Novel de novo intronic variant of SYNGAP1 associated with the neurodevelopmental disorders

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Pathogenic variants in SYNGAP1 have been associated with a spectrum of neurodevelopmental disorders characterized by developmental delays, intellectual disabilities, epilepsy, hypotonia, and 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 (WGS) 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 SYNGAP1-related neurodevelopmental disorders, providing novel insights into the genetic basis and diagnosis of these disabilities. Medical Genetics Medical Genetics Whole exome sequencing minigene SYNGAP1 intronic variation variant interpretation Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Disruptions in the molecular mechanisms that govern the structure and function of glutamatergic synapses are thought to be a key factor in the development of certain neurodevelopmental disorders (Bourgeron, 2015 ; Grant, 2012 ; Zoghbi and Bear, 2012 ). Deleterious mutations in synaptic proteins have been associated with the related neurodevelopmental disorders, and numerous animal models exhibit deficits in synapse structure and/or function (Guilmatre et al., 2009 ; Hamdan et al., 2011b ; Sudhof, 2008 ). Mutations in SYNGAP1 resulted in a neurodevelopmental disorder termed Mental retardation-type 5 (MRD5, OMIM #612621) consisting of intellectual disabilities (ID), developmental delay, autism spectrum disorders (ASD), motor impairments, hypotonia, epilepsy and maladaptive behaviors (Berryer et al., 2013 ; Clement et al., 2012 ; Mignot et al., 2016 ). Neuroimaging in these affected individuals is typically normal, though rare cases have reported brain atrophy and delayed myelination (Hamdan et al., 2011a ; Vissers et al., 2010 ). However, a significant number of individuals display ataxia, presenting with a broad-based and unsteady gait. Seizure management in epilepsy varies significantly between individuals. Some patients achieve control with one or more antiepileptic drugs, while others have epilepsy that remains resistant to medication (Berryer et al., 2013 ). With the growing identification of the affected individuals, a consistent clinical syndrome associated with SYNGAP1 variants has been delineated. De novo mutations in SYNGAP1, particularly loss-of-function varients have been identified as one of the more prevalent genetic causes of MRD5, with an estimated prevalence of 0.7-1% in specific populations (Deciphering Developmental Disorders, 2015). The genetic architecture of SYNGAP1-related neurodevelopmental disorder is complex, as evidenced by various studies employing cytogenetics, linkage analysis, association studies, and whole-genome or exome sequencing. The human SYNGAP1 gene on chromosome 6p21.3 consists of 19 exons that encodes the synaptic RAS-GTPase-activating protein (SYNGAP)(Kim et al., 1998a ). This gene is primarily found localized to dendritic spines in the postsynaptic density (PSD) of excitatory glutamatergic neurons, where it suppresses signaling pathways linked to NMDA receptor (NMDAR) mediated synaptic plasticity and AMPA receptors (AMPAR) membrane insertion (Kim et al., 2005 ; Krapivinsky et al., 2004 ). The encoded protein regulates the trafficking of the different receptors, as well as Ras and Rap to the postsynaptic, thereby influencing synaptic plasticity and neuronal homeostasis (Vazquez et al., 2004 ). In the SYNGAP1 sequence, the N-terminal pleckstrin homology (PH) domains act as membrane recruitment modules by binding to phospholipids (Lemmon, 2004 ). C2 domains are known to function as Ca 2+ -dependent phospholipidbinding modules (Nalefski and Falke, 1996 ). Various C-terminal exons of the SYNGAP1 undergo alternative splicing, leading to the generation of multiple protein isoforms for postsynaptic scaffold protein interaction (Chen et al., 1998 ). Depending on the location of different expressed variants, SYNGAP1 can either or suppress the dendritic spine synapse function (Clement et al., 2012 ). Many of these variants are nonsense or frameshift mutations that cause nonsense-mediated decay of SYNGAP1 mRNA or dysfunctional truncated protein products, likely resulting in haploinsufficiency. Intellectual disabilities-causing SYNGAP1 mutations have been identified along the entire length of the SYNGAP1 gene, with most of the mutations occurring from exons 3–17 (Gamache et al., 2020 ). Mutations can also occur in the intronic regions (non-coding regions between exons) of SYNGAP1 with MRD5 as detailed in table1. The intronic mutations, especially those near the exon-intron boundaries (splice sites), can affect splicing and lead to aberrant transcripts that are either degraded or translated into non-functional proteins (Anna and Monika, 2018 ; Borras et al., 2019 ). In this study, a young boy from the Chinese family was diagnosed with the SYNGAP1-related neurodevelopmental disorders, identifying a likely pathogenic intronic variant (c.3582 + 2T > G) through Whole-Exome and Sanger sequencing. Minigene analysis confirmed the pathogenicity of the SYNGAP1 intronic variant, indicating its importance in clinical genomics. Our findings highlighted other reports of pathogenic intronic variants in the junctional regions of SYNGAP1 exons 16 and 17, which are a relatively common cause of this neurodevelopmental disorder, with significant implications for the clinical diagnosis, prognosis, and management of patients with SYNGAP1 pathogenic intronic variants. Materials and methods Subjects An 9-year-old Chinese boy, diagnosed with autism spectrum disorder, moderate intellectual disability, mild developmental delay and speech impairment, was thoroughly evaluated at the Department of Pediatrics, Ganzhou Maternal and Child Health Hospital. Comprehensive assessments were conducted including a detailed medical history, physical examinations, skull magnetic resonance imaging (MRI), electroencephalogram (EEG), and Wei’s intelligence assessment. The boy was an only child of the healthy, non-consanguineous parents. The boy's medical history was unremarkable during the prenatal, delivery, and postpartum periods. Sample Collection This research was approved by the Ethics Committee of the Ganzhou Maternal and Child Health Hospital. Peripheral venous blood samples were taken from the boy and his parents following the acquisition of informed consent from all participants. RNA was isolated using the PAXgene Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland). DNA was extracted from the peripheral blood using the QIAamp DNA Mini Kit for blood (Qiagen, Hilden, Germany). Trio-based WES was performed by Kangxu Diagnostics (Beijing, China), including exome library preparation, sequencing and data analysis. Variant screening was based on clinical phenotypes of the affected subjects, population database (dbSNP, 1,000 Genome, ExAC), disease database (OMIM, HGMD, Clinvar) and biological information prediction tools (SIFT, Polyphen2, Mutation Taster and Splice AI). A novel variant of SYNGAP1 were indentified in the boy (c.3582 + 2T > G) while his parents displayed normal. The SYNGAP1 variant were confirmed by Sanger sequencing. To assess the potential impact of the c.3582 + 2T > G variant in SYNGAP1, the RDDCSC ( https://rddc.tsinghua-gd.org/search-middle?to=SplitToolModel , accessed on 23 April 2024) online in silico splice site prediction software was used. SYNGAP1 Expression Constructs Following prior confirmation of SYNGAP1 expression in blood, genomic DNA was extracted from the blood sample of patients and controls by using the TIANamp Genomic DNA Mini Kit for blood (TIANGEN). The extracted DNA were used as templates to amplify exons 15, 16, 17 and introns therein via reverse transcription with primers Forward (F): 5’-CCGGAATTCCTCTATGGACATGGCTCGC-3’ and Reverse (R):5’- GGGTTCGAAGATCAAGAGCATCATTGGCAG-3’. The PCR products were purified by the HiPure Gel Pure Micro Kit (Magen). The wt and mutant cDNAs were then cloned into the mammalian expression vector pCDNA3.4 (Addgene) using nucleic acid endonuclease EcoRI-HF, HindIII-HF and T4 DNA ligase (New England Biolabs). Plasmids were isolated from individual clones and verified by restriction digestion to contain an insert with the expected size. Both WT and mutant plasmids contained the whole sequence of exon 15–17 and a portion of the upstream and downstream intron sequence futher confirmed by the sequencing. Cell Culture and Transfection B16-F1 and HEK293 cell lines (American Type Culture Collection, ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) sourced from GIBCO Life Technologies. The medium was supplemented with 10% fetal bovine serum (FBS) from Invitrogen, 100 U/ml penicillin-streptomycin (GIBCO Life Technologies), and 2.5 µg/ml Plasmocin (InvivoGen). Cells were incubated at 37°C in a humidified