Genotypic and Phenotypic Spectrum of PRRT2-Related Variations: Clinical Analysis and Treatment Response in Fourteen Unrelated Chinese Patients

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This gene encodes a proline-rich transmembrane protein 2, which plays a crucial role in neuronal development and synaptic transmission. Mutations in this gene can result in a variety of clinical phenotypes. Methods: We retrospectively analyzed 14 pediatric patients from unrelated families with epilepsy or paroxysmal motor disorders, all genetically confirmed to harbor PRRT2 mutations. Comprehensive clinical data were collected, including seizure semiology, EEG, MRI, treatment response, and developmental outcomes. Genetic analysis involved whole-genome sequencing or a customized epilepsy gene panel, with single nucleotide variants validated by Sanger sequencing. The pathogenesis of both mutations((c.189delC p.Lys64Argfs*26) and c.971G>C p.Gly324Ala)) was studied by in vitro experiments. Results: The cohort comprised 6 females and 8 males with onset ages ranging from 3 to 7 months. We identified 13 patients with point mutations and one with an exon 2-4 deletion.The hotspot mutation c.649dupC p.Arg217Profs*8 was most prevalent (11/14). Eleven patients exhibited the BFIE phenotype, one had PKD, and two were unclassifiable. Most patients (12/14, 85.7%) achieved seizure freedom, showing favorable responses to oxcarbazepine or levetiracetam. Developmental delays were observed in 5 patients, potentially associated with non-c.649dup mutations (e.g., c.189delC p.Lys64Argfs*26). Functional studies confirmed that the c.189delC p.Lys64Argfs*26 mutation significantly reduced mRNA and protein expression, while the c.971G>C p.Gly324Ala variant did not affect splicing but was predicted pathogenic. PRRT2 vitro experiments variants BFIE PKD Figures Figure 1 Figure 2 INTRODUCTION Chromosomal 16P11.2 copy number variation is one of the most common genetic causes of neurodevelopmental disorders. In addition to intellectual disabilities and autism spectrum disorders, the clinical phenotype of the variation is also characterized by seizures, and some patients only exhibit seizures with no other signs of disorder. Most scholars believe that the main cause of epilepsy is the inclusion of an important epilepsy-related gene, PRRT2. PRRT2 is a profile-rich protein that interacts with the synaptic-associated protein SNAP25 to interfere with glutamate signaling and glutamate receptor activity, leading to increased glutamate release and increased neuronal excitability [1-2]. Heterozygous variations in PRRT2 can cause four different neurological disorders, including benign familial infantile epilepsy (BFIE), paroxysmal motor disorders (PKD), PKD with infantile convulsions, and familial hemiplegic migraine (FHM). Among the diseases related to PRRT2 mutations, the majority of patients have BFIE characterized by self-limited epilepsy; this disorder typically begins to develop at 6 months of age, but is accompanied by normal psychomotor development and a good prognosis [3]. However, variations in different loci of the same gene still exhibit different clinical manifestations. Luo et al. further expanded the disease phenotype by studying PRRT2 mutations in 44 epilepsy patients [3]. However, genetic analysis of the phenotypic differences caused by PRRT2 mutations is still needed. Here, we analyzed the genetic characteristics of Fourteen children with neurological disorders caused by PRRT2 mutations. Most patients present with BFIE . Different loci correspond to different phenotypes, and based on the severity of their phenotypes, we further conducted in vitro functional validation, revealing the pathogenesis of this locus, and discussed the relationships among the PRRT2 genotype and phenotype, treatment, and prognosis. MATERIALS AND METHODS Patients In this study, we retrospectively analyzed Fourteen children from unrelated families with epilepsy or paroxysmal motor disorders, all of whom had previously been confirmed to have PRRT2 gene mutations through genetic testing. Clinical data, including age of onset, family history, developmental history, clinical manifestations, electroencephalogram (EEG) signals, magnetic resonance imaging (MRI), treatment, and follow-up, were collected and analyzed. All Eleven patients were diagnosed according to the diagnostic criteria for BFIE, with reference to [4] and [5], respectively. One patient was diagnosed with PKD, while the other two patients had an episode type that could not be classified as a certain syndrome.This study was approved by the Ethics Committee of Dongguan Children's Hospital. Genetic analysis Three milliliters of peripheral blood was extracted from each patient for DNA analysis. Some patients underwent whole-genome sequencing, while others selected a custom-designed gene panel targeting the coding exons of 1,170 epilepsy-associated genes, including PRRT2, which contained 16402 coding regions and a total of 2682931 bases. Using bioinformatics analysis techniques, the sequencing results were paired with reference sequences and combined with multiple databases and egg function prediction tools to determine clinically significant variations in the sample DNA. The reference genome GRCh37 was paired with the sequences, after which the results of single nucleotide variants were validated via Sanger sequencing. The large deletion encompassing exons 2–4 was confirmed by a custom-designed gene panel targeting the coding exons of 1,170 epilepsy-associated genes. In Vitro Functional Studies Plasmid Construction Expression vectors for wild-type (WT) and mutant (mut1, mut2) human PRRT2 were constructed based on the p3XFlag-CMV-7.1 backbone. The codon-optimized full-length PRRT2 coding sequence (CDS) was used as the template. (a) p3XFlag-CMV-7.1-WT: The wild-type (WT) PRRT2 coding sequence was amplified by PCR using a synthetic full-length PRRT2 CDS as the template, with primers CMV-PRRT2-EcoRI-F and CMV-PRRT2-SalI-R（Table 2）. The resulting PCR product and the p3XFlag-CMV-7.1 vector were double-digested with EcoRI and SalI, followed by ligation to construct the p3XFlag-CMV-7.1-wt expression vector. (b) p3XFlag-CMV-7.1-mut1: The mut1 mutation was introduced using a two-step overlap PCR strategy. First, two fragments (mut1-1 and mut1-2) were generated using primer pairs CMV-PRRT2-EcoRI-F/PRRT2-mut1-R and PRRT2-mut1-F/CMV-PRRT2-SalI-R, respectively. These fragments served as templates for a second PCR with the flanking primers CMV-PRRT2-EcoRI-F and CMV-PRRT2-SalI-R to generate the full-length EcoRI-mut1-SalI fragment. (c) p3XFlag-CMV-7.1-mut2: The mut2 fragment was directly amplified using primers CMV-PRRT2-EcoRI-F and PRRT2-mut2-SalI-R. All PCR-amplified fragments (WT, mut1, mut2) were sequenced verified, digested with EcoRI/SalI, and cloned into the correspondingly digested p3XFlag-CMV-7.1 vector. The final constructs were validated by colony PCR and Sanger sequencing. Cell Culture and Transfection HEK293T cells were cultured in DMEM（Gibco） supplemented with 10% fetal bovine serum at 37°C with 5% CO₂. For transfection, cells were seeded in 6-well plates and transfected at 70-90% confluence using Lipofectamine™ 3000 reagent (Invitrogen) according to the manufacturer's instructions. Briefly, 2.5 µg of plasmid DNA was diluted in 125 µL Opti-MEM medium, mixed with 5 µL Lipofectamine™ 3000 reagent (pre-diluted in 125 µL Opti-MEM), and incubated for 10-15 min at room temperature before adding to the cells. RNA Extraction and Quantitative RT-PCR (qPCR) Total RNA was extracted from transfected cells 48 hours post-transfection using TRIzol reagent (Invitrogen) following the standard protocol. RNA concentration and purity were determined spectrophotometrically. Genomic DNA was removed using DNase I (Takara). First-strand cDNA was synthesized using a PrimeScript™ RT reagent Kit (Takara). Quantitative real-time PCR (qPCR) was performed under the following conditions: 95°C for 3 min, followed by 45 cycles of 95°C for 15 s and 60°C for 30 s, and a final melt curve analysis. The expression levels of target genes were normalized to GAPDH. Primers used are listed in Table 2. Western Blot Analysis Transfected cells were harvested 48 hours post-transfection and lysed in RIPA buffer containing protease inhibitors. Protein concentration was determined using a BCA assay kit (Yeasen Biotechnology). Equal amounts of protein (20-30 µg) were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). Membranes were blocked with 5% non-fat milk for 1.5 hours at room temperature and then incubated overnight at 4°C with primary antibodies (anti-FLAG, Mabnus, 1:2000; anti-GFP, DIAAN, 1:1000; anti-GAPDH, CST, 1:1000). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1.5 hours at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system. RESULTS Clinical and Genetic Profile of Pediatric Patients with PRRT2 Variants In this study, we summarized the gender, age at onset, seizure type, disease severity epilepsy syndrome, family history, electroencephalogram (EEG), magnetic resonance imaging (MRI) of the brain, medication and prognosis, PRRT2 gene mutation sites, and parental verification information of these pediatric patients. There were 6 females and 8 males, with onset ages ranging from 3 to 7 months.Among them, 13 patients had PRRT2 point mutations. One of the mutations was a hotspot mutation (c.649dupC p.Arg217Profs*8), and the other two mutations had been previously reported (c.189delC p.Lys64Argfs*26 and c.971G>C p.Gly324Ala), but no functional validation was conducted One patient had a PRRT2 gene fragment deletion (2-4 exon deficient). 11 patients exhibited the BFIE phenotype, one patient had PKD, and the remaining two patients could not be classified as having any type of epilepsy syndrome. In terms of treatment, most patients responded well to levetiracetam and oxcarbazepine, with an overall good prognosis. Eleven pediatric patients (P1-P5, P7, P10-P14) carried a frameshift mutation, (c.649dupC p.Arg217Profs*8), in the PRRT2 gene, which was clearly classified as "pathogenic (Path)" or "likely pathogenic (Likely Path)" and was the most prevalent genetic etiology in this study cohort. The other variants included two point mutations (c.971G>C p.Gly324Ala (P6); c.189delC p.Lys64Argfs*26 (P9) and a frameshift deletion of exons 2-4 (P8)). Six cases (57.1%) had a clear positive family history, with one parent or family member (grandfather, sisters) having a history of epilepsy. The remaining cases were considered to have de novo mutations or unknown family history. Generalized tonic-clonic seizures (GTCS) are the most common type of seizures (12 cases), and focal seizures are also relatively common (4 cases). Nine pediatric patients (71.4%) exhibited normal intellectual and motor development. Five pediatric patients (P2, P5, P8, P9, P13) showed language or motor developmental delays. Notably, none of these patients carried the classic c.649dupC p.Arg217Profs*8 mutation or more severe mutation types (such as c.189delC p.Lys64Argfs*26, exon deletions), suggesting that the type of genetic variation may be associated with the severity of neurodevelopmental outcomes. Eleven pediatric patients exhibited abnormal electroencephalograms (EEGs). Abnormal discharges were predominantly observed during sleep stages. The discharge locations predominantly occurred in the posterior regions of the brain (occipital and posterior temporal lobes), but could also involve the central and frontal regions. Three pediatric patients had completely normal EEGs, which is consistent with the diagnostic characteristics of BFIE. Thirteen pediatric patients showed no significant structural abnormalities on cranial MRI. Only one pediatric patient (P13) had abnormal MRI findings (abnormal signal in the tentorial cisterns and dural enhancement in the left temporal pole). This patient also exhibited developmental delay, and its clinical significance requires further investigation. The most commonly used antiepileptic drugs are oxcarbazepine (OXC), levetiracetam (LEV), and valproic acid (VPA). The overall prognosis is excellent: among the 14 pediatric patients, 12 (85.7%) achieved seizure-free status. OXC has demonstrated good efficacy and tolerability, and has been used effectively by multiple pediatric patients. Some pediatric patients exhibited poor initial response to LEV or VPA (P4, P7, P12, P13, P14) or developed adverse reactions (such as constipation caused by VPA, P1). After switching to another medication (especially OXC), seizures were mostly controlled. Construction of the wild-type and mutant expression vectors A. The mutation c.971G>C p.Gly324Ala is located in exon 3, and the c.971 base is mutated from G to C. The mutation of the amino acid glycine (G) at position 324 to alanine (A) B. The c.189delC p.Lys64Argfs*26 mutation is located at Exon 2, the c.189 base is missing, and the amino acid lysine (K) at position 64 is mutated to arginine (R), resulting in a termination codon at position 26.(Figure 2A) Q PCR test results The results of three replicates showed that there was no significant change in the mRNA expression level of mutant mut1 compared to wt, while the mRNA expression level of mutant mut2 was significantly downregulated compared to wt (Figure 2B). Western blot results The results of three replicates all showed that bands were detected at the 65 kDa position for the wild-type wt and mutant mut2 (consistent PMID reported in the literature: 22832103). The expression level of the mutant mut2 protein was significantly lower than that of the wild-type wt protein, and no bands were detected near the target position for the mutant mut1 (no bands were detected even after prolonged tablet pressing).(Figure 2C) DISCUSSION The PRRT2 gene is located at chromosomal region 16p11.2. This gene has a total length of 3794 bp and contains 4 exons; this gene encodes a transmembrane protein for proline. PRRT2 is expressed in brain regions such as the cortex, hippocampus, basal ganglia, and cerebellum, increasing the extent of its clinical phenotype. This gene was first reported by Chen et al. [6] and was detected as a pathogenic gene for PKD in 8 PKD families through whole-exome sequencing. Subsequently, Heron et al. [7] also reported this gene mutation in BFIE patients. With the improvement of gene detection technology, additional phenotypes are gradually being reported. Currently, the most common phenotype known is BFIE, followed by PKD [8], and a very small number of patients exhibit migraines. This study further expands our understanding of the genotype-phenotype correlation in PRRT2-related diseases by conducting clinical and genetic analyses on 14 unrelated Chinese pediatric epilepsy patients carrying PRRT2 gene variations, combined with functional experimental verification. This study not only reconfirmed that the hotspot mutation c.649dup p.Arg217ProfsTer8 in the PRRT2 gene is the main pathogenic factor for benign familial infantile epilepsy (BFIE), but more importantly, through functional experiments, it revealed the pathogenicity of two variations (c.189delC p.Lys64Argfs*26) and c.971G>C p.Gly324Ala), providing new evidence for understanding the phenotypic differences caused by different mutations in this gene. High consistency between PRRT2 c.649dup hotspot mutation and typical BFIE phenotype The vast majority of patients in this cohort (11/14) carry the c.649dupC p.Arg217Profs*8) hotspot mutation in the PRRT2 gene, and their clinical phenotypes are highly consistent with the classic characteristics of benign familial infantile epilepsy (BFIE) [9]. These patients all exhibited onset in infancy, manifesting as clusters of generalized tonic-clonic seizures (GTCS) or focal seizures. Most of them had normal neurological development, and although some showed abnormal discharges in the posterior or central regions of the electroencephalogram, most did not have severe background activity abnormalities. More importantly, they responded extremely well to drug treatment. This finding strongly supports the central role of c.649dup as the primary pathogenic mutation in BFIE and suggests that this specific genotype may indicate a relatively benign clinical course and excellent treatment outcomes. Non-classical mutations and phenotypic expansion: from benign epilepsy