C Foussier

P A Barral

M Jerosh-Herold

V Gariboldi

Stanislas Rapacchi

A Gallon

A Bartoli

Z Bentatou

Maxime Guye

Monique Bernard

M Jerosh Herold

S Rapacchii

Z Bentamou

A Jacquier
email: alexis.jacquier@ap-hm.fr

Quantification of diffuse myocardial fibrosis using CMR extracellular volume fraction and serum biomarkers of collagen turnover with histologic quantification as standard of reference

Keywords: Cardiac magnetic resonance imaging, Aortic valve stenosis, Myocardial fibrosis, Extracellular volume fraction, T1 mapping CITP: C-terminal telopeptide of type 1 collagen CMR: cardiac magnetic resonance CVF: collagen volume fraction ECV: extracellular volume fraction interobserver correlation coeficient LGE: late gadolinium-chelate enhancement LVEF: Left ventricular ejection fraction MMPs: Matrix metalloproteinases MMP-2: Matrix metalloproteinases 2

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Diffuse myocardial fibrosis is a pathophysiological process causing the impairment of myocardial function and electrical disorganization in valvulopathies. Progress in cardiac magnetic resonance (CMR) now permits the assessment of diffuse myocardial fibrosis in clinical practice by detecting the expansion of extracellular volume (ECV) using T1-mapping techniques [START_REF] Messroghli | Clinical recommendations for cardiovascular magnetic resonance mapping of T1, T2, T2* and extracellular volume: a consensus statement by the Society for Cardiovascular Magnetic Resonance (SCMR) endorsed by the European Association for Cardiovascular Imaging (EACVI)[END_REF]. Aortic valvulopathies are characterized not only by progressive valve dysfunction but also by the left ventricular remodeling response [START_REF] Dweck | Calcific aortic stenosis: a disease of the valve and the myocardium[END_REF]. Valvular disease causes pressure overload of the left ventricle and triggers ventricular remodeling response that maintains myocardial performance for years. However, with time, this process decompensates to heart failure, heralded clinically by the development of symptoms and adverse events, leading to consideration of aortic valve replacement.

Current guidelines recommend intervention in patients with severe aortic stenosis (AS) and evidence of symptoms related to the valvular disease, but other markers include a reduction in ejection fraction < 50%, an abnormal exercise tolerance test or a rise in brain natriuretic peptide level [START_REF] Baumgartner | ESC/EACTS Guidelines for the management of valvular heart disease[END_REF][START_REF] Nishimura | AHA/ACC Guideline for the management of patients with valvular heart disease: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines[END_REF]. Unfortunately, symptoms are often difficult to identify in the elderly comorbid patients encountered in clinical practice, and many of the other changes appear only late in the course of the disease after irreversible myocardial damage has become established. Consequently, there is extensive interest in identifying novel, objective markers of early left ventricular decompensation to optimize the timing of aortic valve replacement.

The development of such markers requires to improve understanding of the pathophysiology underlying left ventricular decompensation in AS. Histological studies have suggested that myocardial fibrosis and cell death are both important drivers of this process [START_REF] Krayenbuehl | Left ventricular myocardial structure in aortic valve disease before, intermediate, and late after aortic valve replacement[END_REF][START_REF] Hein | Progression from compensated hypertrophy to failure in the pressure-overloaded human heart: structural deterioration and compensatory mechanisms[END_REF] Myocardial fibrosis can range from focal replacement fibrosis caused by myocardial infarction to diffuse interstitial fibrosis, characterized by the accumulation of collagen in myocardial interstitial tissue. The serum biomarkers of collagen turnover are based on measuring the circulating molecules of extracellular matrix breakdown or remodeling. Matrix metalloproteinases (MMPs) play pivotal roles in the development of fibrosis by controlling extracellular matrix degradation. The tissue inhibitor of metalloproteinases (TIMPs) inhibits MMPs activity [START_REF] Bonnans | Remodelling the extracellular matrix in development and disease[END_REF]. Several studies have validated the correlation between diffuse interstitial fibrosis and the serum biomarkers of collagen turnover but there is very little in the literature concerning the correlation between serum biomarkers, CMR and the histological measurement of fibrosis [START_REF] Querejeta | Serum carboxyterminal propeptide of procollagen type I is a marker of myocardial fibrosis in hypertensive heart disease[END_REF][START_REF] Izawa | Mineralocorticoid receptor antagonism ameliorates left ventricular diastolic dysfunction and myocardial fibrosis in mildly symptomatic patients with idiopathic dilated cardiomyopathy: a pilot study[END_REF][START_REF] Klappacher | Measuring extracellular matrix turnover in the serum of patients with idiopathic or ischemic dilated cardiomyopathy and impact on diagnosis and prognosis[END_REF][START_REF] López | Galectin-3 and histological, molecular and biochemical aspects of myocardial fibrosis in heart failure of hypertensive origin[END_REF][START_REF] López | Alterations in the pattern of collagen deposition may contribute to the deterioration of systolic function in hypertensive patients with heart failure[END_REF]. The up regulation of MMPs and the inadequate inhibition by TIMPs alters the balance between proteolysis/antiproteolysis resulting in the proliferation of fibroblasts and the progression of diffuse myocardial fibrosis [START_REF] Polyakova | Matrix metalloproteinases and their tissue inhibitors in pressure-overloaded human myocardium during heart failure progression[END_REF].

The purpose of this study was to compare the assessment of diffuse interstitial myocardial fibrosis in aortic valve diseases using extracellular volume fraction (ECV) and serum biomarkers of collagen turnover using results of myocardial biopsy as standard of reference.

Material and methods

Patients

This study was approved by the national ethics committee (ID RCB: 2011-A01395-36 31) and all patients gave their informed consent to participate. We prospectively included all patients with severe AS or aortic regurgitation (AR), referred to our Institution for valvular replacement. Non-inclusion criteria were: history of myocardial infarction, hemodynamic instability requiring catecholamine treatment, liver failure, tumor, renal insufficiency (creatinine clearance < 40 mL/min/1.73 m²), high risk of acute coronary syndrome before CMR, history of allergy to gadolinium chelate, pregnancy, breastfeeding, age lower than 18 years and claustrophobia. Exclusion criteria were refusal to undergo CMR and history of cardiac arrhythmia. During a period of 7 days, all patients underwent CMR for ECV quantification, blood samples for collagen turn over biomarker quantification and surgery with myocardial biopsy. New York Heart Association (NYHA) dyspnea classification was recorded for all patients

CMR imaging and image analysis

All CMR examinations were performed on a 1.5T MR system (Avanto ® , Siemens Healthineers) with a 32-channel receiver array. Images were acquired in decubitus position with cardiac gating and inspiration breath-hold. Heart rate and blood pressure were recorded during imaging. CMR protocol included two different sequences: Steady state free precession cine sequences were performed in the long axis, 4 chambers, 2 chambers, LVOT and short axis to cover the whole LV with the following parameters: slice thickness, 7 mm; field of view, 320 mm; temporal resolution, 28.9 ms; TE/TR, 1.6/3.6 ms; flip angle, 35°; image resolution, 256 × 144 -222. Images were postprocessed to evaluate left-ventricular end diastolic (EDV) and end systolic volume (ESV), myocardial mass and LV ejection fraction (LVEF) using vendor-provided software (Argus ® , Siemens Healthineers). T1 mapping using a modified "Look-Locker" technique (MOLLI) [START_REF] De Meester De Ravenstein | Histological Validation of measurement of diffuse interstitial myocardial fibrosis by myocardial extravascular volume fraction from Modified Look-Locker imaging (MOLLI) T1 mapping at 3 T[END_REF] with reconstruction including motion correction, was performed before and 17 min after intravenous injection of 0.2 mmoL/kg of gadoterate meglumine (Dotarem ® , Guerbet) with the following parameters: slice thickness, 6 mm; field of view, 360 mm; TE/TR, 1.13/2.26 ms; flip angle, 35°; matrix resolution, 256 × 144 -222. For pre-contrast T1 mapping a 5 (3) 3 MOLLI acquisition scheme was used, and for the post-contrast map a 4 (1) 3 (1) 2 scheme.

