Analyze Of Clinical Phenotype Of The Hereditary Nonpolyposis Colorectal Cancer In Northerner Of China And Basic Research Of Its Molecular Genetics

PrefaceColorectal cancer is a type of cancer with evident genetic predisposition whose onset was affected by genetic factor and environmental factor. Herediatery nonpopsis colorectal cancer(HNPCC) is an autosomal dominant inheritance syndrome , its penetrance is as high as 70 - 80% , and occupy about 5-15% of the colorectal cancer. The molecular genetic basis of the disease is germ line mutation of the mismatch repair gene in which the hMLHl and hMLH2 played an important role and occupy 90% of the mutations. They are nucleonic acid hydratase and can hydrolyse the mismatched base pairs and so to correct the mi-spairing of the nucleotide bases in the process of replication of DNA and the possibly insertion or depletion of small piece of bases, so to ensure the accurate replication of DNA and the stability and conservatism of human hereditary. Once the mutation of the gene happened, the expression of the mismatch repair protain would decrease and lose, so the mismatched base pairs would increase and the simple repeat sequence of DNA would amplified or lose, so called MSI which results in the happening of tumor. MSI is the phenotype of MMR, the expression of hMLHl/ hMLH2 protain and detection of microsatellite instability can reflect the gene mutation of MMR gene. Our research analysed the clinic phenotype of northerner of China with HNPCC or probands with ordinary genetic colorectal cancer and detected the expression of hMLHl/ hMLH2 protain and microsatellite instability to forecast the gene mutation of MMR gene, and so to provide clinical evidence for identification of genetic colorectal cancer family.Material and methods1. Clinical data(1) The patients with colorectal cancer who were treated at the department of oncosurgery in the 1 st Affiliated Hospital of China Medical University from Jan 2000 to Dec 2004 were investigated . Among them 22 families belonged to HNPCC and 20 families to the ordinary hereditary colorectal cancer family. 14 families accord with Amsterdam criteria II and 8 families accord with Japan criteria, all of the families accord with the Chinese criteria of HNPCC. In accordance with the front standard, we instituted the criteria for the ordinary hereditary colorectal cancer: 0 three or more persons of the family who suffered with the colorectal correlated cancer confirmed by pathology in three generations of the family, (g) at least 2 persons of the family who suffered with the colorectal cancer and was not accord with the above 2 criteria. (3) the patients must be parents and children or brothers and sisters. @ not confined with ages. (5) the patients with familial adenomatous polyposis were excluded.2. Grouping(l)HNPCC group (A group) 20 patients (12 men, 8 women, and middle age 48 years, range 32 - 70 years) who fulfilled the criteria for HNPCC of Chinese people were selected and their family history were obtained by follow - up study. Among them 9 cases with carcinoma of ascending colon, 2 cases with carcinoma of transverse colon, 1 case with carcinoma of descending colon, 2 cases with carcinoma of sigmoid colon and 6 cases with carcinoma of rectum.(2)The ordinary hereditary colorectal cancer group{B group) 20 patients (13 men, 7 women, and middle age 61 years, range 30 83 years) who fulfilled the criteria for ordinary hereditary colorectal cancer of Chinese people were selected. Among them 5 cases with carcinoma of ascending colon, 3 cases with carcinoma of transverse colon, 1 case with carcinoma of descending colon, 2 cases with carcinoma of sigmoid colon and 9 cases with carcinoma of rectum.(3) The sporadic colorectal cancer group(C group) 20 patients{10 men, 10 women, and middle age 65 years, range 42 - 80years) who were final diag-nosed of colorectal cancer by pathology and with no family history were selected. Among them 5 cases with carcinoma of ascending colon, 4 cases with carcinoma of sigmoid colon and 11 cases with carcinoma of rectum.(4)The colorectal polyps group. (D group) 20 outpatients( 10 men, 10 women, and middle age 45 years, range 31 - 70years) who were final diagnosed of colorectal polyps by pathology were selected, their tissues were obtained by enteroscope electric cutting. Among them 2 cases with polyps of ascending colon, 3 cases with polyps of transverse colon, 6 cases with polyps of descending colon, 5 cases with polyps of sigmoid colon and 4 cases with carcinoma of rectum.