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Rapid, low cost and sensitive detection of Calreticulin mutations by a PCR based amplicon length differentiation assay for diagnosis of myeloproliferative neoplasms.
Trung, Ngo Tat; Quyen, Dao Thanh; Hoan, Nghiem Xuan; Giang, Dao Phuong; Trang, Tran Thi Huyen; Velavan, Thirumalaisamy P; Bang, Mai Hong; Song, Le Huu.
Afiliação
  • Trung NT; Centre for Genetic Consultation and Cancer Screening, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam.
  • Quyen DT; Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam.
  • Hoan NX; Department of Molecular Biology, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam.
  • Giang DP; Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam.
  • Trang TTH; Department of Molecular Biology, 108 Institute of Clinical Medical and Pharmaceutical Sciences, Hanoi, Vietnam.
  • Velavan TP; Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam.
  • Bang MH; Institute of Tropical Medicine, Universitätsklinikum Tübingen, Tübingen, Germany.
  • Song LH; Vietnamese - German Center for Medical Research, 108 Institute of Clinical Medical and Pharmaceutical Sciences, No 1, Tran Hung Dao Street, Hai Ba Trung District, Hanoi, Vietnam.
BMC Med Genet ; 20(1): 115, 2019 06 27.
Article em En | MEDLINE | ID: mdl-31248375
BACKGROUND: Calreticulin (CALR) gene mutations are currently recommended as biomarkers in diagnosis of patients with myeloproliferative neoplasms (MPN) with Jak2 V617F negative phenotype. Our aim was to establish a rapid, low cost and sensitive assay for identification of CALR gene mutations and to validate the diagnostic performance of the established assay in a patient cohort with different clinical MPN phenotypes. METHODS: One hundred five Philadelphia-negative MPN patients, including polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF) were initially screened for JAK2 mutations by amplification-refractory mutation system (ARMS-PCR) methodology and were further subjected to detection of CALR gene mutations by our in-house assay, a PCR based amplicon length differentiation assay (PCR-ALDA). The PCR-ALDA methodology was compared with real time PCR and Sanger sequencing methods. Furthermore, the analytical sensitivity of the assay was established. RESULTS: PCR - ALDA approach was able to detect and discriminate the pseudo-positive samples containing more than 1% CALR mutant alleles. CALR mutations were not detected in 63 Jak2 V617F positive cases in all three methods. In contrast, amongst 42 Jak2 V617F negative cases, both PCR-ALDA and Sanger sequencing coherently identified 12 CALR mutants compared to 10 CALR mutants detected by real-time PCR method. CONCLUSION: PCR-ALDA can be utilized as an easy-to-use, rapid, low cost and sensitive tool in the detection of CALR mutations in Philadelphia-negative MPN patients.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Calreticulina / Reação em Cadeia da Polimerase em Tempo Real / Mutação / Transtornos Mieloproliferativos Tipo de estudo: Diagnostic_studies / Health_economic_evaluation / Prognostic_studies Limite: Adolescent / Adult / Aged / Aged80 / Female / Humans / Male / Middle aged Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Calreticulina / Reação em Cadeia da Polimerase em Tempo Real / Mutação / Transtornos Mieloproliferativos Tipo de estudo: Diagnostic_studies / Health_economic_evaluation / Prognostic_studies Limite: Adolescent / Adult / Aged / Aged80 / Female / Humans / Male / Middle aged Idioma: En Ano de publicação: 2019 Tipo de documento: Article