叶酸代谢通路关键基因单核苷酸多态与早产的相关性研究

发布时间：2018-04-21 23:27

本文选题：单核苷酸多态 + 3’-UTR　；参考：《南京医科大学》2014年硕士论文

【摘要】：早产(preterm birth, P1B)定义为孕龄小于37周或从末次月经第一天开始计算少于259天分娩。早产在我国的发生率约为8%,并且有上升的趋势,是导致围产儿发病和死亡的首要原因。目前,早产的发生机制尚不完全清楚,普遍认为,早产是由遗传因素与环境因素共同作用的结果。流行病学调查显示：妊娠妇女孕前及孕期补充叶酸能够有效降低早产的发生率。因此,叶酸代谢基因的缺陷导致的叶酸利用能力不足成为研究早产遗传病因的重要方向。5,10-二甲基四氢叶酸还原酶(5,10-methylenetetrahydrofolate reductase, MTHFR)作为叶酸代谢的关键酶,其基因单核苷酸多态位点(Single nucleotide polymorphisms, SNPs)成为研究早产遗传病因的热点。但是,对MTHFR基因功能性SNPs的寻找多集中于编码区域,而对基因表达具有重要调控作用的3’非编码区域(3'-UTR)尚无研究；为进一步拓展研究思路,寻找早产的可能遗传病因,本研究着眼于能够调节基因表达水平的3'-UTR,探索MTHFR基因3'-UTR的SNPs与早产的相关性。用1argetScan, DIANA-microT和PicTar在线软件预测作用在MTHFR基因3'-UTR的microRNA (miRNA),选择三个软件预测结果的交集作为候选miRNA,再用miRNASNP在线软件对候选rmiRNA种子序列上是否有SNP位点进行预测,最终筛选到9个SNP作为候选研究位点。运用SNaPShot基因分型技术检测480个早产样本和655个正常对照样本中9个候选SNPs的基因型显示：rs1537515和rs1537516存在连锁不平衡,且为完全连锁。通过关联分析发现：rs1537515和rs1537516的杂合基因型在早产组中的分布频率为12.7%,显著低于正常对照组17.9%,结果提示,rs1537515和rs1537516的杂合基因型可能是早产发生的保护因素,(P=0.017,OR=0.654,95%CI=[0.47,0.91])。为进一步探索这两个SNPs对早产的保护作用是由于调节MTHFR基因的表达的结果,还是由于与位于编码区影响MTHFR酶活性的SNPs连锁的结果,我们用Haploview4.2软件对MTHFR这两个位点区域进行连锁不平衡分析,找出与阳性位点连锁不平衡的位点,并通过SNaPShot基因分型技术进行验证,以期解答rs1537515和rs1537516对早产的作用,并找到与早产具有相关性的其他位点。我们发现：rs13306556,rs2274976,rs1537515和rs1537516之间存在完全连锁,它们的杂合基因型都是早产的保护因素(P=0.017,OR=0.65,95%CI=[0.46,0.91])；-4846048位点与rs1537515和rs1537516不连锁,但是此位点的杂合基因型是早产的易感因素(p=0.001,OR=1.67,95%CI=[1.24,2.25])。进一步分析表明,与rs1537515和rsl537516连锁的rs13306556位于非编码区,而rs2274976位于Exonll可导致谷氨酸转变成精氨酸,这些位点的功能验证以及位点之间的相互作用需要进一步研究。
[Abstract]:Preterm birth (P1B) is defined as gestational age less than 37 weeks or less than 259 days from the first day of the last menstruation. The incidence of premature delivery in China is about 8%, and it is the leading cause of perinatal morbidity and death. At present, the mechanism of preterm birth is not completely clear. It is generally believed that preterm birth is the result of the combination of genetic and environmental factors. Epidemiological investigation showed that folic acid supplementation before and during pregnancy can effectively reduce the incidence of preterm delivery in pregnant women. Therefore, the deficiency of folic acid metabolism gene leads to the deficiency of folic acid utilization ability, which is the key enzyme of folic acid metabolism in the study of preterm genetic disease, I. e., 510- dimethyl-tetrahydrofolate reductase (MTHFRase). Single nucleotide polymorphisms (SNPs) have become a hot spot in the study of preterm genetic diseases. However, the search for functional SNPs of MTHFR gene is mainly focused on the coding region, while the 3'non-coding region, which plays an important role in the regulation of gene expression, has not been studied. The aim of this study was to explore the relationship between SNPs of MTHFR gene 3'-UTR and preterm delivery. Using 1Arget Scan, DIANA-microT and PicTar online software to predict microRNA miRNAs of MTHFR gene 3'-UTR, the intersection of three software prediction results was selected as candidate miRNAs, and then miRNASNP online software was used to predict whether there were SNP loci on the candidate rmiRNA seed sequences. Finally, 9 SNP sites were selected as candidate sites. SNaPShot genotyping technique was used to detect 9 candidate SNPs genotypes in 480 preterm labor samples and 655 normal controls. The results showed that there was linkage disequilibrium and complete linkage between rs1537516 and Rs1537515. The frequency of heterozygous genotypes of 1: rs1537515 and rs1537516 in preterm labor group was 12.775, which was significantly lower than that in normal control group (17.9%). The results suggested that the heterozygous genotype of rs1537515 and rs1537516 might be the protective factor of preterm labor, 0.65495CI= [0.470.91]. In order to further explore whether the protective effect of these two SNPs on preterm labor is due to the regulation of the expression of MTHFR gene, or to the linkage with SNPs, which is located in the coding region, which affects the activity of MTHFR enzyme, We used Haploview4.2 software to analyze the linkage disequilibrium between the two loci of MTHFR, and to find out the linkage disequilibrium with positive loci, and to verify by SNaPShot genotyping technique, in order to explain the effect of rs1537515 and rs1537516 on premature delivery. Other loci associated with preterm delivery were also found. We found that there was a complete linkage between rs13306556rs2274976rs1537515 and rs1537516, and their heterozygous genotypes were all the protective factors for preterm delivery, P0.017OR0.65 / 95CI = [0.460.91] -4846048 was not linked to rs1537515 and rs1537516, but the heterozygote genotype of this locus was the susceptible factor of preterm labor: p0.001 OR1.6795 CI = [1.2452.25]. Further analysis showed that rs13306556 linked to rs1537515 and rsl537516 was located in the non-coding region while rs2274976 located in Exonll could lead to the conversion of glutamate to arginine. The functional verification of these sites and the interaction between these sites needed further study.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.21

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