Determination of CEBPA mutations in pediatric acute leukemia
Received: March 02, 2018
Accepted: September 01, 2018
Published: December 05, 2018
Genet.Mol.Res. 17(4): http://dx.doi.org/gmr16039936
The CCAAT/enhancer-binding protein-alpha (CEBPA) is lineage-specific transcription factor in the hematopoietic system. Several studies have reported the presence and frequency of CEBPA mutations.In this study, we aimed the clinical features and the prognostic significance associated with CEBPA mutations in 30 pediatric patients with acute leukemia.In addition, association between found variants and mutations of Ten-Eleven-Translocation 2 (TET2), Kirsten rat sarcoma viral oncogene homolog (KRAS), and Casitas B-cell lymphoma (CBL), Fms-Related Tyrosine Kinase (FLT3), Januse Kinase-2 (JAK2) and Nucleophosmin 1 (NPM1) were analyzed which are important prognostic risk factors for pediatric acute leukemia. The entire CEBPA coding region was screened using the Next Generation Sequencing (NGS) method. Furthermore, TET2, KRAS and CBL genes were screened using the NGS method, FLT3 gene was analyzed by Real Time Polymerase chain Reaction (Real Time-PCR).JAK2 and NPM1 genes were screened by Sanger DNA sequencing. CEBPA mutations were detected in 16 (53.3%) of 30 patients. In total, ten distinct of nucleotide changes were identified in 30 patients including 6 novel and 4 known mutations by sequencing the entire CEBPA gene. We found 6 frame shift mutations, 1 missense mutation, 3 synonymous variant. The most common mutation was the c.487del G resulting p.Glu163Ser in 5 cases. Three patients carried CEBPA double mutations. The detected variants in this article seem to be the first screening results of genes studied by NGS in pediatric acute leukemia patients. Our results also showed some degree of association between FLT3-ITD, TET2, KRAS, CBL and CEBPA mutations.
Clinical and genetic prognostic markers are important in the classification of leukemia patients. Aberrant chromosomal translocations and gene mutations frequently occur in transcriptional factor that lead to uncontrolled proliferation of lymphoid and myeloid progenitors (Pabst T and Mueller BU, 2007; Kassem N et al., 2013; Roe JS and Vakoc CR, 2014; Preudhomme C et al., 2002). The CEBPA gene is a member of the leucine zipper family of transcription factor family that is essential for the differentiation of myeloid cells (Hollink IH et al., 2011). The CEBPA gene located on chromosome 19 q13.1 encodes the basic leucin zipper (bZIP) family of transcription factors (Fasan A et al., 2014). It is expressed at high levels during myeloid cell differentiation and binds to the promoters of multiple specific genes at different levels of myeloid linage maturation (Kassem N et al., 2013). In AML patients, It has been reported that two types of mutations in CEBPA gene; N terminal and C terminal (Kassem N et al., 2013; Hollink IH et al., 2011; Liss A et al., 2014; Pabst T et al., 2008). The N terminal mutations are located between the major translational start site and a second ATG further downstream lead to premature stop of translation of the wild type p 42 CEBPA protein while preserving translation of a short p30 isoform that suppresses the function of the full length protein. Mutations in the C terminal are usually in frame and deletions that affect homo-hetero-dimerization and DNA binding [6-8]. CEBPA mutations have been reported that are seen in approximately 4.5-6% of pediatric Acute Myeloid Leukemia (AML) patients. In addition CEBPA mutations are found in 5-14% of adult patients with AML (Hollink IH et al., 2011; Pabst T et al., 2008; Leroy H, Roumier C, Huyghe P, Biggio V, Fenaux P, Preudhomme C, 2005). There is no study which investigates CEBPA mutations in Turkish pediatric acute leukemia patients. Therefore in our study, we investigated entire CEBPA coding region in 30 patients with Turkish pediatric acute leukemia patients by using NGS technique and to assess the frequency of CEBPA mutations and its clinico-hematologic correlation, as well as to analyze the cooperating mutations, including FLT3, TET2, CBL, KRAS, JAK2 and NPM1 mutations in pediatric acute leukemia patients.
PATIENTS AND STUDY DESIGN
Study population consisted of 30 patients aged between 1 and 15 years a who were admitted to Losante Children’s and Adult Hospital with the diagnosis of pediatric acute leukemia. Seventeen patients were diagnosed as AML, 4 as mixed leukemia, 9 as ALL. The study is carried out in accordance with the code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The Ankara University, School of Medicine Ethics Committee approved the study protocol (project No.03-107-13/2013) and informed consent was provided by the patients’ parents.
