Viet Huong Nguyen

Abderrahime Sekkat

Carmen Jiménez

Delfina Muñoz

Daniel Bellet

David Muñoz-Rojas

Impact of Precursor Exposure in Spatial Atomic Layer Deposition on Process Efficiency and Film Properties

Keywords: spatial ALD, atmospheric pressure, precursor exposure, process efficiency, substrate velocity

Being able to control the exposure of precursors to the substrate surface is essential towards an optimum Atomic Layer Deposition (ALD) process. Conventional ALD usually requires large excesses of precursors and expensive vacuum chamber in which the precursor exposure can be controlled via the injection pulse duration followed by a delay time. A version of ALD, namely Spatial ALD (SALD), which has the same unique assets as conventional ALD but being faster, vacuum-free, and easier to scale up, has recently attracted much attention. In SALD, precursors are continuously exposed to a moving surface, and thus it is more complicated to tune precursor exposure than with conventional ALD. Here, we present a study on how to control this critical parameter in for the deposition of ZnO films using a close-proximity, open-air SALD. Our results are based on both simulations and experiments. A simple physical model supported by Comsol Multiphysics simulations has been developed to study the effect of the substrate velocity as well as the precursor concentration in the carrier gas on film growth rate. We found that the precursor entrainment by the moving substrate induces an asymmetric precursor concentration profile that affects the obtained GPC. Also, we show that the mass density and structural properties of the deposited films depend closely on the film growth rate. For instance, fast growth does not always produce dense films. Indeed, a compromise between the growth rate and the precursor consumption 2 should be considered to obtain both good process efficiency and high-quality films. Our findings are particularly relevant for using SALD in large-scale coating applications, in which high deposition rate and good coating density should be obtained with a minimal ALD precursor consumption.

Introduction

Atomic layer deposition (ALD) has been considered as one of the best thin-film growth technologies thanks to its capability of depositing pinhole-free, conformal films at relatively low temperatures. [START_REF] George | Atomic Layer Deposition: An Overview[END_REF][START_REF] Van Bui | Atomic and molecular layer deposition: off the beaten track[END_REF][START_REF] Cremers | Conformality in atomic layer deposition: Current status overview of analysis and modelling[END_REF] Over the past decades, research advances have expanded the use of this surfacecontrolled layer-by-layer technology beyond microelectronic applications [START_REF] Johnson | A brief review of atomic layer deposition: from fundamentals to applications[END_REF][START_REF] Mai | Low-Temperature Plasma-Enhanced Atomic Layer Deposition of Tin(IV) Oxide from a Functionalized Alkyl Precursor: Fabrication and Evaluation of SnO 2 -Based Thin-Film Transistor Devices[END_REF], including the development of biosensors, [START_REF] Graniel | Atomic layer deposition for biosensing applications[END_REF] materials for barrier coatings [START_REF] Park | Thin film encapsulation for flexible AM-OLED: a review[END_REF][START_REF] Maindron | Defect analysis in low temperature atomic layer deposited Al2O3 and physical vapor deposited SiO barrier films and combination of both to achieve high quality moisture barriers[END_REF][START_REF] Singh | Development of ALD Coatings for Harsh Environment Applications[END_REF][START_REF] Khan | Stability Enhancement of Silver Nanowire Networks with Conformal ZnO Coatings Deposited by Atmospheric Pressure Spatial Atomic Layer Deposition[END_REF][START_REF] Nguyen | Low-cost fabrication of flexible transparent electrodes based on Al doped ZnO and silver nanowire nanocomposites: impact of the network density[END_REF] or energy conversion and storage. [START_REF] Liu | Water Splitting by Tungsten Oxide Prepared by Atomic Layer Deposition and Decorated with an Oxygen-Evolving Catalyst[END_REF][START_REF] Meng | Emerging Applications of Atomic Layer Deposition for Lithium-Ion Battery Studies[END_REF][START_REF] Bui | Low-temperature atomic layer deposition delivers more active and stable Pt-based catalysts[END_REF][START_REF] Kim | Electrosprayed BiVO4 nanopillars coated with atomic-layer-deposited ZnO/TiO2 as highly efficient photoanodes for solar water splitting[END_REF][START_REF] Grillo | From Single Atoms to Nanoparticles: Autocatalysis and Metal Aggregation in Atomic Layer Deposition of Pt on TiO2 Nanopowder[END_REF][START_REF] Liu | Conformal deposition of atomic TiO2 layer on chalcogenide nanorod with excellent activity and durability towards solar H2 generation[END_REF][START_REF] Fang | Atomic layer deposition in porous electrodes: A pore-scale modeling study[END_REF] However, one of the main drawbacks of ALD is a slow growth rate, which can be attributed to slow purging steps alternatively performed between different precursor injections. [START_REF] Muñoz-Rojas | Speeding up the unique assets of atomic layer deposition[END_REF] Additionally, the use of a vacuum chamber makes conventional ALD technology more difficult and expensive to scale up towards an industrial level. In this context, a variation of ALD technique, namely spatial ALD (SALD), has been recently attracting much attention since this ALD-based technology can be applied to deposit materials at atmospheric pressure and low temperatures.

Additionally, SALD provides very fast deposition rate and in a scalable fashion, while keeping the unique assets of ALD technology. [START_REF] Yersak | Spatial atomic layer deposition for coating flexible porous Li-ion battery electrodes[END_REF][START_REF] Illiberi | Spatial atomic layer deposition of zinc oxide thin films[END_REF][START_REF] Illiberi | Spatial Atmospheric Atomic Layer Deposition of AlxZn1-xO[END_REF][START_REF] Muñoz-Rojas | Spatial Atomic Layer Deposition (SALD), an emerging tool for energy materials. Application to new-generation photovoltaic devices and transparent conductive materials[END_REF] For instance, SALD is compatible with roll-to-roll technology for fast industrial thin film deposition. [START_REF] Musselman | Nanomanufacturing: High-Throughput, Cost-Effective Deposition of Atomic Scale Thin Films via Atmospheric Pressure Spatial Atomic Layer Deposition[END_REF] The key point of an ALD technology is the possibility of thoroughly controlling the half-reactions between precursors and the substrate surface, and at the same time to minimize the precursor use and optimize the ALD growth rate (expressed in the thickness of the layer deposited on the surface after each ALD cycle, i.e. nm/cycle). [START_REF] Cremers | Conformality in atomic layer deposition: Current status overview of analysis and modelling[END_REF][START_REF] Parsons | Progress and future directions for atomic layer deposition and ALD-based chemistry[END_REF] In an ideal ALD process, a complete monolayer of precursor molecules would be formed on the substrate surface after each precursor exposure to the substrate surface. In such an adsorption process, the amount of precursor being exposed to the surface (calculated as the product of the precursor exposure time and the precursor concentration) should be appropriately selected to achieve the saturation state of the precursor-substrate surface reaction. The determination of a critical value of the exposure corresponding to such a complete ALD half-reaction is thus necessary. Indeed, since ALD is a self-limiting process, for a given precursor concentration in the gas phase, an exposure time longer than the critical value is not useful but causes a waste of ALD precursors, which are commonly expensive. In conventional ALD, the exposure time usually depends on the chamber/sample size, the concentration of the precursors in the gas phase, the chamber pressure, as well as the substrate temperature.

