Kibel A S, Schutte M, Kern S E, Isaacs W B, Bova G S
Department of Urology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.
Cancer Res. 1998 Dec 15;58(24):5652-5.
The identification of homozygous deletions in malignant tissue has been a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the PCR to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes such as BRCA2 and PTEN. Twenty RDA clones were generated by comparing genomic DNA from a prostate cancer xenograft to the same patient's normal kidney DNA. Southern blot analysis of the tester and driver and of normal and xenograft DNA, using the differential products as probes, showed the homozygous deletion in 16 of 20 RDA clones. The sequence of one of the differential products overlapped HSU59962, a genomic GenBank sequence on chromosome 12p12-13. Multiplex PCR of the xenograft DNA using polymorphic repeats mapped the deletion to a 1-5-cM region on 12p. Genomic DNA isolated from a panel of cryostat microdissected metastatic prostate adenocarcinomas/normal pairs was screened for loss of heterozygosity using the same polymorphic repeats. Loss of heterozygosity was demonstrated in 9 (47%) of 19 patients. This region may contain, or lie in close proximity to, tumor suppressor genes important in the progression and/or initiation of prostate cancer.
在恶性组织中鉴定纯合缺失一直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，并促进了诸如BRCA2和PTEN等肿瘤抑制基因的鉴定。通过比较前列腺癌异种移植瘤的基因组DNA与同一患者的正常肾DNA，产生了20个RDA克隆。以差异产物为探针，对测试者和驱动者以及正常和异种移植DNA进行Southern印迹分析，结果显示20个RDA克隆中有16个存在纯合缺失。其中一个差异产物的序列与HSU59962重叠，HSU59962是12号染色体p12 - 13上的一个基因组GenBank序列。利用多态性重复序列对异种移植瘤DNA进行多重PCR，将缺失定位到12号染色体短臂上1 - 5厘摩的区域。使用相同的多态性重复序列对一组经冷冻切片显微切割的转移性前列腺腺癌/正常组织对中分离的基因组DNA进行杂合性缺失筛查。在19例患者中有9例（47%）显示杂合性缺失。该区域可能包含对前列腺癌进展和/或起始至关重要的肿瘤抑制基因，或者与这些基因紧密相邻。
