印迹基因在人类卵母细胞与植入前胚胎的表达及印迹疾病PWS分子诊断的研究

发布时间：2018-01-31 06:14

本文关键词： 印迹基因人类卵母细胞植入前胚胎 mRNA表达印迹疾病辅助生殖技术(ART) 单细胞巢式RT-PCR Prader-Willi综合征荧光原位杂交技术(FISH) 甲基化特异性PCR(MS-PCR)　出处：《中南大学》2007年博士论文　论文类型：学位论文

【摘要】： 基因印迹(gene imprinting))是指亲源依赖性单等位基因表达,即只有一个父源性或母源性等位基因表达,也称亲代印迹(parentalimprinting)。具有这种现象的基因被称为印迹基因(imprinted gene)。传统的孟德尔规律指出:胚胎从父亲和母亲遗传的两个拷贝,即等位基因(alleles)均有同等的机会表达,与其亲代来源无关。基因印迹不遵循经典的孟德尔遗传,是一种非遗传性基因调控方式,为一种表观遗传修饰现象,其修饰方式主要为DNA甲基化。基因印迹发生于配子形成过程中,可影响卵泡成熟,胚胎形成、发育、胎儿生长及胎盘分化,印迹中心(imprinting centre,IC)的印迹异常则导致流产、死胎、畸型及智力障碍等一系列遗传性印迹疾病,以及儿童和成人与基因印迹相关的肿瘤。目前,在卵母细胞和胚胎早期印迹基因的表达、印迹形成、辅助生殖技术(assisted reproductive technology,ART)的体外培养和显微操作等技术是否干扰了印迹基因的甲基化印迹状态等问题,已成为生殖医学及遗传学研究的热点。自首例体外受精(in vitro fertilization,IVF)婴儿1978年在英国诞生后,胞浆内单精子注射(intracytoplasmic sperm injection,ICSI)于1992年成功应用于治疗男性不育,ART做为治疗不孕的重要技术已在世界范围内广泛应用。近年来美、英、德等很多国家研究提出ART的体外培养和显微操作可能干扰了卵母细胞或早期胚胎印迹基因的甲基化印迹状态,从而导致基因印迹疾病,如:韦-伯综合征(Beckwith-Wiedemannsyndrome,BWS)和安吉尔曼综合征(Angelman syndrome,AS)等遗传性印迹疾病,Halliday等比较1316500例自然怀孕出生的孩子和14894例经ART出生的孩子的发病率,发现BWS的发病风险ART人群比正常人群高9倍,同时报道7例经ART出生的BWS患者,其中6例存在父源性印迹基因KCNQ1OT1或H19的印迹缺失。Orstavik和Cox等报道经ICSI出生的3例AS患儿,都是由于母源性表达的基因UBE3A在染色体15q11-13区域的印迹丢失而发病。在ART出生患儿中70%的BWS和100%的AS患者存在母源性等位基因甲基化异常,导致印迹丢失,而在普通患者中仅有50%-60%的PWS和5%的AS患者各存在甲基化异常。另外,人类胚胎干细胞体外培养,是否干扰了胚胎干细胞的基因印迹状态也需要进一步研究。由于人类卵母细胞和胚胎的来源及相关伦理问题的限制,且标本仅含单个细胞至数个细胞,mRNA含量极微,实验室条件要求高,技术难度大,其相关研究多以动物为主,对人类的研究,仅国外少量文献报道了IGF2、H19等数条印迹基因在卵母细胞及植入前胚胎中的表达。而与PWS相关的印迹基因SNRPN、NDN,与AS相关的印迹基因UBE3A,与BWS相关的印迹基因KvLQT、MASH2,与SRS相关的印迹基因PEG1等,在人类卵母细胞及植入前胚胎的表达情况报道极少,与SRS相关的候选印迹基因PEG10、ASB4,目前国内外尚未见报道,本研究应用单细胞巢式RT-PCR技术,对SNRPN、NDN、UBE3A、KvLQT1、MASH2、PEG1、PEG10、ASB4等与印迹疾病相关的8个印迹基因进行mRNA检测,旨在了解人类印迹基因在卵母细胞和植入前胚胎的表达情况,以对其各种异常表达情况、ART与印迹疾病的关系和印迹基因调控机制进行初步探讨,为遗传性印迹疾病的PGD开展奠定理论和实验基础。