Thomas Duminy
email: thomas.duminy@insa-lyon.fr

Ronan Henry

Jérôme Adrien

Aurélien Doitrand

Sylvain Meille

Anisotropic fracture in nacre-like alumina

Keywords: Finite fracture mechanics, nacre-like alumina, crack initiation, anisotropy

Fracture in nacre-like alumina is studied experimentally and numerically with alumina platelets main alignment direction perpendicular (along-configuration) to the one commonly tested (acrossconfiguration) in the literature. In along-configuration, brittle failure is noted after crack initiation with no macroscopic crack deviation, unlike what is observed in across-configuration. However, NLA exhibits in along configuration initiation critical stress intensity factor K Ic = 5.3 ± 0.5 MPa.m 1/2 , in the same order of magnitude as in across-configuration. The increase in K Ic compared to monolithic alumina can be explained by a phenomenon of crack initiation containment at the interface between platelets. Unstable crack propagation occurs just after initiation, mainly following the platelet interfaces which results in crenelated fracture surfaces with crack decentering with respect to the initial notch tip.

Introduction

Ceramics usually show limited fracture toughness associated with a brittle behavior at ambient temperature, which is a severe limitation to a wider use as structural materials. Increasing crack resistance of ceramics has therefore been a major theme of research, mainly by the development of extrinsic mechanisms, i.e. mechanisms that act behind the crack tip, to hinder crack propagation Natural nacre is composed of 95 vol.% of staggered stiff calcium carbonate platelets (CaCO 3 ) embedded in 5 vol.% of a compliant secondary phase made of proteins [START_REF] Jackson | The Mechanical Design of Nacre[END_REF] [START_REF] Raj | Mechanical Properties of Nacre-Like Composites: A Bottom-Up Approach[END_REF]. Consequently, its structure is often referred to as 'brick-and-mortar' by analogy with masonry. Toughening in nacre is explained by several extrinsic mechanisms that act at different length scales, namely breaking of mineral bridges that form between platelets [START_REF] Meyers | Biological materials: Structure and mechanical properties[END_REF], interlocking of platelets [START_REF] Espinosa | Tablet-level origin of toughening in abalone shells and translation to synthetic composite materials[END_REF] [START_REF] Barthelat | On the mechanics of mother-of-pearl: A key feature in the material hierarchical structure[END_REF], platelet pull-out, and crack deflection in the secondary phase [START_REF] Barthelat | On the mechanics of mother-of-pearl: A key feature in the material hierarchical structure[END_REF] [START_REF] Song | Structural and mechanical properties of the organic matrix layers of nacre[END_REF] [15] [START_REF] Loh | Nacre toughening due to cooperative plastic deformation of stacks of co-oriented aragonite platelets[END_REF].

Efforts have been made in the last two decades to manufacture artificial nacres that replicate the brick-and-mortar microstructure of natural nacre. Such materials consist of micron-sized ceramic platelets embedded in a secondary phase. The platelets are generally made of mono-crystalline alumina [START_REF] Bouville | Strong, tough and stiff bioinspired ceramics from brittle constituents[END_REF] [18] [START_REF] Evers | Nacre-like alumina with unique high strain rate capabilities[END_REF] while the secondary phase can be for instance a metal [START_REF] Launey | A novel biomimetic approach to the design of high-performance ceramic-metal composites[END_REF] or a polymer [START_REF] Oner Ekiz | An efficient hybrid conventional method to fab-ricate nacre-like bulk nano-laminar composites[END_REF] [START_REF] Launey | Designing highly toughened hybrid composites through nature-inspired hierarchical complexity[END_REF].

To avoid the decrease in fracture strength associated with the use of ductile secondary phases and to envision using these materials at elevated temperatures, full mineral solutions have been developed [START_REF] Bouville | Strong, tough and stiff bioinspired ceramics from brittle constituents[END_REF] [START_REF] Pelissari | Refractory interphase and its role on the mechanical properties of boron containing nacre-like ceramic[END_REF]. Recent works focused on the manufacturing and on the mechanical characterization of nacre-like alumina (NLA), made of alumina platelets embedded in an amorphous glassy phase [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF] [25] [START_REF] Bouville | Strong and tough nacre-like aluminas: Process-structure-performance relationships and position within the nacre-inspired composite landscape[END_REF]. This configuration results in a material having, along across direction, a strength typical of dense polycrystalline alumina (430 ± 30 MPa), but with a crack initiation stress intensity factor more than twice higher (5.8 ± 0.2 MPa.m 1/2 ). Using a mineral secondary phase thus leads to a trade-off between toughness and strength for a ceramic material [START_REF] Bouville | Strong and tough nacre-like aluminas: Process-structure-performance relationships and position within the nacre-inspired composite landscape[END_REF].

Heretofore the knowledge of the mechanical properties of NLA remains fragmentary, especially when considering the anisotropic character of NLA. Indeed, resistance to crack propagation in these materials has almost exclusively been characterized using Single Edge Notched Beam (SENB) in bending in a configuration resulting in extensive crack deflection at the interfaces between platelets, i.e. with alumina platelet c-axis being mainly collinear with the SENB height (x-axis) (both axes are defined on Fig. 1) [START_REF] Bouville | Strong, tough and stiff bioinspired ceramics from brittle constituents[END_REF] [18] [27]. This configuration is referred to as across-configuration [START_REF] Currey | Mechanical properties of mother of pearl in tension[END_REF] in this paper (Fig. 1 -Top Left). Some attempts have been made to characterize the anisotropy of mechanical properties in nacre-like materials, but they were mostly limited to compressive tests on nacre-like with ductile secondary phase [START_REF] Akurati | Deformation mechanisms in ice-templated alumina-epoxy composites for the different directions of uniaxial compressive loading[END_REF] [START_REF] Chan | Energy dissipation in composites with hybrid nacrelike helicoidal microstructures[END_REF]. Chan et al. [START_REF] Chan | Energy dissipation in composites with hybrid nacrelike helicoidal microstructures[END_REF] infiltrated alumina platelet scaffolds with different platelet orientations with polydimethylsiloxane. They show that stiffness, strength and toughness vary with the platelet orientation with respect to the loading direction.

Akurati et al. [START_REF] Akurati | Deformation mechanisms in ice-templated alumina-epoxy composites for the different directions of uniaxial compressive loading[END_REF] tested NLA with epoxy as a secondary phase. They show that different deformation mechanisms can be triggered depending on the platelet orientation. In addition, indentation tests on NLA with alumina platelet and amorphous glassy phase were performed to characterize the anisotropy of the material [START_REF] Abando | Anisotropy effect of bioinspired ceramic/ceramic composites: Can the platelet orientation enhance the mechanical properties at micro-and submicrometric length scale?[END_REF]. Both anisotropy in Young's modulus and hardness where assessed for temperature ranging from room temperature to 550°C. All of these studies were focused on NLA in across-configuration (see . However, none of these studies investigated the behaviour of NLA with the platelet c-axis being collinear with the sample z-axis (i.e. obtained by 90°rotation from across-configuration around the y-axis as illustrated in Fig. 1 -Top Right and hereafter referred to as along-configuration [START_REF] Currey | Mechanical properties of mother of pearl in tension[END_REF]).

Understanding and characterizing fracture mechanisms for other material orientations than 'across configuration' appears crucial to envision using NLA as structural materials. The objective of this work is therefore to characterize crack initiation and propagation in NLA in another platelet orientation than the commonly studied one. The experimental characterization of NLA in along-configuration both at macro-and micro-scale is presented in Section 2. Section 3 is dedicated to the modelling of crack initiation using the coupled criterion at both scales, a comparison with experiments is then provided in section 3.3. The results are finally discussed and compared to those obtained in across-configuration.

Experimental characterization of NLA: along-configuration

Methods

Mechanical testing at the macro-scale

4-points bending tests (4PB) are carried out on SENB samples. The composition of these samples is the same as in [START_REF] Bouville | Strong, tough and stiff bioinspired ceramics from brittle constituents[END_REF] [24] [START_REF] Henry | Interface failure in nacre-like alumina[END_REF], namely 95.0 vol% of α-Al 2 O 3 monocrystalline platelets and 5.0 vol% of glass phase (SiO 2 -Al 2 O 3 -CaO (65:25:10 wt.%)). The manufacturing process is the same as in [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF], i.e. field assisted sintering of a freeze-dried powder. This results in a material relative density after sintering of 98% (3.88 g.cm -3 ) [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF]. Mechanical characterization being the main standpoint of the present article, the reader is referred to the previously cited articles for further details on the manufacturing process. Macroscopic bending tests were carried out using a uniaxial testing machine (Instron 8562, High Wycombe UK) with support span distance of 12 mm and a loading span distance of 3 mm (Fig. 1 -Top Right). Critical stress intensity factors K Ic values are computed according to [START_REF]C28 Committee, Test Methods for Determination of Fracture Toughness of Advanced Ceramics at Ambient Temperature[END_REF].

Geometrical parameters of tested samples are illustrated in Fig. 2 and listed in Table 1. A diamondsaw notch of approximately 200 µm width and 1.9 mm depth refined by a razor blade is machined in each sample to act as crack promoter. The notch tip radius lies between 5 and 8 µm depending on the sample (see Table 1).

Imaging technique characterization

Fracture surfaces of tested samples are characterized by different imaging techniques to identify the failure mechanisms. Scanning Electron Microscopy (SEM) observations are assessed to evaluate the interaction of the crack with the microstructure of gold sputter coated samples. Gold coating is applied on the surface of the samples to avoid charging effect, i.e. accumulation of electrons at the surface of the sample that would dramatically affect the observation quality. They are conducted on a VEGA 3 SEM (Tescan, Brno, Czech Republic) under an accelerating voltage of 10kV and using secondary electrons. SEM allow for high quality observation but are rather limited to acquire out-of-plane quantitative information. Micro-computed tomography (µ-CT) is performed with a 2 µm voxel size using an EasyTom Nano tomography system (RX Solutions, Annecy, France). The principle is the following. The sample under investigation is mounted on a rotor. An X-ray beam is sent on the sample. 650 scans are acquired, each of them taken at a different angular position of the sample. The transmitted X-ray beam signal is acquired by a charge coupled device camera.

