Régine Mosser-Ruck

Michel Cathelineau

Damien Guillaume

Delphine Charpentier

D Avy Rousset

Odile Barres

Nicolas Michau

CHLORITE, VERMICULITE, OR SAPONITE Regine M Berthierine

Osser-Ruck

AND N ICOLAS M Ichau

Effects of

Keywords: Berthierine, Chlorite, Fe Metal, Nuclear Waste, Saponite, Smectite, Vermiculite

INTRODUCTION

The interaction between smectites and Fe metal has been investigated in recent years in response to queries by nuclear-waste management agencies about the longterm behavior of engineered barriers after closure of the repository. Previous works, dealing with Fe-clay interaction, report the formation of distinct types of clays (serpentine-like phases, dioctahedral or trioctahedral Fe-rich smectites, chlorite) and distinct mineral sequences, and sometimes appear to be contradictory. The contradiction has encouraged this comparison of previous experimental conditions and results with new data. In close contact with the waste packages ('near-field system') and in reduced conditions, water is unstable, Fe is released, and H 2 is produced. The main parameters to be considered in such a system are shown in Figure 1 and a summary of the experimental conditions and experimental results described in the literature is presented in Table 1.

Experimental temperatures

Depending on countries and concepts, the initial temperature of the overpack in the repository will vary between 80ºC and 100ºC. The main problem which arises when conducting experiments on smectites at such temperatures is the difficulty in reaching equilibrium, especially when the L/C ratio is small. In engineered clay-barrier conditions, L/C is ~0.2Ô0.1, and it depends on the density of the bentonite. To compensate for kinetic effects, one possibility is to increase the temperature by conducting the experiments at greater temperatures, up to 250À300ºC [START_REF] Guillaume | Etude expe ´rimentale du syste `me fersmectite en pre ´sence de solution a `80ºC et 300ºC[END_REF][START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF][START_REF] Wilson | The effect of iron on bentonite stability: an investigation into reactions between native Fe, magnetite, montmorillonite and aqueous solutions[END_REF][START_REF] Wilson | The effect of iron on montmorillonite stability. (II) Experimental investigation[END_REF][START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF]. Another possibility is to increase the reactive surface by increasing the dispersion of particles and the amount of water.

Changes in redox conditions and Fe concentration in the fluids

After closure of the repository, the engineered barrier will be submitted to increasingly reducing conditions (1, 2, and 3 in Figure 1). The scenario includes (1) the consumption of residual oxygen introduced in the pores during the early steps through the oxidation of the Fe 2+ phases such as pyrite; and (2) the production of H 2 due to the instability of water in the presence of Fe metal [START_REF] Garrels | The beidellite-saponite solid-solution: An experimental approach[END_REF]. The Fe metalÀwater interaction results in a drastic reduction in f O 2 to the water instability boundary, e.g. within the field of magnetite + H 2 , and close to the Fe metal + H 2 field. At an f O 2 fixed by the boundary Fe metal/magnetite or less than the f O 2 determined by the equilibrium:

Changes in pH and in the ionic strength

The penetration of alkaline fluids issued from the concrete-water interaction in other engineered parts of the repository must result in an increase in pH of the interstitial fluid within the clay barrier. Alkaline fluids are generally progressively neutralized by the interaction with clays, as shown by experiments (trend a in Figure 1). The pH decreases from values of 12 or 10 to ~8.5 (at 80ºC for instance), when no alkaline reserve is introduced, e.g. when the alkalinity of the incoming fluid is only considered as a starting experimental feature and is not maintained [START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF]. The dissolution of Fe metal may produce an increase in pH (trend b in Figure 1) if no other mineral reaction is involved:

Fe 0 + 2 H 2 O ? H 2 + 2 OH À + Fe 2+
When an allochtonous source of fluids is considered, e.g. alkaline fluids from the concrete parts of the repository, the ionic strength of the incoming fluids in contact with the steel containers will increase. In such a case, the increasing activity of calcium stabilizes other kinds of minerals such as zeolites [START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF].

The reported experiments (Table 1) were carried out in autoclaves in the presence of air, e.g. with an O 2 reserve [START_REF] Lantenois | Re ´activite ´fer me ´tal/smectites en milieu hydrate ´a `80ºC[END_REF][START_REF] Lantenois | Experimental study of smectite interaction with metal Fe at low temperature: 1 À Smectite destabilization[END_REF][START_REF] Perronnet | Re ´activite ´des mate ´riaux argileux dans un contexte de corrosion me ´tallique. Application au stockage des de ´chets radioactifs en site argileux[END_REF][START_REF] Perronnet | Evidence of a critical content in Fe(0) on Foca7 bentonite reactivity at 80ºC[END_REF], under argon atmosphere [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF], or under N 2 atmosphere [START_REF] Wilson | The effect of iron on bentonite stability: an investigation into reactions between native Fe, magnetite, montmorillonite and aqueous solutions[END_REF]. Various amounts of Fe, as powders of metal (Fe 0 ) and/or oxides (Fe 2 O 3 , Fe 3 O 4 ), were added to the experimental media leading to different Fe/C ratios, from 0.1 to 2, according to the authors. Liquid/clay ratios also vary. Experiments were conducted at L/C values of 10 [START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF][START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF]), 16.7 (Perronnet, 2004;[START_REF] Perronnet | Evidence of a critical content in Fe(0) on Foca7 bentonite reactivity at 80ºC[END_REF][START_REF] Lantenois | Experimental study of smectite interaction with metal Fe at low temperature: 1 À Smectite destabilization[END_REF], and - ------------------------Experiment ------------------------ 66.7 [START_REF] Wilson | The effect of iron on bentonite stability: an investigation into reactions between native Fe, magnetite, montmorillonite and aqueous solutions[END_REF][START_REF] Wilson | The effect of iron on montmorillonite stability. (II) Experimental investigation[END_REF]. Experiments carried out at high temperature to compensate for kinetic effects yielded to the formation of Fe-saponite and chlorite after dioctahedral Na-Ca smectite at 300ºC [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF] or berthierine-like minerals at 250ºC [START_REF] Wilson | The effect of iron on bentonite stability: an investigation into reactions between native Fe, magnetite, montmorillonite and aqueous solutions[END_REF][START_REF] Wilson | The effect of iron on montmorillonite stability. (II) Experimental investigation[END_REF]. At lower temperatures (<150ºC), dioctahedral smectite was sometimes preserved [START_REF] Guillaume | Etude expe ´rimentale du syste `me fersmectite en pre ´sence de solution a `80ºC et 300ºC[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF][START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF]. On the other hand, [START_REF] Lantenois | Experimental study of smectite interaction with metal Fe at low temperature: 1 À Smectite destabilization[END_REF], [START_REF] Perronnet | Re ´activite ´des mate ´riaux argileux dans un contexte de corrosion me ´tallique. Application au stockage des de ´chets radioactifs en site argileux[END_REF][START_REF] Perronnet | Evidence of a critical content in Fe(0) on Foca7 bentonite reactivity at 80ºC[END_REF] reported the formation of a 7 A ˚phase (berthierine or a mineral of the odiniteÀcronstedtiteÀgreenalite series) for experiments carried out at 80ºC. An overall consideration of the experimental conditions and experimental results compiled from the available data of the literature (Table 1) shows that the mineralogical assemblages obtained after experiment are probably very dependent on parameters such as the Fe/C and the L/C ratios. The purpose of the present study was to evaluate the role of these parameters by comparing published data (Table 1) to results obtained from a new set of experiments carried out at 80ºC, 150ºC, and 300ºC with different Fe/C ratios (0.1 and 0.5) and L/C ratios (5 and 10). The effects of time and temperature, as well as the impact of an increased availability of the Fe metal, were investigated. Special attention was paid to the impact of the distance to the Fe plate on the nature of the run products.

MATERIALS, METHODS, AND ANALYTICAL TECHNIQUES

Batch experiments were conducted at 80, 150, and 300ºC in dilute chlorine solutions (NaCl, CaCl 2 ) having ionic strength and major cation compositions close to the natural waters present in sediments and similar to that of experiments described by [START_REF] Guillaume | Etude expe ´rimentale du syste `me fersmectite en pre ´sence de solution a `80ºC et 300ºC[END_REF] and [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF]. The starting product was a Wyoming bentonite (MX80) consisting of >85 wt.% of smectite. The smectite is a montmorillonite and the half structural formula (<2 mm fraction of the bentonite) given by [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF] Bentonite was placed in contact with Fe oxides (magnetite) and Fe metal in autoclaves over periods ranging between 3 and 12 months. The L/C mass ratios were 5 or 10 (rather less than those used in previously published works, see Table 1), and Fe (powder of metal Fe + magnetite)/clay ratios were 0.5 or 0.1. A plate of metallic Fe (~1 cm 2 ) was added in all experiments. The starting products were introduced into the autoclave under Ar atmosphere. Details of the experimental procedures have been published elsewhere [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF][START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF]. The mineralogical and chemical evolutions of the clays w e r e s t u d i e d b y X -r a y D i f f r a c t i o n ( X R D ) , Transmission Electron Microscopy (TEM) using High Resolution and Electron Dispersive Spectroscopy (HRand EDS-TEM), and micro-Fourier Transform Infrared (mFTIR) spectroscopy.

