Nadjia Kachenoura
email: nadjia.kachenoura@inserm.fr

PhD Emilie Bollache

PhD Gilles Soulat

MD PhD Stéphanie Clément-Guinaudeau

MD Golmehr Ashrafpoor

MD Ludivine Perdrix

MD Benoit Diebold

PhD Magalie Ladouceur

PhD Elie Mousseaux

Right Ventricular Diastolic Function in Aging: A Head-to-head Comparison between Phase-Contrast MRI and Doppler Echocardiography

Keywords: phase-contrast MRI, right ventricle, diastolic function, aging Declarations

Purpose. To evaluate right ventricle (RV) diastolic function from phase-contrast MRI (PC-MRI) in aging.

Methods. 89 healthy individuals (50 men, 43±15 years) underwent cardiac MRI including 2D PC-MRI (1.5T) and reference Doppler echocardiography of both ventricles on the same day. Conventional echocardiographic parameters were estimated: early (E, cm/s) and atrial (A) peak velocities as well as myocardial early peak longitudinal velocity (E'). PC-MRI images were analyzed using custom software, providing: E', E and A waves along with respective peak flow rates (Ef, Af, mL/s) and filling volume (mL), for both ventricles. Intra-and interobserver reproducibility was studied in 30 subjects and coefficients of variation (CoV) as well as intra-class correlation coefficients (ICC) were provided.

Results. RV diastolic function indices derived from PC-MRI data were reproducible (CoV≤21%, ICC≥0.75) and reliable as reflected by significant associations with left ventricular diastolic function indices assessed using both echocardiography (linear regression Pearson correlation coefficient r≤0.59) and PC-MRI (r≤71). Despite the fair associations between RV echocardiography and PC-MRI (r≤0.25), the highest correlation with age was obtained for MRI Ef/Af ratio (r=-0.64, p<0.0001 vs. r=-0.40, p=0.0001 for echocardiographic E/A). Among PC-MRI E/A ratios, highest correlations with age were observed for flow rate and mean velocity ratios (r=-0.61, p<0.0001) as compared to maximal velocity ratios (r=-0.56, p<0.0001). Associations with age for E' were equivalent between PC-MRI (mean velocity: r=-0.40, p<0.0001; maximal velocity: r=-0.36, p=0.0005) and echocardiography (r=-0.36, p=0.0006). Finally, the significant and age-independent associations between RV mass/end-diastolic volume and E' were stronger for PC-MRI (mean velocity: r=-0.36, p=0.0006; maximal velocity: r=-0.28, p=0.007) than echocardiography (r=-0.09, p=0.38).

Conclusion. PC-MRI tricuspid inflow and annulus myocardial velocity parameters were reproducible and able

to characterize age-related variations in RV diastolic function.

Introduction

Right ventricular (RV) dysfunction has been widely associated with increased mortality in patients with congenital heart disease, heart failure, pulmonary hypertension and coronary artery disease [START_REF] Haddad | Right Ventricular Function in Cardiovascular Disease, Part I: Anatomy, Physiology, Aging, and Functional Assessment of the Right Ventricle[END_REF]. Although the in-depth exploration of the left ventricular (LV) morphology and function using imaging techniques is now commonly performed, the RV has remained the forgotten ventricle for a long time. This might be due to its anatomical location and orientation, its complex shape and geometry as well as its multiple trabeculae. Similar to LV, RV diastolic dysfunction precedes the decrease in ejection fraction and systolic dysfunction. Accordingly, the early and robust detection and quantification of diastolic dysfunction are crucial for optimal patient management. In clinical routine, the evaluation of LV and RV diastolic function is achieved using Doppler echocardiography [START_REF] Horton | Assessment of the Right Ventricle by Echocardiography: A Primer for Cardiac Sonographers[END_REF]. For both chambers, several diastolic parameters are conventionally measured: peak velocities of the early (E) and late (A) filling inflow, annular early longitudinal peak velocity (E'), as well as the associated E/A and E/E' ratios.

MRI cine images are commonly used for the evaluation of both LV and RV volumes, myocardial mass and systolic function. Attempts to evaluate RV diastolic function have been achieved while evaluating RV volumes variations throughout the cardiac cycle [START_REF] Maceira | Reference right ventricular systolic and diastolic function normalized to age, gender and body surface area from steady-state free precession cardiovascular magnetic resonance[END_REF]. In addition, while cine MRI has been recently proposed to assess RV strain using feature tracking techniques [START_REF] Kempny | Quantification of biventricular myocardial function using cardiac magnetic resonance feature tracking, endocardial border delineation and echocardiographic speckle tracking in patients with repaired tetralogy of fallot and healthy controls[END_REF], again such evaluation in the RV remains less common than in the LV or left atrium [START_REF] Scatteia | Strain imaging using cardiac magnetic resonance[END_REF]. Furthermore, despite the excellent anatomical coverage provided by MRI, measurements of the abovementioned morphological and functional quantitative indices were shown to be less reproducible on the RV than the LV [START_REF] Mooij | Reproducibility of MRI measurements of right ventricular size and function in patients with normal and dilated ventricles[END_REF][START_REF] Backhaus | Cardiovascular magnetic resonance imaging feature tracking: Impact of training on observer performance and reproducibility[END_REF].

Phase-contrast MRI (PC-MRI) has been successfully used for the evaluation of LV diastolic function, revealing high agreement with Doppler echocardiography as well as ability of MRI measures to detect diastolic dysfunction with a high reliability [START_REF] Hartiala | Velocity-encoded cine MRI in the evaluation of left ventricular diastolic function: Measurement of mitral valve and pulmonary vein flow velocities and flow volume across the mitral valve[END_REF][START_REF] Paelinck | Feasibility of tissue magnetic resonance imaging[END_REF][START_REF] Paelinck | Tissue cardiovascular magnetic resonance demonstrates regional diastolic dysfunction in remote tissue early after inferior myocardial infarction[END_REF][START_REF] Marsan | Comparison Between Tissue Doppler Imaging and Velocity-Encoded Magnetic Resonance Imaging for Measurement of Myocardial Velocities, Assessment of Left Ventricular Dyssynchrony, and Estimation of Left Ventricular Filling Pressures in Patients With Ischemic Cardiomyopathy[END_REF][START_REF] Rathi | Routine evaluation of left ventricular diastolic function by cardiovascular magnetic resonance: A practical approach[END_REF][START_REF] Rubinshtein | Comparison of Magnetic Resonance Imaging Versus Doppler Echocardiography for the Evaluation of Left Ventricular Diastolic Function in Patients With Cardiac Amyloidosis[END_REF][START_REF] Bollache | Automated left ventricular diastolic function evaluation from phase-contrast cardiovascular magnetic resonance and comparison with Doppler echocardiography[END_REF][START_REF] Buss | Classification of diastolic function with phase-contrast cardiac magnetic resonance imaging: validation with echocardiography and age-related reference values[END_REF][START_REF] Suzuki | Validity and Reliability of Three-chamber-View Threedirectional Encoded Phase-contrast Magnetic Resonance Velocity-Vector Mapping for Transmitral Velocity Measurements: Comparison with Doppler Echocardiography and Intra-and Inter-observer Variability[END_REF][START_REF] Seemann | Assessment of diastolic function and atrial remodeling by MRI -validation and correlation with echocardiography and filling pressure[END_REF][START_REF] Fyrdahl | Sector-wise golden-angle phase contrast with high temporal resolution for evaluation of left ventricular diastolic dysfunction[END_REF][START_REF] Thavendiranathan | Mitral annular velocity measurement with cardiac magnetic resonance imaging using a novel annular tracking algorithm: Validation against echocardiography[END_REF]. We hypothesise that velocity imaging through the tricuspid valve as well as at the RV basal slice along with a shape-independent segmentation technique would help for the assessment of RV diastolic function. Accordingly, we first adapted a software that was previously designed for LV diastolic function quantification [START_REF] Bollache | Automated left ventricular diastolic function evaluation from phase-contrast cardiovascular magnetic resonance and comparison with Doppler echocardiography[END_REF], to the evaluation of tricuspid flow patterns and tricuspid annulus longitudinal motion from PC-MRI data, while evaluating the intra-and inter-observer reproducibility of the derived RV diastolic function measurements.. We also performed a head-to-head comparison of PC-MRI RV diastolic function indices against those estimated using the reference Doppler echocardiography performed on the same day in healthy volunteers.

Finally, physiological associations with age and RV mass to end-diastolic volume ratio were studied, and we further tested the reliability of PC-MRI-derived indices of RV diastolic function through their associations with echocardiographic and PC-MRI indices of LV diastolic function while assuming an interrelated biventricular aging.