Then, a reconstruction software gathers all scans to obtain a three dimensional-distribution of the linear X-ray attenuation coefficient within the sample. This 3D distribution forms an image from which one can extract 2D sections (see Fig. 6 -Left). The distribution of X-ray attenuation can be related to a density variation, thus allowing for the identification of cracks inside a sample, providing their dimensions are larger than the spatial resolution, i.e. approximately 2 to 3 times the voxel size. With the set-up used, the major cracks could be identified. However, the interaction between a crack and its neighbouring platelets cannot be assessed as the platelet characteristic dimensions are below the µ-CT resolution.

Therefore, confocal observations of the fracture surface are carried out using an NPS-NP1 con-focal sensor (Hirox, Tokyo, Japan) with a lateral resolution of 1 µm and a height resolution of 20 nm. Confocal microscopy allows for a 3-dimensional topological reconstruction of the surface and for height profile measurement, only a fraction of the total fracture surface being observed to limit the acquisition time at such high resolution. This surface reconstruction first enables to determine the exact location of the notch tip as the junction between the razor-blade machined notch and the crack surface. Then, 2-dimensional profiles are measured on the crack surface close to the notch tip corresponding to the crack initiation site.

Bending tests on micro-cantilever beams

In addition to macroscale four-points bending tests, we also analyse a micro-cantilever test carried out on NLA in along-configuration on samples processed using the same manufacturing route as in [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF] [25], resulting in similar composition and microstructure. The sample was prepared using a Focalized Ion Beam (FIB) (NVision 40, Carl Zeiss, Oberkochen, Germany) with gallium ions at an accelerating voltage of 30 kV. Ionic currents from 3 nA down to 300 pA were used for the milling steps [START_REF] Henry | Interface failure in nacre-like alumina[END_REF]. The obtained micro-cantilever has a pentagonal section. The force was applied ex situ using a nano-indenter under displacement control (G200, Keysight, Santa Rosa, USA). Fig. 3 -Left is a schematic representation of the micro-cantilever beam before testing.

4-points bending tests 115

The force-displacement curves of the macroscopic NLA samples tested in along-configuration are shown in Fig. 5. All samples exhibit an elastic brittle behavior. No stable crack propagation was observed. The crack path observed on the specimen surface seems at first sight similar to that of a homogeneous isotropic material -crack propagating parallel to the notch direction (see Fig. 4). The forces at crack initiation are listed in Table 1. Critical stress intensity factor at 7

In across-configuration on samples of the same composition and process conditions, Saad et al.

obtained an apparent critical stress intensity factor at initiation equal to 5.8 ± 0.2 MPa.m 0.5 [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF].

Micro-computed tomography

SEM observations

The fracture surface roughness is evidenced in Fig. 7, which can be explained by cracking mainly occurring along the interfaces between the platelets. Platelet misalignment with respect to their main orientation direction is also observed, as already characterized by Saad et al., i.e.

15°mean platelet misalignment with respect to the plane perpendicular to the pressing direction during processing. Only two broken platelets are observed as shown inside white ellipses. Broken platelets can be identified by the presence of spherical pores on the fractured surface (corresponding to initially close porosity inside the platelets). Indeed, porosity is not observed on the surface of unbroken platelet [25] [33].

Confocal microscopy

As confocal microscopy has a better spatial resolution as compared to X-ray tomography, it enables the quantification of surface roughness. It can be concluded from the different imaging technique results (SEM, XRay tomography, profilometry) that crack initiates along the interface between platelets, with local deviations around platelets. However, no macroscopic crack deviation is observed (see Fig. 4) which is a major difference with NLA tests in across-configuration where a crack deviation of typically 70°to 80°f rom the notch plane can be observed at the macroscopic scale [START_REF] Doitrand | Determination of interface fracture properties by micro-and macro-scale experiments in nacre-like alumina[END_REF]. Two hypotheses on the crack initiation location in along-configuration can thus be proposed:

• The crack initiates on a transverse interface at the scale of one platelet -i.e. approximately 0.5 µm in the z-direction (see the corresponding yellow interface in Fig. 1).

• The crack initiates on a basal interface with a larger surface (see the corresponding green interface in Fig. 1-bottom).

Bending test on microcantilever

We observed a linear elastic behavior up to brittle fracture. The force at crack initiation is 3.0 mN. The microcantilever tested in bending as shown in Fig. 3 exhibits a non-flat fracture surface, with crenellations of one to four platelets thick. As compared to the macro-scale tests, the fraction of broken platelets in the fracture surface is larger at the microscale (see surfaces

showing small spherical pores in Fig. 3). However, these pores are mainly located on the beam area loaded under compression. Therefore, this may indicate that crack initiation occurs at the interface between platelets and subsequent unstable crack propagation causes platelets fracture. The coupled criterion (CC) is a method developed by Leguillon [START_REF] Leguillon | Strength or toughness? A criterion for crack onset at a notch[END_REF] for the prediction of crack initiation. It states that the stress must be larger than the tensile strength over the whole area corresponding to the crack before initiation, and that the potential energy released by crack initiation must be larger than the energy required for the creation of the crack surfaces. These conditions write:

   σ nn ≥ σ c -∆W ≥ G c ∆S (1)
with σ nn the opening stress along the path of the newly created crack before initiation, σ c the tensile strength, ∆W the potential energy variation due to crack opening, ∆S the crack surface, and G c the material critical energy release rate.

This can also be written as:

   σ nn ≥ σ c , -∆W ∆S = G inc ≥ G c , (2)
where G inc is the incremental energy release rate.

Both conditions can be normalized:

   σnn σc ≥ 1, G inc Gc ≥ 1. (3)
Since the stress criterion is generally a decreasing function of the crack surface, it provides an upper bound for the admissible range of crack initiation surface S c . On the contrary the energy criterion is generally an increasing function of the crack surface [START_REF] Weißgraeber | Crack nucleation in negative geometries[END_REF]. Consequently, it yields a lower bound for the admissible initiation crack surface range. For a too small imposed loading, the upper bound obtained with the stress criterion is smaller than the lower bound obtained with the energy criterion, which means there is no crack surface for which both criteria are fulfilled (Fig. 10). Therefore, loading must be increased in order to increase the upper bound given by the stress criterion while decreasing the lower bound given by the energy criterion until both bounds match, which enables determining the imposed loading at crack initiation. As a consequence, the criterion induces that a crack can only initiate for a finite length, contrary to infinitesimal crack extensions obtained in LEFM [START_REF] Griffith | VI. The phenomena of rupture and flow in solids[END_REF]. It is worth noting that in the particular case of a pre-existing crack under displacement control, the coupled criterion is consistent with LEFM as it also admits infinitesimal crack propagation [39] [40].

The above explanation is schematically illustrated in Fig. 10, the two criteria from (3) being plotted as a function of the crack surface for two different loadings.

When the applied loading is high enough (i.e. F = F c , dotted lines on Fig. 10), for crack surfaces smaller than the one corresponding to the intersection of both curves, the stress criterion is fulfilled but not enough energy would be released by the creation of such cracks. On the contrary, a too large crack cannot initiate since the stress criterion would not be fulfilled on the whole crack path before initiation. The intersection of the two curves represents the crack surface for which the two criteria are fulfilled simultaneously.

NLA exhibits linear elastic behavior before crack initiation. Consequently, under the hypothesis of small deformations, the proportionality between stress and applied displacement U 0 and between potential energy and square of applied displacement writes:

   σ nn (S) = k(S)U 0 ≥ σ c , G inc (S) = A(S)U 2 0 ≥ G inc , (4)
With k(S) and A(S) the two proportionality functions.

The aim is thus to find, among all possible value for the applied displacement U , a critical displacement U c , which corresponds to the smallest imposed displacement that fulfils both conditions of the CC:

     U (S) = max( G inc A(S) ; σc k(S) ), U c = min S (U (S)). (5)
As can been seen from all equations above, determining U c requires the knowledge of the stress field -and thus the function k -and of the potential energy released as a function of the crack length -and thus the function A. Then, the predicted initiation force is derived from the obtained value of the critical displacement and can be compared to forces measured experimentally. The stress and potential energy are obtained by FE calculations as explained in next section. Functions A and k depend on the crack geometry. Assuming a given crack geometry, U c can be determined using the above-mentioned method. If the crack path is unknown, it may be determined as the one minimizing U c among all possible cracks, which may be computationally costly. In the sequel, the possible crack configurations are based on experimental observations.

2D implementation of the coupled criterion at the macroscale

A 2D model of SENB sample is shown in Fig. 2. The numerical sample dimensions are set to the mean values of the 4 tested samples. NLA displays a transversely isotropic behavior at the macroscopic scale and its elastic properties have been determined using ultrasonic contact technique. The shear and longitudinal wave speeds in the material are directly related to E and ν and are calculated using a transmitter/receiver sensor working at a frequency of 5 MHz. We obtained E aa = E bb = 368 GPa Young's modulus in the direction of the platelet plane, E cc = 320 GPa Young's modulus in the transverse direction (along platelet c-axis), ν ab = 0.24 and ν ac = 0.24 Poisson's ratio in the platelet plane and transverse direction respectively [START_REF] Doitrand | Determination of interface fracture properties by micro-and macro-scale experiments in nacre-like alumina[END_REF].

In Fig. 2-Left, the red circles indicate a vertical imposed displacement along the x-axis and the grey circles indicate a vertical displacement set to 0.0 mm along the x-axis to model the fixed spans. Quadratic triangular elements are chosen with a minimum mesh size of 0.2 µm around the crack, whose path is modelled as a segment. Such a configuration results in about 7 000 degrees of freedom. Mesh convergence is ensured so that initiation force differences smaller than 1% are obtained when using a finer mesh discretization.

