Programmpunkt

10:15–10:30

Titel: FBXO11 haploinsufficiency also stems from de novo missense variants and impairs neuronal differentiation and migration in an iPSC-based neuronal model
ID: W2-002
Art: Invited talk
Redezeit: 15 min
Session: Workshop 2
Neurodevelopmental Disorders

Referent: Anne Gregor (Bern/CH)


Abstract - Text

Abstract-Text

Recently, we and others identified de novo FBXO11 variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpressing mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. Together with the mutational data our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs.


To better understand the molecular mechanisms resulting from FBXO11 haploinsufficiency, we created a neuronal disease model. We generated FBXO11 knockout induced pluripotent stem cells using CRISPR/CAS9 technology and differentiated those cells into neuronal precursor cells and neurons using a dual SMAD inhibition protocol. As FBXO11 functions as a nuclear E3-ubiquitin ligase subunit, we hypothesized that target proteins may be involved in transcriptional regulation and performed whole transcriptome analysis on FBXO11 deficient neurons. Our data of decreased expression of differentiation genes and increased expression of stemness genes suggest that neuronal differentiation might be impaired in these neurons. We confirmed the known stemness factor NANOG to interact with FBXO11 by mass-spectrometry and subsequent co-immunoprecipitation. In line with our results from transcriptomic analysis, we found that cell proliferation rates during neuronal differentiation are increased in FBXO11 knockout cells. Additionally, neuronal migration is impaired in the neurosphere assay. Our data therefore suggest that impaired neural differentiation and migration may be key factors in the pathogenesis of FBXO11-associated NDDs.