Duc-Hung Pham1, Ramesh Kudira1, Lingfen Xu2, C Alexander Valencia3, Jillian L Ellis1, Tiffany Shi1, Kimberley J Evason4, Immaculeta Osuji1, Nelson Matuschek1, Liva Pfuhler1, Mary Mullen1, Sujit K Mohanty5, Ammar Husami6, Laura N Bull7, Kejian Zhang8, Sami Wali9, Chunyue Yin10, Alexander Miethke11. 1. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. 2. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Pediatric Gastroenterology, Shengjing Hospital of China Medical University, Shenyang, China. 3. Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; Department of Preclinical Education, Lake Erie College of Osteopathic Medicine, Erie, Pennsylvania. 4. Department of Pathology and Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah. 5. Department of Pediatric and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. 6. Division of Human Genetics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. 7. Liver Center Laboratory, Department of Medicine and Institute for Human Genetics, University of California, San Francisco, San Francisco, California. 8. Mount Sinai Genomics, New York, New York. 9. Pediatric Gastroenterology, Prince Sultan Military Medical City, Riyadh, Saudi Arabia. 10. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio. Electronic address: chunyue.yin@cchmc.org. 11. Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio. Electronic address: alexander.miethke@cchmc.org.
Abstract
BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.
BACKGROUND & AIMS: The etiology of cholestasis remains unknown in many children. We surveyed the genome of children with chronic cholestasis for variants in genes not previously associated with liver disease and validated their biological relevance in zebrafish and murine models. METHOD: Whole-exome (n = 4) and candidate gene sequencing (n = 89) was completed on 93 children with cholestasis and normal serum γ-glutamyl transferase (GGT) levels without pathogenic variants in genes known to cause low GGT cholestasis such as ABCB11 or ATP8B1. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing was used to induce frameshift pathogenic variants in the candidate gene in zebrafish and mice. RESULTS: In a 1-year-old female patient with normal GGT cholestasis and bile duct paucity, we identified a homozygous truncating pathogenic variant (c.198delA, p.Gly67Alafs∗6) in the ABCC12 gene (NM_033226). Five additional rare ABCC12 variants, including a pathogenic one, were detected in our cohort. ABCC12 encodes multidrug resistance-associated protein 9 (MRP9) that belongs to the adenosine 5'-triphosphate-binding cassette transporter C family with unknown function and no previous implication in liver disease. Immunohistochemistry and Western blotting revealed conserved MRP9 protein expression in the bile ducts in human, mouse, and zebrafish. Zebrafish abcc12-null mutants were prone to cholangiocyte apoptosis, which caused progressive bile duct loss during the juvenile stage. MRP9-deficient mice had fewer well-formed interlobular bile ducts and higher serum alkaline phosphatase levels compared with wild-type mice. They exhibited aggravated cholangiocyte apoptosis, hyperbilirubinemia, and liver fibrosis upon cholic acid challenge. CONCLUSIONS: Our work connects MRP9 with bile duct homeostasis and cholestatic liver disease for the first time. It identifies a potential therapeutic target to attenuate bile acid-induced cholangiocyte injury.
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