MD Dario Saracino
email: dario.saracino@icm-institute.org

MD, PhD Isabelle Le Ber

How can we define the presymptomatic C9orf72 disease in 2022? An overview on the current definitions of preclinical and prodromal phases Authors

Keywords: Frontotemporal dementia, frontotemporal lobar degeneration, amyotrophic lateral sclerosis, C9orf72, biomarker, presymptomatic disease

come

How can we define the presymptomatic C9orf72 disease in 2022? An overview on the current definitions of preclinical and prodromal phases

Dario Saracino, Isabelle Le Ber

Introduction

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases lying along a clinical continuum and sharing common pathophysiological and genetic mechanisms. One of the most relevant advances in the understanding of how these two diseases are related has been the identification, in 2011, of a hexanucleotide repeat expansion in C9orf72 in families concerned by FTD, ALS, or the combination of the two [START_REF] Dejesus-Hernandez | Expanded GGGGCC Hexanucleotide Repeat in Noncoding Region of C9ORF72 Causes Chromosome 9p-Linked FTD and ALS[END_REF][START_REF] Renton | A Hexanucleotide Repeat Expansion in C9ORF72 is the Cause of Chromosome 9p21-Linked ALS-FTD[END_REF].

C9orf72 expansions turned out to be the most frequent cause of genetic FTD and ALS in most countries, explaining up to 25% of familial FTD cases and around 40% of familial ALS cases [START_REF] Majounie | Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study[END_REF][START_REF] Ber | C9ORF72 Repeat Expansions in the Frontotemporal Dementias Spectrum of Diseases: A Flow-chart for Genetic Testing[END_REF][START_REF] Cammack | Prospective natural history study of C9orf72 ALS clinical characteristics and biomarkers[END_REF]. When both disorders coexist, C9orf72 expansions can be found in up to 80% of cases [START_REF] Majounie | Frequency of the C9orf72 hexanucleotide repeat expansion in patients with amyotrophic lateral sclerosis and frontotemporal dementia: a cross-sectional study[END_REF][START_REF] Ber | C9ORF72 Repeat Expansions in the Frontotemporal Dementias Spectrum of Diseases: A Flow-chart for Genetic Testing[END_REF]. Besides, they can occur also in FTD or ALS patients without overt family history of neurodegenerative diseases, at a frequency estimated between 6% and 20% [START_REF] Cammack | Prospective natural history study of C9orf72 ALS clinical characteristics and biomarkers[END_REF][START_REF] Mahoney | Frontotemporal dementia with the C9ORF72 hexanucleotide repeat expansion: clinical, neuroanatomical and neuropathological features[END_REF] thus underscoring the importance of genetic testing even in apparently sporadic cases. Notably, no such overlap exists with other relatively frequent genes after C9orf72, such as progranulin gene (GRN) and microtubule associated protein tau gene (MAPT), identified in FTD phenotypes, or superoxide dysmutase 1 (SOD1), responsible of pure ALS. Less common disease-causing genes can be involved in both cognitive, motor, or complex phenotypes [START_REF] Sellami | Genetic forms of frontotemporal lobar degeneration: Current diagnostic approach and new directions in therapeutic strategies[END_REF].

The age at onset in C9orf72 disease is extremely variable, ranging between the 2 nd and the 9 th decade, with a peak at 58 years [START_REF] Moore | Age at symptom onset and death and disease duration in genetic frontotemporal dementia: an international retrospective cohort study[END_REF]. There is increasing evidence about the heterogeneity of clinical phenotypes, encompassing cognitive, behavioral and motor syndromes. In addition to the behavioral variant of FTD (bvFTD), ALS and the association FTD/ALS, C9orf72 patients may occasionally present with psychiatric phenotypes, mainly qualifying as atypical, late-onset psychoses [START_REF] Snowden | Distinct clinical and pathological characteristics of frontotemporal dementia associated with C9ORF72 mutations[END_REF][START_REF] Galimberti | Autosomal Dominant Frontotemporal Lobar Degeneration Due to the C9ORF72 Hexanucleotide Repeat Expansion: Late-Onset Psychotic Clinical Presentation[END_REF][START_REF] Devenney | Psychiatric disorders in C9orf72 kindreds: Study of 1,414 family members[END_REF]. Less common presentations, identifiable in less than 5% of carriers, include primary progressive aphasia (PPA) variants [START_REF] Moore | Age at symptom onset and death and disease duration in genetic frontotemporal dementia: an international retrospective cohort study[END_REF][START_REF] Saracino | Primary progressive aphasias associated with C9orf72 expansions: Another side of the story[END_REF], and parkinsonian syndromes (corticobasal syndrome, progressive supranuclear palsy, and, rarely, typical parkinsonism) [START_REF] Boeve | Characterization of frontotemporal dementia and/or amyotrophic lateral sclerosis associated with the GGGGCC repeat expansion in C9ORF72[END_REF][START_REF] Gasca-Salas | Characterization of Movement Disorder Phenomenology in Genetically Proven, Familial Frontotemporal Lobar Degeneration: A Systematic Review and Meta-Analysis[END_REF][START_REF] Carneiro | Isolated parkinsonism is an atypical presentation of GRN and C9orf72 gene mutations[END_REF]. Knowledge about the implications of C9orf72 repeat expansion in disease pathophysiology has been continuously increasing since the discovery of the gene. The first intron of C9orf72 contains a G4C2 sequence which in healthy individuals mostly ranges between 2 and 8 repeats, and in any case below 30, which has been conventionally fixed as a pathogenic threshold [START_REF] Renton | A Hexanucleotide Repeat Expansion in C9ORF72 is the Cause of Chromosome 9p21-Linked ALS-FTD[END_REF][START_REF] Beck | Large C9orf72 Hexanucleotide Repeat Expansions Are Seen in Multiple Neurodegenerative Syndromes and Are More Frequent Than Expected in the UK Population[END_REF].

The majority of affected individuals carry a pathologic expansion in the range of hundreds or thousands of repeats [START_REF] Snowden | Distinct clinical and pathological characteristics of frontotemporal dementia associated with C9ORF72 mutations[END_REF]; interestingly, the repeat length does not significantly affect the disease phenotype or the age at onset [START_REF] Fournier | Relations between C9orf72 expansion size in blood, age at onset, age at collection and transmission across generations in patients and presymptomatic carriers[END_REF]. In addition, expansions of intermediate length (between 20 and 30 repeats), have been suggested to increase the risk of developing parkinsonian syndromes or ALS [START_REF] Nuytemans | C9ORF72 Intermediate Repeat Copies Are a Significant Risk Factor for Parkinson Disease[END_REF][START_REF] Iacoangeli | C9orf72 intermediate expansions of 24-30 repeats are associated with ALS[END_REF]. The biological functions of the C9ORF72 protein are not completely understood, but it has been determined to act as a GTPase activating protein (GAP), in partnership with two other subunits [START_REF] Tang | The C9orf72-SMCR8-WDR41 complex is a GAP for small GTPases[END_REF].