From Section 3.1.1, we know that solving the CC requires the knowledge of the functions k(S) and A(S) defined in Eq 4. The function k(S), which links the imposed displacement and the stress field, is obtained based on FE simulation without crack. Then, the nodes along the crack path are successively released. For each crack surface, the elastic strain energy is computed which enables calculating the function A(S).

Crack initiation and propagation in NLA in across-configuration is an interfacial process [START_REF] Bouville | Strong, tough and stiff bioinspired ceramics from brittle constituents[END_REF] [START_REF] Henry | Interface failure in nacre-like alumina[END_REF]. Doitrand et al. [START_REF] Doitrand | Determination of interface fracture properties by micro-and macro-scale experiments in nacre-like alumina[END_REF] determined NLA interface tensile strength σ c and critical energy release rate G c for across-configuration from macro-and micro-scale bending tests. Additionally, strength and toughness for a single alumina platelet was determined in [START_REF] Doitrand | Revisiting the strength of micron-scale ceramic platelets[END_REF] from microbending tests [START_REF] Feilden | Micromechanical strength of individual Al 2 O 3 platelets[END_REF]. These material parameters are listed in Table 2.

Strength (MPa) Toughness (J.m -2 ) Al 2 O 3 platelets [41] 1100 22
Interface [START_REF] Doitrand | Determination of interface fracture properties by micro-and macro-scale experiments in nacre-like alumina[END_REF] 438 1.9

Table 2: Fracture parameters used in the 2D model.

3D implementation of the coupled criterion at the macroscale

• Transverse interface

Transverse interfaces have a normal collinear with the y-axis. Fig. 11-a illustrates the crack surface along with its characteristic dimensions c and T ci . The solution of the CC gives the crack initiation surface S c . In the 3D models we introduce the parameter T ci , which represent the crack dimension along the z-axis (see Fig. 11). If T ci = B the crack initiates along the whole sample thickness. On the contrary, T ci < B means the crack initiates only in a fraction of the sample thickness (for instance a single interface between two platelets or the interface between two stacks of platelets).

• Basal interface

Such interfaces are oriented with their normal collinear with the z-axis. Fig. 11-b shows a possible crack shape at initiation following a stress isocontour. The initiation crack front shape can be described only by one parameter, namely the crack surface, S.

3D micro-scale validation model

The micro-scale sample tested in along-configuration is modelled with the same features as the three-dimensional macro-scale model, e.g. a minimum mesh size of 0.1 µm and quadratic tetrahedron elements. The mesh contains 30.000 degrees of freedom. A negative displacement is imposed along the x-axis on one side of the micro-cantilever on exactly one node (see Fig. 3 -Left). Furthermore, the crack surface location can be varied using the parameter L i which defines the position of the crack from the base of the cantilever, and the parameter T i , which defines the position of the interface along the z-axis.

Results

2D model

Fracture surface observations mainly indicate that crack propagates inside the platelet interface, but also reveal a few broken platelets. The CC enables calculating the force corresponding to crack initiation inside a platelet or at the platelet interface. The crack initiation force assuming that crack initiates inside alumina platelets equals 470 N for a notch tip radius of 5 µm. In the case of crack initiation at the interface, the corresponding crack initiation force determined using the CC in 2D is F 2D init = 60 N for the same notch tip radius. In the two previously mentioned cases, the crack initiation happens at the notch apex, where the opening stress is maximum. Therefore, crack initiation is more likely to occur at the platelet interface than inside a platelet. However, the crack initiation force at the interface predicted using the CC underestimates the initiation force measured experimentally (237 ± 24 N), which may be explained by several reasons:

• The notch tip radius ρ i can have a non-negligible influence on the force at crack initiation.

This has been extensively shown in the study of the influence of notch tip radius on stress intensity factor considering a crack perfectly centered with respect to a U-notch [START_REF] Damani | Critical notch-root radius effect in SENB-S fracture toughness testing[END_REF] [45]

[46] [47] [START_REF] Carpinteri | A Finite Fracture Mechanics approach to the asymptotic behaviour of U-notched structures[END_REF]. Fig. 13 shows the influence of both the razor-blade notch radius and the crack decentering on the crack initiation force for several configurations. The cyan-star µm, the underestimation of the crack initiation force obtained numerically cannot only be explained by uncertainties on the notch tip radius.

• In NLA, since crack initiation occurs at the platelet interface, the crack may possibly be shifted with respect to the notch center as shown on µ-CT slices (see Fig. 6). The blue-squares, green-pentagons and red-triangles curves in Fig. 13 represent the crack initiation force variation as a function of the notch radius for different crack decentering configurations.

It can be seen that the influence of the crack decentering characterized by the parameter D i (see Fig. 2 -Right) on the crack initiation force remains small for notches with large tip radius -i.e. ρ i > 20 µm. When decreasing the notch radius, the influence of the decentering becomes more significant. For a notch tip radius lying in the range of experimentally observed notch tip radii, F 2D init matches the experimental values for D i 4.0 µm. For D i equals to 7.5 µm the numerical crack initiation force is one order of magnitude higher than the experimental force at crack initiation. Experimentally, the mean of the absolute D i value across the SENB thickness (along the z-axis) equals 7.0 µm. Thus, D i has a considerable influence, and variates across the sample thickness. This variation cannot be assessed with 2D models.

3D model

• Crack initiation in transverse interface: Fig. 14 shows the displacement to apply to the SENB sample to fulfill the CC as a function of the crack surface for two different values of T ci , the crack initiation thickness. On the left part of the figure, T ci = 0.5 µm, which is the thickness of one platelet. On the right part, T ci is equal to 4 µm, equivalent to the thickness of 8 platelets stacked together with no local misalignment within the stack, resulting in a smooth 4 µm long interface.

For small crack T ci , only the energy part of the CC drives crack initiation since whatever the crack surface, the required imposed displacement to fulfill the energy criterion is larger than the one to fulfill the stress criterion. For larger T ci , the imposed displacement to fulfill the stress criterion is larger (respectively smaller) than the displacement to fulfill the energy criterion for large (small) enough crack surfaces. Therefore, the minimum imposed displacement for which both criteria are fulfilled is attained for the crack surface corresponding to the intersection of both curves. The crack thickness at initiation for which we observe a transition between both cases is equal to 2.5 µm.

The initiation force as a function of T ci is shown in Fig. 15. The curve can be divided into two distinct parts. For small T ci , the crack initiation force obtained with the 3D model F 3D init increases as T ci decreases. When T ci increases from 0.4 µm to 30 µm, F 3D init decreases from 230 to 36 N. For T ci ranging from 30 to 1500 µm, no notable variation is noted in the force, reaching a plateau. F 2D init value obtained with the 2D model for the same razor-blade notch radius is slightly higher than F 3D init in 3D for T ci in the plateau regime. This indicates an overestimation of the 2D model F 2D init as compared to the 3D model. This overestimation of F 2D init has already been described on scarf adhesive joints [START_REF] Doitrand | Comparison between 2D and 3D applications of the coupled criterion to crack initiation prediction in scarf adhesive joints[END_REF] and on rhombus hole PMMA specimens under compression [START_REF] Doitrand | Experimental characterization and numerical modeling of crack initiation in rhombus hole PMMA specimens under compression[END_REF].

• Crack initiation in basal interface: 3D FE calculations show that the maximum opening stress at the notch tip σ zz (the opening stress for basal interfaces) is 4.4 times lower than σ yy (the opening stress for transverse interfaces, see Fig. 12). Fig. 16 -Left shows the distribution of σ zz at mid-sample thickness, corresponding to the basal interfaces opening stress distribution around the notch, which decreases with increasing distance from the notch tip.

Provided the incremental energy release rate is an increasing function of the crack area (Fig. 16 -Right), the crack surface can be determined based on the stress iso-contours. It gives a crack initiation force obtained with a 3D model F 3D init of 221 N to initiate a basal interface crack. This value is similar to the one found earlier for a transverse interface with T ci = 0.4 µm, and is close to the initiation force measured experimentally (237 ± 24 N). The initiation crack surface for basal interfaces obtained with the CC is 18.8 µm 2 , which corresponds to a 2.8 µm crack extension along the x-axis and 7.5 µm crack extension along the y-axis.

Modelling of the micro-cantilever test

Similarly, to the macro-scale, we model two possible configurations for the micro-cantilever test (see Section 2.2.5). match the experimental force.

Discussion

K Ic obtained in nacre-like alumina tested in along-configuration is 5.3 ± 0.5 MPa.m 0.5 . This value is similar to the stress intensity factor at initiation in across-configuration on similar samples (5.8 ± 0.2 MPa.m 0.5 ) [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF]. It is larger than the typical critical stress intensity factor of polycrystalline alumina (≈ 3.5 MPa.m 0.5 ). These results are listed in Table 3. It shows the efficiency of the brick-and-mortar structure in improving the toughness of NLA in both across-configuration and along-configuration. In these configurations, critical stress intensity factors at initiation are almost equivalent. However, depending on the platelet orientation, the platelets influence the composite crack initiation differently.

Material

K Ic NLA -across 5.8 ± 0.2 MPa.m 0.5

NLA -along 5.3 ± 0.5 MPa.m 0.5

Table 3: Sum-up of the experimental results obtained for across-and along-configuration.

The reinforcement mechanism involved in across-configuration is based on crack deviation by the microstructure at the macroscale. This deviation leads to a reinforcement for both crack initiation and propagation [25] [35] .

In along-configuration, no crack deviation is observed at the macro-scale (Fig. 4). SENB observations at the micro-scale show local crack deviation at the interfaces between platelets, as shown with µ-CT tomography and confocal microscopy. The maximum amplitude of this crack deviation is in the range of tens of micrometers, i.e. within the same order of magnitude than the platelet dimensions along the a or b axis (see Fig. 1). This behavior is further emphasized by the fracture surface observation of micro-cantilever beam tested in bending (Fig. 3). The fracture surface exhibits crenelations resulting from crack deviation by platelets.

The variation of crack decentering across the sample thickness observed experimentally (see Section 2.2.2 and Section 2.2.4) indicates that 3-dimensional effects exist in crack initiation in NLA in along-configuration. The load required to initiate a decentered crack has to be higher than for a crack centered with respect to the notch tip as the stress field decreases when moving away from the notch tip in the linear elastic regime.

