【作者】 张磊；

【导师】 陈子江；

【作者基本信息】 山东大学 ， 妇产科学， 2019， 博士

【摘要】 第一章全基因组测序在复发性流产人群中对染色体异常病因的扩大诊断复发性流产(recurrent pregnancy loss,RPL)是常见的产科并发症,约影响1%的育龄女性。RPL病因复杂,遗传因素、抗磷脂抗体综合征和子宫解剖结构异常是明确的致病原因,感染、自身免疫因素、内分泌或代谢异常比如糖尿病或甲状腺疾病等病因虽与流产相关,但与RPL的关系不甚明确。排除这些因素,约50%的RPL病人经检测无异常,称为不明原因复发性流产(unexplained RPL,uRPL)。因uRPL人群病因不明确,只能提供经验治疗,但无法获得满意的治疗效果。外周血G显带是染色体核型分析的金标准,约5%的RPL夫妇中至少一方的染色体为异常,常染色体相互易位是最常见的染色体异常,其次为罗伯逊易位和倒位。染色体异常的RPL人群可以通过植入前遗传学检测筛选正常/平衡胚胎,以获得活产结局。但是目前G显带分辨率为320400条带,存在分辨率较低的缺点,若能够在RPL人群中增加诊断精度,进一步获得明确病因的诊断,则RPL病人能够直接临床受益。有文章显示,全基因组测序(whole-genome sequencing,WGS)有分辨率高的特点,能够额外检测出常规显带染色体分析未发现的染色体异常。目的:应用一项经过验证的低深度WGS,对uRPL病人的外周血DNA进行测序分析,以期检测出常规G显带核型分析未发现的染色体异常,进一步扩大诊断RPL人群中染色体异常的发生率。方法:共纳入1024对uRPL夫妇应用WGS进行外周血DNA测序,纳入标准为:发生2次或以上孕早期临床妊娠自然流产,夫妇双方外周血常规G显带无异常,女方无解剖结构异常,无抗磷脂抗体综合征或自身抗体阳性,无内分泌异常或代谢性疾病。测序发现的平衡易位或倒位,应用聚合酶链式反应(polymerase chain reaction,PCR)和Sanger测序验证断裂位点,同时收集病人的新鲜外周血进行高分辨显带和荧光原位杂交(fluorescence in-situ hybridization,FISH)确认衍生染色体;对测序发现的拷贝数变异(copy number variants,CNVs)应用染色体微阵列分析芯片(chromosomal microarray analysis,CMA)进行验证。结果:1024对夫妇的2048份外周血DNA样本,13份没有通过质量控制,因此1011对(98.7%)uRPL夫妇纳入最终WGS结果分析。测序结果显示,48对(4.75%)夫妇中发现了额外的49例染色体重排,包括22例平衡易位和27例倒位。在22例平衡易位中,14例在初始显带核型或高分辨显带中得到确认,8例易位因为交换片段隐匿或衍生染色体与正常染色体带型类似而不能在显带核型中得到确认;27例倒位中,4例在显带核型中得到确认,剩余23例因片段隐匿或倒位片段低于显带分辨率而不能通过G显带检测。WGS报告的染色体重排均通过PCR和Sanger测序得到验证,部分异常通过FISH进一步确认。另外,WGS检测出1例致病CNV和5例可能致病CNVs,均通过CMA技术得到验证。结论:WGS能够检测出常规G显带核型分析无法检测出的染色体异常,推荐将WGS应用于RPL病人病因学筛查中,以提高染色体异常病因检出率,为后续治疗提供临床指导。第二章 染色体易位对减数分裂模式的影响第一节 易位涉及近端着丝粒染色体与携带者性别相互作用影响常染色体相互易位的减数分裂模式相互易位是一种常见的染色体结构异常,指的是两条非同源染色体分别发生一次断裂,断裂片段相互交换后重接,形成两条衍生染色体,在普通人群中的发生率为0.74-1.25例每1000新生儿,在复发性流产人群中发生率约为2.4%。在配子形成过程中,减数分裂I的前期,两条衍生染色体和相应的两条正常染色体形成四射体,在I的后期,四射体有对位、邻位-1、邻位-2、3:1、4:0这几类分离模式,其中只有对位分离模式才能产生染色体正常或平衡的配子,其他分离模式产生的配子均含有不平衡染色体结构异常。由于这些染色体不平衡配子的产生,相互易位携带者会面临一系列生育相关问题,包括不孕,流产,新生儿先天畸形等。已有研究表明,四射体的分离模式受到一些比如携带者性别、年龄和涉及染色体类型等因素的影响,但是在所有的报道中,受到明显影响的是邻位-1、邻位-2、3:0这些产生染色体不平衡配子的分离模式,只有一项研究显示产生染色体正常或平衡配子的对位分离模式受到明显影响,同时,影响四射体分离的这些因素之间是否有相互作用不清楚。目的:探究相互易位携带者性别与易位涉及染色体类型之间是否相互作用,影响四射体的分离模式。方法:回顾性分析在我院行植入前遗传学检测(preimplantation genetic testing for structural rearrangement,PGT-SR)的相互易位携带者的囊胚结果。在2013年1月至2016年9月共473对夫妇通过PGT-SR助孕,包括243例女性相互易位携带者(76例易位涉及近端着丝粒染色体)和230例男性携带者(88例易位涉及近端着丝粒染色体),活检的滋养外胚层细胞应用基于微阵列芯片的比较基因组杂交技术检测细胞团的染色体组成,根据易位涉及染色体类型、携带者性别和年龄分析四射体的分离模式和囊胚的整倍体率。结果:本次研究共分析了2101枚囊胚的四射体分离模式。在涉及近端着丝粒染色体的相互易位亚组中,男性携带者产生的对位分离模式比例(53.9 vs 33.4%,P<0.0001)明显高于女性携带者,同时3:1分离模式(6.8 vs 16.3%,P<0.0001)比例明显降低。在易位未涉及近端着丝粒染色体亚组中,男性携带者的四射体分离模式与女性携带者相似。同时,在女性携带者亚组中,若易位涉及近端着丝粒染色体,与未涉及近端着丝粒染色体的易位相比,对位分离的模式明显减少(33.4 vs 45.2%,P<0.001)且3:1分离模式明显增加(16.3 vs 8.2%,P<0.001);在男性携带者亚组中,与未涉及近端着丝粒染色体的易位相比,对位分离模式比例(53.9 vs 46.9%,P=0.031)在涉及近端着丝粒染色体的易位中增加,同时邻位-1分离模式明显减少(27.1 vs 37.3%,P<0.001)。易位携带者的性别不影响四射体的分离模式。囊胚的整倍体率与女性年龄相关,高龄女性囊胚整倍体率明显低于年轻女性。结论:在常染色体相互易位减数分裂过程中,易位涉及近端着丝粒染色体与携带者性别相互作用影响四射体的对位分离模式比例,进而影响染色体正常或平衡配子的产生。这项研究的发现可以为相互易位携带者提供更详细的遗传咨询,明确其能产生整倍体配子的可能性。第二节携带者性别和年龄对罗伯逊易位减数分裂模式的影响罗伯逊易位由两条近端着丝粒染色体发生着丝粒融合形成,其发生率在普通人群为0.74-1.23例每1000新生儿,在复发性流产患者中为3.1%。在减数分裂I前期,新形成的一条衍生染色体与相应的两条正常近端着丝粒染色体形成三价体,在I后期,三价体按照以下模式进行分离:对位、邻位和3:0分离,其中只有对位分离才能形成染色体正常或平衡配子。