摘要
1.研究目的
(1)应用高分辨熔解曲线分析(HRMA)技术建立髓系肿瘤中相关基因(C-KIT、IDH、IDH2、DNMT3A、U2AF1和JAK2)突变的分子诊断平台。
(2)探讨这些基因突变的临床意义。
2.研究方法
(1)临床病例收集、细胞分离及DNA提取：收集按FAB和WHO标准诊断的初诊急性髓系白血病(AML)221例、骨髓增生异常综合征(MDS)88例和骨髓增殖性肿瘤(MPN)142例骨髓标本,用人淋巴细胞分离液(Ficoll-Hypaque)分离骨髓单个核细胞,DNA抽提试剂盒提取基因组DNA。
(2)PCR扩增：针对不同基因(包括C-KIT、IDH1、IDH2、DNMT3A、U2AF1和JAK2)的基因突变位点建立相应的PCR扩增反应体系,在ABI7300 PCR扩增仪上分别对这些基因进行扩增。
(3) HRMA检测：将PCR扩增产物在LightScanner分析仪上进行荧光扫描,应用分析软件进行处理,分析HRMA检测不同基因突变的灵敏度,并进一步检测待测标本的基因突变情况。
(4)DNA测序：首先评估DNA测序检测不同基因突变的灵敏度,然后对HRMA扫描鉴定为突变的标本进行测序。
(5)突变的临床意义分析：用Spearman秩和相关检验进行突变与性别、年龄、血液学参数、染色体分组、FAB分型、WHO分型等的相关性分析；应用卡方检验或Fisher确切检验对突变阳性组和阴性组之间的分类变量(如性别、染色体分组、FAB分型和WHO分型等)进行差异性比较,应用Mann-WhitneyU检验对突变阳性组和阴性组之间的连续变量(如年龄和血液学参数等)进行差异性比较。应用Kaplan-Meier和Cox分析分别对生存时间进行单因素和多因素分析。
3.研究结果
(1) C-KIT突变：①HRMA检测C-KIT突变的灵敏度达5%,高于DNA测序的灵敏度(10%)；②对160例AML患者进行检测发现9例(6%)存在C-KIT基因突变,进一步测序证实4例为D816V突变、4例为N822K突变、1例为D816H和N822K双位点突变；9例C-KIT突变主要发生于FAB-M2亚型,其中7例患者伴有t(8;21)易位,2例患者为正常核型。在25例t(8；21)易位AML患者中,C-KIT突变发生率为28%,突变者与非突变者比较,两组在性别、年龄、血象方面并无差异。
(2) IDH1/IDH2突变：①HRMA检测IDH1 R132H、IDH2 R140Q和IDH2 R172K突变的灵敏度分别为5%、2%和5%,均高于DNA测序的灵敏度(10%)；②对198例AML患者的进一步检测发现IDH1和IDH2突变发生率分别为2.0%(4例)和5.0%(10例),均为杂合性突变、且均为单独发生。IDH1突变包括3例R132H和1例R132S突变,IDH2突变包括7例R140Q和3例R172K突变。伴IDH1/IDH2突变的AML患者年龄明显高于无突变者(P=0.002)。两组患者在性别、血细胞计数和FAB分型方面并无统计学差异(P>0.05)。染色体核型正常的AML患者中IDH1/IDH2突变率(12/85,14.1%)明显高于核型异常患者(2/99,2.0%)(P=0.004)。③82例MDS患者分别检出2例(2.4%)和3例(3.6%)存在IDH1(R132H和R132S各1例)和IDH2突变(均为R140Q)。IDH1和IDH2突变均为杂合性、且均为单独发生。伴或不伴IDH1/IDH2突变者在性别、年龄、血红蛋白含量和血小板计数方面并无显著性差异(P>0.05),但伴突变者的白细胞计数明显高于无突变者(P=0.010)虽然全部突变均见于核型良好组,但并无统计学意义(P=0.407)。正常核型患者IDH1/IDH2突变率(5/52,9.6%)高于异常核型患者(0/30,0%)(P=0.153)。此外,根据IPSS分组,所有IDH突变均发生于低危组或中危-1组患者。多变量Cox分析揭示年龄和IPSS分组是MDS患者独立的预后因素,而IDH1/IDH2突变对生存时间并无影响。④85例慢性髓系白血病(CML)和57例Ph-MPN患者中均未检测出IDH1/IDH2突变。
(3) DNMT3A突变：①HRMA检测DNMT3A R882H突变的灵敏度为2%,高于DNA测序的灵敏度(10%)；②182例AML患者中12例(6.6%)检出DNMT3A突变,均为杂合性突变,包括8例R882H、3例R882C和1例R882P。R882突变与患者的性别和白细胞计数并无相关性,突变者年龄高于无突变者、且突变者血小板计数明显高于无突变者DNMT3A R882突变更常见于单核类AML (M4和M5,7/52例,13.4%)而非单核类AML (M1、M2、M3和M6,5/130,3.8%)(P=0.041)76例正常核型患者中10例(13.2%)存在R882突变,显著高于核型异常患者(2/92例,2.2%)(P=0.007)。③51例MDS患者检出4例(7.8%)DNMT3A R882突变,均为杂合性,包括3例R882H和1例R882C；伴或不伴DNMT3A突变者在年龄、血细胞计数、WHO分型等方面并无显著性差异；4例突变均发生于正常核型患者及IPSS中危组。伴突变者中位生存时间为9个月,而不伴突变者中位生存时间为25个月(P=0.047)。④79例CML和57例Ph-MPN患者中均未检测出DNMT3A突变。
(4)U2AF1突变：①HRMA检测U2AF1 S34和Q157突变的灵敏度均为5%,高于DNA测序的灵敏度(10%)；②221例AML患者中4例(1.8%)检出U2AF1突变,均为杂合性突变,包括2例S34Y和2例Q157(1例Q157P和1例Q157R)突变。突变患者与非突变患者在性别、年龄、血细胞、FAB分型和核型分组之间并无差异。伴U2AF1突变的患者生存时间明显短于无突变者(中位时间分别为1和7个月,P=0.034)③88例MDS患者检出4例(4.5%)S34突变,均为杂合性,包括3例S34F和1例S34Y。伴或不伴U2AF1突变者在性别、血细胞计数、WHO分型和IPSS分组方面并无显著性差异。伴U2AF1突变者年龄较无突变者年轻(P=0.059)。72例患者具有随访结果,突变患者和无突变患者在生存时间上并无显著性差异(P=0.954)。④79例CML和57例Ph-MPN患者中均未检测出U2AF1突变。
(5)JAK2突变：①HRMA检测JAK2 V617F突变的灵敏度为5%,高于DNA测序的灵敏度(10%)；②48例Ph-的MPN患者中,JAK2 V617F突变可见于3例(100%,均为杂合子突变)真性红细胞增多症(PV)34例(60%,6例纯合子突变、28例杂合子突变)原发性血小板增多症(ET)和3例(60%,1例纯合子突变、2例杂合子突变)原发性骨髓纤维化(PMF)。而57例CML患者均为阴性。HRMA的检测结果均经直接测序证实。诊断的特异性和灵敏度均为100%。
