An Study Of Methylation Of Mismatch Repair Gene In Hereditary Nonpolyposis Colorectal Cancer

Background Hereditary nonpolyposis colorectal cancer is an autosomal dominant genetic disease, caused by mismatch repair gene mutations, and accounts for about 5% -10% of all colorectal cancers. The most effective treatment of HNPCC is endoscopic and surgical removal of the tumor. If the primary and recurrent tumor can be detected earlier and radical surgery can be carried out, which can significantly improve the prognosis. MMR gene mutation of the detection has become the gold standard HNPCC confirmed, but the some researchers found that known species of MMR gene mutation can't be found in some families(including some microsatellite instability-high tumor family), and further study showed that mismatch repair gene promoter CpG island methylation is the the cause of gene inactivation and tumor. So far, genomic DNA in patients with HNPCC, reports about the methylation of MLH1, MSH2 are less. Detection of genomic DNA methylation may be HNPCC screening and monitoring to provide a means for HNPCC and the diagnosis, treatment, to provide a new way of thinking.Objective To study the methylationg status of CpG island on promoter region mismatch repair gene MLH1 and MSH2 in Chinese in hereditary nonpolyposis colorectal cancer, and to explore the relations between MLH1, MSH2 promoter region of the CpG island methylation status of mismatch repair gene inactivation and hereditary non-polyposis colorectal cancer occurrence and development of the relations, So as to HNPCC screening and monitoring to provide a means for HNPCC and the diagnosis, treatment, to provide a new way of thinking.Materials and MethodsSpecimens* Surpported by the Nature Science Foundation of Beijing(NO.7062064) 47 samples without micromutation and large fragment deletion are from 106 samples which have been confirmed HNPCC. Later, four cases from the same family( Amsterdam Criteria II) were added, and MSH2 gene mutation has been confirmed.Methods1)DNA extraction: follow step description of the kit, and the extraction of genomic DNA was detected by agarose gel electrophoresis, the concentration of the DNA was assayed using spectrophotometry, then stored at -20℃.2)Genomic DNA sulfurize treatment: follow step description of the kit, at least 1μg genomic DNA was transformed, then stored at -20℃.3)MSP: PCR amplifications were carried out in the same sample, using methylation primers and non-methylation primers respectively. PCR products were detected on the 2% agarose gel electrophoresis, observe the strap under UV lamps. Criteria: the specific fragment M illustrates methylation: complete methylation (fragment M), partial methylation(fragment M and U); non-methylation (fragment U).Results1) In 47 examples, hypermethylation of MLH1 promoter was detected in 6 examples(12.8%), by analyzing the electropherogram of methylation, 4 examples were completely methylated, and 2 examples were partial methylated;2)In 36 examples, hypermethylation of MSH2 promoter was detected in 27 examples(75.0%), and all of them were complete methylation; the remain (25.0%) were non-methylation.3) Complete methylation of MSH2 promoter was detected in the four augmented examples.Conclusions(1)In HNPCC, there is a high frequency of methylation in MSH2 gene promoter region, and it's an important factor in the course of development associated with HNPCC. MLH1 gene promoter methylation is relative rare;(2)In the experiment, DNA methylation occurred mainly in the AC-II family, and DNA methylation of HNPCC may shows genetic characteristics;(3)Methylation was detected in a family which has confirmed MSH2 gene mutation, it is confirmed that gene methylation has inheritance as genetic mutation. Both may play synergies to the incidence of HNPCC.