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ECFS standards of care on CFTR-related disorders: Diagnostic criteria of CFTR dysfunction

  • I. Sermet-Gaudelus
    Correspondence
    Corresponding author at: INSERM U 1151, Institut Necker Enfants Malades, Paris, France.
    Affiliations
    Centre de Reference Maladies Rares Mucoviscidose et Maladies Apparentées, Hôpital Necker Enfants Malades, Paris, France

    INSERM U 1151, Institut Necker Enfants Malades, Paris, France

    Pediatric Pulmonology, University Hospital UZ Leuven, Gasthuisberg, Leuven, Belgium
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  • E. Girodon
    Affiliations
    INSERM U 1151, Institut Necker Enfants Malades, Paris, France

    APHP Centre, Service de Médecine Génomique des Maladies de Système et d'Organe, Hôpital Cochin, Université de Paris, Paris, France
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  • F. Vermeulen
    Affiliations
    CF Centre, University Hospital, Gasthuisberg, Leuven, Belgium
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  • G.M. Solomon
    Affiliations
    Department of Internal Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine and the Gregory Fleming James CF Research Center, University of Alabama at Birmingham, Birmingham, AL USA
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  • P. Melotti
    Affiliations
    Cystic Fibrosis Centre, Azienda Ospedaliera Universitaria Integrata, Verona, Italy
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  • S.Y. Graeber
    Affiliations
    Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine and Cystic Fibrosis Center, Charité – Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany

    German Center for Lung Research (DZL), Associated Partner Site, Berlin, Germany

    Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany
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  • I. Bronsveld
    Affiliations
    University Medical Centre Utrecht, Utrecht, the Netherlands
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  • S.M. Rowe
    Affiliations
    Departments of Medicine, Pediatrics, and Cell Developmental and Integrative Biology, The Gregory Fleming James CF Research Center, University of Alabama, Birmingham, AL, USA
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  • M. Wilschanski
    Affiliations
    Hadassah Hebrew University Medical Center, Jerusalem, Israel
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  • B. Tümmler
    Affiliations
    Klinik für Pädiatrische Pneumologie, Allergologie und Neonatologie, Medizinische Hochschule Hannover, Hannover, Germany

    Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research, Hannover Medical School, Germany
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  • Author Footnotes
    1 Co last authors
    G.R. Cutting
    Footnotes
    1 Co last authors
    Affiliations
    Department of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA
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  • Author Footnotes
    1 Co last authors
    T. Gonska
    Footnotes
    1 Co last authors
    Affiliations
    Department of Paediatrics, Hospital for Sick Children, University of Toronto, Toronto, Canada
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  • Author Footnotes
    1 Co last authors
Published:October 05, 2022DOI:https://doi.org/10.1016/j.jcf.2022.09.005

      Highlights

      • CFTR-related disorders are clinical entities with features of CF and evidence for CFTR dysfunction but not meeting criteria for diagnosis of CF.
      • The spectrum of disorders involving CFTR dysfunction correlates with a continuous gradient of CFTR function.
      • The level of CFTR dysfunction may be measured directly in vivo or ex vivo by CFTR bioassays or indirectly by CFTR variants.
      • Parameters are lacking to differentiate CF from CFTR-RD activity in CFTR bioassays.

      Abstract

      The spectrum of disorders involving CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction correlates with a continuous gradient of CFTR function defined by the combination of two allelic CFTR variants. CFTR-related disorders are clinical entities with features of cystic fibrosis (CF) and evidence for presence of CFTR dysfunction but not meeting criteria for diagnosis of CF. Individuals with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. The level of CFTR dysfunction may be measured in vivo (sweat testing, nasal potential difference measurements) and/or by ex vivo tests (intestinal current measurement), or indirectly indicated by CFTR variants, as alteration in sequence of the CFTR gene translates into CFTR dysfunction. CFTR bioassays can aid in the diagnosis of individuals with CF, but we lack parameters to differentiate CF from CFTR-RD. In the era of the CFTR modulators and their potential clinical benefit, it is of utmost importance to diagnose CFTR-RD as unambiguously as possible. We therefore propose the following to define compatible CFTR dysfunction in a person with a suspected diagnosis of CFTR-RD :
      (1) evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or
      (2) One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types, or
      (3) Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant.

      1. Introduction

      Since the first descriptions of cystic fibrosis (CF) in the 1930s and following the cloning of the CFTR (cystic fibrosis transmembrane conductance regulator) gene in 1989, there have been continuous efforts to improve diagnostic tools in order to identify CF promptly and accurately [
      • Kerem B.
      • Rommens J.M.
      • Buchanan J.A.
      • Markiewicz D.
      • Cox T.K.
      • Chakravarti A.
      • et al.
      Identification of the cystic fibrosis gene: genetic analysis.
      ,
      • Bell S.C.
      • Mall M.A.
      • Gutierrez H.
      • Macek M.
      • Madge S.
      • Davies J.C.
      • et al.
      The future of cystic fibrosis care: a global perspective.
      ]. Diagnosis of individuals with classic forms of CF is straightforward in most cases, either after newborn screening (NBS) or based on symptoms. Nevertheless, as physicians also face challenging diagnostic dilemmas in individuals with inconclusive sweat test results and/or mono-organ clinical presentations, analysis of sequence variants in the CFTR gene has become part of diagnostic criteria, making diagnosis easier and faster in previously unsolved cases when it results in the discovery of 2 CF-causing variants [
      • Rosenstein B.J.
      • Cutting G.R.
      The diagnosis of cystic fibrosis: a consensus statement. Cystic fibrosis foundation consensus panel.
      ]. Since then, many CFTR gene variants have been identified in the context of clinical situations evocative of CF that do not meet the standard CF diagnosis criteria. Some of these conditions have been named CFTR-related disorders (CFTR-RD) and are part of the spectrum of diseases associated with CFTR dysfunction [
      • Bombieri C.
      • Claustres M.
      • De Boeck K.
      • Derichs N.
      • Dodge J.
      • Girodon E.
      • et al.
      Recommendations for the classification of diseases as CFTR-related disorders.
      ].
      Ten years after the publication of the international recommendations for the classification of CFTR-RD [
      • Bombieri C.
      • Claustres M.
      • De Boeck K.
      • Derichs N.
      • Dodge J.
      • Girodon E.
      • et al.
      Recommendations for the classification of diseases as CFTR-related disorders.
      ], the diagnosis of these disorders still poses significant challenges, where attributing some cases to the CFTR-RD spectrum of disease can be extremely difficult. Besides congenital bilateral absence of the vas deferens (CBAVD) and some cases of acute recurrent or chronic pancreatitis and disseminated bronchiectasis, and other clinical disease manifestations may also be “CFTR-related” and need an appropriate clinical evaluation. Moreover, extensive genetic analysis in some individuals with CF-like symptoms has led to an increasing number of cases where a CFTR variant is encountered on only one allele [
      • Wang X.
      • Moylan B.
      • Leopold D.A.
      • Kim J.
      • Rubenstein R.C.
      • Togias A.
      • et al.
      Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population.
      ,
      • Miller P.W.
      • Hamosh A.
      • Macek Jr., M.
      • Greenberger P.A.
      • MacLean J.
      • Walden S.M.
      • et al.
      Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis.
      ,
      • Miller A.C.
      • Comellas A.P.
      • Hornick D.B.
      • Stoltz D.A.
      • Cavanaugh J.E.
      • Gerke A.K.
      • et al.
      Cystic fibrosis carriers are at increased risk for a wide range of cystic fibrosis-related conditions.
      ]. Finally, some asymptomatic infants detected by NBS, labelled ‘CRMS/CFSPID’ (CFTR related metabolic syndrome or CF screen positive, inconclusive diagnosis) may be at risk of developing CFTR-RD later in life (8). Some infants remain asymptomatic, while others manifest symptoms later in life, resulting in the criteria for a diagnosis of CF. Alternatively, the presence of limited disease may warrant a diagnosis of CFTR–RD. The observation that CFTR variants associated with CFTR-RD can be observed in trans of a CF-causing variant in infants with CRMS/CFSPID provides a molecular rationale for follow up of these patients [
      • Levy H.
      • Nugent M.
      • Schneck K.
      • Stachiw-Hietpas D.
      • Laxova A.
      • Lakser O.
      • et al.
      Refining the continuum of CFTR-associated disorders in the era of newborn screening.
      ].
      The diagnosis of CFTR-RD requires biological criteria that include evidence of CFTR dysfunction and/or the presence of CFTR variants with clinical consequences. However, diagnostic criteria, including sweat test, genotype and diagnostic cut-offs for adjunctive, mainly electro-physiologically based measurements of CFTR function are not as standardized as they were developed for CF diagnosis. Moreover, in or ex vivo CFTR functional tests such as Nasal Potential Difference or Intestinal Current Measurement in Ussing chambers are not available at most centers or to all patients, especially those with mono-organ disorders, who may not be referred to CF centers. Likewise, the extent of CFTR gene studies vary, depending on countries or regions. Finally, and most importantly, the level of residual CFTR function qualifying for CFTR-RD has not yet been clarified.
      To seek clarification of these issues, the European Cystic Fibrosis Society gathered experts from Europe and North America. Here, we present the consensus recommendations for the evaluation of CFTR dysfunction according to different biomarkers that may be used for standardized classification of CFTR-RD. This manuscript is part of a series of papers aiming to describe the CFTR-RD spectrum.
      To ensure review of relevant literature, a comprehensive search strategy was undertaken. The search yielded 3140 references, of which 2466 were discarded as not relevant or overlapping. The remaining 674 references were reviewed by the core group and an additional 540 discarded. Reviews and case reports were rejected. The papers were independently assessed for quality by respective expert ranking them according a standardized SOP (https://www.has-sante.fr › docs › pdf › analiterat), including (i) clear statement of objectives and inclusion criteria, (ii) definition of the study population, (iii) presence of a control population, (iv) phenotypic characterization of the patients, (v) description of the statistical analysis, (vi) mutation analysis for all the groups, (vii) extensive mutation analysis.

