CEBPA Gene Mutational Status
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CEBPA Gene Mutational Status
A Complete Screening Using High-Resolution Melt Curve Analysis Filip Ra´zga,1 Dana Dvor˘a´kova´,1 Toma´s˘ Jurc˘ek,1 Ivana Jezˇ´ıs˘kova´,1 Zlatus˘e Kr˘´ıstkova´1 and Jir˘´ı Mayer1,2 1 Center of Molecular Biology and Gene Therapy, Department of Internal Medicine-Hemato-Oncology, University Hospital Brno and Masaryk University, Brno, Czech Republic 2 Department of Internal Medicine-Hemato-Oncology, University Hospital Brno and Masaryk University, Brno, Czech Republic
Abstract
In recent years, several independent prognostic factors in cytogenetically normal acute myeloid leukemia (CN-AML) have been reported. Mutations or the expression levels of certain genes have been often used as molecular markers for prediction of a patient’s outcome or for evaluation of treatment outcome. One of them, the gene encoding CCAAT/enhanced binding protein alpha (CEBPA), plays an important role in myeloid differentiation and, when mutated, confers a favorable prognosis for patients with CN-AML. Complete mutation screening of the CEBPA gene is therefore beneficial and requires fast, precise, and sensitive diagnostic tools. Thus, for routine diagnostics, we developed a screening method using highresolution melt curve analysis prior to direct sequencing, where only positive samples (according to reference) are further sequenced. With this approach, all positive and negative patients were successfully distinguished, and the results obtained were in absolute concordance with the direct sequence analysis.
Acute myeloid leukemia (AML) is a heterogenous group of neoplastic disorders characterized by overproduction of hematopoetic precursor cells harboring genetic and epigenetic alterations.[1] Besides the major cytogenetic abnormalities, such as reciprocal translocations or inversions, individual gene mutations also play an important role in the pathogenesis of AML. During the last decade, several clinically significant genes with mutations that directly affect proliferation, myeloid differentiation, cell cycle regulation, or apoptosis, have been reported.[2] One of them, the CCAAT/enhancer-binding protein alpha (C/EBPa) gene (CEBPA; chromosome 19q13.1), encodes a transcription factor that plays an essential role in the differentiation of myeloid hematopoietic cells. CEBPA consists of two N-terminal transactivation domains (TAD1 and TAD2), a DNA binding domain, and a C-terminal ‘leucine zipper’ domain, and is translated into two major proteins, p42 (42 kDa) and p30 (30 kDa). Its role in leukemogenesis[3,4] has been confirmed by many experiments to date, and it has been found that the loss of C/EBPa function leads to blocking of the granulocytic differentiation in AML.[5,6] Three mechanisms of the CEBPA gene inactivation have been observed: (i) downregulation of C/EBPa expression;[7] (ii) inhibition of CEBPA mRNA
translation;[8] and (iii) mutations affecting the constitution of the CEBPA gene itself.[6] Many of the CEBPA mutations (occurr
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