Dysregulation of muscle cholesterol transport in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons, with a typical lifespan of 3-5 years. Altered metabolism is a key feature of ALS that strongly influences prognosis, with an increase in whole-body energy expenditure and changes in skeletal muscle metabolism, including greater reliance on fat oxidation. Dyslipidemia has been described in ALS as part of the metabolic dysregulation, but its role in the pathophysiology of the disease remains controversial. Among the lipids, cholesterol is of particular interest as a vital component of cell membranes, playing a key role in signal transduction and mitochondrial function in muscle. The aim of this study was to investigate whether motor dysfunction in ALS might be associated with dysregulation of muscle cholesterol metabolism. We determined cholesterol content and analyzed the expression of key determinants of the cholesterol metabolism pathway in muscle biopsies from thirteen ALS patients and ten asymptomatic ALS-mutation gene carriers compared to sixteen controls. Using human control primary myotubes, we further investigated the potential contribution of cholesterol dyshomeostasis to reliance on mitochondrial fatty acid. We found that cholesterol accumulates in the skeletal muscle of ALS patients and that cholesterol overload significantly correlates with disease severity evaluated by the Revised ALS Functional Rating Scale. These defects are associated with overexpression of the genes of the lysosomal cholesterol transporters Niemann-Pick type C1 (NPC1) and 2 (NPC2), which are required for cholesterol transfer from late endosomes/lysosomes to cellular membranes. Most notably, a significant increase in NPC2 mRNA levels could be detected in muscle samples from asymptomatic ALS-mutation carriers, long before disease onset. We found that filipin-stained unesterified cholesterol accumulated in the lysosomal compartment in ALS muscle samples, suggesting dysfunction of the NPC1/2 system. Accordingly, we report here that experimental NPC1 inhibition or lysosomal pH alteration in human primary myotubes was sufficient to induce the overexpression of NPC1 and NPC2 mRNA. Finally, acute NPC1 inhibition in human control myotubes induced a shift towards a preferential use of fatty acids, thus reproducing the metabolic defect characteristic of ALS muscle. We conclude that cholesterol homeostasis is dysregulated in ALS muscle from the presymptomatic stage. Targeting NPC1/2 dysfunction may be a new therapeutic strategy for ALS to restore muscle energy metabolism and slow motor symptom progression.

Transactive response DNA-binding protein 43 is enriched at the centrosome in human cells.

The centrosome, as the main microtubule organizing centre, plays key roles in cell polarity, genome stability and ciliogenesis. The recent identification of ribosomes, RNA-binding proteins and transcripts at the centrosome suggests local protein synthesis. In this context, we hypothesized that TDP-43, a highly conserved RNA binding protein involved in the pathophysiology of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, could be enriched at this organelle. Using dedicated high magnification sub-diffraction microscopy on human cells, we discovered a novel localization of TDP-43 at the centrosome during all phases of the cell cycle. These results were confirmed on purified centrosomes by western blot and immunofluorescence microscopy. In addition, the co-localization of TDP-43 and pericentrin suggested a pericentriolar enrichment of the protein, leading us to hypothesize that TDP-43 might interact with local mRNAs and proteins. Supporting this hypothesis, we found four conserved centrosomal mRNAs and 16 centrosomal proteins identified as direct TDP-43 interactors. More strikingly, all the 16 proteins are implicated in the pathophysiology of TDP-43 proteinopathies, suggesting that TDP-43 dysfunction in this organelle contributes to neurodegeneration. This first description of TDP-43 centrosomal enrichment paves the way for a more comprehensive understanding of TDP-43 physiology and pathology.

Neurofilament accumulations in amyotrophic lateral sclerosis patients' motor neurons impair axonal initial segment integrity.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease in adults with no curative treatment. Neurofilament (NF) level in patient' fluids have recently emerged as the prime biomarker of ALS disease progression, while NF accumulation in MNs of patients is the oldest and one of the best pathological hallmarks. However, the way NF accumulations could lead to MN degeneration remains unknown. To assess NF accumulations and study the impact on MNs, we compared MNs derived from induced pluripotent stem cells (iPSC) of patients carrying mutations in C9orf72, SOD1 and TARDBP genes, the three main ALS genetic causes. We show that in all mutant MNs, light NF (NF-L) chains rapidly accumulate in MN soma, while the phosphorylated heavy/medium NF (pNF-M/H) chains pile up in axonal proximal regions of only C9orf72 and SOD1 MNs. Excitability abnormalities were also only observed in these latter MNs. We demonstrate that the integrity of the MN axonal initial segment (AIS), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of pNF-M/H accumulations in C9orf72 and SOD1 MNs. We establish a strong correlation between these pNF-M/H accumulations, an AIS distal shift, increased axonal calibers and modified repartition of sodium channels. The results expand our understanding of how NF accumulation could dysregulate components of the axonal cytoskeleton and disrupt MN homeostasis. With recent cumulative evidence that AIS alterations are implicated in different brain diseases, preserving AIS integrity could have important therapeutic implications for ALS.

