Jean Baudry

Emanuel Bertrand

Catherine Rouzeau

Olivier Greffier

Anne Koenig

Rémi Dreyfus

Laetitia Cohen-Tannoudji

Cécile Goubault

Lydie Bressy

Laure Vincent

Nicolas Lequeux

Jérôme Bibette

Colloids for studying molecular recognition

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

INTRODUCTION

Organic and inorganic colloids have been used as biosensors for many years. A biosensor is aimed at determining various parameters associated to biological species such as single bio-molecules, molecular complexes, virus, cells and tissues. The understanding of mechanism from a molecular scale up to the cell function is certainly one of the most important scientific issues in molecular biology. It simultaneously requires information concerning concentrations, structures, conformations and dynamics, both at different length and time scales. The possibility of measuring such parameters requires the development of as much as possible sensitive, selective, real-time and multi-scale biosensors. After reviewing most of the biosensors that have already been used, we will show how the colloidal organic / inorganic composites that we use give access to new biophysical tools and are able to improve our quantitative knowledge of molecular recognition phenomena. In particular, we will explain their ability to improve considerably the sensitivity of immunological diagnosis agglutination tests. Tirés à part : E. Bertrand, LCMD, ESPCI, 10 rue Vauquelin, 75231 Paris Cedex 05 One crucial aspect of bio-molecules concerns their ability to build reversible complexes through specific molecular recognition mechanisms. Indeed, a major property of bio-molecules is to bind a variety of ligands in order to fulfill a specific function such as mediating cell adhesion, triggering receptor mediated cell activation or regulating intracellular networks and immuno-response. Probing recognition phenomena has given rise to various types of sensors focusing on rupturing or binding, single as opposed to multi-molecular events as encountered in adhesion of mesoscopic objects (vesicles, cells or drops), and finally thermodynamic or kinetic aspects of bulk ligand-receptor reactions.

PROBING MOLECULAR RECOGNITION

Classical biochemistry methods give the thermodynamic and kinetic constants when both ligand and receptor are in solution, or when one of them is immobilized on a surface [START_REF] Lauffenburger | Receptors: Models for binding, trafficking, and signalling[END_REF]. These constants can be readily determined with new tools, measuring for instance the variation of evanescent wave intensity upon binding of antigens on antibody-coated surface (Biacore S.A.). However, in lots of relevant situations, both macromolecules are supported by cells or tissues, so their fluctuations become strongly restricted, as a result of anchoring itself, crowding from neighbors and eventually compression due to surface forces… An important challenge in biophysics concerns the characterization of these effects, which certainly requires setting new tools.

Using red cell sensors and vesicles, both handled by micro-pipettes, rupturing of single molecular complexes has been precisely studied [START_REF] Evans | [END_REF]. It was shown that the separation of a ligand from its receptor under force followed a unidimensionnal path, with an energydistance curve determined by a so-called energy landscape, displaying sequential maxima or barriers. The plot of unbinding force versus the logarithm of the loading rate is found to be composed of sequential straight segments, the slope of which might be written as kT/x i , where x i represents the distance between the location of the energy minimum and the i th barrier on the energy landscape. The surface force apparatus (SFA) is also a means to probe some collective binding processes. The measurement of macroscopic adhesion as a function of surface distance, after a reaction time of about one second allows the rate of binding to be inferred, which was attributed to the predicted molecular spacers dynamics [3]. Reflectance interference contrast microscopy (RICM) follows the formation of specific adhesive patches between a model solid grafted surface and a vesicle or a cell; the role of macromolecular repellers coupled to the presence of specific linkers has been largely addressed [4]. The laminar flow chamber operated at low shear rate can provide additional information on the fine properties of ligand-receptor association and rupture. Binding frequencies are obtained by pooling all colloidal trajectories in a given experiment and calculating the ratio of the number of steps followed by an arrest, divided by the total number of steps. In the same way, from the statistics of arrest duration, the unbinding frequency is computed. This technique gives valuable information about the role of shear rate, and can evidence the existence of transient states during the process of binding as in the case of streptavidin-coated micro-beads interacting with a biotin-derivatized surface [5].

BIOTECHNOLOGICAL IMPLICATIONS

Recognition has also many biotechnological implications. A major one concerns biological diagnosis or sorting of bio-molecules, virus and cells. In most cases, a targeted specie is probed using a complementary molecule which forms a complex, such as a ligand and receptor, antigen and antibodies or complementary nucleic acids strands. The challenging aspect of diagnostic technologies is to transform the microscopic scale recognition process into a more macroscopic signal from a quantity of formed complexes as low as possible and within a biological mixture containing many different species. Today, developing simple, low cost, fast, and highly sensitive procedures to measure the concentrations of bio-molecules within serum, plasma, or in environmental conditions is certainly one of the major challenges of life science.

Early immuno-chemistry was based on precipitation of large complexes made of antibodies and antigens. Use of organic colloids, namely latex particles, did significantly improve the detection sensitivity because of the increase in scattered light when aggregation between grafted colloids takes place. Indeed, Brownian colloids will quickly find their target and at the same time optical change due to either agglutination or immobilization will be dramatically enhanced. As the most simple but very generic example, let us consider an antigen Ag having two epitopes for two monoclonal antibodies A and B. To reveal the presence of such an antigen, let us add a mixture of two types of colloids, each one grafted with A or B. The formation of small clusters is then expected, the formation rate depending on many factors. Change in light scattering due to the presence of these small clusters will reveal the existence of sandwich-like structures: A-Ag-B (Latex Agglutination Immunoassay). This homogeneous assay, as opposed to heterogeneous assays, which require washing steps prior to detection, is today by far the most simple and straightforward. Regarding heterogeneous assays, the most extensively used is the ELISA assay (Enzyme-Linked Immunosorbent Assay). In this assay, the latex particles have been commonly replaced by inorganic / organic composite magnetic particles, which are very useful since magnetic separation is rapid and convenient. The last part of this article will focus on the new biophysical and biotechnological applications of these magnetic particles and their exact chemical nature will thus be addressed later on. In an ELISA assay, the capture of antigens is operated through the recognition of only one type of antibody which is grafted onto the magnetic particles; note that there is no possible agglutination in that case. After removing and washing, a labeled-antibody is added to form the sandwich-structure; the label or the socalled tag of this second antibody may be either an enzyme (ELISA), a fluorescent, a radioactive or a chemi-luminescent molecule. After incubation, the sample is washed again to remove the labeled antibodies in excess, the signal being then detected. Because of these two washing steps, sensitivity as low as pmol/l are routinely obtained on automates. The main drawback of this approach concerns its complexity and therefore its associated cost as well as the required time compared to homogeneous assays. It should be noticed that the ELISA method is used in all commercial instruments once high sensibility is required, with test duration varying from 1 to 2 hours.

