Yuchun Tu

Yanyan Yuan

++ Karine Le Guen

Jean-Michel André

Jingtao Zhu

Zhanshan Wang

Françoise Bridou

Angelo Giglia

Philippe Jonnard
email: philippe.jonnard@upmc.fr

X-ray fluorescence induced by standing waves in the grazing-incidence and grazing-exit modes: study of the Mg-Co-Zr system

Keywords: X-ray standing wave, X-ray fluorescence, Multilayer, Interface PACS. X-ray standing waves, 68.49.Uv, Structure and nonelectronic properties of multilayers, 68.65.Ac, Solid-solid interfaces, 68.35.-p, X-ray fluorescence, 78.70.En

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Periodic multilayers alternating two or more layers of nanometer thickness can be used to diffract radiation in the x-ray and extreme ultra-violet ranges [START_REF] Attwood | Soft X-Rays and Extreme Ultraviolet Radiation[END_REF]. Thus, they are used as optical components for numerous applications in photolithography, x-ray microscopy, xray spectroscopy, in space telescopes or on synchrotron beamlines. However, the optical performance of such multilayer stacks greatly depends on the quality of their interfaces. So it is important to characterize them, which is often done by x-ray reflectivity in the hard x-ray range or at the application wavelength or by transmission electron microscopy.

Recently we have demonstrated (Jonnard et al., 2014)that x-ray fluorescence (XRF) generated by x-ray standing waves (XSW), combining both grazing incidence (GI) and grazing exit (GE) modes, is an efficient meansfor the characterization of such stacks. Indeed, the standing wave generated by the incident radiation (GI mode) or characteristic emission (GE mode) has the same period as the multilayer. Then, by rotating the sample in an angular range centered around the Bragg angle of the incident or emitted radiation, it is possible to move the nodes and anti-nodes of the electric field at specific positions within the stack, an interface or the center of a layer, and thusto locate the origin of the generated x-ray signal with great depth sensitivity [START_REF] Bedzyk | The X-Ray Standing Wave Technique[END_REF]. This technique is related to the technique of x-ray standing wave at grazing incidence and exit [START_REF] Sakata | The X-Ray Standing Wave Technique[END_REF].

We apply GI-and GE-XRF to the Mg-Co-Zrtri-layer system whose period thickness, around 9.5 nm, was designed to have constructive interferences and Bragg peak at not extreme grazing incidence. Two tri-layers, Si/[Mg/Co/Zr] x30 and Si/[Mg/Zr/Co] x30 , weredeposited by magnetron sputtering, where the order of the layers in the stack is different. This kind of multilayers had already been studied for optical applications around the 25 nm spectral range (Le Guen et al., 2011a;[START_REF] Predel | [END_REF]. With respect to the present multilayers, the Co and Zr thicknesses were the same whereas Mg layers were much thicker. They have been thoroughly characterized by x-ray reflectivity in the hard and soft x-ray ranges, x-ray emission spectroscopy, nuclear magnetic resonance spectroscopy, secondary ion mass spectrometry and also transmission electron microscopy (Le Guen et al., 2011a;[START_REF] Predel | [END_REF]Zhu et al., 2011;Jonnard et al., 2013). This paper is organized as follows. Firstly, in the experimental section we indicate how the samples are prepared and recall some details of the GI-and GE-XRF experiments. We present the improvement of the angular resolution in the GE mode with respect to our previous study of the Co/Mg bi-layer system, The way simulations are obtained and handledfor comparison with the experimental curves is then described. Finally, we present and discuss comparatively the results for both tri-layer systems, obtained in the GI and GE modes giving the angular variations of the intensity of the Mg K(2p-1s transition) and Co L(3d-2p 3/2 transition) characteristic emissions.

EXPERIMENTAL METHODS

Samples

The deposition of the samples was done by magnetron sputtering ontosilicon wafers used as substrates. After deposition, a 3.5 nm-thick boron carbide (B 4 C) thin layer was added as a capping layer to prevent samples from oxidation. Different multilayers were deposited on the basis of the Si/[Mg(5.45 nm)/Co(2.45 nm)] 30 /B 4 C (3.5 nm) bi-layer system.The Mg/Co bi-layer system had already been studied by GI-and GE-XRF previously (Jonnard et al., 2014) and is used here with the purpose to demonstrate the improvement of the angular resolution and to reveal that the combination of both GI and GE modes is powerful for the characterization of buried interfaces of already very well studied systems. The two tri-layer samples are : (5.45 With this notation the layers are written in the order of their deposition.

 Si / [Mg
Following deposition, all the samples were characterized by grazing incident x-ray reflectivity at the Cu K wavelength(0.154 nm). The thickness, roughness and density of the each layer were determined by fitting the reflectivity curves using the designed one as a model of the stack.

The results are givenin Table 1 for the tri-layer systems.The stack parameters are close to the designed ones. The roughness, or interface width is limited to around 0.5 nm.

Table 1. Parameters of the tri-layersystems as deduced from the fits of their reflectivity curves.

For each layer, are indicated its thickness (nm), density(g.cm -3 ) and interface width (nm).

Schemes of the experiments

The experimental details had already been given (Jonnard et al., 2014) andhere we only recall the main characteristics of our two experimental procedures, both performed on the same samples. Experiments were performed at the BEAR beamline of the ELETTRA synchrotron radiation facility [START_REF] Nannarone | AIP Conf. Proc[END_REF]. Prior to the XSW experiments, we obtain an XRF spectrum of one sample with a silicon drift detector (SDD) to determine in which spectral region the fluorescence emission of an element, Co Lor Mg K in our case, should be integrated. The incident photon energy to excite the Co L emission was 807.6 eV; it was 1332 eV to excite the Mg K emission.

In the GI-XRF mode, the intensity of an emission is measured as a function of the glancing anglei, i.e. the angle between the synchrotron beam and the sample surface, for angles close to the Bragg angle calculated from the period of the sample and the wavelength of the incident radiation. In the GE-XRF mode, the intensity of an emission is measured as a function of the take-off angle of emissiond, i.e. the angle between the detector and the sample surface, for angles close to the Bragg angle calculated from the period of the sample and the wavelength of the characteristic emitted radiation. This is illustrated in Figure 1.In our case there is a fixed angle of 60° between the directions of the incident andthe detected radiations. It results from this mechanical constraint that d = 120 -i (angles en degrees). In both modes, the SDD is located in the incidence plane, i.e. the plane defined by the incident beam and the normal to the sample surface. are the glancing angle and glancing take-off angle respectively.

Angular resolution in GE-XRF mode

In the GI-XRF the angular resolution is governed by the divergence of the incident beam, that is to say of the synchrotron radiation beam. It is quite small, 0.2°in our experimental conditions.

In the GE-XRF the angular resolution is governed by the aperture of the detector. Taking into account the aperture of the SDD, 5 mm, and its distance from the sample, 300 mm, leads to a 0.9° angular aperture. As shown in Figure 2, to improve the angular resolution, we can insert two slits with width respectively of 1.0 or 0.5 mm, at 140 mm from the sample and 160 mm from the SDD. These configurations correspond to angular acceptances of 0.4 and 0.2°, respectively.The rotation of the slit-holder around the azimuthal axis allows putting the desiredslit in the incidence plane.

Figure 2. Experimental setup: the slit holder can be rotated to put one of the two narrow slits in the axis going from the sample to the detector.

We present in Figure 3, the GE-XRF curves showing the variations of the Mg Kα intensity of the Co/Mg sample with the three possible configurations to check the angular resolution: without slit, and slit of 1.0 mm or 0.5 mm. In all three cases, the main feature around 3°, relative to the first order diffraction of the emitted radiation, is clearly observed. However, the feature regarding the second order diffraction of the emitted radiation, located around 6°, is hardly seen without a slit and is better distinguished when the slit width decreases. This is due to the improvement of the angular resolution, as can be seen on the normalized curves (Fig. 3b). We estimate to have gained a factor 2 on the resolution when going from the configuration with no slit to the one with the 0.5 mm slit. This was estimated from the angular distance between the maximum and the dip of the first order feature. Let us note that the improvement of the resolution is detrimental the collected intensity, which decreases by a factor 3, as can be seen in the raw data (Fig. 3a). However, owing to the large incident flux delivered by the synchrotron, we choose in the following to work in the configuration with the narrowest slit. normalized to the maximum of the first diffraction order feature.Measurement with no detection slit (solid line), with a 1.0 mm slit (dotted line) and with a 0.5 mm slit (dashed line).

Fit of the experimental curves

The fit of the experiment relies on simulations of the intensity of the characteristic fluorescence radiation. The model having been already presented [START_REF] Chauvineau | [END_REF]Jonnard et al., 2014), we recall only its main points. First, the intensity of the exciting electromagnetic field at a given depth in the stack and below a given glancing angle i, is calculated. This is done from the recurrent formalism used to calculate the optical properties of stacks from the optical constants and thicknesses of its various layers. The intensity generated at the same depth from an element of a given concentration is proportional to the square of the electric field and to the concentration. Then, the fluorescence intensity arriving below thetake-off angle dfrom the source to be located is calculated by applying the reciprocity theorem (von Laue, 1935;[START_REF] James | The Optical Principle of the Diffraction of X-rays London: Sir Lawrence Bragg[END_REF]. Let us note that the chosen formalism can be applied to both GI and GE modes. Thus, the fits are performed so that a best agreement is obtained for both GI and GE curves at the same time. This imposes a strict constraint on the fitted parameters. They are the same as the ones determined from the reflectivity measurements (thickness, roughness and density of the layers), which are generally used as input values. The roughness of the various layers is introduced and considered fixed at the value given by the reflectivity measurement. The boron carbide capping layer is also considered with its nominal thickness.