      2. Definition of CFTR-RD related CFTR dysfunction

      CFTR-related disorders are clinical entities with features of CF and evidence of CFTR dysfunction but not meeting criteria for a CF diagnosis [
      • Bombieri C.
      • Claustres M.
      • De Boeck K.
      • Derichs N.
      • Dodge J.
      • Girodon E.
      • et al.
      Recommendations for the classification of diseases as CFTR-related disorders.
      ]. The clinical entities suggestive of a diagnosis of CFTR-RD are discussed in the third paper of this serie. In this article, we discuss the levels of CFTR dysfunction whose alteration are consistent with a diagnosis of CFTR-RD. This includes in vivo and ex vivo measurements and/or presence of CFTR variants, whose alteration in sequence is associated with a level of CFTR dysfunction evocative of CFTR-RD.
      This document recommends that CFTR dysfunction compatible with a diagnosis of CFTR-RD may be defined as:
      • (1)
        Evidence of CFTR dysfunction in vivo or ex vivo in at least two different CFTR functional test types
      • or
      • (1)
        One CFTR variant known to reduce CFTR function and evidence of CFTR dysfunction in vivo or ex vivo in at least two functional test types
      • or
      • (1)
        Two CFTR variants shown to reduce CFTR function, with at most one CF-causing variant
      The increasing availability of phenotypic and genotypic data has aided the recognition, understanding and improved description of a spectrum of disorders involving CFTR dysfunction. There is a continuous gradient of CFTR function according to the combination of two allelic CFTR variants (Fig. 1) [
      • Levy H.
      • Nugent M.
      • Schneck K.
      • Stachiw-Hietpas D.
      • Laxova A.
      • Lakser O.
      • et al.
      Refining the continuum of CFTR-associated disorders in the era of newborn screening.
      ]. Although most of the data available rely on cell lines or CFTR transcript level, the following lines of evidence can be drawn. Traditionally, Cystic Fibrosis is characterized by absence or near absence of CFTR function [
      • Levy H.
      • Nugent M.
      • Schneck K.
      • Stachiw-Hietpas D.
      • Laxova A.
      • Lakser O.
      • et al.
      Refining the continuum of CFTR-associated disorders in the era of newborn screening.
      ]. Exocrine pancreatic sufficiency and milder lung disease are observed when CFTR function is increased to 3–5% of Wild Type (WT) CFTR function [
      • Ramalho A.S.
      • Beck S.
      • Meyer M.
      • Penque D.
      • Cutting G.R.
      • Amaral M.D.
      Five percent of normal cystic fibrosis transmembrane conductance regulator mRNA ameliorates the severity of pulmonary disease in cystic fibrosis.
      ,
      • Chillon M.
      • Dork T.
      • Casals T.
      • Gimenez J.
      • Fonknechten N.
      • Will K.
      • et al.
      A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA–>G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.
      ,
      • Van Goor F.
      • Yu H.
      • Burton B.
      • Hoffman B.J
      Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
      ,
      • Highsmith W.E.
      • Burch L.H.
      • Zhou Z.
      • Olsen J.C.
      • Boat T.E.
      • Spock A.
      • et al.
      A novel mutation in the cystic fibrosis gene in patients with pulmonary disease but normal sweat chloride concentrations.
      ,
      • Kerem E.
      • Rave-Harel N.
      • Augarten A.
      • Madgar I.
      • Nissim-Rafinia M.
      • Yahav Y.
      • et al.
      A cystic fibrosis transmembrane conductance regulator splice variant with partial penetrance associated with variable cystic fibrosis presentations.
      ,
      • Chiba-Falek O.
      • Kerem E.
      • Shoshani T.
      • Aviram M.
      • Augarten A.
      • Bentur L.
      • et al.
      The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10kb C–>T mutation.
      ]. In cell cultures, normal transepithelial chloride transport is usually observed when CFTR function measured in human airway epithelial cells ex vivo exceeds 10% of WT [
      • Dannhoffer L.
      • Blouquit-Laye S.
      • Regnier A.
      • Chinet T.
      Functional properties of mixed cystic fibrosis and normal bronchial epithelial cell cultures.
      ,
      • Johnson L.G.
      • Olsen J.C.
      • Sarkadi B.
      • Moore K.L.
      • Swanstrom R.
      • Boucher R.C.
      Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis.
      ,
      • Farmen S.L.
      • Karp P.H.
      • Ng P.
      • Palmer D.J.
      • Koehler D.R.
      • Hu J.
      • et al.
      Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR.
      ]. Beyond this threshold, CFTR-RDs such as CBAVD, acute recurrent/chronic pancreatitis, and chronic sino-pulmonary disease may be observed [
      • Wilschanski M.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Zielenski J.
      • Tullis E.
      • et al.
      Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials.
      ,
      • LaRusch J.
      • Jung J.
      • General I.J.
      • Lewis M.D.
      • Park H.W.
      • Brand R.E.
      • et al.
      Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
      ,
      • Girodon E.
      • Cazeneuve C.
      • Lebargy F.
      • Chinet T.
      • Costes B.
      • Ghanem N.
      • et al.
      CFTR gene mutations in adults with disseminated bronchiectasis.
      ,
      • Davis P.B.
      Cystic fibrosis.
      ]. Above 30% WT, the occurrence of CFTR-RD seems to be rare based on reports from the large database CFTR2 and in vitro experiments [
      • Pagani F.
      • Stuani C.
      • Tzetis M.
      • Kanavakis E.
      • Efthymiadou A.
      • Doudounakis S.
      • et al.
      New type of disease causing mutations: the example of the composite exonic regulatory elements of splicing in CFTR exon 12.
      ,
      • Mak V.
      • Jarvi K.A.
      • Zielenski J.
      • Durie P.
      • Tsui L.C.
      Higher proportion of intact exon 9 CFTR mRNA in nasal epithelium compared with vas deferens.
      ,
      • McCague A.F.
      • Raraigh K.S.
      • Pellicore M.J.
      • Davis-Marcisak E.F.
      • Evans T.A.
      • Han S.T.
      • et al.
      Correlating cystic fibrosis transmembrane conductance regulator function with clinical features to inform precision treatment of cystic fibrosis.
      ,
      • Zhang L.
      • Button B.
      • Gabriel S.E.
      • Burkett S.
      • Yan Y.
      • Skiadopoulos M.H.
      • et al.
      CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium.
      ]. Besides CF and CFTR-RDs, there are clinical conditions, which do not follow Mendelian inheritance pattern, such as bronchial asthma, chronic rhinosinusitis, allergic broncho-pulmonary aspergillosis, recurrent pancreatitis or primary sclerosing cholangitis, where the proportion of CF carriers is higher than in the general population [
      • Miller P.W.
      • Hamosh A.
      • Macek Jr., M.
      • Greenberger P.A.
      • MacLean J.
      • Walden S.M.
      • et al.
      Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations in allergic bronchopulmonary aspergillosis.
      ,
      • Miller A.C.
      • Comellas A.P.
      • Hornick D.B.
      • Stoltz D.A.
      • Cavanaugh J.E.
      • Gerke A.K.
      • et al.
      Cystic fibrosis carriers are at increased risk for a wide range of cystic fibrosis-related conditions.
      ,
      • Groman J.D.
      • Meyer M.E.
      • Wilmott R.W.
      • Zeitlin P.L.
      • Cutting G.R.
      Variant cystic fibrosis phenotypes in the absence of CFTR mutations.
      ,
      • Castellani C.
      • Quinzii C.
      • Altieri S.
      • Mastella G.
      • Assael B.M.
      A pilot survey of cystic fibrosis clinical manifestations in CFTR mutation heterozygotes.
      ,
      • Werlin S.
      • Scotet V.
      • Uguen K.
      • Audrezet M.P.
      • Cohen M.
      • Yaakov Y.
      • et al.
      Primary sclerosing cholangitis is associated with abnormalities in CFTR.
      ,
      • Esposito M.V.
      • Aveta A.
      • Comegna M.
      • Cernera G.
      • Iacotucci P.
      • Carnovale V.
      • et al.
      Extensive CFTR gene analysis revealed a higher occurrence of cystic fibrosis transmembrane regulator-related disorders (CFTR-RD) among CF carriers.
      ,
      • Ooi C.Y.
      • Castellani C.
      • Keenan K.
      • Avolio J.
      • Volpi S.
      • Boland M.
      • et al.
      Inconclusive diagnosis of cystic fibrosis after newborn screening.
      ]. Although some of these patients can be reclassified as having a CFTR-RD based on extensive genetic screening or functional analysis, these observations raise the question whether decrease of CFTR function at 50% of WT can contribute to organ dysfunction in the context of a multifactorial disease mechanism which includes non-CFTR genetic factors and environmental influence. These CFTR-associated disorders are described in the third paper of this serie.
      Fig 1
      Fig. 1Spectrum of CFTR disorders
      The phenotypic classification and the variant combination is expressed according to the level of CFTR activity in vivo. A patient may be asymptomatic with a CFTR activity below 50%. He/she is considered at risk to develop a CFTR-RD later in life.
      The variant combination is shown according to the anticipated level of CFTR function.
      « CF » is a CFTR variant whose CFTR activity is anticipated between 0 and 10% of normal.
      « VVCC » is a CFTR variant with residual CFTR function above 10% of normal CFTR activity. The range of function in these variants may cause CF, CFTR-RD, or no disease depending on the penetrance.
      « WT » is the reference with no CFTR variant.
      Sweat Chloride (Cl) concentration is shown according to the level of CFTR function.

      2.1 Other factors than CFTR related chloride transport involved in CFTR-RD

      CFTR dysfunction, not only impairs Cl secretion or absorption, but also involves a bicarbonate (HCO3-) transport defect. It has been proposed that CFTR variants that selectively alter HCO3- transport preferentially affect the pancreas and the vas deferens [
      • LaRusch J.
      • Jung J.
      • General I.J.
      • Lewis M.D.
      • Park H.W.
      • Brand R.E.
      • et al.
      Mechanisms of CFTR functional variants that impair regulated bicarbonate permeation and increase risk for pancreatitis but not for cystic fibrosis.
      ,
      • Bernardino R.L.
      • Jesus T.T.
      • Martins A.D.
      • Sousa M.
      • Barros A.
      • Cavaco J.E.
      • et al.
      Molecular basis of bicarbonate membrane transport in the male reproductive tract.
      ,
      • Laselva O.
      • Moraes T.J.
      • He G.
      • Bartlett C.
      • Szarics I.
      • Ouyang H.
      • et al.
      The CFTR mutation c.3453G >C (D1152H) confers an anion selectivity defect in primary airway tissue that can be rescued by ivacaftor.
      ]. The evidence that some CF-causing CFTR variants result in normal Cl transport but with a remaining elevated Na+ reabsorption indicates that excessive ENaC activity can also contribute to CFTR related epithelial dysfunction [
      • Donaldson S.H.
      • Boucher R.C.
      Sodium channels and cystic fibrosis.
      ]. Moreover, the consequences of CFTR gene variants may differ according to the sensitivity of an organ to CFTR dysfunction, the abundance of CFTR expression and consequently the amount of mature complex glycosylated CFTR, as well as the specific role of CFTR in that organ and the presence of alternative channels [
      • Rave-Harel N.
      • Kerem E.
      • Nissim-Rafinia M.
      • Madjar I.
      • Goshen R.
      • Augarten A.
      • et al.
      The molecular basis of partial penetrance of splicing mutations in cystic fibrosis.
      ]. For example, Wolffian tissues, which constitutively produce less full-length CFTR mRNAs than the respiratory tissue due to differential splicing efficiency, appear to be more sensitive to CFTR dysfunction, as compared to bronchial epithelium [
      • Rave-Harel N.
      • Kerem E.
      • Nissim-Rafinia M.
      • Madjar I.
      • Goshen R.
      • Augarten A.
      • et al.
      The molecular basis of partial penetrance of splicing mutations in cystic fibrosis.
      ,
      • Chiba-Falek O.
      • Parad R.B.
      • Kerem E.
      • Kerem B.
      Variable levels of normal RNA in different fetal organs carrying a cystic fibrosis transmembrane conductance regulator splicing mutation.
      ,
      • Noone P.G.
      • Pue C.A.
      • Zhou Z.
      • Friedman K.J.
      • Wakeling E.L.
      • Ganeshananthan M.
      • et al.
      Lung disease associated with the IVS8 5T allele of the CFTR gene.
      ].
      At the patient level, CFTR activity and transepithelial fluid transport might be also mitigated by the activity of other transporters (e.g. SLC26A9, ENaC, TMEM16A for ion transport, SLC26A4, SLC26A3 for HCO3- transport) [
      • Sermet-Gaudelus I.
      • Edelman A.
      • Fajac I.
      Channelopathies in bronchiectasis.
      ,
      • Bakouh N.
      • Bienvenu T.
      • Thomas A.
      • Ehrenfeld J.
      • Liote H.
      • Roussel D.
      • et al.
      Characterization of SLC26A9 in patients with CF-like lung disease.
      ]. Other gene variants such as SPINK1 for pancreatitis or ADGRG2 for CBAVD [
      • Masson E.
      • Chen J.M.
      • Audrezet M.P.
      • Cooper D.N.
      • Ferec C.
      A conservative assessment of the major genetic causes of idiopathic chronic pancreatitis: data from a comprehensive analysis of PRSS1, SPINK1, CTRC and CFTR genes in 253 young French patients.
      ,
      • Pagin A.
      • Bergougnoux A.
      • Girodon E.
      • Reboul M.P.
      • Willoquaux C.
      • Kesteloot M.
      • et al.
      Novel ADGRG2 truncating variants in patients with X-linked congenital absence of vas deferens.
      ] may contribute as well as epithelial physiopathology such as aging (senescence) and environmental factors such as exposure to tobacco smoke [
      • Patel S.D.
      • Bono T.R.
      • Rowe S.M.
      • Solomon G.M.
      CFTR targeted therapies: recent advances in cystic fibrosis and possibilities in other diseases of the airways.
      ]. This interplay between genetic and external factors probably modulates the disease phenotype and course along an individual life span, explaining transition from CFTR-RD to CF in some individuals, but not others, though they may carry the same genotype. For example, cigarette smoke decreases the expression of the CFTR gene, causes protein destabilization, and gated channel dysfunction in vitro [
      • Sloane P.A.
      • Shastry S.
      • Wilhelm A.
      • Courville C.
      • Tang L.P.
      • Backer K.
      • et al.
      A pharmacologic approach to acquired cystic fibrosis transmembrane conductance regulator dysfunction in smoking related lung disease.
      ].
      As a whole, CFTR genotyping and CFTR function provide the diagnosis of CFTR-RD [
      • Raraigh K.S.
      • Han S.T.
      • Davis E.
      • Evans T.A.
      • Pellicore M.J.
      • McCague A.F.
      • et al.
      Functional assays are essential for interpretation of missense variants associated with variable expressivity.
      ]. The principal assays to approximate CFTR function testing are performed in vivo (sweat testing, nasal potential difference (NPD) measurements) or ex vivo (intestinal current measurement (ICM)).
      Depending on the level of CFTR dysfunction and/or the evidence of CFTR variants with clinical consequences, the diagnosis of CF and/or CFTR-RD may be confirmed, uncertain or unlikely (Table 1). In case of uncertain diagnosis, -a single functional test in the CFTR-RD range (usually sweat test) and none or a single CFTR variant identified-, additional functional tests of CFTR should be performed. Some CFTR-RD patients may be outside these guidelines and should be discussed case by case.
      Table 1CFTR-RD definition according to genotype and in vivo CFTR dysfunction.
      CFTR genotype
      in vivo evidence of CFTR dysfunction below the threshold of CF0 variant1 disease-causing variant2 allelic disease-causing variants**, at most 1 CF-causing
      Noruled outunlikely*confirmed
      Yes, 1 testuncertain *uncertain *confirmed
      Yes, at least 2 testsconfirmedconfirmedconfirmed
      * depending on the results of the CFTR functional tests performed and the extent of the genetic analysis.
      **: Disease-causing variants include all variants causing CFTR dysfunction, either CF or CFTR-RD.

