Stéphane Poyet
email: stephane.poyet@cea.fr

Gaëtan Touzé

Jean-Luc Adia

Laurent Charpin

Sylvie Michel-Ponnelle

A simple method to measure the isosteric energy of adsorption

Keywords:

The effect of temperature on water vapour desorption isotherms can be described simply using the Clausius-Clapeyron equation and the isosteric energy of adsorption, the latter conventionally estimated using two (or more) isotherms obtained at different temperatures. This approach has some disadvantages, however, and this article puts forward a simple alternative methodology for characterising this isosteric energy experimentally. Our method is based on monitoring the relative humidity of a cementitious material heated under sealed conditions. The evolution of the relative humidity as a function of temperature makes it possible to directly evaluate the isosteric energy. In practice, the tests are performed using a cell of the type that is used for self-desiccation studies.

Introduction

Temperature has a significant influence on the adsorption of water vapour in cementitious materials: for a given relative humidity (RH) value, it induces (inter alia) a drop in the quantity of water retained in the porous network at equilibrium when it increases [START_REF] Hundt | Sorptionsuntersuchungen an zemenstein, zementmörtel und beton[END_REF][START_REF] Radjy | Temperature data for water sorption in hardened cement paste: enthalpy, entropy and sorption isotherms at different temperatures[END_REF][START_REF] Ishida | Enhanced modeling of moisture equilibrium and transport in cementitious materials under arbitrary temperature and relative humidity history[END_REF][START_REF] Poyet | Experimental investigation of the effect of temperature on the first desorption isotherm of concrete[END_REF][START_REF] Brue | Effect of temperature on the water retention properties of two high performance concretes[END_REF][START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF][START_REF] De Burgh | Prediction of water vapour sorption isotherms and microstructure of hardened Portland cement pastes[END_REF][START_REF] Brue | Five year drying of high performance concretes: effect of temperature and cement-type on shrinkage[END_REF][START_REF] Maruyama | Temperature dependency of short-term length-change and desorption isotherms of matured hardened cement[END_REF]. This phenomenon has been attributed to thermal desorption [START_REF] Poyet | Temperature dependence of the sorption isotherms of cementbased materials: heat of sorption and Clausius-Clapeyron formula[END_REF]. According to Le Chatelier's principle [START_REF] Chatelier | Sur un énoncé général des lois des équilibres chimiques (in French)[END_REF], a change in temperature in a system at equilibrium moves the position of equilibrium to counteract the change. Because adsorption is an exothermic process, an increase in temperature displaces the equilibrium between the water vapour and the adsorbed water towards the endothermic reaction, i.e. desorption. As a result, the amount of water retained in the pores decreases, as mentioned above. This effect may be described simply but effectively by means of the Clausius-Clapeyron equation [START_REF] Pan | Examination of the approximations used in determining the isosteric heat of adsorption from the Clausius-Clapeyron equation[END_REF][START_REF] Sircar | Isosteric heat of adsorption: theory and experiment[END_REF][START_REF] Fomkin | Adsorption of gases, vapors and liquids by microporous adsorbents[END_REF][START_REF] Builes | Isosteric heats of gas and liquid adsorption[END_REF] (note that the derivation is carried out with the water content w constant):

q st (w) = -R ∂ln(p v ) ∂ ( 1 T ) ⃒ ⃒ ⃒ ⃒ ⃒ ⃒ ⃒ ⃒ w (1)
where q st is the isosteric energy of adsorption of the water vapour (J/ mol); R the constant of ideal gases (8.3145 J/mol/K); p v the vapour pressure (Pa) at equilibrium with the mass water content w (wt%) and the absolute temperature T (K).

When the isosteric heat q st is known (i.e. when the variations in the q st value as a function of the water content w are known), it is easy to estimate the equilibrium displacement induced by a change in temperature in terms of the relative humidity h or vapour pressure p v by integrating Eq. (1). We then obtain:

p v (w, T) = p v (w, T 0 )exp [ q st (w) (T -T 0 ) RT 0 T ] (2) h(w, T) = h(w, T 0 ) p vs (T 0 ) p vs (T) exp [ q st (w) (T -T 0 ) RT 0 T ] (3)
where h(w, T) is the value of the relative humidity at equilibrium with the water content w and the temperature T; p vs (T) is the saturated vapour pressure (Pa) at temperature T and T 0 is a temperature for which the isotherm is known (i.e. the relation between w and h). It should be noted that T 0 generally corresponds to the ambient temperature (ca. 20 • C). We should emphasise here that this simple approach does not factor in possible changes in the distribution of the size of the pores, linked (for example) to the modification of the calcium-silicate-hydrates (CSH) [START_REF] Aono | Nano-structural changes of C-S-H in hardened cement paste during drying at 50 • C[END_REF][START_REF] Gallucci | Effect of temperature on the microstructure of calcium silicate hydrate (C-S-H)[END_REF][START_REF] Bahafid | Effect of the hydration temperature on the microstructure of Class G cement: C-S-H composition and density[END_REF][START_REF] Bahafid | Effect of the hydration temperature on the pore structure of cement paste: experimental investigation and micromechanical modelling[END_REF][START_REF] Avet | Effect of temperature on the water content of C-A-S-H in plain Portland and blended cements[END_REF] and/or the partial dissolution of the calcium sulfoaluminates [START_REF] Perkins | Solubility of ettringite (Ca6[Al(OH)6]2(SO4)3 26H2O) at 5-75 • C[END_REF][START_REF] Zhou | Thermal stability and decomposition mechanisms of ettringite at <120 • C[END_REF], which could impact the adsorption/desorption isotherms. This in no way implies that these phenomena do not occur; rather that their influence on the isotherms is negligible compared to that of thermal desorption.

From a practical point of view, the isosteric heat of adsorption q st can be evaluated simply by measuring the isotherm at two different temperatures (T 0 and T 1 ); Eq. ( 1) then becomes [START_REF] Poyet | Temperature dependence of the sorption isotherms of cementbased materials: heat of sorption and Clausius-Clapeyron formula[END_REF]:

q st (w) = R ( T 0 T 1 T 0-T 1 ) ln [ p v (w, T 0 ) p v (w, T 1 ) ] (4)
where p v (w, T 0 ) and p v (w, T 1 ) are the vapour pressures at equilibrium with the water content w and temperatures T 0 and T 1 respectively. Even though the above approach is very simple, it nevertheless does have some disadvantages. In the first instance, it requires experimental results at two different temperatures (or more if possible). Although the characterisation of isotherms at ambient temperature may be considered a routine operation for many laboratories, the same cannot be said for tests at other temperatures: we need only note how few results are published in this field to recognise this. Secondly, since the derivation of Eq. ( 1) must be performed at a constant water content, a mathematical function must be used to extrapolate the value of the vapour pressure p v (w, T) based on the experimental results available at temperature T. In practice, different functions can be used such as adsorption models, for example BET [START_REF] Brunauer | Adsorption of gases in multimolecular layers[END_REF], GAB [START_REF] Anderson | Modifications of the Brunauer, Emmett and Teller equation[END_REF][START_REF] Van Den | Vapour Sorption Equilibria and Other Water-Starch Interactions; a Physico-chemical Aproach[END_REF], Pickett [START_REF] Pickett | Modification of the Brunauer Emmett Teller theory of multimolecular adsorption[END_REF] or any other bijective function [START_REF] Poyet | Determination of the intrinsic permeability to water of cementitious materials: influence of the water retention curve[END_REF]. Unfortunately, the choice of function is far from a trivial matter in that the q st (w) values obtained in this way depend on the model used. For example, significant differences were obtained using the GAB and Pickett models [START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF]. These two points highlight the need for an experimental method that can evaluate the isosteric energy more directly while avoiding any descriptive model. The aim of this article is to suggest an alternative methodological approach and to compare it with the available results (desorption isotherms at different temperatures).

Material and methods

First, it should be mentioned that the approach presented here is not new; contrary to what the authors initially thought. A similar approach has recently been proposed [START_REF] Syamaladevi | Water activity change at elevated temperatures and thermal resistance of Salmonella in all purpose wheat flour and peanut butter[END_REF][START_REF] Tadapaneni | A new method to determine the water activity and the net isosteric heats of sorption for low moisture foods at elevated temperatures[END_REF] and used for the study of food products [START_REF] Tadapaneni | Improving design of thermal water activity cell to study thermal resistance of Salmonella in low-moisture foods[END_REF][START_REF] Xu | High temperature water activity as a key factor influencing survival of Salmonella Enteritidis PT30 in thermal processing[END_REF][START_REF] Jin | Food component influence on water activity of lowmoisture powders at elevated temperatures in connection with pathogen control[END_REF][START_REF] Pérez-Reyes | The influence of elevated temperatures and composition on the water activity of egg powders[END_REF]. It may be possible that other similar studies have been published, and especially in other scientific fields, although no other papers could be found by the authors.

Rather than performing tests using a conventional approach -i.e. at a constant temperature measuring the water content at equilibrium with different RH values (regulated with saturated salt solutions, for example) -it seemed more relevant and practical with a view to using Eq. ( 1) to work at a constant water content w while varying the temperature T and measuring the vapour pressure p v (w, T) at equilibrium. This type of approach is commonly used to characterise the hygrothermic coefficient [START_REF] Bažant | Delayed thermal dilatations of cement paste and concrete due to mass transport[END_REF][START_REF] Nguyen | Unsaturated nanoporomechanics[END_REF] that links the increase in RH induced by an increase in temperature [START_REF] Radjy | Temperature data for water sorption in hardened cement paste: enthalpy, entropy and sorption isotherms at different temperatures[END_REF][START_REF] Nilsson | Temperature effects in relative humidity measurements on concretesome preliminary studies[END_REF][START_REF] Persson | Self-desiccation and chloride migration[END_REF][START_REF] Grasley | Thermal dilation and internal relative humidity of hardened cement paste[END_REF][START_REF] Colinart | Temperature dependence of sorption isotherm of hygroscopic building materials. Part 1: experimental evidence and modeling[END_REF][START_REF] Wang | May reversible water uptake/ release by hydrates explain the thermal expansion of cement paste? -arguments from an inverse multiscale analysis[END_REF].

The easiest way to conduct this type of test is to take advantage of the experience gained from studies on self-desiccation: the methods and devices developed have proved their ability to prevent any water loss from the sample tested [START_REF] Copeland | Self desiccation in Portland cement pastes[END_REF] and, it follows, to the water content during the test. For practical reasons, it was decided in this instance to use the device designed by Garcia Boivin [START_REF] Boivin | Retrait au jeune âge du béton : Développement d'une méthode expérimentale et contribution à l'analyse physique du retrait endogène[END_REF]: this consists of a stainless steel body (Fig. 1) which can accommodate a sample in a small cavity equipped with a thermo-hygrometer (Rotronic HygroClip IM-3 capacitive probe), which can be used to measure the RH at equilibrium with the sample. The device is immersed in a thermostated bath during a test to limit temperature fluctuations. Water tightness is achieved by means of a PTFE cylinder and O-rings (as well as screws not shown in Fig. 1).