Analysis of particles was coupled with HRTEM images so that particle edges could be observed. X-ray diffraction, carried out on air-dried and glycolated preparations of samples, was used to investigate the swelling feature of the clays and to discriminate the main run products (saponite, vermiculite, and chlorite or berthierine). Serpentine group minerals and Fe-rich chlorite were extremely difficult to distinguish by XRD (because of the similarities of their chemistry and structure À HRTEM was used to help in the identification; and because of the small amount of newly formed phases in comparison to the unmodified starting product). Due to the number of run samples, crystal-chemical trends are presented as they, in particular, show clearly the increasing incorporation of Fe in newly formed phases, the permanence or otherwise of exchangeable cations typical of smectite-type or vermiculite layers, and the changes in the occupancy of the tetrahedral site by Si which was the best indicator of the smectite to nonswelling (chlorite or berthierine) conversion.

The FTIR spectra were obtained in the mid-IR region (4000À600 cm À1 ) by micro-Fourier Transform Infrared (mFTIR) spectroscopy using a Bruker IFS 55 spectrometer associated with a Bruker A590 microscope. All measurements were conducted in transmission mode on samples deposited on ZnSe slides. The diameter of the beam was 60 mm and the spectral resolution was 2 cm À1 . The recording time was ~1 min. Each spectrum was processed using the OPUS program (#Bruker).

RESULTS

Effect of Fe/clay ratio 80ºC experiments. At 80ºC for a L/C ratio of 10 and an Fe/C ratio of 0.1, run products were characterized by slight decreases in Si content and interlayer charge (Na + K + 2Ca) together with a slight increase in Fe content (Figure 2). The duration of experiment had no effect on the reaction and a possible equilibrium was obtained after 3 months. In contact with the Fe plate (large Fe/C ratio) and for a short duration (3 months), clay particles were richer in Fe (filled triangles in Figure 2) than the bulk run product.

A more significant evolution was observed for a greater Fe/C ratio (0.5) and for a smaller L/C ratio (5) (gray triangles in Figure 2): these experimental conditions favored the crystallization of an Fe-rich and Si-and Ca-poor phase. The duration of this experiment was important (12 months) but equilibrium was quickly obtained for shorter durations. The XRD patterns showed that the fine fraction (<4 mm) of this last run product (Figure 3) was mostly composed of a swelling clay-like smectite. Additional saturations of this fraction with Li and Mg before XRD analysis confirmed that a significant proportion of the run product was fully expandable. The run product could be thus a mixture of a smectite and an Fe-rich 14 A ˚chlorite (chamosite type) or a 7 A ˚clay (berthierine type) since the global interlayer charge of the particles decreases. The disagreement between XRD and EDS-TEM results could be explained by a mechanical enrichment in smectite particles during the separation of the <4 mm fraction for X-ray analysis. A HRTEM image of the bulk of this run product shows both numerous long flakes typical of smectite (10 A interlayer) and several shorter and thicker particles with a 7 A ˚interlayer (Figure 4) and confirmed the coexistence of at least two types of clay in the run sample. The 7 A ˚Fe-rich phase was, therefore, mostly found for experiments with a large Fe/C ratio. The 7 A phase had a composition close to that of berthierine (gray triangles in Figure 2) and its abundance decreased to undetectable amounts in experiments carried out with smaller Fe/C ratios. Clay particles of this run product were also sampled at the contact with the Fe plate and analyzed by FTIR spectroscopy. The IR spectrum (Figure 5) was compared to that of the starting Na-montmorillonite (MX80). In the spectra for both the starting montmorillonite and the run product, bands o b s e r v e d b e t w e e n 3 8 0 0 À3 1 0 0 c m À1 a n d 1700À1500 cm À1 were assigned to OH-stretching vibrations from clay minerals and water and to water-bending vibrations, respectively. The spectrum of the run product showed several new bands at 1410, 1385, 1356, 712, and 667 cm À1 . By comparison with literature data [START_REF] Saumagne | Advances in Molecular Spectroscopy[END_REF][START_REF] Toppani | A 'dry' condensation origin for circumstellar carbonates[END_REF], these bands were assigned to stretching (between 1410 and 1356 cm À1 ) and bending modes (712 and 667 cm À1 ) of the CO 3 2À group. Otherwise, the bending band fitted at 1585 cm À1 was assigned to strongly bound water. This kind of FTIR spectrum was very similar to that obtained by [START_REF] Toppani | A 'dry' condensation origin for circumstellar carbonates[END_REF] for complex hydrated carbonates. The starting MX80 bentonite containing ~2% carbonates would be the source of CO 2 in the experimental system, allowing the formation of bicarbonates at the contact with the Fe plate.

300ºC experiments. The impact of the Fe/C ratio on the transformation of montmorillonite at three different experiment durations (3, 6, and 9 months) was studied (Figure 6). Experiments with a small Fe/C ratio (0.1, diamonds in Figure 6) and with a large Fe/C ratio of 0.5 (squares) were compared. The EDS-TEM analyses showed that the increase in Fe/C ratio enhanced the chemical evolution. The decrease in Si content was more significant (Si <3 for a half formula based on 11 oxygens) in run products obtained in experiments with an Fe/C ratio of 0.5 for all durations and in the 9 month experiment only when the Fe/C ratio was 0.1. A long experiment (9 months) and a small Fe/C ratio had the same impact on the tetrahedral occupancy as a short experiment (3 months) and a large Fe/C ratio. Different Fe/C ratios also yielded distinct octahedral occupancies of the run clays. In the small Fe/C-ratio (0.1) experiments, both Fe and Mg enrichments were observed in run products (arrows in Figure 6) whereas a large Fe/C ratio in the experimental medium led to a significant Fe enrichment of clays only. Interlayer occupancy of the run products was generally depleted both in Ca and Na for all run products. The new data obtained at 300ºC from greater Fe/C-ratio experiments were in good agreement with results obtained by [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF] and [START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF] who demonstrated that the chemical evolution of the montmorillonite in MX80 bentonite depends on the distance to the Fe plate and consequently on the availability of Fe in the medium. Iron enrichment in particles sampled near and at the contact with the Fe plate (where the Fe/C ratio was large) was always far more significant.

Effect of temperature

One of the most important parameters controlling the transformation of smectite is temperature. Its effect was also studied in the presence of a metallic Fe plate and under an Fe/C ratio of 0.5 and a L/C ratio of 5. Three long duration experiments (12 months) were conducted at 80ºC, 150ºC, and 300ºC. They showed that the increase in temperature both enhanced Si depletion and Fe enrichment of the run clays (Figure 7). The interlayer charges were different in newly formed clays yielding to the formation of Fe-rich saponite at 300ºC (Na + K + 2Ca ~0.3 to 0.4) and the crystallization of a 7 A ˚phase mixed with starting smectite at a temperature of <150ºC (Na + K + 2Ca <0.2). In the greater Fe/C-ratio (0.5) experiments, no zeolite byproducts were observed, regardless of the temperature.

DISCUSSION AND CONCLUSIONS

Mineralogical transformations and sequences

From literature data (Table 1) and results presented in the present paper, at 80ºC and 150ºC, in the presence of Fe (powder and plate) and Fe oxides (magnetite), and for specific experimental conditions (large L/C ratio and Fe/C <0.5), montmorillonite evidently remains the predominant clay mineral in run products. Under any pH (neutral or alkaline), similar weak enrichment of Fe and depletion of Si were observed in the clays. At 300ºC, vermiculite was predominant, associated with Fe-rich trioctahedral smectite (saponite-type) in alkaline conditions [START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF] whereas Fe-rich chlorites associated with Fe-rich trioctahedral smectite (saponitetype) were identified in neutral conditions [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF]. The experimental crystallization of Mg-vermiculite after smectite in alkaline solutions was also mentioned by [START_REF] Chermak | Low temperature experimental investigation of the effect of high pH NaOH solutions on the opalinus shale, Switzerland[END_REF]. In the study of [START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF], the formation of Fe-rich vermiculites seemed to be favored by the initially alkaline conditions and the presence of Fe metal, all other conditions being equal in comparison with the 300ºC experiments reported by [START_REF] Guillaume | Experimental synthesis of chlorite from smectite at 300ºC in the presence of metallic Fe[END_REF]. In all cases, chemical zoning was observed from the very near contact with the Fe plate toward the bulk clay, usually a decrease in Fe in the run products sometimes in favor of Mg (at 300ºC).

The occurrence of quartz, feldspars, magnetite, and zeolites as reaction by-products in all experiments is consistent both with thermodynamic modeling predictions [START_REF] Cathelineau | Interactions fluides/argilites en conditions de stockage profond des de ´chets nucle ´aires. Inte ´re ˆt du couplage expe ´rimentation/mode ´lisation dans la compre ´hension des me ´canismes de transformation des argiles et la pre ´diction a long terme du comportement de la barrie `re argileuse[END_REF][START_REF] Kluska | Predictions of the mineralogical transformations in a bentonite barrier surrounding an iron radioactive container[END_REF][START_REF] Montes | Modelling of transport and reaction in an engineered barrier for radioactive waste confinement[END_REF] and data from other experiments on bentonites (Table 1). The simplified conversion of a The production of H + in reaction 1 partly compensates the production of OH À due to the dissolution of Fe (reaction 2), thus explaining the near-neutral pH of most experimental solutions (measured after quenching). The availability of silica and cations in solution led to the formation of feldspars or zeolites. The presence of bicarbonates or carbonates at the contact of the Fe plate in one experiment is analogous to corrosion products of the steel canister reported in the literature (e.g. [START_REF] Schlegel | Metal corrosion and argillite transformation at the water-saturated, high-temperature iron-clay interface: A microscopic-scale study[END_REF] and the results of geochemical modeling of Fe-clay interactions [START_REF] Bildstein | Modelling iron-clay interactions in deep geological disposal conditions[END_REF][START_REF] Savage | Modelling iron-bentonite interactions[END_REF]. Discrete calcite originally present in the bentonite probably dissolved at the beginning of the experiment.