      2.2 CFTR genotype as a surrogate marker for CFTR-RD

      Following international standards for variant interpretation, criteria for considering variants as causing CFTR-RD are based on clinical, epidemiological and functional data (Table 1 supplemental) [
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • et al.
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American college of medical genetics and genomics and the association for molecular pathology.
      ]. It must be clarified initially that (i) disease-causing variants include all variants causing CFTR dysfunction, either CF or CFTR-RD and (ii) variants found in patients with CFTR-RD do not exclusively cause CFTR-RD. Some can also cause CF while others are mainly found in patients with CFTR-RD or in asymptomatic subjects who can become symptomatic later in life. Criteria for considering variants as CFTR-RD-causing include the following lines of evidence [
      • Castellani C.
      • Cuppens H.
      • Macek Jr., M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ,
      • Dequeker E.
      • Stuhrmann M.
      • Morris M.A.
      • Casals T.
      • Castellani C.
      • Claustres M.
      • et al.
      Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders–updated European recommendations.
      ].
      The range of symptoms described in those patients is the first step of characterization. In the CFTR2 database, which collects clinical data and sweat chloride from patients with CF enrolled in national registries and CF Centers [
      • Sosnay P.R.
      • Siklosi K.R.
      • Van Goor F.
      • Kaniecki K.
      • Yu H.
      • Sharma N.
      • et al.
      Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene.
      ], variants causing a CFTR-RD may be found either in the category of Variants of Varying Clinical Consequence (VVCC) or in that of Non CF-Causing Variants. Others may be Variants of Unknown Significance (VUS) and may be reclassified as neutral or “disease causing” after future functional evaluation. Complementary to CFTR2, CFTR-France collects data from patients with CF but also patients with CFTR-RDs and thus individualizes a specific class of CFTR-RD variants [
      • Claustres M.
      • Theze C.
      • des Georges M.
      • Baux D.
      • Girodon E.
      • Bienvenu T.
      • et al.
      CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.
      ]. Other general databases such as ClinVar [
      • Landrum M.J.
      • Chitipiralla S.
      • Brown G.R.
      • Chen C.
      • Gu B.
      • Hart J.
      • et al.
      ClinVar: improvements to accessing data.
      ], and Human Gene Mutation Database ® [
      • Stenson P.D.
      • Mort M.
      • Ball E.V.
      • Chapman M.
      • Evans K.
      • Azevedo L.
      • et al.
      The human gene mutation database (HGMD((R))): optimizing its use in a clinical diagnostic or research setting.
      ], also provide clinical information and variant classification, mostly based on literature data, but numerous variants have been over classified as “pathogenic” [
      • Boussaroque A.
      • Bergougnoux A.
      • Raynal C.
      • Audrezet M.P.
      • Sasorith S.
      • Ferec C.
      • et al.
      Pitfalls in the interpretation of CFTR variants in the context of incidental findings.
      ], without discrimination between CF and CFTR-RD causing, frequently assigned in the CF category. This is an important issue for genetic counselling, because variants with a high penetrance for CFTR-RD are generally not considered for cascade testing in relatives or prenatal diagnosis of CF [
      • Dequeker E.
      • Stuhrmann M.
      • Morris M.A.
      • Casals T.
      • Castellani C.
      • Claustres M.
      • et al.
      Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders–updated European recommendations.
      ].
      The second criterion relies on epidemiological data, thanks to large reference datasets in the general population such as gnomAD [
      • Raraigh K.S.
      • Aksit M.A.
      • Hetrick K.
      • Pace R.G.
      • Ling H.
      • O'Neal W.
      • et al.
      Complete CFTR gene sequencing in 5058 individuals with cystic fibrosis informs variant-specific treatment.
      ,
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alfoldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      ] and for affected individuals such as CFTR2 and CFTR-France. By comparing the prevalence of CFTR variants in the general and affected populations, it is possible to infer the probability of pathogenicity of a variant using a Bayesian approach [
      • Collaco J.M.
      • Raraigh K.S.
      • Betz J.
      • Aksit M.A.
      • Blau N.
      • Brown J.
      • et al.
      Accurate assignment of disease liability to genetic variants using only population data.
      ]. A higher frequency of a variant in patients with a CFTR-RD than in the healthy control population is a strong argument to consider a variant as CFTR-RD-causing. This approach is accurate when applied to variants that individually have a high likelihood of causing CF or CFTR-RD but less accurate when other variants have to be present in the same gene (so called complex alleles) to cause disease [
      • Rohlfs E.M.
      • Zhou Z.
      • Sugarman E.A.
      • Heim R.A.
      • Pace R.G.
      • Knowles M.R.
      • et al.
      The I148T CFTR allele occurs on multiple haplotypes: a complex allele is associated with cystic fibrosis.
      ,
      • Groman J.D.
      • Hefferon T.W.
      • Casals T.
      • Bassas L.
      • Estivill X.
      • Des Georges M.
      • et al.
      Variation in a repeat sequence determines whether a common variant of the cystic fibrosis transmembrane conductance regulator gene is pathogenic or benign.
      ]. Accuracy is also decreased when variants cause modest reductions in CFTR function (so called hypomorphic variants) and are dependent on the presence of other factors such as genetic or environmental modifiers to develop disease, such as the R117H variant. Variants above 2% frequency are very unlikely to cause either CF or CFTR-RD, as is the case for c.1666A>G (p.lle556Val) (allelic frequency of 4.7% in the Asian population). Penetrance analysis can help evaluate variant potential pathogenicity. For example, in individuals carrying the F508del/R117H;T7 compound heterozygous genotype, the penetrance of CFTR-RD has been documented as low as 3% [
      • Boussaroque A.
      • Audrezet M.P.
      • Raynal C.
      • Sermet-Gaudelus I.
      • Bienvenu T.
      • Ferec C.
      • et al.
      Penetrance is a critical parameter for assessing the disease liability of CFTR variants.
      ,
      • Thauvin-Robinet C.
      • Munck A.
      • Huet F.
      • Genin E.
      • Bellis G.
      • Gautier E.
      • et al.
      The very low penetrance of cystic fibrosis for the R117H mutation: a reappraisal for genetic counselling and newborn screening.
      ]. CFTR-France reports observations of compound heterozygous genotypes in asymptomatic subjects, thus providing some insight for penetrance of given variants. However, penetrance estimates are lacking for most CFTR variants, especially those associated primarily with CFTR-RD.
      The third criterion, the level of CFTR dysfunction, is based on evidence of a residual CFTR activity in vivo (e.g.; combining the variants in the two alleles) above 10% and below 50% of the normal [
      • Van Goor F.
      • Yu H.
      • Burton B.
      • Hoffman B.J
      Effect of ivacaftor on CFTR forms with missense mutations associated with defects in protein processing or function.
      ,
      • Dannhoffer L.
      • Blouquit-Laye S.
      • Regnier A.
      • Chinet T.
      Functional properties of mixed cystic fibrosis and normal bronchial epithelial cell cultures.
      ,
      • McCague A.F.
      • Raraigh K.S.
      • Pellicore M.J.
      • Davis-Marcisak E.F.
      • Evans T.A.
      • Han S.T.
      • et al.
      Correlating cystic fibrosis transmembrane conductance regulator function with clinical features to inform precision treatment of cystic fibrosis.
      ,
      • Castellani C.
      • Cuppens H.
      • Macek Jr., M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ]; however these thresholds may be challenged by CFTR functional studies at the individual level. Additionally, the impact on CFTR mRNA expression and splicing efficiency may be evaluated in vitro by minigene experiments and in vivo by assessing mRNA level in cells or tissues [
      • Martin N.
      • Bergougnoux A.
      • Baatallah N.
      • Chevalier B.
      • Varilh J.
      • et al.
      Exon identity influences splicing induced by exonic variants and in silico prediction efficacy.
      ,
      • Raynal C.
      • Baux D.
      • Theze C.
      • Bareil C.
      • Taulan M.
      • Roux A.F.
      • et al.
      A classification model relative to splicing for variants of unknown clinical significance: application to the CFTR gene.
      ,
      • Sharma N.
      • Sosnay P.R.
      • Ramalho A.S.
      • Douville C.
      • Franca A.
      • Gottschalk L.B.
      • et al.
      Experimental assessment of splicing variants using expression minigenes and comparison with in silico predictions.
      ,
      • Felicio V.
      • Ramalho A.S.
      • Igreja S.
      • Amaral M.D.
      mRNA-based detection of rare CFTR mutations improves genetic diagnosis of cystic fibrosis in populations with high genetic heterogeneity.
      ]. The impact on CFTR protein synthesis, maturation and function may be studied in vitro in different cell systems which can be transfected by DNA [
      • McCague A.F.
      • Raraigh K.S.
      • Pellicore M.J.
      • Davis-Marcisak E.F.
      • Evans T.A.
      • Han S.T.
      • et al.
      Correlating cystic fibrosis transmembrane conductance regulator function with clinical features to inform precision treatment of cystic fibrosis.
      ,
      • Dequeker E.
      • Stuhrmann M.
      • Morris M.A.
      • Casals T.
      • Castellani C.
      • Claustres M.
      • et al.
      Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders–updated European recommendations.
      ]. Data on CFTR expression and function derived from primary human epithelial cells can also be informative in this regard, whether derived from the gastrointestinal or respiratory tract. However, virtually no assay reflects the full biological function of the CFTR protein due to its variety of cellular functions and differences in susceptibility to CFTR dysfunction among different organs. Alternatively, CFTR protein abundance or biosynthesis may be assessed in patients’ biopsies retrieved from nasal or rectal mucosa [
      • Joynt A.T.
      • Evans T.A.
      • Pellicore M.J.
      • Davis-Marcisak E.F.
      • Aksit M.A.
      • Eastman A.C.
      • et al.
      Evaluation of both exonic and intronic variants for effects on RNA splicing allows for accurate assessment of the effectiveness of precision therapies.
      ,
      • Schucht S.
      • Minso R.
      • Lex C.
      • Reiss J.
      • Stanke F.
      • Tamm S.
      • et al.
      Functional analysis of the p.[Arg74Trp;Val201Met;Asp1270Asn]/p.Phe508del CFTR mutation genotype in human native colon.
      ,
      • van Barneveld A.
      • Stanke F.
      • Tamm S.
      • Siebert B.
      • Brandes G.
      • Derichs N.
      • et al.
      Functional analysis of F508del CFTR in native human colon.
      ].
      In silico tools may help in situations where there is virtually no available data [
      • van Barneveld A.
      • Stanke F.
      • Ballmann M.
      • Naim H.Y.
      • Tummler B.
      Ex vivo biochemical analysis of CFTR in human rectal biopsies.
      ,
      • Sasorith S.
      • Baux D.
      • Bergougnoux A.
      • Paulet D.
      • Lahure A.
      • Bareil C.
      • et al.
      The CYSMA web server: an example of integrative tool for in silico analysis of missense variants identified in Mendelian disorders.
      ,
      • Baux D.
      • Van Goethem C.
      • Ardouin O.
      • Guignard T.
      • Bergougnoux A.
      • Koenig M.
      • et al.
      MobiDetails: online DNA variants interpretation.
      ]. However, they exhibit significant limitations for CFTR variants with a high degree of uncertainty, and thus their interpretation must be considered with caution, in particular, with regard to CFTR-RD variants [
      • Castellani C.
      • Cuppens H.
      • Macek Jr., M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ,
      • Boussaroque A.
      • Bergougnoux A.
      • Raynal C.
      • Audrezet M.P.
      • Sasorith S.
      • Ferec C.
      • et al.
      Pitfalls in the interpretation of CFTR variants in the context of incidental findings.
      ].
      A CFTR-RD genotype can be considered as a surrogate marker of CFTR dysfunction based on variant pathogenicity. A CF-causing variant is a CFTR variant which causes CF when combined in trans with another CF-causing variant. A variant of Varying Clinical Consequence (VCC) is a CFTR variant with residual CFTR function. When combined in trans with a CF-causing variant, it can cause CF or CFTR-RD but can be also found in asymptomatic subjects. Depending on CFTR function alteration, VCC variants may have a higher penetrance for CF (high risk to develop CF), for CFTR-RD (high risk to develop a CFTR-RD) but may be also identified in asymptomatic subjects (low risk to develop a CFTR-RD and minimal risk to develop CF). Indeed, a number of VCC variants are frequently observed in individuals with CFTR-RD when in trans with a CF-causing variant, with a low risk to develop into CF, e.g.; R117H;T7, TG12T5, F1052V [
      • Bombieri C.
      • Claustres M.
      • De Boeck K.
      • Derichs N.
      • Dodge J.
      • Girodon E.
      • et al.
      Recommendations for the classification of diseases as CFTR-related disorders.
      ,
      • Castellani C.
      • Cuppens H.
      • Macek Jr., M.
      • Cassiman J.J.
      • Kerem E.
      • Durie P.
      • et al.
      Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice.
      ,
      • Dequeker E.
      • Stuhrmann M.
      • Morris M.A.
      • Casals T.
      • Castellani C.
      • Claustres M.
      • et al.
      Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders–updated European recommendations.
      ,
      • Kopanos C.
      • Tsiolkas V.
      • Kouris A.
      • Chapple C.E.
      • Albarca Aguilera M.
      • Meyer R.
      • et al.
      VarSome: the human genomic variant search engine.
      ]. In contrast, another group of variants also found in asymptomatic individuals are associated with a very low risk of developing a symptomatic disease (very low penetrance of CFTR-RD) when in trans with a CF-causing defect e.g.; TG11T5 or Q1352H.
      A list of variants frequently identified in patients with CFTR-RD is provided in Table 2 based on CFTR-2, CFTR-France and gnomAD [
      • Sosnay P.R.
      • Siklosi K.R.
      • Van Goor F.
      • Kaniecki K.
      • Yu H.
      • Sharma N.
      • et al.
      Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene.
      ,
      • Claustres M.
      • Theze C.
      • des Georges M.
      • Baux D.
      • Girodon E.
      • Bienvenu T.
      • et al.
      CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.
      ,
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alfoldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      ]. This list, aiming to be as exhaustive as possible, incorporates VCC variants according to their penetrance for CF or CFTR-RD, as well as some variants called “non disease causing” in CFTR2 because they can be found in patients with CFTR-RD. While there are mechanistic molecular genetic criteria for assessing a variant as CF-causing according to variant types (nonsense, frameshift, large deletion…), criteria for a CFTR-RD-causing defect are lacking. The majority of them are missense variants and splicing variants located outside the canonical acceptor and donor splice sites or sequence variants in deep intron regions [
      • Raraigh K.S.
      • Aksit M.A.
      • Hetrick K.
      • Pace R.G.
      • Ling H.
      • O'Neal W.
      • et al.
      Complete CFTR gene sequencing in 5058 individuals with cystic fibrosis informs variant-specific treatment.
      ]. In addition, there are a number of frequent complex alleles in CFTR-RD that combine at least two variants in cis on the same parental allele [
      • Claustres M.
      • Theze C.
      • des Georges M.
      • Baux D.
      • Girodon E.
      • Bienvenu T.
      • et al.
      CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.
      ]. At the individual level, a genotype compatible with a CFTR-RD typically combines a variant known to cause a CFTR-RD, as listed in Table 2, and on the other allele either a CF-causing or another variant known to cause a CFTR-RD.
      Table 2Variants frequently encountered in patients with CFTR related disorders.
      Legacy nameHGVS name (cDNA, NM_000492.4)HGVS name (protein)rsID
      P5Lc.14C>Tp.Pro5Leurs193922501
      R74Wc.220C>Tp.Arg74Trprs115545701
      D110Ec.330C>Ap.Asp110Glurs397508537
      R117Gc.349C>Gp.Arg117Glyrs77834169
      R117H;T7c.350G>Ap.Arg117Hisrs78655421
      R117Lc.350G>Tp.Arg117Leurs78655421
      A120Tc.358G>Ap.Ala120Thrrs201958172
      621+3A>Gc.489+3A>GNo protein namers377729736
      G194Vc.581G>Tp.Gly194Valrs397508763
      V201M (complex allele with R74W and D1270N)c.601G>Ap.Val201Metrs138338446
      Q237Ec.709C>Gp.Gln237Glurs397508784
      R258Gc.772A>Gp.Arg258Glyrs191456345
      M265Rc.794T>Gp.Met265Argrs148519623
      1002–1113_1002–1110delc.870–1113_870–1110delNo protein nameNot found
      G314Ec.941G>Ap.Gly314Glurs75763344
      R334Qc.1001G>Ap.Arg334Glnrs397508137
      A349Vc.1046C>Tp.Ala349Valrs121909021
      R352Wc.1054C>Tp.Arg352Trprs193922497
      Q359Rc.1076A>Gp.Gln359Argrs397508153
      TG12T5c.1210–34_1210–6TG[12]T
      • Wang X.
      • Moylan B.
      • Leopold D.A.
      • Kim J.
      • Rubenstein R.C.
      • Togias A.
      • et al.
      Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population.
      No protein nameNot found
      TG13T5c.1210–34_1210–6TG[13]T
      • Wang X.
      • Moylan B.
      • Leopold D.A.
      • Kim J.
      • Rubenstein R.C.
      • Togias A.
      • et al.
      Mutation in the gene responsible for cystic fibrosis and predisposition to chronic rhinosinusitis in the general population.
      No protein nameNot found
      D443Y (complex allele with G576A and R668C)c.1327G>Tp.Asp443Tyrrs147422190
      C491Rc.1471T>Cp.Cys491Argrs397508213
      F575Yc.1724T>Ap.Phe575Tyrrs773569201
      D579Gc.1736A>Gp.Asp579Glyrs397508288
      E588Vc.1763A>Tp.Glu588Valrs397508297
      D614Gc.1841A>Gp.Asp614Glyrs201124247
      I618Tc.1853T>Cp.Ile618Thrrs139468767
      G622Dc.1865G>Ap.Gly622Asprs121908759
      P750Lc.2249C>Tp.Pro750Leurs140455771
      2789+2insAc.2657+2_2657+3insANo protein namers397508414
      D924Nc.2770G>Ap.Asp924Asnrs201759207
      R933Gc.2797A>Gp.Arg933Glyrs397508436
      V938Gc.2813T>Gp.Val938Glyrs193922511
      M952Tc.2855T>Cp.Met952Thrrs142773283
      M952Ic.2856G>Cp.Met952Ilers151048781
      L967Sc.2900T>Cp.Leu967Serrs1800110
      S977F (complex allele with TG12T5)c.2930C>Tp.Ser977Phers141033578
      I980Kc.2939T>Ap.Ile980Lysrs397508463
      L997Fc.2991G>Cp.Leu997Phers1800111
      Y1014Cc.3041A>Gp.Tyr1014Cysrs149279509
      F1016Sc.3047T>Cp.Phe1016Serrs397508488
      Y1032Cc.3095A>Gp.Tyr1032Cysrs144055758
      F1052Vc.3154T>Gp.Phe1052Valrs150212784
      G1069Rc.3205G>Ap.Gly1069Argrs200321110
      R1070Wc.3208C>Tp.Arg1070Trprs202179988
      R1070Q (complex allele with S466X)c.3209G>Ap.Arg1070Glnrs78769542
      F1074Lc.3222T>Ap.Phe1074Leurs186045772
      F1099Lc.3297C>Ap.Phe1099Leurs747754623
      D1152Hc.3454G>Cp.Asp1152Hisrs75541969
      V1153Ec.3458T>Ap.Val1153Glurs397508567
      T1246Ic.3737C>Tp.Thr1246Ilers397508600
      D1270Nc.3808G>Ap.Asp1270Asnrs11971167
      Q1291Rc.3872A>Gp.Gln1291Argrs397508621
      Q1291Hc.3873G>Cp.Gln1291Hisrs121909015
      Not applicablec.3874–4522A>GNo protein nameNot found
      N1303Ic.3908A>Tp.Asn1303Ilers397508636
      Q1352H(G>C)c.4056G>Cp.Gln1352Hisrs113857788
      S1455Xc.4364C>Gp.Ser1455Xrs121909043
      Q1476Xc.4426C>Tp.Gln1476Xrs374705585
      L1480Pc.4439T>Cp.Leu1480Prors758818611