4.结论
(1) HRMA是检测基因突变的一个高通量技术,其特异性好、灵敏度高、成本低廉,可有效用于对大批量标本进行基因突变的筛选。
(2)中国人群AML和MDS患者中存在IDH1和IDH2基因突变,均为杂合子突变,主要见于正常核型患者;IDH1/IDH2突变并非是MDS患者的不良预后因素。
(3) DNMT3A R882突变是中国人群AML和MDS患者的一个常见分子事件,该突变均为杂合子突变,主要发生于正常核型患者；DNMT3AR882突变是MDS患者的一个不良预后因素。
(4)中国人群AML和MDS中U2AF1突变发生于密码子S34和Q157,均为杂合子突变,但发生率相对较低；伴U2AF1突变的AML患者生存时间较短。
OBJECTIVE:
(1) Establish the molecular diagnosis platform of common gene mutations in myeloid malignancies using high-resolution melting analysis (HRMA).
(2) Explore the clinical relevance of these gene muations.
METHODES:
(1) Collection of clinical cases, cell separation and DNA extraction: Bone marrows were collected from 221 cases with acute myeloid leukemia (AML),88 myelodysplastic syndrome (MDS) and 142 myeloproliferative neoplasm (MPN) which were diagnosed accoriding to FAB and WHO cirtieria. Bone marrow mononuclear cells were separated using Ficoll-Hypaque and genomic DNA was extracted using DNA Purification Kit.
(2) PCR amplification:Different PCR systems were established to amplify different mutated genes (C-KIT, IDH1, IDH2, DNMT3A, U2AF1 and JAK2) according to the specific mutated sites of different genes in 7300 Thermo cycler.
(3) HRMA detection:PCR products were transferred to the LightscannerTM platform for scanning and were analyzed using software package. The sensitivity of HRMA was evaluated. Gene mutations in the detected samples were further analyzed.
(4) DNA sequencing:The sensitivity of direct DNA sequencing was evaluated for different gene mutations. The samples which were identified positive in clinical cases were further sequenced.
(5) Analysis of clinical relevance of gene mutations:The correlation between gene mutations and sex, age, hematologic parameters, chromosomal groups, FAB subtypes and WHO subtypes was analyzed with Spearman's rank correlation. Chi-square analysis or Fisher exact test was carried out to compare the difference of categorical variables between patient groups. Mann-Whitney's U-test was carried out to compare the difference of continuous variables between patient groups. Survival was analyzed according to the Kaplan-Meier method (univariant analysis) and the Cox regression method (multivariate analysis).
RESULTS:
(1) C-KIT mutation:①HRMA could distinguish C-KIT mutation with the maximal sensitivity of 5% in a background of wild-type DNA, higher than that of direct DNA sequencing (10%).②In 160 AML patients, 9 (6%) cases were identified with C-KIT mutations, which were further confirmed by DNA sequencing, including 4 D816V mutations,4 N822K mutations, and 1 dual muations of D816H and N822K.8 mutations occurred in the patients with AML-M2 subtype, among which 7 cases had the translocation t(8;21); 2 had normal karyotype. Among 25 cases with t(8;21), the frequency of C-KIT mutation was 28%. There was no difference in sex, age and blood parameters between mutated and wild-type groups.