Comparing properly experimental and simulated curves requiresto take into account the effect of the beam imprint on the sample. Indeed, contrary to the case of reflectivity measurements where the specular geometry ensures that the imprint of the incident and reflected beams is the same, this is not the case for GE and GI measurements owing to the asymmetric geometry. Thus in the GI case, a 1/sin(i) factor (Li et al., 2012) is introduced. For the GE mode, no geometrical factor was taken into account since this correction is significant only for the small glancing angles. The simulated curves are then normalized on one point of the experimental curve.

RESULTS AND DISCUSSION

For both Mg K and Co L emissions, we show in Figures 4 and 5the GI-and GE-XRF curves of the Mg/Co/Zr and Mg/Zr/Co multilayers, respectively. The modulationof the intensity is clearly observed on each curve at an angleclose to the one calculated from the Bragg law at the first and seconddiffraction orders.It can be seen that the amplitude of the observed features are quite similar in the case of the Mg K emission of both samples, whereas for the Co L emission, the featuresexhibit a higher contrast for Mg/Co/Zr(Fig. 4c and4d) with respect to the ones of Mg/Zr/Co (Fig. 5c and5d). This is valid for both GI and GEmodes and can be seen in We can deduce that in these multilayers, the interfaces involving the Mg layers, Mg-Co and Mg-Zr, are abrupt. This is in agreement with previous results(Le Guen et al., 2011a;[START_REF] Predel | [END_REF]Zhu et al., 2011) of nuclear magnetic resonance and x-ray emission spectroscopies giving the chemical state of the Co and Mg atoms in the stack, respectively. The analysis of the Co Lα emission is more complicated. First, it must be kept in mind that the emission energy, 776.4 eV (Jonnard & Bonnelle, 2011), is only 2.4 eV away from the Co 2p 3/2 ionization threshold, 778.8 eV (Jonnard & Bonnelle, 2011). That is to say, the Co optical constants used in the simulations have to be chosen in a range where they are strongly varying and also not known with high accuracy. This introduced an uncertainty on the fit of the angular curves. This is in contrast to the case of the Mg Kα emission, where the emission energy, 1253.6 eV (Jonnard & Bonnelle, 2011) is about 50 eV lower than the Mg 1s ionization threshold, 1303.4 eV (Jonnard & Bonnelle, 2011).

For the Co Lα emission of the Mg/Co/Zr system, the simulations reproduce the general shape of the GI and GE curves. These simulations are done with starting parameters deduced from the reflectivity measurements, the optical indices of the CXRO database (http://henke.lbl.gov/optical_constants/) and assuming a stack without inter-diffusion at the interfaces, that is to say by using a tri-layer stack. For the Mg/Zr/Co system, this description fails even to reproduce the contrast of the features, see Fig. 5c and5d. Keeping a tri-layer structure but allowing the variation of the Co optical constants, the agreement between experimental and calculated emission curves is still not satisfying. In consequence, we modify Mg/Zr/Co as a Mg/Co x Zr y bi-layer stack where x and y are the relative numbers of Co and Zr atoms, respectively. The values of x and y, estimated from the number of Co and Zr atoms introduced within the Co and Zr layers in Mg/Zr/Co during its deposition, are found equal to 0.78 and 0.22, respectively, so that x/y is equal to 3.5. Such values are close to the ones of the Co 23 Zr 6 compound present in the Zr-Co binary phase diagram [START_REF] Predel | [END_REF]. The simulations performed by modelling the stack as a Mg/Co x Zr y bi-layer stack greatly improve the agreement with the experiment, see Fig. 5c and5d. A bi-layer stack of the same composition was already used to fit the extreme UV reflectivity curves of a similar system, however with much thicker Mg layers (13 nm), designed to work around 25 nm (Le Guen et al., 2011a). Let us note that regarding the Mg Kcurve, there is no significant difference between the simulations with a trior bi-layer system, as the Mg atoms are not involved in interfacial diffusion.

Thus, the drastic differences between the curves of Mg/Co/Zr and Mg/Zr/Co, allow us to deduce that the Zr-on-Co interfaces in the Mg/Co/Zrmultilayer areabrupt, whereas the Co-on-Zr interfaces in the Mg/Zr/Co multilayer are wide enough to consider the two Co and Zr layers as a single layer. Generally, alternate interfaces in multilayer structure are asymmetric due to the difference in the surface free energy of the constituents of the multilayer. This is analogous to what happens in the particularly well documented for the Mo/Si system (Yulin et al., 2002).

In order to understand the interfacial behaviour of the Mg/Zr/Co system, we calculated the mixing enthalpies of the Co-Mg, Co-Zr and Mg-Zr systems by using the Miedema's"macroscopic atom" model, whose details are given elsewhere (Miedema et al., 1980;Das et al., 2013). This approach already proved useful to explain the interfacial phenomena taking place in annealed Co/Mo 2 C nanometer multilayers (Yuan et al., 2015). The parameters used for the calculations, the electronegativities∅ * , the electron densities at the first Wigner-Seitz boundary 𝑛 𝑤𝑠 and the atomic volumes V, are collected in Table 2. We show in Figure 7

CONCLUSION

Whereas the reflectivity measurement in the hard x-ray range was not enough sensitive to detect the asymmetric behaviour of interfaces in periodic multilayers, XRF generated by soft x-ray XSW, in particular after the improvement of the angular resolution,prove useful to study the structure of periodic multilayer stacks and to characterize their buried interfaces in a nondestructive way.The combination of both GI and GE modes enables to put higher constraints on

Figure 1 .

 1 Figure 1.Experimental configurations for the GI-XRF (a) and GE-XRF (b) experiments.i and d

Figure 3 .

 3 Figure 3. GE-XRF of the Mg Kα emission of the Co/Mg sample: (a) raw data; (b) data

Figures 6

 6 Figures 6(a) and 6(b) where the experimental XSW curves of the Mg/Co/Zr and Mg/Zr/Co

Figure 4 .Figure 5 .

 45 Figure 4.GI-XRF (a) and (c) and GE-XRF (b) and (d) curves for the Mg K (a) and (b) and

Figure 6 .

 6 Figure 6. GI-XRF (a) and GE-XRF (b) curves for the Co L emission ofthe Mg/Co/Zr (full dots) and Mg/Zr/Co (empty dots) multilayers.

  the calculated mixing enthalpies as a function of the mole fraction.The Co-Zr system shows anegative mixing enthalpy over the whole composition range, whereas the Co-Mg and Mg-Zr systems both show only positive values. This indicates that a compound is easy to form at the interfaces between the Co and Zr layers, whichis not possible at the interfaces involving the Mg layers. Moreover, there is a large difference in the surface free energies of Co and Zr, 2.0 J.m -2 and 1.6 J.m -2 respectively[START_REF] Vitos | [END_REF]. Thus, during the deposition of the Co atoms on the Zr layers, since the surface free energy of Zris lower, Co atoms can move on the surface guided by the chemical driving force. This leads to a strong mixing at the interfaces and to the formation of the Co x Zr y compound in the Mg/Zr/Co multilayer. On the other hand, in the Mg/Co/Zr multilayer, during the deposition of the Zr atoms onto the Co layers, no such chemical driving force exists. Therefore the intermixing at Zr-on-Co interface takes place as the result of random thermal motions only and hence the concentration profile is expected to be anabrupt error function.This is consistent with previous results(LeGuen et al., 2011a;[START_REF] Predel | [END_REF]Zhu et al., 2011) on large period samples, demonstrating the good optical performances of Mg/Co/Zr owing to abrupt interfaces, whereas it was shown that Mg/Zr/Co has to be considered as a bi-layer system Mg/Co x Zr y .

Figure 7 .

 7 Figure 7. Calculated mixing enthalpies of the Co-Mg (a), Co-Zr (b) and Zr-Mg (c) systems.

  the fitting parameters of the XSW curves. Working in the soft x-ray range gives sensitivity to the chemical state of the atoms owing to the large variation of the absorption coefficient in the vicinity of an absorption edge. Thus the contrast of the XSW curves depends on the chemical bound of the emitting element. This enables to couple the depth sensitivity of the XSW technique to the elemental and chemical sensitivity of XRF. Also, let us remark, that photon excitation is not mandatory in the GE mode. In this case, XSW intensity measurements can be also made upon electron or ion irradiation, thus do not require a synchrotron facility. The combination of GI-and GE-XRF generated by XSW allows us to determine that Mg/Co/Zr and Mg/Zr/Co multilayers have to be considered as tri-layer and bi-layer stacks respectively, owing to the asymmetric behaviour of the Zr-on-Co and Co-on-Zr interfaces following the positive mixing enthalpy of the Co-Zr system and the different values of the surface free energies of Co and Zr. Thus the Mg/Zr/Co multilayer can be described as Mg/Co x Zr y with x/y around 3.5. ACKNOWLEDGMENT Parts of this work were done in the framework of the international ANR-NSFC COBMUL project (ANR #10-INTB-902-01 and NSFC #11061130549) and of the CaiYuanpei Project (EGIDE PHC No. 30248NF), 973 program (No. 2011CB922203), and National Natural Science Foundation of China (Nos. 11375131 and 11305104). A part of the research leading to these

Table 2 .

 2 Elemental values of ∅ * , 𝑛 𝑤𝑠 and V for calculating the mixing enthalpies of the Co-Mg, Co-Zr, Zr-Mg systems.

	Element ∅ * (V) 𝑛 𝑤𝑠	1/3 (d.u.) 1/3 V(𝑐𝑚 3 /𝑚𝑜𝑙)
	Mg	3.42	1.20	13.97
	Co	5.10	1.77	6.70
	Zr	3.62	1.38	14.10

results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) CALIPSO under grant agreement n°312284.