Three main pathogenic mechanisms have been hypothesized in C9orf72-associated disease, including loss of physiological role of C9ORF72 protein, accumulation of RNA foci in the nuclei and toxicity from dipeptide repeat proteins (DPR) generated from repeat-associated non-ATG (RAN) translation [START_REF] Lagier-Tourenne | Targeted degradation of sense and antisense C9orf72 RNA foci as therapy for ALS and frontotemporal degeneration[END_REF]. Hence, RNA foci and cytoplasmic DPRs accumulations coupled with p62-positive inclusions represent pathological hallmarks specific to C9orf72 disease, in addition to diffuse neuronal and oligodendroglial TDP-43 positive inclusions [START_REF] Mizielinska | C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci[END_REF][START_REF] Mackenzie | The neuropathology associated with repeat expansions in the C9ORF72 gene[END_REF].

Insights in the disease mechanisms have paved the way to the development of diseasemodifying treatments, mainly acting to contrast the deleterious effect of the C9orf72 expansion.

Among them, one of the most developed so far consists of antisense oligonucleotides (ASOs), small DNA or RNA molecules binding to a complementary RNA sequence and leading to its degradation, thus modulating gene expression [START_REF] Jiang | Gain of Toxicity from ALS/FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs[END_REF]. Phase 1 and 2 trials targeting C9orf72associated FTD and ALS have started in recent years (www.clinicaltrials.gov NCT03626012, NCT04288856, NCT04931862, NCT04993755). These interventions should be eventually developed to block the pathological cascade and, ideally, prevent or significantly delay the occurrence of clinical symptoms. Therefore, the presymptomatic disease phase is getting more and more interest as an ideal time window in which innovant therapeutic -or rather preventive -approaches should be tested. At present, the definitions and the time references of the presymptomatic phase, as well as the tools of choice for the longitudinal monitoring of the disease trajectory are a matter of intensive research. This review provides an overview on the current knowledge about the presymptomatic phase of the C9orf72 disease, particularly focusing on: i) the proposed classification of presymptomatic stages, ii) the contribution of biomarkers to trace preclinical disease trajectory, and iii) the earliest clinically relevant changes associated with disease onset, and their interest in ongoing and future clinical trials tackling C9orf72-associated pathology.

Current framework for defining the presymptomatic disease stages

At present there is limited information about the sequential ordering of events occurring in FTD and ALS before clinical onset. In the field of neurodegenerative diseases, studies focusing on Alzheimer's disease (AD), particularly in its monogenic forms, have substantially contributed to define the concept of a presymptomatic disease stage, characterized by progressive lesion accumulation [START_REF] Selkoe | The amyloid hypothesis of Alzheimer's disease at 25 years[END_REF][START_REF] Jack | NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease[END_REF][START_REF] Dubois | Clinical diagnosis of Alzheimer's disease: recommendations of the International Working Group[END_REF], up to a prodromal, oligosymptomatic stage defined as mild cognitive impairment (MCI) [START_REF] Petersen | Practice guideline update summary: Mild cognitive impairment: Report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology[END_REF]. In Huntington's disease (HD), longitudinal studies on presymptomatic carriers allowed to depict the progressive changes occurring during the pre-manifest phase, with the useful contribution of repeat length and other genetic modifiers in the prediction of the age at onset [START_REF] Langbehn | on behalf of an International Huntington's Disease Collaborative Group. A new model for prediction of the age of onset and penetrance for Huntington's disease based on CAG length: Prediction of the age of onset and penetrance for HD[END_REF][START_REF] Biglan | Motor abnormalities in premanifest persons with Huntington's disease: The PREDICT-HD study: Motor Abnormalities in Premanifest HD[END_REF][START_REF] Lee | Identification of Genetic Factors that Modify Clinical Onset of Huntington's Disease[END_REF].

In the case of FTD and ALS, a privileged point of view to study the presymptomatic phase is offered by carriers of disease-causative mutations, identified among first-degrees relatives of genetic patients. Over the recent years, international consortia have been built to assemble and study large cohorts of presymptomatic carriers in a standardized manner, to increase our knowledge on presymptomatic stage. The Genetic Frontotemporal Initiative (GENFI), has been developed in Europe and Canada (www.genfi.org), and the ARTFL (Advancing Research and Treatment for Frontotemporal Lobar degeneration) and LEFFTDS (Longitudinal Evaluation of Familial FrontoTemporal Dementia Subjects) (www.allftd.org), as well as the presymptomatic familial ALS (Pre-fALS) study [START_REF] Benatar | Presymptomatic studies in ALS: Rationale, challenges, and approach[END_REF], have been initiated in US. Besides, multiple national initiatives have assembled country-based cohorts, such as the Australian DINAD (Dominantly Inherited Non-Alzheimer Dementias, www.ecdc.org.au/genetic-ftd-trials); the New Zealand Genetic FTD (FTDGeNZ) [START_REF] Ryan | Diagnosing preclinical dementia: the NZ Genetic Frontotemporal Dementia Study (FTDGeNZ)[END_REF], the French PREV-DEMALS and Predict-PGRN [START_REF] Caroppo | Lateral Temporal Lobe: An Early Imaging Marker of the Presymptomatic GRN Disease?[END_REF][START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF] cohorts, the Belgian presymptomatic C9orf72 cohort [START_REF] Vocht | Use of Multimodal Imaging and Clinical Biomarkers in Presymptomatic Carriers of C9orf72 Repeat Expansion[END_REF], and the Multi-partner Consortium to expand Dementia Research in Latin America (ReDLat) [START_REF] Ibanez | The Multi-Partner Consortium to Expand Dementia Research in Latin America (ReDLat): Driving Multicentric Research and Implementation Science[END_REF]. All these initiatives highlight the important challenge and critical need to better characterize this stage of the disease.

Overall, research results coming from the abovementioned initiatives contributed to generate a conceptual framework useful to define and further classify the presymptomatic phases of FTD and ALS, which could be thus employed in the context of C9orf72 expansions, responsible of both these diseases. Key recommendations have been recently defined in the works by [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF] [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF] and [START_REF] Benatar | Preventing amyotrophic lateral sclerosis: insights from pre-symptomatic neurodegenerative diseases[END_REF] [START_REF] Benatar | Preventing amyotrophic lateral sclerosis: insights from pre-symptomatic neurodegenerative diseases[END_REF], and are summarized below (Figure 1).

