onset. The finding was confirmed in both sensitivity analyses and in Mendelian Randomization. We find that a higher salary is associated with a younger age of ALS onset taking into account sex, occupation, years of education, and clinical presentation.

Objective: While motor symptoms are well-known in ALS, non-motor symptoms are often under-reported and may have a significant impact on quality of life. In this study, we aimed to examine the nature and extent of non-motor symptoms in ALS. Methods: A 20-item questionnaire was developed covering the domains of autonomic function, sleep, pain, gastrointestinal disturbance, and emotional lability, posted online and shared on social media platforms to target people with ALS and controls. Results: A total of 1018 responses were received, of which 927 were complete from 506 people with ALS and 421 unaffected individuals. Cold limbs (p 1.66 × 10-36), painful limbs (p 6.14 × 10-28), and urinary urgency (p 4.70 × 10-23) were associated with ALS. People with ALS were more likely to report autonomic symptoms, pain, and psychiatric symptoms than controls (autonomic symptoms B = 0.043, p 6.10 × 10-5, pain domain B = 0.18, p 3.72 × 10-11 and psychiatric domain B = 0.173, p 1.32 × 10-4). Conclusions: Non-motor symptoms in ALS are common. The identification and management of non-motor symptoms should be integrated into routine clinical care for people with ALS. Further research is warranted to investigate the relationship between non-motor symptoms and disease progression, as well as to develop targeted interventions to improve the quality of life for people with ALS.

Repeat expansions in the C9orf72 gene are the most common genetic cause of (ALS) and frontotemporal dementia (FTD). Like other genetic forms of neurodegeneration, pinpointing the precise mechanism(s) by which this mutation leads to neuronal death remains elusive, and this lack of knowledge hampers the development of therapy for C9orf72-related disease. We used an agnostic approach based on genomic data (n = 41,273 ALS and healthy samples, and n = 1,516 C9orf72 carriers) to overcome these bottlenecks. Our drug-repurposing screen, based on gene- and expression-pattern matching and information about the genetic variants influencing onset age among C9orf72 carriers, identified acamprosate, a γ-aminobutyric acid analog, as a potentially repurposable treatment for patients carrying C9orf72 repeat expansions. We validated its neuroprotective effect in cell models and showed comparable efficacy to riluzole, the current standard of care. Our work highlights the potential value of genomics in repurposing drugs in situations where the underlying pathomechanisms are inherently complex. VIDEO ABSTRACT.

ABSTRACT Primary lateral sclerosis (PLS) is the rarest form of motor neurone disease (MND). It is characterized by upper motor neuron degeneration, leading to progressive weakness, spasticity and functional disability. Although PLS does not typically shorten life substantially, it gradually impacts quality of life as the diseases progresses. There is no established genetic cause of PLS. One of the biggest challenges faced by people with PLS is delayed diagnosis and misdiagnosis, since the initial symptoms can be similar to amyotrophic lateral sclerosis (ALS), the most common form of MND. In the absence of a concrete genetic test that differentiates PLS from other MNDs, this delay in diagnosis is inevitable. Understanding the genetic basis of PLS might help in reducing the time from the onset of symptoms to diagnosis, and it will improve our understanding of the disease biology favouring the development of a treatment. The aim of our study is to collect a large international PLS genetic and clinical dataset to investigate its genetic and phenotypic landscapes as well as to evaluate whether genetic testing should be advised in PLS. Through Project MinE and AnswerALS, we accessed whole-genome sequencing data of 120 PLS, 7405 ALS and 2444 controls. We identified variants in several MND genes such as FIG4, FUS, SPG7, SPG11 and SQSTM1 genes among others and repeat expansions in the ATXN1 (12.2%) and NIPA1 (7.3%) genes, but none in the C9orf72 and ATXN2 genes. Overall PLS patients harboured fewer clinically actionable MND-associated variants than ALS patients (p = 0.0001), however, depending on the panel, up to 11% of people with PLS might benefit from genetic testing. By looking at the clinical characteristics of these cohorts, the age of symptom onset was not younger for people with PLS than for those with ALS in both Project MinE and AnswerALS. On such bases, we advise that the current diagnostic criteria that discourage the use of genetic testing and rely on age of onset should be reconsidered.

