Jarek Wosik

Wei Chen

Kuang Qin

Rafik M Ghobrial

Jacek Z Kubiak

Malgorzata Kloc

Magnetic Field Changes Macrophage Phenotype

Keywords: macrophages, magnetic field, RhoA knockout, cytoskeleton, chronic rejection, mechanotransduction, magnetic force

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INTRODUCTION

Macrophages have several functionally different phenotypes/subtypes. M0 macrophages are naïve/unpolarized macrophages. One of the most common subtypes of activated macrophages are M1 proinflammatory "killer" macrophages, which produce damaging reactive oxygen species and express nitric oxide synthase iNOS, and M2 anti-inflammatory "repair" macrophages, which produce enzyme Arginase-1 that depletes L-arginine and deprives iNOS of its substrate [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF][START_REF] Liu | ROCK inhibition impedes macrophage polarity and functions[END_REF][START_REF] Liu | Dissonant response of M0/M2 and M1 bone-marrow-derived macrophages to RhoA pathway interference[END_REF][START_REF] Liu | Macrophage/monocyte-specific deletion of Ras homolog gene family member A (RhoA) downregulates fractalkine receptor and inhibits chronic rejection of mouse cardiac allografts[END_REF].

Macrophages, like all eukaryotic cells, contain an actin-filament cytoskeleton. Macrophage migration occurs via dynamic rearrangements of actin filaments. Our recent studies showed that pharmacologic or genetic interference with the small GTPase RhoA pathway, which is the master regulator of actin, causes extreme elongation of macrophages (hummingbird phenotype), disrupts the Golgi/endosomal pathway, prevents macrophage migration into the graft (through the clustering of the CX3CR1 receptor) and inhibits chronic rejection in the rodent model system (4-7).

Here we were interested in finding out if an external magnetic field, in conjunction with transduction processes, could induce cytoskeletal rearrangements in macrophages and change their shape and molecular and organellar phenotype.

It is already known that external mechanical force applied to the cell has a direct impact and can affect the cell cytoskeleton [START_REF] Ghibaudo | Substrate Topography Induces a Crossover from 2D to 3D Behavior in Fibroblast Migration[END_REF][START_REF] Ladoux | Physically based principles of cell adhesion mechanosensitivity in tissues[END_REF]. It is also known that nonuniform magnetic fields can create such magnetic force-driven stimuli. The cell responds to the external stimuli by remodeling the cytoskeleton, which is visco-elastic and provides a continuous mechanical coupling throughout the cell as it changes. This, in turn, induces an internal cell stress and changes in certain cellular components and components such as actin-filament polymerization, focal adhesions, etc. Such conversion of mechanical forces to biochemical interactions is referred to as mechanotransduction. There are reports that changes in ion-channel activity at the plasma membrane of cells may convey mechanical stresses from the cell membrane to internal organelles, causing changes in gene transcription and inducing apoptosis [START_REF] Janmey | The cytoskeleton and cell signaling: component localization and mechanical coupling[END_REF]. Other reports show that pathways of mechanically induced cell damage can include activation of the Caspase-3 protease pathway [START_REF] Liu | Cyclic-stretch induces the apoptosis of myoblast by activation of Caspase-3 protease in a magnitude-dependent manner[END_REF] and TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) [START_REF] Oh | Role of elevated pressure in TRAIL-induced apoptosis in human lung carcinoma cells[END_REF] and also cleavage of Caspases 3 and 9 [START_REF] Boccafoschi | Overstressed mechanical stretching activates survival and apoptotic signals in fibroblasts[END_REF].

In spite of outstanding recent progress in research of the influence of electromagnetic fields on the biology of cells and the expanding use of magnetic materials in biomedical applications, surprisingly little is known about the influence of a magnetic field at the cellular level [START_REF] Miyakoshi | Effects of static magnetic fields at the cellular level[END_REF][START_REF] Murabayashi | Application of magnetic field for biological response modification[END_REF].

The nature and strength of interactions of electromagnetic fields with cell or tissue mainly depend on electric-and magnetic-field-produced polarizations. The ability to induce such polarizations is measured by electric and magnetic-field susceptibilities. There are significant differences between interactions of both fields with cells/tissue, because for a typical tissue the electric susceptibility is 10 5 -10 6 times larger than the magnetic-field susceptibility and, as a result, the presence of the electric field can cause significant cell/tissue damage, whereas magnetic-field interactions with cell/tissue are relatively weak.

There are mixed reports about the influence of magnetic fields on cell growth and functions, and most but not all studies suggest that there is no obvious observable effect, even at as high as 10 T or higher values of uniform magnetic fields. In addition, although such fields in some studies have been shown to affect cell differentiation and viability; they did not have longlasting, damaging effects [START_REF] Valiron | Cellular disorders induced by high magnetic fields[END_REF]. Furthermore, the nonuniform magnetic fields, in contrast to the uniform fields, were proven to generate sufficiently large magnetically induced mechanical forces able to affect cell morphology, differentiation and functionality [START_REF] Qian | Large gradient high magnetic fields affect osteoblast ultrastructure and function by disrupting collagen I or fibronectin/αβ1 integrin[END_REF][START_REF] Shi | Effects of microgravity modeled by large gradient high magnetic field on the osteogenic initiation of human mesenchymal stem cells[END_REF]. As a result a few in vitro studies carried out recently for spatially modulated magnetic fields showed a clear magnetic field-cell interaction [START_REF] Kauffmann | Diamagnetically trapped arrays of living cells above micromagnets[END_REF][START_REF] Denegre | Cleavage planes in frog eggs are altered by strong magnetic fields[END_REF][START_REF] Winkleman | A magnetic trap for living cells suspended in a paramagnetic buffer[END_REF]. Although there are many technical challenges to generate sufficiently high fields and gradients, recent progress in fabrication of strong permanent magnets based on rare-earth elements and also use of patterned magnetic microstructures have allowed generation of highly nonuniform magnetic fields, resulting in local gradients' magnitudes reaching up to extremely high, such as 10 6 -T/m, values [START_REF] Zablotskii | Life on magnets: stem cell networking on micro-magnet arrays[END_REF].

In these studies, we have designed a permanent rare-earth magnet setup with well-defined magnetic fields and field-gradient patterns; and we have investigated the effect of these fields and results from the presence of magnetic force on cultured, unpolarized/naïve mouse peritoneal macrophages.

MATERIAL AND METHODS

Magnetic field/field-gradients generation

Experiments with cultured macrophages were carried out, using a pair of permanent neodymium-iron-boron (NdFeB) flat planar magnets to generate the desired magnetic-field patterns. The magnets' dimensions were 5 mm by 10 mm by 1.9 mm, and they were zinc-plated and axially magnetized in a direction perpendicular to the 5-mm by 10-mm plane (Digi-Key, ND105236). Two combinations of the magnetization direction were selected for the planar alignment of two identical magnets: a parallel NS-NS and anti-parallel NS-SN configuration.

The adjacent surfaces of the magnets were highly polished and, by using custom-made nonmagnetic tools, they were framed together with no gaps left between their surfaces. The magnets used in experiments had the following parameters: remanence magnetization B r (min) 1.24 T, coercivity H cB (induction curve) 950 kA/m, and coercivity H cJ (polarization curve) 1750 kA/m. A fiber-epoxy G-10 material was used to make a holder for the chamber slide containing macrophages in culture medium and generating a magnetic-field NdFeB magnet structure. A 0.1mm-thick cover glass separated the macrophages from the magnets. A custom-made chamber slide containing macrophages in culture medium was placed atop the NdFeB magnets. The replacement of a 1-mm-thick standard microscope slide with a much thinner (0.1-mm thick) cover glass allowed us to decrease the distance between the magnetic structure and the macrophages, which settle and grow on the bottom surface of the chamber.

Magnetic-field simulations of the magnetic field, magnetic-field gradient and magnetic-force distribution were carried out to analyze the forces exerted by the gradient field on the macrophages. All calculations were performed using the magnetic-fields module of COMSOL Multiphysics 5.0, an interactive full-wave, finite element-based software package [START_REF] Multiphysics | User's guide[END_REF]. AC/DC and Mathematics software modules were employed for the calculations. Also, Ansoft (Maxwell) similar numerical method software was used for some simulations. The magnets' geometry, material parameters, and field boundary conditions were selected according to the experimental magnet configuration and used magnetic materials. The magnetization strength of the 2 permanent magnets used in experiments was defined through their known remanence and coercivity values. The geometry of the magnets' configuration was modeled so as to be very close to the experimental setup dimensions, as shown in Figures 1 and2.

The magnetic field is calculated in a magnetostatic configuration. The magnetic-field gradient is obtained by taking the derivative of the z-component of the B-field. As the last step, from the formula , the magnetic field was calculated, using the field calculator option in the Maxwell software.

To keep magnets as close as possible to the cells, we modified chamber slides, replacing the existing microscope slide with a much thinner microscope cover glass. The 1-well chamber slides were modified as follows: the chamber was detached from the microscope slide, and all silicone was removed. The indented edges of the chamber were coated, using the wooden end of a Q-tip, with Dow Corning high-vacuum grease; and the chamber was firmly attached to the 24x60-mm microscope cover glass. The thusly constructed chamber slide was placed in a holder containing magnets (or without magnets present for control purposes) and sterilized with 70% ethanol in a cell-culture hood for 3 days before use. Just before seeding the macrophages, we removed the ethanol and rinsed the chamber slides 3 times with sterile PBS and once with Modified Eagle Medium.

