Candidate Genetic Markers Research In Yunnan Lynch Syndrome

Background:Colorectal cancer incidence rates are rapidly increasing in China Colorectal cancer is the forth most commonly diagnosed cancer in China, The highest incidence rates are found in developing countries. Approximately 10～15% of patients with colorectal cancer have a family history. The almost 2～3% colorectal cancer is the Lynch syndrome or hereditary nonpolyposis colorectal cancer （HNPCC） which is characterized by autosomal dominant inheritance. This syndrome is caused by a mutation in one of the following DNA-mismatch repair genes:MSH2, MLH1, MSH6 and PMS2. In a pedigree with classical Lynch syndrome, half of the relatives in successive generations have colorectal, stomach, ovary, endometrial or another cancer. All cancers related to the syndrome are characterized by an early age of onset. The family history is the most important clinical prognostic factor. Therefor screening and intervention the family members of the Lynch syndrome would be reduce incidence and mortality. In 1990, the International Collaborative Group on HNPCC （HNPCC-ICG） proposed the Amsterdam criteria and subsequently the extended Amsterdam Ⅰ criteria and Amsterdam Ⅱ criteria. However, these criteria were limited in clinical practice because of their low sensitivity. Consequently, the National Cancer Institute proposed the Bethesda guidelines, and more recently the revised Bethesda guidelines. Bethesda Guidelines, which were intended to identify CRC tissues that should be targeted for analysis, either looking for MSI or abnormal immunohistochemistry （IHC）. However, there is still lack of diagnosis criteria of molecular,and there is a big difference for the site of mutation of patients with different area. Therefore, the aim of this study is to screen and predict differences biomolecular in Lynch syndrom for gene diagnosis. In detection DNA mismatch repair genes, we found there exists significant difference of gene mutation and mutation sites in different races and different regions. And some patients, who meet the clinical diagnostic standand, have no MMR gene mutation. It is too late to start diagnose and treatment, as well as the basic research for Lynch syndrome, and the difference vary greatly in different regions. In the research, we preliminary discovered the clinical pathylogical features of Yunnan Lynch syndrome, and selected the related molecular biomarkers, which will provide theoretical and research basis for genetic diagnosis and screening family members in hereditary colorectal cancer of Yunnan province.Methods:107 patients colorectal cancer in March 2008-December 2012 were selected from cancer center in the First Affiliated Hospital of Kunming Medical university. In accordance with Bethesda diagnostic criteria with 63 cases, Amsterdam Ⅱ with 44 cases. Collect the patient’s clinical data, as well as microsatellite instability, tumor MMR immunostaining, and germline MMR mutational status data. follow up 7 Lynch syndrome families with MSI-H and dMMR. Extracting blood DNA in Lynch syndrome family. Then using Target capture combined with high deep sequencing technology to detect MLH1, MSH2, MSH6, PMS2 and APC genes in these families. Select a family who is not occur MMR gene mutation using whole exome sequencing to detect other gene mutation.Results:1.107 patients in the right half colon cancer 32 cases （30%）,10 cases of left colon cancer （9%）,12 cases sigmoid colon carcinoma （11%）,53 cases of rectal cancer （50%）. Amsterdam Ⅱ group is priority to right half colon cancer, while in Bethesda group is rectal cancer.In 107 patients of 87 is adenocarcinoma （81%）, mucous adenocarcinoma accounted for 20 cases （19%）.2.107 patients with 66 cases of is MSI-H （62%）, the Amsterdam Ⅱ group （82%） and Bethesda group 47%（P< 0.01）. BAT-26 in the two groups of patients were the highest （P< 0.01）.3.107patients with 39 （36.4%） cases of PMS2 mismatch repair defect; Amsterdam Ⅱ group with 20 （45.5%） cases of MSH2+MSH6 mismatch repair defect; Bethesda group with 37 （58.7%） cases of PMS2 mismatch repair defect.66 patients with MSI-H of MSH2+MSH6 mismatch repair defect 22 （33.3%）.4. Three cases 107 of patients MSI-H and MLHl have BRAFV600E mutation.5. MLH1, MSH2, MSH6, PMS2 and APC genes were sequenced in the 7 famlies with Lynch Syndrome. No variants were found in the family B and G.The possible diseasing variations c.398₃99insA in MLH1 gene was identified in the family A. There are the unknown variations c.1480 T>C （p.Ser494Pro, Het） in MSH2 gene and c.1262G>A, Het （A61/93G） in the PMS2 gene in the family C and F respectively. The possible disease-causing variations c.398₃99insA in the MLH1 gene was identified in the family A. The possible disease-causing variations EX1₈ del （het） in the MSH2 gene was identified in the family D. The pathogenic variation c.1731 G>A（Het A74/73G） was detected in the MLH1 gene in the family E. In the family D, the possible disease-causing variations EX1₈ del （het） in the MSH2 gene was identified.6. The possible disease-causing variations c.571G>A p.V191I in the BRCA1 gene was identified in the family G. We also found the same mutation site in family BConclusions:1. Different clinical diagnostic criterias for Lynch syndrome have some differences in the clinicopathological types. Right adenocarcinoma is the main in Amsterdam Ⅱ Group, and rectal adenocarcinoma is the main in Bethesda Group. In the detection of microsatellite instability, MSI-H patients are more in the Amsterdam Ⅱ Group compared with Bethesda Group. BAT-26 loci is identical to MSI, so BAT-26 can be singly used as the initial evaluation of MSI status.2. The expression loss rate of PMS2 protein is the highest and its consistency with MSI-H is not high, so PMS2 can not be singly used as the index in Lynch syndrome screening.MSH2 and hMSH6 proteins, and MLH1 and PMS2 proteins were lost at the same time, and their consistency with MSI-H is high, so the combination measurement of MSH2 and hMSH6 proteins, and MLH1 and PMS2 proteins would have a better screening and applicable value in Lynch syndrome screening.3. Compared with the detection method of microsatellite instability, the immunohistochemical detection method is more economic, convenient, and beneficial to do the clinical primary screening.4. The detection of BRAFV600E mutation can be used to identify the microsatellite unstable colorectal cancers with genetic germline source to those with sporadic source.5. New suspected pathogenic mutation locus of MMR gene are detected:MLH1 gene c.398₃99insA, Het （W54/M45）, MSH2 gene c.1480 T>C （p.Ser494Pro, Het）, MSH2gene EX1₈ del, het, PMS2 gene c.1262G>A, Het （A61/93G）.A known pathogenic mutation loci of MLH1 gene c.1731 G>A （Het A74/73G） was detected.6. A new suspected pathogenic gene BRCA1 gene c.571G>A p.V191I was found by the technology for exome sequencing, and this loci may be a new mutation loci in Lynch syndrome. This result needs to be further verified by enlarging samples and in terms of functions.