1853 acute myeloid leukemia AML 10 μmol /L 0. 5 μl Hotstart polymerase 1. 0 μl DNA 2. 0 Fms 3 Fms - like tyrosine kinase μl ng RNA 25 3 FLT3 in

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鹃切郝
7 years ago
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1 1852 Chinese Journal of Pathophysiology FLT DHPLC AML Fms 3 FLT3 ITD 14 PCR 121 AML DH- PLC CE 121 PCR DHPLC % 34. 5% 11. 4% % bp CE P > PCR DHPLC AML Fms 3 R A doi /j. issn Establishment of a method to quantitatively detect FLT3 internal tandem duplication in acute myeloid leukemia with denaturing high - performance liquid chromatography CHEN Jian - lan 1 LI Qing - shan 1 WANG Han - ping 1 YING Yi 1 DU Qing - hua 1 XU Yan - li 1 CHEN Xiao - yan 1 XIE Jian - jin 1 MAO Ping 1 LI Zhi - peng 2 1 Department of Hematology Guangzhou First Municipal People's Hospital The Affiliated Hospital of Guangzhou Medical College Guangzhou China 2 Laboratory of Molecular Diagnosis School of Life Sciences Xiamen University Xiamen China. E - mail qingshan_l@ yahoo. com ABSTRACT AIM To establish a relatively - quantitative method to detect the internal tandem duplication ITD mutation of Fms - like tyrosine kinase 3 FLT3 gene in acute myeloid leukemia AML patients using denaturing high - performance liquid chromatography DHPLC. METHODS According to the fact that much more mutations are located in exon 14 we designed the primers and use the method of polymerase chain reaction PCR to specifically amplify mutation gene in 121 cases of AML and relatively quantified the situation of mutant allelic gene of by the method of DHPLC. The effectiveness of DHPLC was verified by the method of capillary electrophoresis CE. The sequenced results from PCR amplified products of 121 samples were compared. RESULTS A characteristic of elution peak was detected by DHPLC with 10. 7% overall positive rate 13 /121 and varied in the proportion of mutant alleles with a single duplicated insert fragment from 21 bp to 87 bp. The median range of mutant alleles was 34. 5% 11. 4% %. No significant difference of the positive rates and mutation proportions between the results with DH- PLC and the results with CE method was observed. The results of mutant gene of 121 samples were consistent with the results using sequencing method. CONCLUSION A relatively - quantitative method to analyze AML patients with mutation by DHPLC is successfully established. KEY WORDS Denaturing high - performance liquid chromatography Leukemia myeloid acute Fms - like tyrosine kinase 3 Internal tandem duplication Genes Tel E - mail qingshan_l@ yahoo. com

039 Establishment of a method to quantitatively detect FLT3 internal tandem duplication in acute myeloid leukemia with denaturing high - performance liquid chromatography CHEN Jian - lan 1 LI Qing -