The preclinical disease stage defines the period between the start of the neurodegenerative process and the appearance of the first signs and symptoms of disease. Theoretically, with C9orf72 expansions this phase should correspond to progressive accumulation of DPR proteins, RNA foci and, sequentially, TDP-43 pathology [START_REF] Troakes | An MND/ALS phenotype associated with C9orf72 repeat expansion: Abundant p62-positive, TDP-43-negative inclusions in cerebral cortex, hippocampus and cerebellum but without associated cognitive decline: p62 proteinopathy[END_REF][START_REF] Mackenzie | Subcortical TDP-43 pathology patterns validate cortical FTLD-TDP subtypes and demonstrate unique aspects of C9orf72 mutation cases[END_REF]. However, the limited information obtainable from pathological biomarkers in vivo does not allow to determine how early these degenerative changes occur. Therefore, it is currently unclear whether a "no disease" stage, characterized by the absence of any pathological lesions exists, and the boundary between this latter and the subsequent preclinical phase is particularly hard to identify [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF]. During the preclinical stage, clinical symptoms are completely absent, and no ongoing denervation changes should be found on EMG [START_REF] Benatar | Preventing amyotrophic lateral sclerosis: insights from pre-symptomatic neurodegenerative diseases[END_REF]. Different biomarkers can variably contribute to inform on preclinical disease trajectory, as it will be discussed further [START_REF] Vocht | Use of Multimodal Imaging and Clinical Biomarkers in Presymptomatic Carriers of C9orf72 Repeat Expansion[END_REF][START_REF] Van Der Ende | A data-driven disease progression model of fluid biomarkers in genetic frontotemporal dementia[END_REF].

The prodromal disease stage is defined by the appearance the first subtle cognitive, behavioral and motor signs, and lasts up to the onset of full-blown disease. Prodromal FTD is characterized by gradual changes affecting social cognition, executive functions or language, as well as recent behavioral modifications including reduced initiative, diminished empathy, change in dietary habits, repetitive or ritualized actions or behaviors [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF]. These changes from the individual's baseline status should be of such intensity as to preserve independence in daily living, albeit a mild impact on close relationships or highly demanding professional tasks cannot be excluded [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF]. From a quantitative approach, the Clinical Dementia Rating (CDR) plus National Alzheimer's Disease Coordinating Center (NACC) Frontotemporal Lobar Degeneration (FTLD) rating scale (CDR+NACC FTLD) is one of the tools of choice to score the severity of symptoms in FTD patients [START_REF] Miyagawa | Use of the CDR® plus NACC FTLD in mild FTLD: Data from the ARTFL/LEFFTDS consortium[END_REF][START_REF] Peakman | Comparison of clinical rating scales in genetic frontotemporal dementia within the GENFI cohort[END_REF]. Preclinical, asymptomatic stage is defined by a CDR+NACC FTLD global score equal to 0, whereas prodromal subjects should present a score of 0.5. Prodromal ALS is characterized by mild motor complaints (cramps, early fatigue), subtle signs at neurological examination (fasciculations, changes in reflexes) or isolated EMG signs of ongoing denervation, without overt muscular weakness. "Phenotransition" is the term proposed by the ALS community to indicate the passage from the preclinical to the prodromal stage [START_REF] Benatar | Preventing amyotrophic lateral sclerosis: insights from pre-symptomatic neurodegenerative diseases[END_REF].

Overall, to account for the heterogeneity of the cognitive/clinical manifestations of the prodromal stage, especially with the occurrence of C9orf72 expansions, the unifying concept of mild cognitive/behavioral/motor impairment (MCBMI) has been proposed [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF].

However, several confounding factors should be ruled out before affirming that a MCBMI is due to underlying FTD or ALS. Cognitive impairment, especially affecting domains which are atypical for FTD, could result from degenerative processes unrelated to mutational status, as well as from non-degenerative conditions (cerebrovascular lesions or sleep disturbances among many others). Subtle changes in behavior or personality are not specific for FTD, and could derive from unrelated psychiatric conditions or substance abuse. When evaluating motor signs and symptoms, common confounding conditions such as radiculopathies should be carefully looked for. Moreover, C9orf72 disease offers another source of uncertainty, due to the existence of long-standing psychiatric phenotypes, hardly distinguishable from primary psychiatric disorders, which could precede the onset of FTD or ALS by years or decades [START_REF] Ducharme | Psychiatric Presentations of C9orf72 Mutation: What Are the Diagnostic Implications for Clinicians?[END_REF][START_REF] Sellami | Schizophrenia Phenotype Preceding Behavioral Variant Frontotemporal Dementia Related to C9orf72 Repeat Expansion[END_REF].

The term "phenoconversion" indicates the transition from the prodromal to the full-blown clinical stage. Clear boundaries between the two stages are barely definable, therefore some operational criteria for phenoconversion have been proposed, including: 1) fulfillment of diagnostic criteria for bvFTD, PPA, ALS or other associated syndromes; 2) CDR+NACC FTLD score equal or greater than 1; 3) loss of independence in daily living; 4) significant impact on social/professional activities despite preserved autonomy (e.g., because of language deficits or inappropriate social behavior) [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF].

It should be kept in mind, however, that some inconsistencies may emerge when applying this general framework to individual disease carriers, because of the differences in the temporal course across distinct genotypes [START_REF] Moore | Age at symptom onset and death and disease duration in genetic frontotemporal dementia: an international retrospective cohort study[END_REF][START_REF] Benatar | Neurofilaments in pre-symptomatic ALS and the impact of genotype[END_REF], the interindividual variability in presymptomatic trajectories and the role of disease modifiers whose role is only partially understood [START_REF] Zhang | A C6orf10/LOC101929163 locus is associated with age of onset in C9orf72 carriers[END_REF][START_REF] Barbier | SLITRK2, an Xlinked modifier of the age at onset in C9orf72 frontotemporal lobar degeneration[END_REF].

The role of biomarkers across the presymptomatic phases

Definition and types of biomarkers

The term "biomarker" indicates an observable and measurable feature whose levels serve as objective indicators of different physiological or pathological states, or are associated to the response to therapeutic interventions [START_REF] Hendrix | Perspectives on statistical strategies for the regulatory biomarker qualification process[END_REF]. In the context of presymptomatic C9orf72 carriers, biomarkers are intended to monitor disease evolution form the presymptomatic to the full-blown stages, predict the proximity to phenoconversion and, eventually, serve as outcome measures in therapeutic trials [START_REF] Taga | Current and emerging ALS biomarkers: utility and potential in clinical trials[END_REF][START_REF] Swift | Fluid biomarkers in frontotemporal dementia: past, present and future[END_REF]. It has to kept in mind that the analytical variability of a biomarker should be appropriate for its context of use. For instance, during the preclinical phase an optimal disease-tracking biomarker should be sensitive enough to capture the evolution of the underlying pathophysiological cascade, which could last several years or decades. On the other hand, a valuable biomarker to predict clinical onset should stay as stable as possible in non-progressing carriers, and display clear and sustained changes close to phenoconversion.