      3. Sweat test as a surrogate marker for CFTR-dysfunction

      Sweat Chloride Concentration (SCC) reflects abnormal transepithelial salt transport resulting from defective chloride reabsorption by CFTR in the sweat duct [
      • Keenan K.
      • Dupuis A.
      • Griffin K.
      • Castellani C.
      • Tullis E.
      • Gonska T.
      Phenotypic spectrum of patients with cystic fibrosis and cystic fibrosis-related disease carrying p.Arg117His.
      ]. While CFTR dysfunction increases SCC, it does not result in inflammation or damage of the sweat gland, making SCC independent of disease progression [
      • Quinton P.M.
      Cystic fibrosis: lessons from the sweat gland.
      ,
      • Munger B.L.
      • Brusilow S.W.
      • Cooke R.E.
      An electron microscopic study of eccrine sweat glands in patients with cystic fibrosis of the pancreas.
      ]. The methodology for sweat collection and Cl measurements is well standardized and commercial equipment is available at a reasonable cost [
      • Cirilli N.
      • Southern K.W.
      • Barben J.
      • Vermeulen F.
      • Munck A.
      • Wilschanski M.
      • et al.
      Standards of care guidance for sweat testing; phase two of the ECFS quality improvement programme.
      ]. For these reasons, sweat Cl concentration is a useful biomarker to assess CFTR function in vivo in patients with CFTR-RD. A SCC >= 60 mmol/L is suggestive of CF, while a SCC below 30 mmol/L makes CF unlikely, leaving an ‘intermediate range’ between 30 and 59 mmol/L [
      • Farrell P.M.
      • White T.B.
      • Ren C.L.
      • Hempstead S.E.
      • Accurso F.
      • Derichs N.
      • et al.
      Diagnosis of cystic fibrosis: consensus guidelines from the cystic fibrosis foundation.
      ].

      3.1 Factors contributing to variability of sweat chloride concentration

      Sweat Chloride Concentration increases slightly with age in both healthy subjects, and in individuals with CF [
      • Mishra A.
      • Greaves R.
      • Smith K.
      • Carlin J.B.
      • Wootton A.
      • Stirling R.
      • et al.
      Diagnosis of cystic fibrosis by sweat testing: age-specific reference intervals.
      ,
      • Collaco J.M.
      • Blackman S.M.
      • Raraigh K.S.
      • Corvol H.
      • Rommens J.M.
      • Pace R.G.
      • et al.
      Sources of variation in sweat chloride measurements in cystic fibrosis.
      ]. Environmental factors may also influence SCC as was demonstrated in a study of 1761 twins/siblings with CF. Inter-subject differences in SCC was ascribed to the type of CFTR mutation (more than 50% of the variance), climate and family diet which contributed to 13% of the differences, and unique individual factors such as modifier genes or individual exposure factors (4% of the difference)[
      • Collaco J.M.
      • Blackman S.M.
      • Raraigh K.S.
      • Corvol H.
      • Rommens J.M.
      • Pace R.G.
      • et al.
      Sources of variation in sweat chloride measurements in cystic fibrosis.
      ]. Variation over time was noted, with 13% of the variability attributed to the within-subject variation over time and 10% to the test variability.
      Test variability on a properly executed sweat collection is typically low, with 95% limits of repeatability below +/- 5 mmol/L for SCC < 30 mmol/L, and +/- 10 mmol/L for SCC > 60 mmol/L in F508del homozygous CF patients [
      • Collaco J.M.
      • Blackman S.M.
      • Raraigh K.S.
      • Corvol H.
      • Rommens J.M.
      • Pace R.G.
      • et al.
      Sources of variation in sweat chloride measurements in cystic fibrosis.
      ,
      • LeGrys V.A.
      • Moon T.C.
      • Laux J.
      • Rock M.J.
      • Accurso F.
      Analytical and biological variation in repeated sweat chloride concentrations in clinical trials for CFTR modulator therapy.
      ,
      • Vermeulen F.
      • Lebecque P.
      • De Boeck K.
      • Leal T.
      Biological variability of the sweat chloride in diagnostic sweat tests: a retrospective analysis.
      ]. The variability is wider for SCC between 30 and 60 mmol/l, around +/- 20 mmol/L and may be more likely be affected by dietary and fluid intake [
      • Collaco J.M.
      • Blackman S.M.
      • Raraigh K.S.
      • Corvol H.
      • Rommens J.M.
      • Pace R.G.
      • et al.
      Sources of variation in sweat chloride measurements in cystic fibrosis.
      ,
      • LeGrys V.A.
      • Moon T.C.
      • Laux J.
      • Rock M.J.
      • Accurso F.
      Analytical and biological variation in repeated sweat chloride concentrations in clinical trials for CFTR modulator therapy.
      ]. In a cohort of patients with SCC in the intermediate range, only half of the sweat tests remained so when repeated [
      • Vermeulen F.
      • Lebecque P.
      • De Boeck K.
      • Leal T.
      Biological variability of the sweat chloride in diagnostic sweat tests: a retrospective analysis.
      ]. Similar results were reported by Cirilli et al., whereby patients with CF displayed a lower variability than healthy subjects (coefficient of variation 20.2% vs 31.1%) [
      • Cirilli N.
      • Raia V.
      • Rocco I.
      • De Gregorio F.
      • Tosco A.
      • Salvadori L.
      • et al.
      Intra-individual biological variation in sweat chloride concentrations in CF, CFTR dysfunction, and healthy pediatric subjects.
      ]. In subjects with CFTR-RD, values ranging from normal to CF were reported when SCC measurements were repeated [
      • Minso R.
      • Schulz A.
      • Dopfer C.
      • Alfeis N.
      • Barneveld A.V.
      • Makartian-Gyulumyan L.
      • et al.
      Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.
      ]. Evidence on when to repeat SCC and how to handle discordances between repeated SCC is lacking. Repeating the sweat test at least once, even if normal, seems warranted. Taking into account the most abnormal SCC value to decide on further assessment (CFTR genetics and bioassays) will optimize the sensitivity of the diagnostic algorithm, but at the cost of testing more individuals.

      3.2 Sweat chloride concentration and the CFTR dysfunction spectrum

      The dynamic range of SCC as a physiologic assay is mainly in the low range of CFTR function. Using data from the CFTR2 project, McCague et al. showed a logarithmic relationship between CFTR function assessed in cell lines transfected with a CFTR variant cDNA and the SCC of individuals with CF carrying this variant. CFTR mutations resulting in a CFTR activity range from 1 to 10% wild type activity translated in a mean change in SCC of roughly 30 mmol/L. Further increase of CFTR function from 10 to 30–50%, the range of CFTR dysfunction expected in CFTR-RD patients, resulted in a smaller change in SCC, around 20 mmol/L [
      • McCague A.F.
      • Raraigh K.S.
      • Pellicore M.J.
      • Davis-Marcisak E.F.
      • Evans T.A.
      • Han S.T.
      • et al.
      Correlating cystic fibrosis transmembrane conductance regulator function with clinical features to inform precision treatment of cystic fibrosis.
      ].Obligate heterozygote subjects may have higher SCC than non-carriers, reported slightly above the 30 threshold (34 ± 16 mmol/L), but this is of course not consistent with a diagnosis of CFTR-RD [
      • Wilschanski M.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Zielenski J.
      • Tullis E.
      • et al.
      Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials.
      ,
      • Sermet-Gaudelus I.
      • Dechaux M.
      • Vallee B.
      • Fajac A.
      • Girodon E.
      • Nguyen-Khoa T.
      • et al.
      Chloride transport in nasal ciliated cells of cystic fibrosis heterozygotes.
      ].
      As stated above, variants associated with CFTR-RD display a large range of CFTR function alteration, reflected in a high proportion of patients with SCC < 60 mmol/L, either intermediate or even below 30 mmol/L [
      • Wilschanski M.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Zielenski J.
      • Tullis E.
      • et al.
      Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials.
      ,
      • Keating C.L.
      • Liu X.
      • Dimango E.A.
      Classic respiratory disease but atypical diagnostic testing distinguishes adult presentation of cystic fibrosis.
      ]. In some CFTR-RD patients, sweat test may be slightly above 60 mmol/L and the diagnosis of CFTR-RD may be considered if all the other diagnosis criteria of CFTR-RD are met. Table 3 reports the scarce prospective data on subjects with CFTR-RD. A selection bias is expected, as patients with a normal or intermediate SCC and/or with a single or no CFTR mutation(s) are less likely to be referred for further evaluation of CFTR-RD. They show that mean SCC and the proportion of subjects with SCC in the intermediate or CF range increase with the number of mutated CFTR alleles, however remaining below the values seen in the CF group [
      • Wilschanski M.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Zielenski J.
      • Tullis E.
      • et al.
      Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials.
      ,
      • Ooi C.Y.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Martin S.
      • Ray P.N.
      • et al.
      Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?.
      ]. The level of sweat Cl is not correlated to the diagnosis as the proportion of subjects finally diagnosed with no CF, CFTR-RD or CF among those with a SCC in the intermediate range is similar [
      • Minso R.
      • Schulz A.
      • Dopfer C.
      • Alfeis N.
      • Barneveld A.V.
      • Makartian-Gyulumyan L.
      • et al.
      Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.
      ,
      • Aalbers B.L.
      • Yaakov Y.
      • Derichs N.
      • Simmonds N.J.
      • De Wachter E.
      • Melotti P.
      • et al.
      Nasal potential difference in suspected cystic fibrosis patients with 5T polymorphism.
      ]. Additionally, as already reported in CF, SCC level does not correlate to severity in patients with CFTR-RD, and some mutations, such as 3849 + 10 kb C >T, are associated with a negative sweat test [
      • Keenan K.
      • Dupuis A.
      • Griffin K.
      • Castellani C.
      • Tullis E.
      • Gonska T.
      Phenotypic spectrum of patients with cystic fibrosis and cystic fibrosis-related disease carrying p.Arg117His.
      ,
      • Thauvin-Robinet C.
      • Munck A.
      • Huet F.
      • de Becdelievre A.
      • Jimenez C.
      • Lalau G.
      • et al.
      CFTR p.Arg117His associated with CBAVD and other CFTR-related disorders.
      ].
      Table 3Sweat Chloride concentrations and results of nasal potential difference reported in different studies including patients with CFTR-RD.
      ReferenceLevel of evidence*Genetic screeningPatient PopulationSCCNPD
      18Level 2Full sequencing of CFTR exons and flanking intronic regions.CBAVD n = 60

      Control group: 25 controls, 21 heterozygotes and 50 CF
      25% of CBAVD subjects have a SCC<30 mmol/L,45% in the intermediate range.

      Most CBAVD subjects with 1 or 2 CFTR mutations had a SCC in the intermediate or CF range.
      CBAVD with 1 or 2 CFTR mutations display intermediate TCC and Δamiloride between obligate heterozygote and PS-CF level.
      28Level 3Full sequencing of CFTR exons.Primary sclerosing cholangitis

      n = 32
      23 patients tested: SCC > 60 mmol/L (n = 6); 30–59 mmol/L (n = 21)4/32 had WI in the CF range
      29Level 4Full sequencing of CFTR exons.1st or 2d degree relatives of patients with CF, genotyped as ‘carriers and assessed for CFTR-RD if a second variant

      353 retrospective cohort

      353 prospective cohort.
      Retrospective cohort: 7/353 diagnosed with CFTR-RD, all with a SCC between 40 and 60 mmol/L

      Prospective cohort:

      CFTR-RD: 32/353 had 2 CFTR variants, 24 with a SCC of 36–56 mmol/L, 8 with a SCC<30 mmol/L

      Asymptomatic: 8/353, with a SCC < 30 mmol/L
      ND
      58Level 4Not stated, registry data.