environment with 5% CO2. Transfections were performed using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada), followed by a 48-hour incubation prior to RNA extraction. RNA analysis Total RNA was extracted with Trizol (TaKaRa) from the transfected cells, and cDNA was acquired with PrimeScriptTMRT reagent Kit (TaKaRa). The RT-PCR products was analyzed by electrophoresis on 2% agarose gels containing ethidium bromide and visualized by exposure to ultraviolet light (Bio-Red). Each DNA band was purified by HiPure Gel Pure DNA Micro Kit (Megen). Direct sequencing of purified RT-PCR products was performed on the Sangon Biotech, Shanghai. Primers used for minigene assay of SYNGAP1 were as follows: SYNGAP1-F1:5’CTCTATGGACATGGCTCGCCTCCCCTCCCC-3’; SYNGAP1-R1: 5’-CTGCCAATGATGCTCTTGATCTGGGAATCC-3’. Protein structure prediction The protein sequence with 1343 amino acid residues of SYNGAP1 was downloaded from uniprot web ( https://www.uniprot.org/ ). The wild-type and mutant-type(SYNGAP1: c.3582 + 2T > G)3D structure of the SYNGAP1 protein were predicted using AlphaFold web server ( https://golgi.sandbox.google.com/about ) (Abramson et al., 2024 ). The best model was selected based on pLDDTs prediction scores. The higher the score, the more confident is the structure. Prediction models editing was performed was visualized using PyMOL program ( https://pymol.org/ ). Bioinformatics analysis The protein-protein interaction (PPI) network associated with SYNGAP1 was constructed using STRING ( https://string-db.org/ ). the amino acid sequence of the SYNGAP1 protein, comprising 1,343 residues, was retrieved from the UniProt database ( https://www.uniprot.org/ ). This sequence was used as the input query for STRING, where the parameters were set to ensure high-confidence interactions were captured. The confidence score threshold was adjusted to include only interactions supported by strong evidence such as experimental data, co-expression, and co-occurrence analyses. Results De novo heterozygous variant were identified A de novo heterozygous pathogenic variants in SYNGAP1, NM_006772.3: c.3582 + 2T > G, was identified in the young male patient. Genetic analysis of both parents revealed that neither carried the variant in SYNGAP1 (Fig. 1 A-B), and both exhibited the normal intellectual abilities and motor development, ruling out inherited transmission. Notably, the c.3582 + 2T > G variant represents a unique intronic mutation in SYNGAP1, identified for the first time in this proband. This variant has not been previously documented in the existing scientific literature or major genetic databases, including ClinVar, Deciphering Developmental Disorders (DDD), PubMed, and the Human Gene Mutation Database (HGMD), making it a significant new addition to the spectrum of SYNGAP1-related pathogenic variants. This discovery adds to the growing understanding of intronic mutations and their possible potential impact on neurodevelopmental disorders. Clinical characteristics of the individual affected by the SYNGAP1 c.3582 + 2T > G variant The proband exhibited no abnormalities at birth. At 4 months of age, he was unable to grasp objects, indicating motor developmental delay. He was noted to be late in achieving independent walking, which occurred at 1 year and 9 months. His language development was significantly delayed. At the age of 9, his verbal communication was limited to simple expressions such as "yes" and "no," and remained unable to say "daddy" or "mommy as well as full phrases. In 2018, a comprehensive assessment for autism spectrum disorder (ASD) were carried on the proband including the psychological tests, clinical examinations, and consideration of the family medical history. We utilized standardized assessment tools such as the Autism Diagnostic Observation Schedule (ADOS) for ASD evaluation. The patient was diagnosed with ASD at Guangdong Provincial Children's Hospital, China. At present, electroencephalogram (EEG) examination during wakefulness revealed abnormal brain activity characterized by a generalized pattern of moderately frequent high amplitude spikes and slow waves, diffuse waves, or bursts (Fig. 2 ). Of note, a brain MRI was read as normal. There is no family history of similar neurodevelopmental disorders or global developmental delay. In vitro minigene splicing assay for the SYNGAP1 c.3582 + 2T > G variant To investigate the impact of the c.3582 + 2T > G variant, a minigene splicing assay was carried on both the control and patient samples. Following confirmation of the sequencing results, the pCDNA3.4-SYNGAP1-WT and pCDNA3.4-SYNGAP1-MUT (c.3582 + 2T > G) plasmids were separately transfected into HEK293T and HeLa cells (Fig. 3 A). After 48 hours of transfection, total RNA was extracted and subsequently reverse-transcribed into cDNA. The complementary DNA fragment covering exons 15–17 was amplified by reverse transcription (RT)-PCR and next analyzed using agarose gel electrophoresis. The agarose gel electrophoresis results demonstrated that a 1458-base pair (bp) fragment was amplified from the cDNAs of both the control subject and patient, whereas a 1559-bp fragment was also detected in the cDNA of patient (Fig. 3 B-C). Cloning and sequencing of the two PCR products generated from patient showed that the 1458-bp fragment corresponded to the wild-type transcript comprising exons 15-16-17, whereas the 1559-bp fragment corresponded to a transcript in which abnormal splicing led to the retention of a 101 bp fragment of intron 16 (Fig. 3 D). This retention causes a frameshift form at amino acid position 1195, resulting in extending for 34 amino acids followed by a premature stop codon (c.3582 + 101_3582 + 201ins101, p.Val1195Glyfs*34) (Fig. 3 C). Effect of mutations on protein structure In order to analyze the effect of the novel variants NP_006763.2:c.3582 + 2T > G, (p.Val1195Glyfs*34) on the structural integrity of the SYNGAP1 protein, AlphaFold web server ( https://golgi.sandbox.google.com/about ) was used to predict changes in SYNGAP1 protein structures. The NP_006763.2:c.3582 + 2T > G variant of SYNGAP1 could induce a frameshift mutation, resulting in a truncated SYNGAP1 protein comprising 1228 of the 3354 amino acids presented in the full-length mature protein (Fig. 4 A-B). For the mutated SYNGAP1 protein, a pTM score of 0.51 reflects moderate confidence in the predicted structure of the truncated protein. The frameshift mutation likely leads to a significant alteration of the protein’s three-dimensional conformation, particularly in the C-terminal region. The frameshift notably disrupts the C-terminal region of SYNGAP1, a domain critical for interacting with key postsynaptic density proteins, such as scaffolding protein PSD-95 (postsynaptic density protein 95). PSD-95 helps anchor NMDA receptors at the synapse organizes synaptic signaling complexes, facilitating the downstream signaling pathways activated by NMDA receptor activation, such as calcium influx that leads to synaptic plasticity (Kennedy, 1998 ). The interaction between PSD-95 and NMDA receptors is crucial for processes like long-term potentiation (LTP), which underlies learning and memory (Ehrlich and Malinow, 2004 ). Disruption of SYNGAP1's interaction with PSD-95 may also in turn, compromise its ability to regulate downstream signaling pathways involving Ras, potentially leading to synaptic dysfunction (Kim et al., 1998b ). We used STING software to predict the interacting proteins with SYNGAP1 and indentified several functional proteins, including DLG2, DLG3, DLG4, SHANK2, SHANK3, ULK1, ULK2, GRIN2B, NRAS and DLGAP2 (Fig. 4 D, Table 2). DLG2 DLG3 and DLG4 are all critical for regulating NMDA receptor signaling, facilitating the synaptic plasticity that is critical for learning, memory formation and cognitive function. SHANK2 and SHANK3 act as a major scaffold protein, interacts with various proteins like NMDA-type receptor to orchestrate dendritic spine and synapse formation. Notably, GRIN2B, a component of NMDA receptor complexes, is distributed throughout the dendrites at extrasynaptic sites. Variants of GRIN2B are commonly found in individuals with neurodevelopmental disorders characterized by mild to profound developmental delays and intellectual disabilities (Myers et al., 2019 ; Sabo et al., 2022 ). Given the importance of the C-terminal domain in synaptic function, this mutation highlights the possible mechanism for the cognitive and developmental deficits observed in patients with SYNGAP1 mutations. Clinical phenotypes of patients with De Novo SYNGAP1 mutation at intron 16/exon 17 We reviewed the clinical profiles of all known patients with de novo intronic mutations in SYNGAP1 at intron 16/exon 17, including the four mutations c.3583-9G > A, c.3583-6G > A, c.3583-1G > T and c.3583 + 3A > T discussed in this study (Table 