Jump from pre-mutation to pathologic expansion in C9orf72.

An expanded G4C2 repeat in C9orf72 represents the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). However, the lower limit for pathological expansions is unknown (the suggested cutoff is 30 repeats). It has been proposed that the expansion might have occurred only once in human history and subsequently spread throughout the population. However, our present findings support a hypothesis of multiple origins for the expansion. We report a British-Canadian family in whom a ∼70-repeat allele from the father (unaffected by ALS or FTLD at age 89 years) expanded during parent-offspring transmission and started the first generation affected by ALS (four children carry an ∼1,750-repeat allele). Epigenetic and RNA-expression analyses further discriminated the offspring's large expansions (which were methylated and associated with reduced C9orf72 expression) from the ∼70-repeat allele (which was unmethylated and associated with upregulation of C9orf72). Moreover, RNA foci were only detected in fibroblasts from offspring with large expansions, but not in the father, who has the ∼70-repeat allele. All family members with expansions were found to have an ancient known risk haplotype, although it was inherited on a unique 5-Mb genetic backbone. We conclude that small expansions (e.g., 70 repeats) might be considered "pre-mutations" to reflect their propensity to expand in the next generation. Follow-up studies might help explain the high frequency of ALS- or FTLD-affected individuals with an expansion but without a familial history (e.g., 21% among Finnish ALS subjects).

DNA methylation age-acceleration is associated with disease duration and age at onset in C9orf72 patients.

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.

C9orf72 and ATXN2 repeat expansions coexist in a family with ataxia, dementia, and parkinsonism.

BACKGROUND: Intermediate 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. METHODS: Blood 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. RESULTS: Both affected siblings carry an uninterrupted 37-repeat expansion in ATXN2 and a methylated G4 C2 -repeat allele in C9orf72 that is typical of large pathogenic expansions. CONCLUSIONS: The 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.

Hypermethylation of the CpG-island near the C9orf72 G4C2-repeat expansion in FTLD patients.

The G4C2-repeat expansion in C9orf72 is a common cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). C9orf72 transcription is reduced in expansion carriers implicating haploinsufficiency as one of the disease mechanisms. Indeed, our recent ALS study revealed that the expansion was associated with hypermethylation of the CpG-island (5'of the repeat) in DNA samples obtained from different tissues (blood, brain and spinal cord). However, the link between FTLD and methylation of the CpG-island is unknown. Hence, we investigated the methylation profile of the same CpG-island by bisulfite sequencing of DNA obtained from blood of 34 FTLD expansion carriers, 166 FTLD non-carriers and 103 controls. Methylation level was significantly higher in FTLD expansion carriers than non-carriers (P = 7.8E-13). Our results were confirmed by two methods (HhaI-assay and sequencing of cloned bisulfite PCR products). Hypermethylation occurred only in carriers of an allele with >50 repeats, and was not detected in non-carriers or individuals with an intermediate allele (22-43 repeats). As expected, the position/number of methylated CpGs was concordant between the sense and anti-sense DNA strand, suggesting that it is a stable epigenetic modification. Analysis of the combined ALS and FTLD datasets (82 expansion carriers) revealed that the degree of methylation of the entire CpG-island or contribution of specific CpGs (n = 26) is similar in both syndromes, with a trend towards a higher proportion of ALS patients with a high methylation level (P = 0.09). In conclusion, we demonstrated that hypermethylation of the CpG-island 5'of the G4C2-repeat is expansion-specific, but not syndrome-specific (ALS versus FTLD).

Hypermethylation of the CpG island near the G4C2 repeat in ALS with a C9orf72 expansion.

The G4C2 repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G4C2 repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5' of the G4C2 repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD).

The C9orf72 repeat expansion itself is methylated in ALS and FTLD patients.

The most common cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a G4C2-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 G4C2-repeat, but only in a fraction of carriers (up to 36 %). Here, we tested the hypothesis that the G4C2-repeat itself could be the main site of methylation. To evaluate (G4C2)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 G4C2-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 (G4C2)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 (G4C2)n -methylation might sometimes spread to the 5'-upstream region, but not vice versa. It is stable over time, since (G4C2)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.

Functional connectivity changes in neurodegenerative biomarker-positive athletes with repeated concussions.