IMPROVING THE SENSITIVITY OF HOMOGENEOUS ASSAYS

The challenge today for physicists, chemists, and immuno-chemists is to make homogeneous assays much more sensitive. In order to do so a detailed understanding of the different steps leading to specific sandwich-structures when starting with Brownian grafted colloids, should be addressed. "Agglutination assays" were introduced almost 50 years ago [6], and today several hundreds different tests based on this principle can be found on the market, mainly for infectious disease detection and protein quantification. These tests are rapid and simple: specific beads and samples to be analyzed are simply mixed, then absorbance after several minutes of incubation is measured. Today most of the ongoing research work about these agglutination tests aims at improving their limited sensitivity (ñ mol/l).

The more stringent limitation is related to colloidal stability in such a complex biological medium, having a very high ionic strength and containing a lot of very adherent proteins such as fibrinogen, which is found in plasma [7]. It is clear that aggregation must only occur in the presence of antigens, and never be due to any attractive colloidal force (van der Waals, hydrophobic or electrostatic). Therefore, some kinds of repulsive forces must exist between particles to ensure the absence of non-specific links, or so called "false positives". However since antigen-antibody recognition involves quasi-contact forces (less than 1nm, see ref. [8]), increasing colloidal stability systematically decreases immuno-reactivity. Chemical and biochemical know-how has been developed [9] about the core and surface of particles (as responsible for colloidal stability and the possibility of having functional groups for further grafting with probes or ligands), about the antibody fixation (type and density, adsorption or covalent coupling, saturation with other protein) and about the medium which affects the optimization of the test (pH, ionic strength, surfactants, depleting agents). It turns out that all parameters are strongly coupled together and therefore conditions must be redefined for any change of antibodies, antigens or particles [10]. However, as long as this simple strategy is concerned, there is always some non-specific aggregation, which is superimposed to the specific one, imposing the limit of the sensitivity. Indeed, the measurable level of antigen in solution that can be detected, can never exceed roughly the number of non-specific links that form between beads during the test. Moreover the variability of biological fluids is adding some more complexity that limits even more the degree of reliability of the detection in the lower concentration limit.

The second limitation is related to the detection of aggregates. Since we are interested in determining low concentrations of antigens, the problem is essentially shifted to quantification of doublet formation. The most widely used technique is turbidimetry, because of its simplicity. As long as beads are small compared to the wavelength of the employed light (which is not exactly the case in practice), a doublet will scatter twice as much light than two separated beads would do. In the low concentration regime of antigens, which means that the bead concentration is larger than the antigen concentration, scattering of singulets (non aggregated single beads) will interfere with doublet signal, by contrast with ELISA-like assays where excess secondary labeled antibodies are washed out prior to detection. Therefore, the number of doublets that can be detected is limited by the total number of beads in solution. One possibility would be to decrease the particle volume fraction φ in order to increase the sensitivity. Kinetics of doublet formation would then become too slow. Let us first assume an ideal system where the immune recognition is diffusion-limited, the average time t b for a doublet to form is [START_REF] Russel | Colloidal Dispersions[END_REF]:

t b = a 2 / (6Dφ)
where a is the particle radius, φ the volume fraction and D the diffusion coefficient. We find t b of about 1s when typical values for a and for φ are respectively 100 nm and 0.1%. But in fact, the specific link formation is most often reaction-limited, due to the intrinsic antibody-antigen association rate [START_REF] Lauffenburger | Receptors: Models for binding, trafficking, and signalling[END_REF] or due to the repulsive colloidal forces, as considered previously. In this regime, several encounter events are necessary for a specific link to form. Therefore dilution will dramatically increase the assay duration, which precludes any optimization in that direction.

Several strategies have been proposed to overcome the noise caused by non-specific links. Single molecule studies are certainly inspiring these strategies. They are based on force differentiation, originating from the fact that with well-designed colloidal surfaces, nonspecific links remain weaker than specific ones. Force differentiation may be applied through the following procedure: first, an immuno-chemical sandwich is expected between a grafted wall and a magnetic bead. This first step would take about 1-hour incubation, the necessary time to allow bead diffusion towards the wall. Second, a magnetic field is applied in a controlled manner, so that the applied force on the particles is enough to break up the nonspecific links only. The beads are then counted with a CCD camera [START_REF] Lee | [END_REF], with a magnetoresistive transducer [13] or a CMOS Hall sensor [START_REF] Aytur | Solid-State Sensor, Actuator and Microsystems Workshop[END_REF].

Another method consists in using a SQUID (Superconducting Quantum Interference Device) "microscope" to measure the relaxation time of the particle magnetization at ambient temperature. Beads in solution undergo Brownian rotation, while for the bound particles only Néel relaxation is allowed. Since the time scales of these two relaxations are quite different [START_REF] Chemla | [END_REF] a precise differentiation is possible. Recently, this idea of force differentiation was extended, using DNA zipper as force probe [16], and better or comparable performance than "ELISA" assays was claimed. It should be noticed that the first washing step in ELISA assay may be considered as helping to remove part of non-specific capture as well and is also participating to a force differentiation strategy.

