A new mutation in TUBB1 associated with thrombocytopenia confirms that C‐terminal part of β1‐tubulin plays a role in microtubule assembly

To the Editor: Inherited macrothrombocytopenia is a heterogeneous group of rare diseases (1). Causative mutations are commonly found in genes that affect proplatelet formation. β1-tubulin, a hematopoietic-specific tubulin isotype, is critical for this function. TUBB1-related diseases are rare autosomal dominant macrothrombocytopenia characterized by mild bleeding symptoms and enlarged platelets. These disorders are caused by mutations in the TUBB1 gene, which encodes the β1-tubulin. To date, only two Japanese families with TUBB1 disorders have been reported, in which two different TUBB1 mutations (R318W and F260S) have been identified (2, 3). Here, we report, in a French family with macrothrombocytopenia, a novel mutation located in the C-terminal part of the β1-tubulin protein, confirming that this domain plays a key role in microtubule assembly. This study was approved by our institutional review board (Comité de Protection des Personnes Sud-Ouest et Outre Mer III, Bordeaux, France).The proband was a 31-year-old pregnant woman who was referred to us for an evaluation of thrombocytopenia. Her platelet number was assessed by manual count and found to be 60× 109 platelets/l. Peripheral blood smears showed that platelets were markedly large. MYH9 disorders, Bernard–Soulier syndrome, αIIbβ3-related macrothrombocytopenia, and type 2B von Willebrand disease were excluded. Macrothrombocytopenia was also detected in two other siblings (father and sister) (Table S1, Supporting information). We then performed a whole exome sequencing analysis in the proband which identified a c.1267C>T nonsense mutation (p.Q423*) occurring in the last exon of the TUBB1 gene (Fig. S1a). This mutation falls within the C-terminal 30 amino acids part of the protein. It was already described in the publically available ExAC database, but with a very rare allele frequency (0.01%). A second heterozygous substitution, p.R307H, previously described as a functional polymorphism was also identified (4). These two mutations were also detected in the patient’s sister (father’s genomic DNA was not available). Platelet mRNAs from the proband and her sister were extracted, retrotranscribed and amplified in order to quantify allelic expression of the TUBB1 gene by PCR-RFLP. mRNA expression of platelet β1-tubulin was comparable between wild-type (WT) and mutant allele (Fig. S1b), confirming that mRNA harboring the c.1267C>T mutation was not degraded by nonsense-mediated decay (NMD), a quality-control mechanism that selectively degrades mRNAs harboring more proximal premature termination codons. So, the mutant mRNA was expected to be stable, but the resulting truncated protein to be non-functional with a dominant-negative effect. This hypothesis is consistent with mouse models showing that the loss of a single Tubb1 allele was not sufficient to cause the phenotype (2). As the anti-β1-tubulin antibody used (SAP.4G5, Thermo Fisher Scientific, Waltham, MA) specifically recognizes a sequence peptide corresponding to the C-terminal part of the protein, immunoblotting cannot demonstrate if the mutated protein is normally expressed (Fig. S1c), and a decreased expression by 50% would be expected, which was not the case here. We then hypothesized that up-regulation of wild-type β1-tubulin partially compensates for an abnormal expression of the mutated β1-tubulin. However, in order to demonstrate that the Q423* mutation has an effect on microtubule assembly, peripheral blood smears were used for immunofluorescence analysis. The circumferential ring staining was virtually absent and microtubules were aggregated in platelets from affected individuals (Fig. 1a.). At the ultrastructural level, most of the patients’ platelets isolated from fixed whole blood displayed a very large size with a round shape and contained atypical membrane complexes (Fig. 1b). Tubulin 3D model shows that the C-terminal domain consists of two antiparallel α-helices, H11 and H12. It is a negatively charged domain rich in glutamate which tends to be less associated with the rest of the protein structure, projecting outward from the subunit (Fig. S2). The Q423 residue falls within the α-helix H12, lying at the outer surface of β1-tubulin. Joe PA et al. have previously studied the role of the C-terminal region in microtubule assembly by generating a series of truncation