Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions

Abstract

RNA binding motif protein X-linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene's importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

Fig. 1. Genetic and molecular characterization of Gustavson syndrome.

A Extended family pedigree of the family affected with Gustavson syndrome (91 individuals). Sanger sequencing of the RBMX variant in 36 individuals (blue squares) revealed that all investigated affected individuals were hemizygous (dark gray) and had inherited the allele from their carrier mothers. Patients with trisomy 18 is highlighted in light gray. B X-chromosome inactivation (XCI) analysis and variant detection on DNA and RNA confirms that asymptomatic carrier females (n = 11) are protected from disease by silencing the pathogenic allele (black arrow). XCI result from all individuals are presented in Supplementary Fig. 1. C Schematic of hnRNP G with known domains. Top: RBMX variants listed in the gnomAD and ClinVar databases. ClinVar variants are underlined, green: homozygous/hemizygous variants (all reported stop gained, start lost, splice acceptor, in-frame insertions/deletions or frameshift insertions/deletions), blue: heterozygous variants (all reported stop gained, start lost, splice acceptor, in-frame deletions or frameshift deletions), black: homozygous/hemizygous missense variants. Red lines: variants associated with Gustavson syndrome (p.(Pro162del)) and Shashi syndrome (p.Glu346fs). GnomaD variants obtained 22-07-28, including variants in the canonical transcript and excluding variants flagged as of poor quality or in low complexity regions. Bottom: Domains of hnRNP G are RNA recognition motif, RNA binding Arg-Gly-Gly-box, Nascent transcripts targeting domain, RNA binding domain (52, 54).

Fig. 2. Clinical manifestations in patient V:7.

A Magnetic resonance imaging showing lissencephaly with very thick cortex and dorsal dominance. As well as, some hypotrophy of vermis, while the frontal part of corpus callosum is well developed and the back part is thinner. B Patient V:7 showing dysmorphic features include bitemporal narrowing, wide mouth, puffy eyelids, broad mouth, high palate, two hemangiomas, microcephaly, short stature and overweight. For more detailed clinical description and phenotype comparison to other patients with Gustavson syndrome, see Supplementary data 1 and Supplementary Table 4B.

Fig. 3. SH3-domain investigation in hnRNP G.

A hnRNP G amino acid sequence (P38159) with the p.(Pro162del) variant highlighted with a star, predicted polyproline II helices highlighted in red (strong prediction) and blue (weaker prediction) using PPIIPRED prediction. B Saturation binding experiments between two SH3 domains and the labeled hnRNP G peptide (amino acids 150–170) revealed binding with moderate affinities to SH3 domains. C Displacement binding experiments between BIN1-SH3 domain and two longer hnRNP G peptides (wildtype and △P162 variant) showed no clear difference in SH3 binding affinity between the wildtype and the mutant peptide. D Displacement experiments between BIN1-SH3 and the shorter hnRNP G peptides (wildtype and △P162 variant), showing a reduced binding affinity to SH3 domains when the p.(Pro162del) variant was introduced.

Fig. 4. Transcriptome analysis of RBMX in neuronal cells.

A Transcriptomics experimental setup with transient transfection of the wildtype vector (pink) or mutant vector (p.(Pro162del): turquoise) in SH-SY5Y cells. B The top 100 most differentially expressed genes between wildtype (pink) and mutant cells (turquoise). Each line represents the expression of one gene, where upregulated genes are red and downregulated genes are blue. The genes (rows) and samples (columns) of the top 100 most differentially expressed genes are hierarchical clustered according to expression patterns. Significantly upregulated genes (star) in the mutant cells (padj<0.05): ZNF805;ENSG00000204524.7, PCDHA10;ENSG00000250120.8, LYSMD3;ENSG00000176018.13. Significantly downregulated genes (star) in the mutant cells (padj<0.05): COL2A1;ENSG00000139219.19, EVPL;ENSG00000167880.8, TTN;ENSG00000120332.17, AC010207.1;ENSG00000260633.1.