【作者】 赵巍；

【导师】 郑杨；

【作者基本信息】 吉林大学 ， 内科学， 2014， 博士

【摘要】 近30年来随着医学技术水平的进步和内、外科纠治水平的提高，85%罹患先天性心脏病（CHD）的儿童患者可以良好成活至生育年龄。在美国大约130万的成年人患有CHD，即成人先天性心脏病（ACHD），而这个数字正以每年5%的速度递增。美国心脏病协会根据ACHD的复杂程度，将ACHD分为简单、中等程度及复杂病变三个类别，其中简单至中等复杂程度的单纯性CHD（如房间隔缺损、室间隔缺损等）约占据ACHD的70%。这些病变的患者大多是在当地医院或者普通的社区医院就诊，并未进行过遗传测试或咨询，而遗传咨询和产前诊断对于这个群体非常重要。在本实验中，我们采用比较基因组杂交芯片技术方法来研究简单至中等复杂程度的的ACHD患者的基因拷贝数变异（CNVs）情况，并根据结果对相关患者进行了可能致病基因的筛查，为CHD的发病机制提供新的线索和理论依据，也有助于该类人群的遗传咨询和产前诊断。目的：研究分析简单-中等复杂程度成人先天性心脏病的基因拷贝数变异，并筛选其可能的致病基因，逐步建立表型与基因型的相关性。研究结果为患者的病因解释、个体化诊疗和遗传咨询等方面给予极大的帮助，以及对进一步阐明先心病的病因学机制研究打下基础。方法：本研究对100个患有不同种类的成人非复杂型的先天性心脏病的患者和65个健康对照组进行了全基因组比较基因组杂交芯片技术（array-CGH）分析，并应用另外一个array-CGH平台对有意义的实验结果进行了验证。根据实验结果对所有VSD患者进行了CRKL基因突变筛查。本研究收集ACHD患者和对照组的外周静脉血，采用酚-氯仿法提取全基因组DNA，标记并保存。基因芯片检测平台为Roche NimbleGen公司的array-CGH检测平台（Human CGH3x720K Whole-Genome Tiling v3.0array芯片，GenePix4000B分析仪，NimbleScanTM v2.6和NimbleGen’sSignalMapTM v1.9分析软件）。此平台共包含720，000个位点，探针之间的平均间隔是2509个碱基对。为了评估拷贝数变异增加或者减少，我们分别应用0.3和-0.3作为log2比率阈值。我们应用该平台检测所有研究对象的的全基因组DNA，并与美国国立医学图书馆的国家生物技术信息中心基因数据库、美国加州大学圣克鲁兹分校基因数据库中公布的基因序列相比较，并参考以上数据库中公布的区段性复制区域区域及正常的CNVs，除外其对数据的影响，并排除染色体片段拷贝数异常仅覆盖基因内含子的区域，从而整理出具有临床意义的染色体片段拷贝数异常的数据。我们又对检测出18q23,22q11.2,3q21.3的3位患者用另一个array-CGH平台（AgilentTechnologies DNA microarray4.2M）进行了验证。根据实验结果对22q11.2微缺失患者及其他VSD患者进行了CRKL基因突变筛查。结果：本研究通过array-CGH分析总共得到45个较大的CNVs，其中包括13个拷贝数缺失和32个拷贝数重复。该45个CNVs来自于36个ACHD样本中的40个独立位点，平均基因组大小是405.3kb。其中有39个CNVs仅存在于唯一一个患者。对照组样本中发现14个稀有而较大的CNVs。研究证明，在ACHD患者中出现稀有而较大的CNVs比健康对照组中的稀有而较大的CNVs频率高，但统计学显示无显著性差异（39/100vs.14/65, p=0.02）。在ACHD组中发现一例患者存在22q11.2微缺失，大小为0.7Mb，与经典3Mb的22q11.2微缺失有少量重叠。我们同既往报道过的有相同拷贝数异常区域的患者表型对比，本研究存在22q11.2微缺失的患者（10085）并不存在典型的DiGeorge综合征、颚心面综合征和圆锥动脉干异常面容综合征的心脏外表型。在45个CNVs中，其中3个CNVs中(22q11.2,18q23,3q21.3)发现有与心脏发育密切相关的基因（CRKL, NFATC1, PLXNA1）。其中CRKL参与心脏流出道和咽弓动脉的重建，NFATC1参与心脏瓣膜和心脏间隔的形态发生过程，而PLXNA1参与神经管的行程和心脏形态发生。在健康对照组的样本中，未发现有包含有与心脏发育相关的基因的CNVs。分析较大的CNVs在相同位点的发生率，我们发现有两位ACHD患者均存在16q23.1微重复。第一位患者100536是一位26岁的男性，他和他的父亲均有相同类型的ASD，且存在相同的16q23.1微重复。患者100183是一位存在PDA的男性患者，他也存在16q23.1的微重复，且与患者100536均包含基因WWOX。另一位也存在遗传性CHD的患者未发现存在拷贝数异常。此外，另两个染色体位点（9q22.33,6q24.1）分别存在于两个CHD病例中，但是无显著性差异（2/100vs.0/65, p=0.366, Fisher确切概率法）。本研究根据实验结果对22q11.2微缺失患者及其他VSD患者进行了CRKL基因突变筛查，结果显示所有VSD患者并无CRKL任何形式的基因突变。结论：本研究首次以简单-中等复杂程度成人先天性心脏病这个群体为研究对象。经Array-CGH分析100例简单-中等复杂程度ACHD患者及65例健康对照组，总共得到45个稀有且较大的CNVs。在ACHD组中发现一例VSD患者存在“非典型”远端22q11.2微缺失，而该患者不存在与22q11.2微缺失综合征相关的心脏外畸形。在其中3个CNVs （22q11.2,18q23,3q21.3）中发现有与心脏发育密切相关的基因（CRKL, NFATC1, PLXNA1）。研究发现了两位ACHD患者及其中一位患者的父亲均存在16q23.1微重复。基因筛查结果显示CRKL不是本例22q11.2微缺失患者的致病基因。研究结果有助于该类人群的遗传咨询和产前诊断，也为先天性心脏病的发病机制提供新的线索和理论依据。更多还原

