Development of DNA diagnosis method of polycystic kidney disease and cloning of genes related to the formation of kidney cysts

1.建立了多囊肾病1（PKD 1）的DNA诊断方法。多囊肾是最常见的单基因疾病之一。由于PKD 1基因第34 ~ 46外显子区域具有独特的序列，我们采用常规的PCR-SSCP方法检测该区域的突变。在第45外显子发现一个由CAG变为TAG的无义突变（Gln 4124 stop），在第43内含子发现一个20 bp的缺失，该缺失是由于内含子较小而引起的剪接突变。在123例患者中发现10例外显子46由GAC突变为GAT（Asp 4234 Asp），107例外显子43由GCC突变为GCA（Ala 3910 Ala）。由于已发现与外显子1至33的序列相似的序列存在于同一染色体上的其他至少三个位点中，因此我们使用锚定PKD 1特异性引物进行了长范围PCR和RT-PCR。我们使用WAVE DNA片段分析系统对PCR产物进行分析.为了克隆与肾囊肿形成相关的新基因，我们采用了两种技术。(1)我们以PKD 1 cDNA为探针筛选了人基因组BAC文库。我们通过FISH分析定位了26个克隆。7个克隆被定位在16 p13上，19个克隆被定位在16号染色体以外的染色体上。酶切后用脉冲场凝胶电泳分离。经Southern印迹杂交分析，我们分离出DNA片段作为阳性条带。我们正在对它们进行亚克隆和测序。(2)从PKD患者和正常人肾组织中提取RNA，合成cDNA。我们采用了差异显示技术。用24个上游引物和9个下游引物进行PCR扩增，产物经PAGE电泳分析，发现正常肾有40条带，多囊肾有25条带。我们正在对这些DNA片段进行测序，并确认不同的表达。

1. We have established the method of DNA diagnosis of polycystic kidney disease1(PKD1). Polycystic kidney disease is one of the most frequent monogenic diseases. Since theregion from exon 34 to exon 46 of PKD1 gene has unique sequnce, we carried out ordinary PCR-SSCP analysis to detect mutations in this region. One nonsense mutation from CAG to TAG in exon45 (Gln4124stop) and deletion of 20bp in intron 43 were found. This deletion caused the splicing mutation because of the size of the intron is small. Three polymorphisms were found : five alleles of different size in intron42,10 among 123 patients mutated from GAC to GAT in exon 46(Asp4234Asp) and 7 among 107 patientsmutated from GCC to GCA in exon 43(Ala 3910Ala). Since the similar sequence to those from exon 1 to 33 has been found to be present in at least three other loci elsewhere on the same chromosome, we carried out the long range PCR and RT-PCR using the anchored PKD1-specific primers. We are analysing the PCR products using WAVE DNA fragment analysis system.2. To clone new genes related to the formation of kidney cyst, wetooe two techniques. (1) We have screened human genomic BAC library using PKD1 cDNA as probes. We have mapped 26 clones by FISH analysis. Seven clones were mapped on 16p13 and nineteen clones on chromosomes other than chromosome 16. After digesting with restriction enzymes we seperated with pulse-field gel electrophoresis. By Southern blot hybridization analysis we have isolated DNA fragments as positive bands. We are subcloning and sequencing them. (2) RNA was isolated from kidney tissues of PKD patient and normal individual and cDNA was synthesized. We carried out Differential Display technique. PCR were performed using 24 upstream primers and 9 down stream primers and the products were analyzed with PAGE.By comparing the patterns we found 40 bands only in normal kidney and 25 bands only in polycystic kidney. We are sequensing these DNA fragments and confirming the different expression.