Background Repetitive head impacts in former contact sport athletes are associated with cognitive impairment, accelerated cerebral atrophy and risk of neurodegenerative disease. Epigenetic clocks derived from age-associated DNA methylation (DNAm) profiles may capture accelerated biological ageing in neurodegenerative conditions; however, their application in former athletes remains unexplored. Here we aim to explore the application of epigenetic clocks as a measure of accelerated biological ageing in a cohort of former athletes. Methods In 126 former athletes (96% male; mean age: 54.5±14.4 years; mean concussions: 6.8±6.7), we examined associations of brain volumes, plasma neurofilament light levels and cognitive/behavioural scores with DNAmAge-acceleration, AgeAccelResidual and DNAmFitAge-acceleration. Results We only found an association between the number of concussions and DNAmFitAge-acceleration (p=0.003, B=0.46, R²=0.063), indicating that every two additional concussions were associated with a 5-year increase in DNAmFitAge-acceleration. There was also a trend towards an association between years of play and DNAmAge-acceleration in older athletes. Conclusions These preliminary findings suggest that specific epigenetic clock measures may serve as early markers of biological ageing related to repetitive head impacts.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are fatal neurodegenerative diseases sharing clinical and pathological features. Both involve complex neuron-glia interactions, but cell-type-specific alterations remain poorly defined. We performed single-nucleus RNA sequencing of the frontal cortex from C9orf72-related ALS (with and without FTLD) and sporadic ALS (sALS). Neurons showed prominent changes in mitochondrial function, protein homeostasis, and chromatin remodeling. Comparison with independent datasets from other cortical regions revealed consistent pathway alterations, including upregulation of STMN2 and NEFL across brain regions and subtypes. We further examined dysregulation of alternative polyadenylation (APA), an understudied post-transcriptional mechanism, uncovering cell-type-specific APA patterns. To investigate its regulation, we developed the alternative polyadenylation network (APA-Net), a multi-modal deep learning model integrating transcript sequences and RNA-binding protein (RBP) expression profiles to predict APA. This atlas advances our understanding of ALS/FTLD molecular pathology and provides a valuable resource for future mechanistic studies.