Contrarily to conventional ALD, in SALD the precursors are continuously injected onto the substrate, for instance from different gas outlets of an injection head. These reactive precursor regions are spatially separated from mixing thanks to inert gas barriers, which are alternatively located between the precursor regions. Given the close distance needed between the injection head and the substrate (~ 200 µm) to avoid precursor cross-talk, this kind of approach is commonly called close-proximity SALD. [START_REF] Levy | Stable ZnO thin film transistors by fast open air atomic layer deposition[END_REF][START_REF] Hoye | Research Update: Atmospheric pressure spatial atomic layer deposition of ZnO thin films: Reactors, doping, and devices[END_REF] When performing SALD with a moving substrate, the distribution of precursors to the substrate surface will be considerably affected. As a result, controlling the precursor exposure is much more complicated in the case of SALD than in ALD since there is no longer a deposition chamber, the purging step is indeed substituted by passing the substrate through an inert gas region. Obviously, the optimization of the exposure is more critical for metal precursors than for oxidizing agents, especially for large-scale applications since the ALD precursors are usually very expensive. In literature, several approaches related to modeling the ALD deposition reaction, such as adsorption kinetics or surface reaction mechanisms have been proposed. [START_REF] Elliott | Atomic-scale simulation of ALD chemistry[END_REF][START_REF] Puurunen | Surface chemistry of atomic layer deposition: A case study for the trimethylaluminum/water process[END_REF][START_REF] Grillo | Modeling the precursor utilization in atomic layer deposition on nanostructured materials in fluidized bed reactors[END_REF][START_REF] Xie | Mechanistic modeling of atomic layer deposition of alumina process with detailed surface chemical kinetics[END_REF] In the case of SALD, Poodt et al. developed a physical model to study the deposition kinetics of Al2O3 when using a rotating SALD injection system, [START_REF] Poodt | On the kinetics of spatial atomic layer deposition[END_REF] in which the exposure time of precursors is simply supposed to be inversely proportional to the rotation frequency. Also, while Deng et al. proposed a model to quantitatively link the film growth throughput and precursor utilization, it is only based on the gas flows and precursor concentration, and other parameter such as the injector-substrate gap or the gas entrainment due to the moving substrate are not taken into account. [START_REF] Deng | Mechanistic modeling study on process optimization and precursor utilization with atmospheric spatial atomic layer deposition[END_REF] Finally, the effect of the deposition parameters on the film properties is not discussed in previous works. Thus, in-depth studies to link the optimization of the film growth rate with ALD precursor consumption, taking into account precursor concentration, gas flow values, substrate velocity and deposition gap, and discuss the properties of the films obtained as a function of the parameters used are still needed.

In this work, we present simulation and experimental results related to tuning the precursor exposure to the substrate surface via the substrate velocity, precursor concentration in the carrier gas and substrate/injection head gap. The effect of these parameters on the growth rate and the physical properties of deposited ZnO thin films is thoroughly discussed. The study has been performed for the case of ZnO deposition using a close-proximity SALD approach. However, it can be easily extended to any material or other SALD approaches.

Experimental section

The deposition of ZnO films was performed with a home-made atmospheric pressure spatial atomic layer deposition (AP-SALD) system. [START_REF] Nguyen | Increasing the Electron Mobility of ZnO-Based Transparent Conductive Films Deposited by Open-Air Methods for Enhanced Sensing Performance[END_REF] Diethylzinc (DEZ, Zn(C2H5)2 from Sigma Aldrich) and water (H2O) were used as zinc precursor and oxidizing agent, respectively, while nitrogen was used as carrying and purging gas. The substrate oscillated at a constant distance of 180 µm from the injection head. All samples were deposited on borosilicate glass substrates at a substrate temperature of 200 °C. Such experimental conditions have been previously used to deposit thin ZnO conformal layers at the top of silver nanowire networks to enhance both the thermal and electrical stability of such networks (see figure S1 in the supporting information). [START_REF] Aghazadehchors | Versatility of bilayer metal oxide coatings on silver nanowire networks for enhanced stability with minimal transparency loss[END_REF] Several simulation results were obtained using Comsol Multiphysics via a combination of three modules: heat transfer in fluids, laminar flow, and transport of diluted species. The details related to the simulations are described in part 4 of the Supporting Information. When studying the effect of the substrate velocity, other experimental parameters such as substrate temperature, the substrate/injection head gap, or nitrogen bubbling flows through zinc precursor and water (which are supposed to be directly related to reactant quantity carried to the injection head) were maintained constant. The thickness, density, and crystalline structure of the deposited films were investigated using an FS-1™ Multi-Wavelength Ellipsometer from Film Sense, an X-ray Reflectometry system (D500 Siemens), and an X-ray Diffractometry system (D8 Advance Bruker). The texture coefficients of the different ZnO films were calculated from the diffraction peak intensities relative to each other and to the standard powder pattern (ICSD 29272), as follows:

          0 0 / 1 / I hkl I hkl TC hkl I hkl I hkl N   Eq (1)
where   TC hkl and   I hkl are the texture coefficient and the peak intensity of the (hkl) reflection peak, respectively.   0 I hkl is the (hkl) peak intensity in the powder pattern (considered from ICSD 29272) and N is the number of reflections present in the powder pattern (N=4 in our case). For a randomly oriented polycrystalline sample, the texture coefficients are equal to unity, as in the case of the powder pattern. Conversely, for samples having a preferential orientation, the corresponding texture coefficient is equal to N while the other coefficients are equal to zero.

Theory

An ideal ALD process should provide a single monolayer of precursor after each successive exposure. However, when precursor molecules from the gas phase impinge on the substrate surface and begin to stick, a complete monolayer may not be formed due to several factors including the steric hindrance of bulky precursors, [START_REF] Kim | A kinetic study of ZnO atomic layer deposition: Effects of surface hydroxyl concentration and steric hindrance[END_REF] or the balance between absorption and desorption kinetics of precursor molecules (which depends on the deposition pressure, substrate temperature, and precursor concentration in the gas phase). For a given precursor and deposition pressure and temperature, the effect of the precursor concentration in the gas phase on the ALD growth of ZnO film can easily be understood. In the case of the ZnO deposition presented here, we maintained sufficient water supply to ensure surface water saturation. The nitrogen bubbling flow through DEZ container was then varied to study the effect of the concentration of DEZ con the growth rate of ZnO films.

Using a simple model based on Langmuir isotherm adsorption and desorption curves, [START_REF] Tien | Introduction to Adsorption: Basics, Analysis, and Applications[END_REF] the growth rate of ZnO at a constant temperature can be theoretically calculated. Be (t)

 the fraction of the surface covered at the time t , the variation of this quantity is simply the difference between the adsorption and desorption rates, expressed as follows:

          1 a d dt k DEZ t k t dt      Eq (2)
where a k , d k and   DEZ refer to the rate constants for the adsorption and desorption processes, and the gas phase concentration of the molecular species (in this case is zinc precursor, DEZ ), respectively. This equation can be easily solved, giving the expression of the fraction of surface covered (with the boundary condition (  

00 t   ):             1 exp a a d a d k DEZ t k DEZ k t k DEZ k           Eq (3)
In the case of SALD, the deposition is usually carried out at low temperatures and under atmospheric pressure. It can therefore be classed as a high-pressure process. As a first approximation, we assumed that the desorption rate is small compared to the adsorption rate during the precursor exposure time. Hence, Eq (3) can be rewritten as:

      1 exp a t k DEZ t     Eq (4)
We assume that the GPC is proportional to the fraction of surface coverage  after each precursor exposure, which is associated with the exposure time, also called residence time res t , calculated from the substrate velocity. Therefore, the dependence of GPC on precursor exposure can be simply expressed as follows:

        1 , 1 exp res a res GPC DEZ t K k DEZ t        Eq (5)
with 1 K is a constant corresponding to the static growth rate, 0 GPC , when using a sufficiently long exposure.

