Single Nucleotide Polymophism Sites Screening Of Hereditary Nonpolyposis Colorectal Cancer And Preliminary Study Of Its Sensitivity With 5-fluorouracil

Background and ObjectivesBecause of the chang of diet and life, incidence of colorectal cancer increased gradually in recent years, colorectal cancer has become one of the major diseases led to death in developed countries and regions. To be Hereditary nonpolyposis colorectal cancer (HNPCC) a important subunit of colorectal cancer, it is account for 5%-15%.HNPCC is an autosomal and dominantly inherited cancer predisposition caused by a constitutional defect in a mismatch repair (MMR) gene, also called Lynch syndrome. The genetic basis of HNPCC is MMR gene mutation. The absolute risk of colorectal cancer who carrying the mutation is 70%-90% before the age of 70, incidence of extracolonic cancers are also significantly higher. There are at least six genes associated with this predisposition:hMLH1, hMSH2, hPMS1, hPMS2, hMSH3 and hMSH6. hMLH1 and hMSH2 genes account for more than 90% of the HNPCC families with identified germline mutations. The MMR gene expression is mismatch repair protein,an nucleic acid hydrolase. In the process of DNA replcation, MMR gene hydrolyse the mismatch repaired bases, so that DNA can be copied accurately to sure the conservative and stability of human genetic. If the gene occurred mutation, the expression of mismatch repair protein will decrease, did not express or appear protein truncation, so that errors of DNA replication are increased and occur miscrosatellite instability(MSI), which lead to colorectal cancer and other extracolonic cancers such as endometral cancer, ovarian cancer, bladder cancer. HNPCC did not attract enough attention and ofen missed diagnosis in China. Therefore, understanding the characteristics, genetic characteristics and clinical treatment of HNPCC play a important role for preventing and treating this disease. Immunohistochemical staining and microsatellite analysis are commonly used as the first diagnostic screening test for HNPCC. The identification of mutations in MMR genes serves as the gold standard in diagnosing HNPCC, playing a forward-looking role. It can get the message that the risk level of occuring HNPCC before the patient and their families occurred the disease. But detection of gene mutation are often expensive, laborious and slow, it is diffcult to used in clinical widely. Therefor, looking for a new genetic markers associated with MMR gene mutation, establishing a convenience (such as peripheral blood), simple, cheap way, become a waiting problem of final diagnosing HNPCC. Single nucleotide polymophism (SNP) is the most abundant genetic variation of human genome, as its role of the third generation of genetic markers, SNP can be a specific signs for screening HNPCC, it can find high risk groups with fast and accurate. To finding some SNP sites associated with common disease is a very important way to reveal the complex reason of human diseases.At present, the treatment of colorectal cancer is still surgery, after surgery, conventional adjuvant chemotherapy were given to prevent the transfer of cancer cells. The most anti-tumor activity of chemotherapy drugs are 5-fluorouracil (5-FU) for colorectal cancer. Because different patients have a significant differences in medicine sensitivity, their prognosis also have a significant differences. This requires that adopting individual treatment with different individuals in order to achieve the best therapy effect. In recent years,clinical chemotherapy of individual forward-looking by the way of medicine-sensitive expriment has been subject to more attention at home and abroad. It can provide the best treatment for the patients, improve survival rates and quality. MMR gene mutation cause HNPCC, and MMR system was involved in a variety of cells which responsed to chemotherapy. Therefor, to MMR gene deletion compare with MMR gene integrity of colorectal cancer can help clinical treatment.My study was based on the preliminary studies,collected recent 193 suspected HNPCC (≤50 years old) to immunohistochemical staining for hMLH1 and hMSH2 proteins and microsatellite analysis, screening for highly suspected patients, then detected SNP genotyping and analysis relevance through the case-control study, to find HNPCC-related SNP sites. Finally, to collect the colorectal cancer cells with deleted MMR gene and integrited MMR gene, respectively. Analysis the differences of diferent cells sensitivity with 5-FU, provide a basis for. guiding drug of clinical screening of suspected HNPCC patients.Methods1. The MMR protein expression of suspected HNPCCImmunohistochemical staining was used to detect expression of hMLH1 and hMSH2 genes in suspected HNPCC patients. With abnormal expression of hMLH1/MSH2 protein was considered as highly suspected HNPCC patients.2. MSI analysis of