Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Pediatric Gastroenterology, Shengjing Hospital of China Medical University, Shenyang, China.
Gastroenterology. 2021 Jul;161(1):287-300.e16. doi: 10.1053/j.gastro.2021.03.026. Epub 2021 Mar 23.
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.
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.
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.
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.
许多儿童的胆汁淤积病因仍不清楚。我们对慢性胆汁淤积症儿童的基因组进行了调查，以寻找以前与肝脏疾病无关的基因中的变异，并在斑马鱼和小鼠模型中验证其生物学相关性。
对 93 名胆汁淤积症且血清 γ-谷氨酰转移酶 (GGT) 水平正常且无 ABCB11 或 ATP8B1 等已知导致低 GGT 胆汁淤积症的基因致病性变异的儿童进行全外显子组 (n=4) 和候选基因测序 (n=89)。CRISPR (成簇规律间隔短回文重复序列)/Cas9 基因组编辑用于在候选基因中诱导斑马鱼和小鼠的移码致病性变异。
在一名 1 岁女性正常 GGT 胆汁淤积和胆管稀少患者中，我们发现 ABCC12 基因 (NM_033226) 存在纯合截短致病性变异 (c.198delA, p.Gly67Alafs∗6)。在我们的队列中还检测到另外 5 种罕见的 ABCC12 变异，包括一种致病性变异。ABCC12 编码多药耐药相关蛋白 9 (MRP9)，属于尚未涉及肝脏疾病的未知功能的腺苷 5'-三磷酸结合盒转运蛋白 C 家族。免疫组织化学和 Western blot 显示人类、小鼠和斑马鱼胆管中存在保守的 MRP9 蛋白表达。斑马鱼 abcc12 基因敲除突变体易发生胆管细胞凋亡，导致幼年期胆管逐渐丢失。与野生型小鼠相比，MRP9 缺陷型小鼠的小叶间胆管数量较少，碱性磷酸酶水平较高。它们在胆酸挑战时表现出更严重的胆管细胞凋亡、高胆红素血症和肝纤维化。
我们的工作首次将 MRP9 与胆管稳态和胆汁淤积性肝病联系起来。它确定了一个潜在的治疗靶点，以减轻胆汁酸诱导的胆管细胞损伤。
