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Mutations in theUBQLN2gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The neuropathology of suchUBQLN2-linked cases of ALS/FTD is characterised by aggregates of the ubiquilin 2 protein in addition to aggregates of the transactive response DNA-binding protein of 43 kDa (TDP-43). ALS and FTD withoutUBQLN2mutations are also characterised by TDP-43 aggregates, that may or may not colocalise with wildtype ubiquilin 2. Despite this, the relative contributions of TDP-43 and ubiquilin 2 to disease pathogenesis remain largely under-characterised, as does their relative deposition as aggregates across the central nervous system (CNS). Here we conducted multiplex immunohistochemistry of threeUBQLN2p.T487I-linked ALS/FTD cases, three non-UBQLN2-linked (sporadic) ALS cases, and eight non-neurodegenerative disease controls, covering 40 CNS regions. We then quantified ubiquilin 2 aggregates, TDP-43 aggregates, and aggregates containing both proteins in regions of interest to determine howUBQLN2-linked and non-UBQLN2-linked proteinopathy differ. We find that ubiquilin 2 aggregates that are negative for TDP-43 are predominantly small and punctate, and are abundant in the hippocampal formation, spinal cord, all tested regions of neocortex, medulla, and substantia nigra inUBQLN2-linked ALS/FTD but not sporadic ALS. Curiously, the striatum harboured small punctate ubiquilin 2 aggregates in all cases examined, while large diffuse striatal ubiquilin 2 aggregates were specific toUBQLN2-linked ALS/FTD. While ubiquilin 2 deposition in frontotemporal regions may enhance cognitive risk inUBQLN2-linked cases, ubiquilin 2 is deposited mainly in clinically unaffected regions throughout the CNS such that overall symptomology in these cases maps best to the aggregation of TDP-43.

Charcot–Marie-Tooth (CMT) is a commonly inherited, non-fatal neurodegenerative disorder that affects sensory and motor neurons in patients. More than 90 genes are known to cause axonal and demyelinating forms of CMT. The p.R158H mutation in the pyruvate dehydrogenase kinase 3 (PDK3) gene is the genetic cause for an X linked form of axonal CMT (CMTX6). In vitro studies using patient fibroblasts and iPSC-derived motor neurons have shown that this mutation causes deficits in energy metabolism and mitochondrial function. Animal models that recapitulate pathogenic in vivo events in patients are crucial for investigating mechanisms of axonal degeneration and developing therapies for CMT. We have developed a C. elegans model of CMTX6 by knocking-in the p.R158H mutation in pdhk-2, the ortholog of PDK3. In addition, we have developed animal models overexpressing the wild type and mutant form of human PDK3 specifically in the GABAergic motor neurons of C. elegans. CMTX6 mutants generated in this study exhibit synaptic transmission deficits, locomotion defects and show signs of progressive neurodegeneration. Furthermore, the CMTX6 in vivo models display energy deficits that recapitulate the phenotype observed in patient fibroblasts and iPSC-derived motor neurons. Our CMTX6 animals represent the first in vivo model for this form of CMT and have provided novel insights into the cellular function and metabolic pathways perturbed by the p.R158H mutation, all the while closely replicating the clinical presentation observed in CMTX6 patients.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and 4 disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild type full length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic C. elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and four disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases, with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene–intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild-type full-length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic Caenorhabditis elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress, which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene–intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

The second most common form of Charcot-Marie-Tooth neuropathy (CMT), X-linked CMT type X1 (CMTX1), is caused by coding and non-coding mutations in the gap junction beta 1 (GJB1) gene. The non-coding GJB1 c.-103C > T mutation (NM_000166.5) has been reported to cause CMTX1 in multiple families. This study assessed the internal ribosomal entry site (IRES) activity previously reported for the rat Gjb1 P2 5’ untranslated region (UTR). Using a bicistronic assay and transfecting RT4 Schwann cells, IRES activity of the human GJB1 P2 5’ UTR was compared to the GJB1 P2 5’ UTR containing either the c.-103C > T mutation or the non-pathogenic c.-102G > A variant. No differences in GJB1 P2 5’ UTR IRES activity were observed between the negative control, the wild-type P2 5’ UTR, the c.-103C > T 5’ UTR or the c.-102G > A 5’ UTR, irrespective of the GJB1 intron being present (p = .429 with intron, and p = .865 without). A theoretical c.-131A > G variant was predicted to result in the same RNA secondary structure as the GJB1 c.-103C > T P2 5’ UTR. However, no significant difference was observed between expression from the wild-type GJB1 P2 5’ UTR and the GJB1 c.-131A > G variant (p = .688). Deletion of the conserved region surrounding the c.-103C > T mutation (c.-108_-103del) resulted in significantly higher expression than the c.-103C > T mutation alone (p = .019), suggesting that the conserved c.-108_-103 region was not essential for translation. The reporter assays in this study do not recapitulate the previously reported GJB1 IRES activity and suggest an alternate pathogenic mechanism for the c.-103C > T CMTX1 non-coding mutation.

