Frequent Ki-ras Mutation In MSI-H Colorectal Cancer Deficient In DNA Mismatch Repair Genes

ObjectiveThe occurrence of the tumor is a long process that cells gradually lose genetic stability. The genes controling the basic biological activities are affected which finally led to the transforming and tumorigenesis. The sporadic mutation rate is insufficient for the thus large - scale variety in time of the life span of an individual. A status which high mutation is prone to occur in genome is essential for the process of carcinogenesis. This hypothesis is widely accepted and nominated as " imitator phenotype" . The mechanisms of keeping the fidelity of heredity information are very complicated. Among them three candidates are thought to be most essential, namely mismatch repair system, polymerease proofreading function and oxidative damage elimination. They can recognize and repair damages caused by different mechanisms respectively or corporately. The deficiency of these mechanisms will cause the emergence of the mutator phenotype.Microsatellites are numerous 1 ~ 5 base paired small repeat sequences extending between genes. DNA polymerases are prone to slip during DNA duplication in such loci. DNA mismatch repair system is one of the systems in maintaining the length of Microsatellites loci. Microsatellite instability can thus reflect the deficiency in DNA mismatch repair system to some extend.The reported rate of microsatellite instability differs greatly. Such controversy might partly be derived from methodological difference for microsatellite instability detection. A double fluorescent marked laser scan based on automatic fragment analysis was developed with high sensitivity and reproducibility. Not only the existence but also the patterns of microsatellite loci can be revealed in detail.Ki - ras mutation is one of the common genetic phenomena in colorectal cancer. The mutation hotspot of codon 12 conveys transforming activity regardless whatever mutations occur. It serves as a marker of the mutation spectrum in the genome in this study. The relationship between the spectrum of mutation in this site and microsatellite status aiming at revealing the complicated mechanisms led to the tumorigenesis of colorectal cancer.Material and method1. SpecimensTissues were collected from consecutive surgically dissected samples of 77 CRC patients (44 male and 33 female) admitted in the Department of Surgery II, Kyushu University Hospital from 1997 to 1998 with ages ranging from 15 to 86 (mean, 62.4). Genomic DNA library of tumours and non - carcinogenic margins was built.2. ReagentsEx — Taq polymerase^ primers and reagents for PCR(Takara, Japan) Cycle sequencing Kit and TS ( ABI, Applied Biosystems, USA) PRINCETON CENTRI -SEP Column (ABI, Applied Biosystems, USA)3. Methods1) HRFMA ( High resolution Fluorescent Microsatellite Analysis assay ) to detect the Microsatellite status in colorectal cancer.2 ) After Genomic PCR, Direct sequencing was employed in detecting mutations in Codon 12, 13 of Ki - ras.3 ) Direct sequencing of the whole exons and exon - intron joints of hMLHl and hMSH2 genes.4)Chi -square test and Fisher exact test for statistic analysis.Result1. By HRFMA assay, 13 (16.9%) MSI-H, 18(23.4%) MSI-L, and 46 (59.7% ) were detected in 77 cases.