The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis.

Mutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS.

Impact of a frequent nearsplice SOD1 variant in amyotrophic lateral sclerosis: optimising SOD1 genetic screening for gene therapy opportunities.

OBJECTIVE: Mutations in superoxide dismutase 1 gene (SOD1), encoding copper/zinc superoxide dismutase protein, are the second most frequent high penetrant genetic cause for amyotrophic lateral sclerosis (ALS) motor neuron disease in populations of European descent. More than 200 missense variants are reported along the SOD1 protein. To limit the production of these aberrant and deleterious SOD1 species, antisense oligonucleotide approaches have recently emerged and showed promising effects in clinical trials. To offer the possibility to any patient with SOD1-ALS to benefit of such a gene therapy, it is necessary to ascertain whether any variant of unknown significance (VUS), detected for example in SOD1 non-coding sequences, is pathogenic. METHODS: We analysed SOD1 mutation distribution after SOD1 sequencing in a large cohort of 470 French familial ALS (fALS) index cases. RESULTS: We identified a total of 27 SOD1 variants in 38 families including two SOD1 variants located in nearsplice or intronic regions of the gene. The pathogenicity of the c.358-10T>G nearsplice SOD1 variant was corroborated based on its high frequency (as the second most frequent SOD1 variant) in French fALS, the segregation analysis confirmed in eight affected members of a large pedigree, the typical SOD1-related phenotype observed (with lower limb onset and prominent lower motor neuron involvement), and findings on postmortem tissues showing SOD1 misaccumulation. CONCLUSIONS: Our results highlighted nearsplice/intronic mutations in SOD1 are responsible for a significant portion of French fALS and suggested the systematic analysis of the SOD1 mRNA sequence could become the method of choice for SOD1 screening, not to miss these specific cases.

Clinical and Molecular Landscape of ALS Patients with SOD1 Mutations: Novel Pathogenic Variants and Novel Phenotypes. A Single ALS Center Study.

Mutations in the copper zinc superoxide dismutase 1 (SOD1) gene are the second most frequent cause of familial amyotrophic lateral sclerosis (ALS). Nearly 200 mutations of this gene have been described so far. We report all SOD1 pathogenic variants identified in patients followed in the single ALS center of Lyon, France, between 2010 and 2020. Twelve patients from 11 unrelated families are described, including two families with the not yet described H81Y and D126N mutations. Splice site mutations were detected in two families. We discuss implications concerning genetic screening of SOD1 gene in familial and sporadic ALS.

Increased prevalence of granulovacuolar degeneration in C9orf72 mutation.

Granulovacuolar degeneration (GVD) is usually found in Alzheimer's disease (AD) cases or in elderly individuals. Its severity correlates positively with the density of neurofibrillary tangles (NFTs). Mechanisms underlying GVD formation are unknown. We assessed the prevalence and distribution of GVD in cases with TDP-43-related frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS-TDP). Consecutively autopsied cases with FTLD/ALS-TDP and C9orf72 mutations (FTLD/ALS-C9; N = 29), cases with FTLD/ALS-TDP without C9orf72 mutations (FTLD/ALS-nonC9; N = 46), and age-matched healthy controls (N = 40) were studied. The prevalence of GVD was significantly higher in the FTLD/ALS-C9 cases (26/29 cases) than in the FTLD/ALS-nonC9 cases (15/46 cases; Fisher exact test; p < 2×10-6) or in the control group (12/40 individuals; p < 1×10-6). Average Braak stages and ages of death were not significantly different among the groups. The CA2 sector was most frequently affected in the FTLD/ALS-C9 group, whereas the CA1/subiculum was the most vulnerable area in the other groups. Extension of GVD correlated with the clinical duration of the disease in the FTLD/ALS-C9 cases but not in the FTLD/ALS-nonC9 cases. The GVD-containing neurons frequently had dipeptide repeat (DPR) protein inclusions. GVD granules labeled with antibodies directed against charged multivesicular body protein 2B or casein kinase 1δ were attached to DPR inclusions within GVD. Our results suggest that development of GVD and DPR inclusions is related to common pathogenic mechanisms and that GVD is not only associated with NFTs seen in AD cases or aging individuals.