Bone marrow samples were collected with Heparin-containing tubes, and chromosome analysis was performed using G-banding. After the slide preparation, G-banding using Giemsa-staining was carried out according to the standard procedures. On each slide 20 metaphases were analyzed by a light microscope (Nikon, JAPAN). Karyotypes were described according to International Standing Committee on Human Cytogenetic Nomenclature (ISCN) (Basel S, 2013).
Fluorescence in situ Hybridization (FISH) was performed on interphase nuclei and metaphase chromosomes of bone marrow cells using dual-color/dual-fusion probes for translocations of inv (16;16), t(9;22), t(15;17), chromosome 19, and dual-color/deletion probe for del 7q, labeled in green and red spectra according to the manufacturer’s protocol provided by Cytocell, UK. Counterstaining was performed with 40, 6-dia midino-2-phenylindole (DAPI). At least 100 nuclei were analyzed under the Fluorescence microscope, and image capture was performed using Nikon Eclipse 80i equipped with a CCD-camera (CoolCube1), appropriate filters and Isis software (MetaSystems).
DNA isolation and next generation sequencing
Blood samples were collected with EDTA-containing tubes and DNA was extracted from peripheral blood and bone marrow leukocytes with Mag-NA Pure automatic DNA isolation instrument (Roche Diagnostics, Manheim, Germany). We used NGS to study the entire CEBPA gene region that was presented by 4 overlapping amplicons. Primer sequences list were performed Table 1. NGS sample preparation and process stages were generated, previously described by Grossmann et al., 2011.
|Primer set||Forward||Tm °C||Reverse||Tm °C||Amplicon Length (bp)|
|CEBPA set 1||5’- GCCATGCCGGGAGAACT 3’||62||5’-CCCGGGTAGTCAAAGTCG-3’||59||357|
|CEBPA set 2||5’- CCTTCAACGACGAGTTCCTG-3’||61||5’-CGGCTGGTAAGGGAAGAGG-3’||62||335|
|CEBPA set 3||5’-GAGGAGGATGAAGCCAAGC-3’||60||5’-CTCGTTGCTGTTCTTGTCCA-3’||60||357|
|CEBPA set 4||5’-TGGCAGCGCCGTCAAG-3’||65||5’- CCAGGGCGGTCCCACA-3’||65||357|
Table 1: Primers used for amplification of CEBPA gene.
NGS was carried out using 454 GS Junior System Instrument Roche Applied Science. Data were analyzed using the GS Amplicon Variant Analyzer software version 2.3. (Roche Applied Science). We used to determination of variances; filters were adjust to show variances in more than 1% of bidirectional reads per amplicon in at least one patient. Figure 1 summaries location of the amplicons on the CEBPA protein.
Detection of FLT3, TET2, CBL, KRAS, JAK2 and NPM1 mutations
FLT3 mutation was analyzed by Real Time PCR on Light Cycler 480 II instrument (Roche Diagnostics, Gmbh, Mannheim, Germany). Results were analyzed with the High Resolution Melting (HRM) method using genotype profiles. Different plots were created by selecting negative controls as the base-line. Therefore, fluorescence of the all other samples was diagramed relative to this sample. Fluorescence signals were analyzed and significant differences used as indicators of mutations (Kohlmann A et al., 2010; Tan AY et al., 2008; Murugesan G et al., 2006; Akin DF et al., 2016).
Hot-spot exons of TET2, KRAS and CBL genes were screened using the NGS method. All coding exons of TET2 (exons 3 and 11) were presented by 27 amplicons. Besides, two primer pairs were amplified known mutational hotspot regions to describe the RING finger domain and linker sequence for CBL (exons 8 and 9) and KRAS (exons 2 and 3). The analyses were performed as previously described by Kohlmann et al. (Kohlmann A et al., 2010). The twelfth of the exons of the JAK2 and NPM-1 genes were amplified by polymerase chain reaction (PCR). PCR products were sequenced using the Beckman DNA Sequencer System. (Beckman Coulter, USA).
Statistical analyses were performed with SPSS 15.0 (SPSS IBM, USA). Chi-square test, independent sample t-test, or Mann–Whitney U-test, as appropriate for the type of data being analyzed, were used to assess the statistical significance of the difference between the two groups. P values less than 0.05 were considered statistically significant.
