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急性髓系白血病患者FLT3与NPM1基因突变检测及其临床意义
【作者】 马亮；
【导师】 黄尚志；
【作者基本信息】 北京协和医学院 ， 医学遗传学， 2011， 硕士
【摘要】 本研究采用PCR技术扩增Fms样酪氨酸激酶-3（fms-like tyrosine kinase 3,FLT3）基因外显子14-15和核仁磷酸蛋白1 （nucleophosmin family, member 1,NPM1）基因外显子11。通过琼脂糖凝胶电泳、变性聚丙烯酰氨凝胶电泳（Denaturing polyacrylamide gel electrophoresis, Denaturing PAGE）与毛细管电泳（Capillary electrophoresis, CE）3种方法对99例初诊急性髓系白血病（acute myeloid leukemia,AML）患者FLT3基因中小片段插入突变（length mutation, FLT3-LM）进行检测；并用变性PAGE和毛细管电泳两种方法对上述患者NPM1基因插入突变进行检测；同时我们还应用PCR-变性PAGE法对44例骨髓增生异常综合征（myelodysplastic syndromes, MDS）患者的FLT3-LM和NPM1插入突变进行检测。采用G显带方法分析了72例AML患者和39例MDS患者的染色体核型。本研究目的是确定FLT3-LM与NPM1突变在初诊AML患者中的发生率、在FAB各亚型患者中的分布、与AML患者染色体核型以及临床疗效之间的关系；通过对部分FLT3或NPM1基因插入突变阳性患者的PCR产物的直接测序,确定该两种基因突变的特点；建立一种检测FLT3-LM与NPM1插入突变灵敏可靠的方法。结果：1.在AML与MDS患者中均可检出大量染色体核型异常,AML患者中染色体核型异常率68.06%（49/72）高于MDS患者染色体核型异常率46.15%（18/39）（p<0.05）。2.AML患者FLT3-LM发生率为30.3%（30/99）。各种电泳技术检测的效率略有不同：琼脂糖凝胶电泳、变性]PAGE、毛细管电泳3种方法的检出率分别为20.2%（20/99）,29.29%（29/99）,30.3%（30/99）。对照组中MDS患者应用变性PAGE方法的检出率为4.55%（2/44）,正常人中未检出。3.AML患者NPM1基因插入突变发生率为15.15%（15/99）。变性PAGE、毛细管电泳两种方法的检出率分别为11.11%（11/99）和15.15%（15/99）,同时检测出2例内含子缺失突变。对照组中MDS患者与正常人应用变性PAGE方法均末检出突变。4.30例FLT3-LM+的AML患者中26例患者突变型与野生型等位基因比值（allelic ratios, AR）大于0.02,4例患者AR小于0.02。FLT3-LM+ M6型患者中,AR均小于0.05。30例FLT3-LM+患者中28例患者（93.33%）突变条带为1条,2例患者（6.67%）突变条带多于1条。插入突变序列长度3-144bp不等,不同亚型的插入片段有差别。5.13例FLT3-LM+患者直接测序结果显示,4例插入碱基序列为FLT3野生型片段的完全重复,2例为完全陌生的碱基插入,7例为部分碱基序列重复。所有重复或插入序列发生在p.E573位至p.P606位之间,多集中于p.F590-p.R595之间。6.10例NPM1基因插入突变阳性患者PCR产物直接测序结果显示,10例均为A型突变（c.860863dupTCTG）。突变导致NPM蛋白羧基末端读码框移,末尾7个氨基酸WQWRKSL被11个氨基酸CLAVEEVSLRK所代替。2例缺失突变直接测序结果显示,1例为IVS10-18-15delCTTT,另外1例IVS10-17-15delTTT.该2例患者为新发现的内含子缺失突变。12例患者中有4例存在3’UTR区缺失1个碱基T（*165delT,rs34351976）的多态性改变。7.49例核型异常AML患者中FLT3-LM发生率为24.49%（12/49）,23例核型正常AML患者中FLT3-LM为43.48%（10/23）（p>0.05）；49例核型异常AML患者中NPM1突变为4.08%（2/49）,23例核型正常AML患者中NPM1突变为26.09%（6/23）（p<0.05）。8.99例AML患者中79例有治疗记录,6例早期死亡（生存期小于14天）,可供疗效评价的患者为73例。FLT3-LM+患者中完全缓解（CR）率为36.36%（8/22）,低于FLT3-LM患者CR率62.75%（32/51）（p<0.05）。早期死亡6例患者中FLT3-LM+占50%（3/6）。73例可评价疗效患者中,CR患者中FLT3-LM+占20%（8/40）、部分缓解（PR）患者中为38.1%（8/21）、未缓解（NR）患者中为50%（6/12）。49例非M3型患者中FLT3-LM+17例,FLT3-LM32例,达到CR的患者共17例,其中FLT3-LM患者CR率为43.75%（13/32）,FLT3-LM+患者CR率17.65%（4/17）（p>0.05）。9.15例NPM1阳性患者中FLT3-LM发生率为40%（6/15）,高于NPM1阴性患者FLT3-LM发生率28.57%（24/84）,但无统计学差异（p>0.05）。10.FLT3-LM-/NPMl+患者的CR率为80%（4/5）,FLT3-LM-/NPM1患者的CR率为60.87%（28/46）,FLT3-LM+/NPM1患者的CR率为41.18%（7/17）FLT3-LM+/NPM1+患者CR率为20%（1/5）。非M3亚型AML患者的治疗结果为：FLT3-LM-/NPM1+患者CR率为80%（4/5）,FLT3-LM-/NPM1患者CR率为37.04%（10/27）,FLT3-LM+/NPM 1患者CR率为16.67%（2/12）,FLT3-LM+/NPM1+患者CR率为20%（1/5）。11.在17例FLT3-LM+患者的63份标本中,复查时仍有11例患者为阳性（共20份标本）。出现的阳性条带均与初诊时阳性条带大小相同。结论：1.AML与MDS患者中存在大量染色体核型异常,对诊断预后具有重要意义。2.在FLT3-LM基因突变检测中,相对于琼脂糖凝胶电泳,变性PAGE和毛细管电泳具有较高的分辨率和灵敏度。3.FLT3-LM与NPM1突变为AML患者中常见基因改变。NPM1突变与FLT3-LM无相关性。4.FLT3插入基因序列突变长度范围变动较大,部分导致插入点的氨基酸发生取代,其他为整框插入；突变型/野生型AR范围波动较大。5.部分FLT3-LM+患者插入序列为陌生碱基,FLT3基因突变位置相对较集中,所有重复或插入发生在p.E573位至p.P606位之间,多集中于p.F590-p.R595之间。6.AML患者中正常核型患者FLT3-LM发生率与异常核型患者无统计学差异,正常核型患者NPM1基因突变发生率高于异常核型患者。7.NPM1插入突变均为A型突变,突变导致NPM蛋白羧基末端读码框移,末尾7个氨基酸WQWRKSL被11个氨基酸CLAVEEVSLRK所代替。首次在NPM1基因内含子区发现2例缺失突变,突变类型分别为IVS10-18-15delCTTT和IVS10-17-15delTTT。8.FLT3-LM对CR率有不利影响,NPM1突变单独发生的患者有较高的CR率,NPM1和FLT3-LM同时发生突变的患者CR率较低,预后不好。9.FLT3-LM或可作为AML患者微小残留病检测标记。