Amyotrophic Lateral Sclerosis (ALS) is recognised to be a complex neurodegenerative disease involving both genetic and non-genetic risk factors. The underlying causes and risk factors for the majority of cases remain unknown; however, ever-larger genetic data studies and methodologies promise an enhanced understanding. Recent analyses using published summary statistics from the largest ALS genome-wide association study (GWAS) (20,806 ALS cases and 59,804 healthy controls) identified that schizophrenia (SCZ), cognitive performance (CP) and educational attainment (EA) related traits were genetically correlated with ALS. To provide additional evidence for these correlations, we built single and multi-trait genetic predictors using GWAS summary statistics for ALS and these traits, (SCZ, CP, EA) in an independent Australian cohort (846 ALS cases and 665 healthy controls). We compared methods for generating the risk predictors and found that the combination of traits improved the prediction (Nagelkerke-R-2) of the case-control logistic regression. The combination of ALS, SCZ, CP, and EA, using the SBayesR predictor method gave the highest prediction (Nagelkerke-R-2) of 0.027 (P value = 4.6 x 10(-8)), with the odds-ratio for estimated disease risk between the highest and lowest deciles of individuals being 3.15 (95% CI 1.96-5.05). These results support the genetic correlation between ALS, SCZ, CP and EA providing a better understanding of the complexity of ALS. Refereed/Peer-reviewed

A large 78 kb insertion from chromosome 8q24.3 into Xq27.1 was identified as the cause of CMTX3 in three families of European descent from Australia (CMT193, CMT180) and New Zealand/United Kingdom (CMT623). Using the relatedness tool XIBD to perform genome-wide identity-by-descent (IBD) analysis on 16 affected individuals from the three families demonstrated they all share the CMTX3 disease locus identical-by-descent, confirming the mutation arose in a common ancestor. Relationship estimation from IBD segment data has genetically linked all three families through 6th and 7th degree relatives.

Background and objectivesCharcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy and often presents during childhood. Guidelines for the optimal management of common problems experienced by individuals with CMT do not exist, for either children or adults. We formed the Paediatric CMT Best Practice Guidelines Consortium to develop evidence and consensus-based recommendations for the clinical management of children and adolescents with CMT, with the primary objective of promoting optimal, standardised care globally.MethodsDevelopment of this clinical practice guideline involved a series of systematic reviews covering 10 clinical questions, modified Delphi methodology involving an international panel of clinicians to generate consensus where evidence did not exist, and application of the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach to evaluate the body of literature and formulate recommendations.ResultsThe final guideline includes three evidence-based and 31 consensus-based recommendations. They encompass the management of muscle weakness, balance and mobility impairment, sensory symptoms, muscle cramps, impaired upper limb function, respiratory impairment, maintenance of joint range of motion and non-surgical management of joint deformity. Consensus was not achieved in some management areas, reflecting differences in practice between clinicians and healthcare settings, and highlighting the need for further research.ConclusionsThis clinical practice guideline provides practical and implementable guidance on the management of common clinical problems experienced by children with CMT and advocates for improved access to multidisciplinary care. Successful dissemination and implementation of these recommendations will be critical in ensuring their application across multiple healthcare settings.

Background Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. Methods We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. Results We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. Conclusions This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.

Machado-Joseph disease (MJD/SCA3) is the most common form of dominantly inherited ataxia worldwide. The disorder is caused by an expanded CAG repeat in the ATXN3 gene. Past studies have revealed that the length of the expansion partly explains the disease age at onset (AO) variability of MJD, which is confirmed in this study (Pearson's correlation coefficient R2 = 0.62). Using a total of 786 MJD patients from five different geographical origins, a genome-wide association study (GWAS) was conducted to identify additional AO modifying factors that could explain some of the residual AO variability. We identified nine suggestively associated loci (P < 1 × 10-5). These loci were enriched for genes involved in vesicle transport, olfactory signaling, and synaptic pathways. Furthermore, associations between AO and the TRIM29 and RAG genes suggests that DNA repair mechanisms might be implicated in MJD pathogenesis. Our study demonstrates the existence of several additional genetic factors, along with CAG expansion, that may lead to a better understanding of the genotype-phenotype correlation in MJD. FA and CL were funded by the Fonds de Recherche du Québec–Santé. SM is funded by FCT (CEECIND/00684/2017) and by NORTE-01-0145- FEDER-000008, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). FM and LI are funded by Fundaçao de Amparo a Pesquisa do Estado de São Paulo (FAPESP, 2013/07559-3). MLSP and LBJ were funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq) and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). GAR holds a Canada Research Chair in Genetics of the Nervous System and the Wilder Penfield Chair in Neurosciences.

