Khaisa Avchukir
email: khaisa.avchukir@cfhma.kz

Bibissara Dzhalmukhamedovna Burkitbayeva

Florence Vacandio

Akmaral Mukhambetovna Argimbayeva

Gulmira Rakhymbay

Influence of tetrabutylammonium chloride on the electrodeposition of indium from chloride solution on a glassy carbon electrode

Keywords: Indium, Electrodeposition, Nucleation and growth, Electrolyte additive, Tetrabutylammonium choride

Influence of tetrabutylammonium chloride on the electrodeposition of indium from chloride solution on a glassy carbon electrode

Khaisa Avchukir, Bibissara Dzhalmukhamedovna Burkitbayeva, Florence

Vacandio, Akmaral Mukhambetovna Argimbayeva, Gulmira Rakhymbay

Introduction

Electrodeposition of indium has been comprehensively investigated on solid electrodes such as platinum, glassy carbon electrode, nickel, tungsten, molybdenum and copper [START_REF] Huang | Electrodeposition of Indium on Copper for CIS and CIGS Solar Cell Applications[END_REF][START_REF] Liu | Electrochemical Study of the Properties of Indium in Room Temperature Chloroaluminate Molten Salts[END_REF][START_REF] Valderrama | Electrodeposition of indium onto Mo/Cu for the deposition of Cu(In,Ga)Se2 thin films[END_REF][START_REF] Yang | Electrochemical Study of Indium in a Water-Stable 1-Ethyl-3-Methylimidazolium Chloride/Tetrafluoroborate Room Temperature Ionic Liquid[END_REF][START_REF] Chung | Nucleation Process of Indium on a Copper Electrode[END_REF][START_REF] Pettit | Electrodeposition of indium on molybdenum studied with optical second harmonic generation and electrochemical impedance spectroscopy[END_REF][START_REF] Rakhymbay | Electrochemical deposition of indium: nucleation mode and diffusional limitation[END_REF]. This great interest is due to the practical application of indium, namely, the production of an ultrapure metal with subsequent use in the preparation of various semiconductor alloys. The most complete review of the electroreduction of indium on various electrodes is given in [START_REF] Chung | Electrochemical behaviors of Indium[END_REF][START_REF] Piercy | The electrochemistry of indium[END_REF]. Recently, in the electrodeposition of indium, ionic liquids were used as an electrolyte, which makes it possible to obtain compact coatings of indium and its alloys [START_REF] Estager | Electrochemical synthesis of indium(0) nanoparticles in haloindate(III) ionic liquids[END_REF][START_REF] Traore | New insight into indium electrochemistry in a Tf2N-based room-temperature ionic liquid[END_REF][START_REF] Hsieh | Electrodeposition of Stoichiometric Indium Antimonide from Room-Temperature Ionic Liquid 1-Butyl-1-Methylpyrrolidinium Dicyanamide[END_REF][START_REF] Hsiu | Electrodeposition of palladium-indium from 1-ethyl-3methylimidazolium chloride tetrafluoroborate ionic liquid[END_REF][START_REF] El Abedin | Electrodeposition of selenium, indium and copper in an air-and water-stable ionic liquid at variable temperatures[END_REF]. This effect is explained by the increase of the number of active sites during electrodeposition of indium on tungsten and glassy carbon electrodes [START_REF] Yang | Electrochemical Study of Indium in a Water-Stable 1-Ethyl-3-Methylimidazolium Chloride/Tetrafluoroborate Room Temperature Ionic Liquid[END_REF]. From aqueous chloride electrolytes, high purity indium is obtained, but forms dendritic coatings [START_REF] Kozin | Electrochemical refining of indium in aqueous solutions with solid electrodes[END_REF].

Earlier studies [START_REF] Mun Õz | Electrodeposition of Indium onto Vitreous Carbon from Acid Chloride Solutions[END_REF][START_REF] Gunawardena | The electrodeposition of indium onto vitreous carbon from acidic chloride media[END_REF] have established the influence of chloride ions on the electrocrystallization of indium on a glassy carbon electrode, and the limiting stage of electrochemical reduction of indium and the nucleation mechanism were determined. It has been established that as the concentration of chloride ions increases, the nucleation rate increases too.

To improve the quality of the coatings, it is necessary to add surfactants to the electrolyte, which show have a leveling action in indium electrodeposition. In [START_REF] Wen | Lead ion and tetrabutylammonium bromide as inhibitors of the growth of spongy zinc in single flow zinc/nickel batteries[END_REF][START_REF] Mele | Silver electrodeposition from water-acetonitrile mixed solvents in the presence of tetrabutylammonium perchlorate : PPart II-A SERS study of acetonitrile reactivity and tetrabutylammonium adsorption[END_REF][START_REF] Mele | Silver electrodeposition from water-acetonitrile mixed solvents and mixed electrolytes in the presence of tetrabutylammonium perchlorate. Part I-electrochemical nucleation on glassy carbon electrode[END_REF][START_REF] Zhang | The effect of quaternary ammonium-based ionic liquids on copper electrodeposition from acidic sulfate electrolyte[END_REF], additions of quaternary ammonium salts were used to inhibit dendritic formation during electrodeposition of zinc, copper and silver; these additives showed a high leveling effect. Formation of compact coatings is possible when surfactants are added to the electrolyte. So, in [START_REF] Leung | Zinc deposition and dissolution in methanesulfonic acid onto a carbon composite electrode as the negative electrode reactions in a hybrid redox flow battery[END_REF][START_REF] Wang | Effects of bismuth ion and tetrabutylammonium bromide on the dendritic growth of zinc in alkaline zincate solutions[END_REF], for electrodeposition of zinc on carbon steel (AISI 1018), additives of quaternary ammonium compounds were used to obtain compact coatings. Nbenzyltriethylammonium chloride and tetrabutylammonium hydroxide additives inhibit the growth of dendrites and can be used as a leveling additive, whereas in the case of tetraethylammonium hydroxide this effect is negligible [START_REF] Ortiz-Aparicio | Electrodeposition of zinc in the presence of quaternary ammonium compounds from alkaline chloride bath[END_REF].

In this paper, we studied the electroreduction of indium on a glassy carbon electrode in the presence of tetrabutylammonium chloride (TBACh) using a rotating disk electrode and chronoamperometry. In addition, the kinetics of the initial stages of electrocrystallization and local deposition of fragments of a new solid phase has been studied. Calculations for the study of nucleation were carried out using threedimensional nucleation models of Scharifker-Hills, Scharifker-Mostany and Mirkin-Nilov-Heerman-Tarallo. The theoretical calculations used in this work are described in detail in the works of the above authors [START_REF] Mirkin | Three-dimensional nucleation and growth under controlled potential[END_REF][START_REF] Scharifker | Three-dimensional nucleation with diffusion controlled growth[END_REF][START_REF] Milchev | Electrochemical nucleation and growth of nano-and microparticles: Some theoretical and experimental aspects[END_REF][START_REF] Heerman | Theory of the chronoamperometric transient for electrochemical nucleation with diffusion-controlled growth[END_REF][START_REF] Scharifker | Theoretical and experimental studies of multiple nucleation[END_REF].