Background and Objectives There is no disease-modifying treatment of corticobasal syndrome (CBS) and progressive supranuclear palsy (PSP), 2 disorders characterized by their striking phenotypic, and, in CBS, pathologic heterogeneity. Seed amplification assays (SAAs) could enable the detection of neuropathologic processes, such as alpha-synuclein (alpha Syn) copathology, that affect the success of future disease-modifying treatment strategies. The primary objective was to assess possible alpha Syn copathology in CBS and PSP, as detected in CSF using an alpha Syn SAA (alpha Syn-SAA). Secondary objectives were to evaluate the association of alpha Syn-SAA positivity with other biomarkers including of Alzheimer disease (AD), and with clinical presentation. We hypothesized that alpha Syn-SAA positivity would be detectable in CBS and PSP and that it would be associated with AD biomarker positivity and beta-amyloid (A beta) 42 levels, neurodegeneration as assessed by neurofilament light chain (NfL) levels, and symptoms associated with synucleinopathies. Methods This cross-sectional observational study included patients clinically diagnosed with CBS and PSP who underwent a lumbar puncture between 2012 and 2021 (Toronto Western Hospital, Canada). CSF was tested for alpha Syn-SAA positivity, AD biomarkers, and NfL levels. Clinical data were derived from medical records. Results We tested the CSF of 40 patients with CBS (19 female patients, 65.9 +/- 8.6 years) and 28 with PSP (13 female patients, 72.5 +/- 8.7 years old), mostly White (n = 50) or Asian (n = 14). alpha Syn-SAA positivity was observed in 35.9% patients with CBS and 28.6% with PSP. In young-onset, but not late-onset patients, alpha Syn-SAA positivity and AD positivity were associated (odds ratio [OR] 8.8, 95% CI 1.2-82.6, p < 0.05). A multivariable linear regression analysis showed a significant interaction of alpha Syn-SAA status by age at onset on CSF A beta 42 levels (beta = 0.3 +/- 0.1, p < 0.05). Indeed, age at onset was positively related to A beta 42 levels only in alpha Syn-SAA-positive patients, as shown by slope comparison. A logistic regression analysis also suggested that REM sleep behavior disorder was associated with alpha Syn-SAA positivity (OR 60.2, 95% CI 5.2-1,965.8; p < 0.01). Discussion We detected a frequency of alpha Syn-SAA positivity in CBS and PSP in line with pathologic studies, highlighting the usefulness of SAAs for in vivo detection of otherwise undetectable neuropathologic processes. Our results also suggest that AD status (specifically low A beta 42) and older age at onset may contribute to alpha Syn-SAA positivity. This opens new perspectives for the stratification of patients in clinical trials.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two age-related and fatal neurodegenerative disorders that lie on a shared disease spectrum. While both disorders involve complex interactions between neuronal and glial cells, the specific cell-type alterations and their contributions to disease pathophysiology remain incompletely understood. Here, we applied single-nucleus RNA sequencing of the orbitofrontal cortex, a region affected in ALS-FTLD, to map cell-type specific transcriptional signatures in C9orf72-related ALS (with and without FTLD) and sporadic ALS cases. Our findings reveal disease- and cell-type-specific transcriptional changes, with neurons exhibiting the most pronounced alterations, primarily affecting mitochondrial function, protein homeostasis, and chromatin remodeling. A comparison with independent datasets from different cortical regions of C9orf72 and sporadic ALS cases showed concordance in several pathways, with neuronal STMN2 and NEFL showing consistent up-regulation between brain regions and disease subtypes. We also interrogated alternative polyadenylation (APA) as an additional layer of transcriptional regulation, demonstrating that APA events are not correlated with identified gene expression changes. To interpret these events, we developed APA-Net, a deep learning model that integrates transcript sequences with RNA-binding protein expression profiles, revealing cell type-specific patterns of APA regulation. Our atlas illuminates cell type-specific pathomechanisms of ALS/FTLD, providing a valuable resource for further investigation.

The long-term consequences of concussions may include pathological neurodegeneration as seen in Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). Tau-PET showed promise as a method to detect tau pathology of CTE, but more studies are needed This study aimed (1) to assess the association of imaging evidence of tau pathology with brain volumes in retired athletes and (2) to examine the relationship between tau-PET and neuropsychological functioning. Former contact sport athletes were recruited through the Canadian Football League Alumni Association or the Canadian Concussion Centre clinic. Athletes completed MRI, [18F]flortaucipir tau-PET, and a neuropsychological battery. Memory composite was created by averaging the Rey Auditory Verbal Learning Test and Rey Visual Design Learning Test z-scores. Grey matter (GM) volumes were age/intracranial volume corrected using normal control MRIs. Tau-PET

Multimodal biomarkers may identify former contact sports athletes with repeated concussions and at risk for dementia. Our study aims to investigate whether biomarker evidence of neurodegeneration in former professional athletes with repetitive concussions (ExPro) is associated with worse cognition and mood/behavior, brain atrophy, and altered functional connectivity. Forty-one contact sports athletes with repeated concussions were divided into neurodegenerative biomarker-positive (n = 16) and biomarker-negative (n = 25) groups based on positivity of serum neurofilament light-chain. Six healthy controls (negative for biomarkers) with no history of concussions were also analyzed. We calculated cognitive and mood/behavior composite scores from neuropsychological assessments. Gray matter volume maps and functional connectivity of the default mode, salience, and frontoparietal networks were compared between groups using ANCOVAs, controlling for age, and total intracranial volume. The association between the connectivity networks and sports characteristics was analyzed by multiple regression analysis in all ExPro. Participants presented normal-range mean performance in executive function, memory, and mood/behavior tests. The ExPro groups did not differ in professional years played, age at first participation in contact sports, and number of concussions. There were no differences in gray matter volume between groups. The neurodegenerative biomarker-positive group had lower connectivity in the default mode network (DMN) compared to the healthy controls and the neurodegenerative biomarker-negative group. DMN disconnection was associated with increased number of concussions in all ExPro. Biomarkers of neurodegeneration may be useful to detect athletes that are still cognitively normal, but with functional connectivity alterations after concussions and at risk of dementia.

