The Study Of BRCA1/2 Mutations In Chinese Han Women And The Associations Between Moderate-Penetrant Susceptibility Genes Polymorphisms And The Disease

Background and Objection:Breast cancer is the most common cancer among women.Compared with western countries,China has lower incidence of breast cancer.With the development of economy, society and lifestyle changes,the incidence of breast cancer in China is rising and the onset age is younger.Acccording to the data from International Agency for Research on Cancer（IARC） in 2008, the standardized incidence Chinese female breast cance is 21.6/100 000,ranked 99th in the global 184 countries. Standardized mortality rate 5.7/100 000, ranked 145th, were significantly lower than average of the world.But because of the large population of China,cases newly diagnosed are accounting for 12.2% of the world,and the deaths 9.6%.Breast cancer is a heterogeneous tumor and its development is a complicated process involving interactions of multiple factors such as genetic background,level of hormone,immune status,physical condition,lifestyle,and the environment.Lifestyle is an important player in tumorigenesis.The major risk factors that can be modified to decrease risk for breast cancer include improving diet,controlling weight,limiting alcohol consumption and cessation of smoking.However,higher risks for breast cancer are attributed to age,gender and having a family history of the disease.Simply being female increases the risk 100 foldrwomen have a higher chance to develop breast cancer than men because female breast cells are constantly exposed to the growth-promoting effects of the hormones estrogen and progesterone. In addition,the risk to develop breast cancer is age related:breast and ovarian cancer incidence is much higher among post-menopausal women.This suggests that in the majority of breast cancer patients an accumulation of genetic defects is required for initiation and progression of the tumor. About 15% of breast cancer patients have a family history for the disease.In this group of patients there is a higher chance of an underlying inherited genetic defect in a breast cancer susceptibility gene.These cases will develop breast cancer at an earlier age.Breast cancer development is a carcinogens resulted from multi-gene and multi-stage and multi-gene synergy,from carcinogen function to the formation of tumor involved in a variety of different penetrant susceptibility genes.Cancer predisposing genes can be divided into three types:high penetrant genes,intermediate-penetrant and low-penetrant genes.BRCA1 and BRCA2 genes are the most commonly high penetrant genes.Additional rare,but highly penetrant genes include PTEN,TP53,CDHl,and STK11.BRCA1 and BRCA2 mutations are inherited in an autosomal dominant fashion,but act recessively on the cellular level as tumor suppressor genes involved in double-stranded DNA （dsDNA） break repair. Female carriers of mutations in BRCA1 or BRCA2 have a risk of breast cancer of 45-87%.Male carries of BRCA1 have an increased risk of breast cancer,though to a lesser degree than carriers of BRCA2.Most notably,there is an increased risk of other femal cancer.There is an increased risk of ovarian cancer,with an estimated risk of 45-60% for BRCA1 carriers and 11-35% for BRCA2 carriers.Some developed countries have carried out the genetic counseling and genetic testing for BRCAl/2,early screening for mutation carriers and prevention interventions.Mutations and rearrangements or deletions in BRCA1 and BRCA2 are estimated to explain only 15% of familial breast cancers.Collectively with highly penetrant genes, it is estimated that the known high-penetrance genes account for no more than 25% of cases.Linkage studies have failed to demonstrate additional reproducible loci for highly penetrant genes predisposing to breast cancer.A number of studies have focused on genes proposed to increase the risk of breast cancer based on their known cellular functions in families with pedigrees suggestive of a predisposition to breast cancer. Studies have identified a number of additional DNA repair genes that interact with BRCAl,BRCA2,and/or the BRCA pathways,and confer about a twofold increase in breast cancer risk, including CHEK2,BRiP1,ATM,and PALB2.Human RAD51 is one of the key proteins for homologous recombination.we commonly studied polymorphisms of RAD51 gene are G135C （rs1801320） and G172T （rsl801321）.Both of them are located in the 5’untranslated region and seem to be of functional relevance.These two polymorphisms were shown to affect mRNA stability or translational efficiency,leading to altered polypeptide product levels and altering the function of encoding RAD51 protein,and influenced the DNA repair capacity to some extent.Intermediate-penetrant could explain 20-30% of hereditary breast cancer,suggesting s that there are other the susceptibility genes associated with breast cancer.With the development of experimental techniques to enhance and sequencing technology,Genome-wide association study found that other breast cancer susceptibility genes may form the role of multiple genes and the occurrence of cancer. For breast cancer such complex diseases,mainly from foreign population genetics （large,medium,small populations and analysis of genome-wide association scan analysis）,find a large number of susceptibility genes and risk loci,such as FGFR2, TNRC9,MAP3Kl,LSPl,RAD51,ESR1,TOX and so on.Currently,genome-wide analysis found more than 20 genes （ABHD8,ABCC4,ACTL7A,ANKLE1,ANKRD16, CCND1,CDKN2A,CDKN2B,COL1A1,ECHDC1,ESR1,FBN1,FBXO18,FGF19,FGF 3,FGF4,FGFR2,GLG1,GRIK1,KLF4,LOC643714,LSP1,MAP3K1,MRPS30,MYEO V,ORAOV1,RAD23B,RAD51L1,RNF146,ROPN1L,SLC4A7,TNRC9,TOX3,ZMIZ1 ,ZNF） and more than 50 SNPa closely related with breast cancer.These low-penetrance susceptibility genes and susceptibility locus has not been directly applied in clinical practice,more used to screen with building a risk