微阵列比较基因组杂交技术检测儿童急性淋巴细胞白血病的遗传学异常

发布时间：2018-04-28 09:09

本文选题：急性淋巴细胞白血病 + 微阵列比较基因组杂交　；参考：《广州医学院》2012年硕士论文

【摘要】：儿童急性淋巴细胞白血病是最常见的儿童恶性肿瘤，占儿童癌症的30%。临床上划分治疗风险组依赖发病年龄、诊断时白细胞计数、免疫学、细胞遗传学、泼尼松诱导反应等参数。采用不同的化疗强度可使预后明显改善，尤其是急性淋巴细胞白血病（acutelymphoblastic leukemia，ALL）的临床治愈率接近80%,但15-20%的ALL最终会复发，且复发后的5年无病生存率（Disease FreeSurvival，EFS）仅仅10-40%,是最常见的癌症相关死亡原因。遗憾的是，，部分最初是低风险或者良好细胞遗传学标记的患儿也会面临治疗失败或复发，这说明白血病隐藏了更隐匿更显微的遗传物质改变。可见传统的细胞和分子遗传学技术虽然在检测染色体数目或结构变异上各有优势，但对亚显微的遗传物质改变的检测却受到种种限制，例如传统的染色体检查方法G显带核型分析技术，虽然可以从宏观上分析全套染色体的数目和结构异常，但是其分辨率低，最好约为5-10Mb，白血病细胞培养失败和正常细胞过度生长占40%比例，且不同实验室之间对核型解释也有差异。荧光原位杂交（Fluorescence insitu Hybridization，FISH）虽然可以检测到染色体的亚显微结构异常（分辨率由探针大小决定，可以达到100Kb），但却要已知明确或可疑异常的染色体异常，所以仅有少数几个有针对性的探针用于检测几个DNA变异区域，不能做到全基因组筛查。微阵列比较基因组杂交（array comparative genomic hybridization，array CGH）是一种高分辨率、高通量、高效率的全基因组筛查技术，能检测到亚显微的染色体异常（微重复或微缺失）、精确定位断裂点，分辨率高，达100kb，有效地弥补了现有方法的局限性，对于非平衡染色体畸变是一个强有力的工具。本研究采用该技术检测急性淋巴细胞性白血病患儿的遗传学异常，评估其在儿童急性淋巴细胞性白血病遗传学诊断上的应用价值。一、目的 1利用微阵列比较基因组杂交技术分析儿童急性淋巴细胞性白血病患儿的遗传学异常，探讨其在白血病遗传学异常检测中的应用价值。 2对儿童急性淋巴细胞白血病患儿进行全基因组DNA拷贝数异常（CNVs）分析，发现常见的染色体异常区域。二、方法 1将19例患儿提取骨髓基因组DNA后按Affymetrix公司的CytoScanHD Array实验操作指南进行全基因组杂交与扫描，再用ChromosomeAnalysis Suite分析软件进行全基因组拷贝数变异分析。 2数据库及文献比对分析（1）查询database of genomic variants(http://pro ects.tcag.ca/variation; GRCh37, Feb2009),排除多态性CNVs。（2）查询OMIM、NCBI、Ensembl数据库了解涉及的染色体片段及基因的是否与白血病相关。（3）通过检索词“leukemia”、“cancer”、“Acute lymphoblasticleukemia”、“children”、“copy number aberration”、“arrayComparative Genomic Hybridization”、“genetics”、涉及CNVs的染色体或基因，如“1q44”、“IKZF1”等相互组合在pubmed上搜索文献，与实验结果比较。三、结果 1、每个患儿均检出涉及4-11条染色体的拷贝数变化，最常涉及的有拷贝数变化最小共同区域（minimal common regions，MCR）的有1q44、8p11.22、9p21.3、12p13.31、12q21.3、14q32.33、22q11.22。 2、几个已经证实的白血病相关基因及癌症候选基因包括CDKN2A、CDKN2B、PAX5、 BTG1、IKZF1、OR2C3、VPREB1在少量的样本中被检测到，对于ETV6（虽然常涉及易位，但也会有缺失），JAK2（致病性与基因突变相关），ARID5B等未能检出可能与样本数量少有关。 3、患儿0819251临床诊断为ALL-L2,Pro-B,HR，BCR-ABL1阴性，array-CGH发现同时有CDKN2A/CDKN2B（9p21.3），PAX5（9p）和IKZF1（7p12.2）的CNL，且这几个拷贝数缺失分别为101Kb，118Kb，109Kb。这几个基因的拷贝数减少与临床进展快，早期复发有关。而该患儿复发早，进展快，与之相符，拷贝数大小均在200Kb以下，极大的体现了array-CGH的高敏感性高分辨率。四、结论 1olig-array CGH在白血病遗传学分析上有重要价值，它能轻易发现隐藏在5Mb的片段里的疾病候选基因或相关基因，并能精确定位断裂点。 2olig-芯片在肿瘤中的的应用也有局限性，若以白血病拷贝数变化为研究目的，应改进分析软件的计算方法或减少样本的生物学复杂性。
[Abstract]:Children with acute lymphoblastic leukemia is the most common malignant tumor of children. The 30%. clinical division of cancer in children depends on the age, leukocyte count, immunology, cytogenetics, prednisone induced reaction and other parameters. The use of different intensity of chemotherapy can improve the prognosis, especially in acute lymphocytes. The clinical cure rate of acutelymphoblastic leukemia (ALL) is close to 80%, but 15-20% ALL will eventually relapse, and the 5 year disease-free survival (Disease FreeSurvival, EFS) after recurrence is only 10-40%, which is the most common cause of cancer related death. In the face of failure or recurrence of treatment, this suggests that leukaemia conceals a more occult and more microscopic genetic change. Although traditional cell and molecular genetic techniques have advantages in detecting the number of chromosomes or structural variations, the detection of submicroscopic genetic changes, such as traditional chromosome tests, is limited. Methods G banding karyotype analysis technique, although the number and structural abnormalities of the whole set of chromosomes can be analyzed macroscopically, but the resolution is low, the best is about 5-10Mb, the failure of the leukemia cell culture and the normal cell overgrowth are 40%, and the karyotype interpretation between different laboratories is also different. Fluorescence in situ hybridization (Fluorescence insit) U Hybridization, FISH), although the submicroscopic structure of chromosomes can be detected (resolution is determined