Rapid and sensitive detection of internal tandem duplication and activating loop mutations of FLT3

• Published in: British journal of haematology Vol. 130; no. 2; pp. 203 - 208
• Main Authors: Mills, Ken I., Gilkes, Amanda F., Walsh, Val, Sweeney, Marion, Gale, Rosemary
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01.07.2005; Blackwell; Blackwell Publishing Ltd
• Subjects: activating loop mutations; Acute Disease; acute myeloid leukaemia; Age Distribution; Base Sequence; bioanalyser; Biological and medical sciences; Biomarkers, Tumor - genetics; Female; FLT3 mutations; fms-Like Tyrosine Kinase 3; Hematologic and hematopoietic diseases; Hematology; Humans; internal tandem duplication; Leukemia, Myeloid - genetics; Leukemias. Malignant lymphomas. Malignant reticulosis. Myelofibrosis; Male; Medical sciences; Middle Aged; Molecular Sequence Data; Mutation; Polymerase Chain Reaction - methods; Proto-Oncogene Proteins - genetics; Receptor Protein-Tyrosine Kinases - genetics; Sex Distribution; Tandem Repeat Sequences - genetics; FLT3 mutations; Duplication; Hematology; Acute; Malignant hemopathy; internal tandem duplication; activating loop mutations; bioanalyser; acute myeloid leukaemia; Genetics; Fms-like tyrosine kinase 3; Diagnosis; Mutation; Acute myelocytic leukemia
• ISSN: 0007-1048; 1365-2141
• DOI: 10.1111/j.1365-2141.2005.05589.x

Summary Mutations of the FLT3 gene, a receptor tyrosine kinase, are the most frequent genetic alteration reported in acute myeloid leukaemia, with internal tandem duplications (ITD) or mutations within the activating loop (AL) reported at a frequency of around 24% and 6%, respectively. ITD mutations have associated with a poor prognosis. In this study we have used polymerase chain reaction (PCR), combined with restriction enzyme digestion for the detection of AL mutations, with the DNA products separated on the Agilent 2100 Bioanalyser using a DNA‐500 kit. This analysis enabled the rapid identification of mutations in FLT3, approximate sizing of the ITD, an estimate of the proportion of mutant RNA and in some cases, specific heteroduplex patterns associated with triplet deletions. Our data shows that approximately 16% of the patients examined had an ITD mutation and over 13% had a mutation in the AL including triplet deletions involving codons 835/836 and point mutations in codon D839. Based on the sensitivity and speed of the bioanalyser, we suggest that this method is invaluable and provides an improvement to the current use of agarose gels for the analysis of FLT3 PCR products.