2D FE models show that the opening stress tens of micrometers away from the notch tip is significantly lower than at the notch tip. Consequently, the numerical crack initiation force obtained by applying the CC on decentered configuration show that this micro-scale crack deviation cannot be neglected. Moreover, 2D models enable accounting only for crack decentering with respect to the notch center. However, the crack tortuosity in z-direction is not considered in 2D. Consequently, 3D models must be considered.

CC results from the 3D macroscopic FE models show a competition between two crack opening configurations at the interface between platelets. The results are summarized in Table 4. A crack can initiate from the opening of:

• a transverse interface (see Fig. 1-bottom). Relative location of the transverse interface to the notch tip is critical. When the interface is perfectly centered with respect to the notch tip, it is submitted to the largest opening stress. Consequently, if the interface is decentered, the opening stress is lower and crack initiation is less favored. The thinner the interface (i.e.

the thinner the platelet), the larger the crack initiation force. We found that the transverse interface thickness for which the numerical crack initiation force matches the experimental failure force under 4PB, d init char is equal to 0.4 µm (see Table 4). At such scale, the CC predicts that crack initiation is only driven by energy. Indeed, the applied displacement for which stress criterion is fulfilled is always lower than the displacement to fulfill the energy criterion.

• a basal interface (see Fig. 1-bottom). It must be long enough (at least 7.5 µm along the y-axis, see Fig. 16) to store enough energy for crack creation. Replacing the platelets in the composite by similar monocrystalline alumina platelets, with the same thickness, but with different dimensions would result in a difference in crack initiation force. This 7. Numerically, we find that crack initiation force for transverse interface and basal interface are equals when the platelet c dimension (i.e. thickness, see Fig. 1-bottom) equals 0.4 µm (providing sufficiently large basal surface). Thus, using platelets with larger c dimension, crack should initiate for smaller experimental load, resulting in smaller K Ic and be located on transverse interface. On the contrary, decreasing c dimension below 0.4 µm should not have an effect on crack initiation force. In that case, crack initiation will be located on basal interface. This clearly emphasized the competition between basal and transverse interfaces for crack initiation, which primarily depends on the platelet c dimension. It should be noted that the platelet thickness of the SENB samples and micro-cantilever beam is evaluated to approximately 0.5 µm (d exp char , see Table 4), which is closed to the above-defined optimum thickness. It should be kept in mind that these findings are based on the study of model that does not consider defects or imperfections in the material, e.g. platelet misalignment [START_REF] Saad | Toughening mechanisms in nacre-like alumina revealed by in-situ imaging of stress[END_REF]. These imperfections in the microstructure also play a role, thus modifying the extent to which the platelet dimensions play a role in the composite reinforcement.

Both SENB and micro-cantilever beam 3D model emphasize the importance of describing crack initiation using an approach relying on both stress and energy considerations such as the CC. The crack initiation containment reduces strongly the influence of the stress level on crack initiation which ultimately reduces here to an energetic phenomenon. Consequently, it would make the determination of the tensile strength uncertain in an inverse identification procedure, as previously shown by Jimenez and Leguillon [START_REF] Alfaro | Finite Fracture Mechanics at the micro-scale. Application to bending tests of micro cantilever beams[END_REF] and Doitrand et al. [START_REF] Doitrand | Revisiting the strength of micron-scale ceramic platelets[END_REF] on micro-samples.

In macroscopic SENB samples, the z-direction (i.e. along sample thickness) dimension encompass thousands of platelets. Consequently, it is likely that at least one transverse interface is well positioned with respect to the notch tip. However, for the micro-cantilever beam, less than 10 platelets are encompassed in the z-direction. Consequently, both basal and transverse interfaces might not be positioned near the beam maximum opening stress zones. This might explain the differences between numerical and experimental crack initiation force for both basal and transverse interfaces.

Conclusion

Crack initiation in NLA has been studied experimentally and numerically on bending samples (SENB samples and micro-cantilever beam) with a platelet orientation, denoted as alongconfiguration, that differs from the literature most-studied configuration [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF] [25] [START_REF] Bouville | Strong and tough nacre-like aluminas: Process-structure-performance relationships and position within the nacre-inspired composite landscape[END_REF], denoted as across-configuration. NLA exhibits an elastic brittle behavior in along-configuration with no stable crack propagation after initiation. The brick-and-mortar microstructure induces a micro-scale interaction with the crack as shown by local crack deviation at the interfaces, but no macroscopic crack deviation from the notch plane. 3D models using the coupled criterion show the crack is contained at initiation at the interface between two platelets. Crack initiation containment leads to a reinforcement of the NLA critical stress intensity factor in along-configuration at initiation when compared to polycrystalline alumina up to values closes to those reported for NLA in acrossconfiguration [START_REF] Saad | A simple approach to bulk bioinspired tough ceramics[END_REF].

Figure 1 :

 1 Figure 1: (Top): Schematic representation of a NLA SENB sample with platelets in (Left) across-configuration, (Right) along-configuration. The crack is schematized by the blue lines. (Bottom) Schematic representation of platelet local arrangement. Two kinds of interfaces can be depicted: green interfaces are labelled as basal interfaces and yellow interfaces are labelled as transverse interfaces.

Figure 2 :

 2 Figure 2: Schematic representation of the samples and definition of geometrical parameters. Left: general view of the specimen. Right: magnification of the notch tip. The crack is schematized in red. The dimension of the sample along the z-axis (thickness) is depicted by the parameter B inTable1.

Figure 3 : 6 2

 36 Figure 3: (Left): Schematic of a micro-cantilever sample in along-configuration prepared by FIB. Some platelets are illustrated in brown. The black arrow indicates the loading location. (Middle): Micro-cantilever fracture surface after testing in along-configuration. (Right): Definition of geometric features and of the possible crack initiation sites. The corresponding color code will be explained in Section 3

Figure 4 :

 4 Figure 4: Macroscopic observation of the crack propagation direction on a sample tested in four-points bending.

Figure 5 :

 5 Figure 5: Force-displacement curves of NLA samples tested in 4-points bending in along-configuration.

Fig. 6 -

 6 Fig.6-Left shows four tomography slices selected at different locations along the thickness of a beam tested in 4-points bending, as illustrated in Fig.6-Right. We observe that the crack is not always centered with respect to the notch bisector, resulting in some crack decentering as illustrated by the parameter D i (Fig 2-Right). This decentering is not constant for one sample, but varies across the sample thickness (along the z-axis direction) (see Fig.3). D i values at different locations along the thickness cannot be accurately determined from the µ-CT data since the spatial resolution is not sufficient.

Figure 6 :

 6 Figure 6: µ-CT slices at different locations along the thickness of a NLA sample tested in 4-points bending, showing the deviation of the crack location with respect to the notch plane. The image on the right gives the distance of each µ-CT observation from the sample side.

Figure 7 :

 7 Figure 7: (Left): SEM image (bottom view) of a SENB sample after bending test showing interactions of the crack with the microstructure. The razor blade notch lies in the foreground and the crack surface in the background. (Right): SEM image view of a SENB sample after test showing the presence of two broken platelets (as shown with white ellipses). The axis systems are defined in accordance with Fig. 1. On both images, the red dashed lines depict the notch tip.

  Fig 8 shows a 3D reconstructed fracture surface near the notch tip, evidencing the fracture surface roughness due to crack propagation along the interface between platelets. A 2D profile is captured at the junction between the notch and the

Figure 8 :

 8 Figure 8: Partial confocal reconstruction of a SENB sample fracture surface.

Figure 9 :

 9 Figure 9: Di evolution as a function of the position along the sample thickness.

Fig. 3 -Fig 1 .

 31 Fig. 3 -Middle shows that 4 interfaces are possible crack initiation interfaces. They are shown on Fig. 3 -Right.

Figure 10 :

 10 Figure 10: Schematic explanation of the CC: energy (light blue) and stress criteria (red) as a function of crack surface for imposed loadings equal to (dotted lines) or lower (full lines) than the crack initiation loading.

Fig. 11

 11 Fig.11shows a schematic illustration of the SENB 3D model. The upper cylinders illustrate the roller location where displacement is imposed along the x-axis. The lower cylinders depict the support location. Quadratic tetrahedron are used, with a 0.1 µm minimum mesh size around the crack. This model contains about 700 000 degrees of freedom. The same mesh convergence procedure than in Section 3.1.2 is applied.Adding a third dimension to the model enables modelling a planar crack of finite thickness. The crack is always assumed to initiate at mid-thickness of the crack plane. The possible crack shapes are determined after a first FE simulation by assuming they coincide with the stress isocontours,

Figure 11 :

 11 Figure 11: Macroscopic 3-dimensional model. The material is considered homogeneous. A magnification of the microstructure is represented on the front-left part of the sample. The yellow rectangle represents the crack shape at initiation under transverse interface assumption. The green circular shape represents the crack shape at initiation under basal interface assumption.

Figure 12 :

 12 Figure 12: Schematic representation of some platelets lying around the crack surface, with the basal and transverse interfaces depicted in green and yellow respectively. The black arrows represent the loading direction during bending.

Figure 13 :

 13 Figure 13: 2D-simulated force at crack initiation as a function of the razor-blade notch radius, for different crack decentering.

Figure 14 :

 14 Figure 14: Displacement predicted for Tci equal to (Left) 0.5 µm and (Right) 4.0 µm by each criterion of the CC. The black circle represents the smallest displacement and corresponding crack surface for which both criteria are fulfilled.

Figure 15 :

 15 Figure 15: 3D-simulated force at crack initiation for a 4-pts bending sample with Gc = 1.9 J.m -2 and σc = 438 MPa. The dashed black line represents the value obtained with a 2-dimensional model.

Figure 16 :

 16 Figure 16: (Left) Macroscopic SENB basal interface stress field σzz around the razor-blade notch at z = B/2. The coordinate system is the same as in Fig. 11. (Right) incremental energy release rate associated with the initiation of a crack in a basal interface as a function of the crack surface.