Materials and Methods

Study population and data acquisition

A group of 100 healthy volunteers (58 men, 42±15 years) who had Doppler echocardiography and MRI exams on the same day for the evaluation of LV and RV function, was included. All subjects were asymptomatic and free of overt cardiovascular disease defined as a clinical history of hypertension, diabetes mellitus, dyslipidaemia, renal disease, known inflammatory conditions, and malignancy. The study protocol was approved by the institutional review board and informed consent was obtained from all participants. Doppler echocardiography was performed by a 15-year experienced echocardiographer using a GEMS Vivid 7 system (GE Healthcare, Chicago, IL, USA). Both transmitral and tricuspid early filling and late atrial filling (E and A, respectively) peak velocities as well as LV mitral and RV tricuspid annular early peak longitudinal velocities (E') were measured. MRI was performed using a 1.5 T system (Signa HDx, GE Healthcare, Chicago, IL, USA). First, standard cine steady-state free precession (SSFP) sequences were acquired in long and short axis views during consecutive breath-holdings to cover the whole heart using the following averaged scan parameters: echo time TE = 1.5 ms, repetition time TR = 3.7 ms, flip angle = 50°, acquisition matrix = 260×192, pixel size = 0.74 mm, slice thickness = 8 mm, views per segment = 12, reconstructed temporal resolution = 15ms after applying a view sharing technique. Then, 2-chamber and 4-chamber views helped position planes in the LV and RV perpendicular to the inflow and located below the annulus at the level of the tips of the opened mitral and tricuspid valve leaflets, respectively, to acquire trans-mitral and trans-tricuspid through-plane velocity series using a retrospectively ECGgated PC pulse sequence during breath-holding and the following scan parameters: encoding velocity Venc = 180 cm/s, TE = 3.1 ms, TR = 7.6 ms, views per segment = 2, view sharing was used resulting in an effective temporal resolution of 15 ms. Moreover, a myocardial longitudinal velocity series was acquired on the most basal slice location during breath-holding using retrospective ECG gating and the following scan parameters: Venc = 15 or 20 cm/s, TE = 5 ms, TR = 9.5 ms, views per segment = 2, view sharing was used resulting in an effective temporal resolution of 20 ms. These additional acquisition parameters were used for both flow and tissue velocity-encoded series: flip angle = 20°, acquisition matrix 256x128, pixel size = 1.6-1.9 mm, slice thickness = 8 mm. Immediately after MRI acquisitions, central systolic (SBP) and diastolic (DBP) blood pressures were measured at the right carotid artery using the applanation tonometry PulsePen device (DiaTecne, Milano, Italy).

Cine SSFP images were analyzed using QMASS ® software (Medis, Leiden, the Netherlands), resulting in RV end-diastolic (EDV) and end-systolic (ESV) volumes as well as RV mass. RV ejection fraction as well as its mass normalized by EDV were further derived.

Analysis of MRI data and extraction of diastolic function indices

PC-MRI data analysis was performed by 10-year experienced operators blinded to patient characteristics and echocardiographic findings. PC-MRI modulus images were challenging to segment because of the flow-related contrast variations throughout time. Therefore, we preferred to process phase velocity images, which presented connected areas in terms of velocity sign within the inflow region. A custom software previously designed for trans-mitral flow quantification [START_REF] Bollache | Automated left ventricular diastolic function evaluation from phase-contrast cardiovascular magnetic resonance and comparison with Doppler echocardiography[END_REF] was used. Processing steps are briefly summarized below.

The segmentation algorithm was based on the connectivity properties of mitral and tricuspid velocities and comprised three main steps. First, a region of interest (ROI) was manually placed around the trans-valvular inflow on a single phase at the middle of the cardiac cycle, to ensure valve opening. Then, mean velocity was calculated within this ROI for each time phase, and the cardiac phase corresponding to its highest absolute value was used for segmentation initialization. Such automated initialization was based on the detection of the largest connected inflow region, in terms of velocity sign. The centre of mass of this region was then calculated and reported to the neighbouring time phases. Third, the largest connected region around this centre of mass was automatically detected in these neighbouring phases, and their centre of mass was used to repeat the process phase by phase towards the beginning and the end of the cardiac cycle.

After time-resolved valvular orifice segmentation, velocity and flow-rate waveforms were derived (Figure 1.A).

The mitral and tricuspid flow mean and maximal velocity curves were used to estimate velocity-related parameters (Emean, Amean and Emax, Amax, respectively, in cm/s), by automatically detecting the two highest local peaks over the diastolic period. Similar processing was applied to the mitral and tricuspid flow-rate curve to detect the early (Ef, in mL/s) and late (Af, in mL/s) RV peak filling rate. The filling volume (FV, in mL) was defined as the area under the mitral or tricuspid flow-rate curve comprised between the beginning and the end of the filling period (Figure 1.A). Emean/Amean, Emax/Amax and Ef/Af ratios as well as peak filling rates normalized by the filling volume (Ef/FV and Af/FV, in s -1 ), were further calculated.

Myocardial longitudinal motion analysis was also performed on velocity images. However, the connectivity process was not adapted because of the basal bi-directional (up and down) longitudinal motion throughout the cardiac cycle, inducing positive and negative velocities. Accordingly, a classification based on the k-means algorithm was applied to velocity profiles of fixed pixels over the whole cardiac cycle, within an ROI manually drawn around the LV and RV on a single phase. This classification allowed isolating a myocardial cluster on the LV lateral wall and RV free wall, respectively, which was then used to derive time-resolved maximal and mean myocardial longitudinal velocity waveforms (Figure 1.B). E'mean and E'max were extracted from both mean and maximal myocardial velocity curves, respectively, as the first local peak during the RV filling period. Finally, Emean/E'mean and Emax/E'max ratios were also reported. Of Note, quality of PC velocity images and the derived velocity and flow-rate waveforms was rated visually as good or poor (in case of absence of RV free wall myocardial cluster or noisy velocity profiles), and data with poor quality were excluded from further analysis.

Variability study

Intra-and inter-operator variability of RV diastolic function indices was studied on a subgroup of 30 randomly selected subjects (43±18 years, 15 males), while repeating the analysis by the first rater (EB) and then by two independent raters (EB and NK), respectively, and extracting the abovementioned functional velocity and flowrate quantitative parameters measurements from PC-MRI data (Emean, Amean, Emax, Amax, Ef, Af, FV, E'mean, E'max).

Statistical analysis

The study group was divided into 2 subgroups according to age median: group 1 ≤ 40 years and group 2 > 40 years. Mean value and standard deviation were provided for basic characteristics, MRI RV volumes and mass, as well as Doppler echocardiography and PC-MRI RV diastolic function indices. A Wilcoxon rank-sum test was used to study significance of the differences between the 2 age groups. Linear regression was used to study physiological associations between indices of RV diastolic function and age, as well as the agreement between imaging modalities both on the RV and LV. For associations with age, further multivariate models were used while adjusting for cofounding effects such as gender and body mass index (BMI). Associations between Doppler echocardiography-derived LV E, A waves, mitral annulus E' velocity and their PC-MRI-derived counterparts on the RV extracted from either mean, maximal velocity or flow rate, as well as associations of paired indices between the LV and RV as assessed by Doppler and PC-MRI, were studied using linear regressions. Finally, linear regressions were also used to investigate the relationship between RV mass to EDV ratio and RV diastolic relaxation-related E', and multivariate models were used to adjust for age. Pearson correlation coefficients were provided. Intra-and inter-operator variability of PC-MRI tricuspid blood flow and myocardial parameters calculation was assessed using linear regressions, intra-class correlation coefficients (ICC), coefficients of variation (CoV) and Bland-Altman analyses. Mean bias and limits of agreement, as defined as mean bias ± 1.96 x standard deviation, were reported. A p value < 0.05 was considered as significant. Statistical analyses were performed using JMP Pro v14.0.0 (SAS Institute, Cary, NC, USA).

Results

Processing time for the evaluation from PC-MRI of RV diastolic function for one subject was less than 3 minutes, on a personal computer (CPU 2.67 GHz, 3 Gb RAM). While PC-MRI tricuspid flow data were analyzed and inflow-related indices were extracted successfully in all subjects, 11 subjects were excluded from further analyses due to myocardial dataset poor quality. Table 1 summarizes basic characteristics, MRI RV volumes and mass according to age group, for the remaining 89 healthy volunteers (50 males, age = 43±15 [20-79] years) who had all measures. We found a significant age-related decrease in end-systolic and end-diastolic RV volumes along with a slight increase in RV ejection fraction and unchanged RV mass, resulting in a significant elevation of RV mass to EDV ratio with age.

RV PC-MRI data analysis was reproducible, as reflected by high intra-(Figure 2) and inter-(Figure 3) observer intra-class correlation coefficients (ICC), low mean biases and narrow limits of agreement, as well as low coefficients of variation (CoV) for the estimation of trans-tricuspid flow rate and velocity as well as RV basal myocardial longitudinal velocity parameters: CoV=4.6% and 18% for Emax, CoV=6.7% and 9.9% for Amax, CoV=3.1% and 7.6% for Emean, CoV=5.9% and 8.2% for Amean, CoV=3.3% and 2.8% for Ef, CoV=5.2% and 2.3% for Af, CoV=7.7% and 8.6% for FV, CoV=21% and 21% for E'max, CoV=15% and 14% for E'mean, respectively.

Only fair associations were found between PC-MRI and echocardiography RV diastolic function indices. Such correlations did not exceed r=0.28, p=0.006 for E, r=0.22, p=0.04 for A, r=0.45, p<0.0001 for E/A, whether calculated in PC-MRI from tricuspid mean, maximal velocity or flow rate; r=0.30, p=0.01 for E' whether calculated in PC-MRI from RV myocardial mean or maximal velocity.

On the other hand, stronger correlations were found between both modalities on the LV: r≥0.42, p<0.0001 for E, r≥0.30, p≤0.004 for A, r≥0.61, p<0.0001 for E/A, r≥0.30, p≤0.006 for E'. In addition, significant associations were obtained between all PC-MRI-derived RV diastolic function indices and LV indices, as assessed by either Doppler echocardiography or PC-MRI (Table 2), as well as between RV and LV Doppler paired indices: E: r=0.53, p<0.0001; A: r=0.35, p=0.0007; E': r=0.36, p=0.0007. Distribution of Doppler echocardiography and PC-MRI RV diastolic function indices according to age group are summarized in Table 3, along with their linear regressions with age. As expected, a decrease in E wave and an increase in A wave were observed with age when using both modalities. Such trends reached significance for echocardiographic A wave (r=0.40, p<0.0001) and PC-MRI E wave, whether calculated from mean (r=-0.57, p<0.0001), maximal (r=-0.58, p<0.0001) velocity or flow rate (r=-0.53, p<0.0001; r=-0.47, p<0.0001 when normalized by filling volume) curves. The increase in PC-MRI A wave with age was significant when derived from mean velocity (r=0.22, p=0.03) or flow rate curve normalized by filling volume (Af/FV: r=0.60, p<0.0001).