Víctor González-Castro

Johan Debayle
email: debayle@emse.fr

Yanal Wazaefi

Mehdi Rahim

Caroline Gaudy-Marqueste

Jean-Jacques Grob

Bernard Fertil

Texture descriptors based on adaptive neighborhoods for classification of pigmented skin lesions

Keywords: General adaptive neighborhoods, Local binary patterns, Mathematical morphology, Self-organizing maps, Texture description

In this paper, different texture descriptors are proposed for the automatic classification of skin lesions from dermoscopic images. They are based on color texture analysis obtained from (1) color mathematical morphology (MM) and Kohonen Self-Organizing Maps (SOM) or (2) Local Binary Patterns (LBPs), computed with the use of local adaptive neighborhoods of the image. Neither of these two approaches need a previous segmentation process. In the first proposed descriptor, the adaptive neighborhoods are used as structuring elements to carry out adaptive mathematical morphology operations which are further combined by using Kohonen SOM, and it has been compared with a non-adaptive version. In the second one, the adaptive neighborhoods enable geometrical feature maps to be defined, from which Local Binary Patterns (LBP) histograms are computed. Also, it has been compared with a classical LBP approach. A ROC analysis of the experimental results shows that the adaptive neighborhood-based LBP approach yields the best results. It outperforms the non-adaptive versions of the proposed descriptors and the dermatologists' visual predictions.

Introduction

In 2012 there were more than 11150 new cases of skin cancer in France (3.1% of all detected cancers), 15% of which were mortal. Late diagnosis of skin cancer makes treatments much less efficient (i.e. a melanoma may become very aggressive in just a few months). Therefore, its early detection becomes essential to improve the chances of curing the skin cancer and, thus, the patient's chances of survival. 1 This is not an easy task for a non-experienced observer, as it is shown in figure 1. To carry out the detection of melanoma, dermatologists use several state-of-the-art methods, often called rules, such as the ABCD rule [START_REF] Nachbar | The ABCD rule of dermatoscopy: High prospective value in the diagnosis of doubtful melanocytic skin lesions[END_REF] (Asymmetry, Border irregularity, Color irregularity and Differential structure, i.e. the size and number of structural features) or the Menzies scoring namely the 7-point checklist, [START_REF] Johr | Dermoscopy: alternative melanocytic algorithms-the ABCD rule of dermatoscopy, menzies scoring method, and 7-point checklist[END_REF] which are based on the presence of certain texture patterns. However, although dermatologists could use such kind of analytical pattern recognition rules to predict malignancy, they appear to use more a non-analytical reasoning, derived through experience of prior examples to identify skin lesions. [START_REF] Aldridge | Novice identification of melanoma: Not quite as straightforward as the abcds[END_REF] Some studies suggest that experienced clinicians make a diagnosis intuitively and then they alter their analytical assessment to fit in with their preconceptions about the relationship between these features (e.g., the ABCD) and the diagnosis (e.g., melanoma). [START_REF] Mclaughlin | Is analytic information processing a feature of expertise in medicine?[END_REF][START_REF] Norman | Building on experience the development of clinical reasoning[END_REF] An automation of individual analyses of nevi would be very helpful not only for dermatologists, as they would make more accurate diagnoses and thus make better decisions about the need of surgery, but also for general practitioners, who could make better decisions about sending patients to the specialist more appropriately. Therefore, it would save economic and time resources to the Social Security system. Automatic melanoma detection, based on the analysis of dermoscopy images, has been re-ceiving an increasing attention in the literature. [START_REF] Korotkov | Computerized analysis of pigmented skin lesions: A review[END_REF] Most works reproduce the classic rules used by dermatologists, extracting features linked to them. [START_REF] Tanaka | A study on the image diagnosis of melanoma[END_REF][START_REF] Tenenhaus | Detection of melanoma from dermoscopic images of naevi acquired under uncontrolled conditions[END_REF][START_REF] Isasi | Melanomas non-invasive diagnosis application based on the ABCD rule and pattern recognition image processing algorithms[END_REF][START_REF] Amelard | High-level intuitive features (HLIFs) for intuitive skin lesion description[END_REF] Others go beyond and combine them with other types of characteristics. For example, Rastgoo et al. combine them with bag of words (BoW) descriptors. [START_REF] Rastgoo | Automatic differentiation of melanoma from dysplastic nevi[END_REF] However, such kind of feature extraction methods need a previous segmentation of the lesion, which is often a very tricky step for computers, as it may not be clear, even for specialists, where the boundaries of the lesion are. Indeed, even trained dermatologists differ significantly when delineating the same lesion in separate incidents. [START_REF] Zortea | Automatic segmentation of dermoscopic images by iterative classification[END_REF] There are numerous papers devoted to automatic boundary detection of skin lesions. [START_REF] Glaister | Segmentation of skin lesions from digital images using joint statistical texture distinctiveness[END_REF][START_REF] Celebi | Lesion border detection in dermoscopy images[END_REF][START_REF] Erkol | Automatic lesion boundary detection in dermoscopy images using gradient vector flow snakes[END_REF] Other methods have been investigated in the literature where dermoscopic images do not need any segmentation process as proposed in, [START_REF] Wazaefi | Contribution of a classifier of skin lesions to the dermatologist's decision[END_REF] where a texture descriptor computed by means of the Local Binary Patterns [START_REF] Ojala | A comparative study of texture measures with classification based on featured distributions[END_REF][START_REF] Ojala | Multiresolution gray-scale and rotation invariant texture classification with local binary patterns[END_REF][START_REF] Pietikäinen | Computer Vision Using Local Binary Patterns[END_REF] (LBPs) is used. Nevertheless, only intensity information of the color components are taken into consideration.

In this paper some methods for discriminating automatically between melanoma and benign skin lesions from dermoscopic images with no need of segmentation are proposed. They are based on the General Adaptive Neighborhood Image Processing (GANIP) framework. [START_REF] Debayle | General Adaptive Neighborhood Image Processing: Part I: Introduction and Theoretical Aspects[END_REF][START_REF] Pinoli | General adaptive neighborhood mathematical morphology[END_REF] The first one is based on a texture descriptor using color mathematical morphology (MM) [START_REF] González-Castro | Color Adaptive Neighborhood Mathematical Morphology and its application to pixel-level classification[END_REF] and Kohonen selforganizing maps. [START_REF] Kohonen | Self-Organizing Maps[END_REF] The second one is based on a combination of LBPs and local geometrical measurements. Specifically, the lesions are described by means of the LBPs calculated from maps created by means of geometrical features of the General Adaptive Neighborhoods (GANs) [START_REF] Debayle | General Adaptive Neighborhood Image Processing: Part I: Introduction and Theoretical Aspects[END_REF] of pixels.

The rest of the paper is organized as follows: First of all, a detailed explanation of the required methods and tools, and how these are used to create the descriptors is given in sections 2 and 3, respectively. Afterwards, the performance of these descriptors is assessed on a real dataset of skin lesions, as stated in section 4. Finally, conclusions and future perspectives are shown in section 5.

The required methods and tools

General Adaptive Neighborhoods (GANs)

The GANIP approach [START_REF] Debayle | General Adaptive Neighborhood Image Processing: Part I: Introduction and Theoretical Aspects[END_REF][START_REF] Pinoli | General adaptive neighborhood mathematical morphology[END_REF] provides a general framework for multiscale, local and adaptive image processing and analysis of gray-level images. It is based on extracting spatial neighborhoods called General Adaptive Neighborhoods (GANs) from the points of the image, whose size and shape are adapted to the local features of the image. Specifically, a GAN is a subset of the spatial support D ⊆ R 2 constituted by connected points whose values in relation to a selected criterion (luminance, contrast, etc.) fit within a homogeneity tolerance. As a result, GANs are adaptive with the spatial structures and self-defined from the image.

Let f be an image defined in D with range in R, and let h be a criterion mapping, also defined in D and valued in R, based on local measurements such as luminance, contrast, etc. For each point x ∈ D, the GANs (denoted V h m (x)) are subsets in D built upon h in relation to a homogeneity tolerance m ∈ R + . More precisely, V h m (x) fulfills two conditions: (i) its points are close to x in relation to the criterion mapping and (ii) the GAN is a path-connected set.

Thus, the GANs are formally defined as:

V h m (x) = C {y∈D: |h(y)-h(x)|≤m} (x), (1)
where However, these GANs do not satisfy the symmetry property -which is relevant for visual, topological, morphological and practical reasons [START_REF] Debayle | General Adaptive Neighborhood Image Processing: Part I: Introduction and Theoretical Aspects[END_REF] -, defined as:

C X (x) denotes the path connected component of X ⊆ D containing x ∈ D. Therefore, it is ensured that ∀x ∈ D x ∈ V h m (x).
∀ x, y ∈ D y ∈ A(x) ⇐⇒ x ∈ A(y), (2)
where {A(x)} x∈D is a collection of subsets A(x) ⊆ D. For this reason, GANs defined in equation

(1) are called Weak General Adaptive Neighborhoods (W-GANs).

In order to get this property satisfied, a new set of GANs, called Strong General Adaptive Neighborhoods (S-GANs) is defined as:

N h m (x) = z∈D {V h m (z) : x ∈ V h m (z)}. (3)
The reader interested in further theoretical aspects on GANs is referred to. [START_REF] Debayle | General Adaptive Neighborhood Image Processing: Part I: Introduction and Theoretical Aspects[END_REF]

Color Mathematical Morphology

Morphological operators 24 need the sets of the intensities to be processed to hold a total order relationship. However, in the case of color images this is not straightforward due to the vectorial nature of their points.