by the size of the probe, can reach 100Kb), but a clear or suspicious abnormal chromosome abnormality is known, so only a few targeted probes are used to detect several DNA variation regions and can not be screened for whole genome. Array comparative genomic hybridization (array CGH) is a high resolution, high throughput, high efficiency full genome screening technique, which can detect submicroscopic chromosomal abnormalities (microduplication or microdeletion), accurately locate breakpoints, and reach a high resolution, and reach 100kb, which effectively compensates for the limitations of existing methods. Nonbalanced chromosome aberration is a powerful tool. This study used this technique to detect genetic abnormalities in children with acute lymphoblastic leukemia and evaluate its application in the genetic diagnosis of acute lymphoblastic leukemia in children.
First, the purpose
1 the genetic abnormalities of children with acute lymphoblastic leukemia were analyzed by microarray comparative genomic hybridization, and the value of their application in the detection of genetic abnormalities in leukemia was discussed.
2 a total genomic DNA copy number (CNVs) analysis was performed in children with acute lymphoblastic leukemia, and common chromosomal abnormalities were found.
Two, method
1 after extracting the bone marrow genomic DNA from 19 children, the whole genome hybridization and scanning were carried out according to the CytoScanHD Array experimental guidelines of Affymetrix company and the whole genome copy number variation analysis was carried out by ChromosomeAnalysis Suite analysis software.
Comparison and analysis of 2 databases and Literature
(1) query database of genomic variants (http://pro ects.tcag.ca/variation; GRCh37, Feb2009) to exclude polymorphism CNVs.
(2) query the OMIM, NCBI, Ensembl database to find out whether the chromosome segments and genes are related to leukemia.
(3) by searching the words "leukemia", "cancer", "Acute lymphoblasticleukemia", "children", "copy number aberration", "arrayComparative Genomic Hybridization", "Genetics", the chromosomes or genes involved in it, such as "", "Genetics", and so on, search each other on the literature and compare with the experimental results. Compared to.
Three, the result
1, the number of copies of 4-11 chromosomes was detected in each child, and the most frequently involved minimal common regions, MCR, with 1q44,8p11.22,9p21.3,12p13.31,12q21.3,14q32.33,22q11.22., was 1q44,8p11.22,9p21.3,12p13.31,12q21.3,14q32.33,22q11.22.
2, several confirmed leukemia related genes and cancer candidate genes, including CDKN2A, CDKN2B, PAX5, BTG1, IKZF1, OR2C3, VPREB1, were detected in a small number of samples, for ETV6 (although often involved in translocation, but also missing), JAK2 (pathogenicity associated with gene mutation), ARID5B and other failure detection may be associated with fewer samples.
3, 0819251 of the children were diagnosed as ALL-L2, Pro-B, HR, and BCR-ABL1 negative. Array-CGH found that there were CDKN2A/CDKN2B (9p21.3), PAX5 (9p) and IKZF1 (7p12.2) CNL. The number of copies is below 200Kb, which greatly reflects the perceptual high resolution of Gao Min's array-CGH.
Four. Conclusion
1olig-array CGH is of great value in the genetic analysis of leukemia. It can easily detect the disease candidate genes or related genes hidden in the 5Mb fragments, and can accurately locate the breakpoints.
The application of 2olig- chip in the tumor is also limited. If the copy number of leukemia is studied, the computational method of the analysis software should be improved or the biological complexity of the sample should be reduced.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R733.71

【参考文献】