Figure 1
Patient characteristics, BCR-ABL breakpoint sequence, and genotyping of single colonies. (A) Clinical diagnoses, treatment history, and diagnostic workup of a 56-year-old female patient with coexisting JAK2-V617F mutation and BCR-ABL rearrangement. BM, bone marrow; CMR4.5, complete molecular remission with a log4 to log5 reduction of BCR-ABL transcripts; GRA, granulocytes; n.d., not determined; Ph, Philadelphia chromosome. (B) Sequencing chromatogram of a 157-bp amplicon derived from granulocyte DNA revealed the breakpoint sequence in the intron 14 of BCR and intron 1 of ABL. The same sequences were obtained in colonies positive for BCR-ABL. (C) Analysis of single colonies for mutation in JAK2-V617F and BCR-ABL rearrangement in the genomic DNA before and after tyrosine kinase (dasatinib) therapy. Mononuclear cells from peripheral blood were grown in methylcellulose in the presence of erythropoietin. Single burst-forming units erythroid (BFU-E) and non–BFU-Es (ie, CFU-G and CFU-GM) were picked and analyzed individually for the presence of JAK2-V617F mutation by allele-specific PCR. Each colony is represented by a dot that is placed into 1 of 6 quadrangles representing the 6 possible genotypes: wild-type (WT), heterozygous (het) and homozygous (hom) for JAK2-V617F on the vertical axis, and for BCR-ABL on the horizontal axis. Percentages are shown in red for each possible genotype. (D) Genotyping of DNA from single colonies. Ethidium-bromide–stained PCR fragments for the BCR-ABL breakpoint and for BCR (loading control) were separated by agarose gel electrophoresis (top panel). The chromatograms of the allele-specific PCR assay (AS-PCR) show the presence or absence of the wild-type “G” or mutant sequence “T” in codon 617 of JAK2. The results of individual colonies marked with small letters in panel C are shown. Results of microsatellite analysis for a marker on chromosome 9p are shown below. Note that all colonies tested retained both alleles (1 and 2), excluding uniparental disomy as the reason for the loss of BCR-ABL/JAK2-V617F double-mutant colonies. n.a., not available.

Patient characteristics, BCR-ABL breakpoint sequence, and genotyping of single colonies. (A) Clinical diagnoses, treatment history, and diagnostic workup of a 56-year-old female patient with coexisting JAK2-V617F mutation and BCR-ABL rearrangement. BM, bone marrow; CMR4.5, complete molecular remission with a log4 to log5 reduction of BCR-ABL transcripts; GRA, granulocytes; n.d., not determined; Ph, Philadelphia chromosome. (B) Sequencing chromatogram of a 157-bp amplicon derived from granulocyte DNA revealed the breakpoint sequence in the intron 14 of BCR and intron 1 of ABL. The same sequences were obtained in colonies positive for BCR-ABL. (C) Analysis of single colonies for mutation in JAK2-V617F and BCR-ABL rearrangement in the genomic DNA before and after tyrosine kinase (dasatinib) therapy. Mononuclear cells from peripheral blood were grown in methylcellulose in the presence of erythropoietin. Single burst-forming units erythroid (BFU-E) and non–BFU-Es (ie, CFU-G and CFU-GM) were picked and analyzed individually for the presence of JAK2-V617F mutation by allele-specific PCR. Each colony is represented by a dot that is placed into 1 of 6 quadrangles representing the 6 possible genotypes: wild-type (WT), heterozygous (het) and homozygous (hom) for JAK2-V617F on the vertical axis, and for BCR-ABL on the horizontal axis. Percentages are shown in red for each possible genotype. (D) Genotyping of DNA from single colonies. Ethidium-bromide–stained PCR fragments for the BCR-ABL breakpoint and for BCR (loading control) were separated by agarose gel electrophoresis (top panel). The chromatograms of the allele-specific PCR assay (AS-PCR) show the presence or absence of the wild-type “G” or mutant sequence “T” in codon 617 of JAK2. The results of individual colonies marked with small letters in panel C are shown. Results of microsatellite analysis for a marker on chromosome 9p are shown below. Note that all colonies tested retained both alleles (1 and 2), excluding uniparental disomy as the reason for the loss of BCR-ABL/JAK2-V617F double-mutant colonies. n.a., not available.

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