MD Pascale Fanen
email: pascale.fanen@inserm.fr

Adeline Wohlhuter-Haddad

Alexandre Hinzpeter

Genetics of Cystic Fibrosis: CFTR Mutation Classifications towards Genotype-Based CF Therapies

Keywords: CFTR, mutation, CFTR-RD, therapy, Variants of Unknown significance CBAVD, congenital bilateral absence of the vas deferens, CF, cystic fibrosis, CFTR, Cystic Fibrosis Transmembrane conductance Regulator, CFTR-RD, CFTR related disease, COPD, chronic obstructive pulmonary disease, EJC, exon junction complex, NBD, nucleotide-binding domains, NGS, next generation sequencing, NPD, nasal potential difference measurements, NMD, Nonsense-Mediated mRNA Decay, PABP, Poly(A)-binding protein, PI, pancreatic insufficiency, PTC, premature termination codon, PS, pancreatic sufficiency, VUs,

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel.

Since the identification of the disease in 1938 and up until 2012, CF patients have been treated exclusively with medications aimed at bettering their respiratory, digestive, inflammatory and infectious symptoms. The identification of the CFTR gene in 1989 gave hopes of rapidly finding a cure for the disease, for which over 1,950 mutations have been identified. Since 2012, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly or its key processing steps, giving rise to novel promising therapeutic tools. This review presents the current CFTR mutation classifications according to their clinical consequences and to their effect on the structure and function of the CFTR channel. How these classifications are essential in the establishment of mutation-targeted therapeutic strategies is then discussed. The future of CFTR-targeted treatment lies in combinatory therapies that will enable CF patients to receive a customized treatment.

Introduction

Mutations disrupting the function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene, also referred to as ABCC7, cause Cystic Fibrosis (CF), a severe autosomal recessive disease. CF affects 1 in 2,500 to 4,500 newborns among the Caucasian population. All other ethnic groups are affected to a lesser extent. In 1989, Francis Collins, Lap-Chee Tsui and John R. Riordan [START_REF] Kerem | Identification of the cystic fibrosis gene: genetic analysis[END_REF] identified the CFTR gene and its most frequent mutation, F508del, as being the cause of CF. The CFTR gene is located on the long arm of chromosome 7 (7q31.2) and its structure comprises 27 exons, spanning over 190kb (Fig. 1A). After splicing of the large introns, the CFTR mRNA is 6.5kb and all of its exons are necessary to produce a functional CFTR protein.

Since 1990, the laboratories dedicated to CF genetic diagnosis and research worldwide have been grouped into the international Cystic Fibrosis Genetic Analysis Consortium (CFGAC). This consortium identifies and describes mutations of the CFTR gene. Inputs are then deposited on the CF mutation database "CFTR1" (http://www.genet.sickkids.on.ca/cftr/) for public access. Since the discovery of the CFTR gene, extensive sequencing enabled the identification of over 1,950 different mutations. Recently, a second CF mutation database "CFTR2" for "Clinical and Functional Translation of CFTR", has been created (http://www.cftr2.org/). It gathers up-to-date information about newly discovered CFTR gene mutations, classifies them as disease-causing, neutral or mutation of varying clinical consequences, and provides clinical information about specific mutation combinations.

To better our understanding of CF pathogenesis, as well as to facilitate diagnosis and choice of treatment, two classification systems for CFTR mutations have been created, one according to clinical consequences, the other according to CFTR structure-function assessment. Currently available CF therapies mainly treat the symptoms of the disease, and have increased the mean life expectancy of CF patients from 5 years in the 1970s to about 35 to 40 years of age today. To address the underlying cause of the disease, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly, or its key processing steps. These novel therapeutic tools correct specific defects responsible for CFTR protein loss-of-function and need to be adapted to each patient's genotype. This highlights the importance of thoroughly characterizing each patient at the molecular level. This review first presents the clinical spectrum of Cystic Fibrosis, as well as the two existing classification systems for CFTR mutations. How CFTR mutations are assigned to CF patients is then described. The use of this essential information in the development of genotype-based therapeutic strategies is then discussed.

Classification of CFTR mutations

The clinical spectrum of CF

The first description of the disease was done in 1938 by Dorothy Hansine Andersen, describing an abnormal pancreas, which presented with cysts and fibrosis [START_REF] Andersen | Cystic fibrosis of the pancreas and its relation to celiac disease[END_REF]. The clinical spectrum of CF has since greatly expanded, giving rise to diagnoses of classic and nonclassic CF presented in Figure 2 (De [START_REF] Boeck | Cystic fibrosis: terminology and diagnostic algorithms[END_REF][START_REF] Farrell | Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report[END_REF].

Although classic CF most often presents with a severe multi-organ phenotype, and non-classic CF with milder single-organ phenotypes, this is not always the case. Whether the phenotype is "severe/mild" or "multi/single-organ" is not intrinsically linked to the classic or non-classic CF diagnosis. These are subjective terms, which should be used for descriptive purposes exclusively.

The great phenotypic variability of CF has been shown to implicate not only the type of CFTR mutations, but also other genetic factors such as modifier genes, (see chapter from Guillot and Corvol in this special issue) and environmental factors (lifestyle, treatment) (De [START_REF] Boeck | Cystic fibrosis: terminology and diagnostic algorithms[END_REF]. In the diagnostic algorithms proposed by the European Consensus Group, patients with one or more phenotypic characteristic suggestive of CF are classified according to their pilocarpine sweat test result (chloride >60 mmol/L, 30-60 mmol/L or <30 mmol/L) (Castellani et al., 2008). To date, no test has proven to be as practical or reliable as the sweat test for clinical diagnostic purposes.

Classic CF -The majority of CF patients suffer from classic CF, yet their organs are affected to varying degrees. Patients are diagnosed with classic CF if they have one or more phenotypic characteristic and a sweat chloride concentration of >60 mmol/L (Fig. 2). They may have exocrine pancreatic insufficiency (PI) or pancreatic sufficiency (PS). The disease can have a severe course with rapid progression of symptoms or a milder course with very little deterioration over time. Classic CF patient mortality is mainly due to progressive respiratory disease. From a genetic standpoint, classical CF is characterized by one established CF-causing mutation on each CFTR allele.

Non-classic CF -Non-classic CF describes individuals with at least one CF phenotypic characteristic and a normal (<30 mmol/L) or borderline (30-60 mmol/L) sweat chloride level, in whom detection of one mutation on each CFTR allele, or direct quantification of CFTR dysfunction by NPD measurement has been confirmed [START_REF] Boyle | Nonclassic cystic fibrosis and CFTR-related diseases[END_REF][START_REF] Rosenstein | Nonclassic cystic fibrosis: a clinical conundrum[END_REF] (Fig. 2). Non-classic CF patients have either multi-or single-organ involvement. Most of them have exocrine PS and milder lung disease. As described by the CF Diagnostic Working Group, "some patients with single-organ involvement resulting from CFTR dysfunction may be more appropriately given an alternative diagnostic label, as recommended in the World Health Organization diagnostic list" [START_REF] Who | Classification of cystic fibrosis and related disorders. Report of a joint WHO/ICF(M)[END_REF]. These alternative diagnoses for non-classic CF, or CFTR-related disorders, include isolated obstructive azoospermia, chronic pancreatitis, disseminated bronchiectasis, allergic bronchopulmonary aspergillosis, diffuse panbronchiolitis and sclerosing cholangitis, and all have at least one identified associated CFTR mutation, some of which are discussed hereafter.

Classification of CFTR mutations based on clinical consequences

In 2007, the Consensus Conference, organized by the European Cystic Fibrosis Society with the partnership of the European Society of Human Genetics and the EuroGentest Network of Excellence, allowed for the establishment of a classification of CFTR mutations, into the following four groups, according to their clinical consequences (Table 1). Group A comprises mutations that cause classic CF (CF-causing), Group B includes mutations that cause non-classic CF (associated to CFTR-related disorders), Group C comprises mutations with no known clinical consequences, and Group D consists of mutations with unknown or uncertain clinical relevance (also referred to as VUs) [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF].

Only a few of the hundreds of CFTR mutations identified to date have been shown to result in classic CF. Within a given ethnic group, specific mutations may be more frequent than others.