同时,本研究针对临床疑为PWS患儿,对该患儿及其父母从染色体及基因水平对15q11-13区带的印迹基因SNRPN是否微缺失、易位、单亲二倍体等进行检测,对其病因从分子水平做出精确诊断,以对其父母再次妊娠作出遗传指导,避免PWS患儿再次出生,从产前水平甚至从胚胎植入前水平阻断PWS的发生提供实验依据。第一章印迹基因在人类卵母细胞及植入前胚胎mRNA表达目的:研究印迹基因在人类卵母细胞和植入前胚胎中的mRNA表达,对其生物学意义、印迹调控机制、ART与印迹疾病的关系的探讨,并为开展胚胎植入前遗传学诊断提供理论和实验依据。方法:选择北大深圳医院辅助生殖医学中心2005年6-9月IVF、ICSI废用的GV、MⅠ、MⅡ的卵母细胞,2、4、8细胞胚胎及囊胚。或者经IVF/ICSI-ET治疗后已出生健康儿、自愿捐献多余冻存的胚胎。无遗传病家族史。经患者同意及医院医学伦理协会同意。在倒置显微镜下去除透明带,细胞裂解,cDNA制备,应用单细胞巢式PCR技术,对SNRPN、NDN、UBE3A、KvLQT1、MASH2、PEG1、PEG10、ASB4等与印迹疾病相关的8个印迹基因进行mRNA检测,并对其中SNRPN、UBE3A基因进行PCR产物纯化、连接转化、摇菌提取质粒测序验证。结果: 1.SNRPN、PEG1基因卵母细胞在GV、MⅠ、MⅡ期,胚胎在2、4、8细胞及囊胚阶段均有mRNA的表达 2.UBE3A基因自卵母细胞MⅠ期开始表达,维持表达至整个植入前胚胎 3.NDN基因表达于卵母细胞GV、MⅠ、MⅡ及胚胎8细胞与囊胚阶段 4.KvLQT1基因各期卵母细胞及植入前胚胎均无mRNA表达 5.MASH2基因仅MⅡ期卵母细胞、8细胞胚胎及囊胚中出现表达 6.PEG10、ASB4基因在MⅡ期卵母细胞、4、8细胞胚胎及囊胚中有表达结论: 1.国内首次证实与PWS相关的染色体15q11-13上父源性印迹基因SNRPN在卵母细胞GV、MⅠ、MⅡ期及植入前胚胎有表达,NDN表达于8细胞与囊胚及卵母细胞GV、MⅠ、MⅡ期,提示SNRPN、NDN促进卵母细胞成熟,早期胚胎生长发育。 2.国内首次检测了与AS密切相关的染色体15q11-13上母源性印迹基因UBE3A表达于卵母细胞MⅠ、MⅡ期及植入前胚胎各期,提示其印迹状态受ART某些环节的干扰导致AS发病率增高提供理论依据。 3.首次证实7号染色体父源表达的原癌基因PEG10表达于MⅡ期卵母细胞及4、8细胞与囊胚中,提示ART可能干扰其印迹状态,导致PEG10的活化,发生SRS。并督促我们必需长期随访ART出生人群,监测SRS及肿瘤的发生率。 4.首次证实7号染色体上新近证实的父源印迹的基因ASB4表达于MⅡ期卵母细胞及除2细胞外的植入前胚胎。 5.国内首次证实在11号染色体上母源表达、与胎盘发育有关的印迹基因MASH2在人类卵母细胞MⅡ期和8胚胎细胞阶段出现表达。 6.证实了印迹基因表达在卵母细胞和胚胎的不同阶段,具有时间的特异性,提示各个基因组的印迹抹除、重建及维持也具有时间的特异性。 7.单细胞巢式RT-PCR扩增成功率为81.0%(64/79),证实该技术准确性较高、稳定性较强,为进一步开展印迹疾病的PGD打下牢固基础。第二章印迹疾病PWS分子诊断的研究目的:旨在从染色体及基因水平对临床疑为PWS的患儿及其父母进行印迹基因SNRPN的检测,以作出PWS及其病因的确诊,为其父母再次生育提供理论依据,指导临床遗传咨询、治疗及产前诊断。方法:采用甲基化特异性PCR(methylation specific PCR,MS-PCR)技术研究染色体上15q11-13区与PWS密切相关的印迹基因SNRPN外显子alpha区上的19个CG位点的父母等位基因的甲基化状况。其作用原理基于DNA经亚硫酸氢钠修饰后,来自父方非甲基化序列的胞嘧啶(C)转变为尿嘧啶(U),而来自母方甲基化序列保持不变,随后用甲基化和非甲基化两对具有高度特异性的引物进行扩增,根据有无相应的特异产物对其病因做出诊断,并采用FISH技术从染色体水平上很直观的进一步精确确诊。结果: 1.患儿 MS-PCR结果:无父源非甲基化扩增条带,证实SNRPN基因外显子alpha区父源等位基因缺失或为母源单亲二倍体(maternal uniparentaldiplont,matUPD),确诊患儿为PWS。 