急性巨細胞遽增性白血病首次在1931年由 Von Boros 與 Karangi 所提出，隨著超顯微鏡細胞化學與免疫學表現型技術的發展，增加了診斷出在細胞形態上是屬於巨細胞型急性白血病的機率，也因此 French-American-British Co-operative Group 在1985年正式將急性巨細胞遽增性白血病列為急性骨髓性白血病的第七種亞型，而在患有急性骨髓性白血病的嬰兒當中，最常見的亞型則為第四種、第五種與第七種。一位25天大的嬰兒有白血球增生、肝脾腫大與貧血的情形，再進一步檢查後，被診斷出患有急性巨細胞遽增性白血病，並發現帶有異常染色體組型 46,XY,t(1;1)(p13;p36)。本研究的主要目標是利用目標基因研究方法，找到這位病人位在染色體斷裂點 1p13 與 1p36 上的可能致病基因。在搜尋過 NCBI Map Viewer Database、OMIM Morbid Map、OMIM Gene Map 等等資料庫後，我們找到了位在染色體 1p13 上的有三個可能的候選基因，位在染色體 1p36 上的則有 15 個可能的候選基因。由於 Mitelman Database of Chromosome Aberrations in Cancer 與其他相關的文獻都有關於 RBM15-MKL1 融合基因是為 t(1;22)(p13;q13) 型急性巨細胞遽增性白血病相關基因的報導，因此我們推測在這位 t(1;1)(p13;p36) 型的病人，RBM15 也是位在染色體 1p13 這端的疾病相關基因，在比對了 MKL1 與位在染色體 1p36 上的15 個可能候選基因的 Gene Ontology term 後，我們將 SKI 列為位在染色體 1p36 這端最有可能的目標基因。為了進一步確認 RBM15 與 SKI 是否為 t(1;1)(p13;p36) 型病人的疾病相關基因，我們分別從 cDNA 與 基因體 DNA 兩方面去進行分子特性分析，而根據分析的結果，RBM15 與 SKI 可能為新的 t(1;1)(p13;p36) 型急性巨細胞遽增性白血病相關基因。

Acute megakaryoblastic leukemia (AMKL) was first described by Von Boros and Karangi in 1931, was a result of developments in ultrastructural cytochemistry and immunologic phenotyping acute myeloid leukemia (AML) of megakaryocytic lineage have been diagnosed increasingly. The French-American-British (FAB) Co-operative Group established the criteria for the diagnosis and added this category as a distinct subtype of AML (M7) in 1985. The main subtypes of AML in the infants are M4, M5, and M7. One 25-day-old infant was referred to the hospital for further examination of white blood cell. Hepatosplenomegaly and anemia were physically examined, and he was diagnosed to be an AMKL case. Abnormal karyotype 46,XY,t(1;1)(p13;p36) was observed in this patient. This study aims to identify the AMKL potentially related genes on the breakpoints of Homo sapiens autosomal (HSA) 1p13 and 1p36 in this case by candidate gene approaches. Data-mining of the AMKL potentially related genes on breakpoints of HSA1p13 and 1p36 through NCBI Map Viewer Database, OMIM Morbid Map, and OMIM Gene Map were performed. We identified three candidate genes on HSA1p13 and 15 candidate genes on HSA 1p36. RBM15-MKL1 fusion on t(1;22)(p13;q13) was reported to be AMKL genes by Ma et al., Mercher et al., and the Mitelman Database of Chromosome Aberrations in Cancer. We anticipated RBM15 is also a related gene on HSA1p13 in this AMKL case, and compared the Gene Ontology terms between MKL1 and these 15 candidate genes on HSA1p36. SKI becomes our first candidate gene on 1p36 in this case. To identify candidate genes locating at HSA1p13 and 1p36, including RBM15 and SKI were screened at both cDNA and genomic DNA levels. According to these results, RBM15 and SKI are more likely to be candidate genes. Thus RBM15 and SKI may be the novel AMKL genes in t(1;1)(p13;p36) AMKL patients.

