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

1.建立了多囊肾病的DNA诊断方法。多囊肾病是最常见的单基因疾病之一。由于PKD1基因外显子34至外显子46的区域具有独特的序列，我们进行了常规的PCR-SSCP分析来检测该区域的突变。发现45号外显子CAG突变为Tag无义突变(Gln4124 Stop)，43号内含子缺失20bp。由于内含子的大小较小，这种缺失导致了剪接突变。在43号外显子(Ala 3910Ala)上发现了3种不同大小的等位基因：内含子42有5个大小不等的等位基因，外显子46(Asp4234Asp)由GAC突变为GAT(Asp4234Asp)的患者有10例，外显子43(Ala 3910Ala)由GCC突变为GCA的患者有7例。由于与外显子1至33的序列相似的序列已被发现存在于同一染色体的其他至少三个基因座中，因此我们使用锚定的PKD1特异性引物进行了长距离PCR和RT-PCR。我们正在使用WAVE DNA片段分析系统对扩增产物进行分析。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)以PKD1cDNA为探针，筛选出人基因组BAC文库。通过FISH分析，我们已经定位了26个克隆。将7个克隆定位在16p13上，19个克隆定位在16号染色体以外的染色体上。用限制性内切酶酶切后，用脉冲场凝胶电泳分离。经Southern印迹杂交分析，分离到阳性条带。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。我们进行了差异显示技术。用24条上游和9条下游引物进行聚合酶链式反应，扩增产物经PAGE分析，仅在正常肾组织中扩增出40条带，在多囊肾组织中仅扩增出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.