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. 2017 Jan 15;26(2):258-269.
doi: 10.1093/hmg/ddw383.

Uner Tan Syndrome Caused by a Homozygous TUBB2B Mutation Affecting Microtubule Stability

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Free PMC article

Uner Tan Syndrome Caused by a Homozygous TUBB2B Mutation Affecting Microtubule Stability

Martin W Breuss et al. Hum Mol Genet. .
Free PMC article

Abstract

The integrity and dynamic properties of the microtubule cytoskeleton are indispensable for the development of the mammalian brain. Consequently, mutations in the genes that encode the structural component (the α/β-tubulin heterodimer) can give rise to severe, sporadic neurodevelopmental disorders. These are commonly referred to as the tubulinopathies. Here we report the addition of recessive quadrupedalism, also known as Uner Tan syndrome (UTS), to the growing list of diseases caused by tubulin variants. Analysis of a consanguineous UTS family identified a biallelic TUBB2B mutation, resulting in a p.R390Q amino acid substitution. In addition to the identifying quadrupedal locomotion, all three patients showed severe cerebellar hypoplasia. None, however, displayed the basal ganglia malformations typically associated with TUBB2B mutations. Functional analysis of the R390Q substitution revealed that it did not affect the ability of β-tubulin to fold or become assembled into the α/β-heterodimer, nor did it influence the incorporation of mutant-containing heterodimers into microtubule polymers. The 390Q mutation in S. cerevisiae TUB2 did not affect growth under basal conditions, but did result in increased sensitivity to microtubule-depolymerizing drugs, indicative of a mild impact of this mutation on microtubule function. The TUBB2B mutation described here represents an unusual recessive mode of inheritance for missense-mediated tubulinopathies and reinforces the sensitivity of the developing cerebellum to microtubule defects.