Experimental

All electrochemical experiments were carried out in a conventional three-electrode electrochemical cell in a volume of 100 mL using Autolab ® PGSTAT 302 N and Bio-Logic ® VSP 300 potentiostat/galvanostats. A platinum plate (an indium plate in case of long-term chronoamperometry) and an Ag/AgCl (Metrohm ® ) were used as the counter and reference electrodes, respectively. The working electrode was GCE produced by Metrohm ® with the geometric area of about 0.07 cm 2 .

A 2 M NaCl solution containing 0.05 M InCl3 with pH 1.5 was used as a blank solution.

Tetrabutylammonium chloride with different concentrations was then added into blank solution. InCl3 (Sigma Aldrich, 99.99%) and TBACh (Sigma Aldrich, 99.99%) were used without further purification.

The experiments were performed with variation of the temperature, overpotential and rotation speed.

The scan rate was 10 mV s -1 . Before each measurement the working electrode was polished and rinsed with distilled water. From the voltammetric study, reduction potentials for chronoamperometric experiments were chosen in order to study the mechanism of nucleation from current transient and then obtain bulk indium deposits with longer deposition times. The chronoamperometric measurements at 35 ° were chosen because of the optimal temperature of electrochemical refining of indium which is ~ 40 °C. Voltammetric researches were then conducted in a temperature range of 25 -55 °C.

The electrical double layer impedance was measured using an Autolab ® PGSTAT 302 N potentiostat/galvanostat with a FRA 32 M module at the potential of -1.0 V in the frequency range of 100 kHz to 210 Hz at the amplitude of 10 and the system was calibrated using a standard equivalent cell produced by Metrohm. Impedance measurements were carried out in 2 M NaCl solutions (pH 1.5) without and with 1× 10 -4 M of TBACh. The interpretation of the experimental data of impedance measurements was carried out using the Frumkin -Melik-Gaikazyan (FMG) circuit, which is presented in the work of Laes [START_REF] Laes | Adsorption kinetics of tetrabutylammonium cations on Bi( 0 1 1̄ ) plane[END_REF] on the kinetics of tetrabutylammonium ion adsorption on a Bi(011) plane. A good correlation was obtained between the experimental data and their fitting using this circuit. Criteria for estimating the correlation were such parameters as the χ 2 -Chi-square function and Δ 2 -weighted sum of squares.

Scanning Electron Microscopy (SEM Philips XL-30 FEG) was used to study the morphology of the indium deposits.

Results and discussion

Kinetics of indium electroreduction

The current-time curves of indium electroreduction from the following aqueous acidic chloride solutions with and without TBACh at the cathodic potential E = -0.95V are shown in Fig. 1. At this potential, all current can be attributed to indium deposition, given the high overvoltage of hydrogen reduction on indium (j0=8 10 -12 A cm -2 ) [31].

<Figure 1>

Fig. 1 shows the limiting diffusion current at small times (1500 s) of the I -t curve. It can be seen from Fig. 1 (curves 1,3) that at large times (3000-7500s), there is a significant increase in the cathodic deposition current of indium for the blank electrolyte and blank solution with 10 -3 M TBACh (from about 7.2 to 20.1 mA). Since any rise in cathodic current during a potentiostatic electrodeposition is assumed to be due to an increase in true electrode surface area, the variation in current versus time at constant applied potential reflects the change in surface morphology (roughness, often dendritic growth) with a high sensitivity [START_REF] Wen | Lead ion and tetrabutylammonium bromide as inhibitors of the growth of spongy zinc in single flow zinc/nickel batteries[END_REF]. Thus, in this manner, the efficiency of morphology-controlling additive and its concentration can be readily assessed by their effect on the current [START_REF] Wilcox | Electrolyte additives for zinc-anoded secondary cells. I. Brighteners, levellers and complexants[END_REF]. The addition of 10 -4 M TBACh in the electrolyte of electrodeposition produces flat current-time responses (curve 2). If the concentration of TBACh increases 10-fold, no effect is observed (curve 3). Visual observation of the sample in blank solution after the experiment showed needle -shaped dendritic indium. Same observations were made for samples obtained in the presence of 10 -3 M TBACh deposits. But with addition of 10 -4 M TBACh in the electrolyte, the current remains constant, indicating the inhibitory effect on formation of dendrites during indium electrodeposition. This is also confirmed by visual observation.

To explain the inhibitory effect of TBACh on the process of dendritic formation during electrodeposition of indium, the kinetics of electroreduction and phase-formation of indium on a GCE was studied. Inset) It was found that the rate-determining stage of the electroreduction of indium on a glassy carbon electrode is diffusion.

Electroreduction of indium from chloride solutions can be represented as follows:

<Figure 2>

The above presented dependence (Fig. 2. Inset) is characterized by a high correlation coefficient (in all cases R 2 > 0.99), which makes it possible to calculate the diffusion coefficient of In 3+ ions at different temperatures (Table 1). As the temperature increases, an increase in the limiting cathode currents is observed (Fig. 3), which is associated with an increase in the diffusion coefficient (

<Figure 3>

The effect of TBACh on the discharge-ionization of indium is shown in Fig. 4 (A). Adsorption of surfactants on the surface of the electrode can change the rate of electrode reactions within wide limits.

In this case, the slowdown is most often observed. One can see a significant decrease in the cathodic currents in the presence of the additive, and a decrease in the slope angle of the j -E curve is observed at the anode branch of the cyclic voltammogram, which is associated with an increase in the polarization resistance. Without TBACh it is observed that the limiting diffusion current is the same for reverse and forward course of the voltammogram. But addition of TBACh shows a significant difference between the forward and reverse course. This difference could be due to the different adsorption of tetrabutylammonium ions on the glassy carbon electrode and on the surface covered with indium. One can see the decrease in the rate of the process in the region of high surface coverages of glassy carbon electrode with adsorbate (more negative than -1.1V) and the influence of surface nature on limiting cathode current is insignificant. The difference in the cathodic current density in the potential range from -0.64V to -0.9V in both cases (curve 1 and curve 2) is related to the nucleation overpotential.

The given dependence ("current crossover") is typical for the processes of 3D electrodeposition of metals. In addition, the observed shift in the potential of the initiation of deposition (En is the nucleation potential) is due to the inhibition of the electroreduction process of indium (Fig. 4A). Adsorption, which is preferable at fractures, blocks them, which leads to a reduction in the number of growth sites and the formation of new nuclei and clusters on a flat surface regardless of the existing steps [START_REF] Gamburg | Theory and Practice of Metal Electrodeposition[END_REF].

<Figure 4>

The adsorption of TBACh on the surface of the glassy carbon electrode is confirmed by impedance measurements (Fig. 4 (B)). The impedance spectroscopy diagrams are shown in Fig. 4 (B). The data have been perfectly fitted using a Frumkin-Melik-Gaykazyan circuit, where Rel and Cdl are respectively the resistance of solution and the double layer capacitance and W is the Warburg element that describes the diffusion of species. According to classical models, this circuit includes an additional capacitance B.B. Damaskin and al [START_REF] Korotkov | Tetrabutylammonium ion adsorption on indium electrodes[END_REF], also studied the adsorption behavior of tetrabutylammonium iodide (TBAI) on polycrystalline indium in a solution of Na2SO4. They pointed out from the capacitance measurements that with an addition of 10 -3 M TBAI, the double layer capacitance is ~ 10 μF cm -2 . This decrease of the double layer capacitance means a decrease in the surface charge of the electrode and an inhibition of the process. In this case, adsorbed molecules or ions can directly participate in all stages of the electrode process, including charge transfer. The influence of adsorbate is described by the amount of adsorbed ions coverage -θ. This function f(θ) describes not only the partial blocking of the surface by the adsorbate, but also the overcoming of the additional potential barrier ΔG(θ) by the depolarizer, which includes a quantity reflecting interactions in the adsorbed layer such as the bridging effect. In addition, the above function describes the change in the ψ ' -potential in the presence of adsorbed ions.