Pre and post contrast maps were transferred to a CMR 42 workstation (Circle). ECV was measured directly on the ECV map calculated by the software from pre and post T1 maps.

ECV measures were carried out in the 12 segments at the basal ( 6) and mid (6) myocardial levels. The sub-endocardial and sub-epicardial area were excluded (cut off value, 5%) to avoid partial volume effect with blood or epicardial fat.

A late gadolinium-chelate enhancement (LGE) sequence was acquired 10 min after the beginning of intravenous administration of contrast material with the following parameters TR/TE, 650/1.56 ms; slice thickness, 6 mm; number of slabs, 14 (to encompass the left ventricles in the short axis in one breath-hold); flip angle, 10° ; field of view, 350 × 400 mm; matrix size, 152 × 134; and inversion time, 270 -325 ms (set to null normal myocardium at the end-diastolic phase).

A qualitative analysis of LGE was performed to detect the presence of LGE and the location of myocardial enhancement. CMR images were anonymized and analyzed by two experienced observers blinded to clinical data (A. J. with 10 years of experience in CMR and C.F. with 2 years of experience in CMR).

Blood sample analysis

Blood samples of two serum tubes (6 mL each) and a heparin plasma tube (6 mL) were taken during the week before aortic valve replacement. Standard bioassays were performed with hematocrit. For specific collagen turnover serum biomarkers, samples were centrifuged within 30 min after the samples were taken. Aliquots were then frozen and stored at -80 °C. The serum biomarkers measured were: carboxy terminal procollagen type 1 propeptide (PICP), C-terminal telopeptide of type 1 collagen (CITP), N-terminal propeptide of type III procollagen (PIIINP), metalloproteinase matrix 2 (MMP-2), tissue inhibitor of metalloproteinase (TIMP-1), galectin, osteopontin, bone specific alkaline phosphatase, and cardiac troponin 1.

Myocardial biopsies and histological study

At the time of surgery, a biopsy of the interventricular septum was performed using an 18 Gauge needle-biopsy (SuperCore™ Biopsy Instrument, Argon Medical Devices, Inc.).

Biopsies were collected from the basal antero septum under direct visual control during the aortic valve replacement procedure, then fixed in 10% buffered formalin and embedded in paraffin, and finally sliced and stained with collagen-specific picrosirius red. The histological measurements of myocardial fibrosis were limited to one sampling site in the LV and were obtained using a 18G needle biopsy which resulted in small myocardial sampling volumes.

Histopathological analysis was performed blinded to clinical and imaging data. Two pathologists performed histological measurements and one performed twice after a period of one month. With respect to the measurements of histological myocardial fibrosis, the collagen volume fraction (CVF) of myocardium was determined by quantitative morphometry (CellˆD, Olympus Soft Imaging Solutions) after exclusion of the subendocardial areas. Fibrosis was quantified on stained sections and expressed as a percentage of area, tagged by picrosirius over total myocardial area (Figure 1).

Statistical analysis

Quantitative variables were expressed as means ± standard deviations (SD) and ranges. Categorical variables were expressed as raw numbers, proportions and percentages.

Results

Study population

Thirty-one patients were prospectively included from May 2012 to October 2013. 5 patients were excluded from CMR (claustrophobia, 4 and severe arrhythmia, and analysis comparing CMR with blood biomarker were performed on 26 patients. Five patients (all suffering from AS) refused to undergo surgical myocardial biopsy but had the whole protocol, so analysis comparing histology with other parameters were performed on 21 patients. Figure 2 shows the study flow chart.

Patients characteristics are summarized in Table 1. Seventeen patients had severe AS and four patients had severe AR. The mean delay between CMR, serum biomarkers and surgery was 4 ± 1.5 (SD) days (range: 2 -5 days). Myocardial biopsies were performed without any complications in all patients. Mean global native T1 was 984.

CMR versus CVF

Neither basal anteroseptal native T1 nor global native T1 correlated with CVF (r = 0.22; P = 0.33). A positive moderate correlation was found between basal anteroseptal ECV and CVF (r = 0.65; P = 0.002) (Fig. 3). No significant correlation was found between global ECV and CVF (r = 0.46; P = 0.101. Global ECV strongly correlated with basal anteroseptal ECV (r = 0.79; P < 0.0001)) (Fig. 4). Bland-Altman analysis revealed good agreement between global and basal anteroseptal ECV (Fig. 5). Overall ECV was not significantly greater than basal anteroseptal ECV but showed a larger SD of the mean and segmental ECV value ranged from 21% to 48% considering all measured segments with a positive bias of 0.014 ± 0.017 (SD) %for the difference between global ECV and basal anteroseptal ECV and with a positive bias of 5.37 ± 6.15 (SD) % for the percent of difference between the two measurements.

CMR versus serum biomarkers of collagen turnover

There was no significant correlation between native T1 map and serum biomarkers.

Poor correlation was found between overall ECV and TIMP-1 (r = 0.41; P = 0.04) (Fig. 6) and overall ECV and MMP-2 (r = 0.39; P = 0.04) (Fig. 7). No significant associations were found between global ECV and the expression of PICP (P = 0.12), CITP (P = 0.72), Galectin (P = 0.99) and PIINP (P = 0.44).

Serum biomarkers versus ECV, CVF and clinical data

No correlation was found between the basal anteroseptal ECV or global ECV and these parameters. CITP moderately correlated with EDVi (r = 0.54; P = 0.007), ESVi (r = 0.55; P =0.01) and myocardial mass (r = 0.47; P = 0.02). PIC-P poorly correlated with EDVi (r = 0.47; P = 0.02) and ESVi (r = 0.41; P = 0.04) (Table 2). Galectin, TIMP-1 and MMP-2 did not correlate with these functional parameters. No significant relationship was found between the blood biomarkers and histologic fibrosis.

Reproducibility of measurements

The interobserver reproducibility of the histological measurements (ICC) was 0.80 with a bias of 0 ± 4 (SD) % and the intraobserver reproducibility was 0.89 with a bias of 0 ± 2%. The inter and intraobserver reproducibility for ECV assessment were ICC: 0.61 with a bias of -0.8 ± 3.6 (SD) % and 0.71 with a bias of -0.8 ± 3.0 (SD) %.

Discussion

In this study, we found that the basal anteroseptal ECV derived from 1.5 T CMR MOLLI T1 mapping, 17 min after bolus injection of a gadolinium chelate moderately correlates with the magnitude of diffuse interstitial myocardial fibrosis measured by picosirius red staining on histopathological samples taken from patients undergoing valve surgery. In addition, we observed that basal anteroseptal ECV is strongly correlated with global ECV.

Finally, global ECV poorly correlated with TIMP-1 and MMP-2 which are known serum biomarkers of collagen turnover.

The global ECV measured in the present study is within the normal range published elsewhere [START_REF] Vo | Pooled summary of native T1 value and extracellular volume with MOLLI variant sequences in normal subjects and patients with cardiovascular disease[END_REF]. This was in line with previous publication showing that ECV is slightly increased in patients with valvular disease [START_REF] De Meester De Ravenstein | Histological Validation of measurement of diffuse interstitial myocardial fibrosis by myocardial extravascular volume fraction from Modified Look-Locker imaging (MOLLI) T1 mapping at 3 T[END_REF][START_REF] Treibel | Reappraising myocardial fibrosis in severe aortic stenosis: an invasive and non-invasive study in 133 patients[END_REF]. De Meester et al. included patients with AS and patients with AR and found a mean global ECV of 28.9 ± 5.5 (SD) % and a mean CVF of 6.1 ± 4.3 (SD) % at 3T [START_REF] De Meester De Ravenstein | Histological Validation of measurement of diffuse interstitial myocardial fibrosis by myocardial extravascular volume fraction from Modified Look-Locker imaging (MOLLI) T1 mapping at 3 T[END_REF]. Treibel et al. who included only patients with AS, found a mean global ECV of 28.4 ± 2.4 (SD) % at 1.5T CMR and a mean CVF of 11.5 ± 8.6 (SD) % [START_REF] Treibel | Reappraising myocardial fibrosis in severe aortic stenosis: an invasive and non-invasive study in 133 patients[END_REF].

