【作者】 刘洋；

【导师】 谢建生；

【作者基本信息】 南方医科大学 ， 临床检验诊断学（专业学位）， 2016， 硕士

【摘要】 研究背景：先天性心脏病(Congenital heart diseases,简称CHD)是一类最常见的出生缺陷,约占出生缺陷总数的1/3。在活产儿中其发生率约为6-8‰。在先天性结构畸形中其是导致新生儿及儿童死亡的最常见原因。同时,先天性心脏畸形也被认为是导致胚胎早期流产的重要原因之一,至少约10%的早期流产胚胎存在严重先天性心脏畸形。随着产前诊断技术,心脏外科技术及护理技术的快速发展,目前超过75%的CHD患儿可存活至成年。据估计,先天性心脏畸形在成人中的发生率约为3‰,且还在不断增长。探索先天性心脏畸形的发病原因能够使我们进一步从本质上认识和了解先天性心脏病。不仅对成年先天性心脏病人群的健康管理显得至关重要,同时也能够促进先天性心脏病的产前诊断和干预,从而预防严重先天性缺陷儿的出生。先天性心脏病病因复杂,目前只有大约20%左右的CHD病因已知,其余80%左右均为病因未知病例。能够导致先天性心脏病的主要原因有环境因素、遗传因素及环境因素和遗传因素的共同作用。目前已经明确的能够导致CHD的环境因素主要包括风疹病毒感染,化学致畸剂的暴露如维甲酸、锂、苯妥英钠等,母亲孕期酗酒,母体疾病如糖尿病、系统性红斑狼疮等。遗传因素中如染色体异常变异(包括数目异常和结构异常)、单个基因突变等均可导致先天性心脏畸形的发生。染色体数目异常中最常见的能够导致先天性心脏畸形的异常有21三体,约50%的患者会出现CHD; Turner综合征,50%以上会出现CHD；18三体综合征和13三体综合征,几乎90%以上的患者会出现CHD。染色体结构异常中染色体局部异常导致的染色体微缺失微重复综合征是引起CHD的一类常见原因。目前已发现超过40余种染色体微缺失微重复综合征与CHD相关。单基因突变中目前已知的与先天性心脏畸形相关的基因大约有200多种,常见的有PTPN11等基因突变导致的Noonan综合征,TBX5基因突变导致的Holt-Oram综合征(HOS), FBLN等基因突变导致的马凡综合征(Marfan’s syndrome)等。目前越来越多的研究发现遗传因素在先天性心脏畸形的发生中发挥着主要作用,越来越多的在胚胎心脏形态发育过程中发挥着重要作用的结构基因、信号分子及转录因子等已被鉴定出。阐明CHD发生的分子机制有助于理解CHD的发病原因,从而为探索CHD的病因提供可靠依据,有助于早期诊断CHD,并为进一步的治疗和管理提供可靠依据。基因组拷贝数变异(copy number variants CNVs)是指存在于染色体上某一区域的局部缺失或重复,通常影响到相邻的一串基因,大小一般介于1kb到几Mb不等。CNV属于染色体结构异常的一种常见类型,它是导致染色体微缺失微重复综合征的致病原因。染色体微缺失微重复综合征种类繁多,已有超过40余种该类综合征被证实与先天性心脏畸形的发生相关,从这些致病区域中已鉴定出大量与心脏发育相关的基因,如NKX2.5、ETS-1、 ELN、TBX1等等。目前大量研究表明,CNV是导致先天性心脏畸形的一个重要原因,其不仅可以引起综合征型先天性心脏畸形如Digeorge综合征和Williams综合征等,而且还可以引起非综合征型先天性心脏畸形。据估计,约10%左右的先天性心脏畸形患者存在稀有的新发CNV,其发生率远远高于在正常人群中的发生率。对这些稀有的新发CNV区域进行研究,可能会为进一步揭示先天性心脏畸形的发病机制提供线索。经典的细胞遗传学分析技术G显带染色体核型分析一直被认为是检测染色体异常的金标准。但其分辨率不高,只能检测出大于4Mb以上的染色体异常,且容易漏诊4-10Mb之间的染色体异常变异。同时染色体核型分析的异常检出率与实验室制片技术及分析者的经验存在着很大关系。随着分子遗传学技术的飞速发展,FISH技术、MLPA技术、array-CGH技术、SNP-array技术等一大批分子遗传学检测技术的应用大大提高了检测染色体异常的分辨率。特别是随着CMA技术(主要包括array-CGH技术和SNP-array技术)的成熟,使得一次性针对整个基因组进行CNV分析得以实现,且分辨率可以达到30kb以上。目前CMA技术已被推荐用作不明原因发育障碍／智力障碍、癫痫、孤独症谱系障碍及先天性畸形等患儿的一线检测技术。在产前诊断领域CMA技术也被推荐作为一线检测方法应用于存在结构畸形的胎儿中。但由于CMA技术存在价格昂贵,对实验室及操作者水平要求高等不足,导致其目前尚未在国内得到普及。而MLPA技术凭借其成本低,操作简便,实验周期短等诸多优点目前已在很多地方得以应用,但它是一种靶向检测技术,无法发现探针没有覆盖的区域的染色体异常。研究目的：本次研究以有先天性心脏畸形的胎儿作为研究对象,能够覆盖因为先天性心脏畸形导致的宫内死亡胎儿及因严重先天性心脏畸形而终止妊娠的胎儿,从而弥补了目前国内外先天性心脏畸形遗传因素研究主要以活产儿为研究对象存在的病种较少的局限。从而能够更广泛和深入的研究心血管畸形的类型及其发病机制。寻找胎儿期导致先天性心脏畸形的主要遗传学变异类型,同时以期发现新的导致先天性心脏畸形的遗传学变异类型,进一步揭示先天性心脏畸形发病的分子机制。通过对常规G显带染色体核型分析技术、MLPA技术和CMA技术相互比较从而探讨不同染色体异常检测技术在检测胎儿先天性心脏畸形遗传学变异的临床应用过程中的优劣。研究方法：1、收集产前胎儿III级超声检查提示存在先天性心脏畸形的胎儿的羊水或终止妊娠后的脐带组织作为研究标本。其中标本主要是在孕中期收集。2、羊水标本常规行G显带染色体核型分析,同时提取羊水细胞基因组DNA应用MLPA P095试剂盒检测21、18、13、X、Y五条染色体的非整倍体异常,P036、P070试剂盒检测23对染色体亚端粒微缺失微重复异常,P245试剂盒检测21种常见染色体微缺失综合征(1p36 deletion syndrome,2p16 microdeletion, 2q23 microdeletion,2q33 microdeletion,3q29 microdeletion,9q22.3 microdeletion, 15q24 deletion syndrome, 17q21 microdeletion,22q13/Phelan-McDermid syndrome, Cri du Chat syndrome, DiGeorge syndrome, Langer-Giedion syndrome, Miller-Dieker syndrome, NF1 microdeletion syndrome, Prader-Willi/Angelman syndrome, MECP2/Xq28 duplication, Rubinstein-Taybi syndrome, Smith-Magenissyndrome, Sotos syndrome, Williams syndrome, Wolf-Hirschhorn syndrome)。若MLPA检测发现21、18、13、X染色体非整倍体异常,则检测终止,若MLPA未发现上述四种染色体异常,则继续对羊水标本行array-CGH或SNP-array分析。脐带标本除不进行常规G显带染色体核型分析外,其余检测方法及步骤均与羊水标本相同。结果：本次研究共收集先天性心脏畸形标本107例,其中共检出异常31例,包括22例目前已知的致病性异常和9例临床意义未明变异。异常结果比例为29%(31/107),致病性异常比例为20.6%(22/107)。在73例单纯性心脏畸形中共发现16例异常结果(11例致病性异常和5例临床意义未明变异),致病性异常结果比例为15.1%(11/73)；在34例合并心外异常的CHD中,共检出15例异常(11例致病性异常和4例临床意义未明变异),致病性异常结果比例为32.4%(11/34)。