Study Of Association Between B-cell Lymphoma-2 And Tumor Necrosis Factor Gene Polymorphisms And Endometriosis

BackgroundEndometriosis (EM) is defined as the presence of endometrial tissue (glands and stroma) outside the uterus. Ectopic endometrium of endometriosis may invade all parts of the body. But the most common site is the pelvis, especially the ovaries and uterosacral ligaments. It is estimated to occur in 10-15% of women, especially reproductive-age, and often is associated with pelvic pain and infertility. Although it is a benign disease, endometriosis does have cancer clinical behavior characteristics, such as planting, invasion and distant metastasis. Etiology and pathogenesis of endometriosis has been widely studied in recent years, but such studies have not yet entirely clear.At present, the theory of pathogenesis of endometriosis focuses on the following aspects:Transplantation Theory, Coelomic Metaplasia, Induction Theory, Immunologic Factors and Inflammation, Environmental Factors and Dioxin, Genetic Factors, but any of the above theory can not fully explain the pathogenesis of endometriosis. Studies have shown that endometriosis may be caused by the interaction of genes and environment. The risk of first-degree relatives of patients with endometriosis is seven times as women without family history. What is more, when one of monozygotic twin sisters suffering from endometriosis, another is seventy-five percent. Single nucleotide polymorphism (SNP) is a polymorphism caused by a single nucleotide mutation, which is one of the most common human genetic variations. Studies have shown that there is an association between endometriosis risk and cytokines and inflammatory factors, steroidogenic enzymes and receptors, estrogen receptors, adhesion molecules and matrix enzyme gene polymorphism.Although it is a benign disease, endometriosis does have cancer clinical behavior characteristics. Studies have shown that blocked apoptosis and inflammation plays an important role in endometriosis. B lymphoma -2 (Bcl-2) genes can inhibit apoptosis. At the same time, studies have shown that there is association between Bcl-2 gene polymorphism and thyroid cancer, throat cancer, chronic lymphocytic leukemia, premature ovarian. KORMAZ D suggested that the protein expression of Bcl-2 in ectopic endometrial tissue of endometriosis patients is higher than normal endometrial tissue. At present, the studies focused on the association between Bcl-2 gene polymorphisms and endometriosis risk is rare. To this end, we genotyped Bcl-2 gene(rs1088477A/G and rs2279115A/C) single nucleotide polymorphism of 302 patients with endometriosis (case group) and 337 women without suffering from endometriosis by allele-specific PCR (AS-PCR) and DNA sequencing, to study the association between Bcl-2 gene single nucleotide polymorphisms and endometriosis risk.Pelvic inflammatory and immune dysfunction plays an important role in the occurrence and development of endometriosis, and tumor necrosis factor (TNF), interleukin (IL) and other cytokines have the same function. Studies have shown that the peritoneal fluid TNF concentration of patients with endometriosis was significantly higher than healthy women, and the peritoneal fluid TNF concentration of patients with active endometriosis was significantly higher than inactive persons. At the same time, the highest TNF concentration of peritoneal fluid appeared in patients with moderate and severe endometriosis. The association between endometriosis and tumor necrosis factor (TNF) gene polymorphism has been studied widely. However, the result is far from conclusive. In order to investigate the association between endometriosis and TNF gene(TNF-α-238G/A、TNF-α-308G/A、 TNF-α-857C/T、TNF-α-863C/A、TNF-α-1031 T/C、TNF-β+252G/A) polymorphism, 8 studies (including 1221 patients with endometriosis and 1376 healthy women) were collected and a meta-analysis was conducted.ObjectiveThis study is mainly focused on the association between and Bcl-2 and TNF gene(TNF-α-238G/A、TNF-α-308G/A、TNF-α-857C/T、TNF-α-863C/A TNF-α-1031T/C、TNF-β+252G/A) polymorphisms and endometriosis risk.Subjects and methods1. Subjects1.1 Subjects of Bcl-2 genotyping 302 endometriosis patients in this study were received surgical operation (abdominal or laparoscopic) and pathological examination in Nanfang Hospital, Zhujiang Hospital and Foshan First People’s Hospital from October 2008 to November 2009.337 controls in this study were received surgical operation due to ectopic pregnancy, cesarean from the same period and hospital., they were confirmed without endometriosis. There is no significant difference among case group and control group of subjects with age (P> 0.05). Each study subjects were from southern Chinese Han population, and unrelated to each other. All subjects without family history of endometriosis, and each subject was informed consent.1.2 Subjects of meta-analysis According to inclusion and exclusion criteria of literature, there were eight relevant