Most CF patients are Caucasian, however, other ethnicities are also affected, and p.Phe508del remains the most frequent classic CF mutant allele, accounting for approximately 70% of worldwide chromosomes [START_REF] Lucotte | Complete map of cystic fibrosis mutation DF508 frequencies in Western Europe and correlation between mutation frequencies and incidence of disease[END_REF]. As more and more patients present with nonclassic phenotypes, DNA screens for common CFTR mutations have become essential for CF diagnosis. To that effect, the American College of Medical Genetics (ACMG) has recommended a core panel of 23 mutations to assess CF risks in diagnosis, carrier screening and sequencing analyses (Table 2). These 23 mutations fall into Group A, as classic CF-causing mutations, with the exception of p.Arg117His, which is discussed later in a paragraph dedicated to complex alleles. This core panel may be complemented with the screening of additional ethnic-specific mutations found in European and/or other selected population. Some mutations may be associated with different clinical presentations. Groups A and B are therefore not mutually exclusive. For example, the p.Asp1152His mutation may result in isolated congenital bilateral absence of the vas deferens (CBAVD), or in a fully expressed CF lung disease with pancreatic sufficiency. Non-classic CF disorders have a pathophysiology that may be attributed to CFTR defect, as these patients are characterized by a prevalence of CFTR mutation higher than that expected in the general population.

Group D mutations have unknown or uncertain clinical relevance to CF phenotypic characteristics. It is often difficult to assess the association between CFTR mutations and clinical entities that, in some cases, may have other non-CFTR related aetiologies. Other factors, such as the environment, modifier genes and the progression of the disease with age, can also affect the clinical heterogeneity of patients carrying these large spectrum mutations.

It is important to note that CFTR genotype is not a useful predictor of the severity of lung disease in the individual patient, and should not be used as an indicator of prognosis (Castellani et al., 2008).

Classification of CFTR mutations based on CFTR structure-function

Five major classes of CFTR mutations were created according to their consequences on CFTR function [START_REF] Welsh | Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis[END_REF]: mutations interfering with protein synthesis, mutations affecting protein maturation, mutations altering channel regulation, mutations affecting chloride conductance, and mutations reducing the level of normally functioning CFTR at the apical membrane [START_REF] Welsh | Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis[END_REF] (Fig. 3). A sixth class consisting of mutations decreasing the stability of CFTR present at the plasma membrane or affecting the regulation of other channels was proposed, but has been poorly investigated [START_REF] Haardt | C-terminal truncations destabilize the cystic fibrosis transmembrane conductance regulator without impairing its biogenesis. A novel class of mutation[END_REF]. These class VI mutations are now combined with class V, as mutations leading to a reduced amount of functional CFTR protein [START_REF] Welsh | Cystic fibrosis[END_REF].

Class I: mutations preventing the production of a full-length CFTR protein

Class I mutations result in the total or partial lack of production of a functional CFTR protein (Fig. 3). Such mutations may arise either due to (1) a nucleotide substitution introducing an inframe premature termination codon (PTC) -UAA, UAG or UGA-, (2) frame-shifting insertions or deletions, (3) mutations at the invariant dinucleotide splice junctions, or introduction of a PTC, resulting in complete skipping of an out of frame exon (Fig. 1B), (4) a complete or partial deletion of the CFTR gene or (5) a rearrangement in the gene altering the exon sequence.

Mutations that generate PTCs can reduce the steady-state level of mRNA via nonsense-mediated decay (NMD), which degrades the abnormal PTC-containing mRNA [START_REF] Maquat | When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells[END_REF]. Such a phenomena has been shown with p.Gly542*, p.Arg553*, p.Trp1282*, and one frameshift mutation (p.Phe316LeufsX12), but a notable exception is p.Arg1162*, which produces a normal CFTR mRNA level as compared to wild-type CFTR [START_REF] Will | Transcript analysis of CFTR nonsense mutations in lymphocytes and nasal epithelial cells from cystic fibrosis patients[END_REF].

The p.Gly542* mutation is the most frequent worldwide mutation of its class, affecting at least one allele of up to 4% of CF patients. Other mutations reach a higher frequency in specific populations, due to a founder effect; for example 48% of all alleles in Ashkenazi Jews in Israel carry the p.Trp1282* mutation and 24% carry the p.Tyr122* mutation in the French Reunion Island [START_REF] Dugueperoux | Cystic fibrosis at the Reunion Island (France): spectrum of mutations and genotype-phenotype for the Y122X mutation[END_REF][START_REF] Shoshani | Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease[END_REF].

Class II: mutations altering the cellular processing of the protein

Class II mutations are associated with defective processing due to misfolding of the protein (Fig. 3) and may be found within any domain of the CFTR protein. The misfolded protein is retained in the Endoplasmic Reticulum (ER), retro-translocated to the cytoplasm, where it is degraded by the ubiquitin/proteasome pathway [START_REF] Jensen | Multiple proteolytic systems, including the proteasome, contribute to CFTR processing[END_REF][START_REF] Ward | Degradation of CFTR by the ubiquitin-proteasome pathway[END_REF]. CFTR maturation through the cellular compartments can be assayed by western blot. The coreglycosylated CFTR (band B) migrates at a lower molecular weight than the fully-glycosylated CFTR protein (band C). Depending on the mutation, one may observe either a partial reduction (p.Leu206Trp, third transmembrane segment) or a complete absence (p.Arg1066Cys, fourth intracellular loop or p.Phe508del, NBD1) of mature CFTR. The most frequent CF mutation, p.Phe508del, has been well studied. It has been shown that p.Phe508del leads to energetic and kinetic instability of the NBD1 domain due to improper local folding [START_REF] Protasevich | Thermal unfolding studies show the disease causing F508del mutation in CFTR thermodynamically destabilizes nucleotide-binding domain 1[END_REF].

Additionally, CFTR domain assembly is altered, namely the interaction between NBD1 and the fourth cytoplasmic loop (CL4) [START_REF] Cui | Domain interdependence in the biosynthetic assembly of CFTR[END_REF][START_REF] He | Restoration of domain folding and interdomain assembly by second-site suppressors of the DeltaF508 mutation in CFTR[END_REF]. Correction of both NBD1 energetics and interface instability is therefore required to restore p.Phe508del processing [START_REF] Mendoza | Requirements for efficient correction of DeltaF508 CFTR revealed by analyses of evolved sequences[END_REF][START_REF] Rabeh | Correction of both NBD1 energetics and domain interface is required to restore DeltaF508 CFTR folding and function[END_REF]. Initially, mutations belonging to this class were thought to cause severe CF similarly to p.Phe508del or p.Arg1066Cys, but p.Leu206Trp was shown to be associated with variable phenotype (Clain et al., 2005a).

Class III: mutations disturbing the regulation of the Cl -channel

These mutations are frequently located in the ATP binding domains (NBD1 and NBD2) and are referred to as gating mutations (Fig. 3). They are missense mutations producing a protein efficiently inserted in the membrane at normal levels, but resistant to activation by protein kinase A. The main example is p.Gly551Asp, which abolishes ATP-dependent gating, resulting in an open probability that is ~100-fold lower than that of the wild type channel [START_REF] Anderson | Regulation by ATP and ADP of CFTR chloride channels that contain mutant nucleotide-binding domains[END_REF]. Other class III mutations such as the frequent mutations p.Arg560Thr or p.Gly970Arg are listed in Figure 3 [START_REF] Seibert | Cytoplasmic loop three of cystic fibrosis transmembrane conductance regulator contributes to regulation of chloride channel activity[END_REF]. Noteworthy, this latter mutation is located in the third cytoplasmic loop (CL3) of the protein, highlighting the fact that knowing the localization of the amino acid substitution is insufficient to predict the resulting CFTR structural and functional anomalies.

Class IV: mutations altering the conduction of the Cl -channel

These mutations are mostly located within membrane spanning domains implicated in the constitution of the channel pore (Fig. 3). The missense mutations located in these regions produce a protein efficiently inserted in the membrane, which retains a cAMP-dependent Cl -channel activity, but with a reduced channel conductance. Alleles in this class are typically associated with a milder form of the disease. The p.Arg117His mutation is the best-characterized class IV mutation. While this mutant is correctly processed and generates cyclic AMP-regulated apical Cl - currents, patch-clamp analysis demonstrated a reduction in both channel open probability and conductance [START_REF] Reddy | Selective activation of cystic fibrosis transmembrane conductance regulator Cl-and HCO3-conductances[END_REF]Quinton, 2001, Sheppard et al., 1993).

Recently, a subclass IVb (b stand for bicarbonate) has been proposed for mutations affecting CFTR bicarbonate conductance specifically, without reducing its chloride conductance [START_REF] Schneider | Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis[END_REF]. As of today, only a few of such mutants have been identified, namely p.Arg75Gln and p.Ile148Thr [START_REF] Choi | Aberrant CFTR-dependent HCO3-transport in mutations associated with cystic fibrosis[END_REF][START_REF] Schneider | Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis[END_REF]. While p.Ile148Thr appears not to be deleterious [START_REF] Claustres | Are p.I148T, p.R74W and p.D1270N cystic fibrosis causing mutations?[END_REF], p.Arg75Gln was found in some studies at a higher frequency in cohorts of patients presenting CFTR-RD [START_REF] Divac | High frequency of the R75Q CFTR variation in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Schneider | Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis[END_REF]. Other mutations affecting the CFTR bicarbonate conductance are bound to be identified, probably in patients presenting with a CFTR-RD.

