Identification of novel TUBB1 variants in patients with macrothrombocytopenia

Background/aim: Macrothrombocytopenia is an autosomal-dominant disorder characterized by increased platelet size and a decreased number of circulating platelets. The membrane skeleton and the link between actin filaments of the skeleton and microtubules, which consist of alpha and beta tubulin [including the tubulin beta-1 chain (TUBB1)] heterodimers, are important for normal platelet morphology, and defects in these systems are associated with macrothrombocytopenia. Materials and methods: In this study, we sequenced the exons of the TUBB1 gene using DNA isolated from the peripheral blood samples of healthy controls (n = 47) and patients with macrothrombocytopenia (n = 37) from Turkey. The TUBB1 expression levels in fractioned blood samples from patients and healthy controls were analyzed by RT-qPCR and Western blot. Microtubule organization of the platelets in the peripheral blood smears of patients, and in mutant TUBB1-transfected HeLa cells, were analyzed by immunofluorescence staining. Results: A new TUBB1 c.803G>T (p.T178T) variant was detected in all of the control and patient samples. Importantly, we found 3 new heterozygous TUBB1 variants predicting amino acid substitutions: G146R (in 1 patient), E123Q (in 1 patient), and T274M (in 4 patients); the latter variant was associated with milder thrombocytopenia in cancer patients treated with paclitaxel. Ectopic expression of TUBB1 T274M/R307H variant in HeLa cells resulted in irregular microtubule organization. Conclusion: Further clinical and functional studies of the newly identified TUBB1 variants may offer important insights into their pathogenicity in macrothrombocytopenia.Key words: TUBB1, macrothrombocytopenia, MYH9, platelets

PDF

___

1. Peyvandi F, Spreafico M. National and international registries of rare bleeding disorders. Blood Transfusion 2008; 6 (2): s45- 48. doi: 10.2450/2008.0037-08
2. Kunishima S, Okuno Y, Yoshida K, Shiraishi Y, Sanada M et al. ACTN1 mutations cause congenital macrothrombocytopenia. The American Journal of Human Genetics 2013; 92 (3): 431- 438. doi: 10.1016/j.ajhg.2013.01.015
3. Bolton-Maggs PH, Chalmers EA, Collins PW, Harrison P, Kitchen S et al. A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO. British Journal of Haematology 2006; 135 (5): 603-633. doi: 10.1111/j.1365-2141.2006.06343.x
4. Rao AK, Songdej N. Inherited thrombocytopenias: the beat goes on. Blood 2015; 125 (5): 748-750. doi: 10.1182/ blood-2014-11-610675
5. Italiano JE Jr., Bergmeier W, Tiwari S, Falet H, Hartwig JH et al. Mechanisms and implications of platelet discoid shape. Blood 2003; 101 (12): 4789-4796. doi: 10.1182/blood-2002-11-3491
6. Burley K, Westbury SK, Mumford AD. TUBB1 variants and human platelet traits. Platelets 2018; 29 (2): 209-211. doi: 10.1080/09537104.2017.1411587
7. Schwer HD, Lecine P, Tiwari S, et al. A lineage-restricted and divergent beta-tubulin isoform is essential for the biogenesis, structure and function of blood platelets. Curr Biol 2001; 11 (8): 579-586. doi: 10.1016/S0960-9822(01)00153-1
8. Kunishima S, Nishimura S, Suzuki H, Imaizumi M, Saito H. TUBB1 mutation disrupting microtubule assembly impairs proplatelet formation and results in congenital macrothrombocytopenia. European Journal of Haematology 2014; 92 (4): 276-282. doi: 10.1111/ejh.12252
9. Ravid K. Platelet marginal bands: not so marginal. Blood 2008; 111 (9): 4423-4423. doi: 10.1182/blood-2008-02-137786
10. Freson K, De Vos R, Wittevrongel C, Thys C, Defoor J et al. The TUBB1 Q43P functional polymorphism reduces the risk of cardiovascular disease in men by modulating platelet function and structure. Blood 2005; 106 (7): 2356-2362. doi: 10.1182/ blood-2005-02-0723
11. Poster Communications. Thrombosis Research 2016; 141: S67- 68. doi: 10.1016/S0049-3848(16)30387-5
12. Basciano PA, Bussel J, Hafeez Z, Christos PJ, Giannakakou P. The beta-1 tubulin R307H single nucleotide polymorphism is associated with treatment failures in immune thrombocytopenia. British Journal of Haematology 2012; 160 (2): 237-243. doi: 10.1111/bjh.12124
13. Leandro-Garcia LJ, Leskela S, Inglada-Perez L, Landa I, de Cubbas AA et al. Hematologic beta-tubulin VI isoform exhibits genetic variability that influences paclitaxel toxicity. Cancer Research 2012; 72 (18): 4744-4752. doi: 10.1158/0008-5472. CAN-11-2861
14. D’Andrea G, Chetta M, Margaglione M. Inherited platelet disorders: thrombocytopenias and thrombocytopathies. Blood Transfusion 2009; 7 (4): 278-292. doi: 10.2450/2009.0078-08
15. Mhawech P, Saleem A. Inherited giant platelet disorders. Classification and literature review. American Journal of Clinical Pathology 2000; 113 (2): 176-190. doi: 10.1309/FC4HLM5V-VCW8-DNJU
16. Kunishima S, Kobayashi R, Itoh TJ, Hamaguchi M, Saito H. Mutation of the beta1-tubulin gene associated with congenital macrothrombocytopenia affecting microtubule assembly. Blood 2009; 113 (2): 458-461. doi: 10.1182/ blood-2008-06-162610
17. Davis B, Toivio-Kinnucan M, Schuller S, Boudreaux MK. Mutation in beta1-tubulin correlates with macrothrombocytopenia in Cavalier King Charles Spaniels. Journal of Veterinary Internal Medicine 2008; 22 (3): 540-545. doi: 10.1111/j.1939-1676.2008.0085.x
18. Basciano PA, Matakas J, Pecci A, Civaschi E, Cagioni C et al. Beta-1 tubulin R307H SNP alters microtubule dynamics and affects severity of a hereditary thrombocytopenia. Journal of Thrombosis and Haemostasis 2015; 13 (4): 651-659. doi: 10.1111/jth.12824
19. Qu H, Wang X, Tian L, Hakonarson H. Application of ACMG criteria to classify variants in the human gene mutation database. Journal of Human Genetics 2019; 64: 1091-1095. doi: 10.1038/s10038-019-0663-8