MOLECULAR BASIS OF HNPCC--HEREDITARY NONPOLYPOSIS COLORE

我们研究的长期目标是了解遗传性非息肉病性结直肠癌（HNPCC）的分子基础。我们之前发现90%的HNPCC肿瘤具有微卫星不稳定性（MSI）。各种模型系统的遗传、生化和功能研究表明，MSI是由细胞DNA错配修复缺陷（MMR）引起的。人类MMR需要六种不同的基因。据信，遗传性种系缺陷的四个基因，hMSH2, hMLH1， hPMS1和hPMS2，导致HNPCC。为了全面了解HNPCC，我们分析了109个符合阿姆斯特丹标准的HNPCC种类，通过DNA测序寻找四个基因的整个编码区存在遗传突变的证据。根据我们的实验结果，我们将这些HNPCC分为三个不同的组。第一组由37种确定的种系MMR突变组成。第二组由24种异常MMR cDNA缺失或插入组成，但没有检测到基因组突变。第三组由剩余的48种没有显示任何可检测的种系MMR突变组成。然而，在所有II组和48种III组中的37种肿瘤中均存在MSI，表明MMR基因发生纯合突变。我们的假设是，像I组一样，MSI阳性的II组和III组HNPCC也是由有缺陷的种系MMR基因引起的。为了验证这一假设，我们建议继续我们目前对II和III组HNPCC的研究，使用各种方法，包括遗传、生化和功能研究，如下所述的具体目标。目的1：确定观察到的异常II组cDNA缺失或插入是否与疾病分离；目的II：检查四种MMR基因的基因组缺失和非结合区突变是否在这些HNPCC中起作用；目的III：确定其他MMR基因hMSH3和hMSH6是否参与这些HNPCC；目的IV：确定II组和III组HNPCC引起的肿瘤中是否存在MMR基因突变；目的V：使用基于单等位基因的功能分析（MAFA）来确定来自II组和III组HNPCC的易感种系MMR等位基因的功能是否失活，其中假定突变的HNPCC等位基因通过与无MMR的仓鼠细胞系杂交融合从野生型等位基因中分离出来。综合起来，这些相互关联的方法将导致对HNPCC分子基础的新见解，对该疾病的临床诊断和治疗具有重要意义。

The long term goal of our research is to understand the molecular basis of hereditary nonpolyposis colorectal cancer (HNPCC). We previously showed that tumors arising from 90% of HNPCC had microsatellite instability (MSI). Genetic, biochemical, and functional studies in a variety of model systems have demonstrated that MSI is caused by defective cellular DNA mismatch repair (MMR). Human MMR requires six different genes. It is believed that inherited germline defects in four of these genes, hMSH2, hMLH1, hPMS1, and hPMS2, cause HNPCC. To gain a comprehensive understanding of HNPCC, we have analyzed 109 HNPCC kindreds meeting the Amsterdam Criteria for the evidence of an inherited mutation in the entire coding region of the four genes by DNA sequencing. Based on the results of our experiment, we have divided these HNPCC into three different groups. The first group consists of 37 kindreds with defined germline MMR mutations. The second group consists of 24 kindreds with abnormal MMR cDNA deletions or insertions but without detectable genomic mutations. The third group consists of the remaining 48 kindreds that do not show any detectable germline MMR mutations. Nevertheless, MSI was present in tumors arising from all the group II and 37 of the 48 group III kindreds, indicating homozygous mutation in the MMR gene. Our hypothesis is that like group I, MSI positive group II and III HNPCC is also caused by a defective germline MMR gene. To test this hypothesis, we propose to continue our current investigation on group II and III HNPCC using a variety of approaches, including genetic, biochemical, and functional studies as outlined in the following specific aims. AIM 1: To determine if the observed abnormal group II cDNA deletions or insertions segregate with the disease; AIM II: To examine if genomic deletions and nonbonding region mutations of the four MMR genes play any role in these HNPCC; AIM III: To determine if other MMR genes, hMSH3 and hMSH6, are involved in these HNPCC; AIM IV: To determine if MMR gene mutation is present in tumors arising from group II and III HNPCC; and AIM V: To determine if the function of the predisposed germline MMR allele from group II and III HNPCC is inactivated using mono- allelic-based-functional-analysis (MAFA), in which the presumed mutant HNPCC allele is separated from the wild type allele by hybrid fusion with an MMR-null hamster cell line. In combination, these interconnected approaches should lead to new insights into the molecular basis of HNPCC, with important implications for the clinical diagnosis and therapy of the disease.