共有72例标本进行了染色体核型分析,共检出14例异常,13例致病性异常,1例临床意义未明变异,异常比例为19.4%(14/72)；107例标本进行了MLPA检测,共检出22例异常,21例致病性异常,]例临床意义未明结果,异常比例为20.6%(22/107)；共93例标本进行了CMA检测,共检出20例异常,其中12例致病性异常,8例临床意义未明变异。其中在染色体核型分析和MLPA结果均正常的85例标本中CMA共检出9例异常结果(1例致病性异常、8例临床意义未明变异)。经CMA检测出的12例致病性异常中经MLPA检测共检出11例异常,漏诊1例,8例临床意义未明变异经MLPA检测只发现1例异常。检出的22例目前已知的致病性异常主要包括3例21三体,3例18三体,2例13三体,1例Turner综合征嵌合体,3例染色体不平衡易位(5号和15号不平衡易位、11号和14号不平衡易位、7号和8号不平衡易位),5例22q11.2微缺失综合征,4例Jacobsen综合征,1例8p23.1微重复。本次研究中共发现9例临床意义未明结果,发生率约为8.4%。9例临床意义未明变异主要涉及到的染色体区域有3q13.13q13.2,5q12.1,14q21.2q21.3,15q13.3,18p11.31p11.22,18p11.31、Xq27.2、 Xp11.3p11.23、7q。其中有5例胎儿表现为单纯性先天性心脏畸形,4例胎儿除心脏畸形外还合并有其他心外异常。结论：1、107例胎儿先天性心脏畸形标本中有31例存在染色体数目或结构异常(29.0%)。其中在染色体结构异常中22q 11.2微缺失综合征和.lacobsen综合征是两类最常见的导致胎儿心脏畸形的遗传学变异。2、基因组拷贝数变异(CNV)是导致先天性心脏畸形的一类常见原因,其中临床意义未明结果在先天性心脏畸形胎儿中的发生率约为8.4%,提示临床意义未明的CNV可能在先天性心脏畸形中发挥着一定作用。3、多重连接依赖探针扩增技术(MLPA)是一种简便快速的方法用于发现先天性心脏畸形胎儿染色体异常。对于一些无法开展CMA检测的医疗机构,MLPA技术可以作为一种替补方法用于检测常见染色体变异。更多还原

【Abstract】 Research Background:Congenital heart malformations,also called congenital heart disease(CHD), are the most common birth defects. They account for one third of the birth defects and occurs in almost 6-8‰ live births. It is the leading cause of neonatal and childhood death. CHD is also the major cause of miscarriages and at least 10% fetuses aborted within the first trimester have congenital heart malformations, so it is regarded as an important reason leading to early abortion. So far, over 75% patients who have CHD can survive to adulthood depending on the advance of prenatal diagnosis,cardiac surgical techniques and cardiac care. It is estimated that the incidence of CHD in adults is 3‰ with growing trend. By investigating the evolution and morphologic of embryo heart, it can help us to understand the pathogenesis of CHD, to improve prenatal diagnosis and health management, to provide better treatment of CHD and to prevent the severe birth defects.The etiology of CHD is complex. So far, only 20% of CHD patients can be found to have clear pathogenesis and the rest of 80% CHD patients have unknown reason. The main causes leading to CHD include environmental and genetic factors as well as the interaction between environmental and genetic factors. The environmental factors associated with CHD include fetal infection such as TORCH pathogenic microorganisms, teratogens exposures(retinoic acid, lithium, phenytoin sodium, alcohol), maternal diseases(diabetes, systemic lupus erythematosus). The genetic factors such as chromosomal numerical or structural anomalies, Mendelian syndrome and non-syndromal single gene mutations are the most common reasons of CHD.50% patients with Down syndrome and Turner syndrome, more than 90% patinets with Edwards syndrome and Patau syndrome have congential heart malformations. Over forty different kinds of chromosomal microdeletion or microduplication syndromes were reported to be associated with CHD. More than 200 genes are linked to CHD, which include syndromic and non-syndromic CHD. Examples of Mendelian syndromes that have heart defect include Noonan syndrome (the affected gene is PTPN11), Holt-Oram syndrome (the affected gene is TBX5), Marfan’s syndrome (the affected gene is FBLN). Epidemiologic studies show that genetic factors are