In our case, the electroreduction of In occurs via the electron bridge mechanism as a result of adsorption of chloride ions on indium ad-atoms and the further interaction of these ions with [InCl2(H2O)n] + ions from the solution. The electron bridge mechanism of indium electroreduction was investigated by A.G. Munoz, S.B. Saidman and J.B. Bessone in the work [START_REF] Mun Õz | Electrodeposition of Indium onto Vitreous Carbon from Acid Chloride Solutions[END_REF]. The change in the surface of the glassy carbon electrode on the reverse course of the current-voltage curve leads to the appearance above-mentioned effect and to a change in the surface coverage (θ). This apparently explains the difference in the limiting diffusion current of indium electroreduction in the forward and reverse course of the potential scan (Fig.

4A, curve 2).

Similarly, it can be seen from Fig. 4 (A) that in the presence of the TBA + ion, with decreasing potential, the cathode current density decreases gradually, and at a potential of -1.2 V it approaches zero. This effect can be explained by the surface coverage (θ) by adsorbed TBA + ions. Shifting the potential into the negative region can increase θ and the repulsive interaction energy between the [N(C4H9)4] + and [In(H2O)n] 3+ ions, thereby inhibiting the electrode process. From the literature [START_REF] Afanas'ev | Influence of ions of surface active substance on the kinetics of electrochemical reduction of ions and neutral molecules[END_REF][START_REF] Krylov | The Present State and Problems of the Theory of the Kinetics of Electrode Reactions Accompanied by the Adsorption of Inactive Substances and Reagents[END_REF] it is known that the change in the free energy of the repulsive interaction depends nonlinearly on the surface coverage, and it increases sharply at θ > 0.6. The nature of the interaction of adsorbed TBA + ions and the depolarizer is very complex, and deserves special study.

Analogous voltammetric measurements and calculations have been performed for the determination

+ 3
In D in the presence of TBACh. Fig. 5 shows the CVA at different temperatures and the rotation speed of the electrode.

<Figure 5>

To assess the correctness of the determination of the diffusion coefficient of indium ions under the investigated conditions by the RDE method, the universal Stokes-Einstein equation was used, which is applicable both for organic and for aqueous media. Here:

+ 3
In D is the diffusion coefficient of In (III) ions (m 2 s -1 ); k is the Boltzmann constant (1.381 × 10 -10 J K -1 ), ƞ is the dynamic viscosity of the solution (Pa s), a is the radius of the ion (m), and T is the absolute temperature (K).

It is known from [START_REF] Lindqvist-Reis | Structure of solvated metal ions: Solution and crystal structure of gallium, indium, scandium, yttrium, lanthanum and calcium ions with water and non-aqueous oxygen donor solvents[END_REF][START_REF] Lindqvist-Reis | The Structure of the Hydrated Gallium(III), Indium(III), and Chromium(III) Ions in Aqueous Solution. A Large Angle X-ray Scattering and EXAFS Study[END_REF][START_REF] Persson | Hydrated metal ions in aqueous solution: How regular are their structures?[END_REF] that in aqueous solutions, the hydrated indium ion -In(H2O)6 3+ has a regular octahedral shape and a radius of 21.4 nm. The values ƞ of the dynamic viscosity of the solution were taken from the literature [START_REF] Kestin | Tables of the Dynamic and Kinematic Viscosity of Aqueous KCl Solutions[END_REF]. The possibility of applying the Stokes-Einstein model to calculate the theoretical radius of indium ions from the experimentally determined values of the diffusion coefficient is shown in [START_REF] Kityk | Choline chloride based ionic liquids containing nickel chloride: Physicochemical properties and kinetics of Ni(II) electroreduction[END_REF]. The values of the diffusion coefficient of In 3+ , calculated from equation (1) and experimentally found, and the activation energy (Ea) are presented in Table 1.

<Table 1>

The is a slight difference in the calculated and experimentally determined values which could be due to the lack of consideration of the solvation process and various interactions in solution with the participation of background electrolyte components, in obtaining the calculated values of the diffusion coefficient. Similar results were obtained by Andrew P. Abbott and his colleagues in [START_REF] Abbott | Electrodeposition of copper composites from deep eutectic solvents based on choline chloride[END_REF]. Taking into account all of the above, as well as the simplified nature of the model, it can be noted that the difference in the values of the indium ion diffusion coefficient, calculated and experimentally determined, is entirely permissible.

To confirm the reliability of the values

+ 3
In D obtained by the RDE method, we found the diffusion coefficient of In 3+ ions at 35°C from chronoamperometric measurements based on the Cottrell equation.

Its numerical value was 6.35× 10 -6 cm 2 s -1 , which agrees well with the result obtained by the RDE method (6.14× 10 -6 cm 2 s -1 ). Thus, it is established from the results obtained that the electroreduction of indium on a glassy carbon electrode proceeds in a diffusion mode. The addition of 10 -4 M TBACh leads to a shift in the potential of the onset of indium deposition into the region of negative potentials and the inhibition of the cathodic process. This is confirmed by a significant increase in the activation energy of the process (by 17 kJ mol -1 ).

Nucleation studies

In order to determine the kinetic parameters of electrocrystallization and the type of nucleation, potentiostatic transients for the electrodeposition of indium on GC electrode in 0.05 M InCl3 containing solutions were obtained. It can be seen from the chronoamperograms that, at a potential of -0.65 V, indium electrodeposition is not observed (Fig. 6 A,B). At a potential of -0.70 V, electroreduction of In 3+ ions occurs and the current maxima are observed (jmax = -6.3 mA cm -2 ; tmax = 10.61 s), which is associated with the overlapping of the diffusion zones of individual crystallites and hemispherical masstransfer gives way to linear mass-transfer to a effectively planar surface. The further course of the current transients is explained by the diffusion limitation. Additions of tetrabutylammonium to deposition electrolyte significantly shift the chronoamperogram maxima to the region of small times (tmax = 0.93 s; jmax = -23.3 mA cm -2 ).

<Figure 6>

The shape of the experimental potentiostatic transients in the non-dimensional coordinates ((j/jmax) 2t/tmax) depends on the nature of the rate-determining stage and the type of nucleation. In the case of diffusion-controlled processes, the approach to analyzing the above transients has been developed by B.R. Scharifker and G.J. Hills. Experimental transients are compared in Fig. 7 A, B to transients calculated according to the Scharifker-Hills (SH) model for instantaneous and progressive threedimensional nucleation.

<Figure 7>

The experimental curves obtained without tetrabutylammonium chloride are in good agreement with a 3D progressive nucleation (Fig. 7A). Same type of nucleation has been pointed out with TBACh additives.

