Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder
Data files
Jul 20, 2024 version files 24.11 MB
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CFTR.gookin.vcf.gz
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README.md
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Supplemental_Figure_S1.tif
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Supplemental_Table_S1.csv
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Supplemental_Table_S2.csv
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Supplemental_Table_S3.csv
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Abstract
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.
https://doi.org/10.5061/dryad.2rbnzs7xq
This dataset includes supplementary materials for the manuscript entitled Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder.
Description of the data and file structure
Supplemental Figure S1 illustrates sample procurement and appearance of gallbladder from each of 9 dogs having mucosal RNA extracted for targeted gene expression analysis. Samples of lumen mucosa were obtained by excision from regions devoid of mucus or from which mucus could be gently removed. During sampling (panel A) and after removal of sample (panel B). Remaining panels show each of 9 individual mucocele gallbladders used for mucosal RNA sample collection. Pictures are immediately post-cholecystectomy followed by opening of the gallbladder to expose the lumen.
Supplemental Table S1 documents the location and frequency of variants identified by whole genome sequencing in the upstream and downstream non-coding sequence of CFTR in dogs at low-risk, high-risk, and definitively diagnosed with gallbladder mucocele formation.
Canine CFTR gene sequence data was obtained by means of whole genome sequencing performed on blood collected from 8 Shetland sheepdogs with gallbladder mucocele formation (column confirmed disease) and the location and frequency of variants in the coding region of CFTR were compared to 115 dogs from 12 breeds at high risk for mucocele formation (column high risk) and 2,519 dogs from 340 breeds considered at low risk for diagnosis of mucocele formation (column low risk). Location and frequencies of CFTR variants in the non-coding 5' and 3' UTR of CFTR in each group of dogs is shown in supplemental Table S1.
Supplemental Table S2 documents the location and frequencies of CFTR variants in the intronic regions of CFTR in each group of dogs at low-risk, high-risk, and definitively diagnosed with gallbladder mucocele formation.
Canine CFTR gene sequence data was obtained by means of whole genome sequencing performed on blood collected from 8 Shetland sheepdogs with gallbladder mucocele formation (column confirmed disease) and the location and frequency of variants in the coding region of CFTR were compared to 115 dogs from 12 breeds at high risk for mucocele formation (column high risk) and 2,519 dogs from 340 breeds considered at low risk for diagnosis of mucocele formation (column low risk). Location and frequencies of CFTR variants in the intronic regions of CFTR in each group of dogs is shown in supplemental Table S2.
Supplemental Table S3 Metadata for dogs undergoing canine CFTR gene sequence analysis.
VCF File Tab-delimited variant call file for dogs undergoing CFTR gene sequence analysis.
Code/Software
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. Since VEP used the dog NM_001007143.1 CFTR transcript to make predictions, we codon aligned this sequence with the human CFTR transcript NM_000492.4, used by the cftr2 database (https://cftr2.org/ updated April 7, 2023), to map homologous coding sequence positions between the human and dog CFTR gene.
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.