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.

ER/Golgi trafficking is facilitated by unbranched actin filaments containing Tpm4.2

“…Animal experiments were performed in accordance with UNSW Australia Animal Care and Ethics Committee approval and Australian National Health and Medical Research Council (NHMRC) guidelines. The Tpm3.1 [B6‐ Tpm3 tm2(Δ9d)Pgun ] and Tpm4.2 [B6‐ Tpm4 tm1(Δ1b)Hrd ] knock‐out (KO) mouse (C57Bl/6 background) lines and Tpm4.2 functional null mouse ( Tpm4 Plt53/Plt53 ) (BALB/c background) lines have been reported previously (Pleines et al, ; Schevzov et al, ; Vlahovich et al, ). The Tpm4.2 functional null mouse line carries a single base pair mutation that disrupts splicing, resulting in the expression of a mutant Tpm4.2 protein that cannot form a polymer on the actin filament (Schevzov et al, ).…”

“…In contrast, Tpm4 Plt53/Plt53 cells had reduced recovery of compact Golgi structure with BFA washout; 33% of Tpm4 Plt53/Plt53 cells had compact Golgi with vehicle‐treatment and only 21% of cells had compact Golgi at 150 min of BFA washout (Figure b). To verify that these results were due to loss of Tpm4.2 function, we performed the same measurements on MEFs from an independent Tpm4.2 knockout (KO) mouse line (Pleines et al, ). Similar to the Tpm4 Plt53/Plt53 cells, the majority of vehicle‐treated Tpm4.2 KO cells had abnormal Golgi morphology (22% with compact Golgi), were disrupted with BFA treatment (0% cells with compact Golgi) and with BFA wash‐out only 25% of Tpm4.2 KO cells had regained compact Golgi (Supporting Information Figure S2).…”

“…This effect is independent of the overall surface area of the cells, as there is no size difference between the WT and Figure 2b). To verify that these results were due to loss of Tpm4.2 function, we performed the same measurements on MEFs from an independent Tpm4.2 knockout (KO) mouse line (Pleines et al, 2017). Similar to the Figure S2).…”

“…Tpm4.2 has been shown to be associated with specialized membrane structures, such as the resorptive membranes in osteoclasts (McMichael, Kotadiya, Singh, Holliday, & Lee, 2006;McMichael and Lee, 2008), as well as the regions of the muscle sarcoplasmic reticulum (SR) in close contact with the t-tubule membranes (terminal SR) (Vlahovich et al, 2009). Recently, Tpm4.2 was shown to regulate the pinching off of platelets from the megakaryocyte plasma membrane (Pleines et al, 2017). Taken together, the results of the present study together with published findings raise the possibility that Tpm4.2 has a general role in the specialisation and budding of membrane domains.…”

Tropomyosin 1 genetically constrains in vitro hematopoiesis

“…[6][7][8][9] Because most GWAS SNPs are non-coding, likely influencing transcriptional expression of key genes, 10,11 it has been challenging to derive functional biochemical understanding of the key genes of action, [11][12][13] and few studies have elucidated biochemical mechanisms for platelet trait variability loci. [14][15][16][17][18] One strategy to narrow focus on candidate genes is to link non-coding variation to expression of nearby genes. 1,19,20 However, platelet trait variation GWAS have thus far implicated >6700 expression quantitative trait loci (eQTL) affecting expression of >1100 genes (Methods), highlighting a need to more specifically identify putatively functional sites.…”