The stationary nucleation rate -∞ AN and saturation nucleus densitysat N for 3D progressive nucleation with diffusion control are calculated from the current maxima of the potentiostatic transients, according to the well-known equations [START_REF] Scharifker | Theoretical and experimental studies of multiple nucleation[END_REF]. In the SH model, the estimation of the nucleation parameters is based only on one point (tmax and jmax). For a more accurate assessment of the kinetic parameters of indium crystallization on glassy carbon, more appropriate models have been used, such as the Scharifker-Mostany (SM) model and the Mirkin-Nilov-Heerman-Tarallo (MNHT) model. According to the literature [START_REF] Matsushima | Electrodeposition of Cu Nanoparticles on BDD Electrodes: Reactions and Nucleation Mechanism[END_REF], the accuracy of the SM model depends on the time interval, and this model well describes theoretical dependence of j vs. t just in the limit of a longer time. The MNHT model includes a correction to the SM model for short periods of time. The values of the stationary nucleation rate, obtained by fitting the theoretically calculated jct curve (Fig. 8) using the SM and MNHT models are presented in Table 2.

<Figure 8> <Table 2>

As can be seen from Table 2 and Fig. 9, in case of indium electrodeposition on a glassy carbon electrode as the polarization increases, the stationary nucleation rate and the saturation nucleus density increase.

At a potential of -0.7 V in a blank electrolyte, the stationary nucleation rate calculated from the SH model was 1.25 × 10 4 cm -2 s -1 , and at a potential of -0.95 V the nucleation rate is 2.76× 10 7 cm -2 s -1 . This effect is enhanced in the presence of tetrabutylammonium additives. Thus, at a potential of -0.95 V in the presence of TBACh, the value ∞ AN increases from 2.76× 10 7 cm -2 s -1 to 1.05× 10 8 cm -2 s -1 . From the values sat N found the average radius of grains of electrodeposited indium was calculated from equation 7 in [START_REF] Yang | Electrochemical Study of Indium in a Water-Stable 1-Ethyl-3-Methylimidazolium Chloride/Tetrafluoroborate Room Temperature Ionic Liquid[END_REF] (Fig. 9).

<Figure 9>

Increase in overvoltage significantly reduces the average radius grains of the cathode deposit in the absence of TBACh. With an increase in the overvoltage from 60 mV to 310 mV, the average grain radius decreases from 65.2 μm to 9.5 μm, respectively. Additions of tetrabutylammonium even with small overvoltages lead to the formation of finely dispersed deposits of indium. At overvoltage of 310 mV in the presence of TBACh, a deposit with a grain size of 5.7 μm is formed. In the presence of 10 -4 M TBACh in the electrolyte, the difference in the grain size with increasing overvoltage decreases.

To confirm these calculations, the morphology of indium coatings obtained in an electrolyte containing 10 -4 M TBACh was observed by Scanning Electron Microscopy (Fig. 10), and EDX analysis results are shown in inset. For each deposit, EDX analysis confirmed the presence of indium metal on the glassy carbon substrate.

<Figure 10>

The comparison of the morphology of indium coatings in electrolytes with and without tetrabutylammonium chloride shows that in presence of additive, the grains have a smaller radius. This is in good agreement with the previously calculated average radii of grains and confirms the levelling effect of TBACh, leading to the formation of dense and uniform coatings.

Conclusion

In order to obtain dense, uniform indium deposits during electrorefining, indium was deposited on a glassy carbon electrode in chloride electrolytes containing tetrabutylammonium chloride. It has been established that small additions of tetrabutylammonium in the electrolyte cause a leveling effect.

The diffusive nature of the rate-determining stage of electroreduction of indium on a glassy carbon electrode from chloride electrolytes by the method of a rotating disk electrode has been established. The diffusion coefficient of In 3+ ions was determined in chloride electrolytes with and without TBACh. In an electrolyte containing 10 -4 M TBACh, the diffusion coefficient decrease and the activation energy of the process increases by 17 kJ mol -1 , indicating an inhibition of the electrode process.

The experimentally determined diffusion coefficient is in agreement with that theoretically calculated from Stokes-Einstein equations. Table 1 The diffusion coefficients of In 3+ ions and the activation energy of diffusion for a blank electrolyte (BE) and blank electrolyte containing 10 -4 M tetrabutylammonium chloride solution (ME), respectively.

Fig. 2 2 / 1 ω

 221 Fig. 2 shows cyclic voltammograms of the discharge-ionization of indium on a glassy carbon electrode

  the Arrhenius equation (Fig.3. Inset).

(

  Cad) related to adsorption of surfactant. From the figure (Fig. 4 (B) inset), a significant reduction of the double layer capacitance is observed in electrolyte containing TBACh. This indicates clearly the adsorption of TBACh on the electrode surface in the range of the electrodeposition potentials of indium. With a TBACh concentration of 10 -4 M, the capacitance is ~ 7.4 μF cm -2 .

From

  chronoamperometric measurements a three-dimensional progressing nucleation with diffusion control has been established, in agreement to Scharifker-Hills model. The presence of tetrabutylammonium increases the nucleation rate and the saturation density of the number of active centers. This leads to a decrease in the average grain radius of electrodeposited indium at an overvoltage of 310 mV in 10 -4 M TBACh electrolyte, from 12.5 μm to 5.7 μm. The results of SEM analysis confirm the formation of compact coatings of indium.

Fig. 1

 1 Fig.1 Current -time curves at a constant applied potential -0.95 V vs. Ag/AgCl for electrodeposition of indium in a blank electrolyte (1): 0.05 M InCl3 + 2 M NaCl; with 10 -4 M TBACh (2); with 10 -3 M TBACh (3), at 35°C. Inset: images for indium coatings after electrolysis.

Fig. 2

 2 Fig. 2 Cyclic voltammograms obtained at 25°C on GC electrode in 0.05 M InCl3 + 2 M NaCl solution for various rotating rates (250, 500, 750, 1000, 1500, 2000 and 2500 rpm). Scan rate of 10 mV s -1 . Inset: Levich plot performed at various temperatures: 1 -25°C; 2 -35°C; 3 -45°C; 4 -55°C.

Fig. 3 3 In D versus 1 -T

 331 Fig. 3 Cyclic voltammograms obtained at the rotation rate 2000 rpm on GC electrode in 0.05 M InCl3 + 2 M NaCl solution for various temperatures. Scan rate of 10 mV s -1 . Inset: evolution of + 3 In D versus 1 -T (R 2 =0.97).

Fig. 4 (

 4 Fig. 4 (A) Cyclic voltammograms performed at the rotation rate 2000 rpm on GC electrode in a blank electrolyte (1) and in the blank electrolyte containing 10 -4 M TBACh (2). Scan rate of 10 mV s -1 , at 25°C; (B) Nyquist plots for the glassy carbon electrode in 2 M NaCl aqueous solutions (a) and with additions of 1× 10 -4 M TBACh (b) at E= ̶ 1.0 V (vs. Ag|AgCl), pointsexperimental data; solid lines -fitting according to the Frumkin -Melik-Gaikazyan model (FMG), at 25°C. Inset: FMG circuit and the data of the fitting.