Electrophysiological Characterization of C9ORF72-Associated Amyotrophic Lateral Sclerosis: A Retrospective Study.

OBJECTIVE: C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS). The aim of the present study was to determine whether C9ORF72-associated ALS (C9-ALS) patients present distinctive electrophysiological characteristics that could differentiate them from non C9ORF72-associated ALS (nonC9-ALS) patients. METHODS: Clinical and electrodiagnostic data from C9-ALS patients and nonC9-ALS patients were collected retrospectively. For electroneuromyography, the mean values of motor conduction, myography, and the mean values of sensory conduction were considered. Furthermore, the proportion of ALS patients with electrophysiological sensory neuropathy was determined. RESULTS: No significant difference was observed between 31 C9-ALS patients and 22 nonC9-ALS patients for mean motor conduction and myography. For sensory conduction analyses, mean sensory conduction was not significantly different between both groups. In total, 38% of -C9-ALS patient and 21% of nonC9-ALS patients presented electrophysiological sensory neuropathy (p = 0.33). In -C9-ALS patients with electrophysiological sensory neuropathy, 80% (8/10) were male and 67% (6/9) presented spinal onset compare to 25% (4/16, p = 0.014) male and 25% (4/16, p = 0.087) with spinal onset in those without electrophysiological sensory neuropathy. CONCLUSION: Although not different from nonC9-ALS, these results suggest that sensory involvement is a frequent feature of C9-ALS patients, expanding the phenotype of the disease beyond the motor and cognitive domains.

Axonal transport deficits and neurodegenerative diseases.

The intracellular transport of organelles along an axon is crucial for the maintenance and function of a neuron. Anterograde axonal transport has a role in supplying proteins and lipids to the distal synapse and mitochondria for local energy requirements, whereas retrograde transport is involved in the clearance of misfolded and aggregated proteins from the axon and the intracellular transport of distal trophic signals to the soma. Axonal transport can be affected by alterations to various components of the transport machinery. Here, we review the current state of knowledge about axonal transport defects that might contribute to the pathogenesis of particular neurodegenerative diseases.

System xC- is a mediator of microglial function and its deletion slows symptoms in amyotrophic lateral sclerosis mice.

Amyotrophic lateral sclerosis is the most common adult-onset motor neuron disease and evidence from mice expressing amyotrophic lateral sclerosis-causing SOD1 mutations suggest that neurodegeneration is a non-cell autonomous process where microglial cells influence disease progression. However, microglial-derived neurotoxic factors still remain largely unidentified in amyotrophic lateral sclerosis. With excitotoxicity being a major mechanism proposed to cause motor neuron death in amyotrophic lateral sclerosis, our hypothesis was that excessive glutamate release by activated microglia through their system [Formula: see text] (a cystine/glutamate antiporter with the specific subunit xCT/Slc7a11) could contribute to neurodegeneration. Here we show that xCT expression is enriched in microglia compared to total mouse spinal cord and absent from motor neurons. Activated microglia induced xCT expression and during disease, xCT levels were increased in both spinal cord and isolated microglia from mutant SOD1 amyotrophic lateral sclerosis mice. Expression of xCT was also detectable in spinal cord post-mortem tissues of patients with amyotrophic lateral sclerosis and correlated with increased inflammation. Genetic deletion of xCT in mice demonstrated that activated microglia released glutamate mainly through system [Formula: see text]. Interestingly, xCT deletion also led to decreased production of specific microglial pro-inflammatory/neurotoxic factors including nitric oxide, TNFa and IL6, whereas expression of anti-inflammatory/neuroprotective markers such as Ym1/Chil3 were increased, indicating that xCT regulates microglial functions. In amyotrophic lateral sclerosis mice, xCT deletion surprisingly led to earlier symptom onset but, importantly, this was followed by a significantly slowed progressive disease phase, which resulted in more surviving motor neurons. These results are consistent with a deleterious contribution of microglial-derived glutamate during symptomatic disease. Therefore, we show that system [Formula: see text] participates in microglial reactivity and modulates amyotrophic lateral sclerosis motor neuron degeneration, revealing system [Formula: see text] inactivation, as a potential approach to slow amyotrophic lateral sclerosis disease progression after onset of clinical symptoms.

EVolution in ALS diagnosis: molecular markers in extracellular vesicles.

