Mutated RAP1GDS1 causes a new syndrome of dysmorphic feature, intellectual disability & speech delay

Abstract

Background: RAP1GDS1 (RAP1, GTP-GDP dissociation stimulator 1), also known as SmgGDS, is a guanine nucleotide exchange factor (GEF) that regulates small GTPases, including, RHOA, RAC1, and KRAS. RAP1GDS1 was shown to be highly expressed in different tissue types including the brain. However, mutations in the RAP1GDS1 gene associated with human diseases have not previously been reported.

Methods: We report on four affected individuals, presenting intellectual disability, global developmental delay (GDD), and hypotonia. The probands' DNA was subjected to whole-genome sequencing, revealing a homozygous splice acceptor site mutation in the RAP1GDS1 gene (1444-1G > A). Sanger sequencing was performed to confirm the segregation of the variant in two Saudi families. The possible aberrant splicing in the patients' RNA was investigated using RT-PCR and changes in mRNA expression of the patients were confirmed using qRT-PCR.

Results: The identified splice variant was found to segregate within the two families. RT-PCR showed that the mutation affected RAP1GDS1 gene splicing, resulting in the production of aberrant transcripts in the affected individuals. Quantitative gene expression analysis demonstrated that the RAP1GDS1 mRNA expression in all the probands was significantly decreased compared to that of the control, and Sanger sequencing of the probands' cDNA revealed skipping of exon 13, further strengthening the pathogenicity of this variant.

Conclusion: We are the first to report the mutation of the RAP1GDS1 gene as a potential cause of GDD and hypotonia. However, further investigations into the molecular mechanisms involved are required to confirm the role of RAP1GDS1 gene in causing GDD and hypotonia.

Figure 1

Diagrammatic representation of Clinical features, pedigree, and sequence chromatograms of the identified pathogenic variant in the RAP1GDS1 gene. (A) Pedigree of the analyzed family members. The allelic status is given below each tested individual. Symbols are as follows: filled, affected; empty, unaffected; dotted, heterozygous carrier; black arrow, individuals subjected to whole genome sequencing (WGS). (B) Photographs showing the phenotypes of the patient. The pictures show the facial dysmorphic, global developmental delay, and hypotonia features. Written informed consent was obtained from the patient’s parents for the publication of images. (C) Segregation of the identified splice site variant (c.1444‐1G > A) in both families.

Figure 2

Molecular characterization of c.1444‐1G > A splicing variant in RAP1GDS1 gene. (A) Schematic representation of the RAP1GDS1 genomic region: exons (Yellow), intron (Black), UTRs (Blue). Red arrow represents the identified splice acceptor site variant. (B) Agarose gel (2%) demonstrating the results of the RT‐PCR performed on the RNA extracted from the peripheral blood mononuclear cells of the unrelated normal control and the family members. PCR amplification of the region around the splice site variant showed a decrease in the size of the RAP1GDS1 gene product in all probands from families 1 and 2 (Red arrow, 115 bp) following the skipping of exon 13 compared the control sample (Green arrow, 242 bp). The carrier individuals of the same mutation (heterozygous) from both families showed two bands. No template control (NTC). (C) Representing Sanger sequencing of the wild‐type control and affected individual’s cDNA. Chromatograms sequence analysis showing a complete skipping of exon 13 (blue arrow) in the affected individuals compared to the controls. (D) Quantitative real time‐ PCR showed that the RAP1GDS1 mRNA expression level was significantly inhibited in the proband IV‐9 and IV‐10 from family 1 and proband IV‐4 and IV‐2 from family 2 as compared to the normal control. There was also a significant decrease in the mRNA level of RAP1GDS1 expression in all carrier members from both families compared to the wild type control, as they are heterozygous for the mutation. Results are represented as the mean ± SD of three independent experiments (one‐way ANOVA). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Crystal shape protein model of the RAP1GDS1. (A) Model of the wild type RAP1GDS1 structure consisting monomer A and B. (B) Model of RAP1GDS1 protein with the location of the effected armadillo motif highlighted in green (from Asp 361 to Leu 402). (C) Expected protein model for the mutated RAP1GDS1 (c.1444‐1G > A) showed complete skipping for the armadillo motif (circled in red) with significant change in the protein structure. The images were created by SWISS‐MODEL.