2 1853 acute myeloid leukemia AML 10 μmol /L 0. 5 μl Hotstart polymerase 1. 0 μl DNA 2. 0 Fms 3 Fms - like tyrosine kinase μl ng RNA 25 3 FLT3 internal tandem duplication ITD min s 55 1 min 72 1 min μl ABI Veriti PCR AML 2 FLT3 7 min 1 PCR 95 5 min - ITD 25 1 min relapse rate RR disease - free survival 2. 3 DHPLC WAVE 3500 DFS overall survival OS Buffer A 0. 1 mol /L TEAA 50 ml 2 mmol /L TEAA μl ACN HPLC 1 LBuffer B 0. 1 mol /L TEAA 25% ACN 50 ml 2 mmol /L TEAA ml capillary electrophoresis CE WAVE ACN HPLC 1 L PCR 3 15 μl PCR denaturing high - performance liquid chromatography DHPLC chronic myeloid leukemia 260 nm A CML ABL 4 DHPLC DHPLC DHPLC DHPLC CE /2 100% CE 5 AML bp AML PCR 25 M1 11 M2 35 μl 5 μl 10 PCR buffer 100 mmol /L Tris - HCl M3 35 M4 9 M5 26 M6 4 M ph mmol /L KCl 15 mmol /L MgCl mmol /L dntps 1 U Taq HS DNA 150 nmol /L 5 μl gdna T3 Thermocycler PCR 1. 2 EZHigh TM DNA kit Texas BioGene min 18 Touchdown buffer 2. 5 mmol /L dntps 2. 5 mmol /L Mg 2 + PCR s s 0. 5 RNase H 2 O TaKaRa s 17 PCR s LCGreen plus dye Hotstart polymerase Qiagen FLT3 s s 72 7 min - ITD NCBI TaKaRa 1 μl PCR DHPLC TEAA ACN Transgenomic CEQ 600 size standard mixture Beckman Coulter p /n SLS DNA 1 CEQ8800 EZHigh TM DNA kit DNA Frag - 2 A 260 /A 280 DNA Fragment Analysis DNA PCR 14 DNA PCR 367 bp 328 bp 5' - AGC AAT 367 bp 367 bp TTA GGT ATG AAA GCC AGC TA - 3' 5' - GGT DNASep 0. 9 ml /min % = μl SLS 2. 5 μl S1 TGC CGT CAA AAT GCT GAA AG - 3' 5 S2 buffer 5. 0 μl 10 μmol /L 0. 5 μl % = S2 / S1 + S2 100%

min relapse rate RR disease - free survival 2. 3 DHPLC WAVE 3500 DFS overall survival OS Buffer A 0. 1 mol /L TEAA 50 ml 2 mmol /L TEAA + 250 μl ACN HPLC 1 LBuffer B 0.

3 DNA DNA 1 DHPLC CE DNAMAN NCBI FLT3 Table 1. The quantitative analysis of mutation in 2 positive samples examined with DHPLC and CE methods %. the average for three repeative 3 experiments SPSS Sample's No. DHPLC t CE AML DHPLC / % % 12. 4% % 1 10 PCR 3 2 DHPLC 1 2 AML CE DNA CE 367 bp 121 AML bp bp 367 bp / % bp 3 DHPLC CE % 11. 4% % P > < 25% 5 25% 50% 5 > 50% Figure 1. The DHPLC characteristic elution peaks. From top to bottom represent sample of wildtype the 28th sample mutation in the low percentage 12. 4% the 8th sample mutation in the moderate percentage 42. 3% the 7th sample FLT3 - ITD mutation in the high proportion 87. 0%. 1 DHPLC AML AML WHO 40% - 50% 6 7 DHPLC 60 AML AML CE 1 2 RR DFS OS RR DFS

0 1 121 4 5 7 8 11 14 28 79 85 93 107 110 28 79 12. 4 79. 8 11. 4 76. 5 118 2 3 85 1 13 /121 10. 7% 93 3 107 13 110 16. 3 16. 3 23. 5 76. 3 15. 8 16. 6 25. 2 73. 5 34. 0% 12. 4% 118 16. 2 15. 0-87.

4 1855 Figure 2. The analysis result of one sample using CE method. 2 CE OS 25% DHPLC 25% - 50% 10. 7% 50% 13 /121 AML 20% - 30% Bullinger 8 AML 30% - 40% 9 AML Thiede 2 FLT3 - ITD CE FLT3 DHPLC CE AML 10 CE PCR FLT3 DHPLC 1 - ITD CE DHPLC DHPLC CE DHPLC DNA CE 9 PCR DHPLC DNA FLT3 - ITD ITD FLT3 CE 121 AML 13 DH- 1 Gale RE Green C Allen C et al. The impact of FLT3 internal tandem duplication mutant level number size and PLC CE 1 2 P > interaction with NPM mutations in a large cohort of young

- ITD CE DHPLC DHPLC CE DHPLC DNA CE 9 PCR DHPLC DNA FLT3 - ITD ITD FLT3 CE 121 AML 13 DH- 1 Gale RE Green C Allen C et al.

