A Hexanucleotide Repeat Expansion in C9ORF72 Is the Cause of Chromosome 9p21-Linked ALS-FTD

There is increasing evidence that frontotemporal dementia and amyotrophic lateral sclerosis are part of a disease continuum. Recently, a hexanucleotide repeat expansion in C9orf72 was identified as a major cause of both sporadic and familial frontotemporal dementia and amyotrophic lateral sclerosis. The aim of this study was to investigate clinical and neuropathological characteristics of hexanucleotide repeat expansions in C9orf72 in a large cohort of Dutch patients with frontotemporal dementia. Repeat expansions were successfully determined in a cohort of 353 patients with sporadic or familial frontotemporal dementia with or without amyotrophic lateral sclerosis, and 522 neurologically normal controls. Immunohistochemistry was performed in a series of 10 brains from patients carrying expanded repeats using a panel of antibodies. In addition, the presence of RNA containing GGGGCC repeats in paraffin-embedded sections of post-mortem brain tissue was investigated using fluorescence in situ hybridization with a locked nucleic acid probe targeting the GGGGCC repeat. Hexanucleotide repeat expansions in C9orf72 were found in 37 patients with familial (28.7%) and five with sporadic frontotemporal dementia (2.2%). The mean age at onset was 56.9 ± 8.3 years (range 39-76), and disease duration 7.6 ± 4.6 years (range 1-22). The clinical phenotype of these patients varied between the behavioural variant of frontotemporal dementia (n = 34) and primary progressive aphasia (n = 8), with concomitant amyotrophic lateral sclerosis in seven patients. Predominant temporal atrophy on neuroimaging was present in 13 of 32 patients. Pathological examination of the 10 brains from patients carrying expanded repeats revealed frontotemporal lobar degeneration with neuronal transactive response DNA binding protein-positive inclusions of variable type, size and morphology in all brains. Fluorescence in situ hybridization analysis of brain material from patients with the repeat expansion, a microtubule-associated protein tau or a progranulin mutation, and controls did not show RNA-positive inclusions specific for brains with the GGGGCC repeat expansion. The hexanucleotide repeat expansion in C9orf72 is an important cause of frontotemporal dementia with and without amyotrophic lateral sclerosis, and is sometimes associated with primary progressive aphasia. Neuropathological hallmarks include neuronal and glial inclusions, and dystrophic neurites containing transactive response DNA binding protein. Future studies are needed to explain the wide variation in clinical presentation.

A large hexanucleotide (GGGGCC) repeat expansion in the first intron of C9ORF72, a gene located on chromosome 9p21, has been recently reported to be responsible for ~40% of familial amyotrophic lateral sclerosis cases of European ancestry. The aim of the current article was to describe the phenotype of amyotrophic lateral sclerosis cases carrying the expansion by providing a detailed clinical description of affected cases from representative multi-generational kindreds, and by analysing the age of onset, gender ratio and survival in a large cohort of patients with familial amyotrophic lateral sclerosis. We collected DNA and analysed phenotype data for 141 index Italian familial amyotrophic lateral sclerosis cases (21 of Sardinian ancestry) and 41 German index familial amyotrophic lateral sclerosis cases. Pathogenic repeat expansions were detected in 45 (37.5%) patients from mainland Italy, 12 (57.1%) patients of Sardinian ancestry and nine (22.0%) of the 41 German index familial amyotrophic lateral sclerosis cases. The disease was maternally transmitted in 27 (49.1%) pedigrees and paternally transmitted in 28 (50.9%) pedigrees (P = non-significant). On average, children developed disease 7.0 years earlier than their parents [children: 55.8 years (standard deviation 7.9), parents: 62.8 (standard deviation 10.9); P = 0.003]. Parental phenotype influenced the type of clinical symptoms manifested by the child: of the 13 cases where the affected parent had an amyotrophic lateral sclerosis-frontotemporal dementia or frontotemporal dementia, the affected child also developed amyotrophic lateral sclerosis-frontotemporal dementia in nine cases. When compared with patients carrying mutations of other amyotrophic lateral sclerosis-related genes, those with C9ORF72 expansion had commonly a bulbar onset (42.2% compared with 25.0% among non-C9ORF72 expansion cases, P = 0.03) and cognitive impairment (46.7% compared with 9.1% among non-C9ORF72 expansion cases, P = 0.0001). Median survival from symptom onset among cases carrying C9ORF72 repeat expansion was 3.2 years lower than that of patients carrying TARDBP mutations (5.0 years; 95% confidence interval: 3.6-7.2) and longer than those with FUS mutations (1.9 years; 95% confidence interval: 1.7-2.1). We conclude that C9ORF72 hexanucleotide repeat expansions were the most frequent mutation in our large cohort of patients with familial amyotrophic lateral sclerosis of Italian, Sardinian and German ancestry. Together with mutation of SOD1, TARDBP and FUS, mutations of C9ORF72 account for ~60% of familial amyotrophic lateral sclerosis in Italy. Patients with C9ORF72 hexanucleotide repeat expansions present some phenotypic differences compared with patients with mutations of other genes or with unknown mutations, namely a high incidence of bulbar-onset disease and comorbidity with frontotemporal dementia. Their pedigrees typically display a high frequency of cases with pure frontotemporal dementia, widening the concept of familial amyotrophic lateral sclerosis.

