【作者】 熊夏思；

【导师】 陈士岭；

【作者基本信息】 南方医科大学 ， 妇产科， 2008， 硕士

【摘要】 研究背景和目的辅助生殖技术通过近几十年的发展,已经成为治疗各种原因不孕的重要手段。全世界已有三百多万儿童是经辅助生殖技术出生的,在发达国家已占所有出生人口比重的1-3%。对于辅助生殖技术安全性的问题成为了研究的焦点和热点。辅助生殖技术操作一般认为是相对安全的,但近来的研究表明经辅助生殖技术增加了出生缺陷、印记紊乱和儿童期肿瘤的发病风险。有几个小规模的临床实验报道了经辅助生殖技术出生儿普-威综合征(Prader- Willi syndrome,PWS)和安吉尔曼综合征(Angelman syndrome,AS)的发病风险增高。尽管经辅助生殖技术出生儿发生印记基因疾病的绝对风险很小,但对动物的研究发现经体外培养的胚胎印记基因的表达异常,表观遗传学发生了改变,导致印记表达的紊乱。因此有必要通过对辅助生殖技术出生儿和ART相关标本的实验研究来调查辅助生殖技术的安全性。普-威综合征(PWS)和安吉尔曼综合征(AS)是两种先天性的神经行为发育异常综合症,也是基因印记最为典型的例子。两者具有不同的临床症状和遗传形式,但致病因素都与15号染色体长臂11-13区(15q11-13)印记基因聚集区有关。PWS主要表现为肥胖、身材矮小、肌张力减退和轻度智力发育迟缓。AS表现为共济失调、重度精神发育障碍、少语、表情愉悦。约70%的PWS患者的致病原因为染色体15q11-13缺失,且缺失主要发生在父源染色体。25%患者的染色体核型可见母源单亲二体型(uniparental disomy UPD)和约5%的患者发生了印记突变(imprinting mutation)。与PWS不同,70-80%的AS患者为母源染色体缺失,父源二体型占2%,另外在无染色体缺失的AS患者中约20%存在此区母源表达的UBE3A的截断突变。UBE3A主要在脑组织中表达,编码一种泛素蛋白酶,参与蛋白的更新过程,脑形成过程中UBE3A表达异常与AS有关。由此可见,PWS是由于15q11-13父源基因表达欠缺所引起,而AS为母源基因表达缺陷所致,两者均与基因组印记的调节有重要关系。15q11-13区域存在多个印记基因(如SNRPN、NDN、ZNF127等),其中父源表达的SNRPN(smallnuclear riboprotein-associated polypeptide N)在PWS中起到了关键性作用。SNR-PN编码一核蛋白相关的小核蛋白N,在调节mRNA拼接过程中起重要作用。无论是某一亲本的缺失还是单亲二体其结果均是失去了某一亲本上的活性等位基因而造成PWS及AS,说明父源与母源的15q11-q13功能不同。这两类综合征明显有印记的参与,但至今人们对这些基因在疾病中的具体作用还不够清楚,这些印记基因与印记相关的疾病发病机制之间的关系还需进一步研究。辅助生殖技术与印记基因疾病之间不寻常的相关性是一个尚未解开的迷团。辅助生殖技术将人类精卵受精和胚胎早期育过程置于实验室条件下进行,为受精和胚胎早期发育的表观遗传学研究提供了最佳模型。表观遗传学(epigenetics)是主要不涉及DNA序列的情况下改变基因组的修饰,如DNA甲基化、基因组印记、组蛋白修饰、染色体重塑和非编码RNA。这种修饰不仅可以影响个体的发育,而且还可以在细胞分裂过程中遗传下去。表观遗传学是阐述基因组功能及基因表达的关键研究领域之一。基因印记是其中一个重要的组成部分。基因组印记(genomic imprinting)又称亲本印记(parentalimprinting)是20世纪80年代提出的一种非孟德尔遗传方式。胎盘类哺乳动物继承父源和母源的两套染色体组,其中大多数基因进行双等位基因表达,但有一部分却只由特定的来源于某一亲本的单一等位基因进行表达,约占基因总量的0.1%,这种表观遗传修饰的现象称为基因印记。其中父(母)源等位基因不表达者,称父(母)源印记。印记基因所产生的效应在胚胎和胎盘的发育过程起着重要的作用,特别是对胎盘发育极为重要,其正确表达与胚胎、胎盘和行为的正常发育相关。印记会影响某些遗传疾病与某些肿瘤的发生,及其发病的年龄、外显率、表现度。基因组印记功能的紊乱将引起多种发育异常、死胎和儿童肿瘤。与印记相关疾病在染色体层次上可见到单亲二体染色体、杂交后丢失及基因印记丢失现象。由此可见,基因组印记是一种全新的遗传机制,此种非孟德尔遗传现象的发生,揭示人类胚胎发育的更深层次的机制。DNA甲基化是基因表观遗传学的重要机制之一,目前已知与人类的胚胎发育分化和肿瘤细胞的发生发展有关。它是一种DNA复制后的酶促反应过程。DNA复制以后,在DNA甲基化酶作用下,将S-腺苷酰甲硫氨酸(SAM)分子上的甲基转移到DNA子中胞嘧啶残基的5位碳原子上。甲基的引入可使DNA分子空间结构改变,影响了DNA与蛋白质分子的结合,从而导致某些基因转录失活,表达受到抑制,甚至丧失功能。CpG双核苷酸中胞嘧啶5位碳原子是甲基化的靶位点。甲基化特异性聚合酶链反应(methylation-specific PCR,MS-PCR)是基因甲基化分析在速度和敏感性上的突破。其原理是将DNA先用重亚硫酸盐处理,使未甲基化的胞嘧啶转变为尿嘧啶,而甲基化的不变,基于这种差异性,设计两对特异性的引物:一对甲基化特异性引物和一对非甲基化特异性引物,就可将甲基化等位基因化学修饰的DNA序列与非甲基化等位基因区分。在正常情况下SNRPN基因的CpG岛在父源性染色体不被甲基化,而在母源性染色体则发生甲基化。针对SNRPN基因启动子区域的MS-PCR,在检测正常人基因组DNA时既可得以父源性SNRPN未被甲基化位点为模板的特异性PCR产物,又可得以母源性SNRPN甲基化位点为模板的特异性PCR产物,帮助判断SNRPN基因是否存在的甲基化位点异常。研究内容本文采用MS-PCR来检测与ART相关的标本如经ART妊娠早孕绒毛和早期自然流产绒毛、ART出生儿的脐血基因组DNA的PWS/AS印记调控区SNRPN甲基化状况,并与自然妊娠绒毛和出生儿脐血进行对照,借此发现辅助生殖技术与印记基因疾病PWS/AS之间可能存在的关联性,探讨辅助生殖技术对表观遗传学的影响及印记基因疾病之间可能存在的关系,为进一步验证ART技术的安全性奠定初步的理论基础。材料与方法对象在南方医院2007年1月份至12月份期间就诊患者中收集,经ART妊娠早孕绒毛(10例)和自然流产绒毛(6例);自然妊娠早孕绒毛(10例)和自然流产绒毛(6例)及经体外受精-胚胎移植技术出生儿脐血标本(12例)和正常自然妊娠新生儿脐血标本(10例)。本文通过采用甲基化特异性聚合酶链反应(MSP)来检测上述绒毛和脐血基因组DNA中的印记调控区SNRPN基因的甲基化表达状况。主要结果1.10例经ART妊娠的早孕绒毛基因组DNA经MS-PCR后均出现母系甲基化174bp阳性产物和父系非甲基化100bp阳性产物条带。2.10例自然妊娠早孕绒毛基因组DNA,经MS-PCR电泳后均出现母系甲基化174bp阳性产物和父系非甲基化100bp阳性产物条带。3.经ART妊娠自然流产绒毛基因组DNA行MSP电泳后4例仅出现母系甲基化174bp阳性产物条带,发生父系非甲基化产物的丢失;自然妊娠自然流产的绒毛经MSP电泳后3例仅出现父系非甲基化100bp阳性产物条带,发生了母系甲基化产物的丢失。4.12例经ART技术出生儿脐血基因组DNA经MSP电泳后均出现母系甲基化174bp阳性产物和父系非甲基化100bp阳性产物。主要结论1.本文在国内首次使用甲基化特异性PCR来检测早孕绒毛和自然流产绒毛PWS/AS印记调控区15q11-13的SNRPN基因甲基化状态。2.经辅助生殖技术出生儿脐血和早孕绒毛基因组DNA的PWS/AS印记调控区SNRPN基因甲基化状态与自然妊娠相同;未发现辅助生殖技术有增加印记基因疾病的发病风险。3.自然流产绒毛不管是经辅助生殖技术妊娠还是自然妊娠,其基因组DNA的PWS/AS印记调控区SNRPN基因甲基化表达的异常,提示表观遗传学方面的异常可能是导致自然流产的一个重要因素。4.经辅助生殖技术妊娠后发生自然流产的部分绒毛标本基因组DNA的PWS/AS印记调控区SNRPN基因发生了父系非甲基化产物表达的丢失,可能与遗传、表遗传、内分泌、感染、免疫和辅助生殖技术的各个操作环节相关,但无明显的证据表明是由ART相关操作直接导致的。更多还原