Frontotemporal dementia and amyotrophic lateral sclerosis are clinically and pathologically overlapping disorders with shared genetic causes. We previously identified a disease locus on chromosome 16p12.1-q12.2 with genome-wide significant linkage in a large European Australian family with autosomal dominant inheritance of frontotemporal dementia and amyotrophic lateral sclerosis and no mutation in known amyotrophic lateral sclerosis or dementia genes. Here we demonstrate the segregation of a novel missense variant in CYLD (c.2155A>G, p.M719V) within the linkage region as the genetic cause of disease in this family. Immunohistochemical analysis of brain tissue from two CYLD p.M719V mutation carriers showed widespread glial CYLD immunoreactivity. Primary mouse neurons transfected with CYLDM719V exhibited increased cytoplasmic localization of TDP-43 and shortened axons. CYLD encodes a lysine 63 deubiquitinase and CYLD cutaneous syndrome, a skin tumour disorder, is caused by mutations that lead to reduced deubiquitinase activity. In contrast with CYLD cutaneous syndrome-causative mutations, CYLDM719V exhibited significantly increased lysine 63 deubiquitinase activity relative to the wild-type enzyme (paired Wilcoxon signed-rank test P = 0.005). Overexpression of CYLDM719V in HEK293 cells led to more potent inhibition of the cell signalling molecule NF-κB and impairment of autophagosome fusion to lysosomes, a key process in autophagy. Although CYLD mutations appear to be rare, CYLD’s interaction with at least three other proteins encoded by frontotemporal dementia and/or amyotrophic lateral sclerosis genes (TBK1, OPTN and SQSTM1) suggests that it may play a central role in the pathogenesis of these disorders. Mutations in several frontotemporal dementia and amyotrophic lateral sclerosis genes, including TBK1, OPTN and SQSTM1, result in a loss of autophagy function. We show here that increased CYLD activity also reduces autophagy function, highlighting the importance of autophagy regulation in the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis.

EGR2 (early growth response 2) is a crucial transcription factor for the myelination of the peripheral nervous system. Mutations in EGR2 are reported to cause a heterogenous spectrum of peripheral neuropathy with wide variation in both severity and age of onset, including demyelinating and axonal forms of Charcot-Marie Tooth (CMT) neuropathy, Dejerine-Sottas neuropathy (DSN/CMT3), and congenital hypomyelinating neuropathy (CHN/CMT4E). Here we report a sporadic de novo EGR2 variant, c.1232A > G (NM_000399.5), causing a missense p.Asp411Gly substitution and discovered through whole-exome sequencing (WES) of the proband. The resultant phenotype is severe demyelinating DSN with onset at two years of age, confirmed through nerve biopsy and electrophysiological examination. In silico analyses showed that the Asp411 residue is evolutionarily conserved, and the p.Asp411Gly variant was predicted to be deleterious by multiple in silico analyses. A luciferase-based reporter assay confirmed the reduced ability of p.Asp411Gly EGR2 to activate a PMP22 (peripheral myelin protein 22) enhancer element compared to wild-type EGR2. This study adds further support to the heterogeneity of EGR2-related peripheral neuropathies and provides strong functional evidence for the pathogenicity of the p.Asp411Gly EGR2 variant.

ObjectiveSince the first report of CHCHD10 gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in CHCHD10 has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia.MethodsWhole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in CHCHD10. CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology.ResultsNo causal, novel or disease-associated variants in CHCHD10 were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease.ConclusionsGenetic variation in CHCHD10 is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.

Objectives Hereditary sensory neuropathy type 1 (HSN1) is a rare progressive neuropathy characterised by profound sensory dysfunction, often accompanied by significant weakness. Muscle magnetic resonance (MR) imaging with determination of intramuscular fat accumulation has been proposed as a marker of progression in this condition and we aimed to investigate this further. Methods Calf-level muscle MR images were acquired longitudinally over three years in patients with genetically confirmed HSN1 due to SPTLC1 and SPTLC2 mutations. These patients were part of a larger trial of L-serine supplementation as a candidate therapy and MR images were acquired at baseline and while on treatment. Individual muscles and muscle groups were manually segmented at two cross-sectional levels in the proximal calf. Intramuscular fat accumulation and muscle atrophy were assessed. Results Detailed MR analysis was performed in a preliminary series of three patients. We demonstrated an average annual change in MR-based intramuscular calf fat fraction of 3.2%, 1.0% and 3.7% at a cross-sectional level 130mm below the tibial plateau and 2.0%, 0.6% and 1.2% at a cross-sectional level 25% of the tibial plateau-medial malleolus distance. The degree of muscle atrophy did not significantly change. There appeared to be ongoing progression of disease during this short duration of L-serine supplementation. Conclusion MR-based intramuscular calf fat fraction can be used to monitor progression in HSN1 and has potential utility in clinical trials. Technical limitations to this technique may be overcome using volumetric imaging with automated muscle segmentation in the future. Further investigation of L-serine supplementation is required.

Objective To report clinical stabilisation and improved summated compound motor action potentials (CMAP) in a patient with Hereditary sensory and autonomic neuropathy type I (HSAN-1) following high dose serine therapy. Case A 52-year-old male presented in 2006 with a typical HSAN-1c phenotype and over the ensuing years had progressive distal to proximal sensory disturbance, associated lancinating pains, and mild progressive distal predominant limb weakness. A Ser384Phe mutation in the SPTLC2 gene located on chromosome 14q24 was identified in this patient in 2017. In late 2018 high dose serine therapy (11 grams TDS) was commenced, resulting in stabilisation of clinical weakness. Lower limb motor and sensory responses were absent at presentation in 2006 with initial summated bilateral upper-limb median and ulnar nerve CMAP being 31.2mV and steadily declining to 4.2mV in March 2019. Following Serine therapy, the summated CMAP increased to 5.4mV in August 2020 and 9.5mV in February 2021, furthermore previously absent upper-limb sensory nerve responses are now recordable. Conclusions High dose Serine replacement therapy may lead to clinical stabilisation and improved neurophysiological parameters in HSAN-1. HSAN-1, an autosomal dominant sensory neuropathy occurs secondary to mutations in the enzyme Serine-Palmitoyltransferase (SPT), an essential enzyme in the de-novo synthesis of sphingolipids. The administration of high dose Serine may overcome altered SPT substrate specificity in HSAN-1, which preferentially uses L-alanine and L-glycine instead of L-serine and allow the formation of typical 1-deoxysphingolipids as opposed to atypical 1-deoxysphingolipids generally seen in this condition, with early treatment possibly preventing clinical progression.