In the present study, the histological measurements of myocardial fibrosis were limited to one sampling site in the LV using a 18-Gauge needle biopsy. In AS and valvular regurgitation, the diffuse reactive myocardial fibrosis is more ubiquitous than focal fibrosis [START_REF] Mewton | Assessment of myocardial fibrosis with cardiovascular magnetic resonance[END_REF]. Child et al. demonstrated that ECV values show a significant association with CVF when sampled by septal ROI instead of entire coverage of the midventricular short-axis slice [START_REF] Child | T1 values by conservative septal postprocessing approach are superior in relating to the interstitial myocardial fibrosis: findings from patients with severe aortic stenosis[END_REF]. In fact, diffuse myocardial fibrosis is a heterogeneous process [START_REF] Kazbanov | Effects of heterogeneous diffuse fibrosis on arrhythmia dynamics and mechanism[END_REF]. Myocardial fibrosis is categorized according to distinct patterns: compact, patchy, interstitial and diffuse [START_REF] Nguyen | Cardiac fibrosis and arrhythmogenesis: the road to repair is paved with Perils[END_REF].

These different patterns of fibrosis may not have equivalent arrhythmogenic profiles [START_REF] Morita | Increased susceptibility of aged hearts to ventricular fibrillation during oxidative stress[END_REF][START_REF] Mcdowell | Susceptibility to arrhythmia in the infarcted heart depends on myofibroblast density[END_REF].

LGE is an accurate method of assessing focal replacement fibrosis, but only partially reflects the complete burden of myocardial fibrosis in cases of severe aortic valvulopathy.

Nevertheless focal replacement fibrosis, measured by LGE, worsens the prognosis for adverse cardiovascular events in dilated and hypertrophic cardiomyopathy substantially [START_REF] Becker | The prognostic value of late gadolinium-enhanced CMR in nonischemic dilated cardiomyopathy[END_REF][START_REF] Weng | Prognostic value of LGE-CMR in HCM: a meta-analysis[END_REF]. One the other hand, diffuse myocardial fibrosis, measured by ECV, is also significantly associated with cardiovascular mortality: death, higher hospitalization rate for heart failure, or both. [START_REF] Schelbert | Myocardial fibrosis quantified by extracellular volume is associated with subsequent hospitalization for heart failure, death, or both across the spectrum of ejection fraction and heart failure stage[END_REF].

In the future, more works may be required to assess the heterogeneity of myocardial fibrosis in order to understand precisely which parameters will be the best surrogate markers for myocardial prognosis.

MMPs play a pivotal role in the development of fibrosis by controlling extra cellular matrix degradation [START_REF] Polyakova | Matrix metalloproteinases and their tissue inhibitors in pressure-overloaded human myocardium during heart failure progression[END_REF]. Recent studies have shown that MMPs are not only involved in extra cellular matrix degradation, but also in the progression of heart failure [START_REF] Spinale | Membrane associated matrix proteolysis and heart failure[END_REF]. The cardiac expression of TIMP-1 and TIMP-2 is significantly increased in chronic pressure-overloaded cardiopathy compared with controls and is related to the degree of interstitial fibrosis [START_REF] Bonnans | Remodelling the extracellular matrix in development and disease[END_REF].

Nevertheless propeptide of procollagen which correlates with histologic CVF [START_REF] Izawa | Mineralocorticoid receptor antagonism ameliorates left ventricular diastolic dysfunction and myocardial fibrosis in mildly symptomatic patients with idiopathic dilated cardiomyopathy: a pilot study[END_REF][START_REF] Klappacher | Measuring extracellular matrix turnover in the serum of patients with idiopathic or ischemic dilated cardiomyopathy and impact on diagnosis and prognosis[END_REF], correlates with excentric and concentric cardiac remodeling in our study. Myocardial fibrosis increases ventricular stiffness and leads to diastolic dysfunction. Zeng et al. showed a link between ECV and diastolic strain rate in diabetic rabbits [START_REF] Zeng | The association between diffuse myocardial fibrosis on cardiac magnetic resonance T1 mapping and myocardial dysfunction in diabetic rabbits[END_REF]. Wu et al. showed a correlation between ECV and diastolic function in patients with hypertrophic cardiomyopathy [START_REF] Wu | Myocardial fibrosis evaluated by diffusion-weighted imaging and its relationship to 3D contractile function in patients with hypertrophic cardiomyopathy[END_REF]. Furthermore, patients with type 2 diabetes mellitus and normal myocardial systolic strain exhibit increased ECV, which correlates with their glycated hemoglobin level [30]. Our ability to evaluate ECV in a variety of cardiomyopathies may enhance our understanding of the pathogenesis and help better assess prognosis of these conditions. There is a gap that must be filled before we are able to define the parameters reflecting myocardial fibrosis that could be of clinical interest.

Our study has limitations: Our prospective experimental study was limited to a single center with a relatively small number of patients. One limitation of any radiological vs histological study is the almost unavoidable mismatch between placement of the local ECV's ROI and the biopsy site, which could lead to a possible miscorrelation, particularly with a fine 18-Gauge coaxial biopsy.

In conclusion, concerning severe aortic valvulopathy, diffuse myocardial fibrosis assessed by anterosepto-basal ECV correlates with histological myocardial fibrosis.

Anteroseptobasal ECV strongly correlates with global ECV, which poorly correlates to TIMP-1 and MMP-2, serum biomarkers involved in the progression of heart failure. Diffuse myocardial fibrosis remains an inhomogeneous process, incompletely depicted by only ECV analysis.

Figures

  Distribution normality was checked using the Shapiro-Wilk test. Patient characteristics were compared with an Anova test for continuous quantitative data, and with Chi-square test for quantitative discrete data. Correlations were estimated by using the Pearson correlation coefficient once normality had been demonstrated, otherwise the Spearman correlation coefficient was used. Local ECV, to be compared with CVF, was measured within a regionof-interest (ROI) in the basal anteroseptal segment and the ROI location was carefully correlated with the location of the targeted surgical biopsy. Global ECV represented the average of the 12 different ECV measures (6 in the basal segments and 6 in the midventricular segments), and was compared with serum biomarkers. Correlations between ECV, histologic fibrosis and serum biomarker of collagen turnover were assessed by the Pearson correlation coefficient (r). The strength of correlation was classified as weak (0.2 < r ≤ 0.5), moderate (0.5 < r ≤ 0.8), strong (0.8 < r < 1) and perfect (r =1). Bland-Altman analysis was used to illustrate agreement between basal anteroseptal ECV and global ECV. Intraclass correlation coefficient (ICC) was used to assess inter and intra observer reproducibility for histological measurements and ECV.Statistical tests were 2-tailed and P < 0.05 was considered to indicate a statistically significant difference. All statistical analysis was performed with available software packages (GraphPad Prism, Graph Pad Software, Inc. La Jolla, USA).

  3 ± 22.5 ms (range: 922.1 -1067.3 ms), mean global ECV was 26.7 ± 2.7 (SD) % (range: 23.4 -32.5%). Mean basal anteroseptal native T1 was 961.2 ± 34.3 (SD) ms (range: 918.4 -1073.7 ms) and mean basal anteroseptal ECV was 26.1 ± 2.6 (SD) % (range: 23.2 -30.8%). The mean percentage of CVF was 12.4 ± 9.7 (SD) % (range: 3.2 -25.7%). No LGE was detected in the interventricular septum, nor in the LV subendocardial layer. Four patients showed subtle areas of LGE on CMR at the insertion points of RV on the LV.

Figure 1 :

 1 Figure 1: Multiparametric fibrosis quantification with CMR and histopathological sample from: A 77-year-old man suffering from severe aortic stenosis (AS) (LGE images are not shown because of normality). The different maps were created with Circle ® software (Calgary, Canada). A: Native T1 map (1028 ms), B: Post contrast T1 map (449 ms), C: Extra cellular volume (0.29), D: Collagen volume fraction (14 %).