to neurodevelopmental disorders It is noteworthy that the cases with more severe phenotypes in this cohort are all associated with non-classical mutations other than c.649dup. For instance, children carrying c.189delC p.Lys64Argfs*26 (P9) and deletions of exons 2-4 (P8) all exhibited language and motor development delays, as well as relatively more difficult-to-control seizures. This aligns with the increasing number of recent research reports[10], indicating that certain PRRT2 mutations (such as c.189delC p.Lys64Argfs*26) or larger deletions may be associated with more complex phenotypes, transcending the scope of traditional BFIE and involving paroxysmal kinesigenic dyskinesia (PKD), episodic dystonia, and varying degrees of neurodevelopmental delay. In this study, there are 1 case of fragment deletion, 1 case of missense mutation, and 7 cases of frameshift mutation. Among the patients with frameshift mutations, 6 (c.649dupC p.Arg217Profs*8) were hotspot mutations. Another frameshift mutation was P9 (c.189delC p.Lys64Argfs*26), and one missense mutation was P6 (c.971G>C p.Gly324Ala). These two mutations have been reported in previous studies, but no functional validation had been conducted before; the P9 phenotype is PKD, and the P6 phenotype is BFIE. Using several prediction algorithm tools, we predict that neither of these mutations will affect splicing. In vitro, we used a microgene strategy to simulate physiological processes and extract mRNA from the designed microgenes. By detecting the expression levels of wild-type and mutant transcripts, we confirmed that the c.971G>C p.Gly324Ala site has an impact on splicing and that this mutation can cause a decrease in mRNA expression. Further analysis and Western blotting revealed that a band at the target band was present, which is consistent with the findings in the literature [1]; however, the expression level was significantly lower than that in the wild-type group. These findings indicate that the abnormal transcript expression altered the expression level of the protein. Although the mutation caused downregulation of mRNA expression, it still produced functional proteins, resulting in a mild clinical phenotype and a good prognosis. However, the c.189delC p.Lys64Argfs*26) mutation did not cause any changes in mRNA expression. After further Western blotting, we did not detect any bands at the target location, suggesting that this mutation may lead to a decrease in the stability of the mutated protein, resulting in undetectable protein degradation bands. The clinical phenotype of this patient was complex, and after years of antiepileptic treatment, the condition could not be completely controlled. In addition, the patient's father had a history of epilepsy, and the patient's mutation had been inherited from his father. The pathogenicity of this patient’s condition is clear, indicating that the mutation at this locus leads to significant protein damage and future adverse effects.This indicates that not all mutations lead to completely consistent "benign" outcomes. Therefore, in clinical genetic counseling, families with detected non-hotspot mutations should be informed of the potential variability in phenotype, and long-term developmental follow-up is recommended. Therapeutic response and drug selection: potential advantages of oxcarbazepine In terms of treatment, the observations from this cohort provide important clinical medication references. Although levetiracetam (LEV) and valproic acid (VPA) are effective for some pediatric patients, we also observed multiple cases (P4, P7, P12, P13, P14) where initial use of LEV or VPA yielded poor efficacy or intolerance. In contrast, oxcarbazepine (OXC) demonstrated excellent efficacy and good tolerability in this cohort, with multiple pediatric patients successfully controlling seizures after switching to or directly using OXC. This is consistent with OXC's role as a sodium channel blocker [11], which may more effectively inhibit the pathophysiological mechanism of neuronal hyperexcitability caused by loss of function of PRRT2 [12]. Although larger prospective studies are still needed for verification, the real-world data from this study suggest that for PRRT2-related epilepsy, OXC may be considered as one of the first-line or preferred antiepileptic drugs. CONCLUSION This study has several limitations. Firstly, it is a retrospective study with a limited sample size. Secondly, the lack of long-term video electroencephalogram monitoring and neuropsychological development assessment follow-up for patients may prevent the full capture of subtle clinical changes and developmental trajectories. Future research is needed to conduct multicenter, large-sample prospective cohort studies, combined with functional experiments, to more deeply reveal the molecular mechanisms underlying genotype-phenotype associations. In summary, this study, through the analysis of Fourteen children with PRRT2-related diseases, consolidates the strong association between the c.649dup mutation and typical benign BFIE, while revealing that non-classical mutations may lead to a broader and more severe phenotypic spectrum, including neurodevelopmental disorders. In terms of treatment, oxcarbazepine (OXC) shows promising application prospects. Our results emphasize the value of genetic testing in infantile epilepsy, which not only provides crucial information for diagnosis and prognosis but also serves as an important basis for guiding individualized treatment and genetic counseling. Abbreviations BFIE benign familial infantile epilepsy PKD paroxysmal motor disorders FHM familial hemiplegic migraine EEG electroencephalogram MRI magnetic resonance imaging GTCS Generalized tonic-clonic seizures OXC oxcarbazepine LEV levetiracetam VPA valproic acid Declarations Acknowledgments We are extremely grateful to the patient and her family for their full cooperation throughout the study. Author Contributions JL and QL designed the study. DF and SL performed the clinical materials collections. QX and SH did the literature search and wrote the paper. FL and JH reviewed and edited the manuscript. All authors read and approved the manuscript. Ethics approval and consent to participate This study has been approved by the Ethics Review Committee of Dongguan Children’s Hospital Affiliated to Guangdong Medical University.（Ethics number：LL2022092218）. The study strictly followed the relevant provisions of the declaration of Helsinki. Consent for publication The patients or their parents have signed a written informed consent to publish their clinical and genetic details. We explained that patients' names and photos would not be published. This consent form was given directly to the research coordinator during the submission of the manuscript. Competing interests This manuscript has not been published and is not under consideration for publication elsewhere. We have no conflicts of interest to disclose. Acknowledgments The authors thank the patient for sample contribution. Funding Information This work was supported by The 2023 Dongguan Social Development Science and Technology Key Project Fund（grant numbers: 20231800935362）. 