發(fā)布時間：2018-04-22 17:39

  本文選題：非梗阻性無精癥 + 線粒體DNA�。� 參考：《南京醫(yī)科大學(xué)》2015年博士論文

【摘要】：不孕不育是生殖健康領(lǐng)域一項亟需解決的重大科學(xué)問題。育齡夫婦中約10-15%存在不同程度的生育障礙。導(dǎo)致不孕不育的原因很多,其中男方因素約占50%。前人研究表明,在過去的半個世紀里,男性精液質(zhì)量顯著下降,精子生成障礙已成為男性不育最常見的病因之一。精子生成障礙可能與環(huán)境化學(xué)污染物、遺傳因子改變、表觀遺傳修飾異常等密切相關(guān)。大約30%的精子生成障礙患者是由于遺傳學(xué)的異常引起的。線粒體作為細胞核外唯一含有遺傳物質(zhì)的細胞器,其參與的氧化磷酸化過程為精子生成、分化及活力維持提供所必需的能量。而且線粒體參與多種生物學(xué)過程,在ROS(Reactive Oxygen Species)平衡、細胞凋亡以及多種信號通路調(diào)節(jié)中都具有重要作用。線粒體基因組(Mitochondrial DNA,mt DNA)編碼氧化磷酸化體系的蛋白亞基和其自身的RNA翻譯元件,mt DNA變異會導(dǎo)致線粒體功能改變,進而引起精子生成障礙,導(dǎo)致男性不育。目前關(guān)于mt DNA遺傳變異對精子生成或精液質(zhì)量的影響已有相關(guān)報導(dǎo)。然而由于受到mt DNA變異檢測技術(shù)、樣本量大小以及種群差異等多種因素的限制,線粒體基因組遺傳變異對精子生成的影響也不盡相同。為此,我們提出以下研究假設(shè):(1)人類線粒體基因組在精子生成過程中發(fā)揮重要作用,其遺傳變異可導(dǎo)致精子生成障礙;(2)在精子生成障礙人群中,一些特定的線粒體DNA單倍群會產(chǎn)生富集(3)氧化應(yīng)激損傷或精子分化異常,導(dǎo)致線粒體基因組含量代償性增加。為了全面、系統(tǒng)地評估人類mt DNA遺傳變異在精子生成中的作用,本項目擬以前期提出的“胞質(zhì)遺傳”理論為基礎(chǔ),以大樣本人群關(guān)聯(lián)研究為切入點,應(yīng)用分子生物學(xué)等研究手段(高通量測序、中通量基因分型等),結(jié)合線粒體DNA單倍群分析,探討是否存在與精子生成障礙相關(guān)的功能性遺傳變異;特定線粒體DNA單倍群是否會增加精子生成障礙的風(fēng)險以及線粒體DNA拷貝數(shù)變異是否與精液質(zhì)量異常相關(guān),明確人類線粒體基因組遺傳變異在精子生成中的作用,揭示可能的分子機制,為男性不育的分子診斷和治療提供新的途徑和方法。第一部分人類線粒體基因組遺傳變異在非梗阻性無精癥(NOA)發(fā)生中的作用目的為了闡明人類線粒體基因組在非梗阻性無精癥(NOA)中的作用,我們對NOA不育男性與健康生育對照的線粒體全基因組進行測序,篩選與精子生成障礙相關(guān)的線粒體DNA遺傳變異,并在大樣本人群中進一步確認。方法采用兩階段病例對照研究。第一階段,應(yīng)用高通量測序技術(shù)對96例無精癥病例和96例對照進行mt DNA全測序,鑒定mt DNA單倍群,篩選出常見mt DNA單倍群以及與精子生成障礙相關(guān)的mt DNA遺傳變異。第二階段,針對536例無精癥病例和489例對照,利用Sna Pshot測序技術(shù),基于13個東亞地區(qū)常見單倍群的編碼區(qū)特征突變點分型結(jié)果,分析人群遺傳背景。隨后,利用SNPscan測序技術(shù),對測序階段篩選出的遺傳變異進行驗證。為了進一步闡明線粒體遺傳變異導(dǎo)致的線粒體損傷的機制,我們應(yīng)用總抗氧化能力(total antioxidant capability,T-AOC)和超氧化物歧化酶(superoxide dismutase,SOD)檢測試劑盒檢測并比較病例和對照以及不同單倍群之間精漿抗氧化能力。結(jié)果根據(jù)第一階段測序所得的線粒體DNA全序列,鑒定每個個體mt DNA單倍群分型,結(jié)合mt DNA系統(tǒng)發(fā)生樹以及東亞地區(qū)常見單倍群,篩選了13個東亞地區(qū)常見單倍群及其定義位點,對比了病例和對照兩組間的遺傳背景。同時篩選了10個線粒體DNA潛在的功能性遺傳變異位點,包括6個編碼區(qū)的可能導(dǎo)致非同義突變的遺傳變異(m.3394TC,m.6881AG,m.8684CT,m.11696GA,m.12358AG,m.13135GA),1個遺傳變異位于t RNA基因上(m.15968TC),另外3個遺傳變異位于第一高變區(qū)(HVRI)(m.16224TC,m.16319GA,m.16497AG)。對第二階段大樣本獨立人群的遺傳背景進行分析,結(jié)果提示單倍群M8*在病例組的比例顯著高于對照組(OR 2.61,95%CI 1.47-4.61)(P=6.76×10-4),提示單倍群M8*會增加精子生成障礙的風(fēng)險。此外,在獨立人群中驗證潛在遺傳變異位點,結(jié)果發(fā)現(xiàn)m.8684CT在病例組中顯著高發(fā)(OR 4.14,95%CI 1.56-11.03)(P=2.09×10-3),提示m.8684CT同樣會增加精子生成障礙的風(fēng)險。同時由于m.8684CT是單倍群M8a的遺傳標(biāo)記位點,因此,我們進一步提出假設(shè),遺傳背景單倍群M8a導(dǎo)致了遺傳變異m.8684CT在無精癥人群中富集。為了驗證上述假設(shè),我們進一步分析單倍群M8*的2個亞單倍群,M8a和Z,比較單倍群M8a和單倍群Z在病例和對照之間的分布,發(fā)現(xiàn)單倍群M8a在病例組中的比例顯著高于對照組(OR 4.14,95%CI 1.56-11.03)(P=2.09×10-3),而單倍群Z在兩組間的分布并無顯著統(tǒng)計學(xué)差異(OR 1.86,95%CI 0.92-3.77)(P=7.88×10-2)。結(jié)果提示,遺傳背景單倍群M8a導(dǎo)致了遺傳變異m.8684CT在無精癥人群中富集,增加了其導(dǎo)致精子生成障礙的風(fēng)險。為進一步研究線粒體遺傳變異致線粒體損傷的水平,我們對精漿抗氧化能力進行檢測。與對照組相比,病例組T-AOC顯著下降(P0.05),提示病例組線粒體功能受損,而SOD活性沒有差異。在不同單倍群之間,由于樣本量的限制,T-AOC和SOD并沒有發(fā)現(xiàn)明顯差異。結(jié)論 遺傳背景單倍群M8a導(dǎo)致了遺傳變異m.8684CT在無精癥人群中富集,增加了其導(dǎo)致精子生成障礙的風(fēng)險。同時,mt DNA遺傳變異可導(dǎo)致精漿抗氧化能力下降,提示線粒體損傷,從而能夠引起精子生成障礙。第二部分人類線粒體基因組遺傳變異在少弱精癥發(fā)生中的作用目的為了全面研究線粒體基因組與少弱精癥病因?qū)W之間的關(guān)聯(lián),通過深度測序線粒體全基因組,篩選與精液質(zhì)量下降相關(guān)的線粒體DNA遺傳變異,并在獨立人群中進行驗證。方法采用兩階段病例對照研究。篩選階段,采用下一代測序技術(shù)對233例少弱精癥病例和233例對照進行mt DNA全測序。鑒定樣本單倍群并分析主要單倍群分布。