The overall decrease in E and increase in A waves resulted in a significant decrease in E/A ratio, which showed a higher correlation with age when considering PC-MRI (Emax/Amax: r=-0.56, p<0.0001; Emean/Amean: r=-0.61, p<0.0001; Ef/Af: r=-0.64, p<0.0001) as compared to echocardiography (r=-0.40, p=0.0001; Figure 4). Of note, highest correlations with age among PC-MRI E/A ratios were observed for flow rate and mean velocity as compared to maximal velocity. We also obtained the expected significant decrease in myocardial longitudinal velocities with age, when using both echocardiography (r=-0.36, p=0.0006) and PC-MRI (E'mean: r=-0.40, p<0.0001; E'max: r=-0.36, p=0.0005; Figure 4). The decrease in E' resulted in a slight increase in E/E', revealing an elevation of RV filling pressures with age, although remaining within a normal range for both modalities.

Finally, most significant associations with age were further independent of the cofounding effect of gender or increased BMI.

Significant and stronger associations between RV mass/EDV and E' were found for PC-MRI (E'max: r=-0.28, p=0.007; E'mean: r=-0.36, p=0.0006) as compared to echocardiography (r=-0.09, p=0.38; Table 4). Such association remained significant after adjustment for age only when considering PC-MRI E' derived from mean velocity curve.

Discussion

Quantification of RV diastolic function remains particularly challenging for both MRI and echocardiography given the complex shape, thin wall and anatomical location of the RV. Indeed, while LV diastolic function has been widely explored using Doppler echocardiography, 2D PC-MRI and more recently 4D flow MRI, fewer echocardiographic studies have reported RV exploration, highlighting its complex evaluation [START_REF] Litwin | Should We Test for Diastolic Dysfunction? How and How Often?[END_REF]. Even fewer studies were found in the MRI literature and most of them focused on cine anatomical images [START_REF] Suzuki | Assessment of right ventricular diastolic and systolic function in patients with dilated cardiomyopathy using cine magnetic resonance imaging[END_REF][START_REF] Maceira | Reference right ventricular systolic and diastolic function normalized to age, gender and body surface area from steady-state free precession cardiovascular magnetic resonance[END_REF][START_REF] Liu | Normal values for myocardial deformation within the right heart measured by feature-tracking cardiovascular magnetic resonance imaging[END_REF][START_REF] Sato | The impact of ambrisentan and tadalafil upfront combination therapy on cardiac function in scleroderma associated pulmonary arterial hypertension patients: cardiac magnetic resonance feature tracking study[END_REF].

Age-related variations in RV diastolic indices

In the present work, a semi-automated and fast analysis including segmentation of the tricuspid valve orifice, myocardial clustering and quantification of RV diastolic function indices was applied to PC-MRI data.

Quantitative indices were found to be reproducible, especially when estimated from flow rate and mean velocity curves, which might be less prone to noise and to the complex geometry and flow patterns of the RV than maximal velocities. Such indices were then compared to echocardiography in 89 healthy volunteers who had both exams on the same day. Discrepancies we observed between echocardiography and PC-MRI might be explained by several factors which can be related to: 1) technical differences between such modalities, including temporal resolution which has a major impact on the measurement of peak velocities, or also the fact that PC-MRI measurements are based on velocities perpendicular to a 2D plane while Doppler echocardiographic measurement is based along a unidirectional beam; 2) inherent challenges to image the RV because of the irregular shape including for proper alignment between slice or beam positioning and the flow or myocardial motion direction; 3) physiological differences between the two non-simultaneous exams, including free breathing vs. breath-holding which can have an impact on diastolic function; 4) patient body size which might technically affect echocardiographic more than MRI measures. This might be corroborated by the fact that MRI-to-MRI associations were stronger than echocardiography vs. MRI when comparing RV and LV diastolic function indices, which further demonstrated the reliability of our measurements. Indeed, as expected, parallel changes in RV and LV diastolic function were observed in our healthy population. In addition, differences between MRI and Doppler echocardiography were toned down on the LV with a less complex geometry when compared to the RV. Despite the fair correlation between PC-MRI and echocardiography, associations of RV diastolic function indices as assessed by both imaging modalities with age were in line with previous echocardiographic findings [START_REF] Kukulski | Normal regional right ventricular function and its change with age: a Doppler myocardial imaging study[END_REF][START_REF] Watanabe | Influence of Aging on Cardiac Function Examined by Echocardiography[END_REF][START_REF] Innelli | The impact of ageing on right ventricular longitudinal function in healthy subjects: a pulsed tissue Doppler study[END_REF][START_REF] Chiha | Does Normal Ageing Alter Right Ventricular Relaxation Properties? A Tissue Doppler Study[END_REF][START_REF] Vriz | The impact of age and gender on right ventricular diastolic function among healthy adults[END_REF], and consistent with the knowledge on LV filling and relaxation that has been extensively reported in both MRI and echocardiographic literature [START_REF] Aquaro | Diastolic dysfunction evaluated by cardiac magnetic resonance: the value of the combined assessment of atrial and ventricular function[END_REF][START_REF] Zile | Diastolic heart failure--abnormalities in active relaxation and passive stiffness of the left ventricle[END_REF]. Indeed, we found a decrease in tricuspid early filling wave E and myocardial longitudinal peak velocity E', as the myocardium might stiffen with aging due to changes in its composition, as well as an increase in A wave, reflecting a compensatory contribution of the right atrium to maintain appropriate RV filling. This resulted in a significant decrease in E/A and a slight increase in E/E' ratios. Such associations with age were slightly stronger for PC-MRI indices when compared to echocardiography.

Superiority of PC-MRI mean velocity-and flow rate-derived RV diastolic indices over maximal velocities

PC-MRI diastolic function indices were extracted from maximal velocities (Emax, Amax, E'max), as usually performed using Doppler echocardiography, but also from mean velocities (Emean, Amean, E'mean) and flow rates (Ef, Af), in order to reduce PC-MRI errors due to both velocity noise and changes, throughout diastole, in transtricuspid flow jet orientation relative to the acquisition plane positioning. Indeed, PC-MRI when combined with an automated segmentation enables the delineation of the tricuspid orifice through time, providing time-resolved RV filling flow rate which is an instantaneous volume and thus less sensitive to jet orientation than maximal velocity waveform, which can be underestimated if the acquisition plane is not positioned exactly perpendicular to the tricuspid inflow. As such, PC-MRI diastolic function indices derived from mean velocity and flow rate curves resulted in overall higher or equivalent correlations with age and were found to be more reproducible than maximal velocity indices. One might also emphasize the consistency of PC-MRI flow rate indices when normalized to filling volume to account for preload, which is a major concern when evaluating RV diastolic function.

Association between RV PC-MRI relaxation-related E' and mass to end-diastolic volume

Our RV cine MRI data analysis also revealed the expected [START_REF] Fiechter | Age-related normal structural and functional ventricular values in cardiac function assessed by magnetic resonance[END_REF][START_REF] Kawel-Boehm | Normal values for cardiovascular magnetic resonance in adults and children[END_REF] decrease in RV volumes as well increase in ejection fraction and RV mass to end-diastolic volume ratio with age. This latter index of RV remodeling was significantly associated with RV relaxation, as quantified by E', though only for PC-MRI. Such association was independent of age and was again stronger for mean velocity than maximal velocity-derived E', consistently with the aforementioned findings.

Diastolic dysfunction is a subclinical alteration underlying heart failure, before the apparition of decreased ejection fraction and has been widely described in the LV. In addition, it has shown [START_REF] Rommel | Load-Independent Systolic and Diastolic Right Ventricular Function in Heart Failure With Preserved Ejection Fraction as Assessed by Resting and Handgrip Exercise Pressure-Volume Loops[END_REF] in patients during the compensated phase of heart failure with preserved ejection fraction, that RV diastolic dysfunction was impaired while RV systolic function remained stable, suggesting that diastolic function alteration might be a biventricular phenomenon. Accordingly, MRI, which is already the modality of choice to evaluate volumes, ejection fraction and myocardial mass on both ventricles, along with the addition within the same cardiac exam of two 15 to 20second acquisitions and 3-minute analysis might be of major clinical usefulness in the early detection, management and follow-up of patients with incipient heart failure.

Limitations

While the analysis of tricuspid inflow images was successful in all cases, E' could not be measured from PC-MRI in 11% of subjects, indicating the need for better-resolved techniques that are more robust to the RV thin wall such as feature tracking, which can be used on conventionally acquired cine MRI long axis data in clinical routine, to track the tricuspid annulus over the cardiac cycle and derive its longitudinal velocity as previously done in the LV [START_REF] Seemann | Assessment of diastolic function and atrial remodeling by MRI -validation and correlation with echocardiography and filling pressure[END_REF]. Another limitation of our study is the lack of a gold standard that would be independent of both MRI and echocardiography imaging modalities. However, invasive data were not available in our healthy volunteers.

Conclusion

A fast and reproducible analysis of PC-MRI data provided reliable quantitative indices of tricuspid valve inflow and annular myocardial longitudinal motion, which were able to detect in 89 healthy volunteers the expected agerelated changes in RV diastolic function, as well as independent association between diastolic relaxation and RV remodeling, evaluated through mass to end-diastolic volume ratio. The addition of such data acquired within a breath-hold to a routine MRI exam might be of clinical usefulness in disease involving the RV, such as pulmonary hypertension, heart failure or congenital heart disease.