A typical 3D scheme of a SALD injection head is shown in Figure 1a. The head, which is placed at a short distance from the moving substrate, delivers the reactive precursors onto the substrate surface to reproduce ALD cycles, i.e. DEZ/ N2/ H2O/ N2 and so on. If the substrate is immobile and placed close enough to the injection head, Comsol Multiphysics simulation shows that the reactants can be adequately separated from mixing, as shown by gas velocity and precursor concentration profile in Figure 1b and Figure 1c, respectively, and by previous reports. [START_REF] De La Huerta | Influence of the Geometric Parameters on the Deposition Mode in Spatial Atomic Layer Deposition: A Novel Approach to Area-Selective Deposition[END_REF] In the close-proximity SALD approach, the precursor exposure depends on several parameters including substrate velocity (ν), the distance between two adjacent exhaust channels next to the precursor outlets (noted As a first approximation, at a given   DEZ , when the substrate moves at a speed ν (cm/s), every point on the substrate will be exposed to DEZ-containing gas during a so-called residence time,

/ res DEZ t d v 
, where dDEZ is the distance between two adjacent exhaust channels, as shown in Figure 1c (dDEZ = 2.5 mm in the design shown in Figure 1a). Therefore, GPC versus substrate velocity (ν) can be expressed as follows:

      1 , 1 exp a DEZ k DEZ d GPC DEZ v K v           Eq (6)
a) a)

10

In practice, the precursor concentration in the gas phase,   DEZ , is not easily accessible but it is assumed to be proportional to the nitrogen bubbling flow through DEZ, denoted DEZ f . Therefore, Eq (6) can be rewritten as:

  2 1 , 1 exp DEZ DEZ DEZ K f d GPC f v K v          Eq (7)
With 2 K is a constant proportional to the rate constant for the adsorption reaction. This simple equation can be used to study the effect of the substrate velocity or DEZ bubbling flow on the ZnO growth while keeping the other parameters constant. We will see later that the DEZ delivered to the substrate surface is significantly affected by the substrate velocity, leading to a spatial redistribution of the precursor along the substrate surface. The gas molecules at the substrate surface are indeed entrained by the movement of the latter. This effect will be discussed in the next part with simulation and experimental results.

Results and discussion

Effect of precursor concentration

As mentioned previously, ALD precursors being usually expensive, an excess injection of precursors to the substrate surface is not desirable. It may also lead to unexpected effects such as precursor condensation or etching, as schematically represented in Figure 2a. The condensation effect is more favorable at high deposition pressure and low-temperature, [START_REF] Tien | Introduction to Adsorption: Basics, Analysis, and Applications[END_REF] which is indeed the case of AP-SALD. The structural properties of the deposited ZnO films with varying precursor concentration were investigated using X-ray diffraction (XRD). Figure 3a shows an increase of the XRD peak intensities of ZnO films versus DEZ f , which is mainly due to the thickness effect. We remark that the samples deposited with small DEZ f values ([10 sccm, 30 sccm]) exhibit similar crystalline nanostructure with (100) reflection as the dominant peak. The texture coefficients corresponding to (110) and (002) reflection peaks are rather close to each other, while the value for the (100) peak is almost twice higher, as shown in Figure 3b. In the cases of DEZ f > 30 sccm, the (002) reflection peak appears more intense compared to the (100) peak, which can also be visualized by an abrupt variation in the texture coefficient. In summary, the precursor concentration in the gas phase, which is related to DEZ f in our case, has a significant impact not only on the film growth rate but also on the nanostructure of the deposited films. Thus, while it has been shown that close-proximity SALD can be used in CVD mode, [START_REF] De La Huerta | Influence of the Geometric Parameters on the Deposition Mode in Spatial Atomic Layer Deposition: A Novel Approach to Area-Selective Deposition[END_REF][START_REF] Musselman | Rapid open-air deposition of uniform, nanoscale, functional coatings on nanorod arrays[END_REF] care must be taken to ensure that the properties of the deposited films are still acceptable for the targeted applications. The ALD condition can thus be achieved by placing the DEZ f value in an appropriate range ([25 sccm, 30 sccm]) to reach the ALD saturation condition while avoiding any precursor condensation.

DEZ DEZ K f d GPC K v                b)

Effect of substrate velocity on GPC and film properties

Figure 4a shows a 2D Comsol Multiphysics simulation of the DEZ concentration profile at the corresponding injection channel for different values of substrate velocity, i.e. from 0 cm/s to 16 cm/s. In the case of an immobile substrate, the DEZ concentration profile is symmetric and centered between the two DEZ exhaust channels adjacent to the DEZ channel. When the substrate is moving beneath the gas injector (to the right in this illustration), the precursor molecules are entrained by the moving substrate, creating an asymmetric concentration profile, as observed in Figure 4a. x N x 0

x i+1

x i x i+2

[DEZ] i+2 [DEZ] i+1

[DEZ] at substrate surface (mol/m As a consequence, the simple relationship between the precursor exposure and the substrate velocity previously shown Eq ( 6) is no longer appropriate to describe the dependence of the GPC versus the substrate velocity and therefore needs a better approach. Although the precursor concentration in the gas phase is not constant in the vertical direction, the fraction of precursor molecules, that efficiently participate in ALD half-reactions, concerns only the first layers in contact with the substrate. Figure 4b shows this fraction for various substrate velocities. As the substrate velocity increases, both an asymmetry and a decrease in DEZ concentration are observed. This can be attributed to two main reasons: i) the gas-entraining effect caused by the moving substrate, and ii) the presence of two adjacent exhaust channels located next to the DEZ outlet. Although the high velocity of the substrate could facilitate the mixture of DEZ and H2O in the gas phase, thus enhancing a CVD contribution to the film growth, our simulations show that this is not the case (see part 3 of the Supporting Information for details).

In an ideal case, the substrate surface would be exposed to a constant concentration of DEZ, namely   input DEZ as shown in Figure 4c, during a constant exposure time that is simply equal to the ratio of the distance between two adjacent exhaust channels, DEZ d , to the substrate velocity, v .

In such a case, the precursor exposure to the substrate surface would be calculated as follows:

  input DEZ DEZ d Ex v   Eq (8)
Where   .s) Substrate velocity (cm/s) A corresponds to the symmetric   DEZ distribution under the precursor channel when the substrate is immobile ( = 0 cm/s).

   0 2 0 N x x effective ss DEZ x dx A k v Ex v v      a)
ss k is a constant depending on many parameters including the DEZ concentration in the gas phase, the viscosity of the mixed gas, as well as the substrate temperature. According to this simulation result, the exposure shown in the Eq (6) is not simply inversely proportional to the substrate velocity, but there exists an effective exposure, as shown in Figure 5a. Hence, a new physical approach towards   GPC  can be calculated as follows:

  0 1 1 exp a ss A GPC v K k k v v              Eq (10)
The right side of Eq [START_REF] Khan | Stability Enhancement of Silver Nanowire Networks with Conformal ZnO Coatings Deposited by Atmospheric Pressure Spatial Atomic Layer Deposition[END_REF] can be simplified by setting . Figure 5b shows the experimental data and corresponding fitting curves using Eq [START_REF] Khan | Stability Enhancement of Silver Nanowire Networks with Conformal ZnO Coatings Deposited by Atmospheric Pressure Spatial Atomic Layer Deposition[END_REF] In general, experimental parameters should be optimized to obtain a fast deposition rate, which is indeed a compromise between a high substrate velocity and a long enough residence time.