Animals and peritoneal macrophages

Generation of RhoA-deficient Lyz2 Cre+/-RhoA flox/flox mice was described previously [START_REF] Liu | Dissonant response of M0/M2 and M1 bone-marrow-derived macrophages to RhoA pathway interference[END_REF]. Committee and animal protocol AUP-0317-0006 (IS00003962), entitled, "Tolerance Induction in a Rodent System" was used. Mice euthanasia was performed according to the TMHRI Euthanasia of Rodents Procedure, by isoflurane overdose via a vaporizer inhalation, followed by cervical dislocation and thoracotomy to ensure death.

Peritoneal macrophages were collected in PBS from the peritoneal cavity of euthanized C57BL/6 (wild-type) or RhoA-deficient Lyz2 Cre+/-RhoA flox/flox (RhoA KO) mice. The collected cell suspension was centrifuged at 1700 RPM for 5 min, supernatant was discarded, and the cells' pellet was resuspended in Dulbecco Modified Eagle Medium supplemented with 10% FBS, 100U/mL penicillin and 100ug/mL streptomycin. The macrophages from C57BL/6 mice were seeded in the magnets' chamber slides. For control, the C57BL/6 macrophages were grown on slides without magnets. The macrophages from RhoA KO mice were seeded only on chamber slides without magnets.

After overnight incubation at 37° and 5% CO 2, the medium was replaced with fresh medium.

After another overnight incubation, macrophages were fixed in chamber slides.

Fixation, actin, vinculin, Golgi and TRPM2 staining

Macrophages were fixed in chamber slides in 1% formalin in 1 x PBS with 0.05% Triton for

Western blotting

Macrophages collected from chamber slides were pelleted for 5 min at /3200-rpm centrifugation, resuspended in 1× loading buffer with Halt™ Protease Inhibitor Cocktail and boiled for 5 min. The lysates were resolved on SDS-PAGE and blotted (Trans-Blot® Turbo™)

to an LF PVDF membrane. Blots were blocked in 5% fat-free milk in TBST at room temperature for 2 hrs. Subsequently blots were incubated with primary antibodies (GAPDH, Cell Signaling

Technology at 1:1000 dilution, Arg-1, R&D systems, at 1:5000 and iNOS, eBioscience, at 1:5000 dilution) in blocking solution, overnight at 4°. After washing 3 times (20 min each wash) in TBST, blots were incubated with 1:5000 dilution of secondary antibodies (Goat anti rabbit HRP, Santa Cruz, Rabbit anti-Sheep IgG (H+L) HRP, Invitrogen™, and Goat antirat HRP, Santa Cruz), washed 3 times (20 min each wash) in TBST; and protein bands were visualized on X-ray film, using GAPDH-SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher) or Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore).

RESULTS

Magnetic-field generation

Two different configurations of each pair of the magnets were used for experiments: NS-NS and NS-SN, where N and S are north and south magnetic poles, respectively. However, results presented in this article, for consistency, were selected only for the NS-SN configuration. In this configuration, due to a much higher gradient above the interface between the 2 magnets, a proportionally stronger influence of the magnetic field compared with the parallel configuration was observed. In Figure 1, 2D simulations of the magnetic field, magnetic-field gradient and magnetic force are shown.

Calculations were done for the NS-SN configuration, along the y-axis. Simulations presented in Figures 1A, 1B and 1C indicate that, above the interface of the 2 polarized in opposite directions magnets, there is a very steep gradient due to a large change of field magnitude along only a 200-300-m distance. Also, steep gradients exist on both magnets' edges. A plot of mechanical force acting on the macrophages placed above the magnets is shown in Figure 1D. It can be seen that the force distribution map follows the gradient magnitude change.

Figure 2 shows the locations of the most characteristic areas above magnets, where highly elongated and magnetic-field-unaffected macrophages were observed.

For the elongated case, 2 such locations were selected, the first above the magnets' interface, where the maximum gradient was created, and the second close to the magnet corners along the diagonal line of the whole structure. In the first and second locations, the longest macrophages and waves of long-range, aligned, elongated macrophages were observed. The calculated distribution of the magnetic field-induced-force component present above the magnets is also marked.

Analysis of macrophage phenotype

For our experiments isolated peritoneal M0 macrophages from C57BL/6 mice were seeded on modified chamber slides with the cover glass bottom placed on magnets (see Material and Methods) and grown for 48 hr. For control the C57BL/6 macrophages were grown on slides without magnets. Subsequently, macrophages were fixed; stained for actin, vinculin (focal adhesion marker) and Golgi complex with GM130 protein Golgi marker; and analyzed by fluorescence microscopy. The morphology of macrophages and Golgi and vinculin staining were compared to that of RhoA-deleted macrophages, which were grown without magnets.

Phenotype of macrophages exposed to a magnetic field

When we looked at distribution of actin-stained macrophages on the slides, we found that, while macrophages grown without magnets had uniform distribution on the slide surface (Figure 3A, B), the macrophages grown on magnets were aligned in distinct rows or waves and extremely elongated within these rows (Figure 3C,D,E).

The calculation of the number of elongated macrophages within 2 mm 2 areas (from 5 different magnet setups) within the rows shows that there is a statistically significant difference in the number of elongated macrophages counted on the magnet and outside the magnet (Figure 3F).

Analysis of macrophage morphology showed that macrophages grown in control chambers without magnets were slightly elongated, with an average length around 50 m (Figure 3A,B and Figure 4B). In contrast, macrophages grown on magnets had an extremely elongated tail and average length > 150 m (Figure 3E, F; Figure 4 A,C). A degree of this extreme elongation was very similar to the elongation of the hummingbird phenotype macrophages with deleted RhoA (Figure 4 A,D).

Analysis of focal adhesions and Golgi complex in magnetic field-exposed macrophages

We showed previously that the hummingbird phenotype of RhoA-deleted macrophages was caused by inability to disassemble vinculin-rich focal adhesions in the macrophage tail [START_REF] Liu | Dissonant response of M0/M2 and M1 bone-marrow-derived macrophages to RhoA pathway interference[END_REF]. To see if a similar mechanism was responsible for elongation of macrophages grown on magnets, we immunostained them with anti-vinculin antibody.

Figure 5 shows that in control macrophages vinculin-positive staining was concentrated in the macrophage body, around the nucleus and in the area occupied by the podosomes (Figure 5A-C).

In magnet-grown macrophages vinculin staining was present in the macrophage body; in the podosome area; and, surprisingly, also in the nuclei (Figure 5 Golgi staining showed that in control macrophages the Golgi complex was very compact and situated in close vicinity to the nucleus (Figure 6A). In contrast, macrophages grown on magnets showed vesicular GM130 staining in the cytoplasm of the cell body and complete absence of a defined Golgi complex (Figure 6B). In comparison, in the RhoA-deleted macrophages Golgi staining was highly diminished, but some of the staining was still present in the vicinity of the nucleus (Figure 6C).

All these observations indicate that the applied magnetic field had a profound effect on the macrophage actin cytoskeleton and actin-dependent molecules and structures, such as vinculin (focal adhesions) and the Golgi complex.

Molecular-marker expression in magnetic field-exposed macrophages

Western blot analysis with anti-iNOS and anti-Arg-1 antibodies, which are the markers of M1 and M2 macrophage subtypes, respectively, showed that, while control and magnet-grown M0 macrophages did not express the M1 marker iNOS protein, the expression of the M2 marker Arg-1 was highly upregulated in magnet-grown MO macrophages (Figure 7).

This indicates that a magnetic field induced both morphological (elongation) and molecular changes in M0 macrophages toward the M2 (anti-inflammatory) phenotype. Although Arg-1 is the signature marker of M2 macrophages, a low level of Arg-1 expression in MO macrophages is quite common because, although these macrophages are theoretically nonactivated, they often show, depending on the mouse state and environment, some degree of activation.

Cation channel distribution

The TRPM2 (the cation channel transient receptor potential melastatin 2/Transient Receptor Potential Cation Channel Subfamily M Member 2) is a Ca 2+ -permeable cation channel that belongs to the ion transport protein family. Studies in the gastric inflammation mice model showed that TRPM2-deficient macrophages have disrupted Ca 2+ homeostasis and are unable to control intracellular calcium levels, which results in calcium overloading [START_REF] Kashio | Redox signal-mediated sensitization of transient receptor potential melastatin 2 (TRPM2) to temperature affects macrophage functions[END_REF][START_REF] Beceiro | TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection[END_REF]. Our preliminary data show that macrophages exposed to magnetic field gradient cluster TRPM2 in the vicinity of the nucleus (Figure 8).

Such a clustering may render the channels nonfunctional and disrupt normal Ca 2+ homeostasis.

Because it is known that actin polymerization and organization are Ca 2+ -dependent, such a disruption may cause changes in the macrophage actin cytoskeleton and actin-related functions.

In summary, we showed that the exposure of macrophages to the magnetic field causes macrophage elongation and disrupts actin-dependent molecules and structures, such as the Golgi complex, vinculin (focal adhesions) and receptors. These changes mimic the changes caused by macrophage-specific deletion of RhoA (Figure 9).

DISCUSSION

Two possible forces acting on macrophages can be defined: (i) the Lorentz force, as sketched in Figure 10A, and (ii) the susceptibility buoyance volume force (from Archimedes' principle)

acting on a cell suspended in a buffer medium and subjected to a nonuniform magnetic field, as is shown in Figure 10B.

The Lorentz force will be created due to the magnetic-field interaction with ionic currents in the membrane. Such force will be present in both uniform and nonuniform (inhomogeneous) fields. There are reports about the influence of static magnetic fields on cells where modified cell shape, structural changes in plasma membrane and increased level of intracellular Ca 2+ have been observed. One example of such changes was reported by Chionni et al., [START_REF] Chionna | Cell shape and plasma membrane alterations after static magnetic fields exposure[END_REF] where lymphocytes and U937 cells in the presence or absence of apoptosis-inducing drugs were strongly affected by the presence of a static magnetic field.

