摘 要

子痫前期是一种妊娠期特征性疾病，是导致孕产妇和新生儿死亡的主要原因之一。此外，诊断为子痫前期的孕产妇产后罹患心血管疾病的概率更高。目前该疾病尚无可靠的治疗方法，仅有的临床干预措施是终止妊娠。本研究目的是通过深入的生物信息学的分析，从遗传学的角度探讨可能有预测应用价值的子痫前期DNA相关生物标志物。

方法：按照孕周、年龄、籍贯因素1：1个体匹配选取子痫前期患者与同期正常孕产妇各10名。提取外周血样本DNA，用Affymetrix CytoScan HD细胞遗传学芯片进行杂交；使用Affymetrix的Chromosome Analysis Suite（ChAS）分析软件处理芯片原始数据获得全部拷贝数变异（CNV）和单核苷酸多态性（SNP）信息；筛选两组间差异基因后进一步进行GO和Pathway富集分析，得到差异的CNV。SNP研究则先对位点进行注释，然后结合Clinvar等多个数据库注释的结果筛选差异位点及基因。

结果：CNV分析结果显示：共44个全基因组拷贝数变异区域在两组孕妇CNV事件发生差异高于30%，其中差异CNV区域与正常人高频拷贝数变异区域（DGV）数据库中无重叠的有27个，分别是1q23.1、3p14.2、5q35.3、6q26、7q36.2等，是在子痫前期患者中发现的新拷贝数变异，涉及到27个差异基因：CHSY1, KRTAP9-7, LPA, LMF1, APOBEC3A, APOBEC3A_B, APOBEC3B, BCAS1, BTNL3, DPP6, EXT1, EYA2, FBRS, FHIT, IL1RAPL1, MALT1, MIR4756, NTRK1, OR52N1, OR52N5, PPP2R3B, PREX2, PRR14, SPATA6L,TBC1D3F, TBC1D3, LOC440434。GO/Pathway富集分析结果涉及的生物学功能包括：蛋白质水解、B细胞分化、发展性程序性细胞死亡和调节甘油三酯的代谢过程等，通路包括：粘多糖生物合成、细胞凋亡和B细胞受体信号等。SNP位点分析结果显示：共30个差异突变位点在两组孕妇SNP事件发生差异高于30%，涉及26个突变基因：KIR3DL2,MUC16, ZNF888, OR5B3, XIRP2,NLRP5,ERC1,PLEKHG2,NIPAL1, LAMB4,ERICH6B,FSIP1,APOBEC2,VPS13C,TPPP2,KRT77,EFHB,FHAD1, NPSR1, KCNU1,AP3B1,OR6C68,ANXA4,EFCAB5, FAM71A和MALRD1。上述基因中除 LPA, KIR3DL2和MUC16以外，均未见与子痫前期发病有关的报道。

结论：本研究提示孕妇外周血DNA分析所获得的差异CNV 和SNP可能成为临床子痫前期预测的潜在生物标志物。

关键词：子痫前期；生物标志物；CNV；SNP

Abstract

Pre-eclampsia remains an important cause of maternal and perinatal morbidity and mortality. In addition, women diagnosed with preeclampsia have a greater risk of cardiovascular disease after giving birth. There is no effective treatment of the disease but the early termination of pregnancy. The purpose of this study was to investigate the potential clinically relevant DNA biomarkers in predicting preeclampsia from a genetic perspective through intensive bioinformatical analysis.

Methods: Ten preeclampsia patients and ten normal pregnant women were individually matched by the gestational age, age and native place factors. DNA were extracted from peripheral blood samples and hybridized by Affymetrix CytoScan HD cytogenetic chip. The chip raw data are processed to obtain full copy number variation (CNV) and single nucleotide polymorphism (SNP) information by Affymetrix's Chromosome Analysis Suite (ChAS) analysis software. Further GO and Pathway enrichment analyses were performed to obtain differential CNV after screening differentially expressed genes between two groups. The mutation loci for SNPs were annotated and then the differential loci and genes were screened by combining the results of multiple database annotations such as Clinvar.

Results: CNV analysis showed: A total of 44 genome-wide copy number variation regions were observedwhen the difference in CNV events between the two groups was greater than 30%. There were 27 CNVs i.e. 1q23.1、3p14.2、5q35.3、6q26、7q36.2 and so on, which have no overlap between difference CNV regions and DGV database of normal high frequency area of copy number variation. The 27 different genes were new copy number variants found in preeclampsia patientsincluding CHSY1, KRTAP9-7, LPA, LMF1, APOBEC3A, APOBEC3A_B, APOBEC3B, BCAS1, BTNL3, DPP6, EXT1, EYA2, FBRS, FHIT, IL1RAPL1, MALT1, MIR4756, NTRK1, OR52N1, OR52N5, PPP2R3B, PREX2, PRR14, SPATA6L,TBC1D3F, TBC1D3 and LOC440434. GO/Pathway enrichment analysis indicated that the biological function of these genes include protein hydrolysis, B cell differentiation, developmental programmed cell death, and regulation of triglyceride metabolism and so on. and pathways include glycosaminoglycan biosynthesis, apoptosis, and B cell receptor signaling and so on. The SNP loci analysis showed that a total of 30 differential mutation loci were observed in the two groups of pregnant women when the difference in SNPevents wasgreater than 30%. It involved 26 mutant genes: KIR3DL2, MUC16, ZNF888, OR5B3, XIRP2, NLRP5, ERC1, PLEKHG2, NIPAL1, LAMB4, ERICH6B, FSIP1, APOBEC2, VPS13C, TPPP2, KRT77, EFHB, FHAD1, NPSR1, KCNU1, AP3B1, OR6C68, ANXA4, EFCAB5, FAM71A and MALRD1. , No genes have been reported to be involved in the pathogenesis of preeclampsia except for LPA, KIR3DL2 and MUC16 among the 26 mutant genes and 27 CNVs.

Conclusion:This study suggests that the differential CNV and SNP obtained from the analysis of the blood DNA analysis of pregnant women may be a potential biomarker for preeclampsia.

Key Words：Preeclampsia; Biomarker; CNV; SNP

目 录

1 绪论.........................................................................................................................................1

1.1子痫前期概述 1

1.2 子痫前期的临床预测指标......................................................................................... 1

1.3 拷贝数变异与疾病研究............................................................................................. 2

2 材料与方法 4

2.1研究对象 4

2.2 生物样本的采集与保存..............................................................................................4

2.3 基因芯片检测..............................................................................................................4

2.4 基因数据处理与分析..................................................................................................7

2.5 对相关基因及功能通路的探索..................................................................................7

3 结果 9

3.1 研究对象统计描述 9

3.2 拷贝数变异分析结果..................................................................................................9

3.2.1病例组与对照组间拷贝数变异差异 9