(2) IDH1/IDH2 mutation:①HRMA could distinguish IDH1 R132H, IDH2 R140Q and IDH2 R172K mutations with the maximal sensitivity of 5%,2% and 2%, respectively, higher than that obtained by direct DNA sequencing (10%).②IDH1 mutations were found in 4 of 198 AML patients (2.0%) and IDH2 mutations were found in 10 cases (5.0%). All mutations were heterozygous. IDH1 and IDH2 mutations were mutually exclusive. IDH1 mutations included 3 R132H and 1 R132S. while IDH2 mutations were R140Q (n=7) and R172K (n=3). AML patients with IDH1/IDH2 mutations had significantly older age than those with wild-type IDHs (P=0.002). There were no difference in sex, blood parameters and FAB subtypes between cases with and without mutations (P>0.05). IDH mutations were significantly more frequently observed in cytogenetically normal AML (12/85,14.1%) than in cytogenetically abnormal AML (2/99, 2.0%) (P=0.004).③2 of 82 (2.4%) MDS cases were identified with IDH1 mutations (one R132H and one R132S) and 3 (3.6%) with IDH2 mutations (all R140Q). IDH1 and IDH2 mutations were also heterozygous and mutually exclusive. No significant defference in sex, age, hemoglobin and platelets was observed between MDS patients with and without IDH1/IDH2 mutations (P>0.05). MDS cases with IDH1/IDH2 mutations had higher white blood cell (WBC) counts than those without mutations (P=0.010). All IDH1/IDH2 mutations were observed in cases with favorable risks according to karyotype classification, however, statistical difference was not observed (P=0.407). The frequency of IDH1/IDH2 mutations were higher in MDS patients with normal karyotype (5/52,9.6%) than those with abnormal karyotype (0/30,0%) (P=0.153). In addition, all IDH mutations were identified in patients at Low and Int-1 risks according to IPSS classification. Multivariate analysis showed that age and IPSS grouping were independent prognostic factors. The overall survival (OS) of MDS patients with and without IDH1/IDH2 mutations did not differ.④No IDH1 or IDH2 mutations were detected in 85 chronic myeloid leukemia (CML) and 57 Ph-MPNs.
(3) DNMT3A mutation:①HRMA could easily distinguish R882H mutation with the maximal sensitivity of 2%, higher than that obtained by DNA sequencing (10%).②R882 mutations were identified in 12 (6.6%) of 182 AML patients. All mutations were heterozygous, including R882H (n=8), R882C (n=3) and R882P (n=1). No correlation was observed between R882 mutations and gender or WBC counts. AML patients with DNMT3A R882 mutations were more prevalent at older age and present with significantly higher median platelet counts at diagnosis compared to those without mutations. DNMT3A R882 mutations were found more frequently among monoblastic leukemia (M4 and M5,7 of 52,13.4%) compared to non-monoblastic leukemia (M1, M2, M3 and M6,5 of 130, 3.8%) (P=0.041). Among the patients with normal karyotypes,13.2%(10 of 76) cases showed R882 mutation, significantly higher than 2.2%(2 of 92) in those with chromosomal abnormalities (P=0.007).③4 (7.8%) heterozygous DNMT3A R882 mutations were also identified in MDS, including 3 R882H and 1 R882C mutations. The difference in age, hematologic parameters and WHO classifications was not seen between patients with and without mutations. All 4 patients were identified with normal karyotype and were classified in intermediate-risk group according to IPSS classification. The OS of MDS patients with DNMT3A mutation (median 9 months) was shorter than those without mutation (median 25 months) (P=0.047).④No DNMT3A R882 mutations were detected in 79 CML and 57 Ph-MPNs.
(4) U2AF1 mutation:①HRMA could easily distinguish U2AF1 S34 and Q157 mutations with the maximal sensitivity of 5%, higher than that obtained by DNA sequencing (10%).②U2AF1 mutations were identified in 4 (1.8%) of 221 AML patients. All mutations were heterozygous, including 2 S34Y and 2 Q157 (1 Q157P and 1 Q157R) mutations. There were no difference in sex, age, blood parameters, FAB subtypes, and karyotype classification between cases with and without mutations (P>0.05). The OS of AML patients with U2AF1 mutation (median 1 months) was shorter than those without mutation (median 7 months) (P=0.034).③4 (4.5%) cases were identified with heterozygous S34 mutations (3 S34F and 1 S34Y). No significance in sex, blood parameters, WHO subtypes, and IPSS classification was observed between MDS patients with and without U2AF1 mutations (P>0.05). The cases with U2AF1 mutations had younger age than those without mutations (.P=0.059). No difference in OS was observed between patients with and without U2AF1 mutations (P=0.954).④No U2AF1 mutations were detected in 79 CML and 57 Ph-MPNs.
(5) JAK2 mutation:①HRMA assay was able to distinguish 5% of JAK2 V617F mutant, more sensitive than that obtained by DNA sequencing (10%).②Out of 48 cases with Ph-negative MPN, all 3 (100%) with polycythemia vera (PV) were positive for the presence of JAK V617F heterozygous mutation,34 (70%) with essential thrombocythemia (ET) were positive for JAK V617F mutation (6 homozygous and 28 heterozygous), and 3 (60%) with primary myelofibrosis (PMF) were positive (1 homozygous and 2 heterozygous). JAK2 V617F mutation was not found in all patients with CML. HRMA detection of all cases was fully concordant with the results of direct sequencing. Both the diagnostic specificity and sensitivity were 100%.