The second force leading to interaction of a static magnetic field with cells is caused by the difference between physical characteristics between cells and the cell medium. From Archimedes' principle in the presence of a magnetic -field gradient, there will be force acting on cells. The magnetic energy experienced by a cell placed in a magnetic field can be written as ,

where is the dipole moment, with  and V representing the susceptibility and volume of the cell, respectively, and μ 0 being the permeability of free space equal to 4π x 10 7

H/m. Since the volume of a biological cell is relatively small, we assume that the magnetic field and susceptibility are constant over the cell volume. Rewriting the magnetic energy equation as , and using the magnetic-force equation, defined as , results in the following formula:

(2)

As can be seen from this equation, the magnetic force is dependent on the product of the magnetic induction (in T units) with the magnetic-field gradient (in T/m). Since it is directly proportional to the product of the two, it is often referred to as the "force product"; and it is given in T 2 /m units. This force is a result of magnetic buoyancy and the diamagnetic repulsive forces. The close-to-interface gradient magnitude is estimated as 10 4 T/m, and it is relatively large, but smaller than gradients achieved for periodically patterned structures [START_REF] Kauffmann | Diamagnetically trapped arrays of living cells above micromagnets[END_REF][START_REF] Zablotskii | High-Field Gradient Permanent Micromagnets for Targeted Drug Delivery with Magnetic Nanoparticles[END_REF][START_REF] Gassner | Magnetic forces produced by rectangular permanent magnets in static microsystems[END_REF].

The nature and strength of interactions of electromagnetic fields with cell or tissue mainly depend on electric-and magnetic-field-produced polarizations. The ability to induce such polarizations is measured by electric and magnetic-field susceptibilities. There are significant differences between interactions of both fields with cells/tissue, because for a typical tissue the electric susceptibility is 10 5 -10 6 times larger than the magnetic-field susceptibility; and, as a result, the electric-field presence can cause significant cell/tissue damage, whereas magnetic field interactions with cell/tissue are relatively weak. Cells are mostly diamagnetic and, in principle, when suspended in a nonmagnetic medium, they are expelled by the magnetic field.

However, buffer medium can be either dia-or paramagnetic; and in such a case the response of cells to the presence of a magnetic field [START_REF] Denegre | Cleavage planes in frog eggs are altered by strong magnetic fields[END_REF][START_REF] Eguchi | Cleavage and survival of Xenopus embryos exposed to 8 T static magnetic fields in a rotating clinostat[END_REF] mainly depends on the difference between susceptibilities of the cell and medium. Most biological cells are weakly diamagnetic. The force direction will depend on the sign of the magnetic susceptibility difference . For positive ,

U = V cB 2 / 2m 0 F(x, y, z) = ÑU B 16
cells will be attracted to the highest gradient lines; whereas for the negative sign, repulsion will take place. Our experiments showed that cells are attracted to the highest gradient line (as defined in Figure 10), clearly indicating that macrophages are more diamagnetic than medium.

In Figure 11 we present a plot of the magnetic force calculated for two reverse-polarized (NS-SN) magnets as a function of the x, y position. A good correlation between the calculated directions of magnetic field-induced forces above magnets and experimentally observed alignment of elongated macrophages is shown. Two characteristic locations were selected: the first one above the maximum of the gradient lines between two magnets and the second one close to the magnet corner, where two force components perpendicular to each other exist. These forces are related to the magnets' edge-introduced gradients. Elongated macrophages were observed along the maximum gradient lines and along a diagonal line very close to corners.

The cellular response to external mechanical stimuli results in several induced internal forces, and such forces for the actin network are believed to be in the range of 10 pN to a few nN [START_REF] Giardini | Compression forces generated by actin comet tails on lipid vesicles[END_REF][START_REF] Noireaux | Growing an Actin Gel on Spherical Surfaces[END_REF]. One other force is the adhesion force exerted by the cell to the substrate through focal adhesion, and the magnitude of this force is in the range of 1-100 nN [START_REF] Balaban | Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates[END_REF][START_REF] Stricker | Spatiotemporal Constraints on the Force-Dependent Growth of Focal Adhesions[END_REF]. A single protein stretching is reported to require 2-10 pN [START_REF] Grashoff | Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics[END_REF], whereas a single stress fiber stretching needs a larger force of 10 nN [START_REF] Deguchi | Tensile properties of single stress fibers isolated from cultured vascular smooth muscle cells[END_REF][START_REF] Zablotskii | Effects of high-gradient magnetic fields on living cell machinery[END_REF]. High magnetic-field gradients interacting with diamagnetic materials cause an effect analogous to microgravity [START_REF] Beaugnon | Levitation of organic materials[END_REF][START_REF] Kuznetsov | Intracellular magnetophoresis of amyloplasts and induction of root curvature[END_REF][START_REF] Yamaguchi | Magnetic Field Effects on Materials: Fundamentals and Applications[END_REF] and produce forces of an order of pN. Estimation of magnitude of such forces in our system is in the same range.

The nonhomogeneous magnetic field generated by our magnets was sufficiently high that the resulting magnetic force applied to peritoneal mouse macrophages caused extreme elongation (hummingbird phenotype) and acquisition of the anti-inflammatory M2 phenotype. This elongated M2-like phenotype mimics the phenotype resulting from the pharmacological (Y27632 inhibitor) or genetic (macrophage-specific deletion of RhoA) interference with the RhoA pathway (4-7). This suggests that application of a magnetic-field gradient may potentially replace the RhoA interference approach to change macrophage phenotype, functions and migration.

Although the elongated phenotype of RhoA-deleted and magnet-grown macrophages is very similar, the mechanism of the extreme elongation in magnetic-versus RhoAinterference seems to be different. In the RhoA interference situation, the elongated macrophages permanently accumulated vinculin-rich focal adhesions in the tail, which caused inability of the tail to detach from the substrate while the front of the macrophage was moving forward [START_REF] Liu | Macrophage/monocyte-specific deletion of Ras homolog gene family member A (RhoA) downregulates fractalkine receptor and inhibits chronic rejection of mouse cardiac allografts[END_REF]. In contrast, the magnetic interference did not result in accumulation of focal adhesions in the tail. Surprisingly, magnetic-field exposure caused aggregation of vinculin in macrophage nuclei. It is known that the cell nucleus contains a pool of nuclear actin that participates in regulation of chromatin status and gene transcription. Thus, it is possible that magnet-induced changes in actin distribution affect not only cytoplasmic but also nuclear actin and by doing so also influence actin-binding molecules such as vinculin, causing its influx into the nucleus. In addition, the effect of a magnetic field on macrophage Golgi differs from the effect of RhoA deletion. Magnetic interference seems to cause dispersion of Golgi staining, while the RhoA-deletion highly reduces Golgi staining. Thus, further studies are needed to establish what cytoskeletal changes are responsible for extreme macrophage elongation during magnetic interference and how they affect subcellular architecture and organelles.

Our preliminary hypothesis is that magnetic-field forces have changed the ionic currents and/or distorted the macrophage membrane. Our preliminary results indicate that magnet-exposed macrophages cluster the cation channel receptor TRPM2. Receptor clustering may render it nonfunctional, which disrupts Ca +2 homeostasis. This, in turn, will affect actin polymerization (which is ion current dependent) and lead to macrophage elongation. Similar clustering of the CX3CR1 receptors (which direct macrophages to the transplanted organs) was observed in RhoA-deleted macrophages) (4-7). Our previous studies showed that, under control conditions, polarization of the M0 macrophage toward the M2 phenotype is accompanied by macrophage elongation, which in turn induces an M2-specific geneexpression pattern [START_REF] Liu | Mouse macrophage polarity and ROCK1 activity depend on RhoA and nonapoptotic Caspase 3[END_REF][START_REF] Liu | ROCK inhibition impedes macrophage polarity and functions[END_REF][START_REF] Liu | Dissonant response of M0/M2 and M1 bone-marrow-derived macrophages to RhoA pathway interference[END_REF][START_REF] Liu | Macrophage/monocyte-specific deletion of Ras homolog gene family member A (RhoA) downregulates fractalkine receptor and inhibits chronic rejection of mouse cardiac allografts[END_REF]. This suggests that, when a macrophage is forced, by application of a magnetic field, to elongate, it will switch on the expression of M2-specific genes such as Arg-1.

We also observed that macrophages were aligned according to the magnetic forces' pattern, as shown in Figures 10 and11. We believe that the rotational magnetic-field forces, due to high diamagnetic anisotropy of elongated macrophages, are responsible for such behavior. In addition, we observed that macrophages were arranged in distinctive rows/waves, in which macrophages closely followed each other. The wound-healing studies showed that fibroblasts form the rows of cells with special ''leader'' cells that drag a column of "follower" cells behind them [START_REF] Omelchenko | Rho-dependent formation of epithelial "leader" cells during wound healing[END_REF][START_REF] Gov | Collective cell migration patterns: follow the leader[END_REF]. These studies showed that the leader cell, which is the first cell that had elongated, reorganized the actin cytoskeleton. These cytoskeletal changes in the leader cell induced cytoskeletal changes and directional movement of the followers. It is possible that a similar mechanism is responsible for the creation of macrophage long rows/waves in response to the magnetic-field force.

The cellular cytoskeleton undergoes extensive rearrangements, not only in healthy cells but also in cancer cells. The deregulation of cytoskeleton structure in many types of human cancer is responsible for the increased divisions and migratory activity of tumor cells and is linked with poor patient outcome. Thus, a magnetic field-initiated transduction technique of introducing local mechanical forces on cells, while developed for macrophages can be adapted and implemented for cancer cells.

