Mutation of the β1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly

Abstract

Congenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. We identified the first TUBB1 mutation, R318W, in a patient with congenital macrothrombocytopenia. The patient was heterozygous for Q43P, but this single-nucleotide polymorphism (SNP) did not relate to macrothrombocytopenia. Although no abnormal platelet ␤1-tubulin localization/marginal band organization was observed, the level of ␤1-tubulin was decreased by approximately 50% compared with healthy controls. Large and irregular bleb protrusions observed in megakaryocytes derived from the patient's peripheral blood CD34 ؉ cells suggested impaired megakaryocyte fragmentation and release of large platelets. In vitro transfection experiments in Chinese hamster ovary (CHO) cells demonstrated no incorporation of mutant ␤1-tubulin into microtubules, but the formation of punctuated insoluble aggregates. These results suggested that mutant protein is prone to aggregation but is unstable within megakaryocytes/platelets. Alternatively, mutant ␤1-tubulin may not be transported from the megakaryocytes into platelets. W318 ␤1-tubulin may interfere with normal platelet production, resulting in macrothrombocytopenia. IntroductionCongenital macrothrombocytopenia is a genetically heterogeneous group of rare disorders. [1][2][3] The most frequent forms include MYH9 disorders, such as May-Hegglin anomaly, and Bernard-Soulier syndrome. In approximately half of the cases the pathogenesis remains unknown; thus, a definite diagnosis is not possible. The linkage between the membrane skeleton and cytoskeletal actin filaments as well as the marginal microtubule band maintains normal platelet morphology. 4,5 Defects in these systems may result in macrothrombocytopenia. The microtubules are assembled from ␣-and ␤-tubulin heterodimers. ␤1-Tubulin expression is restricted in the megakaryocyte/platelet lineage. 6 Tubb1 knockout mice show thrombocytopenia and spherical platelets. 7 TUBB1 Q43P functional polymorphism has been reported. However, it may not be relevant to macrothrombocytopenia. 8 We identified the first TUBB1 mutation affecting microtubule assembly in congenital macrothrombocytopenia. Methods PatientThe patient was a 7-year-old boy who was incidentally found to have thrombocytopenia (platelets, 40-60 ϫ 10 9 /L). He was diagnosed with immune thrombocytopenic purpura. Peripheral blood smears showed the prominent appearance of giant platelets. Electron microscopy showed no other abnormalities ( Figure 1A,B). There were no leukocyte inclusion bodies, confirmed by myosin IIA localization. 9 The platelets aggregated normally with adenosine diphosphate (ADP), collagen, and ristocetin. Flow cytometry showed normal expression of platelet GPIb/IX. An initial bone marrow examination revealed normal megakaryocyte number and morphology. The mother of the patient also had macrothrombocytopenia. Peripheral blood samples were obtained after the mother gave informed consent in accordance with the Declaration of Helsinki for the study, which was approved by the...

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects

“…However, some protein functions currently remain unknown (SLFN14 and GNE). [4][5][6][7][8][9] . Although our knowledge of the causes of IT continues to grow, presently a genetic diagnosis is only reported in approximately 50% of individuals.…”

Novel point mutations in β-tubulin gene for carbendazim resistance maintaining nematode pathogenicity of Paecilomyces lilacinus

“…The principle of benzimidazole resistance is closely associated with point mutations in the β-tubulin genes that change the structure of the fungicide-binding point (Gafur et al 1998;Albertini et al 1999;Peres et al 2004;Chung et al 2006;Davidson et al 2006;Ziogas et al 2009). T274I, R282Q, and Q292E mutations significantly weakened the interactions and binding of the drugs taxol and epothilone (Natarajan and Senapati 2012), while Q43P and R318W mutations had a significant impact on platelet physiology in immune thrombocytopenia (Freson et al 2005;Kunishima et al 2009;NavarroNunez et al 2011). The genetics of carbendazim resistance in Aspergillus nidulans (van Tuyl 1977), Ustilago maydis (Ziogas and Girgis 1993) and other fungal species revealed that in most cases it was based on a single gene.…”

Pathogenesis and management of inherited thrombocytopenias: rationale for the use of thrombopoietin-receptor agonists

“…Despite the key role of microtubules in PPF, mutation of a component of the microtubule system was identified in only one family with IT [3]. The p.R318W change in b1-tubulin was predicted to affect microtubule assembly by disrupting interaction between a-and b-tubulin to form heterodimers.…”

“…2). BM examination is not required for diagnosis of these forms: however, in the few analyzed cases, it demonstrated normal or increased number of mature Mks with normal morphology [3,44,45].…”

“…In the last 5 years, nine new genes whose mutations are responsible for thrombocytopenia have been identified [1][2][3][4][5][6][7][8][9], and this led to the recognition of 6 novel nosographic entities [1]. To date, ITs encompass 20 forms caused by mutations of 21 genes ( Table 1).…”