CONCLUSIONS:
(1) HRMA is a high-throughput technique for the detection of gene mutation and can be utilized to screen gene mutation in a large scale samples for good specificity, high sensitivity and low cost.
(2) Heterozygous IDH1 and IDH2 mutations are present in Chinese AML and MDS patients and mainly observed in cytogenetically normal cases. IDH1/IDH2 mutations may have no prognostic impact on patients with MDS.
(3) DNMT3A R882 mutations are recurrent molecular aberrations in AML and MDS. R882 mutations are all heterozygous and mainly observed in cytogenetically normal patients. DNMT3A R882 muation may be an adverse prognostic event in MDS.
(4) U2AF1 mutations, a molecular event of low frequency in Chinese AML and MDS, occur at S34 and Q157 and are all heterozygous. The overall survival of AML patients with U2AF1 mutation was shorter than those without mutation.
引文
[1]Mrozek K, Marcucci G, Paschka P, et al. Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics:are we ready for a prognostically prioritized molecular classification? Blood,2007,109(2):431-448.
[2]Dohner H. Implication of the molecular characterization of acute myeloid leukemia. Hematology Am Soc Hematol Educ Program.2007:412-419.
[3]Marcucci G, Haferlach T, Dohner H. Molecular genetics of adult acute myeloid leukemia:prognostic and therapeutic implications. J Clin Oncol.2011,29(5): 475-486.
[4]Renneville A. Roumier C, Biggio V, et al. Cooperating gene mutations in acute myeloid leukemia:a review of the literature. Leukemia,2008,22(5):915-931.
[5]Falini B. Mecucci C, Tiacci E. et al. Gimema acute leukemia working party. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med,2005.352(3):254-266.
[6]Reilly JT. Class Ⅲ receptor tyrosine kinases:role in leukaemogenesis. Br J Haematol,2002,116(4):744-757.
[7]Miettinen M, Lasota J. KIT (CD117):a review on expression in normal and neoplastic tissues, and mutations and their clinicopathologic correlation. Appl Immunohistochem Mol Morphol.2005,13(3):205-220.
[8]Schnittger S, Kohl TM, Haferlach T, et al. KIT-D816 mutations in AMLl-ETO-positive AML are associated with impaired event-free and overall survival. Blood,2006,107(5):1791-1799.
[9]Kindler T, Lipka DB, Fischer T. FLT3 as a therapeutic target in AML:still challenging after all these years. Blood,2010,116(24):5089-5102.
[10]Pratz KW, Levis MJ. Bench to bedside targeting of FLT3 in acute leukemia. Curr Drug Targets,2010,11(7):781-789.
[11]Pabst T, Mueller BU. Complexity of CEBPA dysregulation in human acute myeloid leukemia. Clin Cancer Res,2009,15(17):5303-5307.
[12]Ernst T. Chase AJ, Score J. et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet. 2010,42(8):722-726.
[13]Nikoloski G, Langemeijer SM, Kuiper RP, et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet, 2010,42(8):665-667.
[14]Delhommeau F, Dupont S, Delia Valle V, et al. Mutation in TET2 in myeloid cancers. N Engl J Med,2009,360(22):2289-2301.
[15]Mardis ER, Ding L, Dooling DJ, et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med,2009,361(11):1058-1066.
[16]Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med,2010,363(25):2424-2433.
[17]Corey SJ, Minden MD, Barber DL, et al. Myelodysplastic syndromes:the complexity of stem-cell diseases. Nat Rev Cancer,2007,7(2):118-129.
[18]Bernasconi P. Molecular pathways in myelodysplastic syndromes and acute myeloid leukemia:relationships and distinctions-a review. Br J Haematol, 2008,142(5):695-708.
[19]Boultwood J, Wainscoat JS. Gene silencing by DNA methylation in haematological malignancies. Br J Haematol,2007,138(1):3-11.
[20]Nimer SD. Myelodysplastic syndrome. Blood,2008,111 (10):4841-4851.
[21].Malumbres M, Barbacid M. RAS oncogenes:the first 30 years. Nat Rev Cancer, 2003,3(6):459-465.
[22]Watanabe-Okochi N, Kitaura J. Ono R, et al. AML1 mutations induced MDS and MDS/AML in a mouse BMT model. Blood,2008,111(8):4297-4308.
[23]Harada H, Harada Y, Niimi H, et al. High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia. Blood,2004,103(6):2316-2324.
[24]Harada Y, Harada H. Molecular pathways mediating MDS/AML with focus on AML1/RUNXI point mutations. J Cell Physiol,2009,220(1):16-20.
[25]Chen CY. Lin LI. Tang JL, et al. RUNX1 gene mutation in primary myelodysplastic syndrome--the mutation can be detected early at diagnosis or acquired during disease progression and is associated with poor outcome. Br J Haematol.2007,139(3):405-414.