In the literature several order relationships have been proposed (i.e., marginal, lexicographical, partial or reduced ordering). In this work the so-called Ω-ordering -denoted as ≺ Ω -, proposed by Angulo, [START_REF] Angulo | Morphological colour operators in totally ordered lattices based on distances: Application to image filtering, enhancement and analysis[END_REF] has been used:

c 1 ≺ Ω c 2 =                                d RGB (c 1 , c 0 ) > d RGB (c 2 , c 0 ) or d RGB (c 1 , c 0 ) = d RGB (c 2 , c 0 ) and                c R 1 < c R 2 or c R 1 = c R 2 and c G 1 < c G 2 or c R 1 = c R 2 and c G 1 = c G 2 and c B 1 < c B 2 (4)
where

d RGB (c 1 , c 2 ) represents the distance between two points c 1 = (c R 1 , c G 1 , c B 1 ) and c 2 = (c R 2 , c G 2 , c B 2 )
in the color space RGB (see equation ( 5)) and c 0 stands for the reference color, which in this work has been set to c 0 = (255, 255, 255). Regarding the definition in equation ( 4), the reference color is an upper bound for the colors and, in the RGB color space, the intuitive "biggest color" is the white, i.e., (255, 255, 255). That is the reason why that choice for c 0 has been made. It is easy to prove that ≺ Ω is a total order relationship. This order can be used with any color space. More details about color ordering can be found in. 22

d RGB (c 1 , c 2 ) = (c R 1 -c R 2 ) 2 + (c G 1 -c G 2 ) 2 + (c B 1 -c B 2 ) 2 (5)
Let x be a point of the spatial support D ⊆ R 2 , the resulting classical color erosion and dilation of an image f : D → R 3 at point x by means of a disk B r of radius r as structuring element (SE)

is given respectively by:

E r (f )(x) = inf Ω {f (w) : w ∈ B r (x)} (6) D r (f )(x) = sup Ω {f (w) : w ∈ Br (x)}, (7)
where sup Ω and inf Ω stand for the supremum and infimum according to the total order relationship ≺ Ω . It is possible to define more advanced operators by combining dilations and erosions such as openings, closings, alternate filters, toggle contrast, top hat, etc.

Color Adaptive Neighborhoods (CANs) and Mathematical Morphology

The idea behind adaptive mathematical morphology is to replace the classical spatially invariant (i.e. with fixed shape and size) SE by spatially variant (i.e. adaptive) SEs. In this way, the Color Adaptive Neighborhoods (CANs) -an extension to color of the General Adaptive Neighborhoods (GANs) [START_REF] Debayle | General Adaptive Neighborhood Image Processing: Part I: Introduction and Theoretical Aspects[END_REF][START_REF] Debayle | General Adaptive Neighborhood Image Processing: Part II: Practical Application Examples[END_REF] -, defined in, [START_REF] González-Castro | Color Adaptive Neighborhood Mathematical Morphology and its application to pixel-level classification[END_REF] can be used as adaptive structuring elements for color morphological operators. The Color Adaptive Neighborhood (CAN) of a point x, denoted V f m (x), is defined as a spatial region included in D built upon the color image f in relation with a homogeneity tolerance value m ∈ R + . More precisely, V f m (x) must fulfill the same conditions as the GANs (section 2.1), i.e., (i) its points must have a color value close to the one of x, and (ii) the set has to be path-connected, considering the usual Euclidean topology on D ⊆ R 2 .

Therefore, CANs are formally defined as:

V f m (x) = C {y∈D; d RGB (f (y),f (x))≤m} (x), (8)
where C X (x) denotes the path-connected component of X which contains x ∈ D, and d RGB (f (y), f (x))

stands for the distance between the color points f (y) and f (x) in the color space RGB.

However, if they were used directly as Adaptive Structuring Elements (ASEs), the symmetry property, i.e. x ∈ V f m (y) ⇐⇒ y ∈ V f m (x) may not be satisfied. Therefore, the so-called Strong

Color Adaptive Neighborhoods (S-CANs) are used as ASEs:

N f m (x) = z∈D {V f m (z)| x ∈ V f m (z)} (9)
Thus, the elementary adaptive morphological operators of erosion and dilation are defined respectively as:

E m (f )(x) = inf Ω {f (w) : w ∈ N f m (x)} (10)
D m (f )(x) = sup Ω {f (w) : w ∈ N f m (x))} (11)

GAN-based Minkowski functionals

Integral geometry provides a suitable family of geometrical and topological descriptors of 2-D and 3-D spatial patterns, known as Minkowski functionals. [START_REF] Michielsen | Integral-geometry morphological image analysis[END_REF] In 2-D, there are three Minkowski functionals: area, perimeter and Euler number, denoted respectively A, P and χ.

These functionals are defined on the class of nonempty compact convex sets in R 2 . They have been extended to the convex ring, [START_REF] Mecke | Statistical Physics and Spatial Statistics[END_REF] i.e. the set of all finite unions of convex bodies, which may be considered as a realistic Euclidean model for digital images. In this paper, the densities of these functionals are used (i.e. the functionals are normalized by the area of the spatial support D). The densities of the area, perimeter and Euler number are denoted A A , P A and χ A , respectively.

The GAN-based Minkowski maps [START_REF] Rivollier | Integral geometry and general adaptive neighborhood for multiscale image analysis[END_REF] are defined by assigning to each point x ∈ D the Minkowski density functional of its GAN V h m (x). More explicitly, the GAN-based Minkowski map of a graylevel image, denoted by µ h m , is defined by:

µ h m (x) = µ(V h m (x)), (12)
where µ denotes a Minkowski density functional (i.e. µ ≡ A A , µ ≡ P A or µ ≡ χ A ).

Local Binary Patterns

Local Binary Patterns (LBP) were introduced by Ojala et al. [START_REF] Ojala | A comparative study of texture measures with classification based on featured distributions[END_REF] This original version worked in a 3 × 3 pixel block of an image, and was later generalized [START_REF] Ojala | Multiresolution gray-scale and rotation invariant texture classification with local binary patterns[END_REF] to remove any limitations on size of the neighbor or the number of sampling points. LBP is a grey level texture descriptor that extracts the local spatial structure of an image. Given a pixel, a pattern code is computed by comparing it with the value of its neighbors, as stated in equation (13).

LBP P,R (x c , y c ) = P -1 p=0 s(g p -g c )2 p , (13)
where g c is the value of the central pixel (x c , y c ), g p is the value of its p th neighbor, P is the number of neighbors and R is the radius of the neighborhood. The functions(z) is defined as:

s(z) =        1, z ≥ 0 0, z < 0 (14)
Finally, the whole image is described by means of a histogram of LBP values of all pixels.

The proposed descriptors

In this work we propose two texture descriptors of the color images: the first one is based on color mathematical morphology and Self-Organising Maps, and the other one is based on Local Binary Patterns computed on the GAN-Minkowski maps of the R, G and B components of the images.

CANMM-based descriptor

The first Color Adaptive Neighborhood Mathematical Morphology (CANMM)-based descriptor is computed in two steps. First of all, the images are described locally, based on the chromatic information of each point in several successive dilations and erosions. Afterwards, these local descriptors are used to describe the image globally by means of a Self-Organizing Map. [START_REF] Kohonen | Self-Organizing Maps[END_REF]

Pixel-level description

The descriptors of each pixel consist on a concatenation of (i) its color components in the original image and (ii) the color components of the same point in multiscale dilations and erosions (i.e., using structuring elements of different sizes p i ). For classical (resp. adaptive) mathematical morphology, p i denotes the radius (resp. homogeneity tolerance) of the SE (resp. adaptive structuring element).

Therefore, the general expression (i.e. for either classical or adaptive mathematical morphology) of the descriptor of each pixel x ∈ D is:

X n (x) = [E pn (f )(x), ..., E p 1 (f )(x), f (x), D p 1 (f )(x), ..., D pn (f )(x)] (15)
where n stands for the number of erosions and dilations which are carried out, f (x) represents the values of the color components of the image f at x and E p i (f )(x) (resp. D p i (f )(x)) represents the values of the color components at x of the erosions (resp. dilations) of f with a structuring element with size or tolerance p i . Figure 4 shows an illustration of such concatenation.

Global image descriptor

Once the pixels of an image are described, a global descriptor of that image is built. This is done by means of a Kohonen Self-Organising Map. [START_REF] Kohonen | Self-Organizing Maps[END_REF] This map has been generated by means of 20 images selected by experts (i.e., 10 representative images of each class), from which the lesions were roughly segmented manually. Thereafter, the map was generated in a training step using the pixels of the lesions and 5% of the other pixels (randomly selected). Note that this segmentation is only used for these 20 training images.

Afterwards, the pixels of the image are projected onto the neurons of the map in order to cluster them. Finally, the final image descriptor is the histogram of the hits of the neurons, i.e. the number of pixels that have been projected on each neuron of the map.

A diagram showing the description process is shown in figure 5.

GAN-Minkowski LBP descriptor

The proposed texture descriptor based on LBP is built in two steps.

First of all, the GAN-based Minkowski map µ h m of the different color components R, G and B of the original color image is computed. Thereafter, the LBP P,R operator (eq. 7) of each of these maps is computed, and the three histograms are concatenated. A graphic description of this process is shown in figure 6. 4 Experiments and results

Experiments

Image database

The image dataset that has been used in this experiment was composed of 1097 dermoscopic images of pigmented skin lesions, 88 of them being histopathologically confirmed melanomas.

Thus, two classes were considered in this experiment: on the one hand confirmed melanomas and on the other hand the remaining benign lesions.

All images were acquired by several dermatologists equipped with a digital camera (SONY W120) combined with a Heine Delta 20 dermoscope. After the acquisition was carried out, the images were normalized so that they have the same pixel size.

Image descriptors

Three different experiments have been done in this work, using either the CANMM-based descriptor (section 3.1), the GAN-Minkowski LBP descriptor (section 3.2) or a combination of both.

First of all, concerning the descriptor based on color mathematical morphology, the local pixel descriptor described in section 3.1.1 has been computed using (i) the adaptive CAN-based erosions and dilations and (ii) classical (i.e. non adaptive) erosions and dilations, for the sake of comparison.

In this case, a square-shaped structuring element has been used, with width sizes varying between 5 and 50 in steps of 5. In the case of the adaptive CAN-based erosions and dilations, the values for the tolerance m varied from 5 to 50 in steps of 5. Therefore, in both cases 10 different erosions and dilations were carried out, so each pixel was described by means of 63 features (i.e. the RGB components of the pixel in the original image and its components after the erosions and dilations). After that, the LBP P,R was computed from each of these three Area maps. The number of samples P was fixed to 8, but different possible values for the radius R were assessed (specifically, they varied from 1 to 6). Thus, the LBP histogram of each component has a length of 256. The final descriptor is a concatenation of the three LBP histograms (i.e. the final descriptor has 768 features).