3)(Bayat et al., 2022 ; Brimble et al., 2019 ; Redin et al., 2014 ; Strauss et al., 2018 ). All patients exhibited mild global developmental delay with varying degrees of language development disorders. Three patients lacked speech ability, while one could produce short phrases accompanied by gestures. Additionally, two patients were diagnosed with autism, including patient 1 (c.3583-9G > A). Data for one patient are unavailable. Autism was confirmed in three patients using the Autism Diagnostic Observation Schedule (ADOS). Notably, patient 2 (c.3583-6G > A) did not meet the criteria for autism spectrum disorder according to the ADOS-2, but delays in social communication were documented. our patients with intronic mutations in SYNGAP1 experienced epilepsy, with variable seizure types including absence seizures, tonic-clonic seizures, or myoclonic seizures. Seizure control was poor in one patient, who was resistant to treatment with lamotrigine and cannabidiol, while the remaining four patients had unspecified seizure control. We investigated whether these patients exhibited similar electroencephalographic features. Review of their EEGs revealed generalized abnormal slow waves, diffuse waves, or bursts. The EEG of patient 5 was not available for review. Interestingly, brain MRI results varied: some patients had normal scans, while others showed cerebral atrophy and cerebellar syndrome.. Discussion As next-generation sequencing technologies become more prevalent in modern medicine, an increasing number of genetic causes for SYNGAP1-related neurodevelopmental disorders are being identified clinically. A novel de novo intronic variant in SYNGAP1 was confirmed outside the canonical splice site in our research, which significantly impacts the mature SYNGAP1 transcript. This variant is predicted to result in a truncated SYNGAP1 protein, specifically distrution of crucial C-terminal domains. These domains are essential for binding to postsynaptic density (PSD) proteins, including components of the NMDAR complex (Kornau et al., 1995 ; Niethammer et al., 1996 ) and play a pivotal role in neuronal signaling pathways critical for normal synaptic function (Kim et al., 1998a ), further reinforcing our conclusion that pathogenic mutations in SYNGAP1 are associated with a loss of its function (Komiyama et al., 2002 ). The phenotypes of the patients described here are similar to those reported previously with SYNGAP1 variants affecting the junction between intron 16 and exon 17 (Table 2). Although our case represents a clinical presentation, all those described have the neurodevelopmental phenotypes consistent with the SYNGAP1-related neurodevelopmental disorders. All patients with SYNGAP1 mutations show moderate ID with some structural anomalies reported on brain imaging except for one patient for whom data were unavailable. Large-scale studies have shown that most SYNGAP1 variants associated with ID, ASD, and epilepsy are loss-of-function, resulting in SYNGAP1 haploinsufficiency (Berryer et al., 2013 ). This loss of SYNGAP1 disrupts the excitatory/inhibitory balance, accelerates synapse maturation, and hinders synaptic plasticity during crucial stages of development, leading to impaired cognitive and behavioral functions (Clement et al., 2012 ). The loss-of-function phenotypes of SYNGAP1 seem to be conserved across various vertebrate species, making animal models with SYNGAP1 haploinsufficiency valuable for studying the role of SYNGAP1 in disease contexts (Kilinc et al., 2018 ). Mice heterozygous for alleles containing intragenic deletions in SYNGAP1 show impaired synaptic plasticity, dendritic spine morphogenesis, and learning (Muhia et al., 2010 ). In addition to aberrant synaptic plasticity, SYNGAP1 hypofunction may lead to broad dysregulation of brain development. SYNGAP1 heterozygous mice exhibit disrupted development of brain sensory systems, affecting regions from the thalamus to the primary somatosensory cortex (Barnett et al., 2006 ; Carvill et al., 2013 ; Hamdan et al., 2011a ). The identification of a balanced translocation involving SYNGAP1, along with deletions that include SYNGAP1 in patients with ID, further supports the idea that SYNGAP1 haploinsufficiency leads to ID. However, some of these rearrangements are harder to interpret due to the involvement of additional genes (Krepischi et al., 2010 ; Pinto et al., 2010 ). The reported features in these SYNGAP1 intronic variants individuals vary somehow in different type of epilepsy. One patient among them are being treated refractory as lamotrigine and the ketogenic diet but continues to experience inadequate seizure control (Brimble et al., 2019 ). SYNGAP1 disruption in mice leads to increased recruitment of AMPAR to postsynaptic glutamatergic synapses, heightening excitatory transmission and potentially leading to seizures (Clement et al., 2012 ; Khlaifia et al., 2023 ). Notably, valproate or topiramate exerted its antiepileptic effects in the patients, as both drugs inhibit AMPA receptor activity (Bai et al., 2022 ; Zarate-Lopez et al., 2024 ). Interestingly, the patient with intronic mutations in SYNGAP1 indentified in our research exhibit some form of ID and are also diagnosed with ASD, similar to those reported in published studies (Agarwal et al., 2019 ; Mignot et al., 2016 ). This finding supports the hypothesis that mutations leading to disrupted SYNGAP function could result in behavioral phenotypes reminiscent of ASDs, including repetitive and obsessive behaviors, abnormal social interaction and communication. The connection between SYNGAP1 dysfunction and ASD was significantly reinforced by evidence indicating that SYNGAP1 haploinsufficiency leads to early maturation of dendritic spines (Clement et al., 2012 ). Abnormal regulation of spine maturation during development is believed to play a major role in the underlying causes of other neurodevelopmental disorders associated with ASD (He and Portera-Cailliau, 2013 ). While the impact of intronic variant indentified in our research is predicted protein truncation through reading frameshift, the application of this dogma to variants outside the canonical splice sites is less certain. Our research demonstrates the value of clinical genetic sequencing for identifying intronic mutations in SYNGAP1, especially when interpreting candidate variants of uncertain significance near intron-exon junction. The implications are significant to confirm a suspected diagnosis can directly affect medical treatment, while also reducing the necessity for further diagnostic investigations. Conclusion Our study identified a novel de novo pathogenic heterozygous variant in SYNGAP1, specifically c.3582 + 2T > G, and supports the pathogenicity of intronic variants between SYNGAP1 exon 16 and exon 17. Given the rarity of genetically confirmed SYNGAP1 patients, the small sample size limits the potential to draw definitive conclusions, such as establishing a clear genotype-phenotype correlation with any developmental parameters. Although Experimental validation in animal models, particularly mice, have not yet been conducted to validate this specific variant, such experiments would be crucial to further understanding the functional consequences and detailed mechanisms of SYNGAP1 disruptions. Future studies involving larger patient cohorts and more comprehensive neuropsychological and developmental assessments will better define developmental progression and provide a foundation for further targeted clinical trials in individuals with pathogenic SYNGAP1 intronic variants. Declarations Statement of Ethics This research was approved by the Ethics Committee of the Ganzhou Maternal and Child Health Hospital, Ganzhou, China. Informed consents were obtained from the family for all clinical studies and for publication. Acknowledgments The authors are grateful to the patient and their family members for their participation in this study. No funding is applicable for this study. Contributions BL, WX, and MS collected the sequencing data and clinical information. RL, QZ, and MH performed the data analysis and made the figure 1. BL and WX prepared the initial draft of the manuscript. SH wrote, revised and finalized the manuscript. All authors contributed to the article and approved the final version for submission. Contact for reagent and resource sharing Requests for further information and requests for resources should be directed to Shuwen He ( [email protected] ). Conflict of interest The authors declare that this research was conducted without any commercial or financial relationships that could be interpreted as a potential conflict of interest. References Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A.J., Bambrick, J., et al. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630 , 493-500. 