Figure 2 :

 2 Figure 2: Study flowchart.

Figure 3 :

 3 Figure 3: Graph shows correlation between basal anteroseptal extracellular volume (ECV) and collagen volume fraction (CVF) (r = 0.65; P = 0.0026).

Figure 4 :

 4 Figure 4: Graph shows correlation between global extracellular volume (ECV) and basal anteroseptal extracellular volume (ECV) (r = 0.79; P < 0.0001).

Figure 5 :

 5 Figure 5: Graph shows Bland-Altman plots for comparison between global extracellular volume (ECV) and basal anteroseptal extracellular volume (ECV). The top and bottom line shows +1.96 standard deviation (SD) and -1.96 SD.Positive bias of 0.0141 (SD of bias 0.0173) for the difference between global ECV and basal anteroseptal ECV and with a positive bias of 5.37% (SD of bias of 6.15%) for the % of difference between the both measures.

Figure 6 :

 6 Figure 6: Graph shows correlation between global extracellular volume (ECV) and tissue inhibitor of metalloproteinase (TIMP-1) (r = 0.41; P = 0.0378).

Figure 7 :

 7 Figure 7: Graph shows correlation between global extracellular volume (ECV) and Matrix metalloproteinase 2 (MMP-2) (r = 0.39; P = 0.0475).

Table 1 :

 1 Patients characteristics.

Table 2 :

 2 Correlation between functional CMR variables and serum biomarkers in 26 patients.

Funding

This research received PHRC grant: PHRC N= 2011-A00887-34

Ethics approval and consent to participate

This study was approved by the national ethical committee (ID RCB: 2011-A01395-36 31).

All patients gave their informed consent to participate. All authors agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work are appropriately investigated, resolved, and the resolution documented in the literature

Authors' Contributions

Competing Interests

The authors declare that they have no competing interests Galectin-3 (ng/mL) r = 0.08; P = 0.56 r = 0.04; P = 0.61 r = -0.15; P = 0.31 r = 0.14; P = 0.27 TIMP-1 (ng/mL) r = 0.17; P = 0.39 r = -0.05; P = 0.57 r = 0.01; P = 0.59 r = 0.21; P = 0.18 MMP-2 (ng/mL) r = 0.02; P = 0.47 r = 0.12; P = 0.41 r = 0.08; P = 0.37 r = -0.


Tommaso Lo Barco

Anna Kaminska

Roberta Solazzi

Claude Cancés

; Giulia Barcia

Nicole Chemaly

Elena Fontana

Isabelle Desguerre

Laura Canafoglia

Caroline Hachon

Le Camus

Emma Losito

Laurent Villard

Monika Eisermann

Bernardo Dalla Bernardina

Nathalie Villeneuve

Rima Nabbout
email: rimanabbout@yahoo.com

Prof Rima Nabbout

SYNGAP1-DEE: a visual sensitive epilepsy

Keywords: SYNGAP1, Reflex epilepsy, Fixation-off sensitivity, Eye-closure sensitivity, Photosensitivity, Myoclonic seizures

Objective

To further delineate the electroclinical features of individuals with SYNGAP1 pathogenic variants.

Methods

Participants with pathogenic SYNGAP1 variants and available video-electroencephalogram (EEG) recordings were recruited within five European epilepsy reference centers. We obtained molecular and clinical data, analyzed EEG recordings and archived video-EEGs of seizures and detailed characteristics of interictal and ictal EEG patterns for every patient.

Results

We recruited 15 previously unreported patients and analyzed 72 EEGs. Two distinct EEG patterns emerged, both triggered by eye closure. Pattern 1 (14/15 individuals) consisted of rhythmic posterior/diffuse delta waves appearing with eye-closure and persisting until eye opening (strongly suggestive of fixation-off sensitivity). Pattern 2 (9/15 individuals) consisted of diffuse polyspikeand-wave discharges triggered by eye closure (eye-closure sensitivity). Both patterns presented in 8/15. Including archived video-EEG clips of seizures from 9/15 patients, we analyzed 254 seizures.

Of 224 seizures experienced while awake, 161 (72%) occurred at or following eye closure. In 119/161, pattern 1 preceded an atypical absence, myoclonic seizure or myoclonic absence; in 42/161, pattern 2 was associated with eyelid myoclonia, absences and myoclonic or atonic seizures.

Conclusions

Fixation-off and eye closure were the main triggers for seizures in this SYNGAP1 cohort.

Significance

Combining these clinical and electroencephalographic features could help guide genetic diagnosis.

Highlights

-We analyzed 72 EEGs from 15 unreported individuals exhibiting developmental encephalopathy with pathogenic SYNGAP1 variants.

-72% of awake seizures occurred at eye-closure or with eyes closed.

-Fixation-off and eye closure were the main triggers for seizures in this SYNGAP1 cohort.

INTRODUCTION

SYNGAP1 is a Ras-GTPase activating protein that is selectively expressed in the brain and involved in the modulation of excitatory synaptic transmission by N-methyl-D-aspartate (NMDA) receptors and neurotrophins [START_REF] Kim | SynGAP: A synaptic RasGAP that associates with the PSD-95/SAP90 protein family[END_REF]. SYNGAP1 haploinsufficiency disrupts the excitatory/inhibitory balance in the developing hippocampus and cortex, resulting in accelerated glutamatergic synapse maturation and altered synaptic plasticity [START_REF] Clement | Pathogenic SYNGAP1 mutations impair cognitive development by disrupting maturation of dendritic spine synapses[END_REF][START_REF] Aceti | Syngap1 haploinsufficiency damages a postnatal critical period of pyramidal cell structural maturation linked to cortical circuit assembly[END_REF].

SYNGAP1 pathogenic variants are reported in individuals with intellectual disability (ID), with or without seizures or autism spectrum disorder (ASD). Recently, a distinct developmental and epileptic encephalopathy (DEE) was described (SYNGAP1-DEE) [START_REF] Kim | The role of synaptic GTPase-activating protein in neuronal development and synaptic plasticity[END_REF][START_REF] Hamdan | Mutations in SYNGAP1 in autosomal nonsyndromic mental retardation[END_REF][START_REF] Pinto | Functional impact of global rare copy number variation in autism spectrum disorders[END_REF][START_REF] Clement | Pathogenic SYNGAP1 mutations impair cognitive development by disrupting maturation of dendritic spine synapses[END_REF][START_REF] Berryer | Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF] as characterized by psychomotor delay preceding epilepsy onset, with seizures appearing at a mean age of two years. Individuals present mostly generalized seizures, namely: myoclonic, atonic, and myoclonic-atonic seizures; atypical absences; eyelid myoclonia and myoclonic absences [START_REF] Hamdan | Mutations in SYNGAP1 in autosomal nonsyndromic mental retardation[END_REF][START_REF] Zollino | Integrated analysis of clinical signs and literature data for the diagnosis and therapy of a previously undescribed 6p21.3 deletion syndrome[END_REF][START_REF] Berryer | Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency[END_REF][START_REF] Von Stülpnagel | SYNGAP1 Mutation in Focal and Generalized Epilepsy: A Literature Overview and A Case Report with Special Aspects of the EEG[END_REF][START_REF] Von Stülpnagel | Chewing induced reflex seizures ("eating epilepsy") and eye closure sensitivity as a common feature in pediatric patients with SYNGAP1 mutations: Review of literature and report of 8 cases[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF]. Some individuals have photosensitivity and fixation-off sensitivity (FOS) [START_REF] Berryer | Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency[END_REF][START_REF] Mignot | Genetic and neurodevelopmental spectrum of SYNGAP1 -associated intellectual disability and epilepsy[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF], and FOS was also reported to trigger seizures in two individuals [START_REF] Mignot | Genetic and neurodevelopmental spectrum of SYNGAP1 -associated intellectual disability and epilepsy[END_REF]. Other reflex seizure triggers have included eating, sounds and touch [START_REF] Mignot | Genetic and neurodevelopmental spectrum of SYNGAP1 -associated intellectual disability and epilepsy[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF][START_REF] Von Stülpnagel | Chewing induced reflex seizures ("eating epilepsy") and eye closure sensitivity as a common feature in pediatric patients with SYNGAP1 mutations: Review of literature and report of 8 cases[END_REF], and eye closure sensitivity (ECS) has been identified as likely involved in the mechanism of reflex seizures [START_REF] Von Stülpnagel | Chewing induced reflex seizures ("eating epilepsy") and eye closure sensitivity as a common feature in pediatric patients with SYNGAP1 mutations: Review of literature and report of 8 cases[END_REF].

