Aileen Azari-Yam - Department of Medical Genetics, Tehran university of Medical Sciences, Tehran, Iran
Javad Tavakkoly-Bazzaz - Department of Medical Genetics, Tehran university of Medical Sciences, Tehran,Iran
Sirus Zeinali - Department of molecular medicine, Pasteur Institute of Iran, Tehran, Iran

Introduction Cytogenetically normal (CN) acute myeloid leukemia (AML) represents a heterogeneous disease entity in which mutations in certain genes have been linked toclinical outcome. The presence of CCAAT/enhancer binding protein alpha (CEBPA) gene mutations in patients with CN-AML confers a favorable prognosis. AML with mutated CEBPA is a provisional disease entity in the 2008 World Health Organization classification of leukemias. Routine screening of all CN-AML patients for CEBPA mutations is therefore important for individual risk-adapted therapy but is challenging due to technical difficulties related with high GC content of the gene. Detecting CEBPA mutations as either biallelic (biCEBPA) or monoallelic (moCEBPA) forms which is of prognostic significance is another challenge. The distribution of mutations through the entire CEBPA gene confers another challenge. We set out to explore the frequency of CEBPA mutations in a cohort of 50 cytogenetically normal adult AML patients. Material and methods:To amplify and direct sequencing of the entire coding region of CEBPA, five primer pairs were used in four PCR reactions. To determine whether in patients with more than one mutation in the CEBPA gene the different mutations were on the same allele or on different alleles, the entire coding region of patients with two mutations was amplified. The PCR products were subcloned into TOPO cloning vector and 16 clones from the patient were sequenced. Analysis of variants were performed using cancer genome databases and Polyphen and SIFT online tools.Results Sixteen sequence variants were found including 1 (6%) known polymorphism, 4 (25%) silent variants, 2 (12%) benign variants, 3 (18%) inframe insetion/deletions, 1 (6%) frameshift mutation, 4 (25%) damaging variants. Only one patient had a double mutation which were proved to be biallelic. Conclusion CEBPA gene mutation detection is important for risk-adopted therapy of AML patients, but high cost of the conventional methods such as Sanger sequencing due to technical difficulties, impedes its routine application in clinical settings especially in low-income societies.

Acute myeloid leukemia, CEBPA mutation, sequencing