Therefore, according to Eq [START_REF] Park | Thin film encapsulation for flexible AM-OLED: a review[END_REF] and Eq [START_REF] Maindron | Defect analysis in low temperature atomic layer deposited Al2O3 and physical vapor deposited SiO barrier films and combination of both to achieve high quality moisture barriers[END_REF], high values of indicates that the growth rate of ZnO is less affected by the substrate velocity if higher DEZ bubbling flow is used. The opposite trend is observed for c v . A higher value of this characteristic velocity is expected given the fact that a fast substrate velocity can be used while maintaining a constant eff res t . From another point of view, the optimization of the SALD process concerns the improvement of the growth rate with as little as possible precursor consumption. Three pivotal parameters should be taken into account: growth rate GPC (nm/cycle), precursor consumption that is directly proportional to DEZ f (sccm) and deposition time dep t (s), of which the latter is related to the substrate velocity v (cm/s). Let dep  denote a parameter combining these factors, namely process efficiency, and be written as follows: Apart from the growth rate, other important features that have to be considered such as film density. Figure 7a shows the XRR patterns of ZnO films deposited at various substrate velocities in the range of [3.3, 8.3 cm/s], from which two important parameters can be extracted, i.e. film mass density and thickness. This range of substrate velocities generates a variation in film thickness from 27.5 nm to 60 nm, which corresponds to GPC values in between [0.15, 0.33 nm/cyc]. Figure 7b shows the dependence of the mass density of the deposited ZnO films on the growth rate. An optimum range of GPC values corresponding to a maximum mass density of 5.42 g/cm 3 can be appreciated, while the bulk value is expected to be 5.61 g/cm 3 . For high GPC values (≥ 0.3 nm/cycle), the lower mass density of the deposited films can be related to excess precursor physisorbed on the substrate surface, making somehow a mixed-CVD-ALD deposition mode rather than pure ALD mode. In the case of slower growth rates (< 0.2 nm/s), corresponding to nonsaturating ALD surface reaction, the low mass density values could be related to the film thickness effect. Indeed, ZnO thin films prepared by SALD at 200 °C using diethylzinc result in polycrystalline structure, in which the grain size (and therefore the film density) significantly depends on the film thickness. The dependence of the optical properties of ZnO films deposited by SALD on the substrate velocity is also discussed in part 2 of the Supporting Information.

dep DEZ GPC v f    Eq (7)

Conclusions

In this work, simulation and experimental studies have been conducted to study the effect of a critical parameter, namely, precursor exposure in the case of a close-proximity spatial atomic layer deposition (SALD) approach. This parameter depends directly on the substrate velocity, the geometric design of the injection head, as well as the carrier gas flow rate. A simple physical model has been developed to study the effect of the substrate velocity and the precursor concentration in the carrier gas (   DEZ ) on the growth rate of the deposited ZnO films. Comsol Multiphysics results

show that the entrainment of the precursor molecules by the moving substrate induces an asymmetry in   DEZ profile that affects the obtained growth per cycle (GPC). When studying thoroughly the experimental data based on the effect of   DEZ and the substrate velocity at constant   DEZ , we found that an excess of physisorbed precursor molecules on the substrate surface in the case of high   DEZ can be indirectly observed via the clear variation of the film growth rate when either increasing   DEZ or decreasing the substrate velocity beyond a certain value. The agreement between experimental observations and the model appears very good.

Finally, we have also shown that the mass density of the ZnO deposited film depends closely on the substrate velocity, and that a fast growth rate does not always produce dense films. Indeed, a compromise between the growth rate and the precursor consumption should be considered to obtain both a high film density and a good process efficiency, of which the latter is proposed for the first time to quantify such analysis in SALD. The proposed simple and original approach can pave the way to better understand thin film growth using any SALD system, since it provides a comprehensive guide on optimizing growth rate, film density, and precursor consumption. This is indeed crucial when using SALD for large-scale coating applications.

  Figure 1c), the concentration of precursor in the gas phase (   DEZ or   2 HO ), the gas flow through each channel, as well as the distance between the injection head and the substrate ( gap d ).

Figure 1

 1 Figure 1: a) 3D scheme of the close-proximity SALD injection head used in this work, represented along with a glass substrate

Figure 2 :

 2 Figure 2: a) a schematic representation of growth per cycle (GPC) versus exposure time, b) Experimental GPC as a function of N2 bubbling flow through DEZ (sccm). The fit using the simple Langmuir adsorption kinetics model (Eq (7)) is shown by the dashed line for N2 bubbling flow through DEZ in the range of [0 sccm, 33 sccm]. The substrate velocity, substrate temperature, and injector/substrate gap were fixed at 5 cm/s, 200 °C, and 180 µm, respectively.

Figure 2b shows thev

  Figure 2b shows the experimental data of GPC as a function of DEZ fand the fitting curve using the simple Langmuir adsorption kinetics model presented in Eq[START_REF] Park | Thin film encapsulation for flexible AM-OLED: a review[END_REF]. In this experiment, the

Figure 3 :

 3 Figure 3: a) X-ray diffraction patterns and b) texture coefficients of ZnO films deposited with different N2 bubbling flows through DEZ, DEZ f .

3 )Figure 4 :

 34 Figure 4: a) DEZ 2D concentration profile at its injection channel for different values of substrate velocity, b) the corresponding DEZ concentration profiles projected at the substrate surface level. In this case, the substrate temperature and the gap between the substrate and the injection head were fixed at 200 °C and 180 µm, respectively, c) schematic illustration of the variation of [DEZ] as a function of position for a substrate velocity of 8 cm/s, as implemented in the proposed model. The data shown here were simulated with Comsol Multiphysics.

1 ,

 1 of the ideal rectangle shown in Figure4c. However, the simulation shows that the precursor concentration profiles are not simply constant but quite complex and shifted to the direction of substrate motion. In Figure4c, we suggest a more meticulous estimation of the effective exposure of DEZ to the substrate surface by discretizing the   DEZ profiles into N small segments indicated by coordinates 0x , 1 x ... N x . If N is large enough, the precursor concentration in each segment ii xx    can be considered constant and the exposure of the precursor to this portion of the substrate would indeed be equal to     i DEZ x dx  . Therefore, we define an effective exposure, effective Ex, which can be calculated as follows:

Figure 5 :

 5 Figure 5: a) Simulation result of the effective exposure (

  easily the DEZ flow can be entrained by the moving substrate, the observed decreasing trend of ss k versus DEZ f

   dep Time required to perform 100 ALD cycles (s)

Figure 6 :

 6 Figure 6: Process efficiency as a function of the time required to perform 100 ALD cycles in the case of ZnO deposition by SALD.

Figure 6 Figure 7 :

 67 Figure 6 describes the variation of process efficiency dep  as a function of the time required

Table 1 :

 1 for the GPC of ZnO films deposited at different   DEZ values as a function of substrate velocity,  . Our model properly fits the data of the high and intermediate substrate velocities for various DEZ bubbling flow rates (15, 30 and 50 sccm). Conversely, it can be observed that the growth rates deviate from the theoretical model at low substrate velocities, which correspond to the longer exposure time. As discussed in Figure2, an extra injection of precursors to the substrate surface can create condensation if the subsequent purging step is not sufficiently long. This fact was not taken into account in our simple model, yielding an underestimation of the growth rate at low substrate velocities. The same argument could also explain the increase of the static growth rate, 1 The summary of the parameters extracted from the fits is shown in Table1. The static growth rate obtained when using the value of Some critical parameters extracted or calculated from the fits shown in Figure5b.

	K ,

Acknowledgments

This work was funded by the Agence Nationale de Recherche (ANR, France) via the programs ANR-16-CE05-0021 (DESPATCH), ANR-18-CE09-0041 (Meaning), ANR-18-CE09-0036 (Panassé) and ANR-17-CE05-0034 (Oxygen). This work was supported by the European Joint Doctorate FunMAT program (H2020-MSCA-ITN-2014, Project ID 641640). This project was also supported by the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02) through the project Eco-SESA. This work was also performed within the framework of the Centre of Excellence of Multifunctional Architectured Materials "CEMAM" no. AN-10-LABX-44-01. This work was as well supported by Région Auvergne Rhône-Alpes through the project Pack Ambition Recherche 2018 Eternité. The Carnot Energies du Futur is acknowledged through the project FREE. David Muñoz-Rojas acknowledges funding through the Marie Curie Actions (FP7/2007-2013, Grant 631111). The authors would like to warmly thank L. Rapenne for her technical assistance, and Dr. César Masse de la Huerta and Dr. Hao Van Bui for fruitful discussions.