BACKGROUND:Postoperative outcome following deep brain stimulation (DBS) of the subthalamic nucleus is variable, particularly with respect to axial motor improvement. We hypothesized a genetic underpinning to the response to surgical intervention, termed "surgicogenomics". OBJECTIVE:We aimed to identify genetic variants associated with clinical heterogeneity in DBS outcome of Parkinson's disease (PD) patients that could then be applied clinically to target selection leading to improved surgical outcome. METHODS:Retrospective clinical data was extracted from 150 patient's charts. Each individual was genotyped using the genome-wide NeuroX array tailored to study neurologic diseases. Genetic data were clustered based on surgical outcome assessed by comparing pre- and post-operative scores of levodopa equivalent daily dose and axial impairment at one and five years post-surgery. Allele frequencies were compared between patients with excellent vs. moderate/poor outcomes grouped using a priori defined cut-offs. We analyzed common variants, burden of rare coding variants, and PD polygenic risk score. RESULTS:NeuroX identified 2,917 polymorphic markers at 113 genes mapped to known PD loci. The gene-burden analyses of 202 rare nonsynonymous variants suggested a nominal association of axial impairment with 14 genes (most consistent with CRHR1, IP6K2, and PRSS3). The strongest association with surgical outcome was detected between a reduction in levodopa equivalent daily dose and common variations tagging two linkage disequilibrium blocks with SH3GL2. CONCLUSION:Once validated in independent populations, our findings may be implemented to improve patient selection for DBS in PD.

Age at onset of amyotrophic lateral sclerosis (ALS) is highly variable (eg, 27–74 years in carriers of the G 4 C 2 -expansion in C9orf72 ). It might be influenced by environmental and genetic factors via the modulation of DNA methylation (DNAm) at CpG-sites. Hence, we combined an epigenetic and genetic approach to test the hypothesis that some common single nucleotide polymorphisms (SNPs) at CpG-sites (CpG-SNPs) could modify ALS age of onset. Our genome-wide DNAm analysis suggested three CpG-SNPs whose DNAm levels are significantly associated with age of onset in 249 ALS patients (q < 0.05). Next, genetic analysis validated the association of rs4970944 with age of onset in the discovery (n = 469; P = 0.025) and replication (n = 4160; P = 0.007) ALS cohorts. A meta-analysis of the cohorts combined showed that the median onset in AA-carriers is two years later than in GG-carriers (n = 4629; P = 0.0012). A similar association was observed with its tagging SNPs, implicating a 16 Kb region at the 1q21.3 locus as a modifier of ALS age of onset. Notably, rs4970944 genotypes are also associated with age of onset in C9orf72 -carriers (n = 333; P = 0.025), suggesting that each A-allele delays onset by 1.6 years. Analysis of Genotype-Tissue Expression data revealed that the protective A-allele is linked with the reduced expression of CTSS in cerebellum (P = 0.00018), which is a critical brain region in the distributed neural circuits subserving motor control. CTSS encodes cathepsin S protein playing a key role in antigen presentation. In conclusion, we identified a 16 Kb locus tagged by rs4970944 as a modifier of ALS age of onset. Our findings support the role of antigen presenting processes in modulating age of onset of ALS and suggest potential drug targets (eg, CTSS ). Future replication studies are encouraged to validate the link between the locus tagged by rs4970944 and age of onset in independent ALS cohorts, including different ethnic groups.

