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目的检测p53基因第五外显子“CCGG”序列第二个胞嘧啶(C)的甲基化状况,探讨p53基因在地方性砷中毒(地砷病)发生发展中的作用;同时检测DNA甲基化转移酶(DNA methytransferase,DNMT)DNMT3B基因启动子区-149位点C46359T单核苷酸多态性(Single Nucleotide Polymorphism,SNP)的情况,探讨DNMT3B启动子区-149位点SNP与地砷病易感性是否具有一定的关联。方法采用甲基化敏感性限制性内切酶(Methylation-sensitive restriction Endonuclease,MS-RE)-PCR法,检测118例砷中毒患者和126例正常对照p53基因第五外显子甲基化状况;采用聚合酶链反应-限制性片段长度多态性分析技术(PCR-RFLP),检测123例砷中毒患者与126例正常对照DNMT3B启动子区-149位点SNP情况。结果 (1)p53基因第五外显子甲基化率砷中毒组为50.85%,对照组为88.89%,砷中毒组明显低于对照组(P<0.01);在砷中毒不同病情组,甲基化率均低于对照组,差异均有统计学意义(P<0.01),且随着病情加重,甲基化率有降低的趋势,但差异无统计学意义(P>0.05);(2)砷中毒人群DNMT3B启动子区CT杂合型频率为4.89%,TT纯合型频率为95.11%,未检出CC纯合型;对照组人群中DNMT3B启动子区CT杂和型频率为4.76%,TT纯合型频率为95.24%,未检出CC纯合型;该基因该多态位点基因型分布在砷中毒人群和对照组人群中差异无统计学意义(P>0.05);经性别、年龄校正,未发现-149多态位点不同基因型与罹患地砷病有关联OR=1.138(95%CI:0.309~4.198,P=0.846);(3)根据文献报道的数据,将对照组人群与中国北部人群、美国白种人群、英国人群基因型分布进行比较,发现对照组人群与中国北部人群差异无统计学意义(P>0.05),与美国白人、英国人比较,差异有统计学意义(P<0.01)。结论 (1)燃煤砷污染可致砷中毒组人群p53基因第五外显子低甲基化;(2)本次实验未观察到DNMT3B基因启动子区-149位点C46359T SNP与罹患地砷病存在关联;(3)对照组人群DNMT3B基因启动子区-149位点SNP与北方人群报道基因型分布一致,在该基因该位点SNP检测方面为中国人群基因库提供了基础数据。
Objective To detect the methylation status of the second cytosine (C) in the fifth exon “CCGG” of p53 gene and to explore the role of p53 gene in the development of endemic arsenism (Arsenicosis) To investigate the single nucleotide polymorphism (SNP) of C46359T at the -149 site of DNA methytransferase (DNMT) promoter in DNMT3B gene promoter and explore the relationship between SNP at -149 in DNMT3B promoter and Whether or not arsenic susceptibility has a certain relationship. Methods methylation-sensitive restriction endonuclease (MS-RE) -PCR method was used to detect the methylation status of exon 5 in 118 arsenism patients and 126 normal controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect SNPs at -149 in 123 cases of arsenism and 126 cases of DNMT3B promoter. Results (1) The methylation rate of exon 5 in p53 gene was 50.85% in arsenic poisoning group and 88.89% in control group, and the arsenic poisoning group was significantly lower than that in control group (P <0.01) (P <0.01). As the disease progressed, the rate of methylation decreased, but the difference was not statistically significant (P> 0.05); (2) ). The frequencies of CT heterozygous frequency in DNMT3B promoter region were 4.89%, TT homozygote frequency was 95.11% in arsenism patients, and no CC homozygote was detected in control group. The frequency of CT miscellaneous type in DNMT3B promoter region was 4.76% , TT homozygous frequency was 95.24%, CC homozygosis was not detected; the genotype distribution of the polymorphism loci in the arsenic poisoning group and the control group had no statistical significance (P> 0.05) (95% CI: 0.309 ~ 4.198, P = 0.846). (3) According to the data reported in the literature, the control was not associated with the a-disease of arsenic in -149 polymorphic loci. The comparison of the genotypes between the Chinese population and the northern population, the American Caucasian population and the British population showed that there was no significant difference between the control group and northern China (P> 0.05) People had statistical significance (P <0.01) differences. Conclusions (1) Arsenic contamination of coal can cause hypomethylation of exon 5 of p53 gene in arsenic poisoning group. (2) No SNP of C46359T at -149 site in promoter region of DNMT3B gene is associated with arsenic (3) SNP at -149 in promoter region of DNMT3B gene in control group was consistent with the reported genotype distribution in northern China. The SNP detection of this gene provided the basic data for Chinese population gene pool.