Deep learning modeling of rare noncoding genetic variants in human motor neurons defines CCDC146 as a therapeutic target for ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study, we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs, this method prioritizes functional noncoding variants using deep learning, links cis-regulatory elements (CREs) to target genes using epigenomics data, and integrates these data through gene-level burden tests to identify survival-modifying variants, CREs, and genes. We apply this approach to analyze 6,715 ALS genomes, and pinpoint four novel rare noncoding variants associated with survival, including chr7:76,009,472:C>T linked to CCDC146. CRISPR-Cas9 editing of this variant increases CCDC146 expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes, including TDP-43 mislocalization. Suppressing CCDC146 with an antisense oligonucleotide (ASO), showing no toxicity, completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within C9ORF72. ASO targeting of CCDC146 may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases.

Mendelian Randomization Study With Clinical Follow-Up Links Metabolites to Risk and Severity of Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) exhibits phenotypic heterogeneity and variable response to therapy. The metabolome has been implicated in the pathogenesis of PAH, but previous works have lacked power to implicate specific metabolites. Mendelian randomization (MR) is a method for causal inference between exposures and outcomes. METHODS AND RESULTS: Using genome-wide association study summary statistics, we implemented MR analysis to test for potential causal relationships between serum concentration of 575 metabolites and PAH. Five metabolites were causally associated with the risk of PAH after multiple testing correction. Next, we measured serum concentration of candidate metabolites in an independent clinical cohort of 449 patients with PAH to check whether metabolite concentrations are correlated with markers of disease severity. Of the 5 candidates nominated by our MR work, serine was negatively associated and homostachydrine was positively associated with clinical severity of PAH via direct measurement in this independent clinical cohort. Finally we used conditional and orthogonal approaches to explore the biology underlying our lead metabolites. Rare variant burden testing was carried out using whole exome sequencing data from 578 PAH cases and 361 675 controls. Multivariable MR is an extension of MR that uses a single set of instrumental single-nucleotide polymorphisms to measure multiple exposures; multivariable MR is used to determine interdependence between the effects of different exposures on a single outcome. Rare variant analysis demonstrated that loss-of-function mutations within activating transcription factor 4, a transcription factor responsible for upregulation of serine synthesis under conditions of serine starvation, are associated with higher risk for PAH. Homostachydrine is a xenobiotic metabolite that is structurally related to l-proline betaine, which has previously been linked to modulation of inflammation and tissue remodeling in PAH. Our multivariable MR analysis suggests that the effect of l-proline betaine is actually mediated indirectly via homostachydrine. CONCLUSIONS: Our data present a method for study of the metabolome in the context of PAH, and suggests several candidates for further evaluation and translational research.

Rare and common genetic determinants of mitochondrial function determine severity but not risk of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving selective vulnerability of energy-intensive motor neurons (MNs). It has been unclear whether mitochondrial function is an upstream driver or a downstream modifier of neurotoxicity. We separated upstream genetic determinants of mitochondrial function, including genetic variation within the mitochondrial genome or autosomes; from downstream changeable factors including mitochondrial DNA copy number (mtCN). Across three cohorts including 6,437 ALS patients, we discovered that a set of mitochondrial haplotypes, chosen because they are linked to measurements of mitochondrial function, are a determinant of ALS survival following disease onset, but do not modify ALS risk. One particular haplotype appeared to be neuroprotective and was significantly over-represented in two cohorts of long-surviving ALS patients. Causal inference for mitochondrial function was achievable using mitochondrial haplotypes, but not autosomal SNPs in traditional Mendelian randomization (MR). Furthermore, rare loss-of-function genetic variants within, and reduced MN expression of, ACADM and DNA2 lead to ∼50 % shorter ALS survival; both proteins are implicated in mitochondrial function. Both mtCN and cellular vulnerability are linked to DNA2 function in ALS patient-derived neurons. Finally, MtCN responds dynamically to the onset of ALS independently of mitochondrial haplotype, and is correlated with disease severity. We conclude that, based on the genetic measures we have employed, mitochondrial function is a therapeutic target for amelioration of disease severity but not prevention of ALS.

Causal factors in primary open angle glaucoma: a phenome-wide Mendelian randomisation study.