Facioscapulohumeral muscular dystrophy (FSHD) is a common genetic disease of the skeletal muscle with a characteristic pattern of weakness. Facioscapulohumeral muscular dystrophy type 2 (FSHD2) accounts for approximately 5% of all cases of FSHD and describes patients without a D4Z4 repeat contraction on chromosome 4. Phenotypically FSHD2 shows virtually no difference from FSHD1 and both forms of FSHD arise via a common downstream mechanism of epigenetic derepression of the transcription factor DUX4 in skeletal muscle cells. This results in expression of DUX4 and target genes leading to skeletal muscle toxicity. Over the past decade, major progress has been made in our understanding of the genetic and epigenetic architecture that underlies FSHD2 pathogenesis, as well as the clinical manifestations and disease progression. These include the finding that FSHD2 is a digenic disease and that mutations in the genes SMCHD1, DNMT3B, and more recently LRIF1, can cause FSHD2. FSHD2 is complex and it is important that clinicians keep abreast of recent developments; this review aims to serve as an update of the clinical, genetic, and molecular research into this condition.

Spinocerebellar ataxia 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by expansion of the trinucleotide repeat region within theATXN3/MJDgene. Mutation ofATXN3causes formation of ataxin-3 protein aggregates, neurodegeneration and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model SCA3. SCA3 SH-SY5Y cells were found to contain ataxin-3 oligomeric species and protein aggregates. Treatment with SB increased activity of the autophagy protein quality control pathway in the SCA3 cells, decreased presence of ataxin-3 aggregates and presence of ataxin-3 oligomers in an autophagy-dependent manner. Treatment with SB was also beneficialin vivo,improving swimming performance, increasing activity of the autophagy pathway and decreasing presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on the zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signalling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway through a PKA/AMPK-dependent process and that this has beneficial effectsin vitroandin vivo. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.

Hereditary sensory and autonomic neuropathy type I (HSAN-1) is an autosomal dominant sensory neuropathy occurring secondary to mutations in the SPTLC1 and SPTLC2 genes. We present two generations of a single family with Ser384Phe mutation in the SPTLC2 gene located on chromosome 14q24 characterized by a typical HSAN-1c presentation, with additional findings upper motor neuron signs, early demyelinating features on nerve conduction studies, and type II juxtafoveal retinal telangiectasias also known as macular telangiectasias (MacTel II). Although HSAN1 is characterized as an axonal neuropathy, demyelinating features were identified in two subjects on serial nerve conduction studies comprising motor conduction block, temporal dispersion, and prolongation of F-waves. MacTell II is a rare syndrome characterized by bilateral macular depigmentation and Muller cell loss. It has a presumed genetic basis, and these cases suggest that the accumulation of toxic sphingoplipids may lead to Muller cell degeneration, subsequent neuronal loss, depigmentation, and progressive central macular thinning.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. ALS can be modeled in zebrafish (Danio rerio) through the expression of human ALS-causing genes, such as superoxide dismutase 1 (SOD1). Overexpression of mutated human SOD1 protein causes aberrant branching and shortening of spinal motor axons. Despite this, the functional relevance of this axon morphology remains elusive. Our aim was to determine whether this motor axonopathy is correlated with impaired movement in mutant (MT) SOD1-expressing zebrafish. Transgenic zebrafish embryos that express blue fluorescent protein (mTagBFP) in motor neurons were injected with either wild-type (WT) or MT (A4V) human SOD1 messenger ribonucleic acid (mRNA). At 48 hours post-fertilization, larvae movement (distance traveled during behavioral testing) was examined, followed by quantification of motor axon length. Larvae injected with MT SOD1 mRNA had significantly shorter and more aberrantly branched motor axons (p

Background People with neurodegenerative disorders show diverse clinical syndromes, genetic heterogeneity, and distinct brain pathological changes, but studies report overlap between these features. DNA methylation (DNAm) provides a way to explore this overlap and heterogeneity as it is determined by the combined effects of genetic variation and the environment. In this study, we aim to identify shared blood DNAm differences between controls and people with Alzheimer’s disease, amyotrophic lateral sclerosis, and Parkinson’s disease. Results We use a mixed-linear model method (MOMENT) that accounts for the effect of (un)known confounders, to test for the association of each DNAm site with each disorder. While only three probes are found to be genome-wide significant in each MOMENT association analysis of amyotrophic lateral sclerosis and Parkinson’s disease (and none with Alzheimer’s disease), a fixed-effects meta-analysis of the three disorders results in 12 genome-wide significant differentially methylated positions. Predicted immune cell-type proportions are disrupted across all neurodegenerative disorders. Protein inflammatory markers are correlated with profile sum-scores derived from disease-associated immune cell-type proportions in a healthy aging cohort. In contrast, they are not correlated with MOMENT DNAm-derived profile sum-scores, calculated using effect sizes of the 12 differentially methylated positions as weights. Conclusions We identify shared differentially methylated positions in whole blood between neurodegenerative disorders that point to shared pathogenic mechanisms. These shared differentially methylated positions may reflect causes or consequences of disease, but they are unlikely to reflect cell-type proportion differences.

