微阵列比较基因组杂交技术在出生缺陷中的运用

发布时间：2018-02-27 12:14

  本文关键词： 腹裂 脐膨出 微阵列比较基因组杂交技术 拷贝数变异 定制基因芯片 拷贝数变异 腭心面综合征 猫叫综合症 定制基因芯片 圆锥动脉干畸形 22q11微缺失 22q11微重复　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分圆锥动脉干畸形患者22q11.2微缺失和微重复的定制基因芯片检测第一节利用已知病因的病例验证定制基因芯片目的：用Agilent定制基因芯片对已知染色体缺陷的病例进行检测,比较实验结果,明确定制基因芯片的准确性。方法：选出以往用多重连接依赖探针扩增技术(multiplex ligation-dependent probe amplification, MLPA)确诊的10例腭心面综合征患者和Cytogenetic Whole-Genome 2.7M基因芯片确诊的2例猫叫综合症患者的血清,采用本研究的Agilent 8×15K定制基因芯片方法检测,并用Agilent Genomic Workbench Lite Edition 6.5版进行结果分析。结果：12例病例中,定制基因芯片检测出了与MLPA及全基因组芯片一致的基因组拷贝数变异(copy number variations, CNVs),其中8例腭心面综合征患者为22q11.2区域的微缺失,2例腭心面综合征患者为22q11.2区域的微重复,而2例猫叫综合症患者为5q的部分缺失。结论：这款8×15K定制基因芯片能够快速而准确的检测出22q11.2微缺失或微重复、猫叫综合征等11种疾病的染色体畸变,从而提供可靠的遗传学信息。第二节应用定制基因芯片检测27例CTDs患者的染色体畸变目的：分析27名圆锥动脉干畸形(conotruncal defects, CTDs)患者的遗传学病因,特别是22q11.2区域的致病性CNVs发生概率及比例,为下一步的遗传学咨询、风险评估、后续治疗等提供理论基础。方法：选择单纯的CTDs患者27例,利用德国QIAGEN公司gDNA抽提试剂盒提取gDNA。采用Agilent 8×15K定制基因芯片方法进行CNVs检测,尤其关注22q11.2区域的染色体畸变。利用德国Applied Biosystems公司生产的ViiA 7 Realtime PCR仪进行SYBR Green实时定量荧光PCR对芯片结果进行验证分析。结果：在27例CTDs患者中,芯片检测发现1例22q11.2区域的微缺失,3例22q11.2区域的微重复。实时定量荧光PCR验证结果与芯片一致。结论：通过8×15K定制基因芯片检测技术对CTDs患者进行检测,为14.8%(4/27)的患者明确了病因,其中3.7%(1/27)为22q11.2微缺失,11.1%(3/27)为22q11.2微重复。这款定制基因芯片作为一种快速、准确的染色体病研究新手段,在先天畸形病因诊断中有重大意义。第二部分 微阵列比较基因组杂交技术在检测腹壁缺损染色体不平衡畸变的运用目的：腹裂和脐膨出是两类常见但发病机制不同的腹壁缺损畸形,本实验旨在用array-CGH技术探索两类疾病中新的及相同的基因组拷贝数变化,探讨array-CGH技术在诊断和产前诊断不平衡染色体畸变的中的应用价值。方法：5例腹裂及5例脐膨出引产胎儿,利用德国QIAGEN公司gDNA抽提试剂盒提取gDNA。利用Agilent 244K array-CGH技术进行全基因组CNVs检测。利用德国Applied Biosystems公司生产的ViiA 7 Real-time PCR仪进行SYBR Green实时定量荧光PCR对芯片结果进行验证分析。结果：在5例腹裂胎儿中,array-CGH检测未发现CNVs。在5例脐膨出胎儿中,array-CGH检测1例胎儿未发现CNVs; 1例胎儿为18三体；编号562胎儿存在4个染色体区域缺失,分别为del(5) (q.13.1-13.2), del(10)(q21.3-22.1), del(14)(q13.1-13.2)和del(14)(q21.3-22.1);编号569胎儿存在4个染色体区域重复,分别为dup(4)(p16.3-16.1), dup(7)(p22.1), dup(8)(q24.3)和dup(9)(q34.3);编号598胎儿存在6个染色体区域重复,分别为dup(2)(p22.1), dup(2)(q31.2),dup(2)(q34), dup(3)(q28-29), dup(10)(q21.3)和dup(22)(q12.3)。2例胎儿中存在相同区域的CNVs,即10q21.3。所有array-CGH技术检测结果均经实时定量荧光PCR技术验证,验证结果与上述array-CGH结果一致。结论：1.Array-CGH技术是一种全新的现代化分子核型分析技术,是遗传学研究领域里的一项重大突破,将染色体病的诊断水平精确提高到基因水平上。其分辨率和准确性极高,有效地克服或弥补了现有的染色体诊断技术的局限性。2.腹裂畸形发生机制中染色体畸变的可能性小。3.脐膨出畸形发生机制中染色体畸变的可能性大,未发现明显的共同致病性CNVs。产前发现脐膨出畸形是进一步遗传学诊断和遗传咨询的标志。
[Abstract]:Patients with 22q11.2 microdeletion and microduplication malformation the gene chip detection section by using the known causes were verified gene chip to the first part conotruncus: detection of known chromosomal defect cases with Agilent gene chip, comparing the experimental results, the accuracy of specific gene chip. Methods: select the past with multiple connections dependent probe amplification (multiplex ligation-dependent probe amplification, MLPA) in 10 cases with velocardiofacial syndrome diagnosed in patients with Cytogenetic and Whole-Genome 2.7M gene chip confirmed 2 cases of syndrome patients, this research adopts Agilent 8 * 15K gene chip detection method, and the results were analyzed by Agilent Genomic Workbench Lite Edition version 