TP53, FLT3, KIT, NRAS and WT1 gene point mutations detection in acute myeloid leukemia with abnormal karyotype

Vinogradov А. V., Rezaykin A. V., Sergeev A. G.

Sverdlovsk Regional Ministry of Health, Ural State Medical University, Ekaterinburg, Russian Federatian

Abstract. Aim: to estimate the frequency of mutations in FLT3 gene exons 12–15 and 19–21, KIT gene exons 7–12 and 16–19, NRAS gene exons 1–4, TP53 gene exons 4–11, WT1 gene exons 6-9 in acute myeloid leukemia (AML) patients (pts) with abnormal karyotype using direct sequencing technique.
Materials and methods. Bone marrow and peripheral blood samples were obtained from 55 AML pts aged 20 to 79, who were treated in Sverdlovsk Regional Hematological Centre during the period 2008 – 2014. Distribution of the pts according to FAB-classification was as follows: AML M0 – 3, M1 – 2, M2 – 29, M4 – 12, M4eo – 3, M5 – 1, M6 – 4, blastic plasmacytoid dendritic cell neoplasm — 1. Total RNA was extracted from leukemic cells and subjected to reverse transcription. FLT3 gene exons 12–15 and 19–21, KIT gene exons 7–12 and 16–19, NRAS gene exons 1–4, TP53 gene exons 4–11 and WT1 gene exons 6–9 were amplified by PCR. Detection of mutations in FLT3, KIT, NRAS, TP53 and WT1 genes was performed by direct sequencing. Sequencing was realized using an automatic genetic analyzer ABI Prism 310.
Results. The average frequency of functionally significant mutations in all investigated genes among the treated AML pts was 38.2 %, and varied in different morphologic, cytogenetic subgroups and in the presence of other genetic abnormalities. Average frequency in AML with abnormal karyotype of NRAS mutations was 19.2 %, TP53 – 18.4 %, KIT – 14.3 %, FLT3 – 9.6 %, WT1 – 3.6 %. There was no molecular damage in TP53 and WT1 tumor suppressor genes in acute myeloid leukemia with abnormal karyotype were accompanied by or mutations of genes, encoding cell signaling proteins (FLT3, KIT and NRAS). In 23.8 % cases AML pts with abnormal karyotype and co-existing mutations in proto-oncogenes and anti-oncogenes characterized by more dismal prognosis and poor response to standard chemotherapeutic regimens.
Key words: point mutation, acute myeloid leukemia, TP53 gene, FLT3 gene, KIT gene, NRAS gene, WT1 gene, sequencing

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Authors
Vinogradov Alexander V.
Sverdlovsk Regional Ministry of Health, Department of Specialized and Hi-Tech Medical Care
MD, chief therapist, scientific advisor
Russian Federation, 620014, Ekaterinburg, Wainer str., 34b
vinogradov-av@russia.ru
Rezaykin Alexey V.
Ural State Medical University, Department of microbiology, virology and immunology, Laboratory of Molecular Diagnostic
MD, assistant
Russian Federation, 620028, Ekaterinburg, Repin str., 3
alexrez@usma.ru

Sergeev Alexander G.
Ural State Medical University, Department of microbiology, virology and immunology, Laboratory of Molecular Diagnostic
MD, professor, chief of the department, scientific chief of the laboratory
Russian Federation, 620028, Ekaterinburg, Repin str., 3
sergeev@usma.ru