Machado-Joseph disease (MJD, also known as spinocerebellar ataxia-3) is a fatal neurodegenerative disease that impairs control and coordination of movement. Here we tested whether treatment with the histone deacetylase inhibitor sodium valproate (SV) prevented a movement phenotype that develops in larvae of a transgenic zebrafish model of the disease. We found that treatment with SV improved the swimming of the MJD zebrafish, increased levels of acetylated histones 3 and 4, but also increased expression of polyglutamine expanded human ataxin-3. Proteomic analysis of protein lysates generated from the treated and untreated MJD zebrafish also predicted that SV treatment had activated the sirtuin longevity signaling pathway and this was confirmed by findings of increased SIRT1 protein levels and sirtuin activity in SV treated MJD zebrafish and HEK293 cells expressing ataxin-3-84Q, respectively. Treatment with resveratrol (another compound known to activate the sirtuin pathway), also improved swimming in the MJD zebrafish. Co-treatment with SV alongside EX527, a SIRT1 activity inhibitor, prevented induction of autophagy by SV and the beneficial effects of SV on the movement in the MJD zebrafish, indicating that they were both dependent on sirtuin activity. These findings provide the first evidence of sodium valproate inducing activation of the sirtuin pathway. Further, they indicate that drugs that target the sirtuin pathway, including sodium valproate and resveratrol, warrant further investigation for the treatment of MJD and related neurodegenerative diseases.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an estimated heritability of around 50%. DNA methylation patterns can serve as biomarkers of (past) exposures and disease progression, as well as providing a potential mechanism that mediates genetic or environmental risk. Here, we present a blood-based epigenome-wide association study (EWAS) meta-analysis in 10,462 samples (7,344 ALS patients and 3,118 controls), representing the largest case-control study of DNA methylation for any disease to date. We identified a total of 45 differentially methylated positions (DMPs) annotated to 42 genes, which are enriched for pathways and traits related to metabolism, cholesterol biosynthesis, and immunity. We show that DNA-methylation-based proxies for HDL-cholesterol, BMI, white blood cell (WBC) proportions and alcohol intake were independently associated with ALS. Integration of these results with our latest GWAS showed that cholesterol biosynthesis was causally related to ALS. Finally, we found that DNA methylation levels at several DMPs and blood cell proportion estimates derived from DNA methylation data, are associated with survival rate in patients, and could represent indicators of underlying disease processes.

Hannu Laaksovirta konsortion jäsenenä. The Genomics England Research Consortium, The International ALS/FTD Genomics Consortium (iAFGC), The International FTD Genetics Consortium (IFGC), The International LBD Genomics Consortium (iLBDGC), The NYGC ALS Consortium, The PROSPECT Consortium,17 James B. Rowe,17 Luisa Benussi,18 Giuliano Binetti,18,19 Roberta Ghidoni,18 Edwin Jabbari,20,21 Coralie Viollet,22 Jonathan D. Glass,23 Andrew B. Singleton,24 Vincenzo Silani,25,26 Owen A. Ross,27 Mina Ryten,8,28,29 Ali Torkamani,30 Toshiko Tanaka,31 Luigi Ferrucci,31 Susan M. Resnick,32 We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered. We examined the role of repeat expansions in the pathogenesis of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) by analyzing whole-genome sequence data from 2,442 FTD/ALS patients, 2,599 Lewy body dementia (LBD) patients, and 3,158 neurologically healthy subjects. Pathogenic expansions (range, 40?64 CAG repeats) in the huntingtin (HTT) gene were found in three (0.12%) patients diagnosed with pure FTD/ALS syndromes but were not present in the LBD or healthy cohorts. We replicated our findings in an independent collection of 3,674 FTD/ALS patients. Postmortem evaluations of two patients revealed the classical TDP-43 pathology of FTD/ALS, as well as huntingtin-positive, ubiquitin-positive aggregates in the frontal cortex. The neostriatal atrophy that pathologically defines Huntington?s disease was absent in both cases. Our findings reveal an etiological relationship between HTT repeat expansions and FTD/ALS syndromes and indicate that genetic screening of FTD/ALS patients for HTT repeat expansions should be considered.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by the progressive degeneration of motor neurons. Recently, genetic variants in GLT8D1 and ARPP21 were associated with ALS in a cohort of European descent. A synergistic relationship was proposed between ALS associated variants in GLT8D1 and ARPP21. We aimed to determine the prevalence of genetic variation in GLT8D1 and ARPP21 in an Australian cohort of familial (n = 81) and sporadic ALS (n = 618) cases using whole-exome and whole-genome sequencing data. No novel mutations were identified in either gene, nor was there significant enrichment of protein-altering sequence variation among ALS cases. GLT8D1 and ARPP21 mutations are not a common cause of ALS in Australian familial and sporadic cohorts.

