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Tubulinopathies are rare neurological disorders caused by alterations in tubulin structure and function, giving rise to a wide range of brain abnormalities involving neuronal proliferation, migration, differentiation and axon guidance. TUBB is one of the ten β-tubulin encoding genes present in the human genome and is broadly expressed in the developing central nervous system and the skin. Mutations in TUBB are responsible for two distinct pathological conditions: the first is characterized by microcephaly and complex structural brain malformations and the second, also known as “circumferential skin creases Kunze type” (CSC-KT), is associated to neurological features, excess skin folding and growth retardation. We used a combination of immunocytochemical and cellular approaches to explore, on patients’ derived fibroblasts, the functional consequences of two TUBB variants: the novel mutation (p.N52S), associated with basal ganglia and cerebellar dysgenesis, and the previously reported variant (p.M73T), linked to microcephaly, corpus callosum agenesis and CSC-KT skin phenotype. Our results demonstrate that these variants impair microtubule (MT) function and dynamics. Most importantly, our studies show an altered epidermal growth factor (EGF) and transferrin (Tf) intracellular vesicle trafficking in both patients’ fibroblasts, suggesting a specific role of TUBB in MT-dependent vesicular transport.

Purpose To estimate diagnostic yield and genotype-phenotype correlations in a cohort of 811 patients with lissencephaly or subcortical band heterotopia. Methods We collected DNA from 756 children with lissencephaly over 30 years. Many were tested for deletion 17p13.3 and mutations of LIS1 , DCX , and ARX , but few other genes. Among those tested, 216 remained unsolved and were tested by a targeted panel of 17 genes ( ACTB , ACTG1 , ARX , CRADD , DCX , LIS1 , TUBA1A , TUBA8 , TUBB2B , TUBB , TUBB3 , TUBG1 , KIF2A , KIF5C , DYNC1H1 , RELN , and VLDLR ) or by whole-exome sequencing. Fifty-five patients studied at another institution were added as a validation cohort. Results The overall mutation frequency in the entire cohort was 81%. LIS1 accounted for 40% of patients, followed by DCX (23%), TUBA1A (5%), and DYNC1H1 (3%). Other genes accounted for 1% or less of patients. Nineteen percent remained unsolved, which suggests that several additional genes remain to be discovered. The majority of unsolved patients had posterior pachygyria, subcortical band heterotopia, or mild frontal pachygyria. Conclusion The brain-imaging pattern correlates with mutations in single lissencephaly-associated genes, as well as in biological pathways. We propose the first LIS classification system based on the underlying molecular mechanisms.

Purpose TUBA1A and TUBB2B tubulinopathies are rare neurodevelopmental disorders characterized by cortical and extracortical malformations and heterogenic phenotypes. There is a need for quantitative clinical endpoints that will be beneficial for future diagnostic and therapeutic trials. Methods Quantitative natural history modeling of individuals with TUBA1A and TUBB2B tubulinopathies from clinical reports and database entries of DECIPHER and ClinVar. Main outcome measures were age at disease onset, survival, and diagnostic delay. Phenotypical, neuroradiological, and histopathological features were descriptively illustrated. Results Mean age at disease onset was 4 ( TUBA1A ) and 6 months ( TUBB2B ), respectively. Mortality was equally estimated with 7% at 3.2 ( TUBA1A ) and 8.0 years ( TUBB2B ). Diagnostic delay was significantly higher in TUBB2B (12.3 years) compared with TUBA1A tubulinopathy (4.2 years). We delineated the isotype-dependent clinical, neuroradiological, and histopathological phenotype of affected individuals and present brain malformations associated with epilepsy and an unfavorable course of disease. Conclusion The natural history of tubulinopathies is defined by the genotype and associated brain malformations. Defined data on estimated survival, diagnostic delay, and disease characteristics of TUBA1A and TUBB2B tubulinopathy will help to raise disease awareness and encourage future clinical trials to optimize genetic testing, family counseling, and supportive care.

