Advantages of routine next-generation sequencing over standard genetic testing in the amyotrophic lateral sclerosis clinic.

BACKGROUND: Next-generation sequencing has enhanced our understanding of amyotrophic lateral sclerosis (ALS) and its genetic epidemiology. Outside the research setting, testing is often restricted to those who report a family history. The aim of this study was to explore the added benefit of offering routine genetic testing to all patients in a regional ALS centre. METHODS: C9ORF72 expansion testing and exome sequencing was offered to consecutive patients (150 with ALS and 12 with primary lateral sclerosis [PLS]) attending the Oxford Motor Neuron Disease Clinic within a defined time period. RESULTS: A total of 17 (11.3%) highly penetrant pathogenic variants in C9ORF72, SOD1, TARDBP, FUS and TBK1 were detected, of which 10 were also found through standard clinical genetic testing pathways. The systematic approach resulted in five additional diagnoses of a C9ORF72 expansion (number needed to test [NNT] = 28), and two further missense variants in TARDBP and SOD1 (NNT = 69). Additionally, 3 patients were found to carry pathogenic risk variants in NEK1, and 13 patients harboured common missense variants in CFAP410 and KIF5A, also associated with an increased risk of ALS. We report two novel non-coding loss-of-function splice variants in TBK1 and OPTN. No relevant variants were found in the PLS patients. Patients were offered double-blinded participation, but >80% requested disclosure of the results. CONCLUSIONS: This study provides evidence that expanding genetic testing to all patients with a clinical diagnosis of ALS enhances the potential for recruitment to clinical trials, but will have direct resource implications for genetic counselling.

Correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell-derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by CRISPR/Cas9 genome editing using homology-directed repair.

The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.

Genetic testing in motor neurone disease.

A minority (10%-15%) of cases of amyotrophic lateral sclerosis (ALS), the most common form of motor neurone disease (MND), are currently attributable to pathological variants in a single identifiable gene. With the emergence of new therapies targeting specific genetic subtypes of ALS, there is an increasing role for routine genetic testing for all those with a definite diagnosis. However, potential harm to both affected individuals and particularly to asymptomatic relatives can arise from the indiscriminate use of genetic screening, not least because of uncertainties around incomplete penetrance and variants of unknown significance. The most common hereditary cause of ALS, an intronic hexanucleotide repeat expansion in C9ORF72, may be associated with frontotemporal dementia independently within the same pedigree. The boundary of what constitutes a possible family history of MND has therefore extended to include dementia and associated psychiatric presentations. Notwithstanding the important role of clinical genetics specialists, all neurologists need a basic understanding of the current place of genetic testing in MND, which holds lessons for other neurological disorders.

Two case reports of distal upper limb weakness following influenza-like illness: an emerging pattern of para-infectious myositis in adults.

BACKGROUND: Myositis is a recognised complication of numerous systemic viral infections including influenza. In adults the typical pattern is characterised by myalgia and marked proximal muscle weakness in upper and lower limbs and resolves slowly over weeks rather than days. CASE PRESENTATION: Here, we describe two male patients with myositis with an unusual distribution of weakness in the distal upper limbs, which both followed a flu-like illness and resolved spontaneously. Both patients had moderate elevations in creatine kinase, extensive negative serological investigations, normal nerve conduction studies and myopathic changes on electromyography. CONCLUSIONS: In the para-infectious context, myositis is an important differential of acute distal upper limb weakness. This unusual pattern of acute muscle weakness should be recognised to avoid unnecessary in treatments. Similar cases in the recent literature in male patients between the ages of 25 to 55 are reviewed and suggest an emerging pattern of para-infectious myositis.

Single-copy expression of an amyotrophic lateral sclerosis-linked TDP-43 mutation (M337V) in BAC transgenic mice leads to altered stress granule dynamics and progressive motor dysfunction.

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of 'sporadic' cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

C9orf72 and RAB7L1 regulate vesicle trafficking in amyotrophic lateral sclerosis and frontotemporal dementia.

A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

C9orf72 Hexanucleotide Expansions Are Associated with Altered Endoplasmic Reticulum Calcium Homeostasis and Stress Granule Formation in Induced Pluripotent Stem Cell-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons, decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis, reduced levels of the antiapoptotic protein Bcl-2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063-2078.

