Establishment of a diagnostic procedure and preliminary scanning for length mutations in cebpa/bzip domain in vietnamese acute myeloid leukemia patients - Trinh Le Phuong

TAP CHI SINH HOC 2015, 37(1se): 40-44 DOI: 10.15625/0866-7160/v37n1se. ESTABLISHMENT OF A DIAGNOSTIC PROCEDURE AND PRELIMINARY SCANNING FOR LENGTH MUTATIONS IN CEBPA/BZIP DOMAIN IN VIETNAMESE ACUTE MYELOID LEUKEMIA PATIENTS Trinh Le Phuong, Bui Huong Quynh, Do Thi Thanh Trung, Pham Bao Yen* University of Science, VNU, *cinaus@gmail.com ABSTRACT: Acute Myeloid Leukemia (AML) is a hematological disorder triggered by the development of abnormal myeloblasts from myeloid stem cells in bone marrow. There is strong clinical evidence to prove that AML is related to mutations in several genes such as Nucleophosmin 1 (NPM1), FMS-like tyrosine kinase 3 (FLT3), and CCAAT/enhancer binding protein alpha (CEPBA) and support the use of these mutations as molecular markers to detect AML in normal karyotype (NK) cases. CEBPA mutations, contributing to approximately 10% of NK-AML cases, occur along the intron-free gene, but are highly concentrated on the N terminal, transactivation domain (TAD) and C terminal, bZIP regions. To complete the set of genetic tests required in AML diagnosis, this study aimed to establish a standard, simple, and effective procedure to detect CEBPA/bZIP length mutations in the C terminus for regular use in the molecular laboratories of Vietnamese hospitals. Starting materials used were total DNA extracted from AML patients, which served as template for PCR with specific primers to amplify a fragment of the bZIP region. The PCR products were then analysed by a single-strand conformation polymorphism (SSCP) protocol for mutation detection. To facilitate the procedure establishment, a synthetic mutation in which a tri-nucleotide sequence was inserted was successfully generated and utilized in PCR-SSCP. Scanning of 156 Vietnamese AML patients using the set-up protocol revealed several mutants and the characteristics of the respective mutations were preliminarily analysed. Keywords: Acute Myeloid Leukemia (AML), CCAAT/enhancer binding protein alpha (CEBPA), diagnosis, mutation, Polymerase chain reaction- single-strand conformation polymorphism (PCR- SSCP). INTRODUCTION The CCAAT enhancer binding protein alpha (CEBPA) is a typical basic region leucine zipper transcription factor involving in regulation of myelopoiesis and granulopoiesis, especially myeloid differentiation [‎5]. The protein, 358 amino acid long, consists of the two N terminal transactivation domains, TAD1 and TAD2 and C terminal leucine zipper required for dimerization and a basic region adjacent to the zipper necessary for DNA-binding, called bZIP. CEBPA is encoded by an intronless gene and mapped to human chromosome 19q13.1 [‎4]. The CEBPA mutations occur along the gene, and reportedly contribute to 7-15% of Acute Myeloid Leukemia (AML) [‎2, ‎3]. AML is a severe and rapidly developing blood cancer, which is triggered by the formation of abnormal myeloblasts from myeloid stem cells in bone marrow [‎6]. There is a strong evidence to prove that CEBPA mutations used as molecular marker for AML diagnosis and prognosis, in addition to mutations in other genes such as Nucleophosmin 1 (NPM1), FMS-like tyrosine kinase 3 (FLT3) [‎6]. There are various techniques to detect CEBPA/bZIP length mutations including electrophoresis and direct sequencing. However, a rapid and well established diagnostic procedure has not yet been set up for regular use in hospitals in Vietnam. In this study, we investigated different methods to establish a rapid diagnostic procedure based on PCR amplification followed by modified SSCP to detect mutation in CEBPA/bZIP. Moreover, we preliminarily screened the mutations in 156 Vietnamese patients. MATERIALS AND METHODS Study subjects and genomic DNA isolation: Blood specimens of 156 patients diagnosed