Mutations in the tail and rod domains of the neurofilament heavy-chain gene increase the risk of ALS.

OBJECTIVE: Neurofilament heavy-chain gene (NEFH) variants are associated with multiple neurodegenerative diseases, however, their relationship with ALS has not been robustly explored. Still, NEFH is commonly included in genetic screening panels worldwide. We therefore aimed to determine if NEFH variants modify ALS risk. METHODS: Genetic data of 11,130 people with ALS and 7,416 controls from the literature and Project MinE were analysed. We performed meta-analyses of published case-control studies reporting NEFH variants, and variant analysis of NEFH in Project MinE whole-genome sequencing data. RESULTS: Fixed-effects meta-analysis found that rare (MAF <1%) missense variants in the tail domain of NEFH increase ALS risk (OR 4.55, 95% CI 2.13-9.71, p < 0.0001). In Project MinE, ultrarare NEFH variants increased ALS risk (OR 1.37 95% CI 1.14-1.63, p = 0.0007), with rod domain variants (mostly intronic) appearing to drive the association (OR 1.45 95% CI 1.18-1.77, pMadsen-Browning = 0.0007, pSKAT-O = 0.003). While in the tail domain, ultrarare (MAF <0.1%) pathogenic missense variants were also associated with higher risk of ALS (OR 1.94, 95% CI 0.86-4.37, pMadsen-Browning = 0.039), supporting the meta-analysis results. Finally, several tail in-frame deletions were also found to affect disease risk, however, both protective and pathogenic deletions were found in this domain, highlighting an intricated architecture that requires further investigation. INTERPRETATION: We showed that NEFH tail missense and in-frame deletion variants, and intronic rod variants are risk factors for ALS. However, they are not variants of large effect, and their functional impact needs to be clarified in further studies. Therefore, their inclusion in routine genetic screening panels should be reconsidered.

Hippocampal aggregation signatures of pathogenic UBQLN2 in amyotrophic lateral sclerosis and frontotemporal dementia.

Pathogenic variants in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis and/or frontotemporal dementia characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial amyotrophic lateral sclerosis and/or frontotemporal dementia cases not caused by UBQLN2 pathogenic variants, particularly C9orf72-linked cases. This makes the mechanistic role of mutant ubiquilin 2 protein and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 44 genotypically diverse amyotrophic lateral sclerosis cases with or without frontotemporal dementia, including eight cases with UBQLN2 variants (resulting in p.S222G, p.P497H, p.P506S, p.T487I (two cases), and p.P497L (three cases)). Using multiplexed (5-label) fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43, dipeptide repeat aggregates, and p62, in the hippocampus of controls (n = 6), or amyotrophic lateral sclerosis with or without frontotemporal dementia in sporadic (n = 20), unknown familial (n = 3), SOD1-linked (n = 1), FUS-linked (n = 1), C9orf72-linked (n = 5), and UBQLN2-linked (n = 8) cases. We differentiate between i) ubiquilin 2 aggregation together with phosphorylated TDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation promoted by UBQLN2 pathogenic variants that cause amyotrophic lateral sclerosis/and frontotemporal dementia. Overall, we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in amyotrophic lateral sclerosis with or without frontotemporal dementia, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene variants and to understand the mechanisms of UBQLN2-linked disease.

Mutations in FUS lead to synaptic dysregulation in ALS-iPSC derived neurons.

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset neurodegenerative disorder characterized by progressive muscular weakness due to the selective loss of motor neurons. Mutations in the gene Fused in Sarcoma (FUS) were identified as one cause of ALS. Here, we report that mutations in FUS lead to upregulation of synaptic proteins, increasing synaptic activity and abnormal release of vesicles at the synaptic cleft. Consequently, FUS-ALS neurons showed greater vulnerability to glutamate excitotoxicity, which raised neuronal swellings (varicose neurites) and led to neuronal death. Fragile X mental retardation protein (FMRP) is an RNA-binding protein known to regulate synaptic protein translation, and its expression is reduced in the FUS-ALS lines. Collectively, our data suggest that a reduction of FMRP levels alters the synaptic protein dynamics, leading to synaptic dysfunction and glutamate excitotoxicity. Here, we present a mechanistic hypothesis linking dysregulation of peripheral translation with synaptic vulnerability in the pathogenesis of FUS-ALS.

SOD1-ALS-Browser: a web-utility for investigating the clinical phenotype in SOD1 amyotrophic lateral sclerosis.

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.