Tubulins are the highly conserved subunit of microtubules which involve in various fundamental functions including brain development. Microtubules help in neuronal proliferation, migration, differentiation, cargo transport along the axons, synapse formation, and many more. Tubulin gene family consisting of multiple isotypes, their differential expression and varied post translational modifications create a whole new level of complexity and diversity in accomplishing manifold neuronal functions. The studies on the relation between tubulin genes and brain development opened a new avenue to understand the role of each tubulin isotype in neurodevelopment. Mutations in tubulin genes are reported to cause brain development defects especially cortical malformations, referred as tubulinopathies. There is an increased need to understand the molecular correlation between various tubulin mutations and the associated brain pathology. Recently, mutations in tubulin isotypes ( TUBA1A , TUBB , TUBB1 , TUBB2A , TUBB2B , TUBB3 , and TUBG1 ) have been linked to cause various neurodevelopmental defects like lissencephaly, microcephaly, cortical dysplasia, polymicrogyria, schizencephaly, subcortical band heterotopia, periventricular heterotopia, corpus callosum agenesis, and cerebellar hypoplasia. This review summarizes on the microtubule dynamics, their role in neurodevelopment, tubulin isotypes, post translational modifications, and the role of tubulin mutations in causing specific neurodevelopmental defects. A comprehensive list containing all the reported tubulin pathogenic variants associated with brain developmental defects has been prepared to give a bird’s eye view on the broad range of tubulin functions.

Microtubules are dynamic cytoskeletal polymers that mediate numerous, essential functions such as axon and dendrite growth and neuron migration throughout brain development. In recent years, sequencing has revealed dominant mutations that disrupt the tubulin protein building blocks of microtubules. These tubulin mutations lead to a spectrum of devastating brain malformations, complex neurological and physical phenotypes, and even fatality. The most common tubulin gene mutated is the α-tubulin gene TUBA1A, which is the most prevalent α-tubulin gene expressed in post-mitotic neurons. The normal role of TUBA1A during neuronal maturation, and how mutations alter its function to produce the phenotypes observed in patients, remains unclear. This review synthesizes current knowledge of TUBA1A function and expression during brain development, and the brain malformations caused by mutations in TUBA1A.

Heterozygous, missense mutations in both alpha- and beta-tubulin genes have been linked to an array of neurodevelopment disorders, commonly referred to as “tubulinopathies”. To date, tubulinopathy mutations have been identified in three beta-tubulin isotypes and one alpha-tubulin isotype. These mutations occur throughout the different genetic domains and protein structures of these tubulin isotypes, and the field is working to address how this molecular-level diversity results in different cellular and tissue-level pathologies. Studies from many groups have focused on elucidating the consequences of individual mutations; however, the field lacks comprehensive models for the molecular etiology of different types of tubulinopathies, presenting a major gap in diagnosis and treatment. This review highlights recent advances in understanding of tubulin structural dynamics, the roles microtubule-associated proteins (MAPs) play in microtubule regulation, and how these are inextricably linked. We emphasize the value of investigating interactions between tubulin structures, microtubules, and MAPs to understand and predict the impact of tubulinopathy mutations at the cell and tissue levels. Microtubule regulation is multifaceted and provides a complex set of controls for generating functional cytoskeleton at the right place and right time during neurodevelopment. Understanding how tubulinopathy mutations disrupt distinct subsets of those controls, and how that ultimately disrupts neurodevelopment, will be important for establishing mechanistic themes among tubulinopathies that may lead to insights in other neurodevelopment disorders and normal neurodevelopment.

Background The TUBA1A -associated tubulinopathy is clinically heterogeneous with brain malformations, microcephaly, developmental delay and epilepsy being the main clinical features. It is an autosomal dominant disorder mostly caused by de novo variants in TUBA1A. Results In three individuals with developmental delay we identified heterozygous de novo missense variants in TUBA1A using exome sequencing. While the c.1307G > A, p.(Gly436Asp) variant was novel, the two variants c.518C > T, p.(Pro173Leu) and c.641G > A, p.(Arg214His) were previously described. We compared the variable phenotype observed in these individuals with a carefully conducted review of the current literature and identified 166 individuals, 146 born and 20 fetuses with a TUBA1A variant. In 107 cases with available clinical information we standardized the reported phenotypes according to the Human Phenotype Ontology. The most commonly reported features were developmental delay (98%), anomalies of the corpus callosum (96%), microcephaly (76%) and lissencephaly (agyria-pachygyria) (70%), although reporting was incomplete in the different studies. We identified a total of 121 specific variants, including 15 recurrent ones. Missense variants cluster in the C-terminal region around the most commonly affected amino acid position Arg402 (13.3%). In a three-dimensional protein model, 38.6% of all disease-causing variants including those in the C-terminal region are predicted to affect the binding of microtubule-associated proteins or motor proteins. Genotype-phenotype analysis for recurrent variants showed an overrepresentation of certain clinical features. However, individuals with these variants are often reported in the same publication. Conclusions With 166 individuals, we present the most comprehensive phenotypic and genotypic standardized synopsis for clinical interpretation of TUBA1A variants. Despite this considerable number, a detailed genotype-phenotype characterization is limited by large inter-study variability in reporting.