The identification of biomarkers for amyotrophic lateral sclerosis (ALS) is a central issue in disease research. In a recent article, Chatterjee et al. show that blood extracellular vesicles (EVs) with high levels of transactive response DNA-binding protein 43 (TDP-43) accurately discriminate patients with ALS from controls and correlate with disease severity, providing a promising biomarker for early diagnosis and monitoring.

Mutations in SQSTM1 encoding p62 in amyotrophic lateral sclerosis: genetics and neuropathology.

Mutations in SQSTM1 encoding the sequestosome 1/p62 protein have recently been identified in familial and sporadic cases of amyotrophic lateral sclerosis (ALS). p62 is a component of the ubiquitin inclusions detected in degenerating neurons in ALS patients. We sequenced SQSTM1 in 90 French patients with familial ALS (FALS) and 74 autopsied ALS cases with sporadic ALS (SALS). We identified, at the heterozygote state, one missense c.1175C>T, p.Pro392Leu (exon 8) in one of our FALS and one substitution in intron 7 (the c.1165+1G>A, previously called IVS7+1 G-A, A390X) affecting the exon 7 splicing site in one SALS. These mutations that are located in the ubiquitin-associated domain (UBA domain) of the p62 protein have already been described in Paget's disease and ALS patients carrying these mutations had both concomitant Paget's disease. However, we also identified two novel missense mutations in two SALS: the c.259A>G, p.Met87Val in exon 2 and the c.304A>G, p.Lys102Glu in exon 3. These mutations that were not detected in 360 control subjects are possibly pathogenic. Neuropathology analysis of three patients carrying SQSTM1 variants revealed the presence of large round p62 inclusions in motor neurons, and immunoblot analysis showed an increased p62 and TDP-43 protein levels in the spinal cord. Our results confirm that SQSTM1 gene mutations could be the cause or genetic susceptibility factor of ALS in some patients.

Phenotype difference between ALS patients with expanded repeats in C9ORF72 and patients with mutations in other ALS-related genes.

BACKGROUND: Expanded GGGGCC hexanucleotide repeats in the promoter of the C9ORF72 gene have recently been identified in frontotemporal dementia (FTD), Amyotrophic Lateral Sclerosis (ALS) and ALS-FTD and appear as the most common genetic cause of familial (FALS) and sporadic (SALS) forms of ALS. METHODS: We searched for the C9ORF72 repeat expansion in 950 French ALS patients (225 FALS and 725 SALS) and 580 control subjects and performed genotype-phenotype correlations. RESULTS: The repeat expansion was present in 46% of FALS, 8% of SALS and 0% of controls. Phenotype comparisons were made between FALS patients with expanded C9ORF72 repeats and patients carrying another ALS-related gene (SOD1, TARDBP, FUS) or a yet unidentified genetic defect. SALS patients with and without C9ORF72 repeat expansions were also compared. The C9ORF72 group presented more frequent bulbar onset both in FALS (p<0.0001 vs SOD1, p=0.002 vs TARDBP, p=0.011 vs FUS, p=0.0153 vs other FALS) and SALS (p=0.047). FALS patients with C9ORF72 expansions had more frequent association with FTD than the other FALS patients (p<0.0001 vs SOD1, p=0.04 vs TARDBP, p=0.004 vs FUS, p=0.03 vs other FALS). C9ORF72-linked FALS patients presented an older age of onset than SOD1 (p=0.0139) or FUS mutation (p<0.0001) carriers. Disease duration was shorter for C9ORF72 expansion carriers than for SOD1 (p<0.0001) and TARDBP (p=0.0242) carriers, other FALS (p<0.0001) and C9ORF72-negative SALS (p=0.0006). CONCLUSIONS: Our results confirm the major role of expanded repeats in C9ORF72 as causative for ALS and provide evidence for specific phenotypic aspects compared to patients with other ALS-related genes.

Integrative genetic analysis illuminates ALS heritability and identifies risk genes.

Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). We show that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk (P = 3.71.10-03; odds ratio = 3.29; 95%CI, 1.37 to 7.87) in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, our study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

Recurrent G41S mutation in Cu/Zn superoxide dismutase gene (SOD1) causing familial amyotrophic lateral sclerosis in a large Polish family.