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.

Single-nucleus multiomic atlas of frontal cortex in amyotrophic lateral sclerosis with a deep learning-based decoding of alternative polyadenylation mechanisms.

The understanding of how different cell types contribute to amyotrophic lateral sclerosis (ALS) pathogenesis is limited. Here we generated a single-nucleus transcriptomic and epigenomic atlas of the frontal cortex of ALS cases with C9orf72 (C9) hexanucleotide repeat expansions and sporadic ALS (sALS). Our findings reveal shared pathways in C9-ALS and sALS, characterized by synaptic dysfunction in excitatory neurons and a disease-associated state in microglia. The disease subtypes diverge with loss of astrocyte homeostasis in C9-ALS, and a more substantial disturbance of inhibitory neurons in sALS. Leveraging high depth 3'-end sequencing, we found a widespread switch towards distal polyadenylation (PA) site usage across ALS subtypes relative to controls. To explore this differential alternative PA (APA), we developed APA-Net, a deep neural network model that uses transcript sequence and expression levels of RNA-binding proteins (RBPs) to predict cell-type specific APA usage and RBP interactions likely to regulate APA across disease subtypes.

Axial Impairment Following Deep Brain Stimulation in Parkinson's Disease: A Surgicogenomic Approach.

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.

Combined epigenetic/genetic study identified an ALS age of onset modifier.

Age at onset of amyotrophic lateral sclerosis (ALS) is highly variable (eg, 27-74 years in carriers of the G4C2-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.

Interaction of APOE4 alleles and PET tau imaging in former contact sport athletes.

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 G2019S LRRK2 mutation in Brazilian patients with Parkinson's disease: phenotype in monozygotic twins.

Mutations in the Leucine-Rich Repeat Kinase 2 gene (LRRK2) are mainly responsible for idiopathic Parkinson's disease (PD) with either a dominant pattern of transmission or a sporadic occurrence due to the reduced penetrance. A majority of LRRK2 kindreds demonstrate an extremely variable age-at-onset in affected members of the same family. The G2019S is the most common LRRK2 mutation, which accounts for 1-5% PD patients in North America, and up to 40% of patients from an isolated Arab population. We assessed the frequency of the G2019S mutation in 83 Brazilian PD patients originally preselected for having an early age-at-onset (<50 years) and/or a positive family history. The mutation was detected in three probands (3.5%). Our clinical findings in these kindreds include the first description of the phenotype in identical twins discordant for handedness (a general phenomenon found in approximately 25% monozygotic twins). However, both twins developed right asymmetric PD. The clinical presentation of twins was strikingly similar including an identical PD onset at age 60. This observation may suggest that genetic factors predominantly determine age-at-onset.

Unaffected mosaic C9orf72 case: RNA foci, dipeptide proteins, but upregulated C9orf72 expression.

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.

Novel GRN Mutations in Patients with Corticobasal Syndrome.

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.

Mutation analysis of CHCHD2 in Canadian patients with familial Parkinson's disease.

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.

Mutation analysis of C9orf72 in patients with corticobasal syndrome.

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.

Mutation analysis of patients with neurodegenerative disorders using NeuroX array.

Genetic analyses of patients with neurodegenerative disorders have identified multiple genes that need to be investigated for the presence of damaging variants. However, mutation analysis by Sanger sequencing is costly and time consuming. We tested the utility of a recently designed semi-custom genome-wide array (NeuroX; Illumina, Inc) tailored to study neurodegenerative diseases (e.g., mutation screening). We investigated 192 patients with 4 different neurodegenerative disorders for the presence of rare damaging variations in 77 genes implicated in these diseases. Several causative mutations were identified and confirmed by Sanger sequencing, including PSEN1 p.M233T responsible for Alzheimer's disease in a large Italian family, as well as SOD1 p.A4V and p.I113T in patients with amyotrophic lateral sclerosis. In total, we identified 78 potentially damaging rare variants (frequency <1%), including ABCA7 p.L400V in a family with Alzheimer's disease and LRRK2 p.R1514Q in 6 of 98 patients with Parkinson's disease (6.1%). In conclusion, NeuroX appears to be helpful for rapid and accurate mutation screening, although further development may be still required to improve some current caveats.