      Subjects carrying the R117H variant
      179 R117H subjects with symptoms or family history of CF (3% R117H-5T, others R117H-7T).

      4 severe lung disease, 83 isolated CBAVD, 44 CBAVD with pulmonary/pancreatic symptoms, 23 other CFTR-RD phenotypes, 12 asymptomatic
      SCC intermediate (40–59 mmol/L) or >60 mmol/L in most subjects.

      No correlation between SCC and disease manifestations

      ND
      70Level 4Not stated.

      Extracted from registry/clinical files
      50 subjects with R117H variant diagnosed after symptomatic presentation or after newborn screening

      20 R117H-5T, 30 R117H-7T

      Most with F508del in trans
      R117H-5T subjects: 83% had a SCC>60 mmol/L and 17% an intermediate SCC

      R117H-7T subjects: 23% had a SCC>60 mmol/L (CF) and 60% an intermediate SCC
      ND
      80Level 2Sequencing of CFTR exons and flanking intronic regions.Symptoms suggestive of CFTR-RD (n = 219).

      Final classification was CFTR-RD in 63, CF in 17.
      16% of the CFTR-RD subjects had a SCC<30 mmol/L; 56% in the intermediate range.

      Proportion of subjects with intermediate SCC similar in the non-CF, CFTR-RD and CF groups.
      37 of the 63 CFTR-RD patients have a SS score below the CF range.
      83Level 2

      CFTR variant panel.

      Sequencing and search for large deletions.
      Chronic respiratory disease n = 68

      Chronic or recurrent pancreatitis n = 42

      Azoospermia n = 92

      Control group: 104 non-CF, 52 heterozygotes, 64 CF-PS, 43 CF-PI.
      60% of CFTR-RD patients had a SCC<40 mmol/L. Proportion with intermediate SCC similar across the different clinical presentations, heterozygotes and CF-PS.ND
      84Level 4Subjects with a 5T allele, TGm status available only for 38 patients79/86 5T patients with known SCC and classed as CF (n = 29), CFTR-RD (n = 16), non-CF (n = 34)

      Most subjects had a SCC in the intermediate (30–59 mmol/L) or >60 mmol/L. Intermediate SCC in almost all CFTR-RD and ¾ CF subjects.

      SCC higher when severe mutations in trans, and presence of 5T-TG13.
      33% of the 5T/5T patients had abnormal NPD (WI >0.7)
      88Level 2Sequencing of CFTR exons/flanking introns and promotor, search for large deletionsAcute recurrent pancreatitis or idiopathic chronic pancreatitis n = 56One third of patients had a SCC in the intermediate or CF range, independently of the number of CFTR mutations nor with abnormal NPD values6/56 of Recurrent/chronic pancreatitis have 2 CFTR variant; TCC abnormal in all the patients when done

      18/56 have 1 CFTR variant; TCC abnormal in all the patients when done
      89Level 2CFTR variant panel, sequencing if 1 variant foundAcute recurrent pancreatitis n = 67More than half of the patients had a SCC in the intermediate or CF range, most without CFTR variants

      SCC ≥ 60 mmol/L; n = 5

      SCC between 40 and 60 mmol/L: n = 22
      4/56 tested patients had abnormal TCC and WI index
      90Level 2Full sequencing of CFTR exons.Non-CF bronchiectasis and SCC<60 mmol/L: 13 with 2 CFTR variants and a genotype compatible with a CFTR-RD (CF/VVCC), 107 with 0 or 1 variant.

      No matched cohort
      Intermediate SCC found more often in subjects with 2 CFTR mutations (53% vs 18% if 0 variant and 9% if 1 CFTR variant)TCC and WI with intermediated values in subjects with a genotype compatible with a CFTR-RD
      CBAVD: bilateral absence of the vas deferens; CF: Cystic Fibrosis; CFTR: cystic fibrosis transmembrane conductance regulator; CFTR-RD: CFTR Related Disorders; ND: no data; NPD: Nasal Potential Difference; PS: pancreatic sufficient; SCC: Sweat Chloride Concentration: TCC: total chloride conductance; VVCC: Variant of Varying Clinical Consequence; WI: Wilschanski Index. Grading : adapted from « Guide d'analyse de la littérature et gradation des recommandations ». Agence Nationale d'Accréditation et d'Evaluation en Santé. Update 2020. https://www.has-sante.fr › docs › pdf › analiterat.
      All these studies show that subjects with CFTR-RD have higher SCC than non-diseased controls, and a lower SCC compared to patients with CF, but with a considerable overlap, especially between CFTR-RD and heterozygotes. Moreover, the correlation between intermediate SCC and phenotype is weak and inconsistent.

      3.3 Sweat chloride concentration threshold between CFTR-RD and heterozygote

      Prospective cohort studies to establish the normal range for SCC are scarce. Thus the cut-off for SCC to distinguish CFTR-RD from CF heterozygotes remains controversial and imperfect, reflecting the spectrum of disease despite having similar molecular defects between individuals. In the largest reported prospective cohort, SCC between 30 and 59 mmol/L was seen in up to one third of clinically (but not fully genotyped) non-CF subjects above the age of 10 years [
      • Mishra A.
      • Greaves R.
      • Smith K.
      • Carlin J.B.
      • Wootton A.
      • Stirling R.
      • et al.
      Diagnosis of cystic fibrosis by sweat testing: age-specific reference intervals.
      ]. Other cohort studies found lower proportions of SCC in the intermediate range in the non-CF subjects [
      • Vermeulen F.
      • Lebecque P.
      • De Boeck K.
      • Leal T.
      Biological variability of the sweat chloride in diagnostic sweat tests: a retrospective analysis.
      ]. Ooi et al. reported 11 of 52 non-diseased heterozygotes with a SCC in the intermediate range defined as 40–59 mmol/L, illustrating that shifting the thresholds for CFTR dysfunction to higher SCC is unlikely to solve the issue with the overlap between CFTR-RD and heterozygote patients [
      • Ooi C.Y.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Martin S.
      • Ray P.N.
      • et al.
      Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?.
      ]. Agreement between CFTR dysfunction established by SCC and NPD was also not improved using a 40 mmol/L cut-off for SCC rather than 30 mmol/L [
      • Ooi C.Y.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Martin S.
      • Ray P.N.
      • et al.
      Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?.
      ].
      Altogether, sweat chloride concentration remains the most straightforward tool to evaluate CFTR function in vivo. SCC is correlated to the CFTR genotype and, to a lesser extent, to the clinical manifestations of CFTR dysfunction [
      • Wilschanski M.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Zielenski J.
      • Tullis E.
      • et al.
      Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials.
      ,
      • McCague A.F.
      • Raraigh K.S.
      • Pellicore M.J.
      • Davis-Marcisak E.F.
      • Evans T.A.
      • Han S.T.
      • et al.
      Correlating cystic fibrosis transmembrane conductance regulator function with clinical features to inform precision treatment of cystic fibrosis.
      ,
      • Werlin S.
      • Scotet V.
      • Uguen K.
      • Audrezet M.P.
      • Cohen M.
      • Yaakov Y.
      • et al.
      Primary sclerosing cholangitis is associated with abnormalities in CFTR.
      ,
      • Esposito M.V.
      • Aveta A.
      • Comegna M.
      • Cernera G.
      • Iacotucci P.
      • Carnovale V.
      • et al.
      Extensive CFTR gene analysis revealed a higher occurrence of cystic fibrosis transmembrane regulator-related disorders (CFTR-RD) among CF carriers.
      ,
      • Keating C.L.
      • Liu X.
      • Dimango E.A.
      Classic respiratory disease but atypical diagnostic testing distinguishes adult presentation of cystic fibrosis.
      ,
      • Ooi C.Y.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Martin S.
      • Ray P.N.
      • et al.
      Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?.
      ,
      • Aalbers B.L.
      • Yaakov Y.
      • Derichs N.
      • Simmonds N.J.
      • De Wachter E.
      • Melotti P.
      • et al.
      Nasal potential difference in suspected cystic fibrosis patients with 5T polymorphism.
      ,
      • Thauvin-Robinet C.
      • Munck A.
      • Huet F.
      • de Becdelievre A.
      • Jimenez C.
      • Lalau G.
      • et al.
      CFTR p.Arg117His associated with CBAVD and other CFTR-related disorders.
      ,
      • Treggiari D.
      • Tridello G.
      • Menin L.
      • Borruso A.
      • Pintani E.
      • Iansa P.
      • et al.
      Role of sweat ion ratios in diagnosing cystic fibrosis.
      ,
      • Casals T.
      • Bassas L.
      • Egozcue S.
      • Ramos M.D.
      • Gimenez J.
      • Segura A.
      • et al.
      Heterogeneity for mutations in the CFTR gene and clinical correlations in patients with congenital absence of the vas deferens.
      ,
      • Bishop M.D.
      • Freedman S.D.
      • Zielenski J.
      • Ahmed N.
      • Dupuis A.
      • Martin S.
      • et al.
      The cystic fibrosis transmembrane conductance regulator gene and ion channel function in patients with idiopathic pancreatitis.
      ,
      • Werlin S.
      • Konikoff F.M.
      • Halpern Z.
      • Barkay O.
      • Yerushalmi B.
      • Broide E.
      • et al.
      Genetic and electrophysiological characteristics of recurrent acute pancreatitis.
      ,
      • Bienvenu T.
      • Sermet-Gaudelus I.
      • Burgel P.R.
      • Hubert D.
      • Crestani B.
      • Bassinet L.
      • et al.
      Cystic fibrosis transmembrane conductance regulator channel dysfunction in non-cystic fibrosis bronchiectasis.
      ]. It is reported below 60 mmol/L in CFTR-RD (e.g.; mainly in the intermediate range between 30 and 59 mmol/L), however, its contribution to the diagnosis of CFTR-RD is limited by (1) the high variability of SCC over time in the intermediate range (2) lower dynamic range of the SCC for CFTR dysfunction at intermediate ranges compared to the most severe end of the spectrum of CFTR dysfunction and (3) the overlap of SCC between heterozygotes and subjects with CFTR-RD, even with adapted cut-off.

      4. Nasal potential difference (NPD)

      Nasal potential difference (NPD) estimates the ion conductance based on transepithelial voltage changes across nasal surface epithelial cells in vivo. The loss of CFTR-dependent anion conductance causes hyperpolarization, which is associated with a characteristic pattern of voltage changes in response to specific transport inhibitors and agonists. North American and European NPD Standard Operating Procedures (SOPs) were published with new and better quantitative and discriminatory parameters for using the NPD as a bio-marker of CFTR function [
      • Solomon G.M.
      • Bronsveld I.
      • Hayes K.
      • Wilschanski M.
      • Melotti P.
      • Rowe S.M.
      • et al.
      Standardized measurement of nasal membrane transepithelial potential difference (NPD).
      ,

      Network) UC-TCFF-TDNatE-CECFSCT. Standardized Measurement of nasal membrane transepithelial potential difference (NPD). Standard Operating Procedure 528012014.

      ].

      4.1 NPD analytical interpretation

      Several strategies for quantitative interpretation have been evaluated. In addition to the reliance on measures of CFTR-dependent chloride conductance during perfusion of the nasal mucosa with a low chloride solution containing isoproterenol to activate CFTR (Total Chloride Conductance, TCC) response [
      • Solomon G.M.
      • Bronsveld I.
      • Hayes K.
      • Wilschanski M.
      • Melotti P.
      • Rowe S.M.
      • et al.
      Standardized measurement of nasal membrane transepithelial potential difference (NPD).
      ,

      Network) UC-TCFF-TDNatE-CECFSCT. Standardized Measurement of nasal membrane transepithelial potential difference (NPD). Standard Operating Procedure 528012014.

      ], other investigations have yielded comparable results in resolving challenging diagnoses using integrated measures of both sodium flux (ΔAmiloride) and TCC. The most widely accepted are the Wilschanski Index [
      • Wilschanski M.
      • Famini H.
      • Strauss-Liviatan N.
      • Rivlin J.
      • Blau H.
      • Bibi H.
      • et al.
      Nasal potential difference measurements in patients with atypical cystic fibrosis.
      ] and Sermet scores [
      • Sermet-Gaudelus I.
      • Girodon E.
      • Sands D.
      • Stremmler N.
      • Vavrova V.
      • Deneuville E.
      • et al.
      Clinical phenotype and genotype of children with borderline sweat test and abnormal nasal epithelial chloride transport.
      ].
      NPD workflow shown in Fig. 2 is suitable to determine a provisional diagnosis of CFTR dysfunction, but has not been studied as a singular diagnostic test for CFTR-RD. There is no defined diagnostic NPD parameter, and more specifically no validated thresholds for CFTR-RD.
      Fig 2
      Fig. 2Workflow of interpretation of NPD tracing.
      Single center investigation suggests that scores combining sodium and chloride transport clarify more patients into a provisional diagnosis of CF than TCC alone, but multi-center validation is lacking.
      Interpretation of NPD must be performed by experienced personnel. Solomon et al. showed that specialized interpreters have excellent agreement about the quantitative values of tracings across the diagnosis spectrum, with all tracings meeting a rigorous “interpretability” standard [
      • Solomon G.M.
      • Liu B.
      • Sermet-Gaudelus I.
      • Fajac I.
      • Wilschanski M.
      • Vermeulen F.
      • et al.
      A multiple reader scoring system for nasal potential difference parameters.
      ]. Confidence in tracing analysis, however, showed greater variability in “indeterminate’ tracings, but did not affect quantitative analysis of metrics such as TCC. Therefore, confidence and interpretability analysis is necessary to interpret diagnosis based on single measurement.

