Out-of-frame Translation Rescues a Loss-of-function Variant in a Novel TBCE Phenotype

Abstract

Context: Pathogenic variants in the TBCE gene, encoding tubulin-specific chaperone E crucial for tubulin folding, are linked to three severe neurodevelopmental disorders: Hypoparathyroidism-retardation-dysmorphism syndrome, Kenny-Caffey syndrome type 1, and progressive encephalopathy with amyotrophy and optic atrophy.

Objective: We identified patients with a novel, milder TBCE-associated phenotype and aimed to characterize it at the clinical and molecular levels.

Materials and methods: We conducted splicing analysis using deep next-generation sequencing of RT-PCR products and detected TBCE through Western blotting. Translation efficiency was measured using a luciferase reporter assay. Overexpression experiments were performed in Hela cells with tubulin staining. Immunofluorescence analysis was used for Golgi complex assessment, while microtubule dynamics were studied post-nocodazole treatment. Electron microscopy facilitated ultrastructural studies.

Results: We report 7 patients with a novel, milder TBCE phenotype, presenting with amyotrophy, testicular failure, and mild intellectual disability, with or without short stature. All patients were homozygous or compound-heterozygous for the NM_003193.5:c.100 + 1G > A variant, which causes a splicing alteration and early frameshift. However, we found that the mild phenotype arises due to translation from an alternative open reading frame, producing a partially functional protein. Dermal fibroblasts showed reduced Golgi compactness but normal microtubule dynamics. Electron microscopy revealed varying levels of acto-myosin degradation. The c.100 + 1G > A variant was found to be 10 times more frequent in Slavic samples than in gnomAD, suggesting underdiagnosis of this phenotype.

Conclusion: This study uncovers complex molecular mechanisms contributing to the milder phenotype in patients with the c.100 + 1G > A variant, providing insights into a new TBCE-related disorder.

Keywords: TBCE; microtubule; new phenotype; out-of-frame translation; splicing; tubulin; tubulin-specific chaperone E.