An insertion mutation in ABCB4 is associated with gallbladder mucocele formation in dogs

Mealey et al. Comparative Hepatology 2010, 9:6 http://www.comparative-hepatology.com/content/9/1/6 Open Access RESEARCH An insertion mutation in ABCB4 is associated with gallbladder mucocele formation in dogs Research Katrina L Mealey*1,4, Jonathan D Minch1, Stephen N White2,3,4, Kevin R Snekvik3 and John S Mattoon1 Abstract Background: ABCB4 functions as a phosphatidylcholine translocater, flipping phosphatidylcholine across hepatocyte canalicular membranes into biliary canaliculi. In people, ABCB4 gene mutations are associated with several disease syndromes including intrahepatic cholestasis of pregnancy, progressive familial intrahepatic cholestasis (type 3), primary biliary cirrhosis, and cholelithiasis. Hepatobiliary disease, specifically gallbladder mucocele formation, has been recognized with increased frequency in dogs during the past decade. Because Shetland Sheepdogs are considered to be predisposed to gallbladder mucoceles, we initially investigated ABCB4 as a candidate gene for gallbladder mucocele formation in that breed, but included affected dogs of other breeds as well. Results: An insertion (G) mutation in exon 12 of canine ABCB4 (ABCB4 1583_1584G) was found to be significantly associated with hepatobiliary disease in Shetland Sheepdogs specifically (P < 0.0001) as well as other breeds (P < 0.0006). ABCB4 1583_1584G results in a frame shift generating four stop codons that prematurely terminate ABCB4 protein synthesis within exon 12, abolishing over half of the protein including critical ATP and a putative substrate binding site. Conclusions: The finding of a significant association of ABCB4 1583_1584G with gallbladder mucoceles in dogs suggests that this phospholipid flippase may play a role in the pathophysiology of this disorder. Affected dogs may provide a useful model for identifying novel treatment strategies for ABCB4-associated hepatobiliary disease in people. Background Bile is produced by the collective actions of a number of transporters located on the canalicular membrane of hepatocytes [1]. Active transport of biliary solutes creates an osmotic force that attracts water through tight junctions and aquaporins in the hepatocyte membrane [2,3]. Bile salts are the most important biliary solute. Other important solutes of bile include cholesterol and phospholipids. The presence of phospholipids, phosphatidylcholine (PC) in particular, in the biliary lumen is crucial for protecting the epithelial cell membranes lining the biliary system from the cytotoxic detergent actions of bile salts [3-5]. Bile salt cytotoxicity is substantially reduced in the presence of PC owing to the formation of mixed micelles (PC + bile salts) rather than simple micelles (bile salts only). Thus, a decrease in the amount of biliary PC * Correspondence: 1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-6610, USA Full list of author information is available at the end of the article leads to injury of epithelial cells lining the biliary system [6]. ABCB4 functions exclusively as a phospholipid translocator [6]. ABCB4 is expressed on cannalicular membranes of hepatocytes where it translocates PC from the hepatocyte to the biliary canalicular lumen [7]. Proper function of ABCB4 is critical for maintaining hepatobiliary homeostasis as evidenced by the myriad of diseases that occur when polymorphisms of ABCB4 cause complete or partial protein dysfunction. ABCB4 deficiency is associated with a variety of hepatobiliary disorders in people including progressive familial intrahepatic cholestasis (PFIC type 3), cholelithiasis, and cholestasis of pregnancy [4,8-10]. Abcb4-/- mice, in which Abcb4 function is lacking entirely, also develop severe hepatobiliary disease that starts at a few weeks of age and progresses throughout life [11,12]. Hepatobiliary disease in dogs has been recognized with increased frequency during the past several years. In particular, gallbladder mucoceles (mucinous hyperplasia or mucinous cholecystitis) have been documented to be an © 2010 Mealey et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mealey et al. Comparative Hepatology 2010, 9:6 http://www.comparative-hepatology.com/content/9/1/6 increasingly important cause of hepatobiliary disease in dogs [13-15]. Histopathologic findings associated with ABCB4 associated diseases in people, including intrahepatic cholestasis, cholecystitis, and periportal inflammation [13,16,17], are not commonly reported in dogs with gall bladder mucoceles. Additionally, gallbladder mucoceles are not a component of ABCB4 linked syndromes in people or mice. Gallbladder mucoceles, which occur rarely in people, are often associated with extrahepatic bile duct obstruction. The etiology of gallbladder mucoceles in dogs has not yet been identified, but extrehepatic bile duct obstruction is not commonly associated with this disorder [14,15]. Gallbladder mucoceles may result from chronic injury to the epithelial lining of the biliary system since hypersecretion of mucin is the typical physiologic response of any epithelial lining to injury. Recently Shetland Sheepdogs were identified as a breed that is predisposed to gallbladder mucocele formation, suggesting a genetic predisposition [13]. Because ABCB4 dysfunction is associated with hepatobiliary disease in people and mice, we postulated that a defect in canine ABCB4 might be responsible for gallbladder mucocele disease in dogs, and Shetland Sheepdogs in particular. Therefore, we sequenced canine ABCB4 in affected and unaffected Shetland Sheepdogs as well as affected and unaffected dogs of other breeds. Methods Collection of DNA from affected and unaffected individuals All work was approved by the institutional Animal Care and Use Committee. Collection of DNA from affected Shetland Sheepdogs was accomplished by soliciting owners' cooperation. In order to cast a wide net, owners of dogs with confirmed (ultrasound, surgery, or histopathology) or suspected (elevated liver enzymes - alkaline phosphatase, alanine aminotransferase and/or gamma glutamyl transferase -, total bilirubin, cholesterol and/or triglycerides) gallbladder disease were asked to submit a cheek swab, copy of the dog's pedigree, and copy of the dog's medical record. Contact of Shetland Sheepdog owners was made through the American Shetland Sheepdog Association. For collection of unaffected Shetland Sheepdogs, an additional request for DNA from healthy Shetland Sheepdogs (with confirmatory medical records) was made. For collection of DNA from affected dogs of any breed, records from the Washington Animal Disease Diagnostic Laboratory were searched for canine patients with his (...truncated)