Cellular characterization of fibroblasts harboring variants in TRAPPC2L linked to neurodevelopmental disorders.

In this thesis, I focus on TRAPPC2L, a recently discovered core subunit of the TRAnsport Particle Protein (TRAPP) complexes, which is not well studied. I performed biochemical and cell biological functional studies to characterize the cellular phenotype of the first identified homozygous missense variants (p.Asp37Tyr and p.Ala2Gly) in TRAPPC2L linked to neurodevelopmental delays and intellectual disabilities. In this study, I show that only the p.(Ala2Gly) variant, but not the p.(Asp37Tyr) variant, disrupted the interaction betweenTRAPPC2L and TRAPPC6a, another core TRAPP protein. I also show by using size exclusion chromatography that both TRAPPC2L variants disrupted the assembly and stability of the TRAPP complexes in lysates from fibroblasts harbouring the two variants. In addition, we used two different membrane trafficking assays on fibroblasts from individuals harboring the variants in TRAPPC2L and we found delays in endoplasmic reticulum-to-Golgi and post-Golgi trafficking. In this study, I better characterized the role of TRAPPC2L in the function and assembly of TRAPP and supported the pathogenicity of the two TRAPPC2L variants, p.(Asp37Tyr) and p.(Ala2Gly)

Bi-allelic mutations in TRAPPC2L result in a neurodevelopmental disorder and have an impact on RAB11 in fibroblasts

Background: The combination of febrile illness-induced encephalopathy and rhabdomyolysis has thus far only been described in disorders that affect cellular energy status. In the absence of specific metabolic abnormalities, diagnosis can be challenging. Objective: The objective of this study was to identify and characterise pathogenic variants in two individuals from unrelated families, both of whom presented clinically with a similar phenotype that included neurodevelopmental delay, febrile illness-induced encephalopathy and episodes of rhabdomyolysis, followed by developmental arrest, epilepsy and tetraplegia. Methods: Whole exome sequencing was used to identify pathogenic variants in the two individuals. Biochemical and cell biological analyses were performed on fibroblasts from these individuals and a yeast two-hybrid analysis was used to assess protein-protein interactions. Results: Probands shared a homozygous TRAPPC2L variant (c.109G>T) resulting in a p.Asp37Tyr missense variant. TRAPPC2L is a component of transport protein particle (TRAPP), a group of multisubunit complexes that function in membrane traffic and autophagy. Studies in patient fibroblasts as well as in a yeast system showed that the p.Asp37Tyr protein was present but not functional and resulted in specific membrane trafficking delays. The human missense mutation and the analogous mutation in the yeast homologue Tca17 ablated the interaction between TRAPPC2L and TRAPPC10/Trs130, a component of the TRAPP II complex. Since TRAPP II activates the GTPase RAB11, we examined the activation state of this protein and found increased levels of the active RAB, correlating with changes in its cellular morphology. Conclusions: Our study implicates a RAB11 pathway in the aetiology of the TRAPPC2L disorder and has implications for other TRAPP-related disorders with similar phenotypes