Mechanisms of carcinogenesis by CRC-susceptibility genes

描述（由申请人提供）： 我们正在开发一个标本库，其中包括来自1,500多个连续的德系犹太人结直肠癌（CRC）病例的基因组DNA样本。该储存库将与基本的临床病理学参数和肿瘤标本本身相关联。储存库的一个主要应用是鉴定CRC易感基因，与相同数量的健康、年龄和性别匹配的德系犹太人对照相比，这些基因在病例中的频率更高。一个这样的CRC易感基因是blmAsh突变，这是DNA修复蛋白BLM中的蛋白质终止突变，它使患结直肠癌的风险增加三倍。我们在这里提出了一个试点项目，利用肿瘤标本通过存储库访问调查机制（S）的大肠癌的blmAsh载体。blmAsh突变导致突变等位基因的BLM蛋白表达减少> 95%。假设有两种机制：1）癌症易感性由BLM的单倍不足引起，产生所有结肠细胞转化的组成性风险，以及2）BLM的体细胞缺失是致癌作用的早期步骤。为了检验这些假设，我们将通过使用BLM抗体的免疫组织化学研究与非携带者相比，在blmAsh携带者的肿瘤细胞中是否可检测到BLM蛋白表达，以及通过杂合性丢失研究，正常BLM等位基因是否在遗传上丢失。此外，我们将研究blmAsh对结直肠癌发生的已知机制的影响。通过比较来自blmAsh携带者和来自非携带者的配对肿瘤-正常材料，我们将测试i）通过蛋白质截短测试的APC基因的突变状态，ii）微卫星不稳定性，包括BLM本身中的A9运行，iii）MLH 1和MSH 2免疫组织化学，iv）通过直接DNA测序和免疫组织化学的TP 53的突变状态，v）通过直接测序的RAS的突变状态和vi）通过免疫组织化学的DCC。本申请描述了DNA库和相关数据库的设计，我们将用于识别突变载体的方法，以及用于测试致癌机制的程序。

DESCRIPTION (provided by applicant): We are developing a repository of specimens consisting of genomic DNA samples from over 1,500 consecutive Ashkenazi Jewish colorectal cancer (CRC) cases. This repository will be linked to essential clinico-pathological parameters and to the tumor specimens themselves. A major application of the repository is the identification of CRC-susceptibility genes that are present in the cases are at greater frequency compared to the same number of healthy, age- and sex- matched Ashkenazi Jewish controls. One such CRC-susceptibility gene is the blmAsh mutation, which is protein-terminating mutation in the DNA-repair protein BLM, that confers a three fold increased risk of developing colorectal cancer. We propose here a pilot project to utilize the tumor specimens accessible through the repository to investigate mechanism(s) of colorectal carcinogenesis in blmAsh carriers. The blmAsh mutation causes a >95 percent reduction of BLM protein expression from the mutant allele. Two mechanisms are hypothesized: 1) cancer susceptibility is caused by haploinsufficiency of BLM, generating a constitutive risk of transformation of all colon cells and 2) somatic oss of BLM is an early step in carcinogenesis. To test these hypotheses, we will investigate whether BLM protein expression is detectable in the tumor ceils of blmAsh carriers compared to noncarriers by immunohistochemistry using BLM antibodies and whether or not the normal BLM allele is lost genetically by loss of heterozygosity studies. In addition, we will investigate the effect of blmAsh on known mechanisms of colorectal carcinogenesis. By comparison of paired tumor-normal material from blmAsh carriers and from noncarriers, we will test i) mutational status of the APC gene by protein truncation test, ii) microsatellite instability, including the A9 run in BLM itself, iii) MLH1 and MSH2 immunohistochemistry, iv) mutational status of TP53 by direct DNA sequencing and immunohistochemistry, v) mutational status of RAS by direct sequencing and vi) DCC by immunohistochemistry. This application describes the design of the DNA repository and associated database, methods we will use for the identification of mutation carders, and the procedures for testing mechanisms of carcinogenesis.