  330

Figure 18 :

 18 Figure 18: Micro-cantilever basal interface on zone 4 (see Fig. 3). (Left) Stress field σzz. The coordinate system is the same as in Fig. 3. (Right) Incremental energy release rate as a function of the crack surface.

Table

  .

Table 1 :

 1 Sample L (mm) W (mm) B (mm) ρ s (µm) F Samples geometrical dimensions, notch tip radius and experimental force at crack initiation F exp init .

	exp init (N)

Table 4 :

 4 5 µm length is called d init char . It is the characteristic numerical microstructure length for which crack initiation happens. It has to be compared with d exp char , which is the characteristic dimension of the interface, induced by the platelet dimensions (see Table 4). As long as d exp char > d init char , the microstructure does not constrain crack initiation. The two quantities being approximately equal means that a change in d expchar , e.g. by changing the platelets dimensions, would lead to a change in fracture mechanism. Numerical and experimental characteristic lengths for each interface at the micro-and macro-scale, along with the experimental force at failure.

	Scale Interface type	F exp init	d init char (µm) d exp char (µm)
	macro	transverse	237 ± 24 N	0.4	0.5
	macro	basal	237 ± 24 N	7.5	10.0
	micro	transverse	3 mN	0.1	0.5
	micro	basal	3 mN	1.8	10.0

Acknowledgement

This work was supported by the LABEX MANUTECH-SISE (ANR-10-LABX-0075) of Université de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). Moreover, we thank The French Ministry of Education and Research for funding this work, through a PhD scholarship CRediT Thomas Duminy: Conceptualization, Methodology, Software, Validation, Investigation, Formal analysis, Writing -Original Draft, Writing -Review & Editing, Visualization, Project administration; Ronan Henry: Methodology, Investigation; Jérôme Adrien: Investigation, Resources; Aurélien Doitrand: Conceptualization, Methodology, Software, Validation, Writing -Review & Editing,Supervision, Project administration, Funding acquisition; Sylvain Meille: Conceptualization, Methodology, Validation, Writing -Review & Editing, Supervision, Project administration, Funding acquisition

• Crack initiation in transverse interfaces

Assuming crack initiation occurs in one transverse interface only, F 3D init is determined for each possible zone 1, 2 and 3 as defined in Fig. 3. The values obtained are respectively equals to 1.5, 1.7, 1.6 mN for these three zones, around half of the experimental crack initiation force (3.0 mN). Fig. 17 shows the influence of T ci on F 3D init for zone 3 (see Fig. 3). The force obtained increases as T ci decreases. However, the numerical force equals the experimental one for a T ci equals to 0.1 µm, which is inconsistent with the experimental observation (e.g. zone 3 has a thickness of 0.49 µm). We can note that for T ci values below 0.75 µm in Fig. 17, energy only has an influence on crack initiation, as could be observed at the macro-scale for small values of T ci (see Fig. 14 -Left).

• Crack initiation in basal interfaces

Maximum values for σ zz are obtained in the specimen middle plane perpendicular to the z-axis and close to the clamped zone (y ≈ 0)(see the coordinate system in Fig. 3). Consequently, among all the basal interfaces in Fig. 3, zone 4 depicted in green is the basal interface on which σ zz is maximum. Fig. 18 -Left shows the stress field on zone 4. In this configuration the CC gives an initiation loading F 3D init of = 9.5 mN. The comparison of the experimental force (3.0 mN -see Section 2.2.5) to the F 3D init computed for the basal and transverse interfaces shows crack initiation in transverse interfaces is more favorable than in basal interface. However, both fail to predict accurately crack initiation as none of the crack initiation force values


PhD Samir Bouasker

PhD Nisha Patel

BSc, PhD Rebecca Greenlees

MD Diana Wellesley

PhD Lucas Fares Taie

Ph.D Naif A Almontashiri

PhD Júlia Baptista

Malak Ali Alghamdi

PhD Sarah Boissel

MD Jelena Martinovic

BSc, PhD Ivan Prokudin

MD Samantha Holden

FRCPath, PhD Hardeep-Singh Mudhar

BSc, PhD Lisa G Riley

MSc Christina Nassif

MD, PhD Tania Attie-Bitach

MD Marguerite Miguet

PHD Marion Delous

PhD Sylvain Ernest

MD Julie Plaisancié

MD, PhD Patrick Calvas

PhD Jean-Michel Rozet

MD Arif O Khan

MSc Fadi H Hamdan

PhD, FRACP Robyn V Jamieson

MD Fowzan S Alkuraya
email: falkuraya@kfshrc.edu.sa

MD Jacques L Michaud
email: jacques.michaud.med@ssss.gouv.qc.ca

MD, PhD Nicolas Chassaing
email: chassaing.n@chu-toulouse.fr

Bi-allelic variants in WNT7B disrupt the development of multiple organs in humans

Keywords: Diaphragmatic anomalies, Anophthalmia/Microphthalmia, and WNT7B, microphthalmia, lung agenesis, tracheomalacia, diaphragmatic eventration, cardiac defect, hypoplastic kidneys, PDAC syndrome, Matthew-Wood syndrome P6 F Bilateral hypoplasia Thin diaphragmatic cupula F Unilobular and dysplastic left lung No Microphthalmia Yes Renal cysts, semicircular canals hypoplasia Ventricular septal defect Schizencephaly, ureteral duplication, vertebral abnormalities Bilateral microphthalmia No Coelosomia, vesical extrophy, kidney hypoplasia, angulation of right femur, anal anteposition, polysplenia, absent pancreas lung Bilateral diaphragmatic cupula herniation Right colobomatous microphthalmia Ventricular septal defect, pulmonary artery agenesis Cleft palate, unilateral agenesis Panhypopituitarism, meningocele, right carotid artery agenesis Unilateral hypoplasia Unilateral hernia Unilateral microphthalmia with retinal dysplasia and anterior chamber abnormalities Bilateral cryptorchism, spleen hypoplasia BayesDel_addAF, DANN, DEOGEN2, EIGEN

Background: Pulmonary hypoplasia

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KEY MESSAGES

What is already known on this topic: The combination of Pulmonary hypoplasia/agenesis, Diaphragmatic hernia/eventration, Anophthalmia, and Cardiac defects delineates the PDAC syndrome. Pathogenic variants in two genes involved in the retinoic acid pathway, STRA6 and RARB, were previously known to be involved in this syndrome but explained only a subset of cases.

What this study adds:

We identified WNT7B bi-allelic pathogenic variants in one newborn and two fetuses, from two families, displaying features overlapping with the PDAC syndrome. In a third family, molecular autopsy by proxy in a couple that lost two babies due to lung hypoplasia revealed that both parents where heterozygous for a WNT7B nonsense variation.

How this study might affect research, practice or policy:

This study identified a third PDAC syndrome gene and raises the possibility that PDAC syndrome involves developmental processes that are regulated by an interaction between the retinoic acid and WNT pathways.

INTRODUCTION

Pulmonary agenesis is a congenital anomaly in which there is complete absence or severe hypoplasia of one or both lungs, estimated to complicate around 1 per 15,000 pregnancies. 1 Very little is known about its genetic basis in humans. Moreover, even if up to 50% of fetuses or newborns with pulmonary agenesis have associated congenital abnormalities, 1 there are very few recognizable syndromes associated with this developmental anomaly. The combination of Pulmonary hypoplasia/agenesis, Diaphragmatic hernia/eventration, Anophthalmia, and Cardiac defects delineates one such syndrome: the PDAC syndrome, also known as Matthew-Wood syndrome (MWS), Spear syndrome or MCOPS9 (MIM 601186). Pathogenic variants in two genes, namely STRA6 (MIM 610745; autosomal recessive inheritance) and RARB (MIM 615524; autosomal dominant and recessive inheritance) have previously been identified in PDAC-affected individuals. 2 3 Many cases of PDAC syndrome, however, remain unexplained. By using exome sequencing (ES), we identified biallelic likely deleterious variants in WNT7B in 3 families with phenotypes consistent with PDAC syndrome. In order to confirm the link between WNT7B pathogenic variants, we studied wnt7ba and wnt7bb mutant zebrafish lines.

We finally developed in vitro functional analyses to confirm the deleterious effect of the identified variants in our patients. Our results suggest that bi-allelic variants in WNT7B cause a polymalformative disorder that overlaps with the PDAC syndrome.

METHODS

Patients and molecular screening

We first performed ES in three patients with features of the PDAC syndrome (patients 1 to 3). To identify potentially pathogenic variants in exomes, we filtered out synonymous variants, intronic variants other than those affecting the consensus splice sites, and variants with a minor allele frequency greater than 0.5% in public (1000 Genomes, EVS, dbSNP138, GnomAD) and local (CHU Sainte-Justine Research Center exome dataset) databases. We next query variants affecting the same genes within the 3 screened individuals, as de novo, homozygous and compound heterozygous variants for each individual.

Seventeen additional patients (patients 4 to 20) with features of the PDAC syndrome and no identified pathogenic variants in STRA6 and RARB were also screened by Sanger sequencing of all WNT7B coding exons using the primers listed in Table S1 (patients 4 to 20). Finally, four patients from two families were identified by GeneMatcher 4 as they harbor rare variants in WNT7B (Patients 21 to 24). In these families, molecular screening was performed by ES for patient 21 and the parents of patients 23 and 24. Sanger sequencing was used to investigate the presence of the familial variants in patient 22. Phenotype of these 24 patients are summarized in Table S2. Variants numbering refers to the NM_058238.2 WNT7B isoform.