Primary open angle glaucoma (POAG) is a chronic, adult-onset optic neuropathy associated with characteristic optic disc and/or visual field changes. With a view to identifying modifiable risk factors for this common neurodegenerative condition, we performed a 'phenome-wide' univariable Mendelian randomisation (MR) study that involved analysing the relationship between 9661 traits and POAG. Utilised analytical approaches included weighted mode based estimation, the weighted median method, the MR Egger method and the inverse variance weighted (IVW) approach. Eleven traits related to POAG risk were identified including: serum levels of the angiopoietin-1 receptor (OR [odds ratio] = 1.11, IVW p = 2.34E-06) and the cadherin 5 protein (OR = 1.06, IVW p = 1.31E-06); intraocular pressure (OR = 2.46-3.79, IVW p = 8.94E-44-3.00E-27); diabetes (OR = 5.17, beta = 1.64, IVW p = 9.68E-04); and waist circumference (OR = 0.79, IVW p = 1.66E-05). Future research focussing on the effects of adiposity, cadherin 5 and angiopoietin-1 receptor on POAG development and progression is expected to provide key insights that might inform the provision of lifestyle modification advice and/or the development of novel therapies.

A Y374X TDP43 truncation leads to an altered metabolic profile in amyotrophic lateral sclerosis fibroblasts driven by pyruvate and TCA cycle intermediate alterations.

A p.Y374X truncation in TARDBP was recently shown to reduce expression of TDP43 in fibroblasts isolated from ALS cases. In this follow up study focused on assessing the downstream phenotypic consequences of loss of TDP43 in the context of the truncation, we have shown a striking effect on the fibroblast metabolic profile. Phenotypic metabolic screening uncovered a distinct metabolic profile in TDP43-Y374X fibroblasts compared to controls, which was driven by alterations in key metabolic checkpoint intermediates including pyruvate, alpha-ketoglutarate and succinate. These metabolic alterations were confirmed using transcriptomics and bioenergetic flux analysis. These data suggest that TDP43 truncation directly compromises glycolytic and mitochondrial function, identifying potential therapeutic targets for mitigating the effects of TDP43-Y374X truncation.

Clinical testing panels for ALS: global distribution, consistency, and challenges.

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.

A cell-penetrant peptide blocking C9ORF72-repeat RNA nuclear export reduces the neurotoxic effects of dipeptide repeat proteins.

Hexanucleotide repeat expansions in C9ORF72 are the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Studies have shown that the hexanucleotide expansions cause the noncanonical translation of C9ORF72 transcripts into neurotoxic dipeptide repeat proteins (DPRs) that contribute to neurodegeneration. We show that a cell-penetrant peptide blocked the nuclear export of C9ORF72-repeat transcripts in HEK293T cells by competing with the interaction between SR-rich splicing factor 1 (SRSF1) and nuclear export factor 1 (NXF1). The cell-penetrant peptide also blocked the translation of toxic DPRs in neurons differentiated from induced neural progenitor cells (iNPCs), which were derived from individuals carrying C9ORF72-linked ALS mutations. This peptide also increased survival of iNPC-differentiated C9ORF72-ALS motor neurons cocultured with astrocytes. Oral administration of the cell-penetrant peptide reduced DPR translation and rescued locomotor deficits in a Drosophila model of mutant C9ORF72-mediated ALS/FTD. Intrathecal injection of this peptide into the brains of ALS/FTD mice carrying a C9ORF72 mutation resulted in reduced expression of DPRs in mouse brains. These findings demonstrate that disrupting the production of DPRs in cellular and animal models of ALS/FTD might be a strategy to ameliorate neurodegeneration in these diseases.

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.