(5) The other two slides of normal intestine tissue were taken as positive and negative controls.Methods1. Follow - up visit: At first we look up the illness record and put the data into computer. Then set up follow - up records for all the cases be selected and carry out follow - up visit by means of telephone, letter or visiting face to face. The contents including (1 ) the age and sex of patients when they were first dignosed of cancer. (2) the location of cancer(including cancers of non - colorectal cancer). The splenic flexure of colon were taken as boundary, the proxi-mum colon to it was defined as right hemicolon cancer and the distal colon to it was defined as left hemicolon cancer which including rectal cancer. ( 3 ) multi -cancer: more than 2 independent cancers of colon and rectum were named multi- cancer, among which dignosed by pathology less than half a year named syn-chronia multi - cancer and more than hah0 an year named heterochronia multi -cancer. (4) the clinical manifestation of the probands. At last draw the family maps of A and B group which the probands were made the core and do pedigree analysis to affirm the mode of inheritance of the family.2. For MSI analyse, normal and tumor tissues of the three groups embedded with paraffin, 4-5 slides of tissue with thickness of 4ujn was cut and stained with HE. Normal and tumor tissues were selected with microscope, theywere transferred to the EP tubes which contained 150jxl cell lysate. Then DNA of the normal and tumor tissues were extracted with DNA extraction kit.The primers of the 5 microsatellite sites of HNPCC (BAT26, BAT25, D2S123, D5 S 346 ,D17S250) were selected according to recommend of the international cooperation organization{ table 1). The primers were synthesized by TAKARA company.The PCR products were analyzed by electrophoresis on 1.5 % agarose gels containing ethidium bromide. The difference of the two clinical phenotype was compared.Detection of MSI single strand conformation polymorphism ( SSCP) was used to analyze, the PCR product of normal and turner tissues was mixed with the same volume of alkaline buffer, then started with a 971 denaturation forlO min and be put in the mixture of water and ice for 5 min. Then electrophoresis vertically on the 10% nondenaturing polyacrylamide gel (constant power;60w) for about 4 hours, when the indicating straps reached the inferior margin of the gel or the tracer agent disappeared, doing silver staining, coloration, fixing and terminating. When the film dried 24 h later, the imaging was observed.3. Detection of expression of hMLHl/ hMLH2 protain. normal and tumor tissues of the four groups embedded with paraffin, 4-5 slides of tissues with thickness of 4 m was cut. Take off paraffin in xylol and hydrated by gradient alcohol and then put in the 3%H202for 15 min, and then dipped in the lOmol/L buffer solution of citrate and boiled in the electric rice cooker to modify the antigen. The first antibody was added, the working concentration is 1:50, incubated at 4°C for a night and washed with PBS and the second antibody was added, sealed for 15 min and washed with PBS, coloration with DAB and terminated when buffy granules were seen. Afterstain, anhydration, transparent, mounting and observing. PBS was used to substitute the first antibody as negative control, two slides of normal intestine were taken as positive control in every experiment.4. Results assessment(1) MSI was defined by the presence of novel bands following PCR ampli-cation of tumor DNA, which were not present in PCR products of the corresponding normal DNA. More than 2 of them showed positive results was thoughthigh frequency microsatellite instability ( MSI - H) , only 1 of them showed positive result was thought low frequency microsatellite instability ( MSI -L) while none of them showed positive results was named microsatellite stable. (MSS)(2) Results assessment of immunohistochemistry: in accordance with the immunohistochemical criteria of Friedrichs'. The positive stained cell and the degree of staining were observed. Methods as follows: count scores according to the percentage of positive cells