Ornsiri Aungsupravate

Noorman David Lucas

Abu Hassan

Matthew P Tonge

Garry Warrender

Patrice Castignolles

Marianne Gaborieau

Robert G Gilbert
email: b.gilbert@uq.edu.au

Noorman Abu Hassan

Ansell Shah

Water vapour transmission in butadiene-MMA-methacrylic acid latex films

Keywords: Emulsion polymerization, Barrier products, Water vapour transmission, Vulcanization, Elastomer

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Barrier protection products (industrial gloves, surgical gloves and condoms) are made from synthetic and natural rubber latexes. The latex is then coagulated onto a mould ('dipping') and the resulting film vulcanized (lightly crosslinked) to form the final barrier product.

Natural rubber latex gives softness, adequate tensile strength, breathability and good elastic recovery, but a significant number of users develop allergic responses to the proteins found in natural rubber latex [START_REF] Vandenplas | Occupational asthma caused by natural rubber latex[END_REF][START_REF] Llatser | Anaphylaxis to natural rubber latex in a girl with food allergy[END_REF][START_REF] Kujala | Glove-related rhinopathy among hospital personnel[END_REF][4][START_REF] Nutter | Contact urticaria from Rubber[END_REF]. Although methods for reduction of the allergen content of natural rubber latex have been developed [START_REF] Perrella | Enzyme treated natural rubber latex: an emerging technology[END_REF][START_REF] Perrella | Natural rubber latex protein reduction with an emphasis on enzyme treatment[END_REF][START_REF] Beezhold | Methods to remove proteins from natural rubber latex[END_REF][START_REF] Klinklai | Hyperdeproteinized natural rubber prepared with urea[END_REF][10][START_REF] Sebastian | Chlorination of latex gloves[END_REF][START_REF] Vivaygananthan | A novel method for preparing low-allergen natural rubber latex[END_REF], they are expensive to implement and/or diminish desirable properties. Another disadvantage of natural rubber latex is the inability to 'finetune' the raw polymer in a way that effectively permits the engineering of infinite variations of the properties of synthetic latexes, a problem that has been only partly solved (e.g. [START_REF] Hourston | Modification of natural rubber latex. III. Natural rubberpolystyrene composite latexes synthesized using azobisisobutyronitrile as initiator[END_REF][START_REF] Wang | Surface modification of natural rubber latex films by graft copolymerization[END_REF][START_REF] Lamb | Modification of natural and artificial polymer colloids by "topology-controlled" emulsion polymerization[END_REF][START_REF] Lehrle | Modification of natural rubber: a study to assess the effect of vinyl acetate on the efficiency of grafting MMA on rubber in latex form, in the presence of AIBN[END_REF][START_REF] Lee | Structure-Property Relationships in Modified Natural Rubber Latexes Grafted with Methyl Methacrylate and Vinyl neodecanoate[END_REF]). Because of this problem, synthetic latexes are often used (e.g. [START_REF] Urban | Polymer Dispersions and their Industrial Applications[END_REF], but even then allergenicity is not entirely absent, for reasons just becoming clear [START_REF] Chipinda | Oxidation of 2-Mercaptobenzothiazole in Latex Gloves and Its Possible Haptenation Pathway[END_REF]). These latexes contain relatively high amounts of a conjugated diene monomer (e.g. butadiene, which is comparable to natural rubber in terms of elasticity), a carboxylic-acid-containing monomer (e.g. methacrylic acid, MAA) and other monomers. Vulcanization is an essential part of the process of manufacturing a barrier product, by generating a crosslinked network in the polymer matrix. There are two types of curing agents used for vulcanization. A traditional sulfur-curing agent can be used to covalently crosslink with the butadiene units in the polymer. Alternatively, the carboxyl groups can be ionically crosslinked via zinc ions, resulting in a high tensile strength in the formed films; the zinc oxide also acts as an activator for sulfur crosslinking reactions [START_REF] Coran | Chemistry of the vulcanization and protection of elastomers: A review of the achievements[END_REF]. Furthermore, the carboxylic monomer enhances curing and wettability characteristics during the dipping process [START_REF] Blackley | Polymer Latices. 3. Applications of latices[END_REF]. While many polymeric materials, such as natural rubber latex, poly(vinyl chloride), neoprene and "nitrile" (a carboxylated butadiene/acrylonitrile copolymer) have been successfully used in making gloves, all current materials suffer from one or more disadvantages, such as poor breathability or poor polar solvent resistance. There is no single material that can be used for all purposes.

The particular property of interest in the present paper is breathability, which is a measure of the rate at which water vapour passes through a polymer film: water vapour transmission (WVT). This property is important for comfortable usage. Despite its importance, breathability has been the subject of only a few publications in the open literature [START_REF] Hayashi | High Moisture Permeability Polyurethane for Textile Applications[END_REF][START_REF] Sun | Sorption/desorption properties of water vapour in poly(2hydroxyethyl methacrylate): 1. Experimental and preliminary analysis[END_REF][START_REF] Schuman | Dispersion coating with carboxylated and cross-linked styrene-butadiene latices. 1. Effect of some polymer characteristics on film properties[END_REF][START_REF] Yang | nipam[END_REF][START_REF] Yang | Evaluation of poly(N-isopropylacrylamide) modified hydroxyl-terminated polybutadiene based polyurethane membrane[END_REF][START_REF] Stroeks | The moisture vapour transmission rate of block co-poly(ether-ester) based breathable films. 2. Influence of the thickness of the air layer adjacent to the film[END_REF][START_REF] Stroeks | Modeling the moisture vapor transmission rate through segmented block co-poly(ether-ester) based breathable films[END_REF]. The chemical structure and film thickness of the polymer are the main determinants of the permeability of a film [START_REF] Stroeks | The moisture vapour transmission rate of block co-poly(ether-ester) based breathable films. 2. Influence of the thickness of the air layer adjacent to the film[END_REF]. Five informative studies reveal some general principles, which guide the present work.

(1) Hayashi and co-workers [START_REF] Hayashi | High Moisture Permeability Polyurethane for Textile Applications[END_REF] reported studies of multi-block polyurethane composed of poly(ethylene glycol) and hard segments of poly(4,4'-diphenylmethane diisocyanate) and poly(ethylene glycol)/poly(oxytetra methylene glycol). These polymers have high WVT rate (WVTR) above the glass transition temperature (T g ) and low WVTR below T g . The WVTR was dependent on the ethylene glycol concentration, the ethylene glycol being the softer and more hydrophilic segment.

(2) Sun and Lee [START_REF] Sun | Sorption/desorption properties of water vapour in poly(2hydroxyethyl methacrylate): 1. Experimental and preliminary analysis[END_REF] reported sorption/desorption properties of water vapour on the hydrophilic poly(2-hydroxyethyl methacrylate), poly(HEMA). The sorption of water was postulated to involve hydrogen bond formation between a water molecule and two hydroxyl page 4 groups in the polymer. However, the data suggested that the process was a complex one even for this homopolymer, depending on the state of water, the polymer relaxation behaviour (related to T g ), and the interactions between water and polymer.

(3) Schuman et al. [START_REF] Schuman | Dispersion coating with carboxylated and cross-linked styrene-butadiene latices. 1. Effect of some polymer characteristics on film properties[END_REF] investigated the influence of characteristics such as the degree of crosslinking, T g and degree of carboxylation in carboxylated styrene-butadiene latexes on the tensile properties and WVTR. There was a slight tendency for WVTR to decrease with increasing crosslinking. The degree of carboxylation had no pronounced influence on the barrier properties, but there was a slight tendency for WVTR to decrease with increasing amounts of carboxylic groups. One expects that increasing the amount of hydrophilic units, as carboxylic groups, would increase the WVTR. The slight decrease of WVTR in this work suggests that a low WVTR was because of a rather high gel content. There may be then an optimum degree of carboxylation to ensure that the material is hydrophilic enough but not overly crosslinked.

(4) Yang et al. [START_REF] Yang | Evaluation of poly(N-isopropylacrylamide) modified hydroxyl-terminated polybutadiene based polyurethane membrane[END_REF] synthesized a block copolymer of hydroxyl-terminated polybutadiene and a polyurethane, which exhibited a higher WVTR than the pure polyurethane. They postulated a permeation of water through the samples in two steps: water vapour adsorbing on the surface and then diffusing through the membrane. They explained the higher WVTR in the copolymer by a more extensive adsorption of water vapour on the surface of the copolymer than on pure polyurethane.

(5) Stroeks et al. [START_REF] Stroeks | The moisture vapour transmission rate of block co-poly(ether-ester) based breathable films. 2. Influence of the thickness of the air layer adjacent to the film[END_REF][START_REF] Stroeks | Modeling the moisture vapor transmission rate through segmented block co-poly(ether-ester) based breathable films[END_REF] studied water permeability of segmented block poly(ether-b-ester) based breathable films by using two methods: ASTM E96B and ASTM E96BW. In the ASTM E96BW test, the film is in direct contact with water while in the ASTM E96B test an air layer exists between film and water. Water permeability was found to depend on film thickness, polymer type, relative humidity and also of the presence or not of stagnant air layers surrounding the film.

In the present study, the WVTR and mechanical properties of latexes of butadiene/methyl methacrylate/methacrylic acid copolymers is investigated. Butadiene is hydrophobic, methyl methacrylate (MMA) is slightly hydrophilic, and MAA is hydrophilic. The recipe is based on page 5

a patent [START_REF] Suddaby | Compositions suitable for making elastomeric film and glove articles[END_REF] and has similarities with the latexes of Schuman et al. [START_REF] Schuman | Dispersion coating with carboxylated and cross-linked styrene-butadiene latices. 1. Effect of some polymer characteristics on film properties[END_REF]. Since Schuman et al. observed that WVTR does not increase linearly with the degree of carboxylation, the effects of butadiene to MMA ratio and of MAA concentration over WVTR and mechanical properties are systematically investigated here using a factorial design [START_REF] Montgomery | Design and Analysis of Experiments[END_REF]. The thrust of this work is to relate the end-use properties of butadiene-based latexes to the molecular architecture of the polymer backbone.