Two such devices were used in this study, and were first tested using saturated salt solutions: these tests confirmed the tightness of the devices, making it possible to estimate the uncertainty of the temperature and RH measurements (±0.3 • C and ±2% RH respectively; see Appendix 1).

A CEM I paste (Ordinary Portland Cement, OPC) was selected as the material for this study since it has been analysed in the past and the q st isosteric energy was evaluated on the basis of Eq. ( 4) by Drouet et al. [START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF]. The paste was prepared in 2009 using a 2 L batch and then poured into cylindrical plastic moulds (diameter 50 mm, height 80 mm). The cylinders were demoulded 24 h later and stored in an airtight container and submerged in water. Excess portlandite was added to limit calcium leaching and carbonation. Nine years later (in 2018), cylinders were removed from the containers and thin discs (3 mm thick) were cut using a diamond disc saw on the central part of the cylinders. They were then resaturated under vacuum [START_REF] Hornain | GranDuBé -Grandeurs associées à la durabilité des bétons[END_REF] and placed in desiccation cabinets above saturated salt solutions to equilibrate them to the given RH values [START_REF] Greenspan | Humidity fixed points of binary saturated aqueous solutions[END_REF]. The saturated salt solutions used are summarised in Table 1 together with the RH obtained. Finely-divided calcium oxide (CaO) was added to the desiccation cabinets to prevent the carbonation of the samples; it should be noted that the presence of CaO did not interfere with the maintenance of the RH by the various salt solutions. This was verified from time to time by checking the RH value in the different cabinets.

The discs were left for at least one year in the desiccation cabinets so they could reach water equilibrium. They were then removed, weighed and immediately introduced into the self-desiccation devices. The temperature of the thermostated bath was increased in steps up to a maximum of 50 • C in order to limit the destabilisation of the minerals of Fig. 1. View of the self-desiccation device used in this study. Adapted from [START_REF] Boivin | Retrait au jeune âge du béton : Développement d'une méthode expérimentale et contribution à l'analyse physique du retrait endogène[END_REF].

Table 1

Saturated salt solutions used to dry the samples prior to the tests [START_REF] Greenspan | Humidity fixed points of binary saturated aqueous solutions[END_REF].

Salt

Formula RH at 20 the cement paste. The temperature and RH values were recorded throughout the tests. Following the tests, the discs were dried at 80 • C (with CaCl 2 as a desiccant) in order to estimate their water content. This protocol was chosen so that the results could be compared with the data acquired by Drouet et al. [START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF]. It should also be noted that for Portland cements, this drying protocol essentially gives the same results as drying at 105 • C (see Table 8 of reference [START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF]).

Results

Fig. 2 shows, by way of example, the results obtained with the disc initially dried at 43% RH with the K 2 CO 3 solution (Table 1). The vapour pressures p v were calculated from RH values using Rankine's formula:

p v = RH × p vs with p vs (T) = p a exp ( A + B T ) (5)
where p vs represents the water vapour saturation pressure (Pa), p a the atmospheric pressure (101.3 kPa) and A and B are two constants (A = 13.7 and B = 5120 K). For this test, the measurement device was stored in ambient air (20 • C ± 2 • C) before being immersed in the thermostated bath, which explains the temperature fluctuations observed during the first week of the test. It is interesting to observe that the RH value at equilibrium with the disc at ambient temperature is approximately 44% (i.e. a vapour pressure of 1.05 kPa), consistent with the expected value (Table 1). This indicates that the one-year period in the desiccation cabinet was enough to achieve water equilibrium. When the temperature increases, the vapour pressure at equilibrium also increases: this indicates that the water is released by the cement paste. This water loss does not give rise to a significant variation in the sample's water content, as was verified by weighing the sample after the test. It must be noted here that equilibrium was obviously not reached at each RH-step (Fig. 2). For practical reasons, equilibrium was assumed to be reached when the RH-increase rate was lower than 0.2%/day, just like in sorption balance experiments [START_REF] Poyet | The use of sorption balance for the characterization of the water retention curve of cement-based materials[END_REF]. The use of this criterion made it possible to reduce the duration of the tests while ensuring the measurement stability of the ln (p v ) values (Fig. 3) necessary for the evaluation of the isosteric energy of adsorption q st (see Eq. ( 1)).

Fig. 4 compiles the vapour pressure values (log scale) at the end of each temperature step as a function of the inverse of the temperature (1000 / T). It should be noted that the uncertainty in the values presented is much lower than the size of the points used to represent them. The slope of the regression line is used to estimate the energy q st = 56.2 kJ/mol (as an absolute value).

All the q st values obtained by testing all the dried discs with the salt solutions in Table 1 are presented in Fig. 5 as a function of the RH measured at ambient temperature. The horizontal error bar represents Fig. 2. Example of temperature and vapour pressure results obtained during a test (paste dried using K 2 CO 3 ). Fig. 3. Example of stability of ln(p v ) measurements obtained during a test (paste dried using K 2 CO 3 ). Fig. 4. Assessment of q st average value using a paste sample initially equilibrated at 43% HR (using K 2 CO 3 at ambient temperature). the constant uncertainty of ±2% for the RH measurement while the vertical error bar represents the uncertainty in the q st isosteric energy. The vertical bars represent the q st minimum and maximum values that were determined as explained in the Appendix. The q st uncertainty is not constant and increases when the RH at equilibrium decreases; this point is discussed in Appendix 1. The q st values decrease when the RH increases, and are all higher than the mean value of the latent heat of the water vaporisation (∆H vap ≈ 43.6 kJ/mol between 20 • C and 50 • C); this point is discussed below.

Knowing the water content and RH value at equilibrium at ambient temperature (approximately 20 • C) for each disc meant it was possible to estimate the desorption isotherm of the paste (Fig. 6). As for Fig. 5, the error bars correspond to the uncertainties of the measurements (±2% for the RH and ±0.5% for the water content). The comparison with the desorption isotherm obtained by Drouet et al. [START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF] with larger samples is good. This demonstrates the absence of a significant parasitic effect of atmospheric carbonation [START_REF] Auroy | Impact of carbonation on unsaturated water transport properties of cement-based materials[END_REF] despite the thinness of the discs used here (3 mm thick). Fig. 7 shows the q st isosteric energy as a function of the water content of the discs. As with Fig. 5, when the water content increases, the q st isosteric energy decreases and tends towards the mean value of the latent heat of the water vaporisation (∆H vap ). This indicates that the further a water molecule is from the substrate, the more it behaves like free water. Conversely, the closer it is to the substrate, the higher its adsorption energy. The measurements performed in this study with the self-desiccation device are consistent with the results obtained by Drouet et al. [START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF], even if there are significant differences in the steepest section of the curve (approximately 20 kJ/mol deviation around w = 5%). We should bear in mind that the uncertainty in the isosteric energy assessed by Drouet et al. [START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF] is of the order of ±10 kJ/mol and may, therefore, contribute significantly to the differences observed here.

These results show that the self-desiccation device can be used to assess the values of the q st isosteric energy of adsorption with a level of confidence equivalent to the conventional method (using isotherms at different temperatures). As is seen in Fig. 7, there are no results at low moisture contents (in the monolayer, i.e. for 0% ≤ w ≤ 4.8%). This is due to the form of the isotherm (a very rapid rise in the water content at low RH; see Fig. 6), which does not allow easy access to water content values below 5% (in this particular case) by means of the usual salt solutions. As it stands, this methodology cannot be used to obtain information on the q st variations in the monolayer (and more particularly on the validity of the optimum).

Conclusion

This article presents an alternative methodology for evaluating q st isosteric energy of adsorption, which can be used to simply describe the effect of temperature on water desorption isotherms using the Clausius-Clapeyron relationship. The method consists of introducing a samplepreviously conditioned to a water content -in an airtight container and monitoring the RH variations at equilibrium as the temperature increases. From a practical perspective, this methodology is simple to set up and requires only common laboratory equipment (a thermostated bath and thermo-hygrometer). The self-desiccation cell used here can be replaced by any type of container that is small in volume and that can be sealed effectively.

The results obtained in this study show that this approach can be employed to assess the value of the q st isosteric energy with a level of confidence that is at least equivalent to the usual method (using Clausius-Clapeyron and isotherms at different temperatures). It is difficult, however, to obtain results for low water contents (i.e. for water content values that are smaller than the monolayer). Furthermore, uncertainty in the q st value obtained increases as the RH tested decreases in relation to the uncertainty linked to the thermo-hygrometer used here.

CRediT authorship contribution statement

Declaration of competing interest

No potential conflict of interest was reported by the authors. When the value of the initial RH is above about 20%, the uncertainty in the value of the isosteric energy q st remains limited, i.e. between ±2 kJ/mol and ±5 kJ/mol approximately (Fig. 5). This is due to the fact that the uncertainties in the temperature and RH do not significantly modify the position of the points and, therefore, the value of the slope of the line (0.3 K is low compared to the temperature value and 2% is low compared to the RH value). On the other hand, the more the value of the initial RH drops, the greater the uncertainty becomes in relation to the RH value and modifies the position of the points on Fig. 10. For example, in the case of CaCl 2 (Table 1), the initial RH is of the order of 4% (at 20 • C); the uncertainty therefore represents half of its value, which maximizes the uncertainty in the q st value. In this particular case, the evaluation gives the following result with a high degree of uncertainty: q st = 94.6 -12.0 +21.0 kJ/mol. The uncertainty at low RH could be significantly reduced by limiting the uncertainty relating to the RH measurement by using more suitable sensors: dew point sensors, for instance.

Fig.

  Fig. st versus relative humidity of the disc at ambient temperature.

S

  . Poyet: Conceptualization, Methodology, Validation, Investigation, Data curation, Supervision, Writing -Original draft G. Touze: Investigation, Data curation J.-L. Adia: Validation, Writing -Review & editing, Project administration, Funding acquisition L. Charpin: Validation, Writing -Review & editing, Project administration, Funding acquisition S. Michel-Ponnelle: Validation, Writing -Review & editing, Project administration, Funding acquisition.

Fig. 6 .

 6 Fig.6. desorption isotherm at ambient temperature (approx. 20 • C) and comparison with data from Drouet et al.[START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF].

Fig. 7 .

