Understanding molecular mechanisms and predicting phenotypic effects of pathogenic tubulin mutations

Abstract

Cells rely heavily on microtubules for several processes, including cell division and molecular trafficking. Mutations in the different tubulin-α and -β proteins that comprise microtubules have been associated with various diseases and are often dominant, sporadic and congenital. While the earliest reported tubulin mutations affect neurodevelopment, mutations are also associated with other disorders such as bleeding disorders and infertility. We performed a systematic survey of tubulin mutations across all isotypes in order to improve our understanding of how they cause disease, and increase our ability to predict their phenotypic effects. Both protein structural analyses and computational variant effect predictors were very limited in their utility for differentiating between pathogenic and benign mutations. This was even worse for those genes associated with non-neurodevelopmental disorders. We selected tubulin-α and -β disease mutations that were most poorly predicted for experimental characterisation. These mutants co-localise to the mitotic spindle in HeLa cells, suggesting they may exert dominant-negative effects by altering microtubule properties. Our results show that tubulin mutations represent a blind spot for current computational approaches, being much more poorly predicted than mutations in most human disease genes. We suggest that this is likely due to their strong association with dominant-negative and gain-of-function mechanisms.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules

“…Together these data suggest that these disease-causing variants are acting through non–loss-of-function mechanisms, i.e., through dominant-negative or gain-of-function effects. Indeed, it is difficult to distinguish between these two possibilities ( 30 ), particularly for tubulins ( 31 ). Despite a decreased intrinsic propensity for the PCD-only variants to assemble into microtubules, transcriptional up-regulation of TUBB4B itself and its chaperones still produces mutant TUBB4B protein.…”

“…Indeed, recent cryo-EM maps revealed an interaction between the α-tubulin C-terminal tail that links adjacent dimers to two conserved arginine residues (R391 and R392) on β-tubulin to stabilize the microtubule filament ( 33 ). Moreover, several other pathogenic missense mutations have been reported in mostly neurodegenerative disorders at this position in other β-tubulin isotypes, including TUBB4A p.R391H/L, TUBB3 p.R391L, TUBB2A p.R391H, and TUBB8 p.R391C, where these mutations were predicted to disrupt microtubule stability ( 31 , 34 ).…”

Understanding the heterogeneous performance of variant effect predictors across human protein-coding genes

“…We referred to the gnomAD variants as “putatively benign” because, although this set is likely to contain a small proportion of disease-causing variants, particularly from genes with recessive inheritance or variable clinical penetrance, the vast majority are expected to be neutral or nearly neutral variants. This approach has been widely used in previous studies [25,28,35] and has proven highly useful. Moreover, we suggest that this is more reflective of the actual clinical utilisation of VEPs, whereby the challenge is in distinguishing pathogenic variants from rare, unclassified-but-benign variants, rather than known benign variants, which tend to be common and easy to identify [36,37].…”

MAPping tubulin mutations

“…While some mutations influence MAP binding in a predictable way given their physical proximity, it is evident that tubulin variants can have allosteric effects on microtubule structure thereby influencing MAP binding in unexpected ways. This is perhaps one reason, why the phenotypic prediction of tubulin mutations has proved to be so difficult ( Attard et al, 2022 ).…”