Dynactin-1 mediates rescue of impaired axonal transport due to reduced mitochondrial bioenergetics in amyotrophic lateral sclerosis motor neurons.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons, which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS, and TDP-43 modulates several mitochondrial transcripts. In this study, we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons, linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected, which correlated with downregulation of the motor protein complex, DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport, which can be partially rescued by DCTN1 overexpression.

Cellular and axonal transport phenotypes due to the C9ORF72 HRE in iPSC motor and sensory neurons.

Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) from patients with amyotrophic lateral sclerosis (ALS) and the C9ORF72 hexanucleotide repeat expansion (HRE) have multiple cellular phenotypes, but which of these accurately reflect the biology underlying the cell-specific vulnerability of ALS is uncertain. We therefore compared phenotypes due to the C9ORF72 HRE in MNs with sensory neurons (SNs), which are relatively spared in ALS. The iPSC models were able to partially reproduce the differential gene expression seen between adult SNs and MNs. We demonstrated that the typical hallmarks of C9ORF72-ALS, including RNA foci and dipeptide formation, as well as specific axonal transport defects, occurred equally in MNs and SNs, suggesting that these in vitro phenotypes are not sufficient to explain the cell-type selectivity of ALS in isolation.

C9orf72-ALS human iPSC microglia are pro-inflammatory and toxic to co-cultured motor neurons via MMP9.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, with additional pathophysiological involvement of non-neuronal cells such as microglia. The commonest ALS-associated genetic variant is a hexanucleotide repeat expansion (HRE) mutation in C9orf72. Here, we study its consequences for microglial function using human iPSC-derived microglia. By RNA-sequencing, we identify enrichment of pathways associated with immune cell activation and cyto-/chemokines in C9orf72 HRE mutant microglia versus healthy controls, most prominently after LPS priming. Specifically, LPS-primed C9orf72 HRE mutant microglia show consistently increased expression and release of matrix metalloproteinase-9 (MMP9). LPS-primed C9orf72 HRE mutant microglia are toxic to co-cultured healthy motor neurons, which is ameliorated by concomitant application of an MMP9 inhibitor. Finally, we identify release of dipeptidyl peptidase-4 (DPP4) as a marker for MMP9-dependent microglial dysregulation in co-culture. These results demonstrate cellular dysfunction of C9orf72 HRE mutant microglia, and a non-cell-autonomous role in driving C9orf72-ALS pathophysiology in motor neurons through MMP9 signaling.

Human stem cell models of neurodegeneration: From basic science of amyotrophic lateral sclerosis to clinical translation.

Neurodegenerative diseases are characterized by progressive cell loss leading to disruption of the structure and function of the central nervous system. Amyotrophic lateral sclerosis (ALS) was among the first of these disorders modeled in patient-specific iPSCs, and recent findings have translated into some of the earliest iPSC-inspired clinical trials. Focusing on ALS as an example, we evaluate the status of modeling neurodegenerative diseases using iPSCs, including methods for deriving and using disease-relevant neuronal and glial lineages. We further highlight the remaining challenges in exploiting the full potential of iPSC technology for understanding and potentially treating neurodegenerative diseases such as ALS.

Human iPSC co-culture model to investigate the interaction between microglia and motor neurons.

Motor neuron diseases such as amyotrophic lateral sclerosis are primarily characterized by motor neuron degeneration with additional involvement of non-neuronal cells, in particular, microglia. In previous work, we have established protocols for the differentiation of iPSC-derived spinal motor neurons and microglia. Here, we combine both cell lineages and establish a novel co-culture of iPSC-derived spinal motor neurons and microglia, which is compatible with motor neuron identity and function. Co-cultured microglia express key identity markers and transcriptomically resemble primary human microglia, have highly dynamic ramifications, are phagocytically competent, release relevant cytokines and respond to stimulation. Further, they express key amyotrophic lateral sclerosis-associated genes and release disease-relevant biomarkers. This novel and authentic human model system facilitates the study of physiological motor neuron-microglia crosstalk and will allow the investigation of non-cell-autonomous phenotypes in motor neuron diseases such as amyotrophic lateral sclerosis.

Multicentre appraisal of amyotrophic lateral sclerosis biofluid biomarkers shows primacy of blood neurofilament light chain.

