Susceptibility Genes Orientation And Mutation Screening Of The Preliminary Study In A Rare Multi-cancer Pedigree

Cancer family is different from familial aggregated cancer pedigree with the characteristics which is diverse tumor genesis aggregated in pedigree.The molecular genetics mechanism is involved in a series of molecular abnormity mediated by related tumor suppressor genes or susceptibility genes which always play an important role in early stage of molecular events led to diverse tumorigenesis.Although families about multi-cancer pedigree have been reported in some laboratories around the world,the true molecular genetics mechanism is not well-known. Lacking sufficient propositus who can offer valuable genetic information and lacking high-throughput methods became the bottleneck in this investigation.1.Rescuing protection and collection of a rare multi-cancer pedigreeA rare large multi-cancer pedigree,including six generations and over 103 members,were found in Changsha,Hunan.Altogether there are 10 affected survivals of 15 patients in this family and many kinds of cancers or tumors were referred,such as endometrial cancer, hysteromyoma,breast carcinoma,breast fibroids,colon carcinoma,rectal cancer,gastric cancer,hemangioma,cyst of kidney,et al.Some patients were suffered from several kinds of tumors in his lifetime such as with hysteromyoma and breast carcinoma,Ⅳ-1 with hemangioma and hysteromyoma,Ⅳ-3 with colon carcinoma,rectal cancer and breast fibroids andⅣ-4 with colon carcinoma and endometrial cancer.In this multi-cancer pedigree breast reproductive system and alimentary system tumors are in the majority,separately accounted for 50%and 37.5%of the total man-time.10 affected survivals with continuous distribution in the pedigree are in two generations.Among them,there are 2 male patients and 8 female patients.The gender differences might be existed, but it mainly showed off autosomal dominant inheritance.The average age of onset in this pedigree is 42.5y and the earliest age of onset in this pedigree isⅤ-30(29y) and the oldest age isⅡ-1(61y).There is a trend that the suffered individual age of onset was becoming younger,which is known as anticipation in genetics.This multi-cancer pedigree belongs to very rare and precious genetic resources with the characteristics of many affected survivals and many kinds of tumors.Some patients are in the terminal stage of cancer entered the lives at stake.In order to preserve the most complete pedigree information and genetic samples,we are now racing against time to do our utmost to rescue that acquisition of samples and each one was immortalized lympholeukocyte cell lines in simultaneous.At present,we have collected 24 core members of the peripheral blood samples,8 affected individuals,10 non-affected individuals and 6 spouses,and the average age was 44.91±16.80 years(8-73 years).There are 21 core members in the pedigree with successful completion of the peripheral blood lymphocytes immortalization,including 7 patients,8 non-affected individuals and 6 spouses.All of these efforts have been made enough preparations for deeply and repeatedly researching with the pathogenesis of the multi-cancer pedigree.Completion with the proper protection and collection of the multi-cancer pedigree provided valuable materials for study on the complex etiology and mechanisms of cancer family.In-depth study on them will have strategic importance and practical significance to fill the international blank in this field.2.The purpose of this paperAt present,positional cloning strategy is the effective means for disease susceptibility genes research in pedigrees.Therefore, pedigree-based linkage analysis strategy was used to look forward to locating susceptible genes of the rare multi-cancer pedigree.We have chosen high throughput SNP array to do genome-wide scan,genotyping and linkage analysis in 24 core members of this pedigree to positioning the Section of genetic susceptibility.In this foundation,16 micro-satellites have been used to do fine mapping and haplotype analysis to verify the linkage.Then,the direct sequencing-based mutation screening of susceptibility genes strategies was used to do tumor susceptibility gene mutation screening in the susceptible section and some of the important function of oncogenes,tumor suppressor genes or familial colon cancer related genes in genome-wide scope also have been screened.We hope to find some genetic mutations closely related to the Cancer Etiology of the pedigree.3.genome-wide linkage analysis based on SNP arrayFirst of all,GeneChip(?) Mapping 500K array containing 500,000 SNP