The Covid-19 pandemic has impacted healthcare. Our aim was to identify how amyotrophic lateral sclerosis (ALS) care in the UK has been affected by the pandemic by exploring the experiences of people living with ALS (plwALS), healthcare professionals (HCPs) working with plwALS, and ALS care centers. Three surveys were carried out to explore the experiences of plwALS, HCPs and ALS care centers during the pandemic. Quantitative data were analyzed using descriptive and inferential statistics and triangulated with the qualitative data which were analyzed thematically. Responses from 53 plwALS, 73 HCPs and 23 ALS care centers were analyzed. Five main themes were identified: keeping safe, losses, negative emotions, delivering care and alternative care delivery in a pandemic. PlwALS and HCPs felt that care was sub-optimal as a result of the pandemic. Changes to care included longer waiting times and face-to-face appointments being canceled or replaced by virtual consultations. While benefits of virtual consultations were reported, concerns were raised about incomplete clinical assessments and the disruption of provision of testing and interventions. ALS care has changed as a result of the pandemic. Patients have had a lack of face-to-face contact with HCPs and have experienced delays to investigations and treatments. PlwALS and HCPs were concerned about the impact of this change, but the long-term implications remain unclear. We propose recommendations for HCPs caring for plwALS, that will promote continuity of evidenced based care in the context of a pandemic.

Mutations in the superoxide dismutase 1 (SOD1) gene are the second most common known cause of ALS. SOD1 variants express high phenotypic variability and over 200 have been reported in people with ALS. It was previously proposed that variants can be broadly classified in two groups, 'wild-type like' (WTL) and 'metal binding region' (MBR) variants, based on their structural location and biophysical properties. MBR variants, but not WTL variants, were associated with a reduction of SOD1 enzymatic activity. In this study we used molecular dynamics and large clinical datasets to characterise the differences in the structural and dynamic behaviour of WTL and MBR variants with respect to the wild-type SOD1, and how such differences influence the ALS clinical phenotype. Our study identified marked structural differences, some of which are observed in both variant groups, while others are group specific. Moreover, collecting clinical data of approximately 500 SOD1 ALS patients carrying variants, we showed that the survival time of patients carrying an MBR variant is generally longer (∼6 years median difference, p < 0.001) with respect to patients with a WTL variant. In conclusion, our study highlighted key differences in the dynamic behaviour between WTL and MBR SOD1 variants, and between variants and wild-type SOD1 at an atomic and molecular level, that could be further investigated to explain the associated phenotypic variability. Our results support the hypothesis of a decoupling between mechanisms of onset and progression of SOD1 ALS, and an involvement of loss-of-function of SOD1 with the disease progression.

Objective: Variants in the superoxide dismutase (SOD1) gene are among the most common genetic causes of amyotrophic lateral sclerosis. Reflecting the wide spectrum of putatively deleterious variants that have been reported to date, it has become clear that SOD1-linked ALS presents a highly variable age at symptom onset and disease duration.Methods: Here we describe an open access web tool for comparative phenotype analysis in ALS: https://sod1-als-browser.rosalind.kcl.ac.uk/. The tool contains a built-in dataset of clinical information from 1383 people with ALS harboring a SOD1 variant resulting in one of 162 unique amino acid sequence alterations and from a non-SOD1 comparator ALS cohort of 13,469 individuals. We present two examples of analyses possible with this tool, testing how the ALS phenotype relates to SOD1 variants that alter amino acid residue hydrophobicity and to distinct variants at the 94th residue of SOD1, where six are sampled.Results and conclusions: The tool provides immediate access to the datasets and enables bespoke analysis of phenotypic trends associated with different protein variants, including the option for users to upload their own datasets for integration with the server data. The tool can be used to study SOD1-ALS and provides an analytical framework to study the differences between other user-uploaded ALS groups and our large reference database of SOD1 and non-SOD1 ALS. The tool is designed to be useful for clinicians and researchers, including those without programming expertise, and is highly flexible in the analyses that can be conducted.

Objective: In 2021, the Clinical Genome Resource (ClinGen) amyotrophic lateral sclerosis (ALS) spectrum disorders Gene Curation Expert Panel (GCEP) was established to evaluate the strength of evidence for genes previously reported to be associated with ALS. Through this endeavor, we will provide standardized guidance to laboratories on which genes should be included in clinical genetic testing panels for ALS. In this manuscript, we aimed to assess the heterogeneity in the current global landscape of clinical genetic testing for ALS. Methods: We reviewed the National Institutes of Health (NIH) Genetic Testing Registry (GTR) and members of the ALS GCEP to source frequently used testing panels and compare the genes included on the tests. Results: 14 clinical panels specific to ALS from 14 laboratories covered 4 to 54 genes. All panels report on ANG, SOD1, TARDBP, and VAPB; 50% included or offered the option of including C9orf72 hexanucleotide repeat expansion (HRE) analysis. Of the 91 genes included in at least one of the panels, 40 (44.0%) were included on only a single panel. We could not find a direct link to ALS in the literature for 14 (15.4%) included genes. Conclusions: The variability across the surveyed clinical genetic panels is concerning due to the possibility of reduced diagnostic yields in clinical practice and risk of a missed diagnoses for patients. Our results highlight the necessity for consensus regarding the appropriateness of gene inclusions in clinical genetic ALS tests to improve its application for patients living with ALS and their families.