To tackle the immuno-reactivity/colloidal stability paradigm, a strategy using ultrasonic waves has been proposed [17]. It is still a classical agglutination format, but during the incubation time between beads and antigens, the sample is placed within an ultrasonic apparatus. Sensitivity is increased 10 to 1000 times depending on the nature of antigens, particle size and type. Two phenomena may be responsible for this enhancement. An ultrasonic standing wave-field subjects the particles to a radiation force and drives them towards pressure nodal regions. First, the local bead concentration increases, therefore increasing the collision probability. Second, the applied force can be enough (order of pN [18]), to overcome the repulsive colloidal force barrier.

Finally, several strategies have been proposed to improve the detection, all of them aiming at lowering as much as possible the background signal due to non aggregated beads. Colloids can be synthesized with a high level of refinement including controlled size, surface and content, which allows to implement new adjustable properties. Tiny gold particle will change color upon specific agglutination due to shifts in the plasma resonance [19]. Furthermore, when coupled to temperature change (melting studies), such shifts can reveal single base pair mismatch between complementary DNA strands [20]. In Luminescent Oxygen Channeling Immunoassay (LOCI [21]), "donor" and "acceptor" beads are used to form the sandwich: the donor particle produces excited singlet oxygen under 680 nm light exposition. This oxygen diffuses and initiates a chemi-luminescent reaction in the vicinity of the acceptor bead. Another strategy consists in tracking aggregates or particles one by one. In the Copalis™ technique, as in flow cytometry, a fluidic system aligns particles in a single file. They pass through a laser beam, and from their distinct scattering patterns it is possible to count the number of singulets, doublets etc. Related techniques are based on the formation of a sandwich type structure between a grafted bead and a secondary fluorescent antibody, which recognizes the desired antigen as in ELISA. Moreover, beads may be also tagged allowing multiplexed detection by using a flow cytometer (Luminex Corp.). Two-photon excitations have been also used for the same purpose, because the observation volume is so small that there is virtually no background fluorescence [22]. In both methods, the scattering signal comes from the tiny probed volume while the particles act as local concentrators of antigens, increasing the assay performance by orders of magnitude in terms of concentrations [23].

SELF-ASSEMBLING OF MAGNETIC COLLOIDS: MOLECULAR RECOGNITION AND APPLICATIONS

This part introduces the basics of using super-paramagnetic composite colloids and their spontaneous self-assembling ability under field, as new biosensors. The possibility to vary the magnetic interaction, and therefore the distance between particles within chain-like structures, opens new possibilities first as a new detection tool and second to resolve recognition at a molecular level.

The particles must fulfill some very specific requirements in order to be used for this strategy: beads must be spherical, mono-disperse, strongly magnetic and Brownian which imposes a maximum diameter of about 1 µm. In addition, their surfaces must provide very strong colloidal stability, and possess organic functional groups to be further grafted by macromolecules of biological interest. Such particles are now commercially available and manufactured by Ademtech S.A. (http://www.ademtech.com). At the first stage of their preparation, they consist in calibrated emulsion droplets of an organic ferrofluid in water. The ferrofluid used is a suspension of γ-Fe 2 0 3 ferrimagnetic inorganic nano-particles of radii ranging from 5 to 10 nm in octane. The nano-particles are stabilized by oleic acid. Thanks to a controlled emulsification process [24], the obtained ferrofluid emulsion droplets are very mono-disperse with a diameter ranging from 150 nm to 1 µm. This emulsion undergoes a thermal treatment in order to evaporate most of the octane within the droplets and to obtain very concentrated and thus strongly magnetic spherical beads. The calibrated beads then undergo a surface polymerization in order to avoid the presence of ferrimagnetic particles at their surface and to obtain a homogeneous controlled surface with a high density of carboxylic groups. Surface polymerization leads to highly charged particles at neutral pH, providing a strong colloidal stability through electrostatic repulsion. At this stage, one ends up with colloidal particles that are both inorganic inside and organic at their surfaces and that fulfill all the formerly exposed requirements. Finally, before being used for molecular recognition and its applications, biological molecules, such as antibodies, must be grafted onto carboxylic sites, which involves routine biochemical know-how. The application of a magnetic field to an aqueous suspension of such magnetic beads induces a magnetic dipole in each bead. Due to Brownian motion coupled to anisotropic dipolar interactions, the magnetic particles eventually form chains. These structures are reversible: by switching the external field off, magnetic moments vanish, and the Brownian agitation redisperses the suspension. At low bead concentrations, the chains are one particle thick, well separated and align along the field direction, as can been seen on figure 1. This one-dimensional order is giving rise to a very remarkable interference pattern which is used to measure the inter-particle distance from collecting the Bragg scattered light at 180° [25]. Moreover, the magnetic content of beads and their distance within the chain (deduced from Bragg scattering) enables to deduce the applied magnetic force. Since the colloidal repulsive force strictly balances this attractive magnetic force, the force distance profile is readily determined for various conditions. One example is given in figure 2, where electrostatic forces are characterized as a function of ionic strength. This technique originally offered the first direct measurement of electrostatic repulsion between colloids and the validation of the predicted exponential decay of the Debye-Hückel theory [25]. Steric repulsion induced by an adsorbed polymer was also probed with this technique. It was shown that the repulsion did follow an exponential decay with distance, the decay length being proportional to the radius of gyration of the adsorbed polymer [26].