Study of wnt7ba and wnt7bb mutant zebrafish lines

The zebrafish genome contains two orthologs of WNT7B, wnt7ba and wnt7bb, whose corresponding proteins share an identity of 85 % and 89 %, respectively, with their human homolog. [START_REF] Beretta | All four zebrafish Wnt7 genes are expressed during early brain development[END_REF] The wnt7ba (sa31394) and wnt7bb (sa24585) mutant zebrafish lines were generated at Adult zebrafish (7-9 months old) were analysed by X-ray and histological analysis. Fishes were sacrificed by prolonged immersion (10 min) in Benzocaine, (375µg/l) and X-rayed using a Faxitron (MX-20DC12). Whole fish were fixed overnight in 4% paraformaldehyde at 4°C, decalcified in a 0.5 M EDTA pH 8.0 solution for 10 days at 4°C and embedded in paraffin. Cross (head) or coronal (body) sections of 4 µm were stained with hematoxylin-eosin (H&E ; eyes and heart) or Periodic acid-Schiff (PAS ; kidney).

Functional analyses

In vitro functional analysis. WNT7B cDNA was cloned into the pCMV-SPORT6 vector (Invitrogen) in frame with a green fluorescent protein (gfp) cassette. Site-directed mutagenesis (Invitrogen) was used to introduce the variants into the WNT7B cDNA. HEK293T cells were plated to 60 to 80 % confluency in 96-well plates and subsequently transfected with plasmids expressing WNT7B (WT or mutant; 75 ng), Renilla luciferase (125 pg), TOPFlash/FOPFlash (25 ng) and LRP5 (25 ng). LRP5 cooperatively activates canonical Wnt signaling with WNT7B. [START_REF] Wang | Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5[END_REF] Luciferase activity was assayed 24 hours after transfection, using a dual-luciferase reporter assay system (Promega).

The level of luciferase activity was calculated as a TOPFlash/FOPFlash reporter ratio excluding the effect of the minimal promoter activity. Renilla luciferase was used as a normalizer. The luciferase activity of the empty vector was set to 1. Results are presented as the mean ± SEM of at least three independent experiments performed in triplicate.

RESULTS

Identification of candidate biallelic WNT7B variants

-Family 1: We studied a female fetus who displayed a phenotype consistent with PDAC syndrome but without any pathogenic variant in STRA6 or RARB (Fig. 1A, Table S2, P1). Briefly, the pregnancy was terminated at 35 6/7 weeks of gestation because of the presence of several abnormalities on the obstetrical ultrasound, including polyhydramnios, bilateral microphthalmia, retrognathia, atrial septal defect, poor cortico-medullary differentiation of the kidneys and a large bladder. Nuchal translucency was 1mm at 12 weeks of gestation. The fetus weighed 2060 grams (10 th centile). The autopsy revealed multiple major abnormalities, including microphthalmia with dysplastic retina (Fig. 2), choanal atresia, agenesis of the larynx and trachea, bilateral pulmonary agenesis, atrial septal defect, diaphragmatic eventration, and hypoplastic kidneys (combined weight: 10.3 grams; reference values: 20 +/-4 grams) with pyelocalyceal dilatation and medullary cysts involving distal and collecting tubules (Fig. 2). Dysmorphic features including tall forehead, frontal hirsutism, large anterior fontanel, small palpebral fissures, flat nasal bridge, microretrognathia. Neuropathological examination did not reveal any gross nor microscopic brain abnormalities. The parents were first cousins and originated from Tunisia.

Because of the parental consanguinity in this family, we analyzed the ES dataset of the affected fetus for rare homozygous variants. We identified 11 variants meeting these criteria (Table S3). Because of the presence of microphthalmia, we next focused our attention on variants in genes associated with eye development. By using the integrated Systems Tool for Eye gene discovery (iSyTE), we found that 2 of these variants (p.(Arg98*) in WNT7B and p.(Arg1281Lys) in TRIOBP)

were expressed during eye development. TRIOBP pathogenic variants have previously been [START_REF] Shahin | Mutations in a novel isoform of TRIOBP that encodes a filamentous-actin binding protein are responsible for DFNB28 recessive nonsyndromic hearing loss[END_REF] , and thus, the highest priority variant was the homozygous nonsense variant in WNT7B (c.292C>T, p.(Arg98*)). Consistent with a recessive mode of inheritance, the c.292C>T variant in WNT7B was further confirmed by Sanger sequencing to be homozygous in the proband and heterozygous in the parents (Fig. 1A).

The WNT7B gene includes 4 exons, which encode a 349 amino-acid growth factor that comprises N-and C-terminal domains connected by a linker (Fig 1B). The c.292C>T variant in WNT7B is absent from public SNP databases (1000 Genomes, EVS, dbSNP138, GnomAD) and from CHU Sainte-Justine Research Center exome dataset (n>1000). It is located in the second exon, which is present in all 5 known WNT7B isoforms. The c.292C>T variant is likely inducing a loss of WNT7B function as it is predicted to abolish the C-terminal part of the protein encoded by exons 3 and 4, which binds to the Frizzled receptors (Fig. 1B). [START_REF] Janda | Structural basis of Wnt recognition by Frizzled[END_REF] Indeed, deletion of exon 3 and 4 results in a null allele in mice. [START_REF] Parr | Wnt7b regulates placental development in mice[END_REF] In addition, the c.292C>T variant may induce degradation of the transcript through the non-sense mediated mRNA decay system triggered upon the association of UPF1 with an Exon Junction Complexe. [START_REF] Kurosaki | Nonsense-mediated mRNA decay in humans at a glance[END_REF] We next used Sanger sequencing to search for rare variants in WNT7B in 17 additional individuals with features of the PDAC syndrome. This screen did not reveal any candidate variant in this gene. We then queried WNT7B in GeneMatcher, a freely accessible website that enables the identification of individuals with variants in candidate genes. We identified three matches for WNT7B, which correspond to the families described hereafter (Families 2, and 3):

-Family 2 is non-consanguineous and from the UK. The proband, a female, was born at 37 weeks of gestation following a pregnancy with late polyhydramnios. She died quickly after birth were major abnormalities of the respiratory and cardiovascular systems with marked pulmonary hypoplasia (combined lung weight = 3.66 g, 10% of that expected), tracheomalacia affecting much of the trachea, and a small proximal trachea-oesophageal fistula. Histology of the left lung showed primarily bronchial structures although the right lung was more appropriate for gestational age.

(Fig 1A,
There was total anomalous pulmonary venous drainage (TAPVD) with an absent right pulmonary vein, a small left pulmonary vein of uncertain course, an absent right pulmonary artery and extremely small left pulmonary artery. There was also a large secundum atrial septum defect and the diaphragms appeared elevated although they were not deficient. Kidneys were hypoplastic with scattered sclerosed glomeruli. The eyes were not examined. Brain displays severe autolysis but no specific abnormalities was identified and prenatal ultrasound scans did not reveal any cerebral anomaly.

The second pregnancy was interrupted at 24 weeks of gestation after ultrasound scan evidenced severe lung hypoplasia with highly placed diaphragms and the heart at the apex of the chest. This fetus (Table S2, P22) displayed retromicrognathia as well as heart and lung anomalies similar to those found in P21, including lung hypoplasia (combined pulmonary weight <10% of that expected), total anomalous pulmonary venous drainage, no pulmonary veins attached to the heart and normal pulmonary arteries. No tracheal abnormality was noted. Kidneys were structurally normal but hypoplastic (weighing 3.26 g combined versus 6.5 +/-2.1 g expected). The eyes showed bilateral microphthalmia (axial length of 9.5 mm) with posterior inferior colobomas in both eyes and a dysplastic retina in the right eye (Fig 3). There was no brain significant abnormality. -Family 3, a healthy consanguineous couple from Tunisia gave birth to two newborn babies who died immediately after birth (at 37 and 39 weeks of gestation, respectively) from respiratory distress and edema (Table S2, P23 and P24). Prenatal ultrasound scans did not reveal any cerebral anomaly.

We have little information regarding these newborns. However, severe lung hypoplasia was documented in both newborns and parietal skull bone softening was observed in one of them. Since the DNA was unavailable from either demised babies, we resorted to a molecular autopsy by proxy approach using ES [START_REF] Shamseldin | Molecular autopsy in maternal-fetal medicine[END_REF] , which revealed that both parents were heterozygous for the p.(Arg98*) variant (Fig 1A). Interestingly, this nonsense variant was also found in Family 1.

The three identified WNT7B variants (c.225C>G, c.292C>T, and c.562G>A) could be classified as pathogenic (class 5) based on the ACMG criteria (Table S4).

Functional validation of candidate variants

In order to demonstrate the deleterious effects of the identified variants on protein function, we conducted in vitro functional studies. We studied the functional impact of the variants on the reflecting more marked instability of the p.Arg89Ter peptide. In vivo, both nonsense variants would be expected to be subjected to nonsense mediated decay, which is not modelled in this assay which uses intronless expression clones. This luciferase assay demonstrates that any transcripts that may escape nonsense mediated decay would not result in functional protein.

Zebrafish mutant lines analysis

We next sought to assess the impact of a loss of wnt7ba and/or wnt7bb on the morphology of adult zebrafish. Compared with wild-type fish, wnt7ba -/-and wnt7bb -/-mutants showed no gross morphological anomalies of the eye, heart and kidney (Fig 5a). However, we observed that the swimbladder of the wnt7bb -/-mutant was formed by a single chamber contrary to the presence of anterior and posterior chambers in the wild-type and wnt7ba -/-counterparts (Fig. 5b). No difference of viability was observed between the wild-type, wnt7ba +/-, wnt7bb +/-, wnt7ba -/-, wnt7bb -/-as well as double heterozygous (wnt7ba +/-;wnt7bb +/-) fish. In contrast, the genotyping of 500 fish from wnt7ba +/-x wnt7bb +/-mating showed the absence of the wnt7ba -/-;wnt7bb -/-genotype at two months of age, suggesting that these two genes have redundant functions for development and/or survival in the zebrafish.

DISCUSSION

We identified rare and likely deleterious biallelic variants in WNT7B in 3 patients with severe lung hypoplasia from two unrelated families. These patients showed a pattern of malformations, including microphthalmia, lung hypoplasia and cardiac defects, that is similar to the PDAC syndrome. Interestingly, we also identified two siblings from a third family (Family 3) whose parents were carrying the same nonsense variant in WNT7B as in Family 1. Although phenotyping of these siblings was limited, both showed severe lung hypoplasia. It thus appears likely that these siblings were homozygous for the nonsense variant, further supporting the association between WNT7B disruption and lung hypoplasia. The fact that such variants were independently found in multiple unrelated families with a similar pattern of malformations strongly suggests a causal link.