【Abstract】 Background and objectiveIn the past decades,assisted reproductive technology(ART) have developed rapidly and are important treatment for infertile couples.There are more than three millions of children conceived by ART,which now accout for 1-3%of the total annual births in developed countries.So people take more concerns on the safty of ART.ART have been generally considered to be relatively safe medical treatments.But rencent studies have suggested that there may be links between ART and an increased risk of birth defects,imprinting disorders and childhood cancer.Several clinical studies have reported an increased frequency of ART conceptions among children with Prader-Willi syndrome(PWS) and Angelman syndrome(AS).The absolute risk of an imprinting disorder after ART appears to be very small,but animal study have demonstated that in vitro embryo culture is associated with disordered genomic imprinting and alterations in epigenetics.So it is important to do some researches investigating the safty of ART.PWS and AS are two congenital neurobehavioural disturbances syndrome.They are the most typical model of genomic imprinting and have different clinical characters and genetic patterns.A common region involved in these syndromes is the 15q11-13.PWS is characterized by obesity,short stature,muscular hypotonia and mild mental retardation and AS is characterized by poor balance accompanied by jerky movements,severe mental retardation,absence of speech and happy disposition.PWS is caused by a deficiency of chromosome 15q11-13:mainly a paternal deletion(70%),a maternal disomy(25%) or an imprinting mutation(5%).In contrast,AS genotype is charaterised by a maternal deletion(70-80%),a paternal disomy(2%) or an imprinting mutation of UBE3A(20%) in AS gene located on the same chromosomal regions.UBE3A is mainly expressed in brain and encodes a ubiquitin-protease which involved in renew process of protein.Abonormal expression of UBE3A in encephalization is associated with AS.PWS is caused by deletion of paternal 15q11-13 and AS is caused by deletion of maternal 15q11-13,they have a close relationship with regulation of genomic imprinting.There are several imprinting genes on chromosomal 15q11-13,such as SNRPN(small nuclear riboproteinassociated polypeptide N),NDN and ZNF127.Paternal expressed SNRPN takes an important role in PWS and AS.SNRPN gene encodes a polypeptide of a small nuclear ribonucleoprotein N which is indispensable for pre-mRNA processing.Either any parental deletion or UPD lost one of the active parental allele resulting in PWS or AS,which indicates maternal and paternal 15q11-13 has different functions.Although imprinting is involved in these syndromes,the specific contributions of these genes in imprinting defects were unclear.The mechanisms between imprinting