CLINICS 2011;66(5):793-799 Mutations in Patients with Myelodysplastic Syndromes Machado-Neto JA et al. Figure 3 - JAK2 V617F genotyping. (A) PCR amplification of JAK2: lane 1: 100 bp ladder; lane 2: negative control; lanes 3 to 6 – 460-bp amplicons obtained from the genomic DNA of a patient with PV (3), a CMML patient after disease progression (4) and two MDS patients (with RA) (5 and 6). (B) BsaXI digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in a PV patient (3) and in the CMML patient positive for the JAK2 V617F allele after disease progression (4); lanes 5 and 6: digestion pattern observed in two MDS patients (with RA) with wild-type JAK2 alleles. BigDye terminator version 3.0 cycle sequencing kit, according to the manufacturer’s instructions, using either one of the primers used for amplification (Table 3). After ethanol- sodium acetate precipitation, samples were analyzed on the ABI PRISM 3100 Genetic Analyzer. RESULTS PI3K mutation analysis Samples from 51 MDS patients were screened for PI3K mutations; all 51 samples were screened at diagnosis, and 5 were screened again after disease progression. We exam- ined exons 9 and 20, as a previous report has shown that over 75% of the PI3K mutations found in a large number of cancers are present in these exons.19 The sequencing of PCR products showed the absence of mutations in exons 9 and 20 of the PI3K gene in all MDS patients. PCR products and the sequences of exons 9 and 20 are presented in figure 1. FLT3-ITD and NPM1 mutation analysis Forty-six MDS patients were screened for FLT3-ITD and NPM1 exon 12 mutations at diagnosis, and 5 of these patients were also screened at the time of disease progres- sion. AML patients with the FLT3-ITD or NPM1 mutations were used as positive controls. Analysis of DNA samples from the MDS patients showed that all samples included fragments of normal size, indicating the absence of muta- tions (figure 2). JAK2 V617F and FLT3-D835 mutation analysis Fifty-one MDS patients were screened for JAK2 V617F, and forty-seven were screened for FLT3-D835 at the time of diagnosis. Five patients were screen after disease progression. One PV patient with JAK2 V617F and one AML patient with FLT3-D835 were used as positive controls. RFLP analysis showed the absence of JAK2 and FLT3-D835 mutations in all MDS patients at the time of diagnosis. Interestingly, we Figure 4 - FLT3-D835 genotyping. (A) PCR amplification of FLT3: lane 1: 100 bp ladder; lane 2: negative control; lanes 4 to 5: 114-bp amplicons obtained from the genomic DNA of patients with MDS (3-4) and AML (5). (B) Eco321 digestion: lane 1: 100 bp ladder, lane 2: negative control; lanes 3 and 4: digestion pattern observed in two MDS patients negative for the FLT3-D835 allele, lane 5: digestion pattern observed in an AML patient with the FLT3-D835 mutation. 797