The Ca 2+ ions released can enter the interlayer space of smectite and HCO 3 À can combine with other cations as Fe 2+ at the contact of the Fe plate to form hydroxycarbonate (or carbonate) species. Neutral to high pH values at the contact with the Fe plate (as shown by the presence of magnetite in some experiments) and a small P CO 2 could favor the formation of hydroxycarbonates.

Finally, the mineralogical sequences depend on the availability of Fe in solution and on the starting pH of the solution. Under neutral pH conditions, the sequence of newly formed mineral assemblages is thus as follows:

(1) dioctahedral smectite (di-Sm) > Fe-rich di-Sm for small Fe/C ratios and small L/C ratios, or berthierinelike mineral if the Fe/C ratio is >0.5 at 80ºC or 150ºC;

(2) di-Sm > Fe di-Sm > Fe-saponite > trioctahedral chlorite + feldpars + zeolite, at 300ºC.

Experiments carried out with a starting pH of 12 led to the following mineral sequences:

(1) di-Sm > Fe di-Sm + palygorskite at 80º and 150ºC, (2) di-Sm > vermiculite (Ô Fe saponite) + mordenite at 300ºC

The possible mineral zonings are, therefore, distinct when the fluids come from the geological barrier or from concrete.

Comparison with natural occurrences

An additional line of comparison is provided by the consideration of natural assemblages and the following transitions: the berthierine-to-chlorite conversion [START_REF] Iijima | Berthierine and chamosite in coal measures of Japan[END_REF][START_REF] Hillier | Chlorite interstratified with a 7 A ˚mineral: an example from offshore Norway and possible implications for the interpretation of the composition of diagenetic chlorites[END_REF], the di-to trioctahedral smectite transition [START_REF] Inoue | Conversion of smectite to chlorite by hydrothermal diagenetic alterations, Hokuroku Kuroko mineralization area, northeast Japan[END_REF][START_REF] Inoue | Smectite-to-chlorite transformation in thermally metamorphosed volcanoclastic rocks in the Kamikita Area, north Honshu, Japan[END_REF], and the smectite-to-chlorite conversion [START_REF] Bettison-Varga | The role of randomly mixed-layered chlorite/smectite in the transformation of smectite to chlorite[END_REF][START_REF] Robinson | Reaction pathways and reaction progress for the smectite-to-chlorite transformation: evidence from hydrothermally altered metabasites[END_REF][START_REF] Schiffman | The smectite to chlorite transition in a fossil seamount hydrothermal system: the basement complex of La Palma, Canary Islands[END_REF]. The temperature range of stability of chlorite is rather large, between 110ºC and 420ºC [START_REF] Iijima | Berthierine and chamosite in coal measures of Japan[END_REF][START_REF] Cathelineau | Temperaturecomposition relationships of authigenic micaceous minerals in the Los Azufres geothermal system[END_REF][START_REF] Hillier | Chlorite interstratified with a 7 A ˚mineral: an example from offshore Norway and possible implications for the interpretation of the composition of diagenetic chlorites[END_REF][START_REF] Buatier | Iron in hydrothermal clays from the Galapagos spreading centre mounds: consequences for the clay transition mechanism[END_REF][START_REF] Aagaard | Formation of grain-coating chlorite in sandstones. Laboratory synthesized vs. natural occurrences[END_REF] indicating a potential formation of chlorite minerals at temperatures close to those of interest in waste disposal. The coexistence of dioctahedral smectite and chlorite is frequent above 160ºC in geothermal systems where minerals are directly precipitated from a fluid and do not derive from the alteration of a clay precursor [START_REF] Cathelineau | A chlorite solid solution geothermometer. The Los Azufres (Mexico) geothermal system[END_REF][START_REF] Cathelineau | Temperaturecomposition relationships of authigenic micaceous minerals in the Los Azufres geothermal system[END_REF][START_REF] Bailey | Hydrous Phyllosilicates[END_REF]. Fe-rich chlorites have also been described in sandstone reservoirs at temperatures of 100À120ºC. Berthierine has been observed mostly in sedimentary basins as an early, low-temperature diagenetic phase (15À40ºC), of which the upper limit of stability is within the 65À130ºC temperature range [START_REF] Iijima | Berthierine and chamosite in coal measures of Japan[END_REF]Aagard et al., 2000). Above 160ºC, berthierine is entirely converted to chamosite in natural systems through mixed layering [START_REF] Hillier | Chlorite interstratified with a 7 A ˚mineral: an example from offshore Norway and possible implications for the interpretation of the composition of diagenetic chlorites[END_REF][START_REF] Hillier | Chlorite interstratified with a 7 A ˚mineral: an example from offshore Norway and possible implications for the interpretation of the composition of diagenetic chlorites[END_REF], experimentally, above 200À250ºC (Aagard et al., 2000). Ca-zeolites (laumontite, clinoptilolite, mordenite) are commonly observed associated with smectite and chlorite as alteration products of basic glasses of magmatic rocks submitted to hydrothermal alteration [START_REF] Cathelineau | A chlorite solid solution geothermometer. The Los Azufres (Mexico) geothermal system[END_REF][START_REF] Alt | Hydrothermal alteration of 1 km section through the upper oceanic crust, DSDP Hole 504B: Mineralogy, chemistry and evolution of seawater-basalt interaction[END_REF]Schiffman and Fridleifsson, 1991, among others). For diagenetic settings, Gu ¨ndogdu et al. (1996) suggested that the formation of zeolites and the amount of zeolites being formed could also be controlled by the amount of Fe released during the dissolution of volcanic glass. Those authors showed that a large Fe content favors the formation of smectite rather than zeolite (especially clinoptilolite). The experiments carried out at various Fe/C ratios (Table 1) confirm this suggestion. At 300ºC, zeolites are only observed for experiments with small Fe contents (Fe/C = 0.1) [START_REF] Guillaume | Etude expe ´rimentale du syste `me fersmectite en pre ´sence de solution a `80ºC et 300ºC[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF][START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF]. In lower-temperature experiments (80ºC or 150ºC), zeolites do not form probably because of kinetic effects [START_REF] Guillaume | Etude expe ´rimentale du syste `me fersmectite en pre ´sence de solution a `80ºC et 300ºC[END_REF][START_REF] Guillaume | Experimental study of the transformation of smectite at 80ºC and 300ºC in the presence of Fe oxides[END_REF][START_REF] Perronnet | Re ´activite ´des mate ´riaux argileux dans un contexte de corrosion me ´tallique. Application au stockage des de ´chets radioactifs en site argileux[END_REF][START_REF] Charpentier | Bentonite-iron interactions under alkaline condition: an experimental approach[END_REF][START_REF] Perronnet | Evidence of a critical content in Fe(0) on Foca7 bentonite reactivity at 80ºC[END_REF]. At the same low temperatures but in experiments with large Fe contents (at the contact with the Fe plate and for Fe/C ratios > 0.5), zeolites were not observed.

The experimental mineral assemblages observed in the present experiments and in experiments described in the literature are, therefore, compatible with those found in nature as the upper boundary of berthierine is at ~120À130ºC and the lowest temperature estimated for chlorite is ~110ºC. Authigenic dioctahedral smectite and chlorite are found in the same samples as co-existing phases, possibly at equilibrium, among a large range of temperature from 160 to 240ºC, while saponites are mostly found in the presence of fluids having a greater ionic strength (close to or above sea water) than hydrothermal waters from geothermal systems related to volcanic activity.

Redox conditions and role of intermediate f O 2

The overall consideration of the experimental sets of data indicates that the redox state of the system and the availability of Fe seem to be the two main critical parameters which control the formation of the Fe-rich mineral phases and their relative abundance. In fact, the main changes in physical-chemical parameters concern the redox conditions which dramatically reduce toward the f O 2 close to water dissociation, and the production of H 2 , which has been confirmed experimentally in some Japanese experiments which show the bubbling of H 2 at the Fe/C interface [START_REF] Idemitsu | Diffusion behaviour of iron corrosion products in buffer materials[END_REF]. The subsequent increase in pH due to Fe dissolution is balanced by smectite-to-berthierine or chlorite conversion.