FISH结果:证实为15号染色体15q11-13片段SNRPN基因的微缺失。结合MS-PCR结果,该PWS患儿病因系15号父源染色体15q11-13片段SNRPN基因的微缺失,排除matUPD(15)。 2.患儿父母与正常成人无异常。 3.PCR产物测序结果证实SNRPN基因外显子alpha区扩增的基因片断存在19个甲基化位点(-CG-)。其母源为甲基化,父源为非甲基化。结论: 1.FISH技术从染色体水平及MS-PCR技术从基因水平能对PWS等印迹疾病做出准确、快速的诊断,可作为印迹疾病确诊及产前印迹疾病筛查的常规技术。 2.MS-PCR技术结合其PCR产物测序结果,可以了解某些基因CpG岛的甲基化状况,为在植入前胚胎逆转异常甲基化位点,阻断及治疗印迹疾病带来新的思路。
[Abstract]:Gene imprinting (gene imprinting)) refers to parent of origin dependent monoallelic expression, which is only a paternal or maternal allele expression, also known as parental imprinting (parentalimprinting). This phenomenon is called gene imprinting gene (imprinted gene). It is pointed out that the traditional Mendel rule: embryos from the two copies of the father and the mother is genetic, allele (alleles) expressed the same opportunities related to the parental origin of genetic imprinting. Mendel does not follow the classic, is a kind of non genetic regulation of genes, as an epigenetic modification, the modification is mainly DNA methyl. Gene betides gametogenesis, can affect oocyte maturation, embryo formation, development, growth and differentiation of fetal placenta, imprinting Center (imprinting centre IC) is abnormal imprinting of induced abortion, fetal death, malformation and mental disorders A series of genetic imprinted diseases and tumors in children and adults associated with gene imprinting. At present, the expression in oocytes and early embryos of imprinted gene imprinting, assisted reproductive technology (assisted reproductive technology, ART) in vitro and micro manipulation technology is the problem of interference of imprinted gene methylation the imprinting status, has become a hot topic in medical genetics and reproduction.