Summary We meta-analyze amyotrophic lateral sclerosis (ALS) genome-wide association study (GWAS) data of European and Chinese populations (84,694 individuals). We find an additional significant association between rs58854276 spanning ACSL5-ZDHHC6 with ALS (p = 8.3 × 10−9), with replication in an independent Australian cohort (1,502 individuals; p = 0.037). Moreover, B4GALNT1, G2E3-SCFD1, and TRIP11-ATXN3 are identified using a gene-based analysis. ACSL5 has been associated with rapid weight loss, as has another ALS-associated gene, GPX3. Weight loss is frequent in ALS patients and is associated with shorter survival. We investigate the effect of the ACSL5 and GPX3 single-nucleotide polymorphisms (SNPs), using longitudinal body composition and weight data of 77 patients and 77 controls. In patients’ fat-free mass, although not significant, we observe an effect in the expected direction (rs58854276: −2.1 ± 1.3 kg/A allele, p = 0.053; rs3828599: −1.0 ± 1.3 kg/A allele, p = 0.22). No effect was observed in controls. Our findings support the increasing interest in lipid metabolism in ALS and link the disease genetics to weight loss in patients., Graphical Abstract, Highlights • Cross-ethnic meta-analysis finds an association between the ACSL5-ZDHHC6 locus and ALS • The ACSL5-ZDHHC6 association is replicated in an independent Australian cohort • ACSL5-ZDHHC6 lead SNP is in ACSL5 and is an eQTL of ZDHHC6 in brain tissues • ACSL5 SNPs might have an effect on fat-free mass in ALS patients, Using meta-analysis of European and Chinese ALS GWAS data, Iacoangeli et al. find an association between ACSL5-ZDHHC6 and ALS risk, with replication in an Australian cohort. They identify B4GALNT1, G2E3-SCFD1, and TRIP11-ATXN3 using a gene-based analysis. They also find a suggestive association between ACSL5 SNPs and lower fat-free mass in patients.

The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer’s disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.

Charcot-Marie-Tooth (CMT) is a group of inherited diseases clinically and genetically heterogenous, characterised by length dependent degeneration of axons of the peripheral nervous system. A missense mutation (p.R158H) in the pyruvate dehydrogenase kinase 3 gene (PDK3) has been identified as the genetic cause for an X-linked form of CMT (CMTX6) in two unrelated families. PDK3 is one of four PDK isoenzymes that regulate the activity of the pyruvate dehydrogenase complex (PDC). The balance between kinases (PDKs) and phosphatases (PDPs) determines the extend of oxidative decarboxylation of pyruvate to generate acetyl CoA, critically linking glycolysis and the energy producing Krebs cycle. We had shown the p.R158H mutation causes hyperactivity of PDK3 and CMTX6 fibroblasts show hyperphosphorylation of PDC, leading to reduced PDC activity and ATP production. In this manuscript we have generated induced pluripotent stem cells (iPSCs) by re-programming CMTX6 fibroblasts (iPSCCMTX6). We also have engineered an isogenic control (iPSCisogenic) and demonstrated that genetic correction of the p.R158H mutation reverses the CMTX6 phenotype. Patient-derived motor neurons (MNCMTX6) show increased phosphorylation of the PDC, energy metabolism defects and mitochondrial abnormalities, including reduced velocity of trafficking mitochondria in the affected axons. Treatment of the MNCMTX6 with a PDK inhibitor reverses PDC hyperphosphorylation and the associated functional deficits founds in the patient motor neurons, demonstrating that the MNCMTX6 and MNisogenic motor neurons provide an excellent neuronal system for compound screening approaches to identify drugs for the treatment of CMTX6.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by the loss of upper and lower motor neurons resulting in paralysis and eventual death. Approximately 10% of ALS cases have a family history of disease, while the remainder present as apparently sporadic cases. Heritability studies suggest a significant genetic component to sporadic ALS, and although most sporadic cases have an unknown genetic aetiology, some familial ALS mutations have also been found in sporadic cases. This suggests that some sporadic cases may be unrecognised familial cases with reduced disease penetrance in their ancestors. A powerful strategy to uncover a familial link is identity-by-descent (IBD) analysis, which detects genomic regions that have been inherited from a common ancestor. IBD analysis was performed on 83 Australian familial ALS cases from 25 families and three sporadic ALS cases, each of whom carried one of three SOD1 mutations (p.I114T, p.V149G and p.E101G). We defined five unique 350-SNP haplotypes that carry these mutations in our cohort, indicative of five founder events. This included two founder haplotypes that carry SOD1 p.I114T; linking familial and sporadic cases. We found that SOD1 p.E101G arose independently in each family that carries this mutation and linked two families that carry SOD1 p.V149G. The age of disease onset varied between cases that carried each SOD1 p.I114T haplotype. Linking families with identical ALS mutations allows for larger sample sizes and increased statistical power to identify putative phenotypic modifiers.

We conducted DNA methylation association analyses using Illumina 450K data from whole blood for an Australian amyotrophic lateral sclerosis (ALS) case–control cohort (782 cases and 613 controls). Analyses used mixed linear models as implemented in the OSCA software. We found a significantly higher proportion of neutrophils in cases compared to controls which replicated in an independent cohort from the Netherlands (1159 cases and 637 controls). The OSCA MOMENT linear mixed model has been shown in simulations to best account for confounders. When combined in a methylation profile score, the 25 most-associated probes identified by MOMENT significantly classified case–control status in the Netherlands sample (area under the curve, AUC = 0.65, CI95% = [0.62–0.68], p = 8.3 × 10−22). The maximum AUC achieved was 0.69 (CI95% = [0.66–0.71], p = 4.3 × 10−34) when cell-type proportion was included in the predictor.