Incidence of CEBPA mutations
Among 30 pediatric acute leukemia patients, CEPBA mutations were found in 16 cases (53.3%) Patient characteristics of the 30 pediatric acute leukemia cases are shown in Table 2. Ten distinct of nucleotide changes were identified in 30 patients including 6 novel and 4 known mutations by sequencing the entire gene as shown in Table 3. The frame shift mutation was the most common mutation subtype that was found in 6 types. The remaining mutation subtypes were 3 synonymous variants and 1 missense mutation.
|Characteristics||No CEBPA mutation||CEBPA mutation||p value|
|Female ( n:9)||3||6|
|Biphenotypic Acute Leukemia (n:4)||2||2|
|High Risk (n:19)||11||8||0,20|
|Median Risk (n:2)||1||1|
|Standard Risk (n:9)||2||7|
|Down Syndrome (n:4)||-||4||0,52|
|Fragile X Syndrome (n:1)||-||1|
|Normal Karyotype (n:19)||10||9||1|
|FLT3 mutations (n:8)||5||3|
|JAK2 mutations (n:2)||-||2|
|TET2 mutations (n:5)||-||5|
|CBL mutations (n:1)||-||1|
|KRAS mutations (n:4)||-||4|
|NPM1 mutations (n:1)||1||-|
Table 2: Patient characteristics according to CEBPA mutation status.
AML: Acute Myeloid Leukemia; ALL: Acute Lymphoblastic Leukemia; Ex: Exitus
|Mutation No||Type of mutations||No of patients||Nucleotide change||Amino-acid change||Localization||Mutation Type||Comment||Rs number||Clinical Significance|
|p.Thr230Thr||Between TAD2-DNA binding domain||Synonymous variant||Rs 34529039||Bening|
|p.Ile68LeufsTer41||TAD-1 domain||Frameshift mutation in
|Rs 137852731||Pathogenic for AML|
|p.Phe73SerfsTer35||N-terminal domain||Frameshift mutation||Rs 137852733||Pathogenic for AML|
|p.His191His||Between TAD2-DNA binding domain||Synonymous variant||Rs 192240793||Bening|
|5||Nucleotide change||3||g.5444 C>T
|p.Pro112Pro||Between TAD1-TAD2 domain||Synonymous variant||Novel||NA|
c.382 del C
|p.Pro128ProsTer31||Between TAD1-TAD2 domain||Frameshift mutation||Novel||NA|
|7||Deletion||5|| g. 5597
c.487 del G
|p.Glu163Ser||Between TAD1-TAD2 domain||Frameshift mutation||Novel||NA|
c.300 del C
|p.Gly100GlysTer59||TAD-1 domain||Frameshift mutation||Novel||NA|
c.955-961 ins TTGACC
|p.Ser319TrpsTer13||C-terminal domain||Frameshift mutation||Novel||NA|
|10||Nucleotide change||1||g.5599 C>A
|p.Glu163Asp||Between TAD1-TAD2 domain||Missense variant||Novel||NA|
Table 3: Characterization of CEBPA Mutations in Pediatric Acute Leukemia.
Three patients carried CEBPA double mutations. The other 13 patients carried a single CEBPA mutation. As shown in Table 3, six patients had mutations occurring between TAD-1 domain and TAD2 domain. 4 patients had mutations in TAD1 domain, 2 patients had the combination of a between TAD2 domain and DNA binding domain mutation and a deletion between TAD-1 Domain and TAD-2 Domain. 4 patients had frame shift mutations in TAD1 domain and N-terminal part of protein.
The novel 487 deletion of G variant was observed in 5 patients (16.6%) and 3 (10%) patients presented a novel 336 C>T substitution. The 198_201 duplication of CTAC was detected 3 (10%) patients. Three (10%) of 30 patients had the 690 G>T synonymous variant. Patient 28 had a 573C>T synonymous variant. The patient 29 had a 955_961 insertion of TTGACC in DNA binding domain of CEBPA protein. Patient 10 had combined variants of 300 delG and 487 delG.
Patients with CEPBA mutations were also analyzed for mutations of FLT3, NPM1 and JAK2 Exon 12. In the 30 pediatric acute leukemia patients examined, FTL3 mutations were detected in two of the sixteen patients with CEBPA mutations and 5 of the 14 patients without CEBPA mutations had FLT3 mutations. Two patients had both JAK2 and CEBPA mutations. One patient had NPM1 mutation without CEBPA mutations.