The above-mentioned sequences are distinct from that proposed for greater f O 2 , lower temperatures (<150ºC), and very large L/C ratios where the predominant newly formed phases are serpentine-group minerals (odiniteÀcronstedtite, [START_REF] Lantenois | Re ´activite ´fer me ´tal/smectites en milieu hydrate ´a `80ºC[END_REF][START_REF] Lantenois | Experimental study of smectite interaction with metal Fe at low temperature: 1 À Smectite destabilization[END_REF][START_REF] Perronnet | Re ´activite ´des mate ´riaux argileux dans un contexte de corrosion me ´tallique. Application au stockage des de ´chets radioactifs en site argileux[END_REF][START_REF] Perronnet | Evidence of a critical content in Fe(0) on Foca7 bentonite reactivity at 80ºC[END_REF]. The discrepancy is mostly linked to the role of: (1) the L/C ratio: when <5, the fluid chemistry is buffered by the smectite and its evolution toward 7 A phases seems either impossible or very slow; (2) the redox conditions: under intermediate f O 2 , Fe 2+ /Fe 3+ phases of the serpentine group are favored, while under the conditions of water instability, Fe-rich dioctahedral or trioctahedral smectite forms at low temperatures (<150ºC), and chlorite or vermiculite form at temperatures above 200À250ºC. A schematic impression of mineral zoning taking into account the redox state, the availability of Fe, and the L/C ratio at low temperatures (<150ºC) is presented in Figure 8.

The nature of the non-swelling Fe-rich clay (7 A ˚vs. 14 A ˚phases) is debatable mostly because of the difficulty in determining the exact structure of a phase which is limited in quantity, and mixed mechanically or structurally (mixed-layering) with other minerals such as di-or trioctahedral smectites. A considerable set of arguments exists in favor of the formation of chlorite at high temperature and this mineral is also predicted by geochemical modeling of the smectite-Fe interaction, even at 80ºC [START_REF] Kluska | Predictions of the mineralogical transformations in a bentonite barrier surrounding an iron radioactive container[END_REF][START_REF] Montes | Modelling of transport and reaction in an engineered barrier for radioactive waste confinement[END_REF]. Alternately, a series of smectite-Fe experiments resulted in the formation of a 7 A ˚phase at lower temperatures (80ºC, 150ºC) when the Fe/C and L/C ratios are large. Their stability field seems to have been increased by more oxidizing redox conditions, which favor serpentine-group minerals. The two groups of minerals, chlorite and Fe-serpentine, can thus be formed, but the exact time-temperature pairs delimiting their formation remains poorly known. New data presented here show that for 1 y experiments, the temperature boundary could be between 150 and 300ºC, but these data cannot be extrapolated to other time-temperature pairs close to those of interest for waste disposal (e.g. 1000 to 10,000 y/ 50À80ºC for instance).

Role of the smectite-Fe contact on the smectite conversion rate

A role of the direct contact between Fe metal and clay has been put forward by [START_REF] Perronnet | Re ´activite ´des mate ´riaux argileux dans un contexte de corrosion me ´tallique. Application au stockage des de ´chets radioactifs en site argileux[END_REF] as a necessary condition for the smectite-berthierine conversion. The results of the present study show, however, that 7 A ˚or 14 A ˚phases may form at distinct distances from the Fe plate, but with distinct Fe contents. The clay minerals crystallized at more than a few mm from the Fe plate are Mg-rich whatever their nature (chlorite or vermiculite). In any case, the rate of alteration of the clay barrier will be extremely dependent on the ability of Fe to migrate through the clay barrier as Fe 2+ . This process is controlled by: (1) the rate of corrosion of iron; (2) the rate of formation of a possible crust of magnetite (or siderite when P CO 2 is increased) at the interface with the Fe canister, which may reduce the amount of Fe 2+ released to the system; and (3) the dynamics of migration of Fe through diffusion processes.

Consequences for swelling properties

The transformation of montmorillonite into the association of serpentine-like or chlorite phases might lead to significant modifications of the material properties [START_REF] Devineau | MX80/iron interactions at 80, 150, and 300ºC under alkaline conditions: influence of the mineralogical transformations on texture[END_REF]. On the contrary, the formation of saponite or vermiculite, e.g. minerals having swelling properties has a less critical effect on the swelling properties of the bentonite, than the formation of chlorite or a serpentine-like phase. The vermiculitization of montmorillonite is accompanied by a reduction of the structural Fe in the newly formed clay particles. The reduction in Fe may influence the properties of smectite [START_REF] Stucki | Effects of iron oxidation state on the texture and structural order of Na-nontronite[END_REF]Stucki et al., 1984aStucki et al., , 1984b;;[START_REF] Lear | Effects of iron oxidation state on the specific surface area of nontronite[END_REF]. [START_REF] Lear | Effects of iron oxidation state on the specific surface area of nontronite[END_REF] described, for example, the decrease in swelling capacity and of the specific surfaces of nontronite due to the collapse of some interlayers after Fe reduction. In addition, the proportion of swelling phases decreases as some of the material is converted to a non-swelling phase (zeolite). Besides, zeolites such as mordenite have a large cation exchange capacity and large adsorption capacity [START_REF] Pabalan | Thermodynamics of ion exchange between clinoptilolite and aqueous solutions of Na + /K + and Na + /Ca 2+[END_REF]. The exchange and adsorption capacities of the zeolite + vermiculite assemblage may be thus as important as those of the initial bentonite.

Extrapolation of results to small-water-content bentonite and low temperatures With regard to a nuclear waste repository, the experimental simulations above have demonstrated an evolution toward clay minerals with smaller CEC values. The transformation was observed to its greatest extent nearest to the contact with the added metallic Fe plate and under specific experimental conditions favoring the reactions. Prediction of the potential behavior of smectite in the presence of a steel container and their related alteration products, under nuclear-waste-repository conditions, is made difficult by the likely temperature range of 25À90ºC. Under such low temperatures, very little mineralogical change could be determined from experiments carried out over short durations (<1À2 y) and for liquid/ mineral ratios typical of an engineered barrier (<0.1). Experiments carried out at higher temperatures could, however, be extrapolated to lower temperatures as the type of mineralogical changes observed at high temperature is predicted by numerical modeling either at high (300ºC) or much lower temperatures (80À100ºC). However, kinetic predictions using constraints from temperature-time pairs still require experimental calibration.

H 2 O

 2 ? H 2 + Ý O 2 the Fe metal is transformed into magnetite following the reaction: 3 Fe 0 + 2 O 2 ? Fe 3 O 4 thus yielding the mass balance reaction: 4 H 2 O + 3 Fe 0 ? Fe 3 O 4 + 4 H 2

Figure 1 .

 1 Figure 1. Main parameters taken into account in the experimental modeling of the FeÀclay interactions: (a) pH reduction due to alkaline fluidÀbentonite interaction; (b) pH increase due to Fe dissolution; (c) temperature-pH field chosen for new data in this study: (1) excavation under atmospheric f O 2 ; (2) f O 2 reduction while O 2 stock lasts; and (3) instability of water.

Figure 2 .

 2 Figure 2. Comparison of the crystal-chemistry of the clay phase for experiments carried out at 80ºC with a L/C ratio of 10 or 5, and an Fe/C ratio of 0.1 or 0.5. The durations were 3 months (open triangles), 6 months (open diamonds), 9 months (open squares), or 12 months (gray triangles). Filled triangles correspond to particles sampled on the Fe plate. The circle corresponds to the range of values of the starting MX80 bentonite Si vs. Na + K + 2Ca; Fe (total Fe converted to Fe 3+ ) vs. Mg; Ca vs. Na; and Fe/(Fe + Mg) vs. Si diagrams plotted from EDS-TEM analyses and structural formulae calculated on the basis of 11 oxygens (diagrams after Grauby et al., 1993).

Figure 3 .

 3 Figure 3. XRD patterns recorded from the fine fraction (<4 mm) of the run product obtained in the 12-month experiment carried out at 80ºC in the presence of a large Fe/C ratio (0.5) and L/C ratio of 5: (a) air-dried sample (AD); (b) glycolated sample (EG: solvation with ethylene glycol vapor at room temperature over 24 h); and (c) heated sample (H: 4 h at 550ºC). The index of reflections corresponds to d hkl in A ˚. The XRD data were collected using a D8 Bruker diffractometer with CoKa 1 radiation (l = 1.7902 A ˚). The general operating conditions were 35 kV accelerating voltage, 45 mA intensity, step-scanning at 0.035º2y intervals, 3 s counting time, 3À40º2y for oriented powder.

Figure 4 .

 4 Figure 4. HRTEM analysis of the run product obtained from the 12-month experiment carried out at 80ºC in the presence of a large Fe/C ratio (0.5) and L/C ratio of 5.

Figure 5 .

 5 Figure 5. IR spectra in transmission mode of the starting MX montmorillonite and the run product sampled at the contact of the Fe plate in 80ºC, 12 months, L/C = 5, Fe/C = 0.5 experiment.

Figure 6 .

 6 Figure 6. Comparison of the crystal chemistry of the clay phases for 3-, 6-, and 9-month experiments carried out at 300ºC with a L/C ratio of 10, and different Fe/C ratios (0.1 or 0.5). Si vs. Na + K + 2Ca; Fe (total Fe converted to Fe 3+ ) vs. Mg; Ca vs. Na; and Fe/ (Fe + Mg) vs. Si diagrams are plotted from EDS-TEM analyses and structural formulae calculated on the basis of 11 oxygens (diagrams after Grauby et al., 1993).

Figure 7 .