Nathalie Dorison

Pauline Gaignard

Aurélien Bayot

Antoinette Gelot

Pierre Hadrien Becker

Salma Fourati

Elise Lebigot

Perrine Charles

Timothy Wai

Patrice Therond

Abdelhamid Slama

MITOCHONDRIAL DYSFUNCTION CAUSED BY NOVEL ATAD3A MUTATIONS

Keywords:

Mitochondrial respiratory chain integrity depends on a number of proteins encoded by nuclear and mitochondrial genomes. Mutations of such factors can result in isolated or combined respiratory chain deficits, some of which can induce abnormal morphology of the mitochondrial network or accumulation of intermediary metabolites. Consequently, affected patients are clinically heterogeneous, presenting with central nervous system, muscular, or neurodegenerative disorders. ATAD3A is a nuclear-encoded ATPase protein of the AAA+ family and has been localized to the inner mitochondrial membrane. Recently reported mutations or large deletions in the ATDA3A gene in patients have been shown to induce altered mitochondrial structure and function and abnormal cholesterol metabolism in a recessive or dominant manner. Here, we report two siblings presenting axonal sensory-motor neuropathy associated with neonatal cataract. Genetic analyses identified two novel mutations in ATAD3A; a point mutation and an intronic 15 bp deletion affecting splicing and leading to exon skipping. Biochemical analysis in patient cells and tissues showed abnormal function of the mitochondrial respiratory chain in muscle and abnormal mitochondrial cristae structure. These new cases underline the large spectrum of biochemical and clinical presentations of ATAD3A deficiency and the different modes of inheritance, making it an atypical mitochondrial disorder

Introduction

ATAD3A is an inner mitochondrial membrane protein that interacts with MFN2, OPA1 and DNM1L, small GTPases that perform mitochondrial membrane fusion (MFN2, OPA1) and fission (DNM1L), suggesting that ATAD3A may be another mitochondrial protein with a role in mitochondrial dynamics [START_REF] Fang | ATPase family AAA domain-containing 3A is a novel anti-apoptotic factor in lung adenocarcinoma cells[END_REF]. In addition, ATAD3A has been implicated in mitochondrial DNA maintenance [START_REF] Gilquin | The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes[END_REF] and cholesterol metabolism [START_REF] Rone | Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to steroid hormones[END_REF]. In humans, ATAD3A is a nuclear-encoded ATPase protein of the AAA+ family and is a part of ATAD3 cluster genes constituted by three paralogs: ATAD3A, ATAD3B and ATAD3C which have an extensive sequence homology, but other species have only one ATAD3 locus [START_REF] Li | ATAD3, a vital membrane bound mitochondrial ATPase involved in tumor progression[END_REF]. The first mutations in ATAD3A leading to mitochondrial dysfunction were identified in eight patients from 7 families with distinct neurological syndromes associated to recurrent de novo mono-allelic or biallelic variations [START_REF] Harel | Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes[END_REF]. Five patients from the same family are heterozygous for the c.1582C>T, p.(Arg528Trp) mutation with a dominant-negative mechanism while the others patients have biallelic variants, two siblings with a homozygous c.158C>T p.(Thr53Ile) mutation and one patient with compound heterozygous deletions in the ATAD3 gene cluster, ATAD3A/ATAD3B in one allele and ATAD3A/ATAD3C in the second allele. Later on, Cooper and coworkers [START_REF] Cooper | ATpase-deficient mitochondrial inner membrane protein ATAD3a disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia[END_REF] identified in 2017 another ATAD3A de novo mutation, c.1064G>A p.(Gly355Asp), in a woman presenting with spastic paraplegia and axonal neuropathy. Her son presented a dyskinetic cerebral palsy. Fibroblasts from these patients exhibited fragmented mitochondria and an upregulated autophagy. The authors demonstrated a dominant-negative effect of this mutation on the activity of the ATAD3A protein. The same year, another study [START_REF] Desai | ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism[END_REF] reported five subjects with a more severe phenotype, a fatal pontocerebellar hypoplasia associated with large biallelic deletions resulting from genomic rearrangement affecting the ATAD3 gene cluster leading to altered cholesterol metabolism as then demonstrated in a mouse model of ATAD3 muscle deficiency [START_REF] Peralta | ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels[END_REF]. Very recently, a large recurrent de novo ATAD3 duplication was associated with fatal neonatal cardiomyopathy with corneal clouding and encephalopathy in 21 families [START_REF] Gunning | Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism[END_REF][START_REF] Frazier | Clinical and Translational Article Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the ATAD3 Locus Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the[END_REF]. To date, 48 affected patients have been documented (summarized in Table 1) carrying recessive or dominant mutations and large deletions/duplications affecting the ATAD3 cluster genes [START_REF] Harel | Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes[END_REF][START_REF] Cooper | ATpase-deficient mitochondrial inner membrane protein ATAD3a disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia[END_REF][START_REF] Desai | ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism[END_REF][START_REF] Gunning | Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism[END_REF][START_REF] Al Madhoun | Ketogenic diet attenuates cerebellar atrophy progression in a subject with a biallelic variant at the ATAD3A locus [response to letter[END_REF][START_REF] Peeters-Scholte | Genotype-phenotype correlation in ATAD3A deletions: Not just of scientific relevance[END_REF][START_REF] Peralta | Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities[END_REF].

Here we report the discovery of two new recessively inherited pathogenic variants in the ATAD3A gene, c.1609T>A, p.(Trp537Arg) and c.1614+2_1616+16del, p.(Arg503Profs *11) and related clinical and biochemical data. The patients are brother and sister from unrelated parents, presenting with axonal sensory-motor neuropathy associated with congenital cataracts and abnormal mitochondrial cristae morphology.

Material and methods

Patients: Two siblings from healthy non-consanguineous parents. Informed consent for diagnosis and research studies was obtained from their parents.

Biochemical studies

Respiratory chain activities were assessed in cultured skin fibroblasts and muscle biopsy as previously reported [START_REF] Rustin | Biochemical and molecular investigations in respiratory chain deficiencies[END_REF][START_REF] Chretien | Reference charts for respiratory chain activities in human tissues[END_REF]. Cultured skin fibroblasts were grown in HAM F-10 medium (Eurobio, Les Ulis, France) supplemented with antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin), 50 mg/L uridine and 12% fetal bovine serum (FBS). Cells were cultured at 37°C in a humidified incubator with 5% CO 2 .

Molecular Analysis

Genomic DNA was extracted from muscle or fibroblasts by standard methods. Mitochondrial DNA in muscle was analyzed qualitatively and quantitatively by long range PCR or qPCR respectively [START_REF] Chabi | Quantification of mitochondrial DNA deletion, depletion, and overreplication: Application to diagnosis[END_REF]. The high-throughput sequencing of total mtDNA and of a panel of 185 nuclear genes involved in the most common mitochondrial pathologies (gene list upon request) were performed by NGS on Illumina Miseq instrument. This panel was designed using the Agilent Sure select Design web Application. Preparation of genomic DNA for NGS analysis and bioinformatics workflow were performed as previously described [START_REF] Bouali | New MCM8 mutation associated with premature ovarian insufficiency and chromosomal instability in a highly consanguineous Tunisian family[END_REF]. Sanger sequence was confirmed by PCR with specific primers to confirm detected mutations and allelic segregation from parents.

Total RNA was extracted from cultured skin fibroblasts, with or without emetine, using the EZ1 RNA Tissue mini kit (QIAGEN, Valencia, CA). Reverse-transcription was performed with the Omniscript Reverse Transcriptase kit (QIAGEN, Valencia, CA). The ATAD3A cDNA was amplified using two specific primers which frame exons 14, 15 and 16. After a 35-cycle amplification of the first strand cDNA corresponding to 50 ng of starting RNA, PCR products were subjected to 2% agarose gel electrophoresis. Sequencing reactions were carried out with the Big Dye Terminator cycle sequencing kit and analyzed with an ABI 3100 genetic analyzer (Applied Biosystems).

siRNA transfection

Reverse transfection of skin fibroblasts with siRNA was performed using Lipofectamine RNAiMAX reagent following manufacturer's instructions. Briefly, 160 μL of PBS1X containing 0.8 μL of 50 μM siRNA and 1.6 μL Lipofectamine RNAiMAX was incubated for 1h at room temperature. Then, 20 μL per well of this transfection mixture was distributed in appropriate wells of a 96 well-plate before addition of 80 μL per well of cell suspension at 50 000 cells/mL in complete DMEM medium (DMEM containing 4.5 g/L D-glucose, 2 mM glutamine as GlutaMAX, 1 mM sodium pyruvate, 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin). Every single siRNA was transfected in triplicate in two independent control fibroblast cell lines. Non-Targeting siRNA Pool #2: Dharmacon D-001206-14-05; Human DNM1L1 siRNA SMART Pool: Dharmacon M-012092-01; YME1L siRNA: Dharmacon M-006103-01.