隨后,篩選與少弱精癥相關(guān)的線粒體基因組遺傳變異位點。驗證階段,針對688例少弱精癥患者及533例對照,利用SNa Pshot測序方法,基于13個東亞地區(qū)常見的單倍群定義位點分型結(jié)果,分析mt DNA單倍群遺傳背景。利用SNPscan測序技術(shù),對測序階段篩選的遺傳變異進行驗證。結(jié)果根據(jù)篩選階段測序所得的線粒體DNA全序列,鑒定樣本單倍群并整合入13個東亞地區(qū)常見單倍群,分析少弱精癥和對照兩組的單倍群分布,結(jié)果符合東亞特有mt DNA單倍群的頻率分布。同時篩選出7個可能增加精液質(zhì)量下降的風(fēng)險的遺傳變異位點。其中,4個位點位于編碼區(qū)(m.12338TC,m.12361AG,m.13928GC和m.A15235 AG),1個位點位于t RNA(m.5601CT),2個位點位于第一高變區(qū)(m.16179 CT和m.16291 GA)。對驗證階段大樣本獨立人群的遺傳背景進行分析,發(fā)現(xiàn)少弱精癥組和對照組兩組間線粒體單倍群分布沒有統(tǒng)計學(xué)差異,提示兩組間遺傳背景的一致性。此外,通過獨立人群的驗證發(fā)現(xiàn),位于第一高變區(qū)(HVS-I)的潛在遺傳變異位點m.16179 CT與少弱精癥顯著相關(guān)(OR 3.10,95%CI 1.41-6.79)(P=3.10×10-3)。為了進一步闡明線粒體遺傳變異對精液質(zhì)量的影響,我們分析了精子密度和精子活力兩個指標(biāo)。m.16179 CT和m.12361 AG能顯著增加少精癥的風(fēng)險,P值分別為1.90×10-4(OR 4.18;95%CI 1.86-9.40)和5.50×10-3(OR 3.30;95%CI1.36-8.04)。m.16179 CT同時能顯著增加弱精癥的風(fēng)險(OR 3.17;95%CI1.40-7.16)(P=3.50×10-3)。結(jié)論線粒體DNA遺傳變異m.16179 CT和m.12361 AG可以增加少弱精癥的發(fā)病風(fēng)險。第三部分少弱精癥的線粒體基因組拷貝數(shù)變異研究目的通過比較少弱精癥與健康生育男性對照精子mt DNA拷貝數(shù)水平,分析mt DNA含量與精液質(zhì)量的關(guān)系,探討導(dǎo)致mt DNA拷貝數(shù)差異的可能原因方法利用實時熒光定量PCR技術(shù),采用相對定量的方法,檢測100例少弱精癥病例和80例正常對照的精子mt DNA拷貝數(shù)。結(jié)果少弱精癥組的平均mt DNA拷貝數(shù)79.02±10.07,顯著高于對照人群的mt DNA拷貝數(shù)為21.40±3.69(P0.001)。結(jié)論mt DNA拷貝數(shù)水平與精液質(zhì)量異常顯著相關(guān),mt DNA拷貝數(shù)水平可以作為精液質(zhì)量異常的重要標(biāo)志物。
[Abstract]:Infertility is an important scientific problem that needs to be solved in the field of reproductive health. About 10-15% in couples of childbearing age have different degrees of fertility disorder. There are many reasons for infertility. Among them, the male factor accounts for the previous study of 50%.. In the past half century, the quality of male sperm has decreased significantly, and the disorder of spermatogenesis has become a problem. One of the most common causes of male infertility. The disturbance of spermatogenesis may be closely related to environmental chemical pollutants, genetic changes, and epigenetic modification. About 30% of the patients with spermatogenesis are caused by genetic abnormalities. Mitochondria are the only organelles containing genetic material outside the nucleus, and the oxygen is involved. The process of phosphorylation is necessary for the production of spermatogenesis, differentiation and vitality. Moreover, mitochondria participate in a variety of biological processes and play an important role in the balance of ROS (Reactive Oxygen Species), apoptosis and the regulation of various signal pathways. The mitochondrial gene group (Mitochondrial DNA, MT DNA) encodes the oxidative phosphorylation system. The protein subunit and its own RNA translation element, MT DNA variation can lead to mitochondrial function changes, resulting in sperm formation disorders, causing male infertility. Currently, the effects of MT DNA genetic variation on spermatogenesis or semen quality have been reported. However, the size and population of the MT DNA variation detection technique, the size of the sample and the population The effects of genetic variation of mitochondrial genome on spermatogenesis are different. Therefore, we propose the following hypothesis: (1) human mitochondrial genome plays an important role in spermatogenesis, and