[26]Steensma DP, Gibbons RJ, Mesa RA, et al. Somatic point mutations in RUNX1/CBFA2/AMLI are common in high-risk myelodysplastic syndrome. but not in myelofibrosis with myeloid metaplasia. Eur J Haematol, 2005,74(1):47-53.
[27]Melo JV, Barnes DJ. Chronic myeloid leukaemia as a model of disease evolution in human cancer. Nat Rev Cancer,2007,7(6):441-453.
[28]Cortes J, Hochhaus A, Hughes T, et al. Front-line and salvage therapies with tyrosine kinase inhibitors and other treatments in chronic myeloid leukemia. J Clin Oncol,2011,29(5):524-531.
[29]Calabretta B, Perrotti D. The biology of CML blast crisis. Blood,2004,103(11): 4010-4022.
[30]Radich JP. The biology of CML blast crisis. Hematology Am Soc Hematol Educ Program,2007:384-391.
[31]Campbell PJ, Green AR. The myeloproliferative disorders. N Engl J Med, 2006,355(23):2452-2466.
[32]Swerdlow SH, Campo E, Harris NL, et al (Eds):WHO classification of tumours of haematopoietic and lymphoid tissues. IARC:Lyon 2008.
[33]Frantz C. Sekora D, Henley DC, et al. Comparative evaluation of three JAK2V617F mutation detection methods. Am J Clin Pathol.2007,128(5): 865-874.
[34]Tan AY. Westerman DA. Dobrovic A. A simple, rapid, and sensitive method for the detection of the JAK2 V617F mutation. Am J Clin Pathol,2007,127(6): 977-981.
[35]Kakavas VK, Plageras P, Vlachos TA, et al. PCR-SSCP:a method for the molecular analysis of genetic diseases. Mol Biotechnol,2008,38(2):155-163.
[36]Chen Q, Lu P, Jones AV. et al. Amplification refractory mutation system, a highly sensitive and simple polymerase chain reaction assay, for the detection of JAK2 V617F mutation in chronic myeloproliferative disorders. J Mol Diagn, 2007,9(2):272-276.
[37]Pourzand C, Cerutti P. Genotypic mutation analysis by RFLP/PCR. Mutat Res, 1993,288(1):113-121.
[38]Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography:a review. Hum Mutat,2001.17(6):439-474.
[39]Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome Res. 2001,11(1):3-11.
[40]Jones AV, Kreil S, Zoi K, et al. Widespread occurrence of the JAK2 V617F mutation in chronic myeloproliferative disorders. Blood,2005,106(6): 2162-2168.
[41]Jelinek J, Oki Y, Gharibyan V, et al. JAK2 mutation 1849G>T is rare in acute leukemias but can be found in CMML, Philadelphia chromosome-negative CML, and megakaryocytic leukemia. Blood,2005,106(6):3370-3373.
[42]Steensma DP. JAK2 V617F in myeloid disorders:molecular diagnostic techniques and their clinical utility. J Mol Diagn,2006,8(4):397-411.
[43]Chou LS, Lyon E, Wittwer CT. A comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning:cystic fibrosis transmembrane conductance regulator gene as a model. Am J Clin Pathol,2005,124(3):330-338.
[44]Whittall RA, Scartezini M, Li K. Development of a high-resolution melting method for mutation detection in familial hypercholesterolaemia patients. Ann Clin Biochem.2010,47(Pt1):44-55.
[45]Yarden Y, Kuang WJ, Yang-Feng T, et al. Human proto-oncogene c-kit:A new cell surface receptor tyrosine kinase for an unidentified ligand. EMBO J. 1987,6(11):3341-3351.
[46]Yarden Y,.Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem,1988.57:443-478.
[47]Lennartsson J. Jelacic T, Linnekin D, et al. Normal and oncogenic forms of the receptor tyrosine kinase kit. Stem Cells,2005,23(1):16-43.
[48]Malaise M. Steinbach D, Corbacioglu S. Clinical implications of c-Kit mutations in acute myelogenous leukemia. Curr Hematol Malig Rep,2009.4(2):77-82.
[49]Longley BJ, Reguera MJ, Ma Y. Classes of c-KIT activating mutations: proposed mechanisms of action and implications for disease classification and therapy. Leuk Res,2001,25(7):571-576.
[50]Aguirre-Lamban J. Riveiro-Alvarez R, Garcia-Hoyos M, et al. Comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning in the ABCA4 gene. Invest Ophthalmol Vis Sci,2010,51(5):2615-2619.
[51]Erali M, Voelkerding KV, Wittwer CT. High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol,2008,85(1):50-58.
[52]Kennerson ML, Warburton T. Nelis E. et al. Mutation scanning the GJB1 gene with high-resolution melting analysis:implications for mutation scanning of genes for Charcot-Marie- Tooth disease. Clin Chem,2007,53(2):349-352.
[53]Bennett JM, Catovsky D, Daniel MT, et al. Proposed revised criteria for the classification of acute myeloid leukaemia. A report of the French-American-British Cooperative Group. Ann Intern Med,1985,103(4): 620-625.
[54]Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood,2009,114(5):937-951.