Class V: mutations reducing the amount of functional CFTR protein

Most of the class V mutations reduce the total amount of CFTR protein by affecting pre-mRNA splicing (Fig. 3). These splice site mutations can induce complete or partial exclusion of an exon. In the latter case, production of normal mRNA is maintained, but in lower quantity.

When the skipped exon is in phase (Fig. 1A), the transcript will produce an incomplete nonfunctional channel. On the other hand, when the skipped exon is out of phase, a PTC will rapidly appear and the transcript will be degraded by the NMD pathway. Classically, splicing mutations are located in the introns close to the splice sites, where they alter key splicing signals implicated in proper exon recognition (acceptor site, donor site, branch point or polypyrimidine tract). Yet, a growing number of them are being identified within exons. These exonic nucleotide substitutions can induce exon skipping by either disrupting Exonic Splicing Enhancer (ESE) or creating Exon Splicing Silencer (ESS) motifs [START_REF] Pagani | Genomic variants in exons and introns: identifying the splicing spoilers[END_REF]. Since direct RNA analysis is not routinely performed, the number of mutations causing splicing defects is underestimated.

The most frequent and well-studied is the skipping of exon 10 (formerly named exon 9). The presence or absence of this exon is correlated with a polymorphism within the polypyrimidine tract located upstream of the acceptor splice site (Poly-T tract). While a Poly-T tract of 7T or 9T ensures up to 90% of normal splicing, the shorter 5T Poly-T tract allows no more than 10 to 40% of normal mRNA to be produced [START_REF] Chu | Genetic basis of variable exon 9 skipping in cystic fibrosis transmembrane conductance regulator mRNA[END_REF].

Complex alleles and multi-class mutations

Some CFTR alleles have the particularity of harbouring two distinct mutations. They are referred to as complex alleles. The two mutations can induce additive defects, leading to a more severe phenotype than would each mutation separately [START_REF] Clain | Two mild cystic fibrosisassociated 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], Clain et al., 2005b).

This can be illustrated by the class IV R117H mutation (c.350G>A, p.Arg117His) whose severity is modulated in cis by the 5/7/9T polypyrimidine tract (c.1210-12T(5_9)in intron 9. While the R117H-T7 genotype is associated with milder forms of CF such as CBAVD, and most of the time even absence of symptoms, the R117H-T5 can be identified in patients having elevated sweat chloride and clinical cystic fibrosis, which in some cases is severe [START_REF] Thauvin-Robinet | The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening[END_REF].

Finally, some point mutations have been shown to alter multiple distinct processes, such as splicing and routing or maturation and channel conductance. This is for example the case of the p.Phe508del mutation, which presents, in addition to a processing defect, a gating defect characterized by a lower open probability of the channel and a reduced stability of the protein at the cell surface due to an increased recycling rate [START_REF] Dalemans | Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation[END_REF].

Assigning CFTR mutations to CF patients: a methodological perspective

As of today, only a minority of the 1950 known mutations have been analyzed at the protein level [START_REF] Ferec | Assessing the Disease-Liability of Mutations in CFTR[END_REF] and even fewer at the mRNA level. Studying the DNA/RNA obtained from clinically characterized CF patients has led to the identification of their mutations.

With the upcoming introduction of NGS, the number of identified VUs will be greatly increased [START_REF] Abou Tayoun | A comprehensive assay for CFTR mutational analysis using next-generation sequencing[END_REF][START_REF] Trujillano | Next generation diagnostics of cystic fibrosis and CFTR-related disorders by targeted multiplex high-coverage resequencing of CFTR[END_REF]. The labelling of these VUs, as diseasecausing or as neutral (Group C) mutations, will be challenging. Presented hereafter are some guidelines and available tools enabling such a distinction.

One such tool is the CFTR2 database, initiated to establish disease liability of individual CFTR mutations, as well as to determine the relationships between specific mutations and symptoms of CF. This project (http://www.cftr2.org/) has compiled clinical data from nearly 40,000 CF patients and relates the clinical phenotype (pancreatic status, lung function, pulmonary infection) to the electrophysiological phenotype (sweat chloride and ion transport in epithelial cells transfected with the corresponding mutated CFTR). Among the 1221 mutations reported so far on the CFTR2 database, 190 mutations have already been characterized. For the remaining mutations, indirect evidence of pathogenic potential may be derived from the following (adapted from [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF]):

1) in vivo assessment of abnormal CFTR-mediated chloride transport, as assessed by sweat test or NPD and ex vivo assessment by short circuit measurements using Ussing chambers (nasal brushing or rectal biopsy),

2) molecular assessment of sequence variations CFTR mutations with high confidence for pathogenic potential:

• change in an amino acid residue predicted to affect CFTR synthesis and/or function • premature termination signal (insertion, deletion or nonsense mutation)

• alteration of the invariant dinucleotide of the donor or acceptor splice sites.

CFTR mutations with lower confidence for pathogenic potential:

• sequence variation detected in several unrelated individuals with CF • change in an amino acid residue absent in at least 100 non-CF chromosomes of healthy carriers from the same ethnic group • change in a highly evolutionarily conserved amino acid residue • sequence variation creating/unmasking a cryptic splice site • exonic or intronic sequence variation modifying splicing.

When the observed criteria point to a mutation with lower confidence, additional tools and approaches are necessary to confirm or invalidate its pathogenic potential. The first approach is phenotype-driven, based on the analysis of well-phenotyped subjects with classic CF, thus helping to interpret the clinically relevant mutations [START_REF] Sosnay | Evaluation of the disease liability of CFTR variants[END_REF]. The second are bioinformatic tools, which are now extensively used in clinical diagnosis. However, the computational methods mainly based on evolutionary conservation and protein structure, have limitations [START_REF] Dorfman | Do common in silico tools predict the clinical consequences of amino-acid substitutions in the CFTR gene?[END_REF]. Notably, for the prediction of splicing defects, the use of a combination of tools is more powerful than individual ones [START_REF] Aissat | Combined computational-experimental analyses of CFTR exon strength uncover predictability of exon skipping level[END_REF][START_REF] Raynal | A classification model relative to splicing for variants of unknown clinical significance: application to the CFTR gene[END_REF]. In silico predictions need to be validated by either in vivo measures (when possible) or in vitro assays. Many in vitro assays are available today, exploring the effect of mutations on specific cellular processes. Indeed, splicing defects are reproduced using minigenes, and defects in transcriptional level estimated by reporter gene assays. CFTR processing and stability are monitored by biochemical assays such as western blot and surface biotinylation. Protein localization can be visualized by immuno-cytochemistry and channel function measured by electrophysiological techniques (Ussing chamber, halide-sensitive assays or patch-clamp). While these tools enable to correctly identify major defects, subtle changes in the individual cellular processes tested are more difficult to interpret. Indeed, it must be pointed out that 1-1.5 % of CF alleles remain undetermined in patients with classic CF [START_REF] Castellani | Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice[END_REF]. The classification and assignment of CFTR mutations to CF patients is the basis for current and future genotype-based therapeutic strategies.

Current mutation-targeted therapeutic strategies

As our understanding of the genetics of CF grows, and as technological advances render molecular diagnostic tools more powerful, many CF researchers worldwide now focus on the development of drugs that aim to treat CF by targeting the underlying cause of the disease. These drugs include molecules inducing readthrough of premature termination codons, potentiator molecules, as well as corrector molecules, presented hereafter.

Molecules inducing readthrough of PTC

Premature termination codons usually induce the destruction of the PTC-harbouring transcript through the NMD pathway; however, some PTCs can lead to the production of a truncated protein. In the nucleus, the removal of the introns leaves an exon junction complex (EJC) located molecule was found to suppress nonsense mutations more efficiently than aminoglycosides without nephro-or oto-toxic side effects. A double-blind, placebo-controlled Phase III study is now under evaluation. Novel readthrough molecules are being identified, such as RTC13 and RTC14 [START_REF] Du | Nonaminoglycoside compounds induce readthrough of nonsense mutations[END_REF] or NB124 [START_REF] Rowe | Suppression of CFTR premature termination codons and rescue of CFTR protein and function by the synthetic aminoglycoside NB54[END_REF]. Interestingly, an NMD inhibitor, amlexanox, has recently been shown to induce readthrough [START_REF] Gonzalez | Rescue of nonsense mutations by amlexanox in human cells[END_REF]. Such drugs presenting dual activities could increase readthrough efficiency by increasing the amount of target transcripts.

