Cystic fibrosis transmembrane conductance regulator functional evaluations in a G542X+/- IVS8Tn:T7/9 patient with acute recurrent pancreatitis

Abstract

Background: Acute recurrent pancreatitis (ARP) is characterized by episodes of acute pancreatitis in an otherwise normal gland. When no cause of ARP is identifiable, the diagnosis of "idiopathic" ARP is given. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene increase the risk of ARP by 3- to 4-times compared to the general population, while cystic fibrosis (CF) patients present with a 40- to 80-times higher risk of developing pancreatitis.

Case summary: In non-classical CF or CFTR-related disorders, CFTR functional tests can help to ensure a proper diagnosis. We applied an individualized combination of standardized and new CFTR functional bioassays for a patient referred to the Verona CF Center for evaluation after several episodes of acute pancreatitis. The CFTR genotype was G542X+/- with IVS8Tn:T7/9 polymorphism. The sweat (Cl^-) values were borderline. Intestinal current measurements were performed according to the European Cystic Fibrosis Society Standardized Operating Procedure. Recent nasal surgery for deviated septum did not allow for nasal potential difference measurements. Lung function and sputum cultures were normal; azoospermia was excluded. Pancreas divisum was excluded by imaging but hypoplasia of the left hepatic lobe was detected. Innovative tests applied in this case include sweat rate measurement by image analysis, CFTR function in monocytes evaluated using a membrane potential-sensitive fluorescent probe, and the intestinal organoids forskolin-induced swelling assay.

Conclusion: Combination of innovative CFTR functional assays might support a controversial diagnosis when CFTR-related disorders and/or non-classical CF are suspected.

Keywords: Case report; Controversial diagnosis; Cystic fibrosis; Cystic fibrosis transmembrane conductance regulator function; Intestinal current; Organoids; Recurrent acute pancreatitis; Sweat test.

Figure 1

Results of standardized cystic fibrosis transmembrane conductance regulator functional test. Intestinal current measurement tracings from the controversial clinical case (presented herein), cystic fibrosis (CF) patients with pancreatic insufficiency-CF or pancreatic sufficiency-CF and control (non-CF donor). As shown in the top panel, there were positive tissue responses to forsk/IBMX: Forskolin/3-isobutyl-1-methylxanthine, Carbachol and Histamine clearly visible in acute recurrent pancreatitis and not consistent with CF diagnosis. ARP: Acute recurrent pancreatitis; CF: Cystic fibrosis; PI-CF: Pancreatic insufficiency-cystic fibrosis; PS-CF: Pancreatic sufficiency-cystic fibrosis.

Figure 2

Results of cystic fibrosis transmembrane conductance regulator functional assays. A: Membrane depolarization by single cell fluorescence analysis performed in monocytes with (black trace) and without (white trace) stimulus added at 5 min. The cystic fibrosis (CF) index was calculated as reported[10]; B: Normalized volume increase of individual organoids obtained during Forskolin-induced swelling assay. Organoids were obtained from the acute recurrent pancreatitis patient carrying the mutation G542+/-, a non-CF subject with or without pre-incubation with cystic fibrosis transmembrane conductance regulator-inh72 and with stimulation with the potentiator VX770, and a CF patient. Box and whisker plots (10^th-90^th percentiles) correspond to the normalized volume increase with respect to the baseline for each subject; the midline in boxes indicates median. Symbols indicate significant differences (one way-ANOVA; ^aP < 0.05; ^bP < 0.02) identified by Dunn’s method to compare all groups vs the non-CF control. CFTR: Cystic fibrosis transmembrane conductance regulator; CF: Cystic fibrosis; CFI: Cystic fibrosis index; FIS: Forskolin-induced swelling.