FISH analysis of NF1-associated myeloid malignancies. (A-B) Metaphase spreads of EBV-transformed cells from patient 1 that were hybridized with NF1 probe P1-12 (A) and BAC clone 1000G21, which contains the D17S928 locus at 17q25 (B). Each of 20 metaphase cells examined showed signals on both chromosome 17 homologs, consistent with disomy. The chromosome 17 homologs were identified by Hoechst-actinomycin D staining, which reveals a Q-banding-like pattern. (C) Dual-color FISH performed by cohybridizing a digoxigenin-labeled probe P1-12 (rhodamine signal) and an α-satellite probe specific for the centromere of chromosome 7 (CEP7, SpectrumGreen), which showed monosomy 7 and NF1 disomy in bone marrow cells from patient 3. Interphase nuclei were counter-stained with DAPI, and the slides mounted with PDD antifade solution. Images were visualized using a Zeiss Axioplan microscope equipped with a 63×/1.25 NA oil Plan Neofluar lens, Optivar setting 1.6. Images were acquired using a Photometrics cooled charge-coupled device (CCD) camera (Photometrics, Tucson, AZ) and NIH Image software (National Institutes of Health, Bethesda, MD), and were processed using Adobe Photoshop. (D) Graphic summary of interphase FISH analyses of myeloid leukemia cells with NF1 (red triangle) and control probes. Probe 263P1 is a 70-kb PAC clone containing D5S479 (chromosome band 5q31; yellow squares). CEP17 is a centromere-specific probe for chromosome 17 (green circles), and CEP7 is a centromere-specific probe for chromosome 7 (blue diamond). C1 is a cryopreserved bone marrow sample from a patient with AML-M4 in complete remission. Control samples C2 through C5 are cryopreserved bone marrow samples from 4 children with myeloid leukemias that retained heterozygosity at the NF1 locus. The mean distribution of signals for the chromosome 17 centromere-specific probe was determined by the interphase analysis of bone marrow cells from 10 healthy individuals. This graph is a summary of data given in Table S2.