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理论;第一种也是最被接受的理论是， 个体出生时具有缺陷等位基因，然后正常WT等位基因经历体细胞突变， 首先上升到肾小管扩张，然后是囊肿，即“两次打击”假说。第二个观点是基因剂量 足以引起疾病时，PKD 1基因的蛋白产物多囊蛋白-1（PC 1）的水平降低到低于 阈值和囊肿以随机方式出现。我们希望以一种新颖的方式来研究这两种观点 利用PC 1、PC 2和纤维囊蛋白都存在于外泌体上的事实，我们称之为外泌体样囊泡 （ELV）。我们知道PKD 1突变个体的PC 1水平降低， 每ELV的PC 1与身高调整后的肾脏总体积（HtTKV）成反比。观察总量 PC 1在汇集的外泌体中的含量告诉我们关于该疾病的发病机制的信息很少。然而，一项新技术 允许研究者确定个体ELV上的PC 1的量，NanoView。 我们知道ELV含有与PC 1共定位于外泌体上的CD 133（CD 133）。我们预测，如果 如果“两次击中”假设成立，那么肾脏中PKD 1无效细胞的克隆将产生CD 133+和CD 133+的ELV。 PC 1-，NanoView将在ADPKD中观察到PC 1 + ELV的双峰分布，但在正常尿液中未观察到。 CD 133 +/PC-与CD 133 +/PC 1 + ELV的比值将与HtTKV相关。如果单倍不足是 因此，每个ELV的PC 1平均强度将与HtTKV成反比。NanoView 技术可以区分“两次命中”和单倍不足模型。 为了进一步剖析单倍不足的机制，我们将研究两个主要观点。首先是 人PKD 1基因是不寻常的，因为它在内含子21和22中具有两个长的富含CT的片段， 剪接并产生较小形式的PC 1，我们称之为Trunc_PC 1。我们将研究这种可能性 效率与HtTKV成反比。第二个观点是PKD 1存在人与人之间的差异， 启动子强度和启动子随年龄衰减。我们将使用真实的时间来研究这种可能性 RT-PCR法我们认为，不同的机制对疾病的治疗方式有着深远的影响。 如果单倍不足是正确的，那么设计增加内源性PKD 1 mRNA量的策略 和PC 1蛋白将是适当的，而在“两次打击”的情况下，这些策略将不起作用， 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.