Figures

Figure 1.
Figure 1.
An extended Uner Tan syndrome family harbours a recessive mutation in TUBB2B. (A) Family tree with two branches of the previously reported family (30). Double lines: reported consanguinity. Filled symbols: affected individuals. Family tree was simplified relative to the previously published version and omits unaffected and affected deceased children from generation III. (B) Sagittal MRI image of patient III-4. (C) Image of patient III-4 using all four limbs for locomotion, the diagnostic hallmark of Uner Tan Syndrome. (D,E) Sagittal MRI images of patients III-7 and III-8, respectively. Arrowheads in B, D and E indicate severe cerebellar hypoplasia for all patients. Images similar to B–E have been published previously (30). (F) Upper model depicts the TUBB2B coding sequence spanning four exons and the 5’ and 3’ UTRs. Grey line indicates the position of the c.1169G > A mutation (RefSeq: NM_178012.4). Lower model depicts the TUBB2B protein sequence with the three tubulin domains (N-terminal, intermediate and C-terminal). Grey line indicates the position of the resulting p.390R > Q amino acid substitution (RefSeq: NP_821080.1). (G) Residue p.R390 is conserved in homologs of all vertebrates, D. melanogaster and C. elegans. The budding yeast homolog Tub2p shows conservation of the positive charge by an encoded lysine residue in place of arginine.
Figure 2.
Figure 2.
Location of the R390 residue within the tubulin heterodimer. (A–G) Depiction of the 2D electron crystallography of the tubulin heterodimer using a previously published data set (RCSB PDB: 1JFF) (37). (A–D) Overview of the tubulin heterodimer in lateral (A and C) and frontal views (B and D), relative to their position in an assembled microtubule. Shown are a surface model (A,B) and a mixed surface and ribbon model (C,D). Arrowheads indicate the location of the R390 residue. α: α-tubulin; β: β-tubulin; i: heterodimer surface facing the inside (lumen) of the microtubule; o: heterodimer surface facing outwards in a microtubule. (E–G) Magnified view of the R390 residue (E) and the adjacent positively charged R391 and K392 residues (F,G). These three positively charged amino acids form a binding pocket that interacts with the α-tubulin of the longitudinally adjacent tubulin-heterodimer.
Figure 3.
Figure 3.
The R390Q mutation does not affect tubulin folding or heterodimer integration into the microtubule lattice (A) Denaturing gel of in vitro transcribed and translated reaction products for wild-type (WT) and the TUBB2B R390Q mutant, in which the 35S-methinonine labelled proteins were detected by autoradiography. Note that the translational efficiency is not affected by the mutation. Arrowhead indicates the β-tubulin band. (B) Kinetic analysis of tubulin folding on non-denaturing gels of wild-type and R390Q translation products. Arrowheads (from top to bottom) denote the following complexes: chaperonin (CCT)/β-tubulin binary complex, the TBCD/β-tubulin co-complex, the prefoldin (PFD)/β-tubulin complex, the native tubulin heterodimer and the TBCA/β-tubulin co-complex. The molecular identities are assigned on the basis of their characteristic electrophoretic mobility. Shown are lanes for different reaction times (20, 40, 60, 90 and 150 minutes) in the rabbit reticulocyte lysate (TNT) and a combination of 90 minutes TNT and 60 minutes of chase with unlabelled wild-type tubulin. The R390Q mutation does not affect the kinetics of tubulin heterodimer formation or endpoint yields. (C–H) Immunofluorescence images of HEK293T cells transfected with FLAG-tagged wild-type Tubb2b (C–E) or mutant R390Q (F–H) and stained with antibodies against α-tubulin and the FLAG-tag. Shown are composite images (C and F) and grey-scale images of the individual channels (D, E, G and H). Wild-type and mutant tubulin heterodimers are both capable of integration into the microtubule lattice. Scale bar in (C) shows 5 µm. Magnifications of the dashed rectangles are shown in the upper right corner of each image.
Figure 4.
Figure 4.
Introduction of the K390Q mutation into the single budding yeast β-tubulin gene results in increased sensitivity to microtubule destabilizing drugs. (A–D) Spot assays showing a logarithmic dilution series for the original wild-type strain (BY4741) and derivatives in which the normal TUB2 locus was replaced by either TUB2.URA3 or tub2-K390Q.URA3. Cells grow normally on YPD (A), but show increased sensitivity to nocodazole (B) and benomyl (C,D), suggesting decreased microtubule stability in the presence of tubulin-heterodimers with the K390Q amino acid substitution.
Figure 5.
Figure 5.
Tubb2b is expressed at high levels in the embryonic cerebellum. (A–C) Overview scan of a sagittal section of an embryonic day (E) 16.5 head of the Tg(Tubb2b-eGFP)GlbDAK line. eGFP is expressed under the control of the endogenous Tubb2b locus on a bacterial artificial chromosome (BAC). (B) shows a magnification of the dashed rectangle encircling the cerebellar anlage in (A); in (C), the GFP channel is shown as a grey-scale image of (B). Hoechst staining for nuclear DNA is shown in white in A and B. (D,E) Confocal images of a control (D) and a transgenic animal (E). Hoechst staining for nuclear DNA is shown in blue in (D) and (E). PCC: Purkinje cell cluster area; EGZ: External Granular Zone. (F) Quantification of the average fluorescence intensity found in the EGZ and the PCC for control (n = 1) and transgenic mice (n = 3). For transgenic mice, mean ± SEM are shown. (G–Z) Fluorescent images of the developing cerebellum of the Tg(Tubb2b-eGFP)GlbDAK line at E16.5 stained for Pax6 (G–J), pH3 (K–N), NeuN (O–R), Calbindin (S–V) and Sox2 (W–Z). (H, L, P, T and X) show magnifications of the dashed rectangle in (G,K, O,S and W) respectively. (I, M, Q, U and Y), as well as (J, N, R, V and Z) show grey-scale images of the GFP and immunostaining channels, respectively. Scale bars show 1000 µm in A, 200 µm in B, 100 µm in (D) and (G) and 20 µm in (H).

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