Molecular diagnosis of von Hippel-Lindau disease (VHL) using VHL tumor suppressor gene and the research for the VHL gene product

为了明确日本人群中VHL患者的分子基础，我们收集了72例无关的日本VHL患者，并用单链构象多态(SSCP)分析和Southern印迹分析对45例VHL患者进行了VHL基因的胚系突变检测。SSCP分析和Southern杂交分别检测到23个(51%)VHL基因内突变和3个(6.7%)缺失。基因内突变包括14个错义突变、7个微缺失或插入和2个剪接点突变。外显子1的10个突变中有9个位于37个核苷酸的短区域。其中包括五个独特的突变位点，这些突变在Prevoius研究中没有发现。与西方VHL患者不同，日本VHL患者中未发现无义突变。在22例无嗜铬细胞瘤的VHL患者中发现的突变包括11个错义突变，6个微缺失或插入，2个剪接点改变和3个缺失。在4例嗜铬细胞瘤VHL患者中发现的突变包括683位错义突变(密码子228)，712位错义突变(38位密码子)和776位错义突变(259位密码子)。我们的结果提示与西方患者相比，VHL基因具有独特的特征。VHL基因探针的原位杂交研究显示，VHL基因在血管母细胞瘤的大脑和小脑神经元细胞和间质细胞中表达。此外，用抗VHL蛋白的单抗表达VHL蛋白，可见VHL蛋白在神经细胞和脑肿瘤细胞中表达。

To define the molecular basis of VHL patients in Japanese populations, we collected 72 unrelated Japanese VHL patients and completely tested for germline mutations of the VHL gene in 45 VHL patients by single-strand conformation polymorphism (SSCP) analysis and Southern blot analysis. We detected 23 (51%) intragenic mutations of the VHL gene and three (6.7%) deletions by SSCP analysis and Southern blot respectively. The intragenic mutations consisted of 14 missense mutations, seven microdeletions or insertions and two splice-site mutations. Nine of 10 mutations in exon 1 are localized in a short region of 37 nucleotides. Five unique sites of mutation were included, which were not seen in prevoius stuidied. Unlike Western VHL patients, nonsense mutations were not found in Japaneses VHL patients. The mutations found in 22 VHL patients without pheochromocytoma consisted of 11 missense mutations, six microdeletions or insertions, two splice-site alterations and three deletions. The mutations found in four VHL patients with pheochromocytomas consisted of one missense mutation at nucleotide 683 (codon 228), two missense mutations at uncleotide 712 (codon 38) and a novel 20 bp insertion at nucleotide 776 (codon 259). Our results suggest that unique features compared with those in Western patients.In situ hybridyzation study with VHL gene probe showed expression of VHL gene at cerebral and cerebellar neuronal cells and stromal cells of hemangioblastoma. In addition, VHL protein expression with anti-VHL protein monoclonal antibody showed expression of VHL protein at neuronal cells and brain tumor cells.