with AML were collected from National Institute of Hematology and Blood Transfusion (NIHBT) following the guidelines and ethical rules of the NIHBT. The total blood was treated with Red Blood Cell (RBC) lysis buffer to isolate leukocytes and genomic DNA was extracted with Magpure Genomic DNA nano kit (Key Laboratory of Enzyme and Protein Technology - KLEPT). The quality and quantity of isolated DNA were examined by electrophoresis on 1% agarose gel and measurement of A260/280 value with Nanodrop 1000c spectrophotometer (Thermo Scientific). BZIP fragment amplification: In order to detect CEBPA/bZIP mutation, a DNA fragment of 143 bp was amplified using CEBPA forward primer: 5’-TCGGTGGACAAGAACAGC-3’; and CEBPA reverse primer: 5’-AGGCGGTCATT GTCAC TGG-3’. PCR mixture (25µl total volume) contained 2.5 µl of 10X Dream Taq Buffer; 0.2 mM for each deoxyribonucleotide triphosphate; 10 pmole of each primer; 1 unit of Dream Taq DNA polymerase (Thermo Scientific); 5% DMSO and 20 ng total DNA. The PCR reaction was performed with the following thermal cycling: 94°C for 5 minutes; 35 cycles of 94°C for 1 minute, 58°C for 45 seconds, 72°C for 1 minute; and finally 72°C for 15 minutes. After that, PCR products were analyzed by 15% nondenaturing polyacrylamide gel. The PCR product was cloned through pGEM T easy kit (Promega), and then sequenced at Macrogen, Inc. (Korea). Generation of a CEBPA/bZIP mutation using Phusion Site-Directed Mutagenesis Kit (Thermo Scientific): The 5’ phosphorylated forward primer CEBPA1407 (5’-CAACGTGG AGAAGACGCAGCAG-3’ with AAG insertion) and 5’ phosphorylated reverse primer CEBPA1407 (5’-CGCTGCTTGGCCTTGTCG-3’) were designed to amplify the whole pGEM T easy plasmid contenting the previously cloned wild type bZIP fragment. The linear plasmid then was re-ligated and transferred into E. coli DH5α competent cells, then grown on ampicillin -LB plates. Positive colonies were selected through SSCP procedure described below. SSCP procedure used for detection of CEBPA mutation PCR product was mixed with 10 ng DNA of 143 bp wild type CEBPA/bZIP fragment ad with a amount of denaturing buffer containing formamide. The mixture was treated at 95oC for 10 minutes and followed by immediate cooling on ice. Finally PCR products were electrophoresis on 10% polyacrylamide gel containing 5% glycerol. RESULTS AND DISCUSSION bZIP fragment amplification and sequencing Figure 1. Amplification of the 143 base pair fragment in the bZIP region of CEBPA gene. Electrophoresis of PCR product on 15% polyacrylamide gel. Template for PCR was the total DNA extracted from AML patients (lane labeled 1 and 2), and distilled water as negative control (lane labeled (-)). M: low range DNA marker in basepair. With the GC percentage of 63% in CEBPA studied region, the condition for PCR was thoroughly optimized adding PCR-enhancing reagents such as DMSO (5%), and Betaine (1 M final concentration) and, using high annealing temperature (58°C), extending annealing step (from 30 to 45 seconds) and increasing number of cycles (from 30 to 45). Final optimized PCR conditions allowed amplification of a sharp DNA band at the position between 100-150 bp bands of the marker on 15% polyacrylamide electrophoresis gel (fig. 1), which matched the theoretical 143 base pair fragment amplified by the designed CEBPA primers. Moreover, the sequencing result confirmed the correctness of the amplified fragment, compared to published CEBPA gene sequence. CEBPA/bZIP mutation generation and diagnostic procedure establishment using SSCP method The trinucleotide insertion at the position 1530-1531 leading to addition of a Lysine (K) at the position 313-314 in CEBPA protein, reported as bZIP-E mutation, was chosen to create synthetic mutant. The mutation generation was done based on the pGEM-T easy plasmid containing the wild type 143 base pairfragment. In detail, three nucleotides of AAG were introduced in the 5’ phosphorylated forward primer CEBPA1407. PCR using the 5’ phosphorylated primers amplified a linear 3161 bp fragment consisting of the whole 3015 bp vector backbone, the 143 bp bZIP fragment and 3 nucleotide insertion (fig. 2A, lane labeled 1). The selection of positive colonies carrying mutation plasmid through direct colony PCR and modified SSCP procedure revealed two different band patterns of PCR product on electrophoresis gel. Sequencing result confirmed that the plasmid giving pattern with a single sharp band was wild type plasmid carrying the 143 bp bZIP fragment (fig. 2b, land labeled 2), while the plasmid giving extra shifted bands was mutated one, carrying 146 bp bZIP fragment (figure 2B, lane labeled 1). The result also suggested that the presence of both wild type and mutation bZIP template sequences in PCR leaded to the appearance of shifted bands in SSCP and PCR/SSCP. a b Figure 2. CEBPA/bZIP-E mutation generation a: Amplification of the whole plasmid pGEM-T easy containing wild type 143 bp bZIP fragment_labeled 1; b: Selection of mutation carrying colonies through PCR/ SSCP method. Colonies was used as template in PCR in the presence of the wild type plasmid _labeled 1 and 2. Distilled water was used in the negative control in PCR lacking of the wild type plasmid_labeled (-). Table 1. The mixture of wild type plasmid and mutation plasmid in different proportion. Mutation level (%) 0 5 10 20 Copy number of wildtype plasmid 7.2 ´ 109 6.84 ´ 109 6.48 ´ 109 5.76 ´ 109 Copy number of mutation plasmid 0 3.58 ´ 108 7.16 ´ 108 1.432 ´ 109 Mutation level (%) 30 50 70 100 Copy number of wildtype plasmid 5.04 ´ 109 3.6 ´ 109 5.012 ´ 109 0 Copy number of mutation plasmid 2.148 ´ 109 3.58 ´ 109 2.16 ´ 109 7.16 ´ 109 The sensitivity of PCR/SSCP method in CEBPA/bZIP mutation detection Figure 3. Sensitivity experiement of PCR/SSCP method. The mixture of wild type plasmid and mutation plasmid in different propotion from 0% to 100% mutation level was used as template for PCR and SSCP analysis. The plasmid contained the wild type CEBPA/bZIP amplicon (called wild type plasmid) and the plasmid contained the mutation CEBPA/bZIP amplicon (called mutation plasmid) were mixed in differentproportions as mentioned in table 1. The PCR/SSCP of these mixtures showed that the distinct extra band at the position of 200 bp which indicated CEBPA/bZIP mutation started appearing in the mixture of 5% mutation (fig. 3). It is concluded that the established method can detect sample with the level of at least 5% mutation. Scanning 156 AML patients through the established PCR/SSCP procedure The set up PCR/SSCP procedure successfully with the wild type and mutation plasmid was then used to scan CEBPA mutations in 156 AML samples. The scanning gave a result of 12 (7.7%) suspected mutation samples. The sequencing result of one suspected sample showed the insertion of three nucleotides CTG, and consequently insertion of amino acid L at position 315 (fig. 4b, Line 3). Moreover, comparing to the mutation level from the sensitivity experiment, it is estimated that this sample probably had the level of 5% to 10% mutation (fig. 4a, sample labled 2). a b Figure 4. Detection of CEBPA/bZIP mutation in 156 AML samples a: Total DNA was used as temple in PCR (sample labeled 1, 2), acccompied with mutation plasmid as positive control (sample labeled (+)) and distilled water as negative control (sample labeled (-)). b: Amino acid sequence of the wild type sample (line 1), generation mution of AAG insertion (line 2), and the mutation sample of CTG insertion (line 3). CONCLUSION A 143 bp fragment of CEBPA/bZIP length mutation containing region was successfully amplified through the optimized PCR condition. A plasmid carrying AAG insertion of CEBPA/bZIP mutation was generated and used for PCR/SSCP procedure establishment. The screening of 156 AML samples using the set up procedure gave the result of 12 