Genetic and functional analysis of TUBB1 variants in congenital hypothyroidism.

Background: Congenital hypothyroidism (CH) is the most common neonatal disorder, primarily caused by thyroid dysgenesis (TD). While the genetic cause has been identified in less than 5% of TD cases, there is an urgent need to investigate additional gene mutations that may be responsible. In 2018, TUBB1 was identified as a novel candidate gene associated with TD. Nevertheless, further research is required to confirm the role of TUBB1 in TD pathogenesis and the association between TUBB1 mutations and TD in humans. Based on the previous genetic analysis of TUBB1 in 289 Chinese TD patients, this study aimed to further validate the association between TUBB1 and TD, and to explore the pathogenic mechanisms of TUBB1 c.952C>T at the cellular level.

Material and methods: We performed real-time polymerase chain reaction (RT-PCR), western blot, Cell Counting Kit 8 (CCK8), and wound healing assay to evaluate the effect of TUBB1 c.952C>T on gene expression, cell proliferation, and migration.

Results: The c.952C>T mutant decreased the expression of TUBB1 in both mRNA and protein level, and inhibited the proliferation of thyroid cells significantly. Also, c.952C>T mutant showed restrain effects on the migration, although there was no stistical significance. Notably, pathogenic TUBB1 variants were not detected in patients with dyshormonogenesis (DH).

Conclusions: TUBB1 variants confer genetic susceptibility to TD but not DH. The pathogenic variant in TUBB1 was identified in 1.38% (4/289) of our Chinese TD patient cohort, and burden test analysis revealed an association between TUBB1 variants and TD. Functional experimental results indicated that the c.952C>T mutant dominantly affects gene expression and proliferation of thyroid cells.