| Background and Objection:Breast cancer is one of the most common cancers among women. There are about 1000,000 people suffering from breast cancer, accounting for about 20% of malignant tumors of the female every year, and 400,000 people will die from it worldwide. Compared with western countries, China has lower incidence of breast cancer. However, the incidence among Chinese women is increasing, and patients with breast cancer are being much younger with the development of industry and the change of lifestyle. In some developed cities such as Shanghai, Beijing, and Tianjin, the incidence has been more than 50/100,000 and Shanghai even reached 52.98/100, 000, ranked as 1st or 2nd cancer among women. The average annual growth rate is 1～ 2 percent higher than Western countries, seriously endangering women’s health. Moreover, the incidence of breast cancer in Chinese women is expected to reach 85.3/100,000 in 2021.Breast cancer has become one of the key programs for cancer prevention in China.Breast cancer is a heterogeneous tumor and it’s 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. It is known that not all women suffered from breast cancer though they live under the same conditions. That may closely related to different genetic background or breast cancer susceptibility genes. Therefore it is needed to carried out gene detections for breast cancer among yong women and establish the database of susceptibility genes and loci suitable for Chinese women. Ultimately, we can prepare the ground for early diagnosis, individualized prevention and treatment foundation of breast cancer to high-risk Han Chinese women.Numbers of breast cancer susceptibility genes have been gradually confirmed by widely application of chromosome linkage analysis, candidate genes analysis and association analysis. These genes can be generally divided into three categories:high penetrance genes, such as BRCA1, BRCA2, TP53, PTEN, STK11, and CDH1. These mutation rate is relatively low in the public but more susceptible to breast cancer. Compared with general population, their risk of breast cancer can be increased by 5-10 times; Moderate penetrance genes, such as ATM, CHEK2, PALB2 and BRIP, whose mutation rate is moderate in public. Compared with general population, their risk of breast cancer can be increased by 2-4 times; Low penetrance genes, such as FGFR2, ESR1, TOX3, LSP1, MAP3K1 and RAD51L1, their mutation rate is widespread in public, but contribute less to breast cancer susceptibility (less than 1.5 times). Nowadays, the genome-wide association studies (GWAS) mainly focus on SNPs of low penetrance susceptibility genes.Single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism which is caused by single nucleotide variation in the genome with mutation rate more than 1%. These mutations include conversion, transversion, deletion and insertion. SNP is the most common human genetic variation form, accounting for more than 90% of all known polymorphisms. Studies found that SNP plays an important role in disease susceptibility and drug sensitivity. SNPs has many advantages such as a great quantity, extensive dispersion, rapid and convenient screening, which make it more suitable for genetic detection of hereditary and complex disease. SNP had became the third generation of genetic markers after the restriction fragment length polymorphism (RFLP) and microsatellite polymorphism. SNP also make itself one of the manifestations of causing cancer susceptibility by susceptible genes. The research of SNP will provide important data and theoretical basis for individualized diagnosis, prevention and treatment of diseases.Estrogen level was recognized to be one of the most important risk factors for breast cancer beside genetic background. Breast cancer is a kind of estrogen-dependent tumor and highly associated with elevated levels or prolonged exposure time of estrogens. Many other risk factors also work through the estrogen directly or indirectly. Hormone levels in vivo are depended on the dynamic equilibrium of the synthesis and metabolism which catalyzed by a series of enzymes including CYP11A1, CYP17, CYP19,170-HSD, STS, CYP1B1, CYP1A1, CYP1A2, CYP3A4, and COMT. Estrogens can play it’s biological function only when combind with the estrogen receptor (ER). The variations of estrogen synthesis, metabolism-related genes might related to the occurrence and development of breast cancer by influencing the activity and function of it’s product, but there’re few data for Han Chinese women.DNA methylation is one of the most classical epigenetic regulations. After DNA replication, methyl groups are transfered from S-adenosylmethionine(SAM) to cytosine or adenine bases catalyzed by DNA Methyltransferase(DNMT) without