- 學位論文
建立診斷唐氏症白血病危險因子之高敏感性分子工具
The development of high sensitive diagnostic methods for Leukemia in Down syndrome
摘要
背景 唐氏症(Down syndrome）是最常見與智能障礙有關的非整倍體(aneuploidy)遺傳疾病，發生率為800分之一。唐氏症患者將會有多重系統徵狀出現，包括身材矮小、智能障礙、畸形、先天性心臟病、先天性胃腸道與泌尿生殖道異常、內分泌功能異常、白血病(Leukemia)及類白血病反應(Leukemoid reaction)。唐氏症患者罹患白血病的機率比一般人高出10到20倍，形成急性巨核細胞白血病(Acute Megakaryoblastic Leukemia；簡稱AMKL)的機率比一般人高出500倍，引發早期造血功能疾病的成因是在體細胞(somatic cell)分裂階段而產生GATA1基因突變(mutation)，造成暫時性骨髓增生疾病(TMD)和急性巨核細胞白血病(AML)。 研究動機 唐氏症白血病大多是與GATA1基因突變有關。由於病人採血不易，因此我們希望能夠建立藉由血片(Dried blood spot；簡稱DBS)萃取DNA來進行GATA1基因定序檢測的方式。 方法 本研究是以血片萃取DNA檢體進行聚合酶鏈鎖反應及Sanger sequencing方法做DNA定序進行變異點檢測，共分析三組病人的GATA1基因是否有突變。第一組為回溯性分析患者24人，其中包含9位有白血球異常(TMD 3人；AMKL 6人)、13位為唐氏症患者但無白血病及2位非唐氏患者但有白血病患者(AMKL 2人)。第二組為前瞻性分析採用本院唐氏確診患者24人，第三組檢體來源為蒙古確診為唐氏症沒有白血病患者39人。 結果 回溯性分析結果呈現24位患者中有8位檢測出GATA1基因突變，結果分別在DS-TMD患者檢出率為100% (3/3)，DS-AMKL患者檢出率為83.3%(5/6)。而前瞻性分析結果顯示本院24位患者當中只有一人年齡約三歲男童檢測出GATA1基因突變，突變偵測率為4.2%。但在蒙古39位患者檢測結果顯示GATA1基因並沒有任何突變(wild type)，突變偵測率為0%。 結論 本研究建立了以血片抽取出的DNA用Sanger定序法針對GATA1基因突變點位檢測的方式。在未來，利用這設計而建立出診斷唐氏症白血病危險因子之高敏感性分子工具，可提供醫師對唐氏症病程的預估及治療做規劃。
並列摘要
Background Down syndrome (DS) is the most common disorders associated with intelligent disabilities in aneuploidy disorders with an incidence of one in 800 live births. Patients with DS present with symptoms of multi-systemic manifestations, including short stature, mental retardation, malformations, congenital heart disease, congenital gastrointestinal and genitourinary tract abnormalities, endocrine dysfunction, Leukemia and Leukemoid reaction. DS patients had 10-20 times higher risk to develop Leukemia, especially the Acute Megakaryoblastic Leukemia (AMKL) which is even 500 times higher. The cause of Leukemia in DS is considered to be related to the GATA1 mutation, resulting in a temporary myeloproliferative disorders and acute megakaryocytic leukemia (AML). DS and leukemia mostly GATA1 gene mutation. Because of patient blood sampling is difficult, so we hope to establish a method to use blood spots (Dried blood spot; referred DBS) to extract DNA sequencing to detect gene GATA1. Method DNA was extracted from dried blood spots were proceeded into polymerase chain reaction and Sanger sequencing method for GATA1 mutation analysis. This study analyzed three groups of patients. The first group was a retrospective analysis of 24 patients, of whom nine had myeloproliferative disorders (TMD 3 people; AMKL 6 people), 13 DS patients without Leukemia and two AMLK patients but not DS. The second group was prospectively analyzed 24 DS patients in our hospital. The third group of the sample was from 39 Mongolia DS patients without Leukemia. Result Retrospective analysis of 24 patients revealed eight DS patients had positive GATA1 mutations, with the detection rate was 100% (3/3) in DS-TMD; and 83.3% (5/6) in DS-AMLK respectively. The prospective analysis of 24 patients in our hospital showed only one positive for GATA1 mutation in a 3 year-old boy with the detection rate of 4.2%. In 39 Mongolian samples, none was detected to have GATA1 mutation (0%). Conclusion From this study, we established the method to analyze GATA1 mutation from DBS samples. In the future, the utilization of this method could help in the early diagnosis of Leukemia in DS patients, providing an early treatment to achieve better outcome.