Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia
Irina Pleines, … , Marloes R. Tijssen, Benjamin T. Kile
Irina Pleines, … , Marloes R. Tijssen, Benjamin T. Kile
Published January 30, 2017
Citation Information: J Clin Invest. 2017;127(3):814-829. https://doi.org/10.1172/JCI86154.
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Research Article Genetics Hematology

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia

Abstract

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea–induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage–dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.

Authors

Irina Pleines, Joanne Woods, Stephane Chappaz, Verity Kew, Nicola Foad, José Ballester-Beltrán, Katja Aurbach, Chiara Lincetto, Rachael M. Lane, Galina Schevzov, Warren S. Alexander, Douglas J. Hilton, William J. Astle, Kate Downes, Paquita Nurden, Sarah K. Westbury, Andrew D. Mumford, Samya G. Obaji, Peter W. Collins, NIHR BioResource, Fabien Delerue, Lars M. Ittner, Nicole S. Bryce, Mira Holliday, Christine A. Lucas, Edna C. Hardeman, Willem H. Ouwehand, Peter W. Gunning, Ernest Turro, Marloes R. Tijssen, Benjamin T. Kile

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Figure 2

TPM4 mutation causes macrothrombocytopenia in humans.

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TPM4 mutation causes macrothrombocytopenia in humans. (A) Schematic rep...
(A) Schematic representation of the major megakaryocyte TPM4 transcript ENST00000300933, which is predicted to encode the 248–amino acid TPM4 protein (UniProt ID P67936). R69 is transcribed from the second exon of the transcript and is 68 amino acids from the amino-terminus of TPM4. R69X is predicted to cause expression of a truncated TPM4 protein. (B) Family trees for pedigree 1 and pedigree 2 with the TPM4 variant are depicted, including the platelet count, platelet volume, and presence/absence (Y/N) of macrothrombocytes when visualized by electron microscopy (shown in blue). Filled symbols: macrothrombocytopenia; gray symbols, unknown; open symbols: normal platelet count and volume and absence of macrothrombocytes. +/M, heterozygous; +/+, WT. (C) Platelet TPM4 RNA levels measured by RT-PCR using GAPDH as housekeeping gene. Graph depicts representative data from a total of n = 3 measurements from 1 (case 1-II-5) or 3 (case 1-III-7) patient samples. (D) Platelet TPM4 protein is reduced in heterozygous carriers of the TPM4 variant. Graph depicts representative data from a total of n = 3 measurements from 1 (case 1-II-5) or 3 (case 1-III-7) samples. Left: Densitometry analysis performed using ImageJ. Right: Protein levels of platelet TPM4 in controls and cases 1-II-5 and 1-III-7. β-Actin was included as an internal loading control. Similar results were obtained when GAPDH was used as a loading control (not shown). (E) Sex-stratified histograms of platelet count (PLT) and mean platelet volume measurements obtained using a Sysmex hematology analyzer from 48,345 blood donors from the INTERVAL study after adjustment for technical artifacts. The red arrows superimposed on the histograms indicate the sex of and values for patients with the truncating variant in TPM4. The green arrows indicate the sex of and values for relatives homozygous for the corresponding WT allele.