Amyotrophic lateral sclerosis (ALS) patients, includingC9orf72-carriers and identical twins [1], have highly variable disease characteristics (e.g., duration and age/site of onset) [7], suggesting the influence of epigenetic variations.DNA methylation (DNAm) is a key epigenetic modification and linked to the risk of several neurodegenerative diseases (Supplementary introduction).The cumulative assessment of DNAm levels at age-related CpGs allows the estimation of multi-tissue DNAm age (epigenetic clock), which could be more accurate for assessing biological age than chronological age.Hypermethylation of the CpG-island 5′ of the C9orf72-repeat and DNAm-age acceleration have been reported to be associated with C9orf72-disease duration and age of onset [9,10].However, epigenetic modifiers in genetically unexplained ALS patients are largely unknown.Hence, we conducted a genome-wide investigation of DNAm (Infinium MethylationEPIC chip) in samples from blood (n = 249) or tissues from the central nervous system (CNS) (n = 18) of mainly sporadic ALS patients (Table 1), and assessed the association of DNAm-age acceleration with disease age of onset and survival (Supplementary methods).Considering the reported 3-year error for estimating DNAm age, we classified the patients into three groups: normal aging (n = 82, DNAm-age acceleration between -3 and 3 years, median = 0.5 years), slow aging (n = 125, DNAm-age acceleration < -3 years, median = -6.3years), and fast aging (n = 42, DNAm-age acceleration > 3 years, median = 5.7 years).Multivariate linear regression analysis detected a highly significant association between blood-based DNAm-age acceleration and ALS age of onset (adjusted p value = 2.2E-16, B = -1.29,R 2 = 0.33, n = 249) (Supplementary methods, Fig. 1a), suggesting that for every 5-year increase in DNAm-age acceleration there is a 6.4-year earlier onset.Cox proportional hazard regression analysis revealed that a faster DNAm-age acceleration is significantly associated with an earlier age of onset [adjusted p value = 9.3E-16, hazard ratio (HR) = 2.12; 95% confidence interval (CI):1.76-2.54],suggesting that the hazard increased by 112% (Fig. 1c).The median age of onset in the fast aging group (48 years; 95%CI: 42-51) was 18 years younger than the slow aging group (66 years; 95%CI: 63-68).Significant results were not

BACKGROUND:Genetic polymorphisms like apolipoprotein E (APOE) and microtubule-associated protein tau (MAPT) genes increase the risk of neurodegeneration. METHODS:38 former players (age 52.63±14.02) of contact sports underwent neuroimaging, biofluid collection, and comprehensive neuropsychological assessment. The [F-18]AV-1451 tracer signal was compared in the cortical grey matter between APOE4 allele carriers and non-carriers as well as carriers of MAPT H1H1 vs non-H1H1. Participants were then divided into the high (N = 13) and low (N = 13) groups based on cortical PET tau standard uptake value ratios (SUVRs) for comparison. FINDINGS:Cortical grey matter PET tau SUVR values were significantly higher in APOE4 carriers compared to non-carriers (p = 0.020). In contrast, there was no significant difference in SUVR between MAPT H1H1 vs non-H1H1 carrier genes (p = 1.00). There was a significantly higher APOE4 allele frequency in the high cortical grey matter PET tau group, comparing to low cortical grey matter PET tau group (p = 0.048). No significant difference in neuropsychological function was found between APOE4 allele carriers and non-carriers. INTERPRETATION:There is an association between higher cortical grey matter tau burden as seen with [F-18]AV-1451 PET tracer SUVR, and the APOE4 allele in former professional and semi-professional players at high risk of concussions. APOE4 allele may be a risk factor for tau accumulation in former contact sports athletes at high risk of neurodegeneration. FUNDING:Toronto General and Western Hospital Foundations; Weston Brain Institute; Canadian Consortium on Neurodegeneration in ageing; Krembil Research Institute. There was no role of the funders in this study.

The repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. C9orf72 patients present with a wide range in disease duration and age of onset. The strongest risk factor for both syndromes is aging, which was linked to DNA methylation (DNAm) age based on the cumulative assessment of the methylation levels of 353 CpGs included on the genome-wide 450k BeadChip. DNAm age may reflect biological age better than chronological age. We conducted a genome-wide blood DNA methylation study of 46 unrelated C9orf72 patients. After correction for multiple testing, none of the CpGs demonstrated association between its methylation level and disease duration or age of onset. However, we detected a significant reverse correlation of DNAm age-acceleration with disease duration and age of onset, suggesting that for every 5-year increase in DNAm age-acceleration there is a 3.2-year earlier age of onset and 1.5-year shorter disease duration. The significant correlations remain after adjusting for gender, TMEM106B genotypes, disease phenotype and C9orf72 5'CpG island methylation status. A similar trend was observed for the blood DNA of affected members of an extended C9orf72 family; and tissues from the central nervous system of C9orf72 autopsy cases. For instance, regression analysis suggested that a 5-year increase in DNAm age-acceleration is linked to an earlier age of onset by 4.7 or 5.5 years for frontal cortex or spinal cord, respectively. Blood DNAm age may be a useful biomarker for biological age, because blood DNAm age-acceleration was similar to all investigated brain tissues, except for cerebellum that ages more slowly. In conclusion, DNA methylation analysis of C9orf72 patients revealed that increased DNAm age-acceleration is associated with a more severe disease phenotype with a shorter disease duration and earlier age of onset.

OBJECTIVE:Suggested C9orf72 disease mechanisms for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration include C9orf72 haploinsufficiency, G4C2/C4G2 RNA foci, and dipeptide repeat (DPR) proteins translated from the G4C2 expansion; however, the role of small expansions (e.g., 30-90 repeats) is unknown and was investigated here. METHODS:We conducted a molecular and pathology study of a family in which the father (unaffected at age 90) carried a 70-repeat allele in blood DNA that expanded to ≈1,750 repeats in his children, causing ALS. RESULTS:Southern blotting revealed different degrees of mosaicism of small and large expansions in the father's tissues from the CNS. Surprisingly, in each mosaic tissue, C9orf72 mRNA levels were significantly increased compared to an ALS-affected daughter with a large expansion. Increased expression correlated with higher levels of the 70-repeat allele (the upregulation was also evident at the protein level). Remarkably, RNA foci and DPR burdens were similar or even significantly increased (in cerebellum) in the unaffected father compared to the daughter with ALS. However, the father did not display TDP-43 pathology and signs of neurodegeneration. CONCLUSION:The presence of RNA foci and DPR pathology was insufficient for disease manifestation and TDP-43 pathology in the mosaic C9orf72 carrier with upregulated C9orf72 expression. It is important to conduct an investigation of similar cases, which could be found among unaffected parents of sporadic C9orf72 patients (e.g., 21% among Finnish patients with ALS). Caution should be taken when consulting carriers of small expansions because disease manifestation could be dependent on the extent of the somatic instability in disease-relevant tissues.

Amyotrophic lateral sclerosis (ALS) is characterised by a loss of motor neurons, leading to paralysis. Several autosomal dominant genes were implicated in ALS pathogenesis, such as SOD1 , with over 180 reported mutations (http://alsod.iop.kcl.ac.uk/), including a p.T137A substitution.1 ,2 Recently, ARHGEF28 , encoding an RNA binding protein involved in the aggregation of light neurofilaments in ALS, was suggested as a novel ALS gene (a heterozygous K280M>fs40X mutation was detected in three patients).3 ,4 Risk of ALS may be modulated by environmental factors, sex and ageing, which could be linked to epigenetic events (eg, DNA methylation). Monozygotic (MZ) twins provide the best opportunity to investigate environmental/epigenetic factors in disease development.5 Hence, we studied ALS-discordant MZ twins, including the evaluation of DNA methylation (DNAm) age, which is an accurate predictor of chronological age across different tissues.6 It is possible that DNAm age better reflects biological age than chronological age does, since age acceleration (DNAm age minus chronological age) was associated with several disorders and mortality. Participants were recruited from the ALS Clinic at the Sunnybrook Health Sciences Centre. Genetic and epigenetic analyses were conducted as described in the online supplementary methods, including mutation analysis of SOD1 and C9orf72 , NeuroX array, reverse transcription PCR (RT-PCR), whole genome DNA methylation array (HumanMethylation450 BeadChip) and bisulfite pyrosequencing. ### Supplementary data [jnnp-2016-313592supp.pdf] The MZ twins from a Canadian PED24 family of Italian origin were 52 years old at last examination and ALS-discordant for 17 years, with the second-born twin (9377) diagnosed with ALS at 35 years (figure 1 …