Dear Editors, Frontotemporal dementia (FTD) is a common form of dementia, characterized clinically by behavioral disturbance, progressive cognitive impairment, and language impairment owing to the degeneration of the frontal and temporal lobes. The discovery that abnormal GGGGCC hexanucleotide repeat expansions (HRE) in C9orf72 was a common cause of FTD and amyotrophic lateral sclerosis (ALS) fueled and speeded up the research in C9orf72-related ALS and FTD worldwide.1, 2 This pathogenic expansion could cause disease by loss of normal function of C9orf72 protein, RNA toxicity, and dipeptide-repeat proteins aggregates.3, 4 Although the mechanisms for different phenotypes of C9orf72 HRE have not been well unveiled, the proteomic cerebrospinal fluid analyses and imaging studies provided potential biomarkers for C9orf72-linked FTD and ALS.5, 6 Previous studies showed the most common symptoms of C9orf72-linked FTD were behavioral disinhibition and psychiatric disorders.7-9 Notably, 60% of C9orf72-related FTD patients developed motor neuron disease symptoms during follow-up.9 C9orf72 HRE was the leading genetic cause of ALS and FTD in Caucasian populations, accounting for 21.7%-47.0% in familial ALS, 13.8%-48.1% in familial FTD, 4.1%-21.1% in sporadic ALS, and 2.2%-18.8% in sporadic FTD.10, 11 However, the C9orf72 HRE was rare in the Asian population, which could only explain around 0.4% ALS in Japan, 3.5% family ALS, and 0.5% sporadic ALS in Chinese populations.12, 13 The prevalence of C9orf72-related FTD was not clear in China, which calls for a large-scale multicenter study. To date, only two Chinese cases of C9orf72-related FTD patients were reported.14, 15 We herein reported a 41-year-old female FTD Chinese patient, who had a familial history of ALS and carried pathogenic C9orf72 HRE (>93, normal <30). In March 2017, a 41-year-old female was admitted to the Department of Neurology with the complaint of progressive speech disturbance and memory impairment for one year. During this time, she presented hesitant speech and word-finding difficulty, accompanied by poor articulation. Also, she had difficulties in recalling. The neuropsychological assessment demonstrated cognitive dysfunction, shown as 14/30 points in Mini-Mental State Examination, 1/3 point in Clinical Dementia Rating Scale, and 38/80 points in Activity of Daily Living Scale. The neuropsychiatric inventory test revealed intact psychological status. Neurological examinations were unremarkable. Notably, her father and two uncles were diagnosed with ALS in their 50s and passed away several years later (Figure 1A). Laboratory tests and electroencephalogram (EEG) showed no obvious abnormality. Cerebrospinal fluid examinations including basic constituents, Aβ1–42, total tau, and phosphorylated tau were within the normal range. Brain Magnetic resonance imaging (MRI) revealed significant frontal and temporal lobe atrophy (Figure 1B). Electromyography (EMG) test revealed distal peripheral neuropathy. In consideration of her obvious family history, we performed the whole exon sequencing and repeat-primed PCR to screen GGGGCC repeat expansion in the C9orf72. The genetic investigation revealed more than 93 times repeat expansions (normal <30) in C9orf72 (Figure 1C). Two years later, the patient’s cognition was further declined. Moreover, the patient exhibited limbs weakness, progressive muscular dystrophy, and dysphagia. Neurological examinations revealed hypermyotonia, fasciculations, hyperreflexia in the affected limb, muscle weakness, and severe muscle atrophy (Figure 1D). These symptoms could be explained by C9orf72-related ALS, but the patient denied EMG examination. We also screened the C9orf72 HRE in other 36 FTD patients and reviewed previous studies implicated in C9orf72 HRE in Chinese FTD patients (Table S1). Though C9orf72 pathogenic expansion is quite rare in Chinese FTD patients, it is still necessary to screen C9orf72 in Chinese FTD patients, especially those with FTD or ALS family history. Also, follow-up efforts need to be strengthened when FTD patients carry common gene mutations of FTD and ALS. FTD patients with severe dementia might have difficulties in communicating with caregivers and are more likely to miss timely and proper treatment when attacked by ALS. There are currently no FDA-approved drugs for FTD, but several off-label medications can be used to manage the symptoms of FTD. Selective serotonin uptake inhibitors and Trazodone proved to attenuate the neuropsychiatric symptoms.16 However, these atypical antipsychotics should be used slowly and cautiously in case of potential extrapyramidal and cognitive side effects. The pharmacologic treatment including acetylcholinesterase inhibitors and NMDA antagonists for Alzheimer’s disease are unlikely to benefit FTD patients. EGb 761® has been recommended in multiple guidelines for the treatment of MCI and dementia, which could improve not only the cognitive performance but also the neuropsychiatric symptoms of the patients.17, 18 EGb 761® might be a potential drug for early treatment of FTD, which needs further studies. Speech therapy may have some efficacy on language dysfunction. Moreover, antisense oligonucleotide target is considered a potential target for patients with C9orf72 HRE. Collectively, this is the first description of C9orf72-related FTD patients manifesting both dementia and ALS-like symptoms in mainland China, which broaden the genetic and clinical features of FTD. The authors declare that there is no conflict of interest. The patient provided written consent for participation. The patient provided written consent for disclosure of medical information and images. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic ALS in Taiwan

