Novel De Novo Intronic Variant of SYNGAP1 Associated With the Neurodevelopmental Disorders

Abstract

Background: SYNGAP1 encodes a Ras/Rap GTPase-activating protein that is predominantly expressed in the brain with the functional roles in regulating synaptic plasticity, spine morphogenesis, and cognition function. Pathogenic variants in SYNGAP1 have been associated with a spectrum of neurodevelopmental disorders characterized by developmental delays, intellectual disabilities, epilepsy, hypotonia, and the features of autism spectrum disorder. The aim of this study was to identify a novel SYNGAP1 gene variant linked to neurodevelopmental disorders and to evaluate the pathogenicity of the detected variant.

Methods: A novel de novo intronic variant in SYNGAP1 was identified by Whole exome sequencing (WES) and confirmed by Sanger sequencing. Minigene assays were conducted to assess whether the intronic variant in SYNGAP1 influenced the normal splicing of mRNA.

Results: A novel de novo intronic variant in SYNGAP1 (c.3582+2T>G) was indentified with clinical features suggestive of neurodevelopmental related disorders. Minigene splicing analysis demonstrated that this noncanonical splice site variant led to the activation of a cryptic acceptor splice site. Consequently, 101 base pairs of intron 16 were aberrantly retained in the mRNA, leading to a frameshift. This frameshift resulted in the introduction of a premature stop codon (TGA) in the coding sequence and the production of a truncated SYNGAP1 protein, potentially leding to loss of function and subsequent disruption of its biological roles.

Conclusion: Our findings highlight the significance of de novo pathogenic SYNGAP1 variants at the intron 16/exon 17 junction in the SYNGAP1-related neurodevelopmental disorders, providing novel insights into the genetic basis and diagnosis of these disabilities.

Keywords: SYNGAP1; intronic variation; minigene; variant interpretation; whole exome sequencing.

FIGURE 1

A de novo variants of SYNGAP1 were identified in the patients. (A) The pedigrees and genotypes of the families are presented, with probands having undergone whole‐exome sequencing (WES). Filled symbols represent affected individuals. (B) Sanger sequencing chromatograms display the SYNGAP1 variants identified in the families. (C) Localization of the SYNGAP1: C.3582+2T>G variant found in the study. All variant annotations are based on the GenBank reference sequence NM_006772.4.

FIGURE 2

Electroencephalographic features of patients with c.3582+2T>G mutation in SYNGAP1 at rest. Generalized spike‐and‐wave discharges with anterior maximum (blue rectangle) and bilateral spike‐and‐wave discharges with posterior maximum, indicated by red arrows in the corresponding electrode pairs.

FIGURE 3

Characterization of the effect of c.3582+2T>G mutation on SYNGAP1 splicing. (A) Construction of the pCDNA3.4‐SYNGAP1‐WT/MUT vector, which contain exon 15–17 and flanking intronic sequences of (a) WT or mutant type (b) (c.3582+2T>G) of the SYNGAP1 gene. (B) Minigene assay performed in HEK 293T and Hela cells transfected with pCDNA3.4‐SYNGAP1‐WT/MUT vector. Agarose gel electrophoresis of the reverse transcription (RT)‐PCR products are obtained from the transfected HEK293T and Hela cells. (C) Schematic representation of the SYNGAP1 genomic region between exon‐15 and exon‐17 on the basis of RefSeq number NM_006772 is shown at the top. The primers (Ex15F and Ex17R) used for polymerase chain reaction (PCR) are represented by arrows. (a) schematic diagram of wide type exon 15–17. (b) The c.3582+2T>G mutation at the exon‐16 consensus donor splice site is indicated. (D) Schematic diagram of sanger sequencing of PCR products from WT and MUT.

FIGURE 4

Potential impact of the c.3582+2T>G mutations on SYNGAP1 structure. (A) The wide‐type structure of the SYNGAP1 protein was downloaded from the PDB database. The mutant protein structure shown in (B) contained a truncated SYNGAP1 protein that consist 1228 of the 1343 amino acids of the mature protein. (C) Diagram of the wild type and mutant SYNGAP1 proteins, as well as the location of mutation. Various predicted SYNGAP1 domains are showed: PH, pleckstrin homology domain (amino acid positions 150–251), C2 domain (amino acid positions 263–362), Ras‐GAP (amino acid positions 392–729), SH3 (amino acid positions 785–815), coiled coil (CC; amino acid positions 1189–1262). SYNGAP1 protein was aligned among different species. (D) The protein–protein interaction (PPI) network associated with SYNGAP1 protein.