人类线粒体基因组遗传变异在精子生成中的作用及其机制

发布时间：2018-04-22 17:39

本文选题：非梗阻性无精症 + 线粒体DNA　；参考：《南京医科大学》2015年博士论文

【摘要】：不孕不育是生殖健康领域一项亟需解决的重大科学问题。育龄夫妇中约10-15%存在不同程度的生育障碍。导致不孕不育的原因很多,其中男方因素约占50%。前人研究表明,在过去的半个世纪里,男性精液质量显著下降,精子生成障碍已成为男性不育最常见的病因之一。精子生成障碍可能与环境化学污染物、遗传因子改变、表观遗传修饰异常等密切相关。大约30%的精子生成障碍患者是由于遗传学的异常引起的。线粒体作为细胞核外唯一含有遗传物质的细胞器,其参与的氧化磷酸化过程为精子生成、分化及活力维持提供所必需的能量。而且线粒体参与多种生物学过程,在ROS(Reactive Oxygen Species)平衡、细胞凋亡以及多种信号通路调节中都具有重要作用。线粒体基因组(Mitochondrial DNA,mt DNA)编码氧化磷酸化体系的蛋白亚基和其自身的RNA翻译元件,mt DNA变异会导致线粒体功能改变,进而引起精子生成障碍,导致男性不育。目前关于mt DNA遗传变异对精子生成或精液质量的影响已有相关报导。然而由于受到mt DNA变异检测技术、样本量大小以及种群差异等多种因素的限制,线粒体基因组遗传变异对精子生成的影响也不尽相同。为此,我们提出以下研究假设:(1)人类线粒体基因组在精子生成过程中发挥重要作用,其遗传变异可导致精子生成障碍;(2)在精子生成障碍人群中,一些特定的线粒体DNA单倍群会产生富集(3)氧化应激损伤或精子分化异常,导致线粒体基因组含量代偿性增加。为了全面、系统地评估人类mt DNA遗传变异在精子生成中的作用,本项目拟以前期提出的“胞质遗传”理论为基础,以大样本人群关联研究为切入点,应用分子生物学等研究手段(高通量测序、中通量基因分型等),结合线粒体DNA单倍群分析,探讨是否存在与精子生成障碍相关的功能性遗传变异;特定线粒体DNA单倍群是否会增加精子生成障碍的风险以及线粒体DNA拷贝数变异是否与精液质量异常相关,明确人类线粒体基因组遗传变异在精子生成中的作用,揭示可能的分子机制,为男性不育的分子诊断和治疗提供新的途径和方法。第一部分人类线粒体基因组遗传变异在非梗阻性无精症(NOA)发生中的作用目的为了阐明人类线粒体基因组在非梗阻性无精症(NOA)中的作用,我们对NOA不育男性与健康生育对照的线粒体全基因组进行测序,筛选与精子生成障碍相关的线粒体DNA遗传变异,并在大样本人群中进一步确认。方法采用两阶段病例对照研究。第一阶段,应用高通量测序技术对96例无精症病例和96例对照进行mt DNA全测序,鉴定mt DNA单倍群,筛选出常见mt DNA单倍群以及与精子生成障碍相关的mt DNA遗传变异。第二阶段,针对536例无精症病例和489例对照,利用Sna Pshot测序技术,基于13个东亚地区常见单倍群的编码区特征突变点分型结果,分析人群遗传背景。随后,利用SNPscan测序技术,对测序阶段筛选出的遗传变异进行验证。为了进一步阐明线粒体遗传变异导致的线粒体损伤的机制,我们应用总抗氧化能力(total antioxidant capability,T-AOC)和超氧化物歧化酶(superoxide dismutase,SOD)检测试剂盒检测并比较病例和对照以及不同单倍群之间精浆抗氧化能力。结果根据第一阶段测序所得的线粒体DNA全序列,鉴定每个个体mt DNA单倍群分型,结合mt DNA系统发生树以及东亚地区常见单倍群,筛选了13个东亚地区常见单倍群及其定义位点,对比了病例和对照两组间的遗传背景。同时筛选了10个线粒体DNA潜在的功能性遗传变异位点,包括6个编码区的可能导致非同义突变的遗传变异(m.3394TC,m.6881AG,m.8684CT,m.11696GA,m.12358AG,m.13135GA),1个遗传变异位于t RNA基因上(m.15968TC),另外3个遗传变异位于第一高变区(HVRI)(m.16224TC,m.16319GA,m.16497AG)。对第二阶段大样本独立人群的遗传背景进行分析,结果提示单倍群M8*在病例组的比例显著高于对照组(OR 2.61,95%CI 1.47-4.61)(P=6.76×10-4),提示单倍群M8*会增加精子生成障碍的风险。此外,在独立人群中验证潜在遗传变异位点,结果发现m.8684CT在病例组中显著高发(OR 4.14,95%CI 1.56-11.03)(P=2.09×10-3),提示m.8684CT同样会增加精子生成障碍的风险。