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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-7941362","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560056637,"identity":"6805687a-1bd4-43a0-ae68-fdc847ba4339","order_by":0,"name":"Fen Liu","email":"","orcid":"","institution":"Dongguan Children’s Hospital Affiliated to Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fen","middleName":"","lastName":"Liu","suffix":""},{"id":560056638,"identity":"f72eccb3-2798-41e1-b291-595e14f464d2","order_by":1,"name":"Juhua Huang","email":"","orcid":"","institution":"Dongguan Children’s Hospital Affiliated to Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Juhua","middleName":"","lastName":"Huang","suffix":""},{"id":560056639,"identity":"9ebfbb9f-9cd6-4c05-9a4b-42a98846e473","order_by":2,"name":"Qiong Xiao","email":"","orcid":"","institution":"Dongguan Children’s Hospital Affiliated to Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Xiao","suffix":""},{"id":560056640,"identity":"81a64590-0c49-48cc-ad02-beebb183bd97","order_by":3,"name":"Shan Huang","email":"","orcid":"","institution":"Dongguan Children’s Hospital Affiliated to Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Huang","suffix":""},{"id":560056641,"identity":"fc5455b6-04a5-43f8-9d1f-b1b28aff47b1","order_by":4,"name":"Simei Lin","email":"","orcid":"","institution":"Dongguan Children’s Hospital Affiliated to Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Simei","middleName":"","lastName":"Lin","suffix":""},{"id":560056642,"identity":"804d8b11-c180-4a04-aeba-5772ba494dca","order_by":5,"name":"Danna Fang","email":"","orcid":"","institution":"Dongguan Children’s Hospital Affiliated to Guangdong Medical University","correspondingAuthor":false,"prefix":"","firstName":"Danna","middleName":"","lastName":"Fang","suffix":""},{"id":560056643,"identity":"fba17722-6022-42fd-890d-e92b8ff96a33","order_by":6,"name":"Jianwei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACPgYGxgMJFRJ2/OyNjQ8/EKOFDYgPJJyxSJbsOdxsLEG0Fsa2CsYNN9LbBHiI0sJ++MCBB2ckmBluPmxjkGCwk9NtIKSFJy0B5Bc+xtmJbQ8KGJKNzQ4QdFiOAdAvEszM0ontBhIMBxK3EdTC/8bgQGKbBGOb5ME2CR6itEjkQLT0AHURq+VZAshhyRI8icBANiDCL/z8yQcf/qios7M/fvzhww8VdnIEtaABA9KUj4JRMApGwSjAAQBB+EKQF9QEywAAAABJRU5ErkJggg==","orcid":"","institution":"Dongguan Children’s Hospital Affiliated to Guangdong Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Li","suffix":""},{"id":560056644,"identity":"6fe8ab6f-68f5-4ae9-ae2e-f012a78b0a50","order_by":7,"name":"Qingming Luo","email":"","orcid":"","institution":"Dongguan Maternal and Child Health Care Hospital","correspondingAuthor":false,"prefix":"","firstName":"Qingming","middleName":"","lastName":"Luo","suffix":""}],"badges":[],"createdAt":"2025-10-25 12:37:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7941362/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7941362/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12920-026-02379-6","type":"published","date":"2026-04-30T15:58:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":98246340,"identity":"8e702630-2b76-4219-bafa-700cbc1d8447","added_by":"auto","created_at":"2025-12-15 16:19:00","extension":"jpg","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":63613,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/1aa9b3894a440a6e872906fa.jpg"},{"id":98246343,"identity":"b3e3f545-c699-4ade-b85d-fa6f2807cd5b","added_by":"auto","created_at":"2025-12-15 16:19:00","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":452608,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript2025.12.8.doc","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/b8b0b4ac853c122b4d4621fd.doc"},{"id":98246441,"identity":"c3eef83b-b6ff-4025-b8fb-dcf0da97460b","added_by":"auto","created_at":"2025-12-15 16:19:03","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":668808,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/fc2e45239e5c52d5845d5af1.jpg"},{"id":98246298,"identity":"c3e4c715-2957-4c36-bd3b-320fa25f4802","added_by":"auto","created_at":"2025-12-15 16:18:57","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":68096,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.doc","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/ff13960157e09209b98250fd.doc"},{"id":98246445,"identity":"ec146770-94bf-49af-a85f-81b22baf5d12","added_by":"auto","created_at":"2025-12-15 16:19:03","extension":"doc","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20992,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.doc","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/e808604563b4149773ffba10.doc"},{"id":98246295,"identity":"cd5b534d-6ca3-4f1e-b4cf-eb3e469d6782","added_by":"auto","created_at":"2025-12-15 16:18:57","extension":"json","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9685,"visible":true,"origin":"","legend":"","description":"","filename":"ea38b7344efd4606975a3e86a9207b68.json","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/d973fc11b8958cc8b22b9334.json"},{"id":98246325,"identity":"1f9db159-3359-4941-82da-959b3064181f","added_by":"auto","created_at":"2025-12-15 16:18:59","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2906987,"visible":true,"origin":"","legend":"","description":"","filename":"unprocessedversionsofallblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/59e6ba06c14f8842c8280dc4.pdf"},{"id":98246456,"identity":"85270c14-19ad-4d2b-a3cb-409c784e7b2d","added_by":"auto","created_at":"2025-12-15 16:19:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30046,"visible":true,"origin":"","legend":"\u003cp\u003eThree types of point mutations were detected in 14 patients (Figure 1): 11 had a mutation of c.649dupC (p.Arg217Profs*8), 1 had a mutation of c.189delC (p.Lys64Argfs*26), and 1 had a mutation of c.971G\u0026gt;C (p.Gly324Ala).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/08cfba36714b5357290a1131.jpg"},{"id":98246255,"identity":"5e1ac45a-2cf3-4e32-83e2-ebd4264e185a","added_by":"auto","created_at":"2025-12-15 16:18:54","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":668808,"visible":true,"origin":"","legend":"\u003cp\u003eA: Results of the construction of the PRRT2 wt/mut vector;\u003c/p\u003e\n\u003cp\u003eB: Relative mRNA expression levels between the PRRT2 mutant and wild-type (QPCR) strains. The primers PRRT2-qPCR-F and PRRT2-qPCR-R were used to detect the expression levels of the wild-type and mutant transcripts in the p3XFlag CMV-7.1 vector. With respect to the p3XFlag CMV-7.1 series of vectors (p3XFlag CMV-7.1 PRRT2), the results of three replicates showed that the mRNA expression level of the mutant mut1 did not significantly change compared to that of the wt, while the mRNA expression level of the mutant mut2 was significantly downregulated compared to that of the wt.\u003c/p\u003e\n\u003cp\u003eC: Relative expression levels (WBs) of the PRRT2 mutant and wild-type proteins. The expression levels of the wt and mut proteins in the p3XFlag CMV-7.1 vector were detected using flag-labeled antibodies. With respect to the p3XFlag CMV-7.1 series of vectors (p3XFlag CMV-7.1 PRRT2), the theoretical size of the wild-type protein is 36.9 kDa, the theoretical size of the mutant mut1 protein is 11.7 kDa, and the theoretical size of the mutant mut2 protein is 36.9 kDa. The results of three replicates all showed that bands were detected at the 65 kDa position for the wild-type wt and mutant mut2 (consistent PMID reported in the literature: 22832103). After the gene mutation, the expression level of the mutant mut2 protein was significantly downregulated compared to that of the wild-type wt protein. No bands were detected near the target position for mutant mut1 (no bands were detected even after prolonged tablet pressing). It is speculated that the stability of the mutant mut protein decreased after the gene mutation, leading to protein degradation and the formation of undetectable bands.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/9fbead46609aae5beea603a6.jpg"},{"id":108442337,"identity":"d9663ca2-d1a2-4bf7-a5a5-3036f83272ac","added_by":"auto","created_at":"2026-05-04 17:05:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":872360,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/354558f4-e1c7-4764-a4f4-f0b205b6208f.pdf"},{"id":98246326,"identity":"d55381bb-1f9b-4db3-bac3-761485e6602c","added_by":"auto","created_at":"2025-12-15 16:18:59","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":68096,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.doc","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/c7007c25fdcefb054852fd18.doc"},{"id":98246459,"identity":"bf6cfcb5-e453-4238-80d9-3707725b38d7","added_by":"auto","created_at":"2025-12-15 16:19:04","extension":"doc","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20992,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.doc","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/8f5839ab3731802c67fd8e33.doc"},{"id":98246455,"identity":"7beb6841-dfee-4ab9-b753-ba60c5875cb9","added_by":"auto","created_at":"2025-12-15 16:19:04","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2906987,"visible":true,"origin":"","legend":"","description":"","filename":"unprocessedversionsofallblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7941362/v1/fe24f2a00c2c4ffa12db0fc6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genotypic