any changes in DNA sequence. DNA methylation plays an important role in cell proliferation and differentiation, interactions of DNA and protein, tissue-specific gene expression, maintenance of normal cell function. Researches have shown that abnormal levels of DNA methylation has a direct impact on embryonic development, and induce a variety of diseases even cancer. There are five kinds of methyltransferase in mammals:DNMT1, DNMT3A, DNMT3B, DNMT3L and DNMT2, the first three play a major role. SNPs on DNMTs may alter it’s transcription thus affecting the expression and activity of the enzymes, and then participate in the occurrence and development of breast cancer.In summary, SNPs in estrogen synthesis, metabolism-related genes, and DNMTs may associated with the susceptibility of breast cancer among Han Chinese women through different pathways. Researches have shown that SNPs in these genes were closely related to breast cancer susceptibility. However, genetic architecture is different among populations and the data reported to date concentrate mainly on Western populations. In this study, we try to evaluate the association of SNPs in estrogen synthesis, metabolism-related genes and DNMTs with the susceptibility of breast cancer among Han Chinese women. We hope to provide a theoretical and data basis for early diagnosis, individualized prevention and treatment of breast cancer appropriate for Han Chinese women.Methods:1. Collection of study subjects The subjects participated in our study were recruited mainly from three areas:Guangdong, Chongqing and Shandong. Breast cancer cases and 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.2. Selection of SNPs Potential 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. All the SNPs can be divided into two parts:one was located in estrogen biosynthesis, metabolism-related genes, and the other in DNA methyltransferase genes.3. Screening of SNPs for breast cancer susceptibility Genotyping of the selected SNPs were performed for all subjects via Sequenom MassARRAY(?)-IPLEX platform. Statistical analysis was accomplished with SPSS13.0 software. Five inheritance models (codominant, dominant, recessive, overdominant, and log-additive) were chosen to evaluate the association between each SNP and breast cancer risk.4. For SNPs in estrogen biosynthesis, metabolism-related genes, we also conducted a correlation analysis of genotype distribution and ER or PR status of breast cancer tissue. As subjects for DNMTs polymorphisms and breast cancer risk were selected from three different regions, association study was carried for each region respectively to assess the difference caused by geographical area.ResultsPart 1 SNPs in estrogen synthesis, metabolism-related genes and breast cancer susceptibilityA total of 38 SNPs in 17 genes were picked out. Genotyping and statistical analysis were for 530 breast cancer patients and 546 healthy controls from Guangdong Han women. It was shown that all SNPs was in Hardy-Weinberg equilibrium (P> 0.05) and exhibited polymorphism in our samples except rs2830, rs4680, rs707762, rs17268974 and rs1695.5 positive SNPs were identified to be related to breast cancer risk by the case-control study.1) rs2279357 was fit in codominant, recessive and log-additive inheritance models. Compared with genotype C/C and C/C-C/T, T/T could increase the breast cancer risk with 1.558 and 1.442 of OR (95% CI:1.092-2.222, P=0.014; 1.052-1.976, P=0.023) respectively. T was also a risk allele for breast cancer with the OR 1.234 (95% CI:1.037-1.469, P=0.018); 2) rs2959003 was fit in codominant and log-additive models. Compared with T/T, Individuals carrying the C/C genotype had a lower risk of breast cancer (OR=0.668,95% CI:0.460-0.971, P=0.034). The alele C showed a protective effect on breast cancer (OR=0.820,95% CI:0.687-0.980, P=0.029).3) rs2959008 was fit in codominant, dominant and log-additive models. Compared with T/T genotype, C/C and C/T-C/C were able to decrease the susceptibility of breast cancer with 0.669 and 0.749 of OR (95% CI:0.452-0.991, P=0.045; 0.584-0.961, P=0.023) respectively. C was also a protective allele (OR=0.808,95% CI:0.675-0.966, P=0.020).4) rs700519 was fit in codominant, dominant, overdominant, and log-additive models. Compared with C/C, C/T and C/T-T/T genotypes might protect individuals from the risk of breast cancer (OR=0.723,95% CI:0.541-0.965, P=0.028; OR=0.731,95% CI:0.552-0.968, P=0.029 respectively). The heterozygotes C/T was also related to breast cancer risk (OR=0.726,95% CI:0.554-0.968, P=0.029) compared with homozygotes. There was a similar tendency for allele T(OR=0.772,95% CI:0.600-0.993, P=0.044).5) rs2069522 was fit in codominant, dominant and overdominant