The Genetic Basis of Pediatric Cholestasis

小儿胆汁淤积的遗传基础

基本信息

批准号：
9565392
负责人：
LAURA N BULL
金额：
$ 39.42万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-24 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9565392
关键词：
3-Dimensional ATP8B1 gene Address Adult Alleles Animal Model Bile fluid Biochemical Biological Biological Models CRISPR/Cas technology Cells Child Childhood Cholestasis Clinical Clinical Data Clinical assessments Collaborations Collection Complement Complex Country DNA DNA sequencing Data Databases Development Diagnosis Disease Disease Management Europe Family Feasibility Studies Functional disorder Funding Gene Mutation Genes Genetic Genetic Diseases Genetic Predisposition to Disease Genetic study Goals Health Hepatocyte Histologic Hospitals Human Impairment In Vitro Inherited Institutes Investigation Knowledge Laboratories Light Liver Liver diseases London Medical Records Methodology Methylation Mission Molecular Molecular Biology Techniques Mutate Mutation National Institute of Diabetes and Digestive and Kidney Diseases Patients Permeability Phenotype Physiology Poland Progressive intrahepatic cholestasis RNA Splicing Research Resources Sampling Serum System Testing Tight Junctions Tissue Banks Tissues Transmembrane Transport Transplantation United States National Institutes of Health Update Variant cholangiocyte cholestatic liver disease college data resource disease phenotype exome exome sequencing experience genetic disorder diagnosis genetic technology genome sequencing human model improved in vitro Model induced pluripotent stem cell innovation liver function liver transplantation next generation sequencing novel patient screening promoter public health relevance trafficking transcriptome sequencing whole genome

项目摘要

SUMMARY/ABSTRACT Bile flow is essential for normal liver function. Cholestasis is the term for all phenomena where bile flow is impaired. Over the last few years, great progress has been made in our understanding of the mechanisms underlying pediatric cholestatic liver disease, including by the current applicants. Despite this, in 40% of children, the primary genetic etiology of cholestasis has not been identified, after targeted next-generation sequencing (tNGS) of known cholestasis genes. Furthermore, our understanding of these diseases, and our ability to treat them, is still limited. This proposal continues the collaboration between Drs. Bull and Thompson. It brings together the unmatched sample and data resources of the 2 largest pediatric liver centers in Europe (King's College London and Children's Memorial Institute in Warsaw), Dr Bull's laboratory, and the NIH-funded Childhood Liver Disease Research Network (ChiLDReN). This collection of resources and experience will enable the following challenges to be addressed. In Aim 1, genetic studies will be performed to identify novel cholestasis genes, and novel mutations in known cholestasis genes. This aim will be accomplished using state-of-the-art genetic technologies including: tNGS, whole exome sequence (WES), whole genome sequencing, RNA-sequencing and methylation analysis. In Aim 2, we will characterize the features of genetically distinct forms of cholestasis, through assessment of clinical and biochemical data from patients. Extensive tissue collections are available and histological and immunohistochemical analysis will be undertaken. In Aim 3, we will investigate the pathophysiological mechanisms underlying genetic cholestasis. Animal models of known genetic forms of cholestasis typically fail to replicate human phenotypes well, so in vitro model systems are required to investigate disease mechanisms and test potential treatments. In vitro systems may be used to assess promoter function, splicing, trafficking and membrane transport, as appropriate. Moreover, CRISPR/Cas9 technology will be used to introduce homozygous mutations into induced pluripotent stem cells (iPSCs), to generate mutated hepatocyte and cholangiocyte-like cells. These cells will be used to explore the consequences of mutations in disease-causing genes, initially focused on increased understanding of disease mechanism in TJP2 deficiency. Preliminary data have shown these cells form canaliculi with tight junctions between cells. Use of such cells will allow us, for example, to test the permeability of tight junctions in vitro. In conclusion, these proposed studies will result in identification of new disease genes and mutations, illuminate the commonalities and differences between different genetic forms of cholestasis, and shed light on basic physiology and disease mechanisms. These aims fulfill key goals of ChiLDReN and are central to the mission of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), as stated in its Action Plan for Liver Disease Research.

摘要/摘要胆汁流动对于正常的肝功能至关重要。胆汁淤积是胆汁流动受阻的所有现象的总称。受损。在过去的几年里，我们对机制的理解取得了很大的进展潜在的小儿胆汁淤积性肝病，包括当前申请人。尽管如此，在 40% 的儿童胆汁淤积的主要遗传病因尚未确定，针对下一代已知胆汁淤积基因的测序（tNGS）。此外，我们对这些疾病的了解以及我们的治疗他们的能力仍然有限。该提案延续了博士之间的合作。公牛和汤普森.它汇集了 2 个最大的儿科肝脏中心无与伦比的样本和数据资源欧洲（伦敦国王学院和华沙儿童纪念研究所）、Bull 博士的实验室以及 NIH 资助的儿童肝病研究网络 (ChiLDReN)。本次资源收集和经验将有助于解决以下挑战。在目标 1 中，将进行基因研究鉴定新的胆汁淤积基因以及已知胆汁淤积基因的新突变。这个目标将是使用最先进的遗传技术完成，包括：tNGS、全外显子序列 (WES)、全基因组测序、RNA 测序和甲基化分析。在目标 2 中，我们将描述通过评估临床和生化数据，了解遗传上不同形式的胆汁淤积的特征患者。可以收集大量的组织，并进行组织学和免疫组织化学分析进行。在目标 3 中，我们将研究遗传性胆汁淤积的病理生理机制。已知胆汁淤积遗传形式的动物模型通常无法很好地复制人类表型，因此需要体外模型系统来研究疾病机制并测试潜在的治疗方法。体外系统可用于评估启动子功能、剪接、运输和膜运输，如合适的。此外，CRISPR/Cas9技术将用于将纯合突变引入诱导多能干细胞（iPSC），产生突变的肝细胞和胆管细胞样细胞。这些细胞将用于探索致病基因突变的后果，最初侧重于增加了解 TJP2 缺乏症的疾病机制。初步数据显示这些细胞形成细胞间有紧密连接的小管。例如，使用此类细胞将使我们能够测试体外紧密连接的渗透性。总之，这些拟议的研究将导致确定新的疾病基因和突变，阐明不同遗传形式之间的共性和差异胆汁淤积，并阐明基本生理学和疾病机制。这些目标实现了以下关键目标儿童是国家糖尿病、消化和肾脏研究所使命的核心疾病 (NIDDK)，如其肝病研究行动计划所述。