 7 Fig.7. energy q st versus water content w and comparison with the results of Drouet et al.[START_REF] Drouet | Temperature influence on water transport in hardened cement pastes[END_REF].

Fig. 10 .

 10 Fig. 10. Illustration of the uncertainty assessment of q st values.

Acknowledgments

This study received financial support from Électricité de France (EDF, grant F35158) and the French Alternative Energies and Atomic Energy Commission CEA (CEA, BETON/TH-ACCID).

Appendix 1. Estimation of uncertainties

The two self-desiccation cells were tested prior to the tests: cups containing saturated salt solutions (KNO 3 , KI, K 2 CO 3 , CH 3 COOK and LiBr) were introduced into the devices, which were sealed and then immersed in the thermostated bath. The temperature of the bath was increased from 20 • C to 65-70 • C in 5 • C steps. The temperature and RH in the gaseous atmosphere above the salt solutions were continuously recorded. It became clear that both hygrometers return similar temperature values (Fig. 8-a) but that the difference between the two probes can reach ±0.3 • C (Fig. 8-b). In terms of the RH, the measured values (Fig. 9) correspond satisfactorily to the values available in the literature [START_REF] Greenspan | Humidity fixed points of binary saturated aqueous solutions[END_REF][START_REF] Kracek | P-T-X relations for systems of two or more components and containing two or more phases[END_REF][START_REF] Carr | Solutions for maintaining constant relative humidity[END_REF][START_REF] Tang | Controlling suction by vapour equilibrium technique at different temperatures, application to the determination of the water retention properties of MX80 clay[END_REF][START_REF] Apelblat | The vapour pressures over saturated aqueous solutions of sodium and potassium acetates, chlorates, and perchlorates[END_REF] although the deviation may reach ±2% at the two extremes of the RH range (11% at 20 • C with the LiBr solution and 93% at 20 • C with the KNO 3 solution). The uncertainty in the isosteric energy value was estimated by considering that the position of the points in Fig. 4 -line ln (p v ) as a function of 1000 RTmay vary depending on the uncertainties outlined above. By only taking into account the two points corresponding to the lowest and highest temperature, and by considering the suitable combinations of uncertainties, we maximize the impact of the slope on the line (Fig. 10) and can estimate the minimum and maximum q st values.


Peter Gergics
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Cathy Smith
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Hironori Bando
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Pérez Millán

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency

Keywords: alternative splicing, growth hormone deficiency, multiplexed assays of variant effects, transcriptional regulation

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

Introduction

POU1F1 (formerly PIT-1) is a signature pituitary transcription factor that directly regulates the transcription of growth hormone (GH), prolactin (PRL), and both the alpha (CGA) and beta (TSHB) subunits of thyroid stimulating hormone{Ingraham, 1990 #608;Gordon, 1993 #609}. In mice, Pou1f1 is expressed after the peak expression of Prop1 at E14.5 and remains expressed into adulthood{Davis, 2016 #79;Li, 1990 #139}. A well-characterized mutant of Pou1f1 (Pou1f1 dw/dw ) carries a spontaneous missense mutation (p.W251C) in the homeodomain that disrupts DNA binding{Li, 1990 #139;Camper, 1990 #137}. The homozygous mutant mice have no somatotrophs, lactotrophs or thyrotrophs except for the Pou1f1-independent rostral tip thyrotrophs{Li, 1990 #139; Simmons, 1990 #611;Slabaugh, 1981 #612;Lin, 1994 #66}. In humans, loss of POU1F1 function typically results in GH, TSH and PRL deficiency{Pfaffle, 2011 #610}.

POU1F1 undergoes an evolutionarily conserved program of alternative splicing{Wallis, 2018 #649;Schanke, 1997 #650}, resulting in a predominant isoform, alpha, that acts as a transcriptional activator and a minor isoform, beta, that acts as a transcriptional repressor{Konzak, 1992 #613;Haugen, 1993 #614;Jonsen, 2009 #615}. In the human pituitary gland, the beta isoform comprises approximately 1-3% of POU1F1 transcripts {Wallis, 2018 #649;Consortium, 2020 #658}. The POU1F1 beta isoform transcript is created by utilization of an alternative splice acceptor sequence for exon 2, located 78 bp upstream of the alpha acceptor, resulting in a 26 amino acid insertion that encodes an interaction domain for the transcription factor ETS1. This insertion, which is absent in the alpha isoform, disrupts the POU1F1 transactivation domain at amino acid 48. The POU1F1 alpha and beta isoforms have different activities depending on the context of the target gene{Konzak, 1992 #613}. For example, the POU1F1 alpha isoform activates its own expression, but the beta isoform does not, and the beta isoform interferes with alpha isoform mediated activation{Jonsen, 2009 #615}. Although alternative splicing of POU1F1 is evolutionarily conserved among vertebrates, the functional roles of the minor, beta isoform remain unclear{Wallis, 2018 #649}.

The first case of a recessive POU1F1 loss of function was described in a patient with combined pituitary hormone deficiency (CPHD) born to consanguineous parents{Tatsumi, 1992 #140}; since then, many unique variants in POU1F1 have been reported in patients with CPHD or isolated growth hormone deficiency (IGHD){Fang, 2016 #23;Gergics, 2019 #616;Birla, 2019 #617;Bas, 2018 #618;Blum, 2018 #301;Bertko, 2017 #619;Birla, 2016 #620}. A few dominant negative mutations have been reported that likely act by interfering with the function of POU1F1 dimers. The p.P76L variant alters the transactivation domain and causes completely penetrant IGHD{Sobrier, 2016 #148}, p.K216E interferes with the ability of POU1F1 to interact with retinoic acid receptors and p300{Cohen, 1999 #15}, and p.R271W interferes with the ability of POU1F1 to be tethered to the nuclear matrix through MATR3, SATB1 and CTNNB1{Skowronska-Krawczyk, 2014 #147}. All of the reported mutations are located in domains shared by the alpha and beta isoforms of POU1F1 and were functionally tested using the alpha isoform only.

We found four missense variants, in four independent families, that shift splicing to favor the POU1F1 beta isoform almost exclusively, while retaining its transcriptional repressor activity on the POU1F1 enhancer. We used a high throughput assay to identify in total 132 variants in and around exon 2 that cause exon skipping, isoform switching, or cryptic isoform use. With this splicing effect catalog, we evaluated additional families with hypopituitarism and identified two unrelated patients carrying synonymous POU1F1 variants that affect its splicing without changing the amino acid sequence, and another patient in which disruption of the alpha isoform acceptor leads to in-frame deletion of two amino acids. This study underscores the importance of evaluating splicing defects as a disease mechanism.

Methods

Patients

The studies were approved by ethical committees: the local Comite de Eticae Pesquisa da Faculdade de Medicina da Universidade de São Paulo (CEP-FMUSP) and the national Comite nacional de etica em pesquisa (CONEP) CAAE, 06425812.4.0000.0068; the Ethics Committee of the Faculty of Medicine, University of Leipzig (UL), Karl-Sudhoff-Institute for Medical History and Natural Sciences, Käthe-Kollwitz-Straße 82, 04109 Leipzig, Germany; and the Comité de Ética en Investigación (Research Ethics Committee) of the Hospital de Niños Ricardo Gutierrez (HNG), Gallo 1330, Ciudad autónoma de Buenos Aires, Argentina (CEI Nº 16.06). The GENHYPOPIT network collected anonymized information in a database declared to health authorities in accordance with local regulations at Aix-Marseille Université (AMU) -Conception Hospital (Assistance Publique -Hôpitaux de Marseille, AP-HM), and a declaration was made to the National Commission for Data Protection and Liberties (CNIL-France): 1991429 v 0. Patients or their parents signed a written informed consent to participate. Families 1, 3, and 6 are historical cases that were referred to the GENHYPOPIT network for genetic testing. Limited information is available for Families 1 and 3, and they were lost for follow up. The University of Michigan Institutional Review Board (UM) found the study exempt because patient DNA samples were anonymized before exome sequencing at UM.

DNA sequencing of patient samples

Individuals from Families 1, 2, 4, and 5 underwent whole exome sequencing (WES). Representative POU1F1 variants in Family 3 and 6 were discovered in a traditional CPHD candidate gene screening using Sanger sequencing (PROP1, POU1F1, LHX3 and LHX4). WES of Families 1 and 5 was carried out at University of Michigan as previously described{Fang, 2016 #23}. WES of Family 2 was performed at the Broad Institute as previously described{Guo, 2014 #621}. WES of Family 4 was performed at the Institute of Human Genetics at University of Leipzig.

Expression vectors and cell culture

The open reading frame of either POU1F1 isoform alpha (NM_000306.3) or beta (NM_001122757.2) was cloned into pcDNA3.1+/C-(K)-DYK. Site directed mutagenesis was used to obtain each of the variant POU1F1 beta isoforms: p.S50A, p.I51S, p.L52W, and p.S53A (Genscript). A firefly luciferase reporter gene was constructed in pNBm81-luc with 14 kb of the mouse Pou1f1 5' flanking sequences that includes early and late enhancers and the promoter, and 13 bp of the 5'UTR. Cloning was performed with Infusion HD (Clontech) or NEBuilder HiFi DNA Assembly (New England Biolabs). Plasmid sequences were confirmed by Sanger sequencing. The pRL-TK renilla (Promega) was used as a normalization control and pcDNA3.1(-) (Thermo-Fisher) to keep the total DNA constant. COS-7 cells were purchased from the American Type Culture Collection. Cells were maintained in Dulbecco's modified eagle medium (DMEM, Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum and penstrep (Gibco). Plasmids were transiently transfected into COS-7 cells using ViaFect Transfection Reagent (Promega, Madison, WI, USA). Luciferase activities were measured as suggested by the manufacturer (Dual-luciferase assay system; Promega).

Exon trapping assay

Human POU1F1 exon 2, flanked by partial intron 1 (85 bp upstream) and intron 2 (178 bp downstream), was cloned into the BamHI cloning site of the pSPL3 plasmid (Invitrogen) to create an exon trapping plasmid with a total insert size of 413 bp. Similarly, a minigene exon trapping plasmid was constructed that included the last 85 bp of intron 1 and the first 85 bp of intron 5, for a total insert size of 3,442 bp including exons 2, 3, 4, and 5. Site directed mutagenesis was used to create the desired variants. Plasmids were transiently transfected into COS-7 cells. Total RNA was purified with RNeasy mini (Qiagen). After reverse transcription, we analyzed exon trapping using RT-PCR with following primers; Primers SD6 Forward (5'-TCT GAG TCA CCT GGA CAA CC-3') and SA2 reverse (5'-ATC TCA GTG GTA TTT GTG AGC -3'){Nisson, 1994 #655}.