Making the surrounding medium/tissue more paramagnetic can increase magnetic force acting on cells. Magnetic resonance imaging contrast agents such as paramagnetic gadolinium can be used to increase magnetic force on cells due to its paramagnetism [START_REF] Winkleman | A magnetic trap for living cells suspended in a paramagnetic buffer[END_REF]. Correlation between biochemical cell processes and cell deformation induced by forces generated by a magnetic-field gradient will lead us to a better understanding of the magnetic-field interaction with the cells.

Figure 2. Experimental setup.

A sketch describing an experimental configuration of two permanent magnets covered by a very thin glass plate is presented. Three of the most characteristic areas above the magnets, where highly elongated and magnetic-field unaffected macrophages were present, are marked as #1, #2 and #3 rectangular shapes, respectively. #1 refers to the interface where the maximum of the gradient was created, #2 marks an area close to the magnet corners along the diagonal line of the whole structure. #3 marks an area outside of the magnetic field. A calculated distribution of the magnetic field-induced force component present above the magnets is also included here.

  Breeding and all experiments were performed according to Methodist Hospital Research Institute's animal care protocol and met NIH standards in concordance with the "Guide for the Care and Use of Laboratory Animals" (DHHS publication No. (NIH) 85-23 Revised 1985), the PHS "Policy on Humane Care and Use of Laboratory Animals" and the NIH "Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training." All studies carried out here were approved by Houston Methodist Institutional Animal Care and Use

  30 min at room temperature, washed 3x15 min with PBS-0.05% Tween 20, and blocked overnight at 4 º C in Casein blocker in PBS (Biorad) with 0.05% Tween 20. Subsequently, macrophages were incubated with 1:200 dilution of anti-vinculin-FITC conjugated antibody (Sigma, USA), anti-GM130 (Golgi marker) antibody (Biorbyt or Sigma), anti-TRPM2 antibody ThermoFisher, USA) and rhodamine-phalloidin (Molecular Probes, Eugene, OR; 3 L of methanolic stock solution of 200 U/mL per 200 L of blocking buffer) in casein blocking buffer, in the dark, at room temperature for 3 hr. After washing, the GM130-and TRPM2-bound macrophages were incubated with FITC-conjugated secondary antibodies in blocking solution for several hours. Next, macrophages were washed 3 times, 1 hr each, in PBS-0.05% Tween 20, at room temperature, in the dark, mounted in Antifade with Dapi (Molecular Probes) and observed with a (wide-field nonconfocal) Nikon fluorescent microscope.

  F-I), but in contrast to the RhoAdeleted macrophages (Figure5D, E), was absent in the tail (Fig.5 H, I). Calculation of the number of cells with the nuclear vinculin showed that in magnet-exposed macrophages 95 of the total 136 counted had vinculin present in the nuclei. In contrast, only 2 control macrophages of a total 119 counted had vinculin in the nuclei. The extreme elongation of magnet-grown macrophages and the disruption of their vinculin localization indicate the disruption of the normal actin cytoarchitecture. Because one of the organelles whose organization depends on actin is the Golgi complex, we also stained macrophages with Golgi marker anti-GM130 protein antibody.

Figure 1 .

 1 Figure 1. Magnetic field and magnetic field-induced mechanical force patterns. (A) Theoretically calculated magnetic induction vector component B z in the YZ plane; (B) magnetic-

Figure 3 .

 3 Figure 3. Distribution and phenotype of macrophages on magnets.Distribution and phenotype of M0 macrophages grown on control (A, B) and magnet (C, D, E) slides are shown. Macrophage actin is stained with rhodamine-phalloidin (red). Macrophages grown on slides without a magnetic field applied exhibit roundish or only slightly elongated shapes (A, B). Grown-onmagnets macrophages are significantly elongated, some with an unusually thin tail and overall length

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 4. Hummingbird phenotype of magnet-exposed and RhoA-deleted macrophages.Macrophages stained with rhodamine-phalloidin (red) to visualize actin. (A) Graph shows that the average length of elongated magnet-exposed and RhoA-deleted macrophages (grown without magnets) is very similar; the length differences are statistically nonsignificant (P=0.8425). Control macrophage (B) is slightly elongated, while magnet-grown (C) and RhoA-deleted, grown without magnets (D) macrophages

Figure 7 .

 7 Figure 7. Macrophage markers expression.Western blot analysis of M1 (inflammatory) and M2 (anti-inflammatory) markers' expression with anti-iNOS and anti-Arg-1 antibodies. The control and magnet-grown (Mag 1, Mag 2) M0 macrophages did not

Figure 8 .

 8 Figure 8. TRPM2 distribution in macrophages. Macrophages were stained with rhodamine-phalloidin (red) for actin and with anti-TRPM2 antibody and FITC-conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). (A, B) The control macrophage shows uniform distribution of TRPM2 (arrows) in the cytoplasm of the macrophage body and tail. (C, D) In macrophages exposed to the magnetic-field gradient, the TRPM2 is clustered around the nucleus (arrows) and absent in the tail. Panels A, C are merged images of actin and DAPI

Figure 9 .

 9 Figure 9. The summary of magnet and RhoA-deletion effects on actin and actin-related structures in the M0 macrophages.Diagram of actin and actin-dependent structure changes caused by magnetic-field exposure in

ACKNOWLEDGMENTS

The authors gratefully acknowledge support by the William Stamps Farish Fund, the Donald D. Hammill Foundation, and the State of Texas through the Texas Center for Superconductivity at the University of Houston. We also acknowledge very helpful conversations with Dmitri Litvinov and Ivan Nekrashevich.

AUTHOR CONTRIBUTIONS

M.K. and J.W. designed research, analyzed data and wrote the paper; W.C. and K.Q. conducted experiments, KQ also simulated magnetic field forces; RMG and JZK analyzed data. in contrast to the RhoA-deleted macrophages, there is no noticeable aggregation of focal adhesions in the tail, and vinculin translocates to the nuclei. The second force (susceptibility buoyance) acting, in the presence of a nonuniform magnetic field, on a diamagnetic cell in less diamagnetic buffer is shown. This is an experimentally observed case, and the cells are attracted to the highest-gradient area.


Pharm.D Christèle Dubourg

Artem Kim

Erwan Watrin

Marie De Tayrac

MD, PhD is Sylvie Odent

Pharm Véronique David
email: veronique.david@univ-rennes1.fr

PhD Valérie Dupé

Recent advances in understanding inheritance of Holoprosencephaly

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

INTRODUCTION

Holoprosencephaly is a severe developmental disorder classically defined as incomplete cleavage of the forebrain that originates from failed midline delineation during early development. There are several degrees of severity defined by the extent of the brain malformations. For the most severe cases, malformations are divided into alobar, semilobar or lobar forms. These brain abnormalities are associated with facial anomalies that are also of varying severity ranging from cyclopia to milder signs such as ocular hypotelorism. The full spectrum of HPE also includes microforms characterized by facial midline defects (e.g., single median incisor) without brain malformations typical of HPE [START_REF] Cohen | Holoprosencephaly: clinical, anatomic, and molecular dimensions. Birth Defects Research[END_REF][START_REF] Dubourg | Holoprosencephaly[END_REF][START_REF] Hahn | Septopreoptic holoprosencephaly: a mild subtype associated with midline craniofacial anomalies[END_REF][START_REF] Muenke | Genetics of ventral forebrain development and holoprosencephaly[END_REF]. HPE occurs in most ethnic groups worldwide. Although implication of maternal diabetes in HPE has been reported [START_REF] Barr | Holoprosencephaly in infants of diabetic mothers[END_REF], the well-established origin of HPE remains almost exclusively genetic and consists of chromosomal abnormalities and nucleotide-based variants [START_REF] Dubourg | Holoprosencephaly[END_REF][START_REF] Dubourg | Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway[END_REF][START_REF] Solomon | Genotypic and phenotypic analysis of 396 individuals with mutations in Sonic Hedgehog[END_REF]. So far, 17 genes have been implicated in HPE, all of which encode A c c e p t e d m a n u s c r i t p t 3 proteins belonging to brain development pathways. Sonic Hedgehog (SHH) was the first discovered HPE gene [START_REF] Roessler | Mutations in the human Sonic Hedgehog gene cause holoprosencephaly[END_REF] and its alterations remain the most common cause of nonchromosomal HPE [START_REF] Dubourg | Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway[END_REF]. SHH has been extensively studied and its functions during early brain development are now well described. A morphogenetic gradient of SHH is established from the ventral midline of the diencephalon to induce appropriate cleavage of both forebrain and eyefield. Remarkably, all HPE genes described so far are involved in the regulation of SHH activity [START_REF] Sun | Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons[END_REF]Xavier et al., 2016).

Initially described as an autosomal dominant trait with incomplete penetrance and variable expressivity, the mode of inheritance of HPE has been progressively redefined. The apparent autosomal dominant transmission with incomplete penetrance observed in a few HPE families may well be due to the cumulative effects of rare variants in two genes or more. Undeniably, the prevalence of oligogenicity has increased for several developmental pathologies since Next Generation Sequencing (NGS) technologies became accessible [START_REF] Bamshad | Exome sequencing as a tool for Mendelian disease gene discovery[END_REF]. Despite technical advances, defining the causative gene for HPE remains a difficult task, and even when one underlying variant is known, prenatal prediction remains uncertain.

Here, we describe clinical features and inheritance aspects of this disease with examples from our experience.

Clinical and genetics features of the cohort The European HPE cohort

In 1996, a European HPE network was established in Rennes, France. Patients are recruited by clinicians from the different French centers of reference for rare developmental diseases as well as from several European clinical centers in the UK, Belgium, Italy, Spain and Portugal. Half of collected samples were fetuses, which enriched our cohort for severe HPE phenotypes [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF].

Over the years, we have collected over 2,700 blood DNA samples or frozen fetal tissues, including patients and relatives, and gathered clinical data and DNA for 1,420 HPE probands. Our cohort contains both apparently sporadic and familial cases, excluding those associated with other malformation syndromes or with known chromosomal abnormalities that could be revealed by standard cytogenetic analysis (e.g., trisomies 18 and 13).