10.1038/s41586-024-07487-w. Agarwal, M., Johnston, M.V., and Stafstrom, C.E. (2019). SYNGAP1 mutations: Clinical, genetic, and pathophysiological features. Int J Dev Neurosci 78 , 65-76. 10.1016/j.ijdevneu.2019.08.003. Anna, A., and Monika, G. (2018). 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Kim, M.J., Dunah, A.W., Wang, Y.T., and Sheng, M. (2005). Differential roles of NR2A- and NR2B-containing NMDA receptors in Ras-ERK signaling and AMPA receptor trafficking. Neuron 46 , 745-760. 10.1016/j.neuron.2005.04.031. Komiyama, N.H., Watabe, A.M., Carlisle, H.J., Porter, K., Charlesworth, P., Monti, J., Strathdee, D.J., O'Carroll, C.M., Martin, S.J., Morris, R.G., et al. (2002). SynGAP regulates ERK/MAPK signaling, synaptic plasticity, and learning in the complex with postsynaptic density 95 and NMDA receptor. J Neurosci 22 , 9721-9732. 10.1523/JNEUROSCI.22-22-09721.2002. Kornau, H.C., Schenker, L.T., Kennedy, M.B., and Seeburg, P.H. (1995). Domain Interaction between Nmda Receptor Subunits and the Postsynaptic Density Protein Psd-95. Science 269 , 1737-1740. DOI 10.1126/science.7569905. Krapivinsky, G., Medina, I., Krapivinsky, L., Gapon, S., and Clapham, D.E. (2004). 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Disruption of hippocampus-regulated behavioural and cognitive processes by heterozygous constitutive deletion of SynGAP. Eur J Neurosci 31 , 529-543. 10.1111/j.1460-9568.2010.07079.x. Myers, S.J., Yuan, H., Kang, J.Q., Tan, F.C.K., Traynelis, S.F., and Low, C.M. (2019). Distinct roles of GRIN2A and GRIN2B variants in neurological conditions. F1000Res 8 . 10.12688/f1000research.18949.1. Nalefski, E.A., and Falke, J.J. (1996). The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci 5 , 2375-2390. 10.1002/pro.5560051201. Niethammer, M., Kim, E., and Sheng, M. (1996). Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. Journal of Neuroscience 16 , 2157-2163. Pinto, D., Pagnamenta, A.T., Klei, L., Anney, R., Merico, D., Regan, R., Conroy, J., Magalhaes, T.R., Correia, C., Abrahams, B.S., et al. (2010). Functional impact of global rare copy number variation in autism spectrum disorders. Nature 466 , 368-372. 10.1038/nature09146. Redin, C., Gerard, B., Lauer, J., Herenger, Y., Muller, J., Quartier, A., Masurel-Paulet, A., Willems, M., Lesca, G., El-Chehadeh, S., et al. (2014). Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing. J Med Genet 51 , 724-736. 10.1136/jmedgenet-2014-102554. Sabo, S.L., Lahr, J.M., Offer, M., Weekes, A., and Sceniak, M.P. (2022). GRIN2B-related neurodevelopmental disorder: current understanding of pathophysiological mechanisms. Front Synaptic Neurosci 14 , 1090865. 10.3389/fnsyn.2022.1090865. Strauss, K.A., Gonzaga-Jauregui, C., Brigatti, K.W., Williams, K.B., King, A.K., Van Hout, C., Robinson, D.L., Young, M., Praveen, K., Heaps, A.D., et al. (2018). Genomic diagnostics within a medically underserved population: efficacy and implications. Genet Med 20 , 31-41. 10.1038/gim.2017.76. Sudhof, T.C. (2008). Neuroligins and neurexins link synaptic function to cognitive disease. Nature 455 , 903-911. 10.1038/nature07456. Vazquez, L.E., Chen, H.J., Sokolova, I., Knuesel, I., and Kennedy, M.B. (2004). SynGAP regulates spine formation. J Neurosci 24 , 8862-8872. 10.1523/JNEUROSCI.3213-04.2004. Vissers, L.E., de Ligt, J., Gilissen, C., Janssen, I., Steehouwer, M., de Vries, P., van Lier, B., Arts, P., Wieskamp, N., del Rosario, M., et al. (2010). A de novo paradigm for mental retardation. Nat Genet 42 , 1109-1112. 10.1038/ng.712. Zarate-Lopez, D., Torres-Chavez, A.L., Galvez-Contreras, A.Y., and Gonzalez-Perez, O. (2024). Three Decades of Valproate: A Current Model for Studying Autism Spectrum Disorder. Curr Neuropharmacol 22 , 260-289. 10.2174/1570159X22666231003121513. Zoghbi, H.Y., and Bear, M.F. (2012). Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities. Cold Spring Harb Perspect Biol 4 . 10.1101/cshperspect.a009886. Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files table1.png Splicing mutations described in the SYNGAP1 gene, with their nucleotide position and associated phenotype tabe2.png Names and functions of predicted SYNGAP1-interacting proteins. table3.png Clinical Features of Patients with De Novo SYNGAP1 mutation at intron 16/exon 17 junction Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5133555","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":357522831,"identity":"8acb326b-fdc1-4ffe-9641-ff1694ad27d2","order_by":0,"name":"Baoqiong Liao","email":"","orcid":"","institution":"1Ganzhou Maternal and Child Health Hospital, Ganzhou, Jiangxi, China.2Fujian Medical University, Fuzhou, Fujian, China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Baoqiong","middleName":"","lastName":"Liao","suffix":""},{"id":357522832,"identity":"a36b27d3-3cbb-41c5-ad87-13fd0437e44c","order_by":1,"name":"Wuming Xie","email":"","orcid":"","institution":"3Ganzhou People’s Hospital, Ganzhou, Jiangxi, China","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wuming","middleName":"","lastName":"Xie","suffix":""},{"id":357522833,"identity":"788efdd5-7d36-44bc-940e-73fe9a60884a","order_by":2,"name":"Mei Shuai","email":"","orcid":"","institution":"Ganzhou Maternal and Child Health Hospital, Ganzhou, Jiangxi, China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mei","middleName":"","lastName":"Shuai","suffix":""},{"id":357522834,"identity":"cfb7e973-ab86-43df-ab58-35a152bb2982","order_by":3,"name":"Rutian Liu","email":"","orcid":"","institution":"Ganzhou Maternal and Child Health Hospital, Ganzhou, Jiangxi, China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rutian","middleName":"","lastName":"Liu","suffix":""},{"id":357522835,"identity":"199fc5f8-4f5d-4bad-9009-024edaac25f8","order_by":4,"name":"Qi Zhang","email":"","orcid":"","institution":"Ganzhou Maternal and Child Health Hospital, Ganzhou, Jiangxi, China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Zhang","suffix":""},{"id":357522836,"identity":"83512acb-c289-4161-bc3f-bf6a4776d80b","order_by":5,"name":"Min Hong","email":"","orcid":"","institution":"Ganzhou Maternal and Child Health Hospital, Ganzhou, Jiangxi, China.","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Hong","suffix":""},{"id":357522837,"identity":"7bdd2edb-9ec9-47b3-81c9-19410d487bf1","order_by":6,"name":"Shuwen He","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYDACCQiVwMDeQLIWngMka5FIIFKH/OweM4mPOxjy+CWfX5Ng+GNDWIvBnTNmkjPPMBRLzs4pk2BsSyNCi0SO2W3eNobEDbdz0iQYGw4T4bAZQC1/gVr23zyTBnTYf8JaGG4AtTCCbJFgPybBwHaACIfdSCv/2dsmkTjjTA6zRWJbMjEOS95s8LPNJrG//fjDGx/+2BHhMAgAxQ6PATB2SAPsD0jUMApGwSgYBSMFAABITDj40a3sAwAAAABJRU5ErkJggg==","orcid":"","institution":"Department of chemistry and molecular biology, Gothenburg university, Gothenburg, Sweden.","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Shuwen","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2024-09-22 17:53:41","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false,"coiExplicitlySet":false},"doi":"10.21203/rs.3.rs-5133555/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5133555/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65143943,"identity":"0b64dd8c-b955-422b-b904-347d7d0a1f3a","added_by":"auto","created_at":"2024-09-24 05:58:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":74381,"visible":true,"origin":"","legend":"\u003cp\u003eA 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\u0026gt;G variant found in the study.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/9a4cc613e622b62c29150e4e.jpg"},{"id":65143944,"identity":"48dd5824-c3c8-4fb9-822a-a19bbf807c75","added_by":"auto","created_at":"2024-09-24 05:58:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":167781,"visible":true,"origin":"","legend":"\u003cp\u003eElectroencephalographic features of patients with c.3582+2T\u0026gt;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.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/1bc70d72ebfbdb4a4c5a2c11.jpg"},{"id":65143949,"identity":"552538a3-f9d7-4ae2-8ae3-0742153a2c72","added_by":"auto","created_at":"2024-09-24 05:58:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":424322,"visible":true,"origin":"","legend":"\u003cp\u003eA 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\u0026gt;G variant found in the study.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/6b419f282b4ab56077b19cc9.png"},{"id":65143948,"identity":"e358d341-6358-42f5-b204-648cd61b288d","added_by":"auto","created_at":"2024-09-24 05:58:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":917104,"visible":true,"origin":"","legend":"\u003cp\u003ePotential impact of the c.3582+2T\u0026gt;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 1,343 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).