Mechanisms of CFTR Functional Variants That Impair Regulated Bicarbonate Permeation and Increase Risk for Pancreatitis but Not for Cystic Fibrosis

Authors

Jessica LaRusch, University of Pittsburgh
Jinsei Jung, Yonsei University College of Medicine
Ignacio J. General, University of Pittsburgh
Michele D. Lewis, Mayo Clinic
Hyun W. Park, Yonsei University College of Medicine
Randall E. Brand, University of Pittsburgh
Andres Gelrud, University of Pittsburgh
Michelle A. Anderson, University of Michigan
Peter A. Banks, Brigham and Women's Hospital, Boston
Darwin Conwell, Brigham and Women's Hospital, Boston
Christopher Lawrence, Medical University of South Carolina
Joseph Romagnuolo, Medical University of South Carolina
John Baillie, Duke University Medical Center
Samer Alkaade, St. Louis University School of Medicine
Gregory Cote, Indiana University School of Medicine
Timothy B. Gradner, Dartmouth-Hitchcock Medical Center
Stephen T. Amann, North Mississippi Medical Center
Adam Slivka, North Mississippi Medical Center
Bimaljit Sandhu, Virginia Commonwealth University Medical Center
Amy Aloe, University of Pittsburgh
Michelle L. Kienholz, University of Pittsburgh
Dhiraj Yadav, University of Pittsburgh
M. Michael Barmada, University of Pittsburgh
Ivet Bahar, University of Pittsburgh
Min G. Lee, Yonsei University College of Medicine
David C. Whitcomb, University of Pittsburgh

Document Type

Article

Original Publication Date

2014

Journal/Book/Conference Title

PLOS Genet

Volume

10

DOI of Original Publication

10.1371/journal.pgen.1004376

Comments

Originally Published at http://dx.doi.org/10.1371/journal.pgen.1004376

Date of Submission

November 2014

Abstract

CFTR is a dynamically regulated anion channel. Intracellular WNK1-SPAK activation causes CFTR to change permeability and conductance characteristics from a chloride-preferring to bicarbonate-preferring channel through unknown mechanisms. Two severe CFTR mutations (CFTRsev) cause complete loss of CFTR function and result in cystic fibrosis (CF), a severe genetic disorder affecting sweat glands, nasal sinuses, lungs, pancreas, liver, intestines, and male reproductive system. We hypothesize that those CFTR mutations that disrupt the WNK1-SPAK activation mechanisms cause a selective, bicarbonate defect in channel function (CFTRBD) affecting organs that utilize CFTR for bicarbonate secretion (e.g. the pancreas, nasal sinus, vas deferens) but do not cause typical CF. To understand the structural and functional requirements of the CFTR bicarbonate-preferring channel, we (a) screened 984 well-phenotyped pancreatitis cases for candidate CFTRBD mutations from among 81 previously described CFTR variants; (b) conducted electrophysiology studies on clones of variants found in pancreatitis but not CF; (c) computationally constructed a new, complete structural model of CFTR for molecular dynamics simulation of wild-type and mutant variants; and (d) tested the newly defined CFTRBD variants for disease in non-pancreas organs utilizing CFTR for bicarbonate secretion. Nine variants (CFTR R74Q, R75Q, R117H, R170H, L967S, L997F, D1152H, S1235R, and D1270N) not associated with typical CF were associated with pancreatitis (OR 1.5, p = 0.002). Clones expressed in HEK 293T cells had normal chloride but not bicarbonate permeability and conductance with WNK1-SPAK activation. Molecular dynamics simulations suggest physical restriction of the CFTR channel and altered dynamic channel regulation. Comparing pancreatitis patients and controls, CFTRBD increased risk for rhinosinusitis (OR 2.3, p<0.005) and male infertility (OR 395, p<<0.0001). WNK1-SPAK pathway-activated increases in CFTR bicarbonate permeability are altered by CFTRBD variants through multiple mechanisms. CFTRBD variants are associated with clinically significant disorders of the pancreas, sinuses, and male reproductive system.

Author Summary

Genetic disorders of ion channels can affect the body's ability to function properly in many ways. CFTR, an ion channel regulating movement of chloride and bicarbonate across cell membranes, is important for absorbing and secreting fluids. If the gene responsible for the CFTR channel is mutated severely, the result is cystic fibrosis, a hereditary disorder in which the patient develops thick mucus, especially in the lungs, as well as scarring (fibrosis) in the pancreas. Cystic fibrosis also affects the sweat glands, nasal sinuses, intestines, liver, and male reproductive system. Mutations to the CFTR gene that do not cause cystic fibrosis have been considered benign. However, we discovered 9 CFTR mutations that do not cause cystic fibrosis but do cause inflammation and scarring of the pancreas (chronic pancreatitis). These mutant CFTR channels secrete chloride, which is important in the sweat glands, lungs, and intestines, but not bicarbonate, which is important in the pancreas, sinuses, and male reproductive tract. We found patients with any of these 9 mutations had chronic pancreatitis, and often sinus infections, and male infertility, but not other symptoms of cystic fibrosis. Our computer models and data will help researchers develop better drugs and help physicians treating patients with chronic pancreatitis.

Rights

Copyright: © 2014 Whitcomb et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Is Part Of

VCU Internal Medicine Publications