Abstract
Chinese…………………………………………………………………………I
English…………………………………………………………………………II
Abbreviations……………………………………………………………………III
Chapter 1. Acute megakaryoblastic leukemia (AMKL)………………1
1.1. A review of AMKL…………………………………………………………1
1.1.1. Introduction…………………………………………………………………1
1.1.2. The incidence of AMKL……………………………………………………1
1.1.3. The formation, causes, and risk factors of AMKL…………………………3
1.1.4. The association of chromosome abnormalities and AMKL…………5
1.1.4.1. The association of Down syndrome and AMKL………………………8
1.1.4.2. The chromosome breakpoint t(1;22)(p13;q13) of AMKL……………9
1.1.5. The clinical presentation of AMKL………………………………………10
1.1.6. The diagnosis of AMKL……………………………………………………11
1.2. Case study……………………………………………………………………13
Chapter 2. Data-mining for AMKL potentially related loci on breakpoints of HSA1p13 and 1p36….………………………15
2.1. Introduction…………………………………………………………………15
2.2. Materials, methods and results……………………………………………15
2.2.1. Materials…………………………………………………………………15
2.2.2. Methods and results………………………………………………………16
2.2.2.1. Data-mining of AMKL potentially related genes on HSA1p13………………………………………………………………16
2.2.2.2. Data-mining of AMKL potentially related genes on HSA1p36………………………………………………………………20
2.2.2.3. Comparison of the Gene Ontology terms between AMKL related gene MKL1 and 15 AMKL potentially related genes on 1p36……………26
Chapter 3. Molecular characterization on t(1;1)(p13;p36) AMKL…29
3.1. Introduction……………………………………………………………29
3.2. Materials and methods………………………………………………………29
3.2.1. Materials…………………………………………………………………29
3.2.2. cDNA synthesis and amplification………………………………………29
3.2.3. Test of the expression of material cDNA…………………………………33
3.2.4. PT-PCR examination of RBM15 and SKI expression………………37
3.2.5. Quantitative PCR……………………………………………………39
3.2.6. Construction of a BAC library………………………………………42
3.2.7. Test of the coverage of the BAC library.…………………………………45
3.2.8. PCR……………………………………………………………………48
3.3. Results and Discussions……………………………………………………51
3.3.1. cDNA synthesis and amplification………………………………………51
3.3.2. Test of the expression of material cDNA…………………………………52
3.3.3. PT-PCR examination of RBM15 and SKI expression………………54
3.3.4. Quantitative PCR……………………………………………………57
3.3.5. Construction of a BAC library………………………………………60
3.3.6. Test of the coverage of the BAC library……………………………61
3.3.7. PCR……………………………………………………………………63
References………………………………………………………………………67

Athale U. H., Razzouk B. I., Raimondi S. C., Tong X., Behm F. G., Head D. R., Srivastava D. K., Rubnitz J. E., Bowman L., Pui C. H., and Ribeiro R. C. Biology and outcome of childhood acute megakaryoblastic leukemia: a single institution's experience. Blood. 2001; 97(12): 3727-32.
Bader J. L. and Miller R. W. US cancer incidence and mortality in the first year of life. Am J Dis Child. 1979; 133(2): 157-9.
Bennett J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A., Gralnick H. R., and Sultan C. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. Br J Haematol. 1976; 33(4): 451-8.
Bennett J. M., Catovsky D., Daniel M. T., Flandrin G., Galton D. A., Gralnick H. R., and Sultan C. Criteria for the diagnosis of acute leukemia of megakaryocyte lineage (M7). A report of the French-American-British Cooperative Group. Ann Intern Med. 1985; 103(3): 460-2.
Bernstein J., Dastugue N., Haas O. A., Harbott J., Heerema N. A., Huret J. L., Landman-Parker J., LeBeau M. M., Leonard C., Mann G., Pages M. P., Perot C., Pirc-Danoewinata H., Roitzheim B., Rubin C. M., Slociak M., and Viguie F. Nineteen cases of the t(1;22)(p13;q13) acute megakaryblastic leukaemia of infants/children and a review of 39 cases: report from a t(1;22) study group. Leukemia. 2000; 14(1): 216-8.
Biondi A., Cimino G., Pieters R., and Pui C.-H. Biological and therapeutic aspects of infant leukemia. Blood. 2000; 96(1): 24-33.
Bloomfield C. D. and Brunning R. D. FAB M7: acute megakaryoblastic leukemia--beyond morphology. Ann Intern Med. 1985; 103(3): 450-2.
Dastugue N., Lafage-Pochitaloff M., Pages M. P., Radford I., Bastard C., Talmant P., Mozziconacci M. J., Leonard C., Bilhou-Nabera C., Cabrol C., Capodano A. M., Cornillet-Lefebvre P., Lessard M., Mugneret F., Perot C., Taviaux S., Fenneteaux O., Duchayne E., and Berger R. Cytogenetic profile of childhood and adult megakaryoblastic leukemia (M7): a study of the Groupe Francais de Cytogenetique Hematologique (GFCH). Blood. 2002; 100(2): 618-26.
Dongen J. J. M. v. and Adriaansen H. J. Immunobiology of leukemia. In "Leukemia" (E. S. Henderson, T. A. Lister, and M. F. Greaves, Eds.), 1996; pp. 83-8, W.B. Saunders Company, Philadelphia, USA.
Gassmann W. and Loffler H. Acute megakaryoblastic leukemia. Leuk Lymphoma. 1995; 18(Suppl 1): 69-73.
Goasguen J. E., Bennett J. M., and Henderson E. S. Biologic diagnosis of leukemias. In "Leukemia" (E. S. Henderson, T. A. Lister, and M. F. Greaves, Eds.), 1996; pp. 8-15, W.B. Saunders Company, Philadelphia, USA.
Heaney M. L. and Golde D. W. Critical evaluation of the World Health Organization classification of myelodysplasia and acute myeloid leukemia. Curr Oncol Rep. 2000; 2(2): 140-3.
Hirt A., Luethy A. R., Mueller B., Gugler E., and Wagner H. P. Acute megakaryoblastic leukemia in children identified by immunological marker studies. Am J Pediatr Hematol Oncol. 1990; 12(1): 27-33.
