Analysis of disease associated genes in polycystic kidney disease

为了鉴定与人类多囊肾病（PKD）发生有关的疾病相关基因，我们构建了人类主要组织相容性复合体（MHC）着丝粒侧250-kb片段的物理图谱，并在该片段内寻找新的基因，包括小鼠Ke 6基因的人类同源物HKE 6。小鼠Ke 6基因是PKD发病的候选基因，通过对YAC克隆和跨越HLA II类着丝粒侧的粘粒克隆的分析，鉴定出8个新基因。HKE 6基因（酒精脱氢酶样基因）定位于HLA-DP亚区的100 kb着丝粒侧。通过鸟枪法测定RING 1、HKE 6和HKE 4亚区中由一个粘粒克隆编码的42，801 bp长区域的基因组序列。HKE 6基因由8个外显子和7个内含子组成，其外显子-内含子结构与小鼠Ke 6基因相同。RING 1基因近端的25 kb区域包括广泛密集的Alu重复簇（约1.2 Alu/kb），迄今为止尚未鉴定出其中的基因。在PKD患者的肾脏中检测到来自HKE 6基因的1.0 kb微弱信息。在这些患者中，没有大规模的缺失或易位，检测到基因组Southern杂交分析的HKE 6 cDNA插入作为探针。此外，通过对HKE 6基因5'上游640-bp区域和外显子1-4区域的PCR产物进行直接测序，未检测到核苷酸缺失、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.