Fig. 1 :

 1 Fig. 1: Right ventricular tricuspid filling inflow (a) and myocardial annulus (b) phase-contrast (PC)-MRI data analysis. Top: color-coded (see scale) through-plane velocity images on a diastolic phase. Results from the segmentation are illustrated with green contours. Bottom: extracted tricuspid flow rate (a) and myocardial longitudinal mean velocity (b) waveforms throughout the cardiac cycle, along with quantitative diastolic function indices: early (Ef) and late (Af) trans-tricuspid peak filling rate and filling volume (FV); basal myocardial early peak longitudinal velocity (E').

Fig. 2 :

 2 Fig. 2: Variability of repeated extraction by the same rater of PC-MRI RV diastolic function parameters derived from maximal (a) and mean (b) velocities as well as flow rate (c), in terms of linear regression (top rows) and Bland-Altman analysis (bottom rows). Intra-class correlation coefficient (ICC) and mean bias (solid horizontal lines) along with limits of agreement (dotted lines), are provided.

Fig. 3 :

 3 Fig. 3: Variability of extraction by two independent raters of PC-MRI RV diastolic function parameters derived from maximal (a) and mean (b) velocities as well as flow rate (c), in terms of linear regression (top rows) and Bland-Altman analysis (bottom rows). Intra-class correlation coefficient (ICC) and mean bias (solid horizontal lines) along with limits of agreement (dotted lines), are provided.

Fig. 4 :

 4 Fig. 4: Linear associations with age of echocardiographic (a) and PC-MRI (b) indices of tricuspid flow and myocardial longitudinal velocities.

FiguresFig. 1 :Fig. 2 :

 12 Figures

Fig. 3 :

 3 Fig. 3: Variability of extraction by two independent raters of PC-MRI RV diastolic function parameters derived from maximal (a) and mean (b) velocities as well as flow rate (c), in terms of linear regression (top rows) and Bland-Altman analysis (bottom rows). Intra-class correlation coefficient (ICC) and mean bias (solid horizontal lines) along with limits of agreement (dotted lines), are provided.

Table 1 .

 1 Basic characteristics, MRI-derived RV end-diastolic (EDV) and end-systolic (ESV) volumes, ejection fraction (EF) and mass for both age groups.

	Tables.
		≤ 40 years	> 40 years	p value
	Number of subjects	47	42
	Gender (Males: N, %)	N=27, 57%	N=23, 55%
	Age (years)	31±5.9	57±9.3	<0.0001
	BMI (kg/m²)	24±4.1	25±3.3	0.02
	Heart rate (bpm)	67±9.0	67±9.9	0.83
	SBP (mmHg)	92±11	102±14	0.0003
	DBP (mmHg)	70±9.8	81±11	<0.0001
	MRI RV volumes and mass indices
	EDV (ml)	148±34	129±46	0.02
	EDV/BSA (ml/m²)	80±15	69±21	0.003
	ESV (ml)	62±20	50±23	0.003
	ESV/BSA (ml/m²)	34±9.4	26±11	0.0003
	EF (%)	58±8.1	62±7.0	0.02
	Mass (g)	37±9.7	38±12	0.85
	Mass/BSA (g/m²)	20±4.1	20±5.2	0.96
	Mass/EDV (g/ml)	0.25±0.06	0.30±0.08	0.0006
	BMI: body mass index; BSA: body surface area; DBP: central diastolic blood pressure; EDV: end-
	diastolic volume; EF: ejection fraction; ESV: end-systolic volume; RV: right ventricular; SBP:
	central systolic blood pressure.

Table 2 .

 2 Associations between PC-MRI-derived RV diastolic function indices and LV diastolic function indices as extracted from either echocardiography (left) or PC-MRI (right). Correlation coefficients (r) and p values are provided.

		LV diastolic function indices
		echocardiography		PC-MRI
		r	p	r	p
	RV PC-MRI diastolic function indices
	Derived from maximal velocity
	Emax	0.47	<0.0001	0.64	<0.0001
	Amax	0.24	0.02	0.38	0.0003
	E'max	0.39	0.0002	0.45	<0.0001
	Derived from mean velocity
	Emean	0.53	<0.0001	0.65	<0.0001
	Amean	0.30	0.005	0.36	0.0007
	E'mean	0.43	<0.0001	0.43	<0.0001
	Derived from flow rate
	Ef/FV	0.35	0.0009	0.55	<0.0001
	Af/FV	0.59	<0.0001	0.64	<0.0001
	FV	-		0.71	<0.0001

Table 3 .

 3 Doppler echocardiographic and PC-MRI RV diastolic function indices for both age groups (left), along with their linear associations with age over the whole group (right).

		≤ 40 years	> 40 years Between groups	Linear associations with age
		n=47	n=42	p value		n=89
	Doppler echocardiography RV diastolic function indices Correlation coefficients (r ) p value	Significant correlates
	E (cm/s)	59±18	55±14	0.20	-0.15	0.17	-
	A (cm/s)	40±10	50±19	0.02	0.40	0.0001*	Age
	E' (cm/s)	14±3.2	12±3.0	0.008	-0.36	0.0006*	Age
	E/A	1.54±0.52	1.21±0.45 0.0005	-0.40	0.0001*	Age, BMI
	E/E'	4.30±1.33	4.76±1.77 0.42	0.17	0.12	-
	MRI RV diastolic function indices
	Derived from maximal velocity
	Emax (cm/s)	48±9.5	38±7.6	<0.0001	-0.58	<0.0001*	Age, BMI
	Amax (cm/s)	36±8.7	38±7.4	0.09	0.17	0.10	-
	E'max
	(cm/s)	9.8±4.4	7.6±4.4	0.02	-0.36	0.0005*	Age, BMI
	Emax/Amax	1.41±0.39	1.04±0.28 <0.0001	-0.56	<0.0001*	Age, BMI
	Emax/E'max	6.01±2.99	7.73±6.43 0.59	0.20	0.05	-
	Derived from mean velocity
	Emean
	(cm/s)	28±6.7	22±5.4	<0.0001	-0.57	<0.0001* Age, gender, BMI
	Amean
	(cm/s)	20±4.8	22±4.6	0.03	0.22	0.03	-
	E'mean
	(cm/s)	7.5±3.1	5.9±3.4	0.02	-0.40	<0.0001*	Age, BMI
	Emean/Amean	1.46±0.39	1.06±0.34 <0.0001	-0.61	<0.0001*	Age, BMI
	Emean/E'mean	4.43±2.23	5.54±4.02 0.68	0.21	0.046	-
	Derived from flow rate
	Ef (ml/s)	326±98	234±84	<0.0001	-0.53	<0.0001*	Age, BMI
	Af (ml/s)	216±72	247±90	0.11	0.20	0.06	-
	FV (ml)	95±29	79±28	0.009	-0.33	0.001*	Age, gender
	Ef/FV (s -1 )	3.49±0.64	2.99±0.58 0.0002	-0.47	<0.0001* Age, gender, BMI
	Af/FV (s -1 )	2.35±0.73	3.20±0.84 <0.0001	0.60	<0.0001*	Age, BMI
	Ef/Af	1.63±0.56	1.00±0.35 <0.0001	-0.64	<0.0001* Age, gender, BMI
	* indicates that the relationship remained significant after adjustment for gender and BMI, and significant independent
	correlates among age, gender and BMI are provided in the last column.

Table 4 .

 4 Linear associations of Doppler echocardiographic and PC-MRI RV E' with RV mass to end-diastolic volume ratio over the whole group.

		Correlation coefficients (r )	p value
	Doppler echocardiography RV diastolic function indices
	E' (cm/s)	-0.09	0.38
	MRI RV diastolic function indices
	E'max, maximal velocity (cm/s)	-0.28	0.007
	E'mean, mean velocity (cm/s)	-0.36	0.0006*
	* indicates that the relationship remained significant after adjustment for age.


MD, MSc Leila Sellami

MD Dario Saracino

MD, PhD Isabelle Le Ber
email: isabelle.leber@upmc.fr

Affiliations

Genetic forms of Frontotemporal lobar degeneration: current diagnostic approach and new directions in therapeutic strategies Authors

Keywords: frontotemporal lobar degeneration, frontotemporal dementia, next-generation sequencing, progranulin (GRN), C9orf72, clinical trial

Recent advances in the genetics of neurodegenerative diseases have substantially improved our knowledge about the genetic causes of Frontotemporal lobar degeneration (FTLD). Three major genes, namely progranulin (GRN), C9orf72 and MAPT, as well as several less common genes, are responsible of the majority of familial cases and of a significant proportion of sporadic forms, including FTLD with or without associated amyotrophic lateral sclerosis and some rarer clinical presentations. Plasma progranulin dosage and next-generation sequencing are currently available tools which allow the detection of a genetic cause in a more rapid and efficient way. This has important consequences for clinical practice and genetic counselling for patients and families. The ongoing investigations on some therapeutic candidates targeting different biological pathways involved in the most frequent genetic forms of FTLD, as well as a better understanding of the early pathophysiological modifications occurring during the presymptomatic phase of the disease could hopefully contribute to develop effective disease modifying therapies. The identification of a causal mutation in a family is of outmost importance indeed to propose to presymptomatic carriers the inclusion in clinical trials with the aim to prevent or delay the onset of disease.

Introduction

Frontotemporal lobar degeneration (FTLD) designates a spectrum of degenerative dementias with remarkable heterogeneity from a clinical, pathological and genetic point of view. FTLD is considered the second cause of adult-onset dementia after Alzheimer's Disease (AD). Its estimated incidence is 1.6 new cases/100 000 subjects/year and its prevalence is as high as 10-15/100 000 subjects between 45 and 65 years [START_REF] Onyike | The epidemiology of frontotemporal dementia[END_REF][START_REF] Olney | Frontotemporal Dementia[END_REF].