The first case of in vitro fertilization (in vitro, fertilization, IVF) the baby was born in Britain in 1978 after intracytoplasmic sperm injection (intracytoplasmic sperm, injection, ICSI) in 1992 successfully used in the treatment of male infertility, ART as an important technique in the treatment of infertility has been in the world wide application. In recent years the United States, Britain, Germany many other countries of the in vitro culture and micromanipulation ART may interfere with the methylation imprinting status of oocytes or early embryos of imprinted genes, resulting in gene imprinting diseases, such as: Beckwith Wiedemann syndrome (Beckwith-Wiedemannsyndrome, BWS) and Angelman syndrome (Angelman syndrome, AS) and other genetic imprinted diseases, Halliday comparison of 1316500 cases of pregnant naturally born children and 14894 cases of ART children born in the incidence of BWS, found that the risk of ART than those of normal group was 9 times higher, while 7 cases reported by ART The birth of the BWS patients, including 6 cases of loss of imprinting of.Orstavik exist paternally imprinted gene KCNQ1OT1 or H19 and Cox reported the 3 cases of children with AS ICSI was born, are due to maternal expression of UBE3A gene in chromosome 15q11-13 loss of imprinting and disease. Born in ART were 70% BWS and 100% the AS patients have maternal allele methylation, resulting in loss of imprinting, and in common with only 50%-60% PWS and 5% AS patients with abnormal methylation. In addition, in vitro culture of human embryonic stem cells, embryonic stem cells are interfering with gene imprinting status also need further research.
As the source of human oocytes and embryos and related ethical issues, and the specimens containing only single cell or a plurality of cells, the mRNA content is minimal, laboratory conditions, technical difficulty, the related research on animal based research on humans, only a small amount of foreign literature reported that IGF2, H19 etc. a number of imprinted gene expression in embryonic development of oocytes and preimplantation. Imprinted genes SNRPN, PWS and NDN related imprinted genes UBE3A and AS related to the imprinted gene KvLQT associated with BWS, MASH2, and SRS related imprinted genes PEG1, in human oocytes and preimplantation embryos the expression is rarely reported, candidate imprinted genes PEG10, SRS and related ASB4, has not been reported so far, the research and application of single cell nested RT-PCR technique, SNRPN, NDN, UBE3A, KvLQT1, MASH2, PEG1, PEG10, ASB4 and other 8 related imprinting and imprinting diseases The gene was detected by mRNA, in order to understand the expression of imprinted genes in human oocytes and preimplantation embryos, with the abnormal expression of imprinted genes, and the regulatory mechanism of ART and imprinting disease was discussed, lay a theoretical and experimental basis for genetic imprinted diseases PGD development. At the same time, this study aimed at clinical suspected PWS children of the imprinted gene SNRPN in children and their parents with the area of 15q11-13 from the chromosome and gene level whether the deletion, translocation, uniparental disomy was detected on the etiology and make accurate diagnosis from the molecular level, genetic guidance to pregnancy on their parents to avoid PWS children from birth again. The level of prenatal even from preimplantation level block to provide experimental basis for the occurrence of PWS.
Chapter 1 the expression of imprinted gene in human oocyte and preimplantation embryo mRNA
Objective: To study the expression of mRNA in human oocytes and preimplantation embryos, and to explore its biological significance, imprinting regulation mechanism, and the relationship between ART and imprinted diseases, and to provide theoretical and experimental evidence for preimplantation genetic diagnosis.
Methods: North Shenzhen hospital assisted reproductive medical center in 2005 6-9 months IVF, ICSI waste with the GV, M I and M II oocytes, 2,4,8 cell embryos and blastocyst. Or after the treatment of IVF/ICSI-ET have been born healthy, voluntary donation of extra frozen embryos. No family history of genetic disease. With the consent of the medical patient consent and hospital ethics Association. Under inverted microscope, zona free, cell lysis, cDNA preparation, application of single cell nested PCR technique, SNRPN, NDN, UBE3A, KvLQT1, MASH2, PEG1, PEG10, mRNA, ASB4 were detected in 8 imprinted genes associated with imprinting diseases, and the SNRPN. UBE3A gene PCR product purification, ligation and transformation, shaked bcteria andextracted plasmids sequencing.