Before these molecules can be used in routine therapy, certain aspects of their mechanisms of action still need to be understood. Among these, what will be their consequence on normal stop codons? It appears that the rapid dissociation of the terminating ribosome at the normal stop codon does not enable efficient readthrough. This is comforted by the absence of elongated proteins after PTC124 treatment [START_REF] Welch | PTC124 targets genetic disorders caused by nonsense mutations[END_REF]. Additionally, if the ribosome decodes the 3' UTR sequence, it will either rapidly encounter an in-frame stop codon or will decode the polyA-tail, leading to the synthesis of a poly-Lysine tail at the end of the nascent protein. This, in turn, initiates a quality control mechanism named non-stop decay, leading in fine to the degradation of both the polypeptide and the transcript [START_REF] Graille | Surveillance pathways rescuing eukaryotic ribosomes lost in translation[END_REF].

Efficiency of readthrough therapy is linked to various parameters, some of which are predictable while others are measurable. Predictions can be made knowing the identity of the stop codon (UAA<UAG<UGA) and the local nucleotide sequence, where nucleotides in position -1 and +4 appear to have the greatest effect [START_REF] Floquet | Statistical analysis of readthrough levels for nonsense mutations in mammalian cells reveals a major determinant of response to gentamicin[END_REF]. The amount of target transcripts, dependent on the level of NMD, also directly influences readthrough efficiency; hence a positive correlation has been obtained between Cl -transport modification and the level of increase in CFTR transcript after gentamicin nasal application [START_REF] Linde | Nonsensemediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin[END_REF]. Skipping of the PTC harboring exon should also be taken into account as it will also reduce the amount of target transcripts [START_REF] Hinzpeter | Alternative splicing of in-frame exon associated with premature termination codons: implications for readthrough therapies[END_REF]. Indeed, taking into account these two parameters, it appeared that 8% of the CFTR transcripts would be targeted by readthrough therapy in a patient homozygous for the p.Glu831X mutation, which would be predictive of poor treatment efficiency [START_REF] Hinzpeter | Alternative splicing at a NAGNAG acceptor site as a novel phenotype modifier[END_REF].

Finally, readthrough of a PTC leads to the incorporation of a random amino acid at the considered position, leading to the production of a pool of full-length proteins bearing a maximum of one single amino acid substitutions. These substitutions will lead to either functional or non-functional proteins. Therefore, PTCs located in key regions implicated in protein processing or function would be less efficiently corrected, even if high levels of expression can be achieved.

Potentiator therapies or activation of CFTR channels

Molecules enhancing channel activity, such as molecules belonging to the phytoestrogen family (genistein and isoflavone), have been identified. A high-throughput screen enabled the identification of VX-770, Ivacaftor (trade name Kalydeco, Vertex Pharmaceuticals). This molecule specifically targets the p.Gly551Asp gating mutation by increasing the channel open probability and consequently the flow of ions transported through the channel [START_REF] Ramsey | A CFTR potentiator in patients with cystic fibrosis and the G551D mutation[END_REF], [START_REF] Eckford | Cystic fibrosis transmembrane conductance regulator (CFTR) potentiator VX-770 (ivacaftor) opens the defective channel gate of mutant CFTR in a phosphorylation-dependent but ATP-independent manner[END_REF]. It also has the ability to increase the open probability of the wildtype channel and other mutants, namely p.Phe508del [START_REF] Van Goor | Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770[END_REF] and p.Arg117His [START_REF] Van Goor | Ivacaftor potentiates multiple mutant CFTR forms[END_REF]. This molecule will thus also benefit patients retaining low level wildtype CFTR expression, e.g. class V mutations. Finally, it has recently been proposed that a reduction of CFTR activity could lead to a CF-like acquired disease. Cigarette smokers presenting with or without Chronic Obstructive Pulmonary Disease (COPD) showed an increased sweat chloride concentration and reduced intestinal CFTR currents [START_REF] Raju | Cigarette Smoke Induces Systemic Defects in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function[END_REF]. This can be explained by a reduced transcriptional expression of CFTR [START_REF] Cantin | Cystic fibrosis transmembrane conductance regulator function is suppressed in cigarette smokers[END_REF] and a reduced stability of the protein [START_REF] Clunes | Cigarette smoke exposure induces CFTR internalization and insolubility, leading to airway surface liquid dehydration[END_REF]. As CFTR potentiators have been shown to increase wild-type CFTR activity, it would be tempting to increase CFTR activity under such conditions.

Corrector therapies or routing CFTR to the plasma membrane

Bringing misfolded p.Phe508del protein to the cell surface is a major goal for the treatment of CF. Some chemical compounds have been reported to allow the protein to evade endoplasmic reticulum quality control, enabling surface expression. These molecules named correctors, have been identified by high-throughput screening, and include molecules such as benzo[c]quinolizinium (MPB) compounds, Corr4a or VX-809 [START_REF] Grove | Mechanisms for rescue of correctable folding defects in CFTRDelta F508[END_REF][START_REF] Pedemonte | Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening[END_REF][START_REF] Pedemonte | Dual activity of aminoarylthiazoles on the trafficking and gating defects of the cystic fibrosis transmembrane conductance regulator chloride channel caused by cystic fibrosis mutations[END_REF][START_REF] Ye | Pyrazolylthiazole as DeltaF508-cystic fibrosis transmembrane conductance regulator correctors with improved hydrophilicity compared to bithiazoles[END_REF]. The most promising, VX-809 can restore up to 15% of non-CF channel activity in primary respiratory epithelia [START_REF] Van Goor | Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809[END_REF][START_REF] Van Goor | Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules[END_REF]. Recently, VX-809 has been shown to correct processing mutations located within the N-terminus of the protein more efficiently. Indeed, some of these processing mutants could be fully corrected under the tested conditions as compared to a maximum 15% correction obtained for p.Phe508del [START_REF] Ren | VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1[END_REF]. Certain correctors could therefore be best suited for specific genotypes.

It must be pointed out that all these correctors have a ceiling efficacy in restoring chloride conductance of approximately 10-15 % [START_REF] Pedemonte | Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening[END_REF][START_REF] Van Goor | Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules[END_REF].

Efficiency could be increased by either combining molecules, by targeting distinct folding steps or by identifying new more potent correctors. Interestingly, some correctors have been shown to be active on other misfolded ABC proteins, namely on the p.Gln141Lys gout-causing mutation in the ABCG2 gene [START_REF] Woodward | Goutcausing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules[END_REF]. This knowledge opens the application of these drugs to other genetic syndromes.

Combinatory therapies to optimize efficiency

As mutations may induce several CFTR dysfunctions, distinct drugs could correct individual defects. For example, p.Phe508del CFTR, if located at the plasma membrane, also presents a gating defect [START_REF] Dalemans | Altered chloride ion channel kinetics associated with the delta F508 cystic fibrosis mutation[END_REF]. Therefore, potentiators aimed at normalizing defective p.Phe508del CFTR Cl -channel gating may increase the effect of correctors, which rescue the traffic of the protein to the membrane. More generally, potentiators would be beneficial for many classes of mutation. Indeed, they could activate channels produced after suppression therapies for class I mutations or residual wild-type channels expressed in class V mutations. Splicing modulators, such as antisense oligonucleotides, could increase the amount of these latter channels. Combinatory therapies may also be considered for compound heterozygotes.

Conclusion

CF is a complex disease with a broader clinical and genetic spectrum than previously thought. This is making CF diagnosis (classic vs. non-classic CF), carrier screening and prenatal screening decisions more and more difficult.

New technologies and research approaches currently enable CF researchers to work at developing drugs that target the dysfunction of the CFTR protein directly. Our knowledge of the existing CFTR mutations and their consequences, both at the structure-function level (how and why the CFTR protein is non-functional) and at the clinical level (what organ(s) are most affected by the mutation), will be essential in order to perfect the design of the new molecules that have mainly been identified by high-throughput screening to date. This information is needed not only for the development of the molecules themselves, but also for the optimization of their delivery to the patient (pill vs. nebulizer etc.).

With our current knowledge, it is still very difficult to associate a specific CFTR mutation to a specific dysregulation of the structure and/or function of the CFTR protein. It is very unlikely that most CF patients may be treated with a single drug. As advances in CF research and development will provide solutions to these challenges, the future gold standard for CF treatment strategy will be a customized combinatory one, targeting both the clinical symptoms and the underlying genetic cause for each CF patient.