The most frequent clinical phenotype is the behavioral variant of frontotemporal dementia (bvFTD), characterized by predominant frontal lobe dysfunction which manifests as behavioral changes, social cognition deficits and dysexecutive syndrome [START_REF] Rascovsky | Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia[END_REF]. Predominant expressive and/or receptive language deficits at onset define the primary progressive aphasias (PPA) [START_REF] Gorno-Tempini | Classification of primary progressive aphasia and its variants[END_REF]. PPA is further distinguished into a nonfluent/agrammatic variant (nfvPPA, previously known as progressive non-fluent aphasia, PNFA) and a semantic variant (svPPA, previously known as semantic dementia, SD). A third presentation, the logopenic variant (lvPPA) is more often associated with underlying AD pathology [START_REF] Rogalski | Aphasic variant of Alzheimer disease: Clinical, anatomic, and genetic features[END_REF].

Other syndromes mainly characterized by atypical parkinsonism are included in the FTLD spectrum: the Richardson's syndrome, which is predictive of underlying progressive supranuclear palsy (PSP) pathology (as well as other clinical syndromes associated with underlying PSP, such as progressive gait freezing) [START_REF] Höglinger | Clinical diagnosis of progressive supranuclear palsy: The movement disorder society criteria: MDS Clinical Diagnostic Criteria for PSP[END_REF], and corticobasal syndrome (CBS), which may be due to underlying corticobasal degeneration (CBD) but also other pathological substrates (e.g. AD) [START_REF] Armstrong | Criteria for the diagnosis of corticobasal degeneration[END_REF]. Finally, amyotrophic lateral sclerosis (ALS) can be present in up to 15% of FTLD patients -mostly in those with bvFTD phenotype -or their relatives [START_REF] Pottier | Genetics of FTLD: overview and what else we can expect from genetic studies[END_REF].

Similar to other neurodegenerative conditions, the pathological substrate of FTLD is the aggregation of insoluble proteins forming pathological inclusions within the neurons. A pathological classification of FTLD is based on the immunohistochemical identification of those proteins. The majority (50-60%) of FTLD cases are characterized by the accumulation of the TAR DNA-binding protein of 43 kDa (TDP-43), and are designated as FTLD-TDP.

Four different subtypes of FTLD-TDP (from A to D) have been established mainly depending on the relative abundance of TDP-43 deposits within the various cortical layers, on the shape of the stained neurites and on the presence of intranuclear inclusions [START_REF] Mackenzie | A harmonized classification system for FTLD-TDP pathology[END_REF]. The existence of a fifth subtype (FTLD-TDP type E) with distinctive pathological features and associated with a more severe clinical course has been recently proposed [START_REF] Lee | Expansion of the classification of FTLD-TDP: distinct pathology associated with rapidly progressive frontotemporal degeneration[END_REF]. The accumulation of cleaved and hyperphosphorylated Tau protein characterizes FTLD-TAU, the second most common pathological variant (30-40% of cases) [START_REF] Irwin | Frontotemporal lobar degeneration: defining phenotypic diversity through personalized medicine[END_REF]. In FTLD-FET/FUS (<10% of cases), other proteins including FUS, EWS and/or TAF15 are present. These proteins, like TDP-43, are located within the nuclei and play a role in RNA metabolism under physiological conditions [START_REF] Neumann | FET proteins TAF15 and EWS are selective markers that distinguish FTLD with FUS pathology from amyotrophic lateral sclerosis with FUS mutations[END_REF]. In very rare cases, the accumulations are solely composed of ubiquitin and other proteasomal components (FTLD-UPS) (Figure 1). Genetic factors play a crucial role in the FTLD-ALS continuum. A family history is present in 30-40% of patients, with evidence of autosomal dominant transmission in the majority of them. This has to be intended in the broadest sense, because, for instance, the presence of one or more cases of ALS in the pedigree of a patient affected by bvFTD should still raise the suspicion of a genetic etiology. An impressive amount of knowledge on FTLD genetics has developed during the past two decades. Twenty-two genes are currently associated with FTLD-ALS and a causative mutation is nowadays identifiable in more than 80% of familial cases and in 10-15% of sporadic forms approximately [START_REF] Pottier | Genetics of FTLD: overview and what else we can expect from genetic studies[END_REF][START_REF] Desmarais | Therapeutic trial design for frontotemporal dementia and related disorders[END_REF] (Figure 2). In this vast genetic landscape, three genes appear by far the most relevant ones due to their frequency in genetic FTLD: progranulin (GRN), chromosome 9 open reading frame 72 (C9orf72) and microtubule associated protein tau (MAPT) [START_REF] Cruts | Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21[END_REF][START_REF] Baker | Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17[END_REF][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][START_REF] Hutton | Association of missense and 5'-splice-site mutations in tau with the inherited dementia[END_REF].

In this review, we will focus on the major FTLD genes, their associated phenotypes and disease mechanisms, and propose an updated diagnostic algorithm to include the less common genes, in order to define a paradigm for the optimal use of genetic analyses. We will also give an overview on the ongoing clinical trials involving pathology-specific disease-modifying approaches in FTLD-related genetic mutations and highlight some promising therapeutic strategies that target the underlying pathology.

The major FTLD genes

Progranulin gene

GRN gene (formerly known as PGRN) mutations were identified in 2006 as responsible of autosomal dominant FTLD [START_REF] Cruts | Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21[END_REF][START_REF] Baker | Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17[END_REF]. They cause approximately 15-20% of familial FTLD and up to 5% of sporadic cases worldwide [START_REF] Rademakers | Recent advances in the molecular basis of frontotemporal dementia[END_REF]. GRN encodes progranulin, a glycosylated secretory protein. Progranulin is highly localized within the lysosomes, where it is cleaved into smaller peptides called granulins with complementary biological activities [START_REF] Paushter | The lysosomal function of progranulin, a guardian against neurodegeneration[END_REF]. Its functions in the brain, though not completely elucidated, relate to neuronal proliferation and survival, axonal growth and neuroinflammation [START_REF] Ahmed | Progranulin in frontotemporal lobar degeneration and neuroinflammation[END_REF]. Progranulin is also expressed in peripheral tissues, mostly in epithelial cells and lymphocytes, and its pleiotropic functions include major roles in wound repair, tumorigenesis and metabolic homeostasis [START_REF] Cenik | Progranulin: A Proteolytically Processed Protein at the Crossroads of Inflammation and Neurodegeneration[END_REF].

Heterozygous GRN mutations cause FTLD via haploinsufficiency, but the exact disease mechanism remains largely speculative. Currently, more than 70 pathogenic null mutations are known, mainly consisting in non-sense variations, exon deletions or small insertions/deletions causing frameshift (www.molgen.ua.ac.be/FTDmutations/) [START_REF] Cruts | Locus-specific mutation databases for neurodegenerative brain diseases[END_REF]. Most lead to a truncated mRNA which is rapidly degraded, thus reducing the level of functional progranulin by about 50% [START_REF] Cruts | Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21[END_REF][START_REF] Baker | Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17[END_REF]. Polymorphisms in TMEM106B gene, encoding another lysosomal protein, have been shown to modulate progranulin levels, likely altering the susceptibility to the disease [START_REF] Lattante | Defining the association of TMEM106B variants among frontotemporal lobar degeneration patients with GRN mutations and C9orf72 repeat expansions[END_REF]. Of note, GRN mutations when present at the homozygous state lead to neuronal ceroid-lipofuscinosis type 11 (CLN11), a multi-systemic lysosomal storage disorder [START_REF] Smith | Strikingly Different Clinicopathological Phenotypes Determined by Progranulin-Mutation Dosage[END_REF][START_REF] Huin | Homozygous GRN mutations: new phenotypes and new insights into pathological and molecular mechanisms[END_REF].

GRN-associated FTLD displays remarkable inter-and intra-familial variability of the age at onset and the clinical phenotype. The age at onset ranges from the late fourth to the ninth decade, with a peak between 60 and 65 years [START_REF] Ber | Phenotype variability in progranulin mutation carriers: a clinical, neuropsychological, imaging and genetic study[END_REF][START_REF] Moore | Age at symptom onset and death and disease duration in genetic frontotemporal dementia: an international retrospective cohort study[END_REF]. The main clinical presentation is bvFTD, in which apathy commonly prevails over disinhibition among the behavioral manifestations. Besides, 15-20% of patients develop a language disorder at onset, characterized by expressive deficits with reduced verbal output, resulting in nfvPPA, lvPPA or a mixed PPA variant [START_REF] Rohrer | Progranulin-associated primary progressive aphasia: A distinct phenotype?[END_REF][START_REF] Kim | Asymmetric pathology in primary progressive aphasia with progranulin mutations and TDP inclusions[END_REF]. Parkinsonism can be associated with these syndromes or can be found in the context of a CBS, another clinical presentation of GRN mutations [START_REF] Ber | Phenotype variability in progranulin mutation carriers: a clinical, neuropsychological, imaging and genetic study[END_REF]. Up to half of the patients show apraxia, dyscalculia and other parietal lobe dysfunctions, about 25% present visual hallucinations and 10-30% display an episodic memory deficit at the neuropsychological assessment [START_REF] Ber | Phenotype variability in progranulin mutation carriers: a clinical, neuropsychological, imaging and genetic study[END_REF].

Neuroimaging studies reveal mostly asymmetric brain atrophy and/or hypometabolism involving frontal, insular, temporal and parietal regions. Subcortical and periventricular white matter hyperintensities of likely degenerative nature are visible in T2-weighted MRI sequences [START_REF] Caroppo | Extensive White Matter Involvement in Patients With Frontotemporal Lobar Degeneration: Think Progranulin[END_REF]. The neuropathological substrate is FTLD-TDP type A, with characteristic intranuclear "cat's-eye" inclusions [START_REF] Mackenzie | A harmonized classification system for FTLD-TDP pathology[END_REF].

