Novel missense mutations in the activation loop of FLT3. (A) Sequencing chromatograms of the N841I and N841Y missense mutations. The black arrows indicate the position of the heterozygote variant base. The forward (F) and the reverse (R) reads are shown. (B) Mass spectrometry–based genotyping detects the N841I and N841Y variants. Primer extension assays were developed for each variant nucleotide and extension reaction was carried out after PCR amplification of the target sequence. Wild-type and variant extension products, differing in mass as a result of differences in the extended length, were detected by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF). The white arrows indicate the variant peaks. The negative control represents the spectra of the extension probe in the absence of input DNA. (C) Schematic diagram of the FLT3 tyrosine kinase and the position of the novel missense mutations within the kinase activation loop. EC indicates extracellular domain; TM, transmembrane; JM, juxtamembrane; and KIN, kinase domain. The position of D835 is also shown for comparison.