ACMG recommended panel of classic CF-causing mutations

G85E R117H R334W R347P A455E I507del F508del G542X G551D R553X R560T R1162X W1282X N1303K 621+1G>T 711+1G>T 1717-1G>A 1898+1G>A 2184delA 2789+5G>A 3120+1G>A 3659delC 3849+10kbC>T
Additional or alternative mutations present at significant frequencies in an ethnic population served by a newborn screening program may be assessed.

Figure 2 Fig. 2 .

 22 Figure 1

Figure 3 Fig. 3 .

 33 Figure 3

Table 1 . Classification of CFTR mutations according to their clinical consequences.

 1 Examples of each of the four groups of mutations are given. * Mutations that may belong either to Group A or to Group B. ** Mutations that may belong either to Group B or to Group C.

Table 2 . ACMG recommended panel of 23 classic CF-causing mutations.

 2 These mutations include missense, stop, splicing and frameshift mutations.

Acknowledgments

The authors thank Dr. Fabrice Lejeune for the critical reading of this manuscript and helpful discussions. The authors thank the French association Vaincre la Mucoviscidose for its continuous support.

at each exon-exon boundary. The removal of these complexes by the ribosome during the first round of translation serves to monitor the pre-mRNA. When a transcript harbours a PTC, the ribosome will stall at this particular position and leave downstream EJCs. The EJC will then recruit UPF1, via its interaction with other UPF proteins (UPF2 and UPF3/3X (or UPF3a or UPF3b)). This in turn will lead to the degradation of the PTC containing transcript by the NMD pathway [START_REF] Amrani | Aberrant termination triggers nonsense-mediated mRNA decay[END_REF], Bidou et al., 2012[START_REF] Maquat | Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics[END_REF]. This model predicts the recognition of PTCs located in all exons, with the exception of the last exon. Additionally, PTCs located in the last 50 nucleotides of the penultimate exon were shown to bypass NMD recognition, probably due to steric hindrances with the EJC. Hence, PTCs have been classified as either NMD-sensitive or NMD-irrelevant depending on their position. More recent data challenged this model, as some NMD-irrelevant transcripts were found to be degraded, whereas some NMD-sensitive transcripts were not. This led to the Faux-3' UTR model, where mRNA degradation by NMD is dependent on the distance between the PTC and the Poly(A)-binding proteins (PABPs) [START_REF] Eberle | Posttranscriptional gene regulation by spatial rearrangement of the 3' untranslated region[END_REF][START_REF] Silva | Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay[END_REF]. According to this model, the latter distance will influence the kinetics of dissociation of the ribosome/polypeptide complex, affecting the recruitment of UPF1 [START_REF] Muhlemann | Recognition of nonsense mRNA: towards a unified model[END_REF]. In any case, most transcripts harbouring PTCs will be degraded, preventing the production of deleterious truncated proteins.

Small molecules have been shown to induce readthrough of PTCs. Readthrough therapy targets a molecular defect common to all genetic diseases. It could therefore treat a substantial proportion of patients as in-frame nonsense mutations account for 12% of all hereditary diseasecausing mutations [START_REF] Kellermayer | Translational readthrough induction of pathogenic nonsense mutations[END_REF]. Studies have been performed in both in vitro models (over 45 different genes in more than 80 studies) and in clinical trials (8 different genes in 16 studies) (reviewed in [START_REF] Lee | Pharmaceutical therapies to recode nonsense mutations in inherited diseases[END_REF]).

Compounds inducing readthrough favor an improper recognition of the PTC and the recruitment of a near cognate tRNA in the A site of the ribosome in place of the termination complex. If enough PTCs are recoded into sense-codons, enough full-length, functional protein may then be restored to provide a therapeutic benefit.

The best characterized drugs active against PTCs are aminoglycosides. Studies demonstrated the restoration of CFTR-dependent Cl -secretion and protein expression with improved clinical endpoints [START_REF] Sermet-Gaudelus | In vitro prediction of stop-codon suppression by intravenous gentamicin in patients with cystic fibrosis: a pilot study[END_REF][START_REF] Wilschanski | Gentamicin-induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations[END_REF]. PTC124 (trade name Ataluren) was more recently identified in a high throughput screen [START_REF] Welch | PTC124 targets genetic disorders caused by nonsense mutations[END_REF]. This


Raphael Belmin

François Casabianca

Jean Marc Meynard

A crop model as an "intermediary object": Lessons from a participatory research on the agronomical bases of PGI Corsican Clementine typicity

Keywords: Clementine, acidity, cropping systems, participatory research, intermediary object

In this article, we build on an on-going participatory research conducted in Corsica to report the use of a heuristic cropping system model as a mediating tool to assist researchers and actors in the understanding of a challenging scientific problem: The agronomical determinism of the acidity of Corsican Clementine, acidulous taste being a strategic typicity attribute for this terroir product. The collaborative research included the participative conception and refinement of an acidity model, the use of the model to design an empirical validation method called Regional Agronomical Diagnosis (RAD), and the up-coming use of RAD's first results to refine the model. Iterations between actors' knowledge, model, and farm survey may continue in the 2 following Clementine campaigns, leading to a step-by-step convergence between model and reality. Our acidity model opens up perspectives for Corsican stakeholders, since it could be used in further participatory research to design innovative cropping systems in adaptation to emerging challenges such as climate change, or new varieties. However, the use of a model to understand an agronomical variable or to orientate field observation is not new. What is new is the fact of using such model as an intermediary objet in a participative device. Through its intrinsic infrastructure, the model structured interactions between actors, researchers, and experimental device, and it enabled convergence of representations between opposed epistemological postures. If the model appeared to be predictive, it would suggest that in localized agri food systems, local knowledge are crucial resources which can be channeled by researchers by using intermediary objects as mediating tools in the perspective of addressing complex scientific problems.

1) Introduction

Understanding agronomical bases of typicity: a challenge for SYAL stakeholders

Localized agri-food systems' sustainability relies on the capacity of its stakeholders to emphasize and maintain products typicity through qualification processes. A poor understanding of agronomical bases of typicity can put localized agri-food systems into a position of vulnerability, especially when broad changes such as climate upheaval affect GI production area. In the case of non transformed agricultural products, such links are difficult to establish because products quality depends on numerous and complex factors (e.g. climate, soil, agricultural practices). This article introduces methods and first results from a participatory approach employed by researchers to build a shared understanding of the typicity of a terroir product: the Corsican Clementine.

1.2) Interest of participatory approaches in agricultural science

An epistemological and social revolution led to the recognition of stakeholders' knowledge and skills as legitimate to contribute to research problems resolution [START_REF] Gasselin | Pourquoi conduire une recherche action en partenariat ? 31-39[END_REF]. This idea has spread in agricultural and rural development research, and it is increasingly recognized that participatory approaches based on local knowledge are an option if sustainability and development goals are to be reached (IAASTD, 2008). Thus, participatory approaches are mobilized in a diversity of thematic contexts. To give just a few examples, such approaches are often mobilized in farming system research, livelihood systems studies, participatory plant breeding, or diagnosis and design of cropping systems [START_REF] Sellamna | La recherche action des origines à nos jours. 23-29[END_REF]. Collaborative approaches are often combined with cropping system research in the understanding of unsatisfactory agronomical performances or in the design of innovative technical itineraries [START_REF] Lavigne Delville | Jachères, fertilité, dynamiques agraires, innovations paysannes et collaborations chercheurs/paysans. Fondement pour des recherches-action en milieu paysan sur la fertilité des terres[END_REF].

1.3) A heuristic model as tool of participatory research structuration

Researchers mobilize a diversity of tools in participatory approaches, ranging from simple artifacts (cross-tabulation, map, black board) to complex models. In this article, we report the use of a heuristic crop model as a mediating tool to assist researchers and stakeholders in the understanding of a challenging scientific problem: The agronomical determinism of the acidity of Corsican Clementine, acidulous taste being a strategic typicity attribute for this terroir product. The article shows that the model acted as an "intermediary object" contributing to knowledge co-elaboration, and to the convergence of actors' representations on the acidity determinism. The main function of the model was to structure mutual exchanges between actors, researchers, and an experimental device. It did so by providing a normativeand evolving -framework channeling heterogeneous actors' inputs, driving empirical observation, incorporating field observations, and by allowing constructive dialogue between holists and reductionists.

1.4) Analytical framework of the model's role in the participatory process

To understand the model's role, we analyzed our participatory research as a process along which multiple interactions between and among actors, artifacts, and institutions led to the convergence of actors' representations on fruit acidity determinism, and to the generation of new knowledge.