同时由于m.8684CT是单倍群M8a的遗传标记位点,因此,我们进一步提出假设,遗传背景单倍群M8a导致了遗传变异m.8684CT在无精症人群中富集。为了验证上述假设,我们进一步分析单倍群M8*的2个亚单倍群,M8a和Z,比较单倍群M8a和单倍群Z在病例和对照之间的分布,发现单倍群M8a在病例组中的比例显著高于对照组(OR 4.14,95%CI 1.56-11.03)(P=2.09×10-3),而单倍群Z在两组间的分布并无显著统计学差异(OR 1.86,95%CI 0.92-3.77)(P=7.88×10-2)。结果提示,遗传背景单倍群M8a导致了遗传变异m.8684CT在无精症人群中富集,增加了其导致精子生成障碍的风险。为进一步研究线粒体遗传变异致线粒体损伤的水平,我们对精浆抗氧化能力进行检测。与对照组相比,病例组T-AOC显著下降(P0.05),提示病例组线粒体功能受损,而SOD活性没有差异。在不同单倍群之间,由于样本量的限制,T-AOC和SOD并没有发现明显差异。结论遗传背景单倍群M8a导致了遗传变异m.8684CT在无精症人群中富集,增加了其导致精子生成障碍的风险。同时,mt DNA遗传变异可导致精浆抗氧化能力下降,提示线粒体损伤,从而能够引起精子生成障碍。第二部分人类线粒体基因组遗传变异在少弱精症发生中的作用目的为了全面研究线粒体基因组与少弱精症病因学之间的关联,通过深度测序线粒体全基因组,筛选与精液质量下降相关的线粒体DNA遗传变异,并在独立人群中进行验证。方法采用两阶段病例对照研究。筛选阶段,采用下一代测序技术对233例少弱精症病例和233例对照进行mt DNA全测序。鉴定样本单倍群并分析主要单倍群分布。随后,筛选与少弱精症相关的线粒体基因组遗传变异位点。验证阶段,针对688例少弱精症患者及533例对照,利用SNa Pshot测序方法,基于13个东亚地区常见的单倍群定义位点分型结果,分析mt DNA单倍群遗传背景。利用SNPscan测序技术,对测序阶段筛选的遗传变异进行验证。结果根据筛选阶段测序所得的线粒体DNA全序列,鉴定样本单倍群并整合入13个东亚地区常见单倍群,分析少弱精症和对照两组的单倍群分布,结果符合东亚特有mt DNA单倍群的频率分布。同时筛选出7个可能增加精液质量下降的风险的遗传变异位点。其中,4个位点位于编码区(m.12338TC,m.12361AG,m.13928GC和m.A15235 AG),1个位点位于t RNA(m.5601CT),2个位点位于第一高变区(m.16179 CT和m.16291 GA)。对验证阶段大样本独立人群的遗传背景进行分析,发现少弱精症组和对照组两组间线粒体单倍群分布没有统计学差异,提示两组间遗传背景的一致性。此外,通过独立人群的验证发现,位于第一高变区(HVS-I)的潜在遗传变异位点m.16179 CT与少弱精症显著相关(OR 3.10,95%CI 1.41-6.79)(P=3.10×10-3)。为了进一步阐明线粒体遗传变异对精液质量的影响,我们分析了精子密度和精子活力两个指标。m.16179 CT和m.12361 AG能显著增加少精症的风险,P值分别为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同时能显著增加弱精症的风险(OR 3.17;95%CI1.40-7.16)(P=3.50×10-3)。结论线粒体DNA遗传变异m.16179 CT和m.12361 AG可以增加少弱精症的发病风险。第三部分少弱精症的线粒体基因组拷贝数变异研究目的通过比较少弱精症与健康生育男性对照精子mt DNA拷贝数水平,分析mt DNA含量与精液质量的关系,探讨导致mt DNA拷贝数差异的可能原因方法利用实时荧光定量PCR技术,采用相对定量的方法,检测100例少弱精症病例和80例正常对照的精子mt DNA拷贝数。结果少弱精症组的平均mt DNA拷贝数79.02±10.07,显著高于对照人群的mt DNA拷贝数为21.40±3.69(P0.001)。结论mt DNA拷贝数水平与精液质量异常显著相关,mt DNA拷贝数水平可以作为精液质量异常的重要标志物。
[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.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698.2

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