【Abstract】 The aim of this study was to analyze the frequencies of fms-like tyrosine kinase-3（FLT3） and nucleophosmin family, member 1 （NPM1） gene mutations in de novo acute myeloid leukemia（AML） patients, to investigate the relationship between FLT3 length mutation （FLT3-LM） or NPM1 mutations and chromosome alterations, FAB subgroups, as well as efficacy of therapy. We also studied the mutant features of FLT3 and NPMl and evaluated the sensitivibility and reliability of the procedure developed for identifying both FLT3 and NPM1 mutations. Genomic DNA was amplified by PCR. The PCR products were seperated with 2% agarose gel electrophoresis,8% denaturing PAGE or capillary electrophoresis to detect the length mutations of FLT3 gene in 99 de novo AML patients.8% denaturing PAGE and capillary electrophoresis were used to detect the mutations of NPM1 gene. Denaturing PAGE was also used to detect the mutations of FLT3 gene and NPM 1 gene in 44 myelodysplastic syndromes （MDS） patients as controls. Karyotyping was performed in 72 AML patients and in 39 MDS patients by G banding techniques. Some of the PCR products of FLT3 and/or NPMl positive patients were sequenced.Results:1. There were various aberrational karyotypes in AML and MDS patients, and the frequencies of abnormal karyotype in AML（68.06%） were higher than that in MDS（46.15%）（p<0.05）.2. The frequencies of FLT3-LM were identified in 30.3%（30/99） of AML patients. The detection rates were different with the electrophoresis techniques employed, 20.2%（20/99） with agarose gel electrophoresis,29.9%（29/99） with denaturing PAGE, and 30.3%（30/99） with capillary electrophoresis, respectively. In control group, 4.55%（2/44） was detected by denaturing PAGE in MDS patients, and none was detected in normal subjects.3. The frequencies of NPM1 mutations were detected in 15.15%（15/99） of AML patients,11.11%（11/99） with denaturing PAGE, and 15.15%（15/99） with capillary electrophoresis. We also identified 2 intronic deletion mutations. In control group, neither was detected by denaturing PAGE in MDS patients nore in normal subjects.4. The ratios of mutant alleles to wild alleles of FLT3 allele（AR） in 26 patients were above 0.02, with 4 patients below 0.02. In FLT3-LM+ M6 patients, all of their AR were below 0.05. Twenty eight patients（93.33%） had a single duplicated allele, while there were 2 patients had 2 or more duplicated alleles. The length of inserted sequeces ranged from 3bp to 144bp in all FLT3-LM+ patients.5. DNA sequences analysis of FLT3-LM+ were performed in 13 patients. Four patients were of pure duplications, and 2 patients had foreign sequences inserted, and the other 7 patients were partial duplications. The duplication or insertion occured in the segment from p.E573 to p.P606 of the FLT3 protein, with the majority clustered in a stretch between codons 590 and 595.6. DNA sequences analysis of NPM1 mutation were performed on 10 patients, all of them were of A type, which was a 4 bp insertion（c.860863dupTCTG）. The C-terminal portion of the NPM protein by replacing the last seven amino acids（WQWRKSL） with 11 residues（CLAVEEVSLRK）. Two new intronic deletion mutations were identified, one case was IVS10-18-15delCTTT, the other was IVS10-17-15delTTT.7. In AML patients with abnormal karyotype, the frequencies of FLT3-LM