Different approaches can be used to provide biomarkers, and those which contribute the most to monitor presymptomatic C9orf72 disease are summarized in Table 1.

Biomarkers to define the preclinical disease trajectory

As already stated, it is hard to define the beginning of lesional accumulation in most neurodegenerative diseases. However, C9orf72 pathophysiology offers a privileged point of view, as DPR proteins, and in particular poly(GP) proteins (originated from both sense and antisense expanded transcripts) can be detectable in the CSF of asymptomatic carriers [START_REF] Lehmer | Poly-GP in cerebrospinal fluid links C9orf72-associated dipeptide repeat expression to the asymptomatic phase of ALS/FTD[END_REF][START_REF] Meeter | Poly(GP), neurofilament and grey matter deficits in C9orf72 expansion carriers[END_REF], without being associated with biomarkers of ongoing degeneration. There is a modest but significant increase of poly(GP) over time, and overall their levels are higher in patients than in presymptomatic carriers [START_REF] Meeter | Poly(GP), neurofilament and grey matter deficits in C9orf72 expansion carriers[END_REF][START_REF] Gendron | Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis[END_REF]. Autoptic studies have shown that accumulation of RNA foci and DPR-positive inclusions precede TDP-43 nuclear delocalisation and cytoplasmic deposition, underscoring that these changes are more closely related to the expansion itself, than to downstream degenerative events [START_REF] Vatsavayai | Timing and significance of pathological features in C9orf72 expansion-associated frontotemporal dementia[END_REF].

The model of the preclinical cascade in AD suggests that brain metabolic changes may antedate structural modifications [START_REF] Jack | NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease[END_REF][START_REF] Dubois | Preclinical Alzheimer's disease: Definition, natural history, and diagnostic criteria[END_REF]. A limited number of studies have explored the role of fluorodeoxyglucose (FDG) PET in the preclinical phase of genetic FTD; for what concerns C9orf72 carriers, clusters of significant hypometabolism have been found in frontotemporal cortices, basal ganglia and thalami, at a time in which volume loss is already detectable [START_REF] Vocht | Use of Multimodal Imaging and Clinical Biomarkers in Presymptomatic Carriers of C9orf72 Repeat Expansion[END_REF].

However, information about temporal dynamics of these alterations is currently lacking.

Similarly, few studies have investigated the potential of fMRI to detect functionally compensated network dysregulations in asymptomatic carriers [START_REF] Lee | Network degeneration and dysfunction in presymptomatic C9ORF72 expansion carriers[END_REF][START_REF] Waugh | Longitudinal changes in network homogeneity in presymptomatic C9orf72 mutation carriers[END_REF]. Salience network and thalamic-seeded network alterations are identifiable early, and are somewhat reminiscent of what observed in C9orf72-associated bvFTD [START_REF] Lee | Network degeneration and dysfunction in presymptomatic C9ORF72 expansion carriers[END_REF][START_REF] Shoukry | Resting State Functional Connectivity Is Decreased Globally Across the C9orf72 Mutation Spectrum[END_REF]. The profile of connectivity changes extends over time, spreading towards the areas mostly affected in the symptomatic phase, which could herald impending neurodegeneration [START_REF] Waugh | Longitudinal changes in network homogeneity in presymptomatic C9orf72 mutation carriers[END_REF][START_REF] Agosta | Structural and functional brain signatures of C9orf72 in motor neuron disease[END_REF].

A number of structural MRI studies elegantly depicted the profile of brain changes identifiable at different points of the preclinical phase and, more recently, investigated their rate of change over time [START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF][START_REF] Lee | Network degeneration and dysfunction in presymptomatic C9ORF72 expansion carriers[END_REF][START_REF] Rohrer | Presymptomatic cognitive and neuroanatomical changes in genetic frontotemporal dementia in the Genetic Frontotemporal dementia Initiative (GENFI) study: a cross-sectional analysis[END_REF][START_REF] Panman | Gray and white matter changes in presymptomatic genetic frontotemporal dementia: a longitudinal MRI study[END_REF][START_REF] Blanc | Faster Cortical Thinning and Surface Area Loss in Presymptomatic and Symptomatic C9orf72 Repeat Expansion Adult Carriers[END_REF][START_REF] Bocchetta | Differential early subcortical involvement in genetic FTD within the GENFI cohort[END_REF][START_REF] Papma | Cognition and gray and white matter characteristics of presymptomatic C9orf72 repeat expansion[END_REF]. Overall, C9orf72 carriers display significant and widespread cortical volume loss compared to non-carriers, including frontal areas, temporo-insular cortices, associative parieto-temporal regions, and hippocampi approximately 20 to 25 years before their estimated disease onset [START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF][START_REF] Lee | Network degeneration and dysfunction in presymptomatic C9ORF72 expansion carriers[END_REF][START_REF] Rohrer | Presymptomatic cognitive and neuroanatomical changes in genetic frontotemporal dementia in the Genetic Frontotemporal dementia Initiative (GENFI) study: a cross-sectional analysis[END_REF][START_REF] Blanc | Faster Cortical Thinning and Surface Area Loss in Presymptomatic and Symptomatic C9orf72 Repeat Expansion Adult Carriers[END_REF][START_REF] Papma | Cognition and gray and white matter characteristics of presymptomatic C9orf72 repeat expansion[END_REF]. At the subcortical level, there is a diffuse and massive volume loss in the thalamus, with prominent involvement of the pulvinar subnucleus [START_REF] Bocchetta | Differential early subcortical involvement in genetic FTD within the GENFI cohort[END_REF], a profile which is coherent with what observed in the clinical phase [START_REF] Bocchetta | Thalamic nuclei in frontotemporal dementia: Mediodorsal nucleus involvement is universal but pulvinar atrophy is unique to C9orf72[END_REF]. Early cerebellar involvement has been also evidenced, in particular in lobules VIIa-Crus II and VIIb, connected to the dorsolateral prefrontal cortex via the thalamus [START_REF] Bocchetta | Differential early subcortical involvement in genetic FTD within the GENFI cohort[END_REF]. Notably, the precocity of grey matter alterations and the degree of subcortical involvement are more important in C9orf72 carriers compared to MAPT or GRN carriers [START_REF] Rohrer | Presymptomatic cognitive and neuroanatomical changes in genetic frontotemporal dementia in the Genetic Frontotemporal dementia Initiative (GENFI) study: a cross-sectional analysis[END_REF][START_REF] Bocchetta | Differential early subcortical involvement in genetic FTD within the GENFI cohort[END_REF]. Individual baseline atrophy at the asymptomatic phase (CDR+NACC FTLD = 0) may be implemented in predictive models of progression towards the prodromal and clinical disease stages [START_REF] Staffaroni | Individualized atrophy scores predict dementia onset in familial frontotemporal lobar degeneration[END_REF].