[55]Lu L, Heinrich MC, Wang LS, et al. Retroviral-mediated gene transduction of c-kit into single hematopoitic progenitor cells from cord blood enhances erythroid colony formation and decrease sensitivity to inhibition by tumor necrosis factor-a and transforming growth factor-β1. Blood,1999,94(7): 2319-2332.
[56]Cairoli R. Beghini A, Grillo G, et al. Prognostic impact of c-kit mutations in core binding factor leukemias:an Italian retrospective study. Blood.2006,107(9): 3463-3468.
[57]Wang YY, Zhou GB, Yin T, et al. AML1-ETO and C-KIT mutation over expression in t(8;21) leukemia:implication in stepwise leukemogenesis and response to Gleevec. Proc Natl Acad Sci U S A,2005,102(4):1104-1109.
[58]Ma Y. Zeng S. Metcalfe DD, et al. The C-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. Blood, 2002,99(5):1741-1744.
[59]Kohl TM, Schnittger S, Ellwart JW, et al. KIT exon 8 mutations associated with core-binding factor (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the receptor in response to stem cell factor. Blood,2005, 105(8):3319-3321.
[60]李渭阳,孙爱宁,吴德沛,等.伴t(8；21)M2型急性髓系白血病患者c-kit和JAK2基因突变分析.中华血液学杂志,2008.29(12)：797-801.
[61]Xu X, Zhao J, Xu Z, et al. Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity. J Biol Chem,2004,279(32):33946-33957.
[62]Sjoblom T, Jones S, Wood LD, et al. The consensus coding sequences of human breast and colorectal cancers. Science,2006,314(5797):268-274.
[63]Parsons DW, Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme. Science,2008,321(5897):1807-1812.
[64]Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med,2009,360(8):765-773.
[65]Zhao S, Lin Y, Xu W, et al. Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-lalpha. Science,2009, 324(5924):261-265.
[66]Ho PA, Alonzo TA, Kopecky KJ. et al. Molecular alterations of the IDH1 gene in AML:a Children's Oncology Group and Southwest Oncology Group study. Leukemia,2010,24(5):909-913.
[67]Paschka P, Schlenk RF, Gaidzik VI, et al. IDH1 and IDH2 mutations are frequent genetic alterations in acute myeloid leukemia and confer adverse prognosis in cytogenetically normal acute myeloid leukemia with NPM1 mutation without FLT3 internal tandem duplication. J Clin Oncol,2010, 28(22):3636-3643.
[68]Marcucci G, Maharry K, Wu YZ, et al. IDH1 and IDH2 gene mutations identify novel molecular subsets within de novo cytogenetically normal acute myeloid leukemia:a Cancer and Leukemia Group B study. J Clin Oncol,2010, 28(14):2348-2355.
[69]Abbas S, Lugthart S, Kavelaars FG, et al. Acquired mutations in the genes encoding IDH1 and IDH2 both are recurrent aberrations in acute myeloid leukemia:prevalence and prognostic value. Blood,2010,116(12):2122-2126.
[70]Kosmider O. Gelsi-Boyer V, Slama L. et al. Mutations of IDH1 and IDH2 genes in early and accelerated phases of myelodysplastic syndromes and MDS/myeloproliferative neoplasms. Leukemia,2010,24(5):1094-1096.
[71]Rocquain J, Carbuccia N, Trouplin V, et al. Combined mutations of ASXL1, CBL. FLT3. IDH1, IDH2, JAK2, KRAS, NPM1, NRAS, RUNX1, TET2 and WT1 genes in myelodysplastic syndromes and acute myeloid leukemias. BMC Cancer,2010,10:401.
[72]Thol F, Weissinger EM, Krauter J, et al. IDH1 mutations in patients with myelodysplastic syndromes are associated with an unfavorable prognosis. Haematologica,2010,95(10):1668-1674.
[73]Tefferi A, Lasho TL, Abdel-Wahab O, et al. IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic-or blast-phase essential thrombocythemia, polycythemia vera or myelofibrosis. Leukemia,2010,24(7):1302-1309.
[74]Pardanani A, Lasho TL, Finke CM, et al. IDH1 and IDH2 mutation analysis in chronic-and blast-phase myeloproliferative neoplasms. Leukemia,2010,24(6): 1146-1151.
[75]Gelsi-Boyer V, Trouplin V, Roquain J, et al. ASXL1 mutation is associated with poor prognosis and acute transformation in chronic myelomonocytic leukaemia. Br J Haematol,2010,151 (4):365-375.
[76]Chou WC, Lei WC, Ko BS. et al. The prognostic impact and stability of Isocitrate dehydrogenase 2 mutation in adult patients with acute myeloid leukemia. Leukemia,2011,25(2):246-253.
[77]Dang L, White DW, Gross S, et al. Cancer-associated IDH1 mutations produce 2-hydroxyglutarate. Nature,2009,462(7274):739-744.
[78]Ward PS, Patel J, Wise DR. et al. The common feature of leukemia-associated IDH1 and 1DH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell,2010.17(3):225-234.
[79]Gross S, Cairns RA, Minden MD, et al. Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations. J Exp Med,2010,207(2):339-344.
[80]Esteller M. Epigenetics in cancer. N Engl J Med.2008,358(11):1148-1159.
[81]Chen J, Odenike O, Rowley JD. Leukaemogenesis:more than mutant genes. Nat Rev Cancer,2010,10(1):23-36.
