N6AMT1多态性、叶酸对砷代谢影响及砷致DNA甲基化改变的研究

发布时间：2018-01-16 13:22

本文关键词：N6AMT1多态性、叶酸对砷代谢影响及砷致DNA甲基化改变的研究　出处：《中山大学》2017年博士论文　论文类型：学位论文

【摘要】：砷是自然界中广泛存在的一类致癌物,饮水型慢性砷暴露已经成为威胁人类健康的全球性公共卫生问题。我国内蒙古自治区的河套平原是世界上最严重的饮水型砷暴露地区之一。长期慢性砷暴露可导致相关非癌症疾病(如皮肤损伤、高血压、糖尿病)和癌症(如皮肤癌、肾癌、肺癌、膀胱癌)发生率和死亡率的上升。在人体内,无机砷(inorganic arsenic,iAs)主要由(+3价氧化态)砷甲基转移酶(arsenic(+3 oxidation state)methyltransferase,As3MT)催化生成一甲基砷酸化合物(monomethylarsonic acid,MMA)和二甲基砷酸化合物(dimethylarsinic acid,DMA),通过尿液排出体外。之前我们研究组利用酵母基因突变筛选系统,发现N-6-腺嘌呤特异性DNA甲基转移酶1(N6 adenine-specific DNA methyltransferase1,N6AMT1)参与砷的甲基化代谢,并通过体外细胞实验发现N6AMT1与As3MT在砷代谢过程中存在相互作用,N6AMT1是一种有潜力的新型砷甲基化转移酶。许多研究证实,遗传多态性是导致个体之间对砷毒性和致癌性作用产生易感性差异的因素之一,但目前还没有以中国人群为基础评价N6AMT1基因多态性对砷代谢影响的研究。在本研究中,我们以289名生活在内蒙古自治区河套平原五原县农村地区、受饮水型慢性砷暴露(水砷浓度㧐10μg/L)影响的居民为研究对象,运用iPLEX Gold技术对挑选的N6AMT1基因和As3MT的基因多态性位点进行分型检测,并以高效液相色谱-电感耦合等离子体质谱检测个体尿液中砷代谢产物含量,经过数据统计分析,发现5个N6AMT1基因多态性位点(rs1003671、rs7282257、rs2065266、rs2738966、rs2248501)和N6AMT1基因单倍体型GGCCAT与砷代谢相关,特别体现在%iAs的显著差异上。N6AMT1基因单倍体型GGCCAT与As3MT基因单倍体型GCAC的组合,表现出砷代谢能力和效率的叠加效应,携带该单倍体型组合的个体有更强的砷代谢能力和效率。流行病学研究发现,膳食营养素(如叶酸、维生素B12、胆碱、蛋氨酸等)的摄入情况也会影响人体内砷的代谢和基因组DNA甲基化水平,其中作为一碳循环底物的叶酸的影响尤为突出。但目前基于中国人群探讨叶酸补充对砷体内代谢情况及DNA甲基化影响的研究比较少。在本研究中,我们把研究对象(与基因多态性研究部分相同的群体)随机分组成3组,进行为期8周的双盲、安慰剂对照叶酸补充试验,经过8周的叶酸补充,与安慰剂对照组相比,低剂量(400μg/d)和高剂量(800μg/d)叶酸补充组个体的血清叶酸水平明显地升高,而血浆同型半胱氨酸水平则明显地降低。叶酸补充组个体对无机砷甲基化代谢能力明显提高,他们尿液中%iAs、%MMA更低而%DMA更高,而且高剂量组在各指标上都表现更优。此外,叶酸的补充对机体外周血全基因组DNA甲基化水平有提高的作用,提示其对维持砷暴露个体正常DNA甲基化水平有积极的意义。无机砷不会引起基因组的点突变,普遍认为表观遗传改变在无机砷致癌作用中担当重要角色,扰乱正常DNA甲基化模式会导致体内原癌基因和抑癌基因的表达失控,从而诱发病变的发生。但是目前对于DNA甲基化模式紊乱与砷诱导癌症发生的相关性,以及变化的甲基化模式在多代之间的保留情况研究得很少。在本研究中,我们以直系亲属3代人为基础,从内蒙古自治区河套平原招募到代表砷暴露组、非砷暴露组和皮肤损伤病人组的119人,通过全基因组DNA甲基化水平分析和Illumina Infinium Human Methylation450微珠芯片甲基化位点检测,发现砷暴露组每一代个体的全基因组DNA甲基化水平都比非暴露组同代个体的低,皮肤损伤病人组的水平比暴露组和非暴露组所有的代数亚组都明显要低。经过分层分位数标准化法和Combat相结合的方式对甲基化数据进行标准化和批次校正处理,暴露组和非暴露组的3代之间有超过5000个基因组位点的甲基化水平存在明显差异(P值0.0001),第2代人甲基化显著差异的位点数量最多,提示生命早期阶段(胎儿期/幼年期)可能对砷诱导的甲基化改变更为敏感。基于暴露组和非暴露组3代之间共同存在显著甲基化差异的964个探针,进行等级聚类分析,皮肤损伤病人的甲基化模式和砷暴露组个体更为相似,但与有砷暴露历史但没发病的个体又存在区别,而非暴露组个体的甲基化模式与皮肤损伤病人和暴露组则区别明显。综上所述,本研究发现在中国人群中N6AMT1基因多态性与砷的生物甲基化代谢存在联系,并且与As3MT基因的特定单倍体型组合存在相互作用关系;叶酸的补充对砷的甲基化代谢和全基因组DNA甲基化水平的维持有积极的作用;砷暴露会导致DNA甲基化模式发生改变,这种改变在多代之间有一定的重叠性,而且与相关病变的发生存在关联。这些发现可以为寻找人群对砷毒性和致癌作用的易感性差异标志物,以及寻找一种经济、有效预防地方性砷中毒的途径,提供一定的理论基础和实践依据。
[Abstract]:Arsenic is a kind of widely exists in the nature of carcinogen, has become a global public health threat to human health of chronic arsenic exposure. Drinking water in the Inner Mongolia Autonomous Region Hetao Plain is one of the regions exposed to arsenic in drinking water is the most serious in the world. Chronic arsenic exposure can lead to non cancer diseases (such as hypertension, skin damage. Diabetes) and cancer (such as skin cancer, renal carcinoma, lung cancer, bladder cancer incidence and mortality rise). In the human body, inorganic arsenic (inorganic arsenic, iAs (+3) is mainly composed of oxidation state) arsenic methyl transferase (arsenic (+3 oxidation state) methyltransferase, As3MT) catalyze the formation of methyl arsenate compounds (monomethylarsonic acid, MMA) and two