Kibel A S, Freije D, Isaacs W B, Bova G S
James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, Maryland, USA.
Genes Chromosomes Cancer. 1999 Jul;25(3):270-6.
The identification of homozygous deletions in malignant tissue is a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the polymerase chain reaction (PCR) to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes, such as BRCA2 and PTEN. We have recently identified a 1-5-cM homozygous deletion on 12p12-13 in a prostate cancer xenograft and found that 47% of patients who died of prostate carcinoma demonstrate focal loss of heterozygosity (LOH) in this region in metastatic deposits. We have now characterized the region of interest by assembling a yeast artificial chromosome (YAC) contig spanning the homozygous deletion and identifying which known genes and expressed sequence tags (EST) lie within the homozygous deletion. A rib metastasis was harvested at autopsy and placed subcutaneously in a male SCID mouse. Genomic DNA from this xenograft and from the patient's normal renal tissue was extracted. Multiplex PCR, with the xenograft and normal DNA used as template, was performed using primers for loci on the Whitehead contig 12.1 believed to be near our region of interest. We found that our deletion lay in a 1-2-Mb interval between WI-664 and D12S358. We then used the same primers to construct a YAC contig across the homozygous deletion. PCR amplification of YAC DNA, using primers for the genomic sequences of known genes and ESTs reported to lie on 12p12-13, was used to identify candidate genes that lay within the deletion. Duplex PCR, with control primers known not to be deleted in the xenograft, was used to confirm that both the CDKN1B and ETV6 genes were homozygously deleted in the xenograft. Mutations in either or both of these genes may play an important role in metastatic prostate carcinoma.
在恶性组织中鉴定纯合缺失是定位肿瘤抑制基因的有力工具。代表性差异分析（RDA）利用选择性杂交和聚合酶链反应（PCR）来分离染色体缺失区域，促进了诸如BRCA2和PTEN等肿瘤抑制基因的鉴定。我们最近在一个前列腺癌异种移植模型中鉴定出12p12 - 13上一个1 - 5厘摩的纯合缺失，并发现47%死于前列腺癌的患者在转移灶中该区域显示杂合性缺失（LOH）。我们现在通过构建跨越纯合缺失的酵母人工染色体（YAC）重叠群并确定哪些已知基因和表达序列标签（EST）位于纯合缺失内，对感兴趣区域进行了特征描述。在尸检时获取一个肋骨转移灶并皮下植入一只雄性SCID小鼠。提取该异种移植模型和患者正常肾组织的基因组DNA。以异种移植模型和正常DNA为模板，使用针对怀特黑德重叠群12.1上被认为靠近我们感兴趣区域的位点的引物进行多重PCR。我们发现我们的缺失位于WI - 664和D12S358之间1 - 2兆碱基的区间内。然后我们使用相同的引物构建跨越纯合缺失的YAC重叠群。利用针对据报道位于12p12 - 13上的已知基因和EST的基因组序列的引物对YAC DNA进行PCR扩增，以鉴定位于缺失内的候选基因。使用已知在异种移植模型中未被缺失的对照引物进行双重PCR，以确认CDKN1B和ETV6基因在异种移植模型中均为纯合缺失。这两个基因中任何一个或两个的突变可能在转移性前列腺癌中起重要作用。