0.3mM 5mM

Another fundamental application of the self-assembling properties of these composite organic/inorganic colloidal materials lies in the determination of the micro-mechanical properties of biological complexes. Once the particles are grafted with ligands and put in the presence of receptors able to recognize two of them, the chains are no more reversible, and instead, specific recognition eventually leads to a sandwich-type molecular construct that can link adjacent particles together. Goubault et al. have just shown that the rigidity of the obtained magnetic chains is quantitatively linked with the bending rigidity of the molecular structure between the magnetic beads [27]. The authors therefore extend the exploration of biomechanics much further than the knowledge of rod-like biopolymers such as actin, DNA and tubulin. This technique should, on the basis of these first results, give a clearer understanding of the mechanical properties of most of the biomolecules involved in cell biology [27]. In terms of biotechnological applications, agglutination assays can be greatly improved using these composite magnetic particles. The basic idea is to exploit chaining as a catalyzer for molecular recognition to occur. Nonetheless, the strategy remains very similar to that of classical agglutination tests except that a magnetic field is used. The antibody-grafted beads are first mixed with the sample to be probed, so the antigens are captured. Second, magnetic field is applied to form chains so that antigens will irreversibly link particles. Third, the field is relaxed, and the size or amount of the remaining chains, which reflects the antigen concentration, is detected using simple turbidimetry, as usually done for detecting aggregates. It has been shown that assay sensitivity can be easily improved by a factor of 1000 [START_REF] Bibette | Méthode de detection d'analytes à l'aide de particules colloidales[END_REF]. As an example, figure 3 shows the kinetics of aggregation of streptavidin-coated magnetic particles under a magnetic field of 9 mT, in the presence of biotinylated BSA (Bovine Serum Albumin). The experiment is done in a square capillary tube of 50 µm width and the aggregate mean length is determined by optical microscopy, as a function of the incubation time under the magnetic field. As can be seen on figure 3, the data points are well fitted by a mono-exponential, characteristic of a first-order like kinetics. The experimental characteristic time is approximately 5 minutes whereas no aggregation can be seen without field after one hour of observation. The origin of the enhancement of this sensitivity is still to be determined, and might be similar to the one responsible for the already mentioned "ultrasonic agglutination assay": since a force is also applied on the beads, the repulsive colloidal barrier can be overcome. Moreover, they are also brought in close contact for quite a long time compared to what is expected from Brownian collisions, greatly enhancing the encountering probability between grafted macromolecules.

The main underlying idea is therefore to apply suitable mechanical and geometrical conditions, within a sufficient time, to induce specific molecular recognition. By contrast to bond rupturing, very little is known on binding, when the macromolecules are grafted on colloids, cells or drops. It seems that a "long" contact time, more than a strong applied force, is efficient [5]. The major goal, from a fundamental point of view, is to determine the rules which govern binding, both for specific (recognition) and non-specific (eventually protein mediated) routes.

CONCLUSION

To summarize, the use of colloidal organic / inorganic composites, namely surfacepolymerized super-paramagnetic particles, offers new tools for studying molecular recognition. The organic part of these particles enables to graft them with all sorts of molecules of biological interest. Besides, their inorganic -and magnetic -part gives a way of manipulating these new biosensors with an external magnetic field. This novel approach has already proved to be applicable and successful in the domain of immunological diagnosis by considerably improving the sensitivity of agglutination assays.

Figure 1 .

 1 Figure 1. Chains of super-paramagnetic particles aligned with the magnetic field in a square capillary tube of 50 µm (optical microscopy, X100 objective)

Figure 2 .

 2 Figure 2.Evolution of the colloidal force between adjacent magnetic beads with the centreto-centre distance, for two ionic strengths. Points correspond to experimental values, the solid line to theoretical predictions (the surface potential is the only fitting parameter).

Figure 3 .

 3 Figure3. Kinetics of aggregation of streptavidin-coated magnetic particles (volume fraction: 0.02 %) in presence of biotinylated BSA (concentration: 5 nmol/l) in a 50 mmol/l phosphate buffer, after incubation under a 9 mT magnetic field (circles), and without magnetic field (diamonds); the solid line is an exponential fit.


A Billet

C Ka

M Heller

F Degrugillier

M.-L Vuillaume

V Thoreau

S Sasorith

C Bareil

C Thèze

C Ferec

G Le Gac

T Bienvenu

E Bieth

V Gaston

G Lalau

A Pagin

M.-C Malinge

F Dufernez

L Lemonnier

M Koenig

P Fergelot

M Claustres

M Taulan- Cadars

A Kitzis

M.-P Reboul

F Becq

P Fanen

C Mekki

M.-P Audrezet

E Girodon

C Raynal

The multi-faceted nature of 15 CFTR exonic variations: Impact on their functional classification and perspectives for therapy

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

Introduction

Classifying missense variants as deleterious or benign still re-mains challenging for geneticists, since the numerous computational algorithms available show variable performance [START_REF] Li | Performance evaluation of pathogenicity-computation methods for missense variants[END_REF] , and in vitro assays are not always available. If an impairment on the resulting protein structure or function is the most expected out-come of such variants, an impact on splicing should also be considered for a comprehensive insight into their pathogenic mechanism. Indeed, at least 10% of so-called missense variants in all genes involved in genetic diseases are actually splicing variants [ 2 , 3 ]. Pathogenic variations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene may cause Cystic fibrosis (CF) and a wide spectrum of milder phenotypes called CFTR-Related Disorders (CFTR-RD) [ 4 , 5 ]. To date, more than 2100 sequence variations have been reported worldwide in the CFTR gene ( http://www. genet.sickkids.on.ca/ ). An efficient international network of geneti-cists, physicians and researchers have built the CFTR2 database that aims to determine the molecular impact and the severity of the most frequent CFTR variants identified in CF patients worldwide [START_REF] Castellani | CFTR2: how will it help care?[END_REF] . Complementarily, the CFTR -France database is a locus-specific database dedicated to rare CFTR variants [START_REF] Claustres | CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants[END_REF] that currently contains a total of 905 different variants, of which 70% are reported in five individuals or fewer. Among these variants, 442 are so-called mis-senses, of which 147 are classified as disease-causing, 4 as non-disease causing, but the majority ( n = 291) is still annotated as variants with unknown clinical significance (VUCS), due to the lack of available data and reliable resources. The functional classification (I-VI) [ 8 , 9 ] and the characterization of a minor ( i.e. , totally disrupted) or residual ( i.e. , partially disrupted) function of these rare variants now condition the patients' eligibility to highly effective modulator therapies.