Interictal EEG exhibits some common features with slow background activity [START_REF] Carvill | Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1[END_REF][START_REF] Mignot | Genetic and neurodevelopmental spectrum of SYNGAP1 -associated intellectual disability and epilepsy[END_REF][START_REF] Jimenez-Gomez | Phenotypic characterization of individuals with SYNGAP1 pathogenic variants reveals a potential correlation between posterior dominant rhythm and developmental progression[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF]) and intermittent rhythmic delta activity in few individuals with mainly occipital predominance [START_REF] Jimenez-Gomez | Phenotypic characterization of individuals with SYNGAP1 pathogenic variants reveals a potential correlation between posterior dominant rhythm and developmental progression[END_REF].

Both generalized and focal discharges, mainly posterior, have been recorded [START_REF] Berryer | Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency[END_REF][START_REF] Von Stülpnagel | SYNGAP1 Mutation in Focal and Generalized Epilepsy: A Literature Overview and A Case Report with Special Aspects of the EEG[END_REF][START_REF] Mignot | Genetic and neurodevelopmental spectrum of SYNGAP1 -associated intellectual disability and epilepsy[END_REF][START_REF] Jimenez-Gomez | Phenotypic characterization of individuals with SYNGAP1 pathogenic variants reveals a potential correlation between posterior dominant rhythm and developmental progression[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF].

We aimed to further delineate the electroclinical patterns of 15 previously unreported individuals with SYNGAP1 pathogenic variants, with the goal of characterizing interictal and ictal EEG patterns.

MATERIALS AND METHODS

Individuals were referred through a network of collaborating geneticists and child neurologists and recruited within five European epilepsy reference centers in France (Paris, Toulouse, Marseille) and Italy (Verona and Milano). Inclusion criteria specified individuals with a) developmental encephalopathy with pathogenic SYNGAP1 variants and b) at least one available video-EEG recorded during infancy or childhood.

Referring physicians were provided with a standardized phenotyping table to assess clinical characteristics, gene pathogenic variants, neuroimaging findings, date of acquisition of key developmental milestones, and detailed epilepsy characteristics. Individuals were classified into three categories according to the International League Against Epilepsy (ILAE) Classification of the Epilepsies (Scheffer et al. 2017): developmental encephalopathy without epilepsy, developmental encephalopathy with epilepsy (in which seizures were sporadic) and developmental and epileptic encephalopathy (in which seizures were often frequent, hindering development beyond the impact of gene dysfunction).

Seizure types were classified using the 2017 ILAE classifications (Fisher et al. 2017).

EEG recordings, with archived video-EEGs of seizures, were centralized via the network of the reference center for rare epilepsies at Necker Hospital in Paris. EEGs were obtained using 19 electrodes placed according to the 10/20 international system, or using 11 electrodes (FP1, FP2, C3, C4, F7, F8, T5, T6, O1, O2, and Cz) and a reference. Signal was acquired at 256 Hz sampling, amplified (1,000 times) and filtered to the 0.01-97 Hz band. Polygraphic recordings included electrocardiograms and surface electromyograms of deltoid muscles. EEGs were analyzed by a team of neurophysiologists and epileptologists (TLB, RN, ME, BDB, AK). Interictal EEG features and ictal patterns of each seizure with an available video-EEG clip were detailed for every patient.

Informed consent was obtained from participants' caregivers. This study had the approval of Necker Hospital ethic committee.

RESULTS

Fifteen new individuals with SYNGAP1-encephalopathy were recruited (10 females and five males).

Genetics findings

Pathogenic variants were de novo in 14/15; in one (case 14), the variant was absent in the mother while no sample from the father was available. Four mutations were missense (cases 3, 5, 7, 9, 11, and 13), while nine were truncating (patients 1, 2, 4, 6, 8, 10, 12, 14, and 15). Three individuals shared the mutation c.1685C>T (p.Pro562Leu), already reported in the literature [START_REF] Berryer | Mutations in SYNGAP1 Cause Intellectual Disability, Autism, and a Specific Form of Epilepsy by Inducing Haploinsufficiency[END_REF]. We report seven new mutations (Table 1).

Clinical data and epilepsy features

Mean age at last follow-up was 7.3 years, ranging from 2.1 to 14 years. Except for one (patient 13) exhibiting mild cognitive impairment, all individuals had moderate to severe developmental delay.

Autistic traits were present in 10/15. Eleven individuals (cases 1-11) presented with developmental and epileptic encephalopathy. Three had developmental encephalopathy with sporadic seizures (cases 12-14) mainly triggered by fever, with one (case 14) having presented only three febrile seizures. One patient had developmental encephalopathy without epilepsy (case 15). All three patients who presented the same pathogenic variant (c.1685C>T (p.Pro562Leu)) exhibited moderate to severe developmental delay and ASD. However, they displayed varying epilepsy phenotypes and varying motor skills development. Main clinical characteristics are summarized in Table 1.

EEG Analysis

Seventy-two EEGs were analyzed (range: 1-10 per patient), including four long-duration video-EEG monitoring sessions, for a total of 139 hours of registration. The median age at first EEG was 3.2 years (range 2.3-12.3 years) and median age during EEG recordings was 5.1 years (range 2.2-13 years).

Archived video-EEG clips of seizures were available for nine individuals (cases 1-9), for a total of 254 seizures (median 20, range 2-135 per patient); 224 seizures occurred during wakefulness and 30 during sleep. Atypical absences were seen in eight individuals (cases 1, 2, 3, 4, 5, 6, 8, and 9) and could be associated with atonic phenomena (cases 1 and 5), oculoclonic movements (cases 5 and 6) or rhythmic axial myoclonia (cases 2, 8, and 9); one patient exhibited an atypical absence status (case 7). Other seizure types included myoclonic seizures (cases 2, 7, and 8), eyelid myoclonia with absences (EMA, cases 5 and 8), atonic seizures (cases 7 and 8), myoclonic absences (case 6), myoclonic atonic seizures (case 8) and myoclonic-atonic-tonic seizures (case 8). Eyelid myoclonia without absences (EM) were not included in the count of total seizures and were found in cases 2, 4, 5, 8, and 9. Of 224 seizures during wakefulness, 161 occurred at eye closure or with eyes closed (72%).

With eyes open, background activity showed no abnormalities, except for in case 7 who presented a diffuse slow activity. Interictal epileptiform abnormalities were observed sporadically, mainly as multifocal low voltage spikes with occipital predominance (cases 1, 2, 3, 5, 6, 10, and 14) or generalized spike-and-wave or polyspike-and-wave discharges (cases 2, 4, 5, 7, 8, 9, and 13). At eye closure and/or keeping eyes closed, two distinct EEG patterns were recorded.

Pattern 1

This pattern was found in 14/15 individuals (93%), including one patient who presented only three febrile seizures (FS) without further epilepsy, and in another patient who did not experience any seizure.

EEGs revealed trains of rhythmic delta waves of 3-3.5 Hz with moderate or high amplitudes bilaterally involving the posterior regions, with a superimposed low voltage spike and often displaying a so-called "notched" morphology. Typically, it appeared with eye closure or within mere seconds of eye closure and persisted until eye opening (Figure 1). This pattern often became subcontinuous while falling asleep and during the initial sleep phases (Figure 2) or at the awakening phase.

