Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders

Abstract

Purpose: Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability.

Methods: By international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro.

Results: In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well.

Conclusion: By identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.

Keywords: Developmental and epileptic encephalopathy; Intellectual disability; Neurodevelopmental disorder; RHOBTB2; Seizures.

Figure 1. Overview of published and novel variants in RHOBTB2.

A. Schematic drawing of RHOBTB2 with domains and identified missense variants clustering in the GTPase or BTB domain region (GenBank: NM_001160036.2) above the scheme and biallelic splice-site and truncating variants below the scheme. Domains were identified and recolored based on SMART prediction.^, Variants in gray were described previously^,-,; variants in black have been identified in this cohort. Recurrent variants are underlined. p.(Arg154*) has been published before, and compound heterozygous variants are marked by a +. Possible genotype-phenotype correlations based on phenotype severity and variant location are indicated by blue, gray, and red boxes. # indicates variants included in experiments. The cancer variant p.(Tyr306Asn), marked with a C, results in impaired binding to Cullin3. B. Conserved positions of the affected amino acids in the GTPase domain according to the UCSC Genome Browser.^, C. Structural model of the GTPase domain. The domain is shown in ribbon presentations with α-helices in red and β-sheets in green. The variant sites (Asp114, Arg116, Arg154, and Arg183) and 1 interacting glutamate are shown in space-filled presentation (atom-type coloring) and are labeled. The potential GEF binding site is exemplarily illustrated for PDZ-RhoGEF (white-space-filled presentation). NDD, neurodevelopment disorder.

Figure 2. Consequences of missense variants regarding binding to Cullin3 and proteasomal degradation of RHOBTB2.

A. Co-immunoprecipitation of His-cMyc-tagged RHOBTB2 and HA-tagged Cullin3 shows equal coprecipitation of Cullin3 for both wild type and RHOBTB2 carrying the missense variants, except for the cancer variant p.(Tyr306Asn). Cells were treated with the proteasomal inhibitor MG132, and co-immunoprecipitation wa8s performed with an antibody against Myc. A representative image from 5 independent experiments is shown. For quantification, Cullin3 bands after co-immunoprecipitation were normalized to the corresponding RHOBTB2 bands and compared with RHOBTB2 wild type. Error bars represent the standard error. Statistical analysis was performed using the one-sample t test with the hypothetical mean set to 1 followed by Bonferroni correction (*P < .05, **P < .01, and ***P < .001). Uncropped blots are provided in Supplemental Figure 4. B. Representative western blot from 3 independent experiments after transfection of wild type and His-cMyc-tagged RHOBTB2 carrying a variant shows impaired proteasomal degradation for both cancer and BTB domain region variants but not for the others. Experiments were performed with (top) and without (bottom) proteasomal inhibitor MG132. For quantification, RHOBTB2 bands were normalized to the loading control GAPDH and compared with RHOBTB2 wild type. Error bars represent the standard error. For statistical analysis, all values >5 were set to 5. Statistical analysis was performed using the one-sample t test with the hypothetical mean set to 1 followed by Bonferroni correction (*P < .05, **P < .01, and ***P < .001). Uncropped blots are provided in Supplemental Figure 5. GAPDH, glyceraldehyde 3-phosphate dehydrogenase; WT, wild-type.