Human-specific Abnormal Alternative Splicing of the Wild-type PKD1 Gene Induces Premature Termination of Polycystin-1

项目摘要 关于ADPKD的发病机制有两种主要的学说；第一种也是最被接受的一种学说是 个体先天携带有缺陷的等位基因，然后正常的WT等位基因经历体细胞突变，从而产生 首先是肾小管扩张，然后是囊肿，这就是“两击”假说。第二种观点是基因剂量是 足以导致疾病，PKD1基因的蛋白产物多囊蛋白-1(PC1)的水平降低到以下 阈值和包囊以随机方式出现。我们希望以一种新颖的方式来研究这两个想法 利用PC1、PC2和纤维囊藻蛋白都存在于外切体上的事实，我们将其称为外切体样囊泡 (ELV)。我们知道，在带有PKD1突变的个体中，PC1的水平降低，并且 每个ELV的PC1与身高调整总肾体积(HtTKV)成反比。观察总量 在汇集的外切体中PC1的表达几乎不能告诉我们疾病的发病机制。然而，一项新技术已经 它允许研究人员确定单个ELV上的PC1的量，NanoView。 我们知道ELV含有CD133(显著蛋白)，它与PC1共同定位在外切体上。我们预测，如果 两次命中假说成立后，肾脏中PKD1缺失细胞的克隆将产生CD133+的ELV和 而且NanoView将在ADPKD中看到PC1+ELV的双峰分布，但在正常尿液中不会。 CD133+/PC-/CD133+/PC1+ELV比值与HtTKV相关。如果单倍性不足是 机制，那么每ELV的平均PC1强度将与HtTKV成反比。因此，NanoView 技术可以区分“两次命中”和单倍体不足模型。 为了进一步剖析单倍体不足的机制，我们将研究两个主要观点。第一个是 人类PKD1基因的不同寻常之处在于它在第21和22内含子上有两个长的富含CT的区域，这两个区域干扰了 拼接并生成较小形式的PC1，我们称之为Trunc_PC1。我们将调查拼接的可能性 效率与HtTKV呈负相关。第二种观点认为，PKD1在人与人之间存在差异 启动子强度和启动子随着年龄的增长而减弱。我们将使用实时技术调查这种可能性 RT-PCR法。我们认为，不同的机制对这种疾病的治疗方式有深远的影响。 如果单倍性不足是正确的，那么旨在增加内源性PKD1mRNA数量的策略 和PC1蛋白将是适当的，而在‘两次击中’的情况下，这些策略将不起作用，并且可能是 有害的，因为两个等位基因都有缺陷。

Project Summary There are two major theories concerning the pathogenesis of ADPKD; the first and most accepted is that an individual is born with a defective allele and then the normal WT allele undergoes a somatic mutation that gives rise first to a tubular dilation and then a cyst, the `two hit' hypothesis. The second idea is that gene dosage is enough to cause disease, the level of the protein product of the PKD1 gene, polycystin-1 (PC1) decreases below a threshold and cysts appear in a stochastic manner. We wish to investigate these two ideas in a novel manner using the fact that PC1, PC2 and fibrocystin are all present on an exosome we term the exosome-like vesicle (ELV). We know that the level of PC1 is decreased in individuals with PKD1 mutations and that the amount of PC1 per ELV is inversely proportional to height adjust total kidney volume (HtTKV). Observing the total amount of PC1 in pooled exosomes tells us little about the pathogenesis of the disease. However, a new technology has become available which allows an investigator to determine the amount of PC1 on individual ELVs, NanoView. We know that ELVs contain CD133 (prominin) which colocalizes on exosomes with PC1. We predict that if the `two hit' hypothesis is true, then clones of PKD1 null cells in the kidney will produce ELVs that are CD133+ and PC1- and that the NanoView will see a bimodal distribution of PC1+ ELVs in ADPKD but not in normal urine. The ratio of CD133+/PC- to CD133+/PC1+ ELVs will correlate with HtTKV. If haploinsufficiency is the mechanism, then the mean intensity of PC1 per ELV will be inversely proportional to HtTKV. Thus, NanoView technology can distinguish between the `two hit' and haploinsufficiency models. To further dissect the mechanism of haploinsufficiency we will investigate two major ideas. The first is that the human PKD1 gene is unusual in that it has two long CT rich tracts in introns 21 and 22 which interfere with splicing and generate a smaller form of PC1, we term Trunc_PC1. We will investigate the possibility that splicing efficiency correlates inversely with HtTKV. The second idea is that there is person to person variation in PKD1 promoter strength and that the promoter attenuates with age. We will investigate this possibility using real time RT-PCR. We think that the different mechanisms have a profound effect on the way the disease may be treated. If haploinsufficiency is correct, then strategies designed to increase the amount of endogenous PKD1 mRNA and PC1 protein will be appropriate whereas in the `two hit' scenario these strategies will not work and may be deleterious as both alleles are defective.