At times, these posterior rhythmic delta waves gradually spread to the anterior regions, organizing into generalized spike-and-wave discharges and keeping the same rhythmicity without clear ictal semiology witnessed by family or recorded on video. In some instances, these generalized spikeand-wave discharges were concomitant with a loss of awareness (cases 2, 3, 4, 5, 6, and 8) and to rhythmic upward movements of the eyes (cases 5 and 6), single or rhythmic axial myoclonia (patients 2 and 8), loss of postural tone (case 5), or rhythmic myoclonic jerks of upper limbs (myoclonic absences, patient 6, Figures 3 and4). In one case (patient 2), pattern 1 persisted with eyes open for 1 minute on the left temporo-occipital region, with the patient looking troubled and crying, mimicking a focal seizure.

Of 224 seizures during wakefulness, 119 (53%) showed this ictal pattern. Repeated myoclonic or atypical absence seizures were seen in three individuals (cases 2, 6, and 8) during episodes of crying with eye closure (Videos 1 and 2), evolving into status epilepticus.

Pattern 2

This pattern consisted of brief discharges of fast activity triggered by eye closure or by simple blinking, lasting until eye opening or for 1-3 seconds if eyes remained closed. This pattern was diffuse or prominent in frontal regions, sometimes followed by irregular polyspikes or polyspikeand-waves, with frontal or occipital predominance and increased frequency and duration during drowsiness and in the first stages of sleep (Figure 5).

Five individuals (2, 4, 5, 8, and 9), exhibited concomitant eyelid fluttering (EM) or sudden axial myoclonia or atonia (cases 5, 8, and 9). In some individuals, a generalized discharge of rhythmic spike-and-waves could follow this pattern while eyes were still closed, determining an atypical absence (EMA; cases 5, 8 and 9; Figure 5). Of 224 seizures during wakefulness, 42 (19%) showed this ictal pattern. Both patterns were found in 8/15 individuals (53%), either during the same or in different recordings (Figure 6). Both EEG patterns were observed after self-stimulation induced by eye closing in 4/9 individuals (cases 2, 4, 5, and 8).

Seventy seizures were recorded while crying (cases 2, 5, 6, 8, and 9), three while eating (cases 2 and5) andfour during perioral self-stimulation (cases 2 and8). In all of these episodes, seizures began at eye closure, at simple blinking (showing pattern 2 as ictal pattern) or while eyes were closed (with pattern 1 initializing the seizure in these cases). Consequently, the main triggering factors observed for seizures appearing during crying, eating or during tactile perioral selfstimulation, were blinking and keeping eyes closed (Videos 1 and 2). The conditions characterized by repeated blinking or persistence of eyes closed could induce subcontinuous paroxysmal events prompting a status epilepticus (Videos 1, 3).

Photoparoxysmal responses during intermittent light stimulation (ILS) were seen in 5/15 (cases 2, 5, 6, 9, and 14), being associated with atypical absence with rhythmic myoclonias (cases 2, 5, and 9), atypical absence with rhythmic upward movements of the eyes (case 6) or eyelids myoclonia with absences (cases 5 and 9). In these cases, ictal events did not appear concomitantly with the start of ILS, but at the moment of eye closure (showing ictal pattern 2) or while keeping eyes closed (with pattern 1 trigging the seizure). No seizures were noted during ILS with eyes open.

EEG findings are summarized in Table 2.

DISCUSSION

We report 15 new cases of SYNGAP1 encephalopathy with SYNGAP1-DEE as the most common phenotype. Individuals showed generalized epilepsy, with atypical absences, myoclonic absences, and myoclonic and atonic seizures as the most represented seizure types. Three patients presented the same mutation but exhibited various epilepsy phenotypes for ID and ASD. EEG analysis in this cohort showed epileptiform discharges, mainly in the form of two recurrent distinctive patterns evoked by eye closure and keeping eyes closed. These patterns were mostly interictal, though in some cases they could precede (pattern 1) or be associated with (pattern 2) a seizure. However, differentiation between ictal and interictal was often hard to define, especially for brief absence seizures.

Pattern 1 appeared with closed eyes and persisted until eye opening, replacing physiological background activity in most cases. Although no patient in this study has been specifically tested by impeding central vision (Frenzel lenses or Ganzfeld stimulation), largely due to the known behavior disturbance, pattern 1 is strongly suggestive of FOS [START_REF] Duncan | The differentiation of '"eye closure"' from '"eyes-closed[END_REF][START_REF] Koutroumanidis | Fixation-off sensitivity in epilepsies other than the idiopathic epilepsies of childhood with occipital paroxysms: A 12-year clinical-video EEG study[END_REF][START_REF] Koutroumanidis | The role of EEG in the diagnosis and classification of the epilepsy syndromes: a tool for clinical practice by the ILAE Neurophysiology Task Force (Part 2)[END_REF]. In our patients, FOS triggered atypical absences, myoclonic absences or atonic or myoclonic seizures in six out of nine patients (66%) with available video-EEG clips of seizures.

In pattern 2, epileptic discharges appeared with eye closure and rapidly disappeared within seconds and before eye opening or with eye opening, in accordance with the definition of ECS [START_REF] Koutroumanidis | Fixation-off sensitivity in epilepsies other than the idiopathic epilepsies of childhood with occipital paroxysms: A 12-year clinical-video EEG study[END_REF][START_REF] Koutroumanidis | The role of EEG in the diagnosis and classification of the epilepsy syndromes: a tool for clinical practice by the ILAE Neurophysiology Task Force (Part 2)[END_REF][START_REF] Karkare | Electroclinical characteristics and syndromic associations of "eye-condition" related visual sensitive epilepsies-A crosssectional study[END_REF]In this cohort, ECS was found to trigger seizures in four of the nine individuals (44%) with available video-EEG clips of seizures.

FOS involves EEG paroxysms that may begin either immediately or seconds after eye closure, typically persisting for as long as the eyes remain closed, and are activated by complete darkness where fixation is impeded [START_REF] Koutroumanidis | Fixation-off sensitivity in epilepsies other than the idiopathic epilepsies of childhood with occipital paroxysms: A 12-year clinical-video EEG study[END_REF][START_REF] Koutroumanidis | The role of EEG in the diagnosis and classification of the epilepsy syndromes: a tool for clinical practice by the ILAE Neurophysiology Task Force (Part 2)[END_REF][START_REF] Karkare | Electroclinical characteristics and syndromic associations of "eye-condition" related visual sensitive epilepsies-A crosssectional study[END_REF]. In contrast, ECS is defined as the appearance of EEG paroxysms within 1 to 3 seconds after positive deflection of the eye closure artifact, lasting for 3 seconds or less, and suppressed by complete darkness [START_REF] Koutroumanidis | Fixation-off sensitivity in epilepsies other than the idiopathic epilepsies of childhood with occipital paroxysms: A 12-year clinical-video EEG study[END_REF][START_REF] Karkare | Electroclinical characteristics and syndromic associations of "eye-condition" related visual sensitive epilepsies-A crosssectional study[END_REF].

FOS is reported in all types of epilepsies: focal or generalized; of structural, genetic, or unknown etiology; with or without photosensitivity. It can occur in drug-responsive or drug-resistant epilepsies, even in individuals without obvious epileptic seizures [START_REF] Koutroumanidis | Fixation-off sensitivity in epilepsies other than the idiopathic epilepsies of childhood with occipital paroxysms: A 12-year clinical-video EEG study[END_REF][START_REF] Koutroumanidis | The role of EEG in the diagnosis and classification of the epilepsy syndromes: a tool for clinical practice by the ILAE Neurophysiology Task Force (Part 2)[END_REF][START_REF] Karkare | Electroclinical characteristics and syndromic associations of "eye-condition" related visual sensitive epilepsies-A crosssectional study[END_REF]. ECS is mainly found in genetic generalized epilepsies, and in genetic or structural focal epilepsies involving the occipital lobe [START_REF] Fabian | Epileptic discharge after eye closure : relation to photosensitivity[END_REF][START_REF] Kurth | Fixation-off sensitivity in an adult with symptomatic occipital epilepsy[END_REF][START_REF] Sevgi | Eye closure sensitivity and epileptic syndromes: A retrospective study of 26 adult cases[END_REF][START_REF] Tassinari | With Myoclonic Absences and Epilepsy With Eyelid Myoclonia and Absences[END_REF][START_REF] Karkare | Electroclinical characteristics and syndromic associations of "eye-condition" related visual sensitive epilepsies-A crosssectional study[END_REF].