methyl arsenate compound (Dimethylarsinic acid, DMA), excreted through the urine. Before we study group using yeast gene mutation screening system, N-6- gland Purine specific DNA methyltransferase 1 (N6 adenine-specific DNA methyltransferase1, N6AMT1) involved in the methylation of arsenic, and through in vitro experiments showed that N6AMT1 interacted with As3MT in arsenic metabolism process, N6AMT1 is a new type of arsenic methylation potential transferase. Many studies have confirmed that genetic polymorphism is the result one of the factors of arsenic toxicity and carcinogenicity of susceptibility differences between individuals, but there is no study on the effect evaluation of N6AMT1 gene polymorphism on arsenic metabolism in China population. In this study, I have to live in the countryside 289 Wuyuan County, the Inner Mongolia Autonomous Region Hetao plain area, affected by chronic arsenic exposure in drinking water (water the concentration of arsenic? 10 g/L) affected residents as the research object, using the polymorphism of iPLEX gene and As3MT N6AMT1 Gold technology to choose the type of detection, and to High performance liquid chromatography inductively coupled plasma mass spectrometry detection of individual urinary arsenic metabolites after statistical analysis of the data, found that 5 N6AMT1 gene polymorphisms (rs1003671, rs7282257, rs2065266, rs2738966, rs2248501) and N6AMT1 gene haplotype GGCCAT and arsenic metabolism, especially in the significant difference on%iAs gene.N6AMT1 single type GGCCAT and As3MT gene GCAC haplotype combinations showed additive effect of arsenic metabolism capability and efficiency, with the combination of the individual haplotype has stronger arsenic metabolism capability and efficiency. Epidemiological studies have found that dietary nutrients (such as folic acid, vitamin B12, choline, methionine) intake will affect the metabolism the body of arsenic and DNA methylation, which affect the carbon cycle as the substrate of folic acid is particularly prominent. But based on the current population of Chinese leaves Study on effect of arsenic metabolism and DNA methylation of acid is relatively small. In this study, we take the research object (and the study of gene polymorphism groups in the same part) were randomly divided into 3 groups, a 8 week double-blind, placebo-controlled trials of folic acid supplementation, after 8 weeks of folic acid. Compared with the placebo group, low dose (400 g/d) and high dose (800 g/d) received folic acid individual serum folic acid levels significantly increased, and plasma homocysteine levels were significantly decreased. Folic acid supplementation group on arsenic methylation capacity of individuals increased significantly,%iAs in their urine, lower%MMA while the%DMA is high, and the high dose group in each index are better performed. In addition, folic acid supplement on the peripheral blood genomic DNA methylation level enhanced the activity, suggesting that the individuals are often exposed to arsenic in maintaining DNA methylation