Immunofluorescence of mitochondria and microscopy

Skin fibroblasts were plated in a 96 well-plate at 6000 cells/well and grown in complete DMEM medium at 37°C in a humidified atmosphere containing 5% CO 2 . After 24h, cells were washed once with warm PBS1X and immediately fixed with warm PFA 4% for 15 min, followed by permeabilization in PBS1X-triton 0.1% for 5 min at room temperature and incubation overnight at 4°C in PBS1X-FBS 10%. Immunostaining of mitochondria was performed using an anti-TOM40 antibody (Proteintech: 18409-1-AP) diluted 1/1000 in PBS1X-FBS 5%, followed by incubation with an appropriate Alexa Fluor 488 fluorescent antibody. Immunofluorescence of transfected fibroblasts was performed after 72h of transfection in similar conditions as described above. Cells were imaged using an automated Operetta CLS confocal spinning disc microscope (PerkinElmer) in the confocal mode at the 20X magnification.

Quantification of mitochondrial morphology

Mitochondrial morphology was quantified using a machine learning approach developed with the Harmony 4.9 software (PerkinElmer) and based on a proprietary algorithm (PerkinElmer).

Briefly, using texture parameters inherent to the software, the system was trained to discriminate 3 different classes of mitochondrial shape: Fragmented, Normal and Hyperfused. To this purpose, control fibroblast cells from two different healthy donors, exhibiting normal mitochondrial morphology, were transfected with siRNA directed against specific gene regulators of mitochondrial dynamics to induce either mitochondrial fragmentation (YME1L1 siRNA), mitochondrial hyperfusion (DNM1L siRNA), or no effect (non-targeting NT siRNA). After 3 days of transfection, the 3 different classes of transfected cells were used for supervised machine learning, with at least 400 cells of each class originating from two independent control fibroblast cell lines from three independent transfected wells. After supervised training, cells were classified automatically into the 3 defined classes. To ensure the validity of the approach, all fibroblast cells (those used for machine learning and those of interest) were seeded, grown and immunostained in parallel in a single 96 well-plate.

Western-blotting

Total cell lysates were prepared on ice using RIPA buffer [150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0] (Sigma) supplemented with a protease inhibitor cocktail (Roche). Protein samples prepared in Laemmli buffer (BioRad) supplemented with 2-mercaptoethanol were separated by SDS-PAGE on a 4-20% Mini-PROTEAN® TGX™ Precast Protein Gel (BioRad). 50 μg of proteins were then electrotransferred onto a Hybond nitrocellulose membrane (GE Healthcare). Primary antibody binding was detected by incubation with a peroxidase-conjugated secondary antibody using Clarity™ Western ECL Substrate (BioRad). Details on primary antibodies are listed below: anti-ATAD3A polyclonal antibody is from Thermo Fisher Scientific (PA5-66727); anti-TUBULIN mouse monoclonal antibody is from Proteintech (HRP-66031); Total OXPHOS Human WB Antibody Cocktail, mouse monoclonal, is from abcam (ab110411).

Results

:

Clinical and biochemical description

The first child of the siblings (II-1) was born to healthy non-consanguineous parents without perinatal problems and normal mensurations. He presented with bilateral congenital cataracts operated at 4 months, a strabismus operated at 6 months with residual nystagmus and ophthalmoplegia related to his visual defect (corrected visual acuity tends to 4 or 5/10 at adult age). Hypotonia with hyporeflexia was noticed since the first months of life; he learned to walk around the age of 3 years and axonal sensory-motor neuropathy was confirmed by electrophysiological studies. At 10 years of age, the child presented ataxic walking with foot deformities and used a wheelchair for extended mobility. By 26 years of age, he required the use of a cane for walking. He manifested pes equines and pes cavus with several foot surgeries in childhood. Arthrodesis was performed at 26 years of age to manage foot deformities.

He had a progressive growth delay (-2ds after 18 months) and at 19 years old weighed 41kg and stood 168 cm tall with a cranial perimeter of 56 cm (no endocrine investigation); at 26 years he weighed 60 kg, which is within the normal range. Clinical examination at this age showed cerebellar syndrome with ataxic gait, pes cavus, amyotrophy of the lower limbs with distal motor deficit, myoclonic tremor of upper limbs, generalized areflexia and distal lower limbs hypopallesthesia, complex severe ophthalmoplegia in all gaze positions with low visual acuity. Neither cardiac dysfunction nor hepatosplenomegaly were observed. He displayed normal cognitive and psychomotor development with normal MRI. Echocardiography was unremarkable with no indication of impaired cardiac function but cardiac MRI showed moderate enlargement of the left ventricle. The dentist reported enamel faults.

His sister (II-2) has a similar but more pronounced phenotype. She was born after an uneventful pregnancy with intrauterine growth retardation (height 47.5 cm; weight 3390 g). A bilateral congenital cataract was noted before 2 months of age and operated at the age of 10 months with residual strabismus, and low visual acuity. The motor delay with hypotonia and hyporeflexia was diagnosed within the first months of life; sitting was acquired at 12 months and autonomous walking at the age of 4 years. Electrophysiological studies were performed at 6 months of age and confirmed the sensory-motor neuropathy. She had progressive amyotrophy and deformities of the feet, instability in walking with frequent falls and partial dependence on a wheelchair (independent walk 10 minutes / climb 2 floors). She underwent several orthopedic procedures for pes cavus. After 18 months she evolved with a growth delay of -3ds for weight and height and had a weak adipose panicle, repeated vomiting and tendency to anorexia. At 19 years old, her height was 145 cm and her weight was 34 kg. She reported normal menstrual cycles. The endocrine and nutritional assessments did not show any abnormalities (normal TSH, GH and IGF1 levels).

Clinical examination showed a severely ataxic gait, failure to sustain a stable standing position, pes cavus, recurvatum of the knees, distal motor deficits with amyotrophy, generalized areflexia, hypopallesthesia of the distal lower limbs, nystagmus and complete ophthalmoplegia in all gaze positions. Corrected visual acuity was 2/10 left and right eyes with bilateral visual correction. Elocution and cognition were reported to be normal.

Brain MRIs were performed at 6 months, 8 and 13 years of age and remained normal although an optic neuropathy was suspected.

Cardiac ultrasound found a discreet enlargement of the posterior wall of the left ventricle at the 18 years of age but without clinical impact (annual monitoring). The dentist reported enamel faults (not reported in the unaffected brother).

Molecular analysis

To identify the cause of the mitochondrial defect, we performed a mitochondrial DNA sequence analysis. We did not find any mutations, neither large deletions nor depletions of

Western blot analysis:

Immunoblot analyses showed a profound reduction of ATAD3A protein in patient fibroblasts consistent with either protein instability or non-sense mediated decay of mRNA and loss of function. ATAD3B protein level was not affected (Fig. 2A). Analysis of subunits of mitochondrial respiratory chain complexes in patient fibroblasts also identified reduced steady state levels of both MTCO2 (complex IV subunit) and NDUFB8 (complex I subunit), whereas ATP5A (complex V subunit), UQCRC2 (complex III subunit) and SDHB (complex II subunit) were maintained at control levels (Fig. 2A).