First, to analyze interactions between actors, researchers, and experimental device, we considered the acidity model as an "intermediary object" carrying embedded rules, which acted as a frame enabling and constraining perceptions, interactions and inputs during the participatory construction. The concept of "intermediary object" forms part of a research tradition putting emphasis on the materiality of things in social sciences. It especially inherits from the actor-network theory [START_REF] Callon | Eléments pour une sociologie de la traduction: La domestication des coquilles Saint-Jacques et des marins-pêcheurs dans la baie de Saint-Brieuc[END_REF][START_REF] Latour | Une sociologie sans objet? Remarques sur l'interobjectivité[END_REF], and the notion of "boundary object" [START_REF] Star | Institutional ecology, translations' and boundary objects: Amateurs and professionals in Berkeley's Museum of Vertebrate Zoology[END_REF]. According to [START_REF] Vinck | De l'objet intermédiaire à l'objet-frontière. Vers la prise en compte du travail d'équipement[END_REF], intermediary objects are material or immaterial artifacts that are produced, mobilized, and exchanged by actors, and which participate in framing action and interactions through processes of representation, translation, and mediation. Recent works have reported the use of intermediary objects to analyze or monitor situations of mediations between heterogeneous actors (see [START_REF] Mcintyre | Représentations spatiales et développement territorial[END_REF] for an illustration). In this article, we analyze how the rules embedded in an intermediary object (a crop model) could structure mobilization of local knowledge by researchers in a participative process. Then, to assess to what extent the model had contributed to the convergence of actors' representations on fruit acidity determinism, we compared the initial perception of each actors' groups with the final shared understanding synthesized in the model. Initial perception of each actors group was analyzed in the light of sociology and institutional theory. For each group, we identified patterns of institutions which shape awareness and interpretation of reality, and coordinate in return actors' perception of and knowledge on acidity determinism. We used the distinction proposed by [START_REF] Scott | Institutions and organizations[END_REF], between regulative rules (i.e. explicit and formal rules such as laws), normative rules (i.e. internalized through social process such as norms or professional identity) and cognitive rules (i.e. the frame through which meaning or sense is made, such as paradigms and ontological posture).

The development of this article is organized as follow: In part 2, we explain how has emerged the Corsican Clementine acidity issue. In part 3, we describe the construction of the participative research (actors, main steps, and experimental device). We then introduce the outcoming acidity model (part 4).

In part 5, we analyze model's contribution to the structuration of interactions between actors, researchers, and experimental device, and to the convergence of actors's representations on fruit acidity determinism. In part 6, we discuss possible implication of the use of crop models to strengthen participatory approaches.

2) Emergence of the Corsican Clementine acidity issue

2.1) Acidity: A crucial but poorly understood quality attribute for Corsican Clementine

Corsican clementine has been recognized as a PGI in 2007, leading to a revival of the sector after a period of decline in the 90s. The reputation of the "Clementine of Corsica" is due to several criteria of fruit quality such as the presence of long leaves in fruit crates, a specific color, and a slightly acidulous taste. As foreign competition increasingly imitates external attributes of Corsican clementine (e.g. presence of leaves in crates), the long term success of the PGI relies on the capacity of local stakeholders to strengthen and sustain other attributes of specific quality, first and foremost the high acidity level. Moreover, the process the defense and management organization recently embarked to obtain a red label makes it all the more urgent to understand the bases of fruit acidity, since the red label specification requires even lower variability and higher threshold in fruit acidity. Although local stakeholders agree that "slightly acidulous taste" is a crucial typicity factor for Corsican Clementine, fruit acidity has remained an unchecked parameter in cropping systems and individual farmers' strategies. This paradox results from a lack of knowledge on the agronomical bases of fruit quality among farmers, extensions, and scientists. This is probably explained by the historical trajectory of local agricultural innovation system, which partitioned fruit quality questions into the field of varietal innovation, leaving aside cropping systems aspects. Moreover evidences show that there are a significant spatial, intra and inter annual fluctuations in fruit acidity within the PGI zone, as well as an effect of agricultural practices on acidity (Belmin, 2013).

2.2) The Corsican Clementine GI area under broad changes

In a context of global changes affecting the PGI zone, this difficulty in objectifying the combined role of climate, soil and practices in the construction of fruit acidity challenges Corsican stakeholders. First and foremost, Mediterranean zone is affected by climate change, with suspected consequences on Corsican clementines' acidity (Pailly; 2014). The 2013 IPCC report indicates that in this geographical area, observed changes since the middle of the 20th century include an increase in droughts' frequency and intensity, extreme precipitations, as well as a decrease in cold nights and frosts. On their side, local farmers and experts assert that fruit acidity is trending down for around 15 years, due to climate upheaval. However, as far as we know, no available scientific data could demonstrate a decrease in Corsican clementine acidity, nor any link between climate change and clementine quality variation. The fact remains that a possible trend reduction in fruit acidity caused by climate change is a rising issue in the PGI area. Climate is not the only broad change affecting production basin and (possibly) fruit quality. There is a quick evolution of agricultural practices with yield and fruit size as the main cropping system performance criterion. These technical changes include annual pruning, weed management, fertilization, as well as harvesting methods. PGI Clementine quality change may also be driven by new varieties and root stocks such as SRA535 or tetraploid root stocks. Such changes are also possible because the PGI specification provides a flexible frame, leaving room for much variability in agricultural practices.

3) A participatory research to understand Clementine acidity variability

Researchers implemented in 2013 a participatory research aiming at better understanding the variability of Corsican Clementine acidity. The participatory research included actors' mobilization, the co-conception of a heuristic acidity model, the use of the model to design an empirical validation method called Regional Agronomical Diagnosis (RAD), and the use of RAD's first results to refine the model. Thus, knowledge on fruit acidity was constructed and refined through organized iterations between model, actors, and on farm survey.

3.1) Creation of a participatory device

We began by designing a participatory research device involving a diversified panel of actors chosen according to their current or historical function in the clementine production basin (Table 1). The core of the device was a working group involving citrus consultant, technicians, agronomists, and researchers from 2 disciplines (ecophysiologists, and geneticists). Interactions with working group's members included 3 workshops as well as individual interviews and field visits. In parallel we consulted 13 farmers through individual interviews, field visits, and participant observation.

3.2) Participatory conception and refinement of a heuristic acidity model

In a first workshop, we identified possible acidity drivers through brainstorming. Participants were asked to say what are, according to their empirical or theoretical knowledge, the main factors involved in the variability of Clementine acidity. The information gathered during the workshop was complemented through individual interviews and field visits with farmers, technicians, and experts. In a second step, we integrated data into a first conceptual crop model. The various data collected so far were graded and organized in an agronomical model, mobilizing and adapting concepts such as yield components [START_REF] Boiffin | Elaboration du rendement et fertilisation azotée du blé d'hiver en Champagne crayeuse. I. -Protocole et méthode d'étude d'un problème technique régional[END_REF][START_REF] Meynard | Diagnostic sur les causes de variation du rendement du blé dans une petite région. La fatigue des sols[END_REF] and yield elaboration compartments (Sebillotte, 1995). Inspired by the notion of yield component, we divided the elaboration of final fruit acidity into simple components whose formative is shorter than the one of final production, and that only depend on a limited number of factors. Acidity components were considered as (semi) independent variables, well identified in the tree cycle. The various factors influencing acidity components were identified, and placed together with the components themselves into 5 interacting compartments: climate, permanent environment, non-permanent environment, agricultural practices, and physiological status of the tree. This first-generation model was then confronted with stakeholders' knowledge through a second workshop and using bilateral meetings. The information collected so far led us to build a second-generation model by introducing new acidity components, and by clarifying the factors influencing each component.

Researchers 2

Researchers are geneticist and ecophysiologist employed by public "publish-oriented" organizations. Geneticist produces scientific knowledge on citrus genome and phylogeny, and selects new citrus rootstocks and varieties responding to identified challenges (disease resistance, yield, caliber, fruit quality). Ecophysiologist produces scientific knowledge on complex interactions between genotypes, phenotypes and environment in citrus.

