Defective binding of ETS1 and STAT4 due to a mutation in the promoter region of THPO as a novel mechanism of congenital amegakaryocytic thrombocytopenia

Abstract

Congenital amegakaryocytic thrombocytopenia (CAMT) is a recessive disorder characterized by severe reduction of megakaryocytes and platelets at birth, which evolves toward bone marrow aplasia in childhood. CAMT is mostly caused by mutations in MPL (CAMT-MPL), the gene encoding the receptor of thrombopoietin (THPO), a crucial cytokine regulating hematopoiesis. CAMT can be also due to mutations affecting the THPO coding region (CAMT-THPO). In a child with the clinical picture of CAMT, we identified the homozygous c.-323C>T substitution, affecting a potential regulatory region of THPO. Although mechanisms controlling THPO transcription are not characterized, bioinformatics and in vitro analysis showed that c.-323C>T prevents the binding of transcription factors ETS1 and STAT4 to the putative THPO promoter, impairing THPO expression. Accordingly, in the proband the serum THPO concentration indicates defective THPO production. Based on these findings, the patient was treated with the THPO-mimetic agent eltrombopag, which induced a significant increase in platelet count and stable remission of bleeding symptoms. Herein, we report a novel pathogenic variant responsible for CAMT and provide new insights into the mechanisms regulating transcription of the THPO gene.

Figure 1.

Identification of c.-323C>T in the promoter region of the THPO gene in a patient with congenital amegakaryocytic thrombocytopenia. (A) Pedigree of the family. The black symbol indicates the subject carrying the homozygous c.-323C>T (NM_000460.2) mutation with severe thrombocytopenia, and the gray symbols the carriers with mild thrombocytopenia. (B) Electropherograms of the THPO gene showing the c.-323C>T mutation in the proband and in his mother. (C) Genomic structure of the human THPO gene and its transcript variants 5 and 1. The genomic structures of the gene in different species are also reported for evolutionary comparison. The black boxes represent the open reading frame. The red boxes show the regions orthologous to the 119 bp human region surrounding the c.-323C nucleotide aligned in (D). The relative genomic positions of the orthologous regions upstream of the ATG translation start site are also indicated. (D) Multiple alignments of the red squared regions indicated in (C) from different species. The c.-323C nucleotide (bold case), as well as the surrounding positions, are well conserved among species. The asterisks indicate conserved nucleotides. Plt: platelet count x 10⁹/L.

Figure 2.

Variant c.-323C>T prevents STAT4 and ETS1 binding on the THPO promoter. (A) Schematic representation of the putative binding sites for transcription factors in the regions (wild-type and mutant form of transcript variant 1) upstream of the transcription initiation site (arrow). The c.-323C>T substitution is in bold case. (B) Luciferase assays were performed in HEK293T cells with pGL3-THPO in either the wild type or c.-323C>T forms. Renilla luciferase co-transfected with the reporter was used to normalize for transfection efficiency. (C) Luciferase assays were performed in HEK293T cells upon silencing endogenous STAT4 and ETS1 alone or in combination with specific short interfering RNA for 48 h. (D) Lysates of HEK293T cells transfected with pGL3-THPO either wild-type or c.-323C>T were subjected to chromatin immunoprecipitation analysis with antibodies recognizing STAT4 or ETS1, or with Protein A/G PLUS-Agarose as a negative control. Binding to the THPO promoter region was calculated as the fraction of the input chromatin bound. Binding to non-specific chromatin is shown in the right panel. wt: wild-type; TF: transcription factor; RLU: relative luminescence units; ns: not significant;si: short interfering; ChIP Ab: chromatin immunoprecipitation antibody; CTRL: control.

Figure 3.

STAT4 and ETS1 regulate THPO expression. (A) THPO, STAT4 and ETS1 expression was evaluated by real-time quantitative polymerase chain reaction, normalized to b-actin RNA expression level. (B) Western blot analysis of THPO, STAT4, and ETS1 expression, using HSP90 and GAPDH as a loading control. Right: the graph shows the quantification of western blot bands measured by densitometry, normalized to GAPDH. (C) Proximity ligation assay (PLA) with primary antibodies against STAT4 and ETS1. (D) Lysates of HepG2 cells were subjected to co-immunoprecipitation analysis with antibodies recognizing STAT4 or IgG as a negative control. All the above experiments were performed in HepG2 cells upon silencing of endogenous STAT4 and ETS1, as indicated with specific short interfering (si) RNA for 48 h. Graphs present the mean ± standard error of mean of three independent experiments. Blots are representative of three biological replicates. P values were calculated by a two-tailed unpaired Student t test: *P<0.05, **P<0.01,***P<0.001.