Altered splicing consecutive to DNA changes in exonic regions is still widely under-evaluated and this neglected mechanism could change the eligibility to personalized CFTR modulators. In this study, after in silico analyses on 319 missense variants reported in the CFTR -France database, we selected 15 a priori missense variants for which bioinformatics tools suggest an impact of nucleotide changes on splicing of the CFTR gene ( Table 1 ). Here, we assessed their molecular impact with a combination of approaches: mini-gene studies, mRNA quantification, protein maturation and traffic ing, protein cellular localization and chloride channel function. We then proposed to classify the variants using the functional classification [ 8 , 9 ] and the clinical classification [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF] in regard with epidemiological data, as the two classification are of complementary interest, to decipher the severity of the variants and the molecular defect that can be targeted by the new therapies ( Fig. 1 ).

Methods

In silico predictions

We used Human Splicing Finder [START_REF] Desmet | Human Splicing Finder: an online bioinformatics tool to predict splicing signals[END_REF] , MaxEntScan [START_REF] Yeo | Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals[END_REF] and NNSplice [START_REF] Kulp | A generalized hidden Markov model for the recognition of human genes in DNA[END_REF] (https://www.fruitfly.org/seq _ tools/splice.html) to perform predictions on all variants reported in the CFTR -France database prior to the start of the project (data update: 2014). We focused on the predictions of acceptor and donor splice sites to select the variants that possibly have an impact on splicing of CFTR pre-mRNA [START_REF] Raynal | A classification model relative to splicing for variants of unknown clinical significance: application to the CFTR gene[END_REF] .

Cell cultures

BEAS-2B cells were cultured in Dulbecco's modified Ea-gle's medium (DMEM, Invitrogen). HeLa cells were cultured in DMEM + GlutaMAX (Invitrogen). Media were complemented with 10% fetal bovine serum (FBS) or 5% FBS and 1% Ultroser, and 1% penicillin/streptomycin. Cells were maintained at 37 °C in 5% CO 2 humidified atmosphere. For all experiments, cells were transfected with the WT and mutant constructs using the jetPEI®transfection reagent (Polyplus transfection) according to manufacturer's protocol. B) Functional classification of variants refers to the molecular defect observed or assumed ( e.g. , class I premature stop codons leading to the synthesis of a truncated protein and its early degradation) [ 8 , 9 ]. The nature of the molecular defect can be correlated to the residual CFTR function from 0% of WT for class I to higher than 30% for some class IV to V variants. In general, patients carrying two variants of class I to III exhibit a CF phenotype, related to the very low level of CFTR function compared to WT-CFTR. Patients carrying at least a variant of class IV to VI, with a residual CFTR function show, in general, milder clinical phenotypes.

Minigene assays

CFTR exons and their flanking intronic regions were introduced in pcDNA3.1 and pSPL3 minigene systems for splicing analysis. Mutants were obtained by directed mutagenesis with specific primers (Quick Change II Site-Directed Mutagenesis Kit, Agilent technologies). The wild-type (WT) and mutant constructs were transfected in BEAS-2B The splicing patterns were visualized on agarose gels and confirmed by Sanger sequencing after RT-PCR on total RNA from cells, as previously described [ 14 , 15 ].

CFTR cDNA expression plasmids

We used the expression vector pTracer-CMV containing the CFTR-wild-type (WT) cDNA (called pTracer-CFTR) [START_REF] Clain | Misprocessing of the CFTR protein leads to mild cystic fibrosis phenotype: misprocessing of cftr and mild phenotype[END_REF] . Single Nucleotides Variants (SNVs), that lead to the amino acid changes (so called "missense variants"), and deletions consecutive to in-frame splicing alterations (called "CFTR-delEx4", "CFTR-del57bpEx5"and "CFTR-delEx9") were generated by directed mutagenesis (Quick Change II Site-Directed Mutagenesis Kit, Agilent). The WT and mutants were transiently expressed in BEAS-2B (for mRNA quantification, western blot protein analysis and immunofluorescence (IF) assays) and HeLa cells (for western blot and patch-clamp protein analysis).

mRNA quantification

Quantitative PCR (qPCR) was performed using CFTR specific primers that hybridize on exon 21 and exons 22-23 boundary. Total cellular RNA was extracted from cells transfected with WT or mutants pTracer-CFTR using the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's instructions. For each transfection, two independent reverse transcriptions with 1 μg of total RNA were conducted using SuperScript®III First-Strand Synthesis System (Invitrogen). qPCR experiments were run in triplicate with 10 ng of cDNA using SYBR Green I PCR Master Mix on the Light Cycler 480 (Roche Diagnostics®). HPRT (Hypoxanthine Guanine Phosphoribosyltransferase) and beta-actin were used as reference genes and T84 cells endogenous CFTR expression was used as a calibrator for normalization. Relative differences in transcripts levels were quantified using the Ct method [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and the 2 -CT method[END_REF] .

Western blot

Western blotting was performed on protein lysates from cells transfected with WT or mutants pTracer-CFTR as previously de-scribed [START_REF] Viart | Transcription factors and miRNAs that regulate fetal to adult CFTR expression change are new targets for cystic fibrosis[END_REF]. Relative intensities of equal areas were compared using the Quantity One®analysis software (Bio-Rad). A ratio C/(C + B) between mutant and WT band intensities was used for Western Blot normalization, relative to laminA/C; B band corresponding to the Endoplasmic Reticulum (ER) sequestered immature CFTR and C band to the transgolgian or transmembrane mature CFTR.

Immunofluorescence

BEAS-2B cells were grown on coverslips, transfected with the WT or mutants pTracer-CFTR, and treated 24 h later as previously described [START_REF] Degrugillier | Phosphorylation of the Chaperone-Like HspB5 Rescues Trafficking and Function of F508del-CFTR[END_REF] . Acquisitions were performed with an Axio Imager 2 and the Zen 2012 software. Fluorescence was collected with a X40/0.75 planneofluar lens. The DAPI and A488/FITC fluorescence filter set were fitted to optimize data acquisition and limit bleedthrough. At least, ten representative immunostaining images were taken by transfection.