models. Compared with T/T genotype, individuals with T/C and T/C-C/C have increased risk (OR=1.500, 95% CI:1.078-2.087, P=0.016; OR=1.421,95% CI:1.029-1.961, P=0.033 respectively). The heterozygotes T/C was also associated with breast cancer risk (OR=1.510,95% CI:1.085-2.101, P=0.015). Statistical analysis for the five positive SNPs shown that individuals carrying more risk genotypes displayed higher risk of breast cancer. If one carried all the five risk genotypes, the susceptibility of breast cancer was significantly higher (OR=2.286,95% CI:1.187-4.399, P=0.013).The relation of gene polymorphisms and hormone receptor status verified 3 SNPs were associated with breast cancer ER and 2 SNPs were with PR. The T/T genotype of rs2279357 and A/A of rs 17268974 were both related to ER(-) of breast cancer tissue (OR=0.574,95% CI:0.366-0.899,P=0.015;OR=0.468,95% CI: 0.243-0.900, P=0.023 respectively). On the contrary, genotype A/A and allele A of rs2446405 were trend to ER(+) breast cancer (OR=2.072,95% CI:1.147-3.742, P=0.016; OR=1.341,95% CI:1.030-1.746, P=0.030 respectively). Genotype T/T and allele T of rs2279357 were associated with breast tissue PR(-)and G/A of rs130021 were associated with PR(+). The OR were 0.529 (95% CI:0.312-0.898, P=0.018), 0.741 (95% CI:0.571-0.963, P=0.025) and 1.756 (95%.CI:1.164-2.648, P=0.007) respectively.Part 2 SNPs in DNMTs and breast cancer susceptibilityIn this study,1413 samples from three regions were recruited including 678 breast cancer patients and 735 healthy controls.11 SNPs in DNMTs were detected among all these samples. The results of statistical analysis indicate that all SNPs was in Hardy-Weinberg equilibrium (P>0.05) and exhibited polymorphism in our samples except rs2228612 and rs11254413. The SNP rs16999593 was fit in codominant, dominant, overdominant and log-additive models. C/T and C/T-C/C were both associated with breast cancer risk (OR=1.319,95% CI:1.046-1.662, P=0.019; OR=1.309,95% CI:1.048-1.634, P=0.018 respectively). The heterozygotes C/T and allele C was also related to breast cancer susceptibility (OR=1.302,95% CI: 1.035-1.638, P=0.024; OR=1.235,95% CI:1.022-1.493, P=0.029 respectively).Stratified analysis on samples from different geographical regions was performed subsequently.1) Among Guangdong women, genotype G/G of rs2228612 and C/T of rsl6999593 were risk genotypes for breast cacner (OR=1.713,95% CI: 1.057-2.775,P=0.029;OR=1.775,95% CI:1.235-2.553, P=0.002 respectively). However, C/T genotype of rs2424908 palyed a protective role in breast cancer susceptibility (OR=0.624,95% CI:0.426-0.913, P=0.015).2) Among Chongqing women, T/C of rs2114724 and A/G of rs2228611 could increase the risk of breast cancer (OR=1.03,95% CI:1.031-2.814,P=0.038), and genotype C/T of rs2424908 might decrease the risk (OR=0.585,95% CI:0.346-0.989, P=0.045).3) Among Shandong women, the C/T of rs 11887120 and G/A of rs 11254413 were related to reduced breast cancer risk (OR=0.553,95% CI:0.360-0.851, P=0.007;OR=0.072, 95% CI:0.040-0.132, P<0.001 respectively).ConclusionsPart 1 SNPs in estrogen synthesis, metabolism-related genes and breast cancer susceptibility1. Five SNPs, rs2279357, rs2959003, rs2959008, rs700519 and rs2069522, were identified to be associated with breast cancer susceptibility in Guangdong Han Chinese women. They could affect the risk of breast cancer in varying degrees and showed a accumulative effect.2. The stratified analysis validated that three SNPs, rs2279357ã€rs17268974〠rs2446405 were interrelated with ER status of breast cancer tissue. Furthermore two SNPs, rs2279357 and rs130021 were associated with PR status.3. The T/T genotype of rs2279357, located in gene CYP11A1, could increase the susceptibility of breast cancer and related to both ER(-) and PR(-) of breast cancer tissue. It suggested that this SNP was a susceptive SNPs possibly associated with breast cancer and not sensitive to endocrine therapy with poor treatment effect and prognosis.Part 2 SNPs in DNMTs and breast cancer susceptibility1. Statistical analysis for all samples showed that genotype C/T and allele C of rs 16999593, in exon 4 of the DNMT1 gene, could increase the susceptibility of breast cancer and the risk of individuals who carried them was 1.319 and 1.235 times compared with general population. However, the results from a Heilongjiang study was contrary to ours.2. There was a significant difference between genotype distribution and breast cancer susceptibility in different regions. Three SNPs, rs2228612, rs 16999593 and rs2424908 were validated as susceptive SNPs for breast cacner among Guangdong womne. Rs2114724, rs2228611 and rs2424908 were susceptive SNPs among Chongqing women. For Shandong women, rs11887120 and rs11254413 were positive loci. |