Mitochondrial morphology:

Analysis of the mitochondrial network in patient fibroblasts by immunofluorescence and confocal microscopy did not show any evidence of abnormal mitochondrial morphology (Fig. 2B). Quantification of mitochondrial shape using a supervised machine learning approach showed no significant increase in fragmented mitochondria in fibroblasts from patients II-1 and II-2 as compared to controls (Fig. 2C). By contrasts, electron microscopy analysis of skeletal muscles showed abnormal mitochondria (Fig. 3,A, B andC). The mitochondria were slightly increased in number with an abnormal morphology, Their size was increased and their diameter was irregular due to the existence of constriction zones; in addition the cristae network was highly disturbed with elongated, non-parallel, branched crests, drawing alveoli in the cytoplasm.

Discussion:

Here we extend the number of patients with ATAD3A mutations and confirm the high clinical variability of the phenotype expressed in affected patients. The patients we described here are siblings from healthy unrelated parents and were identified to have novel biallelic variations: a novel c.1609T>A, p.(Trp537Arg) point mutation and an intronic 15 bp deletion c.1614+2_1616+16del, p.(Arg503Profs*11) resulting in exon 15 skipping and a premature stop codon. Western blot analyses showed that ATAD3A protein was undetectable in fibroblasts and the pedigree argues for a recessive mode of inheritance, excluding a dominant-negative effect of the truncated protein p.(Arg503Profs*11). Therefore, we can assume that the amino acid change p.( Trp537Arg) is an hypomorphic variant that cannot form stable ATAD3A oligomers.

These patients presented early ophthalmic abnormalities in the form of bilateral congenital cataracts, ophthalmoplegia and nystagmus, hypotonia and sensory-motor neuropathy, lateonset cardiac abnormalities without cognitive impairment. Up to now 48 patients from 38 families have been identified to carry pathogenic variants in ATAD3A; 29 patients have dominant variants, 9 patients with recessive single nucleotide variants and 10 patients have recessive deletions in the ATAD3 gene cluster. Indeed, patients with ATAD3A disease, whether dominantly or recessively inherited, highlight the tremendous clinical heterogeneity and wide phenotypic spectrum caused by mutations in this gene (Table1). As observed for patients II-1 and II-2 described in this study, neuromuscular and ophthalmic signs are progressive and constantly present in patients with later onset. Our results suggest that congenital cataracts, which have previously been ascribed to the core phenotype of ATAD3A deficiency [START_REF] Harel | Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes[END_REF][START_REF] Peralta | Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities[END_REF] may be an early sign of disease manifestation caused by recessive ATAD3A mutations. Congenital cataracts have previously been associated with pathogenic mutations in MFN2 [START_REF] Zhao | Embryonic Surface Ectoderm-specific Mitofusin 2 Conditional Knockout Induces Congenital Cataracts in Mice[END_REF], OPA1 [START_REF] Lee | Recessive optic atrophy, sensorimotor neuropathy and cataract associated with novel compound heterozygous mutations in OPA1[END_REF] and other inborn errors of metabolism, but the underlying mechanism remains to be elucidated.

The recent description of recurrent de novo 68 kb duplication between exon 11 of ATAD3A and intron 7 of ATAD3C (with various breakpoints) has revealed two main clinical presentations associated with ATAD3 cluster copy number variations: neonatal pontocerebellar hypoplasia caused by recessive homozygous deletions and hypertrophic or dilated cardiomyopathy caused by de novo dominant duplications. In the both cases, corneal clouding or cataract and encephalopathy were diagnosed and the death occurred in the first months of life.

Regarding point mutations, a genotype-phenotype correlation is more difficult to distinguish. Thr84Met)), and it is consistent with the protein structure, since the C-terminal region with Walker A and Walker B domains constitutes the ATPase core of the protein whereas the N-terminal region contains two coiled-coil regions and two transmembrane domains TM1 and TM2, [START_REF] Hubstenberger | Topological analysis of ATAD3A insertion in purified human mitochondria[END_REF]. However, our description of two new patients with milder phenotypes and biallelic mutations in the C-terminal domain underlines that genotype-phenotype correlations are not straightforward. Moreover, normal ATAD3A protein levels have been observed in fibroblasts from patients with a non-fatal phenotype (patients with dominant-negative variants p.(Arg528Trp) and p.(Gly355Asp), [START_REF] Harel | Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes[END_REF][START_REF] Cooper | ATpase-deficient mitochondrial inner membrane protein ATAD3a disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia[END_REF]) whereas ATAD3A was profoundly depleted in fibroblasts from patients that succumbed to their illness during the neonatal period (homozygous variant p.(Leu406Arg), [START_REF] Peralta | Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities[END_REF]), leading to the notion that protein level of ATAD3A may be correlated to the disease severity. Our data do not support this hypothesis, as we demonstrate that ATAD3A is undetectable in patients II-1 and II-2 fibroblasts even though they present a moderate phenotype.

The precise roles fulfilled by ATAD3 in mitochondria have yet to be fully clarified.

Experimental studies show that ATAD3 is implicated in several functions within mitochondria: organization and structure of the inner mitochondrial membrane, mtDNA nucleoid organization, mitochondrial dynamics, and respiratory chain activity [START_REF] Fang | ATPase family AAA domain-containing 3A is a novel anti-apoptotic factor in lung adenocarcinoma cells[END_REF][START_REF] Gilquin | The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes[END_REF][START_REF] Harel | Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes[END_REF][START_REF] Peralta | ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels[END_REF] and cholesterol import, which constitutes the first step of mitochondrial steroidogenesis [START_REF] Rone | Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to steroid hormones[END_REF][START_REF] Desai | ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism[END_REF]. How these pleotropic effects are connected is not understood. These dysfunctions do not manifest concomitantly in all ATAD3-deficient patients, pointing to mutation-specific effects. Indeed, our study confirms that the mitochondrial structure is affected in ATAD3-deficiency patients: muscle biopsy of patients II-1 and II-2 showed giants mitochondria with severe cristae disorganization; abnormal cristae morphology with either small or swollen mitochondria have also been described in ATAD3-deficient fibroblasts [START_REF] Gunning | Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism[END_REF][START_REF] Peralta | Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities[END_REF]. mtDNA copy number was normal in muscle of patients II-1 and II-2, indicating that the severe alterations of the inner mitochondrial membrane do not necessarily lead to mtDNA depletion, as already underlined [START_REF] Peralta | Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities[END_REF]. Our study did not examine nucleoid structure and thus we cannot exclude mtDNA aberrant organization as reported by Desai et al [START_REF] Desai | ATAD3 gene cluster deletions cause cerebellar dysfunction associated with altered mitochondrial DNA and cholesterol metabolism[END_REF]. ATAD3 has been implicated in the regulation of mitochondrial dynamics, [START_REF] Gilquin | The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes[END_REF], although we did not observed significant alterations in mitochondrial morphology in patients II-1 and II-2 fibroblasts. In human fibroblasts, overexpression of p.Gly355Asp or the p.Lys358Arg variants, also located in the C-terminal domain, leads to increased mitochondrial fragmentation [START_REF] Cooper | ATpase-deficient mitochondrial inner membrane protein ATAD3a disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia[END_REF], although the effects on mitochondrial morphology caused by mutant ATAD3A proteins expressed at endogenous levels appears to be far more subtle. Our enzymatic studies showed that the respiratory chain function is sensitive to alterations of ATAD3: complex I deficiency (patient II-1) or combined deficiencies of complexes I and III (patient II-2) were identified in mitochondria isolated from muscle, as already described for some patients, but not all [START_REF] Harel | Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes[END_REF][START_REF] Frazier | Clinical and Translational Article Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the ATAD3 Locus Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the[END_REF][START_REF] Peralta | Novel ATAD3A recessive mutation associated to fatal cerebellar hypoplasia with multiorgan involvement and mitochondrial structural abnormalities[END_REF]. In contrast, we and others showed that ATAD3 defect did not induced a decrease of the respiratory chain activity in fibroblasts, even though the quantity of some subunits was reduced which implies compensatory mechanism [START_REF] Harel | Recurrent De Novo and Biallelic Variation of ATAD3A, Encoding a Mitochondrial Membrane Protein, Results in Distinct Neurological Syndromes[END_REF][START_REF] Cooper | ATpase-deficient mitochondrial inner membrane protein ATAD3a disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia[END_REF][START_REF] Peralta | ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels[END_REF][START_REF] Frazier | Clinical and Translational Article Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the ATAD3 Locus Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the[END_REF] and/or tissue-specific or cell type-specific thresholds for biochemical dysfunction. The recent study of Frazier et al highlighted well this tissue-specificity: complex I activity was more profoundly decreased in the heart than in skeletal muscle or in fibroblasts of ATAD3-deficient patients suffering from severe cardiomyopathy [START_REF] Frazier | Clinical and Translational Article Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the ATAD3 Locus Fatal Perinatal Mitochondrial Cardiac Failure Caused by Recurrent De Novo Duplications in the[END_REF]. It stands to reason that the respiratory chain activity may be most severely compromised in the most clinically affected tissue.

