ALS Genes in the Genomic Era and their Implications for FTD
Authors: Hung Phuoc Nguyen, Christine Van Broeckhoven, Julie van der Zee
Journal: Trends in Genetics (2018)

High-throughput DNA sequencing, including whole-genome and -exome sequencing, has proven a successful strategy for gene identification in ALS. Substantial progress in gene identification revealed recurrent key molecular mechanisms in ALS, including proteostasis and autophagy, RNA processing, cytoskeleton dynamics, mitochondrial dysfunction, and DNA damage response. ALS and FTD are partners of one disease continuum, consequently gene identification in ALS is impacting FTD genetic etiology and vice versa. The emerging concept of oligogenic inheritance in ALS, and possibly also in FTD, has implications on gene identification, genetic testing, and genetic counseling, as well as therapy development. Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease, characterized genetically by a disproportionately large contribution of rare genetic variation. Driven by advances in massive parallel sequencing and applied on large patient–control cohorts, systematic identification of these rare variants that make up the genetic architecture of ALS became feasible. In this review paper, we present a comprehensive overview of recently proposed ALS genes that were identified based on rare genetic variants (TBK1, CHCHD10, TUBA4A, CCNF, MATR3, NEK1, C21orf2, ANXA11, TIA1) and their potential relevance to frontotemporal dementia genetic etiology. As more causal and risk genes are identified, it has become apparent that affected individuals can carry multiple disease-associated variants. In light of this observation, we discuss the oligogenic architecture of ALS. To end, we highlight emerging key molecular processes and opportunities for therapy. Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease, characterized genetically by a disproportionately large contribution of rare genetic variation. Driven by advances in massive parallel sequencing and applied on large patient–control cohorts, systematic identification of these rare variants that make up the genetic architecture of ALS became feasible. In this review paper, we present a comprehensive overview of recently proposed ALS genes that were identified based on rare genetic variants (TBK1, CHCHD10, TUBA4A, CCNF, MATR3, NEK1, C21orf2, ANXA11, TIA1) and their potential relevance to frontotemporal dementia genetic etiology. As more causal and risk genes are identified, it has become apparent that affected individuals can carry multiple disease-associated variants. In light of this observation, we discuss the oligogenic architecture of ALS. To end, we highlight emerging key molecular processes and opportunities for therapy. Amyotrophic lateral sclerosis (ALS) is a devastating progressive adult-onset neurodegenerative disease, affecting both lower and upper motor neurons in the central nervous system. Core clinical symptoms include weakness in limbs and bulbar muscles, respiratory failure, hyperreflexia, and spasticity of arms or legs. Disease onset occurs on average between 40 and 70 years of age, although younger patients have been reported. Disease progression is often aggressive, with patients dying within 3–5 years postdiagnosis. The estimated annual incidence is from one to three cases per 100 000 people worldwide [1Marin B. et al.Variation in worldwide incidence of amyotrophic lateral sclerosis: a meta-analysis.Int. J. Epidemiol. 2017; 46: 57-74PubMed Google Scholar]. ALS is most often sporadic (see Glossary) but about 5% of patients have a positive family history. Currently, mutations in more than 25 genes have been associated with ALS, with the C9orf72 repeat expansion mutation and SOD1 mutation as the most common genetic causes (please see Table 1 for list of gene/protein abbreviations used throughout the manuscript).Table 1List of Gene and Protein Abbreviations Used Throughout the ManuscriptGenesANXA1Annexin A11ATXN2Ataxin-2C9orf72Chromosome 9 open reading frame 72C21orf2Chromosome 21 open reading frame 2 proteinCCNFCyclin FCHCHD10Coiled-coil-helix-coiled-coil-helix domain-containing protein 10FUSFUS RNA-binding proteinGRNgranulin precursorhnRNPA1Heterogeneous nuclear ribonucleoprotein A1hnRNPA2B1Heterogeneous nuclear ribonucleoprotein A2/B1MAPTMicrotubule-associated protein tauMATR3Matrin 3NEK1NIMA-related kinase 1PFN1Profilin 1SOD1Superoxide dismutase 1TARDBPTAR DNA-binding proteinTBK1TANK-binding kinase 1TIA1T cell-restricted intracellular antigen-1TUBA4ATubulin alpha 4A proteinVCPValosin