For the sake of comparison, other descriptor where the LBP P,R operator was computed directly on the intensities of the color components R, G and B has been assessed.

Finally, a concatenation of these two proposed descriptors has been considered, both the adaptive (i.e., the CANMM-based concatenated with the GAN-Minkowski LBP descriptors) and the classic versions (i.e., the first descriptor computed with non-adaptive mathematical morphology concatenated with the LBP P,R of the intensities of the color components R, G and B). Therefore, this combined descriptor has 1168 features per image.

Classification

Images were subsequently classified by means of a feed-forward Artificial Neural Network (ANN)

working on their feature vectors. The data were normalized before classification, so that they had mean zero and standard deviation one. In this experiment a network with one hidden layer and a logistic sigmoid activation function for the hidden and output layers have been employed. The learning of the network was carried out with the momentum and adaptive learning rate algorithm.

Different combinations of training cycles and neurons in the hidden layer have been used, in order to assess the impact of this configuration on the results.

The classification was carried out using stratified 10-fold cross validation, and the process was repeated 10 times, in order to avoid possible random effects (e.g. due to the random initialization of the network) and over-fitting. The presented results are an average of these 10 runs.

Results

It is necessary to remark that no pre-processing of the data was done in order to prevent the bias of the classifier towards the majority class. However, as the dataset is highly imbalanced towards the the performance of a classifier than the accuracy of the classification. [START_REF] Fawcett | An introduction to ROC analysis[END_REF] It is also widely used in visualizing and analyzing the behavior of diagnostic systems. It summarizes the classifier performance over a range of tradeoffs (i.e., decision thresholds) between benefits (i.e., true positives or sensitivity) and costs (i.e., false positives or 1-specificity). [START_REF] Chawla | Data mining for imbalanced datasets: An overview[END_REF] More details about ROC curves can be found in. [START_REF] Fawcett | An introduction to ROC analysis[END_REF] A measure that summarizes how good this curve is is the area under the ROC curve (AUC). In the case of the descriptors based on mathematical morphology, the neural network configurations (i.e. neurons in the hidden layer and training cycles) that achieved the best results were 300 cycles and 7 neurons, and 400 cycles and 9 neurons for the classical and adaptive approaches with AUCs of 0.859 and 0.854, respectively. In addition, tables 1 Figure 7 depicts the ROC curves of these classifiers along with a mean ROC curve estimated in [START_REF] Wazaefi | Contribution of a classifier of skin lesions to the dermatologist's decision[END_REF] from the predictions carried out by nine dermatologists with this same image dataset. The AUC of of this curve is 0.792. Other works that deal with the problem of melanoma detection in the literature report similar sensitivities and higher specificities than the ones achieved in this work. For example, Tenenhaus et al. [START_REF] Tenenhaus | Detection of melanoma from dermoscopic images of naevi acquired under uncontrolled conditions[END_REF] reported a sensitivity of 95% and a specificity of 60%, although the overall AUC that their classifier achieved was 0.84. Amelard et al. [START_REF] Amelard | High-level intuitive features (HLIFs) for intuitive skin lesion description[END_REF] is more important, the dataset is highly imbalanced -only 8% of the images are melanoma -, while in the case of these works the datasets are either balanced or the proportion of melanoma is 30%. By the way, Wazaefi et al. [START_REF] Wazaefi | Contribution of a classifier of skin lesions to the dermatologist's decision[END_REF] did use the same dataset that was used in this paper, and their classifier achieved an AUC of 0.885. Anyway, as Koratov and García pointed out, [START_REF] Korotkov | Computerized analysis of pigmented skin lesions: A review[END_REF] the absence of benchmark datasets for standardized algorithm evaluation makes difficult to compared different works.

Results with combined descriptors

The concatenation of both the "adaptive" and "classical" (i.e. non adaptive) descriptors were classified following the same experimental configuration as the proposed ones.

As done in previous section, tables 4 and 3 show the AUC in the best classification scenario achieved by the concatenated descriptors in their adaptive and non adaptive versions, respectively, depending on the different assessed values of R. Once again, the highest AUC is shown in bold.

It is remarkable than the non adaptive version of these concatenated descriptors performs better than the adaptive one. A possible explanation is that, although the adaptive version of LBP shows better performance than the "classic" one (AUCs 0.9115 against 0.8948), the non-adaptive MM- Processing high dimensional data is computationally expensive and, due to possible correlations in the data (e.g. the colors of some dilations and erosions might be similar), it is also inefficient. Therefore, a dimensionality reduction using Principal Component Analysis 32 (PCA) has been carried out, with a number of principal components up to 20 (more than 10 were enough in all cases to explain more than 99% of the variance of the dataset. However, the results using these "reduced" data were worse than the "non reduced" version. and the radius R varied, being the best results achieved for R = 6. This GAN approach achieved an AUC of 0.912, while the classical approach achieved an AUC of 0.895. The descriptors based on mathematical morphology achieved similar performances, getting AUCs of 0.859 and 0.854 in the case of the non-adaptive and adaptive approaches, respectively. Any of these descriptors outperformed the dermatologists' predictions on the same image dataset, which obtained an average ROC curve with an AUC of 0.792. [START_REF] Wazaefi | Contribution of a classifier of skin lesions to the dermatologist's decision[END_REF] For future work, other GAN-based geometrical and/or morphometrical features [START_REF] Rivollier | Adaptive shape diagrams for multiscale morphometrical image analysis[END_REF] applied to this problem will be investigated.

Fig 1

 1 Fig 1 Example of an image of a benign lesion (a) and a melanoma (b).

Figure 2

 2 Figure2shows the GAN computed for one pixel of a dermoscopic color image using the

Fig 2

 2 Fig 2 The GAN (b) of one pixel (a) of a dermoscopic image computed on the red color component, using the luminance criterion with the homogeneity tolerance value m = 20.

Figure 3

 3 Figure 3 shows an example of GAN-based Minkowski map of an image using the area as

Fig 3

 3 Fig 3 The GAN-based area map (b) of the green component (a) of a dermoscopic image computed with the luminance criterion and the homogeneity tolerance value m = 20.

Fig 4

 4 Fig 4 Illustration of the concatenation of color components to form the pixel descriptor using color adaptive mathematical morphology.

Fig 5

 5 Fig 5 Diagram of the image description process.

Fig 6

 6 Fig 6 Image description process.

Afterwards, a Kohonen map of size 20 ×

 20 20 was used as explained in section 3.1.2. Therefore, each image has been described by means of a feature vector of 400 elements. Considering the descriptor based on the LBP of the CAN-based Minkowski maps, as it was explained in section 3.2, each of the color components R, G and B of the images of skin lesions has been considered as a grey-level image. First of all, the GAN-based Minkowski map Area with tolerance 20 was computed from each of them (i.e. A R 20 , A G 20 and A B 20 ). The other two Minkowski functionals P and χ, as well as different tolerances m were assessed, but they yielded worse results.

  and 2 show the AUC in the best classification scenario (i.e. the best configuration of the neural network) achieved by the descriptors based on the LBP computed directly on the intensities of the color components and computed on the GAN-Area maps, respectively, depending on the different assessed values of R. The highest AUC is shown in bold.

Fig 7 ROC

 7 Fig 7 ROC Curves of the classifiers generated by means of the Classic Mathematical Morphology, CAN-based Mathematical Morphology, "classical" and GAN-Area-based LBP, together with the mean ROC curve observed for the dermatologists.

5

  Conclusions and future perspectives In this work, two texture descriptors has been introduced and applied to the description and classification of images of naevi as benign lesions or melanoma. The first one is built from the local color data of each pixel, obtained from mathematical morphology operations. It is afterwards used to describe the image globally, by means of Kohonen Self-Organizing Maps. Two approaches of this descriptor has been assessed: (i) the local data has been obtained using the classic (i.e. non adaptive) color mathematical morphology operations and (ii) getting this local data by means of adaptive color mathematical morphology based on the CAN framework. The second descriptor is computed from LBP and local geometrical features (the Minkowski functionals) computed from the GANs of each color component of the image. Its performance has been compared with the classical LBP computed from the intensities of each color component. The best performance has been obtained by the GAN-Minkowski based LBPs, using the area and a tolerance for the GANs equals to 20. The number of samples of the LBPs was fixed to 8

Table 1

 1 AUC of the best combination cycles-neurons in the ANN for the descriptors based on the "classical" LBP.

	R Num. neurons Num. cycles AUC
	1	10	300	0.8726
	2	7	300	0.8948
	3	7	400	0.8934
	4	5	500	0.8946
	5	7	400	0.8895
	6	10	400	0.8898
	benign lesion class, the receiver operating characteristics (ROC) curve is more suitable to illustrate

Table 2

 2 AUC of the best combination cycles-neurons in the ANN for the descriptors based on the GAN-Area-based LBP.

	R Num. neurons Num. cycles AUC
	1	10	500	0.8547
	2	10	500	0.8780
	3	10	500	0.8934
	4	7	300	0.8976
	5	10	500	0.9052
	6	10	500	0.9115

  obtained a sensitivity and a specificity of 92.52% and 73.45%, respectively and Rastgoo et al.[START_REF] Rastgoo | Automatic differentiation of melanoma from dysplastic nevi[END_REF] 98% and 70% of sensitivity and specificity. However, let us remember that in this case previous segmentation of the lesions is not carried out and, what

Table 3

 3 AUC of the best combination cycles-neurons in the ANN for the adaptive concatenated descriptors.

	R Num. neurons Num. cycles AUC
	1	7	300	0.8604
	2	5	300	0.8660
	3	5	300	0.8699
	4	5	300	0.8729
	5	5	400	0.8799
	6	5	300	0.8771

Table 4

 4 AUC of the best combination cycles-neurons in the ANN for the "classical" concatenated descriptors.

	R Num. neurons Num. cycles AUC
	1	5	300	0.8790
	2	7	300	0.8928
	3	10	300	0.8946
	4	5	300	0.8968
	5	5	300	0.8877
	6	10	300	0.8865
	based descriptors slightly outperformed the adaptive one (0.859 and 0.854, respectively). Conse-

quently, it seems that the MM-based descriptor has more weight for the classifier. Anyway, both versions of the concatenated descriptors obtained better results than the dermatologists, the mathematical morphology based descriptors (both the adaptive and non adaptive) and the "classical" LBP-based descriptors. Still they do not outperform the adaptive LBP-based descriptors

Acknowledgments

This work has been supported by the project with reference ANR-12-EMMA-0046 from the French National Research Agency.