若罗伯逊易位涉及1 3号或21号染色体,则有可能生育1 3-三体综合征或者21-三体综合征的新生儿,所以对罗伯逊易位携带者在生育前进行遗传咨询非常有必要。在上一节中,我们发现在涉及近端着丝粒染色体的常染色体相互易位中,不同携带者性别之间减数分离模式存在显著差异,而这种差异在未涉及近端着丝粒染色体的相互易位中不存在。目的:分析携带者的年龄与性别是否影响三价体的分离模式,探究罗伯逊易位的减数分离模式是否与相互易位相同。方法:回顾性分析在我院行PGT-SR助孕的罗伯逊易位携带者囊胚检测结果。在2013年1月至2017年7月共154对罗伯逊易位夫妇通过PGT-SR助孕,包括77例女性携带者和77例男性携带者。活检的滋养外胚层细胞应用基于微阵列芯片的比较基因组杂交技术检测细胞团的染色体组成,根据携带者性别和年龄分析三价体的分离模式,并根据女性年龄分析囊胚的整倍体率。结果:604枚囊胚结果纳入分析。与女牲罗伯逊易位携带者相比,男性携带者中对位分离的比例(82.9 vs 55.2%,P<0.001)明显增加,邻位分离的比例(16.8 vs 42.6%,P<0.001)明显减少,而3:0分离模式的比例没有区别。根据易位涉及染色体分析,在同一携带者性别中,三价体的分离模式是相似的。携带者的年龄对三价体的分离模式没有影响。囊胚的非整倍体率与女性年龄相关,高龄女性的囊胚非整倍体率明显高于年轻女性,且与易位未涉及的其他染色体的新发异常有关,尤其是整条染色体异常。结论:与涉及近端着丝粒染色体的相互易位的分离模式相似,在罗伯逊易位中,男性携带者产生染色体正常或平衡配子的对位分离模式的比例明显高于女性携带者,且三价体的分离模式与携带者的性别无关。这两项研究的联合分析可以为理解染色体易位在不同携带者性别之间的分离模式提供帮助,进而评估携带者的生育风险和进行适当的遗传咨询。更多还原

【Abstract】 Chapter Ⅰ Whole-genome sequencing expands diagnosis of chromosomal abnormalities for couples with recurrent pregnancy lossRecurrent pregnancy loss(PRL)is a common obstetric complication,which adversely affects about 1%of couples seeking pregnancy.The causes of RPL are complicated,among them genetic factors,antiphospholipid syndrome(APS)and congenital uterine malformations being directly related.Factors like infections,autoimmunity and hormonal or metabolic disorders(diabetes mellitus,thyroid dysfunction)have been associated with miscarriage,whereas their roles in RPL are unclear.About half of patients has no identifiably causative factor,which is classified as idiopathic or unexplained RPL(uRPL).To these uRPL patients,some prophylactic treatments are provided,but the effectiveness is unsatisfied.Currently,peripheral blood G-banded chromosome analysis is the standard assay to detect chromosomal rearrangements in couples with RPL despite its limited resolution(320-400 bands).The prevalence of chromosomal abnormalities in RPL couples is approximately 5%,among which autosomal reciprocal balanced translocations being the most commonly observed,followed by Robertsonian translocations and inversions.These patients could yield a livebirth outcome by using preimplantation genetic testing for structural rearrangement to select normal or balanced embryos.Recently,whole-genome sequencing(WGS)shows its capability in yielding additional etiologic diagnoses with improved resolution.Objective:We applied a well-established WGS approach in uRPL couples to expand etiology of chromosomal abnormality among uRPL couples and to determine the extent of additional chromosomal structural rearrangements that would provide valuable information for clinical management.Methods:We performed low coverage WGS retrospectively for 1024 uRPL couples,all of whom had routine G-banding chromosome analysis.Inclusion criteria included:(1)two or more first trimester clinical pregnancy losses;(2)normal karyotypes for both partners via G-banding chromosome analysis;(3)no anatomical malformations;(4)no APS or auto-antibodies positive;(5)no endocrine disorders or metabolic disorders.Chromosomal translocations and inversions were confirmed by polymerase chain