Comparison of clonality and the presence of the JAK2-V617F mutation in MPDs. (A) Design of allele-specific PCR assays for detecting the JAK2-V617F mutation and assessing clonality using the IDS and MPP1 X-chromosomal gene polymorphisms. Horizontal arrows indicate primer positions; fluorescently labeled primers are marked by asterisks. Note that one of the labeled primers is extended by 3 nonhomologous nucleotides to allow separation of the PCR products by size. (B) Analysis of the linearity of the JAK-V617F allele-specific PCR. Quadruplicate reactions were performed on genomic DNAs with known ratios of wild-type and mutant JAK2 alleles. The reactions were performed using homozygous wild-type (G) and homozygous mutant (T) genomic DNA dilutions with increasing proportion of the homozygous mutant DNA. The G and T peak fluorescence ratios were determined and plotted for each genomic DNA dilution. Error bars indicate standard deviation. (C) The chromatograms of 5 DNA samples from panel B are shown. (D) Comparison of clonality determined by X-chromosome inactivation assays with clonality derived from the JAK2-V617F allele-specific PCR in granulocytes (GRA) of one female ET patient (P099). The relative abundance of the mutant JAK2 allele (10%) was calculated by comparing the G and T peak intensities (left). The allelic ratio of 10% T translates to 20% of granulocytes heterozygous for the JAK2-V617F (middle, black bar), or 10% of granulocytes homozygous for the mutation (not shown). Allele-specific PCR assay for the X-chromosomal gene MPP1 is shown at the right. The genomic DNA from patient P099 was heterozygous for a G/T single nucleotide polymorphism (not shown). The relative expression of the 2 MPP1 alleles was determined by comparing the G and T peak intensities obtained by the allele-specific RT-PCR assay in T cells (top chromatogram) and granulocytes (bottom chromatogram). The skewing of expression toward the T allele (96%) translates to the presence of 92% of clonal cells expressing only the T allele (gray bar + black bar), since the remaining 8% of cells that are of polyclonal origin (white bar) express equal amounts of both alleles. (E) Comparison of clonality in patients with ET and PV. Black bars, clonal granulocytes that carry the JAK2-V617F mutation; gray bars, clonal granulocytes that carry the wild-type JAK2 allele; white bars, polyclonal granulocytes. Analysis of T-cell RNA was performed where material was available: *Polyclonal T cells; **Skewed T cells (Figure S2). (F) Comparison of clonality determined by microsatellite PCR for loss of heterozygosity (LOH) on chromosome 20q and clonality defined by the presence of the JAK2-V617F mutation. Allele-specific PCR for JAK2-V617F for 2 patients (P115 and CA71) revealed that only 14% of granulocytes carry the JAK2-V617F mutation (allelic ratio of the T allele = 7%, left panel). In the same 2 patients, LOH (arrows) in the minimal deleted region on chromosome 20q was found in the vast majority of granulocyte DNA (right panel).