and Phenotypic Spectrum of PRRT2-Related Variations: Clinical Analysis and Treatment Response in Fourteen Unrelated Chinese Patients","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eChromosomal 16P11.2 copy number variation is one of the most common genetic causes of neurodevelopmental disorders. In addition to intellectual disabilities and autism spectrum disorders, the clinical phenotype of the variation is also characterized by seizures, and some patients only exhibit seizures with no other signs of disorder. Most scholars believe that the main cause of epilepsy is the inclusion of an important epilepsy-related gene, PRRT2. PRRT2 is a profile-rich protein that interacts with the synaptic-associated protein SNAP25 to interfere with glutamate signaling and glutamate receptor activity, leading to increased glutamate release and increased neuronal excitability [1-2]. Heterozygous variations in PRRT2 can cause four different neurological disorders, including benign familial infantile epilepsy (BFIE), paroxysmal motor disorders (PKD), PKD with infantile convulsions, and familial hemiplegic migraine (FHM). Among the diseases related to PRRT2 mutations, the majority of patients have BFIE characterized by self-limited epilepsy; this disorder typically begins to develop at 6 months of age, but is accompanied by normal psychomotor development and a good prognosis [3]. However, variations in different loci of the same gene still exhibit different clinical manifestations. Luo et al. further expanded the disease phenotype by studying PRRT2 mutations in 44 epilepsy patients [3]. However, genetic analysis of the phenotypic differences caused by PRRT2 mutations is still needed.\u003c/p\u003e\n\u003cp\u003eHere, we analyzed the genetic characteristics of Fourteen children with neurological disorders caused by PRRT2 mutations. Most patients present with BFIE . Different loci correspond to different phenotypes, and based on the severity of their phenotypes, we further conducted in vitro functional validation, revealing the pathogenesis of this locus, and discussed the relationships among the PRRT2 genotype and phenotype, treatment, and prognosis.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we retrospectively analyzed Fourteen children from unrelated families with epilepsy or paroxysmal motor disorders, all of whom had previously been confirmed to have PRRT2 gene mutations through genetic testing. Clinical data, including age of onset, family history, developmental history, clinical manifestations, electroencephalogram (EEG) signals, magnetic resonance imaging (MRI), treatment, and follow-up, were collected and analyzed. All Eleven patients were diagnosed according to the diagnostic criteria for BFIE, with reference to [4] and [5], respectively. One patient was diagnosed with PKD, while the other two patients had an episode type that could not be classified as a certain syndrome.This study was approved by the Ethics Committee of Dongguan Children\u0026apos;s Hospital.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree milliliters of peripheral blood was extracted from each patient for DNA analysis. Some patients underwent whole-genome sequencing, while others selected a\u0026nbsp;custom-designed gene panel targeting the coding exons of 1,170 epilepsy-associated genes, including PRRT2,\u0026nbsp;which contained 16402 coding regions and a total of 2682931 bases. Using bioinformatics analysis techniques, the sequencing results were paired with reference sequences and combined with multiple databases and egg function prediction tools to determine clinically significant variations in the sample DNA. The reference genome GRCh37 was paired with the sequences, after which the results of single nucleotide variants\u0026nbsp;were validated via Sanger sequencing. The large deletion encompassing exons 2\u0026ndash;4 was confirmed by a custom-designed gene panel targeting the coding exons of 1,170 epilepsy-associated genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Functional Studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid Construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExpression vectors for wild-type (WT) and mutant (mut1, mut2) human\u0026nbsp;PRRT2\u0026nbsp;were constructed based on the p3XFlag-CMV-7.1 backbone. The codon-optimized full-length\u0026nbsp;PRRT2\u0026nbsp;coding sequence (CDS) was used as the template.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) p3XFlag-CMV-7.1-WT:\u0026nbsp;\u003c/strong\u003eThe wild-type (WT) PRRT2 coding sequence was amplified by PCR using a synthetic full-length PRRT2 CDS as the template, with primers CMV-PRRT2-EcoRI-F and CMV-PRRT2-SalI-R（Table 2）. The resulting PCR product and the p3XFlag-CMV-7.1 vector were double-digested with EcoRI and SalI, followed by ligation to construct the p3XFlag-CMV-7.1-wt expression vector.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(b) p3XFlag-CMV-7.1-mut1:\u0026nbsp;\u003c/strong\u003eThe mut1 mutation was introduced using a two-step overlap PCR strategy. First, two fragments (mut1-1 and mut1-2) were generated using primer pairs CMV-PRRT2-EcoRI-F/PRRT2-mut1-R and PRRT2-mut1-F/CMV-PRRT2-SalI-R, respectively. These fragments served as templates for a second PCR with the flanking primers CMV-PRRT2-EcoRI-F and CMV-PRRT2-SalI-R to generate the full-length EcoRI-mut1-SalI fragment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(c) p3XFlag-CMV-7.1-mut2:\u0026nbsp;\u003c/strong\u003eThe mut2 fragment was directly amplified using primers CMV-PRRT2-EcoRI-F and PRRT2-mut2-SalI-R.\u003c/p\u003e\n\u003cp\u003eAll PCR-amplified fragments (WT, mut1, mut2) were sequenced verified, digested with EcoRI/SalI, and cloned into the correspondingly digested p3XFlag-CMV-7.1 vector. The final constructs were validated by colony PCR and Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture and Transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK293T cells were cultured in DMEM（Gibco）\u0026nbsp;supplemented with 10% fetal bovine serum at 37\u0026deg;C with 5% CO₂. For transfection, cells were seeded in 6-well plates and transfected at 70-90% confluence using Lipofectamine\u0026trade; 3000 reagent (Invitrogen) according to the manufacturer\u0026apos;s instructions. Briefly, 2.5 \u0026micro;g of plasmid DNA was diluted in 125 \u0026micro;L Opti-MEM medium, mixed with 5 \u0026micro;L Lipofectamine\u0026trade; 3000 reagent (pre-diluted in 125 \u0026micro;L Opti-MEM), and incubated for 10-15 min at room temperature before adding to the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Extraction and Quantitative RT-PCR (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from transfected cells 48 hours post-transfection using TRIzol reagent (Invitrogen) following the standard protocol. RNA concentration and purity were determined spectrophotometrically. Genomic DNA was removed using DNase I (Takara). First-strand cDNA was synthesized using a PrimeScript\u0026trade; RT reagent Kit (Takara). Quantitative real-time PCR (qPCR) was performed under the following conditions: 95\u0026deg;C for 3 min, followed by 45 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 30 s, and a final melt curve analysis. The expression levels of target genes were normalized to GAPDH. Primers used are listed in Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransfected cells were harvested 48 hours post-transfection and lysed in RIPA buffer containing protease inhibitors. Protein concentration was determined using a BCA assay kit (Yeasen Biotechnology). Equal amounts of protein (20-30 \u0026micro;g) were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). Membranes were blocked with 5% non-fat milk for 1.5 hours at room temperature and then incubated overnight at 4\u0026deg;C with primary antibodies (anti-FLAG, Mabnus, 1:2000; anti-GFP, DIAAN, 1:1000; anti-GAPDH, CST, 1:1000). After washing, membranes were incubated with HRP-conjugated secondary antibodies for 1.5 hours at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eClinical and Genetic Profile of Pediatric Patients with PRRT2 Variants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we summarized the gender, age at onset, seizure type, disease severity epilepsy syndrome, family history, electroencephalogram (EEG), magnetic resonance imaging (MRI) of the brain, medication and prognosis, PRRT2 gene mutation sites, and parental verification information of these pediatric patients. There were 6 females and 8 males, with onset ages ranging from 3 to 7 months.Among them, 13 patients had PRRT2 point mutations. One of the mutations was a hotspot mutation (c.649dupC p.Arg217Profs*8), and the other two mutations had been previously reported (c.189delC\u0026nbsp;p.Lys64Argfs*26 and c.971G\u0026gt;C p.Gly324Ala), but no functional validation was conducted One patient had a PRRT2 gene fragment deletion (2-4 exon deficient).