6.5 results: in 12 cases, gene chip was detected with MLPA and genomic core Between the genomic copy number variation (copy number, variations, CNVs), including 8 cases of velocardiofacial syndrome patients for microdeletions of 22q11.2 region, 2 cases with velocardiofacial syndrome patients with micro repeat 22q11.2 regions, and 2 cases of patients with 5q syndrome. Conclusion: the absence of part of the 8 x 15K gene chip can quickly and accurately detect the 22q11.2 deletion or duplication of chromosome aberration, criduchat syndrome and other 11 kinds of diseases, so as to provide reliable genetic information. The second section application of gene chip to detect 27 cases of chromosomal aberration in CTDs patients with variable Objective: to analyze 27 conotruncal defects (conotruncal defects, CTDs) genetic etiology of patients, especially the pathogenicity of CNVs 22q11.2 region occurrence probability and proportion for genetic counseling, the next step of risk assessment, and provide a theoretical basis for the follow-up treatment. Methods: simple 27 cases of patients with CTDs, the German company QIAGEN gDNA extraction kit were used to extract gDNA. Agilent 8 * 15K gene chip method for CNVs detection, especially on chromosome aberrations in 22q11.2 region. The German Applied Biosystems company production of ViiA 7 Realtime PCR SYBR Green for real-time quantitative PCR analysis to validate the results of microarray. Results: in 27 CTDs patients, 1 cases of chip detection deletion in 22q11.2 region, 3 cases of 22q11.2 microsatellite repeat regions. Real time fluorescence quantitative PCR results consistent with chip. Conclusion: the 8 * 15K gene chip detection technology for the detection of CTDs patients was 14.8% (4/27) of the patients with the etiology. 3.7% of them (1/27) for the 22q11.2 deletion, 11.1% (3/27) 22q11.2 microsatellite repeat. This gene chip is a fast and accurate method to study the new chromosome disease, in congenital malformation There is great significance in etiological diagnosis. Comparative genomic hybridization microarray in detection of second part of unbalanced chromosome aberration by abdominal wall defect Objective: gastroschisis and omphalocele are two common but different pathogenesis of abdominal wall defect deformity, explore the new changes in the number and the same genomic copy and two kinds of diseases by using array-CGH technology to the the application of array-CGH technology in diagnosis and prenatal diagnosis of unbalanced chromosome aberration in 5 cases. Methods: 5 cases of omphalocele and gastroschisis of fetus, the German company QIAGEN gDNA extraction kit to extract gDNA. genome CNVs detected by Agilent 244K array-CGH technology. Using the German Applied Biosystems company ViiA 7 Real-time PCR SYBR Green for real-time quantitative PCR analysis to validate the results of microarray. Results: in 5 cases of fetal gastroschisis, array- CGH妫，

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