本文選題：急性淋巴細胞白血病 + 微陣列比較基因組雜交��； 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：兒童急性淋巴細胞白血病是最常見的兒童惡性腫瘤，占兒童癌癥的30%。臨床上劃分治療風(fēng)險組依賴發(fā)病年齡、診斷時白細胞計數(shù)、免疫學(xué)、細胞遺傳學(xué)、潑尼松誘導(dǎo)反應(yīng)等參數(shù)。采用不同的化療強度可使預(yù)后明顯改善，尤其是急性淋巴細胞白血�。╝cutelymphoblastic leukemia，ALL）的臨床治愈率接近80%,但15-20%的ALL最終會復(fù)發(fā)，且復(fù)發(fā)后的5年無病生存率（Disease FreeSurvival，EFS）僅僅10-40%,是最常見的癌癥相關(guān)死亡原因。遺憾的是，，部分最初是低風(fēng)險或者良好細胞遺傳學(xué)標(biāo)記的患兒也會面臨治療失敗或復(fù)發(fā)，這說明白血病隱藏了更隱匿更顯微的遺傳物質(zhì)改變。可見傳統(tǒng)的細胞和分子遺傳學(xué)技術(shù)雖然在檢測染色體數(shù)目或結(jié)構(gòu)變異上各有優(yōu)勢，但對亞顯微的遺傳物質(zhì)改變的檢測卻受到種種限制，例如傳統(tǒng)的染色體檢查方法G顯帶核型分析技術(shù)，雖然可以從宏觀上分析全套染色體的數(shù)目和結(jié)構(gòu)異常，但是其分辨率低，最好約為5-10Mb，白血病細胞培養(yǎng)失敗和正常細胞過度生長占40%比例，且不同實驗室之間對核型解釋也有差異。熒光原位雜交（Fluorescence insitu Hybridization，F(xiàn)ISH）雖然可以檢測到染色體的亞顯微結(jié)構(gòu)異常（分辨率由探針大小決定，可以達到100Kb），但卻要已知明確或可疑異常的染色體異常，所以僅有少數(shù)幾個有針對性的探針用于檢測幾個DNA變異區(qū)域，不能做到全基因組篩查。微陣列比較基因組雜交（array comparative genomic hybridization，array CGH）是一種高分辨率、高通量、高效率的全基因組篩查技術(shù)，能檢測到亞顯微的染色體異常（微重復(fù)或微缺失）、精確定位斷裂點，分辨率高，達100kb，有效地彌補了現(xiàn)有方法的局限性，對于非平衡染色體畸變是一個強有力的工具。本研究采用該技術(shù)檢測急性淋巴細胞性白血病患兒的遺傳學(xué)異常，評估其在兒童急性淋巴細胞性白血病遺傳學(xué)診斷上的應(yīng)用價值。 一、目的 1利用微陣列比較基因組雜交技術(shù)分析兒童急性淋巴細胞性白血病患兒的遺傳學(xué)異常，探討其在白血病遺傳學(xué)異常檢測中的應(yīng)用價值。 2對兒童急性淋巴細胞白血病患兒進行全基因組DNA拷貝數(shù)異常（CNVs）分析，發(fā)現(xiàn)常見的染色體異常區(qū)域。 二、方法 1將19例患兒提取骨髓基因組DNA后按Affymetrix公司的CytoScanHD Array實驗操作指南進行全基因組雜交與掃描，再用ChromosomeAnalysis Suite分析軟件進行全基因組拷貝數(shù)變異分析。 2數(shù)據(jù)庫及文獻比對分析 （1）查詢database of genomic variants(http://pro ects.tcag.ca/variation; GRCh37, Feb2009),排除多態(tài)性CNVs。 （2）查詢OMIM、NCBI、Ensembl數(shù)據(jù)庫了解涉及的染色體片段及基因的是否與白血病相關(guān)。 （3）通過檢索詞“l(fā)eukemia”、“cancer”、“Acute lymphoblasticleukemia”、“children”、“copy number aberration”、“arrayComparative Genomic Hybridization”、“genetics”、涉及CNVs的染色體或基因，如“1q44”、“IKZF1”等相互組合在pubmed上搜索文獻，與實驗結(jié)果比較。 三、結(jié)果 1、每個患兒均檢出涉及4-11條染色體的拷貝數(shù)變化，最常涉及的有拷貝數(shù)變化最小共同區(qū)域（minimal common regions，MCR）的有1q44、8p11.22、9p21.3、12p13.31、12q21.3、14q32.33、22q11.22。 2、幾個已經(jīng)證實的白血病相關(guān)基因及癌癥候選基因包括CDKN2A、CDKN2B、PAX5、 BTG1、IKZF1、OR2C3、VPREB1在少量的樣本中被檢測到，對于ETV6（雖然常涉及易位，但也會有缺失），JAK2（致病性與基因突變相關(guān)），ARID5B等未能檢出可能與樣本數(shù)量少有關(guān)。 3、患兒0819251臨床診斷為ALL-L2,Pro-B,HR，BCR-ABL1陰性，array-CGH發(fā)現(xiàn)同時有CDKN2A/CDKN2B（9p21.3），PAX5（9p）和IKZF1（7p12.2）的CNL，且這幾個拷貝數(shù)缺失分別為101Kb，118Kb，109Kb。這幾個基因的拷貝數(shù)減少與臨床進展快，早期復(fù)發(fā)有關(guān)。而該患兒復(fù)發(fā)早，進展快，與之相符，拷貝數(shù)大小均在200Kb以下，極大的體現(xiàn)了array-CGH的高敏感性高分辨率。 四、結(jié)論 1olig-array CGH在白血病遺傳學(xué)分析上有重要價值，它能輕易發(fā)現(xiàn)隱藏在5Mb的片段里的疾病候選基因或相關(guān)基因，并能精確定位斷裂點。 2olig-芯片在腫瘤中的的應(yīng)用也有局限性，若以白血病拷貝數(shù)變化為研究目的，應(yīng)改進分析軟件的計算方法或減少樣本的生物學(xué)復(fù)雜性。
[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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R733.71

【參考文獻】

相關(guān)期刊論文 前2條

1 欒佐;;造血干細胞移植在遺傳代謝病治療中的應(yīng)用[J];臨床兒科雜志;2006年12期

2 朱照輝;錢軍;林江;錢震;姚冬明;王雅麗;陳芹;肖高飛;;慢性髓系白血病中prame基因轉(zhuǎn)錄本的定量研究[J];中國實驗血液學(xué)雜志;2010年04期

本文編號：1814645