To assess the frequency and phenotype of hexanucleotide repeat expansions in C9ORF72 in a large cohort of patients of Dutch descent with familial (fALS) and sporadic (sALS) amyotrophic lateral sclerosis (ALS), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS). Included were 78 patients with fALS, 1,422 with sALS, 246 with PMA, and 110 with PLS, and 768 control subjects. Repeat expansions were determined by a repeat primed PCR. Familial aggregation of dementia and Parkinson disease (PD) was examined among patients with ALS who carried the repeat expansion. The expanded repeat was found in 33 (37%) of all patients with fALS, in 87 (6.1%) patients with sALS, in 4 (1.6%) patients with PMA, and in 1 (0.9%) patient with PLS. None of the controls carried the mutation. Patients with ALS with the repeat expansion had an earlier age at onset (median 59.3 vs 61.9 years, hazard ratio 1.55, p = 5 × 10(-5)) and shorter survival (median 2.5 vs 2.7 years, hazard ratio 1.46, p = 8 × 10(-4)). Dementia, but not PD, occurred nearly twice as often in relatives of patients with the expansion compared to all patients with ALS or controls (p = 9 × 10(-4)). The hexanucleotide repeat expansion in C9ORF72 is a major cause of fALS and apparently sporadic ALS in the Netherlands. Patients who carry the repeat expansion have an earlier onset, shorter survival, and familial aggregation of dementia. These results challenge the classic definition of fALS and may justify genetic testing in patients with sALS.

