Clinical characterization and in silico structural analysis of microtubule-related gene variants in eleven patients

Objective This study aimed to analyze associations of Microtubules (MTs) mutations with nervous system phenotypes, summarize mutation characteristics of genes including TUBA1A , TUBB2A , TUBB4A , and TUBGCP6 , explore clinical phenotypic diversity and molecular mechanisms, and expand the MTs genotype-phenotype spectrum. Methods Eleven patients with MTs gene mutations were enrolled, and their clinical data were collected. Protein structure prediction was performed using AlphaFold3, and FoldX was employed to calculate mutation-induced energy changes, thereby evaluating protein stability and conformational impacts. Results While global developmental delay was present in all individuals (11/11, 100%), epileptic seizures and structural brain abnormalities were variable phenotypic features, observed in 81.8% (9/11; including 8 with unprovoked epilepsy and 1 with an isolated febrile seizure) and 63.6% (7/11) of patients, respectively, with normal cranial MRI findings documented in four patients (36.4%). Four patients with TUBA1A were associated with severe cortical developmental malformations (lissencephaly, pachygyria), with two exhibiting dynamic developmental regression; four patients with TUBB2A consistently presented with severe developmental impairment and intractable epilepsy (including infantile epileptic spasm syndrome and Lennox-Gastaut syndrome), some displaying sound-induced reflex seizures, while none manifested developmental regression during the follow-up period. Two patients with TUBB4A showed marked differences in age of onset, and one presented with an isolated co-occurrence of systemic lupus erythematosus; one patient with TUBGCP6 was mainly characterized by microcephaly and short stature without epilepsy. Across the 11 patients, a total of 12 variants were identified, comprising 10 missense variants, one non-canonical intronic/splice variant, and one copy number duplication. Prediction of protein structure and energy changes for the 10 missense variants showed varying degrees of increased free energy. Based on in silico structural predictions, we hypothesize that these mutations may potentially impair microtubule function by altering hydrogen bonds and conformation, with representative examples including TUBA1A Y172D (5.15 kcal/mol energy increase) and TUBB4A G10S (introduction of new hydrogen bond interactions). Conclusion This study delineates the diverse clinical spectrum across patients harboring MTs variants: TUBA1A variants were predominantly observed in individuals with cortical malformations and potential developmental regression, TUBB2A with severe static neurodevelopmental delay and drug-resistant epilepsy, TUBB4A with motor impairment (with systemic lupus erythematosus noted in one individual as an unusual co-observation), and TUBGCP6 with primary growth and developmental delay. In silico structural modeling (AlphaFold3 and FoldX) indicates that missense substitutions may perturb protein stability and local conformation (with ΔΔG values varying across sites); however, because these variants are predominantly identified in single individuals, these computational insights represent theoretical biophysical hypotheses rather than validated variant-specific phenotypic signatures. Further functional assays and larger cohorts are required to establish definitive genotype-phenotype correlations.