Mutations in the superoxide dismutase-1 (SOD1) gene have been found in 12-23% of patients with a diagnosis of ALS. Here we describe a large ALS Polish family with a branch in France, carrying a G41S mutation in the SOD1, and characterized by an early onset of the disease and extremely short survival time. The mutation has been initially detected in Italian ALS families with common founder effect. However, in the Polish population the G41S mutation most probably originated from an independent mutation event, as indicated by haplotype analysis. Collected data support the hypothesis that a SOD1 mutation is not the sole factor determining the clinical ALS phenotype.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

Fronto-temporal lobar degeneration: neuropathology in 60 cases.

Sixty cases of frontotemporal lobar degeneration (FTLD) were collected over 22 years. Brain weight was negatively correlated with disease duration. The neuronal and/or glial inclusions were labeled by anti-TDP, anti-FUS or anti-TAU antibodies, respectively, in 40, 3 and 12 cases. In the FTLD-TDP group, mutation of the progranulin gene was found in four cases (FTD-GRN), with nuclear, cat eye inclusions and severe neuronal loss in CA1 and subiculum. The motor neurons were involved in 27 cases (fronto-temporal dementia with amyotrophic lateral sclerosis = FTD-ALS). Familial FTD-ALS cases lived longer than sporadic ones. In nine cases, there was no ALS, no GRN mutation (FTD-NAP). The cases in the FTD-ALS and FTD-NAP subgroups were of Sampathu type 2 (TDP-positive inclusions located mostly in cell bodies and short neurites) with the exception of five cases which belonged to type 1 (long TDP-positive neurites in the superficial layers of the cortex). All of the FTLD-FUS of this series cases were affected by neuronal intermediate filament inclusion disease (NIFID). They were young. The survival was short. In the FTLD-tau group, mutations P301P (previously not recognized as pathogenic), P301L and S305N were identified. Pick disease (n = 5) appeared as a homogeneous sporadic disorder. The current nomenclature allows the neuropathological classification of nearly all the cases of FTD. The prevalence of the different types of FTD is tightly linked to the recruitment. This series was enriched in motor neuron disease (explaining the overall predominance of type 2 TDP inclusions).

SOD1, ANG, VAPB, TARDBP, and FUS mutations in familial amyotrophic lateral sclerosis: genotype-phenotype correlations.

BACKGROUND: Mutations in SOD1, ANG, VAPB, TARDBP and FUS genes have been identified in amyotrophic lateral sclerosis (ALS). METHODS: The relative contributions of the different mutations to ALS were estimated by systematically screening a cohort of 162 families enrolled in France and 500 controls (1000 chromosomes) using molecular analysis techniques and performing phenotype-genotype correlations. RESULTS: 31 pathogenic missense mutations were found in 36 patients (20 SOD1, 1 ANG, 1 VAPB, 7 TARDBP and 7 FUS). Surprisingly two FUS mutation carriers also harboured ANG variants. One family of Japanese origin with the P56S VAPB mutation was identified. Seven novel mutations (three in SOD1, two in TARDBP, two in FUS) were found. None of them was detected in controls. Segregation of detected mutations with the disease was confirmed in 11 families including five pedigrees carrying the novel mutations. Clinical comparison of SOD1, TARDBP, FUS and other familial ALS patients (with no mutation in the screened genes) revealed differences in site of onset (predominantly lower limbs for SOD1 and upper limbs for TARDBP mutations), age of onset (younger with FUS mutations), and in lifespan (shorter for FUS carriers). One third of SOD1 patients survived more than 7 years: these patients had earlier disease onset than those presenting with a more typical course. Differences were also observed among FUS mutations, with the R521H FUS mutation being associated with longer disease duration. CONCLUSIONS: This study identifies new genetic associations with ALS and provides phenotype-genotype correlations with both previously reported and novel mutations.

Phenoconversion from Spastic Paraplegia to ALS/FTD Associated with CYP7B1 Compound Heterozygous Mutations.

Biallelic mutations in the CYP7B1 gene lead to spastic paraplegia-5 (SPG5). We report herein the case of a patient whose clinical symptoms began with progressive lower limb spasticity during childhood, and who secondly developed amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) at the age of 67 years. Hereditary spastic paraplegia (HSP) gene analysis identified the compound heterozygous mutations c.825T>A (pTyr275*) and c.1193C>T (pPro398Leu) in CYP7B1 gene. No other pathogenic variant in frequent ALS/FTD causative genes was found. The CYP7B1 gene seems, therefore, to be the third gene associated with the phenoconversion from HSP to ALS, after the recently described UBQLN2 and ERLIN2 genes. We therefore expand the phenotype associated with CYP7B1 biallelic mutations and make an assumption about a link between cholesterol dyshomeostasis and ALS/FTD.