MOLECULAR BASIS OF HNPCC--HEREDITARY NONPOLYPOSIS COLORE

HNPCC的分子基础--遗传性非息肉病颜色

• 批准号: 6377139
• 负责人: BO LIU
• 金额: $ 26.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6377139
• 关键词: CHO cells; alleles; cell line; clinical research; colorectal neoplasms; complementary DNA; family genetics; gene deletion mutation; genetic susceptibility; genetic techniques; human subject; immunocytochemistry; loss of heterozygosity; molecular oncology; neoplasm /cancer genetics; southern blotting

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.

本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。我们以前的研究表明，90%的HNPCC发生的肿瘤具有微卫星不稳定性（MSI）。 在各种模型系统中的遗传、生物化学和功能研究已经证明MSI是由有缺陷的细胞DNA错配修复（MMR）引起的。 人类MMR需要六种不同的基因。 据信，这些基因中的四个基因hMSH 2、hMLH 1、hPMS 1和hPMS 2的遗传性生殖系缺陷导致HNPCC。 为了全面了解HNPCC，我们分析了109 HNPCC激酶满足阿姆斯特丹标准的证据遗传突变的四个基因的整个编码区的DNA测序。 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个具有确定的生殖系MMR突变的激酶组成。 第二组由24个具有异常MMR cDNA缺失或插入但没有检测到基因组突变的激酶组成。 第三组由未显示任何可检测的生殖系MMR突变的剩余48个激酶组成。 然而，MSI存在于所有II组和48个III组激酶中的37个肿瘤中，表明MMR基因的纯合突变。我们的假设是，与I组一样，MSI阳性的II组和III组HNPCC也是由有缺陷的生殖系MMR基因引起的。为了验证这一假设，我们建议继续我们目前的调查组II和III HNPCC使用各种方法，包括遗传，生化和功能的研究概述了以下具体目标。 目标1：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HNPCC中起作用;目的III：检测其他MMR基因hMSH 3和hMSH 6是否参与HNPCC;目的IV：检测MMR基因突变是否存在于II和III型HNPCC的肿瘤中;目的IV：检测MMR基因突变是否存在于HNPCC的肿瘤中;目的IV：检测HNPCC的肿瘤中是否存在MMR基因突变。AIM V：使用基于单等位基因的功能分析（MAFA）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，其中通过与无MMR仓鼠细胞系的杂交融合将推定的突变HNPCC等位基因与野生型等位基因分离。 结合起来，这些相互关联的方法应导致新的见解HNPCC的分子基础，具有重要意义的临床诊断和治疗的疾病。