      4.2 NPD reproducibility

      Multicenter studies in CF patients carrying mutations with minimal function showed a wide variability both in ΔAmiloride and TCC, attributed to gender and inflammation but also largely to the expertise of the test technician [
      • Kyrilli S.
      • Henry T.
      • Wilschanski M.
      • Fajac I.
      • Davies J.C.
      • Jais J.P.
      • et al.
      Insights into the variability of nasal potential difference, a biomarker of CFTR activity.
      ] . On the other hand, single center studies provide provisional confidence in reproducibility. Yaakov et al. repeated NPD measurements in 68 CF patients divided in a classical and non-classical CF group and showed acceptable variability between measurements [
      • Yaakov Y.
      • Kerem E.
      • Yahav Y.
      • Rivlin J.
      • Blau H.
      • Bentur L.
      • et al.
      Reproducibility of nasal potential difference measurements in cystic fibrosis.
      ]. All differences were within the +/−1 standard deviation which was defined as a clinically significant difference. Only the basal PD in the non-classical was different between measurements possibly due to the fact that basal PD can be influenced by non-CFTR dependent factors. Jaron et al. used NPD to diagnose CF in a group of patients with questionable CF [
      • Jaron R.
      • Yaakov Y.
      • Rivlin J.
      • Blau H.
      • Bentur L.
      • Yahav Y.
      • et al.
      Nasal potential difference in non-classic cystic fibrosis-long term follow up.
      ]. In repeated NPD measures in the group with NPD outside the CF range, there was no significant difference between first and follow up measurement, providing evidence that intra-patient variability is acceptable in expert centers.

      4.3 NPD limitations

      While NPD is a challenging technique, it can be completed in the clinical research setting. Since NPD has been described for ∼40 years, there has been widespread adoption of this technique. As a result, there are ∼30 qualified centers in North America, 15 in Europe and Israel, and 5 in Oceania. Therefore, regional referral allows for access to highly qualified centers but this is not the case everywhere in Europe.
      While there is long standing experience using NPD for diagnosing CF, there are some noted limitations to the technique for its universal role in establishing diagnosis. Factors of variability include: acute upper respiratory tract infection, extensive nasal polyps, prior sinus surgery and CF-related inflammation, which decreases its specificity and sensitivity. Potential biological effects of active or previous tobacco exposure should also be considered [
      • Patel S.D.
      • Bono T.R.
      • Rowe S.M.
      • Solomon G.M.
      CFTR targeted therapies: recent advances in cystic fibrosis and possibilities in other diseases of the airways.
      ,
      • Sloane P.A.
      • Shastry S.
      • Wilhelm A.
      • Courville C.
      • Tang L.P.
      • Backer K.
      • et al.
      A pharmacologic approach to acquired cystic fibrosis transmembrane conductance regulator dysfunction in smoking related lung disease.
      ].
      Secondly, patients have to remain very still and refrain from talking to minimize artifacts and catheter dislodgement. This makes this test difficult in non-cooperative patients and the technique has only been reported in one study in children below 6 years of age [
      • Sermet-Gaudelus I.
      • Girodon E.
      • Roussel D.
      • Deneuville E.
      • Bui S.
      • Huet F.
      • et al.
      Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis.
      ]. Finally, discrepancy between measurements in the two nostrils can occur which may be an issue for making a definitive conclusion [
      • Jaron R.
      • Yaakov Y.
      • Rivlin J.
      • Blau H.
      • Bentur L.
      • Yahav Y.
      • et al.
      Nasal potential difference in non-classic cystic fibrosis-long term follow up.
      ].
      Therefore, a considerable international effort aimed at standardization of the test is ongoing. Both the US CFF-TDN (Cystic Fibrosis Foundation-Therapeutics Development Network) and the ECFS-CTN (European Cystic Fibrosis Society-Clinical Trials Network) created a Standard Operating Procedure (SOP) for use in multicenter and research trials. This recent collaborative work by the CTN and TDN has resulted in a combined, international SOP, bringing together the expertise of the CTN and TDN [

      Network) UC-TCFF-TDNatE-CECFSCT. Standardized Measurement of nasal membrane transepithelial potential difference (NPD). Standard Operating Procedure 528012014.

      ]. With these recent standardizations, combined with comprehensive technical training, ongoing efforts to conduct such multi-center validations are in progress.
      NPD measures are both repeatable and reproducible using current SOPs and interpretation strategies. Possible future ideas for research directions should explore international consensus on NPD interpretation for CFTR dysfunction.

      4.4 NPD in CFTR-RD patients

      The quantitative methods discussed above have mainly been used to demonstrate NPD performance in diagnosing classic CF and normal (no CFTR dysfunction), but not clearly in discriminating CFTR-RD patients both from heterozygotes and patients with CF. The literature shows that quantitative interpretation of NPD allows for a greater detection of CFTR dysfunction than sweat chloride alone, which is often intermediate. A summary of the relevant studies are listed in Table 3.
      Data on obligate heterozygotes are lacking and the limited number of published studies suggest that the TCC is reduced but remains within the normal range [
      • Wilschanski M.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Zielenski J.
      • Tullis E.
      • et al.
      Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials.
      ,
      • Sermet-Gaudelus I.
      • Dechaux M.
      • Vallee B.
      • Fajac A.
      • Girodon E.
      • Nguyen-Khoa T.
      • et al.
      Chloride transport in nasal ciliated cells of cystic fibrosis heterozygotes.
      ,
      • Tucker T.A.
      • Fortenberry J.A.
      • Zsembery A.
      • Schwiebert L.M.
      • Schwiebert E.M.
      The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa.
      ,
      • Schulz A.
      • Tummler B.
      Non-allergic asthma as a CFTR-related disorder.
      ]. This observation is supported by in vitro data suggesting that P.Phe508del CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia [
      • Tucker T.A.
      • Fortenberry J.A.
      • Zsembery A.
      • Schwiebert L.M.
      • Schwiebert E.M.
      The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa.
      ]. Studies of NPD in patients with CFTR-RD listed in Table 3 show intermediate values between CF and heterozygotes with a large range of CFTR dysfunction, reflecting the residual activity of the genotype [
      • Wilschanski M.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Zielenski J.
      • Tullis E.
      • et al.
      Mutations in the cystic fibrosis transmembrane regulator gene and in vivo transepithelial potentials.
      ,
      • Minso R.
      • Schulz A.
      • Dopfer C.
      • Alfeis N.
      • Barneveld A.V.
      • Makartian-Gyulumyan L.
      • et al.
      Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.
      ,
      • Aalbers B.L.
      • Yaakov Y.
      • Derichs N.
      • Simmonds N.J.
      • De Wachter E.
      • Melotti P.
      • et al.
      Nasal potential difference in suspected cystic fibrosis patients with 5T polymorphism.
      ,
      • Bishop M.D.
      • Freedman S.D.
      • Zielenski J.
      • Ahmed N.
      • Dupuis A.
      • Martin S.
      • et al.
      The cystic fibrosis transmembrane conductance regulator gene and ion channel function in patients with idiopathic pancreatitis.
      ,
      • Werlin S.
      • Konikoff F.M.
      • Halpern Z.
      • Barkay O.
      • Yerushalmi B.
      • Broide E.
      • et al.
      Genetic and electrophysiological characteristics of recurrent acute pancreatitis.
      ,
      • Bienvenu T.
      • Sermet-Gaudelus I.
      • Burgel P.R.
      • Hubert D.
      • Crestani B.
      • Bassinet L.
      • et al.
      Cystic fibrosis transmembrane conductance regulator channel dysfunction in non-cystic fibrosis bronchiectasis.
      ,
      • Gonska T.
      • Choi P.
      • Stephenson A.
      • Ellis L.
      • Martin S.
      • Solomon M.
      • et al.
      Role of cystic fibrosis transmembrane conductance regulator in patients with chronic sinopulmonary disease.
      ,
      • Pradal U.
      • Castellani C.
      • Delmarco A.
      • Mastella G.
      Nasal potential difference in congenital bilateral absence of the vas deferens.
      ,
      • Hirtz S.
      • Gonska T.
      • Seydewitz H.H.
      • Thomas J.
      • Greiner P.
      • Kuehr J.
      • et al.
      CFTR Cl- channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis.
      ,
      • Veeze H.J.
      • Sinaasappel M.
      • Bijman J.
      • Bouquet J.
      • de Jonge H.R.
      Ion transport abnormalities in rectal suction biopsies from children with cystic fibrosis.
      ]. There is a lack of fully validated thresholds discriminating between CFTR-RD and CF heterozygotes. This may be partially due to differences between availability of clinical facilities and the techniques employed, emphasizing the need for standardization. It could also be the result of heterogeneity within this population, with genotypes displaying a wide range of CFTR dysfunction. Multicenter studies collecting data from patients with CFTR-RD are needed to gain deeper insight into the dynamic range of this biomarker for discriminating patients with CFTR-RD.

      5. ICM relevance in CFTR-RD

      Intestinal current measurement (ICM) is a biomarker of CFTR function measuring the short circuit current of freshly excised rectal tissue in Ussing chambers [
      • Bronsveld I.
      • Mekus F.
      • Bijman J.
      • Ballmann M.
      • Greipel J.
      • Hundrieser J.
      • et al.
      Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings with cystic fibrosis. The European CF twin and sibling study consortium.
      ,
      • Mall M.
      • Kreda S.M.
      • Mengos A.
      • Jensen T.J.
      • Hirtz S.
      • Seydewitz H.H.
      • et al.
      The DeltaF508 mutation results in loss of CFTR function and mature protein in native human colon.
      ,
      • Mall M.
      • Wissner A.
      • Seydewitz H.H.
      • Kuehr J.
      • Brandis M.
      • Greger R.
      • et al.
      Defective cholinergic Cl(-) secretion and detection of K(+) secretion in rectal biopsies from cystic fibrosis patients.
      ]. ICM assesses the chloride secretory responses to cholinergic agonists and cAMP increase after adenyl cyclase activation [
      • Mall M.
      • Kreda S.M.
      • Mengos A.
      • Jensen T.J.
      • Hirtz S.
      • Seydewitz H.H.
      • et al.
      The DeltaF508 mutation results in loss of CFTR function and mature protein in native human colon.
      ]. Rectal tissue expresses high levels of CFTR protein and compared to the airways, alternative chloride channels seem to rarely contribute to chloride conductance [
      • Hirtz S.
      • Gonska T.
      • Seydewitz H.H.
      • Thomas J.
      • Greiner P.
      • Kuehr J.
      • et al.
      CFTR Cl- channel function in native human colon correlates with the genotype and phenotype in cystic fibrosis.
      ]. CFTR and other chloride channels can be differentiated by their sensitivity to inhibition by the Cl channel blocker DIDS (4,4′-Diisothiocyano-2,2′-stilbenedisulfonic Acid) [
      • Mall M.
      • Kreda S.M.
      • Mengos A.
      • Jensen T.J.
      • Hirtz S.
      • Seydewitz H.H.
      • et al.
      The DeltaF508 mutation results in loss of CFTR function and mature protein in native human colon.
      ].
      ICM detects very low amounts of active CFTR and, therefore, is sensitive to discriminate between non CF controls, pancreatic insufficient CF patients and pancreatic sufficient CF patients expressing a residual CFTR activity [
      • Mall M.
      • Kreda S.M.
      • Mengos A.
      • Jensen T.J.
      • Hirtz S.
      • Seydewitz H.H.
      • et al.
      The DeltaF508 mutation results in loss of CFTR function and mature protein in native human colon.
      ,
      • Derichs N.
      • Sanz J.
      • Von Kanel T.
      • Stolpe C.
      • Zapf A.
      • Tummler B.
      • et al.
      Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data.
      ,
      • Clancy J.P.
      • Szczesniak R.D.
      • Ashlock M.A.
      • Ernst S.E.
      • Fan L.
      • Hornick D.B.
      • et al.
      Multicenter intestinal current measurements in rectal biopsies from CF and non-CF subjects to monitor CFTR function.
      ]. In a multicenter study a good agreement across different sites discriminating between non CF controls and CF patients was observed [
      • Roth E.K.
      • Hirtz S.
      • Duerr J.
      • Wenning D.
      • Eichler I.
      • Seydewitz H.H.
      • et al.
      The K+ channel opener 1-EBIO potentiates residual function of mutant CFTR in rectal biopsies from cystic fibrosis patients.
      ]. Likewise, ICM is a sensitive CFTR biomarker to assess the in vivo response to CFTR modulators within the context of clinical trials or of tests of efficacy in individual patients [
      • Graeber S.Y.
      • Hug M.J.
      • Sommerburg O.
      • Hirtz S.
      • Hentschel J.
      • Heinzmann A.
      • et al.
      Intestinal current measurements detect activation of mutant CFTR in patients with cystic fibrosis with the G551D mutation treated with ivacaftor.
      ,
      • Graeber S.Y.
      • Dopfer C.
      • Naehrlich L.
      • Gyulumyan L.
      • Scheuermann H.
      • Hirtz S.
      • et al.
      Effects of lumacaftor-ivacaftor therapy on cystic fibrosis transmembrane conductance regulator function in Phe508del homozygous patients with cystic fibrosis.
      ,
      • Masson A.
      • Schneider-Futschik E.K.
      • Baatallah N.
      • Nguyen-Khoa T.
      • Girodon E.
      • Hatton A.
      • et al.
      Predictive factors for lumacaftor/ivacaftor clinical response.
      ]. A standardized SOP for ICM was developed by the ECFS CTN Diagnostic Working Group in 2011, but has not yet been published in an academic journal. Hence, custom protocols for ICM remain in use for diagnostics in several CF centers [

      Group E-CECFSCTNaEDW. Ion transport in rectal biopsies for diagnosis and clinical trials in cystic fibrosis. Intestinal Current Measurement, ICM) Version 27 2011.