Gray matter changes are associated with, and often preceded by, white matter tracts degeneration. In C9orf72 carriers, diffusion tensor imaging (DTI) analyses identified reduced fractional anisotropy (FA), a marker of microstructural integrity, in thalamic radiation, corpus callosum, frontotemporal and corticospinal tracts (CST) [START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF][START_REF] Lee | Network degeneration and dysfunction in presymptomatic C9ORF72 expansion carriers[END_REF][START_REF] Panman | Gray and white matter changes in presymptomatic genetic frontotemporal dementia: a longitudinal MRI study[END_REF][START_REF] Papma | Cognition and gray and white matter characteristics of presymptomatic C9orf72 repeat expansion[END_REF][START_REF] Wen | Neurite density is reduced in the presymptomatic phase of C9orf72 disease[END_REF], which are critically involved in the development of FTD and ALS.

Structural changes predictably appear more pronounced and diffuse among the older carriers, namely over 40 years of age [START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF][START_REF] Papma | Cognition and gray and white matter characteristics of presymptomatic C9orf72 repeat expansion[END_REF]. However, a greater progression compared to noncarriers is barely detectable [START_REF] Panman | Gray and white matter changes in presymptomatic genetic frontotemporal dementia: a longitudinal MRI study[END_REF], and only one longitudinal study found a trend towards accelerated cortical thinning in presymptomatic carriers, tough not reaching statistical significance [START_REF] Blanc | Faster Cortical Thinning and Surface Area Loss in Presymptomatic and Symptomatic C9orf72 Repeat Expansion Adult Carriers[END_REF]. This is in line with the modest rates of atrophy observed during the clinical phase of C9orf72 disease [START_REF] Staffaroni | Rates of Brain Atrophy Across Disease Stages in Familial Frontotemporal Dementia Associated With MAPT, GRN, and C9orf72 Pathogenic Variants[END_REF].

Spinal cord imaging features have been proposed as biomarkers for the development of ALS treatments [START_REF] Mendili | Multi-Parametric Spinal Cord MRI as Potential Progression Marker in Amyotrophic Lateral Sclerosis[END_REF], with measurable progression throughout the disease course [START_REF] Van Der Burgh | Crosssectional and longitudinal assessment of the upper cervical spinal cord in motor neuron disease[END_REF].

Preysmptomatic C9orf72 carriers display white matter atrophy at the cervical level, with progressive reduction of FA in CST occurring in individuals over 40 years old [START_REF] Querin | Presymptomatic spinal cord pathology in c9orf72 mutation carriers: A longitudinal neuroimaging study[END_REF]. This is particularly relevant taking into account that no neurophysiological measures proved usefulness to detect motor neuron degeneration in asymptomatic C9orf72 carriers [START_REF] Geevasinga | Cortical Function in Asymptomatic Carriers and Patients With C9orf72 Amyotrophic Lateral Sclerosis[END_REF].

In summary, the preclinical phase of C9orf72 disease is marked by diffuse changes, mostly identifiable by means of neuroimaging approaches, which appear early and progress smoothly over the years. However, there is no sufficient evidence to ascertain if and how these changes could predict the subsequent clinical phenotype an individual carrier will manifest.

Biomarkers to support proximity to clinical onset

In neurodegenerative disorders, one of the most relevant contribution of biomarkers is the aid to identify those individuals who are going to develop the first symptoms and signs of the disease, hence predicting phenoconversion [START_REF] Jack | NIA-AA Research Framework: Toward a biological definition of Alzheimer's disease[END_REF][START_REF] Van Der Ende | A data-driven disease progression model of fluid biomarkers in genetic frontotemporal dementia[END_REF][START_REF] Benatar | Validation of serum neurofilaments as prognostic and potential pharmacodynamic biomarkers for ALS[END_REF]. This information could enhance the stratification of carriers for clinical trials, and possibly provide outcome measures for treatment response [START_REF] Boxer | New directions in clinical trials for frontotemporal lobar degeneration: Methods and outcome measures[END_REF].

Among the fluid-based biomarkers investigated for this purpose, neurofilaments turned out to be particularly useful [START_REF] Van Der Ende | Serum neurofilament light chain in genetic frontotemporal dementia: a longitudinal, multicentre cohort study[END_REF][START_REF] Rojas | Plasma Neurofilament Light for Prediction of Disease Progression in Familial Frontotemporal Lobar Degeneration[END_REF][START_REF] Saracino | Plasma NfL levels and longitudinal change rates in C9orf72 and GRN-associated diseases: from tailored references to clinical applications[END_REF][START_REF] Wilke | Stratifying the Presymptomatic Phase of Genetic Frontotemporal Dementia by Serum NfL and pNfH: A Longitudinal Multicentre Study[END_REF]. Neurofilaments are structural proteins highly expressed in axons, composed of three main subunits, heavy (NfH), medium and light chain (NfL). Both NfL and NfH are released in extracellular fluids in proportion to neuronal loss in several neurological disorders, including neurodegenerative conditions [START_REF] Khalil | Neurofilaments as biomarkers in neurological disorders[END_REF][START_REF] Bridel | Diagnostic Value of Cerebrospinal Fluid Neurofilament Light Protein in Neurology: A Systematic Review and Meta-analysis[END_REF][START_REF] Wilke | Neurofilaments in spinocerebellar ataxia type 3: blood biomarkers at the preataxic and ataxic stage in humans and mice[END_REF]. With the development of the highly sensitive Simoa technique, neurofilament dosage can be easily performed in plasma or serum, whose levels are extremely correlated to CSF ones, thus allowing less invasive, repeatable dosages [START_REF] Meeter | Neurofilament light chain: a biomarker for genetic frontotemporal dementia[END_REF][START_REF] Alirezaei | Neurofilament Light Chain as a Biomarker, and Correlation with Magnetic Resonance Imaging in Diagnosis of CNS-Related Disorders[END_REF]. NfL levels in preclinical C9orf72 carriers are comparable to controls at a group level [START_REF] Van Der Ende | Serum neurofilament light chain in genetic frontotemporal dementia: a longitudinal, multicentre cohort study[END_REF][START_REF] Meeter | Neurofilament light chain: a biomarker for genetic frontotemporal dementia[END_REF], with steady, low-amplitude increases over the years [START_REF] Saracino | Plasma NfL levels and longitudinal change rates in C9orf72 and GRN-associated diseases: from tailored references to clinical applications[END_REF][START_REF] Wilke | Stratifying the Presymptomatic Phase of Genetic Frontotemporal Dementia by Serum NfL and pNfH: A Longitudinal Multicentre Study[END_REF]. NfL levels and their annualized rates of change increase during or just before the prodromal phase [START_REF] Benatar | Neurofilaments in pre-symptomatic ALS and the impact of genotype[END_REF][START_REF] Van Der Ende | Serum neurofilament light chain in genetic frontotemporal dementia: a longitudinal, multicentre cohort study[END_REF][START_REF] Saracino | Plasma NfL levels and longitudinal change rates in C9orf72 and GRN-associated diseases: from tailored references to clinical applications[END_REF], thus allowing to identify those who are at risk of short-term progression to clinical disease, in the subsequent 2 to 5 years [START_REF] Rojas | Plasma Neurofilament Light for Prediction of Disease Progression in Familial Frontotemporal Lobar Degeneration[END_REF]. NfH are particularly stable during the presymptomatic phase, while mostly increasing at the moment of the phenoconversion and during the symptomatic stage [START_REF] Wilke | Stratifying the Presymptomatic Phase of Genetic Frontotemporal Dementia by Serum NfL and pNfH: A Longitudinal Multicentre Study[END_REF]. Their changes occur earlier and are more pronounced in individuals displaying a phenotype of ALS [START_REF] Benatar | Neurofilaments in pre-symptomatic ALS and the impact of genotype[END_REF][START_REF] Benatar | Validation of serum neurofilaments as prognostic and potential pharmacodynamic biomarkers for ALS[END_REF].

