Figure 1.
Figure 1. THPO mutation inactivates the intron 2 splice donor and correlates with elevation of thrombopoietin level. (A) Pedigree of the 6 family members with serum thrombopoietin concentrations (pg/mL) in the 1st row and platelet counts (/mm3) in the 2nd row. The proband (II-1) and her 2 children (III-1, III-2) exhibit thrombocytosis and high serum thrombopoietin levels. The proband's parents (I-1, I-2) and husband (II-2) have normal platelet counts and thrombopoietin levels. (B) A novel heterozygous T > C point mutation at the splice donor site of THPO gene intron 2 was identified through Sanger sequencing in the proband and her children, but not in her parents or husband. (C) The serum thrombopoietin concentrations of family members with the THPO mutation were significantly higher than those family members without the mutation (P < .01), or healthy controls (P < .001). The thrombopoietin levels in the latter 2 groups showed no statistically significant difference. This analysis is performed with 1-way ANOVA followed by the Student-Newman-Keuls multiple comparisons test and the data are presented in the mean with SEM format. D) Basal phosphorylated STAT5 (pSTAT5) levels in CD3−/CD66−/CD14− myeloid progenitors were evaluated and the 95th percentile is presented in the mean with SEM format. An un-paired t test revealed that pSTAT5 levels are significantly higher in the affected group (*P = .024). (E) Top panel: schematic of the cloned inserts used for exon trapping. These inserts were amplified from patient genomic DNA, cloned into the pCR2.1-TOPO Vector, and then subcloned into the pSPL3b exon trapping vector. Middle panel: expected splicing products from the constructs of the cloned sequence within pSPL3b. In the presence of a mutation in intron 2, exon 2 is expected to be spliced out of the resulting product. Bottom panel: electrophoretic visualization of cDNA-PCR products amplified from the constructs after transfection into COS-7 cells. RNA was extracted and reverse transcribed to cDNA 48 hours after the transfection of the pSPL3b-Insert construct into COS-7 cells. PCR was performed using primers SD6 and SA2, and products were resolved on a 2% agarose gel. Splicing of the vector alone yields a 261bp fragment resulting from the flanking vector exons. Splicing of the wild-type Insert 1 and Insert 2 constructs results in 547bp and 634 bp fragments, respectively. The mutant splice products display fragments that are 158bp shorter, indicating complete splicing-out of exon 2.

THPO mutation inactivates the intron 2 splice donor and correlates with elevation of thrombopoietin level. (A) Pedigree of the 6 family members with serum thrombopoietin concentrations (pg/mL) in the 1st row and platelet counts (/mm3) in the 2nd row. The proband (II-1) and her 2 children (III-1, III-2) exhibit thrombocytosis and high serum thrombopoietin levels. The proband's parents (I-1, I-2) and husband (II-2) have normal platelet counts and thrombopoietin levels. (B) A novel heterozygous T > C point mutation at the splice donor site of THPO gene intron 2 was identified through Sanger sequencing in the proband and her children, but not in her parents or husband. (C) The serum thrombopoietin concentrations of family members with the THPO mutation were significantly higher than those family members without the mutation (P < .01), or healthy controls (P < .001). The thrombopoietin levels in the latter 2 groups showed no statistically significant difference. This analysis is performed with 1-way ANOVA followed by the Student-Newman-Keuls multiple comparisons test and the data are presented in the mean with SEM format. D) Basal phosphorylated STAT5 (pSTAT5) levels in CD3−/CD66−/CD14− myeloid progenitors were evaluated and the 95th percentile is presented in the mean with SEM format. An un-paired t test revealed that pSTAT5 levels are significantly higher in the affected group (*P = .024). (E) Top panel: schematic of the cloned inserts used for exon trapping. These inserts were amplified from patient genomic DNA, cloned into the pCR2.1-TOPO Vector, and then subcloned into the pSPL3b exon trapping vector. Middle panel: expected splicing products from the constructs of the cloned sequence within pSPL3b. In the presence of a mutation in intron 2, exon 2 is expected to be spliced out of the resulting product. Bottom panel: electrophoretic visualization of cDNA-PCR products amplified from the constructs after transfection into COS-7 cells. RNA was extracted and reverse transcribed to cDNA 48 hours after the transfection of the pSPL3b-Insert construct into COS-7 cells. PCR was performed using primers SD6 and SA2, and products were resolved on a 2% agarose gel. Splicing of the vector alone yields a 261bp fragment resulting from the flanking vector exons. Splicing of the wild-type Insert 1 and Insert 2 constructs results in 547bp and 634 bp fragments, respectively. The mutant splice products display fragments that are 158bp shorter, indicating complete splicing-out of exon 2.

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