| Background & Objective: Sporadic colorectal cancer is one of the most common malignant tumors threatening people's health seriously and its incidence has been rising obviously in recent years in our country. It is necessary to identify the mechanisms of occurrence and metastasis for achieving early diagnosis and therapy of tumor. Inactivation of tumor suppressor genes is one of the major events involved in carcinogenesis and the most common representation is loss of heterozygosity (LOH).Generally, it contains one or more tumor suppressor gene(s) in the chromosome regions with high frequency of LOH. It is possible to find tumor related gene(s) though searching the regions with high frequency of allele LOH. Finding the regions with high frequency of allele LOH by applying polymorphic microsatellite markers to amplify the corresponding loci of the genome DNA can map tumor related gene(s) to a narrow interval on chromosome of sporadic colorectal cancer. Then the tumor related gene(s) can be identified through filtrating and examining the genes contained in this region. This is one of the major strategies for systemic tumor related genes searching in genome-wide range. Adopting the positional candidate cloning strategy mentioned above, LOH on chromosome 3,16p,16q and 17p was analyzed in this study. Relatively denser polymorphic microsatellite markers were employed to put up fine mapping analysis so that the smallest allelic deletion segments could be found. And the tumor related gene(s) were refinedly mapped to average l-2cM range(s). In addition, two loci which had been found to be involved in tumor differentiation and metastasis on chromosome 2 and 16 in sporadic colorectal cancer were applied fine mapping analysis. This study try to map the tumor related gene(s) to regions as small as possible, and filtrate candidate genes glancingly ,thereby serving basis for further gene filtrating and cloning.Methods: 13,5,5 and 5polymorphic microsatellite markers (average genetic space is approximately 10cM) were analyzed on chromosome 3, 16p,16q, 17p respectively by "touch-down" PCR method for 83 samples of colorectal cancer and relative normal DNA. The corresponding loci of the genome DNA were amplified. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan3.7 and Genotype 3.7 software were employed for genotyping and LOH analysis. The regions with highfrequency LOH was mapped, and relatively denser polymorphic microsatellite markers were employed to refine them. In addition, two loci involved in tumor differentiation and metastasis on chromosome 2 and 16 in sporadic colorectal cancer were applied fine mapping analysis which used above.Result: One distinct locus with high LOH frequency D3S1300 was observed on chromosome 3, and another 5 polymorphric microsatellite markers were employed to refine this region; thereby localizing a minimal region of allelic deletion between markers D3S1547 and D3S1312, genetic space was 1.9cM. One distinct locus D16S404 with high LOH frequency was observed on chromosome 16p, and another 5 polymorphric microsatellite markers were employed to refine this region; thereby localizing a minimal region of allelic deletion between markers D16S406 and D16S3126, genetic space was 1.1cM. One distinct locus D16S503 with high LOH frequency was observed on chromosome 16q,and another 8 polymorphric microsatellite markers were employed to refine this region; thereby localizing two minimal regions of allelic deletion, one was between D16S3143 and D16S3050, the space was 2cM;the other was between D16S3094 and D16S3143 ,the space was 1.6cM.Four loci with high LOH frequencies were defined on chromosome 17,D17S1857 was the farest locus from TP53.Another 10 polymorphric microsatellite markers were employed to refine the region around it. The whole region had high LOH frequency ,and the consecutive interval with the highest LOH frequency was 4Mb, containing three loci D17S2196, D17S953 and D17S1871 in 17pll.2. The region around D2S142 locus on chromosome 2 which involved in tumor differentiation was analyzed by 8 polymorphric microsatellite markers, then localizing two candidate regions of tumor related genes: one was between D2S142 and D2S284,and the other was between D2S2275 and D2S2299.The genetic space were 3.8cM and 3.2cM, respectively. 7 polymorphric microsatellite markers was employed to fine map the region involved in tumor metastasis on chromosome 16q, then identifying two candidate regions of tumor related genes: between D16S498 and D16S3123,1.3cM; between D16S402 and D16S3061, 2cM. Database searching indicated that FHn,PTPRG on chromosome3, USP7 on chromosome 16p, CDH11, CDH8 on chromosome 16q and FLCN, COPS3, PMP22 in 17p11.2 may be involved in tumorigenesis of sporadic colorectal cancer. And NR4A2 , GALNT5, ACVR1C, ACVR1 , LOC344332, NMI, RIF1 in tumor differentiation associated region and CDH13, MBTPS1, OKL38, HSD17B2 in tumor metastasis associated region may bepotential tumor related genes in the candidate regions. Otherwise, there was no genes seemed having relationship with tumorigenesis in the 1.3cM interval between D16S498 and D16S3123.Conclusion: (l)This study provides evidence for the involvement of several putative tumor suppressor genes in tumorigenesis of sporadic colorectal cancer. And some candidate tumor related genes in candidate regions were never identified to be related to colorectal cancer. Examining the expression and mutations of them in colorectal cancers may find new colorectal cancer related gene(s).(2)There were no genes in the 1.3cM candidate region between D16S498 and D16S3123 in 16q24.2 showed any relationship with tumorigenesis. Further study on this region may clone new tumor related gene(s).(3)This study demonstrated that positional candidate cloning was efficient and feasible in cancer research of sporadic colorectal cancer. This strategy remarkably decreased the number of candidate genes, and served scientific basis forfurther gene filtrating and cloning.
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