reaction(PCR)and Sanger sequencing,with some cases being confirmed by resubmitted fresh blood using fluorescence in-situ hybridization(FISH)and high-resolution chromosome analysis.Copy number variants(CNVs)were validated using chromosomal microarray analysis(CMA)chips.Results:Overall,low-coverage WGS yielded results for 98.7%(1011/1024)of the uRPL couples as 13 samples were excluded because of poor DNA quality,resulting in a final cohort of 1011 couples.Among this cohort,49 chromosomal rearrangements were detected in 48(4.75%)couples including 22 balanced chromosomal translocations and 27 inversions.Among these 22 balanced translocations,14 cases were established in original or high-resolution chromosome analysis,leaving 8 cases only identified by WGS due to cryptic translocation or the similar size and banding pattern of the reciprocally exchanged segments.Among the 27 inversions,four cases were established by chromosome analysis while 23 cases were not due to cryptic segment(one case)or size of involved segments below the resolution limits of G-banding chromosome analysis.All chromosomal translocations and inversions were validated by PCR and Sanger sequencing,with FISH being performed in some fresh samples.Besides,one pathogenic and five likely pathogenic CNVs were reported by low-coverage WGS,all being validated using CMA chips.Conclusion:In the context of chromosome analysis for RPL couples,low-coverage WGS identified additional etiologic chromosomal abnormalities,including balanced translocations,inversions and CNVs,compared to standard karyotype results.It is highly recommended for patients with RPL to pursue WGS as this study demonstrates its ability to expand diagnosis of chromosomal abnormalities.Chapter Ⅱ The impacts of chromosomal abnormalities on meiotic segregation patterns Section Ⅰ Interaction of acrocentric ch romosome involved in translocation and sex of the carrier influences the proportion of alternate segregation in autosomal reciprocal translocationsBalanced reciprocal translocation is defined as the result of an exchange of segments between two non-homologous chromosomes.As one type of commonly structural chromosomal rearrangements in human beings,the prevalence of balanced reciprocal translocation is 0.74-1.52 per 1000 newborns and 2.4%in couples with recurrent pregnancy loss.During meiosis,the two translocated chromosomes and their two homologous normal chromosomes form a quadrivalent and subsequently segregate at anaphase I.There are five types of segregation patterns:alternate segregation,adjacent-1 segregation,adjacent-2 segregation,3:1 or 4:0 segregation.Theoretically,gametes with 36 different karyotypes are produced in carriers of a reciprocal translocation.Normal or balanced gametes are produced by an alternate mode of segregation.Gametes produced by the other segregation patterns have unbalanced karyotypes.Carriers of reciprocal translocations are at a significantly increased risk of fertility problems such as infertility,pregnancy loss and newborns with congenital