 7 Figure 7. Comparison of the crystal chemistry of clay particles obtained from the 12-month experiments carried out at 80ºC (open circles), 150ºC (gray diamonds), and 300ºC (gray squares) in the presence of an Fe/C ratio of 0.5 and an L/C ratio of 5.

Table 1 .

 1 List of published results as a function of different physical chemical experimental parameters.

  is: (Si 3.98 Al 0.02 )(Al 1.55 Mg 0.28 Fe II 0.08 Fe III 0.09 )O 10 (OH) 2 Na 0.18 Ca 0.10

  3.98 Al 0.02 )(Al 1.55 Mg 0.28 Fe II

	0.08 Fe III 0.09 )O 10 (OH) 2 Na 0.18 Ca 0.10 +
	Montmorillonite MX80 (Sm)
	7.039 Fe 2+ +10.76 H 2 O + 0.18 e À ?
	1.57 (Si 3 Al)(Al )(Fe II 4.7 Mg 0.3 )O 10 (OH) 8 +
	Chamosite or 3.14 berthierine (chl)
	0.2 Ca 2+ + 0.36 Na + + 3.25 SiO 2 (aq.) +
	0.089 Mg 2+ + 12.96 H +	(1)
	As the dissolution of Fe metal is expressed as:
	Fe 0 + 2 H 2 O ? H 2 + 2 OH À + Fe 2+	(2)
	and reduction of Fe 3+ to Fe 2+ is expressed as:
	Fe 3+ + ÝH 2 ? Fe 2+ + H +	(3)
	the combination of reactions 1, 2, and 3 results in:
	7.039 Fe 0 + 11.698 H 2 O + 2 Sm ?
	1.57 Chl + 0.2 Ca 2+ + 0.36 Na + +
	3.25 SiO 2 (aq.) + 0.089 Mg 2+ + 6.949 H 2 + 0.938 OH À

Vol. 58, No. 2, 2010 Effects of pH and Fe/C and L/C ratios on the transformation of smectite

ACKNOWLEDGMENTS

The experiments were conducted at the Ge ´ologie et Gestion des Ressources Mine ´rales et Energe ´tiques laboratory (G2R, Nancy-Universite ´, CNRS, CREGU, Vandoeuvre-le `s-Nancy, France). The XRD and FTIR spectroscopy data were collected at the Laboratoire Environnement et Mine ´ralurgie (LEM, CNRS-INPL, Vandoeuvre-le `s-Nancy, France), and the EDS and HRTEM data at Nancy Universite ´(Vandoeuvre-le `s-Nancy, France).

The authors thank J. Ghanbaja (UHP, Vandoeuvre-le `s-Nancy, France) for the EDS analyses, and K. Devineau and I. Bihannic (LEM, Vandoeuvre-le `s-Nancy, France) for their help and technical assistance with the XRD. This research was financially supported by ANDRA -Agence Nationale pour la gestion des De ´chets RadioActifs (French national agency for the management of radioactive waste).


Céline René
email: celine.rene@inserm.fr

Damien Paulet

Emmanuelle Girodon

Catherine Costa

Guy Lalau

Julie Leclerc

Faïza Cabet-Bey

Thierry Bienvenu

Martine Blayau

Albert Iron

Hervé Mittre

Delphine Feldmann

Caroline Guittard

Mireille Claustres

Marie Des Georges

p.Ser1235Arg should no longer be considered as a Cystic Fibrosis mutation: results from a large collaborative study

Keywords: cystic fibrosis, diagnosis, genetic counseling, mutation

Among the 1700 mutations reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, a missense mutation, p.Ser1235Arg is a relatively frequent finding. To clarify its clinical significance, we collected data from 104 subjects heterozygous for the mutation p.Ser1235Arg from the French CF network, addressed for various indications including classical CF, atypical phenotypes or carrier screening in subjects with or without a family history. Among them, twenty-six patients (five having CF, ten CBAVD -Congenital Absence of the Vas Deferens-and eleven with CFlike symptoms) and fourteen healthy subjects were compound heterozygous for a second CFTR mutation. An exhaustive CFTR gene analysis identified a second mutation in cis of p.Ser1235Arg in all CF patients and in 81.8% CBAVD patients.

Moreover, epidemiological data from more than 2100 individuals found a higher frequency of p.Ser1235Arg in the general population than in CF or CBAVD patients. These data, added to the fact that in silico analysis and functional assays suggest a benign nature of this substitution gives several lines of evidence against an association of p.Ser1235Arg with CF or CBAVD.

Introduction

Cystic Fibrosis (CF, MIM≠219700), one of the most frequent life-shortening recessively inherited diseases, is caused by alterations in the CFTR gene (Cystic Fibrosis Transmembrane Conductance Regulator; MIM≠602421), that result in loss or impairment of CFTR-mediated ion transport across epithelial cell membrane. The clinical expression and severity of CF are highly variable. Severe phenotypes are usually associated with high concentrations of sweat chloride, early onset of pancreatic insufficiency (PI), meconium ileus at birth and severe lung disease, whereas mild phenotypes are associated with lower sweat chloride concentrations, pancreatic sufficiency (PS) and variable lung disease. Mutations in the CFTR gene are also involved in atypical phenotypes [START_REF] Kerem | Atypical CF and CF related diseases[END_REF] such as CBAVD (Congenital Bilateral Absence of the Vas Deferens; MIM # 277180), mild pulmonary diseases or ICP (Idiopathic Chronic Pancreatitis).

Since the cloning of the CFTR gene, a wide spectrum of molecular abnormalities (more than 1700 mutations, variants or polymorphisms) has already been reported to the Cystic Fibrosis Genetic Analysis Consortium (http://www.genet.sickkids.on.ca/cftr/). However, a clear statement on the pathogenicity of a mutation is difficult to obtain, in particular for missense mutations [START_REF] Cotton | Proof of "disease causing" mutation[END_REF][START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF] . Moreover, the existence of at least two mutations or sequence variations on the same allele, named complex alleles, complicates genetic counseling [START_REF] Savov | Double mutant alleles: are they rare?[END_REF][START_REF] Fanen | Structure-function analysis of a doublemutant cystic fibrosis transmembrane conductance regulator protein occurring in disorders related to cystic fibrosis[END_REF][START_REF] Romey | Complex allele [-102T>A+S549R(T>G)] is associated with milder forms of cystic fibrosis than allele S549R(T>G) alone[END_REF][START_REF] Clain | Two mild cystic fibrosis-associated mutations result in severe cystic fibrosis when combined in cis and reveal a residue important for cystic fibrosis transmembrane conductance regulator processing and function[END_REF][START_REF] Rohlfs | The I148T CFTR allele occurs on multiple haplotypes: a complex allele is associated with cystic fibrosis[END_REF][START_REF] Clain | A neutral variant involved in a complex CFTR allele contributes to a severe cystic fibrosis phenotype[END_REF][START_REF] Niel | A new large CFTR rearrangement illustrates the importance of searching for complex alleles[END_REF][START_REF] Claustres | Are p.I148T, p.R74W and p.D1270N cystic fibrosis causing mutations?[END_REF] . p.Ser1235Arg (3837T>G or c.3705T>G), initially reported by Cuppens et al. [START_REF] Cuppens | Detection of 98.5% of the mutations in 200 Belgian cystic fibrosis alleles by reverse dot-blot and sequencing of the complete coding region and exon/intron junctions of the CFTR gene[END_REF] with a second mutation on the same allele, p.Gly628Arg, is located in a poorly conserved region in the second nucleotide binding fold (NBF2). Earlier reports failed to establish a clear impact of p.Ser1235Arg on the phenotype severity [START_REF] Wei | Suppressive interactions between mutations located in the two nucleotide binding domains of CFTR[END_REF][START_REF] Monaghan | Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: results from a collaborative study[END_REF][START_REF] Dequeker | Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders-updated European recommendations[END_REF][START_REF] Oca | Amniotic fluid digestive enzyme analysis is useful for identifying CFTR gene mutations of unclear significance[END_REF] . The aim of this study was to determine whether we should consider p.Ser1235Arg as a possible CF-associated mutation or reclassify it as a neutral polymorphism. Thus, we implemented a large collaborative study to repertory the patients or individuals bearing the p.Ser1235Arg mutation from nine French laboratories and compared their phenotype, genotype and associated CFTR haplotypes. To predict the potential effect of this sequence variation on the CFTR protein, in silico analyses using various programs and functional studies were performed. Here, we gather lines of evidence that p.Ser1235Arg should be no longer considered as a CF mutation.

Materials and Methods

Patients and individuals

Data were collected from one hundred and four subjects heterozygous or compound heterozygous for the p.Ser1235Arg mutation registered from the French CF network of molecular genetics laboratories. Among them, sixty-seven were referred for diagnosis:

Molecular epidemiological study

To evaluate the p.Ser1235Arg frequency in the general population, we screened for 2114 samples: 929 anonymized dried-blood spot of neonates presenting positive or negative IRT (Immuno Reactive Trypsinogen) at day 3 obtained from the center for neonatal screening in Montpellier, South of France and 1185 genomic DNA from CF patient's or relative's partners from the cohort of the Southern France (Laboratory of molecular genetics of Montpellier) or from the cohort of the Northern France (Laboratory of molecular genetics of Lille).