Generation of an Open-Access Patient-Derived iPSC Biobank for Amyotrophic Lateral Sclerosis Disease Modelling.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting the upper and lower motor neurons, causing patients to lose control over voluntary movement, and leading to gradual paralysis and death. There is no cure for ALS, and the development of viable therapeutics has proved challenging, demonstrated by a lack of positive results from clinical trials. One strategy to address this is to improve the tool kit available for pre-clinical research. Here, we describe the creation of an open-access ALS iPSC biobank generated from patients carrying mutations in the TARDBP, FUS, ANXA11, ARPP21, and C9ORF72 genes, alongside healthy controls. To demonstrate the utilisation of these lines for ALS disease modelling, a subset of FUS-ALS iPSCs were differentiated into functionally active motor neurons. Further characterisation revealed an increase in cytoplasmic FUS protein and reduced neurite outgrowth in FUS-ALS motor neurons compared to the control. This proof-of-principle study demonstrates that these novel patient-derived iPSC lines can recapitulate specific and early disease-related ALS phenotypes. This biobank provides a disease-relevant platform for discovery of ALS-associated cellular phenotypes to aid the development of novel treatment strategies.

Large-scale analyses of CAV1 and CAV2 suggest their expression is higher in post-mortem ALS brain tissue and affects survival.

Introduction: Caveolin-1 and Caveolin-2 (CAV1 and CAV2) are proteins associated with intercellular neurotrophic signalling. There is converging evidence that CAV1 and CAV2 (CAV1/2) genes have a role in amyotrophic lateral sclerosis (ALS). Disease-associated variants have been identified within CAV1/2 enhancers, which reduce gene expression and lead to disruption of membrane lipid rafts. Methods: Using large ALS whole-genome sequencing and post-mortem RNA sequencing datasets (5,987 and 365 tissue samples, respectively), and iPSC-derived motor neurons from 55 individuals, we investigated the role of CAV1/2 expression and enhancer variants in the ALS phenotype. Results: We report a differential expression analysis between ALS cases and controls for CAV1 and CAV2 genes across various post-mortem brain tissues and three independent datasets. CAV1 and CAV2 expression was consistently higher in ALS patients compared to controls, with significant results across the primary motor cortex, lateral motor cortex, and cerebellum. We also identify increased survival among carriers of CAV1/2 enhancer mutations compared to non-carriers within Project MinE and slower progression as measured by the ALSFRS. Carriers showed a median increase in survival of 345 days. Discussion: These results add to an increasing body of evidence linking CAV1 and CAV2 genes to ALS. We propose that carriers of CAV1/2 enhancer mutations may be conceptualised as an ALS subtype who present a less severe ALS phenotype with a longer survival duration and slower progression. Upregulation of CAV1/2 genes in ALS cases may indicate a causal pathway or a compensatory mechanism. Given prior research supporting the beneficial role of CAV1/2 expression in ALS patients, we consider a compensatory mechanism to better fit the available evidence, although further investigation into the biological pathways associated with CAV1/2 is needed to support this conclusion.

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases.

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.

The SOD1-mediated ALS phenotype shows a decoupling between age of symptom onset and disease duration.

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.

A shortened surface electromyography recording is sufficient to facilitate home fasciculation assessment.

INTRODUCTION/AIMS: Fasciculations are an early clinical hallmark of amyotrophic lateral sclerosis (ALS), amenable to detection by high-density surface electromyography (HDSEMG). In conjunction with the Surface Potential Quantification Engine (SPiQE), HDSEMG offers improved spatial resolution for the analysis of fasciculations. This study aims to establish an optimal recording duration to enable longitudinal remote monitoring in the home. METHODS: Twenty patients with ALS and five patients with benign fasciculation syndrome (BFS) underwent serial 30 min HDSEMG recordings from biceps brachii and gastrocnemii. SPiQE was independently applied to abbreviated epochs within each 30-min recording (0-5, 0-10, 0-15, 0-20, and 0-25 min), outputting fasciculation frequency, amplitude median and amplitude interquartile range. Bland-Altman plots and intraclass correlation coefficients (ICC) were used to assess agreement with the validated 30-min recording. RESULTS: In total, 506 full recordings were included. The 5 min recordings demonstrated diverse and relatively poor agreement with the 30 min baselines across all parameters, muscles and patient groups (ICC = 0.32-0.86). The 15-min recordings provided more acceptable and stable agreement (ICC = 0.78-0.98), which did not substantially improve in longer recordings. DISCUSSION: For the detection and quantification of fasciculations in patients with ALS and BFS, HDSEMG recordings can be halved from 30 to 15 min without significantly compromising the primary outputs. Reliance on a shorter recording duration should lead to improved tolerability and repeatability among patients, facilitating longitudinal remote monitoring in patients' homes.

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord.

Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce. We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases. Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.

Structural variation analysis of 6,500 whole genome sequences in amyotrophic lateral sclerosis.

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.

Telomere length analysis in amyotrophic lateral sclerosis using large-scale whole genome sequence data.

Background: Amyotrophic 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. Methods: Samples 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. Results: There 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). Discussion: Although telomeres shorten with age, longer telomeres are a risk factor for ALS and worsen prognosis. Longer telomeres are associated with ALS.

Common Genetic Variants Contribute to Risk of Transposition of the Great Arteries.