level There is positive significance. Do not cause a point mutation in the genome of inorganic arsenic, is generally believed that epigenetic changes play an important role in arsenic carcinogenesis, disrupting the normal DNA methylation patterns can lead to loss of control in vivo expression of oncogenes and tumor suppressor genes, which induce the disease. But the correlation between cancer and disorder induced by arsenic DNA methylation patterns, and retention of methylation pattern changes in multiple generations between very little research. In this study, we take immediate family members of 3 generations of human based, from the Inner Mongolia Autonomous Region to recruit on behalf of the Hetao Plain arsenic exposed group, non arsenic exposed group and 119 patients with skin injury group, through the whole genome the level of DNA methylation analysis and Illumina Infinium Human Methylation450 bead chip methylation detection, found that arsenic exposed group of each individual generation of whole genome DNA methyl The level of all non exposed group with individual low skin injury patients than the level of the exposed group and the control group were all significantly lower algebraic subgroups. Through hierarchical quantile standardization method and Combat method of combining the methylation data standardization and batch correction processing, and exposure group non exposed group of the 3 generation have obvious differences between the methylation level of more than 5000 genomic loci (P = 0.0001), the number of loci of second generations of the most significant differences in methylation, suggesting that the early stages of life (infant / juvenile) on methylation changes induced by arsenic is more sensitive. 964 probe the exposed group and non exposed group between 3 generations together significant methylation differences were based on hierarchical clustering analysis, methylation patterns and patient skin damage exposed group of individuals is more similar, but with arsenic exposure history but not the incidence of the individual And there is a difference, and the non exposed group of individual methylation patterns and skin injury patients and exposed group was obvious difference. In summary, this study found that in Chinese crowd biological methylation N6AMT1 gene polymorphism and the presence of arsenic, and there is interaction between specific haplotype combinations with As3MT gene; folic acid supplement the arsenic methylation and DNA methylation level in maintenance has a positive effect; arsenic exposure can cause DNA methylation pattern changes, this change has certain overlaps in between generations, are connected and related lesions. These findings can find differences on susceptibility to arsenic toxicity and the carcinogenic effect of markers, and find an economic, effective prevention of endemic arsenic poisoning and provide a theoretical basis and practical basis.

【学位授予单位】：中山大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R599.1

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