\u0026nbsp;11\u0026nbsp;patients exhibited the BFIE phenotype, one patient had PKD, and the remaining\u0026nbsp;two\u0026nbsp;patients could not be classified as having any type of epilepsy syndrome. In terms of treatment, most patients responded well to levetiracetam and oxcarbazepine, with an overall good prognosis.\u003c/p\u003e\n\u003cp\u003eEleven pediatric patients (P1-P5, P7, P10-P14) carried a frameshift mutation, (c.649dupC p.Arg217Profs*8), in the PRRT2 gene, which was clearly classified as \"pathogenic (Path)\" or \"likely pathogenic (Likely Path)\" and was the most prevalent genetic etiology in this study cohort. The other variants included two point mutations\u0026nbsp;(c.971G\u0026gt;C\u0026nbsp;p.Gly324Ala\u0026nbsp;(P6); c.189delC\u0026nbsp;p.Lys64Argfs*26\u0026nbsp;(P9) and a frameshift deletion of exons 2-4 (P8)). Six cases (57.1%) had a clear positive family history, with one parent or family member (grandfather, sisters) having a history of epilepsy. The remaining cases were considered to have de novo mutations or unknown family history. Generalized tonic-clonic seizures (GTCS) are the most common type of seizures (12 cases), and focal seizures are also relatively common (4 cases). Nine \u0026nbsp;pediatric patients (71.4%) exhibited normal intellectual and motor development.\u0026nbsp;Five\u0026nbsp;pediatric patients (P2, P5, P8, P9, P13) showed language or motor developmental delays. Notably, none of these patients carried the classic c.649dupC\u0026nbsp;p.Arg217Profs*8 mutation or more severe mutation types (such as c.189delC\u0026nbsp;p.Lys64Argfs*26, exon deletions), suggesting that the type of genetic variation may be associated with the severity of neurodevelopmental outcomes. Eleven\u0026nbsp;pediatric patients exhibited abnormal electroencephalograms (EEGs). Abnormal discharges were predominantly observed during sleep stages. The discharge locations predominantly occurred in the posterior regions of the brain (occipital and posterior temporal lobes), but could also involve the central and frontal regions.\u0026nbsp;Three\u0026nbsp;pediatric patients had completely normal EEGs, which is consistent with the diagnostic characteristics of BFIE. Thirteen pediatric patients showed no significant structural abnormalities on cranial MRI. Only one pediatric patient (P13) had abnormal MRI findings (abnormal signal in the tentorial cisterns and dural enhancement in the left temporal pole). This patient also exhibited developmental delay, and its clinical significance requires further investigation. The most commonly used antiepileptic drugs are oxcarbazepine (OXC), levetiracetam (LEV), and valproic acid (VPA). The overall prognosis is excellent: among the 14 pediatric patients, 12 (85.7%) achieved seizure-free status. OXC has demonstrated good efficacy and tolerability, and has been used effectively by multiple pediatric patients. Some pediatric patients exhibited poor initial response to LEV or VPA (P4, P7, P12,\u0026nbsp;P13, P14) or developed adverse reactions (such as constipation caused by VPA, P1). After switching to another medication (especially OXC), seizures were mostly controlled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe wild-type and mutant expression vectors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The mutation c.971G\u0026gt;C p.Gly324Ala is located\u0026nbsp;in exon 3, and the c.971 base is mutated from G to C. The mutation of the amino acid glycine (G) at position 324 to alanine (A)\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;The c.189delC p.Lys64Argfs*26\u0026nbsp;mutation is located at Exon 2, the c.189 base is missing, and the amino acid lysine (K) at position 64 is mutated to arginine (R), resulting in a termination codon at position 26.(Figure 2A)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQ\u003c/strong\u003e\u003cstrong\u003ePCR test results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of three replicates showed that there was no significant change in the mRNA expression level of mutant mut1 compared to wt, while the mRNA expression level of mutant mut2 was significantly\u0026nbsp;downregulated compared to wt (Figure 2B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of three replicates all showed that bands were detected at the 65 kDa position for the wild-type wt and mutant mut2 (consistent PMID reported in the literature: 22832103). The expression level of the mutant mut2 protein was significantly lower than that of the wild-type wt protein, and no bands were detected near the target position for the mutant mut1 (no bands were detected even after prolonged tablet pressing).(Figure 2C)\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe PRRT2 gene is located at chromosomal region 16p11.2. This gene has a total length of 3794 bp and contains 4 exons; this gene encodes a transmembrane protein for proline. PRRT2 is expressed in brain regions such as the cortex, hippocampus, basal ganglia, and cerebellum, increasing the extent of its clinical phenotype. This gene was first reported by Chen et al. [6] and was detected as a pathogenic gene for PKD in 8 PKD families through whole-exome sequencing. Subsequently, Heron et al. [7] also reported this gene mutation in BFIE patients. With the improvement of gene detection technology, additional phenotypes are gradually being reported. Currently, the most common phenotype known is BFIE, followed by PKD [8], and a very small number of patients exhibit migraines. This study further expands our understanding of the genotype-phenotype correlation in PRRT2-related diseases by conducting clinical and genetic analyses on 14 unrelated Chinese pediatric epilepsy patients carrying PRRT2 gene variations, combined with functional experimental verification. This study not only reconfirmed that the hotspot mutation c.649dup p.Arg217ProfsTer8 in the PRRT2 gene is the main pathogenic factor for benign familial infantile epilepsy (BFIE), but more importantly, through functional experiments, it revealed the pathogenicity of two variations (c.189delC p.Lys64Argfs*26) and c.971G\u0026gt;C p.Gly324Ala), providing new evidence for understanding the phenotypic differences caused by different mutations in this gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh consistency between PRRT2 c.649dup hotspot mutation and typical BFIE phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe vast majority of patients in this cohort (11/14) carry the c.649dupC \u0026nbsp;p.Arg217Profs*8) hotspot mutation in the PRRT2 gene, and their clinical phenotypes are highly consistent with the classic characteristics of benign familial infantile epilepsy (BFIE)\u0026nbsp;[9]. These patients all exhibited onset in infancy, manifesting as clusters of generalized tonic-clonic seizures (GTCS) or focal seizures. Most of them had normal neurological development, and although some showed abnormal discharges in the posterior or central regions of the electroencephalogram, most did not have severe background activity abnormalities. More importantly, they responded extremely well to drug treatment. This finding strongly supports the central role of c.649dup as the primary pathogenic mutation in BFIE and suggests that this specific genotype may indicate a relatively benign clinical course and excellent treatment