The routine clinical integration of individualized objective markers of disease activity in those diagnosed with the neurodegenerative disorder amyotrophic lateral sclerosis is a key requirement for therapeutic development. A large, multicentre, clinic-based, longitudinal cohort was used to systematically appraise the leading candidate biofluid biomarkers in the stratification and potential therapeutic assessment of those with amyotrophic lateral sclerosis. Incident patients diagnosed with amyotrophic lateral sclerosis (n = 258), other neurological diseases (n = 80) and healthy control participants (n = 101), were recruited and followed at intervals of 3-6 months for up to 30 months. Cerebrospinal fluid neurofilament light chain and chitotriosidase 1 and blood neurofilament light chain, creatine kinase, ferritin, complement C3 and C4 and C-reactive protein were measured. Blood neurofilament light chain, creatine kinase, serum ferritin, C3 and cerebrospinal fluid neurofilament light chain and chitotriosidase 1 were all significantly elevated in amyotrophic lateral sclerosis patients. First-visit plasma neurofilament light chain level was additionally strongly associated with survival (hazard ratio for one standard deviation increase in log10 plasma neurofilament light chain 2.99, 95% confidence interval 1.65-5.41, P = 0.016) and rate of disability progression, independent of other prognostic factors. A small increase in level was noted within the first 12 months after reported symptom onset (slope 0.031 log10 units per month, 95% confidence interval 0.012-0.049, P = 0.006). Modelling the inclusion of plasma neurofilament light chain as a therapeutic trial outcome measure demonstrated that a significant reduction in sample size and earlier detection of disease-slowing is possible, compared with using the revised Amyotrophic Lateral Sclerosis Functional Rating Scale. This study provides strong evidence that blood neurofilament light chain levels outperform conventional measures of disease activity at the group level. The application of blood neurofilament light chain has the potential to radically reduce the duration and cost of therapeutic trials. It might also offer a first step towards the goal of more personalized objective disease activity monitoring for those living with amyotrophic lateral sclerosis.

Pattern of spread and prognosis in lower limb-onset ALS.

Our objective was to establish the pattern of spread in lower limb-onset ALS (contra- versus ipsi-lateral) and its contribution to prognosis within a multivariate model. Pattern of spread was established in 109 sporadic ALS patients with lower limb-onset, prospectively recorded in Oxford and Sheffield tertiary clinics from 2001 to 2008. Survival analysis was by univariate Kaplan-Meier log-rank and multivariate Cox proportional hazards. Variables studied were time to next limb progression, site of next progression, age at symptom onset, gender, diagnostic latency and use of riluzole. Initial progression was either to the contralateral leg (76%) or ipsilateral arm (24%). Factors independently affecting survival were time to next limb progression, age at symptom onset, and diagnostic latency. Time to progression as a prognostic factor was independent of initial direction of spread. In a regression analysis of the deceased, overall survival from symptom onset approximated to two years plus the time interval for initial spread. In conclusion, rate of progression in lower limb-onset ALS is not influenced by whether initial spread is to the contralateral limb or ipsilateral arm. The time interval to this initial spread is a powerful factor in predicting overall survival, and could be used to facilitate decision-making and effective care planning.

Impairment of Mitochondrial Calcium Buffering Links Mutations in C9ORF72 and TARDBP in iPS-Derived Motor Neurons from Patients with ALS/FTD.

TDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9ORF72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9ORF72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9ORF72 iPSC-MNs have higher Ca2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca2+ release. We identify here, by RNA sequencing, that both C9ORF72 and TARDBP iPSC-MNs have upregulation of Ca2+-permeable AMPA and NMDA subunits and impairment of mitochondrial Ca2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca2+ uniporter, indicating that impaired mitochondrial Ca2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9ORF72 or TARDBP mutations.

Embryonic progenitor pools generate diversity in fine-scale excitatory cortical subnetworks.

The mammalian neocortex is characterized by a variety of neuronal cell types and precise arrangements of synaptic connections, but the processes that generate this diversity are poorly understood. Here we examine how a pool of embryonic progenitor cells consisting of apical intermediate progenitors (aIPs) contribute to diversity within the upper layers of mouse cortex. In utero labeling combined with single-cell RNA-sequencing reveals that aIPs can generate transcriptionally defined glutamatergic cell types, when compared to neighboring neurons born from other embryonic progenitor pools. Whilst sharing layer-associated morphological and functional properties, simultaneous patch clamp recordings and optogenetic studies reveal that aIP-derived neurons exhibit systematic biases in both their intralaminar monosynaptic connectivity and the post-synaptic partners that they target within deeper layers of cortex. Multiple cortical progenitor pools therefore represent an important factor in establishing diversity amongst local and long-range fine-scale glutamatergic connectivity, which generates subnetworks for routing excitatory synaptic information.

