The Genetic Basis of Pediatric Cholestasis

小儿胆汁淤积的遗传基础

• 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
• 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

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 胆汁流对于正常的肝功能至关重要。胆汁淤积是胆汁流量的所有现象的术语 受损。在过去的几年中，我们对机制的理解取得了巨大进步 基本的小儿胆汁淤积性肝病，包括当前申请人。尽管如此，在40％ 儿童是胆汁淤积的主要遗传病因，尚未确定为下一代后 已知胆汁淤积基因的测序（TNG）。此外，我们对这些疾病的理解和我们的理解 治疗它们的能力仍然有限。该建议继续DRS之间的合作。公牛和 汤普森。它汇集了两个最大的小儿肝脏中心的无与伦比的样本和数据资源 在欧洲（伦敦国王学院和华沙儿童纪念研究所），公牛博士的实验室和 NIH资助的儿童肝病研究网络（儿童）。这些资源集和 经验将使以下挑战得到解决。在AIM 1中，将进行遗传研究 鉴定新的胆汁淤积基因和已知胆汁淤积基因中的新突变。这个目标将是 使用最先进的遗传技术完成的完成，包括：TNG，整个外显子组序列（WES）， 整个基因组测序，RNA测序和甲基化分析。在AIM 2中，我们将表征 通过评估临床和生化数据的遗传不同形式的胆汁淤积形式的特征 患者。可以提供广泛的组织收集，组织学和免疫组织化学分析将是 进行。在AIM 3中，我们将研究遗传胆汁淤积的基础的病理生理机制。 胆汁淤积的已知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和测试潜在治疗。体外 系统可用于评估启动子功能，剪接，运输和膜运输，作为 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），以产生突变的肝细胞和胆管细胞样细胞。这些细胞 将用于探索引起疾病基因突变的后果，最初侧重于增加 了解TJP2缺乏症中疾病机制。初步数据显示了这些单元格 细胞之间有紧密连接的关节。这样的细胞的使用将使我们可以测试 紧密连接在体外的渗透性。总之，这些提出的研究将导致对新的识别 疾病基因和突变，阐明不同遗传形式的共同点和差异 胆汁淤积，并阐明了基本的生理和疾病机制。这些目标实现了 儿童，是国家糖尿病和消化研究所和肾脏的任务的核心 疾病（NIDDK），如其肝病研究计划中所述。