[82]Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell,2010,18(6):553-567.
[83]Christensen BC, Smith AA, Zheng S, et al. DNA methylation, isocitrate dehydrogenase mutation, and survival in glioma. J Natl Cancer Inst, 2011,103(2):143-153.
[84]Lu C, Ward PS, Kapoor GS, et al. IDH mutation impairs histone demethylation and results in a block to cell differentiation. Nature,2012,483(7390):474-478.
[85]Chou WC, Hou HA, Chen CY, et al. Distinct clinical and biologic characteristics in adult acute myeloid leukemia bearing the isocitrate dehydrogenase 1 mutation. Blood,2010,115(14):2749-2754.
[86]Kang MR, Kim MS, Oh JE, et al. Mutational analysis of IDH1 codon 132 in glioblastomas and other common cancers. Int J Cancer,2009,125(2):353-355.
[87]Setty P, Hammes J, Rothamel T, et al. A pyrosequencing-based assay for the rapid detection of IDH1 mutations in clinical samples. J Mol Diagn, 2010,12(6):750-756.
[88]Felsberg J, Wolter M, Seul H, et al. Rapid and sensitive assessment of the IDH1 and 1DH2 mutation status in cerebral gliomas based on DNA pyrosequencing. Acta Neuropathol,2010,119(4):501-507.
[89]Bujko M, Kober P, Matyja E, et al. Prognostic value of IDH1 mutations identified with PCR-RFLP assay in glioblastoma patients. Mol Diagn Ther, 2010,14(3):163-169.
[90]Meyer J, Pusch S, Balss J, et al. PCR-and restriction endonuclease-based detection of IDH1 mutations. Brain Pathol,2010,20(2):298-300.
[91]Andrulis M, Capper D, Luft T. et al. Detection of isocitrate dehydrogenase 1 mutation R132H in myelodysplastic syndrome by mutation-specific antibody and direct sequencing. Leuk Res,2010,34(8):1091-1093.
[92]Wittwer CT, Reed GH, Gundry CN, et al. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem,2003,49(6 Pt1):853-860.
[93]Ugo V, Tondeur S, Menot ML, et al. Interlaboratory development and validation of a HRM method applied to the detection of JAK2 exon 12 mutations in polycythemia vera patients. PLoS One,2010,5(1):e8893.
[94]Reed GH, Kent JO, Wittwer CT. High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics,2007,8(6): 597-608.
[95]Zou Y, Zeng Y, Zhang DF, et al. IDH1 and IDH2 mutations are frequent in Chinese patients with acute myeloid leukemia but rare in other types of hematological disorders. Biochem Biophys Res Commun,2010,402(2):378-383.
[96]Boisselier B, Marie Y. Labussiere M, et al. COLD PCR HRM:a highly sensitive detection method for IDH1 mutations. Hum Mutat,2010,31(12):1360-1365.
[97]Qian J, Xue Y, Pan J, et al. Refractory thrombocytopenia, an unusual myelodysplastic syndrome with initial presentation mimicking an idiopathic thrombocytopenic purpura. Int J Hematol,2005,81(2):142-147.
[98]Caramazza D, Lasho TL, Finke CM, et al. IDH mutations and trisomy 8 in myelodysplastic syndromes and acute myeloid leukemia. Leukemia,2010, 24(12):2120-2122.
[99]Mizuno S, Chijiwa T, Okamura T, et al. Expression of DNA methyltransferases DNMT1,3A, and 3B in normal hematopoiesis and in acute and chronic myelogenous leukemia. Blood,2001,97(5):1172-1179.
[100]Walter MJ, Ding L, Shen D. et al. Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia,2011,25(7):1153-1158.
[101]Gowher H, Loutchanwoot P. Vorobjeva O. et al. Mutational Analysis of the Catalytic Domain of the Murine Dnmt3a DNA-(cytosine C5)-methyltransferase. J-Mol Biol,2006,357(3):928-941.
[102]Turek-Plewa J, Jagodzinski PP. The role of mammalian DNA methyltransferases in the regulation of gene expression. Cell Mol Biol Lett, 2005,10(4):631-647.
[103]Jia D, Jurkowska RZ, Zhang X. et al. Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature,2007, 449(7159):248-251.
[104]Deng T, Kuang Y, Wang L. et al. An essential role for DNA methyltransferase 3a in melanoma tumorigenesis. Biochem Biophys Res Commun,2009, 387(3):611-616.
[105]Yamashita Y, Yuan J, Suetake I. et al. Array-based genomic resequencing of human leukemia. Oncogene,2010,29(25):3723-3731.
[106]Zhao Z, Wu Q, Cheng J. et al. Depletion of DNMT3A suppressed cell proliferation and restored PTEN in hepatocellular carcinoma cell. J Biomed Biotechnol,2010,2010:737535.
[107]Oka M, Meacham AM, Hamazaki T, et al. De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2'-deoxycytidine. Oncogene,2005,24(19):3091-3099.
[108]Yan XJ, Xu J, Gu ZH, et al. Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia. Nat Genet,2011,43(4):309-315.