Machado-Joseph disease (MJD/SCA3) is the most common form of dominantly inherited ataxia worldwide. The disorder is caused by an expanded CAG repeat in the ATXN3 gene. Past studies have revealed that the length of the expansion partly explains the disease age at onset (AO) variability of MJD, which is confirmed in this study. Using a total of 786 MJD patients from five different geographical origins, a genome-wide association study (GWAS) was conducted to identify additional AO modifying factors that could explain some of the residual AO variability. We identified nine suggestively associated loci (P < 1 × 10−5). These loci were enriched for genes involved in vesicle transport, olfactory signaling, and synaptic pathways. Furthermore, associations between AO and the TRIM29 and RAG genes suggests that DNA repair mechanisms might be implicated in MJD pathogenesis. Our study demonstrates the existence of several additional genetic factors, along with CAG expansion, that may lead to a better understanding of the genotype-phenotype correlation in MJD.

We describe an infant with an early-onset demyelinating neuropathy who presented with an upper extremity monoplegia and progressive asymmetric weakness. Neurophysiologic testing revealed a generalized severe neuropathy with marked slowing of nerve conduction. The disproportionate severity and asymmetry of upper extremity involvement at presentation was atypical of inherited neuropathies, and an initial diagnosis of chronic inflammatory demyelinating polyneuropathy was considered. Nerve biopsy showed severe depletion of large myelinated fibers without inflammatory cells, and focally folded myelin sheaths were seen on electron microscopy. Genetic testing revealed a de novo heterozygous mutation in the myelin protein zero gene.

Objective: Hemiplegic migraine in both familial (FHM) and sporadic (SHM) forms is a rare subtype of migraine with aura that can be traced to mutations in the CACNA1A, ATP1A2 and SCN1A genes. It is characterised by severe attacks of typical migraine accompanied by hemiparesis, as well as episodes of complex aura that vary significantly between individuals. Methods: Using a targeted next generation sequencing (NGS) multigene panel, we have sequenced the genomic DNA of 172 suspected hemiplegic migraine cases, in whom no mutation had previously been found by Sanger sequencing (SS) of a limited number of exons with high mutation frequency in FHM genes. Results: Genetic screening identified 29 variants, 10 of which were novel, in 35 cases in the three FHM genes ( CACNA1A, ATP1A2 and SCN1A). Interestingly, in this suspected HM cohort, the ATP1A2 gene harboured the highest number of variants with 24/35 cases (68.6%), while CACNA1A ranked the second gene, with 5 variants identified in 7/35 cases (20%). All detected variants were confirmed by SS and were absent in 100 non-migraine healthy control individuals. Assessment of variants with the American College of Medical Genetics and Genomics guidelines classified 8 variants as pathogenic, 3 as likely pathogenic and 18 as variants of unknown significance. Targeted NGS gene panel increased the diagnostic yield by fourfold over iterative SS in our diagnostics facility. Conclusion: We have identified 29 potentially causative variants in an Australian and New Zealand cohort of suspected HM cases and found that the ATP1A2 gene was the most commonly mutated gene. Our results suggest that screening using NGS multigene panels to investigate ATP1A2 alongside CACNA1A and SCN1A is a clinically useful and efficient method.

Inherited peripheral neuropathies (IPNs) are a clinically and genetically heterogeneous group of diseases affecting the motor and sensory peripheral nerves. IPNs have benefited from gene discovery and genetic diagnosis using next‐generation sequencing with over 80 causative genes available for testing. Despite this success, up to 50% of cases remain genetically unsolved. In the absence of protein coding mutations, noncoding DNA or structural variation (SV) mutations are a possible explanation. The most common IPN, Charcot‐Marie‐Tooth neuropathy type 1A (CMT1A), is caused by a 1.5 Mb duplication causing trisomy of the dosage sensitive gene PMP22. Using genome sequencing, we recently identified two large genomic rearrangements causing IPN subtypes X‐linked CMT (CMTX3) and distal hereditary motor neuropathy (DHMN1), thereby expanding the spectrum of SV mutations causing IPN. Understanding how newly discovered SVs can cause IPN may serve as a useful paradigm to examine the role of topologically associated domains (TADs), chromatin interactions, and gene dysregulation in disease. This review will describe the growing role of SV in the pathogenesis of IPN and the importance of considering this type of mutation in Mendelian diseases where protein coding mutations cannot be identified.

Introduction: Chloride conductance disturbances contribute to sarcolemmal dysfunction in type 1 (DM1) and 2 (DM2) myotonic dystrophy. Studies using muscle velocity recovery cycles (MVRCs) suggest Na+/K+-ATPase activation becomes defective in advanced DM1. We used MVRCs to investigate muscle excitability in DM1 and DM2. Methods: MVRCs were measured for patients with mild (n = 8) and advanced DM1 (n = 11), DM2 (n = 4) and normal controls (n = 30). Results: Residual supernormality after multiple conditioning stimuli was increased in DM2 and advanced DM1. Advanced DM1 was distinguished by increases in muscle relative refractory period and reduced early supernormality, as well as peak amplitude decrements for the first and last responses in train during repetitive stimulation. Discussion: Prolongation of the muscle relative refractory period indicates that depolarization of the resting muscle membrane potential occurs in advanced DM1, with possible implications for future therapeutic approaches. This article is protected by copyright. All rights reserved.