its genetic variation can lead to spermatogenesis obstacle; (2) some specific human spermatogenesis disorder population In order to comprehensively and systematically assess the role of human MT DNA genetic variation in spermatogenesis, this project is based on the "cytoplasmic inheritance" theory, which is based on the earlier "cytoplasmic inheritance" theory, and is based on a large sample population. With the use of molecular biology, such as high throughput sequencing, flux genotyping, and so on, combined with mitochondrial DNA haploid analysis, the presence of functional genetic variation associated with spermatogenesis disorders is explored. Whether the specific mitochondrial DNA haploid group increases the risk of spermatogenesis disorder and the mitochondrial DNA copy number change. Whether it is related to the abnormality of semen quality, to clarify the role of the genetic variation of the human mitochondrial genome in spermatogenesis, to reveal the possible molecular mechanism, to provide new ways and methods for the diagnosis and treatment of male infertility. Part 1 the role of genetic variation in the human mitochondrial genome in the occurrence of non obstructive azoospermia (NOA) Objective to elucidate the role of the human mitochondrial genome in non obstructive azoospermia (NOA), we sequenced the complete mitochondrial genome of NOA male infertility and healthy birth control, screened the mitochondrial DNA genetic variation associated with the dysfunction of spermatogenesis, and further confirmed it in the large sample group. The two stage case was used. In the first stage, 96 cases of azoospermia and 96 cases of control were sequenced with MT DNA, and MT DNA unploploid was identified, the common MT DNA haploid group and the genetic variation of MT DNA related to spermatogenesis barrier were screened. The second stage, 536 cases of azoospermia and 489 cases, using Sna Pshot sequencing technology. In order to further clarify the mechanism of mitochondrial genetic variation caused by mitochondrial genetic variation, we apply the total antioxidant capacity (TOT), based on the analysis of the genetic background of the population based on the mutation points of the coding region of the common population of 13 common populations in East Asia. Then, the genetic variation screened by sequencing is verified by SNPscan sequencing. Al antioxidant capability, T-AOC) and superoxide dismutase (superoxide dismutase, SOD) detection kit were used to detect and compare the antioxidant capacity of the seminal plasma between cases and controls and between different populations. Results according to the complete sequence of mitochondrial DNA from the first stage sequencing, the MT DNA of each individual was identified, combined with the MT DNA system. The common unhaploid population of 13 East Asian regions and its definition loci were screened, and the genetic background between the two groups of cases and the control group was compared. The potential functional genetic variation loci of 10 mitochondrial DNA were screened, including the genetic variation (m.3394TC, m.6881A) that could lead to unsynonymous