Due to its secretion, progranulin can be measured in the plasma, and a reduction of plasma progranulin levels is highly predictive of GRN mutations [START_REF] Ghidoni | Optimal Plasma Progranulin Cutoff Value for Predicting Null Progranulin Mutations in Neurodegenerative Diseases: A Multicenter Italian Study[END_REF]. Progranulin dosage is performed in symptomatic patients as part of the diagnostic work-up (see below), while it has no indications for their at-risk relatives.

C9orf72 repeat expansion

One of the major advances in the genetics of the FTLD-ALS continuum has been the identification, in 2011, of the GGGGCC hexanucleotide repeat expansion in the first intron of C9orf72 gene in families concerned by FTLD, ALS or a combination of both [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]. This mutation explains approximately 25% of familial FTLD and up to 80% of cases with the FTLD-ALS association [START_REF] Ber | C9ORF72 Repeat Expansions in the Frontotemporal Dementias Spectrum of Diseases: A Flow-chart for Genetic Testing[END_REF]. Moreover, it is the first genetic cause of ALS, being responsible for 40% of familial and 6% of sporadic 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].

The protein encoded by C9orf72 is a guanine nucleotide exchange factor (GEF) interacting with various GTPases which is involved in several cellular functions including vesicular trafficking and phagosome formation [START_REF] Webster | The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy[END_REF]. Most individuals in control populations harbor less than 23 GGGGCC repeats, and most often only 2 to 8 repeats, in the first intron of the gene.

An expansion above 30 is considered pathological, even if the majority of patients have many hundreds or thousands of repeats. Three different pathogenic mechanisms, not mutually exclusive, have been proposed: a) loss of physiological functions of the C9ORF72 protein; b) toxicity of mutant RNA that aggregates into nuclear foci; c) accumulation of dipeptide repeats proteins (DPR) generated by repeat-associated non-ATG (RAN) translation [START_REF] Cruts | Current insights into the C9orf72 repeat expansion diseases of the FTLD/ALS spectrum[END_REF].

The mean age at onset in C9orf72 carriers is around 59 years, but it can vary, even in the same family, from 30 to more than 80 years [START_REF] Moore | Age at symptom onset and death and disease duration in genetic frontotemporal dementia: an international retrospective cohort study[END_REF]. The most common cognitive phenotype is bvFTD, characterized by remarkably slow progression of behavioral alterations and executive deficits in a subset of the mutation carriers. Rarer clinical presentations include PPA (nfvPPA or svPPA) or parkinsonian syndrome [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]. In ALS patients, the disease has no particular distinguishing features; bulbar onset and co-occurring cognitive deterioration are overall more frequent with respect to non-mutated cases. The appearance of ALS drastically shortens the disease course [START_REF] Ber | C9ORF72 Repeat Expansions in the Frontotemporal Dementias Spectrum of Diseases: A Flow-chart for Genetic Testing[END_REF]. Interestingly, psychiatric symptoms and syndromes (such as auditory hallucinations, delusions, obsessive-compulsive disorder, bipolar disorder and schizophrenia) may be present in up to around 50% of C9orf72 patients [START_REF] Snowden | Distinct clinical and pathological characteristics of frontotemporal dementia associated with C9ORF72 mutations[END_REF]. They can coexist with the neurological deficits or even appear years or decades before their onset [START_REF] Devenney | Psychiatric disorders in C9orf72 kindreds: Study of 1,414 family members[END_REF]. In C9orf72 disease frontal and temporal atrophy is almost bilateral and symmetrical [START_REF] Whitwell | Neuroimaging signatures of frontotemporal dementia genetics: C9ORF72, tau, progranulin and sporadics[END_REF].

A mosaicism consisting of different sizes of the GGGGCC expansion amongst the different tissues, and among different central nervous system (CNS) regions, has been evidenced

C9orf72 carriers [START_REF] Mcgoldrick | Unaffected mosaic C9orf72 case: RNA foci, dipeptide proteins, but upregulated C9orf72 expression[END_REF]. Additionally, the number of GGGGCC repeats in peripheral lymphocytes appears to unpredictably vary over time in subjects with multiple blood samples, as well as through generations in parents-offspring pairs [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]. Consequently, no reliable correlations can be established between the size of the expansion in lymphocytes and the severity of the disease or its age of onset, differently from some other repeat expansion disorders [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]. The role of short expansions (e.g. a few dozen hexanucleotides) is even more controversial, as they have been found in symptomatic patients as well as in some old unaffected first-degree relatives [START_REF] Mcgoldrick | Unaffected mosaic C9orf72 case: RNA foci, dipeptide proteins, but upregulated C9orf72 expression[END_REF]. Research on putative genetic modifiers, including but not limited to TMEM106B, is currently underway [START_REF] Lattante | Defining the association of TMEM106B variants among frontotemporal lobar degeneration patients with GRN mutations and C9orf72 repeat expansions[END_REF][START_REF] Barbier | Factors influencing the age at onset in familial frontotemporal lobar dementia: Important weight of genetics[END_REF].

The underlying pathological substrate is FTLD-TDP type A or occasionally type B.

Widespread p62-positive inclusions, most notably in the cerebellum where TDP-43 pathology is absent, has been reported in C9orf72 patients, especially with predominant motor phenotypes [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]. Additionally, intranuclear RNA foci and cytoplasmic DPR inclusions are present in the pyramidal cells of the hippocampus, the granular layer of the cerebellum and in several neocortical regions [START_REF] Mackenzie | The neuropathology associated with repeat expansions in the C9ORF72 gene[END_REF].

MAPT gene mutations

MAPT was the first gene identified in families affected by FTLD, in 1998, and the frequent occurrence of parkinsonism together with cognitive and behavioral manifestations in those kindreds led to the descriptive term of "Frontotemporal dementia and parkinsonism linked to chromosome 17" (FTDP-17) [START_REF] Hutton | Association of missense and 5'-splice-site mutations in tau with the inherited dementia[END_REF]. This acronym should be avoided however, considering the frequency of parkinsonism in other genetic forms of FTLD, and the absence of homogeneity in pathological subtypes of FTLD-TAU [START_REF] Forrest | Retiring the term FTDP-17 as MAPT mutations are genetic forms of sporadic frontotemporal tauopathies[END_REF].

In France, the frequency of MAPT mutations is 5-10% in familial forms and about 3% in sporadic cases, but it can as be as high as 20% in some countries, as in northern Europe, due to founder effects [START_REF] Pickering-Brown | Evidence of a founder effect in families with frontotemporal dementia that harbor the tau +16 splice mutation[END_REF].

The tau (tubulin-associated unit) protein encoded by MAPT exists in two different splice variants, 3R and 4R, and is involved in microtubule assembly, cytoskeleton stabilization and axonal transport. Currently more than 50 pathogenic mutations are known, mainly consisting of missense or non-sense base changes, which act by altering the physiological balance of tau isoforms, impairing its binding to axonal cytoskeleton and eventually leading to tau hyperphosphorylation and accumulation [START_REF] Pickering-Brown | The complex aetiology of frontotemporal lobar degeneration[END_REF][START_REF] Ghetti | Invited review: Frontotemporal dementia caused by microtubule-associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging[END_REF].

The age at onset ranges between the third and the seventh decade, with a peak around 50 years, thus earlier than in the other principal genetic forms. The main clinical phenotype is bvFTD with prevailing disinhibition, at times associated with obsessive-compulsive symptoms, episodic memory disturbances and semantic impairment [START_REF] Van Swieten | Hereditary Frontotemporal Dementia Caused by Tau Gene Mutations[END_REF]. SvPPA (without any behavioral troubles), as well as nfvPPA with prominent apraxia of speech, are rarer presentations [START_REF] Van Swieten | Hereditary Frontotemporal Dementia Caused by Tau Gene Mutations[END_REF][START_REF] Henz | A Pair of Siblings with a rare R5H-Mutation in Exon 1 of the MAPT-Gene[END_REF]. In addition to the atypical parkinsonism associated with bvFTD, other motor phenotypes include CBS and PSP [START_REF] Van Swieten | Hereditary Frontotemporal Dementia Caused by Tau Gene Mutations[END_REF].

Neuroimaging studies show predominant bilateral frontal and anterior temporal involvement [START_REF] Whitwell | Neuroimaging signatures of frontotemporal dementia genetics: C9ORF72, tau, progranulin and sporadics[END_REF]. The neuropathology associated with MAPT mutations is FTLD-TAU. Neuronal tau accumulations mainly consist of fibrillary tangles, straight filaments and Pick body-like inclusions. A glial pathology in form of tufted astrocytes, astrocytic plaques or oligodendroglial coiled bodies (somewhat reminiscent of PSP or corticobasal degeneration) often coexists [START_REF] Forrest | Retiring the term FTDP-17 as MAPT mutations are genetic forms of sporadic frontotemporal tauopathies[END_REF][START_REF] Ghetti | Invited review: Frontotemporal dementia caused by microtubule-associated protein tau gene (MAPT) mutations: a chameleon for neuropathology and neuroimaging[END_REF][START_REF] Van Swieten | Hereditary Frontotemporal Dementia Caused by Tau Gene Mutations[END_REF].