gene and imprinting disorders need to be further investigated.The unusual relationship of ART and impinting disorders is poorly understanded.ART put fertilization and early embryo culture in laboratory condition which provide the best model for researching epigenetics of fertilization and early embryo development.Epigenetics is a genomic modification that is not involved in DNA sequence such as DNA methylation,genomic imprinting,histone modification,chromatin remodeling and noncoding RNA.This modifications not only affect the individual development,but also are heritable during cell division.Epigentics is one of the key researches in genomic functions and gene expressions,genomic imprinting is an important part of it.Genomic imprinting or parental imprinting is a non-Mendelian inheritance which was described in 80s twentieth century.Mammals inherit paternal and maternal chromosomes,most of genes express two parental alleles,one is from paternal,another is from maternal.Only a small number of genes express predominantly from one parental allele,accouting for about 0.1%in the mammalian genomes.This epigentics modification phenomenon is called genomic imprinting. Paternal(maternal) imprinting means paternal(maternal) allele is silenced.Many imprinted genes are essential for regulating embryonic and placenta growth,especially for the placenta growth.Correct expression of imprinting genes is associated with normal development of embryo,placenta and behaviors.Disordered imprinting has been implicated in the pathogenesis of genetic diseases and cancer.Disregulation of the expression of genomic imprinting gives rise to a variety of development abnormalities,stillbirths and children tumor.A uniparental disomy, a deletion after hybridisation,a point mutation or an loss of imprinting can be seen on chromosome levels of imprinting associated diseases.It is so clear that genomic imprinting is a new non-Mendelian heredity elucidating the profoud mechanisms underlying human embryonic development.DNA methylation is a crucial epigenetic modification of genome that is involved in regulating differentiation of embryonic development and tumorigenesis.DNA methylation is a enzymatic reaction after DNA duplication which transfer methylium of S-adenosylmethionine(SAM) to cytosine at position C~5 by DNA methyltransferases.The tranfered methylium result in changes of spatial structure what affect DNA binding to protein.This will result in the inactivation of gene transcriptions and silence of gene expression,even lose gene functions.Cytosine at position C~5 in CpG dinucleotides are target sites of methylation.Methylation-specific polymerase chain reaction is a breakthrough in methylation analysis.Its principle is as below:genomic DNA is first subjected to a bisulfite treatment,which serves to deaminate unmethylated cytosines,thus converting them to uracils,whereas methylated cytosines are not converted.Two different PCR are then performed per individual using primers specific for either the unmethylated or the methylated alleles(allele-specific PCR).In this way,we can distinguish methylated alleles from unmethylatd alleles.In normal situations, the human