Figure 2.
Genetic alterations and clonal lymphocytes patterns in these patients. (A) A list of the pathogenic blood cell mutations and clonality of T and B cells detected in each patient. (B) Sanger sequencing data of the MPL c.1210G>A (G404R) heterozygous mutation in CT1 using both blood and hair DNA samples. (C) The MPL G404R mutation failed to support TPO-stimulated growth of Ba/F3 cells. Ba/F3 cells expressing WT MPL, mutant MPL, or vector were incubated in medium without IL-3 but containing 10 ng/mL TPO. Cell numbers were counted daily using hemocytometer and displayed as mean plus or minus SD. (D) The mutant MPL is impaired in TPO uptake, a process involving TPO-MPL binding at the cell surface and its subsequent internalization. Ba/F3 cells expressing WT or mutant MPL were seeded in 6-well plates containing equal amounts of TPO with 5 × 106 cells per well. TPO uptake was monitored by measuring the remaining TPO concentration in cell supernatant after designated incubation time. After 60 minutes, the remaining TPO in WT cells was significantly lower than that of mutant cells (P = .0108) based on paired Student t test. Mean plus or minus SD of 2 separate experiments is displayed. (E) Mutant MPL leads do reduced JAK-STAT pathway activation upon TPO stimulation. Compared with WT MPL (WT), Ba/F3 cells expressing the mutant MPL (G404R) had reduced phosphorylation of JAK-STAT pathway proteins such as p-STAT3, p-STAT5, and p-ERK1/2 after 15 to 120 minutes of TPO stimulation as revealed by western blot. Ba/F3 cells transfected with the expression vector (Vector) was a negative control. β-Actin was a loading control for western blot. (F) Clonality of T cells in CT1 and CT2. Histograms show the frequencies of the top 10 clones of T-cell receptor β locus (TRB) in a sample at platelet count descending phase. Both CT1 and CT2 had clonal TRB. (G) The top clones of TRB in 6 sequential samples in CT1 and 5 in CT2 were identical. Their clonal frequencies were plotted and linked to show their stable longitudinal patterns over a platelet count cycle. IL-3, interleukin-3; SD, standard deviation; VAF, variant allele frequency.

Genetic alterations and clonal lymphocytes patterns in these patients. (A) A list of the pathogenic blood cell mutations and clonality of T and B cells detected in each patient. (B) Sanger sequencing data of the MPL c.1210G>A (G404R) heterozygous mutation in CT1 using both blood and hair DNA samples. (C) The MPL G404R mutation failed to support TPO-stimulated growth of Ba/F3 cells. Ba/F3 cells expressing WT MPL, mutant MPL, or vector were incubated in medium without IL-3 but containing 10 ng/mL TPO. Cell numbers were counted daily using hemocytometer and displayed as mean plus or minus SD. (D) The mutant MPL is impaired in TPO uptake, a process involving TPO-MPL binding at the cell surface and its subsequent internalization. Ba/F3 cells expressing WT or mutant MPL were seeded in 6-well plates containing equal amounts of TPO with 5 × 106 cells per well. TPO uptake was monitored by measuring the remaining TPO concentration in cell supernatant after designated incubation time. After 60 minutes, the remaining TPO in WT cells was significantly lower than that of mutant cells (P = .0108) based on paired Student t test. Mean plus or minus SD of 2 separate experiments is displayed. (E) Mutant MPL leads do reduced JAK-STAT pathway activation upon TPO stimulation. Compared with WT MPL (WT), Ba/F3 cells expressing the mutant MPL (G404R) had reduced phosphorylation of JAK-STAT pathway proteins such as p-STAT3, p-STAT5, and p-ERK1/2 after 15 to 120 minutes of TPO stimulation as revealed by western blot. Ba/F3 cells transfected with the expression vector (Vector) was a negative control. β-Actin was a loading control for western blot. (F) Clonality of T cells in CT1 and CT2. Histograms show the frequencies of the top 10 clones of T-cell receptor β locus (TRB) in a sample at platelet count descending phase. Both CT1 and CT2 had clonal TRB. (G) The top clones of TRB in 6 sequential samples in CT1 and 5 in CT2 were identical. Their clonal frequencies were plotted and linked to show their stable longitudinal patterns over a platelet count cycle. IL-3, interleukin-3; SD, standard deviation; VAF, variant allele frequency.

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