HPE presents a wide continuous spectrum of clinical malformations ranging from severe to milder forms. Our cohort is representative of the full clinical spectrum of HPE phenotypes (Mercier et al ). Within this cohort, the most severe brain malformations are categorized into alobar, semilobar or lobar form. Middle interhemispheric fissure (syntelencephaly) -incomplete separation of the posterior frontal and parietal regions -also belongs to the HPE spectrum (Figure 1). These brain abnormalities are significantly correlated with a variety of distinct facial anomalies ranging from cyclopia, the most severe form, to milder signs such as ocular hypotelorism. Severe facial phenotypes, such as cyclopia, ethmocephaly (proboscis) and cebocephaly are more highly associated with alobar HPE. Similarly, premaxillary agenesis, cleft lip or palate and milder ocular abnormalities (coloboma, retinal dysplasia) are mostly associated with semilobar HPE while the mild midface malformations, such as pyriform sinus stenosis and choanal stenosis are mostly found in lobar HPE.

Patients who present the mildest facial abnormalities, such as hypotelorism, cleft lip or single median incisor, generally do not present easily detectable brain malformations [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF]. These HPE microforms can nonetheless be associated with microcephaly and intellectual disability and their molecular diagnosis is therefore important for proper patient care [START_REF] Bruel | Fifteen years of research on oral-facial-digital syndromes: from 1 to 16 causal genes[END_REF][START_REF] Solomon | Analysis of genotype-phenotype correlations in human holoprosencephaly[END_REF]. Notably, most of these microforms have been diagnosed only because they were relatives of patients with severe HPE. Some families manifest a wide range of phenotypes, from typical alobar HPE with perinatal lethality to microforms such as microcephaly, hypotelorism or both (Mercier et al., 2011). These different observations and clinical correlations made on a European cohort can be extended to all HPE cohorts, as they are very similar to those of North American patients [START_REF] Lacbawan | Clinical spectrum of SIX3-associated mutations in holoprosencephaly: correlation between genotype, phenotype and function[END_REF][START_REF] Solomon | Analysis of genotype-phenotype correlations in human holoprosencephaly[END_REF].

As the frequency of microform HPE is underestimated, we are currently expanding our diagnostic approach to mildly affected relatives of a classical HPE patient. When a typical HPE patient is diagnosed in a family, we routinely perform a careful examination of all family members including neuroimaging techniques (MRI) and determination of clinical features that are not traditionally considered as a part of the HPE spectrum. Our goal is to expand our cohort to a larger number of HPE microforms to ensure that we cover the entire HPE spectrum.

The evolution of genetic strategies for HPE diagnosis

Sanger sequencing and detection of microdeletions in the major HPE genes

From the discovery of the first genes responsible for HPE until recently, genetic analysis of HPE patients has mainly relied on a Sanger sequencing approach [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF]. During that period (1997 to 2010), screening our cohort for nucleotide-based variants in the four HPE genes -SHH, Zinc Finger Protein 2 (ZIC2), Six Homeobox 3 (SIX3) and the Homeobox protein TGIF1 -provided a global variant detection rate of 20 % (8.2 % for SHH, 7.4 % for ZIC2, 3.9 % for SIX3 and 1.1 % for TGIF1 [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF]. All variants in these genes were detected heterozygously, and were shown to be loss-of-function variants [START_REF] Roessler | The mutational spectrum of holoprosencephaly-associated changes within the SHH gene in humans predicts loss-of-function through either key structural alterations of the ligand or its altered synthesis[END_REF]Roessler, Lacbawan, et al., 2009). These A c c e p t e d m a n u s c r i t p t 5 genes have constituted the four major genes of HPE. Meanwhile, novel genes have been implicated in sporadic cases of HPE (Table II and Roessler, El-Jaick, et al., 2009). The implication of each gene represents less than 1 % of HPE patients and they are therefore referred to as minor genes.

In 2003, variants in GLI2, one crucial effector of SHH signaling pathway (Ruiz i Altaba, Palma, & Dahmane, 2002), had been described in HPE patients [START_REF] Roessler | Loss-of-function mutations in the human GLI2 gene are associated with pituitary anomalies and holoprosencephaly-like features[END_REF], and screening of our cohort (Figure 1) revealed GLI2 variants in 3.2 % of the 302 patients tested [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF], thus placing GLI2 as a major gene.

Identification of variants combined with detailed clinical assessment of HPE patients has allowed establishing some genotype-phenotype correlations [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF][START_REF] Solomon | Analysis of genotype-phenotype correlations in human holoprosencephaly[END_REF]. The most severe types of HPE (alobar and semilobar) tend to be associated with ZIC2 and SIX3 alterations while SHH tends to be more frequently associated with microforms. Remarkably, in SHH and SIX3 cases, the facial dysmorphism is associated with brain anomalies while the probands of the ZIC2 group tend to have a combination of severe HPE with few of the facial features. GLI2 variants were preferentially found in patients presenting HPE microforms together with secondary specific features such as pituitary anomalies [START_REF] Bear | Pathogenic mutations in GLI2 cause a specific phenotype that is distinct from holoprosencephaly[END_REF]. These genotype-phenotype correlations have contributed to facilitate molecular analysis and genetic counseling for HPE.

Initially, Sanger sequencing has successfully allowed establishing molecular diagnoses for about 20 % of HPE patients present in our cohort [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF]. In order to explain, at least part of the remaining 80 % of unsolved cases, we have searched for microdeletions in the major HPE genes (SHH, ZIC2, SIX3 and TGIF1) first by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and then by multiplex ligation-dependent probe amplification (MLPA). Deletions in HPE genes were thereby shown to be a common cause of HPE in up to 8.5 % of fetuses and in 5% in our whole cohort [START_REF] Bendavid | Molecular evaluation of foetuses with holoprosencephaly shows high incidence of microdeletions in the HPE genes[END_REF].

In 2006, the combination of these different approaches allowed us to diagnose molecularly 25 % of our patients, very similar to the success rate of our American colleagues [START_REF] Roessler | The mutational spectrum of holoprosencephaly-associated changes within the SHH gene in humans predicts loss-of-function through either key structural alterations of the ligand or its altered synthesis[END_REF][START_REF] Solomon | Analysis of genotype-phenotype correlations in human holoprosencephaly[END_REF][START_REF] Solomon | Genotypic and phenotypic analysis of 396 individuals with mutations in Sonic Hedgehog[END_REF]. As 75 % of cases remained unsolved, we and others in the field have considered alternative genetic causes of HPE.

Chromosomal abnormalities and copy number variants in HPE

Since 2006, a pangenomic technique named comparative genomic hybridization (CGH) array has been used to screen the entire genome for copy number variations (CNVs). The first study we carried out during 2006-2009 revealed an impressively high rate of chromosomal rearrangements in HPE patients (22%), of which 14 % occurred de novo and 8 % were inherited [START_REF] Bendavid | Array-CGH analysis indicates a high prevalence of genomic rearrangements in holoprosencephaly: an updated map of candidate loci[END_REF][START_REF] Bendavid | Holoprosencephaly: An update on cytogenetic abnormalities[END_REF].

Furthermore, the observation of these CNVs can also lead to the detection of parental-balanced In addition, these CNVs involved novel potential HPE loci. Despite relatively low recurrence rates of CNVs, overlapping 6qter region deletions among 4 unrelated patients allowed identification of a ligand of the NOTCH signaling pathway, Delta-like 1 (DLL1) as candidate gene [START_REF] Dupé | NOTCH, a new signaling pathway implicated in holoprosencephaly[END_REF].

Subsequent detection of a nucleotide-based variant in a distinct patient provided further evidence for DLL1 as HPE gene, and, together with expression and functional studies in vertebrates [START_REF] Ratié | Novel genes upregulated when NOTCH signalling is disrupted during hypothalamic development[END_REF]Ware, Hamdi-Rozé, & Dupé, 2014), allowed defining Notch as a novel signaling pathway involved in HPE.

In addition, microrearrangements found in unique cases, pointed to candidate genes such as SIX6 and OTX2, which are both implicated in early brain development [START_REF] Jean | Six6 (Optx2) is a novel murine Six3-related homeobox gene that demarcates the presumptive pituitary/hypothalamic axis and the ventral optic stalk[END_REF][START_REF] Jin | Otx2 and HNF3beta genetically interact in anterior patterning[END_REF]. Another study using a similar approach has also highlighted OTX2 as a candidate gene [START_REF] Rosenfeld | Clinical characterization of individuals with deletions of genes in holoprosencephaly pathways by aCGH refines the phenotypic spectrum of HPE[END_REF].

Since this work, CGH-array has been and still is part of our systematic molecular screening of HPE patients. The novel detected CNVs are listed in Table II. The proportion of CNVs observed in this second period (2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017) is reduced as compared to that of our previous study [START_REF] Bendavid | Array-CGH analysis indicates a high prevalence of genomic rearrangements in holoprosencephaly: an updated map of candidate loci[END_REF]. The rate of disease-relevant CNVs is now shown to be 10 %, half of which being de novo.