WNT7B belongs to a large family of growth factors that regulate diverse aspects of embryonic development by binding to specific receptors on the cell surface. [START_REF] Nusse | Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities[END_REF] In different tissues, WNT7B functions via distinct signaling pathways, including the canonical ß-catenin pathway 9 13 , the non-canonical planar cell polarity pathway [START_REF] Rosso | Wnt signaling through Dishevelled, Rac and JNK regulates dendritic development[END_REF] and the G-protein-linked PKC delta pathway [START_REF] Tu | Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation[END_REF] . In the WNT-ß-catenin pathway, WNT7B binds to a Frizzled receptor and to one of the low-density lipoprotein-related receptor 5 or 6 (LRP5/6), which leads to the stabilization of the transcriptional activator ß-catenin and activation of target genes. Mice with a loss of Wnt7b function die at embryonic day 9.5, before the onset of organogenesis, as Wnt7b is essential for placental development. [START_REF] Parr | Wnt7b regulates placental development in mice[END_REF] Mice homozygous for hypomorphic mutations or for a conditional null mutation induced in cells of the epiblast survive until birth but show severe lung hypoplasia. [START_REF] Shu | Wnt7b regulates mesenchymal proliferation and vascular development in the lung[END_REF] proliferation in both the epithelium and mesenchyme. 17 18 The presence of a similar phenotype of lung hypoplasia in our cases provides some support for the pathogenic involvement of WNT7B.

The swimbladder is an air-filled organ that is a structural homolog of the mammalian lung [START_REF] Torday | Deconvoluting lung evolution using functional/comparative genomics[END_REF] and a suite of genes, including Wnt7b, Nkx2.1, FoxA2, and Gata6, have been shown to be coexpressed during the development of the swimbladder in the zebrafish and of the lung in mice. [START_REF] Rajagopal | Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme[END_REF][START_REF] Cass | Expression of a lung developmental cassette in the adult and developing zebrafish swimbladder[END_REF] We found that the knock-out of wnt7bb, but not of wnt7ba, disrupts the development of the swimbladder, suggesting that WNT7B function has been conserved through evolution, being required for the proper development of the swimbladder in fish and of the lung in mammals. We also found that wnt7ba and wnt7bb functions were redundant in the zebrafish. Detailed characterization of double mutants will be necessary to fully understand the function of these genes in this model. Not only is WNT7B required for the development of the lung in mice but it also plays a role in the development of the trachea, eyes and kidneys, all of which are affected in at least some of our patients. For instance, the fetus from Family 1 showed agenesis of the larynx and trachea, and one fetus from Family 2 had tracheal and bronchi cartilage abnormalities. The trachea of mice with a conditional null allele of Wnt7b appears normal in length and diameter but their cartilaginous rings are incomplete. [START_REF] Rajagopal | Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme[END_REF] There is additional evidence from organotypic culture studies that canonical WNT signaling is required for tracheal chondrogenesis. [START_REF] Snowball | Endodermal Wnt signaling is required for tracheal cartilage formation[END_REF] WNT7/β-catenin signaling has also been demonstrated to be essential for eye development by controlling morphogenesis of the optic cup and patterning of the ocular tissue, promoting the differentiation of the retinal pigment epithelium, and maintaining the dorsal retinal identity. [START_REF] Fujimura | WNT/beta-Catenin Signaling in Vertebrate Eye Development[END_REF] Wnt7b is strongly expressed in the developing retina and lens in mice. [START_REF] Liu | Characterization of Wnt signaling components and activation of the Wnt canonical pathway in the murine retina[END_REF] 15 implicated in the pathogenesis of microphthalmia and anophthalmia. [START_REF] Williamson | The genetic architecture of microphthalmia, anophthalmia and coloboma[END_REF] Studies performed in mice with a hypomorphic variant in Wnt7b showed that macrophage WNT7B is a short-range paracrine signal required for programmed cell death in the vascular endothelial cells of the temporary hyaloid vessels of the post-natal eye. [START_REF] Lobov | WNT7b mediates macrophage-induced programmed cell death in patterning of the vasculature[END_REF] Moreover, WNT7B variants have previously been associated with myopia and central corneal thickness. 26 27 Our work indicates that WNT7B is also required at earlier stages of eye development. Additional studies in animal models will be necessary to dissect this role at the molecular and cellular levels.

Wnt7b also functions in the developing kidney where it is expressed in the future collecting duct network. [START_REF] Yu | A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney[END_REF] In Wnt7b conditional null mice, the cortical epithelial development is normal but the medullary zone fails to develop properly, being characterized by the presence of dilated collecting tubules and the poor growth of the loops of Henle. [START_REF] Yu | A Wnt7b-dependent pathway regulates the orientation of epithelial cell division and establishes the cortico-medullary axis of the mammalian kidney[END_REF] The kidneys of the 3 fetuses in Family 1 and 2 were hypoplastic. In addition, the kidneys of the fetus from Family 1 were characterized by the presence of medullary cysts involving distal and collecting tubules dilatation without any involvement of the cortical domain. Overall, these observations suggest that the function of WNT7B in the developing kidney has been conserved in humans.

WNT7B belongs to a family of 19 genes that play critical roles for the development of multiple organs and systems. Less than half of these genes have been associated with rare human disorders, mainly developmental defects affecting the bones, teeth, and skin. [START_REF] Nusse | Wnt/beta-Catenin Signaling, Disease, and Emerging Therapeutic Modalities[END_REF] By involving WNT7B in the pathogenesis of PDAC syndrome, our study thus broadens the spectrum of human developmental disorders caused by disruption of WNT genes. Pathogenic variants in STRA6 and RARB have previously been shown to cause PDAC syndrome. 2 3 Indeed, the phenotypes associated with the disruption of STRA6, RARB and WNT7B are similar, with the involvement of the lungs and kidneys being more frequent in patients with S5). Both STRA6 and RARB are implicated in retinoic acid signaling. Interestingly, some studies indicate that lung hypoplasia associated with retinoic acid deficiency is mediated at least in part by the disruption of WNT signalling. [START_REF] Chen | A retinoic acid-dependent network in the foregut controls formation of the mouse lung primordium[END_REF] All together, these observations thus raise the possibility that some of the features of PDAC syndrome involve developmental processes that are regulated by an interaction between the retinoic acid and WNT pathways. Further studies, however, will be needed to confirm a functional link between STRA6, RARB and WNT7B. . Histological and X-ray analysis of adults zebrafish. (A) Sections of WT and mutants (wnt7ba -/-/ wnt7bb -/-) zebrafish adult kidneys, hearts and eyes (7-9 months old) were stained with PAS and H&E. Homozygous mutants did not appear to have major abnormalities in kidneys, heart and eyes morphology. (B) X-rays reveal severe anomalies of the swimbladder (absence of the posterior chamber indicated by a star) in the wnt7bb -/-mutant (n=10) but not in the wnt7ba -/- paralog. Abbreviations: CD (Collecting duct) ; G (Glomerulus) ; PT (Proximal tubules) ; DT (Distal tubule) ; V (Ventricle) ; BA (Bulbus arteriosus) ; A (Atrium) ; R (Retina) ; L (Lens) ; C (Cornea) ; I (Iris).

  WES in the first affected patient (P21) in Family 2 revealed the presence of compound heterozygous variants in WNT7B (c.225C>G, p.(Tyr75*) and c.562G>A, p.(Gly188Ser)). Both variants were found by Sanger sequencing in the second affected patient of this family (P22), and https://mc.manuscriptcentral.com/jmedgenet Journal of found to each harbour one of the two identified variants (Fig 1A). The nonsense p.(Tyr75*) is absent from public databases (1000 Genomes, EVS, dbSNP138, GnomAD) and is located in the second exon, as it was also the case for p.(Arg98Ter), found in Family 1. The c.562G>A (p.(Gly188Ser)) is present heterozygous only once among 232162 alleles in GnomAD, and is predicted damaging by several in silico tools (SIFT [score: 0], MutationTaster [p-value:1], Polyphen-2 [HumDiv score: 1; HumVar score: 0.99] and CADD [score:26]). The involved amino acid belongs to a stretch of residues that are conserved in mammals, chicken and zebrafish (Fig 1C).

  -Catenin signaling pathway using a dual luciferase reporter assay. All three WNT7B variants, p.(Tyr75Ter), p.(Arg98Ter), and p.(Gly188Ser) showed reduced WNT7B signalling compared to the wild-type control (P< 0.001), consistent with the proposed deleterious effect of the identified variants (Fig 4). The p.(Arg98Ter) variant WNT7B signaling was lower compared with the p.(Tyr75Ter) variant (P=0.012) and the p.(Gly188Ser) variant (P<0.001), perhaps

Figure 1 .

 1 Figure 1. WNT7B variants identified in this study. (A) Pedigrees of the three families showing segregation of the variants in WNT7B. (B) Localization of the variants with respect to the primary structure of WNT7B. This protein comprises two structural domains, a large N-terminal (NTD) and a C-terminal domain (CTD), which are connected by a linker sequence. (C) Conservation of the Gly188 residue across various species.

Figure 2 .

 2 Figure 2. Histology and immunohistochemical analysis of the eyes and kidneys of patient 1. (A,B) Eye histology. (A) Left eye (H&E, x0,5): Microphtalmia with global retinal detachment. (B) Right eye (H&E, x0,5): Microphtalmia with retinal rings ("rosettes") at the level of optic nerve insertion. (C-H) Renal histology. (C) Right kidney (H&E, x0,5): Cortico-medullar differentiation is preserved in all renicules, although the renal parenchyma is compressed by pyelo-caliceal dilatation. (D) Right kidney (H&E, x2): Number of medullary cysts surrounded by the immature mesenchymal rings. (E): Right kidney (EMA, x0,5): Right kidney showing the diffuse positivity as expected in distal and collecting tubes. (F) Right kidney (EMA, x2): Note positive staining on all medullary cysts. (G): Right kidney (CD10, x0,5): Right kidney showing the diffuse positivity as expected in glomeruli and proximal tubules. (H) Right kidney (CD10, x2): Negative staining on microcysts in medulla.