suspected mutated samples (7.7%). This mutation frequency is similar to the one of 7.3% in the research of Fuster et al, (2011). One suspected mutant was confirmed CTG insertion through cloning and sequencing. Acknowledgement: The research was done at KLEPT (Key Laboratory of Enzyme and Protein Technology). We would like to give special thanks to Vietnam National Institute of Hematology and Blood Transfusion (NIHBT) for sample collection. REFERENCES Fuster O., Barragán E., Bolufer P., Such E., Valencia A., Ibáñez M., Dolz S., de Juan I., Jiménez A., Gómez MT, Buño I., Martínez J., Cervera J., Montesinos P., Moscardó F., Sanz M. Á., 2012. Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia. Ann Hematol., 91(1): 1-7. Fröhling S., Schlenk R. F., Stolze I., Bihlmayr J., Benner A., Kreitmeier S., Tobis K., Döhner H., Döhner K., 2004. CEBPA mutations in younger adults with acute myeloid leukemia and normal cytogenetics: Prognostic relevance and analysis of cooperating mutations. J. Clin. Oncol., 22(4): 624-33. Gombart A. F., Hofmann W. K., Kawano S., Takeuchi S., Krug U., Kwok S. H., Larsen R. J., Asou H., Miller C. W., Hoelzer D., Koeffler H. P., 2002. Mutations in the gene encoding the transcription factor CCAAT/enhancer binding protein a in myelodysplastic syndromes and acute myeloid leukemias. Blood, 99(4): 1332-40. Hendricks-Taylor L. R., Bachinski L. L., Sicilliano M. J., Fertitta A., Trask B., de Jong P. J., Ledbetter D. H., Darlington G. J., 1992. The CCAAT/enhancer bindingprotein (C/EBPα) gene (CEBPA) maps to human chromosome 19q13.1 and the related nuclear factor NF-IL6 (C/EBP ß) gene (CEBPB) maps to human chromosome 20q13.1. Genomics, 14: 12-17. Radomska H. S., Huettner C. S., Zhang P., Cheng T., Scadden D., Tenen D., 2004. CCAAT/Enhancer Binding Protein a Is a Regulatory Switch Sufficient for Induction of Granulocytic Development from Bipotential Myeloid Progenitors. N. Engl J. Med., 350(16): 1605-16. Renneville A., Roumier C., Biggio V., Nibourel O., Boissel N., Fenaux P., Preudhomme C., 2008. Cooperating gene mutations in acute myeloid leukemia: a review of the literature. Leukemia, 22: 915-931. Vardiman J. W., Thiele J., Arber D. A., Brunning R. D., Borowitz M. J., Porwit A., Harris N. L., Le Beau M. M., Hellström-Lindberg E., Tefferi A., Bloomfield C. D, 2009. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood, 10.1182-03-209262. THIẾT LẬP QUY TRÌNH CHẨN ĐOÁN VÀ SÀNG LỌC ĐỘT BIẾN THAY ĐỔI ĐỘ DÀI TRÊN VÙNG GEN CEBPA/BZIP TRÊN BỆNH NHÂN AML VIỆT NAM Trịnh Lê Phương, Bùi Hương Quỳnh, Đỗ Thị Thanh Trung, Phạm Bảo Yên Trường Đại học Khoa học Tự nhiên, ĐHQG Hà Nội TÓM TẮT Bệnh ung thư bạch cầu cấp tính dòng tủy (AML) khởi sinh từ sự phát triển bất thường của các tế bào gốc máu dòng tủy. Phương pháp kiểm tra kiểu hình nhiễm sắc thể có thể chẩn đoàn được 55% số ca bệnh AML. Các nghiên cứu trên thế giới đã chỉ ra những đột biến ở 3 gen: FLT3 (Fms-like tyrosine kinase), NPM1 (Nucleophosmin1) and CEBPA (CCAAT enhancer binding protein α) có liên quan đến AML, đặc biệt là ở nhóm có kiểu hình nhiễm sắc thể bình thường. Trong đó, đột biến trên gen CEBPA chiếm khoảng 10% các ca AML, và tập trung ở 2 vùng TAD và bZIP. Với mục tiêu đề tài là thiết lập quy trình đơn giản để xác định các đột biến ở vùng CEBPA/bZIP, ADN tổng số tách chiết từ máu của những bệnh nhân AML được dùng làm khuôn với các cặp mồi đặc hiệu để nhân lên đoạn gen nghiên cứu. Sản phẩm PCR sau đó được phân tích theo phương pháp SSCP để phát hiện những mẫu có đột biến. Quy trình PCR/SSCP trên được xác lập dựa trên một đột biến nhân tạo có chèn 3 nucleotide, sau đó được sử dụng để sàng lọc 156 bệnh nhân AML. Một số mẫu đã được xác định mang đột biến ở vùng CEBPA/bZIP và được giải trình tự gen. Từ khóa: Bệnh ung thư bạch cầu cấp tính dòng tủy (AML), chẩn đoán phân tử, đa hình cấu trúc mạch đơn (SSCP), khuếch đại ADN (PCR), protein tăng cường gắn CCAAT alpha (CEBPA). Ngày nhận bài: 22-10-2014