suspected HNPCCSelected BAT25, BAT26, D5S346, D2S123 and D17S250 microsatellite loci, PCR-SSCP and DHPLC analysis for detection of microsatellite of suspected HNPCC patients. The patients with MSI-H were highly suspected HNPCC.3. The SNP sites associated with HNPCCSelected 205 colorectal cancer patients as our study object,110 high suspected HNPCC of them,95 old colorectal cancer patients of them. LDR was usded to detect SNP sites of hMLH1 and hMSH2 genes, analysis relevance through the case-control study to determine high-risk HNPCC populations.4. Colorectal cancer with MMR gene deletion associated with 5-FU.Collect four colorectal cancer cells with MMR gene integrity or deletion (SW480,HT29,Lovo,HCT116), HCT116 also with MSI-H, HT29 also with MSS, analysis sensitivity of different colorectal cancer cells through MTT, plate colony formation test, Flow cytometer..Results1. The MMR protein expression of suspected HNPCCWe selected 193 suspected HNPCC patients, and 30 cases old colorectal cancer patients as control. Immunohistochemical staining was used to detect expression of hMLHl and hMSH2 genes in suspected HNPCC patients. Abnormality of immunohistochemical expression for at least one of these MMR proteins was found in 56 of the 193 (29.02%) cases. The abnormal rate of hMLH1/hMSH2 protein in different age group were as follow:younger than 30 years old was 40.00%(4/10),31 to 40 years old was 28.05%(23/82),41 to 50years old was 28.71%(29/101). The abnormal rate of hMLH1/hMSH2 protein in right colon, left colon and rectum cancer were 40.74%,32.65% and 18.89%, respectively. The abnormal rate of hMLHl/hMSH2 protein in family history patients was 46.15%(12/26). The abnormal rate of hMLH1 and hMSH2 protein was 28.85%(30/104) in male and 29.21%(26/89) in female. The abnormal rate of hMLHl and hMSH2 protein in 30 cases of control patients was 6.67%(2/30).2. MSI analysis of suspected HNPCCTo obtain MSI incidence in suspected HNPCC patients, we selected 193 suspected HNPCC patients and 40 cases old CRC patients as control. PCR-SSCP and DHPLC analysis for detection of microsatellite of suspected HNPCC patients.27 cases are not detected because were paraffin-embedded tissues. The rate of MSI-H was 56.63%(94/166)in suspected HNPCC patients. Of patients whose age at diagnosis were younger than 30 years old,66.67%(6/9) showed MSI-H;of those whose age at diagnosis were from 31 to 40,61.43%(43/70) showed MSI-H. Of those whose age at diagnosis were from 41 to 50,51.72%(45/87) showed MSI-H. Among 40 control patients 37.5%(15/40) showed MSI-H. To compare IHC and MSI methods, There are 51 abnormal expression of MMR gene of 94 MSI-H patients,67 normal expression of MMR gene of 72 MSS patients.3. The SNP sites associated with HNPCCSelected 205 colorectal cancer patients as our study object,110 high suspected HNPCC of them,95 sporadic colorectal cancer patients of them. To detect 20 SNP sites of MMR gene. We found a tagger SNPs rs4608577 as a high-related HNPCC SNP site through case-control study. There were 34 heterozygote,67 homozygous in suspected HNPCC group, there were 15 heterozygote,71 homozygous in control group. The odds ratio of rs4608577 was 2.402(OR>1),95%CI was 1.201-4.804. Other SNPs OR<1 and no statistics significant.4. Colorectal cancer with MMR gene deletion associated with 5-FU.We detected the 5-FU sensitivity of four colorectal cancer cells with MMR gene integrity (SW480,HT29) or deletion (Lovo,HCT116), the 5-FU inhibition of four cells was HCT116>Lovo>SW480>HT29 with MTT and plate colony formation test.Flow cytometer showed that growth percent of colorectal cancer cells was HT29 >Lovo.Conclusions1. The mainly abnormal MMR protein was hMLH1 of MMR protein. MMR protein expression was closely related to age and family history, abnormal expression of MMR genes was an important factor to induce incidence of young colorectal cancer.2. The difference among each age groups(≤50 years old) was not significant, however difference was significant between suspected HNPCC and old group.The young colorectal cancer who under 50 years old can be a target of screening HNPCC. MSI analysis combined with MMR protein immunostaining can maximize the detection of MMR deficient tumor and may be a most useful tool for identifying highly suspected HNPCC from suspected patients.3. We found a tagger SNP site rs4608577 as a high-related HNPCC SNP site through case-control study. The conversion or transversion of T base in the rs4608577 locus is a risk factor of occurring HNPCC. It is showed that detecting related-SNPs of suspected HNPCC can be found high-risk patients with simple, easy and fast.4. The sensitivity of MMR gene deletion cells is higher than MMR gene integrity cells, and the sensitivity of MSI-H cells is higher than MSS cells. It is show that using 5-FU can obtain better therapy effect of colorectal caner patients with MMR gene deletion and MSI-H.