Combining standardized and new CFTR functional tests for diagnosis

Introduction: CFTR function measurement in vivo is an actual field of interest for detecting the effects of CFTR genetic variants/rare mutations as well as of drugs targeting the basic defect. A quantitative assay for measur-ing CFTR function in murine and human primary intestinal crypt-based tis-sues was reported (Dekkers JF, et al. Nat Med. 2013;19:939-45); intestinal organoids can be developed after intestinal current measurements (ICM).CFTR functional assays in leukocytes have been previously shown to be capable of clearly discriminating CF and non-CF subjects (Sorio C, et al. PLoS One. 2011;6:e22212). JJ Wine, et al. (PLoS One. 2013;8:e77114) distinguished CF, non-CF and carriers by a ratiometric beta adrenergic/cholinergic sweat test.Methods and Results: European CF Society intestinal current mea-surements (ICM) and nasal potential difference (NPD) standardized oper-ating procedure (SOP) are applied at the CF Centre of Verona for research and diagnosis of atypical cases. We tested an individualized combination of standardized and new CFTR functional bioassays in a patient referred at our center for evaluation after several episodes of acute pancreatitis; CFTR genotype following sequencing analysis was G542X +/- IVS8 T7/T9, borderline sweat [Cl-]values of 41- 45 mEq/L were found by the Gibson and Cooke method. Lung function and sputum cultures were normal; azo-ospermia was excluded. Recent nasal surgery for deviated nasal septum and ensuing scars in both nostrils did not allow NPD measurements. ICM were measured in 4 biopsies with tracings resembling those obtained from non-CF subjects consistent with previously published normal range (Derichs N, et al. Thorax. 2010;65:594-9).CFTR activity by forskolin induced assay (Dekkers, et al.) was consis-tent with that of non-CF organoids; the pre-swollen lumen suggested a non-CF phenotype. CFTR function was tested by the membrane depolarization assay in monocytes. We defined the CF index as outcome of this assay, which was positive in all healthy subjects and negative in all CF patients: in this case the CF index was positive (CF index = +44). The above cited rati-ometric beta adrenergic/cholinergic sweat test provided results overlapping with those of carriers.Conclusions: This study combines relatively simple and robust assays in several tissues and proposes an example of possible individualized appli-cation as support for diagnosis. Missing the possibility to apply standard-ized NPD in this subject, all data were concordant in excluding CF diagno-sis. Such a combination of functional approaches can be valuable for testing drugs targeting the basic defect