Fig. 5 ( 3 InFig. 6

 536 Fig. 5 (A) Cyclic voltammograms obtained at 25°C on GC electrode in a blank electrolyte containing 10 -4 M TBACh for various rotating rates (250, 500, 750, 1000, 1500, 2000 and 2500 Inset: forward course of a cyclic voltammograms; (В) Cyclic voltammograms performed at 2000 rpm on GC electrode in the same solution at various temperatures. Inset: evolution of + 3 In D vs. 1 -T .

Fig. 7

 7 Fig. 7 Dimensionless curves, (j/jmax) 2 vs t/tmax experimental (the symbols) and theoretical (the solid lines) for instantaneous and progressive for the indium nucleation at 35°C on GC electrode in a blank electrolyte (A) and a blank electrolyte + 10 -4 M TBACh solution (B).

Fig. 8

 8 Fig. 8 Experimental (the solid line) and calculated (the symbols) current transients for D = 6.14× 10 -6 cm 2 s -1 , c = 5× 10 -5 mol cm -3 , ρ = 7.3 g cm -3 , M = 114.8 g mol -1 , z = 3 and for SM model ∞ AN = 5.78 × 10 5 cm -2 s -1 , for MNHT model

Fig. 9

 9 Fig.[START_REF] Piercy | The electrochemistry of indium[END_REF] Dependence of the saturation number density of nucleation active sites, sat N , and the average nucleus radius, r , on the applied potential, E, obtained by analysis (SH) of the potentiostatic transients presented in Fig.6, at 35°C in a blank electrolyte (1, 3); a blank electrolyte + 10 -4 M TBACh solution (2, 4).

Fig. 10

 10 Fig. 10 SEM images of indium films deposited at E = -0.90 V vs. Ag|AgCl, the deposition time 15 min, at 35°C from: (A) a blank electrolyte; (B) a blank electrolyte + 10 -4 M TBACh solution. The different magnification: (1) ×500; (2) ×6500. Inset: EDX analysis of indium coating.

Table 2

 2 Calculated values of the stationary nucleation rate, AN for the 3D progressive nucleation according to equations 21, 22[START_REF] Scharifker | Theoretical and experimental studies of multiple nucleation[END_REF] for SH model, equations 12 and 13[START_REF] Milchev | Electrochemical nucleation and growth of nano-and microparticles: Some theoretical and experimental aspects[END_REF] for SM and MNHT models, respectively.

	T, 0 C	BE		ME
		RDE method	Eq. (1)	RDE method
	25	4.93	8.99	2.13
	35	6.14	11.39	4.69
	45	7.67	14.07	6.68
	55	11.41	17.02	9.54
			Ea (kJ mol -1 )
		22.2		39.6

∞

Acknowledgement

Authors gratefully acknowledges the Ministry of Education and Science of the Republic of Kazakhstan for financial support through grant number No. 1580 / GF4.


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Vu

J Erôme Toutain
email: jerome.toutain@chu-bordeaux.fr

David Cappellen

Marie-Ange Delrue

Hussein Daoud

Azza Abd

El Moneim

Pascal Barat

Orianne Montaubin

Franc ¸oise Bonnet

Zong Qi Dai

Christophe Philippe

Cong Toai Tran

Caroline Rooryck

Benoît Arveiler

Robert Saura

Sylvain Briault

Didier Lacombe

Laurence Taine

A homozygous

Keywords: MOMO syndrome, hyperphagia, obesity, intellectual disability, non-coding RNA

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Macrosomia, obesity, macrocephaly, and ocular abnormalities syndrome (MOMO syndrome) has been reported in only four patients to date. In these sporadic cases, no chromosomal or molecular abnormality has been identified thus far. Here, we report on the clinical, cytogenetic, and molecular findings in a child of healthy consanguineous parents suffering from MOMO syndrome. Conventional karyotyping revealed an inherited homozygous balanced reciprocal translocation (16;20)(q21;p11.2). Uniparental disomy testing showed biparental inheritance for both derivative chromosomes 16 and 20. The patient's oligonucleotide array-comparative genomic hybridization profile revealed no abnormality. From the homozygous balanced reciprocal translocation (16;20)(q21;p11.2), a positional cloning strategy, designed to narrow 16q21 and 20p11.2 breakpoints, revealed the disruption of a novel gene located at 20p11.23. This gene is now named LINC00237, according to the HUGO (Human Genome Organization) nomenclature. The gene apparently leads to the production of a non-coding RNA. We established that LINC00237 was expressed in lymphocytes of control individuals while normal transcripts were absent in lymphocytes of our MOMO patient. LINC00237 was not ubiquitously expressed in control tissues, but it was notably highly expressed in the brain. Our results suggested autosomal recessive inheritance of MOMO syndrome. LINC00237 could play a role in the pathogenesis of this syndrome and could

INTRODUCTION

Macrosomia, obesity, macrocephaly, and ocular abnormalities syndrome (MOMO syndrome, OMIM 157980) is an extremely rare syndrome with only four patients reported to date [START_REF] Wallerstein | A new case of MOMO syndrome[END_REF]. The main features of this syndrome are early onset obesity associated with intellectual disability, macrosomia, macrocephaly, ocular abnormalities (retinal coloboma and nystagmus), downward slant of palpebral fissures, and femoral recurvation [START_REF] Moretti-Ferreira | Macrosomia, obesity, macrocephaly and ocular abnormalities (MOMO syndrome) in two unrelated patients: Delineation of a newly recognized overgrowth syndrome[END_REF][START_REF] Zannolli | MOMO syndrome: A possible third case[END_REF][START_REF] Giunco | MOMO syndrome associated with autism: A case report[END_REF][START_REF] Wallerstein | A new case of MOMO syndrome[END_REF]. Although the molecular basis of MOMO syndrome remains unknown, the four patients previously described were from nonconsanguineous families, suggesting autosomal dominant inheritance [START_REF] Moretti-Ferreira | Macrosomia, obesity, macrocephaly and ocular abnormalities (MOMO syndrome) in two unrelated patients: Delineation of a newly recognized overgrowth syndrome[END_REF][START_REF] Zannolli | MOMO syndrome: A possible third case[END_REF].

Cytogenetic results showing homozygous balanced reciprocal translocations are exceptional [START_REF] Zaki | Identification of a novel recessive RELN mutation using a homozygous balanced reciprocal translocation[END_REF]. In patients with abnormal phenotypes, these rearrangements can theoretically help geneticists identify the basis of recessive genetic conditions using positional cloning strategies.

In this report, we present the clinical, cytogenetic, and molecular findings from a patient suffering from MOMO syndrome. Our patient presented a homozygous balanced reciprocal translocation (16;20)(q21;p11.23) inherited from healthy consanguineous parents. We hypothesized that this translocation could disrupt a gene of pathogenic importance for MOMO syndrome. Positional cloning strategies were used to isolate a candidate gene for this syndrome.

