Fig 2: Peripheral blood smears were stained for marginal band detection in healthy or patient’s platelets. Endogenous TUBB1 and a-Tubulin which are subunits of marginal band were marked with specific primary antibodies in combination with Alexa-555 (red) or Alexa-488-conjugated (green) secondary antibodies, respectively. Marginal band was determined as a hollow-ring structure in health platelets but circular microtubule arrangement was disappeared in patient #7’s platelets carrying TUBB1 variant p.T274M/R307H. Merged images (yellowish) were captured in oiled 63X magnification by using LSM 880 (Zeiss) confocal microscope (upper panel). Platelets labeled with an asterisk were zoomed in while image processing (bottom panel).

Fig 3: Sequencing also revealed heterozygous TUBB1 variations identified for the first time in macrothromboytopenia patients in Turkish population. (A) A silent TUBB1 variation c.635C>T (p.H122H), and 3 missense variants (B) c.636C>G (p.E123Q), (C) c.705G>A (p.G146R), and (D) c.821C>T (p.T274M) were detected in patient #30, patient #17, patient #36 and patients #2, #3, #7, and #37, respectively.

Fig 4: TUBB1 expression was detected in both mRNA and protein levels by using blood samples from healthy control (n=4, pooled) and patient #7. RT-qPCR analysis showed relative TUBB1 gene expression in both (A) PBMC and (B) PRP fractions. All Taqman assays were carried out in triplicates and relative fold change was calculated with the ??Ct method. (C) TUBB1 protein band was not detectable in the whole lysate isolated from patient #7 contrary to healthy control.

Fig 5: Entire coding exons were amplified by PCR and sequenced. Heterozygous variations (A) c.130-131AG>CC (p.Q43P), (B) c.803G>T (p.T178T), and (C) c.920G>A (p.R307H) were detected on TUBB1 gene from healthy controls and/or macrothrombocytopenia patients by FinchTV sequence analysis software.