The distribution of the MAA inside or on the surface of latex particles gives different properties to the final end used products. This distribution [START_REF] Slawinski | Seeded emulsion polymerization of styrene: influence of acrylic acid on the particle growth process[END_REF][START_REF] Santos | Partitioning of styrene, butyl acrylate and methacrylic acid in emulsion systems[END_REF][START_REF] Ding | Synthesis and characterization of functionalized polymer latex particles through a designed semicontinuous -Emulsion polymerization process[END_REF][START_REF] Koh | The Influence of Copolymerization with Methacrylic Acid on Poly(butyl acrylate) Film Properties[END_REF] depends upon the reactivity ratios among those monomers, their concentration, the pH and feed regime used in latex synthesis. The amount of MAA in the water phase will also depend on the same factors. If MAA units are located on the surface of the particles, this would be expected to enhance the colloidal stability of the latex and thus to increase the critical coagulation concentration during dipping. On the other hand, if most of the methacrylic acid units are buried inside the particles, the physical crosslinking between Zn ions and MAA units after film formation may be reduced. The spatial distribution of the MAA units in the films probably strongly influences the WVT and hence breathability.

Experimental

Materials

Butadiene (Bd), potassium persulfate, tert-dodecyl mercaptan, tetrapotassium pyrophosphate, naphthalene sulfonic acid, ethylenediaminetetraacetic acid and sodium dodecylbenzene sulfonate (reagent grade, Aldrich) were used without further purification. Methyl methacrylate (MMA), methacrylic acid (MAA) and hydroxyethyl methacrylate (HEMA) (reagent grade, Aldrich) were purified by passing through a basic alumina column to remove inhibitor. Water was demineralized with QPAK 1 columns from Milli-Q. 2-butanone (MEK) was used as a gel content measurement solvent. Nitrile glove (TNT) and polyurethane wound dressing film were used as standard films for WVTR. Compounding chemical ingredients such as a clear coagulant formulation (containing primarily 40 % calcium nitrate) and a 50 % latex compounding curative paste containing conventional sulfur accelerators, were used as supplied by Ansell.

Syntheses

Batch emulsion polymerizations of butadiene, MMA and MAA (with or without hydroxyethyl methacrylate) were performed in a Mettler RC1e reactor under pressure. Potassium persulfate and sodium dodecylbenzene sulfonate (SDBS) were used as initiator and surfactant respectively. Tert-dodecylmercaptan was used as a chain transfer agent. All chemicals except butadiene and potassium persulfate were bubbled with high-purity argon in the reactor to eliminate inherent oxygen. After depressurization, butadiene (a gaseous monomer at room temperature) was transferred to the RC1e reactor with the vessel temperature at 25 °C and stirrer speed at 300 rpm. The temperature was then raised to 52 °C for the reaction. After heater calibration for 1hr 45 min, the initiator solution was pumped into the reactor. The reaction was allowed to run to complete conversion, monitored using the heat flow profile on the RC1e (see Supplementary Information). The standard recipes used throughout this study are based on Suddaby's patent [START_REF] Suddaby | Compositions suitable for making elastomeric film and glove articles[END_REF], varied using factorial design (see below). A standard recipe used for the syntheses is shown in Table 1.

Latex compounding and dipping

Quantities are indicated in parts per hundred dry rubber, phr. Solid content, pH and viscosity of the original latex were measured, and the pH of the latex was adjusted to 8.5 by addition of a 5 wt % potassium hydroxide aqueous solution. Next, 30 wt % SDBS solution (0.72 phr) was added to the latex (100 phr) followed by the addition of the compounding paste (total 3.5 phr accelerator) under slow stirring. The mixture was left for 24 hr under gentle stirring, permitting air bubbles to rise to the surface while preventing creaming and skin formation.

Before dipping, the total solid content of latex was adjusted to 35 -37 % using demineralized water. Solid content, pH, particle size and viscosity of the latex were measured after mixing.

A plate dipping former (mould) (8 cm width, 18 cm length and 0.5 cm depth) was cleaned by sequential dipping into diluted weak base, weak acid and hot water, then dried at 70 °C for at page 7 least half an hour. The former was then dipped into a heated coagulant solution at 55 °C.

After the temperature of the former dropped to around 40 °C, it was dipped into the compounding latex. After air drying for a few minutes, the former coated with the latex was placed in warm water to remove residual latex and serum, then placed in an oven at 70 °C for 15 min to allow pre-curing, and finally transferred to another oven at 120 °C for 30 min to allow complete curing.

Analysis techniques

Experimental design

Factorial design [START_REF] Montgomery | Design and Analysis of Experiments[END_REF] was used to draw conclusions from the experiments and to establish how the input parameters affected the final results. This technique is based on statistical assumptions concerning replication and randomization to minimize experimental variability from run to run. The aim of the present study is to investigate the effect of monomer composition on WVTR and tensile properties (tensile strength and % elongation at break).

The simplest factorial design of type 2 k is used in the present study: the 2 2 factorial design; with two factors A and B, each at two levels. The factors were butadiene to MMA ratio (A), and MAA concentration (B). The total amount of monomers was kept the same for all recipes; thus, total monomer concentration is not an independent factor. The design matrix is shown in Table 2. The low and high levels of Factor A (dimensionless) were 1.375 (70/25) and 2.8 (55/40), respectively. The low level of Factor B was 2 phm (parts per hundred monomer) and the high level was 5 phm. The centre point was 1.92 (62.5/32.5) of Factor A and 3.5 phm of Factor B. The low and high levels of each Factor were selected from the 'preferable' range given in Suddaby's patent [START_REF] Suddaby | Compositions suitable for making elastomeric film and glove articles[END_REF].

The viscosity of the polymer latexes was measured using a Brookfield DV-II+ viscometer. A Malvern HPPS 3.3 light-scattering particle sizer was used to measure the z-average particle size of latex samples. page 8

Solid content

The solid content of the latexes was measured by gravimetry. A few drops of latex were put in an aluminium pan and dried for 24 hr in an oven at 60 ˚C.

Gel content

The gel content is the insoluble fraction of rubber that is crosslinked; however, a reported value of gel content always depends on the details of the technique chosen to measure this quantity. The gel content of the latexes was determined following ASTM D3616-82. Firstly, the latex was cast in a Petri dish and left to dry at room temperature for 5 days to achieve a constant weight of the film. A small piece of about 0.15 g was cut from the dry film and then immersed in 15 mL of methyl ethyl ketone (MEK) for 24 h. Exactly 5 mL of liquid was then pipetted into an aluminium pan and dried to constant weight. The gel content was calculated as the ratio of the dried weight after and before leaching with MEK.

Tensile testing: Tensile strength and percentage elongation at break.

The specimen was prepared by cutting a film in a rectangular shape of 5 mm  8 cm. The length between bench markers (the two marks placed on the specimen and used to measure elongation or strain) was approximately 5 cm. The exact dimensions of the specimen (width, length and thickness) were measured again before carrying out the testing. The specimen was placed in an Instron model 5567 Universal Testing device, using a stretching rate of 500 mm•min -1 until the specimen ruptured. The film sample thickness was ~1.8 -2.5 mm and tensile strength and percent elongation at break measured.

Glass transition temperature

The glass transition temperature (T g ) of the copolymer films were measured by using a DSC 2920 MDSC v. 2.6A. The sample was heated to 80 °C, then cooled to -80 °C, then heated from -80 °C to 80 °C at 5.0 °Cmin -1 under liquid nitrogen. The predicted T g of copolymers was calculated from the Fox equation (e.g. [START_REF] Gedde | Polymer Physics[END_REF]) and also using Pochan et al.'s relation [START_REF] Pochan | Different Approach for the Correlation of the Tg of Mixed Amorphous Systems[END_REF]. page 9

Permeability: water vapour transmission (WVTR)

WVTR was measured based on the ASTM E96-66 procedure, as follows. The environment inside a glove bag was maintained at 50 ± 2 % relative humidity throughout the duration of the experiment by using a saturated aqueous solution of Ca(NO3)2, which gives 51 % humidity at 24.5 °C. The experiment was performed at 23.5 -24.5 °C. A small fan was put inside the glove bag to generate air circulation as recommended in the ASTM procedure and an air velocity of around 0.15 -0.25 ms -1 above the Payne cup (measured with an anemometer); the air velocity specified in the ASTM procedure is between 0.02 and 0.3 ms -1 .

The thickness of a film sample was measured by a micrometer for at least 5 points, near the centre of the film specimen and at the centre of each quadrant. Film thicknesses were around 1.8 -2.5 mm. The dried sample from the oven was put in the glove bag for at least 2 hr before the experiment was conducted in order to equilibrate the sample with the atmosphere moisture inside the glove bag. Sample weight increased initially with time when taken from the oven and placed inside the glove bag (see Supplementary Information). Demineralized water was put into a permeability Payne cup (2.5 cm diameter). The water level was kept 2 cm from the specimen to avoid contact between the specimen and the water. The film then was attached on the Payne cup. The cup with the film was placed on a balance, which was located in the glove bag. The weight change as a function of time was measured every 5 min for at least 2 h. The WVTR rate was calculated as:

WVTR = G t  A (1)
where G is the weight change, t is the time and A is the test area.

Determination of acid distribution in particles

Latex samples were centrifuged at 9  10 4 rpm at 5 °C for 4 hr in a Beckman Coulter Optima L-100 Ultracentrifuge. Three phases were then obtained after ultracentrifugation: a sedimentary latex (located on top of the centrifuge tube), a white liquid (located in the middle) and the aqueous phase (located at the bottom).

page 10

The amount of acid in the discrete phase was measured using Fourier-transform infrared spectroscopy (FTIR). The white liquid obtained from centrifugation was frozen dry under vacuum for 24 h. A dry residual solid was analyzed by FTIR in bulk. Near-IR spectra were taken with a Bruker IFS66v FTIR spectrometer in absorption mode in the region 500 -4000 cm -1 .

The sedimentary latex did not redisperse completely in water or dissolve in common solvents.

Nuclear Magnetic Resonance

The aqueous phase from ultracentrifugation was freeze-dried under vacuum for 24 hr. A dry residual solid was then dissolved in deuterium oxide at 10 mgmL -1 for 24 hr before quantifying the acid using NMR. Quantitative 1 H NMR spectra were recorded at room temperature on a Bruker Avance spectrometer operating at a 1 H Larmor frequency of 300 MHz, with a 5 mm QNP ( 1 H- 19 F/ 31 P/ 13 C) probe. 100 transients were recorded with a 6.

