Identification of novel TUBB1 variants in patients with macrothrombocytopenia

Abstract

Background/aim: Macrothrombocytopenia is an autosomal dominant disorder characterized by increased platelet size and decreased number of circulating platelets. 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 the normal platelet morphology and the defects on these systems are also associated with macrothrombocytopenia. Materials and methods: In this study, we have sequenced the exons of the TUBB1 gene using the DNA isolated from peripheral blood samples of the healthy controls (n = 47) and the patients with macrothrombocytopenia (n = 37) from Turkey. TUBB1 expression levels in fractioned blood samples from the patient and healthy controls were analyzed by RT-qPCR and Western Blot. Microtubule organization of the platelets in the patient's peripheral blood smears and in the mutant TUBB1-transfected HeLa cells was analyzed by using immunofluorescence staining. Results: A new TUBB1 c.803G>T (p.T178T) variant was detected in all of the controls 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 being 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 might give important insights about their pathogenicity in macrothrombocytopenia.

Consequences of pathogenic tubulin-mutations on molecular motor proteins and their implications in tubulinopathies

“…Some thrombocytopenia associated TUBB1 mutants, when transfected in CHO and HeLa cell line, fail to appropriately incorporate in the MTs and is largely diffused in the cytoplasm forming aggregates, thereby suggesting that the pathogenicity of macrothrombocytopenia is mostly occurring through loss-of-function (ÇALIŞKANER et al, 2021;Palma-Barqueros et al, 2021). Even though the TUBB1 mutants do not affect platelet function, they increase platelet size, accompanied by aberrant platelet morphologies, altered Tubb1 tubulin expression, impaired proplatelet formation, platelet maturation, and MT organization in platelets, thereby showing the importance of Tubb1 in these processes (Palma-Barqueros et al, 2021).…”