本文關(guān)鍵詞： 腹裂 臍膨出 微陣列比較基因組雜交技術(shù) 拷貝數(shù)變異 定制基因芯片 拷貝數(shù)變異 腭心面綜合征 貓叫綜合癥 定制基因芯片 圓錐動脈干畸形 22q11微缺失 22q11微重復　出處：《復旦大學》2014年博士論文　論文類型：學位論文

【摘要】：第一部分圓錐動脈干畸形患者22q11.2微缺失和微重復的定制基因芯片檢測第一節(jié)利用已知病因的病例驗證定制基因芯片目的：用Agilent定制基因芯片對已知染色體缺陷的病例進行檢測,比較實驗結(jié)果,明確定制基因芯片的準確性。方法：選出以往用多重連接依賴探針擴增技術(shù)(multiplex ligation-dependent probe amplification, MLPA)確診的10例腭心面綜合征患者和Cytogenetic Whole-Genome 2.7M基因芯片確診的2例貓叫綜合癥患者的血清,采用本研究的Agilent 8×15K定制基因芯片方法檢測,并用Agilent Genomic Workbench Lite Edition 6.5版進行結(jié)果分析。結(jié)果：12例病例中,定制基因芯片檢測出了與MLPA及全基因組芯片一致的基因組拷貝數(shù)變異(copy number variations, CNVs),其中8例腭心面綜合征患者為22q11.2區(qū)域的微缺失,2例腭心面綜合征患者為22q11.2區(qū)域的微重復,而2例貓叫綜合癥患者為5q的部分缺失。結(jié)論：這款8×15K定制基因芯片能夠快速而準確的檢測出22q11.2微缺失或微重復、貓叫綜合征等11種疾病的染色體畸變,從而提供可靠的遺傳學信息。第二節(jié)應用定制基因芯片檢測27例CTDs患者的染色體畸變目的：分析27名圓錐動脈干畸形(conotruncal defects, CTDs)患者的遺傳學病因,特別是22q11.2區(qū)域的致病性CNVs發(fā)生概率及比例,為下一步的遺傳學咨詢、風險評估、后續(xù)治療等提供理論基礎(chǔ)。方法：選擇單純的CTDs患者27例,利用德國QIAGEN公司gDNA抽提試劑盒提取gDNA。采用Agilent 8×15K定制基因芯片方法進行CNVs檢測,尤其關(guān)注22q11.2區(qū)域的染色體畸變。利用德國Applied Biosystems公司生產(chǎn)的ViiA 7 Realtime PCR儀進行SYBR Green實時定量熒光PCR對芯片結(jié)果進行驗證分析。結(jié)果：在27例CTDs患者中,芯片檢測發(fā)現(xiàn)1例22q11.2區(qū)域的微缺失,3例22q11.2區(qū)域的微重復。實時定量熒光PCR驗證結(jié)果與芯片一致。結(jié)論：通過8×15K定制基因芯片檢測技術(shù)對CTDs患者進行檢測,為14.8%(4/27)的患者明確了病因,其中3.7%(1/27)為22q11.2微缺失,11.1%(3/27)為22q11.2微重復。這款定制基因芯片作為一種快速、準確的染色體病研究新手段,在先天畸形病因診斷中有重大意義。第二部分 微陣列比較基因組雜交技術(shù)在檢測腹壁缺損染色體不平衡畸變的運用目的：腹裂和臍膨出是兩類常見但發(fā)病機制不同的腹壁缺損畸形,本實驗旨在用array-CGH技術(shù)探索兩類疾病中新的及相同的基因組拷貝數(shù)變化,探討array-CGH技術(shù)在診斷和產(chǎn)前診斷不平衡染色體畸變的中的應用價值。方法：5例腹裂及5例臍膨出引產(chǎn)胎兒,利用德國QIAGEN公司gDNA抽提試劑盒提取gDNA。利用Agilent 244K array-CGH技術(shù)進行全基因組CNVs檢測。利用德國Applied Biosystems公司生產(chǎn)的ViiA 7 Real-time PCR儀進行SYBR Green實時定量熒光PCR對芯片結(jié)果進行驗證分析。結(jié)果：在5例腹裂胎兒中,array-CGH檢測未發(fā)現(xiàn)CNVs。在5例臍膨出胎兒中,array-CGH檢測1例胎兒未發(fā)現(xiàn)CNVs; 1例胎兒為18三體；編號562胎兒存在4個染色體區(qū)域缺失,分別為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);編號569胎兒存在4個染色體區(qū)域重復,分別為dup(4)(p16.3-16.1), dup(7)(p22.1), dup(8)(q24.3)和dup(9)(q34.3);編號598胎兒存在6個染色體區(qū)域重復,分別為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例胎兒中存在相同區(qū)域的CNVs,即10q21.3。所有array-CGH技術(shù)檢測結(jié)果均經(jīng)實時定量熒光PCR技術(shù)驗證,驗證結(jié)果與上述array-CGH結(jié)果一致。結(jié)論：1.Array-CGH技術(shù)是一種全新的現(xiàn)代化分子核型分析技術(shù),是遺傳學研究領(lǐng)域里的一項重大突破,將染色體病的診斷水平精確提高到基因水平上。其分辨率和準確性極高,有效地克服或彌補了現(xiàn)有的染色體診斷技術(shù)的局限性。2.腹裂畸形發(fā)生機制中染色體畸變的可能性小。3.臍膨出畸形發(fā)生機制中染色體畸變的可能性大,未發(fā)現(xiàn)明顯的共同致病性CNVs。產(chǎn)前發(fā)現(xiàn)臍膨出畸形是進一步遺傳學診斷和遺傳咨詢的標志。
[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媯，

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