assessment for high-risk populations.Breast cancer occurance is the co-working of a multi-stage and multiple genes With the identification of an increasing number of different penetrance breast cancer susceptibility gene for hereditary breast cancer in a more comprehensive understanding of the system,it is can develop more appropriate and accurate genetic testing technology,to better understand and select the appropriate precautions.Methods:1. Collection of study subjects 165 cases（diagnosed before 40 years old） tand 66 cases who have at least one first class or second class relative afflicted with cancer were tested hot-spot mutations in BRCAl/2;46 cases（diagnosed before 35 years old） selected from the preview study were sequenced for all exons of BRCA1 and BRCA2 genes by using capture of sequences intarget regions and next generation sequencing. The subjects participated in the study The Associations of Moderate-Penetrant Susceptibility Genes Polymorphisms and Breast Cancer among Chinese Han Womenwere recruited mainly from two areas:Guangdong and Shandong.524 sporadic breast cancer cases and 542 healthy female controls were collected in the same period according to the case-control study design.Whole blood samples and corresponding clinical information for each subject were also gathered.There were 283 cases from Guangdong and 241 cases from Shandong.542 controls consisted of 312 sampels form Guangdong and 230 samples from Shangdong.2. Detection of BRA 1/2 mutations reported frequently Mutations were selected from the BIC database or mutations found in Asian or Chinese populations.Genotyping of the selected SNPs were performed for all subjects via Sequenom MassARRAY（?）-IPLEX platform.3. BRCA1/2 exome sequencing Gene sequencing was performed for exons of BRCA1 and BRCA2 genes by using capture of sequences intarget regions and high-throughput sequencing technology.4. Detection of moderate-penetrant susceptibility SNPs Potential moderate-penetrant susceptibility loci of breast cancer were picked out from the NCBI SNP database,published genome-wide association studies and positive SNPs reported by other large-scale studies.Genotyping of the selected locis were performed via Sequenom MassARRAY（?）-IPLEX platform.ResultsPart 1 Mutations of BRCA1/2 in hereditary breast cancers among Chinese Han WomenNo BRCA1 mutation was found in 165 early onset breast cancers and 66family breast cancers.Two mutations in BRCA2 were detected, rs80359352 and rs11571661 respectively.1 early onset breast cancer and 1 fimaily carried rs80359352.The mutation was located in exon 11,and four bases ACAA were deletion from 3036 to 3039,resulting frameshift mutaions and the protein was premature termination at condon 958.For rs11571661 of rs11571661,21 early onset breast cancers and 10 family breast cancers were homozygosis; 73 early onset breast cancers and 27 family breast cancers were heterozygosis;73 early onset breast cancers and 27 family breast cancers were wild type.The mutation was in intron 11,deletion from 80th base.Canpturing and sequencing the 46 the early onset breast cancers,we found six mutations in five samples,all of which were frameshift.The rate of BRCA1/2 was 10.87%.One BRCA1 mutation （HlN,p.Leu502_Lys503fs） and four BRCA2 mutations （NFBC70,c.182de1T;NFBC2,c.5164₅165de1AG; 17N,c.6096dupT;NFBC 145,c.9353₉354insTT CTTGA,c.9355₉356insAGACTGCCTTTACC） were detected.We also found 12 BRCA1 rare mutations including two unknow meaning missense mutaions 191T and M119T,the former reported in BIC database.19 rare mutations were found in BRCA2 including 8 unknow meaning missense mutations Y42C, H400R,M784V, K1729T,R2108C, W2619C, K2729N, 13412V.Y42C, H400R,M784V and K2729N were reported in BIC database.Part 2 SNPs in moderate-penetrant genes and breast cancer susceptibilityA total of 11 SNPs in 10 genes or loci were picked out Genotyping and statistical analysis were for 524 sporadic breast cancer patients and 542 healthy controls from Guangdong and Shandong Han women.lt was shown that all SNPs in controls were in Hardy-Weinberg equilibrium（P>0.05） except rs7166081 and exhibited polymorphism in our samples except CHEK2 1100DELC and IVS2+1G>A.The SNP rs10941679 was fit in codominant,dominant,overdominant and log-additive models.G/A or G/A-A/A were both decreased breast cancer risk （OR= 0.63,95% CI:0.45-0.88, P=0.021;OR=0.68,95%Ci:0.49-0.93,p=0.016,respectively）.Stratified analysis on samples from different geographical regions was performed subsequently.1) Among Shandong women,genotype G/C of rsl801320 were risk genotypes for breast cacner （OR=2.03,95% CI:1.30-3.17,P=0.0064）; 2)Among Guangdong women,genotype G/T of rs6138178 were risk genotypes for breast cacner （OR=1.65,95%C/:1.16-2.33,P=0.018）.However,G/A-AA genotype of rs2424908 palyed a protective role in breast cancer susceptibility （OR=0.63,95% C/:0.41-0.97,P=0.034）.ConclusionsPart 1 Mutations of BRCA1/2 in hereditary breast cancers among Chinese Han WomenWe found two common mutations rs80359352 and rs 11571661 in hereditary breast cancer. Six detrimental mutations of BRCA1/2 and some other rare mutations are detected through sequencing of the exons of BRCA 1 and BRCA2 genes in 46 breast cancer patients. This study complements the BRCA1/2 mutation spectrum,and lay the foundation for breast cancer prevention.Part 2 SNPs in moderate-penetrant genes and breast cancer susceptibilityThree SNPs rs10941679,rs1801320 and rs6138178 were identified to be associated with breast cancer susceptibility in Han Chinese women.They could affect the risk of breast cancer in varying degrees and showed a accumulative effect.They can be increase or decrease the risk of breast cancer at different levels.One SNP rs10941679 was associated with breast cacner among all samples.One SNP rs1801320 was validated as susceptive SNP for breast cacner among Shandong women.Rs 10941679 and rs6138178 were susceptive SNPs among Guangdong women.