MATERIALS AND METHODS Clinical Report

A French gypsy boy was admitted for early onset obesity. He was the only child of healthy consanguineous parents. This child was born at term with macrosomia (4,850 g; þ3 SD). Since he was 18 months old, he had significant hyperphagia. At 6 1 = 2 years, he weighed 74 kg (>3 SD) and measured 124 cm (þ1.5 SD; body mass index [BMI]: >48 [>3 SD]; Fig. 1A). His OFC was 57 cm (>3 SD). Other minor dysmorphic features were noted: bulbous nose, almond-shaped eyes, and brachydactyly. He also had hypogenitalism, a marked deformation of the lower limbs with an important femoral recurvation (Fig. 1A), and arterial hypertension. Radiographs of the lower limbs confirmed his femoral recurvation (Fig. 1B). He showed signs of a moderate intellectual disability and underwent special education. At 8 1 = 2 years, he could neither read nor write. At age 11 years, he lost his ability to walk due to bowed legs and obesity. At 14 1 = 2 years, he weighed 170 kg (>3 SD), measured 158 cm (À2 SD; BMI: >68 [>3 SD]), and had an OFC of 60 cm (>3 SD). Ophthalmological examination highlighted bilateral temporal optic atrophy. The child also had a moderate bilateral astigmatism. Molecular studies for Prader-Willi and Fragile X syndrome were negative. A diagnosis of MOMO syndrome was suggested.

Conventional Karyotyping, Uniparental Disomy Testing, and Oligonucleotides Array-Comparative Genomic Hybridization

Lymphocytes were cultured with additional phytohemagglutinin stimulation. Chromosomes were treated with 5-Bromodeoxyuridine, exposed to light and heat, and colored using Giemsa stains to produce good resolution R-bands. Karyotypes were established at a level of resolution of up to 550 bands per haploid set.

Uniparental disomy (UPD) testing was carried out on genomic DNAs extracted from peripheral blood lymphocytes. Microsatellite markers for chromosome 16 (D16S489, D16S672, D16S678, and D16S686) and for chromosome 20 (D20S117, D20S428, D20S446, and D20S453) were studied. Segregation analysis was performed using multiplex fluorescent polymerase chain reaction (PCR) assays and fragment analysis on an ABI prism 310 DNA sequencer (Applied Biosystems, Carlsbad, CA).

Oligonucleotide array-comparative genomic hybridization (array-CGH) was performed using a 105 K oligonucleotide array (Agilent Technologies, Inc., Santa Clara, CA). The patient's genomic DNA was hybridized against a reference DNA. Copy number variations were screened against databases of known variants.

Fluorescence In Situ Hybridization Analysis

Fluorescence in situ hybridization (FISH) analyses were completed on metaphase spreads obtained from Epstein-Barr virus immortalized peripheral lymphocytes. Multiple bacterial artificial chromosome (BAC) and p1-derived artificial chromosome (PAC) clones targeting chromosomes 16 and 20 were used (Invitrogen, Carlsbad, CA). BAC and PAC clones were labeled using the Dig-Nick Translation Mix (Roche Applied Science, Indianapolis, IN) and the BioNick Labelling System (Invitrogen). Centromeric chromosomes 16 and 20 probes were used as controls (Abbott Molecular, Inc., Abbott Park, IL).

Long Range PCR Sub-Cloning and Direct Sequencing

Long range PCR (LR-PCR) was performed for the sub-cloning of a region of about 7 kb using the Expand Long Template PCR System Kit (Roche Applied Science). Primers for the LR-PCR, reverse transcriptase PCR (RT-PCR), and rapid amplification of cDNA ends (RACE) were designed using Primer 3, a web-based interface. Forward and reverse primers for the LR-PCR were designed in order to span regions separated from each other of about 1 kb (Supplementary Table I). LR-PCR products of derivatives chromosomes junctions were purified using MicroSpin S-300 HR Columns (Amersham Biosciences, Piscataway, NJ) and were sequenced using the BigDye Terminator v1.1 Cycle Sequencing Kit on an ABI 3130 Genetic Analyser (Applied Biosystems).

Expression Analysis

Expression of the genes around the two breakpoints was studied by RT-PCR with cDNA from several types of cells including brain, adipose tissue, fibroblast and lymphoblast cell lines (Clontech, Mountain View, CA). Total RNA was extracted using the Totally RNA Kit (Ambion, Carlsbad, CA) and treated with Deoxyribonuclease I-Amplification Grade (Invitrogen). The reverse transcription was completed using SuperScript II Reverse Transcriptase (Invitrogen). PCR reactions were performed using an AmpliTaq Gold DNA Polymerase (Applied Biosystems; primers listed in Supplementary Table II). Quantitative PCR of cDNA was obtained in the lymphoblast cell lines using Brilliant SYBR Green QPCR Master Mix (Stratagene, La Jolla, CA). The results were normalized using the expression level of beta-actin (Clontech).

Full Length Gene Cloning 5 0 and 3 0 RACE was performed in the lymphoblast cell lines with the 5 0 and 3 0 RACE System (Invitrogen). Total RNA was reverse transcribed using the oligo-dT primer. In the 3 0 RACE experiment, we performed a PCR with a gene specific primer in combination with the supplied 3 0 adaptor primer. Nested-PCR was also done using another gene specific primer in combination with the supplied nested primer. In the 5 0 RACE experiment, a 5 0 tailing step preceded the PCR and the nested-PCR which had two gene specific primers in combination with the supplied 5 0 adaptor primer. Full length isoforms in the brain were obtained by 5 0 and 3 0 RACE using the Human Brain Marathon-Ready cDNA (Clontech) in accordance with the manufacturers' instructions. The majority of RACE-PCRs was performed with gene specific primers and different nested gene specific primers in combination with the supplied adaptor primer, AP1, and nested adaptor primer, AP2 (See Supplementary Table I in Supporting Information online).

Mutation Screening of the Candidate Gene

Seven patients with a MOMO-like phenotype were chosen for mutation analysis of the disrupted candidate gene (clinical signs listed in Supplementary Table III in Supporting Information online). We performed direct sequencing as described above, including all exon-intron boundaries.

RESULTS

Conventional karyotyping, uniparental disomy testing, and oligonucleotides array-comparative genomic hybridization.

Conventional karyotyping revealed that our patient had a homozygous apparently balanced reciprocal translocation (16;20)(q21;p11.2; Fig. 2A). Karyotypes of his mother and father were 46,XX,t(16;20)(q21;p11.2) and 46,XY,t(16;20)(q21;p11.2), respectively (Fig. 2A). The bi-parental inheritance of chromosomes 16 and 20 in our MOMO syndrome patient was confirmed by markers D16S6785 (16p13.12), D20S4285 (20q13.2), and D20S446 (20p12.1). The oligonucleotide array-CGH analysis showed no genomic abnormality in our patient.

Fluorescence In Situ Hybridization Analysis

A physical map of 16q21 and 20p11.2 was established using FISH analysis. BAC RP11-467O15 and PAC RP5-872K7 were able to identify that the translocation breakpoints in our patient were on 16q21 and 20p11.23 (Fig. 2B). The signal intensity analysis performed with overlapping FISH probes restricted each breakpoint area to about 60 kb of critical regions.

Long Range PCR Sub-Cloning and Direct Sequencing

The absence of amplification of DNA from MOMO patient lymphocytes indicated that the breakpoints were between primers 7F and 7R on chromosome 16q21 and between primers 5F and 5R on chromosome 20p11.23 (Fig. 3A). The PCR performed on DNA from the MOMO patient with these two pairs of primers, each combining one primer on chromosome 16q21 and one primer on chromosome 20p11.23, allowed amplification of hybrid der(16)-)t(16;20)(q21;20p11.23) and der(20)t(16;20)(q21;20p11.23) DNA fragments, corresponding to each derivative chromosome (Fig. 3B and Supplementary Table IV in Supporting Information online).

