Mechanisms of carcinogenesis by CRC-susceptibility genes

点击翻译按钮获取中文摘要

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.