Analysis Of Npm1 Gene Insertional Mutations In Iranian Acute Myeloid Leukemia (AML) Patients
Recent studies in leukemia patients have revealed cumulative mutations in the genes affecting cell differentiation
and development. Among these genes, IDH2, ASXL1, TET2, FLT3, CEBPA, NAS, IDH1, and NPM1 were investigated in
leukemia. NPM1 mutations have been described as the most frequent molecular alterations in acute myeloid leukemia (AML).
Detection of these mutations has become important for risk stratification and treatment decisions for patients with normal
karyotype AML. Therefore, rapid screening for NPM1 mutations should be available shortly after diagnosis. NPM1 gene is
located on chromosome 5 encoding a nuclear phosphoprotein (nucleophosmin). Frameshift mutations in exon 12 results in the
insertion of four base pairs (TCTG) in the C-terminal region, causing loss of a nucleolar localization signal and aberrant
localization of the protein to the cytoplasm. However, most reports of NPM1 mutations came from Europe and focused mainly
on adults, while report from Asian countries is few. Moreover, at the present time, no data exist in Iranian AML patients with
respect to the incidence and distribution pattern of NPM1 mutations. In the current study, we investigated the exon 12 NPM1
mutations in 40 de novo AML patients, a population of mixed adults and children, and set out to explore the prevalence,
distribution pattern and clinical profile of NPM1 mutations in our population. To this end, genomic DNA was extracted from
peripheral blood samples using QIA amp DNA blood midi kit. A 172bp fragment on NPM1 exon 12 was amplified using
HEX-labeled PCR primers spanning the insertional mutation region. The fluorescent PCR products were undergone capillary
gel electrophoresis on an ABI 3730 instrument and analyzed by associated analysis software. TCTG insertion at positions
956–957 or 960-961 of exon-12 was confirmed in 5 out of 39 AML cases (12.8 %). The results of this assay were confirmed by
PCR-sequencing of the mutation containing fragments. These genetic changes resulted in substitution of tryptophan by other
amino acid residues at either codon 288 or 290 which leads to defective NPM1 protein nuclear localization. This study showed
the remarkable frequency of NPM1 exon 12 mutations in Iranian AML patients as well as the feasibility of capillary gel
electrophoresis analysis for rapid screening of these cases.
Index Terms — AML, Insertional Mutation, Capillary electrophoresis, NPM1.