DNA sequencing of the LR-PCR products allowed us to characterize the two breakpoints and revealed that the translocations occurred at the nucleotide position 62,671,762 of germline chromosome 16 with a deletion of three nucleotides and at the nucleotide position 21,078,333 on germline chromosome 20 with a deletion of eight nucleotides (nucleotide positions determined on chromosome sequences assemblies from the NCBI database: http://www.ncbi.nlm.nih.gov, March 18, 2012; Fig. 4). The breakpoint at 16q21 did not disrupt any known or predicted gene, whereas the breakpoint at 20p11.23 was located within the second intron of a novel gene named LINC00237, predicted to give rise to a non-protein-coding RNA (npcRNA).

Expression Analysis

RT-PCR analysis was performed to evaluate the expression of MOMO syndrome candidate genes (Fig. 5A), surrounding the translocation breakpoints at 16q21 and 20p11.23. Expression of candidate genes was monitored in cells and tissues linked to the disease process (adipocytes, fibroblasts, and brain) obtained from deceased healthy individuals upon autopsy. Expression in these normal tissues and cells was compared to that in MOMO patient lymphoblasts, used as a diseased cellular model, and that in his parents' lymphoblasts, carrying the same balanced chromosomal translocation in a heterozygous state. This analysis revealed that, with one notable exception, candidate genes expressed in normal tissues and cells showed no significant alteration in MOMO syndrome patient cells (Fig. 5B). CDH8 (cadherin 8, type 2), located 601 kb upstream from the 16q21 MOMO syndrome chromosomal breakpoint (Fig. 5A) was expressed in control adipocytes and brain, but was not expressed in control and MOMO patient lymphoblasts (Fig. 5B), preventing its evaluation as a candidate gene. However, its very large distance from the breakpoint did not make it a priority candidate. The 20p11.23 region contains 10 genes (Fig. 5A), in particular several transcription regulator genes (INSM1, XRN2, NKX2-4, NKX2-2, PAX1, and FOXA2), and the breakpoint on chromosome 20 could potentially alter the expression in any of them. INSM1, NKX2-4, NKX2-2, PAX1 were not expressed in normal cells or tissues and in MOMO patient lymphoblasts (Fig. 5B and data not shown), indicating that they are unlikely candidates for the MOMO syndrome. C20orf26, RALGAPA2, PLK1S1, XRN2, and FOXA2 showed similar expression in control and MOMO patient lymphoblasts (Fig. 5B), ruling out their implication as MOMO syndrome genes.

In contrast, RT-PCR analysis showed that LINC00237, not expressed in fibroblasts and adipocytes, was expressed in the brain and in lymphoblasts from control individuals, whereas its transcripts were absent in lymphoblasts from our MOMO syndrome patient (Fig. 5B). This observation was confirmed by quantitative PCR analysis (Fig. 5C). Quantitative PCR analysis also revealed that, as compared to controls, expression of LINC00237 was reduced by around 50% in lymphoblasts from the patient's parents. Both parents carried the translocation in a heterozygous state (Fig. 5C). Altogether, these data indicated that the homozygous MOMO syndrome translocation inactivated LINC00237.

Characterization of LINC00237 and Its Products

Rapid amplification of complementary DNA (cDNA) ends (RACE analysis) was performed on cDNA from control lymphoblasts and identified a variant containing two exons, herein named exon 1b and exon 2 (Fig. 5D). Another variant was reported to initiate from an upstream exon, herein named exon 1a (Fig. 5D), and contain also exon 2 (http://www.ensembl.org and http://blast.ncbi.nlm.nih.gov). This second variant composed of exon 1a and exon 2 was not detected in the tested cells (data not shown). Therefore, we focused our analyses on exon 1b-exon 2 transcripts. For both exon 1a-and exon 1b-initiated transcripts, only one potential 36 amino-acids open reading frame was detected in exon 2, but the candidate ATG initiating codon was not within a Kozak consensus sequence, suggesting that it was unlikely translated into a polypeptide and that LINC00237 was a npcRNA gene (Fig. 6) [START_REF] Altschul | Gapped BLAST and PSI-BLAST: A new generation of protein database search programs[END_REF][START_REF] Altschul | Protein database searches using compositionally adjusted substitution matrices[END_REF]. Further RT-PCR analysis of LINC00237 transcripts confirmed lack of transcript amplification with primers located within exon 2 and also revealed the absence of transcripts joining exons 1b and 2 in MOMO patient cells, and reduction of the latter in his parent's cells, consistent with the position of the translocation breakpoint (Fig. 5E). Transcripts containing exon 1b but not exon 2 were equally amplified in lymphoblasts from healthy individuals and from the MOMO patient, indicating that disruption of LINC00237 upon chromosomal translocation does not alter transcription initiation but prevents synthesis of complete transcripts.

Mutational Screening of LINC00237

Mutation screening of LINC00237 in our cohort of seven patients with MOMO-like syndrome revealed some polymorphisms. However, it did not reveal mutations even when searching for the nonsense, missense or splice-site types.

DISCUSSION

Homozygous balanced reciprocal translocations associated with a morbid phenotype are rare complications due to consanguinity, with only five publications describing the phenomenon to date [START_REF] Leschot | Homozygosity for a Y/22 chromosome translocation: t(Y;22) (q12;p12/13)[END_REF][START_REF] Wilmot | Disomic balanced reciprocal translocation[END_REF][START_REF] Martinet | Fetus with two identical reciprocal translocations: Description of a rare complication of consanguinity[END_REF][START_REF] Baala | Homozygous silencing of T-box transcription factor EOMES leads to microcephaly with polymicrogyria and corpus callosum agenesis[END_REF][START_REF] Zaki | Identification of a novel recessive RELN mutation using a homozygous balanced reciprocal translocation[END_REF]. Chromosomal regions disrupted by these translocations can constitute a way to identify candidate genes for disorders. ). D: Genomic organization of LINC00237, alternative transcription initiation variants (arrows) and resulting splicing events (blue and red dotted lines), and position of the chromosome breakpoint. E: Complementary RT-PCR analysis of LINC00237 transcripts in lymphoblasts from healthy individuals and from the MOMO patient and his parents, showing lack of transcripts joining exon 1b and exon 2 and lack of transcripts containing exon 2 but not exon 1b in MOMO patient cells, consistent with the position of the breakpoint (primers listed in Table ).

In our MOMO syndrome patient, the homozygous balanced reciprocal translocation (16;20) of bi-parental inheritance suggested, contrary to previous beliefs, an autosomal recessive mode of inheritance of this syndrome [START_REF] Moretti-Ferreira | Macrosomia, obesity, macrocephaly and ocular abnormalities (MOMO syndrome) in two unrelated patients: Delineation of a newly recognized overgrowth syndrome[END_REF][START_REF] Zannolli | MOMO syndrome: A possible third case[END_REF].

A positional cloning strategy, combining fine FISH mapping with long range PCR analysis and Sanger sequencing, enabled us to precisely map the translocation breakpoints on both 16q21 and 20p11.23. On 16q21, there was only one possible candidate gene, CDH8 (cadherin 8, type 2), located 601 kb upstream from the breakpoint (Fig. 5A), but expression data and its very large distance from the breakpoint did not make it a likely candidate. On 20p11.23, the translocation disrupted of a novel npcRNA gene, herein named LINC00237. However, nine other genes were located within 1.5 Mb of the breakpoint. As chromosomal breakpoints were in some cases reported to have an effect on the expression of genes located up to 1.3 Mb away [START_REF] Velagaleti | Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia[END_REF], we investigated all 10 genes surrounding the breakpoint on chromosome 20.