In our population, we found a pattern strongly suggestive of FOS (pattern 1) in 14/15 individuals (93%), ECS (pattern 2) in 9/15 individuals (60%), and both in 8/15 (53%). In 66% and 44% of these individuals, respectively, FOS and ECS acted as seizure triggers. Even if the exact ratio of reflex seizures was challenging to determine due to relevant behavior disorders and difficulties in obtaining collaboration and quality video frames during video-EEG recordings, at least 72% of ictal events occurring during wakefulness (with available archived video-EEGs) began when patients' eyes were closed.

Reflex seizures have been reported in SYNGAP1-DEE [START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF][START_REF] Von Stülpnagel | Chewing induced reflex seizures ("eating epilepsy") and eye closure sensitivity as a common feature in pediatric patients with SYNGAP1 mutations: Review of literature and report of 8 cases[END_REF]. In this study, 5/9 (55%) individuals with available archived video-EEG clips exhibited seizures while crying (n=5), eating (n=2) or experiencing orofacial stimuli (n=2); however, eye closure always preceded the stimuli, suggesting that ECS and FOS represent the main seizure trigger mechanism.

ECS was already suggested to be involved in the trigger mechanism of reflex seizures in SYNGAP1 patients [START_REF] Von Stülpnagel | Chewing induced reflex seizures ("eating epilepsy") and eye closure sensitivity as a common feature in pediatric patients with SYNGAP1 mutations: Review of literature and report of 8 cases[END_REF][START_REF] Wolf | Reader response: SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF]. Nonetheless, individuals with SYNGAP1-DEE shares many features of Jeavons Syndrome [START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF], in which eyelid fluttering and absence seizures are triggered by eye-closure [START_REF] Striano | Eyelid myoclonia with absences (Jeavons syndrome): A well-defined idiopathic generalized epilepsy syndrome or a spectrum of photosensitive conditions?[END_REF]). On the other hand, as suggested by Vlaskamp et al, it is likely that more severe cases of Eyelids Myoclonia with Absences Epilepsy may be explained by SYNGAP1 mutations, especially those subjects with earlier onset, poorer intellectual outcome, self-induction and myoclonic or atonic seizures triggered by eyeclosure [START_REF] Caraballo | A study of 63 cases with eyelid myoclonia with or without absences: Type of seizure or an epileptic syndrome?[END_REF][START_REF] Okazaki | Pharmacoresistant epileptic eyelid twitching in a child with a mutation in SYNGAP1[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF]."

FOS was previously anecdotally reported as a seizure trigger in two individuals with SYNGAP1 pathogenic variants within a larger cohort [START_REF] Mignot | Genetic and neurodevelopmental spectrum of SYNGAP1 -associated intellectual disability and epilepsy[END_REF]. A confirmation of FOS through Frenzel lenses or Ganzfeld stimulation should be performed when possible.

FOS as a seizure trigger and the combination of ECS and FOS in the same subject are very rare conditions [START_REF] Panayiotopoulos | Juvenile Myoclonic Epilepsy: A 5-Year Prospective Study[END_REF][START_REF] Koutroumanidis | The role of EEG in the diagnosis and classification of the epilepsy syndromes: a tool for clinical practice by the ILAE Neurophysiology Task Force (Part 2)[END_REF][START_REF] Karkare | Electroclinical characteristics and syndromic associations of "eye-condition" related visual sensitive epilepsies-A crosssectional study[END_REF]) and seem to strongly suggest SYNGAP1 haploinsufficiency.

FOS and ECS are the expression of occipital hyperexcitability, concerning parvocellular pathways (FOS) and the magnocellular system (ECS) [START_REF] Koutroumanidis | Fixation-off sensitivity in epilepsies other than the idiopathic epilepsies of childhood with occipital paroxysms: A 12-year clinical-video EEG study[END_REF][START_REF] Vaudano | The visual system in eyelid myoclonia with absences[END_REF][START_REF] Vaudano | Photosensitive epilepsy is associated with reduced inhibition of alpha rhythm generating networks[END_REF]. SYNGAP1 haploinsufficiency impacts brain function with a range of circuit-specific impairments that disrupt neuronal excitability and function in complex ways [START_REF] Michaelson | SYNGAP1 heterozygosity disrupts sensory processing by reducing touch-related activity within somatosensory cortex circuits[END_REF]). These "circuit-pathologies" interact to disrupt behavior, impair cognition, and promote seizures [START_REF] Michaelson | SYNGAP1 heterozygosity disrupts sensory processing by reducing touch-related activity within somatosensory cortex circuits[END_REF]. A visual cortex hyperexcitability has recently been seen in mouse models with SYNGAP1 haploinsufficiency [START_REF] Katsanevaki | Cortical circuit and behavioural pathophysiology in rodent models of SYNGAP1 haploinsufficiency[END_REF], and the volumes of many cortical areas related to the visual system have found to be changed in SYNGAP1-heterozygous mice, compared to wildtype [START_REF] Kilinc | Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders[END_REF]). These findings, together with our results, suggest a possible involvement of visual circuits in SYNGAP1 haploinsufficiency.

In addition, myoclonic absence seizures were previously reported in 3 individuals carrying SYNGAP1 pathological variants, but EEG iconography was not shown [START_REF] Klitten | A balanced translocation disrupts SYNGAP1 in a patient with intellectual disability, speech impairment, and epilepsy with myoclonic absences (EMA)[END_REF][START_REF] Vlaskamp | SYNGAP1 encephalopathy: A distinctive generalized developmental and epileptic encephalopathy[END_REF]). In the present study, several long-lasting absence seizures associated to rhythmic myoclonia were documented in one patient, mainly, but not exclusively, triggered by eyeclosure. These seizures consisted of long-lasting 2,5-3 Hz spike-and-wave discharge (10 to 30 seconds), bilateral, with higher amplitude on the frontal region and positive transient of the spike.

Excluding the very firsts, each spike-and-wave complex of the discharge was associate to a myoclonia, mainly involving proximal upper limb muscles. Rhythmic myoclonia were temporally separated by a brief interruption of muscular activity. Child did not lose awareness, and he could move and grasp objects, particularly in the second half of the discharge. Even if an increasing tonic activity was not observed on the EMG during the discharges and the spike-and-waves shape was quite irregular (presumably due to antiseizure treatment), these seizures were highly suggestive of the myoclonic absences described by Tassinari in the Epilepsy with Myoclonic Absences [START_REF] Tassinari | Etude des décharges de pointes ondes chez l'homme II. Les aspects cliniques et lectroencephalographiques des absences myocloniques[END_REF].

CONCLUSION

This study further defined the electroclinical features of individuals with SYNGAP1 haploinsufficiency. The occurrence in EEGs of ECS, FOS and, most importantly, combinations of both in children with early onset neurodevelopmental delays and normal brain magnetic resonance imaging (MRI) was indicative of SYNGAP1 haploinsufficiency, especially in the presence of a visual-sensitive epilepsy (Kasteleijn-Nolst [START_REF] Trenité | Visual sensitivity and epilepsy: A proposed terminology and classification for clinical and EEG phenomenology[END_REF]) starting after the first year, with atypical absences and myoclonic and atonic seizures. Combining these specific clinical and electroencephalographic features may help to guide genetic diagnosis and future trials for this genetic condition [START_REF] Kuchenbuch | Add-on cannabidiol significantly decreases seizures in 3 patients with SYNGAP1 developmental and epileptic encephalopathy[END_REF][START_REF] Sullivan | Low-Dose Perampanel Rescues Cortical Gamma Dysregulation Associated With Parvalbumin Interneuron GluA2 Upregulation in Epileptic Syngap1+/-Mice[END_REF].