Tamsin Gannon

Rahat Perveen

Hélene Schlecht

Simon Ramsden

Beverley Anderson

Bronwyn Kerr

Ruth Day

Siddharth Banka

Mohnish Suri

Siren Berland

Michael Gabbett

Alan Ma

Stan Lyonnet

Valerie Cormier-Daire

Rüstem Yilmaz

Guntram Borck

Dagmar Wieczorek

Britt-Marie Anderlid

Sarah Smithson

Julie Vogt

Heather Moore-Barton

Pelin Ozlem Simsek-Kiper

Isabelle Maystadt

Anne Destrée

Jessica Bucher

Brad Angle

Shehla Mohammed

Emma Wakeling

Sue Price

Amihood Singer

Yves Sznajer

Annick Toutain

Damien Haye

Ruth Newbury-Ecob

Melanie Fradin

Julie Mcgaughran

Beyhan Tuysuz

Mark Tein

Katelijne Bouman

Tabib Dabir

Jenneke Van Den Ende

Ming Ho

Luk

Daniela T Pilz

Jacqueline Eason

Sally Davies

Willie Reardon

Livia Garavelli

Orsetta Zuffardi

Koen Devriendt

Ruth Armstrong

Diana Johnson

Martine Doco-Fenzy

Emilia Bijlsma

Sheila Unger

Hermine E Veenstra-Knol

Jürgen Kohlhase

Ivan Fm Lo

Janine Smith

DDD Jill Clayton-Smith
email: jill.clayton-smith@cmft.nhs.uk

Further delineation of the KAT6B molecular and phenotypic spectrum

Keywords: KAT6B, Say-Barber-Biesecker, genitopatellar, blepharophimosis

INTRODUCTION

The Say-Barber-Biesecker syndrome (SBBS, MIM 603736), also referred to as the Say-Barber-Biesecker variant of Ohdo syndrome 1 is a rare multiple congenital anomaly syndrome which is usually diagnosed clinically on the basis of a striking facial phenotype. Typically, patients have a distinctive mask like face with severe blepharophimosis and ptosis, a broad nasal bridge, bulbous nasal tip, small mouth, thin upper lip and small, low set ears. Long thumbs and long great toes may be present along with congenital heart defects, thyroid abnormalities, hypoplastic teeth and absent or hypoplastic patellae 2 . Genital anomalies, usually cryptorchidism are universally present in males and cleft palate has been observed in a number of cases . All individuals with SBBS reported so far have had global developmental delay and severe intellectual disability. Patients with features overlapping those of SBBS were reported by Young and Simpson [START_REF] Young | Unknown syndrome: abnormal facies, congenital heart defects, hypothyroidism, and severe retardation[END_REF] in 1987 and it is now generally agreed that Young-Simpson syndrome and SBBS are in fact the same entity. Young and Simpson were the first to draw attention to the presence of hypothyroidism in this condition. Clayton-Smith et al [START_REF] 'sullivan | Whole-Exome-Sequencing Identifies Mutations in Histone Acetyltransferase Gene KAT6B in Individuals with the Say-Barber-Biesecker Variant of Ohdo Syndrome[END_REF] identified KAT6B, a histone acetyltransferase, as the causative gene for SBBS by a whole exome sequencing approach and reported sequence variants in 13 individuals. Subsequently, Szakszon identified a further two SBBS individuals with KAT6B sequence variants [START_REF] Szakszon | De novo mutations of the gene encoding the histone acetyltransferase KAT6B in two patients with Say-Barber/Biesecker/Young-Simpson syndrome[END_REF] and another individual was reported recently by Yu et al [START_REF] Yu | An individual with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and additional features expands the phenotype associated with mutations in KAT6B[END_REF] . In addition, KAT6B sequence variants have been reported in 11 patients with genito-patellar syndrome (GPS, MIM 606170), a further multiple anomaly phenotype previously considered to be quite distinct from SBBS [START_REF] Campeau | Mutations in KAT6B, Encoding a Histone Acetyltransferase, Cause Genitopatellar Syndrome[END_REF][START_REF] Simpson | De Novo Mutations of the Gene Encoding the Histone Acetyltransferase KAT6B Cause Genitopatellar Syndrome[END_REF] . In GPS the overall clinical picture tends to be more severe than in SBBS [START_REF] Cormier-Daire | Genito-patellar syndrome: a new condition comprising absent patellae, scrotal hypoplasia, renal anomalies, facial dysmorphism and mental retardation[END_REF] . The main diagnostic features of GPS include the presence of large joint contractures, ambiguous genitalia and absent patellae. Agenesis of the corpus callosum, hydronephrosis and congenital heart disease are frequently present and thyroid abnormalities have been reported in rare cases. The facial phenotype of GPS has not been well described but is not considered so distinctive as in SBBS, with the diagnosis usually being made based on the other clinical features.

To further delineate the clinical and molecular spectrum of KAT6B we have studied a new cohort of 57 patients with a clinical diagnosis of SBBS (n = 47), GPS (n = 5), or features overlapping both of these entities (n=5). We have gathered detailed clinical information on this group and screened for sequence variants in the coding exons of KAT6B by Sanger sequencing. Our study aimed to define differences in clinical features between KAT6B variant-positive and KAT6B variant-negative patients and to explore the breadth of the phenotypic spectrum, examining in particular, the clinical overlap between SBBS and GPS.

PATIENTS AND METHODS

Ascertainment of study patients

In 2011 we ascertained a cohort of 19 patients with SBBS for genetic studies and identified likely pathogenic KAT6B sequence variants in 13 of these [START_REF] 'sullivan | Whole-Exome-Sequencing Identifies Mutations in Histone Acetyltransferase Gene KAT6B in Individuals with the Say-Barber-Biesecker Variant of Ohdo Syndrome[END_REF] . For this current study we have gathered a cohort of a further 57 patients with features suggestive of SBBS or GPS who were recruited via clinical geneticists from within the UK or abroad. Forty-five patients were tested for KAT6B sequence variants as part of a research study exploring the genetic basis of blepharophimosis syndromes (REC reference 10/H1016/12). Variants in a further nine patients were identified in the diagnostic laboratory within the Manchester Centre for Genomic Medicine.

Three further individuals (23, 24, 25) were referred to our study after KAT6B variants had been identified in laboratories in Ulm (2) and Freiburg (1). Individuals 23 and 24 had originally been ascertained as part of a study of the UBE3B gene 10 but were consider to have features more suggestive of a KAT6B phenotype. Referring clinicians provided detailed phenotypic information and photographs of participants for review. Consent to publish clinical details was obtained from all participating families and consent to publish photographs was obtained for those included in this report

Sequencing of KAT6B

Samples from the 54 individuals studied in Manchester were screened for sequence variants in all 18 exons of KAT6B by Sanger sequencing. Details of primer design and synthesis are given in Supplementary Information Table 1. The coding exons and the intron-exon boundaries of KAT6B were amplified by polymerase chain reaction. Post-PCR purification was conducted to remove unwanted reaction components using the Agencourt AMpure XP (Beckman Coulter Genomics, Takeley, UK) system on a Biomek NX Liquid Handler (Beckman Coulter). The purified PCR products were then subjected to sequencing using BigDye Terminator v3.1 (Invitrogen). Reactions were purified using Agencourt CleanSEQ solution (Beckman Coulter) by Beckman Coulter NX robotics. Automated capillary electrophoresis was conducted on the ABI 3730XL Genetic Analyzer (Applied Biosystems) using POP-7 polymer (Life Technologies) according to local protocols. The reference sequence of KAT6B NG_032048.1, covering transcript NM_012330.3 was uploaded as a txt.file to Staden package Pregap4 1.4b1. The sequence data was aligned and analysed graphically using the DNA sequence analysis software, STADEN (http://staden.sourceforge.net/). Any variants identified were checked against the NCBI SNP database (dbSNP, http://www.ncbi.nlm.nih.gov/SNP/) and against the Exome Variant Server (http://evs.gs.washington.edu/EVS/) to distinguish common variants from likely pathogenic variants. The variant interpretation software Alamut, (http://www.interactivebiosoftware.com/software/alamut/features) was used to interpret and predict the consequences of identified variants. Sequence variants identified were reported according to the Human Genome Variation Society nomenclature. Where available, DNA from the parents of the patients with possible pathogenic variants was screened to investigate if the change was de novo. Three patients (numbers 23, 24 and 25) were sequenced in other genetic laboratories as reported above using similar methodology, but in cases 23 and 24 only exon 18 was sequenced. Taking into account the clinical context i.e. all patients had been selected based on a clinical diagnosis of SBBS or GPS, sequence variants were classified as causative or not. Variants resulting in a truncated protein were classified as causative. Variants deemed to be likely causative were missense changes which were proven to be de novo or if in silico analysis predicted them to affect splicing or highly conserved amino acids. Sequence changes which were synonymous and did not affect splicing, missense changes reported in EVS or those seen in a parent were classified as being unlikely to cause diasease. Details of causative variants were submitted to the LOVD public database at http://databases.lovd.nl/shared/genes/KAT6B, ( patient IDS 00018483-00018515)