Patch clamp experiments

Cells were transfected with the WT or mutants pTracer-CFTR. Ionic currents were measured in the broken-patch, whole cell con-figuration of the patch clamp method as previously described [START_REF] Billet | Development of automated patch clamp technique to investigate CFTR chloride channel function[END_REF] . All experiments were conducted at room temperature (20-25 °C). Results are expressed as means ±standard error (SE) of n observations.

Chemicals

Forskolin (Fsk) and CFTRinh172 were purchased from Sigma. Stock solutions (10 mM) were prepared in dimethyl sulfoxide and used at a final concentration of 10 μM.

Statistical analysis

Patch Clamp data of CFTR mutants were compared to WT-CFTR using a one-way ANOVA followed by Dunnett's test with Graph-Pad Prism version 7.0 (GraphPad Software). Wilcoxon-test was performed for mRNA level and western blot data interpretation. Differences were considered significant for p values < 0.05.

Results

Five CFTR variants have a major impact on pre-mRNA splicing

Minigene studies showed that the c.523A > G (legacy name I175V) and c.1209G > C (E403D) variants led to aberrant splicing events, since no "full-length"transcripts were observed ( Fig 2A ). For c.523A > G, the reinforcement of an exonic cryptic donor splice site induced a skipping of the last 57-bp of the exon 5, leading to an in-frame deletion in CFTR transcripts. The variant c.1209G > C, located on the last nucleotide of the exon 9, led to an inframe skipping of the whole exon, due to the alteration of the natural donor splice site.

Major aberrant splicing was also observed for three other vari-ants: c.1679G > A (R560K) and c.2908G > C (G970R) that affect the last nucleotide of exon 12 and 17 respectively, and the c.2909G > T (G970V), located on the first nucleotide of exon 18 ( Fig 2B ). They all lead to an out-of-frame deletion of the corresponding exon in CFTR transcripts, suggesting that these three mutants would never lead to amino acid substitution. Nevertheless, as data obtained in vitro should be considered with caution considering quantitative aspects, few residual full-length transcripts, carrying the amino acid substitution, could remain in vivo . We thus performed additional experiments to determine a possible additional impact of all missense variants on protein synthesis, trafficking and activity. We also assessed processing, cellular localization and activity of protein lacking 19 and 31 amino acids, due to c.523A > G (exon 5) and c.1209G > C (exon 9) respectively. Of note, all the SNVs tested and in-frame deletions constructs CFTR-delEx4 and CFTR-del57bpEx5 showed no decrease of CFTR mRNA. Even if minor changes in mRNA quantity may not be detected, or may be conditioned by the cellular background of the heterologous system, we observed that the CFTR-delEx9 construct was associated to a low level (about 50% compared to wild-type) of the shorter aberrant transcript (Suppl. Fig. S1). These results suggest that CFTR-delEx4 and CFTR-del57bpEx5 rather had an impact on protein synthesis or maturation while absence of the entire exon 9 might additionally affect mRNA quantity.

We did not observe any impact on protein synthesis and activity of the mutant proteins carrying I175V and E403D (missense variants). Even if I175 and E403 are two highly conserved amino acids among orthologs, the absence of impact was suspected by the similarity of physicochemical properties of the WT and mutant amino acids in the Venn Diagram and the absence of deleterious effect predicted by the three-dimensional model [START_REF] Sasorith | The CYSMA web server: an example of integrative tool for in silico analysis of missense variants identified in Mendelian disorders[END_REF] . As expected, the two shorter proteins resulting from the aberrant splicing, CFTR-del57bpEx5 and CFTR-delEx9, were not correctly folded (no C band in Western Blot (WB)) (Fig 2A). Of note, IF assays revealed some discrepancies with the results obtained by WB for 2 conditions, CFTR-del57bpEx5 and E403D, with respectively 18% and less than 10% of cells with labeling at the plasma membrane (Fig 2A). However, patch-clamp experiments revealed no chloride channel activity in cells transfected by CFTR-del57bpEx5 and CFTR-delEx9 (containing in-frame deletions), whereas full channel function was conserved for I175V and E403D mutant proteins ( Fig 2C and Suppl. Fig. S3). Thus, the two variants c.523A > G and c.1209G > C may be considered as splicing variants leading to immature proteins and should be classified in the class II of CFTR variants (Table 1).

The amino acid substitutions (missense variants) R560K, G970R and G970V, consecutive to the variants c.1679G > A, c.2908G > C and c.2909G > T respectively, impaired protein folding and/or function. While the R560K induced a complete folding defect of the mutant CFTR, the G970R and G970V rather led to a gating defect without affecting folding of the mutant protein ( Fig 2B ). The total disruption of the channel function in vitro was previously demonstrated for the G970R variant [ 22 , 23 ]

B) Variants c.1679G > A (R560K), c.2908G > C (G970R) and c.2909G > T (G970V).

C) Results of patch clamp experiments for missense variants I175V, E403D, M469V, R560K, G970V and G970D, and the in-frame deletions constructs CFTR-del57bpEx5 and CFTR-delEx9 ( * p < 0.05 and * * p < 0.01 denote statistical significance compared to the WT chloride transport CFTR activity). Note: G970R was not assessed as similar functional assays were available in previous studies [ 22 , 23 , 32 ].

D) Variants c.1405A > G (M469V) and c.2909G > A (G970D)

C) Structural representation of the wild-type E92 (left) and the E92K mutation (right).

The views are centered on the 92 position, highlighted with the yellow label. Residues located at less than 5 ˚A are labeled and depicted with their full side chains. Residues involved in the salt bridge (K95) and the H-bond (Q353) are labeled in blue. The wildtype E92 is forming a salt bridge with K95 (red dotted line) and a hydrogen bond with Q353 (black dotted line). E92K mutation is predicted to cause the loss of the salt bridge with K95.

