Analysis of disease associated genes in polycystic kidney disease

为了确定与人类多囊肾病(PKD)发病相关的疾病相关基因，我们构建了一个250kb片段的物理图谱，并在人类主要组织相容性复合体(MHC)着丝粒一侧的片段中寻找了新的基因，包括小鼠Ke6基因的人类同源基因HKE6。小鼠KE6基因是PKD发生的候选基因。通过对一个YAC克隆和跨越人类白细胞抗原II类区域着丝粒侧的粘粒克隆的分析，鉴定出8个新基因。HKE6基因(酒精脱氢酶类似基因)定位于人类白细胞抗原-DP亚区着丝粒100kb一侧。用鸟枪法测定了RING1、HKE6和HKE4亚区中一个粘粒克隆编码的42,801个核苷酸的基因组序列。HKE6基因由8个外显子和7个内含子组成，具有与小鼠KE6基因相同的外显子-内含子组织。在RING1基因近端的25kb区域包含大量密集的Alu重复序列(约1.2Alu/kb)，到目前为止还没有发现任何基因。在PKD患者的肾脏中检测到来自HKE6基因的1.0kb的微弱信息。在这些患者中，以HKE6基因插入片段为探针进行基因组Southern杂交分析，未发现大规模缺失或易位。此外，通过对HKE6基因5‘上游区和外显子1-4区640-bp的PCR产物的直接测序，未发现核苷酸缺失、插入或碱基替换。

In order to identify a disease associated gene which is involved in the development of human polycystic kidney disease (PKD), we have constructed a physical map of the 250-kb segment and searched new genes within the segment on the centromeric side of the human major histocompatibility complex (MHC) including a human homologue of the mouse Ke6 gene, HKE6.. The mouse Ke6 gene is a candidate gene responsible for the development of PKD.From the analysis of a YAC clone and cosmid clones which span the centromeric side of the HLA class II region, 8 new genes were identified. The HKE6 gene (alcohol dehydrogenase-like gene) was mapped on the 100 kb centromeric side of the HLA-DP subregion. The genomic sequence of a 42,801 bp long region encoded by one cosmid clone in the RING1, HKE6 and HKE4 subregions was determined by the shotgun method. The HKE6 gene is composed of eight exons-and seven introns with the same exon-intron organization as observed in the mouse Ke6 gene. The 25 kb region proximal to the RING1 gene includes an extensive dense cluster of Alu repeats (about 1.2 Alu per kb) and no gene has been identified in this so far. A 1.0-kb faint message from the HKE6 gene was detected in the kidney of PKD patients. In these patients, no large-scale deletion or translocation, was detected using genomic Southern hybridization analysis by the HKE6 cDNA insert as a probe. Additionally, no nucleotide deletion, insertion, or base replacement was detected by the direct sequencing of PCR products from 640-bp of the 5' upstream region and exon 1-4 regions in the HKE6 gene.