Genotype-phenotype analysis

Patients were classified as having either a SBBS phenotype (n =47) a GPS phenotype (n=5) or an overlapping phenotype (n=5) by dysmorphologists from the Manchester Centre For Genomic Medicine on the basis of clinical information and photographs. In accordance with previous literature, large joint contractures, patellar abnormalities and ambiguous genitalia/severe genital anomalies were considered core features of GPS [START_REF] Cormier-Daire | Genito-patellar syndrome: a new condition comprising absent patellae, scrotal hypoplasia, renal anomalies, facial dysmorphism and mental retardation[END_REF] . For SBBS the chracteristic facial features of blepharophimosis and mask-like face were the key diagnostic features. To analyse differences in phenotypic features between variant-positive and variant-negative patients we studied 45 variantpositive patients for which we had detailed clinical information ( Supplementary Table 2a). This group included the 13 patients we had reported previously [START_REF] 'sullivan | Whole-Exome-Sequencing Identifies Mutations in Histone Acetyltransferase Gene KAT6B in Individuals with the Say-Barber-Biesecker Variant of Ohdo Syndrome[END_REF] . We compared their clinical phenotypes with those of 23 variant-negative patients (Supplementary Table 2b) including 5 reported previously (patients 71-75) [START_REF] 'sullivan | Whole-Exome-Sequencing Identifies Mutations in Histone Acetyltransferase Gene KAT6B in Individuals with the Say-Barber-Biesecker Variant of Ohdo Syndrome[END_REF] . Differences between the presence of eleven commonly presenting features in SBBS and GPS were analysed using the Fisher's Exact test. We noted the type and position of the causative and likely causative variants within the SBBS group, the GPS group and the group with the overlapping phenotype. Comparison was also made with sequence data from 28 patients with KAT6B sequence variants previously reported in the literature ( 11 GPS and 17 SBBS).

RESULTS

Sequence analysis

Sequencing of all 18 coding exons and intron-exon boundaries of KAT6B in our new cohort of 57 patients identified 34 causative or likely disease-causing KAT6B variants. Of these, 26/47 SBBS individuals had such variants, 4/5 GPS patients and 4/5 of those with an overlapping phenotype. The majority (29/34) were truncating variants, but 1 was a missense variant, 1 was a synonymous change shared by 3 unrelated SBBS patients and 1 was an in-frame deletion. The sequence variants are summarised in Supplementary Table 3 which also incorporates the findings from our original cohort [START_REF] Young | Unknown syndrome: abnormal facies, congenital heart defects, hypothyroidism, and severe retardation[END_REF] and previous reports [START_REF] 'sullivan | Whole-Exome-Sequencing Identifies Mutations in Histone Acetyltransferase Gene KAT6B in Individuals with the Say-Barber-Biesecker Variant of Ohdo Syndrome[END_REF][START_REF] Szakszon | De novo mutations of the gene encoding the histone acetyltransferase KAT6B in two patients with Say-Barber/Biesecker/Young-Simpson syndrome[END_REF][START_REF] Yu | An individual with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and additional features expands the phenotype associated with mutations in KAT6B[END_REF] . In addition, we observed a recurrent sequence variant which we considered not causative of disease, consisting of a 12bp sequence in exon 16 c. 3252_3263 p.(Glu1086_1089) which was either deleted or duplicated. The majority of the causative sequence variants identified clustered at the C-terminal end of the gene in exon 18, as documented previously. This exon encodes a highly conserved serine and methionine-rich transcriptional activation domain. However, we also identified variants that occurred more proximally in exon 18 and in exons 15, 16 and 17. Three individuals shared an identical de novo synonymous variant in exon 16, c.3147G>A p.(Pro1049Pro). The Alamut 2.2 prediction programme suggested that this change was likely to create a cryptic splice site. All three individuals had the typical SBBS phenotype. One patient had a missense variant in exon 15, c.2959T>C p.(Trp987Arg). The parents of this patient were not available for study so the question as to whether this variant is causative remains but it has not been reported as a SNP on dbSNP and the amino acid 987 is well conserved. In silico analysis predicted that this variant may be causative. In 18/34 cases where parental samples could be tested, all of the sequence changes identified had occurred de novo. Several of the variants were recurrent, having been seen in more than one individual or reported previously in the literature ( see supplementary Table 3).

Genotype/phenotype correlation

The position of the variants in the GPS patients relative to the SBBS patients is shown in Figure 1. In previously reported cases the sequence variants in GPS patients have tended to cluster more proximally to those in SBBS [START_REF] Campeau | Mutations in KAT6B, Encoding a Histone Acetyltransferase, Cause Genitopatellar Syndrome[END_REF][START_REF] Simpson | De Novo Mutations of the Gene Encoding the Histone Acetyltransferase KAT6B Cause Genitopatellar Syndrome[END_REF] . This was the case overall in our four GPS patients who had variants in exon 17 and more proximally in exon 18. However, we observed that patient 54 who had patellar abnormalities, contractures, genital anomalies and agenesis of the corpus callosum (all features which overlap between GPS and SBB) had the c.4205_4206del p.(Ser1402Cysfs*5) variant. This variant has been reported in patients with typical SBBS [START_REF] 'sullivan | Whole-Exome-Sequencing Identifies Mutations in Histone Acetyltransferase Gene KAT6B in Individuals with the Say-Barber-Biesecker Variant of Ohdo Syndrome[END_REF] had truncating variants occurring more proximally to those seen in GPS yet they had a working diagnosis of SBBS and patient 20 who had one of the more proximal variants in exon 17 did not have any abnormalities of the patellae, genitalia, kidneys or corpus callosum. The three individuals (12,13,14) who all shared the same exon 16 de novo synonymous variant fitted better with SBBS as all had the typical facies, normal patellae and none had significant contractures, agenesis of the corpus callosum or renal anomalies. All of the above observations suggest that the clinical distinction between GPS and SBBS is not always clear and it might be more important for the clinician to recognise when a patient has clinical findings within the broader KAT6B spectrum rather than to only consider the possibility of a KAT6B phenotype if the clinical features fit clearly within either the GPS or SBBS entities. This broader presentation of a KAT6B spectrum disorder was the basis upon which testing KAT6B was suggested in case 51, a 24 week fetus with contractures, agenesis of the corpus callosum and hydronephrosis.

Comparison of individuals with and without KAT6B sequence variants

Comparison and analysis of clinical features from variant-positive and variant-negative patients indicated that dysmorphic facial features, in particular blepharophimosis, severe intellectual disability and developmental delay were common to both groups ( Figure 2, Supplementary Figure 1). Genital anomalies, usually cryptorchidism, was universal in males with KAT6B sequence variants and tended to be more severe in GPS, where the genitalia may be ambiguous.

Genital anomalies (hypoplastic labia) were noted in one affected female but we suspect that genital anomalies in females are under-ascertained. Significant features which predicted whether a pathogenic KAT6B variant would be found were the presence of hypotonia, thyroid abnormalities, patellar abnormalities, long thumbs, and great toes, feeding difficulties and dental anomalies. A pathogenic KAT6B sequence variant was much more likely to be identified if the latter features were present (Table 4). The commonest cardiac anomalies observed in the KAT6B positive group were atrial and ventricular septal defects and patent ductus arteriosus, though one patient had a Tetralogy of Fallot, and right-sided aorta and pulmonary stenosis were seen in several patients. Dental anomalies, particularly hypoplastic teeth, emphasised as a salient feature of SBBS in the early literature, were reported as present in only 21/44 individuals with KAT6B variants though they were a significant distinguishing feature. Hearing loss, often remarked upon in the early literature on SBBS was present in around a third of individuals with likely pathogenic KAT6B variants and was usually sensori-neural in origin. Cleft palate was seen in 14/44 patients, drawing attention to the fact that SBBS and GPS deserve consideration as a differential diagnosis for syndromic clefting. Thyroid abnormalities were present in 22/42 KAT6B-positive patients where these had been searched for and only 1/22 KAT6B-negative patients, again making this one of the most important distinguishing features. The commonest abnormality was congenital hypothyroidism with raised TSH and thyroid agenesis was also documented in some patients. There was considerable clinical overlap between the clinical features of the SBBS and the GPS patients. Blepharophimosis was less striking in GPS ( Figure 3) though this group did still have a distinctive, but less obvious facial phenotype. Several patients with SBBS had either agenesis of the corpus callosum, hydronephrosis and joint contractures, features more typical of GPS. In SBBS the joint contractures were less severe and manifested more frequently as talipes and overlapping fingers and toes ( Supplementary Figure 2) rather than large joint contractures. Renal anomalies and agenesis of the corpus callosum were probably under-ascertained in the SBBS patients where MRI brain and renal scans had not always been undertaken. Several GPS patients have had the long, straight thumbs and great toes seen in SBBS, and this was even a feature in the fetus observed at 24 weeks gestation. An interesting observation was the presence of malrotation of the bowel in two patients with KAT6B sequence variants. A further patient had a laryngeal cleft. When analysing the relationship between the position of KAT6B variants and individual clinical features no genotype/phenotype correlation could be established. For patients with thyroid and patellar abnormalities, for example, sequence changes were found across the region from exon 15 to the distal part of exon 18. The three patients who all shared the same exon 16 synonymous change had a similar facial gestalt (Figure 2 f,g,h) which had been easily recognisable as that of SBBS by their referring geneticists, though ptosis was not as prominent in these three patients as others with SBBS. All three had small, low set ears, intellectual disability, global developmental delay, feeding abnormalities and genital anomalies but lacked some of the other structural malformations such as congenital heart defects, cleft palate and patellar abnormalities.

DISCUSSION

This study confirms previous findings that both SBBS and GPS are caused by de novo truncating variants in KAT6B. However, it is clear that not all variants are confined to the terminal exon of this gene, making it important to screen other exons, too, for diagnostic testing. The study identified several novel splice site variants including a common synonymous variant in exon 16.