containing proteinProteinsALS2AlsinCCSCopper chaperone for superoxide dismutaseCHCHD3Coiled-coil-helix-coiled-coil-helix domain-containing protein 3CHCHD6Coiled-coil-helix-coiled-coil-helix domain-containing protein 6CHCHD10Coiled-coil-helix-coiled-coil-helix domain-containing protein 10GLE1Nucleoporin GLE1IKKInhibitor of kappa B kinaseMICOSMitochondrial contact site and cristae organizing systemNFκBNuclear factor kappa-light-chain-enhancer of activated B cellsNIMANever-in-mitosis AOPTNOptineurinRAB8Ras-related protein Rab-8RAB39Ras-related protein Rab-39RRM2Ribonucleoside-diphosphate reductase subunit M2SCFSkp1-Cul1-F-boxSMCR8Smith-Magenis syndrome chromosome region, candidate 8 homologSQSTM1/p62Sequestosome 1TARDBP/TDP-43TAR DNA-binding proteinVAPBVesicle-associated membrane protein-associated protein B/CWDR41WD repeat domain 41 Open table in a new tab ALS is closely related to frontotemporal dementia (FTD). Like ALS, FTD is a progressive neurodegenerative disease characterized by degeneration of the frontal and temporal lobes of the brain, resulting in disturbances of behavior, personality, and language. It is estimated that up to 50% of ALS patients show signs of behavioral dysfunction and/or subtle cognitive impairment, resembling dementia, and up to 15% of ALS patients reach the diagnostic criteria of FTD (referred to as ALS-FTD or FTD-ALS patients) [2Ringholz G.M. et al.Prevalence and patterns of cognitive impairment in sporadic ALS.Neurology. 2005; 65: 586-590Crossref PubMed Scopus (461) Google Scholar, 3Wheaton M.W. et al.Cognitive impairment in familial ALS.Neurology. 2007; 69: 1411-1417Crossref PubMed Scopus (0) Google Scholar, 4Robberecht W. Philips T. The changing scene of amyotrophic lateral sclerosis.Nat. Rev. Neurosci. 2013; 14: 248-264Crossref PubMed Scopus (423) Google Scholar]. Conversely, the same holds true for FTD [5Burrell J.R. et al.Motor neuron dysfunction in frontotemporal dementia.Brain. 2011; 134: 2582-2594Crossref PubMed Scopus (110) Google Scholar, 6Van Langenhove T. et al.Predicting development of amyotrophic lateral sclerosis in frontotemporal dementia.J. Alzheimers Dis. 2017; 58: 163-170Crossref PubMed Scopus (0) Google Scholar]. At the genetic level, mutations in multiple genes contribute to the etiology of both ALS and FTD, as best represented by the C9orf72 repeat expansion, TBK1, VCP, and TARDBP mutations. By contrast, other genes are specifically associated with only one of the diseases, such as SOD1 for ALS or MAPT and GRN for FTD. Although TARDBP mutations are rare in ALS and FTD (<1%), pathologically, aggregation of TAR DNA-binding protein 43 (TDP-43) in affected brain regions and motor neurons are found in the majority of ALS (up to 97%) and FTD (up to 50%) patients [7Ling S.C. et al.Converging mechanisms in ALS and FTD: disrupted RNA and protein homeostasis.Neuron. 2013; 79: 416-438Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 8Mackenzie I.R. Neumann M. Molecular neuropathology of frontotemporal dementia: insights into disease mechanisms from postmortem studies.J. Neurochem. 2016; 138: 54-70Crossref PubMed Scopus (0) Google Scholar]. Owing to this extensive clinical, genetic, and pathological overlap, ALS and FTD are now considered partners of a disease continuum, referred to as the ALS-FTD spectrum, rather than two separate disease entities. Mutations in the major established causal ALS genes (SOD1, TARDBP, FUS, VCP, C9orf72, and PFN1) account for approximately 60%–70% of familial ALS (fALS) and about 10% of apparently sporadic ALS (sALS) cases [9Renton A.E. et al.State of play in amyotrophic lateral sclerosis genetics.Nat. Neurosci. 2014; 17: 17-23Crossref PubMed Scopus (489) Google Scholar], with the GGGGCC hexanucleotide expansion mutation in the 5′ noncoding region of C9orf72 being by far the biggest contributor (Box 1). However, this also indicates that more genes remain to be uncovered. In recent years, advances in massive parallel sequencing approaches such as whole-genome sequencing (WGS) and whole-exome sequencing (WES), hand in hand with large-scale collaborations [which have previously led to the success of genome-wide association studies (GWAS) in ALS] have facilitated a new wave of gene discovery. These studies are specifically designed to identify rare variants that confer disease risk, variants that are typically not picked up by GWAS, which, by design, target common variants. This has led to the recent identification of at least nine genes carrying such rare causal variants, including TBK1, CHCHD10, TUBA4A, MATR3, CCNF, NEK1, C21orf2, ANXA11, and TIA1 (Table 2) [10Johnson J.O. et al.Mutations in the Matrin 3 gene cause familial amyotrophic lateral sclerosis.Nat. Neurosci. 