Physical activity as an exogenous risk factor for amyotrophic lateral sclerosis: a review of the evidence.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder. The only established epidemiological risk factors for ALS are male sex and increasing age. The role of physical activity has been debated as an environmental risk factor. Over the last decade multiple studies have attempted to delineate the architecture of ALS. These have not yet established definite risk factors, often due to low-powered studies, lack of focus on at-risk genotypes and sub-optimal methodology. We have conducted a review of all the studies published between 2009 and December 2021. The free text search terms were [(motor neuron disease) OR (MND) OR (Amyotrophic Lateral Sclerosis) OR (ALS)] AND [(Exercise) or (Physical Activity) or (PA) or (sport)]. We identified common themes, for example soccer, head injury and the physiological mechanisms that differ in ALS patients. We have analysed the relevant, available studies (n = 93), highlighting the underlying reasons for any reported discrepancies. Overall, we have found that the more highly powered studies using validated exposure methodologies, linked strenuous, anaerobic physical activity as a risk factor for ALS. Future large-scale studies focusing on specific at-risk genotypes and physical activity should be conducted to confirm this finding. This will strengthen the evidence already surrounding strenuous physical activity as an environmental risk factor for ALS and allow advice to be given to at-risk family members. Increasing our understanding of the genetic-environmental interactions in the pathophysiology of ALS will allow for the possibility of developing preventative therapeutic approaches.

Phenome-wide Mendelian randomisation analysis identifies causal factors for age-related macular degeneration.

Background: Age-related macular degeneration (AMD) is a leading cause of blindness in the industrialised world and is projected to affect >280 million people worldwide by 2040. Aiming to identify causal factors and potential therapeutic targets for this common condition, we designed and undertook a phenome-wide Mendelian randomisation (MR) study. Methods: We evaluated the effect of 4591 exposure traits on early AMD using univariable MR. Statistically significant results were explored further using: validation in an advanced AMD cohort; MR Bayesian model averaging (MR-BMA); and multivariable MR. Results: Overall, 44 traits were found to be putatively causal for early AMD in univariable analysis. Serum proteins that were found to have significant relationships with AMD included S100-A5 (odds ratio [OR] = 1.07, p-value = 6.80E-06), cathepsin F (OR = 1.10, p-value = 7.16E-05), and serine palmitoyltransferase 2 (OR = 0.86, p-value = 1.00E-03). Univariable MR analysis also supported roles for complement and immune cell traits. Although numerous lipid traits were found to be significantly related to AMD, MR-BMA suggested a driving causal role for serum sphingomyelin (marginal inclusion probability [MIP] = 0.76; model-averaged causal estimate [MACE] = 0.29). Conclusions: The results of this MR study support several putative causal factors for AMD and highlight avenues for future translational research. Funding: This project was funded by the Wellcome Trust (224643/Z/21/Z; 200990/Z/16/Z); the University of Manchester's Wellcome Institutional Strategic Support Fund (Wellcome ISSF) grant (204796/Z/16/Z); the UK National Institute for Health Research (NIHR) Academic Clinical Fellow and Clinical Lecturer Programmes; Retina UK and Fight for Sight (GR586); the Australian National Health and Medical Research Council (NHMRC) (1150144).

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource for treatment of SARS-CoV-2 viral infection. Genome-wide association studies have identified risk loci associated with COVID-19, but many loci are associated with comorbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of the 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins. Aggregating COVID-19 genome-wide association studies statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19. EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. EXOSC2 is a component of the RNA exosome, and here, LC-MS/MS analysis of protein pulldowns demonstrated interaction between the SARS-CoV-2 RNA polymerase and most of the human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression and impeded SARS-CoV-2 replication without impacting cellular viability. Targeted depletion of EXOSC2 may be a safe and effective strategy to protect against clinical COVID-19.

Atypical TDP-43 protein expression in an ALS pedigree carrying a p.Y374X truncation mutation in TARDBP.

We describe an autosomal dominant, multi-generational, amyotrophic lateral sclerosis (ALS) pedigree in which disease co-segregates with a heterozygous p.Y374X nonsense mutation within TDP-43. Mislocalization of TDP-43 and formation of insoluble TDP-43-positive neuronal cytoplasmic inclusions is the hallmark pathology in >95% of ALS patients. Neuropathological examination of the single case for which CNS tissue was available indicated typical TDP-43 pathology within lower motor neurons, but classical TDP-43-positive inclusions were absent from motor cortex. The mutated allele is transcribed and translated in patient fibroblasts and motor cortex tissue, but overall TDP-43 protein expression is reduced compared to wild-type controls. Despite absence of TDP-43-positive inclusions we confirmed deficient TDP-43 splicing function within motor cortex tissue. Furthermore, urea fractionation and mass spectrometry of motor cortex tissue carrying the mutation revealed atypical TDP-43 protein species but not typical C-terminal fragments. We conclude that the p.Y374X mutation underpins a monogenic, fully penetrant form of ALS. Reduced expression of TDP-43 combined with atypical TDP-43 protein species and absent C-terminal fragments extends the molecular phenotypes associated with TDP-43 mutations and with ALS more broadly. Future work will need to include the findings from this pedigree in dissecting the mechanisms of TDP-43-mediated toxicity.

