Figure 2. TPM4 mutation causes macrothrombocytopenia in humans.

(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.