RATIONALE: Dextro-transposition of the great arteries (D-TGA) is a severe congenital heart defect which affects approximately 1 in 4,000 live births. While there are several reports of D-TGA patients with rare variants in individual genes, the majority of D-TGA cases remain genetically elusive. Familial recurrence patterns and the observation that most cases with D-TGA are sporadic suggest a polygenic inheritance for the disorder, yet this remains unexplored. OBJECTIVE: We sought to study the role of common single nucleotide polymorphisms (SNPs) in risk for D-TGA. METHODS AND RESULTS: We conducted a genome-wide association study in an international set of 1,237 patients with D-TGA and identified a genome-wide significant susceptibility locus on chromosome 3p14.3, which was subsequently replicated in an independent case-control set (rs56219800, meta-analysis P=8.6x10-10, OR=0.69 per C allele). SNP-based heritability analysis showed that 25% of variance in susceptibility to D-TGA may be explained by common variants. A genome-wide polygenic risk score derived from the discovery set was significantly associated to D-TGA in the replication set (P=4x10-5). The genome-wide significant locus (3p14.3) co-localizes with a putative regulatory element that interacts with the promoter of WNT5A, which encodes the Wnt Family Member 5A protein known for its role in cardiac development in mice. We show that this element drives reporter gene activity in the developing heart of mice and zebrafish and is bound by the developmental transcription factor TBX20. We further demonstrate that TBX20 attenuates Wnt5a expression levels in the developing mouse heart. CONCLUSIONS: This work provides support for a polygenic architecture in D-TGA and identifies a susceptibility locus on chromosome 3p14.3 near WNT5A. Genomic and functional data support a causal role of WNT5A at the locus.

SCFD1 expression quantitative trait loci in amyotrophic lateral sclerosis are differentially expressed.

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.

Cytoplasmic TDP-43 is involved in cell fate during stress recovery.

Transactive response DNA binding protein 43 (TDP-43) is an RNA processing protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear TDP-43 mislocalizes in patients to the cytoplasm, where it forms ubiquitin-positive inclusions in affected neurons and glia. Physiologically, cytoplasmic TDP-43 is associated with stress granules (SGs). Here, we explored TDP-43 cytoplasmic accumulation and stress granule formation following osmotic and oxidative stress. We show that sorbitol drives TDP-43 redistribution to the cytoplasm, while arsenite induces the recruitment of cytoplasmic TDP-43 to TIA-1 positive SGs. We demonstrate that inducing acute oxidative stress after TDP-43 cytoplasmic relocalization by osmotic shock induces poly (ADP-ribose) polymerase (PARP) cleavage, which triggers cellular toxicity. Recruitment of cytoplasmic TDP-43 to polyribosomes occurs in an SH-SY5Y cellular stress model and is observed in FTD brain lysate. Moreover, the processing body (P-body) marker DCP1a is detected in TDP-43 granules during recovery from stress. Overall, this study supports a central role for cytoplasmic TDP-43 in controlling protein translation in stressed cells.

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis.

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation. Objective: To identify the genetic variants associated with juvenile ALS. Design, Setting, and Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism. Main Outcomes and Measures: De novo variants present only in the index case and not in unaffected family members. Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway. Conclusions and Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.

A recessive S174X mutation in Optineurin causes amyotrophic lateral sclerosis through a loss of function via allele-specific nonsense-mediated decay.

Loss of function (LoF) mutations in Optineurin can cause recessive amyotrophic lateral sclerosis (ALS) with some heterozygous LoF mutations associated with dominant ALS. The molecular mechanisms underlying the variable inheritance pattern associated with OPTN mutations have remained elusive. We identified that affected members of a consanguineous Middle Eastern ALS kindred possessed a novel homozygous p.S174X OPTN mutation. Analysis of these primary fibroblast lines from family members identified that the p.S174X mutation reduces OPTN mRNA expression in an allele-dependent fashion by nonsense mediated decay. Western blotting correlated a reduced expression in heterozygote carriers but a complete absence of OPTN protein in the homozygous carrier. This data suggests that the p.S174X truncation mutation causes recessive ALS through LoF. However, functional analysis detected a significant increase in mitophagy markers TOM20 and COXIV, and higher rates of mitochondrial respiration and ATP levels in heterozygous carriers only. This suggests that heterozygous LoF OPTN mutations may not be causative in a Mendelian manner but may potentially behave as contributory ALS risk factors.

Disease Mechanisms and Therapeutic Approaches in C9orf72 ALS-FTD.

A hexanucleotide repeat expansion mutation in the first intron of C9orf72 is the most common known genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Since the discovery in 2011, numerous pathogenic mechanisms, including both loss and gain of function, have been proposed. The body of work overall suggests that toxic gain of function arising from bidirectionally transcribed repeat RNA is likely to be the primary driver of disease. In this review, we outline the key pathogenic mechanisms that have been proposed to date and discuss some of the novel therapeutic approaches currently in development.

ALS-linked FUS mutants affect the localization of U7 snRNP and replication-dependent histone gene expression in human cells.

Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3'UTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3' end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3' end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.