Other proteomic biomarkers have been investigated in the presymptomatic phase of genetic FTD, including those linked to synaptic function, astrogliosis, inflammation, and complement activation [START_REF] Van Der Ende | A data-driven disease progression model of fluid biomarkers in genetic frontotemporal dementia[END_REF][START_REF] Swift | Fluid biomarkers in frontotemporal dementia: past, present and future[END_REF][START_REF] Heller | Plasma glial fibrillary acidic protein is raised in progranulin-associated frontotemporal dementia[END_REF]. Overall, levels of neuronal pentraxin 2 (NPTX2) decrease, while glial fibrillary acidic protein (GFAP) and complement proteins C3b and C1q sequentially increase, along with NfL and NfH, at the transition between presymptomatic and symptomatic FTD associated with GRN mutations, whereas their trajectory is much less clear for C9orf72 expansions [START_REF] Van Der Ende | A data-driven disease progression model of fluid biomarkers in genetic frontotemporal dementia[END_REF]. Preliminary evidence on novel putative biomarkers identified in the CSF of C9orf72 patients such as chitotriosidase (CHIT1) has not been validated yet in the presymptomatic phase [START_REF] Barschke | Different CSF protein profiles in amyotrophic lateral sclerosis and frontotemporal dementia with C9orf72 hexanucleotide repeat expansion[END_REF].

The expression profile of circulating miRNAs has been found to be altered in several neurodegenerative conditions [START_REF] Grasso | Circulating miRNAs as Biomarkers for Neurodegenerative Disorders[END_REF][START_REF] Schneider | Downregulation of exosomal miR-204-5p and miR-632 as a biomarker for FTD: a GENFI study[END_REF]. In particular, a signature of four miRNA is dynamically altered throughout the presymptomatic and clinical phase of C9orf72 disease, yielding an added value in the prediction of phenoconversion [START_REF] Kmetzsch | Plasma microRNA signature in presymptomatic and symptomatic subjects with C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis[END_REF].

The transition towards the prodromal and clinical disease stage is marked by progressive cognitive and behavioral modifications, and the identification of the most appropriate neuropsychological/behavioral measures is the subject of active research. Longitudinal assessments of cognitive functions in presymptomatic cohorts of FTD mutation carriers provided information about the cognitive trajectories occurring in the main genetic groups [START_REF] Rohrer | Presymptomatic cognitive and neuroanatomical changes in genetic frontotemporal dementia in the Genetic Frontotemporal dementia Initiative (GENFI) study: a cross-sectional analysis[END_REF].

One study showed that C9orf72 carriers display worse scores in verbal fluencies since young age, without relevant changes over time, thus pointing towards a general neurodevelopmental disorder [START_REF] Lulé | Deficits in verbal fluency in presymptomatic C9orf72 mutation gene carriers-a developmental disorder[END_REF]. Episodic memory deficits have been also identified in the presymptomatic phase of C9orf72 disease, with features more closely reflecting a profile of executive impairment rather than a true amnestic syndrome of hippocampal type [START_REF] Papma | Cognition and gray and white matter characteristics of presymptomatic C9orf72 repeat expansion[END_REF][START_REF] Poos | Impairment of episodic memory in genetic frontotemporal dementia: A GENFI study[END_REF].

Among the cognitive functions most closely related to FTD spectrum, social cognition and cognitive inhibition deficits mostly occur in the late presymptomatic phase and are potential predictive biomarkers of phenoconversion [START_REF] Russell | Social cognition impairment in genetic frontotemporal dementia within the GENFI cohort[END_REF][START_REF] Montembeault | Cognitive inhibition impairments in presymptomatic C9orf72 carriers[END_REF][START_REF] Franklin | The Revised Self-Monitoring Scale detects early impairment of social cognition in genetic frontotemporal dementia within the GENFI cohort[END_REF]. This underscores the usefulness of the Social Cognition and Emotional Assessment, shortened version, (mini-SEA) [START_REF] Funkiewiez | The SEA (Social cognition and Emotional Assessment): a clinical neuropsychological tool for early diagnosis of frontal variant of frontotemporal lobar degeneration[END_REF] and the Hayling test [START_REF] Burgess | The Hayling and Brixton Tests[END_REF] to capture relevant, prodromal cognitive changes in C9orf72 carriers.

Additional changes observed in presymptomatic carriers include deficits in the praxis scores [START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF], and impairment in semantic knowledge occurring more closely to onset [START_REF] Moore | A modified Camel and Cactus Test detects presymptomatic semantic impairment in genetic frontotemporal dementia within the GENFI cohort[END_REF], in line with a profile of semantic dysfunction observed in some C9orf72 patients [START_REF] Snowden | Distinct clinical and pathological characteristics of frontotemporal dementia associated with C9ORF72 mutations[END_REF][START_REF] Saracino | Primary progressive aphasias associated with C9orf72 expansions: Another side of the story[END_REF].