Figure 3 .

 3 Figure 3. Histology analysis of the eyes of patient 22. (A) H&E (Haematoxylin and eosin) stained section of the right globe. L=lens; R=retina; S=sclera. The arrow points to a gap in the sclera that indicates the posterior coloboma, through which retina is prolapsing. (B) H&E stained section of the right globe. L=lens; R=retina; S=sclera; ON=optic nerve. This section is parasagittal to the scleral coloboma defect in plate A with the arrow pointing to colobomatous extraocular retinal tissue adjacent to the optic nerve. (C) H&E of the intraocular retina, showing the dysplastic rosettes (arrows). (D) H&E higher power of the colobomatous extraocular retinal tissue adjacent to the optic nerve, showing retinal neuronal cell bodies on the left and the looser eosinophilic glial tissue to the right.

Figure 4 . 23 Figure 5

 4235 Figure 4. Impact of the WNT7B variants on WNT7B signalling in a dual luciferase reporter assay. HEK293T cells were co-transfected with pCMVSPORT6/WNT7B (WT or variant), LRP5, TOPFlash/FOPFlash plasmids, and Renilla plasmid. WNT7B signalling was measured 24 h later by a dual luciferase reporter assay. Experiments were done at least 3 times in triplicate and results are expressed as means ± S.E.M. Asterisks above bars indicate results from a Student's t-test for WNT7B variants or the empty pCMV-SPORT6 vector compared to WT WNT7B (*** p < 0.001). All three variants demonstrated reduced WNT7B signalling compared with the wild-type control.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1. WNT7B variants identified in this study. (A) Pedigrees of the three families showing segregation of the variants in WNT7B. (B) Localization of the variants with respect to the primary structure of WNT7B. This protein comprises two structural domains, a large N-terminal (NTD) and a C-terminal domain (CTD), which are connected by a linker sequence. (C) Conservation of the Gly188 residue across various species. 60x81mm (300 x 300 DPI)

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Table S2 ,

 S2 P21). Post mortem revealed a dysmorphic baby with a very broad nasal bridge, a high arched palate and a weight and head circumference within normal limits. Internally there

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  [START_REF] Rajagopal | Wnt7b stimulates embryonic lung growth by coordinately increasing the replication of epithelium and mesenchyme[END_REF] WNT7B appears to regulate lung development via the canonical WNT-β-catenin pathway by reducing

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  [START_REF] Ang | Spatial and temporal expression of Wnt and Dickkopf genes during murine lens development[END_REF] Interestingly, expression of Wnt7b in the developing brain of the mouse has been shown to be regulated by Pax6, which is

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  STRA6 and WNT7B than in those with variants in RARB (Table

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  18were performed and analysed by FH, CN, NP and FSA. Sanger sequencing were performed by SJ, PC, and NC. Zebrafish studies were performed by SBou, SBoi, LFT, JMR, MM, CN, MD, and SE.In vitro analyses were performed by LGR, RG, DW, IP, and RJ. The first draft of the manuscript was written by SBou, JLM and NC and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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	Supplemental Data
	Supplemental Data include five tables.
	C o C o C o Accession numbers The ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) accession numbers for the variants reported C o C o C o
	n f n f n f in this article are: SCV001976485 (c.225C>G); SCV000598632 (c.292C>T); SCV001976486 n f n f n f
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Table S4 :

 S4 Supplementary TableS3: Variants retains after filtering step used to analyse exome results from Patient 1. ACMG classification of WNT7B (NM_058238.2) variations identified in this study Douzgou S, Tremblay A, Michaud JL. Gain-of-Function Mutations in RARB Cause Intellectual Disability with Progressive Motor Impairment. Hum Mutat 2016;37(8):786-93 doi: 10.1002/humu.23004. 3. Srour M, Chitayat D, Caron V, Chassaing N, Bitoun P, Patry L, Cordier MP, Capo-Chichi JM, Francannet C, Calvas P, Ragge N, Dobrzeniecka S, Hamdan FF, Rouleau GA, Tremblay A, Michaud JL. Recessive and dominant mutations in retinoic acid receptor beta in cases with microphthalmia and diaphragmatic hernia. Am J Hum Genet 2013;93(4):765-72

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						Both parents
	C o n f i d e n t i a l : F o r R e v i e M Right hypoplasia, unilobar lungs No Bilateral anophthalmia cardiovascular Complex malformations M Bilateral agenesis Right diaphragmatic hernia Bilateral anophthalmia Absence of pulmonary veins, pulmonary artery hypoplastic Patients retrieved by GeneMatcher P19 P20 P21 F Bilateral severe lung hypoplasia Bilateral diaphragmatic hernia Not examined Large atrial septum defect, total anomalous pulmonary venous drainage C o n f i d e n t i a l : F o r R e v i e w Pyelocalyceal dilation, cystic kidneys Cleft lip and palate, esophageal atresia, polysplenia, hypoplastic kidney Holoprosencephaly, Cleft lip and palate, Large abnormal trachea and main bronchi cartilage, small trachea-oesophageal fistula, hypoplastic kidneys with scattered sclerosed P23 M Bilateral severe lung hypoplasia Not examined Not examined Not examined Skull bone softening P24 M Bilateral severe lung hypoplasia Not examined Not examined Not examined M: Male; F: Female; TOP: termination of pregnancy; IUGR: intra-uterine growth retardation Chr Position Gene symbol Reference cDNA change Protein change OMIM link SIFT score 1 201180439 IGFN1 NM_001164586 c.6418G>A p.(Ala2140Thr) 147660 0.001 (D) 6 170871046 TBP NM_003194 c.232_234del p.(Gln95del) 600075 11 122722431 CRTAM NM_019604 c.224A>G p.(His75Arg) 612597 0.363 (T) 11 125864848 CDON NM_016952 c.2462G>A p.(Arg821His) 608707 0.009 (D) 15 31521505 LOC283710 NM_001243538 c.75_76del 16 11645607 LITAF NM_001136473 c.431C>T p.(Ser144Phe) 603795 0.00 (D) 20 43836872 SEMG1 NM_003007 c.934A>C p.(Ser312Arg) 182140 0.085 (T) 22 38122405 TRIOBP NM_001039141 c.3842G>A p.(Arg1281Lys) 609761 0.000 (D) 22 46345806 WNT7B NM_058238 c.292C>T p.(Arg98Ter) 601967 22 50589218 MOV10L1 NM_018995 c.2782G>T p.(Ala928Ser) 605794 0.053 (T) 4 156135101 NPY2R NM_000910 c.10A>G p.(Ile4Val) 162642 0.94) (T) Polyphen-2 score: B: probably benign, D: probably damaging, d: possible damaging C o n f i d e n t i a Supplementary cDNA change Protein change MAF (GnomAD) In silico predictions* ACMG 1 criteria ACMG classification c.225C>G p.(Tyr75Ter) 0 PVS1 Very Strong PS3 Strong PM2 Moderate Pathogenic c.292C>T p.(Arg98Ter) 0 PVS1 Very Strong PS3 Strong PM2 Moderate Pathogenic c.562G>A p.(Gly188Ser) 0.0004% 11/12 pathogenic PS3 Strong PM2 Moderate PM3 Moderate PP3 Supporting Pathogenic C o n f i d e n t i a C o n f i d e n t i a C o n f i d e n t i a l : F o r R e v i e w l : F o r l : F o r l : F o r R R R glomeruli P22 F Bilateral severe lung hypoplasia No Bilateral colobomatous microphthalmia, Total anomalous pulmonary venous drainage Hypoplastic kidneys e v e v e v i i i dysplastic retina e e e	No No No No O n l y Yes Yes Polyphen-2 score (HumVar) (gnomAD) Sib of P22 Sib of P24 Sib of P23 MAF 0.138 (B) 0.0036 0 0.017 (B) 0.000036 0.991 (D) 0.0006246 0.0001323 0.071 (B) 0.0007910 0.02 (B) 0.002329 0.967 (D) 0 0 0.874 (d) 0.0001047 0 (B) 0.00002 O n l y No Sib of P21	No No No p.(Tyr75Ter) p.(Gly188Ser) carrying the p.(Arg98Ter) variation Both parents carrying the p.(Arg98Ter) variation Number of homozygous (gnomAD) 1 0 0 1 2 14 0 0 0 0 p.(Tyr75Ter) p.(Gly188Ser)
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Acknowledgments

We acknowledge the generous support from the families published in this article.

Funding

This work was supported by grants from the Fondation Jeanne et Jean-Louis Lévesque (JLM), Fondation Maladies Rares (NC), Retina France (NC, LFT), NHMRC Grant 1099165 (RVJ) and Costco and Luminesce Alliance (LGR). We acknowledge Nicolas Fossat for assistance with vector cloning. We thank the Sequencing and Genotyping Core Facilities at KFSHRC for their technical help. The authors extend their appreciation to the King Salman Center For Disability Research for funding this work through Research Group no RG-2022-011. French patients were recruited through the Rares Diseases Cohorts (RaDiCo) program which is funded by the French National Research Agency under the specific program "Investments for the Future", Cohort grant agreement ANR-10-COHO-0003.

Competing interests

None

Ethics approval statement

Research has been performed in accordance with the Declaration of Helsinki. This study was approved by the CHU Sainte-Justine, by the Sud-Ouest and Outre-Mer II and by the King Faisal Specialist Hospital (KFSRHC RAC#2070023) ethics research boards. Informed consent was obtained from all participants or their legal guardians.

Contributorship Statement

FSA, JLM, RJ and NC contributed to the study conception and design. Patients recruitment and clinical evaluations were performed by NAA, JB, MAA, JM, SH, HSL, TAB, PC and NC. WES Supplementary Table S1: Primers used for WNT7B Sanger sequencing The most frequent features are indicated in bold.