3.3) Use of the model to design a regional agronomic diagnosis

The revised heuristic acidity model was used as an input to co-construct an empirical validation protocol using an approach called Regional Agronomical Diagnosis (RAD). [START_REF] Boiffin | Elaboration du rendement et fertilisation azotée du blé d'hiver en Champagne crayeuse. I. -Protocole et méthode d'étude d'un problème technique régional[END_REF] and [START_REF] Doré | Methodological progress in on-farm regional agronomic diagnosis[END_REF] introduced RAD as a methodological framework for identifying and ranking limiting factors for crop yield (or other variables relating to crop quality and environmental impact) on the regional scale : RAD consists in monitoring a set of measurements in a network of fields cultivated by farmers using current cropping practices. Thus, we mobilized RAD approach in the context of a participatory process, so as to test and refine heuristic model, and to identify the factors explaining the variability of fruit acidity. During the second workshop, stakeholders were introduced to the RAD approach, and they were asked to propose selection criteria's for plots as well as concrete field observations. Researchers structured the discussion using the second-generation acidity model as a starting point. 21 plots were selected and field observations were conceived so as to characterize each acidity component, as well as the main factors suspected to influence them.

3.4) Use of regional agronomic diagnosis' first results to refine the model

In the near future, concrete observations through RAD will lead researchers and working group participants to criticize the second-generation model, and build a third generation model. In turn, this will contribute to the improvement of field observation procedure for the next campaign.

In conclusion, actors were mobilized by researchers in the co-elaboration of 2 successive generations of acidity model: A first model was constructed by incorporating actors' inputs into a crop system framework. A second generation model was then constructed by confronting first model to actors' knowledge, and used as a starting point to conceive an on-farm survey using RAD approach. The upcoming third generation of model is to be constructed through confrontation between second generation model with first results from RAD. These iterations between model, actors knowledge and farm survey may be implemented again in the 2 following Clementine campaigns, and lead to a stepby-step convergence between model and reality. To the top of our knowledge, it is the first time RAD is conceived through a participatory approach, on the basis of a crop model constructed itself through a collaborative work. Nevertheless, the idea that actors' knowledge is valuable in such inductive was already there. [START_REF] Doré | Methodological progress in on-farm regional agronomic diagnosis[END_REF] mentioned that an option for researchers carrying out RAD is to integrate the farmers' knowledge into the identification of relevant measurements, and into data interpretation.

4) Introduction to the model

The second-generation acidity model took the form of a semi coherent set of hypotheses regarding acidity components, their interactions throughout the tree physiological cycle, as well as the various factors influencing the variability of each component. In other words, the variability observed in fruit acidity could be theoretically explained by the condition of elaboration of a limited number of semiindependent processes or "components" (noted C1-C8 following the order in which components were identified in the participatory process) well identified in the tree cycle, each component being driven by specific factors. According to the model, acidity of the fruits from a given parcel results from the meeting of harvest dates (C4) and % fruits harvested at each date (C5) with a long process of acidity decrease (C1) in a diversified cohort of fruits showing a wide range of sizes (C7), maximal acidity reached before ripening (C2), and coloration date (C6). Harvest dates and % fruits harvested at each date are partly constrained by coloration process. Maximal acidity reached before ripening and fruit size repartition are driven by flowering dates (C3), and by the number of fruits per tree (C8). Thus, C1-C8 are observable acidity components, which interact along the tree physiological cycle. The construction of each acidity component is driven by observable stages, processes, or interactions, occurring all into the 5 interacting compartments of acidity elaboration listed below (see Figure 1

C3 -Flowering dates (beginning and end)

C3 determines field-side fruits' acidity because it influences the duration and environmental conditions in which C1, C2, C7, and C8 are elaborated.

C3 is triggered by soil temperature increase in spring (NPE*)

Soil temperature increase in spring (See above "Temperature accumulation")

C4 -Harvest dates

C4 determines field-side fruits' acidity because it interfers with the process of acidity decrease (C1). In Corsica, Harvest is usually done through 2 to 4 stages.

C4 and C5 depend on sociotechnical determinants (e.g. market demand), but also on the percentage and nature of colored fruits at a given moment (PS)

Percentage and nature of colored fruits at a given moment (PS*) are driven by coloring speed (C6)

C5 -Quantity and types of fruits removed at each harvest stage

C5 determines field-side fruits' acidity because fruits from one parcel vary a lot in acidity -mainly according to their specific size (see C7) and degree of ripening (see C1) -, while only a fraction of the available fruits are harvested at each stage. C5 has also effects on the acidity of the fruits harvested at the next stage.

C6 -Coloring date

C6 determines field-side fruits' acidity because it is a driver of harvest date (C4), although decorrelated from the process of acidity decrease (C1). In practice, coloring date often constitutes a limiting factor to the harvest start.

C6 is accelerated by decrease in orchard temperatures, increase in daynight range of temperatures (NPE*), and abundant nitrogen and potassium nutrition during ripening period (PS*).

Orchard temperatures (See above "Temperature accumulation")

Nitrogen and potassium nutrition (See above "Mineral nutrition")

C7 -Size distribution of fruits

C7 determines field-side fruits' acidity because each plot shows a large range of fruit sizes, while large-sized fruits are generally less acid than small fruits. Acid citric content of fruits seems to be diluted as fruit size increases.

C7 is driven by accumulation of temperatures above 13°C (NPE*), and by carbon, mineral, and water nutrition during the filling period (PS*). C7 is also driven by flowering quality, by the number of fruits per tree (see C8) and by nature of fruit-bearing shouts (PS*)

Flowering quality is driven by the nature of flower-bearing shoots (diameter and length) (PS*), by pruning (AP*). Nature of fruit-bearing shouts result from nature of flower-bearing shoots. Carbon nutrition is driven by the relationship between total leaf surface and number of fruits during filling stage, which depends on mineral and water nutrition and temperature accumulation (see above).

C8 -Number of fruits per tree

C7 determines field-side fruits' acidity because it influences size distributuon of fruits (C7) and acidity level of each fruit before maturation (C2).

C8 is driven by flowering intensity and physiological fall (PS*).

5) model as an "intermediary object"

The use of such a crop model to understand variability of agronomical variable or to orientate field observation is not new. Nor is the analytical distinction we made between various components and 5 compartments of acidity elaboration. What is new is the fact of using such representations as a mediating tool between the research team and his partners. In this part, we show that the model acted as an "intermediary object", enabling mutual understanding and representations convergence between opposed epistemological postures. To do so, we considered the acidity model as an "intermediary object" carrying embedded rules, which acted as a frame enabling and constraining perceptions, interactions and inputs during the participatory construction (Figure 2). We can say that two rules are embedded in the model's structure, and have acted together as a normative framework structuring dialogue between actors, researchers, and experimental device. The first rule derives from the compartmental structure of the model (that is to say the analytical distinction between the 5 compartments of climate, permanent environment, non-permanent environment, plant status, and agricultural practices). The second rules stems from the choice of dividing the whole process of fruit acidity elaboration into a limited number of "components" (components are semiindependent processes observable at a specific period in a particular compartment of the cropping system). We show below that these 2 rules have been playing together, by structuring the capitalization of heterogeneous information, the co-conception of an empirical validation device, and the successive model refinement in the light of results from field observation. Models' structure has facilitated the incorporation of heterogeneous pieces of information into a coherent analytical framework. First, actors' inputs were varying from theoretical knowledge on agronomical or physiological mechanisms to account of empirical experiences and isolated observations. Actors' contributions were regarded a priori as valid, provided that the group reached a consensus. Using model's systemic frame, researchers attributed each mentioned mechanism to an acidity component (or to an elements directly or indirectly determining the elaboration of one component). This was especially useful after the first workshop, when we built the first generation model by reviewing consistent quantity of diversified and rambling data. Then, the component analysis helped actors to overcome complexity (at the beginning of the process researchers were confronted to remarks such as: "everything is inextricably connected"), and successively focus actors' attention on well identified processes. Thus, actors were asked to explain which elements of the cropping system were involved in the elaboration of each identified components of fruit acidity. This approach was used to design the second generation model: during the second workshop and bilateral meetings, where actors could easily refine the model because component analysis channeled their inputs and perceptions. Third, models' compartmental structure was helpful because it helped researchers in the overcoming of analytical shortcuts or Fourth, the rules embedded in the model's structure enabled the objectivation of actors' inputs when "colored" by their ontological postures. For instance, it was a mediating tool to encourage actors to translate prescriptions into objective processes. The models' structure has not only facilitated the incorporation of heterogeneous data. It has also directly driven the participative selection of the field observations to be implemented. Acidity components were used so as to identify periods and kind of field observations, and compartments were used to identify where to implement observations. For instance, in the case of C1 (acidity draw down rate during ripening), the representation given by the model encouraged researchers to monitor evolution of fruit quality (sugar, citric acid concentration, and juice percentage) for each fruit size on the 21 plots of the farm network, from 1 rst October to early January. This was combined with other field observation such as meteorological conditions (weather stations), water nutrition state (delta C13 analysis of fruits sampled in October and December, completed with calculation of full water balance), soil permeability, slope, deepness of rootlets, grass cover, and irrigation regimes.