Mutation Nomenclature

Gene variants at the protein level were named as recommended in the Human Genome Variation Society web page (http://www.hgvs.org/mutnomen/). For variations described at the nucleotide level, the A of the ATG translation start codon was numbered as +133 in accordance with the current CFTR gene numbering based on cDNA sequence (GenBank NM_000492.3) and on the CF mutation database. These variations were also given in parentheses following the approved nomenclature format (A of the ATG translation start codon as +1).

CFTR Genotype Analysis

Genomic DNA

Genomic DNA was prepared from peripherical blood leukocytes or from amniotic liquid according to standard protocols. Patients: The CFTR gene coding and flanking regions were analyzed by PCR amplification followed by DGGE (Denaturing Gel Gradient Electrophoresis), SSCA (Single Strand Conformation Analysis), DHPLC (Denaturing High Performance Liquid Chromatography) or heteroduplex analysis followed by sequencing each abnormal pattern detected to characterize the variants. If p.Ser1235Arg was found alone, a screening for large CFTR rearrangements was performed using a semi-quantitative fluorescent multiplex PCR assay. Patients' relatives: A screening for the familial mutations was implemented. Patients' partners:

In primary screening, five to twelve exons, including exons 13 and 19, were screened by laboratories.

To determine the haplotype associated with the p.Ser1235Arg allele, 6 microsatellite markers (IVS1(CA), IVS8(CA), IVS8(TG)m, IVS8T(n), IVS17b(TA), IVS17b(CA)) and one biallelic marker (p.Met470Val or 1540A>G( c.1408A>G)) were investigated.

Guthrie cards

Spots of 3 mm diameter, punched from anonymized cards, were distributed in 96-well plates and DNA was extracted using methanol extraction [START_REF] Caggana | Rapid, efficient method for multiplex amplification from filter paper[END_REF] . p.Ser1235Arg was screened for using DHPLC technology and each abnormal pattern was confirmed by sequencing analysis.

Computer-assisted analysis

To predict the potential pathogenicity of p.Ser1235Arg, we used PolyPhen® and SIFT® softwares. The PolyPhen® program (http://coot.embl.de/PolyPhen/) is based on the sequence homology and the mapping of the substitution site to known protein 3dimensional structures. Results are given as "benign", "possibly damaging", "probably damaging", or "unknown". The SIFT® program (http://blocks.fhcrc.org/sift/) uses sequence homology to predict whether an amino acid substitution will affect protein function and hence, potentially alters phenotype. Results are reported as "deleterious or not". Consequences on the splicing machinery were evaluated by Human Splicing Finder tool (http://www.umd.be/HSF). This program integrates all available matrices to identify ESE, ESS, ISE and ISS motifs: ESE Finder [START_REF] Cartegni | ESEfinder: A web resource to identify exonic splicing enhancers[END_REF] , RESCUE ESE [START_REF] Fairbrother | Predictive identification of exonic splicing enhancers in human genes[END_REF] , Silencer motifs from Sironi et al. [START_REF] Sironi | Silencer elements as possible inhibitors of pseudoexon splicing[END_REF] , ESS decamers from Wang et al. [START_REF] Wang | Systematic identification and analysis of exonic splicing silencers[END_REF] and new algorithms to calculate the consensus values of potential splice sites and search for Branch points. To gain some insight into the potential effect of the p.Ser1235Arg mutation from a structural point of view, we used the experimental structures of MJ0796 (in complex with ATP, pdb identifier 1l2t) as template for modeling as previously described by Eudes et al. [START_REF] Eudes | Nucleotide binding domains of human CFTR: a structural classification of critical residues and diseasecausing mutations[END_REF] . The MJ0796 structure was visualized with MBT SimpleViewer software.

The alignment of sequences of the two CFTR NBDs, other ABC transporters family and MJ0796 was obtained by ClustalW program and visualized using Jalview 2.3 tools.

In vitro functional studies

CFTR expression plasmids, Site-Directed Mutagenesis, minigene constructs and cells

A plasmid expressing the human CFTR gene was constructed by placing the full-length CFTR cDNA coding sequence obtained from pTG5985 plasmid, a gift from Transgene SA, in the pcDNA3(+) expression vector. The p.Ser1235Arg mutation was inserted into the pcDNA3-CFTR vector by site-directed mutagenesis using QuickChange® II Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions.

The human CFTR genomic region encompassing the exon 19 with the flanking intronic sequences, was amplified from a patient heterozygous for the p.Ser1235Arg using primers FOR 5'AATTCTGGAGCTCGAGCAGGCCTATACAGAGCCCATT-3' and REV 5' CTCTTAATTTGCTAGCCATTTTCAAGATGGGAAATCTAAAACA-3' (which introduce NheI and XhoI sites in the PCR product). After enzymatic digestion, the PCR product was subcloned into the XhoI/NheI digested pSPL3 plasmid, kindly gift by Dr. S. Tuffery-Giraud (Laboratory of Molecular Genetics, Montpellier, France). One clone containing the wild-type allele and one with the mutation were retained for expression experiments. All plasmid constructs were fully sequenced.

Beas2B, a human bronchial epithelial cell line expressing CFTR and the monkey kidney fibroblast cells COS-7, which does not express endogenous CFTR, were cultured as previously described [START_REF] Rene | Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway[END_REF] . Cells were seeded at a density of 200, 000 cells/2 ml of medium in 6-well dishes. After 24h, cells were transfected with Polyfect transfection reagent (Qiagen, France) according to the manufacturer's recommendations. All experiments were repeated at least twice.

RNA extraction and RT-PCR

Total RNA was extracted using RNeasy Plus kit (Qiagen, France). Reverse transcription was performed with 1 µg of total RNA as previously described [START_REF] Rene | Binding of serum response factor to cystic fibrosis transmembrane conductance regulator CArG-like elements, as a new potential CFTR transcriptional regulation pathway[END_REF] . 1 μl of the cDNA synthesis reaction was used for PCR analyses using a mixture of 10 pmol of both SD6/SA2 primers and β-actin primers, 0.5 U of Taq DNA polymerase (New England Bio Labs Inc) and 10 mM of dNTPs in a 25 μl volume. Amplification was performed with an initial denaturation step at 94°C for 5 min, 35 cycles of 94°C 30s, 57°C 30s, 72°C 90s, followed by a final extension step 72°C 7 min. PCR products were visualized on 1.5 % agarose gel. Each PCR product was purified and fully sequenced.

Whole cells extracts and Western-Blot

Whole cells extracts from 6-well dishes were extracted and resuspended in Laemmli buffer. Proteins were separated by SDS-PAGE in a 5% acrylamide gel, transferred in a PVDF membrane and immunoblotted with antibody against CFTR (MM13-4 from Millipore).

Statistical analysis

Statistical analyses were performed using Instat® software. Chi-square test was used to compare differences between general population vs CF patients or general population vs CBAVD patients. P-values < 0.05 were considered to indicate statistical significance.

Results

Genotype/phenotype description of individuals carrying p.Ser1235Arg

Among 104 subjects from 91 unrelated families reported by the nine French laboratories, 6 patients are affected with classical CF (Table 1A). Five of them were referred for severe disease with diagnosis before one year, positive sweat test, pancreatic insufficiency and persistent respiratory infections, and one had a mild phenotype with a borderline sweat test. Complete scanning of the 27 exons and their boundaries identified a second mutation on the same allele for all of them: 1) p.Arg785X, located in exon 13, predicting a truncated protein; 2) 875+1G>A (c.743+1G>A) affecting the highly conserved splice donor site sequence; 3) the (TG)13(T)5 allele which promotes CFTR exon 9 skipping in humans [START_REF] Disset | A T3 allele in the CFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with Congenital Bilateral Absence of Vas Deferens (CBAVD)[END_REF][START_REF] Groman | Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign[END_REF][START_REF] Claustres | Molecular pathology of the CFTR locus in male infertility[END_REF] .

Ten patients were referred for CBAVD (Congenital Bilateral Absence of Vas Deferens) and one for CUAVD (Congenital Unilateral Absence of Vas Deferens) without renal anomalies. The (TG)13(T)5 allele was found in 9 subjects with p.Ser1235Arg (Table 1A). Of these, eight were compound heterozygous for p.Phe508del, and two for mutations associated with CF-related diseases [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF][START_REF] Feldmann | CFTR genotypes in patients with normal or borderline sweat chloride levels[END_REF] , [p.Arg117His;T7] and p.Arg1070Trp (http://www.genet.sickkids.on.ca/cftr/). Among 42 patients with monosymptomatic disease carrying p.Ser1235Arg (Table 1A), eight cases (19%) were compound heterozygotes, seven for a severe mutation and one for the intronic variant 406-6T>C (c.274-6T>C). Of these, five harbored the (TG)13(T)5 allele associated in cis with p.Ser1235Arg. Thirty-four individuals (81%) were heterozygous for p.Ser1235Arg: 5 with reduced sperm quality, 16 with sinopulmonary problems such as bronchiectasis, asthma, nasal polyposis, chronic obstructive pulmonary disease, and 13 with digestive symptoms (meconium ileus at birth, pancreatitis, liver disease). One of them, referred for bronchitis and recurrent infections without bronchiectasis, had a positive sweat test (102 mmol/l).