mutations in 6 coding regions. G, m.8684CT, m.11696GA, m.12358AG, m.13135GA), 1 genetic variations were located on the t RNA gene (m.15968TC), and the other 3 were located in the first high variable region (HVRI) (m.16224TC, m.16319GA, m.16497AG). The genetic background of the second stage large sample independent population was analyzed. The results suggested that the proportion of the haploid group in the case group was significantly higher than that of the control. The group (OR 2.61,95%CI 1.47-4.61) (P=6.76 x 10-4) suggested that the haploid group M8* could increase the risk of spermatogenesis disorder. In addition, the potential genetic variation loci were verified in the independent population. The results showed that m.8684CT was significantly higher in the case group (OR 4.14,95%CI 1.56-11.03) (P=2.09 x 10-3), suggesting that m.8684CT would also increase the risk of spermatogenesis disorder. At the same time, since m.8684CT is a genetic marker of the unhaploid group of M8a, we further hypothesized that the genetic background unfold M8a causes the genetic variation of m.8684CT to be enriched in the azoospermia population. In order to verify the above hypothesis, we further analyze 2 subunits of the unhaploid group of M8*, M8a and Z, and compare the unhaploid M8a and the unhaploid Z in the case. The distribution of M8a in the case group was significantly higher than that in the control group (OR 4.14,95%CI 1.56-11.03) (P=2.09 x 10-3), but there was no significant difference in the distribution of Z in the two groups (OR 1.86,95%CI 0.92-3.77) (P=7.88 x 10-2). The accumulation of sperm in azoospermia increased the risk of spermatogenesis disorder. In order to further study the level of mitochondrial damage caused by mitochondrial genetic variation, we detected the antioxidant capacity of the seminal plasma. Compared with the control group, the case group T-AOC decreased significantly (P0.05), suggesting that the mitochondrial function was impaired in the case group, and there was no difference in the activity of SOD. There was no significant difference between T-AOC and SOD among different unhaploid groups. Conclusion the genetic variation of M8a caused the genetic variation of m.8684CT to be enriched in the azoospermia population and increased the risk of spermatogenesis disorder. At the same time, the genetic variation of MT DNA may lead to the decrease of the antioxidant capacity of the seminal plasma, suggesting the mitochondria Second part of the role of genetic variation in the human mitochondrial genome in the pathogenesis of oligoasthenospermia in order to study the association between the mitochondrial genome and the etiology of oligospermia, and to screen the mitochondrial DNA remains related to the drop of sperm mass by deep sequencing of the mitochondrial genome. A two stage case control study was used in a two stage case control study. 