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/542c9bab1aeaedb5ae2c3846.png"},{"id":65144405,"identity":"916a2af7-116c-4fa7-aabe-86887d66d77b","added_by":"auto","created_at":"2024-09-24 06:06:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2124686,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/11418813-c2c5-402e-8d39-7532bab3620e.pdf"},{"id":65144404,"identity":"00b465e0-698b-4591-8227-df28b027673c","added_by":"auto","created_at":"2024-09-24 06:06:30","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":458172,"visible":true,"origin":"","legend":"\u003cp\u003eSplicing mutations described in the SYNGAP1 gene, with their nucleotide\u003c/p\u003e\n\u003cp\u003eposition and associated phenotype\u003c/p\u003e","description":"","filename":"table1.png","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/d8274f55eaa0ed8e5d538971.png"},{"id":65144403,"identity":"e15d0e9e-6a04-40e7-9878-18a86255ff04","added_by":"auto","created_at":"2024-09-24 06:06:30","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":245599,"visible":true,"origin":"","legend":"\u003cp\u003eNames and functions of predicted SYNGAP1-interacting proteins.\u003c/p\u003e","description":"","filename":"tabe2.png","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/a80220ab0ba4e4d77f93789e.png"},{"id":65143947,"identity":"885ab12b-dca2-4ea2-8e33-5270f3b17d85","added_by":"auto","created_at":"2024-09-24 05:58:31","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":268852,"visible":true,"origin":"","legend":"\u003cp\u003eClinical Features of Patients with De Novo SYNGAP1 mutation at intron\u003c/p\u003e\n\u003cp\u003e16/exon 17 junction\u003c/p\u003e","description":"","filename":"table3.png","url":"https://assets-eu.researchsquare.com/files/rs-5133555/v1/bd13a7e84b0c7b5f7c2a4ede.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eNovel de novo intronic variant of SYNGAP1 associated with the neurodevelopmental disorders\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDisruptions in the molecular mechanisms that govern the structure and function of glutamatergic synapses are thought to be a key factor in the development of certain neurodevelopmental disorders (Bourgeron, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Grant, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Zoghbi and Bear, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Deleterious mutations in synaptic proteins have been associated with the related neurodevelopmental disorders, and numerous animal models exhibit deficits in synapse structure and/or function (Guilmatre et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hamdan et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e; Sudhof, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Mutations in SYNGAP1 resulted in a neurodevelopmental disorder termed Mental retardation-type 5 (MRD5, OMIM #612621) consisting of intellectual disabilities (ID), developmental delay, autism spectrum disorders (ASD), motor impairments, hypotonia, epilepsy and maladaptive behaviors (Berryer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Clement et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Mignot et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Neuroimaging in these affected individuals is typically normal, though rare cases have reported brain atrophy and delayed myelination (Hamdan et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e; Vissers et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). However, a significant number of individuals display ataxia, presenting with a broad-based and unsteady gait. Seizure management in epilepsy varies significantly between individuals. Some patients achieve control with one or more antiepileptic drugs, while others have epilepsy that remains resistant to medication (Berryer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). With the growing identification of the affected individuals, a consistent clinical syndrome associated with SYNGAP1 variants has been delineated. De novo mutations in SYNGAP1, particularly loss-of-function varients have been identified as one of the more prevalent genetic causes of MRD5, with an estimated prevalence of 0.7-1% in specific populations (Deciphering Developmental Disorders, 2015). The genetic architecture of SYNGAP1-related neurodevelopmental disorder is complex, as evidenced by various studies employing cytogenetics, linkage analysis, association studies, and whole-genome or exome sequencing.\u003c/p\u003e \u003cp\u003eThe human SYNGAP1 gene on chromosome 6p21.3 consists of 19 exons that encodes the synaptic RAS-GTPase-activating protein (SYNGAP)(Kim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1998a\u003c/span\u003e). This gene is primarily found localized to dendritic spines in the postsynaptic density (PSD) of excitatory glutamatergic neurons, where it suppresses signaling pathways linked to NMDA receptor (NMDAR) mediated synaptic plasticity and AMPA receptors (AMPAR) membrane insertion (Kim et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Krapivinsky et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The encoded protein regulates the trafficking of the different receptors, as well as Ras and Rap to the postsynaptic, thereby influencing synaptic plasticity and neuronal homeostasis (Vazquez et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In the SYNGAP1 sequence, the N-terminal pleckstrin homology (PH) domains act as membrane recruitment modules by binding to phospholipids (Lemmon, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). C2 domains are known to function as Ca\u003csup\u003e2+\u003c/sup\u003e-dependent phospholipidbinding modules (Nalefski and Falke, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Various C-terminal exons of the SYNGAP1 undergo alternative splicing, leading to the generation of multiple protein isoforms for postsynaptic scaffold protein interaction (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Depending on the location of different expressed variants, SYNGAP1 can either or suppress the dendritic spine synapse function (Clement et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Many of these variants are nonsense or frameshift mutations that cause nonsense-mediated decay of SYNGAP1 mRNA or dysfunctional truncated protein products, likely resulting in haploinsufficiency. Intellectual disabilities-causing SYNGAP1 mutations have been identified along the entire length of the SYNGAP1 gene, with most of the mutations occurring from exons 3\u0026ndash;17 (Gamache et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Mutations can also occur in the intronic regions (non-coding regions between exons) of SYNGAP1 with MRD5 as detailed in table1. The intronic mutations, especially those near the exon-intron boundaries (splice sites), can affect splicing and lead to aberrant transcripts that are either degraded or translated into non-functional proteins (Anna and Monika, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Borras et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, a young boy from the Chinese family was diagnosed with the SYNGAP1-related neurodevelopmental disorders, identifying a likely pathogenic intronic variant (c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G) through Whole-Exome and Sanger sequencing. Minigene analysis confirmed the pathogenicity of the SYNGAP1 intronic variant, indicating its importance in clinical genomics. Our findings highlighted other reports of pathogenic intronic variants in the junctional regions of SYNGAP1 exons 16 and 17, which are a relatively common cause of this neurodevelopmental disorder, with significant implications for the clinical diagnosis, prognosis, and management of patients with SYNGAP1 pathogenic intronic variants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubjects\u003c/h2\u003e \u003cp\u003eAn 9-year-old Chinese boy, diagnosed with autism spectrum disorder, moderate intellectual disability, mild developmental delay and speech impairment, was thoroughly evaluated at the Department of Pediatrics, Ganzhou Maternal and Child Health Hospital. Comprehensive assessments were conducted including a detailed medical history, physical examinations, skull magnetic resonance imaging (MRI), electroencephalogram (EEG), and Wei\u0026rsquo;s intelligence assessment. The boy was an only child of the healthy, non-consanguineous parents. The boy's medical history was unremarkable during the prenatal, delivery, and postpartum periods.