Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of CFTR in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR was compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR was quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared to control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR was accompanied by decreases in mRNA for the Cl-/HCO3- exchanger SLC26A3, K+ channels (KCNQ1, KCNN4), and vasoactive intestinal polypeptide receptor (VIPR1) which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low risk, high risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by underlying failure of CFTR function.

Methods
We used the Whole Animal Genome Sequencing (WAGS) pipeline to identify short nucleotide variants in a dataset of 2,642 dogs encompassing both private and public resources including 1,971 genomes from the Dog10K project. Briefly, the WAGS pipeline used Burrows-Wheeler Alignment tool-MEM to map paired-end reads to the UU_Cfam_GSD_1.0 reference genome. Variant calling was executed with Genome Analysis Toolkit (GATK4), and Ensembl’s Variant Effect Predictor (VEP, RRID:SCR_007931) predicted variant annotations and consequences. From the resulting VEP-processed VCF file, we extracted CFTR genic variants plus variants within 1Kb of the flanking sequence that passed filters. Subsequently, non-reference allele frequencies were calculated for each variant within the control, risk, and affected dog groups.