Thus, the three variants c.1679G > A, c.2908G > C and c.2909G > T, should be clearly classified as class V variants due to the out-of-frame aberrant splicing, with insufficient protein synthesis. The experiments performed on R560K, G970R and G970V missense variants showed an impact on protein folding (R560K) or activity (G970R and G970V).

However, if residual full-length transcripts might theoretically be present and translated into variant proteins in vivo, these results are not representative of the major pathogenic mechanism of the nucleotide changes ( i.e. , splicing alteration leading to NMD).

Two variants are exclusive missense variants with an impact on cftr folding and channel function

No significant alteration splicing was observed for the c.1405A > G (M469V) and c.2909G > A (G970D) variants ( Fig 2D ) that can be considered as "authentic" missense variants. The variant M469V mainly impacts the folding of the CFTR protein, with a very weak detection of band C in WB experiments and no measured activity, that sorted it in the class II with minor function. The variant c.2909G > A, located at the first nucleotide of the exon 18, led to the deletion of exon 18 (predicted out-of-frame) in less than 50% of transcripts in the pcDNA3.1 construct only. We also observed a small amount of shorter transcripts in the WT construct ( Fig 2D ). That could be explained by the differences in the strength of the splice sites or the flanking exons used in the two different plasmids. Aberrant transcripts were not visible by agarose gel in the pSPL3 mutant construct but a very small proportion of transcripts lacking the exon 18 was detected by cDNA sequencing, but was considered not-significant. The full-length transcripts containing the c.2909G > A encode for the G970D mutant protein that shows a residual CFTR function, as the channel activity is partially altered without any significant decrease of the mature CFTR ( Fig 2C , 2D and Suppl. Fig. S3). This variant can be classified in the class IV. The minor splicing alteration observed in pcDNA3.1 minigenes should be confirmed by the direct study of transcript patterns from nasal epithelial cells of the patients carrying the c.2909G > A variant.

One variant may have a combined impact on splicing and CFTR folding

The c.274G > A variant, located at the first nucleotide of the exon 4, led to the in-frame deletion of exon 4 in half of the CFTR transcripts in the two minigene systems used in this study ( Fig 3A ). This is concordant with previous observations for another variant at the same position, c.274G > T, that was first considered as a non-sense variant (E92X), but was secondarily reclassified as a splicing variant after the evaluation of transcripts patterns in nasal epithelial cells of a patient [START_REF] Will | A novel exon in the cystic fibrosis transmembrane conductance regulator gene activated by the nonsense mutation E92X in airway epithelial cells of patients with cystic fibrosis[END_REF]. This is concordant with our in vitro results and suggests that this position is critical for the recognition of the acceptor splice site of exon 4. Both E92K and E92-delEx4 mutants blocked the maturation of the protein and the in vitro channel activity was consecutively about null ( Fig 3A , 3B and Suppl. Fig. S3). Regarding the 3D structural model [START_REF] Sasorith | The CYSMA web server: an example of integrative tool for in silico analysis of missense variants identified in Mendelian disorders[END_REF], the amino acid Glu92 is involved in the pore construction and participates in a salt bridge ( Fig 3C ), that may result in a gating defect if a very low proportion of mature protein could be addressed to the plasma membrane in vivo . The variant E92K can therefore concomitantly be considered as a class II variant, due to the splicing defect and the amino acid substitution, and possibly class III of the functional classification.

Seven variants showed no deleterious consequence in vitro

The in vitro experiments performed on the variants R55K, R74Q, T351S, I918M, G1069R, R1070Q and A1364V did not show any deleterious impact on splicing, mRNA expression level, CFTR protein folding and chloride secretion (Suppl. Figs. S2 andS3).

Discussion

The clinical classification of variants is essential to refine genotype-phenotype correlation (CF or CFTR-RD), guide the diagnosis and propose appropriate follow-up to patients [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF] . For the rarest variants, collecting clinical data from patients may be challenging and do not allow to determine the class to which they be-long. In our study, the results of functional assays associated to clinical data from CFTR -France and other available descriptions in the CFTR2 database or in the literature allowed us to refine the phenotypic spectrum of the 15 studied variants. Their clinical and functional classifications ( [START_REF] Boyle | A new era in the treatment of cystic fibrosis: correction of the underlying CFTR defect[END_REF][START_REF] Strub | Transcriptomic and proteostasis networks of CFTR and the development of small molecule modulators for the treatment of cystic fibrosis lung disease[END_REF][START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF] and Fig. 1) based on the data collected by our study are detailed in Table 1 and summarized in Fig. 4 .