Unbiased metabolome screen links serum urate to risk of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disease caused by a combination of genetic and environmental risk factors. The serum metabolome refers to a set of small-molecules which are an important determinant of cellular health. We obtained genome-wide association study (GWAS) summary statistics for serum concentrations of 376 metabolites which were population matched with 2 GWAS studies of AD. For each metabolite we performed 2-sample MR (2SMR) using an inverse variance weighted (IVW) estimate for significance testing. After Bonferroni multiple testing correction one metabolite was causally linked to AD in both GWAS: serum urate. This result was supported by robust 2SMR measures and sensitivity analyses. We applied 2SMR to test for a causal relationship between serum urate and other neurodegenerative diseases: Parkinson disease (PD) and Amyotrophic lateral sclerosis (ALS). In ALS but not PD we identified a nominally significant link between serum urate and disease-risk, although in this case increased serum urate was protective. We conclude that serum urate is a modulator of risk for neurodegeneration. Our work has implications for the design of preventative interventions.

Multiomic analysis reveals cell-type-specific molecular determinants of COVID-19 severity.

The determinants of severe COVID-19 in healthy adults are poorly understood, which limits the opportunity for early intervention. We present a multiomic analysis using machine learning to characterize the genomic basis of COVID-19 severity. We use single-cell multiome profiling of human lungs to link genetic signals to cell-type-specific functions. We discover >1,000 risk genes across 19 cell types, which account for 77% of the SNP-based heritability for severe disease. Genetic risk is particularly focused within natural killer (NK) cells and T cells, placing the dysfunction of these cells upstream of severe disease. Mendelian randomization and single-cell profiling of human NK cells support the role of NK cells and further localize genetic risk to CD56bright NK cells, which are key cytokine producers during the innate immune response. Rare variant analysis confirms the enrichment of severe-disease-associated genetic variation within NK-cell risk genes. Our study provides insights into the pathogenesis of severe COVID-19 with potential therapeutic targets.

Whole-genome sequencing reveals that variants in the Interleukin 18 Receptor Accessory Protein 3'UTR protect against ALS.

The noncoding genome is substantially larger than the protein-coding genome but has been largely unexplored by genetic association studies. Here, we performed region-based rare variant association analysis of >25,000 variants in untranslated regions of 6,139 amyotrophic lateral sclerosis (ALS) whole genomes and the whole genomes of 70,403 non-ALS controls. We identified interleukin-18 receptor accessory protein (IL18RAP) 3' untranslated region (3'UTR) variants as significantly enriched in non-ALS genomes and associated with a fivefold reduced risk of developing ALS, and this was replicated in an independent cohort. These variants in the IL18RAP 3'UTR reduce mRNA stability and the binding of double-stranded RNA (dsRNA)-binding proteins. Finally, the variants of the IL18RAP 3'UTR confer a survival advantage for motor neurons because they dampen neurotoxicity of human induced pluripotent stem cell (iPSC)-derived microglia bearing an ALS-associated expansion in C9orf72, and this depends on NF-κB signaling. This study reveals genetic variants that protect against ALS by reducing neuroinflammation and emphasizes the importance of noncoding genetic association studies.