anomalies,due to the generation of unbalanced gametes in meiotic segregation of a quadrivalent.Previous studies have reported that meiotic segregation patterns of a quadrivalent can be affected by factors such as a carrier’s sex and age and the chromosome type.In these studies,the proportions of adjacent-1,adjacent-2 or 3:1 segregation are significantly different;whereas the reported proportion of alternate segregation does not differ significantly,except in one study,and whether combined effects between these factors exist is unclear.Objective:The aim of this study is to investigate whether meiotic segregation patterns of reciprocal translocations are affected by the combined effect of chromosome type and a carrier’s sex.Methods:A retrospective study of array comparative genomic hybridization outcome data from patients with autosomal reciprocal translocations was conducted to analyze meiotic segregation patterns and blastocyst euploidy rates.We enrolled 473 couples whose embryos were tested between January 2013 and September 2016.Meiotic segregation patterns of 2101 blastocysts from 243 female carriers,including 76 cases with translocations involving an acrocentric chromosome(Acr-ch),and 230 male carriers,including 88 cases with translocations involving Acr-ch,were analyzed according to chromosome type,carrier’s sex and age.Results:In cases with translocations involving the Acr-ch subgroup,the proportion of alternate segregation(53.9 vs 33.4%,P<0.0001)was significantly higher in male carriers than in female carriers,with the proportion of 3:1 segregation(6.8 vs 16.3%,P<0.0001)being significantly lower.The proportions of alternate segregation were similar between sexes in cases with translocations not involving the Acr-ch subgroup.Meanwhile,in the female carrier subgroup,the proportion of alternate segregation(33.4 vs 45.2%,P<0.001)was significantly lower and the proportion of 3:1 segregation(16.3 vs 8.2%,P<0.001)was significantly higher in cases with translocations involving Acr-ch than in those not.In the male carrier subgroup,the proportion of alternate segregation(53.9 vs 46.9%,P = 0.031)was higher and the proportion of adjacent-1 segregation(27.1 vs 37.3%,P<0.001)was significantly lower in cases with translocations involving Acr-ch than in those not.Carrier’s age did not affect the meiotic segregation patterns.However the euploidy rates were significantly lower in couples with advanced compared to young maternal age respectively.Conclusion:Interaction of an acrocentric chromosome involved in the translocation and sex of the carrier influences the proportion of alternate segregation for normal or balanced chromosome contents during meiotic segregation in autosomal reciprocal translocations.The findings of this study provide detailed information for genetic counselling of couples with autosomal reciprocal translocations on their chances of producing euploid gametes.Section Ⅱ Effects of a carrier’s sex and age on segregation patterns of the trivalent of Robertsonian translocationsRobertsonian translocation