Results and Discussion

Effect of monomer composition on physical properties

Monomer concentrations variations were chosen to follow a 2 2 factorial design [START_REF] Montgomery | Design and Analysis of Experiments[END_REF] as a polynomial approximation model, which assumes smooth and continuous output and independent factors. The basic concept is to create homogeneous blocks in which the 'nuisance' factors are held constant and the factors of interest are allowed to vary. A primary page 11

purpose of the factorial design experiments is to select or screen out the few important main effects from the many less important ones. In this study, we investigated the effect of monomer composition on tensile strength, elongation at break, and WVTR. Ranges of monomer concentrations were selected based on the preferable concentration ranges listed in Suddaby's patent [START_REF] Suddaby | Compositions suitable for making elastomeric film and glove articles[END_REF]: see Table 2 (Runs 1 -5).

After vulcanization, an elastomeric poly(butadiene-co-MMA-co-MAA) copolymer film is obtained. Vulcanization introduces dimensional stability, reduced creep and flow, and permits the manufacture of a wide range of rubber articles. The higher is the degree of crosslinking, the higher is the tensile strength and the lower is the extensibility. Polybutadiene has a low T g (-83 °C) [START_REF]Polymer Handbook[END_REF], while poly(MMA) and poly(MAA) have high T g values (105 °C and 162 °C respectively) [START_REF]Polymer Handbook[END_REF]. Table 3 shows the results of tensile strength and elongation at break for various monomer compositions.

A single replicate of the experimental design was run. A design matrix and the response data obtained from a single replicate of the 2 2 experimental designs are shown respectively in Tables 2 and3. Due to only one replicate being performed, there was no internal estimate of error. Using an error mean square is inappropriate in this case. A simple method suggested by Daniel [START_REF] Montgomery | Design and Analysis of Experiments[END_REF] to examine a normal probability plot of the estimates of the effects can be difficult to interpret for small data sets. Trend plots and interactions plots gave the clearest understanding of the causal relationships between input and output variables in this design.

Factorial design results are shown in Table 3 and illustrated graphically in Figures 2 a) and 3 b). The results in Figure 2 a) show that the difference in response for tensile strength between the levels of one factor is not the same at all levels of the other factors. An interaction between these two factors is inferred because the lines at low and high levels are not parallel.

Tensile properties are probably the most frequently considered, evaluated and used throughout the industry. These properties indicate polymer behaviour under loading tension.

Tensile strength is the maximum force when tension stress is applied to polymer in order to elongate it to the point where it ruptures. Crosslinks introduced by vulcanization are chemical crosslinks of pendant double bonds from diene units by sulfur and ionic crosslinks of zinc The assumption of using two-level factorial designs is linearity in the factor effects. A 'centrepoint' run (Run 3, with label "0") was added to check the possible curvature and stability of the process. A reasonable hypothesis is that tensile strength should increase as a function of MAA and butadiene concentrations. There was however no noticeable trend in the data, and inclusion of the centrepoint gives non-monotonic behaviour: a maximum. This nonmonotonic behaviour can arise for a number of reasons [START_REF] Blackley | Polymer Latices. 3. Applications of latices[END_REF]. The total concentration of all crosslinks influences the mechanical properties of the vulcanizates, but changing concentrations of the various species also changes both gel content (See Table 4) and molecular weight and branching distributions. Consistent measurements of molecular weight and branching distributions are hard to obtain consistently in systems of this type [START_REF] Koh | The Influence of Copolymerization with Methacrylic Acid on Poly(butyl acrylate) Film Properties[END_REF][START_REF] Former | Effect of branching and molecular weight on the viscoelastic properties of butyl acrylate[END_REF] because crosslinked polymer does not dissolve in any solvent and was not attempted here. In analyzing the data on latex properties in Table 4, account had to be taken of the observation that the gel content of any batch was not consistent. Furthermore, the magnitude of the tensile strength maximum, and the crosslink concentration at which it occurs, are expected to depend upon the chemical nature of the covalent crosslink introduced during vulcanization. As is well known, the restriction of mobility by crosslinking interferes with film formation. Elongation at break is influenced by the total crosslinking in the vulcanized polymer. Figures 4 c) andd)

show the response data of % elongation at break against Factors A and B. The results showed page 13

the opposite trend to tensile strength, as expected. The two factors seem to be independent of each other.

By looking only at low or high levels of MAA concentration (Figure 3), further inferences could be made as follows. Tensile strength increased with increasing MAA concentration while % elongation at break showed the opposite trend, which corresponds to the ionic crosslink arising from MAA concentration and Zn ions.

Effect of monomer composition on WVTR

Breathability or moisture permeability of film is measured by the amount of moisture that can pass through the film in 24 hr under appropriate conditions. Numerical results are detailed in Table 3. Figure 5 shows the interaction between Factor A and Factor B. High moisture permeability requires the film to exhibit high water absorption and/or a high water-vapour diffusion coefficient. Both functions require a continuous amorphous phase in which T g is well below room temperature (or application temperature). In this case the lack of a dominant effect can be explained as a compromise between rubbery behaviour in the amorphous phase (low T g due to the butadiene content), and mechanical properties (related to MAA content, which gives ionic crosslinking). This is consistent with inferences from a number of studies [START_REF] Stroeks | The moisture vapour transmission rate of block co-poly(ether-ester) based breathable films. 2. Influence of the thickness of the air layer adjacent to the film[END_REF][START_REF] Stroeks | Modeling the moisture vapor transmission rate through segmented block co-poly(ether-ester) based breathable films[END_REF][START_REF] Wicks | Two package waterborne urethane systems[END_REF] which report high WVTR in thermoplastic elastomers composed of alternating polyether and polyester segments, in which variations were made in the hard/soft ratio, the block length of the polyether and the type of polyether. The amount and the type of soft block strongly affect the breathability, and the amount and type of hard block affect the mechanical properties. The WVTR of this type of material (wound dressing film) was measured (Table 3)

and is a decade higher than for glove materials.

Before conducting these experiments, it was hypothesized that increasing the amount and type of hydrophilic monomer should increase the transportation of water vapour through the film.

However, within the experimental range examined here, the WVTR was not significantly increased (Figure 6). Therefore the investigation of where the hydrophilic monomer units are located in the particle and how this affects the WVTR were seen to be important in understanding the present system.

page 14

An appropriate hydrophilic monomer to test the preceding hypothesis was judged to be hydroxyethyl methacrylate (HEMA). Incorporation of this monomer into the butadiene-MMA-MAA copolymer was expected to enhance WVTR. Results reported in Table 3 show that this was not the case, and indeed copolymers containing HEMA seemed to be less effective than those without. The WVTR of commonly used nitrile films, made from carboxylated butadiene acrylonitrile copolymers, is higher than that of the present butadiene-MMA-MAA copolymers (see Table 3). The higher hydrophilicity of polyacrylonitrile compared to poly(MMA) might be one reason for the higher WVTR of nitrile film. However, the difference might also arise from different microstructures.

Investigation of location of MAA by conductimetry and ultracentrifugation

Understanding of the latex morphology, and especially the location of the hydrophilic monomer MAA in the particles and in the film, would provide information about the mechanisms of WVTR. To this end, the distribution of MAA groups is usually investigated by conductometric titration of the latex [START_REF] Ding | Synthesis and characterization of functionalized polymer latex particles through a designed semicontinuous -Emulsion polymerization process[END_REF][START_REF] Koh | The Influence of Copolymerization with Methacrylic Acid on Poly(butyl acrylate) Film Properties[END_REF][START_REF] Tang | Synthesis of well-defined, functionalized polymer latex particles through semicontinuous emulsion polymerization processes[END_REF]. This is indeed the only technique, which allows a direct quantification of the acid groups located at the surface of the particles. Since there were many types of acids in the system (dodecylbenzene sulfonic acid, ethylenediaminetetraacetic acid and naphthalene sulfonic acid as well as MAA), these acids all influence the conductivity of the system, resulting in no clear inferences being able to be drawn from conductance as function of added base (see Supplementary Information). In the case of complex/industrial formulations, it is thus not possible to determine the concentration of acids at the surface of the particle. Advanced characterization techniques such as capillary electrophoresis may offer possibilities in the future. Indirect characterization after separation by ultracentrifugation have been used in this work. It was found that the density of the discrete (copolymer) phase of the untreated latex was very close to that of the continuous phase, so that a good separation between phases by centrifugation could only be achieved if the density of the continuous phase was changed. A 35 % wt of sucrose solution was added in order to increase the aqueous phase density to 1.15 g•mL -1 . Three phases were then obtained after ultracentrifugation: a sedimentary latex, a white liquid and the aqueous phase (located at the bottom).

page 15

Investigation of the aqueous phase by NMR

NMR was used in order to determine the MAA concentration in the aqueous phase. The 1 H NMR spectrum of aqueous phase of Run 5 is shown in Figure 1. All the latexes give spectra comparable to this one. Characteristic signals of sucrose appeared between 3 and 5.5 ppm in D2O. The NMR spectrum of Run 5 (Figure 1) shows the characteristic signals of poly(MAA) at 1.36 and 1.45 ppm corresponding respectively to CH3 and CH2 in D2O at room temperature. Those signals have been reported at 1.0 and 1.1 ppm in D2O at room temperature by Sugai et al [START_REF] Sugai | Proton NMR characterization of syndiotactic poly(methacrylic acid)[END_REF]. However, as PMAA is ionizable, the pH of its solution in D2O has a strong influence on the measured chemical shift s [START_REF] Kay | Conformational transition in poly(methacrylic acid) and butyl vinyl ether-maleic anhydride copolymers studied by 1H page 22 NMR linewidth measurements[END_REF] The discrepancy between the values measured in the present work and by Sugai et al. are postulated to arise from a difference in pH [START_REF] Kay | Conformational transition in poly(methacrylic acid) and butyl vinyl ether-maleic anhydride copolymers studied by 1H page 22 NMR linewidth measurements[END_REF] or from the interaction of the hydroxyl functional group in sucrose and the carboxyl groups in poly(MAA) (in this case there was an excess of sucrose compared to the poly(MAA) concentration) or from a difference in tacticity of the polymers (as already observed for PMMA [START_REF]NMR spectra database of polymers[END_REF]).

