A novel splice donor mutation in the thrombopoietin gene leads to exon 2 skipping in a Filipino family with hereditary thrombocythemia

To the editor:

In contrast to the familial predisposition observed in somatically acquired myeloproliferative neoplasms (low penetrance, clonal hematopoiesis), the hereditary thrombocythemias (HT) are characterized by Mendelian inheritance, high penetrance, and polyclonal hematopoiesis, and appear to only affect the megakaryocytic lineage.¹  All the molecular alterations identified thus far in patients with HT have involved either THPO (thrombopoietin) or its receptor MPL (myeloproliferative leukemia virus oncogene) genes, with 4 and 3 distinct mutations reported, respectively. The HT- associated THPO mutations were either confirmed or expected to increase the translational efficiency of thrombopoietin without altering the sequence of the mature protein.¹  Thrombopoietin, the primary regulator of megakaryopoiesis and platelet production, is produced in the liver, kidney, spleen, and bone marrow.²  Thrombopoietin binds to its receptor and activates the JAK-STAT signaling pathway.³  The presence of multiple upstream AUG codons (uAUG) within the 5′-untranslated region (5′-UTR) precludes efficient translation and prevents harmful overproduction of this potent cytokine.² 

We identified a novel point mutation at the splice donor site of THPO intron 2 (position +2) in a Filipino family with HT. Approval was obtained from the Stanford University institutional review board for these studies and informed consent was provided according to the Declaration of Helsinki. The proband and her 2 children manifested with moderate to severe elevations of the serum thrombopoietin levels and platelet counts, while those were normal in her biologic parents and husband (Figure 1A). The absence of the mutation in either parent suggests the de novo nature of the mutation. Sequencing of the 5′-UTR of THPO in all the family members revealed a heterozygous T > C transition at the splice donor of intron 2 (Figure 1B) in subjects II-1, III-1 and III-2, but not in I-1, I-2 and II-2, indicating the co-segregation of the mutation with the thrombocytosis phenotype. The patients were negative for all known HT-associated MPL mutations. Serum thrombopoietin levels (measured with the human TPO Quantikine kit, R&D Systems) in the affected family members were significantly higher than in the non-affected family members or healthy controls, while there was no statistically significant difference between the latter 2 groups (Figure 1C). To examine the functional effects of increased thrombopoietin levels on downstream JAK-STAT signaling, phospho-specific flow cytometry was performed as previously described⁴  on peripheral blood samples from the family members. As shown in Figure 1D, basal phosphorylated STAT5 (pSTAT5) levels in myeloid progenitors were significantly higher in the affected group (P = .024). Because of the unavailability of the appropriate tissue specimens for direct patient THPO RNA analysis, the in vitro pSLP3b exon trapping system⁵  was used to prove the inactivation of the splice donor and exon 2 skipping as the main consequence of the mutation (Figure 1E). This novel T > C mutation at position +2 involves the same splice donor site as the previously reported G > C mutation at position +1 in a Dutch family.⁶  Thus, it appears that mutations in either position +1 or +2 of the THPO intron 2 splice donor site may result in exon 2 skipping and loss of inhibitory 5′-UTR sequence, leading to increased thrombopoietin expression and thrombocytosis.