Clinic and hematologic correlation with CEBPA mutations
Karyotype analysis was normal in 19 (63%) of the 30 patients. Trisomy 14, Trisomy 19, Trisomy 22, monosomy 7, monosomy 14, inv (16; 16), t(9;22), t(15;17), t(4;11) were found using chromosome banding and FISH analyses. We screened all patients during treatment or relapse phases. All relapse patients (Patient No 1-4, 9.20) had CEBPA mutations. The one patient with t(15;17)/PML-RARα, 1 patient with t(9;22) BCR-ABL, and 1 with inv(16)/ CBFß-MYH11 had CEBPA mutations.
Four patients including CEBPA mutations did not have cytogenetic and molecular aberrations. The presence of CEPBA mutations were no correlation with prognostic classification into “high”, “moderate” and “standard” risk groups of acute leukemia. There was no difference in FAB classification between CEBPA wild type and CEBPA mutant type. The four patients (Patients No 22, 28-30) who had Down syndrome had CEPBA mutations. The two mutations (198_201dupCTAC and 217_218insC) had been reported in previous study were described as pathogenic for AML. The ten (33, 3%) patients died during the treatment or relapse. The clinical and laboratory features of study group were summarized in Table 4. No statistical significant differences were detected in the two groups.
|1||5,5/ M||Biphenotypic (Mixed Phenotypic) Acute Leukemia||HR||Ex-46, XY|| c.690 G>T
|2||2/M||Biphenotypic (Mixed Phenotypic) Acute Leukemia||HR||Ex-46, XY|| c.690 G>T
c.487 del G
|46, XY||c.690 G>T
c.487 del G
|4||11/M||AML-M1||HR||Ex-46, XY|| c.198_201dupCTAC
|5||4/M||Pre B-ALL||SR||46,XY||c.336 C>T
|6||7/M||AML-M4||HR||47, XY,+ 22 Inv (16;16), Fragile X
|8||13/M||AML-M4||HR||46, XY, Ms,Ts 14,||c.336 C>T
|9||4/M||AML-M4||HR||46, XY||c.382 del C||-||-||-||-||-|
|10||5,5/F||AML-M2||SR||46, XX|| c.300 del C
|20||6,5/F||AML-M5||HR||46, XX|| c.487 del G
|22||3/F||AML-NA||HR||Ex, 47, XX (+21) c 21 der(14) (14q1,2→q 3,2ii 1q21→q43),der(19)(19qdel) →p13.3::11q13)11qdel . t(9,22)||c.217_218insC||+||+||p.Pro363Leu
|29||2,5/M||Pre B-ALL||SR||47,XY(+21)||c.955-961 ins TTGACC||-||+||-||-||-|
|30||3/F||Pre B-ALL||SR||47,XX(+21)||c.489 C>A||-||-||-||-||-|
Table 4: CEBPA mutations in sixteen children with acute leukemia.
Acute leukemia is a heterogeneous disorder of hematopoietic stem cells, characterized by multiple genetic events which have an impact on proliferation and differentiation. Some of the genetic and epigenetic alterations play a major role in leukemogenesis; gene mutations, deletions, translocations, and DNA methylation. Compared with adult leukemia, there were fewer studies of gene mutations in pediatric leukemia. We previously reported the frequencies of TET2, CBL, and KRAS mutations by using NGS in pediatric AML patients and the frequencies of FLT3-ITD and FLT3-TKD mutations in acute leukemia patients (Akın DF et al., 2016).
Although CEBPA mutations have been studied for many years in AML, there were no data about its prevalence and prognostic significance in Turkish patients with AML or ALL. In this study, we investigated CEBPA aberrations in pediatric acute leukemia patients to determine their frequency and prognostic impact. In previous studies, several common patterns of CEBPA mutations have been reported in AML. In the N-terminus, small out-of-frame insertions or deletions occur resulting in a premature stop codon, which inhibits transcription of the p42 product. (Preudhomme C et al., 2002; Hollink IH et al., 2011; Fasan A et al., 2014; Fuchs O et al., 2010; Fröhling S et al., 2004; Pabst T et al., 2008; Taskesen E et al., 2011; Leroy H, Roumier C, Huyghe P, Biggio V, Fenaux P, Preudhomme C, 2005; Liang DC et al., 2005).