As for cognitive changes, the earliest, subtle behavioral alterations, far from fulfilling bvFTD criteria, are remarkably difficult to identify. A commonly encountered difficulty is to discriminate between true new-onset behavioral changes and personality traits or attitudes which are typical of the individual and are present since long time. When feasible, repeated assessments with appropriate and sensitive tools, such as the Cambridge Behavioral Inventory (CBI-R) [START_REF] Wear | The Cambridge Behavioural Inventory revised[END_REF], could aid to provide quantitative assessments of this often elusive constellation of symptoms [START_REF] Rohrer | Presymptomatic cognitive and neuroanatomical changes in genetic frontotemporal dementia in the Genetic Frontotemporal dementia Initiative (GENFI) study: a cross-sectional analysis[END_REF]. Among the behavioral dimensions investigated in presymptomatic C9orf72 carriers, apathy scores are higher than controls and increase over time, predicting subsequent cognitive impairment [START_REF] Malpetti | Apathy in presymptomatic genetic frontotemporal dementia predicts cognitive decline and is driven by structural brain changes[END_REF].

Overall, core criteria to define the threshold of cognitive and/or behavioral impairments to comply with a diagnosis of MCBMI due to FTD have not been defined yet, apart from a global score of CDR+NACC FTLD equal to 0.5 [START_REF] Benussi | Conceptual framework for the definition of preclinical and prodromal frontotemporal dementia[END_REF]. This useful tool presents some limitations, however, as it may not reliably identify the core symptoms attributable to prodromal FTD, and discrete cognitive and behavioral impairments (such as those found in a depressed individual showing attentional deficits) are a possible source of bias.

For carriers developing an ALS phenotype, neurophysiological biomarkers support the identification of early signs of motor neuron dysfunction. Isolated denervation signs at EMG, such as positive sharp waves in a single limb muscle or in paraspinal muscles, can indicate early lower motor neuron degeneration, in absence of confounding factors [START_REF] Benatar | Preventing amyotrophic lateral sclerosis: insights from pre-symptomatic neurodegenerative diseases[END_REF]. On the other hand, increased cortical excitability at TMS points towards upper motor neuron involvement [START_REF] Geevasinga | Cortical Function in Asymptomatic Carriers and Patients With C9orf72 Amyotrophic Lateral Sclerosis[END_REF].

Quantitative measures of motor unit loss, such as Motor Unit Number Index (MUNIX) at EMG, only investigated at the clinical stage so far [START_REF] Querin | Biomarkers for C9orf7-ALS in Symptomatic and Presymptomatic Patients: State-of-the-art in the New Era of Clinical Trials[END_REF], could represent a promising approach to better define prodromal ALS.

Finally, other biomarker modalities have contributed to illustrate different pathophysiological changes occurring in the presymptomatic phase of C9orf72 disease, and it is possible that their use will be extended to investigational protocols in the upcoming years. These include a reduction of synaptic density in thalamic and frontotemporal regions observed with UCB-J PET [START_REF] Malpetti | Synaptic density in carriers of C9orf72 mutations: a [11 C]UCB-J PET study[END_REF], executive oculomotor abnormalities at video-oculographic examination [START_REF] Behler | Eye movement alterations in presymptomatic C9orf72 expansion gene carriers[END_REF], and progressive changes in cerebral blood flow measured by arterial spin labeling (ASL) MRI sequences [START_REF] Mutsaerts | Cerebral perfusion changes in presymptomatic genetic frontotemporal dementia: a GENFI study[END_REF].

Long-standing psychopathological and personality features in C9orf72 carriers blur the definition of prodromal stage

One of the difficulties in defining the prodromal stage in C9orf72 carriers is due to the frequent presence of a prominent psychopathological, rather than cognitive, symptomatology at disease onset. Patients with C9orf72-associated bvFTD often show a constellation of psychiatric symptoms and syndromes [START_REF] Ber | C9ORF72 Repeat Expansions in the Frontotemporal Dementias Spectrum of Diseases: A Flow-chart for Genetic Testing[END_REF][START_REF] Snowden | Distinct clinical and pathological phenotypes in frontotemporal dementia associated with MAPT, PGRN and C9orf72 mutations[END_REF][START_REF] Ducharme | Recommendations to distinguish behavioural variant frontotemporal dementia from psychiatric disorders[END_REF][START_REF] Gossink | Early life involvement in C9orf72 repeat expansion carriers[END_REF], which can be inaugural of the cognitive disorder. Even more noteworthy, a subset of C9orf72 carriers may present with isolated psychiatric phenotypes preceding dementia onset by several years or decades [START_REF] Sellami | Schizophrenia Phenotype Preceding Behavioral Variant Frontotemporal Dementia Related to C9orf72 Repeat Expansion[END_REF]. These are usually young adult individuals displaying psychotic syndromes, starting usually at a later age compared to primary psychiatric disorders [START_REF] Galimberti | Autosomal Dominant Frontotemporal Lobar Degeneration Due to the C9ORF72 Hexanucleotide Repeat Expansion: Late-Onset Psychotic Clinical Presentation[END_REF][START_REF] Ducharme | Psychiatric Presentations of C9orf72 Mutation: What Are the Diagnostic Implications for Clinicians?[END_REF][START_REF] Sellami | Schizophrenia Phenotype Preceding Behavioral Variant Frontotemporal Dementia Related to C9orf72 Repeat Expansion[END_REF]. The usually long disease history, in the absence of patent biomarkers of neuronal loss [START_REF] Ducharme | Psychiatric Presentations of C9orf72 Mutation: What Are the Diagnostic Implications for Clinicians?[END_REF][START_REF] Saracino | Plasma NfL levels and longitudinal change rates in C9orf72 and GRN-associated diseases: from tailored references to clinical applications[END_REF], favors the hypothesis of a dysfunctional brain disorder, eventually resulting in a degenerative syndrome. Other psychiatric disturbances display increased frequency among the presymptomatic C9orf72 carriers, including mania [START_REF] Ducharme | Psychiatric Presentations of C9orf72 Mutation: What Are the Diagnostic Implications for Clinicians?[END_REF], depression, substance abuse/dependence, and post-traumatic stress disorder [START_REF] Vocht | Psychopathology in premanifest C9orf72 repeat expansion carriers[END_REF]. Collectively, these findings point to life-long psychiatric vulnerability in the presence of the C9orf72 expansion.

This susceptibility could also translate into several atypical behavioral and personality features present since early life, including fixed behavioral patterns, reduced empathy, tendency to hoarding and excessive sporting [START_REF] Gossink | Early life involvement in C9orf72 repeat expansion carriers[END_REF]. The importance of recognizing these traits in presymptomatic carriers is two-fold: first, it provides a baseline behavioral assessment to be accounted for when evaluating the occurrence of relevant behavioral changes in the prodromal or manifest disease stage; second, it draws attention on the impact C9orf72 expansion may have on brain development and maturation. This is of fundamental importance to correctly define the age of disease onset in patients.