Integration to determine the concentration of poly(MAA) was based on the addition of a known amount of a reference compound. In this study, the signal at 5.4 ppm was chosen as the standard signal, corresponding to sucrose -CH, as shown in Figure 1. The characteristic signals of poly(MAA) at 1.36 and 1.45 ppm were used to determine the concentration of poly(MAA) and poly(HEMA). The characteristic peaks of poly(HEMA) that correspond to the backbone CH3 and CH2 are similar to those of poly(MAA) [START_REF]NMR spectra database of polymers[END_REF]. The concentration of poly(MAA) and poly(HEMA) left in the aqueous phase of any single batch was very small compared to the initial concentration (see Table 5). This means that MAA must be located either on the surface or inside of the sedimentary latex particles. For the copolymer of Bd-MMA-MAA-HEMA, the aqueous-phase polymer concentration includes both MAA and HEMA units but remains negligible compared to their initial concentrations.

Investigation of the white phase by Infrared spectroscopy

The white liquid phase obtained from ultracentrifugation could possibly comprise a homopolymer or copolymer containing hydrophilic monomer as a major component. Such a page 16

hydrophilic copolymer might not be compatible with polybutadiene at the start of polymerization, and thus might not be incorporated into the particles.

FTIR spectra are shown in Figure 7. The FTIR spectrum of sucrose at 20 °C shows the broad band of hydroxyl groups at 3500-3000 cm -1 and other sharp bands in the fingerprint region at 1500-500 cm -1 . Those peaks were found in the FTIR spectra of every white liquid sample.

The characteristic peaks of polybutadiene [START_REF] Pandey | A novel approach for the determination of the physicomechanical properties of polymers by vibrational spectroscopy: the tensile strength of polybutadiene rubber[END_REF][START_REF] Edwards | Raman and FTIR spectroscopic studies of copolymers of methyl methacrylate with butadiene[END_REF] normally appear around 1640-1680 cm -1 for the C=C stretch (the cis-isomer is at 1660 cm -1 , the trans-isomer is at 1675 cm -1 , and the C=C stretch of the pendant group is at 1640 cm -1 ). These peaks were barely apparent in any sample from the white liquid phase; the level of butadiene monomer unit is thus below the detection limit. However, there was a short region between 1800-1500 cm -1 that allowed the other components to be identified (Figure 8). The dominant characteristic peak of poly(MMA) [START_REF] Coleman | A Fourier transform infrared study of polymer blends. I. Poly(vinylidene fluoride)-poly(methyl methacrylate) system[END_REF][START_REF] Schneider | Stretching and deformation vibrations of methylene, C-methyl and methoxy groups of poly(methyl methacrylate)[END_REF] is generally a C=O stretch at 1730 cm -1 . Every spectrum of the white liquid phase showed this type of peak, proving that there was some in this phase. In general, FTIR can distinguish the different functionalities better than other methods. For poly(MAA), the C=O stretching of the carboxyl group [START_REF] Okuda | Graft polymerization of methacrylic acid onto polytetrafluoroethylene initiated by alkyllithium/electron-donating solvents[END_REF] normally appears at around 1700 cm -1 but this peak was not observed here. There were no significant different signals between the films of the copolymers of Bd-MMA-MAA-HEMA and the one of Bd-MMA-MAA. The white liquid thus contains sucrose plus a significant fraction of poly(MMA). Further evidence that supports this conclusion is the ultracentrifugation itself. The density of the sedimentary latex was around 1.03 g•cm -3 (depending on the monomer composition) [START_REF]Polymer Handbook[END_REF] and could thus not be precipitated by ultracentrifugation in pure water. The density of the aqueous phase after adding sucrose was 1.15 g•cm -3 . Most of the latex particles precipitated, except this white phase. The white phase should thus have a density close to 1.15 g•cm -3 . The density of homo poly(MMA) [START_REF]Polymer Handbook[END_REF] is ~1.16 g•cm -3 and is thus consistent with the white phase being mainly composed of poly(MMA). The measured glass transition temperatures are also consistent with this conclusion (see 4.6).

page 17

After separation by ultracentrifugation, no significant amount of MAA can be detected either in the aqueous phase or in the white phase. The MAA groups are thus located in the sedimentary latex. If a significant amount of MAA groups was located on the surface of the particles, they could form hydrophilic domains in the polymer films (the Zn 2+ should contribute to the formation of these domains during the compounding process [START_REF] Kells | Crosslinking in carboxylated nitrile rubber dipped films[END_REF]). Water may then go through the film by moving from one hydrophilic domain to the other. In the mechanism postulated by Wang et al. [START_REF] Wang | Characterization of water-soluble oligomers in acrylic acidstyrene emulsion copolymerization[END_REF], hydrophilic domains at the surface of the film are necessary for a good WVTR because the water needs to adsorb on the surface of the film before diffusing through it. It might be expected that the hydrophilic monomer (MAA) would be primarily located on the particle surface. However, the distribution of acid groups in latexes is influenced by several factors that depend on polymerization conditions [START_REF] Santos | Partitioning of styrene, butyl acrylate and methacrylic acid in emulsion systems[END_REF][START_REF] Koh | The Influence of Copolymerization with Methacrylic Acid on Poly(butyl acrylate) Film Properties[END_REF]. At low pH, MAA is in the unionized, and therefore less hydrophilic, form. Moreover, under certain conditions, high molecular weight poly(MAA) can undergo a coil-to-globule or coilto-rod transition [START_REF] Morawetz | Revisiting some phenomena in polyelectrolyte solutions[END_REF], and in a compact form, it would be unlikely to provide adequate electrosteric stabilization. In the present work, the polymerization was carried out from an initial pH of ca 3.5 to a final pH of ca 4.6; the pKa of MAA is 4.66 at 20 °C [START_REF]Polymer Handbook[END_REF], and thus most of the MAA monomer is unionized and therefore is likely to be mostly buried inside the particle [START_REF] Koh | The Influence of Copolymerization with Methacrylic Acid on Poly(butyl acrylate) Film Properties[END_REF]. This is the most likely explanation for the low WVTR measured on these latexes.

MMA-rich domains

The separation by ultracentrifugation of two types of particles, including some MMA-rich ones is surprinsing. However, it is confirmed by the glass transistion measurements. The glass transition temperatures of the copolymer films are reported in Table 3. Predicted glass transition temperatures were calculated based on two different equations: Fox's equation (based on free-volume changes) and Pochan's equation [START_REF] Koh | The Influence of Copolymerization with Methacrylic Acid on Poly(butyl acrylate) Film Properties[END_REF][START_REF] Pochan | Different Approach for the Correlation of the Tg of Mixed Amorphous Systems[END_REF]. Pochan's equation predicts values noticeably different from the experimental ones (see Supporting Information). The present results fitted well with the predictions of Fox's equation (see Table 3). For polymers 2 and 6, the experimental T g values were however much lower than the theoretical values, which could mean that there was less MMA incorporated into the copolymer than calculated page 18

from the original ratio of the different monomers; this is consistent with the finding that there was a separate MMA-rich phase, discussed below. The applicability of the Fox equation can be inter alia ascribed to the light crosslinking in the samples from the present study having no major effect on short-range chain mobility.

The presence of this MMA-rich phase can be partially explained by the kinetics of polymerization. The reactivity ratios r1 and r2 for butadiene and MMA respectively are reported to be both lower than unity, but with r1 greater than r2 [START_REF]Polymer Handbook[END_REF]. Initial amounts of butadiene and MMA are similar (especially in Run 2). Butadiene should be consumed faster than MMA. The MMA-rich phase (white liquid phase in ultracentrifugation) could then be formed at the end of the polymerization when a negligible amount of butadiene is left and MMA is then the major component. This poly(MMA) phase could well comprise MMA-rich particles formed by secondary nucleation, probably by homogeneous nucleation, which is not unexpected with a monomer such as MMA which has a high propagation rate coefficient [START_REF] Beuermann | Critically evaluated rate coefficients for free-radical polymerization, 2. Propagation rate coefficients for methyl methacrylate[END_REF] and moderate water solubility [START_REF] Coen | Modelling particle size distributions and secondary particle formation in emulsion polymerisation[END_REF][START_REF] Morrison | Conditions for secondary particle formation in emulsion polymerization systems[END_REF].

The presence of these MMA-rich domains was not expected. The hydrophilicity of MMA is probably too low for these domains to contribute significantly to water vapour transmission.

Conclusions

Factorial design experiments on factors affecting usage properties of interest for personal barrier products in poly(butadiene-co-methyl methacrylate-co-methacrylic acid) films, formed after crosslinking with ZnO and other vulcanization reagents, were used to examine how tensile strength of butadiene-MMA-MAA copolymer film is influenced by the MAA concentration. Instead of the zinc ions from zinc oxide ionically crosslinking with the carboxylic groups of MAA during vulcanization to enhance the crosslinking and thus the tensile strength, what was demonstrated was that the butadiene to MMA ratio probably dominates tensile strength through vulcanization with sulfur. On the other hand, the elongation at break of copolymer was independent of both the butadiene to MMA ratio and the MAA concentration but dependent upon the total crosslinking concentration.

page 19

An important property of personal barrier products is breathability, measured as water vapour transmission (WVTR). The WVTR of poly(Bd-co-MMA-co-MAA) was governed by the ratios of the monomer in the system, balanced to have enough rubbery component (butadiene)

as well as good mechanical properties (related to the amount of MMA and MAA).

The relatively low WVTR in the films examined here is not due to any homopoly(MAA) formed during synthesis in the aqueous phase, which was found to be very small.