Tubulinopathies encompass neurodevelopmental disorders caused by mutations in genes encoding for different isotypes of α- and β-tubulins, the structural components of microtubules. Less frequently, mutations in tubulins may underlie neurodegenerative disorders. In the present study, we report two families, one with 11 affected individuals and the other with a single patient, carrying a novel, likely pathogenic, variant (p. Glu415Lys) in the TUBA4A gene (NM_006000). The phenotype, not previously described, is that of spastic ataxia. Our findings widen the phenotypic and genetic manifestations of TUBA4A variants and add a new type of spastic ataxia to be taken into consideration in the differential diagnosis.

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different β-tubulin genes, TUBB2A or TUBB3 . RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of β-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin’s GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

Heterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here, we focus on two mutations at the valine 409 residue of TUBA1A, V409I, and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A -V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG (tumor overexpressed gene) domains. In each assay tested, the TUBA1A -V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A -V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels. Proteins are molecules made up of long chains of building blocks called amino acids. When a mutation changes one of these amino acids, it can lead to the protein malfunctioning, which can have many effects at the cell and tissue level. Given that human proteins are made up of 20 different amino acids, each building block in a protein could mutate to any of the other 19 amino acids, and each mutations could have different effects. Tubulins are proteins that form microtubules, thin tubes that help give cells their shape and allow them to migrate. These proteins are added or removed to microtubules depending on the cell’s needs, meaning that microtubules can grow or shrink depending on the situation. Mutations in the tubulin proteins have been linked to malformations of varying severities involving the formation of ridges and folds on the surface of the brain, including lissencephaly, pachygyria or polymicrogyria. Hoff et al. wanted to establish links between tubulin mutations and the effects observed at both cell and tissue level in the brain. They focused on two mutations in the tubulin protein TUBA1A that affect the amino acid in position 409 in the protein, which is normally a valine. One of the mutations turns this valine into an amino acid called isoleucine. This mutation is associated with pachygyria, which leads to the brain developing few ridges that are broad and flat. The second mutation turns the valine into an alanine, and is linked to lissencephaly, a more severe condition in which the brain develops no ridges, appearing smooth. Hoff et al. found that both mutations interfere with the development of the brain by stopping neurons from migrating properly, which prevents them from forming the folds in the brain correctly. At the cellular level, the mutations lead to tubulins becoming harder to remove from microtubules, making microtubules more stable than usual. This results in longer microtubules that are harder for the cell to shorten or destroy as needed. Additionally, Hoff et al. showed that the mutant versions of TUBA1A have weaker interactions with a protein called XMAP215, which controls the addition of tubulin to microtubules. This causes the microtubules to grow uncontrollably. Hoff et al. also established that the magnitude of the effects of each mutation on microtubule growth scale with the severity of the disorder they cause. Specifically, cells in which TUBA1A is not mutated have microtubules that grow at a normal rate, and lead to typical brain development. Meanwhile, cells carrying the mutation that turns a valine into an alanine, which is linked to the more severe condition lissencephaly, have microtubules that grow very fast. Finally, cells in which the valine is mutated to an isoleucine – the mutation associated with the less severe malformation pachygyria – have microtubules that grow at an intermediate rate. These findings provide a link between mutations in tubulin proteins and larger effects on cell movement that lead to brain malformations. Additionally, they also link the severity of the malformation to the severity of the microtubule defect caused by each mutation. Further work could examine whether microtubule stabilization is also seen in other similar diseases, which, in the long term, could reveal ways to detect and treat these illnesses.