Characterizing the Funtion of Fibbrocystin and Fibbrocystin-L

描述（由申请人提供）：常染色体隐性多囊肾病（ARPKD）是由大而复杂的基因PKHD1突变引起的。临床和分子研究已经揭示了广泛的表型范围与PKHD1突变相关，从围产期死亡到成人发病的肝病。进一步的复杂性是由显著的等位基因异质性提供的，因此只有初步的基因型/表型相关性是可能的。本提案的第一个目标将扩展这些突变研究（以及与表型的关联），以进一步表征典型和非典型ARPKD群体，包括分离的肝脏疾病。基因型在决定表型中的作用将被检查，并强调在功能研究中测试的重要残留物。ARPKD蛋白很大，与膜结合，与其他PKD相关蛋白一样，定位于初级纤毛和基体。Pkhdl缺陷动物纤毛缩短和异常。在人类中发现了一种纤维囊蛋白类似物，纤维囊蛋白样（-L；由PKHDL1编码），尽管有可能的功能提示，但这种蛋白的确切作用尚不清楚（不知道它与ARPKD有关）。纤维囊蛋白- l是蛋白质家族中最古老的成员，与纤维囊蛋白不同的是，它存在于鱼类和衣藻中，它们的沉默导致鞭毛缺陷导致运动性受损；表明纤毛在其他生物中的作用的。第二个目标将使用单克隆抗体和表达构建来确定（并确认）这两种蛋白的亚细胞定位，并观察纤维囊蛋白- l是否与纤毛/基底轴相关。在极化肾上皮细胞中，通过shRNA单独或联合沉默这些基因相关的细胞表型也将被测试。在第三个目标中，原位杂交和morpholino方法将用于定位与发育中的斑马鱼敲除活性相关的表达位点和表型。肾原管的囊性表型是一种可能的结果，正如在一些综合征PKD相关基因的斑马鱼变体中发现的那样，有缺陷的会聚伸展运动也是一种可能的结果。最终目的是敲除小鼠的PkhdH，组成性地（如果必要的话，有条件地在肾脏中）确定纤维囊蛋白- l在哺乳动物中的作用。用现有的Pkhdl - deZ小鼠进行育种，将显示两种纤维囊素的缺失是否会导致加性表型，从而提示相关的作用。总体而言，该项目将揭示纤维囊蛋白家族的功能信息，并确定涉及这些分子的信号传导/发育途径。

DESCRIPTION (provided by applicant): Autosomal recessive polycystic kidney disease (ARPKD) is caused by mutation to the large and complex gene, PKHD1. Clinical and molecular studies have revealed a wide phenotypic range associated with PKHD1 mutation, from perinatal death to adult onset liver disease. Further complexity is provided by marked allelic heterogeneity and so only preliminary genotype/phenotype correlations have been possible. The first aim of this proposal will extend these mutation studies (and associations with phenotype) to further molecularly characterize typical and atypical ARPKD populations, including isolated liver disease. The role of genotype in dictating phenotype will be examined, and important residues highlighted for testing in functional studies. The ARPKD protein is large and membrane bound, and in common with other PKD associated proteins, localized to primary cilia and the basal body. Pkhdl defective animals have shortened and abnormal cilia. A fibrocystin paralog is found in humans, fibrocystin-like (-L; encoded by PKHDL1), and although there are hints at possible function, the precise role of this protein is unclear (it is not known to be associated with ARPKD). Fibrocystin-L is the most ancient member of the protein family and, unlike fibrocystin, present in fish and Chlamydomonas, where silencing results in impaired motility due to defective flagella; suggesting a ciliary role in other organisms. The second aim will use monoclonal antibodies and expression constructs to determine (and confirm) the subcellular localization of both proteins, and see if fibrocystin-L is associated with the ciliary/basal body axis. The cellular phenotypes associated with silencing these genes by shRNA, singly or in combination, in polarized kidney epithelial cells, will also be tested. In the third aim, in situ hybridization and morpholino approaches will be used to localize the sites of expression and the phenotype associated with knocking down activity in the developing zebrafish. Cystic phenotypes in the pronephric duct are one possible outcome, as is defective convergent extension movements, as found in zebrafish morphants of some syndromic PKD associated genes. The final aim will knockout PkhdH in the mouse, constitutively (and, if necessary, conditionally in the kidney) to determine the role of fibrocystin-L in mammals. Breeding with the existing Pkhdl deZ mouse will show whether loss of both fibrocystins results in an additive phenotype, suggesting related roles. Overall the project will reveal functional information about the roles of the fibrocystin protein family and identify signaling/developmental pathways involving these molecules.