The presence of long-standing structural signatures in the brains of C9orf72 carriers, changing little over the years [START_REF] Lee | Network degeneration and dysfunction in presymptomatic C9ORF72 expansion carriers[END_REF][START_REF] Lulé | Deficits in verbal fluency in presymptomatic C9orf72 mutation gene carriers-a developmental disorder[END_REF][START_REF] Caverzasi | Gyrification abnormalities in presymptomatic c9orf72 expansion carriers[END_REF][START_REF] Bede | Consideration of C9orf72-associated ALS-FTD as a neurodevel-opmental disorder: insights from neuroimaging[END_REF], provides additional evidence in favor of a neurodevelopmental hypothesis. The identification of low gyrification, index of immature cortical development, in the regions which are commonly atrophied during the disease also suggests that these abnormalities might confer vulnerability to future degeneration [START_REF] Caverzasi | Gyrification abnormalities in presymptomatic c9orf72 expansion carriers[END_REF]. This is in line with the proposed role for C9ORF72 protein in nervous system maturation and synaptic modelling [START_REF] Xu | C9orf72 Dipeptide Repeats Cause Selective Neurodegeneration and Cell-Autonomous Excitotoxicity in Drosophila Glutamatergic Neurons[END_REF][START_REF] Yeh | C9orf72 is essential for neurodevelopment and motility mediated by Cyclin G1[END_REF].

Overall, several lines of evidence support an influence of C9orf72 repeat expansion on the brain which extends beyond the promotion of neurodegeneration, and the boundaries between developmental and degenerative manifestations appear to be quite blurred. The existence of international initiatives such as the Neuropsychiatric International Consortium on FTD (NIC-FTD) will hopefully expand the knowledge on these dimensions of the disease and raise clinician's awareness to atypical presentations occurring with C9orf72.

Conclusion

Research on genetic FTD and ALS is progressing at an impressive pace. In particular, major advances have been made in the understanding of the pathophysiology of C9orf72 disease since the discovery of the gene, and the events occurring in the presymptomatic phase have been partially elucidated. A shared conceptual framework, and the major acquisitions on the use of biomarkers, are undoubtably helpful for clinicians and researchers to stratify presymptomatic carriers according to their presumed proximity to disease onset. Some unresolved issues still remain, notably for what concerns the translation of evidence collected from large cohort studies to the individual level for clinical purposes. The implementation of those concepts in current practice could hopefully contribute to overcome these obstacles. Additionally, a revision of the currently adopted diagnostic criteria shall be considered, aiming to capture also earlier and milder forms, with obvious impact on disease prevention.

Functional connectivity

Early salience and thalamicseeded network dysregulation [START_REF] Lee | Network degeneration and dysfunction in presymptomatic C9ORF72 expansion carriers[END_REF][START_REF] Waugh | Longitudinal changes in network homogeneity in presymptomatic C9orf72 mutation carriers[END_REF] Cerebral blood flow (ASL) Decreased from 12.5 years before onset [START_REF] Mutsaerts | Cerebral perfusion changes in presymptomatic genetic frontotemporal dementia: a GENFI study[END_REF] Gyrification index Congenitally reduced in C9orf72 carriers [START_REF] Caverzasi | Gyrification abnormalities in presymptomatic c9orf72 expansion carriers[END_REF] Cervical spinal cord WM volume and FA Altered metrics mostly in carriers >40 years [START_REF] Querin | Presymptomatic spinal cord pathology in c9orf72 mutation carriers: A longitudinal neuroimaging study[END_REF] FDG PET uptake Hypometabolism consistent with structural changes [START_REF] Vocht | Use of Multimodal Imaging and Clinical Biomarkers in Presymptomatic Carriers of C9orf72 Repeat Expansion[END_REF] UCB-J PET uptake Reduced synaptic density in preclinical phase [START_REF] Malpetti | Synaptic density in carriers of C9orf72 mutations: a [11 C]UCB-J PET study[END_REF] Neuropsychological Verbal fluencies Reduced scores since young age [START_REF] Lulé | Deficits in verbal fluency in presymptomatic C9orf72 mutation gene carriers-a developmental disorder[END_REF] Episodic memory Preclinical changes reflecting executive dysfunction [START_REF] Poos | Impairment of episodic memory in genetic frontotemporal dementia: A GENFI study[END_REF] Social cognition Deficits in late presymptomatic phase [START_REF] Russell | Social cognition impairment in genetic frontotemporal dementia within the GENFI cohort[END_REF] Cognitive inhibition Deficits mostly in >40 years [START_REF] Montembeault | Cognitive inhibition impairments in presymptomatic C9orf72 carriers[END_REF] Semantic knowledge Deficits in late presymptomatic phase [START_REF] Moore | A modified Camel and Cactus Test detects presymptomatic semantic impairment in genetic frontotemporal dementia within the GENFI cohort[END_REF] Gestural praxis Impaired before 40 years [START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF] Neurophysiological Cortical excitability Increase in ALS patients [START_REF] Geevasinga | Cortical Function in Asymptomatic Carriers and Patients With C9orf72 Amyotrophic Lateral Sclerosis[END_REF]

Figure 1 .

 1 Figure 1. Overview on the presymptomatic and symptomatic stages in C9orf72 disease.

Table legend Table 1 . Main biomarkers studied to monitor C9orf72 disease

 legend1 . ASL: arterial spin labeling; CHIT1: chitotriosidase; FA: fractional anisotropy; FDG: fluorodeoxyglucose; miRNA: micro-RNA; NfH: neurofilament heavy chain; NfL: neurofilament light chain; UCB-J: synaptic vesicle glycoprotein 2A PET tracer; WM: white matter.

	Type of biomarker Measure	Relevance	References
		Poly(GP) proteins (CSF)	High levels since the earliest stages	[53,54]
	Fluid-based	NfL and NfH (CSF and plasma)	Increase in prodromal phase, ≤5 years before phenoconversion	[47,77-80]
		MiRNA	Altered profile, mostly in prodromal carriers	[90]
		CHIT1 (CSF)	Increase in patients	[87]
		Cortical thickness, brain volumes, WM microstructure	Altered from 20-25 years before onset, slowly progressive	[35,58,62-66]
	Neuroimaging

I.L.B. served as a member of advisory boards for Prevail Therapeutics, Denali Therapeutics and Alector, and received research grants from ANR, DGOS, Vaincre Alzheimer Association, ARSla Association, Fondation Plan Alzheimer, JPND outside of the present work.