Superoxide dismutase (SOD1) gene variants may cause amyotrophic lateral sclerosis, some of which are associated with a distinct phenotype. Most studies assess limited variants or sample sizes. In this international, retrospective observational study, we compare phenotypic and demographic characteristics between people with SOD1-ALS and people with ALS and no recorded SOD1 variant. We investigate which variants are associated with age at symptom onset and time from onset to death or censoring using Cox proportional-hazards regression. The SOD1-ALS dataset reports age of onset for 1122 and disease duration for 883 people; the comparator population includes 10,214 and 9010 people respectively. Eight variants are associated with younger age of onset and distinct survival trajectories; a further eight associated with younger onset only and one with distinct survival only. Here we show that onset and survival are decoupled in SOD1-ALS. Future research should characterise rarer variants and molecular mechanisms causing the observed variability.

Background: Genetic penetrance is the probability of a phenotype when harbouring a particular pathogenic variant. Accurate penetrance estimates are important across biomedical fields including genetic counselling, disease research, and gene therapy. However, existing approaches for penetrance estimation require, for instance, large family pedigrees or availability of large databases of people affected and not affected by a disease.Methods: We present a method for penetrance estimation in autosomal dominant phenotypes. It examines the distribution of a variant among people affected (cases) and unaffected (controls) by a phenotype within population-scale data and can be operated using cases only by considering family disease history. It is validated through simulation studies and candidate variant-disease case studies.Results: Our method yields penetrance estimates which align with those obtained via existing approaches in the Parkinson's disease LRRK2 gene and pulmonary arterial hypertension BMPR2 gene case studies. In the amyotrophic lateral sclerosis case studies, examining penetrance for variants in the SOD1 and C9orf72 genes, we make novel penetrance estimates which correspond closely to understanding of the disease.Conclusions: The present approach broadens the spectrum of traits for which reliable penetrance estimates can be obtained. It has substantial utility for facilitating the characterisation of disease risks associated with rare variants with an autosomal dominant inheritance pattern. The yielded estimates avoid any kinship-specific effects and can circumvent ascertainment biases common when sampling rare variants among control populations.

Abstract Amyotrophic lateral sclerosis (ALS) is a heterogeneous neurodegenerative syndrome. In up to 20% of cases, a family history is observed. Although Mendelian disease gene variants are found in apparently sporadic ALS, genetic testing is usually restricted to those with a family history or younger patients with sporadic disease. With the advent of therapies targeting genetic ALS, it is important that everyone treatable is identified. We therefore sought to determine the probability of a clinically actionable ALS genetic test result by age of onset, globally, but using the UK as an exemplar. Blood-derived DNA was sequenced for ALS genes, and the probability of a clinically actionable genetic test result estimated. For a UK subset, age- and sex-specific population incidence rates were used to determine the number of such results missed by restricting testing by age of onset according to UK’s National Genomic Test Directory criteria. There were 6274 people with sporadic ALS, 1551 from the UK. The proportion with a clinically actionable genetic test result ranged between 0.21 [95% confidence interval (CI) 0.18–0.25] in the youngest age group to 0.15 (95% CI 0.13–0.17) in the oldest age group for a full gene panel. For the UK, the equivalent proportions were 0.23 (95% CI 0.13–0.33) in the youngest age group to 0.17 (95% CI 0.13–0.21) in the oldest age group. By limiting testing in those without a family history to people with onset below 40 years, 115 of 117 (98% of all, 95% CI 96%–101%) clinically actionable test results were missed. There is a significant probability of a clinically actionable genetic test result in people with apparently sporadic ALS at all ages. Although some countries limit testing by age, doing so results in a significant number of missed pathogenic test results. Age of onset and family history should not be a barrier to genetic testing in ALS.