Genetic factors are involved in the pathogenesis of familial and sporadic amyotrophic lateral sclerosis (ALS) and constitute a link to its association with frontotemporal dementia (FTD). Gene-targeted therapies for some forms of ALS (C9orf72, SOD1) have recently gained momentum. Genetic architecture in Czech ALS patients has not been comprehensively assessed so far. We aimed to deliver pilot data on the genetic landscape of ALS in our country. A cohort of patients with ALS (n = 88), recruited from two Czech Neuromuscular Centers, was assessed for hexanucleotide repeat expansion (HRE) in C9orf72 and also for genetic variations in other 36 ALS-linked genes via next-generation sequencing (NGS). Nine patients (10.1%) had a familial ALS. Further, we analyzed two subgroups of sporadic patients - with concomitant FTD (n = 7) and with young-onset of the disease (n = 22). We detected the pathogenic HRE in C9orf72 in 12 patients (13.5%) and three other pathogenic variants in FUS, TARDBP and TBK1, each in one patient. Additional 7 novel and 9 rare known variants with uncertain causal significance have been detected in 15 patients. Three sporadic patients with FTD (42.9%) were harbouring a pathogenic variant (all HRE in C9orf72). Surprisingly, none of the young-onset sporadic patients harboured a pathogenic variant and we detected no pathogenic SOD1 variant in our cohort. Our findings resemble those from other European populations, with the highest prevalence of HRE in the C9orf72 gene. Further, our findings suggest a possibility of a missing genetic variability among young-onset patients.

Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins

Amyotrophic lateral sclerosis caused by hexanucleotide repeat expansions in C9orf72: from genetics to therapeutics.

Frontotemporal dementia (FTD) is a highly heritable condition. Up to 40% of FTD is familial and an estimated 15% to 40% is due to single-gene mutations. It has been estimated that the G4C2 hexanucleotide repeat expansions in the C9ORF72 gene can explain up to 37.5% of the familial cases of FTD, especially in populations of Caucasian origin. The purpose of this paper is to evaluate hereditary risk across the clinical phenotypes of FTD and the frequency of the G4C2 expansion in a Colombian cohort diagnosed with FTD.Methods: A total of 132 FTD patients were diagnosed according to established criteria in the behavioral variant FTD, logopenic variant PPA, non-fluent agrammatic PPA, and semantic variant PPA. Hereditary risk across the clinical phenotypes was established in four categories that indicate the pathogenic relationship of the mutation: high, medium, low, and apparently sporadic, based on those proposed by Wood and collaborators. All subjects were also examined for C9ORF72 hexanucleotide expansion (defined as >30 repetitions).Results: There were no significant differences in the demographic characteristics of the patients between the clinical phenotypes of FTD. The higher rate phenotype was bvFTD (62.12%). In accordance with the risk classification, we found that 72 (54.4%) complied with the criteria for the sporadic cases; for the familial cases, 23 (17.4%) fulfilled the high-risk criteria, 23 (17.4%) fulfilled the low risk criteria, and 14 (10.6%) fulfilled the criteria to be classified as subject to medium risk. C9ORF72 expansion frequency was 0.76% (1/132).Conclusion: The FTD heritability presented in this research was very similar to the results reported in the literature. The C9ORF72 expansion frequency was low. Colombia is a triethnic country, with a high frequency of genetic Amerindian markers; this shows consistency with the present results of a low repetition frequency. This study provides an initial report of the frequency for the hexanucleotide repeat expansions in C9ORF72 in patients with FTD in a Colombian population and paves the way for further study of the possible genetic causes of FTD in Colombia.