      ].
      Studies have shown that ICM is safe and feasible in all age groups, especially in the pediatric cohort below 6 years where NPD is not possible, and no confounding effects of chronic inflammation and infection were observed in the rectal tissue. Hemorrhoids are a relative contraindication to perform rectal suction biopsies without optical control. In the Hannover center, bleeding was reported in five out of 400 adults with only one case requiring medical intervention (Data from Hannover 2009–2020). Rectal forcep biopsies with optical control appears safe in all ages and showed no bleeding following 500 procedures (Data from Heidelberg 2006–2020). Limitations include the low number of specialized centers able to perform ICM and the short viability period of freshly excised rectal biopsies.
      The Hannover center has recently published its reference values for 68 healthy non-CF control subjects who were examined by ICM according to the ECFS CTN SOP (80). The cumulative chloride secretory response to the adrenergic agonist forskolin/IBMX and the cholinergic agonists carbachol and histamine, has previously been demonstrated as the most conclusive diagnostic ICM parameter to differentiate a normal response from CFTR dysfunction [
      • Derichs N.
      • Sanz J.
      • Von Kanel T.
      • Stolpe C.
      • Zapf A.
      • Tummler B.
      • et al.
      Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data.
      ]. Applying the SOP cut-off value was determined to be 39 µA/cm2 [
      • Minso R.
      • Schulz A.
      • Dopfer C.
      • Alfeis N.
      • Barneveld A.V.
      • Makartian-Gyulumyan L.
      • et al.
      Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.
      ,
      • Derichs N.
      • Sanz J.
      • Von Kanel T.
      • Stolpe C.
      • Zapf A.
      • Tummler B.
      • et al.
      Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data.
      ]. Having determined this threshold, ICM was used at the Hannover site to make a diagnosis in individuals with a clinical suspicion for CF or CFTR-RD and at most one disease-causing CFTR mutation [
      • Minso R.
      • Schulz A.
      • Dopfer C.
      • Alfeis N.
      • Barneveld A.V.
      • Makartian-Gyulumyan L.
      • et al.
      Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.
      ]. These findings need to be replicated before they can be translated more generally.
      ICM turned out to be a sensitive tool to diagnose CFTR dysfunction in individuals with inconclusive sweat test and CFTR genetics [
      • Minso R.
      • Schulz A.
      • Dopfer C.
      • Alfeis N.
      • Barneveld A.V.
      • Makartian-Gyulumyan L.
      • et al.
      Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.
      ]. Of 74 individuals who had been referred by CF physicians to clarify a diagnosis by ICM, CFTR dysfunction was detected in 28 subjects. Although the positive and negative predictive value of ICM for absence of CFTR activity is close to 1, the differential diagnosis between CFTR-RD and mild pancreatic sufficient (PS) CF needs to be made with consideration of CFTR genetics and/or clinical symptoms. This is clearly shown by the fact that 20% normal CFTR activity is sufficient to normalize trans-epithelial anion secretion in the intestine [
      • Dickinson P.
      • Smith S.N.
      • Webb S.
      • Kilanowski F.M.
      • Campbell I.J.
      • Taylor M.S.
      • et al.
      The severe G480C cystic fibrosis mutation, when replicated in the mouse, demonstrates mistrafficking, normal survival and organ-specific bioelectrics.
      ]. Thus, given the high level of expression of CFTR in intestinal tissue, ICM might not differentiate residual CFTR activity in CFTR-RD patients from normal [
      • Hatton A.
      • Bergougnoux A.
      • Zybert K.
      • Chevalier B.
      • Mesbahi M.
      • Alteri J.P.
      • et al.
      Reclassifying inconclusive diagnosis after newborn screening for cystic fibrosis. Moving forward.
      ].
      Moreover the current SOP for ICM selected concentrations of secretagogues to identify CFTR-mediated chloride secretory activity, but typically fails to differentiate compound heterozygous subjects with R117H-7T and a CF-causing mutation, from a non-CF subject with no CF causing mutation because both show normal chloride secretory activity in the ICM [
      • Hatton A.
      • Bergougnoux A.
      • Zybert K.
      • Chevalier B.
      • Mesbahi M.
      • Alteri J.P.
      • et al.
      Reclassifying inconclusive diagnosis after newborn screening for cystic fibrosis. Moving forward.
      ]. This indicates that future studies comparing different ICM protocols are necessary to determine the most discriminative approach between non-CF subjects and CFTR-RD patients.

      6. Combinations of different biomarkers

      CFTR bioassays have been optimized to make a diagnosis of CF, focusing on the lowest end of the spectrum of epithelial CFTR activity. They are less sensitive to detect high residual activity of mutant CFTR and thus less suitable for detecting CFTR dysfunction in the ranges of milder CFTR dysfunction. Further, the CFTR functional threshold above which disease is unlikely may vary at the individual level. Indeed, a 50% reduction in CFTR expression (e.g. a single gene carrier of a severe CFTR mutation, such as G542X) can have a relatively high degree of CFTR function, ranging from normal to intermediate. In this range, transepithelial anion transport is possibly increased as compared to the degree of expression, although this can vary based on tissue type and assay used to measure functional response. This also reflects the potential contribution of non-genetic factors beyond CFTR, such as environmental factors, or hormonal triggers. This at least partly explains why the relationship between CFTR mRNA expression, protein processing efficiency and CFTR dependent Cl transport is non-linear at the epithelial level [
      • McCague A.F.
      • Raraigh K.S.
      • Pellicore M.J.
      • Davis-Marcisak E.F.
      • Evans T.A.
      • Han S.T.
      • et al.
      Correlating cystic fibrosis transmembrane conductance regulator function with clinical features to inform precision treatment of cystic fibrosis.
      ]. Conversely, it cannot be excluded that the “epithelial function” tools that we use in vivo may not capture differences in CFTR function at the CFTR-RD level. This has been recently shown by comparing CFTR activity at the epithelial level (short circuit current of primary respiratory epithelia) and at the channel level (patch clamp of native cells) where patients with CFTR-RD displayed normal chloride transport at NPD test while the CFTR activity was reduced in patch clamp experiments [
      • Noel S.
      • Servel N.
      • Hatton A.
      • Golec A.
      • Rodrat M.
      • Ng D.R.S.
      • et al.
      Correlating genotype with phenotype using CFTR-mediated whole-cell Cl(-) currents in human nasal epithelial cells.
      ]. Moreover, although Cl transport across the apical membrane of secretory epithelia is the rate-limiting step in fluid and electrolyte transport, the role of CFTR differs according to the organs [
      • Ooi C.Y.
      • Dupuis A.
      • Gonska T.
      • Ellis L.
      • Ni A.
      • Jarvi K.
      • et al.
      Does integration of various ion channel measurements improve diagnostic performance in cystic fibrosis?.
      ]. For example, CFTR is highly expressed at the luminal membrane of enterocytes in the distal gut, where it functions as the principal channel for Cl secretion. As the transepithelial transport reflects both channel dysfunction and the number of proteins expressed, a high number of CFTR channels displaying residual activity results in a more normal intestinal transepithelial transport and a normal or intermediate result in ICM. In other tissues, this relationship can vary based on the degree of constitutive CFTR activity. Thus it is important to keep in mind that a “normal” result for a single bioassay does not necessarily mean a normal CFTR activity in all organs (e.g.; “normalization” of sweat chloride during CFTR modulator treatment does not equate to “restoration” of CFTR normal activity in all organs).
      Finally, bioassays are not interchangeable. This can be explained by the fact that the three tests reflect CFTR activity differently: sweat test assesses Cl reabsorption along the sweat duct; NPD measures changes in the transepithelial voltage caused by relative changes in ion current; whilst intestinal short circuit current measures changes in ion conductance. This is the reason why it is important to consider combinations of different biomarkers to improve diagnostic yield.
      While most of the studies have looked into comparing test performances between different functional tests, a few others have explored the idea of using a scoring system that combines results obtained from various CFTR functional measurements. The Toronto group has undertaken one of the largest studies to evaluate CFTR functional measures in patients presenting with single-organ disease [
      • Ooi C.Y.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Martin S.
      • Ray P.N.
      • et al.
      Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?.
      ]. After demonstrating significant discordance (28%−65% agreement) between the CFTR functional measure of NPD and sweat test to classify these patients into different diagnostic categories, they explored whether combining different measures would more accurately discriminate between those with CF and a CFTR-RD. Ooi et al. used principal component analysis to assay performance of different NPD parameters and sweat test [
      • Ooi C.Y.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Martin S.
      • Ray P.N.
      • et al.
      Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?.
      ]. This analysis identified that a combination of sweat chloride and the NPD parameters Total Chloride Conductance, (TCC) and the combination of TCC with Δamilorie, (which reflects the Na transport) provided the best discrimination between healthy controls, obligate heterozygotes, pancreatic sufficient and pancreatic insufficient CF patients. It also provided additional diagnostic information to further stratify patients according to their level of CFTR dysfunction [
      • Ooi C.Y.
      • Dupuis A.
      • Ellis L.
      • Jarvi K.
      • Martin S.
      • Ray P.N.
      • et al.
      Does extensive genotyping and nasal potential difference testing clarify the diagnosis of cystic fibrosis among patients with single-organ manifestations of cystic fibrosis?.
      ]. Absence of correlation between CFTR function measured as sweat chloride and by NPD was also shown in 50 children presenting with CF-disease like symptoms and an equivocal CF diagnosis arguing against a clear dichotomous classification, but rather favoring a gradient of CFTR functional phenotypes [
      • Sermet-Gaudelus I.
      • Girodon E.
      • Sands D.
      • Stremmler N.
      • Vavrova V.
      • Deneuville E.
      • et al.
      Clinical phenotype and genotype of children with borderline sweat test and abnormal nasal epithelial chloride transport.
      ]. For these children with an equivocal CF diagnosis, Sermet-Gaudelus et al. demonstrated that integrating the chloride secretory parameter with the sodium absorption parameter (“Sermet score”) of NPD measurements can add diagnostic information about presence of CFTR dysfunction. Prior to this, Wilschanski et al., had shown that a NPD composite score based on parameters that assess chloride secretion as well as sodium absorption, both of which are abnormal in CF, better discriminates between CF and non-CF. He suggested that this combined read-out (“Wilschanski score”) better aides in the diagnosis of patients presenting with atypical CF [
      • Wilschanski M.
      • Famini H.
      • Strauss-Liviatan N.
      • Rivlin J.
      • Blau H.
      • Bibi H.
      • et al.
      Nasal potential difference measurements in patients with atypical cystic fibrosis.
      ]. A recent study from Minso et al. performed both ICM and NPD in 26 patients presenting with inconclusive CFTR genetics and sweat tests. They reported discordant test results in 42% of the patients [
      • Minso R.
      • Schulz A.
      • Dopfer C.
      • Alfeis N.
      • Barneveld A.V.
      • Makartian-Gyulumyan L.
      • et al.
      Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusive CFTR genetics and sweat test.
      ]. The authors pointed out that in these cases the abnormal test was associated with the organ disease under investigation- abnormal NPD in those with airway disease and abnormal ICM in those presenting with acute recurrent pancreatitis.
      In summary, observations made in several studies suggest that sweat test, NPD and ICM are not concordant and do not necessarily provide the same diagnostic information. While there is some evidence that combination of various electrophysiological parameters and/or CFTR function measures generated from different tissues more accurately identifies the individual risk for CFTR-RD, large scale studies combining ICM, NPD and sweat test parameters have so far not been performed. These kinds of studies would be of particular interest to further describe the functional spectrum of patients with a CFTR-RD.

      7. General workflow and algorithm proposal

      The diagnosis of CFTR-RD is not straightforward and is based on the CFTR genotype, other CFTR biomarkers, mainly sweat test, NPD and ICM (Fig. 3).
      Fig 3
      Fig. 3Algorithm for CFTR-RD diagnosis
      In the specific cases of “CF/VVCC” or “VVCC/VVCC” genotype observed in patients with sweat test between 30 and 59 mmol/l, functional tests such as NPD or ICM may help to delineate the risk to evolve to CF, e.g.; a patient displaying test results in the CF range is at high risk to evolve to CF even if he/she has symptoms consistent with CFTR-RD at the moment of the evaluation.
      *VVCC variants can be found in .
      After differentiation of the CFTR-RD clinical picture from other conditions not related to CFTR and Pancreatic sufficient CF, combined genotyping and sweat test allows clarification of the following situations.
      • -
        CF is confirmed when two CF variants have been found and/or SCC is above 60 mmol/L
      • -
        CFTR-RD is unlikely when no variant has been found and SCC is below 30 mmol/L. In other cases (none or a single CFTR variant identified and SCC < 60 mmol/L), the diagnosis remains uncertain and both NPD and ICM must be normal to rule out CFTR-RD.
      • -
        Heterozygous non pathogenic condition is very likely when 1 variant is found, SCC < 60 mmol/L and both NPD and ICM are normal.
      • -
        CFTR-RD is diagnosed in 2 different situations: (i) evidence of a CF/VVCC or a VVCC/VVCC genotype (ii) NPD and/or ICM are in the CFTR-RD range and genetic analysis shows either none CFTR variant, or a single CFTR variant, or 2 CFTR variants among those at least 1 VUS.