Several of these rearrangements are recurrently observed in cases of intellectual disability, such as 2.6 Mb-microdeletion of 22q11.21 (proximal deletion) corresponding to DiGeorge syndrome [START_REF] Burnside | 22q11.21 Deletion Syndromes: A Review of Proximal, Central, and Distal Deletions and Their Associated Features[END_REF], 16p11.2 microduplication that confers susceptibility to autism [START_REF] Fernandez | Phenotypic spectrum associated with de novo and inherited deletions and duplications at 16p11.2 in individuals ascertained for diagnosis of autism spectrum disorder[END_REF], 16p13.11 encompassing the NDE1 gene involved in brain neurogenesis and rhombencephalosynapsis [START_REF] Bakircioglu | The essential role of centrosomal NDE1 in human cerebral cortex neurogenesis[END_REF][START_REF] Démurger | Array-CGH Analysis Suggests Genetic Heterogeneity in Rhombencephalosynapsis[END_REF] and 15q11.2 microdeletion emerging as one of the most common cytogenetic abnormalities in intellectual disability and autism spectrum disorder [START_REF] Butler | Clinical and genetic aspects of the 15q11.2 BP1-BP2 microdeletion disorder[END_REF]. These deletions and duplications are thus at the origin of other neurodevelopmental disorders but are not sufficient to fully explain HPE. Nevertheless, CNV detection has increased the diagnostic yield from 25 % to 35 % in our cohort.

Next-generation sequencing (NGS) methods and their use for HPE diagnosis

The discovery of SHH in 1996 was followed by that of other genes -ZIC2, SIX3, TGIF1 and GLI2. Since then, subsequent studies of the pathways implicating these major genes have contributed to the identification of additional HPE genes (Table I). Variants in genes involved in the SHH signaling pathway -PTCH1, DISP1, CDON, GAS1, BOC and SUFU -were described in some HPE patients [START_REF] Bae | Mutations in CDON, encoding a hedgehog receptor, result in holoprosencephaly and defective interactions with other hedgehog receptors[END_REF][START_REF] Dubourg | Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway[END_REF][START_REF] Hong | BOC is a modifier gene in holoprosencephaly[END_REF]Ming et al., 2002;[START_REF] Pineda-Alvarez | Missense substitutions in the GAS1 protein present in holoprosencephaly patients reduce the affinity for its ligand, SHH[END_REF][START_REF] Roessler | Truncating loss-of-function mutations of DISP1 contribute to holoprosencephaly-like microform features in humans[END_REF]. A few variants have been described in NODAL, TDGF1, FOXH1, which encode proteins belonging to the Nodal/TGF-beta pathway (de la [START_REF] De La Cruz | A loss-of-function mutation in the CFC domain of TDGF1 is associated with human forebrain defects[END_REF][START_REF] Roessler | Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly[END_REF][START_REF] Roessler | Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly[END_REF]. The Fibroblast Growth Factor pathway has also been implicated in A c c e p t e d m a n u s c r i t p t 7 HPE through variants in FGF8 and its receptor FGFR1 [START_REF] Arauz | A Hypomorphic Allele in the FGF8 Gene Contributes to Holoprosencephaly and Is Allelic to Gonadotropin-Releasing Hormone Deficiency in Humans[END_REF][START_REF] Simonis | FGFR1 mutations cause Hartsfield syndrome, the unique association of holoprosencephaly and ectrodactyly[END_REF]. More recently, variants in STIL, a gene implicated in the formation of the primary cilia, were also described in HPE families [START_REF] Kakar | STIL mutation causes autosomal recessive microcephalic lobar holoprosencephaly[END_REF][START_REF] Mouden | Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings[END_REF]. These are minor genes as they are reported in less than 1 % of HPE cases. Importantly, all these genes have in common the ability to affect SHH activity [START_REF] Sun | Characterization of the human oncogene SCL/TAL1 interrupting locus (Stil) mediated Sonic hedgehog (Shh) signaling transduction in proliferating mammalian dopaminergic neurons[END_REF]Xavier et al., 2016).

Taking advantage of next-generation sequencing (NGS) in 2010, we established the first gene-panel sequencing method that targeted all known HPE genes [START_REF] Dubourg | Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway[END_REF]. At the time we are writing this manuscript, more than 300 HPE patients have been tested. This study revealed that SHH, ZIC2, SIX3 and GLI2 retain their position of major genes and TGIF1 is relegated to the minor gene group. Furthermore, the identification of numerous variants in FGF8 and FGFR1 strengthens the involvement of FGF signaling in HPE (Figure 1B). Recent functional analysis in Zebrafish has confirmed the contribution of FGF8 variants in HPE [START_REF] Hong | Dominant-negative kinase domain mutations in FGFR1 can explain the clinical severity of Hartsfield syndrome[END_REF][START_REF] Hong | Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly[END_REF] in accordance with the known function of FGF signaling during the specification of the dorso-ventral axis of the forebrain [START_REF] Storm | Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers[END_REF].

From the results we obtained over the years from our 1,420 probands, the 10 first-ranked genes involved are SHH (5.4%), ZIC2 (5.2%), GLI2 (3.2%), SIX3 (3.0%), FGF8 (2.5%), FGFR1 (2.0%), DISP1

(1.2%), DLL1 (1.2%) and TGIF1 (0.9%) (Figure 2; Table I). Some rare deleterious variants have been found in SUFU, a regulator of the Sonic-hedgehog-signaling pathway [START_REF] Dubourg | Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway[END_REF]. By contrast, we did not detect any pathogenic variants in the following minor genes: PTCH1, NODAL, GAS1, TDGF1, CDON, and FOXH1. To date, only a small number of variants have been reported for these genes [START_REF] Bae | Mutations in CDON, encoding a hedgehog receptor, result in holoprosencephaly and defective interactions with other hedgehog receptors[END_REF][START_REF] De La Cruz | A loss-of-function mutation in the CFC domain of TDGF1 is associated with human forebrain defects[END_REF]Ming et al., 2002;[START_REF] Roessler | Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly[END_REF][START_REF] Roessler | Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly[END_REF]. Therefore, more data need to be collected to unambiguously assign these genes to HPE etiology.

As NGS technology evolved and became more accessible, whole exome sequencing (WES) is now routinely used to investigate novel HPE patients (see paragraph below). We expect this new approach will allow us not only to increase the diagnostic yield but also to identify novel HPE genes.

Along the same lines, we are now establishing whole genome sequencing (WGS) approach in order to identify other alterations located in the noncoding part of the genome (~98 %) that has remained largely unexplored until now in the context of HPE.

An important observation has emerged from our experience as clinical reference center for HPE.

When comparing the percentage of positive diagnoses between 2010 and 2017 it appears surprisingly stable (about 35 %), despite the fact that sequencing technology has improved and that A c c e p t e d m a n u s c r i t p t 8 the number of HPE genes has increased. A closer examination of our data indicates that the fraction of patients with a CNV or with a variant in one of the major genes (SHH, ZIC2 and SIX3) has been reduced by half as compared to that of 2010. Paradoxically, this reduction of cases is due to the increase of knowledge in diagnosis of HPE. Indeed, more and more clinical genetic centers are now able to perform their own molecular diagnoses for HPE. When a deleterious variant is found in one of the major HPE genes or when there is a CNV, the patient is not systematically referred to the reference center (i.e., Rennes, France) anymore. In contrast, when a molecular diagnosis could not be established, the patient data are sent to us for further investigation. As a consequence, the proportion of cases with an alteration in the major HPE genes has decreased in our cohort, which explains why our success rate of molecular diagnosis has remained stable over the years.

Modes of inheritance in HPE

In 1996, we initiated an epidemiologic study on 258 HPE cases and concluded that for non-syndromic and non-chromosomal HPE, the most compatible mode of transmission was autosomal dominant with incomplete penetrance and a variable expressivity [START_REF] Odent | Segregation analysis in nonsyndromic holoprosencephaly[END_REF].

In HPE, the analysis of the first four major genes revealed that in most of the cases (70 % for SHH and SIX3) the variants were inherited from a parent who was asymptomatic or only mildly affected (microform HPE). By classic textbook definitions, autosomal dominant inheritance is defined as the transmission of disease from an affected parent to an affected offspring. In this model, half of this parent's offspring is expected to be affected. Our observations show that this model (and ratios of affected offspring) does not appear to apply to HPE. Furthermore, although as many as 17 genes have been linked to HPE, variants in these genes collectively explain only 25 % of all HPE cases. It suggests that HPE is a complex disease with an increasing number of causative genes for which inheritance can vary depending of the affected gene as well as other related factors. This observation stresses the need for clarifying potential modes of its inheritance. In this section, we present different cases from our cohort and discuss their corresponding inheritance pattern.

De novo versus inherited variants in HPE

Systematic sequencing of the major HPE genes has shown a high proportion of de novo variants in ZIC2 (70 %), SHH (30 %) and SIX3 (30 %) (Figure 3 and [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF]. Thus, de novo variants are implicated in numerous sporadic HPE cases. In accordance with the essential role of these genes in early brain development, these de novo variants are loss-of-function and tend to be more deleterious than inherited ones [START_REF] Roessler | The mutational spectrum of holoprosencephaly-associated changes within the SHH gene in humans predicts loss-of-function through either key structural alterations of the ligand or its altered synthesis[END_REF]. In our cohort, 50 % of FGFR1 variants appeared de novo. Adding to complexity, FGFR1 is prone to mosaïcism as we could show in one family in which a patient's father presents an attenuated HPE phenotype [START_REF] Dubourg | Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway[END_REF]. Interestingly, cases of mosaïcism involving FGFR1 were described in other diseases like Hartsfield syndrome and encephalocraniocutaneous lipomatosis [START_REF] Bennett | Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis[END_REF][START_REF] Dhamija | Novel de novo heterozygous FGFR1 mutation in two siblings with Hartsfield syndrome: a case of gonadal mosaicism[END_REF]. It should be noted that mosaic variants could be overlooked depending on the type of tissue tested and on the detection method [START_REF] Braunholz | Hidden mutations in Cornelia de Lange syndrome limitations of sanger sequencing in molecular diagnostics[END_REF], which could artificially increase the proportion of de novo variants.

