Modeling genetic contributions to biliary atresia

项目总结/摘要 胆道闭锁（BA）是一个重要的和复杂的疾病的新生儿，一直回避主要 病因学和病理生理学的发现。最近，NIDDK支持的 ChiLDReN网络对患有心脏和心脏病的BA个体的子集进行了外显子组测序， 腹部偏侧特征-BA脾畸形（BASM）综合征患者 以确定是否有一个遗传病因学在这组多器官发育 畸形发生。BASM外显子组序列分析发现几名参与者具有 纤毛基因PKD 1 L1（一种与心脏偏侧性缺陷相关的基因）的显著突变， 但尚未与胆道疾病联系起来。为了探索机械后果， 在人类PKD 1 L1信号传导受损的情况下，我们开发了一种肝内胆管细胞限制性 Pkd 111 F1/F1;Afp-Cre（LKO）小鼠。初步数据表明，Pkd 1 l1的缺乏， 发育中的小鼠肝脏不仅导致早期胆道畸形， 在成年人的纤维炎症，尤其是在胆管结扎后远端梗阻的情况下 （BDL）。这些组织学特征与人BA肝脏中观察到的特征非常相似。Aim 1探索 在LKO中缺乏Pkd 1 l1信号传导的纤维炎性后果和其他信息 Pkd 1 l1 Fl/Fl杂交系（包括一个具有人胆汁酸库，另一个将删除 pkd 1 l1在整个胆道系统中）和对选择的基于胆汁酸的治疗干预的反应。 目的2：探讨胎儿期和早期发育中胆管的早期形态学特征 出生后肝脏的谱系追踪和多重空间RNA研究。最后，目标3是一个 胆管细胞类器官的体外实验集，极化的Transwell培养物和3D导管- 芯片上研究，以确定分离的Pkd 1 l1 Fl/Fl中的分子和信号传导结果， LKO胆管细胞。总的来说，我们预计这三个目标将提供有史以来第一个基因 BA模型有望发现胆道反应性的新细胞和分子机制 和损害。此外，在提供信息的Pkd 1 l1小鼠中测试基于胆汁酸途径的试剂 模型可能有助于提供支持性的临床前证据，以解决目前缺乏 有效的医学治疗。

Project Summary/Abstract Biliary atresia (BA) is an important and perplexing disease of neonates that has eluded major discoveries of etiology and pathophysiology for decades. Recently, the NIDDK-supported ChiLDReN network performed exome sequencing on a subset of BA individuals with cardiac and abdominal laterality features–those with the BA Splenic Malformation (BASM) syndrome in order to determine if there is a genetic etiology in this group with multi-organ developmental dysmorphogenesis. Analysis of BASM exome sequences found several participants with significant mutations in the ciliary gene PKD1L1, a gene associated with cardiac laterality defects, but not yet linked to biliary tract disease. In order to explore mechanistic consequences to impaired PKD1L1 signaling in humans, we developed an intrahepatic cholangiocyte-restricted Pkd1l1Fl/Fl;Afp-Cre (LKO) mouse. Preliminary data indicates that absence of Pkd1l1 in the developing mouse liver leads not only to early biliary dysmorphology, but an enhanced peribiliary fibroinflammation at adult ages, moreso in the setting of distal obstruction after bile duct ligation (BDL). These histologic features strongly mimic those seen in human BA livers. Aim 1 explores the fibroinflammatory consequences of absent Pkd1l1 signaling in the LKO and other informative Pkd1l1Fl/Fl cross-bred lines (including one with a human bile acid pool and another that will delete Pkd1l1 in the entire biliary tree) and response to select bile acid based therapeutic interventions. Aim 2 explores the delineation of early bile duct dysmorphology in developing prenatal and early postnatal livers with lineage tracing and multiplexed spatial RNA studies. Finally, Aim 3 is an in vitro set of experiments with cholangiocyte organoids, polarized Transwell cultures and 3d duct- on-a-chip studies to define the molecular and signaling consequences in isolated Pkd1l1Fl/Fl and LKO cholangiocytes. Taken together we anticipate that these 3 Aims will provide first-ever genetic models of BA poised to discover new cellular and molecular mechanisms of biliary tract reactivity and damage. In addition, testing of bile acid pathway-based agents in informative Pkd1l1 mouse models may help provide supportive pre-clinical evidence to address the current paucity of effective medical therapeutics in BA.