Association of <scp><i>MPL</i> K39N</scp> and <scp>R102P</scp> heterozygous germline mutations lead to hereditary thrombocytosis
Authors: Emeline Voirin, Anne Bouvier, Isabelle Plo, Léa Durix, Annaëlle Beucher, Stephane Giraudier, Valérie Ugo, Jean‐François Brasme, Damien Luque Paz
Journal: American Journal of Hematology (2024)

Megakaryopoiesis is primarily regulated by the interaction between thrombopoietin (THPO) and its receptor (MPL) located on the surface of cells, ranging from hematopoietic stem cells to megakaryocytes and platelets. MPL signaling is mediated by the JAK2-STAT5 pathway. THPO is constitutively produced by the liver, and its concentration is regulated by clearance through platelet mass. Genetic abnormalities affecting THPO/MPL/JAK2 axis can result in either thrombocytosis or thrombocytopenia. Specifically, somatic gain-of-function mutations in JAK2 and MPL genes are identified in myeloproliferative neoplasms. Conversely, constitutional mutations within THPO, MPL, or JAK2 genes can trigger either hereditary thrombocytosis or thrombocytopenia, depending on whether the mutation is gain or loss of function, respectively.1 Here, we report two cases of severe thrombocytosis in children from two unrelated families with the original association of two heterozygous germline mutations in the MPL gene previously described alone and leading when homozygous to a thrombocytosis for the first one (K39N) and to a thrombocytopenia for the second one (R102P). The first case was a 4-year-old child who presented with isolated thrombocytosis (981×109/L) discovered during an episode of angina with severe hyperthermia. The thrombocytosis persisted at 823×109/L after the resolution of the inflammatory syndrome, with no other reactive cause identified. Molecular screening for myeloproliferative neoplasms was performed, revealing no mutation in JAK2V617F, MPLW515, or CALR genes and absence of BCR::ABL1 transcript. Given this triple-negative thrombocytosis, sequencing of all coding regions of MPL gene and measurement of plasma THPO were performed. Two heterozygous mutations, MPL K39N (Baltimore variant) in exon 2 and MPL R102P in exon 3, were identified. The plasma THPO level was highly increased to 247 pg/mL (normal range < 40 pg/mL). A familial study was conducted showing normal platelet counts in parents and sister (Figure 1A). The MPL K39N mutation was found to be heterozygous in the mother and the young sister, accompanied by a slightly increase of THPO levels. The MPL R102P heterozygous mutation was identified in the father, correlating with a normal THPO level (Figure 1A). These results demonstrated that both variants were located on different alleles and thus were acting in trans. Consequently, the first child received the MPL K39N variant from his mother and the MPL R102P mutation from his father. A second case with a similar molecular pattern was observed in an unrelated family. A premature child (32-week gestation) exhibited high platelet levels at birth (499 × 109/L), which progressively worsened during follow-up to 1069×109/L at 3 months of age. Initial screening for an MPN driver gene mutation was negative, and a next-generation sequencing (NGS) was performed using a custom myeloid panel of 77 genes, revealing the association of MPL K39N and MPL R102P heterozygous variants without additional mutation. The plasma THPO concentration was also increased at 261 pg/mL, mirroring the first case (Figure 1A). The parents had normal platelets counts, and familial genetic study showed MPL K39N heterozygous variant in the mother and MPL R102P heterozygous variant in the father. Plasma THPO levels were not measured in the parents (Figure 1A). The MPL Baltimore (K39N) mutation is found heterozygous in around 9% of African and Afro-American population (gnomAD data) and is known to cause severe thrombocytosis when in a homozygous state (around 800×109/L) but results in mild thrombocytosis when heterozygous (around 500×109/L). In the present families, none of the relatives carrying the heterozygous MPL K39N mutation exhibited thrombocytosis, although plasma THPO levels were slightly increased. The K39N substitution is located in the extracellular domain of MPL, and the mechanism is related to abnormalities in MPL glycosylation leading to partial retention in the Golgi apparatus and subsequent reduced expression of the receptor on the cell membrane. The decreased expression of MPL on mature platelets in the bloodstream leads to increased plasma THPO levels, thereby stimulating megakaryopoiesis.2 The MPL R102P mutation is associated with congenital amegakaryocytosis thrombocytopenia (CAMT) in homozygous state3 and has an allele frequency of 0.03% in the general population (gnomAD data). This mutation is also located in the extracellular domain of MPL. Thrombocytopenia manifests due to receptor retention in the endoplasmic reticulum, causing a complete deficiency in its expression on the cellular membrane in homozygous mutations.4 Thrombocytosis was previously observed in two patients from a same family carrying the MPL R102P heterozygous mutation and exhibiting high levels of THPO.5 Herein, none of the relatives displayed thrombocytosis at diagnosis, suggesting either incomplete penetrance of this described association or the involvement of another variant in the prior report. Familial investigations in the two present cases confirmed that the two mutations were present on different alleles in a trans-configuration. This trans-association of R102P and K39N variants of MPL resulted in the complete retention of the MPL-R102P protein in the endoplasmic reticulum and only the expression of MPL-K39N on the cell surface, as represented in the model in Supplemental Figure S1. Thus, this genotype mimicked an MPL K39N homozygous status, leading to the observed severe thrombocytosis. An earlier association between MPL Baltimore variant and a nonsense MPL mutation has been previously described in a 44-year-old female with thrombocytosis.6 We performed western blot for MPL on purified platelets from the first family (mother, father, and child) (Figure 1B). A reduction in the expression of the MPL protein was found in all three individuals, but only the child with the K39N/R102P association exhibited the presence of an isoform with retarded electrophoretic mobility consistent with incomplete glycosylation. A similar pattern is found in patients homozygous for the MPL K39N mutation, as well as in a cell line engineered with the K39N mutation.2 The absence of such pattern in the mother heterozygous for K39N could be due to an exclusive dimerization of WT isoforms. While there are limited data in the literature, individuals with homozygous MPL K39N mutation typically do not exhibit thrombosis or bleeding syndromes. The two children mentioned in this report underwent annual blood count monitoring without requiring any specific treatment. In conclusion, we report here for the first time the germline association between MPL K39N Baltimore and MPL R102P heterozygous mutations on two different alleles in two children with severe thrombocytosis. The association between pro-thrombocytosis and pro-thrombocytopenia mutations is uncommon but possibly underestimated due to the frequency of these variants in the general population. Finally, this report highlights the importance of sequencing the entire of MPL gene for unexplained thrombocytosis and to consider the trans-association of mutations for biological interpretation. EV collected and analyzed the data and wrote the manuscript. ABo, Abe, and SG performed and analyzed genetics. IP and LD performed the analysis of MPL expression on platelets. VU reviewed the manuscript. JFB took care of patients and supervised the study. DLP supervised the study and wrote the manuscript. All authors declare no competing financial interests related to this work. The data that support the findings of this study are available on reasonable request from the corresponding author. Figure S1. Suggested mechanism to explain thrombocytosis in these patients. the left panel presents a wild type (WT) cell, with normal MPL receptor expression to cell membrane and normal THPO fixation. The right panel shows patient cases with thrombocytosis and the combination of MPLK39N (MPL Baltimore) and MPLR102P mutations: the R102P protein were not expressed in cell surface while the K39N protein is only expressed in few number leading to an increase of THPO level resulting in thrombocytosis. ER, endoplasmic reticulum; THPO, thrombopoietin. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.