POU1F1 Saturation Mutagenesis

The cloned POU1F1 fragment in pSPL3 was divided into four overlapping tiles of 150 bp each, spanning exon 2 plus flanking introns (79 bp upstream to 131 bp downstream). Mutant tile libraries containing every possible single nucleotide variant were synthesized as a single 150mer oligonucleotide pool by Twist Bio. HiFi Assembly was used to replace each wild type tile with the respective mutant tile library amplified from the oligo pool. The resulting mutant minigene library pools were transformed in 10b E. coli (New England Biolabs), with a minimum coverage of 90 clones per mutation.

Mutant library barcoding and sequencing

To tag each mutant minigene clone with a unique barcode, a random barcode sequence (N 20 ) was inserted by HiFi Assembly into the MscI site within the common 3' UTR. Subassembly sequencing{Hiatt, 2010 #622} was used to pair each 3' UTR barcode with its linked variant(s) in cis. Briefly, a fragment starting with the POU1F1 insert and ending at the N 20 barcode (2.2 kb downstream) were amplified from the plasmid library DNA by PCR using 5'-phosphorylated primers. The resulting linear fragment was re-circularized by intramolecular ligation using T4 DNA ligase (NEB), to bring each barcode in close proximity to the mutagenized region. From this re-circularized product, paired-end amplicon sequencing libraries were generated, such that each reverse read contained a plasmid barcode and the paired forward read contained a sequence from the associated POU1F1 insert. Barcode reads were clustered with starcode{Zorita, 2015 #623} (arguments "-d 1 -r 3") to generate a catalog of known barcodes. Variants were called within each barcode group using freebayes{Garrison, 2012 #624} and filtered to require majority support, and read depth ≥4 along the entire region targeted for mutagenesis. Barcode-variant pairing was confirmed by Sanger sequencing of 15 clones selected at random from the POU1F1 library; of those, 13/15 were found in the final catalog of reconstructed sequences and associated barcodes, and all 13 perfectly matched the Sangersequenced clones.

Pooled exon-trap transfection and RNA-seq COS-7 cells were plated at 5x10 6 cells/60 mm plate. Each was transfected with 4 ug of the barcoded mutant exon-trap library using ViaFect reagent (3:1 ratio to DNA). After 24 hrs, RNA was purified as above, and 5 ug of total RNA was used to prepare first-strand cDNA using the SuperScript III First-Strand Synthesis kit (Invitrogen) with oligo dT primers. Spliced transcript was amplified using nested PCR, initially for 6 cycles using the SD6F/SA2R primers, followed by 20 cycles using primers SD2F/jklab0046 (TGTAGTCAGTGCCATCTTGGATCT). Paired-end Illumina sequencing libraries were generated by tailing PCR (6 cycles) with a forward primer within the constant upstream exon (GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT AGGGCATAGTGCCATCTTGGATCT) and a reverse primer immediately downstream of the N 20 barcode (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAGTGAACTGCACTGTG ACA AGCTGC). Unique dual i5/i7 indices were added by a second round of tailing PCR (6 cycles), and the resulting products were purified by SPRI bead cleanup and submitted for Illumina sequencing on a Hiseq 4000 and/or Novaseq instrument.

RNA-seq processing pipeline

Reverse reads containing the plasmid barcode were searched for exact match to a known barcode from the plasmid library. Forward reads containing the spliced sequence were mapped to a variant-specific reference consisting of the POU1F1 exon trap reference sequence with the respective mutation introduced in silico, using GMAP{Wu, 2005 #625} (arguments " -t 8 -f samse --microexon-spliceprob=1.0 --allow-close-indels=2"). From the spliced reads, an isoform catalog was tallied requiring each isoform to be represented by at least three distinct barcodes and nine reads. Spliced reads associated with each barcode were tallied to produce perbarcode isoform usage counts, and percent spliced in (PSI) fractions. Barcodes corresponding to the same POU1F1 variant were then aggregated (weighted by the number of reads obtained) to generate for each variant a mean PSI score for all known isoforms. Isoforms not matching a known isoform (beta, skip, or alpha) were placed in a catch-all category called "OTHER". Barcodes represented by fewer than three reads were discarded from further analyses.

Fold-change and significance testing PSI distributions under the null (no splicing difference) were approximated by bootstrap sampling. For each tested variant, the equivalent number of barcodes was drawn (with replacement) intronic background region variants (defined as intronic variants >20 bp from exon boundaries), repeated 1,000 times, and used to derive a null distribution against which each per-variant observed PSI values was converted to a z-score. For each variant, the z-scores were combined across replicates using Stouffer's test. This process was repeated separately for each isoform. For each of the three tested alternative isoforms (beta, skip, other), a fold-change over background was calculated for each variant. This was taken as the PSI value for that variant and isoform, divided by the sampling mean PSI for that isoform derived from the intron background region barcodes; the median of these values was then computed across replicates. Variants with a z-score > 4.16 (Bonferroni-corrected threshold for = 0.05) and at least a 3-fold change from the average null distribution PSI for the beta, skip, or other isoform in at least half the replicates were nominated as splice disruptive variants (SDV). Variant which fulfilled the z-score threshold but had a fold-change between 2 and 3, or which met the SDV criteria overall but failed to meet it in ≥7 replicates, were labeled as intermediate.

Comparison of bioinformatic predictors

HAL delta_psi scores{Rosenberg, 2015 #659}, SPANR zdelta_psi scores{Xiong, 2015 #660}, SpliceAI ds_max scores{Jaganathan, 2019 #661}, and MMSplice delta_logit_psi scores{Cheng, 2019 #662} were obtained from their original publications without modification. To compute pervariant ESRseq scores{Ke, 2011 #638}, we took the difference between the mean ESRseq zscores of hexamers overlapping a variant position from that of hexamers overlapping the corresponding wildtype position. Precision-recall curves were obtained to summarize each algorithm's ability to predict the experimental determination of splice disruptiveness. For algorithms which output signed scores, area under the curve (prAUC) was separately computed using signed and absolute scores as input and the higher prAUC was taken.

Selection of candidate RNA binding proteins (RBP)

RNACompete z-scores {Ray, 2013 #663} were obtained from the cisBP-RNA database (http://cisbp-rna.ccbr.utoronto.ca). At each position, wild-type and variant-containing z scores were taken as the maximum among the overlapping kmers, and the difference taken between the wild-type and variant scores. Motifs with high scoring matches (wildtype z 3) to the wildtype sequence in the beta variant cluster (c.143 to c.167) were then pursued further.

Results

Mutations in the POU1F1 beta coding region cause hypopituitarism

We initially focused on four cases of hypopituitarism from different cohorts in Europe and South America (Fig. 1A). Affected individuals' presentation was variable, ranging from multiple hormone deficiency with pituitary stalk interruption (Family 1) to isolated GH deficiency (Family 2) (Table 1, Figure S1). The affected individuals had severe short stature and responded well to GH therapy (Figure 1B). To identify causal variants, we performed whole exome sequencing (WES) for individuals in three families. Combined with conventional Sanger sequencing in another family, this revealed four missense variants in exon 2 of the POU1F1 beta isoform, each in an unrelated family (Figure 1A,2A). The four patient POU1F1 missense variants are absent from Genome Aggregation Database (gnomAD) and in-house population-matched exome databases{Lerario, 2020 #656;Vishnopolska, 2018 #657}, and they are predicted to be damaging by several bioinformatic algorithms (Table 1). Remarkably, these variants clustered in four consecutive codons: c.148T>G (p.S50A), c.152T>G (p.I51S), c.155T>G (p.L52W), and c.157T>G (p.S53A) in NM 001122757.3 (Table 1). Only one of these (c.155T>G, Family 3) appears to be de novo; the others were dominantly inherited and co-segregate with hypopituitarism phenotypes, except for c.148T>G which was inherited from the apparently unaffected parent in Family 1, indicating that if causal, this variant is incompletely penetrant. The other parent in Family 1, the two affected children, and one unaffected relative also carried a variant of uncertain significance, SIX3 p.P74R. No other variants in known hypopituitarism genes were detected.

Sequence variants retain POU1F1 beta isoform repressor function

We used a transient transfection assay to determine whether these variants disrupt the ability of POU1F1 to transactivate its own, highly conserved distal enhancer element{Ho, 2015 #191} add ref to Rajas Biochem J 1998 and DiMattia 1993 (Figure 2B). As expected, a Pou1f1 promoter reporter was strongly activated when co-transfected with cDNA of POU1F1 alpha isoform, which does not include the variant sites. Neither WT POU1F1 beta isoform, nor any of the four patient missense variants, showed significant activation of the Pou1f1-luc reporter. Consistent with a repressive role for POU1F1 beta, co-transfection with alpha at a 1:1 ratio significantly suppressed activation compared to the equivalent amount of alpha isoform alone. The four POU1F1 beta variants and WT beta repressed POU1F1 alpha activity to a similar degree.

Patient missense variants disrupt normal POU1F1 splicing to favor the beta isoform

Alpha is normally the predominant POU1F1 isoform, comprising 97-99% of the POU1F1 transcripts in human pituitary gland (ref Wallis), but its splice acceptor is predicted to be much weaker than the beta isoform acceptor 78 bp upstream (MaxEntScan{Yeo, 2004 #653}; scores, alpha: -3.63, beta: 6.96) (Fig. 2C). The beta isoform splice acceptor sequence and coding region are evolutionarily conserved in mammals and birds (Fig. 2D). We reasoned that splice repressor and/or enhancer sequences in POU1F1 may dictate the normal balance of alpha over beta isoforms, and these may be disrupted by the four patient T>G transversions. To test the effect of these variants directly, we cloned POU1F1 exon 2 and portions of the flanking introns into the exon trap splice reporter pSPL3 and introduced each variant by site directed mutagenesis (Fig. 2E). These small minigenes were transfected into COS-7 cells, and RNA was analyzed by RT-PCR. As expected, the wild type minigene produced almost exclusively alpha isoform, while each of the patient variants predominantly produced the beta isoform (Fig. 2F). We also tested splicing with larger minigenes, which contain portions of intron 1 and intron 5 with intact exons 2, 3, 4 and 5 as well as introns 2, 3 and 4, and obtained similar results, indicating the additional sequence context does not strongly influence the observed splicing pattern (Fig. 2G). We also tested two previously reported POU1F1 variants in the longer minigene context. The c.214+1 G>T caused skipping of exon 2, as expected, resulting in an inframe POU1F1 protein that lacks 80% of the transactivation domain{Inoue, 2012 #626}. This variant is associated with mild hypopituitarism. The p.P76L variant is located in the transactivation domain, enhances POU1F1 interaction with other proteins, and is associated with severe, dominant IGHD{Sobrier, 2016 #148}. The effect of this variant on splicing had not been assessed previously, and we found that it produced predominantly alpha isoform expression, indistinguishable from wild type.

