The Abrogated Thrombopoietin (TPO) Signal Transduction In Pediatric Patients Suffering From Thrombocytopenia-Absent Radii Syndrome Is Restored In Adult Patients, Suggesting An Additional, c-Mpl-Jak2-Independent Mechanism for Platelet Biogenesis.
Authors: Fiedler Janine , Strauss Gabriele , Wannack Martin , Fleischhauer Silke , Seidel Kerstin , Klopocki Eva , Mundlos Stefan , Schmugge Markus , Schulze Harald 
Published: October 2019

Abstract
Abstract 1561
Thrombocytopenia-absent radii syndrome (TARS) is a rare congenital disorder defined by low platelet count and bilaterial aplasia of radii. Additionally, patients have perinatal eosinophilia and leukocytosis and are often anemic during the first years of life. At this time, a moderate, but enigmatic increase in platelet counts has been described, but patients remain thrombocytopenic and eventually continue to suffer from severe episodes of bleeding. Megakaryocytes, the immediate precursor cells of platelets, are scarce in the bone marrow and precursor cells fail to produce megakaryocytic colonies in response to thrombopoietin (TPO). Recently, we demonstrated that all TARS patients harbor a microdeletion on chromosome 1q21 which spans 120–200 kb, comprising 12–18 annotated genes. The deletion is also present in some unaffected parents (carriers) indicating that it is essential but not sufficient for generating the TARS phenotype.
We analyzed 158 platelet counts of 33 patients over time and found that platelets increase within 2 years of life in most of our patients, but even in adult patients counts do not reach the lower norm. Thus, we performed an extended analysis of TPO signal transduction in platelets from 20 TARS patients. Overall, Jak2 kinase - despite being expressed in comparable amount - does not become phosphorylated in response to TPO when patients were below age 20, confirming our previous results also performed on young patients. Intriguingly, in platelets isolated from patients over age 20, Jak2 did become phosphorylated. As TPO activates several distinct pathways, we looked for the consequences of this bipartite TPO-responsiveness, including the activation of the alternate januskinase Tyk2, the STAT, the MAPK/ERK, and the Akt pathways. As expected, when Jak2 was not phosphorylated, Tyk2 and all downstream pathways were inactive. In contrast, in the presence of phosphorylated Jak2 (pJak2), all downstream pathways were activated, emphasizing the key role of Jak2 for TPO responsiveness. Platelets from either 20 healthy children or 11 carriers showed normal TPO signaling, excluding that the effect was due to a general age-dependence or a mere consequence of the microdeletion. Densitometric analyses confirmed our overall visual results. Expression levels of the TPO-receptor c-Mpl was not altered in 2 young and 2 adult patients compared to carriers, healthy children and adult controls, arguing against a compensatory upregulation in older patients. Furthermore, we sequenced all coding regions of Jak2 mRNA derived from patient-derived lymphoblastic cell lines (LCL) of one young and one adult patient and could not find any mutations. As bone marrow biopsies are typically not performed, changes in bone marrow cellularity or composition are not directly accessible. Recently, the immature platelet fraction (IPF) has been considered a surrogate marker for megakaryopoiesis. Interestingly, while there was no correlation between platelet count and IPF in 16 patients with TARS, we found a negative correlation between IPF with age. In 9 pediatric patients IPF was elevated (4.6%) compared to the median of 100 pediatric controls (2.7%), while in 7 adult TARS patients the mean IPF was 2.4%. These data provide circumstantial evidence that changes in megakaryopoiesis might drive the change in platelet biogenesis and TPO signaling. Plasma levels of stromal derived factor 1, a chemokine that contributes to restore platelet production in the absence of functional TPO signaling, were within the normal range in 6 patients with TARS. Real-time analysis of mRNA expression in LCL of genes within the microdeleted region indicates comparable expression in 2 unaffected parents with 2 controls, while 3 patients and 2 carriers showed the expected reduced expression. This includes the expression of PIAS3, a negative regulator of the Jak-STAT pathway. PIAS3 protein level, however, was normal in platelet lysates of TARS patients, making a key function for thrombocytopenia in TARS unlikely.
Taken together, our data show an unexpected age-dependent change in TPO-signaling in platelets of TARS patients. As this change occurs much later than the amelioration of platelet counts, we suggest that an unknown factor influences platelet biogenesis during childhood.

Disclosures:
No relevant conflicts of interest to declare.