There is a strong genetic contribution to Amyotrophic lateral sclerosis (ALS) risk, with heritability estimates of up to 60%. Both Mendelian and small effect variants have been identified, but in common with other conditions, such variants only explain a little of the heritability. Genomic structural variation might account for some of this otherwise unexplained heritability. We therefore investigated association between structural variation in a set of 25 ALS genes, and ALS risk and phenotype. As expected, the repeat expansion in the C9orf72 gene was identified as associated with ALS. Two other ALS-associated structural variants were identified: inversion in the VCP gene and insertion in the ERBB4 gene. All three variants were associated both with increased risk of ALS and specific phenotypic patterns of disease expression. More than 70% of people with respiratory onset ALS harboured ERBB4 insertion compared with 25% of the general population, suggesting respiratory onset ALS may be a distinct genetic subtype.

Objectives The use of synthetic data to supplement clinical trial placebo groups or for trial planning is rapidly gaining interest. However, there is not yet an established framework for generating synthetic data for these purposes. In this work we test two approaches to generating synthetic placebo arms for ALS trials with survival being the primary outcome variable. Methods For the first approach, we extracted sample subsets from the UK MND register (n = 308) using an evolutionary algorithm such that the subset baseline variables matched a target trials group, either people enrolled in LiCALS (n = 106) or people included in the PRO-ACT database (n = 171). We also applied trial specific exclusion criteria where possible or alternatively we applied a custom ‘time filter’. For the second approach, survival was predicted for LiCALS participants using the ENCALS model. Survival probabilities from each method were compared to real placebo participants using Kaplan-Meier analysis and the log rank test. Results We found that the synthetic placebo groups derived from the MND register matched the target trials outcomes very well. The ENCALS model produces synthetic placebo groups that are significantly different to the real placebo groups. However, when participants are censored at 6 month intervals, the ENCALS synthetic group matches the target group very well between 24 and 48 months, indicating a possible timeframe that this method could be utilised. Conclusion Both the register based approach and the ENCALS prognostic model generated synthetic placebo groups that matched placebo groups from historical trials. These methods need to be validated in prospective trials.

BackgroundAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of upper and lower motor neurons, leading to progressive weakness of voluntary muscles, with death following from neuromuscular respiratory failure, typically within 3 to 5 years. There is a strong genetic contribution to ALS risk. In 10% or more, a family history of ALS or frontotemporal dementia is obtained, and the Mendelian genes responsible for ALS in such families have now been identified in about 50% of cases. Only about 14% of apparently sporadic ALS is explained by known genetic variation, suggesting that other forms of genetic variation are important. Telomeres maintain DNA integrity during cellular replication, differ between sexes, and shorten naturally with age. Sex and age are risk factors for ALS and we therefore investigated telomere length in ALS.MethodsSamples were from Project MinE, an international ALS whole genome sequencing consortium that includes phenotype data. For validation we used donated brain samples from motor cortex from people with ALS and controls. Ancestry and relatedness were evaluated by principal components analysis and relationship matrices of DNA microarray data. Whole genome sequence data were from Illumina HiSeq platforms and aligned using the Isaac pipeline. TelSeq was used to quantify telomere length using whole genome sequence data. We tested the association of telomere length with ALS and ALS survival using Cox regression.ResultsThere were 6,580 whole genome sequences, reducing to 6,195 samples (4,315 from people with ALS and 1,880 controls) after quality control, and 159 brain samples (106 ALS, 53 controls). Accounting for age and sex, there was a 20% (95% CI 14%, 25%) increase of telomere length in people with ALS compared to controls (p = 1.1 × 10−12), validated in the brain samples (p = 0.03). Those with shorter telomeres had a 10% increase in median survival (p = 5.0×10−7). Although there was no difference in telomere length between sporadic ALS and familial ALS (p=0.64), telomere length in 334 people with ALS due to expanded C9orf72 repeats was shorter than in those without expanded C9orf72 repeats (p = 5.0×10−4).DiscussionAlthough telomeres shorten with age, longer telomeres are a risk factor for ALS and worsen prognosis. Longer telomeres are associated with ALS.