Loss-of-function GRN mutations lead to GRN haploinsufficiency and consequently neurodegeneration with significant heterogeneity in clinical presentation of various syndromes. The aim of this study was to investigate the genetics and clinical features of patients with GRN-related frontotemporal lobar degeneration (FTLD) syndromes. We performed mutation analysis of GRN in 45 unrelated Canadian patients with a broad spectrum of FTLD-like syndromes (mean age at onset of 64.0 ± 11.2 years). In our cohort, two patients were carriers of two novel heterozygous alterations in GRN: 2 bp insertion (c.769–770insCC:p.Q257fs) and 12 bp deletion (c.1009–1020del:p.337–340del). Both patients presented with corticobasal syndrome supported by clinical and radiological findings. The absence of the mutant allele in the RT–PCR product was only observed for the sample with 2 bp insertion in GRN. In contrast, the allele with 12 bp deletion in GRN was not down-regulated at the RNA level and did not segregate with FTLD in the family. Our report extends the evidence for genetic and phenotypic variability in FTLD disorders and detects a novel pathogenic GRN mutation, carriers of which could eventually help to evaluate the efficacy of different treatments at early stages of dementia.

ABSTRACTBackgroundIntermediate interrupted ataxin 2 (ATXN2) alleles (27–33 CAG‐repeats) increase the risk for amyotrophic lateral sclerosis and are reported as modifiers in chromosome 9 open reading frame 72 (C9orf72) carriers, rendering susceptibility to amyotrophic lateral sclerosis rather than frontotemporal lobar degeneration. The clinical presentation of C9orf72 patients with pathogenic ATXN2 alleles (≥35 CAG‐repeats) is unknown.MethodsBlood samples were collected from a family affected by ataxia, dementia, and parkinsonism, but not amyotrophic lateral sclerosis. Mutation analyses of the proband included C9orf72 and 14 ataxia genes, followed by segregation analyses in family members.ResultsBoth affected siblings carry an uninterrupted 37‐repeat expansion in ATXN2 and a methylated G4C2‐repeat allele in C9orf72 that is typical of large pathogenic expansions.ConclusionsThe CAG‐expansion in ATXN2 likely caused the ataxia, whereas the dementia may be linked to both C9orf72 and ATXN2 repeat expansions. The pathological uninterrupted ATXN2 repeat may not have the same modifying effect as intermediate interrupted alleles. © 2016 International Parkinson and Movement Disorder Society.

Recently, several CHCHD2 mutations were reported to be associated with autosomal dominant Parkinson's disease (PD) in a Japanese population. However, an association between CHCHD2 and PD was not observed in 2 Caucasian data sets. The present study searched for CHCHD2 coding variants in Canadian PD patients. Sanger sequencing of all CHCHD2 exons revealed no coding mutations in 155 familial cases. Moreover, 3 coding CHCHD2 polymorphisms available on the NeuroX array (Pro2Leu, Pro14Ser, and Ile118Met) were homozygous for the major allele in an additional 85 PD patients. Our study suggests that CHCHD2 mutations may not account for PD in Canadian patients.

Corticobasal syndrome (CBS) is a neurodegenerative disease characterized by progressive asymmetrical rigidity and apraxia, cortical sensory loss, myoclonus, dystonia, and cognitive impairment. CBS is usually sporadic and associated with tau pathology but there are reports of TDP-43 pathology. We screened 39 CBS cases to determine if any of the cases could be explained by a G4C2-repeat expansion in a noncoding region of C9orf72 gene, the most common genetic cause of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. One patient with CBS had a large (>50 repeats) expansion in C9orf72. Our case features a 63-year-old right-handed woman who developed mild apathy 9 years before presentation, which progressed to include behavioral symptoms, oral stereotypies, significant language impairment, parkinsonism and apraxia. A magnetic resonance imaging acquired at age 60 years, that is, 6 years after disease onset revealed significant asymmetric left > right frontotemporal atrophy, including orbitofrontal and parietal areas. Her father developed a behavioral syndrome and died at an early age. This case highlights the importance of genetic screening for C9orf72 in patients with CBS.