SNRPN gene is expressed from the unmethylated paternal allele,while the silenced maternal copy is methylated.In normal human genomic DNA,We can get PCR specific product of methylated maternal and unmethylated paternal alleles through MS-PCR which aim at SNRPN gene promoter region.We use MS-PCR detecting methylation status on SNRPN imprinting control region in samples which are related to assisted reproductive technologies like early pregnancy villus gotten from mutiplefetal reduction and spontaneous abortion,cord blood of ART births,using nature early pregnancy villus and cord blood of nature birth as control.We want to investigate the correlation between assisted reproductive technology and imprinting diseases to settle preliminary theory foundation in researching the safty of ART.Materials and methodswe have collected early pregnancy villus gotten from mutiplefetal reduction (10 samples) and spontaneous abortion of ART(6 samples),early pregnancy villus(10 samples) and spontaneous abortion villus(6 smaples) of spontaneous pregnancy,cord blood of ART births(12 samples) and spontaneous pregnancy births(10 samples).These collections were finished from January to December 2007 in Nanfang Hospital.We have detected methylation status of SNRPN at imprinting control region in above-mentioned collections.Main results1.MS-PCR on genomic DNA from early pregnancy villus gotten from mutiplefetal pregnancy reduction of ART(10 samples) showed both a 174bp product from the methylated maternal chromosome and a 100bp product from the unmethylated paternal chromosome.2.MS-PCR on genomic DNA from early pregnancy villus of spontaneous pregnancy(10 samples) showed both a 174bp product from the methylated maternal chromosome and a 100bp product from the unmethylated paternal chromosome.3.Four spontaneous abortion villus which got pregnancy by ART only shows a 174bp product from the methylated maternal chromosome;three spontaneous abortion villus of spontaneous pregnancy only shows a 100bp product from the unmethylated paternal chromosome.4.MS-PCR on genomic DNA from cord blood of ART births(12 samples) showed both a 174bp product from the methylated maternal chromosome and a 100bp product from the unmethylated paternal chromosome.Conclusion1.We first use MS-PCR to detect methylation status of SNRPN at imprinting center 15q11-13 in early pregnancy villus and spontaneous abortion villus.2.The MS-PCR of cord blood and mutiplefetal pregnancy reduction villus after ART revealed a methylation status at chromosome 15q11-13 identical to the methylation pattern in the normal control;the results of the present study do not indicate a higher risk of imprinting disorders related to assisted reproductive technologies. 3.The abnormal status of SNRPN in spontaneous abortion villus indicated the abnormality of epigenetics might play an important role in spontaneous abortion.4.Spontaneous abortion villus by ART miss a unmethylated paternal product on imprinting center of PWS/AS,which can be cause by genetics,epigenetics, endocrine,infection,immunization or ART procedures.But there is no evidence to demonstate a direct link between ART and imprinting disorders.更多还原