Monoallelic loss-of-function THPO variants cause heritable thrombocytopenia

Abstract

1. We report rare monoallelic variants of THPO that alter intracellular trafficking and diminish thrombopoietin secretion.

2. Affected cases have autosomal-dominant thrombocytopenia but no other hematological features.

Figure 1.

Rare monoallelic variants in THPO are associated with thrombocytopenia. (A) Associations between rare filtered variants across all genes and case/control groups defined by the presence of thrombocytopenia were inferred using the BeviMed method. Genes with a posterior probability of association >0.4 are shown for dominant and recessive inheritance models. Genes previously associated with thrombocytopenia are shown as blue symbols, whereas those with no confirmed association are shown as gray symbols. (B) Pedigree structures of the 3 index cases (A, II.2; B, I.2; and C, II.1) and pedigree members, indicating the presence (black symbols) or absence (white symbols) of thrombocytopenia and cases with unknown PLT (gray symbols). Genotypes confirmed by Sanger sequencing are annotated against canonical transcript ENST00000204615.7 (GRCh37.p13). wt/wt indicates homozygous for the reference allele. wt/* indicates the monoallelic variant. (C) Schematic diagram of the wt and predicted variant TPO proteins with amino acid numbering indicating the signal peptide (1-21), RBD (22-152), and C-terminal domain (153-353). Black circles indicate the position of consensus N-linked glycan sites. The insertion variants p.E204fs*123 and p.L269Pfs*58 (*) predict altered amino acid sequence (shaded region) downstream of the variant site and chain truncation, both after codon 327. The predicted p.R99W variant (*) is in the RBD. (D) Phylogenetic conservation of R99 across representative mammalian species shown in a sequence line up prepared using Clustal Omega. (E) Ribbon diagram of the TPO RBD derived from PDB:1V7M with direct contact regions between TPO and the TPO receptor indicated in red and the 4 helical regions indicated as A, B, C, and D. In the expanded view (left panel) and detailed vertical view (right panel), the wt R99 (green side chain) occurs in the cross-over region between helices A and B in the wt TPO peptide. Substitution of R99 with a W (blue) residue introduces a amphipathic residue with a bulky aromatic side chain into the space between helices B and C.

Figure 2.

Phenotypic characteristics of monoallelic THPO variants and expression in HEK293T cells. (A) Sex-stratified graphs of PLT and mean platelet volume obtained using a Sysmex XN-Series hematology analyzer from 48 345 blood donors from the INTERVAL study. The arrows indicate the platelet parameters of the cases harboring THPO variants. (B) Representative May-Grünwald-Giemsa–stained peripheral blood films from THPO variant cases confirming thrombocytopenia and indicating that some platelets (arrows) were enlarged. Scale bars, 5 μM. (C) Serum TPO concentration plotted against platelet counts in healthy controls or hemato-oncology patients with thrombocytopenia caused by bone marrow suppression after chemotherapy. Corresponding data from 4 cases with THPO variants (A I.1 and II.2, B I.2, C II.1) are also shown. The solid and dotted lines represent the line of best-fit and 95% confidence intervals, respectively, from a nonlinear regression of control data. (D) Effects of THPO variants in HEK293T cells: Flag-tagged WT, E204Gfs*123, L269P*58, or R99W substituted TPO was expressed in HEK293T cells by transient transfection. Secretion of FLAG-tagged TPO in cell supernatant (D) and lysate (E) was measured using an ELISA and normalized against total protein in the cell lysate. Results are mean ± standard error from ≥5 independent experiments. The P values were calculated using 1-way analysis of variance. (F) Representative images from HEK293T cells transfected with WT and variant THPO cDNAs and stained with an antibody against the FLAG epitope (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (blue). Images were captured using a Leica SP5 II confocal microscope with a 63×/1.4 NA lens and analyzed with ImageJ/Fiji. All images are shown with the same intensity range. (G) Quantification of FLAG-tagged protein by mean fluorescence intensity in representative cells (n = 5).