Saturation mutagenesis screen for splice disruptive effects

We set out to systematically identify splice disruptive variants in POU1F1 exon 2 using a massively parallel splice reporter assay. We designed oligonucleotide pools containing every possible single nucleotide variant across exon 2 (150 bp) and 210 bp of the flanking introns (N=1080), and generated libraries in which these allelic series replaced the wild type POU1F1 fragment in the pSPL3 reporter. To track the splicing outcomes associated with each mutation, we placed a degenerate 20mer barcode in the downstream 3' UTR. The mutant plasmid library was subjected to subassembly sequencing{Hiatt, 2010 #622} to establish the pairing between each unique barcode and its associated POU1F1 mutation. In total, the mutant library contained 255,023 distinct barcoded clones, among which 188,772 (74.0%) had exactly one programmed mutation. Nearly every targeted mutation appeared in this library (1070/1080, 99.1%), with a high degree of redundancy (median 75.0 distinct barcodes/mutation, Figure S2).

The splice reporter library was transfected as a pool into COS-7 cells and processed similarly to the single mutation constructs. Spliced reporter transcripts were read out en masse using paired-end RNA-seq (Fig. 3A), with each forward read measuring an individual splicing outcome and the paired reverse read containing the 3' UTR barcode which identifies the mutation(s) present in the primary transcript. We performed 14 biological replicates, across which 94.2% (81.8-93.4%, mean 87.4%) of barcodes associated with single nucleotide variants in the clone library were detected. As expected, alpha was the predominant POU1F1 isoform (69.2% of reads overall), followed by exon 2 skipping (25.6%), and beta (1.6%). We created a catch-all category ('Other') for the remaining reads (3.6%) derived from the 262 other isoforms detected. Most of those noncanonical isoforms were only scarcely used; among them, the top 20 accounted for >80% of the reads from that category. For each POU1F1 variant, a percent spliced in (PSI) value was computed for each isoform (alpha, skip, beta, other), averaged over the associated barcodes. PSI values were highly reproducible across replicates (median pairwise Pearson's r: 0.92; Figure S3), and the effects measured in the pooled screen were corroborated by individual assays of 17 variants selected for validation (Figure S4).

Splice disruptive variants (SDVs) across POU1F1 exon 2

We measured the impacts upon splicing of 1,070 single nucleotide variants (Figure 3A and Supp. Fig. 5). Of these, 96 (9.0%) were splice disruptive variants (SDVs), which we defined as those which increased usage of beta, skip, or other isoforms by at least three-fold (Bonferronicorrected p<0.05; mean observed fold-change 8.10). SDVs using other isoforms or increasing beta usage were the most frequent (n = 35/96 variants associated with each outcome) followed by those increasing exon skipping (n = 30/96), with some variants (n = 4/96) impacting usage of multiple isoforms (Figure S6). Variants leading to each outcome tended to cluster in distinct regions; notably, the beta-increasing SDVs were located near the 5' end of the beta isoform. Intronic SDVs tended to lead to skipping. A few variants that increased skipping were scattered across exon 2, and there was some enrichment in the 5' end of the beta isoform coding region, but most were enriched near splice donor and acceptor sites: 25 of 26 intronic SDVs were within +/-20 bp of exon 2. We identified an additional 36 intermediate variants which had weaker but still significant effects (2 to 3 fold increase in beta, skip, or other isoforms usage; Bonferroni p<0.05). The majority of these intermediate variants increased exon skipping (n = 22/36; 61.1%) and they clustered similarly to the SDVs associated with each isoform.

We next examined the novel ("other") splicing isoforms. The associated 35 SDVs were nearly all located within the coding region unique to the beta isoform and at the alpha isoform acceptor site (n = 34/35; 97.1%). Of these, most (28/34) create a novel acceptor AG dinucleotide that outcompetes the more distal, native alpha acceptor (Figure S7). Most of these (20/28) result in a frame-shifted transcript with a premature truncation codon predicted to result in non-sense mediated decay. In contrast, every one of the six possible variants in the native alpha acceptor "AG" dinucleotide activate a cryptic acceptor six bases downstream, leading to in-frame deletion of two codons (Figure S8). By contrast to the novel acceptors, of 99 SNVs creating a GT dinucleotide, only one was used as a novel splice donor, c.290:C>T located 4 bp upstream of the native exon 2 donor.

We next checked how the splicing disruption map scored the four POU1F1 missense variants found in Families 1-4. All four showed strongly increased beta isoform usage (beta PSI increased 9.63-11.01 fold over background), as seen in individual minigene assays (Fig. 3B). Our results also recapitulate previously described effects of two variants found in CPHD patients: first, an upstream intronic variant c.143-5:A>G{Takagi, 2017 #628} which led to increased beta usage and intermediately elevated skipping (Figure 3C), and an essential splice donor variant c.292+1 G>T which led to near-complete skipping (Figure S8){Inoue, 2012 #626}.

We also examined the incidence of splice disruptive POU1F1 variants in the general population. The gnomAD database contains 93 of the variants measured here; among those, six (6.5%) are splice disruptive and four (4.3%) are intermediate, with all being individually rare (minor allele frequency ≤ 1.6x10 -5 ; Figure S9). Overall, variants found in gnomAD were not significantly depleted for splice disruptive/intermediate effects relative to randomly selected subsets of the tested single nucleotide variants (p=0.74 Fisher's Exact Test). Thus, POU1F1 SDVs are tolerated to a similar extent as other predicted loss of function variants (stop gain, frameshift, splice site), which are observed throughout POU1F1 at low frequencies in gnomAD.

Additional SDVs, including silent variants, in hypopituitarism patients

We next examined the splicing impacts of synonymous variants, which would typically be given low priority during genetic screening due to their expected lack of coding impact. Of the 108 synonymous variants tested, 14 were splice disruptive and an additional 12 were intermediate (13.0% SDV; 11.1% intermediate; Figure S6). We identified unrelated patients with IGHD carrying two of these synonymous SDVs in the beta isoform coding region near the 5' end of exon 2 (Fig. 3C), both of which were absent in gnomAD and population-matched control databases. The first, c.150T>G (p.Ser50=), was found among an Argentinian cohort (n=171) in a family with two individuals with severe short stature and IGHD (Table 1), for whom WES did not reveal any likely pathogenic variants in known CPHD or IGHD genes. The index case had pituitary hypoplasia, and the patient responded well to recombinant GH treatment. The second, c.153T>A (p.Ile51=), was found in a French family in relatives with severe IGHD. The parent's DNA was not available for testing, and the parent could be an unaffected carrier or an example of gonadal mosaicism. Each of these two silent variants increased beta isoform usage to a degree similar to that of the four patient missense variants (beta fold change=10.7 and 4.15 for c.150T>G and c.153T>A, respectively). In a separate family, we identified a variant disrupting the native alpha splice acceptor (c.219 A>G) co-segregating with hypopituitarism (Figure S8). This variant shifted utilization to a downstream cryptic acceptor causing in-frame deletion of two amino acids, with unknown functional consequences.

Comparison to bioinformatic splicing effect predictions

We examined how scores from splicing effect prediction algorithms compared with these experimental measurements. We scored each single nucleotide variant in the targeted region of POU1F1 using SpliceAI{Jaganathan, 2019 #661}, MMSplice{Cheng, 2019 #662}, SPANR{Xiong, 2015 #660}, HAL{Rosenberg, 2015 #659} and ESRseq scores{Ke, 2011 #638}. Among these, only SpliceAI predicted a high density of SDVs specific to the exon 2 beta region surrounding the patient variants (Figure S10). To benchmark each bioinformatic predictions, we took our SDV calls as a truth set and computed for each algorithm the area under the precision recall curve (Figure S11). SpliceAI was the most highly concordant with our results for both exonic variants (prAUC=0.843 vs other tools' range: 0.251-0.351) and intronic variants (prAUC=0.663 versus other tools' range: 0.549-0.585). Nevertheless, SpliceAI disagreed with our measurements for numerous variants: at the minimum threshold needed to capture all six patient variants as disruptive (SpliceAI deltaMax score≥0.18), it achieved 80.2% sensitivity (n=19 SDVs not predicted by SpliceAI) and 97.3% specificity (n=26 variants predicted by SpliceAI but not identified by our assay) for predicting the SDVs we identified. The degree of concordance with SpliceAI was largely insensitive to the fold change threshold used to call variants as splice disruptive (Figure S11). Additional studies will be required to resolve the discordant predictions for variants observed during clinical screening.

Discussion

We found six unrelated cases with CPHD or IGHD that can be explained by variants that shift splicing to favor the repressive beta isoform POU1F1. The missense variants, p.S50A, p.I51S, p.L52W, and p.S53A, retain repressive function. They act in a dominant negative manner by suppressing the ability of the POU1F1 alpha isoform, expressed from the wild-type allele, to transactivate expression of POU1F1 and other downstream target genes. Using saturation mutagenesis coupled to a high-throughput RNA-seq splicing readout, we systematically tested nearly every possible single nucleotide variant in or near POU1F1 exon 2 for splice disruptive potential (Table S1). We identified 96 SDVs and an additional 36 intermediate SDVs which similarly activate usage of the beta isoform or cause other aberrant splicing outcomes such as exon skipping.

In addition to the four missense variants we identified in patients, this screen also nominated 26 synonymous variants which were SDV or intermediately disruptive, together accounting for nearly a quarter of the possible synonymous variants in POU1F1 exon 2. We identified two of these in unrelated families with IGHD, c.150T>G (p.Ser50=) and c.153T>A (p.Ile51=), each of which increased beta isoform usage similarly to the four patient missense variants that initially drew our attention. These findings underscore the need to closely examine variants for splice disruptive effects, particularly synonymous variants that could be overlooked by traditional exome sequencing filtering pipelines.