Eight fetal samples were referred for risk calculation in the cohort of hyperechogenic fetal bowel. Among them, five carried only p.Ser1235Arg and were considered simple heterozygote because extensive scanning did not reveal a second CF mutation [START_REF] Muller | Predicting the risk of cystic fibrosis with abnormal ultrasound signs of fetal bowel: results of a French molecular collaborative study based on 641 prospective cases[END_REF] . Three cases were compound heterozygous for another mutation. In the first case, a moderate hyperechogenicity was detected at 22 weeks' pregnancy, which decreased at 31. The fetus harbored the p.Phe508del mutation on the other allele and parents decided to continue pregnancy. The second carried the rare missense mutation p.Val920Met and depressed values of digestive enzymes were observed at 17 weeks of pregnancy. In the last case, the fetus carried the complex allele [p.Gly576Ala ;p.Arg668Cys], considered as a mild mutation involved in CBAVD or disseminated bronchiectasis phenotypes in adults [START_REF] Bombieri | Complete mutational screening of the CFTR gene in 120 patients with pulmonary disease[END_REF][START_REF] Casals | Bronchiectasis in adult patients: an expression of heterozygosity for CFTR gene mutations?[END_REF][START_REF] Pallares-Ruiz | Complete mutational screening of the cystic fibrosis transmembrane conductance regulator gene: cystic fibrosis mutations are not involved in healthy men with reduced sperm quality[END_REF] . Although the fetus presented hyperechogenic fetal bowel, the parents were reassured regarding the risk of classical CF. Unfortunately, no data were available on the outcome of these pregnancies. Among 37 individuals tested for cascade carrier screening, 14 were compound heterozygous for p.Ser1235Arg and another CFTR mutation (Table 1B), all being relatives of patients or carriers. Of these, twelve harbored in trans a CF-causing mutation. No symptom of CF was notified although sweat test was not performed.

Notably, cases 4 and 5 were two fertile fathers carrying a Class I mutation (severe) associated in trans of p.Ser1235Arg.

Frequencies of p.Ser1235Arg in the general, CF and CBAVD French populations

Thirty-two p.Ser1235Arg alleles were detected in 2114 subjects of the general population representing an allelic frequency of 0.76% (Table 2). None of them had the p.Arg785X mutation. This allele frequency was compared with those of patients collated in a French study affected with CF (8/7420 alleles) or CBAVD (7/1626 alleles), 0.11% and 0.43%, respectively [START_REF] Claustres | Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France[END_REF] . By χ 2 statistical analysis, the p.Ser1235Arg allelic frequency is significantly higher in the general population than in CF patients (0.76% vs. 0.11%, p< 0.001) but not statistically different from the CBAVD group (0.76% vs. 0.43%, p> 0.1). Recently, six CF patients were re-analyzed. Among them, five with severe disease, harbored the p.Arg785X mutation, while another presenting a mild phenotype had the complex allele [p.Ser1235Arg;T5]. When the p.Ser1235Arg frequencies in CF, CBAVD and the general population are compared to frequencies of p.Phe508del (Table 2), it appears that p.Ser1235Arg is as frequent as p.Phe508del (p=0.91) in the general population (respectively 0.76% vs. 0.82%); however, its implication in classical CF is significantly different (0.11% vs. 67.2% (p<0.0001) and 2.86% (p<0.0001) respectively).

p.Ser1235Arg haplotype backgrounds

Haplotypic markers and familial segregation were completed in thirty-six cases (Table 3). The analysis of haplotypes based on four intragenic CFTR markers (IVS1(CA), IVS8(CA), M470V, IVS17b(CA)) revealed that p.Ser1235Arg is always present on the same haplotype 26-17-M-13. This haplotype associated with the polyvariant (TG)12(T)7 represents 77.8% of cases. Only the IVS17b(TA) microsatellite marker shows a large variability in this study ranging from 7 to 44 repeats. However, three complete haplotypes differing only by one repeat in the IVS17b(TA) (35, 36 and 37) accounted for more than 63% of the cases. The p.Arg785X found in CF patients in cis of the p.Ser1235Arg occurs on a single haplotype 26-17-12-7-M-35-13. The complex allele [p.Ser1235Arg;(TG)13(T)5] characteristic of CBAVD, was also found to be carried on a unique haplotype (26-17-13-5-M-33-13).

In silico predictions and functional impact on the CFTR protein

We evaluated the degree of conservation of serine 1235 residue using alignment of the Nucleotide Binding Domains of the human CFTR with those of ABC transporters, whose the CFTR belongs (Figure 1A). The results show that the serine 1235 is poorly conserved in the ABC transporter family, suggesting that amino acid change in this position is possible. Serine 1235 residue located after a β strand is not involved in anchor or structural points [START_REF] Callebaut | Nucleotide-binding domains of human cystic fibrosis transmembrane conductance regulator: detailed sequence analysis and three-dimensional modeling of the heterodimer[END_REF] . The analysis of its position on the 3D structure of MJ0796 (Figure 1B) previously used as template for modeling the CFTR NBDs [START_REF] Eudes | Nucleotide binding domains of human CFTR: a structural classification of critical residues and diseasecausing mutations[END_REF] , points that the Serine 1235 is located on the exterior surface of the NBD2.

Therefore, this amino acid does not participate to the NBD1/NBD2 heterodimer formation and to the ATP binding and hydrolysis. The limited effect of this amino acid change was confirmed using SIFT® and PolyPhen® programs. To evaluate the functional impact of the mutation on the CFTR maturation, we performed Western-blot analysis on whole cell extracts from COS-7 cells transfected with either wild-type or the p.Ser1235Arg constructs (figure 1C). The results show the presence of the fullyglycosylated CFTR protein (Band C) with the p.Ser1235Arg construct, suggesting that the maturation of the pSer1235Arg-CFTR protein is not altered. These results suggest that p.Ser1235Arg has no functional impact on the CFTR protein.

Functional impact of the pSer1235Arg on the splicing

According to the Splicing Sequences Finder tools [START_REF] Desmet | Human Splicing Finder: an online bioinformatics tool to predict splicing signals[END_REF] , p.Ser1235Arg does not seem to impact the splicing of CFTR transcript. To verify this prediction, we used a minigene-based approach. Constructs containing the wild type and mutant CFTR exon 19 and the intron-exon boundaries were inserted in the pSPL3 vector (figure 2A). After transfection in Beas2B cell line, RNAs were extracted and subjected to RT-PCR. As shown in figure 2B, compared to the wild-type, the presence of the pSer1235Arg alteration does not modify the exon 19 inclusion in the mRNA. Together, these data suggest that pSer1235Arg has no effect on the CFTR mRNA splicing.

Discussion

Since its initial description in 1993, the clinical significance of the p.Ser1235Arg mutation remains unclear. This ignorance makes genetic counseling very difficult, especially in the context of prenatal diagnosis. Through genotype/phenotype analysis of patients or individuals carrying p.Ser1235Arg, analyzed by the French network and functional analysis, we present several lines of evidence that argue against an association of pSer1235Arg with CF or CBAVD although a partial penetrance of p.Ser1235Arg could not be ruled out.

First, the presence of a complex allele [p.Ser1235Arg;class I mutation] in trans of a severe mutation in five patients presenting severe disease (Table 1A) strongly suggests that p.Ser1235Arg alone cannot be considered as a CF-causing mutation.

Moreover, among the eleven patients referred for CAVD, nine are compound heterozygous for a mutation and the complex allele [p.Ser1235Arg;(TG)13(T)5]. The (TG)13(T)5 allele without the contribution of p.Ser1235Arg is sufficient to result in male infertility or in mild phenotypes [START_REF] Disset | A T3 allele in the CFTR gene exacerbates exon 9 skipping in vas deferens and epididymal cell lines and is associated with Congenital Bilateral Absence of Vas Deferens (CBAVD)[END_REF][START_REF] Groman | Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign[END_REF][START_REF] Cuppens | Polyvariant mutant cystic fibrosis transmembrane conductance regulator genes. The polymorphic (Tg)m locus explains the partial penetrance of the T5 polymorphism as a disease mutation[END_REF] . Among the two patients with p.Ser1235Arg as single allele, one has the mutation p.Phe508del in trans while no other mutation was identified in the second one. The possibility of mutations in intronic regions undetectable by our methods cannot be excluded.

Second, we present 14 asymptomatic individuals (Table 1B), of whom 12 carried a severe and two a mild mutation in trans. Three, carrying the complex allele [p.Ser1235Arg; (TG)13(T)5], are sisters or brother of CBAVD or CUAVD patients and no respiratory or digestive symptom was reported. In females, allele (T)5 has been shown to have a low penetrance [START_REF] Sun | CFTR 5T variant has a low penetrance in females that is partially attributable to its haplotype[END_REF] while no urogenital data was available for the male because of absence of parental project. Three adult males are compound heterozygous for p.Ser1235Arg and a severe mutation; two (cases 4 and 5) are biological fathers of CF children and transmitted the severe mutation. Case 7 to 11 are patients' mothers or relatives with no evidence of CF symptoms. Cases 12 and 13 are non-symptomatic sister and first-cousin of a patient with respiratory symptoms whose CF diagnostic could be discussed because symptoms are close to asthma.