233 cases of oligoasthenospermia and 233 cases of control were sequenced by next generation sequencing. The samples were identified and the major monoploid distribution was analyzed. Subsequently, the mitochondrial genome remains associated with oligospermia was screened. In the verification stage, 688 cases of oligoasthenospermia and 533 cases of control were used to analyze the genetic background of MT DNA haploid group based on the SNa Pshot sequencing method, based on the results of the common population defined loci in 13 East Asian regions. The genetic variation screened by sequencing was verified by SNPscan sequencing. The results were based on the screening order. The whole sequence of mitochondrial DNA, which was sequenced, identified the sample haploid group and integrated into 13 common populations in East Asia, and analyzed the distribution of the haploid group in oligoasthenospermia and control two groups. The results were in line with the frequency distribution of the unique MT DNA population of East Asia, and 7 genetic variation loci were screened for the risk of increasing the drop of sperm quality. The 4 loci were located in the coding region (m.12338TC, m.12361AG, m.13928GC and m.A15235 AG), 1 loci in t RNA (m.5601CT) and 2 loci in the first high variable region (m.16179 CT and m.16291 GA). The genetic background of the large sample independent population in the verification stage was analyzed, and the distribution of the mitochondrial haploid group between the oligospermia group and the control group was not found in the two groups. Statistical differences suggested the consistency of genetic background between the two groups. In addition, the potential genetic variation site m.16179 CT located in the first hypervariable region (HVS-I) was significantly correlated with oligospermia (OR 3.10,95%CI 1.41-6.79) (P=3.10 x 10-3) by independent population. In order to further clarify the effect of mitochondrial genetic variation on semen quality, I We analyzed the two indicators of sperm density and sperm motility,.M.16179 CT and m.12361 AG, which could significantly increase the risk of oligospermia. The P value was 1.90 x 10-4 (OR 4.18; 95%CI 1.86-9.40) and 5.50 x 10-3 (OR 3.30; 95%CI1.36-8.04).M.16179 CT could significantly increase the risk of asthenospermia (3.17; 10-3). Conclusions mitochondrial DNA (10-3). NA genetic variation m.16179 CT and m.12361 AG can increase the risk of oligoasthenospermia. Third the study of mitochondrial genome copy number variation of oligoasthenospermia by comparing the level of MT DNA copy number of oligozoospermia and healthy male control sperm, the relationship between the DNA content of MT and the quality of spermatozoa is analyzed, and the copy number of MT DNA is discussed. The possible cause of the difference was used to detect the MT DNA copies of 100 cases of oligoasthenospermia and 80 normal controls by real-time quantitative PCR. Results the average MT DNA copy number of the oligospermia group was 79.02 + 10.07, which was significantly higher than the MT DNA copy number of the control population was 21.40 + 3.69 (P0.001). Conclusion MT DNA Copy number level is significantly correlated with abnormal semen quality. MT DNA copy number level can be used as an important marker of abnormal sperm quality.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R698.2

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