were not different significantly from that in patients with normal karyotype,24.49%（12/49） vs 43.48%（10/23）（p>0.05）. While there was a significant difference in frequencies of NPM1 mutation between patients with abnormal karyotype and with normal karyotype 4.08%（2/49） vs 26.09%（6/23） （p<0.05）.8. The complete remission（CR） rate in FLT3-LM+ patients（36.36%） was lower than that in FLT3-LM- patients（62.75%） in the panel of 73 patients（p<0.05）. The distributions of FLT3-LM+ were 8 out of 40 in CR patients,8 out of 21 in PR patients, and 6 out of 12 in NR patients. Seventeen patients achieved CR among 49 non-M3 AML patients, of which 17 were FLT3-LM+ and 32 were FLT3-LM-, the CR rate was higher in FLT3-LM- patients （43.75%,14/32） than that in FLT3-LM+ patients （17.65%,3/17） （p>0.05）.9. Fourty percents （6/15） of NPM1+ patients were also FLT3-LM+, which is higher than NPMl-FLT3+ patients（28.57%,24/84）, but there was not statistical significance.10. The CR rate in FLT3-LM-/NPM+ patients was 80%（4/5）,60.87%（28/46） in NPMl-/FLT3-LM- patients, and 41.18%（7/17） in FLT3-LM+/NPM1- patients, and 20%（1/5） in FLT3-LM+/NPM1+ patients. In the non-M3 AML patients group, the CR rate was 80%（4/5） with FLT3-LM-/NPM1+, and 37.04%（10/27） with FLT3-LM-/NPM1-,and 16.67%（2/12） with FLT3-LM+/NPM1-, and 20%（1/5） with FLT3-LM+/NPM1+, respectively.11. Twenty samples from 11 patients remained positive in 63 samples of 17 FLT3-LM+ patients followed-up. The lengths of duplication remained the same as that detected when the initial diagnose was made.Conclusions:1. There were plenty of chromosome aberrations in AML and MDS patients, which were important to dignosis and prognosis.2. Denaturing PAGE and capillary electrophoresis were more sensitive than agarose gel electrophoresis in detecting FLT3 length mutations.3. FLT3 length mutation and NPM1 mutation were the common genetic evidences in AML patients. There was no correlation between NPM1 and FLT3-LM mutations.4. The insert sequences of FLT3-LM+ranged from 3bp to 144bp, some insertions led to substitution of amino acids, others remained the reading frame. The ratios of mutant allele to wild allele ranged from 0.01 to 3.5. Some of the insert sequences of FLT3-LM positive patients were foreign bases. The duplication or insertion occured in the region from p.E573 to p.P606 of the FLT3 protein, with the majority clustered in a stretch between codons 590 and 595.6. There was no statistics significance in the frequencies of FLT3-LM+ between AML patients with normal karyotype and with abnormal karyotype, while the frequencies of NPM1 mutation were higher in patients with normal karyotype than that in patients with abnormal karyotype.7. The NPM1 mutations were all of A type, which was a 4 bp insertion （860863dupTCTG）. The C-terminal portion of the NPM protein by replacing the last seven amino acids（WQWRKSL） with 11 residues（CLAVEEVSLRK）.Two intronic deletions were novel, one mutation was IVS10-18-15delCTTT, and the other was IVS10-17-15delTTT.8. FLT3-LM was associated with lower CR rate. FLT3-LM-/NPM1+ was associated with higher CR rate, while FLT3-LM+/NPM1+ was associated with lower CR rate and bad prognosis.9. As a following-up marker, FLT3-LM+ could be used to detect the minimal residual disease for AML patients.