Mutations in TBCK, Encoding TBC1-Domain-Containing Kinase, Lead to a Recognizable Syndrome of Intellectual Disability and Hypotonia

Abstract

Through an international multi-center collaboration, 13 individuals from nine unrelated families and affected by likely pathogenic biallelic variants in TBC1-domain-containing kinase (TBCK) were identified through whole-exome sequencing. All affected individuals were found to share a core phenotype of intellectual disability and hypotonia, and many had seizures and showed brain atrophy and white-matter changes on neuroimaging. Minor non-specific facial dysmorphism was also noted in some individuals, including multiple older children who developed coarse features similar to those of storage disorders. TBCK has been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which is also stimulated by exogenous leucine supplementation. TBCK was absent in cells from affected individuals, and decreased phosphorylation of phospho-ribosomal protein S6 was also observed, a finding suggestive of downregulation of mTOR signaling. Lastly, we demonstrated that activation of the mTOR pathway in response to L-leucine supplementation was retained, suggesting a possible avenue for directed therapies for this condition.

Figure 1

Diagram of TBCK with Reported Variants and Facial Features and Brain Imaging of Affected Individuals (A) A diagram of TBCK includes known domains and the variants present in the individuals described in this report. Abbreviations are as follows: Hom, homozygous; and Comp Het, compound heterozygous. (B–D and G–I) Facial features of individuals 8-1 (B) and 2-1 (G), affected siblings 4-1 (C) and 4-2 (H), and affected siblings 6-1 (D) and 6-2 (I). Individuals 8-1 and 2-1 have coarse features with significant macroglossia, and individuals 4-1 and 4-2 have no significant facial dysmorphisms. Note the mild dysmorphic features, including a wide forehead, bulbous nose, open mouth with macroglossia, thick lips, deep palate, and prognathic mandible, in individual 6-1 and the wide forehead and short neck in individual 6-2. (E, F, and I–K) Brain MRI of individuals 6-1 (E), 6-2 (J), and 7-1 (F and K) demonstrates discrete abnormalities of the periventricular and parietal white matter in both studies.

Figure 2

Western Blot Demonstrates Absent TBCK and Decreased mTOR Signaling in Lympoblastic Cells of Affected Individuals Western blots of immortalized lymphocytes (LCLs) from a control individual and individuals 1-1 and 1-2; both affected individuals have homozygous frameshift variants. (A) Top: TBCK levels were lower in proband LCLs (from individual 1-1 and 1-2) than in control LCLs. Middle: levels of mTOR and non-phosphorylated S6 were unchanged, but levels of PS6 isoform Ser235/236 were lower in LCLs with mutant TBCK than in control cells. Bottom: GAPDH was used as a loading control. (B) Relative densitometry graph shows the levels of PS6 isoform Ser235/236 in fibroblasts with mutant TBCK (from individuals 1-1 and 1-2) and control fibroblasts across independent western blots in triplicate. Lines depict the SE. Cells were provided by Fowzan S. Alkuraya and colleagues of the King Faisal Specialist Hospital and Research Center.

Figure 3

Western Blots Demonstrate Decreased mTOR Signaling in Fibroblasts of Affected Individuals Levels of PS6 isoforms Ser235/236 (A) and Ser240/244 (B) were lower in fibroblasts harboring mutant TBCK (from individual 2-1) than in control cells. The blot demonstrates that levels of PS6 isoform Ser235/236 were, on average, 36% (range 31%–41%, SEM ± 5) lower in fibroblasts with mutant TBCK than in control fibroblasts (p < 0.05). There was no change in the level of non-phosphorylated S6 or mTOR in either LCLs or fibroblasts containing TBCK mutations. GAPDH was used as a loading control.

Figure 4

Western Blot Demonstrates that Leucine Normalizes mTOR Signaling in Fibroblasts of Affected Individuals Enhanced mTOR signaling in TBCK mutant fibroblasts (individual 2-1) with the addition of exogenous L-leucine (600 μg/ml). The western blot demonstrates increased levels of PS6 (24 ± 4%; p < 0.05). GAPDH was used as a loading control.