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TBC1 domain-containing kinase (TBCK) is a pivotal protein essential to brain development, with a complex structure comprising a Protein Kinase domain, a Tre-2 Bub-2 Cdc16 (TBC) domain, and a Rhodanese domain. Mutations in this protein are known to cause TBCK Syndrome, a rare genetic disorder characterized by a spectrum of neurological and developmental abnormalities. In patients, these mutations disrupt cellular pathways, leading to impaired brain development and functioning, which manifest in the diverse and often severe clinical symptoms observed in affected individuals. To further understand how TBCK mutations could affect brain development and behavior, we developed a knockout (KO) mouse model for TBCK. However, our team faced several challenges related to the viability of the mice. Consequently, because there was also a visible phenotype in heterozygous animals, we decided to perform a series of assays to determine if mutations in one allele of TBCK would be sufficient to affect the development and behavior of the animals. Our findings suggest that TBCK haploinsufficiency leads to a subtle but significant neurological phenotype, with observable differences in behavior and mTORC1 signaling pathway in Tbck+/– mice. We extended our study to a human Phenome-Wide Association Study (PheWAS), identifying associations between TBCK heterozygote status and specific neurological manifestations. Notably foot and toe deformities were associated with TBCK mutations. To better understand the impact of these mutations in an unbiased way, we employed the power of multi-omics to dissect different levels of regulation affected in patients. Our comprehensive analysis using RNA sequencing, proteomics, and miRNA sequencing in patient-derived fibroblasts revealed significant alterations in ribosomal function, RNA splicing, and miRNA expression. This holistic view of the cellular landscape in TBCK syndrome uncovers potential therapeutic targets and provides a richer understanding of the disease's complexity. This thesis work offers novel insights into TBCK syndrome, highlighting the nuanced effects of the mutations and laying a foundation for future research into targeted therapies.

TBC1 domain-containing kinase (TBCK) is an important protein with implications in brain development. Biallelic variants in the gene are known to cause TBCK-related neurodevelopmental disorder (OMIM #616900) [1], a rare genetic multisystemic disease characterized by developmental delay, variable developmental regression, seizures, and premature death in late childhood for which no cure is currently available. Though previous work has provided a better understanding of the protein's role, the mechanism for how variants affect gene expression and protein regulation has remained understudied. To better understand the impact of these alterations, and using an unbiased approach, we employed the power of multi-omics to define the cellular consequences at the transcript and protein level. Our comprehensive analysis uncovered significant disruptions in ribosomal and translation-related pathways with widespread alternative splicing defects, and key miRNA changes that validate previously reported molecular findings. This work provides a clearer molecular framework for TBCK dysfunction in cells and offers a valuable foundation to identify potential therapeutic targets.

TBCK Syndrome is a rare Mendelian disorder caused by variants in the gene. Although symptoms affect multiple organ systems, hallmark features include intellectual and developmental disability, craniofacial differences, hypotonia, and premature death. At the cellular level, TBCK has been implicated in mTOR signaling, autophagy, mitophagy, and mRNA trafficking; however, the mechanisms underlying disease onset and progression remain unclear. To address this gap, we characterized a mouse model of TBCK Syndrome. These mice lack exon 5 of the gene, resulting in a whole-body knockout of , modeling the most severe known variant. We performed a comprehensive battery of developmental assays, along with microcomputed tomography and histological analyses, which revealed systemic alterations consistent with those observed in affected individuals. Notably, phenotypic changes arising from Tbck loss emerge early and are detectable in the brain, indicating a primary neurodevelopmental origin of disease pathology. Rigorous characterization of this Tbck-deficient mouse establishes the first platform to investigate disease mechanisms and provides a foundation for preclinical evaluation of gene and targeted pharmacological therapy strategies.