發(fā)布時(shí)間：2018-04-21 23:27

  本文選題：單核苷酸多態(tài) + 3’-UTR　； 參考：《南京醫(yī)科大學(xué)》2014年碩士論文

【摘要】：早產(chǎn)(preterm birth, P1B)定義為孕齡小于37周或從末次月經(jīng)第一天開(kāi)始計(jì)算少于259天分娩。早產(chǎn)在我國(guó)的發(fā)生率約為8%,并且有上升的趨勢(shì),是導(dǎo)致圍產(chǎn)兒發(fā)病和死亡的首要原因。目前,早產(chǎn)的發(fā)生機(jī)制尚不完全清楚,普遍認(rèn)為,早產(chǎn)是由遺傳因素與環(huán)境因素共同作用的結(jié)果。流行病學(xué)調(diào)查顯示：妊娠婦女孕前及孕期補(bǔ)充葉酸能夠有效降低早產(chǎn)的發(fā)生率。因此,葉酸代謝基因的缺陷導(dǎo)致的葉酸利用能力不足成為研究早產(chǎn)遺傳病因的重要方向。5,10-二甲基四氫葉酸還原酶(5,10-methylenetetrahydrofolate reductase, MTHFR)作為葉酸代謝的關(guān)鍵酶,其基因單核苷酸多態(tài)位點(diǎn)(Single nucleotide polymorphisms, SNPs)成為研究早產(chǎn)遺傳病因的熱點(diǎn)。但是,對(duì)MTHFR基因功能性SNPs的尋找多集中于編碼區(qū)域,而對(duì)基因表達(dá)具有重要調(diào)控作用的3’非編碼區(qū)域(3'-UTR)尚無(wú)研究；為進(jìn)一步拓展研究思路,尋找早產(chǎn)的可能遺傳病因,本研究著眼于能夠調(diào)節(jié)基因表達(dá)水平的3'-UTR,探索MTHFR基因3'-UTR的SNPs與早產(chǎn)的相關(guān)性。 用1argetScan, DIANA-microT和PicTar在線軟件預(yù)測(cè)作用在MTHFR基因3'-UTR的microRNA (miRNA),選擇三個(gè)軟件預(yù)測(cè)結(jié)果的交集作為候選miRNA,再用miRNASNP在線軟件對(duì)候選rmiRNA種子序列上是否有SNP位點(diǎn)進(jìn)行預(yù)測(cè),最終篩選到9個(gè)SNP作為候選研究位點(diǎn)。運(yùn)用SNaPShot基因分型技術(shù)檢測(cè)480個(gè)早產(chǎn)樣本和655個(gè)正常對(duì)照樣本中9個(gè)候選SNPs的基因型顯示：rs1537515和rs1537516存在連鎖不平衡,且為完全連鎖。通過(guò)關(guān)聯(lián)分析發(fā)現(xiàn)：rs1537515和rs1537516的雜合基因型在早產(chǎn)組中的分布頻率為12.7%,顯著低于正常對(duì)照組17.9%,結(jié)果提示,rs1537515和rs1537516的雜合基因型可能是早產(chǎn)發(fā)生的保護(hù)因素,(P=0.017,OR=0.654,95%CI=[0.47,0.91])。為進(jìn)一步探索這兩個(gè)SNPs對(duì)早產(chǎn)的保護(hù)作用是由于調(diào)節(jié)MTHFR基因的表達(dá)的結(jié)果,還是由于與位于編碼區(qū)影響MTHFR酶活性的SNPs連鎖的結(jié)果,我們用Haploview4.2軟件對(duì)MTHFR這兩個(gè)位點(diǎn)區(qū)域進(jìn)行連鎖不平衡分析,找出與陽(yáng)性位點(diǎn)連鎖不平衡的位點(diǎn),并通過(guò)SNaPShot基因分型技術(shù)進(jìn)行驗(yàn)證,以期解答rs1537515和rs1537516對(duì)早產(chǎn)的作用,并找到與早產(chǎn)具有相關(guān)性的其他位點(diǎn)。我們發(fā)現(xiàn)：rs13306556,rs2274976,rs1537515和rs1537516之間存在完全連鎖,它們的雜合基因型都是早產(chǎn)的保護(hù)因素(P=0.017,OR=0.65,95%CI=[0.46,0.91])；-4846048位點(diǎn)與rs1537515和rs1537516不連鎖,但是此位點(diǎn)的雜合基因型是早產(chǎn)的易感因素(p=0.001,OR=1.67,95%CI=[1.24,2.25])。進(jìn)一步分析表明,與rs1537515和rsl537516連鎖的rs13306556位于非編碼區(qū),而rs2274976位于Exonll可導(dǎo)致谷氨酸轉(zhuǎn)變成精氨酸,這些位點(diǎn)的功能驗(yàn)證以及位點(diǎn)之間的相互作用需要進(jìn)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.21

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