Ultracentrifugation of the latex led to three distinct phases: a solid, a water phase and a white liquid phase. This last phase contained a large amount of poly(MMA), probably formed by secondary particle formation late in the polymerization. NMR and FTIR analysis suggested that most of the hydrophilic monomers (MAA and HEMA) were located in and/or on the particle, and are probably buried within the particle and do not form extensive hydrophilic domains, which otherwise would enhance water-vapour permeability. The WVTR could thus be improved by locating the hydrophilic monomers at the interface, replacing MAA by a more hydrophilic monomer at low pH (e.g. acrylic acid) or surfmers. The MMA-rich particles proved also non-significant in term of WVTR. MMA could also be replaced by a more slightly more hydrophilic monomer such as HEMA or N-vinyl-pyrrolidone.

page 23

Tables and figures

• Water vapour transmission (WVTR) and tensile properties of films obtained by adding various amounts of ZnO during the compounding

Increasing amounts of ZnO should increase the level of ionic crosslinking; this should make the film more rigid and decrease WVTR. At the same time, increasing amounts of ZnO may enhance the formation of hydrophilic domains; this would increase the WVTR. Varying ZnO concentration lead to small change in WVTR compared to the difference of WVTR between our films and commercial nitrile. Table 4 shows that the highest WVTR was found when the ZnO concentration was intermediate 1.75 phr..

Figure 1 .

 1 Figure 1. The quantity of acid was calculated by integration and comparison of the signals between 1.1 and 1.6 for the poly(MAA) (5 H), and between 5.25 and 5.55 ppm for the sucrose (1 H).

Figure 1 1 H

 1 Figure 1 1 H NMR spectrum of aqueous phase: Run 5

Figure 2

 2 Figure 2 Dependence of response data from Table 4 against factor A for both levels of factor B, a) tensile strength, b) the % elongation at break

Figure 3 31 Figure 4

 3314 Figure 3 Stress against strain at different MAA concentration, a) 2 %, b) 5 %

Figure 5

 5 Figure 5 Dependence of response data in Table 5 on Factor A for both levels of factor, for the water vapour transmission response data

Figure 6 34 wavenumber / cm - 1 Figure 7 35 wavenumber / cm - 1 Figure 8

 634173518 Figure 6 Dependence of response on main effect for both levels and centrepoint, a) Factor A for WVT, b) Factor B for WVT

  page[START_REF] Vivaygananthan | A novel method for preparing low-allergen natural rubber latex[END_REF] ions to acid functional group. Additionally, there are other physical crosslinks in the form of polymer gel, e.g. arising from entanglements. This type of crosslink generally increases the tensile strength of the copolymer film. The ionic crosslinking that occurs during vulcanization is related to ratio of MAA to Zn 2+ . During vulcanization, zinc oxide can act as an activator for sulfur crosslinks and in addition decompose into Zn 2+ , which gives an ionic crosslink. Thus, zinc oxide concentration should have a stronger influence on the tensile strength than sulfur concentration. Vulcanized films showing higher elongation at break generally exhibit lower tensile strength. Elongation at break also depends upon the stretching rate (lower elongation at faster rate). Further independent experiments confirmed that tensile strength increased with increasing ZnO amount (see Supplementary Information).

Table 1

 1 Standard polymerization recipe

	Ingredient	Monomer concentration, phm
	demineralized water	150
	dodecylbenzene sulfonic acid	2.75
	ethylenediaminetetraacetic acid	0.05
	naphthalene sulfonic acid	0.1
	tetrapotassium pyrophosphate	0.1
	tert-dodecylmercaptane	0.6
	potassium persulfate	0.25
	butadiene	70
	methyl methacrylate	25
	methacrylic acid	5
	phm = part per hundred monomer

Table 2

 2 Design matrix and monomer concentration

		Factor A	Factor B Run		Monomer concentration, phm
	Run No.	Bd/MMA		Label	Bd	MMA	MAA	HEMA
			[MAA]
		ratio
	1	1.375	2	(1)	72.21	25.79	2	-
	2	2.8	2	a	56.74	41.26	2	-
	3	1.92	3.5	0	63.49	33.01	3.5	-
	4	2.8	5	b	70	25	5	-
	5	1.375	5	ab	55	40	5
	6	-	-	-	57	39	2	2
		TNT Nitrile glove, a copolymer of Bd, acrylonitrile and MAA, provided by Ansell S
	A
		&T, Malaysia
	B	Wound dressing film, Polyurethane, provided by Ansell S&T, Malaysia

Table 3

 3 Properties of the various polymer films: Tensile strength, elongation at break , WVTR (with the standard deviations SD) and glass transition temperature

	Run	Tensile	strength	Elongation at break	WVT (g/24h/m 2 )	T g (C)
	No.	(MPa)		(%)
		Value	SD	Value	SD	Value	SD	Exper-	Predicted
								imental
	1	16	1.1	1370	28.3	21	5.44	-55	-52
	2	18	1.2	1170	33.1	23	3.43	-38	-31
	3	30	2.4	1010	65.3	20	4.30	-43	-40
	4	15	2.2	974	72.6	21	4.80	-51	-49
	5	25.5	4.2	772	24.2	16	7.09	-28	-27
	6	-	-	-	-	19	-	-39	-31
	A	-	-	-	-	44	-	-18	-
	B	-	-	-	-	792	-	-	-

Table 4

 4 Latex properties

	Polymer			Properties
		Solid	Gel
		content	content	Viscosity		Particle size
		(%)	(%)	(cP)	pH	(nm)
	1	41	62	87.9	4.65	97
	2	38	37	24.3	4.87	95
	3	41	30	51.2	4.61	103
	4	40	47	24.5	4.42	96
	5	37	49	26.9	4.60	82

Table 5

 5 The concentration of poly(MAA) and poly(HEMA) in aqueous phase of various latexes. Concentration is in per litre of continuous phase in latex.

			NMR experiments
		Initial		[MAA] in AQ
		[MAA],	[MAA] in AQ phase,	phase, wt % of the
	Run	mmol•L -1	mmol•L -1	initial concentration
	1	0.198	1.6 × 10 -3	0.8
	2	0.230	2.4 × 10 -3	1.0
	3	0.162	2.7 × 10 -3	1.6
	4	0.092	3.1 × 10 -3	3.4
	5	0.091	4.7 × 10 -3	5.1
	6	0.142	2.6 × 10 -3	1.8

Table 1

 1 WVTR of copolymer film at 0.875 phr of ZnO 0.

875 phr ZnO

	Sample no. Slope (g/h) WVTR, g/h.m 2 WVTR, g/24h.m 2
	1	6.85E-04	0.91	21.79
	2	7.23E-04	0.96	22.97
	3	7.83E-04	1.04	24.90
		Average WVTR =	23.22
		SD		1.57

Table 2

 2 WVTR of copolymer film at 1.75 phr of ZnO 1.

75 phr ZnO

	Sample no. Slope (g/h) WVTR, g/h.m 2 WVTR, g/24h.m 2
	1	1.07E-03	1.42	34.02
	2	9.50E-04	1.26	30.21
	3	5.22E-04	0.69	16.61
		Average WVTR =	26.95
		SD		9.16

Table 3

 3 WVTR of copolymer film at 3.5 phr of ZnO 3.5phr ZnO

	Sample no.	Slope (g/h) WVTR, g/h.m 2 WVTR, g/24h.m 2
	1	7.74E-04	1.02	24.60
	2	7.57E-04	1.00	24.08
	3	6.20E-04	0.82	19.71
		Average WVTR =	22.80
			SD	2.68

Table 4

 4 Tensile properties and WVTR of films at various concentration of ZnO

	Concentration of ZnO, phr Tensile strength, Mpa	% Elongation at break	WVTR, g/24h•m 2
	0.88	11.20	686	23.22
	1.75	17.40	564	26.95
	3.50	19.74	552	22.80

• Conditioning of the polymer films before WVTR measurements

The graph (film from run 6) below show the weight of the film after moved form the oven to the glove box with controlled humidity (Payen Cup is empty, no water, here).

• Water

vapour transmission graphs, weight changing as a function of time, of various polymer films.

	Run 4 Run 5			OS171103 #3 OS171102 #1 OS171102 #3 OS171097 #3 OS171069 #3 OS171088 #3 OS171120 #5
	Run 1 weight ( weight (g Run 3 Run 6 weight ( weight weight (g weight weight weight (g weight ( weight weight (g weight	49.3023 49.3024 49.3025 49.3026 49.3027 49.3028 49.3029 49.3030 49.3031 49.3032 49.3033 49.3034 0.00 48.3062 48.3064 48.3066 48.3068 48.307 48.3072 48.3074 48.3076 48.3078 0.00 49.2566 49.5793 49.5794 49.5795 49.5796 49.5797 49.5798 49.5799 49.5800 49.5801 49.5802 49.601 49.8028 49.3832 50.246 48.75700 49.5803 0.00 49.6142 49.5998 49.6 49.6002 49.6004 49.6006 49.6008 0.00 48.8615 48.8620 48.8038 48.8040 48.8042 48.8044 48.8046 48.8048 49.8014 49.8016 49.8018 49.802 49.8022 49.8024 49.8026 0.00 48.0180 48.0185 48.0190 48.0195 48.0200 48.0205 49.3822 49.3823 49.3824 49.3825 49.3826 49.3827 49.3828 49.3829 49.3830 49.3831 49.6819 49.6820 49.6821 49.6822 49.6823 49.6824 50.2459 49.6826 48.75680 50.2458 49.6825 50.2452 50.2453 50.2454 50.2455 50.2456 48.0198 50.2457 48.0196 48.75660 48.0192 48.0194 48.75620 48.75640 48.75600 48.0190 50.2451 49.3821 0.00 49.6818 0.00 48.75580 48.0188 48.0175 0.00 49.6817 0.00 48.0186 48.75560 48.8036 0.00 48.0184 48.8034 0.00 49.8210 49.8212 0.00 48.6058 48.3426 48.6056 48.2480 48.3424	OS171103 #1 1.00 1.50 time (h) 1.00 time (h) OS171103 #4 OS171069 #1 OS171088 #1 1.00 time (h) OS171102 #2 1.00 time (h) OS171102 #4 OS171097 #1 1.00 time (h) OS171120 #2 1.00 1.50 1.50 1.50 1.50 1.50 1.00 1.50 1.50 2.00 time (h) time (h) 1.00 1.50 OS171097 #4 1.50 2.00 2.50 2.00 2.50 2.00 3.00 2.00 2.50 2.00 2.00 2.00 2.00 2.00 3.00 2.50 3.50 time (h) OS171069 #4 time (h) OS171088 #4 time (h) 1.00 1.50 2.00 time (h) 1.00 1.50 2.00 OS171069 #2 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 time (h) OS171088 #2 OS171120 #6	3.00 2.50 2.50 2.50 2.50 2.50 2.50 3.50 3.00 4.00 2.50 2.50
	weight (g weight weight (g weight weight (g weight weight weight (g weight (g weight ( weight	0.00 49.8782 0.00 49.3190 48.6150 49.1709 49.1710 49.1711 49.1712 49.1713 49.1714 49.1715 49.1716 49.1717 49.1718 49.1719 49.2552 49.2554 49.2556 49.2558 49.2560 49.2562 49.2564 0.00 49.8784 0.00 49.3191 48.6152 49.6128 49.6130 49.6132 49.6134 49.6136 49.6138 49.6140 0.00 48.8590 48.8595 48.8600 48.8605 48.8610 0.00 49.8786 49.8788 49.8790 49.8792 49.8794 49.8796 49.8198 0.00 48.2469 48.2470 48.2471 48.2472 0.00 48.3410 48.3412 0.00 49.3192 48.6315 48.6154 49.3193 48.6316 48.6317 48.6318 48.6042 48.6044 0.00 48.6156 49.3194 48.6158 49.3195 49.8200 49.8202 49.8204 49.8206 49.8208 48.2473 48.2474 48.2475 48.2476 48.2477 48.2478 48.2479 48.3414 48.3416 48.3418 48.3420 48.6324 48.6054 48.3422 48.6319 48.6320 48.6321 48.6046 48.6160 49.3196 48.6048 48.6162 49.3197 48.6322 48.6323 48.6050 48.6052 48.6164 49.3198 49.3199	0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50	OS171103 #2 1.00 1.00 1.00 time (h) time (h) 1.00 time (h) time (h) 1.00 1.00 1.50 time (h) OS171097 #2 1.00 time (h) 1.00 1.50 1.50 1.50 1.50 1.50 1.50 2.00 1.50 1.00 1.50 time (h) time (h) 1.00 1.50 OS171120 #3	2.00 2.00 2.00 2.00 2.00 2.50 2.00 2.00 2.00 2.00	2.50 3.00 2.50 2.50 2.50	2.50 2.50 2.50 2.50 2.50
		48.6148 0.00	0.50	1.00	time (h) time (h) 1.50	2.00		2.50
		0.00	0.50	1.00	1.50	2.00		2.50
					time (h)
	Run 2