Among variants that disrupt CFTR processes in vitro, the classification as CF-causing (class A) was evident for 6 variants, while variants E92K and M469V could be associated with a large spectrum of disorders from CF to CFTR-RD ( i.e. , class A/B). It was previously reported that CF patients with E92K presented milder phenotypes than other CF patients, with lower sweat test values, no pancreatic insufficiency in at least 50% of patients and less Pseudomonas lung colonization [START_REF] Castellani | CFTR2: how will it help care?[END_REF] . CFTR-RD phenotypes have been described in CFTR -France in two patients carrying c.274G > A (E92K) and a CF-causing variant on the second allele. However, we demonstrated that the variant c.274G > A (E92K) may disrupt three molecular CFTR processes: splicing and protein folding, as shown by a recent study [START_REF] Petrova | Spectrum of CFTR mutations in Chechen cystic fibrosis patients: high frequency of c.1545_1546delTA (p.Tyr515X; 1677delTA) and c.274G>A (p.Glu92Lys, E92K) mutations in North Caucasus[END_REF] , but also possibly CFTR function, according to 3D modeling ( Fig. 2C ). It would result in a major functional defect, which is in accordance with other observations [ 6 , 26 ]. Discrepancies between in vitro assays and clinical data were also raised for the variant M469V, for which functional experiments suggested a major pathogenic effect whereas 3 informative patients were reported with a CFTR-RD phenotype, rather suggesting a residual synthesis and activity of the chloride channel ( e.g. , one CBAVD with trans -segregation of a CF-causing variant in CFTR -France). For these two variants, for which in vitro data suggest a more severe impact than the phenotype observed in patients, we could hypothesize that a small fraction of fulllength mutant proteins with some residual function, not detected in vitro , may remain in vivo and reach the plasma membrane in some organs. This could be consistent with the high response of E92K to CFTR modulator therapy that combines VX-809 and VX-770 [START_REF] Ensinck | Phenotyping of rare CFTR mutations reveals distinct trafficking and functional defects[END_REF] CFTR chloride channel function was similar to the WT for R55K, R74Q, T351S, I918M, G1069R, R1070Q and A1364V. As the whole cell patch-clamp assays would have detected a reduced membrane stability of the protein, we concluded that these 7 variants had no impact on gating, chloride conductance, and CFTR stability at the membrane. Thus, these variants clearly do not cause CF, but may present with an incomplete penetrance possibly resulting in a CFTR-RD or no disease were classified as B/C. As bicarbonate secretion was not assessed by our patch-clamp experiments, we can't exclude an impact of these variants on bicarbonate transport activity of the mutant proteins [ 27 , 28 ]. We cannot exclude either the presence of a cryptic disease-causing variant, not detected by the current techniques, that could be in complex allele with the variants, to explain patient's phenotype as previously shown for other variants [ 7 , 29 ]. The R1070Q, was mostly reported in complex allele ( cis -segregation) with the CF-causing variant p.Ser466 * in CF patients but reported alone in milder phenotypes [START_REF] Krasnov | Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships[END_REF]. Despite the absence of functional evidence in favor of the pathogenicity of T351S, G1069R and A1364V, combined with the relative high frequency in the healthy population from specific origins (Variant Allele Frequency of 0.0 0 01 in European and Latino populations for T351S and A1364V respectively), they were also classified as B/C, as they were reported in patients with CFTR-RDs in combination with a second CFTR pathogenic variant (CF or CFTR-RD causing).

In conclusion, our study emphasizes the importance of assessing the impact on splicing of exonic variants to decipher their pathogenic mechanism, which conditions the efficiency of CFTR therapies in vivo [START_REF] Joynt | Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies[END_REF], as illustrated for the G970R, incorrectly considered as a gating mutation [ 23 , 32 ] and well summarized in a very recent communication from Raraigh et al ., [START_REF] Raraigh | Caution advised in the use of CFTR modulator treatment for individuals harboring specific CFTR variants[END_REF] in which the data discussed are in accordance with our results.

Nevertheless, the efficacy of CFTR modulators on splicing alterations that maintain in-frame transcripts should be assessed, as a shorter misfolded protein was detected in vitro . Further experiments on materials derived from patients to confirm the splicing defects observed in vitro as well as testing the efficacy of modulators on primary cultures from the patients carrying those variants must be performed and could support the use of these therapies in previously ineligible patients. Finally, future developments in CF targeted therapy should include splicing modulators that could, in association with correctors and potentiators, correct the basic defect due to out-of-frame splicing alterations related to numbers of variants.

Fig. 1 .

 1 Fig. 1. Two different and complementary classifications for CFTR variants. A) Clinical classification according to the international guidelines of Castellani et al., 2008 [10] . This classification refers to the clinical phenotypes observed in patients carrying the variant of interest and a CF-causing variant in trans ( i.e. , on the other allele): (i) Variants identified only in CF patients are in class A, (ii) variants identified only in CFTR-RD patients are in class B, (iii) variants identified both in CF patients and patients with a CFTR-RD are in class A/B, (iv) variants identified in patients with a CFTR-RD and in healthy individuals are in class B/C, (v) variants identified in healthy individuals and/or with a high frequency in the general population are in class C. Variants of uncertain significance cannot, by definition, be classified in the other classes and belong to class D.

  and our patch clamp experiments showed that the variant G970V conserved a residual function ( i.e. , very low activity) ( Fig 2C and Suppl. Fig. S3).

Fig. 2 .

 2 Fig. 2. Results of the functional experiments in BEAS-2B and/or HeLa cell lines. A)Variants c.523A > G (I175V) and c.1209G > C (E403D).

  From the top to the bottom: pcDNA3.1 and pSPL3 minigene experiments in the BEAS-2B cell line with the schematic representation of aberrant splicing events, representative western blot experiments (molecular weights: B band approximately 131 kDa and C band approximately 160 kDa, lamin A/C used as loading controls) and immunofluorescence assays in HeLa cells.

Fig. 3 .

 3 Fig. 3. Results of the functional experiments in BEAS-2B and/or HeLa cell lines for the variant c.274G > A (E92K). A) From the top to the bottom: pcDNA3.1 and pSPL3 minigene experiments in the BEAS-2B cell line with the schematic representation of aberrant splicing events, representative western blot experiments (molecular weights: B band approximately 131 kDa and C band approximately 160 kDa, lamin A/C used as loading controls) and immunofluorescence assays in HeLa cells for E92K and the in-frame deletion CFTR-delEx4.

Fig. 4 .

 4 Fig. 4. Summary of the functional consequences of the CFTR variants studied and their related functional classification. * Insufficient to no protein synthesis depending on the amount of exon skipping and NMD in vivo (not quantified in this study). * * No deleterious impact in vitro on splicing, CFTR protein synthesis or folding and chloride secretion.

Acknowledgments

We are grateful to the French Association against cystic fibrosis (Vaincre la Mucoviscidose) for its invaluable support and sustained grants for this project and for the CFTR -France database. We ac-knowledge the members of the research teams who implemented the various functional tests in the collaborating laboratories. We thank members of the technical teams for CF molecular diagnosis in all collaborating laboratories and the clinicians who initially referred the patients.

Web Sites

CFTR2 Database -CFTR2, Clinical and Functional Translation of CFTR: https://www.cftr2.org/ (accessed April 13, 2022). CFTR-France database -CFTR-France: https://cftr.iurc.montp. inserm.fr/cftr/ (accessed April 13, 2022).

Conflict of Interest Statement

The authors declare no conflicts of interest.