Sequence changes causing the more severe phenotype of GPS did appear overall to occur more proximally as seen in the previous reports [START_REF] Szakszon | De novo mutations of the gene encoding the histone acetyltransferase KAT6B in two patients with Say-Barber/Biesecker/Young-Simpson syndrome[END_REF][START_REF] Yu | An individual with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) and additional features expands the phenotype associated with mutations in KAT6B[END_REF] . Campeau et al [START_REF] Campeau | The KAT6B-related disorders genitopatellar syndrome and Ohdo/SBBYS syndrome have distinct clinical features reflecting distinct molecular mechanisms[END_REF] have hypothesised that features which occur commonly in both GPS and SBBS are due to either haploinsufficiency or loss of a function mediated by the C-terminal acidic domain with the more severe features seen in GPS being due to alterations in binding capacity of the proteins arising as a result of specific GPS sequence variants. Our results would partially confirm these hypotheses as they suggest that the sequence variants cluster into three domains; a) those occurring more proximally which are subject to nonsense mediated decay (NMD). The phenotype is a milder SBBS phenotype caused by haploinsufficiency. b) variants which are not subject to NMD but cause a more severe GPS phenotype if they affect critical binding sites of KAT6B. c) variants which occur more distally and again escape NMD but cause the SBBS phenotype rather than GPS as they do not interfere with the critical binding sites associated with GPS. All of these three groups could share common features if, as Campeau suggest, haploinsufficiency for the very distal C-terminal region is crucial but it is also possible that the more distal variants are also contributing to a more severe SBBS phenotype due to a gain of function effect. Further studies of the individual sequence variantss are needed to clarify the mechanisms involved.

KAT6B deletions have been documented very rarely in the literature; Tzschach et al. [START_REF] Tzschach | Chromosome aberrations involving 10q22; report of three overlapping interstitial deletions and a balanced translocation disrupting C10orf11[END_REF] described four patients with 10q22 deletions encompassing KAT6B. They had a mild phenotype with common features of hypotonia and developmental delay, especially in the area of speech. Facial features were non-specific, though on scrutiny of the available photographs blepharophimosis did appear to be a feature. Interestingly, one of the patients was noted to have unusually long and straight thumbs. The DDD study (DECIPHER@sanger.uk) [START_REF] Firth | The Deciphering Developmental Disorders (DDD) study[END_REF] identified a patient, DECIPHER number 258813 with a 2.5Mb deletion encompassing KAT6B , HGVS description chr10.hg19:g.75, 971,593_78,526,861del. This deletion involved 9 genes, of which KAT6B is the only one known to be associated with a developmental disorder. This child, a male , weighed 2.1kg at 37 weeks and presented with hypotonia and developmental delay, particularly in the area of expressive speech.

He had short palpebral fissures and prominent epicanthic folds ( Figure 2e) but the facial features were less striking than in SBBS. He had normal patellae and a normal TSH level. He was macrocephalic and an MRI scan showed only mild frontal atrophy. Thiel et al. [START_REF] Kraft | Disruption of the histone acetyltransferase MYST4 leads to a Noonan syndrome-like phenotype and hyperactivated MAPK signaling in humans and mice[END_REF] have previously reported a patient with a translocation disrupting KAT6B and went on to demonstrate that this individual had lowered expression of KAT6B. He had much milder features than patients with SBBS but did have blepharophimosis and was reported as being "Noonan-like." Like the patients reported by Tzschach and the DDD patient there were no major malformations present. The demonstration by Thiel et al. that RAS-MAP pathway gene expression was affected in this patient is interesting in view of the fact that in two of our patients a diagnosis of Cardio-faciocutaneous syndrome had been suggested initially and that pulmonary stenosis and malrotation of the gut, both features seen in RAS-MAP disorders were documented as clinical features in our patient cohort. Only one convincing KAT6B sequence variant has been seen in the proximal part of the gene (exon 3). This was documented in the series by Clayton-Smith et al. [START_REF] 'sullivan | Whole-Exome-Sequencing Identifies Mutations in Histone Acetyltransferase Gene KAT6B in Individuals with the Say-Barber-Biesecker Variant of Ohdo Syndrome[END_REF] in a patient with a much milder blepharophimosis-intellectual disability phenotype. Taken together all of the above observations strengthen Campeau's case that a mild blepharophimosis-intellectual disability phenotype is caused by haploinsufficiency of KAT6B whereas disease-causing variants towards the terminal end interfere with the transcriptional activity of the gene and therefore cause a more severe phenotype through a gain of function effect. These hypotheses remain to be tested in further studies.

From a clinical point of view, clinicians should be aware that the KAT6B phenotypic spectrum is broad, encompassing presentations from mild blepharophimosis and intellectual disability to the severe , life threatening phenotype seen in genitopatellar syndrome. Naming of the KAT6B related disorders has been problematic. Though it is possible to classify some patients as having typical SBBS or GPS, this is not possible in all cases and we propose that considering this whole group as "KAT6B spectrum disorders" may be more helpful. Good diagnostic handles which might indicate a causative KAT6B sequence variant include severe blepharophimosis, patellar abnormalities , thyroid abnormalities, hypothyroidism, long thumbs and toes and agenesis of the corpus callosum . There are some similarities with RAS-MAP disorders and abnormalities of KAT6B may act in part by influencing this pathway. The relatively broad clinical spectrum of KAT6B variants means that clinical diagnosis is not always easy and suggests that affected individuals might be identified in large scale exome and genome sequencing programmes rather than as a result of targeted testing. Apart from one family reported so far where two affected siblings were born to parents who did not carry the variant in blood, and where gonadal mosaicism was presumed, all KAT6B variants have occurred de novo and recurrence risks for most families are low, though prenatal testing should be offered as an option.

No alternative diagnoses have yet been confirmed in those patients who did not have causative variants in KAT6B. On careful clinical review, we considered that the majority of these had some atypical features and that, rather than KAT6B variants or deletions being missed, other diagnoses were more likely. A diagnosis of Cohen syndrome was suggested in patient 57 based on the facial features and additional finding of visual problems. Chromosomal abnormalities, one of the main differential diagnoses have been excluded by microarray analysis. A further differential diagnosis which was excluded in suggestive cases was that of a UBE3B phenotype [START_REF] Basel-Vanagaite | Deficiency for the ubiquitin ligase UBE3B in a blepharophimosis-ptosis-intellectual-disability syndrome[END_REF] .

Verloes et al. reviewed the classification of the group of patients with blepharophimosisintellectual disability syndromes. He classified this group into five main groups, including both SBBS and chromosomal abnormalities . The remaining groups comprised the original patients reported by Ohdo et al. [START_REF] Ohdo | Mental retardation associated with congenital heart disease, blepharophimosis, blepharoptosis and hypoplastic teeth[END_REF] , The X-linked disorder originally reported by Maat-Kievit et al. [START_REF] Maat-Kievit | A case with blepharophimosis resembling Ohdo syndrome[END_REF] and now known to be due to sequence variants within the MED12 gene [START_REF] Anneke | Mutations in MED12 Cause X-Linked Ohdo Syndrome[END_REF] , and a recessive entity associated with microcephaly and multiple structural anomalies. None of our patients had features which were consistent with any of these other groups identified by Verloes. It is likely,

  who lacked the features of large joint contractures, agenesis of the corpus callosum and renal anomalies, although one patient with this variant had patellar abnormalities. The variant occurs more distally to the other GPS variants. Patient 56 whose sequence change occurred more proximally in exon 18 had significant genital anomalies and was also considered to have an overlapping phenotype. The remaining two patients with overlapping phenotypes (53 and 55) had the variants c.4320_4321del p.(Lys1441Glyfs*23) and c.4074_4079del p.Glu1367_Glu1368. Patients 1 and 17

Figure 2 Figure 3

 23 Figure 2

Table 1 . Genotype-phenotype differences between KAT6B mutation-positive and KAT6B mutation-negative patients

 1 Schematic representation of the spectrum of KAT6B mutations identified in this study. All coding exons are shown in purple. Arrows point to the exonic location of the mutations identified. Mutation key: frameshift variants, in-frame deletion, missense variants, nonsense variants. Variants identified in other studies are in italics. The protein structure shows the protein domains. NEMM: highly conserved N-terminal domain with a role in transcriptional repression. PHD: zinc finger domain, MYST: responsible for histone acetyltransferase activity.

		KAT6B + (n1=47)		KAT6B -(n2=23)		p value
		present	not present	not known	present	not present	not known
	Hypotonia	44	2	1	9	14	0	< 0.0001
	Feeding difficulties	42	3	2	15	8	0	0.0051
	Contractures	27	7	13	6	12	5	0.0020
	Dental anomalies	28	14	5	7	16	0	0.0087
	Long thumbs	33	14	0	3	20	0	< 0.0001
	Long great toes	31	16	0	5	18	0	0.0008
	Thyroid abnormalities	23	22	2	1	22	0	0.0001
	Congenital heart defects	27	19	1	12	11	0	0.6177
	Genital anomalies	25	22	0	7	15	1	0.1237
	Abnormal patella	15	31	1	1	22	0	0.0134
	Microcephaly	12	23	12	8	12	3	0.7734
	Cleft palate	14	33	0	2	21	0	0.0690

therefore that there are other causes of blepharophimosis and intellectual disability which remain to be elucidated.

Acknowledgements

Cranio-facial research within the University of Manchester is supported by the Healing Foundation UK Cleft Collective. The DDD study presents independent research commissioned by the Health Innovation Challenge Fund ( grant number HICF-1009-003) a parallel funding partnership between the Wellcome Trust and the Department of Health and the Wellcome Trust Sanger Institute (grant number WT098051). The views expressed in this publication are those of the author(s) and not necessarily those of the Wellcome Trust or the Department of Health. The study has UK research ethics approval (10/H0305/83 granted by the Cambridge South REC and GEN/284/12 granted by the Republic of Ireland REC). The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network.

CONFLICT OF INTEREST

No conflict of interest has been declared by any of the authors.