loci(Affymetrix Inc.) was used to do genome-wide scan,genotyping and linkage analysis in 24 core members of this pedigree.Affymetrix Inc.software Gtype was used to finish SNP genotyping and MERLIN was Chosen to do two-point parameters linkage analysis. We found 3q24-26 was the positive chrosome region that between SNP_A-2277889-SNP_A-19751583(at q24-3q26.1) LOD score was over 1.The maximum LOD was obtained under low-stringency criteria.In dominant model of two-point parameter linkage analysis the maximum logarithm of odds of 2.382 was obtained between SNP_A-2277889-SNP_A-19751583(at q24-3q26.1).LOD scores were computed at following parameters:disease allele frequency(0.0001), penetrance(AA=0.95,Aa=0.95,aa=0),which suggested that this chrosome section linkaged with disease loci.Population allele frequency data was downloaded from the Affymetrix database of the official website. In non-parametric linkage analysis,LOD score was over 1 between SNP_A-2277889-SNP_A-19751583(at q24-3q26.1) and the maximum logarithm of odds of 1.4(p=0.006) was obtained between SNP_A-2053936-SNP_A-2308707(at 3q25.2-3q25.32),which is in accordance with parametric linkage analysis results.After that,we have done LD modeling analysis to the positive region in parametric and non-parametric linkage analysis results.After LD modeling,the maximum LOD score was still over 2(2.061 in before) and the NPL score was still over 1(1.12 in before).So we could rule out the probability of false positive results caused by excessive number of loci. We tried to do multi-point linkage analysis,but the data was too huge to be fulfilled by a general computer.4.Fine mapping to confirm linkage with microsatellitesIn order to achieve multi-point linkage analysis results,and to verify the results of the chip,we focused on chromosome 3q24-26,distance 19.17Mb,using micro-satellite fine positioning scan.By querying the Marshfieldclinic database,19 microsatellites loci which heterozygosity were over 0.7 has been chosen.Ultimately total 16 markers met the requirements of linkage analysis.GENEHUNTER software has been chosen to do both two-point and multi-point linkage analysis.Linkage software has been chosen to do two-point linkage analysis.Under low-stringency criteria and in dominant model,the maximum logarithm of odds for two-point parameter and non-parameter linkage analysis was 1.718(PL) and 2.585(NPL) both at D3S3625(3q25.1), p=0.02161.It did not have too many differences compared with the preliminary results,but in the subsequent multi-point linkage analysis especially non-parametric linkage analysis,we had the exciting discoveries.Between D3S3625-D3S3575(9 loci,4.69-9.09cM),the LOD score was over supported linkage threshold(3.0).At the same time,in this region the NPL score reached extremely significant level of linkage, p＜0.005.The maximum logarithm of odds for multipoint linkage analysis of 10.674(NPL) was obtained including 2 loci,D3S1584 and D3S3710, corresponding with the smallest P value(0.00195).Linkage software got almost entirely consistent results with GENEHUNTER for two-point parameter linkage analysis.In addition,we also have used GENEHUNTER software and Cyrillic software to do haplotype inference.A typical haplotype block (4-7-1-10-4-3-5-8-2-9),10 loci from D3S1555 to D3S3575,was found segregated with disease phonotype in the multi-cancer pedigree.The affected individualsⅣ-40,Ⅳ-1,Ⅳ-3,Ⅳ-4,Ⅳ-5,Ⅳ-18,Ⅴ-31,Ⅴ-30 and non-affected individualsⅢ-7,Ⅳ-6,Ⅴ-2,Ⅴ-3,Ⅴ-4 shared the inferred disease haplotype. 5.Section of the candidate susceptibility gene mutation screeningThrough the UCSC database query and gene annotation,we first have chosen 12 tumor-associated candidate genes in chromosome 3q24-26 to do direct sequencing for mutation screen.Although there is no definite mutation founded in them,but now we have found that in AADAC gene promoter region two particular genotypes of SNP rs 16833935 and rs8193024 was found segregated with disease phonotype in the multi-cancer pedigree.The genotypes distribution of the 2 SNPs had significant difference between patients/carriers and non-carriers/spouse(χ~2 =48,p＜0.0001).It prompted that the 2 particular genotypes might have important relations associated with the incidence of cancer family and we are now carrying out the large sample screen in local population.In addition,we are trying to use the latest technology to do target sequencing in chromosome 3.Furthermore,we have selected some important oncogenes,tumor suppressor gene or genes associated with familial colorectal cancer outside chromosome 3q24-26 to do mutation screen.This part of the work is in progress.