TDP-43 expression in mouse models of amyotrophic lateral sclerosis and spinal muscular atrophy.

BACKGROUND: Redistribution of nuclear TAR DNA binding protein 43 (TDP-43) to the cytoplasm and ubiquitinated inclusions of spinal motor neurons and glial cells is characteristic of amyotrophic lateral sclerosis (ALS) pathology. Recent evidence suggests that TDP-43 pathology is common to sporadic ALS and familial ALS without SOD1 mutation, but not SOD1-related fALS cases. Furthermore, it remains unclear whether TDP-43 abnormalities occur in non-ALS forms of motor neuron disease. Here, we characterise TDP-43 localisation, expression levels and post-translational modifications in mouse models of ALS and spinal muscular atrophy (SMA). RESULTS: TDP-43 mislocalisation to ubiquitinated inclusions or cytoplasm was notably lacking in anterior horn cells from transgenic mutant SOD1G93A mice. In addition, abnormally phosphorylated or truncated TDP-43 species were not detected in fractionated ALS mouse spinal cord or brain. Despite partial colocalisation of TDP-43 with SMN, depletion of SMN- and coilin-positive Cajal bodies in motor neurons of affected SMA mice did not alter nuclear TDP-43 distribution, expression or biochemistry in spinal cords. CONCLUSION: These results emphasise that TDP-43 pathology characteristic of human sporadic ALS is not a core component of the neurodegenerative mechanisms caused by SOD1 mutation or SMN deficiency in mouse models of ALS and SMA, respectively.

Amyotrophic lateral sclerosis: the complex path to precision medicine.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the corticomotorneuronal network responsible for voluntary movement. There are well-established clinical, genetic and pathological overlaps between ALS and frontotemporal dementia (FTD), which together constitute the 'TDP-43 proteinopathies'. An ever-expanding list of genes in which mutation leads to typical ALS have implicated abnormalities in RNA processing, protein homoeostasis and axonal transport. How these apparently distinct pathways converge to cause the characteristic clinical syndrome of ALS remains unclear. Although there are major gaps in our understanding of the essential nature of ALS pathophysiology, the identification of genetic causes in up to 15% of ALS patients, coupled with advances in biotechnology and biomarker research provide a foundation for approaches to treatment based on 'precision medicine', and even prevention of the disease in pre-symptomatic mutation carriers in the future. Currently, multidisciplinary care remains the bedrock of management and this is increasingly being put onto an evidence-based footing.

Initial Identification of a Blood-Based Chromosome Conformation Signature for Aiding in the Diagnosis of Amyotrophic Lateral Sclerosis.

BACKGROUND: The identification of blood-based biomarkers specific to the diagnosis of amyotrophic lateral sclerosis (ALS) is an active field of academic and clinical research. While inheritance studies have advanced the field, a majority of patients do not have a known genetic link to the disease, making direct sequence-based genetic testing for ALS difficult. The ability to detect biofluid-based epigenetic changes in ALS would expand the relevance of using genomic information for disease diagnosis. METHODS: Assessing differences in chromosomal conformations (i.e. how they are positioned in 3-dimensions) represents one approach for assessing epigenetic changes. In this study, we used an industrial platform, EpiSwitch™, to compare the genomic architecture of healthy and diseased patient samples (blood and tissue) to discover a chromosomal conformation signature (CCS) with diagnostic potential in ALS. A three-step biomarker selection process yielded a distinct CCS for ALS, comprised of conformation changes in eight genomic loci and detectable in blood. FINDINGS: We applied the ALS CCS to determine a diagnosis for 74 unblinded patient samples and subsequently conducted a blinded diagnostic study of 16 samples. Sensitivity and specificity for ALS detection in the 74 unblinded patient samples were 83∙33% (CI 51∙59 to 97∙91%) and 76∙92% (46∙19 to 94∙96%), respectively. In the blinded cohort, sensitivity reached 87∙50% (CI 47∙35 to 99∙68%) and specificity was 75∙0% (34∙91 to 96∙81%). INTERPRETATIONS: The sensitivity and specificity values achieved using the ALS CCS identified and validated in this study provide an indication that the detection of chromosome conformation signatures is a promising approach to disease diagnosis and can potentially augment current strategies for diagnosing ALS. FUND: This research was funded by Oxford BioDynamics and Innovate UK. Work in the Oxford MND Care and Research Centre is supported by grants from the Motor Neurone Disease Association and the Medical Research Council. Additional support was provided by the Northeast ALS Consortium (NEALS).