An expression study revealed that none of the expressed candidate genes, except LINC00237, showed significant variation between the lymphoblasts cells of our MOMO syndrome patient and controls. On the contrary, the mature transcripts of LINC00237 detected in normal lymphoblasts were totally absent in MOMO patient lymphoblasts, demonstrating that its disruption by the 20p11.23 breakpoint causes inactivation of this gene. Furthermore, LINC00237 was not ubiquitously expressed in control tissues (not expressed in adipose tissue), but it was notably highly expressed in the brain. Based on these findings, we hypothesize that LINC00237 could play a major role in the pathogenesis of MOMO syndrome. Notably, it was previously shown that npcRNA contribute to the regulation of gene expression in the nervous system [START_REF] Iacoangeli | Regulatory RNAs in brain function and disorders[END_REF]. In particular, we hypothesize that this non-protein-coding gene might be involved in hyperphagia and intellectual disability in MOMO syndrome. However, we cannot completely exclude the possibility that other LINC00237 transcripts, containing additional exons not identified in our RACE assays, might encode proteins whose loss-of-function could contribute to the pathogenesis of the MOMO syndrome.

It would have been very interesting to investigate the translocation (16;20) in other family members. To the best of our knowledge, his parents did not have other children, and there were no cousins or relatives with a MOMO syndrome phenotype.

Mutation screening of LINC00237 in patients with MOMO-like syndrome did not identify alterations. This might be explained by the fact that the patients studied did not present the entire clinical spectrum of MOMO syndrome. [START_REF] Moretti-Ferreira | New mutations in Brazilian patients with overgrowth syndromes[END_REF] recently screened for NSD1 mutations in 49 Brazilian patients with overgrowth syndromes, including three MOMO patients. No alterations of NSD1 were identified in the three MOMO patients [START_REF] Moretti-Ferreira | New mutations in Brazilian patients with overgrowth syndromes[END_REF]. It might be of central interest to screen these patients for LINC00237 alterations.

Due to the difficulties performing functional studies in cultured cells, especially with respect to the cognitive and behavioral nature of some MOMO syndrome features, the complete demonstration of the nature of the gene(s) implicated and the comprehension of the pathogenic mechanisms will certainly require the generation of animal models. An interspecies comparison revealed an 87% similarity level between the mouse and the human sequences of LINC00237. This analysis also revealed a complete conservation on the mouse syntenic chromosomal regions of both 3 Mb regions surrounding the translocation breakpoints on human 16q21 and 20p11.23 (http://www.ensembl.org). These sequence data indicate that it would be feasible, using LoxP site insertion by gene targeting and CRE-mediated recombination, to generate transgenic mouse models carrying translocations equivalent to the MOMO syndrome translocation [START_REF] Smith | A site-directed chromosomal translocation induced in embryonic stem cells by Cre-loxP recombination[END_REF][START_REF] Van Deursen | Cre-mediated site-specific translocation between nonhomologous mouse chromosomes[END_REF]. This type of model, as well as conventional knock-out mouse models, will allow to characterize LINC00237 physiological functions and to investigate its implication, as well as that of other weaker candidate genes surrounding the MOMO translocation breakpoints, in the pathogenesis of the MOMO syndrome.

In summary, this study is the first report identifying a candidate gene for MOMO syndrome. This novel gene, LINC00237, apparently generating a npcRNA, was highlighted after identification and molecular mapping of a homozygous balanced reciprocal translocation affecting 20p11.23. The translocation disrupted LINC00237, resulting in loss of production of complete transcripts. LINC00237 is therefore the best candidate gene so far for MOMO syndrome, at least for the patient investigated in our study. Further mutation screenings in MOMO patients and above-mentioned functional studies in transgenic mouse models are needed to characterize the function of this candidate gene and the consequences of haploinsufficiency.

FIG. 1

 1 FIG. 1. A: Photograph of our MOMO patient at the age 6 1 =2 years with morbid obesity (BMI > 48). B: X-rays of the lower limbs showing an important femoral recurvation.

FIG. 2 .

 2 FIG. 2. Conventional karyotyping and fluorescence in situ hybridization (FISH) analysis. A: Partial conventional karyotyping showing heterozygous t(16;20)(q21;p11.2) translocations in the parental lymphocytes; and homozygous in the proband's lymphocytes. B: FISH analysis on metaphase spreads from proband's lymphocytes. Bacterial artificial chromosomes (BAC) probes RP11-467O15 (16q21) and RP5-872K7 (20p11.23) overlapping the chromosome breakpoints were labeled with Spectrum Red and appear in red, while control centromeric probes on chromosomes 16 and 20 labeled with FITC appear in green. Arrows identify both derivative chromosomes 16 and 20.

FIG. 4 .

 4 FIG. 4. Partial sequences of normal germline chromosomes 16q21 and 20p11.23, and der(16) and der(20) derivative translocated chromosome polymerase chain reaction (PCR) amplified DNA fragments. Arrows indicate breakpoints and junction positions on germline and derivative chromosomes. The breakpoint on 16q21 was mapped between nucleotide positions 62,671,759 and 62,671,760 of germline chromosome 16, and the breakpoint on 20p11.23 was mapped between nucleotide positions 21,078,333 and 21,078,334 of germline chromosome 20 (nucleotide positions determined on chromosome sequences assemblies from the NCBI database: http://www.ncbi.nlm.nih.gov, March 18, 2012).

FIG. 5 .

 5 FIG. 5. Position, respective to breakpoints on chromosomes 16q21 and 20p11.23 (A; from http://www.ncbi.nlm.nih.gov, http://www.ensembl.org, and http://www.genome.ucsc.edu/cgi-bin/hgGateway), and expression status (B-E) of candidate genes in normal cells and tissues and in MOMO patient B lymphoblasts. A: The region at breakpoint on chromosome 16 contains only CDH8 (Cadherin 8, type 2), located 601 kb upstream of the breakpoint. The region at breakpoint on chromosome 20 contains 10 genes and the breakpoint disrupts LINC00237. B: Reverse transcription-polymerase chain reaction (RT-PCR) analysis of candidate genes, and (C) quantitative RT-PCR analysis of LINC00237 in normal cells and tissues and in control and MOMO patient B lymphoblasts. In (B) and (C), LINC00237 was detected using primers within exon 2 (2-F and 2-R2, see Table). D: Genomic organization of LINC00237, alternative transcription initiation variants (arrows) and resulting splicing events (blue and red dotted lines), and position of the chromosome breakpoint. E: Complementary RT-PCR analysis of LINC00237 transcripts in lymphoblasts from healthy individuals and from the MOMO patient and his parents, showing lack of transcripts joining exon 1b and exon 2 and lack of transcripts containing exon 2 but not exon 1b in MOMO patient cells, consistent with the position of the breakpoint (primers listed in Table).

ACKNOWLEDGMENTS

We wish to express our gratitude to the patient and his family for their cooperation and thank the technical staff of the CHU-Bordeaux for its help.