2014; 17: 664-666Crossref PubMed Scopus (140) Google Scholar, 11Bannwarth S. et al.A mitochondrial origin for frontotemporal dementia and amyotrophic lateral sclerosis through CHCHD10 involvement.Brain. 2014; 137: 2329-2345Crossref PubMed Scopus (140) Google Scholar, 12Smith B.N. et al.Exome-wide rare variant analysis identifies TUBA4A mutations associated with familial ALS.Neuron. 2014; 84: 324-331Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 13Cirulli E.T. et al.Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways.Science. 2015; 347: 1436-1441Crossref PubMed Google Scholar, 14Freischmidt A. et al.Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia.Nat. Neurosci. 2015; 18: 631-636Crossref PubMed Scopus (191) Google Scholar, 15Williams K.L. et al.CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia.Nat. Commun. 2016; 711253Crossref PubMed Google Scholar, 16Kenna K.P. et al.NEK1 variants confer susceptibility to amyotrophic lateral sclerosis.Nat. Genet. 2016; 48: 1037-1042Crossref PubMed Scopus (92) Google Scholar, 17van Rheenen W. et al.Genome-wide association analyses identify new risk variants and the genetic architecture of amyotrophic lateral sclerosis.Nat. Genet. 2016; 48: 1043-1048Crossref PubMed Google Scholar, 18Smith B.N. et al.Mutations in the vesicular trafficking protein annexin A11 are associated with amyotrophic lateral sclerosis.Sci. Transl. Med. 2017; 9 (eaad9157)Crossref Scopus (2) Google Scholar, 19Mackenzie I.R. et al.TIA1 mutations in amyotrophic lateral sclerosis and frontotemporal dementia promote phase separation and alter stress granule dynamics.Neuron. 2017; 95: 808-816Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. Here, we will discuss supportive evidence for their respective impact on the genetic architecture of ALS and FTD and linked molecular pathways.Box 1C9orf72 In 2011, the discovery of a noncoding hexanucleotide repeat expansion (GGGGCC) mutation in the 5′ noncoding region of the C9orf72 gene drastically shifted the field, with up to 40% of fALS patients carrying such a repeat expansion. The C9orf72 gene was identified in multiple multigenerational families presenting with FTD-ALS or ALS-FTD and linked to chromosome 9p21 [86Renton A.E. et al.A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.Neuron. 2011; 72: 257-268Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar, 87DeJesus-Hernandez M. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (1849) Google Scholar, 88Gijselinck I. et al.A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study.Lancet Neurol. 2012; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar]. Also in FTD, the C9orf72 repeat expansion is the most common genetic cause, explaining 25% of familial FTD and up to 88% of familial patients with both ALS and FTD [118Van Mossevelde S. et al.Relationship between C9orf72 repeat size and clinical phenotype.Curr. Opin. Genet. Dev. 2017; 44: 117-124Crossref PubMed Scopus (4) Google Scholar].Notably, C9orf72 repeat size is highly polymorphic, and the cut-off to distinguish normal from pathogenic expansions remains somewhat ambiguous (for more on sizing of the repeat and cut-off of pathogenicity, see GeneReviews on C9orf72 [119Cruts M. et al.C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia.in: Adam M.P. GeneReviews. University of Washington, 2015Google Scholar]). In general, in unaffected individuals repeat size varies from two to 24 repeat units, whereas in both ALS and FTD patients the repeat expands from several hundred to several thousand repeats. The smallest repeat with evidence of cosegregation with disease was 50 repeat units [120Gijselinck I. et al.The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter.Mol. Psychiatry. 2016; 21: 1112-1124Crossref PubMed Scopus (24) Google Scholar]. Currently, the relationship between repeat size and disease phenotype (ALS versus FTD) or onset age is being investigated and some studies provide evidence for disease anticipation [120Gijselinck I. et al.The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter.Mol. Psychiatry. 2016; 21: 1112-1124Crossref PubMed Scopus (24) Google Scholar, 121Van Mossevelde S. et al.Clinical evidence of disease anticipation in families segregating a C9orf72 repeat expansion.JAMA Neurol. 