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AimsFrontotemporal lobar degeneration (FTLD) and motor neurone disease are linked by the possession of a hexanucleotide repeat expansion in C9ORF72, and both show neuronal cytoplasmic inclusions within cerebellar and hippocampal neurones which are TDP‐43 negative but immunoreactive for p62 and dipeptide repeat proteins (DPR), these being generated by a non‐ATG RAN translation of the expanded region of the gene.MethodsTwenty‐two cases of FTLD from Newcastle were analysed for an expansion in C9ORF72 by repeat primed PCR and Southern blot. Detailed case note analysis was performed, and blinded retrospective clinical impressions were achieved by review of clinical histories. Sections from all major brain regions were immunostained for TDP‐43, p62 and DPR. The extent of TDP‐43 and DPR pathology in expansion bearers was compared with that in 13 other previously identified cases from the Manchester Brain Bank with established disease.ResultsThree Newcastle patients bearing an expansion in C9ORF72 were identified. These three patients died prematurely, two from bronchopneumonia within 10 months and 3 years of onset, and one from myocardial infarction 3 years after onset. In all three, DPR were plentiful throughout all cerebral cortical regions, hippocampus and cerebellum, but TDP‐43 pathological changes were sparse. The severity of DPR pathological changes in these three patients was similar to that in the Manchester series, although the extent of TDP‐43 pathology was significantly less.ConclusionWidespread accumulation of DPR within nerve cells may occur much earlier than that of TDP‐43 in patients with FTLD bearing expansion in C9ORF72.

Hereditary spastic paraplegia (HSP) is a heterogeneous group of neurodegenerative disorders characterized by a progressive gait disorder, lower limb spasticity, hyper-reflexia, weakness and extensor plantar responses. Recently, large intronic hexanucleotide repeat expansions (GGGGCC) in C9ORF72 have been found to cause frontotemporal dementia (FTD), amyotrophic lateral sclerosis and FTD with motor neuron disease. Owing to the overlapping phenotypes among HSP, amyotrophic lateral sclerosis and FTD with motor neuron disease along with shared pathological findings, we hypothesized that C9ORF72 expansions might be a genetic risk factor or modifier of HSP. Clinically characterized HSP patients were investigated for elongations in the hexanucleotide repeat of C9ORF72. Upon analyses of the repeat lengths in the C9ORF72 gene in a Danish cohort of HSP patients, we found no expansions. We conclude that HSP is most likely not associated with repeat expansions in C9ORF72.

Here we report a family with coexistence of multiple system atrophy (MSA) and amyotrophic lateral sclerosis (ALS) with hexanucleotide repeat expansions in C9orf72. A 65-year-old woman had a 2-year history of ataxia with autonomic dysfunction but without motor neuron signs. She was diagnosed as having MSA based on her clinical history and the hot cross bun sign on brain magnetic resonance imaging. Her 62-year-old brother had progressive weakness, fasciculations, hyperreflexia, and active denervation on electromyography without cerebellar ataxia. He was diagnosed as having ALS. Both patients had a greater than 1000/2 hexanucleotide expansion in C9orf72. Patients with hexanucleotide repeat expansions in C9orf72 can present with MSA as well as ALS or frontotemporal dementia. We report this family with coexisting MSA and ALS, highlighting the phenotypic variability in neurologic presentations with hexanucleotide repeat expansions in C9orf72.

GGGGCC repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). It has been reported that hexanucleotide repeat expansions in C9ORF72 produce five dipeptide repeat (DPR) proteins by an unconventional repeat-associated non-ATG (RAN) translation. Within the five DPR proteins, poly-PR and poly-GR that contain arginine are more toxic than the other DPRs (poly-GA, poly-GP, and poly-PA). Here, we demonstrated that poly-PR peptides transferred into cells by endocytosis in a clathrin-dependent manner, leading to endoplasmic reticulum stress and cell death. In SH-SY5Y cells and primary cortical neurons, poly-PR activated JUN amino-terminal kinase (JNK) and increased the levels of p53 and Bax. The uptake of poly-PR peptides by cells was significantly inhibited by knockdown of clathrin or by chlorpromazine, an inhibitor that blocks clathrin-mediated endocytosis. Inhibition of clathrin-dependent endocytosis by chlorpromazine significantly blocked the transfer of poly-PR peptides into cells, and attenuated poly-PR-induced JNK activation and cell death. Our data revealed that the uptake of poly-PR undergoes clathrin-dependent endocytosis and blockade of this process prevents the toxic effects of synthetic poly-PR peptides.

Analysis of the hexanucleotide repeat in C9ORF72 in Alzheimer's disease