染色体微阵列技术在先天性唇腭裂畸形患者中的应用

发布时间：2018-06-04 13:15

本文选题：先天性唇腭裂 + 染色体微阵列分析　；参考：《广州医科大学》2014年硕士论文

【摘要】：目的 1、探讨全基因组高分辨率染色体微阵列技术(chromosome microarrayanalysis, CMA)在先天性唇腭裂畸形患者中的应用价值。 2、探讨CMA技术在单纯性唇裂(isolated cleft lip, CL)、单纯性腭裂(isolated cleftpalate, CP)以及唇腭裂（cleft lip with palate,CLP）患者之间其致病性拷贝数变异(copy number variants,CNVs)检出率的差异性。 3、为建立先天性唇腭裂畸形的遗传学诊断以及产前诊断的临床操作流程提供理论依据。 4、为探讨应用CMA技术在先天性唇腭裂畸形遗传学诊断及咨询中如何最大程度减少临床意义不明确的CNVs（variants of unknown significance,VOUS）结果解释困惑的策略提供实验室支持。方法 1、选取自2012年8月至2013年8月在广州市妇女儿童医疗中心口腔科确诊、并接受手术矫正治疗的各种类型先天性唇腭裂畸形、伴或不伴发其他畸形的临床患儿共计33例核心家系样本：非综合症型唇腭裂患儿29例，包含了单纯性CL患儿10例，单纯性CP患儿8例，CLP患儿11例；综合征型唇腭裂患儿4例，包含了唇腭裂畸形合并先天性心脏病患儿3例，Van der Woude’s综合症患儿1例。 2、所有病例均事先经常规G显带染色体核型分析，只有染色体核型正常者才进一步行CMA分析。 3、分别使用Qiagen DNA提取试剂盒从患者外周血中提取基因组DNA，并使用NanoDrop分光光度计对DNA的浓度和纯度进行测量。 4、根据美国Affymetrix公司的CytoScan HD芯片平台（195万拷贝数探针和75万SNP探针）的标准实验操作流程对样本DNA进行处理。 5、使用相配套的CHAS软件对扫描芯片产生的.CEL文件进行数据分析。 6、根据DGV（含正常人的CNVs）、DECIPHER（含患者的表型及致病性片段）、OMIM（含已知的致病基因）、CAGdb、ISCA（含良性与致病性的CNVs）、UCSCGenome Browser（显示片段中基因的内容及功能）及PUBMED等以及本实验室的内部数据库对分析结果进行在线比对，判断CNVs的性质。 7、针对临床意义不明确的CNVs结果，进一步行父母样本检测进行综合家系分析，明确CNVs的性质。 8、使用实时荧光定量PCR（real-time polymerase chain reaction, RT-PCR）对致病性CNVs进行验证。结果 1、全部33例患儿均成功进行了全基因组高分辨率CMA技术检测，CMA结果提示6例患儿含有致病性CNVs，致病性CNVs检出率为18.2%(6/33)；26例患儿含有良性CNVs，良性CVNs检出率为78.8%（26/33）；1例患儿含有临床意义不明确的CNVs（VOUS），VOUS检出率为3.0%（1/33）。 2、CMA结果提示6例含致病性CNVs的患儿中，4例（13.8%，4/29）来源于非综合症型唇腭裂患儿，包含了3例单纯性CP患儿（37.5%，3/8），1例CLP患儿（9.1%，1/11）；致病性CNVs分别为10q22.2-q22.3微缺失（1766kb）、20p12.1微缺失（184kb）、22q11.21-q11.23微缺失（3163kb）、8p23.1微重复（198kb）；此外，10q22.2-q22.3区域中的MYST4基因，20p12.1区域中的MACROD2基因是新发现的先天性唇腭裂可疑致病基因。2例（50%，2/4）来源于综合症型唇腭裂患儿，致病性CNVs分别为18q12.3微重复（638kb）、6q26微缺失（389kb）。 3、CMA结果提示26例含良性CNVs的患儿中，，19例患儿的CNVs存在于DGV数据库/CHOP数据库中，最常见的4种（P≥5%）分别为8p11.2微缺失/微重复（11.9%）、14q32.33微重复（18.1%）、14q11.2微缺失（5.8%）、22q11.22微重复（11.9%）；4例患儿的CNVs来源于正常父母一方，分别为9q31.1微重复、10p12.33微缺失、7q31.1微缺失、Xp22.33微重复，为国际上首次报道。 4、CMA结果提示1例患儿含有临床意义仍然不明确CNVs，该VOUS为5q21.1微缺失（110kb）。结论 1、全基因组高分辨率CMA技术在先天性唇腭裂畸形而染色体核型正常的患儿中具有重要的应用价值，该技术能够将致病性CNVs的总体检出率额外提高18.2%，并且具有识别新的致病基因的能力。 2、CMA技术对唇腭裂畸形合并其他结构异常的综合征型患儿其致病性CNVs检出率高于单一唇腭裂畸形的非综合征型唇腭裂患儿（50%vs13.8%）。 3、在非综合征性唇腭裂患儿中，致病性CNVs检出率从高到低排列为单纯性CP(37.5%)CLP(9.1%)单纯性CL（0）。单纯性CL患儿其基因组发生不平衡变异的风险较低。 4、在临床遗传学诊断和产前诊断中，对于常规染色体核型分析未见异常的单纯性CP、CLP、以及先天性唇腭裂畸形合并其他结构异常的临床病例，建议进一步行全基因组高分辨率CMA技术分析。 5、实验室数据分析人员和临床遗传咨询医生之间的充分交流、结合家系综合分析以及内外部数据库之间的数据比对都能够显著降低临床意义不明确的CNVs(VOUS)。
[Abstract]:objective
1, to explore the application value of chromosome microarrayanalysis (CMA) in patients with congenital cleft lip and palate.
2, to explore the difference in the prevalence of CMA in isolated cleft lip (CL), pure cleft palate (isolated cleftpalate, CP) and cleft lip and palate (cleft lip with palate).
3, to provide theoretical basis for establishing genetic diagnosis and prenatal diagnosis of congenital cleft lip and palate.
4, we provide laboratory support for exploring the strategy of using CMA technology to explain the puzzled strategies of CNVs (variants of unknown significance, VOUS) in the genetic diagnosis and consultation of congenital cleft lip and palate deformity.
Method
1, selected from August 2012 to August 2013 in the Department of Stomatology, Guangzhou women and children's Medical Center, all kinds of congenital cleft lip and palate deformity treated by surgical correction, 33 cases of children with or without other malformed clinical children: 29 cases of children with non syndrome cleft lip and palate, including 10 children with simple CL. 8 children with simple CP, 11 children with CLP, 4 children with cleft lip and palate, 3 cases of cleft lip and palate with congenital heart disease, and 1 children with Van der Woude 's syndrome.
2, all cases were analyzed regularly by G chromosome karyotype. Only CMA with normal karyotype was further analyzed.
3, genomic DNA was extracted from peripheral blood of patients by Qiagen DNA extraction kit, and the concentration and purity of DNA were measured by NanoDrop spectrophotometer.