Evidence indicates that common variants found in genome-wide association studies increase risk of disease through gene regulation via expression Quantitative Trait Loci. Using multiple genome-wide methods, we examined if Single Nucleotide Polymorphisms increase risk of Amyotrophic Lateral Sclerosis through expression Quantitative Trait Loci, and whether expression Quantitative Trait Loci expression is consistent across people who had Amyotrophic Lateral Sclerosis and those who did not. In combining public expression Quantitative Trait Loci data with Amyotrophic Lateral Sclerosis genome-wide association studies, we used Summary-data-based Mendelian Randomization to confirm that SCFD1 was the only gene that was genome-wide significant in mediating Amyotrophic Lateral Sclerosis risk via expression Quantitative Trait Loci (Summary-data-based Mendelian Randomization beta = 0.20, standard error = 0.04, P-value = 4.29 × 10-6). Using post-mortem motor cortex, we tested whether expression Quantitative Trait Loci showed significant differences in expression between Amyotrophic Lateral Sclerosis (n = 76) and controls (n = 25), genome-wide. Of 20 757 genes analysed, the two most significant expression Quantitative Trait Loci to show differential in expression between Amyotrophic Lateral Sclerosis and controls involve two known Amyotrophic Lateral Sclerosis genes (SCFD1 and VCP). Cis-acting SCFD1 expression Quantitative Trait Loci downstream of the gene showed significant differences in expression between Amyotrophic Lateral Sclerosis and controls (top expression Quantitative Trait Loci beta = 0.34, standard error = 0.063, P-value = 4.54 × 10-7). These SCFD1 expression Quantitative Trait Loci also significantly modified Amyotrophic Lateral Sclerosis survival (number of samples = 4265, hazard ratio = 1.11, 95% confidence interval = 1.05-1.17, P-value = 2.06 × 10-4) and act as an Amyotrophic Lateral Sclerosis trans-expression Quantitative Trait Loci hotspot for a wider network of genes enriched for SCFD1 function and Amyotrophic Lateral Sclerosis pathways. Using gene-set analyses, we found the genes that correlate with this trans-expression Quantitative Trait Loci hotspot significantly increase risk of Amyotrophic Lateral Sclerosis (beta = 0.247, standard deviation = 0.017, P = 0.001) and schizophrenia (beta = 0.263, standard deviation = 0.008, P-value = 1.18 × 10-5), a disease that genetically correlates with Amyotrophic Lateral Sclerosis. In summary, SCFD1 expression Quantitative Trait Loci are a major factor in Amyotrophic Lateral Sclerosis, not only influencing disease risk but are differentially expressed in post-mortem Amyotrophic Lateral Sclerosis. SCFD1 expression Quantitative Trait Loci show distinct expression profiles in Amyotrophic Lateral Sclerosis that correlate with a wider network of genes that also confer risk of the disease and modify the disease's duration.

Introduction Amyotrophic lateral sclerosis (ALS) has a reported incidence of 1-2/100,000 person-years. It is estimated that there are 5000 people with ALS in the UK at any one time; however, the true figure and geographical distribution, are unknown. In this study, we describe the establishment of a population register for England, Wales, and Northern Ireland and report-estimated incidence.Methods: People with a diagnosis of ALS given by a consultant neurologist and whose postcode of residence is within England, Wales, or Northern Ireland were eligible. The catchment area was based on six data contributors that had been participating since 2016. All centres included in this analysis were in England, and therefore Wales and Northern Ireland are not included in this report. Crude age- and sex-specific incidence rates were estimated using population census records for the relevant postcodes from Office of National Statistics census data. These rates were standardized to the UK population structure using direct standardization.Results: There were 232 people in the database with a date of diagnosis between 2017 and 2018, when missing data were imputed there were an estimated 287-301 people. The denominator population of the catchment area is 7,251,845 according to 2011 UK census data. Age- and sex-adjusted incidence for complete cases was 1.61/100,000 person-years (95% confidence interval 1.58, 1.63), and for imputed datasets was 2.072/100,000 person-years (95% CI 2.072, 2.073).Discussion: We found incidence in this previously unreported area of the UK to be similar to other published estimates. As the MND Register for England, Wales, and Northern Ireland grows we will update incidence estimates and report on further analyses.

Illumina DNA methylation arrays are a widely used tool for performing genome-wide DNA methylation analyses. However, measurements obtained from these arrays may be affected by technical artefacts that result in spurious associations if left unchecked. Cross-reactivity represents one of the major challenges, meaning that probes may map to multiple regions in the genome. Although several studies have reported on this issue, few studies have empirically examined the impact of cross-reactivity in an epigenome-wide association study (EWAS). In this paper, we report on cross-reactivity issues that we discovered in a large EWAS on the presence of the C9orf72 repeat expansion in ALS patients. Specifically, we found that that the majority of the significant probes inadvertently cross-hybridized to the C9orf72 locus. Importantly, these probes were not flagged as cross-reactive in previous studies, leading to novel insights into the extent to which cross-reactivity can impact EWAS. Our findings are particularly relevant for epigenetic studies into diseases associated with repeat expansions and other types of structural variation. More generally however, considering that most spurious associations were not excluded based on pre-defined sets of cross-reactive probes, we believe that the presented data-driven flag and consider approach is relevant for any type of EWAS.