[109]Stegelmann F, Bullinger L, Schlenk RF, et al. DNMT3A mutations in myeloproliferative neoplasms. Leukemia,2011,25(7):1217-1219.
[110]Abdel-Wahab O, Pardanani A, Rampal R, et al. DNMT3A mutational analysis in primary myelofibrosis, chronic myelomonocytic leukemia and advanced phases of myeloproliferative neoplasms. Leukemia,2011,25(7):1219-1220.
[111]Jankowska AM, Makishima H, Tiu RV, et al. Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation:UTX, EZH2 and DNMT3A. Blood,2011,118(14):3932-3941.
[112]Zamore PD, Green MR. Biochemical characterization of U2 snRNP auxiliary factor:an essential pre-mRNA splicing factor with a novel intranuclear distribution. Embo J,1991,10(1):207-214.
[113]Black DL. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem,2003,72:291-336.
[114]Baralle D, Baralle M. Splicing in action:assessing disease causing sequence changes. J Med Genet,2005,42(10):737-748.
[115]Graubert TA, Shen D, Ding L, et al. Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes. Nat Genet,2011,44(1):53-57.
[116]Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature.2011,478(7367):64-69.
[117]Makishima H, Visconte V, Sakaguchi H, et al. Mutations in the spliceosome machinery, a novel and ubiquitous pathway in leukemogenesis. Blood,2012, doi:10.1182/blood-2011-12-399774.
[118]Thol F, Kade S, Schlarmann C, et al. Frequency and prognostic impact of mutations in SRSF2, U2AF1, and ZRSR2 in patients with myelodysplastic syndromes. Blood,2012, doi:10.1182/blood-2011-12-399337.
[119]Grosso AR, Martins S, Carmo-Fonseca M. The emerging role of splicing factors in cancer. EMBO Rep,2008,9(11):1087-1093.
[120]David CJ, Manley JL. Alternative pre-mRNA splicing regulation in cancer: pathways and programs unhinged. Genes Dev,2010,24(21):2343-2364.
[121]Pajares MJ, Ezponda T, Catena R, et al. Alternative splicing:an emerging topic in molecular and clinical oncology. Lancet Oncol,2007,8(4):349-357.
[122]Ding WQ, Kuntz SM, Miller LJ. A misspliced form of the cholecystokinin-B/gastrin receptor in pancreatic carcinoma:role of reduced sellular U2AF35 and a suboptimal 3'-splicing site leading to retention of the fourth intron. Cancer Res,2002,62(3):947-952.
[123]Pacheco TR, Moita LF, Gomes AQ, et al. RNA interference knockdown of hU2AF35 impairs cell cycle progression and modulates alternative splicing of Cdc25 transcripts. Mol Biol Cell,2006,17(10):4187-4199.
[124]Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell,2005,7(4):387-397.
[125]Vainchenker W, Constantinescu SN. A unique activating mutation in JAK2(V617F) is at the origin of polycythemia vera and allows a new classification of myeloproliferative diseases. Hematology Am Soc Hematol Educ Program.2005:195-200.
[126]Zhao R, Xing S, Li Z, et al. Identification of an acquired JAK2 mutation in polycythemia vera. J Biol Chem,2005,280(24):22788-22792.
[127]James C. Ugo V, Le Couedic JP. et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature,2005.434(7037): 1144-1148.
[128]Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med,2005,352(17):1779-1790.
[129]K"roger N, Badbaran A, Holler E, et al. Monitoring of the JAK2-V617F mutation by highly sensitive quantitative real-time PCR after allogeneic stem cell transplantation in patients with myelofibrosis. Blood,2007,109(3): 1316-1321.
[130]Tefferi A, Thiele J, Orazi A, et al. Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia. and primary myelofibrosis:recommendations from an ad hoc international expert panel. Blood,2007,110(4):1092-1107.
[131]Lippert E, Boissinot M, Kralovics R, et al. The JAK2-V617F mutation is frequently present at diagnosis in patients with essential thrombocythemia and polycythemia vera. Blood,2006,108(6):1865-1867.
[132]Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative diseases. Lancet,2005,365(9464): 1054-1061.
[133]Lay M, Mariappan R, Gotlib J, et al. Detection of the JAK2 V617F mutation by LightCycler PCR and probe dissociation analysis. J Mol Diagn,2006,8(3): 330-334.
[134]Vannucchi AM, Pancrazzi A, Bogani C, et al. A quantitative assay for JAK2(V617F) mutation in myeloproliferative disorders by ARMS-PCR and capillary electrophoresis. Leukemia,2006,20(6):1055-1060.
[135]Murugesan G, Aboudola S. Szpurka H, et al. Identification of the JAK2 V617F mutation in chronic myeloproliferative disorders using FRET probes and melting curve analysis. Am J Clin Pathol,2006,125(4):625-633.
[136]Poodt J, Fijnheer R, Walsh IB, et al. A sensitive and reliable semi-quantitative real-time PCR assay to detect JAK2 V617F in blood. Hematol Oncol, 2006,24(4):227-233.
[137]Merker ID, Jones CD, Oh ST, et al. Design and evaluation of a real-time PCR assay for quantification of JAK.2 V617F and wild-type JAK2 transcript levels in the clinical laboratory. J Mol Diagn,2010,12(1):58-64.