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eThis research was approved by the Ethics Committee of the Ganzhou Maternal and Child Health Hospital. Peripheral venous blood samples were taken from the boy and his parents following the acquisition of informed consent from all participants. RNA was isolated using the PAXgene Blood RNA Kit (PreAnalytiX, Hombrechtikon, Switzerland). DNA was extracted from the peripheral blood using the QIAamp DNA Mini Kit for blood (Qiagen, Hilden, Germany). Trio-based WES was performed by Kangxu Diagnostics (Beijing, China), including exome library preparation, sequencing and data analysis. Variant screening was based on clinical phenotypes of the affected subjects, population database (dbSNP, 1,000 Genome, ExAC), disease database (OMIM, HGMD, Clinvar) and biological information prediction tools (SIFT, Polyphen2, Mutation Taster and Splice AI). A novel variant of SYNGAP1 were indentified in the boy (c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G) while his parents displayed normal. The SYNGAP1 variant were confirmed by Sanger sequencing. To assess the potential impact of the c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G variant in SYNGAP1, the RDDCSC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rddc.tsinghua-gd.org/search-middle?to=SplitToolModel\u003c/span\u003e\u003cspan address=\"https://rddc.tsinghua-gd.org/search-middle?to=SplitToolModel\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 23 April 2024) online in silico splice site prediction software was used.\u003c/p\u003e\n\u003ch3\u003eSYNGAP1 Expression Constructs\u003c/h3\u003e\n\u003cp\u003eFollowing prior confirmation of SYNGAP1 expression in blood, genomic DNA was extracted from the blood sample of patients and controls by using the TIANamp Genomic DNA Mini Kit for blood (TIANGEN). The extracted DNA were used as templates to amplify exons 15, 16, 17 and introns therein via reverse transcription with primers Forward (F): 5\u0026rsquo;-CCGGAATTCCTCTATGGACATGGCTCGC-3\u0026rsquo; and Reverse (R):5\u0026rsquo;- GGGTTCGAAGATCAAGAGCATCATTGGCAG-3\u0026rsquo;. The PCR products were purified by the HiPure Gel Pure Micro Kit (Magen). The wt and mutant cDNAs were then cloned into the mammalian expression vector pCDNA3.4 (Addgene) using nucleic acid endonuclease EcoRI-HF, HindIII-HF and T4 DNA ligase (New England Biolabs). Plasmids were isolated from individual clones and verified by restriction digestion to contain an insert with the expected size. Both WT and mutant plasmids contained the whole sequence of exon 15\u0026ndash;17 and a portion of the upstream and downstream intron sequence futher confirmed by the sequencing.\u003c/p\u003e\n\u003ch3\u003eCell Culture and Transfection\u003c/h3\u003e\n\u003cp\u003eB16-F1 and HEK293 cell lines (American Type Culture Collection, ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) sourced from GIBCO Life Technologies. The medium was supplemented with 10% fetal bovine serum (FBS) from Invitrogen, 100 U/ml penicillin-streptomycin (GIBCO Life Technologies), and 2.5 \u0026micro;g/ml Plasmocin (InvivoGen). Cells were incubated at 37\u0026deg;C in a humidified environment with 5% CO2. Transfections were performed using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada), followed by a 48-hour incubation prior to RNA extraction.\u003c/p\u003e\n\u003ch3\u003eRNA analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted with Trizol (TaKaRa) from the transfected cells, and cDNA was acquired with PrimeScriptTMRT reagent Kit (TaKaRa). The RT-PCR products was analyzed by electrophoresis on 2% agarose gels containing ethidium bromide and visualized by exposure to ultraviolet light (Bio-Red). Each DNA band was purified by HiPure Gel Pure DNA Micro Kit (Megen). Direct sequencing of purified RT-PCR products was performed on the Sangon Biotech, Shanghai. Primers used for minigene assay of SYNGAP1 were as follows:\u003c/p\u003e \u003cp\u003eSYNGAP1-F1:5\u0026rsquo;CTCTATGGACATGGCTCGCCTCCCCTCCCC-3\u0026rsquo;;\u003c/p\u003e \u003cp\u003eSYNGAP1-R1: 5\u0026rsquo;-CTGCCAATGATGCTCTTGATCTGGGAATCC-3\u0026rsquo;.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProtein structure prediction\u003c/h2\u003e \u003cp\u003eThe protein sequence with 1343 amino acid residues of SYNGAP1 was downloaded from uniprot web (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The wild-type and mutant-type(SYNGAP1: c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G)3D structure of the SYNGAP1 protein were predicted using AlphaFold web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://golgi.sandbox.google.com/about\u003c/span\u003e\u003cspan address=\"https://golgi.sandbox.google.com/about\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Abramson et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The best model was selected based on pLDDTs prediction scores. The higher the score, the more confident is the structure. Prediction models editing was performed was visualized using PyMOL program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pymol.org/\u003c/span\u003e\u003cspan address=\"https://pymol.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBioinformatics analysis\u003c/h3\u003e\n\u003cp\u003eThe protein-protein interaction (PPI) network associated with SYNGAP1 was constructed using STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). the amino acid sequence of the SYNGAP1 protein, comprising 1,343 residues, was retrieved from the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This sequence was used as the input query for STRING, where the parameters were set to ensure high-confidence interactions were captured. The confidence score threshold was adjusted to include only interactions supported by strong evidence such as experimental data, co-expression, and co-occurrence analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDe novo heterozygous variant were identified\u003c/h2\u003e \u003cp\u003eA de novo heterozygous pathogenic variants in SYNGAP1, NM_006772.3: c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G, was identified in the young male patient. Genetic analysis of both parents revealed that neither carried the variant in SYNGAP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B), and both exhibited the normal intellectual abilities and motor development, ruling out inherited transmission. Notably, the c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G variant represents a unique intronic mutation in SYNGAP1, identified for the first time in this proband. This variant has not been previously documented in the existing scientific literature or major genetic databases, including ClinVar, Deciphering Developmental Disorders (DDD), PubMed, and the Human Gene Mutation Database (HGMD), making it a significant new addition to the spectrum of SYNGAP1-related pathogenic variants. This discovery adds to the growing understanding of intronic mutations and their possible potential impact on neurodevelopmental disorders.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical characteristics of the individual affected by the SYNGAP1 c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G variant\u003c/h2\u003e \u003cp\u003eThe proband exhibited no abnormalities at birth. At 4 months of age, he was unable to grasp objects, indicating motor developmental delay. He was noted to be late in achieving independent walking, which occurred at 1 year and 9 months. His language development was significantly delayed. At the age of 9, his verbal communication was limited to simple expressions such as \"yes\" and \"no,\" and remained unable to say \"daddy\" or \"mommy as well as full phrases. In 2018, a comprehensive assessment for autism spectrum disorder (ASD) were carried on the proband including the psychological tests, clinical examinations, and consideration of the family medical history. We utilized standardized assessment tools such as the Autism Diagnostic Observation Schedule (ADOS) for ASD evaluation. The patient was diagnosed with ASD at Guangdong Provincial Children's Hospital, China. At present, electroencephalogram (EEG) examination during wakefulness revealed abnormal brain activity characterized by a generalized pattern of moderately frequent high amplitude spikes and slow waves, diffuse waves, or bursts (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Of note, a brain MRI was read as normal. There is no family history of similar neurodevelopmental disorders or global developmental delay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro minigene splicing assay for the SYNGAP1 c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G variant\u003c/h2\u003e \u003cp\u003eTo investigate the impact of the c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G variant, a minigene splicing assay was carried on both the control and patient samples. Following confirmation of the sequencing results, the pCDNA3.4-SYNGAP1-WT and pCDNA3.4-SYNGAP1-MUT (c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G) plasmids were separately transfected into HEK293T and HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). After 48 hours of transfection, total RNA was extracted and subsequently reverse-transcribed into cDNA. The complementary DNA