Acknowledgements

The financial support of Ansell is gratefully acknowledged, as is the support of a LIEF grant from the Australian Research Council. We greatly appreciate help from Dr. David Chang for FT-IR measurements. The Key Centre for Polymer Colloids was established and supported by the ARC's Research Centres program.

• Glass transition temperature


PharmD Camille Louvrier

PhD Elma El Khouri

MD Martine Grall Lerosey

MD Pierre Quartier

Anne-Marie Guerrot

PhD Brigitte Bader Meunier

MSc Julie Chican

MSc Malaïka Mohammad

MD Eman Assrawi

PhD Aphrodite Daskalopoulou

MSc Angela Arenas Garcia

Bruno Copin

William Piterboth

Florence Dastot

Le Moal

MSc Sonia A Karabina

PhD Serge Amselem
email: serge.amselem@inserm.fr

PhD Irina Giurgea
email: irina.giurgea@inserm.fr.

De novo gain-of-function variations in LYN

est destinée au dépôt

INTRODUCTION

Lyn, a member of the Src family of protein tyrosine kinases, plays an important role in the regulation of innate and adaptive immune responses [START_REF] Ingley | Functions of the Lyn tyrosine kinase in health and disease[END_REF]. Expressed in immune cells (except T lymphocytes) and in nonhematopoietic cells [START_REF] Ingley | Functions of the Lyn tyrosine kinase in health and disease[END_REF], Lyn is involved in either activation or inhibition of pivotal signaling pathways, such as those dependent on phosphoinositide 3-kinase (PI3K), MAPK, and NF-κB [START_REF] Scapini | Multiple roles of Lyn kinase in myeloid cell signaling and function [review][END_REF][START_REF] Tsantikos | Role of the Lyn tyrosine kinase in the development of autoimmune disease[END_REF]. Lyn is a nonreceptor kinase conserved throughout evolution. Its activity is tightly regulated by key posttranslational modifications. Lyn phosphorylation at Tyr508 keeps the molecule in an inactive closed conformation, whereas dephosphorylation of this residue allows its activation (4) through the subsequent autophosphorylation of Tyr397, which in turn, greatly enhances Lyn enzymatic activity [START_REF] Ingley | Functions of the Lyn tyrosine kinase in health and disease[END_REF]. These consequences on Lyn hyperactivation were initially assessed in vivo in a Lyn up/up knockin mouse model of mice carrying a homozygous mutation at Tyr508 (p.Tyr508Phe, also known as Y508F) [START_REF] Harder | Gain-and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage[END_REF]. Lyn up/up mice developed hemolytic anemia, severe autoimmune glomerulonephritis, and circulating autoreactive antibodies [START_REF] Harder | Gain-and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage[END_REF][START_REF] Slavova-Azmanova | Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling[END_REF][START_REF] Hibbs | Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity[END_REF].

In humans, 2 patients carrying a de novo LYN variation involving Tyr508 (p.Tyr508Phe and p.Tyr508*) and presenting with an earlyonset systemic autoinflammatory disorder (SAID) have been reported [START_REF] Jesus | A de novo nonsense mutation in the tyrosine kinase Lyn in a patient with an early onset autoinflammatory phenotype[END_REF][START_REF] Jesus | Mutations in the tyrosine-protein kinase Lyn cause an early-onset neutrophilic vasculitis syndrome [abstract[END_REF]. While the effect of the missense variation p.Tyr508Phe has been studied for the murine Lyn protein, there is so far no study on the functional impact of the nonsense variation p.Tyr508*.

Herein, we report on a patient carrying a novel de novo LYN missense variation (p.Tyr508His) and investigate the functional consequences (Lyn phosphorylation state and Lyn-dependent NF-κB activation) of all 3 variations affecting Tyr508 in humans (p.Tyr508His, p.Tyr508Phe, and p.Tyr508*). Comparison of the phenotypic features of our patient with those of the 2 patients reported previously allowed us to draw a comprehensive description of the LYN-associated SAID phenotype.

PATIENT AND METHODS

Patient. Clinical features were collected through a standardized form. Written informed consent was obtained from the patient's parents for genetic tests, data sharing, and publication of the patient's photographs according to French law and the principles of the Declaration of Helsinki.

Molecular studies. Genomic DNA was extracted from peripheral blood leukocytes of the patient and her parents. Nextgeneration sequencing (NGS) was performed using a custom sequence capture (SeqCap EZ Choice system; Roche Nimble-Gen) of the exons and flanking intronic sequences of the main SAID-causing genes (i.e., ADA2, AP1S3, CARD14, IL1RN, IL36RN, LACC1, LPIN2, LYN, MEFV, MVK, NCSTN, NLRC4, NLRP1, NLRP12, NLRP3, NOD2, FAM105B, PLCG2, POMP, PSMA3, PSMB4, PSMB8, PSMB9, PSTPIP1, RBCK1, RELA, SH3BP2, SLC29A3, STING1, TNFAIP3, TNFRSF1A, TNFRSF11A, WDR1). Sequencing was performed on a Next-Seq500 (Illumina) platform according to the manufacturer's instructions. The bioinformatics pipeline, previously described [START_REF] Louvrier | NLRP3-associated autoinflammatory diseases: phenotypic and molecular characteristics of germline versus somatic mutations[END_REF], was used to detect germline and somatic variations. The results obtained by NGS were confirmed by Sanger sequencing.

LYN exon 13 was amplified by polymerase chain reaction (PCR) and analyzed by Sanger sequencing using a BigDye Terminator sequencing kit (Applied Biosystems) on an ABI 3730XL automated capillary DNA sequencer (Applied Biosystems). Sequences were analyzed against the LYN reference sequence (NM_002350).

Microsatellite markers (D3 S1292, D1 S249, D2 S117, D4 S406, D5 S426, and DX S991) were amplified by PCR using fluorescent-labeled primers. The PCR products were analyzed on an ABI 3730XL sequencer (Applied Biosystems).

Plasmid constructs. After reverse transcription using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche), the fulllength LYN complementary DNA (NM_002350), which was obtained from the control's immortalized B cells, was subjected to PCR amplification using Q5 Hot Start High-Fidelity 2X Master Mix (BioLabs) and was cloned into a pcDNA3.1/V5-His TOPO TA Expression Kit (Invitrogen) following the manufacturer's instructions. Site-directed mutagenesis was performed on the wild-type (WT) LYN expression vector (pLYN-WT) to generate the plasmids carrying the Lyn missense and nonsense variations pLYN-Y508H, pLYN-Y508F, and pLYN-Y508*.

Cell culture conditions and transfections. A HEK293T cell line was cultured in DMEM GlutaMAX medium (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/ streptomycin (Invitrogen) and maintained at 37 C in a 5% CO 2 atmosphere. HEK293T cells were transfected with different amounts of the above-mentioned plasmids using FuGENE HD transfection reagent (Promega) with a ratio of 1:3 following the manufacturer's instructions. After 24 hours, the transfected cells were collected for immunoblotting or luciferase assay.

Cell lysates preparation. HEK293T cells were lysed in cold lysis buffer (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, phosphatase inhibitor [Roche Diagnostics]) and complete protease inhibitor cocktail (Roche Diagnostics). Lysates were incubated at 4 C under agitation for 1 hour and cleared by 10 minutes of centrifugation at 1,000g and 4 C, then 4× Laemmli buffer (0.2 M dithiothreitol, 0.25 M Tris, 8.4% sodium dodecyl sulfate, 40% glycerol) was added for a final concentration of 1×.

Immunoblotting. Cell lysates were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, blocked in Tris buffered saline-Tween 0.1% with 5% bovine serum albumin (designated as TBST-BSA 5%) for 1 hour and analyzed by Western blotting with the following antibodies: rabbit anti-human Lyn (#2732; Cell Signaling Technology), phospho-(Y508) Lyn (#2731; Cell Signaling Technology), phospho-(Y397) Lyn (#70926; Cell Signaling Technology) and anti-GAPDH-horseradish peroxidase (MA5-15738-HRP; ThermoFisher). All antibodies were incubated overnight at 4 C in TBST-BSA 5%. The secondary antibody (anti-rabbit IgG-horseradish peroxidase; product no. A0545; Sigma-Aldrich) was incubated for 1 hour at room temperature in TBST-BSA 5%. Proteins were detected using Amersham ECL Select Western Blotting Detection Reagent (GE Healthcare) according to the manufacturer's recommendations.

