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。我们先前的研究表明，90%的HNPCC肿瘤存在微卫星不稳定性(MSI)。在各种模型系统中的遗传、生化和功能研究表明，MSI是由缺陷的细胞DNA错配修复(MMR)引起的。人类MMR需要六种不同的基因。人们认为这些基因中的hMSH2、hMLH1、hPMS1和hPMS2中的遗传性胚系缺陷会导致HNPCC。为了全面了解HNPCC，我们通过DNA测序分析了109个符合阿姆斯特丹标准的HNPCC家系，以寻找这四个基因整个编码区遗传突变的证据。根据实验结果，我们将这些HNPCC分为三组。第一组包括37个具有明确的种系MMR突变的家系。第二组包括24个MMR基因异常缺失或插入，但没有检测到基因组突变的家系。第三组由其余48个没有表现出任何可检测到的生殖系MMR突变的家族组成。然而，在所有第II组和第III组48个家族中的37个发生的肿瘤中，MSI都存在，这表明MMR基因存在纯合子突变。我们的假设是，像组I一样，MSI阳性组II和组III HNPCC也是由生殖系MMR基因缺陷引起的。为了验证这一假设，我们建议使用多种方法继续我们目前对第二组和第三组HNPCC的研究，包括以下具体目标中概述的遗传、生化和功能研究。目的1：确定观察到的II组MMR基因的异常缺失或插入是否与疾病分离；目的II：检测4个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：确定在II和III组HNPCC发生的肿瘤中是否存在MMR基因突变；目的V：利用基于单等位基因的功能分析(MAFA)，通过与MMR缺失型仓鼠细胞系的杂交，将假定突变的HNPCC等位基因从野生型等位基因中分离出来，以确定来自II和III组HNPCC的易感胚系MMR等位基因是否被失活。结合起来，这些相互关联的方法应该会带来对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.