fragment covering exons 15\u0026ndash;17 was amplified by reverse transcription (RT)-PCR and next analyzed using agarose gel electrophoresis. The agarose gel electrophoresis results demonstrated that a 1458-base pair (bp) fragment was amplified from the cDNAs of both the control subject and patient, whereas a 1559-bp fragment was also detected in the cDNA of patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C). Cloning and sequencing of the two PCR products generated from patient showed that the 1458-bp fragment corresponded to the wild-type transcript comprising exons 15-16-17, whereas the 1559-bp fragment corresponded to a transcript in which abnormal splicing led to the retention of a 101 bp fragment of intron 16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This retention causes a frameshift form at amino acid position 1195, resulting in extending for 34 amino acids followed by a premature stop codon (c.3582\u0026thinsp;+\u0026thinsp;101_3582\u0026thinsp;+\u0026thinsp;201ins101, p.Val1195Glyfs*34) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of mutations on protein structure\u003c/h2\u003e \u003cp\u003eIn order to analyze the effect of the novel variants NP_006763.2:c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G, (p.Val1195Glyfs*34) on the structural integrity of the SYNGAP1 protein, AlphaFold web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://golgi.sandbox.google.com/about\u003c/span\u003e\u003cspan address=\"https://golgi.sandbox.google.com/about\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict changes in SYNGAP1 protein structures. The NP_006763.2:c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G variant of SYNGAP1 could induce a frameshift mutation, resulting in a truncated SYNGAP1 protein comprising 1228 of the 3354 amino acids presented in the full-length mature protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). For the mutated SYNGAP1 protein, a pTM score of 0.51 reflects moderate confidence in the predicted structure of the truncated protein. The frameshift mutation likely leads to a significant alteration of the protein\u0026rsquo;s three-dimensional conformation, particularly in the C-terminal region. The frameshift notably disrupts the C-terminal region of SYNGAP1, a domain critical for interacting with key postsynaptic density proteins, such as scaffolding protein PSD-95 (postsynaptic density protein 95). PSD-95 helps anchor NMDA receptors at the synapse organizes synaptic signaling complexes, facilitating the downstream signaling pathways activated by NMDA receptor activation, such as calcium influx that leads to synaptic plasticity (Kennedy, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The interaction between PSD-95 and NMDA receptors is crucial for processes like long-term potentiation (LTP), which underlies learning and memory (Ehrlich and Malinow, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Disruption of SYNGAP1's interaction with PSD-95 may also in turn, compromise its ability to regulate downstream signaling pathways involving Ras, potentially leading to synaptic dysfunction (Kim et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1998b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe used STING software to predict the interacting proteins with SYNGAP1 and indentified several functional proteins, including DLG2, DLG3, DLG4, SHANK2, SHANK3, ULK1, ULK2, GRIN2B, NRAS and DLGAP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, Table\u0026nbsp;2). DLG2 DLG3 and DLG4 are all critical for regulating NMDA receptor signaling, facilitating the synaptic plasticity that is critical for learning, memory formation and cognitive function. SHANK2 and SHANK3 act as a major scaffold protein, interacts with various proteins like NMDA-type receptor to orchestrate dendritic spine and synapse formation. Notably, GRIN2B, a component of NMDA receptor complexes, is distributed throughout the dendrites at extrasynaptic sites. Variants of GRIN2B are commonly found in individuals with neurodevelopmental disorders characterized by mild to profound developmental delays and intellectual disabilities (Myers et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sabo et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given the importance of the C-terminal domain in synaptic function, this mutation highlights the possible mechanism for the cognitive and developmental deficits observed in patients with SYNGAP1 mutations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eClinical phenotypes of patients with De Novo SYNGAP1 mutation at intron 16/exon 17\u003c/h2\u003e \u003cp\u003eWe reviewed the clinical profiles of all known patients with de novo intronic mutations in SYNGAP1 at intron 16/exon 17, including the four mutations c.3583-9G\u0026thinsp;\u0026gt;\u0026thinsp;A, c.3583-6G\u0026thinsp;\u0026gt;\u0026thinsp;A, c.3583-1G\u0026thinsp;\u0026gt;\u0026thinsp;T and c.3583\u0026thinsp;+\u0026thinsp;3A\u0026thinsp;\u0026gt;\u0026thinsp;T discussed in this study (Table\u0026nbsp;3)(Bayat et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Brimble et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Redin et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Strauss et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). All patients exhibited mild global developmental delay with varying degrees of language development disorders. Three patients lacked speech ability, while one could produce short phrases accompanied by gestures. Additionally, two patients were diagnosed with autism, including patient 1 (c.3583-9G\u0026thinsp;\u0026gt;\u0026thinsp;A). Data for one patient are unavailable. Autism was confirmed in three patients using the Autism Diagnostic Observation Schedule (ADOS). Notably, patient 2 (c.3583-6G\u0026thinsp;\u0026gt;\u0026thinsp;A) did not meet the criteria for autism spectrum disorder according to the ADOS-2, but delays in social communication were documented. our patients with intronic mutations in SYNGAP1 experienced epilepsy, with variable seizure types including absence seizures, tonic-clonic seizures, or myoclonic seizures. Seizure control was poor in one patient, who was resistant to treatment with lamotrigine and cannabidiol, while the remaining four patients had unspecified seizure control. We investigated whether these patients exhibited similar electroencephalographic features. Review of their EEGs revealed generalized abnormal slow waves, diffuse waves, or bursts. The EEG of patient 5 was not available for review. Interestingly, brain MRI results varied: some patients had normal scans, while others showed cerebral atrophy and cerebellar syndrome..\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs next-generation sequencing technologies become more prevalent in modern medicine, an increasing number of genetic causes for SYNGAP1-related neurodevelopmental disorders are being identified clinically. A novel de novo intronic variant in SYNGAP1 was confirmed outside the canonical splice site in our research, which significantly impacts the mature SYNGAP1 transcript. This variant is predicted to result in a truncated SYNGAP1 protein, specifically distrution of crucial C-terminal domains. These domains are essential for binding to postsynaptic density (PSD) proteins, including components of the NMDAR complex (Kornau et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Niethammer et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and play a pivotal role in neuronal signaling pathways critical for normal synaptic function (Kim et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1998a\u003c/span\u003e), further reinforcing our conclusion that pathogenic mutations in SYNGAP1 are associated with a loss of its function (Komiyama et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phenotypes of the patients described here are similar to those reported previously with SYNGAP1 variants affecting the junction between intron 16 and exon 17 (Table\u0026nbsp;2). Although our case represents a clinical presentation, all those described have the neurodevelopmental phenotypes consistent with the SYNGAP1-related neurodevelopmental disorders. All patients with SYNGAP1 mutations show moderate ID with some structural anomalies reported on brain imaging except for one patient for whom data were unavailable. Large-scale studies have shown that most SYNGAP1 variants associated with ID, ASD, and epilepsy are loss-of-function, resulting in SYNGAP1 haploinsufficiency (Berryer et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This loss of SYNGAP1 disrupts the excitatory/inhibitory balance, accelerates synapse maturation, and hinders synaptic plasticity during crucial stages of development, leading to impaired cognitive and behavioral functions (Clement et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The