is defined as the result of centric fusion of two acrocentric chromosomes.The incidence of Robertsonian translocations is 0.74-1.23 per 1000 newborns and 3.1%in couples with recurrent pregnancy loss.During the pachytene stage of prophase I in gametogenesis of Robertsonian translocations,the derivative chromosome and its two normal homologs form a trivalent structure.At anaphase I,the trivalent segregates in one of the three patterns:alternate segregation,adjacent segregation and 3:0 segregation.Theoretically,there are eight different types of gametes produced with respect to chromosomal constitution,among them two types generated from alternate segregation being normal or balanced.In section one,we have found that a significant difference of meiotic segregation patterns is existed between the carrier’s sex in autosomal reciprocal translocations with an acrocentric chromosome involvement and this difference does not exist in translocations not involving an acrocentric chromosome.Objective:In this study,we further analyze the meiotic segregation patterns of the trivalent of Robertsonian translocations according to a carrier’s sex and age and find some general characteristics shared by these two types of rearrangements.Methods:We designed a retrospective study to analyze the segregation patterns of the trivalent and euploidy rates of blastocyst.Data of 154 couples with Robertsonian translocation were collected.Embryos were detected using array comparative genomic hybridization between January 2013 and July 2017.Segregation patterns of the trivalent of 604 blastocysts from 77 female carriers and 77 male carriers were analyzed according to the carrier’s sex and age.Results:The proportion of alternate segregation was significantly higher(82.9 vs 55.2%,P<0.001)in the male carriers than in the female carriers of Robertsonian translocation,with the proportion of adjacent segregation being significantly lower(16.8 vs 42.6%,P<0.001)and no difference in 3:0 segregation.The segregation patterns were similar in the same sex of carriers when analyzed according to type of the translocations.Carrier’s age had no influence on the segregation patterns of the trivalent.This effect of maternal age on genomic abnormalities was related to abnormality of chromosomes unrelated to the translocations,especially whole chromosomal aneuploidy.Conclusion:The proportion of alternate segregation for normal or balanced chromosome contents is significantly higher in the male carriers than in the female carriers of Robertsonian translocation and the meiotic segregation patterns are independent of a carrier’s age,as observed in autosomal reciprocal translocations with an acrocentric chromosome involvement.The findings from a joint analysis of Robertsonian translocations and autosomal reciprocal ones may provide some clues to understand the underlying mechanisms of meiotic segregation patterns of structural rearrangements in the female and male carriers in order to estimate their reproductive risks and counsel appropriately.更多还原