Aileen Azari-Yam1,2, Samira Dabbagh Bagheri3, Javad Tavakkoly-Bazzaz1, Ameneh Bandehi Sarhaddi3,4, Leili Rejali3,5, Kamran Alimoghaddam6, Marjan Yaghmaie6, Ardeshir Ghavamzadeh6, Sirous Zeinali2. 1. 1 Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences , Tehran, Iran . 2. 2 Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran , Tehran, Iran . 3. 3 Kawsar Human Genetics Research Center , Tehran, Iran . 4. 4 Science and Research Branch, Islamic Azad University , Tehran, Iran . 5. 5 Tehran North Branch, Islamic Azad University , Tehran, Iran . 6. 6 Hematology Oncology, and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences , Tehran, Iran .
Abstract
BACKGROUND: Mutations in the nucleophosmin (NPM1) gene have been used as molecular biomarkers for prognostication of patients with adult acute myeloid leukemia (AML). METHODS: We designed a rapid and sensitive method using the allele-specific-refractory mutation system-polymerase chain reaction (ARMS-PCR) to detect the most common mutations of NPM1 gene, which are mostly four base pair insertions and compared its efficacy with direct sequencing and capillary electrophoresis which served as the gold standards. RESULTS: The incidence of mutation was 22% (33% of patients with normal karyotypes had mutation compared with 16% of patients with abnormal karyotypes) based on the results obtained with capillary electrophoresis analysis and direct sequencing. All of the specimens determined to be mutation-positive by the gold standard tests were also positive by the ARMS-PCR method. Significantly, the ARMS-PCR test also helped determine the mutation status of an extra set of patients who had low call rates on capillary electrophoresis and appeared normal on direct sequencing. DISCUSSION: The low mutation rate in some patients hindered its detection in the gold standard assays because of the interference of the mutation signal by high background noise. The low sensitivity of the gold standard assays for detecting low copy number mutations rates thus increase their risk of producing false negative results that adversely affects prognostication and therapy. Our results suggest that the mutation detection rate of the ARMS-PCR assay is better than existing tests. This is most probably because of the fact that in an ARMS-PCR-based method, the mutated variant is specifically amplified, based on a mutation-specific primer. CONCLUSIONS: We conclude that the high sensitivity of the ARMS-based method together with its rapidity and low expense should make it a suitable choice for clinical laboratories.
BACKGROUND: Mutations in the nucleophosmin (NPM1) gene have been used as molecular biomarkers for prognostication of patients with adult acute myeloid leukemia (AML). METHODS: We designed a rapid and sensitive method using the allele-specific-refractory mutation system-polymerase chain reaction (ARMS-PCR) to detect the most common mutations of NPM1 gene, which are mostly four base pair insertions and compared its efficacy with direct sequencing and capillary electrophoresis which served as the gold standards. RESULTS: The incidence of mutation was 22% (33% of patients with normal karyotypes had mutation compared with 16% of patients with abnormal karyotypes) based on the results obtained with capillary electrophoresis analysis and direct sequencing. All of the specimens determined to be mutation-positive by the gold standard tests were also positive by the ARMS-PCR method. Significantly, the ARMS-PCR test also helped determine the mutation status of an extra set of patients who had low call rates on capillary electrophoresis and appeared normal on direct sequencing. DISCUSSION: The low mutation rate in some patients hindered its detection in the gold standard assays because of the interference of the mutation signal by high background noise. The low sensitivity of the gold standard assays for detecting low copy number mutations rates thus increase their risk of producing false negative results that adversely affects prognostication and therapy. Our results suggest that the mutation detection rate of the ARMS-PCR assay is better than existing tests. This is most probably because of the fact that in an ARMS-PCR-based method, the mutated variant is specifically amplified, based on a mutation-specific primer. CONCLUSIONS: We conclude that the high sensitivity of the ARMS-based method together with its rapidity and low expense should make it a suitable choice for clinical laboratories.
Authors: Florin Tripon; Claudia Bănescu; Adrian P Trifa; Andrei G Crauciuc; Valeriu G Moldovan; Alina Boglis; Istvan Benedek; Smaranda Demian; Carmen Duicu; Mihaela Iancu Journal: Arch Med Sci Date: 2021-03-18 Impact factor: 3.318
Authors: Claudia Bănescu; Florin Tripon; Adrian P Trifa; Andrei G Crauciuc; Valeriu G Moldovan; Alina Bogliş; Istvan Benedek; Delia Dima; Marcela Cândea; Carmen Duicu; Mihaela Iancu Journal: Cancer Med Date: 2019-08-01 Impact factor: 4.452