loss-of-function phenotypes of SYNGAP1 seem to be conserved across various vertebrate species, making animal models with SYNGAP1 haploinsufficiency valuable for studying the role of SYNGAP1 in disease contexts (Kilinc et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Mice heterozygous for alleles containing intragenic deletions in SYNGAP1 show impaired synaptic plasticity, dendritic spine morphogenesis, and learning (Muhia et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition to aberrant synaptic plasticity, SYNGAP1 hypofunction may lead to broad dysregulation of brain development. SYNGAP1 heterozygous mice exhibit disrupted development of brain sensory systems, affecting regions from the thalamus to the primary somatosensory cortex (Barnett et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Carvill et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hamdan et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e). The identification of a balanced translocation involving SYNGAP1, along with deletions that include SYNGAP1 in patients with ID, further supports the idea that SYNGAP1 haploinsufficiency leads to ID. However, some of these rearrangements are harder to interpret due to the involvement of additional genes (Krepischi et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Pinto et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe reported features in these SYNGAP1 intronic variants individuals vary somehow in different type of epilepsy. One patient among them are being treated refractory as lamotrigine and the ketogenic diet but continues to experience inadequate seizure control (Brimble et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). SYNGAP1 disruption in mice leads to increased recruitment of AMPAR to postsynaptic glutamatergic synapses, heightening excitatory transmission and potentially leading to seizures (Clement et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Khlaifia et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, valproate or topiramate exerted its antiepileptic effects in the patients, as both drugs inhibit AMPA receptor activity (Bai et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zarate-Lopez et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, the patient with intronic mutations in SYNGAP1 indentified in our research exhibit some form of ID and are also diagnosed with ASD, similar to those reported in published studies (Agarwal et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mignot et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This finding supports the hypothesis that mutations leading to disrupted SYNGAP function could result in behavioral phenotypes reminiscent of ASDs, including repetitive and obsessive behaviors, abnormal social interaction and communication. The connection between SYNGAP1 dysfunction and ASD was significantly reinforced by evidence indicating that SYNGAP1 haploinsufficiency leads to early maturation of dendritic spines (Clement et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Abnormal regulation of spine maturation during development is believed to play a major role in the underlying causes of other neurodevelopmental disorders associated with ASD (He and Portera-Cailliau, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile the impact of intronic variant indentified in our research is predicted protein truncation through reading frameshift, the application of this dogma to variants outside the canonical splice sites is less certain. Our research demonstrates the value of clinical genetic sequencing for identifying intronic mutations in SYNGAP1, especially when interpreting candidate variants of uncertain significance near intron-exon junction. The implications are significant to confirm a suspected diagnosis can directly affect medical treatment, while also reducing the necessity for further diagnostic investigations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study identified a novel de novo pathogenic heterozygous variant in SYNGAP1, specifically c.3582\u0026thinsp;+\u0026thinsp;2T\u0026thinsp;\u0026gt;\u0026thinsp;G, and supports the pathogenicity of intronic variants between SYNGAP1 exon 16 and exon 17. Given the rarity of genetically confirmed SYNGAP1 patients, the small sample size limits the potential to draw definitive conclusions, such as establishing a clear genotype-phenotype correlation with any developmental parameters. Although Experimental validation in animal models, particularly mice, have not yet been conducted to validate this specific variant, such experiments would be crucial to further understanding the functional consequences and detailed mechanisms of SYNGAP1 disruptions. Future studies involving larger patient cohorts and more comprehensive neuropsychological and developmental assessments will better define developmental progression and provide a foundation for further targeted clinical trials in individuals with pathogenic SYNGAP1 intronic variants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eStatement of Ethics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was approved by the Ethics Committee of the Ganzhou Maternal and Child Health Hospital, Ganzhou, China. Informed consents were obtained from the family for all clinical studies and for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to the patient and their family members for their participation in this study. No funding is applicable for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBL, WX, and MS collected the sequencing data and clinical information. RL, QZ, and MH performed the data analysis and made the figure 1. BL and WX prepared the initial draft of the manuscript. SH wrote, revised and finalized the manuscript. All authors contributed to the article and approved the final version for submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContact for reagent and resource sharing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRequests for further information and requests for resources should be directed to Shuwen He ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that this research was conducted without any commercial or financial relationships that could be interpreted as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A.J., Bambrick, J., et al. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. 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Cold Spring Harb Perspect Biol \u003cem\u003e4\u003c/em\u003e. 10.1101/cshperspect.a009886.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"Tables 1 to 3 are available in the Supplementary Files section."}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Gothenburg","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Whole exome sequencing, minigene, SYNGAP1, intronic variation, variant interpretation","lastPublishedDoi":"10.21203/rs.3.rs-5133555/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5133555/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: 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 features of autism spectrum disorder. The aim of this study was to identify a novel \u0026nbsp;SYNGAP1 gene variant linked to neurodevelopmental disorders and to evaluate the pathogenicity of the detected variant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: A novel de novo intronic variant in SYNGAP1 was identified by Whole exome sequencing (WGS) and confirmed by Sanger sequencing. Minigene assays were conducted to assess whether the intronic variant in SYNGAP1 influenced the normal splicing of mRNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: A novel de novo intronic variant in SYNGAP1 (c.3582+2T\u0026gt;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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: Our findings highlight the significance of de novo pathogenic SYNGAP1 variants at the intron 16/exon 17 junction in SYNGAP1-related neurodevelopmental disorders, providing novel insights into the genetic basis and diagnosis of these disabilities.\u003c/p\u003e","manuscriptTitle":"Novel de novo intronic variant of SYNGAP1 associated with the neurodevelopmental disorders","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-24 05:42:25","doi":"10.21203/rs.3.rs-5133555/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d283098e-0161-4abb-95d7-debfddb899df","owner":[],"postedDate":"September 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38031772,"name":"Medical Genetics"},{"id":38031773,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2024-09-24T05:42:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-09-24 05:42:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5133555","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5133555","identity":"rs-5133555","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}