The clinical features varied amongst the six families, although they were consistent within a family. Families 1, 3, and 4 presented with CPHD, while Families 2, 5, and 6 had IGHD. Moreover, Family 4 developed hypocortisolism. The reason for this variability in presentation is unknown. However, there are precedents for variable clinical features and incomplete penetrance with other cases of hypopituitarism{Gergics, 2019 #616}. Approximately 50% of IGHD progresses to CPHD, and this can even occur when the mutated gene is only expressed in GH-producing cells, i.e. GH1{Cerbone, 2017 #629}. Even individuals with the same POU1F1 mutation (i.e. p.E230K) can present with either IGHD or CPHD{Turton, 2005 #630}, indicating a contributing role for genetic background, epigenetic, and/or environmental factors. Both affected relatives in Family 1 had stalk disruption, a phenotype not currently associated with any other POU1F1 variants. This feature may be due to the presence of an additional variant in SIX3, p.P74R, that was carried by two unaffected relatives. Heterozygous loss of function of SIX3 is associated with incompletely penetrant and highly variable craniofacial abnormalities, including CPHD and holoprosencephaly, and there is precedent in mice for Six3 loss of function to exacerbate the phenotype caused by mutations in other CPHD genes such as Hesx1{Solomon, 2010 #557;Domene, 2008 #633;Gaston-Massuet, 2008 #634}.

Autosomal dominant inheritance is clear in Family 2, in which there were four affected individuals over three generations, as well as Families 4, 5, and 6. POU1F1 acts as a heterodimer{Holloway, 1995 #635}. Some other dominant mutations in POU1F1 act as negative effectors due to the ability of the mutant protein to interfere with the action of the wild type protein produced from the other allele{Rhodes, 1993 #142; Cohen, 2006 #636;[START_REF] Cohen | Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency[END_REF] #15}. The negative effect of POU1F1 beta on the transactivation properties of POU1F1 alpha are context dependent, with differential effects on Gh, Prl and Pou1f1 reporter genes{Theill, 1992 #637}. The strongest effect was reported for autoregulation of POU1F1 expression via the distal, late enhancer; dampening the auto-activation of POU1F1 expression, and adversely affecting differentiation of the entire POU1F1 lineage and result in anterior lobe hypoplasia.

The lack of significant depletion for POU1F1 SDVs among ostensibly healthy adult populations underscores the possibility of variable expressivity and/or penetrance for POU1F1 splicedisruptive variants. This is consistent with the apparently unaffected parents in Families 1 and 6. A subset of these variants, like the patient variant c.219 A>G which disrupts the alpha isoform acceptor and causes a frame-preserving two-codon deletion, may retain partial or complete function. Still others, may cause loss-of-function without dominant negative effects, and would not be expected to be strongly depleted.

In human genes, canonical splice site motifs contain less than half of the information content needed for proper splicing {Lim, 2001 #664}. Additional specificity is provided by short (6-10 nt) motifs termed exonic or intronic silencers and enhancers, which are bound by RNA binding proteins that promote or antagonize splicing{Cartegni, 2002 #665}. Although transcriptomewide atlases have been developed to map these sites{Ke, 2011 #638;Cheung, 2019 #639}, and derive motif models{König, 2012 #640}, it often remains unclear how genetic variants impact their binding and in turn the eventual splicing output. Our splicing effect map identifies a cluster of SDVs at the 5' end of the POU1F1 exon 2, each of which increases the usage of the normally repressed beta isoform. These results suggest the presence of an exonic splice silencer (ESS) which may normally suppress utilization of the beta isoform acceptor. We do not expect any cell type specific factors to be involved because wild type minigene assays in pituitary cell lines and heterologous cell lines mimic the ratios of alpha:beta isoform transcripts found in normal pituitary gland (ref [START_REF] Theill | Differential splicing of the GHF1 primary transcript gives rise to two functionally distinct homeodomain proteins[END_REF]. We mined the cisBP-RNA database {Ray, 2013 #663} and identified eight candidate motifs with strong matches to the U-rich wild-type sequence in this region (c.143 to c.167) corresponding to known splicing factors including ELAVL1 (HuR), RALY, TIA1, and U2AF2 (Figure S12). All six patient variants replaced a U with another base (G in 5 of 6 bases), which may disrupt these motifs at high information content positions (Figure S13). Other variants predicted to disrupt these motifs tended to be beta-promoting more often than intermediate/neutral in our map (p < 0.05, Fisher's Exact test). These trends suggest that U-rich ESS serves to inhibit production of POU1F1 beta and this inhibition is disrupted by CPHDassociated variants, although conclusively identifying the specific cognate binding factor will require further study.

These results extend the breadth of endocrine disorders caused by disrupted splicing. For example, in a large cohort with IGHD from Itabaianinha, Brazil, affected individuals are homozygous for a mutation in the splice donor dinucleotide (c.57 + 1G > A) in the growth hormone releasing hormone receptor gene (GHRHR){Salvatori, 1999 #641}. In addition, most mutations that cause dominant IGHD type II affect splicing of the growth hormone (GH1) gene{Alatzoglou, 2012 #642}. Mutations in splice sites or splice enhancer sequences result in skipping exon 3 and production of a dominant-negative 17.5 kD isoform of growth hormone that lacks amino acids 32-71{Shariat, 2008 #643}. The severity of the disease is variable and correlates inversely with the ratio of to 20 kD GH. Finally, severe short stature associated with Laron syndrome, or GH resistance, can be caused by generation of a cryptic splice site in the GH receptor gene. Individuals from El Oro and Loja in southern Ecuador are homozygous for a p.180E codon variant (GAA to GAG) that do not change the amino acid encoded but create a splice acceptor site 24 nt upstream of the normally utilized site{Berg, 1992 #644}. It is notable that antisense oligonucleotide therapies hold promise for treating diseases caused by abnormal splicing, including IGHD{Miletta, 2012 #645;Kuijper, 2020 #646}.

Splicing disruption accounts for a significant minority of the genetic burden in endocrine disorders, as in human genetic disease more generally {Cummings, 2017 #666;Scotti, 2016 #648}. Some estimates from large-scale screens indicate that 10% of SNV within exons alter splicing, and a third of all disease associated SNVs impact splicing efficiency{Soemedi, 2017 #647}. Variants at or near canonical spice sites are readily recognized as pathogenic{Lord, 2019 #667}, and these can be identified predicted with high accuracy by algorithms such as SpliceAI. However, for exonic variants, particularly those farther from exon junctions, splicing defects may be more challenging to identify bioinformatically{Khan, 2020 #668;Chen, 2020 #669;Dionnet, 2020 #670}. Efforts to interpret these variants will need to account for the functional impacts of changing the encoded protein sequence as well as its splicing. Finally, as our results illustrate, different variants in a single gene may lead to distinct splicing outcomes with diverse consequences ranging from the straightforward loss-of-function to dominant negative effects. A. Schematic of the human POU1F1 gene and protein isoforms produced by use of alternate splice acceptors at exon 2. The Pou1f1 beta isoform has an insertion of 26 amino acids located at amino acid 48 in the transactivation domain. B. COS-7 cells were transfected with a Pou1f1luciferase reporter gene and expression vectors for POU1F1 alpha or beta isoforms either singly or together in the ratios indicated (2N and 1N). WT POU1F1 alpha has strong activation at 2N and 1N dosages. WT and variants of POU1F1 beta isoform have no significant activation over background. A 50:50 mix of alpha and WT beta isoforms exhibited reduced activation. The variant beta isoforms suppress alpha isoform mediated activation to a degree similar to WT. C. Diagram of the splice acceptor site consensus and the genomic DNA sequence at the boundary between intron 1 and splice sites utilized in exon 2 of the POU1F1 gene{Ma, 2015 #652} D. Evolutionary conservation of the genomic sequence encoding POU1F1 beta isoform in mammals and chicken. E. Exon trapping assay with pSPL3 exon trap vector containing exon 2 of POU1F1 and portions of the flanking introns. F. Ethidium bromide-stained gel of exon trap products from cells transfected with the indicated plasmid. Arrowheads indicate the expected products for exon skipping (Blue), alpha isoform (Yellow), and beta isoform (green). G. POU1F1 minigenes spanning from intron 1 to 5, with all of the intervening exons, were engineered with the indicated variants and assayed for splicing. WT and p.P76L POU1F1 splice to produce the alpha isoform, the G>T change in the splice acceptor causes exon skipping (red arrow) and the other patient variants all splice to produce POU1F1 beta isoform.

FiguresFigure 1 .

 1 Figures Figure 1. Clinical characteristics of the variants of POU1F1 beta coding region A. and the sequence of the patients. Family 1-4 have variants in the POU1F1 beta coding region: c.148T>G (p.S50A), c.152T>G (p.I51S), c.155T>G (p.L52W), and c.157T>G (p.S53A). B. Growth curve of the patients from Family 2 and 4. GH replacement therapy was effective in reaching ideal height.

Figure 2 .

 2 Figure 2. Variants in the POU1F1 beta coding region suppress the function of alpha isoform and lead to splicing abnormality.

Figure 3 .

 3 Figure 3. Splicing effect map in POU1F1 exon 2 and flanking introns, and identification of IGHD families with synonymous changes. A. Percent usage of POU1F1 exon 2 alpha (top panel), beta (second panel), skip (third panel), and other isoforms (bottom panel) by variant position, as measured by massively parallel minigene assay. Gray bars denote splicing-neutral variants, while shaded bars indicate the base pair change of each splice disruptive variant (dark colors) and intermediate variant (light colors). Cropped intronic regions are shown in Figure S4. B. A cluster of SDVs near the beta isoform splice acceptor leads to increased usage of the beta isoform, and in some cases, intermediately increased exon skipping. Diamonds colored by the

Acknowledgements

This work was supported by the National Institutes of Health (R01HD097096 to SAC, R01GM129123 to JOK), the Japan Society for Promotion of Science (HB), Grant 2013/03236-5 from the São Paulo Research Foundation (FAPESP) (IJPA), a grant from Pfizer (RP), and the Argentinean National Agency of Scientific and Technical Promotion, PICT 2016-2913 and PICT 2017-0002 (MIPM).

Data availability

Raw sequence reads and processed counts are available at GEO (accession tbd). Jupyter notebooks to reproduce the processed dataset from raw counts are posted at GitHub (URL tbd).

Author Contributions

Transfection and exon trapping: Peter Gergics Brain MRI of patient I.1 (A, B; as a teenager) and II.1 (C-D; as a pre-teen). Thyroid ultrasound of patient I.2 (E) and II.1 (F). 1 Arteria carotis communis. Splice site strength as predicted by MaxEntScan{Yeo, 2004 #653} for novel alternate splice acceptor and donor sites. Upper: P-value corresponds to a t-test comparing the splice site strength at acceptor sites created mutation, comparing accepts not used (n = 117) vs. those used (n = 28). Dashed line (purple) represents the splice site strength of the native beta acceptor site. Solid lines (green) indicate the splice site strength of the native alpha acceptor site and native donor site respectively. Splice site strength is truncated at -20 in the positional plots, but minimum is as low as -31.6 for novel acceptors and donors within this exon.

Figure S10. In silico predictions of splice disrupting variants (SDV).

