*3.3.2.3. Intestinal current measurement (ICM)*

in heterologous cell populations using cell surface markers and selection of cells that display high CFTR function. Technical limitations include the need to perform this assay in specialized

All these methods offer the possibility to evaluate the functional consequences of molecular

Characterization of *CFTR* molecular defect can partially be performed directly after biopsies of tissues that show sufficient CFTR expression. Nasal or lung airway epitheliums are optimal. These tissues are accessible by minimally invasive interventions and display an endogenous expression of CFTR transcripts and protein. Moreover, nasal and bronchial epitheliums show the same cellular composition (ciliated, goblet, columnar and immune cells) although ratios differ slightly [89]. Since quantification and detection of aberrant splicing and quantification or localization of proteins are possible in human tissues, information that they bring is crucial to assess the effect of variants and to propose a functional classification. However, this approach has its limitations and requires other functional assays to perform large-scale genotype–phenotype correlation studies. In addition, especially for nasal tissue, the low quantity of cells collected (out of 500.000 cells per brushing) only allows 'one-shot' tests and hinders mechanistic assays. Moreover, highly variable CFTR expression in heterogeneous cell types, in healthy individuals and in p.Phe508del homozygous patients has been described, varying from 0 to 100% [90]. Other genetic (see below) or environmental parameters could also

Culture of primary cells from CF patients can be performed with brushed nasal or bronchial cells after biopsies. Wild-type endogenous CFTR protein is expressed at the apical mem‐ brane of polarized cells. Therefore, *in vitro* monolayer culture seems no longer adapted. Obtaining polarized cells is promoted by air–liquid interface culture (ALI), proposed since the 2000s, by an *ex vivo* system of collagen-coated porous membrane on which cells are platted after a phase of monolayer amplification or directly after nasal brushing. Basal adherent cells differentiate in all airways epithelial cell types, which organize into a pseudo-

This model offers the opportunity to perform functional assays described above to determine CFTR dysfunction. Molecular defect induced by a specific mutation can be qualitatively

Technical limitations such as bacterial or fungi contaminations or absence of adherence complicate culture of cells obtained from CF patients. Moreover, further studies are needed to determine if extrapolation is possible between observations in primary cells directly after

determined if the cell donor is homozygous for this mutation.

abnormalities on CFTR and finally improve the classification of variants.

centres (using expensive imaging equipment).

*3.3.2.1. Analyses on primary airway cells and tissues*

*3.3.2. Ex vivo CF patients' cells assays*

220 Cystic Fibrosis in the Light of New Research

modify CFTR expression levels.

stratified epithelium [91].

*3.3.2.2. Ex vivo culture of primary airway cells*

ICM was developed as a research tool to assess CFTR function in the 1990s and has been used as a diagnostic test since the early 2000s [92, 93]. At least four superficial rectal biopsies per patient, obtained by suction, are needed and mounted on adequate tissue sliders. ICM consists in blocking epithelial sodium channels by amiloride and stimulates cAMP-mediated CFTRdepending chloride transport in a chloride-free solution with forskolin or IBMX. Cholinergic chloride transport and histaminic reaction were also evaluated and the sum of the response ∆Iscforskolin/IBMX+carbachol+histamine appears to be the best parameter to evaluate CFTR function by ICM, but reference values and ranges have not been established. This combination of ionic responses discriminates patients with CF from healthy subjects but not CF patients with pancreatic insufficiency (PI) or sufficiency (PS). ICM can detect a loss of CFTR function above 80%, therefore CF carriers and CFTR-RD patients may not be identified by this method. Moreover, mild mutations could result in a false-negative ICM.

ICM is not altered by secondary damage on tissue and thus better reveals the primary CFTR dysfunction (compared with nasal potential difference, see below). A new functional CFTR assay using primary CF intestinal organoïds derived from patients and cultured *in vitro* may offer new tools to screen for therapy [94].

Finally, setup and maintenance of dedicated equipment by experienced and trained staff limits its use.

## *3.3.3. In vivo biomarkers assays of CFTR function*

Sweat chloride Test (ST) and Nasal potential difference (NPD) measurement are used as diagnostic tests for CF. In atypical clinical context these *in vivo* tests can give additional arguments to further explore the *CFTR* locus (cf. Figure 2 that describes the molecular diagnostics step-by-step strategy). Furthermore, they provide complementary information for the interpretation of *CFTR* variations.

ST higher than 60 mmol/L is the 'gold standard' and discriminates between healthy and typical CF. Sweat electrolytes are higher in the most severely affected and are lower in those with mild mutation, who have partial rescue of channel function [95-97]. There are two major advantages for the use of sweat test in the evaluation of the *CFTR* mutation severity: (i) stability of the measure throughout life and (ii) non-invasive way of measurement which maintains skin integrity. Indeed, sweat electrolytes levels reflect the primary defect on the CFTR protein and do not highlight secondary consequences of its absence or dysfunction on affected organs. Furthermore, a new means for assessing the secretory function of CFTR has been recently developed based on β-adrenergic-mediated sweating. This method provides a unique evaluation of the purely secretory function of CFTR *in vivo*. It has been shown to be more sensitive in individuals carrying mutations that commonly exhibit normal or borderline sweat chloride rates, such as c.3718-2477C>T (legacy name: 3849+10kbC>T) [98].

The transepithelial NPD measurement estimates the net ion conductance across the nasal airway epithelium and indirectly measure CFTR activity. Sodium conductance is determined after amiloride perfusion (Na+). NPD parameters discriminate CF and CFTR-RD phenotypes and a correlation between NPD and disease severity has been shown [99], although there is an overlap in subjects with mild phenotype [97]. However, other sources of phenotypic variability (modifiers genes or environment) and technical limitations (i.e. maintenance of dedicated equipment and trained personnel) contribute to the inconsistencies of NPD across genotype-phenotype correlation studies. CFTR expression and NPD response can also be modified if nasal epithelium is affected by rhinosinusitis, polyposis or exposure to smoking [100]. However, despite the difficulty of setting up this technique, NPD assessment could be used as a complementary step to support CFTR dysfunction in inconclusive clinical cases and then to confirm the deleterious effect of *CFTR* variants identified in these patients.
