Development of Systems for Comprehensive Genetic Diagnosis of Human Cancers and for Early Detection of Cancer-patients.

1996年，我们在遗传不稳定（MI+）结直肠癌的一个亚群中发现了编码E2F4转录调节因子的基因中的一种新的肿瘤特异性突变。该突变被发现是由于hMSH3基因（一种DNA错配修复基因）的移框突变失活的结果。因此，E2F4基因首次被确定为涉及hMSH3蛋白的缺陷错配修复功能的靶标。这一观点已被许多支持性论文所证实。与此结果相关，我们在一个弥漫性大B细胞淋巴瘤细胞系中发现了人类p107基因的间质缺失，p107基因是肿瘤抑制基因RB的近亲。我们发现，缺失是由两个alu重复序列之间的重组引起的，大约有15 kbp。通过我们的基因组扫描方法，我们在大约30%的肾细胞癌中发现了染色体14q24-31上的一个新的杂合性缺失（LOH）区域，并在这个狭窄的re…More区域鉴定了两个候选肿瘤抑制基因（TSG）。通过传统的微卫星分析，我们发现人类头颈癌中染色体13q34处LOH的发生率很高。已知的候选TSG ING1位于这个位置。我们明确了ING1基因的全基因组结构，并在3例13q34 LOH的原发性头颈癌中发现了3个失活点突变。这是首次有证据表明p107和ING1基因具有TSG功能。此外，我们发现了一个新的蛋白酪氨酸磷酸酶基因HD-PTP作为新的TSG候选基因。该基因位于染色体3p21.3，该区域被认为包含多种人类癌症的TSG。通过类似的方法，我们在7种癌症/肿瘤组的8个染色体区域发现了至少10个TSG候选基因。除了TSG外，我们还分离到了一个编码新的POZ-Zn-Finger蛋白的cDNA，该蛋白的表达导致受体细胞出现恶性表型，这表明该基因是一个新的致癌基因。该基因位于3q26-27，与已知的致癌基因Bcl-6有关。在癌症的分子诊断方面，我们报道了一种在几种骨/软组织肉瘤中检测肿瘤特异性融合基因mrna的系统方法。我们已经开发出一种新的策略来定量血清/血浆中游离DNA的数量，具有非常高的灵敏度（比传统方法高约一万倍）。癌症特异性的基因改变，如K-ras和p53突变，可以在这些血清dna中检测到。因此，利用血浆/血清中的游离DNA来早期检测潜在的癌症患者是很有希望的。少

We have found a novel tumor-specific mutation in the gene encoding E2F4 transcription regulator in a subset of genetically unstable (MI+) colorectal cancers in 1996. The mutation was found to be a result of the inactivating frame-shift mutation of the hMSH3 gene, a DNA mismatch-repair gene. Thus, the E2F4 gene was identified, for the first time, as a target of the defective mismatch repair function involving hMSH3 protein. This notion has been confirmed bv many supporting papers. Relevant to this result, we have found an interstitial deletion within the human p107 gene, a close relative of the tumor suppressor gene RB, in a diffuse-large B cell lymphoma cell line. We found the deletion was raised by a recombination between two Alu-repeats encompassing about 15 kbp. By our genomic scanning method, we found a novel region of loss of heterozygosity (LOH) at chromosome 14q24-31 in about 30% of renal cell carcinomas and identified two candidate tumor suppressor genes (TSG) in this narrow re … More gion. By conventional microsatellite analysis, we found a high incidence of LOH at chromosome 13q34 in human head and neck cancers. The known candidate TSG, ING1, resides at this site. We clarified the whole genomic structure of the ING1 gene and found 3 inactivating point mutations in 3 primary head and neck cancers with 13q34 LOH.These are the first evidence indicatins that the p107 and ING1 genes function as TSG.Further, we discovered a novel protein tyrosine-phosphatase gene, HD-PTP, as a novel TSG candidate. The gene resides at chromosome 3p21.3, the region thought to contain multiple TSG for many types of human cancer. By similar approaches we found at least 10 TSG candidates at 8 chromosomal regions in 7 cancer/tumor groups. In addition to TSG, we isolated a cDNA encoding a novel POZ-Zn-Finger protein, whose expression led the recipient cells to malignant phenotype, suggesting that the gene is a novel oncogene. The gene locates on 3q26-27 and related to the known oncogene, Bcl-6.As to the molecular diagnosis of cancer, we reported a systematic method for detecting tumor-specific, fused-gene mRNAs in several bone/soft-tissue sarcomas. We have developed a new strategy to quantitate the amounts of free DNA in sera/plasma with a very high sensitivity (about ten thousand-fold higher than conventional methods). Cancer-specific genetic alterations, like as K-ras and p53 mutations, could be detected in these serum-DNA.Thus, promising strategies for early detection of potential cancer-patients were developed with free DNA in plasma/serum. Less