5 1856 adult patients with acute myeloid leukemia J. Blood 2 Thiede C Steudel C Mohr B et al. Analysis of FLT3 - activating mutations in 979 patients with acute myelogenous leukemia association with FAB subtypes and identification of subgroups with poor prognosis J. Blood Noguera NI Ammatuna E Zangrilli D et al. Simultaneous detection of NPM1 and mutations by capillary electrophoresis in acute myeloid leukemia J. Leukemia Jiang A Jiang H Brandwein J et al. Prognostic factors in ABL normal karyotype acute myeloid leukemia in the absence of J the mutation J. Leuk Res M Schnittger S Schoch C Dugas M et al. Analysis of FLT3 length mutations in 1003 patients with acute myeloid leukemia correlation to cytogenetics FAB subtype and prognosis in the AMLCG Study and usefulness as a marker for the detection of minmal residual disease J. Blood FLT3 - ITD J Bullinger L D hner K Kranz R et al. An FLT3 gene - expression signature predicts clinical outcome in normal karyotype AML J. Blood DHPLC J S 櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆櫆 Davis JE Braucher DR Walker - Daniels J et al. Absence of Tlr2 protects against high - fat diet - induced inflammation and results in greater insulin - stimulated glucose transport in cultured adipocytes J. J Nutr Biochem Tsuchida A Yamauchi T Ito Y et al. Insulin /Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity J. J Biol Chem Yatagai T Nagasaka S Taniguchi A et al. Hypoadiponectinemia is associated with visceral fat accumulation and insulin resistance in Japanese men with type 2 diabetes mellitus J. Metabolism Ajuwon KM Spurlock ME. Adiponectin inhibits LPS - induced NF - κb activation and IL - 6 production and increases PPARγ2 expression in adipocytes J. Am J Physiol Regul Integr Comp Physiol R R Rahmouni K Fath MA Seo S et al. Leptin resistance TLR4 J. contributes to obesity and hypertension in mouse models of Suganami T Mieda T Itoh M et al. Attenuation of obesity - induced adipose tissue inflammation in C3H / HeJ mice carrying a Toll - like receptor 4 mutation J. Biochem Biophys Res Commun Frantz S Kelly RA Bourcier T. Role of TLR - 2 in the activation of nuclear factor κb by oxidative stress in cardiac myocytes J. J Biol Chem Monaco C Gregan SM Navin TJ et al. Toll - like receptor - 2 mediates inflammation and matrix degradation in human atherosclerosis J. Circulation Vijay - Kumar M Aitken JD Carvalho FA et al. Metabolic syndrome and altered gut microbiota in mice lacking Toll - like receptor 5 J. Science Jiang W Sun R Wei H et al. Toll - like receptor 3 ligand attenuates LPS - induced liver injury by down - regulation of toll - like receptor 4 expression on macrophages J. Proc Natl Acad Sci U S A Vitseva OI Tanriverdi K Tchkonia TT et al. Inducible Toll - like receptor and NF - κb regulatory pathway expression in human adipose tissue J. Obesity Silver Spring Uchikoshi Y. Narrative development in bilingual kindergarteners can Arthur help J. Dev Psychol Bardet - Biedl syndrome J. J Clin Invest Zhang Y Proenca R Maffei M et al. Positional cloning of the mouse obese gene and its human homologue J. Nature Galletti F D'Elia L Barba G et al. High - circulating leptin levels are associated with greater risk of hypertension in men independently of body mass and insulin resistance results of an eight - year follow - up study J. J Clin Endocrinol Metab

3 Noguera NI Ammatuna E Zangrilli D et al. Simultaneous detection of NPM1 and mutations by capillary electrophoresis in acute myeloid leukemia J. Leukemia 2005 19 8 1479-1482. 4.

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr

Syngene GBOXHR Retsch MM400 KQ DE Eppendorf Table 1 The sources of Astragali Radix and Hedysarum Radix No. 1 AMM2011_NMGTY Astr PCR 龙平, 崔占虎, 李虔全, 徐建平, 张春红, 周立社 * *, 李旻辉 ( 包头医学院, 内蒙古包头 014060) DNA 30 28 DNA trnl-trnf PCR PCR 136 bp 323 bp 10% PCR PCR Astragalus membranaceus Fisch. Bge. var. mongho- licus Bge. Hsiao A. membranaceus