Result:
1.SNRPN, PEG1 gene oocyte in GV, M I, M II stage, and the expression of mRNA in 2,4,8 and blastocyst stages
The expression of 2.UBE3A gene from the M I period of oocyte to the whole preimplantation embryo
The expression of 3.NDN gene in oocyte GV, M I, M II, and embryo 8 cells and blastocyst stage
No mRNA expression was found in all stages of oocyte and preimplantation embryo of 4.KvLQT1 gene
5.MASH2 gene is only M II oocyte, and the expression of 8 cell embryos and blastocysts
The expression of 6.PEG10, ASB4 gene in M II oocytes, 4,8 cell embryos and blastocysts
Conclusion:
1. domestic for the first time that chromosome 15q11-13 associated with PWS on the paternal imprinted gene SNRPN in oocytes of GV M, M I, II and preimplantation embryos expressed NDN expression in 8 cells and blastocysts and oocytes of GV M, M I, II, SNRPN, NDN promote oocyte mature, early embryo growth and development.
2., for the first time in China, the maternal imprinting gene UBE3A expressed on chromosome 15q11-13 closely related to AS was expressed in oocyte M I, M II and preimplantation embryos, suggesting that its imprinting status is influenced by some links of ART, which can provide a theoretical basis for the incidence of AS.
For the first time in 3. confirmed the expression of chromosome 7 paternal proto oncogene PEG10 expression in M II oocytes and 4,8 cells and blastocysts, suggesting that ART may interfere with the imprinting status, leads to the activation of PEG10, SRS. and ART must urge us to long-term follow-up of birth population, the incidence of SRS and tumor monitoring.
4. it was first confirmed that the newly confirmed parent imprinted gene ASB4 on chromosome 7 was expressed in M II oocytes and the preimplantation embryos except 2 cells.
5., for the first time in China, the expression of MASH2, a marker related to placenta development, was first identified on chromosome 11, and it was expressed in human M phase II and 8 embryo cell stage.
6., it is confirmed that the expression of imprinted genes in different stages of oocytes and embryos has time specificity, suggesting that the genome blotting, reconstruction and maintenance are time specific.
7. the successful rate of single cell nested RT-PCR was 81% (64/79), which confirmed the accuracy and stability of the technology, and laid a solid foundation for further developing PGD of imprinted diseases.
The second chapter of the study of PWS molecular diagnosis of imprinted disease
Objective: to detect the imprinted gene SNRPN in children and their parents suspected of PWS by chromosome and gene level, so as to make the diagnosis of PWS and its causes, provide theoretical basis for the re birth of their parents, and guide the clinical genetic counseling, treatment and prenatal diagnosis.
Methods: methylation specific PCR (methylation specific PCR, MS-PCR). The methylation status of 19 CG loci in alpha region on the parental alleles of imprinted gene SNRPN on chromosome 15q11-13 region and the technology of PWS is closely related to the outside. Its function is based on the principle of DNA modified by Sodium Bisulfite, from the father of non methylation of cytosine into uracil (C) (U), but the maternal methylation sequence remains unchanged, then amplified by methylation and non methylation of two highly specific primers according to the specific product is there to make a diagnosis of the etiology, diagnosis and further refined by FISH from the technical level of chromosomes is intuitive.
Result:
1. children
MS-PCR results: there was no parental non methylation amplification band, which confirmed that the SNRPN gene exon alpha loci were deleted or the parental maternal alleles were maternal uniparentaldiplont (matUPD). The diagnosis was PWS..
FISH results: the deletion of SNRPN gene was found in chromosome 15q11-13 of chromosome 15. Combined with MS-PCR results, the etiology of PWS was microdeletion of SNRPN gene of paternal chromosome 15q11-13 15, and matUPD (15) was excluded.
2. parents had no abnormality with normal adults.
3.PCR products sequencing confirmed that there were 19 methylation sites (-CG-) in the SNRPN gene exon alpha fragment, and the parent was methylation and the parent was non methylated.
Conclusion:
1.FISH technology can accurately and quickly diagnose the imprinted diseases such as PWS from chromosome level and MS-PCR technology. It can be used as a routine technology for the diagnosis of imprinted diseases and the screening of prenatal imprinted diseases.
2.MS-PCR technology combined with the sequencing results of PCR products, we can understand the methylation status of CpG islands of some genes, and provide new ideas for reversing aberrant methylation sites, blocking and treating imprinted diseases in preimplantation embryos.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R714.8;R394

【共引文献】