Distribution of JAK2 mutations in erythroid progenitors and loss of heterozygosity on chromosome 9p (9pLOH) analysis in patient Vi327. The total number of erythroid colonies analyzed and the percentages of colonies with homozygous or heterozygous JAK2 mutation or wild-type JAK2 are shown. Colony assays in methylcellulose were performed with peripheral blood cells of patients with JAK2 exon 12 mutations (A) or JAK2-V617F mutation (B). Single erythroid colonies were picked and analyzed individually. Horizontal bars indicate the percentages of colonies with homozygous mutation (■), heterozygous mutation (), or wild-type JAK2 (□). For each patient, 2 bars are shown: the upper representing colonies grown in the presence of erythropoietin (Epo⁺) and the lower representing colonies grown without erythropoietin (Epo⁻). The unique patient numbers (UPN) and the allelic ratios of the JAK2 mutations (%mut or %T) in granulocytes (GRA) are shown in the 2 left columns, and the total number of erythroid colonies analyzed is shown in the right column. *Note that in patient p021 colonies positive for exon 12 mutation and colonies with JAK2-V617F were found. None of these colonies carried both mutations simultaneously. (C) Molecular analysis of individual erythroid colonies of patient Vi327. Data from 1 of 4 BFU-E homozygous for the E543-D544del mutation is shown. T-cell DNA from patient Vi327 was used as control (top row). Allele discrimination assay shows the presence of a homozygous E543-D544del mutation (left panel). Two microsatellite markers, D9S1779 and D9S1852, demonstrate loss of heterozygosity on chromosome 9p (9pLOH) in the same colony (middle and right panels). Numbers indicate allele sizes.