DECLARATION OF INTEREST

None. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

1 X X X X X X X X X X

FIGUREFigure 1 .

 1 FIGURE AND VIDEO LEGENDS

Figure 2 .

 2 Figure 2. Two different types of activation during sleep. Left: Pattern 1. Case 8, 3 years 10 months. The rhythmic delta waves seen with eyes closed become subcontinuous on posterior regions while falling asleep and in the first stages of non-rapid eye movement (NREM) sleep. Right: Pattern 2. Case 4, 2 years 10 months. Bursts of diffuse, irregular, spike-and-wave and polyspike-and-wave discharges appear NREM sleep. ECG=electrocardiogram; EMG1= left deltoid.

Figure 3 .

 3 Figure 3. Pattern 1. While keeping eyes closed, posterior interictal activity can gradually spread to the anterior regions and organize in a rhythmic spikes-and-waves sequence that causes an atypical absence without any motor event (left, case 10, 5 years 5 months), or is associated with rhythmic axial myoclonias (middle, case 2, 2 years 5 months) or with a loss of postural tone (right, case 5, 6 years 2 months). DELTD=right deltoid; DELTG=left deltoid; EC=eyes closed; EO=eyes open; ECG=electrocardiogram.

Figure 4 .

 4 Figure 4. Case 6. Top: At the age of 3 years 6 months, eye closure determines the appearance of rhythmic 2.5-3 Hz diffuse spikes and waves, arising from the occipital areas and clinically associated with atypical absence with rhythmic oculoclonic movements. Middle: At the age of 6 years, eye closure leads to the same ictal discharge, this time clinically related to myoclonic absence. Bottom: Same discharge. Note the positive transient of the spike-and-waves on the anterior regions, that clinically relates with a sudden myoclonia of upper limbs. DELTD=right deltoid; DELTG=left deltoid; EC=eyes closed; EO=eyes open; ECG=electrocardiogram.

Figure 5 .

 5 Figure 5. Pattern 2. Simple blinking determines the appearance of low-amplitude spikes, isolated on anterior regions or diffuse (left, case 10, 12 years 4 months); fast generalized spike-and-wave discharges, prominent on posterior and anterior regions and sometimes associated with subtle

Figure 6 .

 6 Figure 6. Pattern 1 and pattern 2 are often found simultaneously in the same electroencephalogram (EEG) recording. Left: case 4, 2 years 6 months. Right: case 5, 5 years 1 month. (*) = blinking; EC=eyes closed; EO=eyes open; ECG=electrocardiogram; EMG(upper)=right deltoid; EMG(bottom)=left deltoid.

Table 1 .

 1 Molecular and clinical data. AA, Atypical absences; AAAt, Atypical absences with atonic phenomena; AAM, Atypical absences with myoclonia; AAOC, Atypical absences with oculoclonic movements; At, Atonic seizures; CLB, Clobazam; CBZ, Carbamazepine; CNZ, Clonazepam; DA, Unspecified drop-attacks; DE, Developmental encephalopathy; DE+E Developmental encephalopathy with epilepsy; DEE, Developmental and epileptic encephalopathy; EM, Eyelids myoclonia; EMA, Eyelids

			myoclonia with absences; ESM; Ethosuximide; FS, Febrile seizures; HYD; Hydrocortisone; KD, Ketogenic
			Diet; LEV, Levetiracetam; LTG, Lamotrigine; M, Myoclonic seizures; MA, Myoclonic absences; MA-T,
			Myoclonic-atonic-tonic seizures; NA, Not available data; PER, Perampanel; RUF; Rufinamide; T, Tonic;
			TC, Tonic-clonic; TPM; Topiramate; VPA, Valproic Acid; VNS; Vagus Nerve Stimulation; ZNS,
			Zonisamide.
	Patient ID	Gender	SYNGAP1	(NM_006772.2:)	mutation	Inheritance	Mutation present in	literature	Diagnosis	Age of	sitting/walking	Age of first words	Cognitive disability	Autism (yes/no)	Other clinical	features	Brain IRM	Age at epilepsy	onset	Seizures types	Age at last FUP	Current language	ability	Seizures frequency	at last FUP	Lifetime	antiepileptic	treament
															Nystagmus ;
	1	F	c.3179dup, p.(Gly1061Trpfs*92)	de novo	-		DEE	9 m / 22 m	41 m	Severe	N	torticollis; spasmus	Normal	28 m	AA, AAAt	4 y 3 m	Words association	Sporadic	VPA, LTG, ESM
															nutans
																										VPA, ESM, ZNS,
	2	M	c.2059C>T, p.(Arg687*) **	de novo	Vlaskamp et al. 2019	DEE	11 m / 24 m	24 m	Moderate / Severe	Y	Behavior disorder	Normal	8 m	AA, AAM, M, FS	3 y 4 m	Single words	Daily	LTG (important worsening), KD (temporary
																										improvement)
	3	F	c.1726T>C, p.(Cys576Arg)	de novo	-		DEE	NA / 25 m	-	Moderate / Severe	Y	Eating disorder	Normal	30 m	AA	3 y 6 m	Absent	Weekly		VPA
															Behavior
															disorder; fine
	4	M	c.3215_3224del, p.(Lys1072Serfs*2)	de novo	-		DEE	11m / 24 m	-	Severe	Y	and gross motor impairment;	Normal	27 m	AA, EM, DA	11 y 3m	Absent	Seizure free	LEV, VPA
															head growth
															slowdown
	5	F	c.922T>C, p.(Trp308Arg)	de novo	Jimenez-Gomez et al. 2019	DEE	9 m / 36 m	-	Moderate / Severe	Y	Drooling; behavioural and sleep issues; growth delay	Bilateral hypersignal of white matter (4 y); Cerebellar atrophy (6 y)	18m	EMA, AA, AAM, AAOC, AAAt, At	6 y 3 m	Absent	Daily	VPA, ESM, ZNS, KD (temporary improvement), CLB, RUF, LTG, VNS
																				EMA,
	6	M	c.1966G>T, p.(Glu656*)	de novo	-		DEE	NA / 19 m	18 m	Moderate	Y	Behavior disorder	Normal	36 m	AA, AAOC,	5 y 4 m	Single words	Daily	VPA, LTG, ESM, LEV, KD
																				MA
															Drooling;
															eating issues
	7	F	c.1685C>T p.(Pro562Leu)	de novo	Berryer et al. 2013; Vlaskamp et al. 2019	DEE	10 m / 26 m	-	Severe	Y	(selectivity); open mouth; low pain sensitivity; hyperacusis;	Normal	24 m	AA, AAAt, T	10 y	Absent	Seizures free	CBZ, VPA, ZNS, TPM, RUF, CLN, CLB, metilprednisolone, HYD
															head growth
															slowdown
	8	M	c.3583-6G>A, p.(Val1195Alafs*27)	de novo	Redin et al. 2014; Bowling et al. 2017	DEE	10 m / 25 m	-	Severe	Y	Nystagmus; feeding issues	Aspecific white matter hypersignal	20 m	AAM, M, T	6 y 6 m	Absent	Daily	LEV, TPM, CLB, CNZ, VPA, ZNS, LTG, PER, KD
															Sleep and
	9	F	c.2798A>G p.(His933Arg)	de novo	-		DEE	NA / 21m	-	Moderate/ Severe	Y	behavioural issues; head growth	NA	20 m	M, AAM, AA	14 y	Absent	Sporadic	VPA, LTG, TPM
															slowdown

Table 2 .

 2 EEG features found. X=found ; AA, atypical absences; AAAt, atypical absences with atonic phenomena; AAM, Atypical absences with myoclonia; AAOC, atypical absences with oculoclonic movements; At, atonic seizures; EM, eyelids myoclonia; EMA, eyelids myoclonia with absences; F, focal; M, myoclonic seizures; MA, myoclonic absences; MA-T, myoclonic-atonictonic seizures; S, Sleep; W, Wake.

	Patient ID	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
	Pattern