2017; 74: 445-452Crossref PubMed Scopus (4) Google Scholar].Little is known about the normal function of the C9orf72 protein, complicating functional characterization, but three major pathological mechanisms have been proposed: (i) loss-of-function and haploinsufficiency. The GGGGCC repeat expansion in the C9orf72 promoter suppresses gene expression, leading to loss of mutant transcript and protein, as seen for other repeat expansion disorders such as fragile X syndrome and Friedrich's ataxia [88Gijselinck I. et al.A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study.Lancet Neurol. 2012; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar]. However, there is evidence challenging the loss-of-function mechanism hypothesis [104Sareen D. et al.Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion.Sci. Transl. Med. 2013; 5 (208ra149)Crossref PubMed Scopus (235) Google Scholar, 105Donnelly C.J. et al.RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.Neuron. 2013; 80: 415-428Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 108Lagier-Tourenne C. et al.Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E4530-E4539Crossref PubMed Scopus (214) Google Scholar, 122Fratta P. et al.Homozygosity for the C9orf72 GGGGCC repeat expansion in frontotemporal dementia.Acta Neuropathol. 2013; 126: 401-409Crossref PubMed Scopus (0) Google Scholar]. (ii) RNA toxicity. Expanded sense (GGGGCC) and antisense (GGCCCC) RNA transcripts form toxic RNA foci, which sequester essential RNA-binding proteins and impair the RNA processing machinery, similar to that of myotonic dystrophy type 1 [87DeJesus-Hernandez M. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (1849) Google Scholar, 123Wheeler T.M. Thornton C.A. Myotonic dystrophy: RNA-mediated muscle disease.Curr. Opin. Neurol. 2007; 20: 572-576Crossref PubMed Scopus (113) Google Scholar]. By contrast, modeling in Drosophila argued against an RNA toxicity mechanism [124Mizielinska S. et al.C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.Science. 2014; 345: 1192-1194Crossref PubMed Scopus (185) Google Scholar, 125Tran H. et al.Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a Drosophila model of C9ORF72 FTD/ALS.Neuron. 2015; 87: 1207-1214Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Flies with a transgene of 160 GGGGCC repeats expressed it, spliced, and formed many sense RNA foci in the nucleus [125Tran H. et al.Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a Drosophila model of C9ORF72 FTD/ALS.Neuron. 2015; 87: 1207-1214Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Yet, no neurodegeneration was observed, suggesting that the accumulation of RNA foci is not sufficient to trigger neurodegeneration. (iii) Proteotoxicity from dipeptide repeat (DPR) aggregates. Repeat-associated non-AUG (RAN) translation of GGGGCC or GGCCCC RNA transcripts generate toxic poly-GA, poly-GP, poly-GR, poly-PA, and poly-PR peptides (in each of the three reading frames), leading to DPR-positive inclusions. These characteristic inclusions are predominant in the cerebellum, hippocampus, and frontotemporal cortex, alongside TDP-43 pathology [126Mori K. et al.Bidirectional transcripts of the expanded C9orf72 hexanucleotide repeat are translated into aggregating dipeptide repeat proteins.Acta Neuropathol. 2013; 126: 881-893Crossref PubMed Scopus (146) Google Scholar]. Several studies demonstrated that C9orf72-derived DPRs are toxic, impair nucleocytoplasmic transport, and can cause neurodegeneration and behavioral deficits [124Mizielinska S. et al.C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.Science. 2014; 345: 1192-1194Crossref PubMed Scopus (185) Google Scholar, 127Kwon I. et al.Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells.Science. 2014; 345: 1139-1145Crossref PubMed Scopus (169) Google Scholar, 128Zhang Y.J. et al.Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress.Acta Neuropathol. 2014; 128: 505-524Crossref PubMed Scopus (106) Google Scholar, 129Yang D. et al.FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions.Acta Neuropathol. 2015; 130: 525-535Crossref PubMed Scopus (31) Google Scholar, 130Freibaum B.D. et al.GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport.Nature. 2015; 525: 129-133Crossref PubMed Scopus (193) Google Scholar, 131Jovicic A. et al.Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.Nat. Neurosci. 2015; 18: 1226-1229Crossref PubMed Scopus (147) Google Scholar, 132Zhang K. et al.The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.Nature. 2015; 525: 56-61Crossref PubMed Scopus (217) Google Scholar, 133Shi K.Y. et al.Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: E1111-E1117Crossref PubMed Scopus (0) Google Scholar, 134Yin S. et al.Evidence that C9ORF72 dipeptide repeat proteins associate with U2 snRNP to cause sis-splicing in ALS/FTD patients.Cell Rep. 2017; 19: 2244-2256Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. By contrast, human postmortem studies found no correlation between DPR protein pathology load and distribution and degree of neurodegeneration or phenotype (ALS, FTD, or mixed ALS-FTD), contesting that DPR protein aggregation is the major pathomechanism in C9orf72 pathogenesis [135Mackenzie I.R. et al.Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers.Acta Neuropathol. 2015; 130: 845-861Crossref PubMed Scopus (42) Google Scholar]. As outlined above, it is clear that the relative contribution of these three molecular mechanisms is still actively debated and further investigated. Nevertheless, it is very likely that a combination of multiple mechanisms is at play.Table 2Overview of Recent ALS Genes with Relative Mutation Frequencies in Different ALS and FTD Cohorts and Associated PathwaysGeneLocusInheritanceLevel of evidenceaLevel of evidence for the recently identified genes in the ALS-FTD spectrum is classified as 'established' or 'to be validated', based on following criteria: established, genes have been replicated by different studies, including supportive evidence from functional studies; to be validated, gene findings based on single study, further validation in different study populations and/or functional studies required.Mutation frequencyTDP-43 pathologyImplicated disease pathwayOverall ALS (%)Familial ALS (%)Sporadic ALS (%)Overall FTD (%)Familial FTD (%)Sporadic FTD (%)ALS-FTD (%)TBK1bMutation frequencies include only loss-of-function mutations.12q14.2ADcAbbreviations: AD, autosomal dominant inheritance; n.d., inheritance mode not determined yet.Established1.33<1<1213–4+Autophagy, inflammationCHCHD1022q11.23ADEstablished<12<1<1<1<1<1+Mitochondrial dysfunction, synaptic integrityTUBA4A2q35ADTo be validated<11<1<1<1––+Cytoskeletal dynamics, axonal transportMATR35q31.2ADEstablished<11–21––––+RNA metabolismCCNF16p13.3ADEstablished<10.6–3.3<14dFrequency was counted in 99 FTD-TDP patients [15].–––+ProteostasisNEK1bMutation frequencies include only loss-of-function mutations.4q33n.d.cAbbreviations: AD, autosomal dominant inheritance; n.d., inheritance mode not determined yet.To be validated11–2<1––––n.d.DNA damage responses, cell cycle control, cytoskeletal organization, mitochondrial membrane regulationC21orf2bMutation frequencies include only loss-of-function mutations.21q22.3n.d.To be validated<1––––––n.d.DNA damage response, cytoskeletal organizationANXA1110q22.3ADTo be validated1.111.7––––+ProteostasisTIA12p13.3ADTo be validated<12.2<1–––<1+RNA metabolisma Level of evidence for the recently identified genes in the ALS-FTD spectrum is classified as 'established' or 'to be validated', based on following criteria: established, genes have been replicated by different studies, including supportive evidence from functional studies; to be validated, gene findings based on single study, further validation in different study populations and/or functional studies required.b Mutation frequencies include only loss-of-function mutations.c Abbreviations: AD, autosomal dominant inheritance; n.d., inheritance mode not determined yet.d Frequency was counted in 99 FTD-TDP patients 15Williams K.L. et al.CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia.Nat. Commun. 2016; 711253Crossref PubMed Google Scholar. Open table in a new tab In 2011, the discovery of a noncoding hexanucleotide repeat expansion (GGGGCC) mutation in the 5′ noncoding region of the C9orf72 gene drastically shifted the field, with up to 40% of fALS patients carrying such a repeat expansion. The C9orf72 gene was identified in multiple multigenerational families presenting with FTD-ALS or ALS-FTD and linked to chromosome 9p21 [86Renton A.E. et al.A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.Neuron. 2011; 72: 257-268Abstract Full Text Full Text PDF PubMed Scopus (1732) Google Scholar, 87DeJesus-Hernandez M. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (1849) Google Scholar, 88Gijselinck I. et al.A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study.Lancet Neurol. 