the predominant reason of CHD although environmental factors may be relevant. A series of structural genes, signaling molecules and transcriptional factors have been found to participate in cardiac differentiation and structure formation. Illustrating the molecular mechanisms of heart development can help us understand the pathogenesis and provide theoretical basis for further treatment and management of CHD.Copy number variants(CNVs) are a form of structural chromosomal variation that manifest as deletions or duplications in the genome. The size of deletion or duplication of chromosomal segments ranges from 1Kb to several Mb often containing multiple contiguous genes. CNVs are the common reasones to cause chromosomal microdeletion or mocroduplication syndromes. There are over 100 different kinds of chromosomal microdeletion or mocroduplication syndromes which have been reported and over 40 of these syndromes have turned out to cause CHD. Based on these CNVs, many genes related to heart development have been identified which includes NKX2.5,ETS-1,ELN,TBX1 and so on. Some CNVs can lead to syndromic CHD such as Digeorge syndrome and Williams syndrome. While others can cause non-syndromic CHD. It is estimated that about ten percent of CHD have rare de novo CNVs. Further studies on these CNVs may provide us with new pathogenic clues of CHD.Classical G-banding chromosomal karyotyping is the gold standard of analyzing chromosomal anomalies. But some structural anomalies less than 4-10Mb are usually been missed by G-Banding karyotyping. The detection rate of chromosomal abnormalities depends on banding quality and the experiences of technologists themselves. With the rapid development of molecular technologies, more and more molecular diagnostic methods such as FISH, MLPA, array-CGH, SNP-array have been applied clinically. Espically with the mature of Chromosomal Microarray Analysis(CMA), we can scan the whole genome in one experiment and have a high resolution. Because of some disadvantages like high expensive, strict standards of laboratory and short of qualified technologists, CMA have not been widely used in China. While MLPA has been widely used in many laboratories because of its low cost, easy operation and short experimental period. But MLPA is a probe targated method, and some chromosomal anomalies would be missed beyond probe targated segments.By collecting samples(amniotic fluid or umbilical cord) from fetuses with congenital heart malformations as our research object, we can overcome some shortages of current researches about CHD, such as missing rare types of CHD when selecting living cases with CHD. We hope that our research can detect new genetic variantions that cause CHD in fetal stage and help us understand comprehensively the pathogenesis of CHD. By comparing three different genetic technologies used in this research(chromosomal karyotyping, MLPA and CMA),we can understand their advantages and disadvantages.Method:(1):Collecting the amniotic fluid samples or umbilical cord samples from fetuses with CHD during the second trimester of pregnancy.