【Abstract】 In past decades, a new population of adults with congenital heart disease(ACHD) emerged who need specialized care. The American College ofCardiology developed a classification pattern to categorize ACHD patients,according to the disease complexity: simple, moderate, and complex lesions.Simple-to-moderate isolated CHD constitute approximately70%of A CHD.Genetic counseling about inheritance, including transmission of CHD tooffspring, is an important issue in this population. Most patients with ACHDhave not had genetic testing or counseling, especially for the simple or moderatelesions. Individuals with these lesions are commonly seen at regional ACHDcenters, or cared for in the general medical community.In this study, we used array-CGH to identify the simple-to-moderateisolated CHD. This study has immediate consequences for genetic counsellingand should pave the way for the elucidation of the pathogenetic mechanismsunderlying CHD.Objective: We assayed submicroscopic imbalances in adults with CHDfocusing on simple-to-moderate phenotypes, with no associated dysmorphisms,a group not previously examined. This CNVs profile provides a spectrum ofgenomic imbalances in this condition, and improves the CNV-phenotype correlations.Methods: A total of100adults with a diverse range of isolated CHD and65ethnically matched controls were screened using whole-genome arraycomparative genomic hybridization（array-CGH）. Blood samples were collectedand genomic DNA was extracted using standard methods. High-resolutionoligonucleotide array comparative genomic hybridization (CGH) was performedusing a Human CGH3x720K Whole-Genome Tiling v3.0array, whichcontains720,000probes with a median probe spacing of2509base pair.Labeling and hybridization were performed following the manufacturer’sprotocol. The patient’s DNA and the reference DNA were labeled with eitherCyanine3(Cy-3) or Cyanine5(Cy-5) by random priming and then hybridizedto the chip via incubation in the MAUI hybridization system. The arrays werescanned (GenePix4000B) and analyzed by NimbleScanTM v2.6andNimbleGen’s SignalMapTM v1.9software. For the assessment of copy numbergains and losses, we used conservative log2ratio thresholds of0.3and-0.3,respectively. Genomic positions refer to human assembly NCBI36/hg18. TheCNVs smaller than20kb were not studied further. We have analyzed the controlpopulation using the same array design.Results: Altogether45large CNVs (13deletions and32duplications) wereobserved at40unique loci from36individuals with an average genomic size of405.3kb. Thirty-nine of the CNVs were unique, each seen in just one patient.Using the same computational algorithm,14putative rare large CNVs were identified. The frequency of rare large CNVs was greater in adult CHD casesthan in control cases, but the difference was not statistically significant (39/100vs.14/65, p=0.02). One patient had a0.7Mb22q11.2deletion, whichmarginally overlapped the common3Mb22q11.2deletion. The clinicalphenotype was examined and findings were compared with those previouslyreported on this genomic region. The patient with22q11.2deletion lacked anyextracardiac phenotype that typically associated with the DiGeorge,velocardiofacial and conotruncal anomaly face syndromes.These variants werenot listed in the Database of Genomic Variants nor found in controls. In three ofthese genomic imbalances (22q11.2,18q23,3q21.3), genes that play animportant role in cardiac development were implicated CRKL, NFATC1,PLXNA1, the latter has not been associated with human CHD before. CRKLwas associated with morphogenesis of cardiac valves and septum, NFATC1wasinvolved in the remodeling of the cardiac outflow tract and pharyngeal archarteries, and PLXNA1was associated with neural tube formation and cardiacmorphogenesis. There was no gene involved in heart development identified inthe CNVs for controls. Studying the occurrence of CNVs at the same loci in theremaining large CNVs, we revealed two patients with16q23.1microduplication.The first patient (100536) was a26-year-old male, both he and his father, whoshared the same cardiac phenotype of atrial septal defect (ASD), carried theidentical duplication of16q23.1. Case100183was a male with patent ductusarteriosus (PDA), he also had a duplication of16q23.1, which encompassed the same gene WWOX with100536. Additionally, CNVs at other two loci (9q22.33,6q24.1) were found in two CHD cases respectively, but the difference was notstatistically significant (2/100vs.0/65, p=0.366, Fisher’s exact test).Conclusion: For the mild-to-moderate phenotypes of isolated ACHD,spare genetic studies have been made.45large (>100kb) rare copy numbervariants (CNVs) were identified in36/100patients. These variants were notlisted in the Database of Genomic Variants nor found in controls. In three ofthese genomic imbalances (22q11.2,18q23,3q21.3), genes that play animportant role in cardiac development were implicated CRKL, NFATC1,PLXNA1, the latter has not been associated with human CHD before. This studydisclosed a0.7Mb22q11.2deletion, which marginally overlapped the common3Mb22q11.2deletions, in one patient with a ventricular septal defect withoutany extracardiac manifestation. Furthermore, we detected one inheritedaberration dup (16q23.1), previously unreported. We make a fine mapping ofchromosomal imbalances and genotype-phenotype comparisons of the clinicallywell-defined patients to seek loci and genes associated with ACHD.更多还原