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Identification of novel TUBB1 variants in patients with macrothrombocytopenia
Zihni Onur Çalışkaner1,2, Abdullah Abdul Waheed1, Merve Tuzlakoğlu Öztürk1
1Department of Molecular Biology and Genetics, Faculty of Science, Gebze Technical University, Kocaeli, Turkey
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.
Insights
Researchers identified new genetic variants in the TUBB1 gene associated with macrothrombocytopenia, a disorder affecting platelet size and count. These findings may improve understanding of the condition's causes.
Area of Science:
- Genetics
- Hematology
- Cell Biology
Background:
- Macrothrombocytopenia is an inherited disorder characterized by enlarged platelets and reduced platelet counts.
- The integrity of the platelet membrane skeleton, involving alpha and beta tubulin heterodimers like TUBB1, is crucial for normal platelet morphology.
Purpose of the Study:
- To investigate the genetic basis of macrothrombocytopenia by sequencing the TUBB1 gene.
- To analyze TUBB1 expression levels and microtubule organization in patients with macrothrombocytopenia.
Main Methods:
- Exome sequencing of the TUBB1 gene in Turkish patients (n=37) and healthy controls (n=47).
- Analysis of TUBB1 expression using RT-qPCR and Western blot.
- Immunofluorescence staining to assess microtubule organization in patient platelets and transfected HeLa cells.
Main Results:
- A previously reported TUBB1 variant (c.803G>T) was found in all samples.
- Three novel heterozygous TUBB1 variants (G146R, E123Q, T274M) were identified in patients.
- The T274M variant showed association with milder thrombocytopenia in cancer patients undergoing paclitaxel treatment and caused irregular microtubule organization in HeLa cells.
Conclusions:
- Newly identified TUBB1 variants may play a role in the pathogenicity of macrothrombocytopenia.
- Further clinical and functional studies are warranted to elucidate the impact of these variants.
