Molecular Genetics of Human ARPKD

描述（由申请方提供）：常染色体隐性遗传性多囊肾病（ARPKD）是儿科发病率和死亡率的重要原因。受影响的儿童患有HTN、肾功能不全和门脉纤维化。ARPKD的临床谱变化很大，大多数病例在婴儿期出现。我们的联盟，由两个以前的奖项支持，使用遗传方法来定义其分子基础。在我们最近的周期中，我们鉴定了PKHD 1基因，并确定它定位于> 400 kb的基因组间隔，编码> 13 kb的mRNA，经历了与67个外显子转录物的复杂剪接模式，该转录物编码最长的ORF和4074个氨基酸的蛋白质。我们已经表明，该基因在所有发育阶段的肾脏中表达最高，尽管它在多种其他组织中也以低水平表达。基因产物，多导管蛋白（PD），是一种I型膜蛋白，被认为可能是一种配体或受体。免疫定位研究已将蛋白质置于基体/初级纤毛中。在使用表位标记的全长重组PD的研究中，我们发现了该分子经历复杂的蛋白水解加工模式的证据。我们的基因型/表型研究发现，具有双等位基因截短PKHD 1突变的个体具有更严重的疾病。我们分离了小鼠直向同源物，确定它也具有复杂的剪接，并产生了5'端具有靶向突变的小鼠。令人惊讶的是，纯合子突变小鼠仅发生胆道和胰腺疾病。初步数据表明，靶向等位基因不是真正的空。在这次更新中，我们力求就这些意见采取后续行动。在目标1中，我们将使用各种表位标记的重组分子、代谢标记、工程突变和突变细胞系来检验PD经历由前蛋白转化酶、TACE或其他金属蛋白酶和分泌酶介导的调节性蛋白水解的假设。我们还将研究内源性PD是否具有相同的性质。在目标2中，我们将开发一个细胞培养模型系统，可用于评估调节膜内蛋白水解的功能后果。目的3检验Pkhdl的完全丧失将导致肾和其他器官功能障碍的假设。我们将描述一种新开发的小鼠品系，其具有靶向外显子3和4的功能性floxed等位基因。如果cre介导的缺失没有导致完全无效的等位基因，我们将通过在整个基因的侧翼引入lox p位点来产生一个。最后一个目标将描述复杂的剪接模式，因为这是基因的一个突出特征，可能解释了最近基因靶向研究的惊人结果，并可能解释了一些观察到的临床变异性。

DESCRIPTION (provided by applicant): Autosomal recessive polycystic kidney disease (ARPKD) is a significant cause of pediatric morbidity and mortality. Affected children suffer from HTN, renal insufficiency and portal tract fibrosis. The clinical spectrum of ARPKD is widely variable with most cases presenting in infancy. Our Consortium, supported by two previous awards, has used genetic approaches to define its molecular basis. In our most recent cycle, we identified the gene, PKHD1, and determined that it maps to a >400kb genomic interval, encodes a >13kb mRNA, undergoes a complicated pattern of splicing with a 67 exon transcript which encodes the longest ORF and a 4074 aa protein. We have shown that the gene is most highly expressed in kidney at all developmental stages, though it is also expressed at low levels in multiple other tissues. The gene product, polyductin, (PD), is a type I membrane protein thought likely to be a ligand or receptor. Immunolocalization studies have placed the protein in the basal body/primary cilia. In studies using epitope-tagged full length recombinant PD, we found evidence that the molecule undergoes a complicated pattern of proteolytic processing. Our genotype/phenotype studies found that individuals with biallelic truncating PKHD1 mutations have more severe disease. We isolated the mouse orthologue, determined that it also has complex splicing and generated mice with targeted mutations of the 5' end. Surprisingly, homozygous mutant mice only developed biliary and pancreatic disease. Preliminary data suggest that the targeted allele is not a true null. In this renewal, we seek to follow-up on these observations. In Aim 1, we will test the hypothesis that PD undergoes regulated proteolysis mediated by proprotein convertases, TACE or other metalloprotease, and secretase using a variety of epitope-tagged recombinant molecules, metabolic labeling, engineered mutations and mutant cell lines. We will also examine whether endogenous PD has the same properties. In Aim 2, we will develop a cell culture model system that can be used to assess the functional consequences of regulated intramembrane proteolysis. Aim 3 tests the hypothesis that complete loss of Pkhdl will result in renal and other organ dysfunction. We will characterize a newly developed line of mice with a functional floxed allele targeting exons 3 and 4. If cre-mediated deletion does not result in a complete null allele, we will generate one by introducing lox p sites flanking the entire gene. The last aim will characterize the complex pattern of splicing since this is such a prominent feature of the gene, likely explains the surprising results of recent gene targeting studies, and may account for some of the observed clinical variability.