outcomes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNon-classical mutations and phenotypic expansion: from benign epilepsy to neurodevelopmental disorders\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is noteworthy that the cases with more severe phenotypes in this cohort are all associated with non-classical mutations other than c.649dup. For instance, children carrying c.189delC p.Lys64Argfs*26 (P9) and deletions of exons 2-4 (P8) all exhibited language and motor development delays, as well as relatively more difficult-to-control seizures. This aligns with the increasing number of recent research reports[10], indicating that certain PRRT2 mutations (such as c.189delC p.Lys64Argfs*26) or larger deletions may be associated with more complex phenotypes, transcending the scope of traditional BFIE and involving paroxysmal kinesigenic dyskinesia (PKD), episodic dystonia, and varying degrees of neurodevelopmental delay. In this study, there are 1 case of fragment deletion, 1 case of missense mutation, and 7 cases of frameshift mutation. Among the patients with frameshift mutations, 6 (c.649dupC p.Arg217Profs*8) were hotspot mutations. Another frameshift mutation was P9 (c.189delC\u0026nbsp;p.Lys64Argfs*26), and one missense mutation was P6 (c.971G\u0026gt;C p.Gly324Ala). These two mutations have been reported in previous studies, but no functional validation had been conducted before; the P9 phenotype is PKD, and the P6 phenotype is BFIE. Using several prediction algorithm tools, we predict that neither of these mutations will affect splicing. In vitro, we used a microgene strategy to simulate physiological processes and extract mRNA from the designed microgenes. By detecting the expression levels of wild-type and mutant transcripts, we confirmed that the c.971G\u0026gt;C p.Gly324Ala site has an impact on splicing and that this mutation can cause a decrease in mRNA expression. Further analysis and Western blotting revealed that a band at the target band was present, which is consistent with the findings in the literature [1]; however, the expression level was significantly lower than that in the wild-type group. These findings indicate that the abnormal transcript expression altered the expression level of the protein. Although the mutation caused downregulation of mRNA expression, it still produced functional proteins, resulting in a mild clinical phenotype and a good prognosis. However, the c.189delC\u0026nbsp;p.Lys64Argfs*26) mutation did not cause any changes in mRNA expression. After further Western blotting, we did not detect any bands at the target location, suggesting that this mutation may lead to a decrease in the stability of the mutated protein, resulting in undetectable protein degradation bands. The clinical phenotype of this patient was complex, and after years of antiepileptic treatment, the condition could not be completely controlled. In addition, the patient's father had a history of epilepsy, and the patient's mutation had been inherited from his father. The pathogenicity of this patient’s condition is clear, indicating that the mutation at this locus leads to significant protein damage and future adverse effects.This indicates that not all mutations lead to completely consistent \"benign\" outcomes. Therefore, in clinical genetic counseling, families with detected non-hotspot mutations should be informed of the potential variability in phenotype, and long-term developmental follow-up is recommended.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTherapeutic response and drug selection: potential advantages of oxcarbazepine\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn terms of treatment, the observations from this cohort provide important clinical medication references. Although levetiracetam (LEV) and valproic acid (VPA) are effective for some pediatric patients, we also observed multiple cases (P4, P7, P12, P13, P14) where initial use of LEV or VPA yielded poor efficacy or intolerance. In contrast, oxcarbazepine (OXC) demonstrated excellent efficacy and good tolerability in this cohort, with multiple pediatric patients successfully controlling seizures after switching to or directly using OXC. This is consistent with OXC's role as a sodium channel blocker [11], which may more effectively inhibit the pathophysiological mechanism of neuronal hyperexcitability caused by loss of function of PRRT2 [12]. Although larger prospective studies are still needed for verification, the real-world data from this study suggest that for PRRT2-related epilepsy, OXC may be considered as one of the first-line or preferred antiepileptic drugs.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study has several limitations. Firstly, it is a retrospective study with a limited sample size. Secondly, the lack of long-term video electroencephalogram monitoring and neuropsychological development assessment follow-up for patients may prevent the full capture of subtle clinical changes and developmental trajectories. Future research is needed to conduct multicenter, large-sample prospective cohort studies, combined with functional experiments, to more deeply reveal the molecular mechanisms underlying genotype-phenotype associations. In summary, this study, through the analysis of\u0026nbsp;Fourteen\u0026nbsp;children with PRRT2-related diseases, consolidates the strong association between the c.649dup mutation and typical benign BFIE, while revealing that non-classical mutations may lead to a broader and more severe phenotypic spectrum, including neurodevelopmental disorders. In terms of treatment, oxcarbazepine (OXC) shows promising application prospects. Our results emphasize the value of genetic testing in infantile epilepsy, which not only provides crucial information for diagnosis and prognosis but also serves as an important basis for guiding individualized treatment and genetic counseling.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBFIE \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; benign familial infantile epilepsy\u003c/p\u003e\n\u003cp\u003ePKD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; paroxysmal motor disorders\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFHM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; familial hemiplegic migraine\u003c/p\u003e\n\u003cp\u003eEEG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; electroencephalogram\u003c/p\u003e\n\u003cp\u003eMRI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; magnetic resonance imaging\u003c/p\u003e\n\u003cp\u003eGTCS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Generalized tonic-clonic seizures\u003c/p\u003e\n\u003cp\u003eOXC \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; oxcarbazepine\u003c/p\u003e\n\u003cp\u003eLEV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;levetiracetam\u003c/p\u003e\n\u003cp\u003eVPA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; valproic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are extremely grateful to the patient and her family for their full cooperation throughout the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eJL and QL designed the study. DF and SL performed the clinical materials collections. QX and SH did the literature search and wrote the paper. FL and JH reviewed and edited the manuscript. All authors read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study has been approved by the Ethics Review Committee of Dongguan Children’s Hospital Affiliated to Guangdong Medical University.（Ethics number：LL2022092218）. The study strictly followed the relevant provisions of the declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patients or their parents have signed a written informed consent to publish their clinical and genetic details. We explained that patients' names and photos would not be published. This consent form was given directly to the research coordinator during the submission of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript has not been published and is not under consideration for publication elsewhere. We have no conflicts of interest to disclose. Acknowledgments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors thank the patient for sample contribution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by The 2023 Dongguan Social Development Science and Technology Key Project Fund（grant numbers: 20231800935362）.