NF-κB luciferase reporter assay. HEK293T cells were cotransfected in 12-well plates with the following plasmids: 200 ng of pcDNA3 empty vector or each of the LYN expression plasmids, 100 ng of pGL4.32[luc2P/NF-κB-RE/Hygro] vector (Promega), and 10 ng of Renilla luciferase control reporter vector (Promega) for normalization. Cells were lysed 24 hours posttransfection and subjected to dual luciferase reporter assays (Promega) using a Tristar LB 941 Multimode Microplate Reader (Berthold Technologies). The firefly luciferase activity was normalized against the Renilla signal. Relative firefly/Renilla signals are reported.

Statistical analyses. Differences were analyzed using the Student's unpaired 2-tailed t-test and were plotted with the Graph-Pad Prism 9 software. A P value less than 0.05 was considered statistically significant. Data are presented as the mean ± SD.

RESULTS

Clinical and biologic phenotype. The patient is a 4-yearold girl born from healthy parents at 26 weeks and 2 days of gestation. Her premature birth was attributable to maternal HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome during pregnancy, and the pregnancy was marked by a second trimester fetal/intrauterine growth restriction (<5th percentile fetal growth size) with cardiomegaly and hepatomegaly associated with persistent Arantius' duct and liver microcalcifications. The patient's birth weight was 925 gm, birth length was 33.6 cm, head circumference was 25.5 cm, and Apgar scores were 5 at 1 minute and 7 at 5 minutes. At birth, she presented with cardiomegaly, a dilated ascending aorta (without bicuspidy), cholestasis, and hepatosplenomegaly with liver calcifications, as well as several complications related to prematurity (hyaline membrane disease in spite of steroids administered to the mother during pregnancy, sensitive abdominal bloating evocative of enteropathy, and a secondary infection with Staphylococcus capitis at 3 weeks of age).

The patient was intubated from birth to day 3, after which noninvasive ventilation (day 3 to day 7) and high-flow oxygen therapy (day 7 to day 21) were required. She received a surfactant (poractant alfa) in the first hours of life due to hyaline membrane disease and received caffeine to prevent apnea. Antibiotic therapy was started due to biologic inflammation (serum C-reactive protein [CRP] level of 40 mg/liter) from birth. Testing for maternal-fetal infection remained negative. The mother was immunized against rubella and cytomegalovirus, and maternal serologic status was negative for toxoplasmosis, syphilis, and HIV. Elevated serum CRP levels (15-63 mg/liter) were reported repeatedly during the patient's first month of life despite the use of several types of antibiotics. She developed recurrent and severe episodes (1 episode per month) of systemic inflammation lasting 3-4 days (serum CRP levels up to 366 mg/liter and hyperleukocytosis [white blood cell count 37 × 10 9 /liter]), with fever, urticarial lesions and atopic dermatitis, arthritis (joint redness and swelling affecting knees and ankles), abdominal pain, and moderate diarrhea. Additionally, acute asthma episodes required several hospitalizations, including 1 hospitalization in intensive care (Table 1, Figure 1A). At 6 months, she presented with growth retardation (-4 SD on the growth curve) and a psychomotor delay. She started walking at 2 years and 4 months. As shown in Figure 1A, at the age of 14 months, she had distinctive facial features with frontal bossing and hypertelorism. Glucocorticoids (1 mg/kg/day) were started at 19 months after a major inflammatory episode (serum CRP level of 238 mg/liter). Because a relapse occurred after reducing the daily dose, anti-interleukin-1 (anti-IL-1) treatment with anakinra (4 mg/kg/day) was introduced but stopped after cutaneous reaction at the injection point; therefore, canakinumab treatment (6 mg/kg every 15 days) was started at the age of 2 years, which was effective on clinical symptoms. However, residual biologic inflammation persisted (CRP levels ranging 6-55 mg/liter and hyperleukocytosis [white blood cell count ranging 12.9 × 10 9 -17.8 × 10 9 /liter]) (Figure 1B).

At the age of 2 years, immunophenotyping of peripheral blood lymphocytes revealed B cell and T cell lymphocytosis (B cell count 2,970/μl and T cell count 9,035/μl), whereas the number of natural killer cells was in normal range. Plasma cytokine measurements performed just before the age of 3 years (blood sampled the day before the next canakinumab injection) revealed a substantial increase in proinflammatory (IL-1β, IL-6, tumor necrosis factor [TNF], IL-18, and interferon-inducible protein 10 [IP-10]) and antiinflammatory (IL-10) cytokines relative to 5 healthy donors (Figure 1C). After starting anti-IL-1 therapy, the patient's growth delay was gradually and partly improved (-4 SD until the age of 2 years, -3.5 SD at the age of 3 years, -2 SD at the age of 4 years).

Identification of a de novo LYN variation. Study of genomic DNA from the patient's peripheral blood cells through an NGS-based approach (SAID gene panel) allowed identification of the c.1522T>C, p.(Tyr508His) missense variation in the LYN gene (herein referred to as Y508H) (Figures 1D andE). Sequencing of parental DNA samples associated with the intrafamilial segregation study of 6 independent microsatellite markers revealed that the Y508H variation occurred de novo. This variation, which is absent from the genome aggregation database (gnomAD), is predicted to be pathogenic according to a Combined Annotation Dependent Depletion (CADD) phred score of 31. Tyr508 is a residue conserved across species as well as among all 7 other members of the Src tyrosine kinase family (Figure 1F), suggesting that amino acid substitutions at this position could be detrimental to Lyn function. In addition, Tyr508 is located in the C-terminal sequence known to be involved in the negative regulation of Lyn activity in mice [START_REF] Harder | Gain-and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage[END_REF]. Notably, the same tyrosine residue is mutated in the mouse model of sustained constitutive activation of Lyn (Lyn up/up ) and in the 2 patients previously reported to be carrying de novo LYN variation involving Tyr508 (Table 1). Taken together, these data strongly suggest that the Y508H variation is a diseasecausing mutation, a conclusion consistent with the classification of this variation according to the guidelines of the American College of Medical Genetics and Genomics (i.e., the PS2, PM1, PM2, PM5, and PP3 criteria can indeed be applied to this de novo mutation, thereby allowing us to classify it as pathogenic) [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF].

Functional characterization of LYN variations identified in SAID patients. To assess the functional consequences of the LYN Y508H, Y508F, and Y508* variations, we studied the phosphorylation state of recombinant forms of human Lyn carrying those variations and compared the results to those obtained with wild-type Lyn (Lyn-WT). Of note, although the Y508* variation introduces a premature stop codon, it should escape the nonsense-mediated messenger RNA (mRNA) decay pathway because of its localization in the last exon of LYN [START_REF] Kurosaki | Nonsense-mediated mRNA decay in humans at a glance [review][END_REF]. After expression of Lyn-WT and each of the mutated Lyn proteins (Lyn-Y508H, Lyn-Y508F, and Lyn-Y508*) in HEK293T cells, we observed an absence of phosphorylation at position 508 and a constitutive phosphorylation of Tyr397 for all mutated proteins (Figure 2A and Supplementary Figure 1, available on the Arthritis & Rheumatology website at https://onlinelibrary.wiley.com/doi/10. 1002/art.42354). Overall, these data clearly show that all 3 variations involving Tyr508 maintain Lyn in its active form, consistent with the gain-of-function effect of the Y508H, Y508F, and Y508* variations.

Furthermore, we sought to evaluate the impact of all 3 LYN variations on the NF-κB signaling pathway by means of a luciferase reporter assay performed under basal conditions. These experiments revealed that, while Lyn-WT did not activate the NF-κB pathway, all 3 variations led to the activation of this pathway (Figure 2B). Phenotypic spectrum of LYN-associated SAID. With the aim of identifying the main phenotypic features associated with Lyn gain of function, we analyzed the phenotype of the patient reported in our study and compared it to the available phenotypic data of the other 2 individuals previously reported as having a de novo LYN variation (Y508F and Y508*) [START_REF] Jesus | A de novo nonsense mutation in the tyrosine kinase Lyn in a patient with an early onset autoinflammatory phenotype[END_REF][START_REF] Jesus | Mutations in the tyrosine-protein kinase Lyn cause an early-onset neutrophilic vasculitis syndrome [abstract[END_REF]. The phenotypic data are summarized in Table 1. Two of the 3 patients were born prematurely, and no birth term information is available for the third patient [START_REF] Jesus | Mutations in the tyrosine-protein kinase Lyn cause an early-onset neutrophilic vasculitis syndrome [abstract[END_REF]. All 3 patients presented with severe inflammatory disease with neonatal onset, characterized by recurrent fever, skin manifestations (such as purpuric skin rash, urticarial lesions, and atopic dermatitis), and arthralgia and/or arthritis, and disease was associated with elevated biologic inflammation markers (high CRP levels and hyperleukocytosis). Other manifestations were observed in 2 of the patients: ocular manifestations (2 of 3 patients), hepatosplenomegaly (2 of 2 patients with available data), abdominal pain (2 of 2 patients with available data), and testicular pain (2 of 2 male patients). Anemia and severe thrombocytopenia requiring splenectomy, as well as circulating autoantibodies and liver damage (increased liver enzymes and, at biopsy a periportal lymphocytic infiltrate, vanishing bile duct disease, and periportal bridging fibrosis) were reported only in the patient the Y508* variation. Of note, our patient presented with facial features reminiscent of CINCA (chronic infantile neurologic, cutaneous, articular) syndrome, which is caused by NLRP3 variations, that were not reported in the 2 previous patients with a de novo LYN variation. Moreover, as mentioned above, the patient reported in the current study also displayed increased plasma levels of proinflammatory (IL-1β, IL-6, TNF, IL-18, IP-10) and antiinflammatory (IL-10) cytokines (Figure 1C); no cytokine data were available for the other patients.