research literatures focused on the association between TNF polymorphism and endometriosis risk.2. Methods2.1 Methods of Bcl-2 genotyping2.1.1 Blood specimen collection 2ml peripheral blood specimen were collected from all cases and controls in the third day after operation, then saved by Sodium Citrate and stored at -20℃.2.1.2 DNA extraction According to the instructions of OMEGA’s EZ 96TM Blood DNA kit, genomic DNA was extracted from peripheral leukocytes.2.1.3 Bcl-2 genotyping We genotyped Bcl-2 gene(rs1088477A/G and rs2279115A/C) single nucleotide polymorphism by allele-specific PCR (AS-PCR) and DNA sequencing.2.2 Methods of meta-analysis The PUBMED, CNKI,EMBASE, and Google Scholar databases were searched for all articles, using the following search statement:("TNF" OR "Tumor Necrosis Factor") AND ("Endometriosis" OR "Endometrioses" OR "Adenomyosis") AND ("Genetic polymorphism" OR "Single nucleotide polymorphism" OR "SNP" OR "Mutant" OR "Gene variation" OR "Gene mutation"). Other relevant documents retrieved by a hand or library research, etc. Our latest searches were conducted on October 8,2013. According to the inclusion and exclusion criteria, eight relevant literatures were extracted, and then we conducted a meta-analysis by STATA12.0 (Stata Corp, College Station, TX, USA) software.2.3 Statistical Analysis2.3.1 Statistical Analysis of the association between Bcl-2 polymorphism and endometriosis risk Genotype and allele frequencies of case and control group were calculated directly. Chi-square test was used to verify Hardy-Weinberg equilibrium. Logistic regression analysis was used to compare genotype frequencies between two groups, OR represents the relative risk,95% CI represent 95% confidence intervals. SPSS21.0 statistical software was used for statistical computation, bilateral P<0.05 was considered statistically significant. OR,95% CI and P were used to determine the association between Bcl-2 gene polymorphisms and endometriosis risk.2.3.2 Statistical Analysis of meta-analysis STATA12.0 software was used for the meta-analysis of association between TNF gene polymorphism and endometriosis risk. First, to assess the heterogeneity of studies, we conducted I2 by STATA12.0 software (I2>50% means heterogeneity, I2<50% means no heterogeneity). Secondly, according to the heterogeneity of studies, we selected corresponding effect mode (I2>50% selects the random effects model, I2<50% selects fixed effects model), then we conducted OR by the selected effect model; Thirdly, different analysis models (such as the dominant model, stealth model, co-dominant model, super-dominant model)were conducted for the association between TNF gene polymorphism and endometriosis risk; Finally, OR,95% CI, P were used to determine the association between Bcl-2 gene polymorphisms and endometriosis risk, and publication bias was conducted by Begg’s test and Egger’s test.Result1. Result of the association between Bcl-2 polymorphism and endometriosis risk1.1 Age distribution of case and control group The average age of control group was 34.3 ± 6.9 years old, while the control group was 33.7 ± 5.5 years old, but the age distribution between the two groups was not statistically significant (P= 0.222).1.2 Result of genotyping For all cases and controls from the selected southern Chinese Han women, Bcl-2 gene rs1088477A/G and rs2279115A/C genotype distribution meet normal distribution and Hardy-Weinberg equilibrium (P= 0.16 and 0.11, respectively).Therefore, the research is representative.1.3 Result of statistical analysis For rs1088477A/G, allele A and G distribution of case group and the control group were 5.0%,95.0% and 4.9%,95.1%, genotype AA, AG, GG distribution were 0.6%,8.6%,90.8% and 1.3%,7.3%,91.4%. The difference of case group and the control group are no statistically significant (P> 0.05). For rs2279115A/C terms, allele A, C distributions of the case group and the control group were 46.9%,53.1% and 44.5%,55.5%, genotype AA, AC, CC, respectively distribution 25.8%,42.1%,32.1% and 22.0%,45.1%,32.9%. The difference of case group and the control group are no statistically significant (P> 0.05).2. Result of meta-analysis The results of this meta-analysis indicated that TNF-α-308G/A, TNF-α-857C/T, TNF-a-863C/A, TNF-β+252G/A polymorphisms were not associated with the risk to endometriosis; However, the TNF-a-1031T/C polymorphism appears to have an influence on endometriosis susceptibility. Compared to the C genotype, the T genotype may decrease the risk(T vs. C: OR=1.057; 95% CI,0.771-1.449;P=0.012); Compared to the C-carriers, the T-carriers may also have a reduced risk to endometriosis (TT vs. C-carriers: OR=1.139; 95% CI,0.814-1.593; P-0.029).Conclusion1. There is no association between endometriosis risk and Bcl-2 gene polymorphism.2. TNF-α-308G/A, TNF-α-857C/T, TNF-α-863C/A and TNF-β+252G/A polymorphisms were not associate with the risk to endometriosis; However, the TNF-α-1031T/C polymorphism appears to have an influence on endometriosis susceptibility.