CEBPA mutations were detected in 30 pediatric acute leukemia cases. (53.3%), with 9 cases combination of mutations and 7 cases single mutations. As the frequency of CEBPA mutations in this study was quite high and the number of patients were not enough to assess between the clinical association and mutations. We found no statistical differences in all of the clinical parameters. The combined mutations (CEBPA, FLT3, NPM1) have been detected in adult AML, their frequencies varied considerably, ranging between 25 and 35% 21. Mutations in CEBPA have been described in approximately 5–14% of adult patients with AML (Preudhomme C et al., 2002; Hollink IH et al., 2011; Barjesteh van Waalwijk van Doorn-Khosrovani S et al., 2003; Gombart AF et al., 2002; Snaddon J et al., 2003). The frequency of CEBPA mutations have been reported to be lower in pediatric AML when compared with the adult AML (Hollink IH et al., 2011; Ho PA et al., 2009; Liang DC et al. 2005). A study by Hollink et al. showed CEBPA mutations in 20 out of the 252 (7.9%) including 14 double mutant and 6 single mutant cases in pediatric AML patients (Hollink IH et al., 2011). Fröhling et al. have shown that frequency of CEBPA was %15 in young adults with AML (Fröhling S et al., 2004) They reported that mutations of CEBPA predict favorable prognosis and improve risk stratification in AML patients with normal cytogenetic.
We identified 10 mutations in CEBPA by NGS. Six novel mutations were detected in present study. The remaining 4 types of CEBPA mutations have been reported in the previous studies. We used an amplicon based sequencing method to find possible new genetic markers for leukemia diagnosis. The c.690G>T, c.198_201dupCTAC, C.217_218insC and c.573C>T variants had been reported in previous studies whereas we detected 6 novel variants in CEBPA gene in this present study. We detected three types of variants in CEBPA; frame-shift, missense and synonymous (Table 3). Totally ten variants were identified involving N-terminal, TADs and C-terminal domains. The most frequent type of variants in CEBPA in pediatric acute leukemia patients is 487 deletion of G which led to a glutamin-serin deletion between the TAD1 and TAD2 domain in CEBPA protein.
In our study we detected that frame-shift mutations (68, 75%) result in premature terminal of the full length 42–kd protein of CEBPA which has been shown in Table 3. The 198_201 duplication of CTAC and 217_218 insertion of C was previously reported as pathogenic mutations for AML. The four of sixteen patients had the pathogenic mutations and one patient had benign mutations which previously reported. Two types of mutations including c.198_201dupCTAC and c.300delC occurred in TAD1 domain. These two types of mutations could be predicted to lose of the transactivation activity of CEBPA protein. The one type mutation (c.955_961 delTTGAC) in the C terminal domain was caused truncated in the b-ZIP domain of CEBPA protein. In our study as well as in previous studies patients with frame shift mutations incoding regions have been showed and this status is associated with favorable clinical outcome (Pabst T et al., 2008; Gombart AF et al., 2002). The only one patient (patient no 22) in our study group has combination mutations of CEBPA, FLT3, TET2, CBL and KRAS genes (Table 4).This patient is three years old girl diagnosed with AML and classified as a high risk group and 18 months later she diagnosed with relapse died during the treatment. The combination of mutations cases has been very few. The combination of mutations carrying patients died in reported studies either during treatment or after relapse (Pabst T et al., 2008; Leroy H, Roumier C, Huyghe P, Biggio V, Fenaux P, Preudhomme C, 2005). We screened 6 patients at relapse: All relapse patients carried CEBPA mutations (Table 4). Fröhling et al. have detected at both types of FLT3 mutations and found no correlation with prognostic influence among AML patients with CEBPA mutations (Fröhling S et al., 2004). In this study we found 3 patients who carried both CEBPA and FLT3 mutations and two of them died during treatment.
A 5-base deletion in the intronic region (1641+179_1641+183delTCTTA-intronic) of JAK2 gene was first reported in a Down syndrome patient associated with B-cell precursor ALL was detected with CEBPA synonymous variant in pediatric biphenotypic acute leukemia and AML patients in this present study. In our study, we found no significant correlation between CEPBA mutations and other gene mutations and clinical parameters. In addition, four patients including CEBPA mutations did not have cytogenetic and molecular aberrations.
This study is the first report of the frequency of CEBPA mutations and its correlation with other genes mutations in Turkey. We think that c.487del G and c.198_201dupCTAC could be important prognostic markers for pediatric acute leukemia patients at relapse. There may be biologic differences existing between adults and children, which should need further study on a larger group of patients to validate the prognostic significance of CEBPA mutations in pediatric acute leukemia.
CEBPA may potentially be genetic markers for pediatric acute leukemia diagnosis. However, these results need to be confirmed by further studies on a larger number of patients.
Conflicts of Interest Statement
The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.
About the Authors
Dilara Fatma Akın Balı
Niğde Ömer Halisdemir University, Faculty of Medicine, Medical Biology, Niğde, Turkey
- [email protected]
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