Identification and functional characterization of de novo variant in the SYNGAP1 gene causing intellectual disability

Abstract

Background: Intellectual disability (ID) is defined by cognitive and social adaptation defects. Variants in the SYNGAP1 gene, which encodes the brain-specific cytoplasmic protein SYNGAP1, are commonly associated with ID. The aim of this study was to identify novel SYNGAP1 gene variants in Chinese individuals with ID and evaluate the pathogenicity of the detected variants. Methods: Whole exome sequencing (WES) was performed on 113 patients diagnosed with ID. In the study, two de novo variants in SYNGAP1 were identified. Sanger sequencing was used to confirm these variants. Minigene assays were used to verify whether the de novo intronic variant in SYNGAP1 influenced the normal splicing of mRNA. Results: Two de novo heterozygous pathogenic variants in SYNGAP1, c.333del and c.664-2A>G, were identified in two ID patients separately. The c.333del variant has been reported previously as a de novo finding in a child with ID, while the c.664-2A>G variant was novel de novo intronic variant, which has not been reported in the literature. Functional studies showed that c.664-2A>G could cause aberrant splicing, resulting in exon 7 skipping and a 16bp deletion within exon 7. Conclusion: We identified two de novo pathogenic heterozygous variants in SYNGAP1 in two patients with ID, among which the c.664-2A>G variant was a novel de novo pathogenic variant. Our findings further enrich the variant spectrum of the SYNGAP1 gene and provide a research basis for the genetic diagnosis of ID.

Keywords: SYNGAP1; intellectual disability; minigene; variant; whole exome sequencing (WES).

FIGURE 1

Two de novo variants of SYNGAP1 were identified in two patients with ID. (A) Families pedigree and genotype are shown. The probands with ID underwent WES. Filled symbols represent affected individuals. (B) Sanger sequencing chromatograms of the SYNGAP1 variants in these families. (C) Localization of the SYNGAP1: c. 333del and c.664-2A>G variant found in the study. The amino acid (aa) positions are referenced to RefSeq number NM_006772.3 (isoform-1: 1343 aa). 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).

FIGURE 2

The effect of the c.664-2A>G variant on splicing was assessed through a minigene assay. (A) Construction of the pcMINI-SYNGAP1-WT/MUT vector, which contain exon 7 and flanking intronic sequences of WT or mutant type (c.664-2A>G) of the SYNGAP1 gene. (B) Minigene assay performed in 293T and Hela cells transfected with the pcMINI-SYNGAP1-WT/MUT vector. The PCR products were isolated by gel electrophoresis. The SYNGAP1 splicing products of wild-type (band a) and variant type (band b and c) are shown. (C, D) Schematic diagram of minigene construction and sanger sequencing of PCR products.

FIGURE 3

A graphical summary of the mechanism of dendritic spine loss caused by the SYNGAP1 c.664-2A>G variant. The SYNGAP1 c.664-2A>G variant causing aberrant splicing and dendritic spine loss.