Third, the frequency of the p.Ser1235Arg in the general population (Table 2) is higher than in CF and CBAVD patients, in accordance with previous reports [START_REF] Monaghan | Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: results from a collaborative study[END_REF][START_REF] Modiano | A large-scale study of the random variability of a coding sequence: a study on the CFTR gene[END_REF] , and is close to the p.Phe508del frequency (0.82%). By contrast, in CF or CBAVD groups, the p.Ser1235Arg frequency is significantly lower than p.Phe508del.

In the functional point of view, results from alignment show that this residue is poorly conserved and occurs in a region not implicated in known CFTR functions.

Moreover, in silico predictions suggest that this nucleotide change is not critical for the mRNA splicing and the CFTR function. These data are confirmed by functional studies, which showed that p.Ser1235Arg does not alter the CFTR mRNA correct splicing and the protein maturation. These data are consistent with previous functional study of this locus in combination with alleles found at p.Met470Val and p.Gly628Arg loci [START_REF] Wei | Suppressive interactions between mutations located in the two nucleotide binding domains of CFTR[END_REF] . Wei et al. demonstrated that the p.Ser1235Arg CFTR protein does not cause change in the chloride transport activity. Besides, the p.Gly628Arg/p.Ser1235Arg mutant protein induces a significantly lower cAMP dependent chloride transport activity than the p.Gly628Arg mutant protein, showing the major importance of genetic background, particularly for missense mutations.

In a second step, we determined the haplotypes linked to p.Ser1235Arg and found the same haplotype in 72.4% of cases. Only the IVS17b(TA) marker presents a large variability although three haplotypes, deriving one from the other by addition or deletion of one dinucleotide (35, 36 or 37 repeats), represent more than 55% of the alleles. Besides, IVS1(CA) 26, a rare allele in the European populations (4.2% vs 80% for alleles 22, 23 ad 24) [START_REF] Mateu | Allele frequencies in a worldwide survey of a CA repeat in the first intron of the CFTR gene[END_REF] , is strictly linked to p.Ser1235Arg. In CBAVD patients, the haplotype 26-17-13-5-M-33-13 was found in 85.7% of p.Ser1235Arg alleles. This data suggests the further change of the (TG)12(T)7 to (TG)13(T)5 on a chromosome bearing the alteration p.Ser1235Arg on a background IVS17b(TA)33. Together, this data suggests that p.Ser1235Arg could derive from a founding event and haplotypes differing by a single microsatellite could depend on slippage phenomena, as already proposed [START_REF] Claustres | CFTR haplotypic variability for normal and mutant genes in cystic fibrosis families from southern France[END_REF] .

In conclusion, there are now strong lines of evidence against a severe deleterious effect of p.Ser1235Arg and its association with classical CF or CBAVD disease.

However, a partial penetrance and/or the presence of other sequence alterations in nonscreened regions particularly in patients with atypical or mild symptoms of cystic fibrosis [START_REF] Feldmann | CFTR genotypes in patients with normal or borderline sweat chloride levels[END_REF][START_REF] Castellani | Analysis of the entire coding region of the cystic fibrosis transmembrane regulator gene in neonatal hypertrypsinaemia with normal sweat test[END_REF][START_REF] Reboul | Iron A: Isolated idiopathic chronic pancreatitis associated with a compound heterozygosity for two mutations of the CFTR gene[END_REF] cannot be ruled-out. This data highlights the importance of searching for a complex allele whenever p.Ser1235Arg is identified. The absence of clinical effects in healthy individuals compound heterozygous for the p.Ser1235Arg and another severe mutation is helpful to provide adequate genetic counseling to the families with regard to its transmission in offspring. B: RT-PCR analysis of mRNA of cell expressing Wild-Type 42 or p.Ser1235Arg(S1235R) constructs. BeaS2B cells were transfected with either empty, wild-type or p.Ser1235Arg pSPL3 vector. After 48 hours, total RNA were extracted and cDNA were synthetized. PCR was performed using SA2 and SD6 primers amplifying the region between exon A and B. On the right, the composition of each transcript was indicated. Each experiment was repeated at least three times.

Titles and legends to figures

  classical CF (six unrelated patients), CAVD (ten individuals with bilateral and one with unilateral Congenital Absence of the Vas Deferens), fetal suspicion of CF (eight fetuses with abnormal ultrasound signs of bowel anomalies) and forty-two individuals presenting CF-related symptoms (according to the International Classification of Disease (ICD) in the section "Cystic Fibrosis and Related Disorders" [Meeting report, 2002]) with normal or borderline sweat chloride values including genital (five individuals), respiratory (twenty-three individuals) or digestive (fourteen individuals) symptoms. Thirty-seven healthy subjects were referred for carrier screening including fourteen individuals with a positive family history and twenty-three partners of CF patients or carriers. Written consents to the genetic study were obtained from the patients and/or their family and from healthy subjects.

Figure1:

  Figure1: Structural and processing impact of p.Ser1235Arg mutation on the CFTR

Figure 2 :

 2 Figure 2: Functional impact on the CFTR mRNA splicing

Table 1A

 1A Genotype and phenotype of CF, CBAVD and CF-like patients carrying the 597 Not Available ; We could only test the mother and an healthy sister (the patient 599 was deceased and the father's DNA was not available) Table1BGenotype and familial history of asymptomatic individuals carrying the p.Ser1235Arg mutation.

	598	p.Ser1235Arg mutation.
		allele 1	Genotype	allele 2	phenotype	No of subjects
		p.Ser1235Arg;p.Arg785X p.Ser1235Arg;p.Arg785X p.Ser1235Arg;875+1G>A (c.743+1C>A) p.Ser1235Arg;p.Arg785X p.Ser1235Arg;(TG)13(T)5 p.Ser1235Arg;(TG)13(T)5 p.Ser1235Arg;(TG)13(T)5 p.Ser1235Arg;(TG)13(T)5 p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg;(TG)13(T)5 p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg;(TG)13(T)5 p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg p.Ser1235Arg Genotype 3629delT (c.3497delT) p.Phe508del NA 1 p.Gly542X p.Gly551Asp p.Phe508del p.Arg1070Trp p.Arg117His; (T)7 p.Phe508del -p.Phe508del --p.Phe508del 406-6T>C (c.274-6T>C) p.Tyr1092X p.Glu831X p.Gln493X p.Ile507del -p.Gly542X -p.Arg668Cys;p.Arg576Ala hyperechogenic fetal bowel severe CF severe CF severe CF severe CF mild CF CBAVD CBAVD CBAVD CBAVD CBAVD CUAVD Suspicion CF/mild phenotype: genital symptoms respiratory symptoms respiratory symptoms respiratory symptoms respiratory symptoms respiratory symptoms respiratory symptoms respiratory symptoms digestive symptoms digestive symptoms hyperechogenic fetal bowel p.Val920Met hyperechogenic fetal bowel p.Phe508del hyperechogenic fetal bowel Familial information allele 1 allele 2 1 p.Ser1235Arg;(TG)13(T)5 p.Phe508del brother of CUAVD (no parental project) 16 13 2 p.Ser1235Arg;(TG)13(T)5 p.Phe508del sister of CUAVD 3 p.Ser1235Arg;(TG)13(T)5 p.Arg1070Trp sister of CBAVD 4 p.Ser1235Arg p.Gly542X father of CF [p.Phe508del]+ [p.Gly542X] and healthy children [p.Ser1235Arg]+ [(TG)11(T)5] 5 p.Ser1235Arg p.Gln493X father of CF [p.Phe508del]+[p.Gln493X] 6 p.Ser1235Arg p.Phe508del uncle of CF (no parental project) 7 p.Ser1235Arg p.Phe508del mother of CF [p.Phe508del]+[1717-1G>A (c.1585-1G>A)] 8 p.Ser1235Arg 2347delG (c.2215delG) mother of CF [p.Phe508del]+[2347delG] 9 p.Ser1235Arg (TG)11(T)5 mother of non CF fetus with hyperechogenic fetal bowel [p.Phe508del]+[(TG)11(T)5] 10 p.Ser1235Arg p.Phe508del sister of CBAVD 11 p.Ser1235Arg p.Phe508del sister of CBAVD 12 p.Ser1235Arg p.Glu831X sister of CF-like patient 13 p.Ser1235Arg p.Phe508del first-cousin of CF-like patient 1 NA: Case # 14 p.Ser1235Arg 18 month-old child with prenatal diagnostic p.Phe508del based on familial history of CF

Table 3 :

 3 CFTR haplotypes for 36 fully haplotyped p.Ser1235Arg chromosomes from 614 CF, CBAVD patients, ruled-out CF (including CF-like symptoms and Fetal echogenic 615

	616	bowel) and general population.
		IVS1(CA)-IVS8(CA)-M470V-IVS17b(CA)	IVS8(TG)m -IVS8(T)n	Mutation in cis	IVS17b(TA )	n	%
							11.
		26-17-M-13	12-7	p.Arg785X	35		1
				875+1G>A
				(c.743+1C>A)	36		2.8
				None	33		2.8
							13.
					35		8
					36		25
							11.
					37		1
					38		2.8
					40		2.8
					44		2.8
					7		2.8
							22.
			13-5		33		2
	617
	618
	619
	620
	621

Acknowledgements

We thank Dr C. Beroud for fruitful discussion in the software analysis and the GREPAM for efficient anonymized sample referral.

Conflict of interest.

The authors declare no conflict of interest.