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLee HY, Huang Y, Bruneau N, Roll P, Roberson ED, Hermann M, Quinn E, Maas J, Edwards R, Ashizawa T, Baykan B, Bhatia K, Bressman S, Bruno MK, Brunt ER, Caraballo R, Echenne B, Fejerman N, Frucht S, Gurnett CA, Hirsch E, Houlden H, Jankovic J, Lee WL, Lynch DR, Mohammed S, M\u0026uuml;ller U, Nespeca MP, Renner D, Rochette J, Rudolf G, Saiki S, Soong BW, Swoboda KJ, Tucker S, Wood N, Hanna M, Bowcock AM, Szepetowski P, Fu YH, Pt\u0026aacute;ček LJ. Mutations in the gene PRRT2 cause paroxysmal kinesigenic dyskinesia with infantile convulsions. Cell Rep. 2012 Jan 26;1(1):2-12. doi: 10.1016/j.celrep.2011.11.001.\u003c/li\u003e\n\u003cli\u003eLi M, Niu F, Zhu X, Wu X, Shen N, Peng X, Liu Y. PRRT2 Mutant Leads to Dysfunction of Glutamate Signaling. Int J Mol Sci. 2015 Apr 23;16(5):9134-51. doi: 10.3390/ijms16059134.\u003c/li\u003e\n\u003cli\u003eLuo HY, Xie LL, Hong SQ, Li XJ, Li M, Hu Y, Ma JN, Wu P, Zhong M, Cheng M, Li TS, Jiang L. The Genotype and Phenotype of Proline-Rich Transmembrane Protein 2 Associated Disorders in Chinese Children. Front Pediatr. 2021 May 10;9:676616. doi: 10.3389/fped.2021.676616.\u003c/li\u003e\n\u003cli\u003eVigevano F. Benign familial infantile seizures. Brain Dev. 2005 Apr;27(3):172-7. doi: 10.1016/j.braindev.2003.12.012.\u003c/li\u003e\n\u003cli\u003eBruno MK, Hallett M, Gwinn-Hardy K, Sorensen B, Considine E, Tucker S, Lynch DR, Mathews KD, Swoboda KJ, Harris J, Soong BW, Ashizawa T, Jankovic J, Renner D, Fu YH, Ptacek LJ. Clinical evaluation of idiopathic paroxysmal kinesigenic dyskinesia: new diagnostic criteria. Neurology. 2004 Dec 28;63(12):2280-7. doi: 10.1212/01.wnl.0000147298.05983.50. \u003c/li\u003e\n\u003cli\u003eChen WJ, Lin Y, Xiong ZQ, Wei W, Ni W, Tan GH, Guo SL, He J, Chen YF, Zhang QJ, Li HF, Lin Y, Murong SX, Xu J, Wang N, Wu ZY. Exome sequencing identifies truncating mutations in PRRT2 that cause paroxysmal kinesigenic dyskinesia. Nat Genet. 2011 Nov 20;43(12):1252-5. doi: 10.1038/ng.1008.\u003c/li\u003e\n\u003cli\u003eHeron SE, Grinton BE, Kivity S, Afawi Z, Zuberi SM, Hughes JN, Pridmore C, Hodgson BL, Iona X, Sadleir LG, Pelekanos J, Herlenius E, Goldberg-Stern H, Bassan H, Haan E, Korczyn AD, Gardner AE, Corbett MA, G\u0026eacute;cz J, Thomas PQ, Mulley JC, Berkovic SF, Scheffer IE, Dibbens LM. PRRT2 mutations cause benign familial infantile epilepsy and infantile convulsions with choreoathetosis syndrome. Am J Hum Genet. 2012 Jan 13;90(1):152-60. doi: 10.1016/j.ajhg.2011.12.003. \u003c/li\u003e\n\u003cli\u003eEbrahimi-Fakhari D, Saffari A, Westenberger A, Klein C. The evolving spectrum of PRRT2-associated paroxysmal diseases. Brain. 2015 Dec;138(Pt 12):3476-95. doi: 10.1093/brain/awv317.\u003c/li\u003e\n\u003cli\u003eLee J, Kim YO, Lim BC, Lee J. PRRT2-positive self-limited infantile epilepsy: Initial seizure characteristics and response to sodium channel blockers. Epilepsia Open. 2023 Jun;8(2):436-443. doi: 10.1002/epi4.12708.\u003c/li\u003e\n\u003cli\u003eScorrano G, Dono F, Corniello C, Evangelista G, Chiarelli F, Sensi SL. Exploring epileptic phenotypes in PRRT2-related disorders: A report of two cases and literature appraisal. Seizure. 2024 Jul;119:3-11. doi: 10.1016/j.seizure.2024.04.019.\u003c/li\u003e\n\u003cli\u003eZhao Q, Hu Y, Liu Z, Fang S, Zheng F, Wang X, Li F, Li X, Lin Z. PRRT2 variants and effectiveness of various antiepileptic drugs in self-limited familial infantile epilepsy. Seizure. 2021 Oct;91:360-368. doi: 10.1016/j.seizure.2021.07.013.\u003c/li\u003e\n\u003cli\u003eFruscione F, Valente P, Sterlini B, Romei A, Baldassari S, Fadda M, Prestigio C, Giansante G, Sartorelli J, Rossi P, Rubio A, Gambardella A, Nieus T, Broccoli V, Fassio A, Baldelli P, Corradi A, Zara F, Benfenati F. PRRT2 controls neuronal excitability by negatively modulating Na+ channel 1.2/1.6 activity. Brain. 2018 Apr 1;141(4):1000-1016. doi: 10.1093/brain/awy051.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PRRT2, vitro experiments, variants, BFIE, PKD","lastPublishedDoi":"10.21203/rs.3.rs-7941362/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7941362/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003ePRRT2 gene mutations are one of the most common genetic factors leading to neurodevelopmental disorders, such as autism spectrum disorder, intellectual disability, and epilepsy. This gene encodes a proline-rich transmembrane protein 2, which plays a crucial role in neuronal development and synaptic transmission. Mutations in this gene can result in a variety of clinical phenotypes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe retrospectively analyzed 14 pediatric patients from unrelated families with epilepsy or paroxysmal motor disorders, all genetically confirmed to harbor PRRT2 mutations. Comprehensive clinical data were collected, including seizure semiology, EEG, MRI, treatment response, and developmental outcomes. Genetic analysis involved whole-genome sequencing or a customized epilepsy gene panel, with single nucleotide variants validated by Sanger sequencing. The pathogenesis of both mutations((c.189delC p.Lys64Argfs*26) and c.971G\u0026gt;C p.Gly324Ala)) was studied by in vitro experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe cohort comprised 6 females and 8 males with onset ages ranging from 3 to 7 months. We identified 13 patients with point mutations and one with an exon 2-4 deletion.The hotspot mutation c.649dupC p.Arg217Profs*8 was most prevalent (11/14). Eleven patients exhibited the BFIE phenotype, one had PKD, and two were unclassifiable. Most patients (12/14, 85.7%) achieved seizure freedom, showing favorable responses to oxcarbazepine or levetiracetam. Developmental delays were observed in 5 patients, potentially associated with non-c.649dup mutations (e.g., c.189delC p.Lys64Argfs*26). Functional studies confirmed that the c.189delC p.Lys64Argfs*26 mutation significantly reduced mRNA and protein expression, while the c.971G\u0026gt;C p.Gly324Ala variant did not affect splicing but was predicted pathogenic.\u003c/p\u003e","manuscriptTitle":"Genotypic and Phenotypic Spectrum of PRRT2-Related Variations: Clinical Analysis and Treatment Response in Fourteen Unrelated Chinese Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 16:17:34","doi":"10.21203/rs.3.rs-7941362/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-27T08:27:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-24T09:40:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145268147110107560908027371660809703278","date":"2026-01-24T09:05:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-05T11:47:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"336795011456794579125337189380034328544","date":"2025-12-15T14:49:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-10T15:22:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-08T14:10:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Medical Genomics","date":"2025-12-08T14:00:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mgnm","sideBox":"Learn more about [BMC Medical Genomics](http://bmcmedgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mgnm/default.aspx","title":"BMC Medical Genomics","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af8fe9a9-ace8-47e1-9155-d6e430239203","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T17:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-7941362","link":"https://doi.org/10.1186/s12920-026-02379-6","journal":{"identity":"bmc-medical-genomics","isVorOnly":false,"title":"BMC Medical Genomics"},"publishedOn":"2026-04-30 15:58:12","publishedOnDateReadable":"April 30th, 2026"},"versionCreatedAt":"2025-12-15 16:17:34","video":"","vorDoi":"10.1186/s12920-026-02379-6","vorDoiUrl":"https://doi.org/10.1186/s12920-026-02379-6","workflowStages":[]},"version":"v1","identity":"rs-7941362","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7941362","identity":"rs-7941362","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}