Currently there is a lack in fundamental understanding of disease progression of most neurodegenerative diseases, and, therefore, treatments and preventative measures are limited. Consequently, there is a great need for adaptable, yet robust model systems to both investigate elementary disease mechanisms and discover effective therapeutics. We have generated a Tol2 Gateway-compatible toolbox to study neurodegenerative disorders in zebrafish, which includes promoters for astrocytes, microglia and motor neurons, multiple fluorophores, and compatibility for the introduction of genes of interest or disease-linked genes. This toolbox will advance the rapid and flexible generation of zebrafish models to discover the biology of the nervous system and the disease processes that lead to neurodegeneration.

Background: Mutations in the genes encoding the heterogeneous nuclear ribonucleoproteins hnRNPA1 and hnRNPA2/B1 have been reported in a multisystem proteinopathy that includes amyotrophic lateral sclerosis (ALS) and inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia. Mutations were also described in the prion-like domain of hnRNPA1 in patients with classic ALS. Another hnRNP protein, hnRNPA3, has been found to be associated with the ALS/frontotemporal dementia protein C9orf72. Objective: To further assess their role in ALS, we examined these hnRNPs in spinal cord tissue from sporadic (SALS) and familial ALS (FALS) patients, including C9orf72 repeat expansion-positive patients, and controls. We also sought to determine the prevalence of HNRNPA1, HNRNPA2B1, and HNRNPA3 mutations in Australian ALS patients. Methods: Immunostaining was used to assess hnRNPs in ALS patient spinal cords. Mutation analysis of the HNRNPA1, HNRNPA2B1, and HNRNPA3 genes was performed in FALS and of their prion-like domains in SALS patients. Results: Immunostaining of spinal motor neurons of ALS patients with the C9orf72 repeat expansion showed significant mislocalisation of hnRNPA3, and no differences in hnRNPA1 or A2/B1 localisation, compared to controls. No novel or known mutations were identified in HNRNPA1, HNRNPA2B1, or HNRNPA3 in Australian ALS patients. Conclusions: hnRNPA3 pathology was identified in motor neurons of ALS patients with C9orf72 repeat expansions, implicating hnRNPA3 in the pathogenesis of C9orf72-linked ALS. hnRNPA3 warrants further investigation into the pathogenesis of ALS linked to C9orf72. This study also determined that HNRNP mutations are not a common cause of FALS and SALS in Australia.

The utility of quantitative muscle ultrasound as a marker of disease severity in Charcot-Marie-Tooth (CMT) disease subtypes was investigated.Muscle ultrasound was prospectively performed on 252 individual muscles from 21 CMT patients (9 CMT1A, 8 CMTX1, 4 CMT2A) and compared to 120 muscles from 10 age and gender-matched controls. Muscle ultrasound recorded echogenicity and thickness in representative muscles including first dorsal interosseus (FDI) and tibialis anterior (TA).Muscle volume of FDI and thickness of TA correlated with MRC strength. Muscle echogenicity was significantly increased in FDI (65.05 vs 47.09; p0.0001) and TA (89.45 vs 66.30; p0.0001) of CMT patients. In TA, there was significantly higher muscle thickness (23 vs 18 vs 16mm; p0.0001) and lower muscle echogenicity (80 vs 95 vs 108; p0.0001) in CMT1A compared to CMTX1 and CMT2A. This corresponded to disease severity based on muscle strength (MRC grading CMT1A vs CMTX1 vs CMT2A: 59 vs 48 vs 44; p=0.002).In CMT, quantitative muscle ultrasound of FDI and TA is a useful marker of disease severity.The current findings suggest that quantitative muscle ultrasound has potential as a surrogate marker of disease progression in future interventional trials in CMT.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterised by the loss of upper and lower motor neurons resulting in paralysis and eventual death. Approximately 10% of ALS cases have a family history of disease, while the remaining cases present as apparently sporadic. Heritability studies suggest a significant genetic component to sporadic ALS, and although most sporadic cases have an unknown genetic etiology, some familial ALS mutations have also been found in sporadic cases. This suggests that some sporadic cases may be unrecognised familial cases with reduced disease penetrance. Identifying a familial basis of disease in apparently sporadic ALS cases has significant genetic counselling implications for immediate relatives. A powerful strategy to uncover a familial link is identity-by-descent (IBD) analysis which detects genomic regions that have been inherited from a common ancestor. We performed IBD analysis on 90 Australian familial ALS cases from 25 families and three sporadic ALS cases, each of whom carried one of three SOD1 mutations (p.I114T, p.V149G and p.E101G). We identified five unique haplotypes that carry these mutations in our cohort, indicative of five founder events. This included two different haplotypes that carry SOD1 p.I114T, where one haplotype was present in one sporadic case and 20 families, while the second haplotype was found in the remaining two sporadic cases and one family, thus linking these familial and sporadic cases. Furthermore, we linked two families that carry SOD1 p.V149G and found that SOD1 p.E101G arose independently in each family that carries this mutation.