Unbiased metabolome screen leads to personalized medicine strategy for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a rapidly progressive neurodegenerative disease that affects 1/350 individuals in the United Kingdom. The cause of amyotrophic lateral sclerosis is unknown in the majority of cases. Two-sample Mendelian randomization enables causal inference between an exposure, such as the serum concentration of a specific metabolite, and disease risk. We obtained genome-wide association study summary statistics for serum concentrations of 566 metabolites which were population matched with a genome-wide association study of amyotrophic lateral sclerosis. For each metabolite, we performed Mendelian randomization using an inverse variance weighted estimate for significance testing. After stringent Bonferroni multiple testing correction, our unbiased screen revealed three metabolites that were significantly linked to the risk of amyotrophic lateral sclerosis: Estrone-3-sulphate and bradykinin were protective, which is consistent with literature describing a male preponderance of amyotrophic lateral sclerosis and a preventive effect of angiotensin-converting enzyme inhibitors which inhibit the breakdown of bradykinin. Serum isoleucine was positively associated with amyotrophic lateral sclerosis risk. All three metabolites were supported by robust Mendelian randomization measures and sensitivity analyses; estrone-3-sulphate and isoleucine were confirmed in a validation amyotrophic lateral sclerosis genome-wide association study. Estrone-3-sulphate is metabolized to the more active estradiol by the enzyme 17ß-hydroxysteroid dehydrogenase 1; further, Mendelian randomization demonstrated a protective effect of estradiol and rare variant analysis showed that missense variants within HSD17B1, the gene encoding 17ß-hydroxysteroid dehydrogenase 1, modify risk for amyotrophic lateral sclerosis. Finally, in a zebrafish model of C9ORF72-amyotrophic lateral sclerosis, we present evidence that estradiol is neuroprotective. Isoleucine is metabolized via methylmalonyl-CoA mutase encoded by the gene MMUT in a reaction that consumes vitamin B12. Multivariable Mendelian randomization revealed that the toxic effect of isoleucine is dependent on the depletion of vitamin B12; consistent with this, rare variants which reduce the function of MMUT are protective against amyotrophic lateral sclerosis. We propose that amyotrophic lateral sclerosis patients and family members with high serum isoleucine levels should be offered supplementation with vitamin B12.

Low expression of EXOSC2 protects against clinical COVID-19 and impedes SARS-CoV-2 replication.

New therapeutic targets are a valuable resource in the struggle to reduce the morbidity and mortality associated with the COVID-19 pandemic, caused by the SARS-CoV-2 virus. Genome-wide association studies (GWAS) have identified risk loci, but some loci are associated with co-morbidities and are not specific to host-virus interactions. Here, we identify and experimentally validate a link between reduced expression of EXOSC2 and reduced SARS-CoV-2 replication. EXOSC2 was one of 332 host proteins examined, all of which interact directly with SARS-CoV-2 proteins; EXOSC2 interacts with Nsp8 which forms part of the viral RNA polymerase. Lung-specific eQTLs were identified from GTEx (v7) for each of the 332 host proteins. Aggregating COVID-19 GWAS statistics for gene-specific eQTLs revealed an association between increased expression of EXOSC2 and higher risk of clinical COVID-19 which survived stringent multiple testing correction. EXOSC2 is a component of the RNA exosome and indeed, LC-MS/MS analysis of protein pulldowns demonstrated an interaction between the SARS-CoV-2 RNA polymerase and the majority of human RNA exosome components. CRISPR/Cas9 introduction of nonsense mutations within EXOSC2 in Calu-3 cells reduced EXOSC2 protein expression, impeded SARS-CoV-2 replication and upregulated oligoadenylate synthase ( OAS) genes, which have been linked to a successful immune response against SARS-CoV-2. Reduced EXOSC2 expression did not reduce cellular viability. OAS gene expression changes occurred independent of infection and in the absence of significant upregulation of other interferon-stimulated genes (ISGs). Targeted depletion or functional inhibition of EXOSC2 may be a safe and effective strategy to protect at-risk individuals against clinical COVID-19.

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.

Genome-wide identification of the genetic basis of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a complex disease that leads to motor neuron death. Despite heritability estimates of 52%, genome-wide association studies (GWASs) have discovered relatively few loci. We developed a machine learning approach called RefMap, which integrates functional genomics with GWAS summary statistics for gene discovery. With transcriptomic and epigenetic profiling of motor neurons derived from induced pluripotent stem cells (iPSCs), RefMap identified 690 ALS-associated genes that represent a 5-fold increase in recovered heritability. Extensive conservation, transcriptome, network, and rare variant analyses demonstrated the functional significance of candidate genes in healthy and diseased motor neurons and brain tissues. Genetic convergence between common and rare variation highlighted KANK1 as a new ALS gene. Reproducing KANK1 patient mutations in human neurons led to neurotoxicity and demonstrated that TDP-43 mislocalization, a hallmark pathology of ALS, is downstream of axonal dysfunction. RefMap can be readily applied to other complex diseases.