(2):Cells from amniotic fluid samples were cultured to carry out G-banding karyotype. DNA were extracted from Cells of amniotic fluid samples to conduct MLPA. The aim of MLPA of kit P095 is to screen aneuploids of chromosomal 21,18,13,X,Y, The aim of MLPA of kits P036 and P070 is to screen subtelomeric deletion or duplication. The aim of MLPA of kit P245 is to screen 21 common microdeletion syndromes. Samples without aneuploid anomalies screened by G-banding karyotype and MLPA were further to carry out array-CGH or SNP-array analysis. The testing process of umbilical cord samples was same as that for amniotic fluid samples except that the umbilical cord samples did not take chromosomal karyotyping analysis.Result:31 anomalies were identified in 107 samples, including 22 pathogenic variations and 9 variants of unknown significance. The rate of total anomalies was 29%(31/107) and the rate of pathogenic variations was 20.6%(22/107). Among 73 isolated CHD samples there were 16 anomalies including 11 pathogenic variations and 5 variants of unknown significance. The rate of pathogenic variations was 15.1%(11/73). Among 34 CHD with eatracardiac abnormal samples,15 cases were found to have chromosomal anomalies, namely 11 pathogenic variations(32.4%,11/34) and 4 variant of unknown significance (VUS).Chromosomal karyotyping analysis was conducted on 72 samples and 14 chromosomal anomalies were found with a 19.4%(14/72) abnormal rate including 13 pathogenic variants and 1 variant of unknown significance. Total 107 samples were screened by MLPA and rate of anomaly was 20.6%(22/107), anomalies including 21 pathogenic variants and 1 variant of unknown significance.93 samples were screened by CMA and 20 anomalies had been found including 12 pathogenic variants and 8 variants of unknown significance. Among the 85 samples that showed no anomalies screened by G-banding karyotype and MLPA,9 samples were identified by CMA to have anomalies, including 1 pathogenic variant and 8 variants of unknown significance(VUS). Among the 20 anomalies identified by CMA,11 pathogenic variations(11out ofl2) and 1 variants of unknown significance(1 out of 8) were found by MLPA.The total 22 pathogenic variations included 3 cases of Down syndrome,3 cases of Edwards syndrome,2 cases of Patau syndrome,1 case of mosaic Turner syndrome,3 cases of interchromosomal unbalanced translocations,5 cases of 22q11.2 deletion syndrome,4 cases of Jacobsen syndrome and lcase of 8p23.1 microduplication. In this research 9 variants of unknown significance were detected with a rate of 8.4%(9/107). The abnomal chromosomal segment includes 3q13.13q13.2,5q12.1,14q21.2q21.3,15q13.3,18p11.31p11.22,18p11.31, Xq27.2, Xp11.3p11.23,7q. Among the 9 cases with variants of unknown significance,5 cases had isolated CHD and 4 cases had extracardiac anomalies.Conclusion:1.31 samples were identified to have chromosomal numerical or structural anomalies in 107 CHD samples with a 29% abnormal rate.22q 11.2 deletion and Jacobsen syndrome are the most common chromosomal abnoemalities.2. CNV is one of the common pathogenic factor that causes CHD. The rate of VUS was 8.4%, suggesting that VUS may play an important role in the development of heart malformations.3. MLPA is an economic and rapid method to investigate chromosomal anomalies in fetuses with CHD. In some medical institutions that can not carry out CMA analysis, MLPA is possible to be an option used to discover chromosomal anomalies.更多还原