Rare examples of autosomal recessive inheritance in HPE patients

In 2007, first case of recessive inheritance was described, involving two compound heterozygous variants in TGIF1 [START_REF] El-Jaick | Functional analysis of mutations in TGIF associated with holoprosencephaly[END_REF]. Then, homozygous variant in FGF8 was identified in one consanguineous HPE family [START_REF] Mccabe | Novel FGF8 mutations associated with recessive holoprosencephaly, craniofacial defects, and hypothalamo-pituitary dysfunction[END_REF]. More recently, another case of recessive inheritance involving FGF8 was described [START_REF] Hong | Loss-of-function mutations in FGF8 can be independent risk factors for holoprosencephaly[END_REF]. This FGF8 variant was functionally validated and shown to be hypomorph, which is consistent with the indispensable role of FGF8 during early development [START_REF] Sun | Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo[END_REF]. A severe loss of function is probably not compatible with embryonic development.

Still, consanguineous families should predispose descendants to autosomal recessive gene combinations. Homozygosity mapping performed on 8 consanguineous HPE families from our cohort did not initially reveal any homozygous variants. Only by complementing mapping with WES we were able to detect in one of these families a homozygous hypomorphic variant for the gene STIL [START_REF] Mouden | Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings[END_REF]. A homozygous nonsense variant of STIL was independently reported by others [START_REF] Kakar | STIL mutation causes autosomal recessive microcephalic lobar holoprosencephaly[END_REF]. STIL is localized to centrioles where it participates in SHH signaling through its function in primary cilia biology and is known to be involved in microcephaly [START_REF] David | Lack of centrioles and primary cilia in STIL(-/-) mouse embryos[END_REF]. Notably, in these two families, HPE is transmitted as a recessive trait associated with severe microcephaly. As no additional variant of STIL was described on more than 100 HPE patients tested [START_REF] Karkera | The genomic structure, chromosomal localization, and analysis of SIL as a candidate gene for holoprosencephaly[END_REF][START_REF] Mouden | Homozygous STIL Mutation Causes Holoprosencephaly and Microcephaly in Two Siblings[END_REF], STIL gene therefore belongs to the minor HPE genes.

We also described a female HPE patient displaying two different mutated DISP1 alleles both inherited from her two healthy parents [START_REF] Mouden | Complex mode of inheritance in holoprosencephaly revealed by whole exome sequencing[END_REF]. These missense variants in the exon 10 were predicted to be deleterious. DISP1 is a protein that mediates the secretion of SHH, that is required for long-range cell to cell signaling (Tian, Jeong, Harfe, Tabin, & McMahon, 2005). This patient presents a localized fusion of forebrain hemispheres (mild form of HPE). We believe that hypomorphic effect of DISP1 missense variant impacts SHH secretion to such an extent that global SHH signaling is decreased to pathological level.

All cases of autosomal recessive inheritance reported so far involve unaffected parents and concern minor HPE genes (Figure 3). Despite systematic sequencing of the major genes (SHH, ZIC2 and SIX3) [START_REF] Roessler | Utilizing prospective sequence analysis of SHH, ZIC2, SIX3 and TGIF in holoprosencephaly probands to describe the parameters limiting the observed frequency of mutant gene×gene interactions[END_REF], no recessive case has been reported for these genes. It suggests that homozygous variants in major genes are not compatible with embryonic development, which is fully consistent with their crucial roles during early developmental stages [START_REF] Geng | Pathogenesis of holoprosencephaly[END_REF][START_REF] Schachter | Murine models of holoprosencephaly[END_REF] and may explain why recessive inheritance is rare in HPE.

Oligogenic inheritance

As presented above, variants in HPE genes are frequently inherited from a parent without a typical HPE phenotype [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF]. Within a given family we observed a large phenotypic heterogeneity between the different variant carriers, illustrating the incomplete expressivity of these variants [START_REF] Kruszka | Expanding the phenotypic expression of Sonic Hedgehog mutations beyond holoprosencephaly[END_REF][START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF][START_REF] Solomon | Analysis of genotype-phenotype correlations in human holoprosencephaly[END_REF][START_REF] Stokes | SIX3 deletions and incomplete penetrance in families affected by holoprosencephaly[END_REF]. These observations support the hypothesis that in these families variant in one HPE-related gene is necessary but not sufficient for the disease to occur, which implies more variants are required for complete phenotypic spectrum. This oligogenic mode of inheritance has already been proposed by our colleagues, who referred to it as "automosal dominant with modifier effects" (S. [START_REF] Hong | Dominant-negative kinase domain mutations in FGFR1 can explain the clinical severity of Hartsfield syndrome[END_REF][START_REF] Hong | BOC is a modifier gene in holoprosencephaly[END_REF]. In this oligogenic model, penetrance and expressivity of existing heterozygous variant is modulated by variants in other genes associated with HPE. Such a synergistic effect between distinct deleterious genetic events is now well-documented in several other hereditary developmental diseases (e.g, Alport, Bardet-Biedl and Kallman Syndromes) [START_REF] Maione | GENETICS IN ENDOCRINOLOGY: Genetic counseling for congenital hypogonadotropic hypogonadism and Kallmann syndrome: new challenges in the era of oligogenism and next-generation sequencing[END_REF][START_REF] Mencarelli | Evidence of digenic inheritance in Alport syndrome[END_REF][START_REF] Redin | Clinical and genetic characterization of Bardet-Biedl syndrome in Tunisia: defining a strategy for molecular diagnosis[END_REF]. In a similar manner, HPE could result from cumulated effects of distinct variants (Mercier, 2013). The use of animal models has reinforced this possibility. Numerous examples of double heterozygous mutant mice displaying HPE-like phenotypes provided evidence for oligogenism by implicating genes controlling either the same or distinct signaling pathways [START_REF] Krauss | Holoprosencephaly: new models, new insights[END_REF].

For years, when a patient presented a deleterious variant in HPE gene, the analysis was interrupted because a likely genetic cause for the disease had been felt to be found. We believe it explains why rare HPE cases compatible with oligogenism have been reported so far (Ming & Muenke, 2002).

High-throughput sequencing and genomic technologies provided a unique opportunity to address this oligogenic inheritance in HPE. Thanks to WES, we have recently started to address the presence of additional events in HPE patients with a known variant in a HPE gene. Some of our most recent unpublished results on the subject are presented below.

Examples of digenic inheritance in HPE

A c c e p t e d m a n u s c r i t p t 11 Digenic inheritance is the simplest form of inheritance for genetically complex diseases. Systematic sequencing of major HPE genes, have described isolated cases with variants in two genes (e.g., SHH/ZIC2; SHH/TGIF) (Ming & Muenke, 2002). We also reported several cases of chromosomal rearrangement (CNV) associated with variant in HPE-related gene [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF].

In one family, presented in figure 2A, deleterious variants in both SHH and DISP1 co-segregated with the disease, while relatives carrying variant only in either SHH or DISP1 presented a mild or no disease phenotype [START_REF] Mouden | Complex mode of inheritance in holoprosencephaly revealed by whole exome sequencing[END_REF]. This first digenic case in a family with several HPE patients prompted us to consider and further investigate digenic inheritance in families with HPE. This hypothesis was significantly reinforced by our experience with HPE genes routinely analyzed by targeted NGS. This study revealed that out of 257 HPE probands, 16 % of the variants used for diagnosis were found in association with a second variant (e.g., FGF8/FGFR1, FGF8/DLL1, DLL1/SHH, DISP1/SUFU) [START_REF] Dubourg | Mutational Spectrum in Holoprosencephaly Shows That FGF is a New Major Signaling Pathway[END_REF]. More recently, two more cases of digenism were reported (ZIC2/BOC and TGIF/BOC) in HPE patients [START_REF] Hong | BOC is a modifier gene in holoprosencephaly[END_REF]. Considering the systematic use of NGS on HPE patients and the increasing number of HPE genes, we expect that digenic cases will continue to accumulate further.

Cases of oligogenic inheritance in HPE

The increasing number of digenic cases provides novel insights into genetic etiology of HPE such as oligogenic inheritance. Oligogenic pattern has never been described in HPE, but is often suggested in other complex diseases such as ciliopathies [START_REF] Reiter | Genes and molecular pathways underpinning ciliopathies[END_REF], retinitis pigmentosa [START_REF] Ali | Genetic characterization and disease mechanism of retinitis pigmentosa; current scenario[END_REF], autism spectrum disorder (ASD) (Yin & Schaaf, 2017), amyotrophic lateral sclerosis (ALS) [START_REF] Nguyen | ALS Genes in the Genomic Era and their Implications for FTD[END_REF], or porphyria [START_REF] Lenglet | From a dominant to an oligogenic model of inheritance with environmental modifiers in acute intermittent porphyria[END_REF]. These disorders share with HPE high genetic and phenotypic heterogeneity as well as incomplete penetrance. We therefore considered oligogenic inheritance as a likely cause of HPE that may account for at least a substantial part of enigmatic cases.

With constantly evolving knowledge of disease genes and emergence of new analysis methods, it has become important to us to reanalyze systematically previous unsolved cases. In that aim, we reevaluated unsolved HPE families by taking into account the possibility of oligogenic inheritance.

The study included families with no identified causal variant as well as families with variants in SHH, ZIC2, SIX3 or TGIF1 inherited from clinically unaffected/mildly affected parent. Parts of this unpublished work are presented and discussed hereafter.

We combined trio-based whole exome sequencing with deep clinical phenotyping of the patients.