Sir, A recent study by Bannwarth et al. (2014) implicated CHCHD10 as a novel gene for amyotrophic lateral sclerosis/frontotemporal lobar degeneration (ALS/FTLD), reporting a p.S59L substitution (c.176C > T; NM_213720.2) in a large French kindred. Affected family members were presented with a complex phenotype that included symptoms of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), cerebellar ataxia, Parkinson’s disease and a mitochondrial myopathy associated with multiple mitochondrial DNA deletions. So far, seven missense CHCHD10 mutations have been reported in patients with a broad phenotypic range, including ALS/FTLD (p.S59L and p.P34S) (Bannwarth et al. , 2014; Chaussenot et al. , 2014), ALS (p.R15L and p.G66V) (Johnson et al. , 2014; Muller et al. , 2014), myopathy (p.R15S and p.G58R) (Ajroud-Driss et al. , 2015) and late-onset spinal motor neuronopathy (p.G66V) (Penttila et al. , 2015). All of them affect exon 2 (a mutational hotspot of CHCHD10 ). Notably, mitochondrial dysfunction has been implicated in several neurodegenerative diseases (Lin and Beal, 2006; Cozzolino et al. , 2013); however, there are no studies evaluating the contribution of CHCHD10 to pure FTLD, Parkinson’s disease or Alzheimer’s disease. Hence, we sequenced CHCHD10 exon 2 in 204 ALS, 153 Parkinson’s disease and 141 Alzheimer’s disease patients from Canada and 158 FTLD patients from Italy in addition to 497 control subjects from USA/UK, Canada and Italy. The cases of ALS and FTLD were free from mutations in SOD1 , GRN , FUS , TARDBP and MATR3 or a repeat expansion in C9orf72 . We identified a known CHCHD10 pathogenic p.R15L mutation (Johnson et al. , 2014; Muller et al. , 2014) in a patient with sporadic ALS (Patient 8807) (Fig. 1A), who developed symptoms involving his upper limb at 54 years of age and remains alive 12 years later. The p.R15L …

The most common cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a G 4 C 2 -repeat expansion in C9orf72. However, the lower limit for pathological repeats has not been established and expansions with different sizes could have different pathological consequences. One of the implicated disease mechanisms is haploinsufficiency. Previously, we identified expansion-specific hypermethylation at the 5′ CpG-island near the G 4 C 2 -repeat, but only in a fraction of carriers (up to 36 %). Here, we tested the hypothesis that the G 4 C 2 -repeat itself could be the main site of methylation. To evaluate (G 4 C 2 ) n -methylation, we developed a novel assay, which was validated by an independent methylation-sensitive restriction enzyme assay. Notably, both assays are qualitative but not quantitative. Blood DNA was available for 270 unrelated individuals, including 71 expansion carriers. In addition, we investigated blood DNA from family members of 16 probands, and 38 DNA samples from multiple tissues of 10 expansion carriers. Finally, we tested DNA from different tissues of an ALS patient carrying a somatically unstable 90-repeat. We demonstrated that the G 4 C 2 -expansion is generally methylated in unrelated carriers of alleles >50 repeats (97 %), while small (<22 repeats) or intermediate (22–90 repeats) alleles were completely unmethylated. The presence of (G 4 C 2 ) n -methylation does not separate the C9orf72 -phenotypes (ALS vs. ALS/FTLD vs. FTLD), but has the potential to predict large vs. intermediate repeat length. Our results suggest that (G 4 C 2 ) n -methylation might sometimes spread to the 5′-upstream region, but not vice versa. It is stable over time, since (G 4 C 2 ) n -methylation was detected in carriers with a wide range of ages (24–74 years). It was identified in both blood and brain tissues for the same individual, implying its potential use as a biomarker. Furthermore, our findings may open up new perspectives for studying disease mechanisms, such as determining whether methylated and unmethylated repeats have the same ability to form a G-quadruplex configuration.