Fig 2: Conservative and tertiary structure prediction of mutant proteins. (A) Cross-species comparison of the region of TUBA3D indicates that the identified mutations affect highly conserved residues. Alignment of sequences surrounding the Gln11stop and Val68Leufs*2 mutations in Human (NP_525125.2), Chimpanzee (NP_001233579.1), Cattle (NP_001039875.1), Dog (XP_005640747.1), Chicken (XP_422851.1) and Clawed frog (NP_001081575.1). (B) The tertiary structure of the mutant and wildtype proteins predicted by the Swiss-Model workspace.

Fig 3: Pedigree and clinical characteristics of the twins’ family. (A) Pedigree of the twins’ family with TUBA3D mutation. The proband is indicated by an arrow. Sanger sequencing confirms the mutations identified by WES. (B) Videokeratograph of the proband. The results showed high central corneal curvature (i.e., Kmax and Kmin of the front surface >60D), significant protrusion of the front and posterior surfaces, and thinning of the corneal thickness of both eyes. OD, right eye. OS, left eye.

Fig 4: TUBA3D expression analysis in human tissues. (A) mRNA expression of TUBA3D in the human cornea, sclera, peripheral blood, human corneal fibroblast cells (HTK), and human corneal epithelial cells (HCEC). (B) a3 Tubulin and GAPDH expression analysis in HTK cell and cornea by Western blotting (cropped from different parts of the same gel). (C) mRNA expression of TUBA3D and genes UPA, UPAR, MMP1, and MMP9 in the blood samples of the twins and their parents.

Fig 5: ROS and SOD1, SOD2 expression levels of the wildtype, c.31 C > T (Gln11stop), and c.201insTT (Val68Leufs*2) mutation cells. HTK cells transiently transfected with constructs encoding HA-tagged wildtype and mutant (c.31 C > T, c.201insTT) TUBA3D proteins. (A) The cell morphology after 72 hours of transfection (B) ROS levels in HTK cells after 48 hours of transfection. n = 3. *P < 0.05. (C) SOD1 and SOD2 expression analysis in HTK cells after transfection by Western blotting. Representative figures of Western blotting (left, cropped from different gels of the same samples) and the relative protein content of each group (n = 3) quantified by Image J software (right). *P < 0.05. (D) The cell survival analysis of the HTK cells after 24, 48, 72 hours of transfection with (right) and without NAC (left).