4, the sample DNA is processed according to the standard experimental operation procedure of CytoScan HD chip platform (1 million 950 thousand copy number probe and 750 thousand SNP probe) of Affymetrix company.
5, use the matching CHAS software to analyze the.CEL files produced by the scanning chip.
6, according to DGV (including normal CNVs), DECIPHER (including the patient's phenotypic and pathogenetic fragments), OMIM (including known pathogenic genes), CAGdb, ISCA (benign and pathogenicity CNVs), UCSCGenome Browser (the contents and functions of the gene in the display segment) and PUBMED and so on, and the internal database of our laboratory to compare the analysis results online, judge the results. The nature of CNVs.
7, in view of the unclear clinical results of CNVs, parents were further tested for comprehensive family analysis to clarify the nature of CNVs.
8, real-time polymerase chain reaction (RT-PCR) was used to verify the pathogenicity CNVs of PCR.
Result
1, all 33 cases had successful complete genome high resolution CMA test. CMA results showed that 6 cases had pathogenicity CNVs, pathogenetic CNVs detection rate was 18.2% (6/33), 26 cases with benign CNVs, benign CVNs detection rate of 78.8% (26/33), 1 children with clinically significant CNVs (VOUS), VOUS detection rate 3% (1 /33).
2, CMA results suggested that of the 6 children with pathogenic CNVs, 4 (13.8%, 4/29) were derived from children with non syndromic cleft lip and palate, including 3 children with simple CP (37.5%, 3/8), 1 cases of CLP (9.1%, 1/11); pathogenic CNVs were 10q22.2-q22.3 microdeletion (1766kb), 20p12.1 microdeletion (184kb), microsatellite microdeletion. (198kb); in addition, the MYST4 gene in the 10q22.2-q22.3 region and the MACROD2 gene in the 20p12.1 region are the newly discovered congenital lip and palate cleft suspected pathogenic gene.2 (50%, 2/4) from children with syndrome type lip and palate. The pathogenic CNVs is 18q12.3 microrepetition (638kb), 6q26 microdeletion (389kb).
3, CMA results showed that of the 26 children with benign CNVs, 19 cases were found in the DGV database /CHOP database, and the most common 4 species (P > 5%) were 8p11.2 microdeletion / micro repetition (11.9%), 14q32.33 microrepetition (18.1%), 14q11.2 microdeletion (5.8%), 22q11.22 microrepetition (11.9%), 4 cases of CNVs from the normal parents, respectively. It is the first report in the world for 9q31.1 duplication, 10p12.33 microdeletions, 7q31.1 microdeletions and Xp22.33 microduplication.
4, CMA results indicate that the clinical significance of 1 cases is still unclear, CNVs, and the VOUS is 5q21.1 microdeletion (110kb).
conclusion
1, the whole genome high resolution CMA technique is of great value in children with congenital cleft lip and palate with normal karyotype. This technique can increase the total physical examination rate of pathogenicity CNVs by an additional 18.2% and have the ability to identify new pathogenic genes.
2, the incidence of pathogenic CNVs in children with cleft lip and palate with other structural abnormalities is higher than that of non syndromic cleft lip and palate children with single cleft palate and cleft palate (50%vs13.8%).
3, in children with non syndromic cleft lip and palate, the incidence of pathogenicity CNVs was from high to low to simple CP (37.5%) CLP (9.1%) simple CL (0). The risk of unbalanced genomes in children with simple CL was low.
4. In clinical genetic diagnosis and prenatal diagnosis, there is no abnormal simple CP, CLP, and congenital cleft lip and Palate Malformation with other structural abnormalities in clinical genetics diagnosis and prenatal diagnosis. It is suggested that the whole genome high resolution CMA technique should be further analyzed.
5, full communication between laboratory data analysts and clinical genetic counselling doctors, combined with family analysis and data comparisons between internal and external databases, can significantly reduce the CNVs (VOUS) that is not clear in clinical significance.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.2

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