Missense Variants in RHOBTB2 Cause a Developmental and Epileptic Encephalopathy in Humans, and Altered Levels Cause Neurological Defects in Drosophila

Abstract

Although the role of typical Rho GTPases and other Rho-linked proteins in synaptic plasticity and cognitive function and dysfunction is widely acknowledged, the role of atypical Rho GTPases (such as RHOBTB2) in neurodevelopment has barely been characterized. We have now identified de novo missense variants clustering in the BTB-domain-encoding region of RHOBTB2 in ten individuals with a similar phenotype, including early-onset epilepsy, severe intellectual disability, postnatal microcephaly, and movement disorders. Three of the variants were recurrent. Upon transfection of HEK293 cells, we found that mutant RHOBTB2 was more abundant than the wild-type, most likely because of impaired degradation in the proteasome. Similarly, elevated amounts of the Drosophila ortholog RhoBTB in vivo were associated with seizure susceptibility and severe locomotor defects. Knockdown of RhoBTB in the Drosophila dendritic arborization neurons resulted in a decreased number of dendrites, thus suggesting a role of RhoBTB in dendritic development. We have established missense variants in the BTB-domain-encoding region of RHOBTB2 as causative for a developmental and epileptic encephalopathy and have elucidated the role of atypical Rho GTPase RhoBTB in Drosophila neurological function and possibly dendrite development.

Keywords: Drosophila melanogaster; RHOBTB2; epileptic Encephalopathy; intellectual disability; proteasom; ubiquitination.

Figure 1

Mutations in RHOBTB2 (A) Clinical pictures of individuals with pathogenic variants in RHOBTB2 show minor and unspecific dysmorphic aspects, and an MRI axial T2 image corresponding to individual 8 demonstrates right hemispheric edema in the setting of acute left hemiparesis. (B) Schematic drawing of RHOBTB2 (longest isoform: GenBank: NM_001160036.1) includes non-coding (gray) and coding (black) exons and encoded domains (colored) according to SMART. The variants identified here and one published variant cluster within the BTB-domain-encoding region. (C) Conservation of the affected amino acids according to the UCSC Browser is depicted with Clustal Omega. (D) Model of the first BTB domain (blue ribbon presentation). Residues Ala474 and Arg483 are highlighted in red and yellow, respectively. (E) Model of the second BTB domain in the homodimeric state. The subunits are shown as blue ribbon and cyan spaced-filled presentations. Residues Arg511 and Asn510 are highlighted in orange and magenta, respectively.

Figure 2

Co-immunoprecipitation and Analysis of Overexpressed RHOBTB2 (A) Co-immunoprecipitation of His-cMyc-tagged RHOBTB2 and HA-tagged CUL3. Precipitation was performed with an antibody against cMyc. HA-CUL3 equally co-precipitates with both mutant and wild-type RHOBTB2. Because RHOBTB2 and CUL3 are the same size, two gels for the same experiment were used and stained separately. (B) Representative western blot after transfection of wild-type and mutant RHOBTB2 with and without co-transfection of CUL3. In each condition, mutant RHOBTB2 was more abundant than the wild-type. (C) Quantification from western blot from four independent experiments, including mean values; error bars represent the standard error of the mean (SEM). (D) Adding proteasome inhibitor to cells transfected with wild-type or mutant RHOBTB2 and co-transfected with CUL3 resulted in equal amounts of wild-type and mutant RHOBTB2.

Figure 3

The Impact of Altered Amounts of RhoBTB on Fruit Fly Behavior (A) Flies overexpressing RhoBTB pan-neuronally (UAS_RhoBTB_2) showed bang-sensitivity phenotypes after vortexing. All ten flies, paralyzed and with spasms, were at the bottom of the vial, whereas flies from the corresponding genetic background line already started to walk up the sides (see also Movie S1). (B) Quantification of the number of flies (either overexpressing RhoBTB or upon knockdown) shaking on the bottom of the vial 5 s after vortexing. The diagram shows the mean value from a minimum number of 100 flies tested per genotype ± SEM. (C–E) Flies overexpressing RhoBTB in motoneurons (C), in all neurons (D), or in glia (E) (black bars) showed significant locomotor impairment in the negative geotaxis assay, as measured by the amount of time that 70% of flies in a vial needed to crawl up 8.8 cm after being tapped down. Only knockdown in all neurons (D), but not in motoneurons or glia (C and E; gray bars), resulted in significant locomotor impairment. Data represent the mean from a minimum of 300 flies tested per genotype ± SEM. Asterisks indicate statistical significance (^∗∗p < 0.01, ^∗∗∗p < 0.001).

Figure 4

The Impact of Altered Amounts of RhoBTB on Fly Synaptic and Dendritic Morphology (A) Representative pictures of a NMJ from a control larva. The white-framed box indicates the cutout on the right side. NMJ area and length and the number of synaptic boutons were determined from staining with anti-dlg (post-synaptic protein discs large), and the number of active zones was determined from staining with anti-nc82 (pre-synaptic protein bruchpilot). Scale bars represent 10 μm. (B) Quantification did not reveal significant alterations in NMJ morphology upon RhoBTB overexpression using the OK6-GAL4 motoneuron driver. (C) Representative class IV da neurons showing smaller size and abnormal dendritic morphology upon knockdown of RhoBTB in comparison with control larvae. In the lower panel, tracing of dendritic branches with NeuronJ is displayed. Scale bars represent 50 μm. (D and E) Quantification of ten da neurons from three to five different animals per genotype. They showed no alteration upon overexpression but did show a significantly reduced number (D) and/or mean length (E) of dendritic branches upon knockdown. Error bars represent the SEM. Asterisks indicate statistical significance (^∗∗p < 0.01, ^∗∗∗p < 0.001). AZ stands for active zone.