Collection And Reservation Of Familial Adenomous Polyps Family Resources In Chinese And Analysis Of Its Clinical And Molecular Characteristics

Familial adenomatous polyposis (FAP) is one of the two commonest familial syndromes that predispose to colorectal cancer and characterized by the occurrence of hundreds to thousands of colorectal polyps. FAP accounts for about 1% of total colorectal cancers and was classified into three types, one is classical FAP(CFAP), the other two are attenuated FAP(AFAP) and MYH associated FAP(MAP). CFAP and AFAP were autosomal dominant diseases and caused by mutations in the adenomatous polyposis coli (APC) tumors suppressor gene. Classically, CFAP is defined clinically by the finding of at least 100 colorectal adenomas. And in AFAP, adenomas was less than one hundreds, and the average age of cancer onset was also later ( 55 years old )compared to that of classical FAP (39 years old) .MAP accounts for about 5% of total FAP ,and was associated with MYH gene mutation as a autosomal ressecive disesease.Clinical features of FAP in foreign population have been revealed. It is characterized by numerous adenomas in colorectum. Its extracolonical manifestation were: Congenital hepertrophy of retinal pigment epithelium (CHRPE), upper gastric polyps, desmoid tumors and dental abnormalities, and APC gene mutation was found about 30-80% in FAP. Some correlation between genotype and phenotype in FAP in oversea population were reported.Although great progress in diagnosis and treatment of FAP have been achived, there are still some ploblems needed to be resolved.Up till now, thediagnosis of FAP is based mainly on clinical phenotype, it is very difficult to distinguish between AFAP adenomas without family history and multiple sporadic adenomas from clinical manifestation. And the carcinogensis of FAP adenomas is also very hard to diagnose at early age, if adenocarcinoma from FAP adenomas can be diagnosed immediatedly at initiation stage, removal of colorectal rectum will be carried timely to extend the patient's survival time significantly. To find new marker to distinguish between FAP adenomas and multiple sporadic adenomas or adenocaicinoma from FAP adenomas is of practical significance such as to determine the colectomy time.Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) as an innovative proteomic technology has been used to analyze numerous proteins and successfully used to distinguish sporadic colorectal cancer patients at early stage from healthy control subjects in our unit. The diagnostic pattern of sporadic colorectal cancer patients at early stage by SELDI was also established with a high sensitivity and specificity. However, there is no reports of proteomic study in FAP using SELDI-TOF.In China, there are some case reports of FAP, but reports about clinical and molecular features of FAP in Chinese are rare, especially with large samples. With the gradual disappear of large families including large FAP families in China, to collect and reserve FAP family resources which can be afforded for future study about its mechanism of development is becoming urgent. In this study, Clinical features about FAP in Chinese is investigated in detail and Denaturing High Performance Liquid Chromatoaphy (DHPLC) is appllied to detect APC gene mutation. The correlations between clinical and molecular features of FAP in Chinese are analyzed. In addition, the pattern and the specific protein finger profiles were explored with SELDI-TOF technology todistinguish FAP adenomas from multiple sporadic adenomas or adenocaicinoma developed from FAP adenomas and healthy control subjects.Methods33 FAP families were investigated by one person and pedigree maps were plotted, the clinical features of patients in one FAP family were investigated by face to face method, if any question, medical records in hospital were needed to verify. CHRPE were checked by the same oculist with indirect ophthalmoscope. Blood sample of family members were taken with informed consent. APC gene mutations were screened by DHPLC followed by sequencer if abnormal profile found. Clinical and molecular characteristics and their correlation were analyzed.Serum protein finger profiles were detected by SELDI with CM 10 protein chip to distinguish FAP adenomas from sporadic adenomas or adenocarcinoma and healthy control subjects. And diagnostic patterns were established with support vector machine(SVM) method.Results1. Collection of FAP family resources in Chinese and analysis of its clinical phenotype:A. 33 FAP families were collected including 26 CFAP, 4 AFAP and 3 SAFAP families, and Blood samples from 132 family members were taken. 