Barplots showing for each variant (color) at every position (x-axis) the splicing effect measurements (top y-axis) and splice disruption as predicted by SpliceAI, ESRSeq, HAL, SPANR, and MMSplice (from second from the top to bottom y-axes). Barplots displaying match scores (y-axis) for selected motifs defined by RNACompete{Ray, 2013 #663} scored against the wild-type POU1F1 sequence beta region (positions c.143 to to c.167).


Rita Maria Ghantous
email: ritamaria.ghantous@oregonstate.edu

Raoul François
email: raoul.francois@insa-toulouse.fr

Stéphane Poyet
email: stephane.poyet@cea.fr

Valérie L'hostis
email: valerie.lhostis@cea.fr

Fabien Bernachy-Barbe
email: fabien.bernachy-barbe@cea.fr

Dietmar Meinel
email: dietmar.meinel@bam.de

Lauriane Portier
email: lauriane.portier@cea.fr

Nhu-Cuong Tran
email: nhu-cuong.tran@edf.fr

Relation between crack opening and extent of the damage induced at the steel/mortar interface

Keywords:

Width of load-induced cracks on concrete surface is correlated to those near rebar.

Introduction

The corrosion of rebars is the main pathology affecting reinforced concrete structures and is therefore a determining factor for their durability. Such structures are subject to unavoidable cracks, which may be due to physical factors (restrained shrinkage, wetting/drying cycles, etc.) and mechanical circumstances (various applied loads and differential settlement). These cracks create pathways for atmospheric carbon dioxide, oxygen, water and chlorides to reach the steel/concrete interface, facilitating the initiation of steel corrosion in affected structures [START_REF] Schießl | Laboratory studies and calculations on the influence of crack width on chloride-induced corrosion of steel in concrete[END_REF][START_REF] Tuutti | Corrosion of Steel in Concrete[END_REF][START_REF] François | Effect of microcraking and cracking on the development of corrosion in reinforced concrete members[END_REF][START_REF] Arya | Influence of crack frequency on reinforcement corrosion in concrete[END_REF][START_REF] Berke | Corrosion of steel in cracked concrete[END_REF][START_REF] Mohammed | Effect of crack width and bar types on corrosion of steel in concrete[END_REF]. Some structural design codes and recommendations [START_REF]EN 1992-1-1, Eurocode 2: Design of concrete structures -Part 1-1 : General rules and rules for buildings[END_REF][START_REF]Control of Cracking in Concrete Structures Reported[END_REF][START_REF] New | Concrete structures standard, part 1-The design of concrete structures[END_REF] impose limits on the crack openings according to the type of concrete and to the exposure class of the structure so as not to affect the structure durability. However, other studies show that the corrosion process is not controlled by the crack widths in the range usually found with reinforcement stresses at service load [START_REF] Darwin | Debate: crack width, cover, and corrosion[END_REF][START_REF] Oesterle | The role of concrete cover in crack control criteria and corrosion protection[END_REF]. Actually, according to the American Code Institute committee 318 [START_REF]ACI Committee 318, Building Code Requirements for Structural Concrete (ACI 318-08[END_REF], surface crack widths in the range usually found with reinforcement stresses at service load levels are not detrimental for structure sustainability. It was also noted in ACI 318-08 [START_REF]ACI Committee 318, Building Code Requirements for Structural Concrete (ACI 318-08[END_REF] that enhancing the concrete cover quality was more important for corrosion protection than controlling the crack width. Additionally, corrosion has been proved to start and develop along the steel/concrete interface zone damaged by the creation of cracks [START_REF] François | Effect of damage in reinforced concrete on carbonation or chloride penetration[END_REF][START_REF] Tremper | The corrosion of reinforcing steel in cracked concrete[END_REF][START_REF] Vesikari | Corrosion of reinforcing steels at cracks in concrete[END_REF][START_REF] Ghantous | Effect of crack openings on carbonation-induced corrosion[END_REF][START_REF] François | Study of the impact of localised cracks on the corrosion mechanism[END_REF]. Several studies [START_REF] Tuutti | Corrosion of Steel in Concrete[END_REF][START_REF] Tremper | The corrosion of reinforcing steel in cracked concrete[END_REF][START_REF] Ghantous | Effect of crack openings on carbonation-induced corrosion[END_REF][START_REF] Millard | Modelling the effects of steel corrosion in concrete, induced by carbon dioxide penetration[END_REF] prove that carbonation-induced corrosion may lead to corrosion products that seal the cracks and limit the access of oxygen and water to the rebar, thus slowing down the propagation of corrosion. Therefore, and according to the literature, the size of the interface area damaged by loading may influence corrosion propagation more than the value of the crack widths. Consequently, a logical question arises: ''Does the size of the mechanically damaged interface increase sharply beyond a given crack width?"

The objective of this paper is to answer this question and determine whether a relationship exists between the value of the crack opening and the size of the corresponding load induced damage at the interface.

The evolution of the crack width was therefore followed from the outer surface of the specimen to the rebar. Additionally, the length of the damaged steel/mortar interface was quantified for different crack openings. The experimental program is presented in the next section and the results obtained are discussed afterwards.

Experimental program

Materials and specimen preparation

The specimens tested were prisms of dimensions 70 Â 70 Â 280 mm. For each specimen, a 6 mm deformed rebar was positioned in the middle of the cross section. The composition of the mortar mixture used for the specimens is shown in Table 1. The yield strength of the steel used was 500 MPa. Mortar was poured into the mold in two layers, each of which was normally vibrated in order to eliminate air voids. After 24 h, the specimens were unmolded and then cured for 28 days immersed in water (with calcium hydroxide). The specimens tested in this study were made from 4 consecutive batches of 60 l each.

Cracking of the prismatic specimens and crack opening control

Cracking protocol

In this study, several crack openings needed to be created in order to determine the effect of the crack opening on the length of the load-induced damage zone at the steel/mortar interface. For that, it was crucial to find a cracking protocol that allowed one crack to be obtained per specimen, with a controllable width in order to avoid possible overlapping of the mechanically damaged interface areas. Several cracking protocols exist in the literature but they were not all suitable for this study. For example, cracking by compression [START_REF] Zhong | Influence of damage degree on self-healing of concrete[END_REF] and freezing/thawing cracking methods [START_REF] Jacobsen | Effect of cracking and healing on chloride transport in OPC conrete[END_REF] generate diffuse cracks. The tensile test performed by imposing direct traction on a steel bar embedded in concrete [START_REF] Alahmad | Traitement des fissurations dans les ouvrages hydrauliques[END_REF] leads to a transversal crack on the whole diameter of the specimen, which is not representative of cracks observed on structures. The expansive core method is used to crack ring shaped mortar specimens [START_REF] Dang | Effects of pre-cracks on both initiation and propagation of re-bar corrosion in pure carbon dioxide[END_REF]. However, this protocol may lead to cracks that are close together, and this may induce an overlap in the lengths of damaged steel/mortar interface. Consequently, it was chosen to crack specimens using three point bending in this study because this allows the crack opening to be controlled.

Thus, after curing, prismatic specimens were cracked using three point bending test equipment. The load direction was perpendicular to the casting direction in order to apply the same mechanical load to the upper and lower surfaces of the specimen with respect to casting direction, as shown in Fig. 1.

By performing loading\unloading cycles and by increasing the maximum applied load from one cycle to another, the residual crack opening (that is to say after unloading) was increased. Therefore, the crack opening could be controlled and a range of values could be obtained (Fig. 2). For this study, three residual crack-opening values, 100 mm, 300 mm and 500 mm were realized on 145 specimens.

Crack opening profile measurements

The LVDT measured the crack opening at one point of the specimen (Fig. 1). Digital image correlation (DIC) was used to obtain the crack opening profile over the entire sample height and to study its evolution with respect to loading/unloading cycles. The set-up of the digital image correlation equipment is illustrated in Fig. 3 (a) and consisted of a CMOS camera equipped with a 35 mm objective and a LED ring lighting apparatus positioned around the center of the specimen, parallel to its surface during the three point bending test. The distance between the specimen surface and the camera determined the area monitored, which was approximately 150  230 mm 2 (height  width) in this study. A reproducible speckle pattern was created using a toothbrush and black ink in order to increase the local contrast and therefore the accuracy of DIC measurements (Fig. 3 (b)). It is known that the size of the speckles in a given speckle pattern is an important parameter in the correlation process [START_REF] Lecompte | Quality assessment of speckle patterns for digital image correlation[END_REF][START_REF] Sutton | Determination of displacements using an improved digital correlation method[END_REF] and should not be bigger than the correlation window (20  20 pixels in this study). The size of the speckles obtained varied between 261 and 750 lm and each covered a few pixels. In this study, the pixel corresponded to approximately 120 lm on the physical sample. The grid of reference points chosen comprised 120  40 points and is shown in Fig. 3 (c).

Camera images were recorded every 1.5 s until the end of the three point bending. Each captured image corresponded to a different loading step. The load applied and the displacements measured by the LVDT were recorded at a frequency of 20 Hz.

Captured images were subsequently processed by the ''CMV" DIC code developed by Bornert et al. [START_REF] Bornert | Multiscale full-field strain measurements for micromechanical investigations of the hydromechanical behaviour of clayey rocks[END_REF] to measure the 2D displacement field at the reference points chosen on the specimen surface (Fig. 4 (b)). The crack opening was equal to the difference of the displacements between two close points chosen at the same height (blue crosses in Fig. 4 (a)) on either side of the crack: the displacement values at these manually selected locations were linearly interpolated from the nearest neighbor points of the DIC grid. The cracks obtained by the three point bending were monitored by the DIC on two specimens only.

Evolution of the crack opening deep in the specimen

The crack openings were determined deep within the specimen using the industrial 3D micro Computed Tomography (CT) scanner at BAM (Federal Institute for Materials Research and Testing in Berlin). This scanner is equipped with a 225 kV micro focus X-ray tube and a flat panel detector with 2048 Â 2048 pixels.

Table 1

Formulations of cementitious materials used for the prismatic specimens.

Measuring the steel/mortar interface damage

The experimental procedure proposed by Ghantous et al. [START_REF] Ghantous | Effect of accelerated carbonation conditions on the characterization of load-induced damage in reinforced concrete members[END_REF] was used to assess the load-induced damage length. In this protocol, the specimens are preconditioned at 25 °C ± 2 °C and 55% ± 5% RH for one month. Then, they are exposed to accelerated carbonation at 3% CO 2 -55% RH -25 °C for 30 days, at the end of which time the carbonated specimens are split in two, rebars are extracted and a dilute solution of phenolphthalein is sprayed on the fresh mortar surface to allow the length of the carbonated interface to be measured. This is representative of the damaged length (Fig. 6). In the present work, the length of the damaged interface was quantified on the upper and lower steel/mortar interfaces with respect to the casting direction (Fig. 7).