Other rare FTLD genes

Rare genes involved in FTLD-ALS association

TARDBP gene, coding the TDP-43 protein, is responsible of ~1% of FTLD and ~3% of ALS cases, and is the most common genetic cause of FTLD-ALS after C9orf72. Almost all pathogenic mutations are located in exon 6 [START_REF] Benajiba | TARDBP mutations in motoneuron disease with frontotemporal lobar degeneration[END_REF]. TANK-binding kinase 1 (TBK1) loss-offunction mutations are associated with late-onset familial ALS, with or without associated FTLD [START_REF] Freischmidt | Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia[END_REF]. It is worth noticing that nfvPPA and svPPA are relatively frequent cognitive phenotypes for both TARDBP and TBK1 mutations [START_REF] Caroppo | Defining the spectrum of frontotemporal dementias associated with TARDBP mutations[END_REF][START_REF] Caroppo | Semantic and nonfluent aphasic variants, secondarily associated with amyotrophic lateral sclerosis, are predominant frontotemporal lobar degeneration phenotypes in TBK1 carriers[END_REF]. Other genes occurring in <1% of familial FTLD-ALS cases each include TUB4A and CHMP2B [START_REF] Perrone | Investigating the role of ALS genes CHCHD10 and TUBA4A in Belgian FTD-ALS spectrum patients[END_REF][START_REF] Skibinski | Mutations in the endosomal ESCRTIII-complex subunit CHMP2B in frontotemporal dementia[END_REF]. Both have been associated with ALS, bvFTD and late-onset atypical parkinsonian syndromes. Notably, the pathology underlying CHMP2B mutations is the rare FTLD-UPS, with ubiquitin-and p62positive inclusions, in absence of TDP-43 accumulation. Additionally, mutations in ANG, OPTN, PFN1, UBQLN1 and MATR3 genes have been mainly reported in ALS families, with or without associated cognitive impairment. FUS/TLS, EWSR1 and TAF15 genes, encoding for three RNA-binding proteins of the FET (Fused in sarcoma (FUS), Ewing's sarcoma, TATA-binding protein associated factor (TAF) 15) family, are all associated with ALS (very rarely co-occurring with dementia), and are characterized by a distinctive underlying pathological substrate (FTLD-FET/FUS) [START_REF] Svetoni | Role of FET proteins in neurodegenerative disorders[END_REF].

Rare genes involved in complex phenotypes

In some families, symptoms of FTLD or ALS are integrated in a more complex phenotypic association including other neurological, musculoskeletal or extra-neurological diseases. This emphasizes the importance of investigating associated features in the family during a thorough clinical interview [START_REF] Al-Obeidi | Genotype-phenotype study in patients with valosin-containing protein mutations associated with multisystem proteinopathy[END_REF].

A variable association of FTLD, inclusion body myopathy (IBM), Paget's disease of bone (PDB) and, rarely, ALS was initially described in some North American families and designated with the acronym IBMPFD (Inclusion-Body Myopathy with Paget's disease of bone and Frontotemporal Dementia) or "multisystem proteinopathy" (MSP) [START_REF] Watts | Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein[END_REF]. VCP is the main gene involved in this complex phenotype. It is responsible of up to 3% of familial FTLD cases, with FTLD-TDP type D as pathological substrate [START_REF] Mackenzie | A harmonized classification system for FTLD-TDP pathology[END_REF]. Approximately 90% of VCP mutation carriers present IBM, 60% PDB and 30% bvFTD [START_REF] Al-Obeidi | Genotype-phenotype study in patients with valosin-containing protein mutations associated with multisystem proteinopathy[END_REF]. Rarer genes leading to MSP include SQSTM1, encoding the p62 protein, and two genes coding for heterogeneous ribonucleoproteins, hnRNPA1 and hnRNPA2B1 [START_REF] Ber | SQSTM1 Mutations in French Patients With Frontotemporal Dementia or Frontotemporal Dementia With Amyotrophic Lateral Sclerosis[END_REF][START_REF] Kim | Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS[END_REF].

Mutations in CSF1R (Colony stimulating factor receptor 1) gene cause a white matter disorder initially known as hereditary diffuse leukoencephalopathy with spheroids (HDLS) [START_REF] Rademakers | Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids[END_REF], and currently designated as adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP), to encompass all its pathological features [START_REF] Konno | Clinical and genetic characterization of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia associated with CSF1R mutation[END_REF]. The onset of symptoms occurs during the fourth decade (range 18-78 years). CSF1R mutations have been found in individuals showing bvFTD or CBS phenotypes [START_REF] Guerreiro | Genetic Analysis of Inherited Leukodystrophies: Genotype-Phenotype Correlations in the CSF1R Gene[END_REF][START_REF] Kim | Analysis of frontotemporal dementia, amyotrophic lateral sclerosis, and other dementia-related genes in 107 Korean patients with frontotemporal dementia[END_REF] but the clinical presentations also variably include seizures, parkinsonism, cerebellar ataxia, spasticity and depression [START_REF] Guerreiro | Genetic Analysis of Inherited Leukodystrophies: Genotype-Phenotype Correlations in the CSF1R Gene[END_REF].

Extensive white matter T2-hyperintensities and thinning of corpus callosum are distinctive MRI findings [START_REF] Konno | Clinical and genetic characterization of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia associated with CSF1R mutation[END_REF][START_REF] Codjia | Adult-Onset Leukoencephalopathy with Axonal Spheroids and Pigmented Glia: An MRI Study of 16 French Cases[END_REF]. A rapid clinical deterioration, the appearance of (mostly generalized) seizures and diffuse white matter involvement with bifrontal atrophy at MRI are important clues for this genetic form. Although rare, CSF1R mutations should be investigated in cases with autosomal dominant disease but also in 'apparently sporadic' cases, as the frequency of de novo mutations can be as high as 40% [START_REF] Konno | Clinical and genetic characterization of adult-onset leukoencephalopathy with axonal spheroids and pigmented glia associated with CSF1R mutation[END_REF].

CHCHD10 gene mutations lead to another complex syndrome associating FTLD-ALS with cerebellar ataxia, mitochondrial myopathy and hearing impairment [START_REF] Perrone | Investigating the role of ALS genes CHCHD10 and TUBA4A in Belgian FTD-ALS spectrum patients[END_REF][START_REF] Chaussenot | Screening of CHCHD10 in a French cohort confirms the involvement of this gene in frontotemporal dementia with amyotrophic lateral sclerosis patients[END_REF]. The mutations of DCTN1 gene, encoding the p150 Glued subunit of dynactin, are associated with ALS, FTLD, parkinsonism and Perry syndrome [START_REF] Wider | Elucidating the genetics and pathology of Perry syndrome[END_REF]. Finally, homozygous and compound heterozygous TREM2 mutations cause polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL, also known as Nasu-Hakola syndrome), but have also been reported in association with an FTLD-like syndrome, without associated bone changes [START_REF] Peplonska | TREM2 variants in neurodegenerative disorders in the Polish population. Homozygosity and compound heterozygosity in FTD patients[END_REF].

Genetic diagnosis in FTLD: new challenges and recommendations

The growing complexity of the genetic landscape of FTLD-ALS, with 22 genes involved thus far, represents an obvious difficulty in identifying the causal mutations. The analysis of all responsible genes with the standard sequencing techniques is time-consuming and expensive.

The development of next generation sequencing (NGS) in the past decade has considerably eased the identification of a causal mutation in FTLD patients and it is nowadays possible to analyze most FTLD genes [START_REF] Goldman | Alzheimer's Disease and Frontotemporal Dementia: The Current State of Genetics and Genetic Testing Since the Advent of Next-Generation Sequencing[END_REF]. However, not all genes undergo mutations diagnosable by means of NGS. This is the case for C9orf72 gene, whose repeat expansions should be looked for separately with repeat primed-PCR or Southern blot, the gold standard methods for repeat expansion detection. Moreover, the interpretation of the huge load of data and the considerable number of variants of uncertain significance (VUS) generated by NGS are new challenges for genetic diagnosis [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF]. Significant diagnostic difficulties arise, for instance, in the presence of missense mutations in FTLD causative genes, such as GRN or TBK1, as they have a variable impact on protein synthesis and their pathogenic role is still unclear in the majority of cases [START_REF] Saracino | The missense p.Trp7Arg mutation in GRN gene leads to progranulin haploinsufficiency[END_REF]. Therefore, caution must be taken when interpreting uncertain results and a good expertise in the genotypes underlying FTLD phenotypes is needed. In this context, studies of familial segregation and additional evidence of pathogenicity (from in silico and/or in vitro models) may be of paramount importance to validate the pathogenicity of genetic variants. A higher level of complexity in the interpretation of NGS data and in the identification of the causal mutation comes from the identification of double mutations -especially involving the rarest FTLD genes -in few FTLD patients [START_REF] Pottier | Wholegenome sequencing reveals important role for TBK1 and OPTN mutations in frontotemporal lobar degeneration without motor neuron disease[END_REF]. Therefore, the extensive analysis of all FTLD genes might be warranted in selected cases, as the identification of a first mutation does not rule out the possibility of a digenic etiology.

To maximize the efficacy of NGS approach, some recommendations for genetic diagnosis in FTLD-ALS patients and families should be proposed. In most genetic forms of FTLD, the core features display a remarkable intra-and interfamilial variability, hence the importance of investigating not only cognitive-behavioral symptoms, but also the existence of ALS, other neurological, musculoskeletal or extra-neurological (e.g. Paget's disease of bone) diseases in the proband or in his family during a thorough clinical interview. The clinical phenotypes of the proband and his affected relatives should be carefully detailed, paying special attention to the heterogeneity in age at onset and clinical presentation.

An algorithm for genetic diagnosis in FTLD-ALS, considering the role of the major genes and the additional contribution from NGS, is presented in Figure 3. Briefly, plasma progranulin dosage should be performed in all FTLD patients (with or without positive family history), and low levels should prompt immediate GRN gene analysis. The association of FTLD with ALS in the same individual or within the family is most often due to the C9orf72 repeat expansion. The research of C9orf72 repeat expansion should be carried out in all subjects with FTLD and FTLD-ALS (both non-familial and familial, considering the presence of ALS not only in the proband but in his family as well). In case of absence of GRN and C9orf72 mutations, a second-level approach for the genetic diagnosis is the implementation of an FTLD-ALS NGS. Atypical FTLD clinical presentations or unusual phenotypic associations should raise the suspicion of the involvement of different, rare genes, possibly prioritizing those more closely associated with the specific clinical context.