We meta-analyze amyotrophic lateral sclerosis (ALS) genome-wide association study (GWAS) data of European and Chinese populations (84,694 individuals). We find an additional significant association between rs58854276 spanning ACSL5-ZDHHC6 with ALS (p = 8.3 x 10(-9)), with replication in an independent Australian cohort (1,502 individuals; p = 0.037). Moreover, B4GALNT1, G2E3-SCFD1, and TRIP11-ATXN3 are identified using a gene-based analysis. ACSL5 has been associated with rapid weight loss, as has another ALS-associated gene, GPX3. Weight loss is frequent in ALS patients and is associated with shorter survival. We investigate the effect of the ACSL5 and GPX3 single-nucleotide polymorphisms (SNPs), using longitudinal body composition and weight data of 77 patients and 77 controls. In patients' fat-free mass, although not significant, we observe an effect in the expected direction (rs58854276: 2.1 +/- 1.3 kg/A allele, p = 0.053; rs3828599: 1.0 +/- 1.3 kg/A allele, p = 0.22). No effect was observed in controls. Our findings support the increasing interest in lipid metabolism in ALS and link the disease genetics to weight loss in patients.

Objective Smoking has been widely studied as a susceptibility factor for amyotrophic lateral sclerosis (ALS), but results are conflicting and at risk of confounding bias. We used the results of recently published large genome-wide association studies and Mendelian randomisation methods to reduce confounding to assess the relationship between smoking and ALS. Methods Two genome-wide association studies investigating lifetime smoking (n=463 003) and ever smoking (n=1 232 091) were identified and used to define instrumental variables for smoking. A genome-wide association study of ALS (20 806 cases; 59 804 controls) was used as the outcome for inverse variance weighted Mendelian randomisation, and four other Mendelian randomisation methods, to test whether smoking is causal for ALS. Analyses were bidirectional to assess reverse causality. Results There was no strong evidence for a causal or reverse causal relationship between smoking and ALS. The results of Mendelian randomisation using the inverse variance weighted method were: lifetime smoking OR 0.94 (95% CI 0.74 to 1.19), p value 0.59; ever smoking OR 1.10 (95% CI 1 to 1.23), p value 0.05. Conclusions Using multiple methods, large sample sizes and sensitivity analyses, we find no evidence with Mendelian randomisation techniques that smoking causes ALS. Other smoking phenotypes, such as current smoking, may be suitable for future Mendelian randomisation studies

Abstract Polymorphisms associated with BIN1 (bridging integrator 1) confer the second greatest risk for developing late-onset Alzheimer’s disease. The biological consequences of this genetic variation are not fully understood; however, BIN1 is a binding partner for tau. Tau is normally a highly soluble cytoplasmic protein, but in Alzheimer’s disease, tau is abnormally phosphorylated and accumulates at synapses to exert synaptotoxicity. The purpose of this study was to determine whether alterations in BIN1 and tau in Alzheimer’s disease promote the damaging redistribution of tau to synapses, as a mechanism by which BIN1 polymorphisms may increase the risk of developing Alzheimer’s disease. We show that BIN1 is lost from the cytoplasmic fraction of Alzheimer’s disease cortex, and this is accompanied by the progressive mislocalization of phosphorylated tau to synapses. We confirmed proline 216 in tau as critical for tau interaction with the BIN1-SH3 domain and showed that the phosphorylation of tau disrupts this binding, suggesting that tau phosphorylation in Alzheimer’s disease disrupts tau–BIN1 associations. Moreover, we show that BIN1 knockdown in rat primary neurons to mimic BIN1 loss in Alzheimer’s disease brain causes the damaging accumulation of phosphorylated tau at synapses and alterations in dendritic spine morphology. We also observed reduced release of tau from neurons upon BIN1 silencing, suggesting that BIN1 loss disrupts the function of extracellular tau. Together, these data indicate that polymorphisms associated with BIN1 that reduce BIN1 protein levels in the brain likely act synergistically with increased tau phosphorylation to increase the risk of Alzheimer’s disease by disrupting cytoplasmic tau–BIN1 interactions, promoting the damaging mis-sorting of phosphorylated tau to synapses to alter synapse structure and reducing the release of physiological forms of tau to disrupt tau function.