In conclusion, our study helps to move our understanding of the physiological and pathophysiological relevance of ATAD3A forward. Identifying novel ATAD3A pathogenic variant will help to establish future genotype-phenotype correlations and delineate pathways of ATAD3A-related disorders.

.

  Electrophysiological studies were performed at 6 months of age and confirmed the sensorymotor neuropathy. At 8 years old the values of motor nerve conduction study (NCS) parameters of the right common peroneal nerve were 1.6 millivolt for the distal amplitude, 1.1 for the proximal amplitude and 37.5 meter/second for the conduction velocity. The left posterior tibial nerve values were 1.5 millivolt for the distal amplitude, 1.1 for the proximal amplitude and 34.8 meter/second for the conduction velocity. The right median nerve values were 5.5 microvolt for the distal amplitude, 5.2 for the proximal amplitude and 51.8 meter/second for the conduction velocity. Distal sensitive latency were normal, for right median nerve values were 2 microvolt/ meter for sensitive distal amplitude, conduction velocity was incalculable. Biochemical investigations of patient II-2 showed increased lactate concentrations in both blood (4.4, 5.7 mmol/l, reference values (RV)<1.8 mmol/l) and CSF (3.8 mmol/l, RV<1.8 mmol/l) with a high lactate/pyruvate ratio (20.7 and 19.8), thus leading us to further study energy metabolism with a particular focus on the mitochondrial respiratory chain activities in muscle and cultured fibroblasts of both patients. A decrease in complex I activity (46 nmol/min/mg of protein, RV: 60-123) for patient II-1 and a decrease in the activities of both complexes I (45 nmol/min/mg of protein, RV: 60-123) and III (386 nmol/min/mg of protein, RV: 1007-2465) for patient II-2 were identified in muscle but normal activities were measured in fibroblasts.

  muscle mitochondrial DNA. High-throughput sequencing of a panel of 185 nuclear genes representing established mitochondrial genetic diseases and Sanger sequencing allowed us to identify, in both affected children, two novel compound heterozygous variants in the ATAD3A gene (NM_001170535): a point mutation c.1609T> A, p.(Trp537Arg) located in a highly conserved region of the exon 15, predicted to induce a deleterious amino acid change, and a 15 bps deletion c.1614+2_1616+16del, p.(Gln503Profs*11) resulting in exon 15 skipping as confirmed by cDNA analysis (Fig.1A, 1B). Parental analysis confirmed allelic segregation in agreement with an autosomal recessive inheritance. The c.1609T> A, p.(Trp537Arg) mutation was inherited from the mother while the c.1614+2_1616+16del, p.(Gln503Profs*11) was inherited from the father.

Peralta

  et al. suggested that mutations affecting the C-terminal domain c.1217T>G, p.(Leu406Arg) could be more severe than those affecting the N-terminal domain (c.158C>T; p.(Thr53Ile), c.251T>C, p.(

FiguresFig. 1 :Fig. 2 :Fig. 3 :

 123 Figures

Table 1 :

 1 ATAD3A-reported cases. F: family, NN: Neonatal period, p: patient, SNV: Single Nucleotide variants.

	SNV	Inheritence
	Allele 1 / allele 2		Outcome	Symptoms	Sources
		Protein domain
	c.1217T>G, p.(Leu406Arg) / c.1217T>G, p.(Leu406Arg)	Recessive	Death in NN period	Axial hypotonia, encephalopathy, cerebellar hypoplasia, congenital cataract,	[11]
	(4p, 1F)			hypertrophic cardiomyopathy, hepatomegaly
		C-terminal
	c.490C>T, p.(Gln164*)/c.230T>G, p.(Leu77Arg)	Recessive	Death in NN period	Calcification in basal ganglia, delayed gyration and white matter	[10]
	(2p, 1F)			myelination, pontocerebellar hypoplasia , developmental delay, ataxia,
		Stop/N-terminal		cerebellar atrophy
	c.158C>T, p.(Thr53Ile) / c.158C>T, p.(Thr53Ile)	Recessive	Alive at 23 and 26 yrs	Developmental delay, seizure, congenital cataract, hypotonia, ataxia, speech	[5]
	(2p, 1F)			delay, optic neuropathy, cerebellar atrophy
		N-terminal
	c.251T>C, p.(Thr84Met) / c.251T>C, p.(Thr84Met)	Recessive	Alive at 4.5 yrs	Developmental delay, ocular motor apraxia, mild cerebellum atrophy. No	[9]
	(1p, 1F)	N-terminal		cardiomyopathy, no optic atrophy
	c.1609T>A, p.(Trp537Arg) / c .1614+2_1616+16del(p.Arg503Profs*11) (2P,	Recessive	Alive at 19 and 26 yrs	Neonatal cataract, axonal sensorimotor neuropathy	This study
	1F)	C-terminal
	c.1582C>T, p.(Arg528Trp) / -	Dominant	Alive at 2 to 6 yrs	Developmental delay (5/5), hypotonia (5/5), optic atrophy (3/5), axonal	[5]
	(5p, 5F)	C-terminal		neuropathy, hypertrophic cardiomyopathy (2/5)
	c.1064G>A, p.(Gly355Asp) / -	Dominant	Alive 35 and 3.5 yrs	Spastic paraplegia, axonal neuropathy and photophobia. No hypertrophic	[6]
	(2p, 1F)	C-terminal		cardiomyopathy, no optic atrophy
	Deletions
	~38 kb del ATAD3B-ATAD3A / ~68 kb del ATAD3C-ATAD3A	Recessive	Death in NN period	Seizures, hypotonia, spasticity, corneal clouding, respiratory failure	[5]
	(1p, 1F)
	38054 bp del ATAD3B-ATAD3A / 38054 bp del ATAD3B-ATAD3A (2p, 1F)	Recessive	Death in NN period	Pontocerebellar hypoplasia	[7]
	38667 bp del ATAD3B-ATAD3A / 38667 bp del ATAD3B-ATAD3A (2p, 2F)	Recessive	Death in NN period	Pontocerebellar hypoplasia	[7]
	38667 bp del ATAD3B-ATAD3A / 38054 bp del ATAD3B-ATAD3A (1p, 1F)	Recessive	Death at 7 months	Pontocerebellar hypoplasia	[7]
	del ATAD3A-ATAD3B / del ATAD3A-ATAD3B (4p, 3F)	Recessive	Death in NN period	Pontocerebellar hypoplasia, delayed gyration	[10]
	Duplications
	68 kb dup ATAD3A-ATAD3B-ATAD3C / -	Dominant	Death in NN period	Hypertrophic or dilated cardiomyopathy, corneal clouding or cataracts,
	(22p, 21F)			hypotonia, encephalopathy, white matter change

Acknowledgments

The authors would like to acknowledge Association des Maladies Mitochondriales (AMMi), France, for the financial support. . T.W. was supported by the European Research Council (ERC) Starting Grant No. 714472 (Acronym "Mitomorphosis").