Variant analysis was further improved by a gene prioritization approach based on clinical ontologies and co-expression networks of known disease-related signaling pathways. In one family, presented in Figure 2B, we identified a variant in SHH, which was inherited from the mother presenting with hypotelorism. Considering oligogenic inheritance, we addressed whether paternal variants could contribute to HPE phenotype in combination with the SHH variant inherited from the mother. Indeed, our analysis revealed paternally inherited variants in two candidate HPE genes -FAT1 and NDST1. Both variants were rare (minor allele frequency below 1 %) and were predicted deleterious by the majority of bioinformatics algorithms (CADD score > 20). FAT1 is a protocadherin and its knockdown in mouse causes severe midline defects including HPE [START_REF] Ciani | Mice lacking the giant protocadherin mFAT1 exhibit renal slit junction abnormalities and a partially penetrant cyclopia and anophthalmia phenotype[END_REF] and NDST1 is an N-deacetylase sulfotransferase and the corresponding mice mutants exhibit reduced SHH signaling and HPE-like phenotype [START_REF] Grobe | Cerebral hypoplasia and craniofacial defects in mice lacking heparan sulfate Ndst1 gene function[END_REF].

Additionally, clinical phenotyping and analysis of cross-species similarities provided further evidence of causality for these genes by revealing a strong overlap of clinical features between the patient and the FAT1 (proboscis) and NDST1 (eye defects) mutant mice. Finally, the segregation analysis showed that the combination of variants in SHH/FAT1/NDST1 was exclusively found in the two affected individuals of this family (Figure 2B). This example nicely illustrates oligogenic inheritance in HPE where the disease results from accumulation of multiple variants in genes associated to HPE phenotypes and/or implicated in SHH signaling (Figure 3). We therefore pursue a systematic reevaluation of all unsolved HPE cases. We believe that numerous other genes will be characterized in patients with oligogenism transmission. An exciting future challenge will be to test experimentally the combined effect of these different variants on early brain development.

Our clinical approach

Over the last two decades, we have followed for prenatal diagnosis 26 HPE families affected by a first case of severe HPE carrying a variant in one of the major HPE gene. In 18 instances, we were able to reassure the parents after establishing the absence of the gene alteration in the fetus. Fetal MRI scan was normal later in pregnancy, and no child had medical problems after birth. A genetic alteration (in SHH, SIX3 or TGIF1) was found in the 8 other cases: 5 children were born, either without brain malformation and asymptomatic, or presenting a less severe form than the proband as predicted by the fetal brain surveillance. Three pregnancies were interrupted after MRI scans showed HPE features.

Nowadays, even when a causative gene for HPE has been found in a patient, the molecular diagnosis is probably not fully established. In order to properly address the molecular diagnosis, it will be necessary to compare the detailed phenotypes of the different family members with the segregation of relevant rare variants. For practitioners involved in counseling, an important consideration is how to communicate results of genetic analysis when potentially deleterious variants are identified but A c c e p t e d m a n u s c r i t p t

13 not yet functionally validated. Ideally, determining the contribution of each variant to the phenotype would be a condition for reliable genetic counseling in HPE families with oligogenic transmission.

Conclusion

In this review, we aimed to present the different patterns of inheritance of HPE in the light of our experience (Figure 3). In some cases, the disease is due to de novo variants; in rare cases the disease exhibits classical Mendelian inheritance with autosomal recessive transmission. In most cases, it emerges that the penetrance and the phenotypic variability have digenic or oligogenic origin. This complex genetic architecture will be better understood by analysis of hundreds of genes with NGS techniques on unsolved HPE cases. Our future challenge will be to differentiate rare variants that have significant impact on the observed phenotype from those with no effect. In blue, number of variants found in 2010. 164/642 (25.4 %) patients were found to harbor variants in SHH, ZIC2, SIX3 or TGIF1. GLI2 variants were identified in 3/208 patients [START_REF] Mercier | New findings for phenotype-genotype correlations in a large European series of holoprosencephaly cases[END_REF].

In red, number of variants found in 2017. Molecular screening of major HPE genes in 1,420 patients revealed 207 variants in SHH, ZIC2, SIX3 and TGIF1. Complementary screenings on a series of 302 patients revealed 32 variants in DLL1, FGF8, FGFR1, DISP1 and GLI2. (A) Family presenting a digenic mode of transmission associating variants in SHH and DISP1 [START_REF] Mouden | Complex mode of inheritance in holoprosencephaly revealed by whole exome sequencing[END_REF]. Minor signs refer to microcephaly.

  subsequently improve prenatal diagnosis in such families.

  ., Jeong, J., Harfe, B. D., Tabin, C. J., & McMahon, A. P. (2005

Figure legends Figure 1 .

 legends1 Figure legends Figure 1. Clinical and molecular details of the European HPE cohort (A) Brain anomalies distribution in European HPE cohort (1,420 probands). (B) Mutational spectrum of HPE.

  (C) Contribution of CGH-array to molecular diagnosis. In 2010, CNVs were detected in 22 % of the 260 patients analyzed by CGH-array, including 36 occurring de novo. In 2017, the screening of our entire cohort (1,420 patients) reported CNVs in 142 patients (10%), including 71 occurring de novo.

Figure 2 .

 2 Figure 2. Examples of complex inheritance in HPE.

(

  B) Family presenting an oligogenic pattern with combined inherited variants in SHH, FAT1 and NDST1. Minor signs refer to hypotelorism (father) and epicanthus (mother).Individuals marked with asterisk were analyzed by whole exome sequencing. NA: Not available for DISP1 sequencing.

Figure 3 .

 3 Figure 3. Different inheritance patterns for HPE are presented together with illustrative cases from our unpublished data.

  De novo del = deletion; dup = duplication; ND = not determined; *CNV (Copy number variation) usually observed in intellectual deficiency

  ). Mouse Disp1 is required in sonic hedgehog-expressing cells for paracrine activity of the cholesterol-modified ligand. Development (Cambridge, England), 132(1), 133-142. https://doi.org/10.1242/dev.01563 Ware, M., Hamdi-Rozé, H., & Dupé, V. (2014). Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus. Frontiers in Neuroanatomy, 8, 140. https://doi.org/10.3389/fnana.2014.00140 Xavier, G. M., Seppala, M., Barrell, W., Birjandi, A. A., Geoghegan, F., & Cobourne, M. T. (2016).

	Hedgehog receptor function during craniofacial development. Developmental Biology, 415(2), 198-
	215. https://doi.org/10.1016/j.ydbio.2016.02.009
	Yin, J., & Schaaf, C. P. (2017). Autism genetics -an overview. Prenatal Diagnosis, 37(1), 14-30.
	https://doi.org/10.1002/pd.4942

TABLE I . List of HPE genes and corresponding percentages of variants found in our HPE cohort.

 I

	Chromosome	Gene	NM	%
	7	SHH	000193.2	5.4
	13	ZIC2	007129.3	5.2
	2	GLI2	005270.4	3.2
	2	SIX3	005413.3	3.0
	10	FGF8	033163.3	2.5
	8 1 6 18 10 1 9 3 11 8 10 3	FGFR1 DISP1 DLL1 TGIF1 SUFU STIL GAS1 TDGF1 CDON FOXH1 NODAL BOC	023110.2 032890.3 005618.3 170695.2 016169.3 001048166.1 002048.2 003212.3 016952.4 003923.2 018055.4 001301861.1 m 2.0 1.2 1.2 0.9 0.4 1 case/375 0 0 0 0 0 Not tested a n u s c r i t p t
	A c c e p t e d

TABLE II : Summary of chromosomal abnormalities in HPE cases

 II

	Patient Cytoband	Type of CNV	Start-End	CNV size	Inheritance
				(GRCh37)
	1	1q43q44	del	242094954_	7 Mb
				249212668		Inherited from
						parental balanced
		3p25.2p22.1	dup	0_	40 Mb	t(1;3)
				39848444
	2	2p15	del	61668439_	109 kb	De novo
				61777447
	3	2p11.2	dup	85824180_	645 kb	De novo
				86469217
				15492317_		Inherited from father
		16p13.11*	dup	16276115	784 kb
	4	3p22;20q11.2	balanced	-	-	Inherited from mother
			translocation
	5	3q25.32	del	157105931_	50 kb	Inherited from mother
				157154842
	6	4q12	del	55193357_	3,00 Mb	ND
				58196685
	7	5q35.3	del	177296851_	1 Mb	Inherited from mother
				178323802
	8	5q35.3	dup	178038828-	1,73 Mb	Inherited
				179766520
	9	7p22.1	del	5399371_	1,4 Mb	De novo
				6871084
	10	7p22.1	dup	5057686_	108 kb	Inherited from father
				5166175
	11	8q23.3q24.11	del	117641330-	410 kb	ND
				118051191
	12	Whole chromosome 8	dup in mosaic	0_	146 Mb	De novo
				146294098
	13	10p15.3p14	del	136361-9946915	9,8 Mb	De novo
	14	11q13.4	dup	74931870-	178 kb	Inherited from mother
				75109882
	15	12q21.32	del	86984993_	672 kb	ND
				87656628
	16	14q23.1	del	60950490_	55 kb	Inherited from mother
				61006021
	17	14q23.1	dup	Unavailable data	900kb	Inherited from mother
	18	15q11.2*	del	22765628-	443 kb	Inherited from mother
				23208901
	19	16p13.11*	del	15492317_	1,3 Mb	ND
				16267306
				29652999_		Inherited from mother
		16p11.2*	dup	30197341	544 kb
	20	18q22.1	dup	64152648_	172 kb	Inherited from mother
				64324336
	21	19q13.42q13.43	dup	56228025_	1,5 Mb	De novo
				57744093
	22	22q11.21*	del	20719112-	745 kb	ND
				21464119
	23	22q11.21*	del	21081060_	424 kb	De novo
				21505558
	24	22q11.21*	del	18894835_	2,6 Mb	De novo
				21464119
	25	22q11.21*	del	18894835_	2,6 Mb	De novo
				21464119
	26	22q11.21*	del	18651614_	3,2 Mb	ND
				21801661
	27	Xq25q28	del	123176394-	29,34 Mb
				152515593