5.2) Contribution of the model to the convergence of actors' representations

The model's infrastructure also allowed researchers to put together different representations of fruit acidity into a single coherent framework. In fact, actors in the participatory device had all significant knowledge on fruit acidity, but this knowledge was always somehow biased by their specific institutional pattern (e.g. epistemological posture, paradigms, cognitive framework…). In this part, we show how the model could act as an "intermediary object", helping mutual understanding between actors, and representations convergence. Each actors' group introduced in part 3.1, was characterized by patterns of institutions which shape awareness and interpretation of reality, and coordinate in return actors' perception of and knowledge on acidity determinism (Table 3). To build this analysis, we used the distinction between regulative rules, normative rules, and cognitive rules introduced in part 1.5 of this paper. To simplify, we found that the participative device opposed actors with reductionists and holists epistemological posture. On the one hand, reductionists envisaged fruit acidity elaboration at the tree level, considered "environment" as a black box, and couldn't upscale analysis at the cropping system level. On the other hand, holists believed that fruit acidity resulted from a wide and inextricable range of inter-linked factors, but incapable of grading factors' relative importance. During workshops and bilateral meetings, the model acted as an "intermediary object", helping dialogue and mutual understanding between reductionists and holists. In fact, this is again the 2 rules Daily routines of orchard observation leading farmers to qualify each single plot according to its caliber, yield, and fruits taste.

No idea on how agricultural practices determine fruit acidity. Rootstock, variety, and "plot effect" as the main fruit acidity drivers.

Fruit acidity results from the year's meteorological condition.

Technicians, agronomists, and citrus consultant

European legislation encourages technicians and agronomists to focus their attention and efforts on phytosanitary pressure reduction.

Most agricultural practices involved in acidity construction are routinised, leading technicians and agronomists to focus on phytosanitary issues.

Holistic epistemological posture leading to consider the complex interactions between soil, plant, climate and practices in fruit quality construction.

Fruit acidity results from a wide range of inter-linked factors.

R&D agents

Research programs and funding focused on varietal innovation and genotype x environment interactions.

Clear disciplinary boundaries leading to reductionist posture

Available genetic diversity as a starting point for any R&D programs

Varietal innovation as a recognized way of improving cultivated citrus.

Reductionist epistemological posture:

plant phenotype results from interactions between genotype and the black-box of plant environment.

Fruit acidity is a physiological process resulting from interactions between genotype and plant environment. On-tree variability of fruit quality is the main level of analysis.

embedded the model (compartmental structure and component analysis) which have allowed putting these different epistemological postures together. Reductionists, who were focused on the tree level and on the interactions between genotype and the black-box of environment, could bring significant inputs by identifying some physiological mechanisms involved in fruit acidity elaboration (mainly C1, C2, C6, C7, and C8 located in physiological status compartment). In other worlds, reductionists were the one identifying the first acidity components. Holists, who had a systemic representation of agro-systems, tried to mobilize reductionists' inputs and upscale the analysis at the cropping system level. Thus, holists could explain to what extent each single components identified by reductionists could be driven by interacting climate, permanent environment, and non permanent environment, and agricultural practices. At the conclusion of the first years' participative process, we noticed a beginning of convergence between actors' representations on fruit acidity determinism. At the end of third workshop, it appeared clearly to everybody that fruit acidity was the result of a limited number of semi-independent processes (the acidity "components"), each observable at a specific period. Although at this stage, precise effect of the various cropping systems elements on the construction of acidity components was unclear, it seemed possible to grade it through a farm survey.

6) Discussion and conclusion

In the purpose of strengthening localized agri-food systems, there is room for innovative participatory research devices and tools able to emphasize and maintain products typicity through qualification processes. This paper reported an on-going participatory initiative designed by researchers to better understanding the variability of Corsican Clementine acidity, an important typicity attribute of this terroir product. The collaborative research included the co-conception of a heuristic acidity model, the use of the model to design an empirical validation method called Regional Agronomical Diagnosis (RAD). The acidity model took the form of a set of hypotheses regarding acidity components, their interactions throughout the tree physiological cycle, as well as the various factors influencing the variability of each component. At the Corsican level, the model opens up perspectives for researchers and stakeholders. It could be used in further participatory research to design innovative "acidity oriented" cropping systems in adaptation to emerging challenges such as climate change, new varieties, or increasing competition of the international market of Clementine. Such technical references could feed the qualification process by allowing the definition of the practiced involved in fruit acidity rather than limiting the PGI specification to product-quality objectives. Apart from the model itself, we could identify other outputs from the participative research process. Although the challenge of uncontrolled fruit acidity was pending, it had not been collectively formulated before the research starts, leading actors to "stick their head in the sand". The participatory research process contributed to the shared recognition that a better agronomical control of fruit acidity is needed (and probably possible) in order to face climate change and to facilitate adoption of a quality label more demanding than the PGI. In other words, the participative process led to a beginning of "construction of the acidity problem" as a medium term challenge for the Corsican basin. More generally, our methodological approach could be used by researchers to strengthen other origin based qualification processes through a better understanding of the complex interactions between, place, people and products.

The effectiveness of our model may be assessed according to 2 criterions, the first one being the ability of the model to act as an intermediary object enabling local knowledge mobilization. We demonstrated that the rules embedded in the model could structure interactions between actors, researchers, and experimental device, channel actors' inputs, and enable representations convergence between opposed epistemological postures. The second effectiveness criterion would be the predictive capacity of the final model. At that point, we couldn't answer yet because the provisional model is still under construction, and may be subjected to other iterative refinements. If the model appeared to be predictive, it would suggest that in localized agri food systems, local knowledge are crucial resources which can be channeled by researchers by using intermediary objects as mediating tools in the perspective of addressing complex scientific problems.

Bibliography

Belmin, R. 2013. Projet Clémentine: Le Diagnostic Agronomique Régional appliqué à l'analyse de la variabilité de l'acidité de la clémentine de Corse. Oral presentation. San Giuliano, 22/03/2013.

  for a schematic representation, and Table 2 for exhaustive model): -Physiological status of tree (PS): This compartment gathers physiological status or processes directly or indirectly involved in the variability of acidity components. -Non permanent environment (NPE): This compartment gathers elements from environment which directly regulate tree's physiological status, and which may fluctuate at the month timescale under the influence of climate and agricultural practices. -Climate (CLIM): Climate influences physiological status of tree through modifications in nonpermanent environment (e.g. rainfall influences soil water availability). -Agricultural practices (AP): Together with climate, agricultural practices influence physiological status of tree through modifications in non-permanent environment (e.g. fertilization modifies mineral availability in soil), or even directly (e.g. pruning). -Permanent environment (PE): This compartment gathers cropping system elements which may not move at the time scale of the research, and which have effect on the interactions between the four other compartments (e.g. density of plantation, soil permeability).

Figure 1 :

 1 Figure 1: Representation of acidity components as described in second-generation model

Flowering

  intensity reflects flower induction, which is driven by orchard low temperature (NPE*) and nutritional stress (PS*) during ripening. Orchard low temperature (See above "Temperature accumulation"). Nutritional stress during ripening results from mineral nutrition (See above). Physiological fall is driven by mineral nutrition during flowering.

Figure 2 :

 2 Figure 2: The acidity model as an "intermediary object"

Table 1 : Main function of the participatory research device's actors Actors type Number Function in the production basin Farmers 13

 1 Farmers run capitalist exploitations of 10-20 hectares of Clementine, often associated with secondary crop such as grapefruit or kiwi.Technicians are employed by cooperatives, farmer groups, or chamber of agriculture. The main part of their work consists in following up individual farmers, and providing technical advice on pest management, fertilization and irrigation regime.Citrus consultant is a highly skilled consultant providing extension to farmers and technicians in Corsica and in all Mediterranean area. He is used to intervene on questions of variety and root stock choice, pruning methods, and pest management. Citrus consultant has developed his skills through variety/root stocks evaluation in experimental or farmers plots.Agronomists are employed by R&D "farmer-oriented" organizations. They implement ex situ experimentation programs to assess the effect of single technical acts on yield, caliber, and fruit quality. They currently develop and spread innovative weed and pest management techniques derived from agro ecological approaches.

	Technicians	6
	Citrus consultant	1
	Agronomists	2

Table 2 :

 2 Acidity components and the factors determining components variability

* PS: Physiological status of tree; NPE: Non permanent environment; CLIM: Climate; AP: Agricultural practices; PE: Permanent environment

Table 3 :

 3 Attempt to analyze how institutions coordinate actors' perception of Clementine acidity.