Identification and detection of colorectal cancer susceptibility genes.

摘要

The genes which cause two forms of colorectal cancer (CRC), FAP and HNPCC, have been identified. The knowledge of these genes makes possible the presymptomatic screening of patients at risk for these disorders. FAP and HNPCC patients are screened using the protein truncation test (PTT). PTT detects truncating APC mutations in 80% of the FAP mutations, and truncating mutations in hMLH1 and hMSH2 accounts for half of all HNPCC cases. In the PTT negative patients it is unclear as to the cause of their disease; missed mutations or other as yet unidentified genes. To explore these possibilities, an analysis of the remaining 20% of FAP patients was performed using MAMA. Of nine patients tested, seven were found to have significantly reduced expression from one of their two alleles, while two patients were found to have full-length expression from both alleles. These results indicate that over 95% of patients with FAP have inactivating mutations in APC, and that a combination of genetic tests can identify the vast majority of patients. This work also suggests that another gene may predispose to FAP in a small fraction of FAP patients. Interestingly this was supported by the identification of a novel APC mutation, I1307K. Analysis of this variant showed it to be associated with a 1.5–2.0 fold increased CRC risk over the general Ashkenazi population. The mechanism by which this caused disease was shown to be hypermutability of this allele in the tumors of these patients. This mutation represents the first familial colorectal cancer gene identified to date. To aid genotyping patients for the I1307K, a genotyping strategy was developed called SOMA, Short Oligonucleotide Mass Analysis. SOMA is performed by PCR amplifying polymorphisms using primers containing type-IIs restriction sequences. Digestion of the PCR products releases an oligonucleotide containing the polymorphism. Mass spectrometry is then performed on this oligo to detect mass differences between each allele. SOMA allows for a rapid, sensitive, and cost effective method to genotype patients and may be used for a high throughput cancer susceptibility gene screen.