Targeted therapies for FTD genetic mutations

The considerable advances in the genetics of FTLD have paved the way to new therapeutic perspectives. Several novel therapies to delay the outbreak and progression of genetic FTD are emerging and some examples of molecules currently being tested are discussed below.

Therapeutic approaches for GRN gene mutations

The main pathogenic mechanism of GRN mutations arises from a loss of functional progranulin, and strategies to restore progranulin haploinsufficiency have emerged as promising avenues of research for GRN-related FTLD. A targeted intervention in GRN mutations might be the replacement of progranulin haploinsufficiency by upregulating expression of the nonmutant GRN allele. Histone deacetylase (HDAC) inhibitors were shown to promote progranulin expression through epigenetic regulation in human neuronal cells [START_REF] She | Selectivity and Kinetic Requirements of HDAC Inhibitors as Progranulin Enhancers for Treating Frontotemporal Dementia[END_REF]. A phase II randomized, double-blind, placebo-controlled trial has tested HDAC inhibitors (FRM-0334) (https://www.clinicaltrials.gov/, NCT02149160), for which results are still pending. Another avenue of research has focused on the Sortilin (SORT1)-progranulin axis. SORT1 is a clearance neuronal receptor via an endocytosis mechanism [START_REF] Lee | Targeted manipulation of the sortilin-progranulin axis rescues progranulin haploinsufficiency[END_REF]. An openlabel phase 2 study in human carriers is currently ongoing (https://www.clinicaltrials.gov/, NCT03987295). Other therapeutic approaches to rescue progranulin haploinsufficiency failed to demonstrate efficacy. A phase one 8-week clinical trial of Nimodipine, a blood-brain barrier-penetrant calcium channel blocker, did not show any significant effect on the concentrations of progranulin in plasma and cerebrospinal fluid [START_REF] Sha | An 8-week, open-label, dose-finding study of nimodipine for the treatment of progranulin insufficiency from GRN gene mutations[END_REF]. Similarly, a small phase 2 clinical trial of Amiodarone led to inconclusive results [START_REF] Alberici | Results from a pilot study on amiodarone administration in monogenic frontotemporal dementia with granulin mutation[END_REF]. Other therapeutic approaches stemming from promising preclinical data in animal models, including oral small molecules, gene therapy and cell-based strategies are currently under development.

Therapeutic approaches for C9orf72 repeat expansion

Since 2011, substantial advances have been achieved in our knowledge regarding C9orf72mediated disease and its underlying pathogenesis. Toxic gain of function from C9orf72 repeat RNA and DPR proteins have been proposed as a crucial disease mechanism [START_REF] Cruts | Current insights into the C9orf72 repeat expansion diseases of the FTLD/ALS spectrum[END_REF]. Hence, main drug discovery efforts aimed at reducing the gain of toxicity caused by the repeatcontaining C9orf72 transcripts. Potential therapies based on this approach consist of antisense oligonucleotides (ASOs). ASOs are short DNA or RNA segments binding a complementary RNA sequence that are already used to modulate gene expression in several neurological diseases. A recent phase 2 clinical trial of ASO therapy targeting RNA-mediated toxicity in human has been conducted in Huntington disease (HTT Rx) another repeat expansions disease. This trial showed its safety and tolerability, and a dose-dependent reduction in levels of the harmful mutant protein in the nervous system [START_REF] Tabrizi | Targeting Huntingtin Expression in Patients with Huntington's Disease[END_REF]. The most important breakthrough in the field of ASO therapy is undoubtedly the recent Food and Drug Administration approval for the Nusinersen, an antisense drug for the treatment of spinal muscular atrophy [START_REF] Finkel | Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy[END_REF]. All these studies pave the way for the development of ASO therapies in neurodegenerative disorders and hold a great promise for the discovery of effective disease-modifying therapies for C9orf72-associated disease. In vivo studies in C9orf72 mice showed a significant reduction of RNA foci, DPR proteins and the development of behavioural deficits after a single dose of ASO therapy [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]. A phase 1 first-in-human study (BIIB078) assessing the safety and tolerability of ASO-mediated therapy administered intrathecally is currently underway in subjects with C9orf72-associated ALS (https://www.clinicaltrials.gov/, NCT03626012).

Several other approaches including therapies based on small molecules and targeting DPR are also under development [START_REF] Desmarais | Therapeutic trial design for frontotemporal dementia and related disorders[END_REF].

Therapeutic approaches for MAPT gene mutations

Several strategies targeting different aspects of tau-related pathogenesis are currently being investigated, including protein kinases inhibitors for modulating tau phosphorylation, Microtubule stabilizers, tau aggregation inhibitors and anti-tau immunotherapy. Potentially relevant clinical trials for different tauopathies include a phase 3 trial of LMTX (TRx0237), a modified derivative of the tau aggregation inhibitor methylthionine chloride in subjects with bvFTD (https://www.clinicaltrials.gov/, NCT01626378) and a phase 2 trial of anti-tau immunotherapy, ABBV-8E12, a humanized recombinant anti-tau antibody, in patients with PSP (https://www.clinicaltrials.gov/, NCT03391765). These studies were completed in 2018, though their results have not been published yet. Gosuranemab (BIIB092), another taudirected monoclonal antibody, demonstrated good tolerability in PSP patients in a phase 1b trial [START_REF] Boxer | Safety of the tau-directed monoclonal antibody BIIB092 in progressive supranuclear palsy: a randomised, placebo-controlled, multiple ascending dose phase 1b trial[END_REF]. So far, these trials have mostly included PSP patients, but it is likely that some of these molecules will be tested in FTLD patients carrying MAPT mutations as well.

At the dawn of a new therapeutic era: the presymptomatic phase as a target window in genetic FTLD

The study of presymptomatic stage in mutation carriers is of outmost importance in light of the advent of promising preventive trials in genetic FTLD families. The presymptomatic phase indeed represents the optimal window for testing therapeutic molecules that target the FTLD-related neurobiological abnormalities at the earliest stage of the disease, before the occurrence of clinical symptoms. The identification of imaging and fluid biomarkers become crucial to monitor therapeutic trials at this stage, and research studies focusing on the presymptomatic phase of genetic FTLD have become of increasing interest over the past few years [START_REF] Bateman | Clinical and Biomarker Changes in Dominantly Inherited Alzheimer's Disease[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]. Emerging data from several studies of well-characterised presymptomatic genetic cohorts, such as GENFI (Genetic Frontotemporal dementia Initiative) [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], LEFFTDS (Longitudinal Evaluation of Familial Frontotemporal Dementia Subjects) [START_REF] Boeve | The longitudinal evaluation of familial frontotemporal dementia subjects protocol: Framework and methodology[END_REF], Predict-PGRN [START_REF] Caroppo | Lateral Temporal Lobe: An Early Imaging Marker of the Presymptomatic GRN Disease?[END_REF] and PREV-DEMALS [START_REF] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF], suggested that the first pathological alterations associated with FTLD gene mutations can be detected 15-10 years before onset of overt clinical symptoms. Accumulating results across these studies have identified early biomarkers in mutation carriers, encompassing neuropsychological, biological and multimodal neuroimaging changes (i.e. structural and functional MRI and PET imaging) [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] Bertrand | Early Cognitive, Structural, and Microstructural Changes in Presymptomatic C9orf72 Carriers Younger Than 40 Years[END_REF][START_REF] Jiskoot | Longitudinal cognitive biomarkers predicting symptom onset in presymptomatic frontotemporal dementia[END_REF][START_REF] Jiskoot | Longitudinal multimodal MRI as prognostic and diagnostic biomarker in presymptomatic familial frontotemporal dementia[END_REF][START_REF] Van Der Ende | Serum neurofilament light chain in genetic frontotemporal dementia: a longitudinal, multicentre cohort study[END_REF][START_REF] Galimberti | Progranulin plasma levels predict the presence of GRN mutations in asymptomatic subjects and do not correlate with brain atrophy: results from the GENFI study[END_REF], but these markers still remain to be validated at the individual level. Once validated, the panel of early biomarkers identified at the presymptomatic stage could be used as efficacy endpoints in clinical trials. The significant advances that have been achieved in the understanding of the biomarkers cascade throughout the presymptomatic stage of genetic FTLD provide insight into the measurement of early disease progression and potential effects of disease-modifying therapies.

Conclusion

In the last few years, a major breakthrough has been achieved in the understanding of the genetics and molecular biology of FTLD. A remaining challenge is translating this substantial knowledge into therapeutic opportunities. Whilst much remains to be done in the field of drug discovery, it is noteworthy that we are now reaching a turning point as regards the development of early biomarkers and preventive therapeutic approaches targeting the prodromal stage of genetic FTLD, before the onset of overt clinical symptoms in causative mutations carriers. The current investigations target different aspects of FTLD pathophysiology and several appealing potential therapeutic candidates are currently being tested in the setting of clinical trials. These studies will hopefully expand the scope of potentially interesting disease modifying therapies in FTLD.

Figure 1 .

 1 Figure 1. Pathological classification of frontotemporal lobar degeneration. TDP:

Figure 2 .

 2 Figure 2. Landscape of the genes associated with the FTLD-ALS continuum. Major

Figure 3 .

 3 Figure 3. Optimal diagnostic algorithm for genetic diagnosis in FTLD, including major

Disclosure of interests I.L.B. is principal investigator for Alector in France and scientific consultant for Prevail Therapeutics. L.S. was supported by a grant from the Société Française de Neurologie -Revue Neurologique.