in the observed cells, 0 to negative results, 1 to positive cells less than 10% , 2 to positive cells between 11 -50% , 3 to positive cells between 51 -80% , 4 to positive cells more than 80%. Then count scores according to the intensity of staining and the assessment mainly depend on the cell staining intensity compared with the color of background, 0 to colorless, 1 to buff , 2 to buffy and 3 to dark brown. The cross product of the above interg-rating of every slides was recorded, cross product of more than 2 for positive expression of protain, both low positive expression and absence expression of pro-tain judged as negative results. The normal epithelium of intestinal mucosa and infiltrative lymph cell were taken as inner positive controls.5. StatisticsThe software SPSS 11.5 was used to analyse. The phenotype, MSI and the weaken or absence expression of protain of every group was compared with chi square test, p <0.05was thought to be significant difference.Results1. The analysis of the clinical phenotype: (1) the ratio of males to females is 1.4;1 (41/30 ) and 1.5:1 ( 38/26 ) separately in the two groups which was no statistical significance. (2) the middle onset age of the two group was 48(32 -70) and 61(30-83) separately, the ratio of morbidity before 50 is 59.2% and 26.6% separately of which the former group was earlier than the latter, and the difference was significant. (p<0.001) (3) the ratio of morbidity of right hemi-colon cancer is 56.9% (29/51) and 29. 2% (7/24) separately, the difference was significant, (p <0. 05) (4) there were 7 cases of multi cancer in the A group and none in the B group. (5) the mean onset age was 64, 56 and 48 yearsold in the first, second and third descendents of the A group and had tendency of be younger while it not happened in the B group.2. The results of detection of MSI: (1) the occurrence rate of MSI - H in the three groups is 85% (17/20) ,40% (8/20) and 10% (2/20) , the difference was significant. ( p < 0. 05 ) ( 2) the mean onset age of MSI - H of the three groups is 43.6,52.2 and 61. 8 years old separately which elevated gradually. The occurrence rate of right hemicolon cancer was 64.7% ,37. 5% and 0 separately which declined gradually, the difference was significant, (p <0. 05) (3 ) among the five loci selected the positive expression rate of BAT26 and BAT25 is highest(94. 1% separately). (4) There was 70.6% (12/17) of poorly differentiated adenocarcinoma in the A group, which was much more than that of B group(50% ,4/8) and C group(50% ,1/2). The difference is significant. (P<0.05)3. The detection results of the expression of hMLHl and hMLH2 protain. (1) the total negative expression rate of hMLHl and hMLH2 protain in the A, B,C and D group is 65% , 30% ,5% and 0 separately, which showed desceas-ing tendency linealy from A group to D group. (2) neither of the two proteins ( hMLHl and hMLH2 protain) showed negative results in the same slide. (3) the negative expression rate of hMLHl protain is higher than that of hMLH2 protain. (p<0.01)Conclusion1. The phenotype of HNPCC was dominant inheritance, the main extro -colorectal cancer was gastric cancer and lung cancer. The synchronia multi -cancer and the heterochronia multi - cancer were commonly seen.2. The genetic location of HNPCC tumors had specificity and represented " anticipation".3. The treatment of HNPCC should abides by individualization principle, the follow - up visit is an important methods to find heterochronia cancer.4. The MSI - H had good conformity with the fairly early onset age, the occurrence rate of right hemicolon cancer and the occurrence of the poorly differen-tiated cancer.5. The detection of MSI is economical and easy to perform and had highly correlation to the clinical patho - characteristics of HNPCC and can be used as a screening method for MMR.6. The negative expression of hMLHl and hMLH2 protain had significant correlations to position and differentiation degree of the colorectal cancers, but had no relationship with the onset age, sex, metastasis of lymph nodes and the stage of Dukes of the patients.7. The negative expression of hMLHl and hMLH2 protain had significant correlations to the possibility of HNPCC, The methods of immunohistochemistry may provide simple, fast, economical and reliable theory proof for discerning and screening of HNPCC.