Therapeutic attempts in LYN-associated SAID. Treatment regimens were different for each patient. The first reported patient with the Y508* variation was treated efficiently with dasatinib (8), a tyrosine kinase inhibitor traditionally used for chronic myeloid leukemia. No response to anti-IL-1 therapy was reported for the patient with the Y508F variation (9), whereas anakinra and canakinumab have shown efficacy on the inflammatory symptoms of our patient who carries the Y508H variation.

DISCUSSION

SAIDs represent a group of diseases that are highly heterogeneous at both the phenotypic and genetic level. The data presented in the current study clearly contribute to the recognition of LYN as a SAID-associated gene that should be screened for variations in patients with unexplained, early-onset inflammatory symptoms and no family history of SAID.

The patient reported here carries a de novo LYN variation (Y508H) affecting a critical tyrosine residue that modulates Lyn activity. Using in vitro functional assays, we demonstrated the gain-of-function effect on Lyn function of the Y508H variation as well as of the other 2 LYN variations (Y508F and Y508*) previously reported in 2 patients [START_REF] Jesus | A de novo nonsense mutation in the tyrosine kinase Lyn in a patient with an early onset autoinflammatory phenotype[END_REF][START_REF] Jesus | Mutations in the tyrosine-protein kinase Lyn cause an early-onset neutrophilic vasculitis syndrome [abstract[END_REF]. Indeed, Y508*, which introduces a premature stop codon, does not result in a loss of protein function. Because of its localization in the last exon of LYN, it is expected to escape nonsense-mediated mRNA decay, and as shown by this study, the resulting truncated protein, which lacks 5 residues of the C-terminus of the protein, is activated. It is also noteworthy that all 3 patients carry variations involving the same amino acid. This striking observation underscores the critical role played by Tyr508 in the regulation of Lyn activity.

By studying Lyn phosphorylation under basal conditions, we demonstrated that all 3 mutated proteins involving Y508 (Lyn-Y508H, Lyn-Y508F, Lyn-Y508*) were phosphorylated at Tyr397, consistent with a hyperactive form of the protein. Moreover, we observed an activation of the NF-κB signaling pathway in basal conditions confirming the constitutive gain of function of the Lyn proteins carrying those variations. It should be noted that these data, which demonstrate the pathogenicity of these 3 Lyn variations, were obtained after expressing the corresponding recombinant Lyn proteins in a human HEK293T cell line. Therefore, it remains possible that, under in vivo conditions, these mutations may have unknown cell type-specific effects.

The 3 patients with LYN gain-of-function variations share clinical and biologic features of systemic inflammation, i.e., recurrent fever, arthralgia, skin manifestations, hepatosplenomegaly, and abdominal pain, as well as hyperleukocytosis and elevated CRP levels. Notably, many of these symptoms, including severe neonatal inflammatory syndrome and urticarial lesions, overlap with those observed in CINCA syndrome, a SAID due to de novo gain-of-function variations in NLRP3 [START_REF] Louvrier | NLRP3-associated autoinflammatory diseases: phenotypic and molecular characteristics of germline versus somatic mutations[END_REF]. In addition, it should be emphasized that the anti-IL-1 treatment canakinumab, effective in our patient, also shows sustained efficacy in CINCA syndrome [START_REF] Sibley | A 24-month open-label study of canakinumab in neonatal-onset multisystem inflammatory disease[END_REF]. Such similarities between CINCA syndrome and LYN-associated SAID should prompt physicians and molecular geneticists to look for LYN variations in patients presenting with symptoms indicative of CINCA syndrome and having no identifiable variations in NLRP3.

It should also be noted that 2 of the 2 patients with a gain-offunction mutation in LYN who had available data for birth term were born prematurely, raising the question as to whether such LYN defects predispose to miscarriage or premature delivery, or contribute to the rarity of LYN-associated SAID. The report of additional patients and families with disease-causing variations in LYN should help answer these questions.

In the Lyn up/up mouse model with a Lyn gain-of-function mutation (Y508F), the adult mice presented with severe autoimmune disease characterized by circulating autoantibodies and severe glomerulonephritis [START_REF] Hibbs | Sustained activation of Lyn tyrosine kinase in vivo leads to autoimmunity[END_REF]. Low titers of circulating autoantibodies have been reported in 1 of the LYN-associated SAID patients [START_REF] Jesus | A de novo nonsense mutation in the tyrosine kinase Lyn in a patient with an early onset autoinflammatory phenotype[END_REF]. As the patient of this study is young (4 years old), follow-up is necessary to detect the possible occurrence of an autoimmune disease.

In conclusion, the functional characterization of the 3 LYN variations so far identified, together with the description of the associated phenotypes, allowed for development of a comprehensive description of the disease entity resulting from LYN gainof-function variations. To improve the severe and persistent inflammatory syndrome associated with LYN variations, sustained treatment (including anti-IL-1 therapy) is required. Such de novo occurrence of sequence variations in LYN also has important implications for genetic counseling.

Figure 1 .

 1 Figure 1. Clinical and genetic description of the patient with a de novo LYN variation. A, Photograph of the patient at age 7 months (left) and 14 months (right) showing urticarial lesions and distinctive facial features. B, Plot showing serum C-reactive protein (CRP) levels measured in the patient at different time points from birth to age 4 years and showing periods of time the patient was receiving glucocorticoids or anti-interleukin-1 treatment. C, Enzyme-linked immunosorbent assay-assessed plasma cytokine levels in the patient just before the age of 3 years (red circles) and in 5 healthy donors (open circles). The horizontal line represents the mean of the 5 interleukin-18 (IL-18) values. D, Genealogic tree of the patient with her LYN genetic status. E, Sanger sequencing electropherogram of genomic DNA extracted from peripheral blood leukocytes of the patient and her parents, showing the LYN c.1522T>C, p.(Tyr508His) variation present in the heterozygous state in the patient and absent in her parents. F, Evolutionary conservation of Lyn protein amino acid Tyr508 across species (left) and in other members of the Src family of protein tyrosine kinases (right). G, Representation of Lyn with domain organization showing the Tyr508His variation.

Figure 2 .

 2 Figure 2. Functional characterization of identified LYN variations. A, Immunoblot analysis of Lyn, phospho-Y508 Lyn [P(Y508)-Lyn], phospho-Y397 Lyn [P(Y397)-Lyn], and GAPDH (as loading control) in HEK293T cells transiently expressing wild-type (WT) Lyn or each of the mutated Lyn proteins (Lyn-Y508H, Lyn-Y508F, or Lyn-Y508*). Immunoblot analysis represents ≥3 independent experiments. B, Impact of each Lyn variation on NF-κB activity. HEK293T cells were transfected with 200 ng of the expression vector encoding wild-type Lyn or each of the mutated Lyn proteins (Lyn-Y508H, Lyn-Y508F, or Lyn-Y508*), or an empty vector (EV) as control, together with an NF-κB reporter construct (100 ng) and a Renilla luciferase control reporter vector for normalization (10 ng). Data represent 5 independent experiments. Values on the vertical axis are the relative firefly/Renilla signal. Circles represent individual experiments. Bars are the mean; whiskers show the SD. * = P ≤ 0.05 and ** = P ≤ 0.01 by Student's unpaired 2-tailed t-test. NT = not transfected.

Table 1 .

 1 Genotype and phenotype of 3 patients identified as carrying a de novo LYN variation resulting in an early-onset autoinflammatory disorder*

			Patient in De Jesus et al 2014	Patient in De Jesus et al 2016
		Patient in the present study	(ref. 8)	(ref. 9)
	LYN variation	c.1522T>C, p.(Tyr508His)	c.1524C>G, p.(Tyr508*)	c.1523A>T, p.(Tyr508Phe)
	Transmission	de novo	de novo	de novo
	Sex	Female	Male	Male
	Birth term	26 weeks, 2 days of gestation	31 weeks of gestation	NA
	Age at onset	Birth	Birth	Birth
	Age at diagnosis	2 years	3 years	15 years
	Fever	Yes	Yes	Yes
	Facial characteristics	Frontal bossing, hypertelorism,	NA	NA
		exophthalmia, low set ears
	Skin involvement	Urticarial lesions, atopic dermatitis	Purpuric skin rash (neutrophilic vasculitis)	Purpuric skin rash
	Hepatosplenomegaly	Yes	Yes	NA
	GI involvement	Abdominal pain, diarrhea	NA	Abdominal pain
	Ocular involvement	No	Periorbital erythema	Conjunctival erythema
	CNS involvement	No	NA	Headaches
	Joint involvement	Arthritis	NA	Arthralgia
	Other clinical signs	Neonatal cardiomegaly, cholestasis	Hydrops fetalis, testicular pain and	Testicular pain, oral ulcers, fatigue
		and liver calcifications, severe	swelling, splenectomy (9 months of age),
		growth delay, asthma	vanishing bile duct disease
	Biologics	Elevated CRP, hyperleukocytosis	Elevated CRP, anemia, severe	Elevated CRP and serum
			thrombocytopenia, increased liver	amyloid A
			enzymes, circulating autoantibodies
	Treatment	Canakinumab 6 mg/kg every	Oral steroids, dasatinib	Unresponsive to anakinra and
		15 days		tocilizumab; partial response to
				colchicine, etanercept

* NA = not available; GI = gastrointestinal; CNS = central nervous system; CRP = C-reactive protein.

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ACKNOWLEDGMENTS

We are grateful to the patient and her parents, whose cooperation made this study possible, and to Dr. Pascale Saugier-Veber for her contribution to collection of samples.

Funded by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Agence Nationale de la Recherche (ANR-17-CE17-0021-01), and ImmunAID, which received funding from the European Union's Horizon 2020 research and innovation programme (grant agreement 779295).

Dr. El Khouri's work was supported by the EMERGENCE-PhenomAID grant from Sorbonne Université.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Giurgea and Amselem had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Louvrier, El Khouri, Karabina, Amselem, Giurgea. Acquisition of data. Louvrier, Grall Lerosey, Quartier, Guerrot, Bader Meunier, Chican, Mohammad, Piterboth, Giurgea. Analysis and interpretation of data. Louvrier, El Khouri, Chican, Assrawi, Daskalopoulou, Arenas Garcia, Copin, Piterboth, Dastot Le Moal, Amselem, Giurgea.