The carbonation of cracked specimens took place inside a climatic chamber where the temperature and the relative humidity were continuously controlled. The standard deviations in the temperature and in the relative humidity inside the chamber were 0.5 °C and 0.2% respectively. The standard deviation detected in the CO 2 pressure value was equal to 0.05% for an operating range from 0 to 100%.

Results

Punctual crack opening values

Fig. 8 shows the reloading force over the cracking force versus the residual crack opening. The cracking force, Fcracking, is the load at which the crack appears while the reloading force, Freload, corresponds to the maximum load applied in the cycle that gives the desired residual crack opening (as indicated in Fig. 2). After testing 145 specimens, it was clear that the residual crack openings had a tendency to increase with the reload ratio despite significant scatter, as shown in Fig. 8. This observed scatter may have been due to a possible difference in the properties of the specimens, which were prepared from 4 consecutive batches. It may also be partly attributable to some variability in the rebar position (± 1 mm from one specimen to another). Additionally, the loading was applied manually during the three point bending and this fact may also have contributed to the observed scatter.

Nevertheless, the scatter observed on the residual crack opening remained limited.

3.2.

Crack opening profile obtained by the DIC over the entire height of the outer surface of the specimen Fig. 9 presents the evolution of the residual crack openings over the entire height of two prismatic specimens (70 Â 70 Â 280 mm) with respect to different loading steps. An increase in the crack width with respect to loading is notable over the entire height of the specimen. The evolution of the crack width with the loading is not the same in both specimens. This is in agreement with the variability detected in Fig. 8. Moreover, it is clearly visible that, once the crack appears, it spreads over 90% of the specimen height with a larger crack width in the region subjected to the higher tensile stress. The crack opening on the outer surface of the specimen at the steel rebar position (3.5 cm height) has an average value of 44% ± 5% of the biggest crack width measured on the lower part of the specimen having 32 mm concrete cover (around 0.5 cm height) as shown in Fig. 10.

Crack opening values deep in the specimen obtained by the X-ray CT measurements

The evolution of the residual crack width at different depths in the specimens is given in Fig. 11. Its evolution with respect to the maximal residual crack width value is shown in Fig. 12. It can be noted that the crack opening decreases with the specimen height but, for a given specimen height, the crack opening values are close to each other even at the level of the rebar position.

In addition, the DIC tendency curve shown in Fig. 10 is drawn in Fig. 12. It can be seen that this tendency curve describes the crack evolution very well on the entire height of the specimen independently of the depth. Therefore, it can be deduced that surface measurements of the crack width can be sufficient to deduce its value inside the specimen, especially near the rebar.

For a specimen showing 100 lm residual crack opening, the crack openings at the rebar position (specimen height = 30 mm approximately) range between 0.25 and 0.4 of the maximum residual crack opening with an average of 0.3 ± 0.06, while the average residual crack openings at the rebar position for specimens showing 300 and 500 lm residual crack openings are 0.36 ± 0.11 and 0.37 ± 0.02 respectively. No difference can be noted between the three residual crack openings and thus the residual crack opening at the rebar position is 35 ± 7% of the maximum residual crack opening. This value is in agreement with the one obtained from the DIC test (44% ± 5%) and not far from the one that could be obtained by applying Thales' theorem (43%).

Quantification of the load-induced steel/mortar interface damage

Fig. 13 shows the lengths of carbonated steel/mortar interfaces measured on 72 prismatic specimens (70 Â 70 Â 280 mm) with respect to the residual crack openings after 30 days of accelerated carbonation (at 3% CO2, 55% RH and 25 °C). As explained previously, in these carbonation conditions, the measured carbonated length is representative of the length of the damaged steel/mortar interface.

A significant de-cohesion between the steel and the surrounding mortar is notable over several centimeters on either side of the crack, independently of the residual crack opening. The length of the damaged interface increases with the increase in the resid- ual crack opening. It is obvious that no sharp variation in the length of the damaged interface exists when the crack openings increase from 100 to 500 mm. Consequently, once a crack appears, even with a small residual crack opening (<100 mm), damage will be present along the steel mortar interface on either side of the crack. Indeed, as described in ( [START_REF] François | A finite macro-element for corroded reinforced concrete[END_REF][START_REF] François | Corrosion and Its Consequences for Reinforced Concrete Structures[END_REF]), before the crack appearance, the cementitious materials bond perfectly to the rebar and the strain deformations of both steel and concrete are similar. In this configuration (non-cracked specimens), the tensile strength is transmitted from the concrete to the rebar due to the perfect bonding. In contrary, after the crack appearance, the mechanism of stress transmission change completely. In a cracked section, tensile stresses are mainly concentrated on the rebar. Then, the strain deformation in the rebar in the cracked area is bigger than the one in the concrete due to the loss of steel/concrete interface bond induced by the mechanical crack. Far from the cracked area, where the bond between the steel and the concrete is not impacted, the concrete transfer a part of the tensile load to the rebar then the strain deformation of rebar and concrete are similar. This mechanism describing the stress transmission is independent from the geometry of the specimen and from the crack opening and it explain why the damage is not sharply increasing with the crack width increase from 100 to 500 lm. This observation is also proven in previous study [START_REF] Ghantous | Effect of accelerated carbonation conditions on the characterization of load-induced damage in reinforced concrete members[END_REF], where 3 different geometrical dimension specimens were tested and it has been shown that the length of the damaged interface remain always limited to several centimeters around the crack independently from its opening (<500 lm).

cm

Crack

Rebar footprint

Carbonated length

Fig. 13 (c) shows that, for the same residual crack opening, the carbonation length along the steel/mortar interface is higher on its lower side than on its upper side, despite both sides being exposed to the same mechanical loading during the three point bending (Fig. 1). In fact, the casting direction leads to a poorer quality of the lower part of the steel/mortar interface than of the upper part [START_REF] Kenny | Statistical relationship between mix properties and the interfacial transition zone around embedded rebar[END_REF][START_REF] Soylev | Quality of steel-concrete interface and corrosion of reinforcing steel[END_REF][START_REF] Mohammed | Chloride-Induced Corrosion of Steel Bars in Concrete with Presence of Gap at Steel-Concrete Interface[END_REF][START_REF] Nelson | Chloride Induced Corrosion of Reinforcement Steel in Concrete, Threshold Values and Ion Distributions at the Concrete-Steel Interface Department of Civil and Environmental Engineering Chloride Induced Corrosion of Reinforcement Steel in Concrete[END_REF]. This may enable the load-induced micro-cracks to propagate for a greater length along the lower interface of the rebar than along its upper interface. It is also visible that the lengths of the damaged steel/mortar interfaces measured on the upper part of rebars intercepting 300 mm and 500 mm residual crack openings correspond to those measured on the lower part of rebars intercepting 100 and 300 mm residual crack openings respectively (blue lines in Fig. 13 (c)). Thus, for a thin crack opening, the length of the damaged steel/mortar interface along the lower part of the rebar could be equivalent to the one encountered along the upper part of a rebar intercepting a larger crack. Since the steel/mortar interface quality has been proved to be a determining factor for corrosion propagation, it can thus be deduced that corrosion risks cannot be limited by simply imposing a threshold on crack openings.

Conclusion

In some structural design codes and recommendations, a threshold is defined for crack opening near the rebar. It is recommended that this value should not be exceeded in order to limit the corrosion process. However, recent research shows that other parameters, such as steel/mortar interface quality, may control the corrosion process more than the crack width does. This study has thus aimed to detect a relation between the crack width and the corresponding size of the load-induced damage at the interface.

Three residual surface crack openings (100, 300 and 500 mm) were produced on reinforced mortar specimens by applying increasingly high loads. The crack opening evolution from the outer surface of the specimen to the rebar position was determined using X-ray CT and its evolution on the outer surface of the specimen was recorded by the DIC. On the other hand, the damage induced by the crack along the steel/mortar interface was quantified on several specimens having different residual crack openings.

For this configuration of specimens (concrete cover of 3 cm with 6 mm deformed rebar positioned in the middle of the specimen), the results indicate that the crack opening is 44 ± 5% smaller near the rebar compared with its maximum value. Additionally, it was observed that, at the rebar height, the crack width measured on the outer surface of the specimen was representative of that at the intersection with the rebar.

It was also observed that the length of damaged steel/mortar interface increased with the residual crack opening. However, there was no notable sharp modification in the size of the damaged interface when the residual crack opening increasing from 100 to 500 mm. Once a crack appears, the steel/mortar interface surrounding this crack will be damaged, independently of its opening. In addition, the length of the mechanically damaged interface on the lower part of a rebar intercepting a thin crack (100 mm) was observed to be equivalent to the length of the damaged interface measured on the upper part of a rebar intercepting a crack with a higher residual width (300 mm). On the basis of all these observations, it was deduced that defining a threshold for the crack opening in order to limit the corrosion risk was not justified, and thus

Fig. 1 .Fig. 2 .

 12 Fig. 1. Three-point bending test on 70 Â 70 Â 280 mm specimen.

Fig. 3 .

 3 Fig. 3. Set-up of digital image correlation equipment.

Fig. 4 .

 4 Fig. 4. Horizontal displacement field of the specimen.

Fig. 5 .

 5 Fig.5. CT -3D visualization of a cracked prismatic mortar specimen.

Fig. 6 .

 6 Fig. 6. Alteration length of the steel/mortar interface for cracked carbonated specimens with a residual crack opening of 0.5 mm.

Fig. 9 .

 9 Fig. 9. Crack-opening profile over the entire height of two specimens (DIC measurements).

Fig. 10 .

 10 Fig. 10. Evolution of the crack opening over the entire height of the specimen with respect to the maximum crack opening (DIC measurements).

Fig. 11 .

 11 Fig.11. Evolution of the residual crack opening vs. the height of the specimen for different depths (X-ray CT measurements).

Fig. 12 .

 12 Fig.[START_REF]ACI Committee 318, Building Code Requirements for Structural Concrete (ACI 318-08[END_REF]. Evolution of the crack opening on the entire height of the specimen with respect to the maximum crack opening at different depths.

R.M. Ghantous et al. / Construction and Building Materials 193 (2018) 97-104

Acknowledgment

The experiments reported in this paper were conducted at the LECBA Laboratory at CEA Saclay and at the LMDC Laboratory of INSA Toulouse. The authors gratefully acknowledge the support of the industrial partner EDF.

crack opening alone could not be used as an indicator of corrosion risk and structure sustainability.

Conflict of interest

None.