92.8% blood samples were effectively used to establish the EBV-transformed immortal lymphocyte cell lines with application of cyclosporin A.B. In addition to numerous colorectal adenomas, CHRPE were found in90.91% FAP families and 91.84% both FAP patients and mutateon gene carrier as the first extracolonic manifestion, stomach polyp and celio-fibroma were the main extra-colonic tumors,C. The difference between CFAP and AFAP were: the average colorectal tumors were more in CFAP families and the mean age of development of colorectal cancer were less about 16 years in CFAP (41 years old) compared to that in AFAP (57 years old), and the appearance of CHRPE was more frequent in CFAP (94.12%) compared to that in AFAP (66.67%).D. Three shapes of CHRPE were found spceifically related to FAP, and 71.1% FAP patients had a pigment larger than 0.5 PD, their location were mainly in Posterior pole. The shape, size and location of CHRPE were different with that in foreign FAP patients.E. A diagnosis criteria of CHRPE in Chinese FAP was set up and its sensitivity and specificity to both FAP and mutation gene carrier were 91.84% and 100% respectively.2. APC gene variations and its correlation to clinical phenotypes:A. APC gene mutations were found in 48.39 FAP families. SNP detection rate was 45.16 % in FAP families, and 4 novel mutations in codon region, 3 cases of mutations in intron region and 1 noval SNP were found.B. Excluded out mutation in intron regions. Mutations were clustered in 15 exon in APC gene and accounting for 86.67%. Frameshift mutation accounting for 86.68% APC gene mutations andnonsense mutation occupied 13.33%.C. 1309 codon mutation resulted in serious phenotype of FAP with one exceptant, and mutations in 5' and 3' region of APC gene were related with slight phenotype, APC gene mutation in FAP patients with CHRPE were clustered in 5' region. These correlationship characteristics were not the same with the results in foreign FAP populations.3. Serum protein profiles by SELDI-TOF to distinguish FAP adenomas from sporadic adenomas or adenocaicinoma and healthy control subjects. A.SELDI-TOF was verified effectively to find some different-expressed serum proteins in FAP adenomas, sporadic adenomas or adenocaicinoma and healthy control subjects:a) Protein of 3.17, 4.18, 4.29, 5.64 and 13.75 kDa m/z were over-expressed in both of FAP adenomas and adenocarcinomas patients developed from FAP adenomas and low-expressed in sporadic adenomas.b) Protein of 2.77, 2.76, 9.40, 9.24 and 4.60kDa m/z were over-expressed in adenocarcinomas patients developed from FAP adenomas and low-expressed in FAP adenomas.c) Protein of 8.47 and 3.40kDa m/z were over-expressed and low-expressed in the subjects with APC gene mutation respectively.B. Some serum protein finger patterns were established to distinguish FAP adenomas from sporadic adenomas or adenocaicinoma from FAPadenomas and healthy control subjects.Conclusion:1. Clinical features of FAP in Chinese revealed, And three special shapes of CHRPE were found related to FAP. The characteristics of CHRPE (including shape, size and location) were different in Chinese FAP and foreign FAP. And CHRPE were verified as a good method with high sensitivity(91.84%) and specificity(100%) to screen asymptomatic FAP patients and mutation gene carrier.2. 4 novel mutations in codon region, 3 cases of mutations in intron region and one novel SNP were found in FAP in Chinese. Frameshift mutation was the main mutation type in APC gene mutation. The correlation between clinical and molecular phenotype in FAP in Chinese was not the same with the results in foreign FAP populations.3. Some different-expressed proteins were screened out by SELDI and serum protein finger patterns were established to distinguish FAP adenomas from sporadic adenomas or adenocaicinoma from FAP adenomas and healthy control subjects.4. Application of cyclosporin A effectively improved the EBV-transformed immortal lymphocyte cell lines rate (92.8%). And 33 rare FAP family recourses in Chinese were Collected and reserved.