      8. Future directions

      8.1 Other “sweat tests”

      Beta-adrenergic sweat secretion tests focus on CFTR dependent sweat secretion in the sweat gland's secretory coil instead of measurement of chloride sweat concentration. Two methods have been established following beta-adrenergic stimulation combining isoprenaline and aminophylline. In contrast to the cholinergic induced sweating, which is used for the standard sweat chloride test, the amount of sweat produced following beta-adrenergic sweat stimulation is minimal so that it cannot be easily collected. One method to assess this low sweat secretion is based on the use of an evaporimeter and the other method is based on imaging the sweat droplet size under a microscope. In both methods, CFTR-independent sweat secretion is induced first by cholinergic stimulation with methacholine, carbachol or pilocarpine. The result is used to normalize the sweat secretion elicited by beta-adrenergic agonists. Although a small number of subjects were included, both methods of beta-adrenergic sweat secretion testing seem to be capable of discriminating heterozygote carriers of one CFTR gene variant from healthy control subjects and CF patients, making this test the first to allow discrimination of CFTR function in this mild CFTR functional spectrum [
      • Quinton P.
      • Molyneux L.
      • Ip W.
      • et al.
      β-adrenergic sweat secretion as a diagnostic test for cystic fibrosis.
      ,
      • Kim J.
      • Farahmand M.
      • Dunn C.
      • Davies Z.
      • Frisbee E.
      • Milla C.
      • et al.
      Evaporimeter and bubble-imaging measures of sweat gland secretion rates.
      ]. Both tests have been successfully translated for use in other CF centers with comparable results, paving the way to standardization for multicenter applications. However, they have been until now performed in a limited number of patients and need further validation for diagnosis setting.
      The evaporimeter-based method records the sweat secretion rate with the help of an evaporimeter, which measures changes in evaporation above a defined skin area. This device was borrowed from the cosmetic industry for this purpose. This test has now been well standardized including the validation of a diagnosis threshold. The second method reports sweat secretion rate by following the change in the size of sweat droplets under a microscope (“bubble test”) with a consistent focus on recording the sweat rate from the same individual sweat glands [
      • Char J.E.
      • Wolfe M.H.
      • Cho H.J.
      • Park I.H.
      • Jeong J.H.
      • Frisbee E.
      • et al.
      A little CFTR goes a long way: cFTR-dependent sweat secretion from G551D and R117H-5T cystic fibrosis subjects taking ivacaftor.
      ,
      • Bergamini G.
      • Tridello G.
      • Calcaterra E.
      • Ceri S.
      • Tagliasacchi M.
      • Bianchi F.
      • et al.
      Ratiometric sweat secretion optical test in cystic fibrosis, carriers and healthy subjects.
      ,
      • Treggiari D.
      • Kleinfelder K.
      • Bertini M.
      • Tridello G.
      • Fedrigo A.
      • Pintani E.
      • et al.
      Optical measurements of sweat for in vivo quantification of CFTR function in individual sweat glands.
      ,
      • Reynaerts A.
      • Vermeulen F.
      • Mottais A.
      • Gohy S.
      • Lebecque P.
      • Frederick R.
      • et al.
      Needle-free iontophoresis-driven beta-adrenergic sweat rate test.
      ,
      • Pallenberg S.T.
      • Junge S.
      • Ringshausen F.C.
      • Sauer-Heilborn A.
      • Hansen G.
      • Dittrich A.M.
      • et al.
      CFTR modulation with elexacaftor-tezacaftor-ivacaftor in people with cystic fibrosis assessed by the beta-adrenergic sweat rate assay.
      ]. Both tests report absolute cholinergic and beta-adrenergic sweat secretion rates as well as the ratio between cholinergic and beta-adrenergic sweat secretion rates as a means to standardize individual and CFTR-independent differences in sweat secretion rates. They have shown that reduced CFTR function can be captured at the intermediate level between heterozygous carriers and CF patients, thus identifying CFTR-RD subjects [
      • Quinton P.
      • Molyneux L.
      • Ip W.
      • et al.
      β-adrenergic sweat secretion as a diagnostic test for cystic fibrosis.
      ,
      • Kim J.
      • Farahmand M.
      • Dunn C.
      • Davies Z.
      • Frisbee E.
      • Milla C.
      • et al.
      Evaporimeter and bubble-imaging measures of sweat gland secretion rates.
      ,
      • Bergamini G.
      • Tridello G.
      • Calcaterra E.
      • Ceri S.
      • Tagliasacchi M.
      • Bianchi F.
      • et al.
      Ratiometric sweat secretion optical test in cystic fibrosis, carriers and healthy subjects.
      ]. Importantly, a recent study suggests that the ”bubble” test allows differentiation of levels of CFTR function in CFTR-RD subjects between healthy carriers and CF patients according to the residual function [
      • Treggiari D.
      • Kleinfelder K.
      • Bertini M.
      • Tridello G.
      • Fedrigo A.
      • Pintani E.
      • et al.
      Optical measurements of sweat for in vivo quantification of CFTR function in individual sweat glands.
      ]. Based on increasing generated data with this test, inclusion of beta-adrenergic sweat secretion into the CF diagnostic algorithm may allow future identification of subjects with CFTR dysfunction below the healthy heterozygote range.

      9. In vitro tests from primary cells

      Recent advances in stem cell technology provided new tools to set up 2D or 3D tissue-derived cultures.
      Human bronchial epithelial (HBE) cells have been used to assess the efficacy of CFTR modulators in vitro. However, these cells cannot be used routinely, as they are derived from explanted lungs. To overcome this problem, human nasal epithelial (HNE) cell cultures have been developed. These are easy to collect by simple nasal brushing and allow quantification of cyclic AMP (cAMP)-mediated Cl transport as a surrogate marker of CFTR function [
      • Pranke I.
      • Hatton A.
      • Masson A.
      • Flament T.
      • Le Bourgeois M.
      • Chedevergne F.
      • et al.
      Might brushed nasal cells be a surrogate for CFTR modulator clinical response?.
      ,

      Golec, A., Pranke I., Hatton A., Dreano E., Hayes K. and Sermet I.. Isolation, cultivation and application of primary epithelial cells obtained by nasal brushing, polyp sample or lung explants. ECFS-CTN/4.1/001 (2021)

      ]. They can be reutilized after cryopreservation in a biobank. HNE cells recapitulate properties of lower airway epithelial cells and are able to distinguish levels of CFTR function according to the CFTR genotype. They allow study of Cl and HCO3- transport and might enable delineation of CFTR dysfunction in CFTR-RD patients, according to baseline CFTR function as well as their response to CFTR modulators [
      • Davis P.B.
      Cystic fibrosis.
      ,
      • Pranke I.
      • Hatton A.
      • Masson A.
      • Flament T.
      • Le Bourgeois M.
      • Chedevergne F.
      • et al.
      Might brushed nasal cells be a surrogate for CFTR modulator clinical response?.
      ,

      Golec, A., Pranke I., Hatton A., Dreano E., Hayes K. and Sermet I.. Isolation, cultivation and application of primary epithelial cells obtained by nasal brushing, polyp sample or lung explants. ECFS-CTN/4.1/001 (2021)

      ]. The correlation between CFTR bioactivity assessed in vitro in this model and in vivo in the nasal mucosa by NPD [
      • Pranke I.M.
      • Hatton A.
      • Simonin J.
      • Jais J.P.
      • Le Pimpec-Barthes F.
      • Carsin A.
      • et al.
      Correction of CFTR function in nasal epithelial cells from cystic fibrosis patients predicts improvement of respiratory function by CFTR modulators.
      ] or ex vivo in native cells by patch clamp has been documented [
      • Noel S.
      • Servel N.
      • Hatton A.
      • Golec A.
      • Rodrat M.
      • Ng D.R.S.
      • et al.
      Correlating genotype with phenotype using CFTR-mediated whole-cell Cl(-) currents in human nasal epithelial cells.
      ].
      Organoids derived from primary tissues of different organs are also usable to determine CFTR function [
      • Dekkers J.F.
      • Wiegerinck C.L.
      • de Jonge H.R.
      • Bronsveld I.
      • Janssens H.M.
      • de Winter-de Groot K.M.
      • et al.
      A functional CFTR assay using primary cystic fibrosis intestinal organoids.
      ]. Interestingly organoids from rectal biopsies can be obtained from samples utilized for intestinal current measurements, thus avoiding multiple biopsies or resampling, and further reducing discomfort for the patients. The parameter that permits evaluation of CFTR function in colonoids is the increase in water transport driven by CFTR activation induced by Forskolin (Forskolin Induced Swelling, FIS), and possibly the baseline morphology [
      • Cuyx S.
      • Ramalho A.S.
      • Corthout N.
      • Fieuws S.
      • Furstova E.
      • Arnauts K.
      • et al.
      Rectal organoid morphology analysis (ROMA) as a promising diagnostic tool in cystic fibrosis.
      ]. As 3D imaging of FIS assay has been reported to correlate with transepithelial current measurements in monolayer cultures obtained from fragmentation of organoids, their use could help to characterize CFTR-RD dysfunction [
      • Sachs N.
      • Papaspyropoulos A.
      • Zomer-van Ommen D.D.
      • Heo I.
      • Bottinger L.
      • Klay D.
      • et al.
      Long-term expanding human airway organoids for disease modeling.
      ]. Furthermore, the use of in vitro expanded cells derived from individual subjects can overcome several limitations associated with ICM measures such as: (i) the absolute need for fresh biopsies for ICM (few hours from collection), a condition severely limiting the possibility of transport to a specialized center (ii) no possibility to repeat or postpone the analysis in case of technical problems of any sort; (iii) the limited number of biopsies that can be obtained from a patient, that reduce the number of tests/conditions. A standardized multicenter protocol for development of these models has been recently published [
      • Vonk A.M.
      • van Mourik P.
      • Ramalho A.S.
      • Silva I.A.L.
      • Statia M.
      • Kruisselbrink E.
      • et al.
      Protocol for application, standardization and validation of the forskolin-induced swelling assay in cystic fibrosis human colon organoids.
      ].
      Altogether, these new tests could be combined to determine CFTR function in different tissues as recently described in a patient with recurrent pancreatitis and possible CFTR-RD showing consistent results in new as well as standardized tests including tests based on monocytes, rectal biopsies and sweat glands [
      • Kelly M.
      • Dreano E.
      • Hatton A.
      • Lepissier A.
      • Golec A.
      • Sermet-Gaudelus I
      • Pranke I.
      Primary human nasal epithelial cells: biobanking in the context of precision medicine.
      ].
      Finally, these novel models can also be used for theratyping, investigating the functional behavior of mutant CFTR to various drug treatments [
      • Pranke I.
      • Hatton A.
      • Masson A.
      • Flament T.
      • Le Bourgeois M.
      • Chedevergne F.
      • et al.
      Might brushed nasal cells be a surrogate for CFTR modulator clinical response?.
      ,
      • Pranke I.M.
      • Hatton A.
      • Simonin J.
      • Jais J.P.
      • Le Pimpec-Barthes F.
      • Carsin A.
      • et al.
      Correction of CFTR function in nasal epithelial cells from cystic fibrosis patients predicts improvement of respiratory function by CFTR modulators.
      ,
      • Caldrer S.
      • Bergamini G.
      • Sandri A.
      • Vercellone S.
      • Rodella L.
      • Cerofolini A.
      • et al.
      Cystic fibrosis transmembrane conductance regulator functional evaluations in a G542X+/- IVS8Tn:T7/9 patient with acute recurrent pancreatitis.
      ]. In the CFTR-RD landscape, the level of correction as well as the drug specificity of a drug rescue effect might give some mechanistic insights into the mutation itself. Secondly, the communication of a diagnosis of CFTR dysfunction together with a therapeutic perspective outlook may make a difference for the patient and clinician alike, in particular in challenging cases such as those presenting with ultra-rare variants and CFTR-RD presentations. Validation of the predictive capacity of these ex vivo assays for CFTR targeted therapies as well as for diagnosis is in progress [
      • Masson A.
      • Schneider-Futschik E.K.
      • Baatallah N.
      • Nguyen-Khoa T.
      • Girodon E.
      • Hatton A.
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      Predictive factors for lumacaftor/ivacaftor clinical response.
      ,
      • Pranke I.
      • Hatton A.
      • Masson A.
      • Flament T.
      • Le Bourgeois M.
      • Chedevergne F.
      • et al.
      Might brushed nasal cells be a surrogate for CFTR modulator clinical response?.
      ,

      Golec, A., Pranke I., Hatton A., Dreano E., Hayes K. and Sermet I.. Isolation, cultivation and application of primary epithelial cells obtained by nasal brushing, polyp sample or lung explants. ECFS-CTN/4.1/001 (2021)

      ,
      • Kelly M.
      • Dreano E.
      • Hatton A.
      • Lepissier A.
      • Golec A.
      • Sermet-Gaudelus I
      • Pranke I.
      Primary human nasal epithelial cells: biobanking in the context of precision medicine.
      ,
      • Ramalho A.S.
      • Furstova E.
      • Vonk A.M.
      • Ferrante M.
      • Verfaillie C.
      • Dupont L.
      • et al.
      Correction of CFTR function in intestinal organoids to guide treatment of cystic fibrosis.
      ,
      • van Mourik P.
      • Beekman J.M.
      • van der Ent C.K.
      Intestinal organoids to model cystic fibrosis.
      ,
      • de Winter-de Groot K.M.
      • Janssens H.M.
      • van Uum R.T.
      • Dekkers J.F.
      • Berkers G.
      • Vonk A.
      • et al.
      Stratifying infants with cystic fibrosis for disease severity using intestinal organoid swelling as a biomarker of CFTR function.
      ,
      • Clancy J.P.
      • Cotton C.U.
      • Donaldson S.H.
      • Solomon G.M.
      • VanDevanter D.R.
      • Boyle M.P.
      • et al.
      CFTR modulator theratyping: current status, gaps and future directions.
      ,
      • Kerem E.
      • Cohen-Cymberknoh M.
      • Tsabari R.
      • Wilschanski M.
      • Reiter J.
      • Shoseyov D.
      • et al.
      Ivacaftor in people with cystic fibrosis and a 3849+10kb C–>T or D1152H residual function mutation.
      ,
      • Graeber S.Y.
      • van Mourik P.
      • Vonk A.M.
      • Kruisselbrink E.
      • Hirtz S.
      • van der Ent C.K.
      • et al.
      Comparison of organoid swelling and in vivo biomarkers of CFTR function to determine effects of lumacaftor-ivacaftor in patients with cystic fibrosis homozygous for the F508del mutation.
      ,
      • Berkers G.
      • van der Meer R.
      • Heijerman H.
      • Beekman J.M.
      • Boj S.F.
      • Vries R.G.J.
      • et al.
      Lumacaftor/ivacaftor in people with cystic fibrosis with an A455E-CFTR mutation.
      ]. The main challenge remains to determine if the level of in vitro correction in these models is correlated to modification of CFTR function in vivo and any eventual translation to clinical change. Recent studies did not find a correlation between range of organoid swelling and respiratory response [
      • Kerem E.
      • Cohen-Cymberknoh M.
      • Tsabari R.
      • Wilschanski M.
      • Reiter J.
      • Shoseyov D.
      • et al.
      Ivacaftor in people with cystic fibrosis and a 3849+10kb C–>T or D1152H residual function mutation.
      ,
      • Graeber S.Y.
      • van Mourik P.
      • Vonk A.M.
      • Kruisselbrink E.
      • Hirtz S.
      • van der Ent C.K.
      • et al.
      Comparison of organoid swelling and in vivo biomarkers of CFTR function to determine effects of lumacaftor-ivacaftor in patients with cystic fibrosis homozygous for the F508del mutation.
      ,
      • Berkers G.
      • van der Meer R.
      • Heijerman H.
      • Beekman J.M.
      • Boj S.F.
      • Vries R.G.J.
      • et al.
      Lumacaftor/ivacaftor in people with cystic fibrosis with an A455E-CFTR mutation.
      ].

      10. Conclusion

      For final diagnostic labeling, the clinician needs to interpret the clinical picture in context with both CFTR genetics and functional assays when the diagnosis remains unclear. CFTR bioassays were initially implemented for the diagnosis of patients with CF, but there is presently a lack of clear cut off/boundary values to differentiate CFTR activity in patients with CFTR-RD from patients with CF and heterozygotes. NPD reflects the spectrum of CFTR activity while ICM is more specific in the delineation of patients with CF. New β-adrenergic sweat rate techniques may prove very useful in ruling out a diagnosis of CF in patients presenting with CF-like symptoms. Patients with CFTR-RDs demonstrate a wide range of CFTR activity and are still under-recognized or misclassified. In the era of the CFTR modulators and their potential clinical benefit, it is of the utmost importance to diagnose them as accurately as possible.

      Declaration of Competing Interest

      All the authors have declared their conflict of interests.

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