2012; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar]. Also in FTD, the C9orf72 repeat expansion is the most common genetic cause, explaining 25% of familial FTD and up to 88% of familial patients with both ALS and FTD [118Van Mossevelde S. et al.Relationship between C9orf72 repeat size and clinical phenotype.Curr. Opin. Genet. Dev. 2017; 44: 117-124Crossref PubMed Scopus (4) Google Scholar]. Notably, C9orf72 repeat size is highly polymorphic, and the cut-off to distinguish normal from pathogenic expansions remains somewhat ambiguous (for more on sizing of the repeat and cut-off of pathogenicity, see GeneReviews on C9orf72 [119Cruts M. et al.C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia.in: Adam M.P. GeneReviews. University of Washington, 2015Google Scholar]). In general, in unaffected individuals repeat size varies from two to 24 repeat units, whereas in both ALS and FTD patients the repeat expands from several hundred to several thousand repeats. The smallest repeat with evidence of cosegregation with disease was 50 repeat units [120Gijselinck I. et al.The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter.Mol. Psychiatry. 2016; 21: 1112-1124Crossref PubMed Scopus (24) Google Scholar]. Currently, the relationship between repeat size and disease phenotype (ALS versus FTD) or onset age is being investigated and some studies provide evidence for disease anticipation [120Gijselinck I. et al.The C9orf72 repeat size correlates with onset age of disease, DNA methylation and transcriptional downregulation of the promoter.Mol. Psychiatry. 2016; 21: 1112-1124Crossref PubMed Scopus (24) Google Scholar, 121Van Mossevelde S. et al.Clinical evidence of disease anticipation in families segregating a C9orf72 repeat expansion.JAMA Neurol. 2017; 74: 445-452Crossref PubMed Scopus (4) Google Scholar]. Little is known about the normal function of the C9orf72 protein, complicating functional characterization, but three major pathological mechanisms have been proposed: (i) loss-of-function and haploinsufficiency. The GGGGCC repeat expansion in the C9orf72 promoter suppresses gene expression, leading to loss of mutant transcript and protein, as seen for other repeat expansion disorders such as fragile X syndrome and Friedrich's ataxia [88Gijselinck I. et al.A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study.Lancet Neurol. 2012; 11: 54-65Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar]. However, there is evidence challenging the loss-of-function mechanism hypothesis [104Sareen D. et al.Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion.Sci. Transl. Med. 2013; 5 (208ra149)Crossref PubMed Scopus (235) Google Scholar, 105Donnelly C.J. et al.RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.Neuron. 2013; 80: 415-428Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 108Lagier-Tourenne C. et al.Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: E4530-E4539Crossref PubMed Scopus (214) Google Scholar, 122Fratta P. et al.Homozygosity for the C9orf72 GGGGCC repeat expansion in frontotemporal dementia.Acta Neuropathol. 2013; 126: 401-409Crossref PubMed Scopus (0) Google Scholar]. (ii) RNA toxicity. Expanded sense (GGGGCC) and antisense (GGCCCC) RNA transcripts form toxic RNA foci, which sequester essential RNA-binding proteins and impair the RNA processing machinery, similar to that of myotonic dystrophy type 1 [87DeJesus-Hernandez M. et al.Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS.Neuron. 2011; 72: 245-256Abstract Full Text Full Text PDF PubMed Scopus (1849) Google Scholar, 123Wheeler T.M. Thornton C.A. Myotonic dystrophy: RNA-mediated muscle disease.Curr. Opin. Neurol. 2007; 20: 572-576Crossref PubMed Scopus (113) Google Scholar]. By contrast, modeling in Drosophila argued against an RNA toxicity mechanism [124Mizielinska S. et al.C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.Science. 2014; 345: 1192-1194Crossref PubMed Scopus (185) Google Scholar, 125Tran H. et al.Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a Drosophila model of C9ORF72 FTD/ALS.Neuron. 2015; 87: 1207-1214Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Flies with a transgene of 160 GGGGCC repeats expressed it, spliced, and formed many sense RNA foci in the nucleus [125Tran H. et al.Differential toxicity of nuclear RNA foci versus dipeptide repeat proteins in a Drosophila model of C9ORF72 FTD/ALS.Neuron. 2015; 87: 1207-1214Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Yet, no neurodegeneration was observed, suggesting that the accumulation of RNA foci is not sufficient to trigger neurodegeneration. (iii) Proteotoxicity from dipeptide repeat (DPR) aggregates. Repeat-associated non-AUG (RAN) translation of GGGGCC or GGCCCC RNA transcripts generate toxic poly-GA, poly-GP, poly-GR, poly-PA, and poly-PR peptides (in each of the three reading frames), leading to DPR-positive inc