Iovino C. S. and Camacho L. H. Acute myeloid leukemia: a classification and treatment update. Clin J Oncol Nurs. 2003; 7(5): 535-40.
Isaacs H. J. Fetal and neonatal leukemia. J Pediatr Hematol Oncol. 2003; 25(5): 348-61.
Lu G., Altman A. J., and Benn P. A. Review of the cytogenetic changes in acute megakaryoblastic leukemia: one disease or several? Cancer Genet Cytogenet. 1993; 67(2): 81-9.
Ma Z., Morris S. W., Valentine V., Li M., Herbrick J. A., Cui X., Bouman D., Li Y., Mehta P. K., Nizetic D., Kaneko Y., Chan G. C., Chan L. C., Squire J., Scherer S. W., and Hitzler J. K. Fusion of two novel genes, RBM15 and MKL1, in the t(1;22)(p13;q13) of acute megakaryoblastic leukemia. Nat Genet. 2001; 28(3): 220-1.
Medrano E. E. Repression of TGF-beta signaling by the oncogenic protein SKI in human melanomas: consequences for proliferation, survival, and metastasis. Oncogene. 2003; 22(20): 3123-9.
Mercher T., Busson-Le Coniat M., Khac F. N., Ballerini P., Mauchauffe M., Bui H., Pellegrino B., Radford I., Valensi F., Mugneret F., Dastugue N., Bernard O. A., and Berger R. Recurrence of OTT-MAL fusion in t(1;22) of infant AML-M7. Genes Chromosomes Cancer. 2002; 33(1): 22-8.
Mercher T., Coniat M. B., Monni R., Mauchauffe M., Khac F. N., Gressin L., Mugneret F., Leblanc T., Dastugue N., Berger R., and Bernard O. A. Involvement of a human gene related to the Drosophila spen gene in the recurrent t(1;22) translocation of acute megakaryocytic leukemia. Proc Natl Acad Sci U S A. 2001; 98(10): 5776-9.
Mrozek K., Heerema N. A., and Bloomfield C. D. Cytogenetics in acute leukemia. Blood Rev. 2004; 18(2): 115-36.
Paredes-Aguilera R., Romero-Guzman L., Lopez-Santiago N., and Trejo R. A. Biology, clinical, and hematologic features of acute megakaryoblastic leukemia in children. Am J Hematol. 2003; 73(2): 71-80.
Pui C. H., Kane J. R., and Crist W. M. Biology and treatment of infant leukemias. Leukemia. 1995; 9(5): 762-9.
Radonic A., Thulke S., Mackay I. M., Landt O., Siegert W., and Nitsche A. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 2004; 313(4): 856-62.
Raimondi S. C., Chang M. N., Ravindranath Y., Behm F. G., Gresik M. V., Steuber C. P., Weinstein H. J., and Carroll A. J. Chromosomal abnormalities in 478 children with acute myeloid leukemia: clinical characteristics and treatment outcome in a cooperative pediatric oncology group study---POG 8821. Blood. 1999; 94(11): 3707-16.
Rozen S. and Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000; 132:365-86.
Russo F., Buda F., Buda C., Aragona P., Tessitori G., Simon G., Chillemi S., Caristi N., and Faiella A. Acute megakaryoblastic leukaemia in a child. Eur J Cancer. 1991; 27(12): 1713-4.
Sande J. E., Arceci R. J., and Lampkin B. C. Congenital and neonatal leukemia. Semin Perinatol. 1999; 23(4): 274-85.
Schoch C. and Haferlach T. Cytogenetics in acute myeloid leukemia. Curr Oncol Rep. 2002; 4(5): 390-7.
Selvey S., Thompson E. W., Matthaei K., Lea R. A., Irving M. G., and Griffiths L. R. Beta-actin--an unsuitable internal control for RT-PCR. Mol Cell Probes. 2001; 15(5): 307-11.
Tallman M. S., Neuberg D., Bennett J. M., Francois C. J., Paietta E., Wiernik P. H., Dewald G., Cassileth P. A., Oken M. M., and Rowe J. M. Acute megakaryocytic leukemia: the Eastern Cooperative Oncology Group experience. Blood. 2000; 96(7): 2405-11.
The Leukemia & Lymphoma Society, Ed. "Acute Myelogenous Leukemia." 2004, The Leukemia & Lymphoma Society, New York, U.S.A.
Weinstein H. J. Acute myelogenous leukemia in infants and children. In "Leukemia" (E. S. Henderson, T. A. Lister, and M. F. Greaves, Eds.), 1996; pp. 509, W.B. Saunders Company, Philadelphia, USA.
Wheeler D. L., Church D. M., Edgar R., Federhen S., Helmberg W., Madden T. L., Pontius J. U., Schuler G. D., Schriml L. M., Sequeira E., Suzek T. O., Tatusova T. A., and Wagner L. Database resources of the National Center for Biotechnology Information: update. Nucleic Acids Res. 2004; 32 Database issue(D35-40.
Zipursky A., Brown E., Christensen H., Sutherland R., and Doyle J. Leukemia and/or myeloproliferative syndrome in neonates with Down syndrome. Semin Perinatol. 1997; 21(1): 97-101.