A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

Causal relationship between female reproductive factors, sex hormones and uterine leiomyoma: a Mendelian randomization study.

RESEARCH QUESTION: Are the observed associations between female reproductive factors and sex hormones with the risk of uterine leiomyoma truly causal associations? DESIGN: The putative causal relationships between female reproductive factors and sex hormones with uterine leiomyoma were investigated using two-sample Mendelian randomization. Statistics on exposure-associated genetic variants were obtained from genome-wide association studies (GWAS). The uterine leiomyoma GWAS from the FinnGen and FibroGENE consortia were used as outcome data for discovery and replication analyses, respectively. Results were pooled by meta-analysis. Sensitivity analyses ensured robustness of the Mendelian randomization analysis. RESULTS: When FinnGen GWAS were used as outcome data, a causal relationship was found between age at menarche (OR 0.84, P < 0.0001), age at menopause (OR 1.08, P < 0.0001), number of live births (OR 0.25, P < 0.001) and total testosterone levels (OR 0.90, P < 0.001) with the risk of uterine leiomyoma. When FibroGENE GWAS were used as outcome data, Mendelian randomization results for age at menopause, the number of live births and total testosterone levels were replicated. In the meta-analysis, a later age at menopause (OR 1.08, P < 0.0001) was associated with an increased risk of uterine leiomyoma. A higher number of live births (OR 0.25, P < 0.0001) and higher total testosterone levels (OR 0.90, P < 0.0001) were associated with a decreased risk of uterine leiomyoma. CONCLUSIONS: A causal relationship between later age at menopause, lower number of live births and lower total testosterone levels with increased risk of uterine leiomyoma was found.

Structural and mechanistic insights into the complexes formed by Wolbachia cytoplasmic incompatibility factors.

Wolbachia bacteria, inherited through the female germ line, infect a large fraction of arthropod species. Many Wolbachia strains manipulate host reproduction, most commonly through cytoplasmic incompatibility (CI). CI, a conditional male sterility, results when Wolbachia-infected male insects mate with uninfected females; viability is restored if the female is similarly infected (called "rescue"). CI is used to help control mosquito-borne viruses such as dengue and Zika, but its mechanisms remain unknown. The coexpressed CI factors CifA and CifB form stable complexes in vitro, but the timing and function of this interaction in the insect are unresolved. CifA expression in the female germ line is sufficient for rescue. We report high-resolution structures of a CI-factor complex, CinA-CinB, which utilizes a unique binding mode between the CinA rescue factor and the CinB nuclease; the structures were validated by biochemical and yeast growth analyses. Importantly, transgenic expression in Drosophila of a nonbinding CinA mutant, designed based on the CinA-CinB structure, suggests CinA expressed in females must bind CinB imported by sperm in order to rescue embryonic viability. Binding between cognate factors is conserved in an enzymatically distinct CI system, CidA-CidB, suggesting universal features in Wolbachia CI induction and rescue.

The promoting effect and mechanism of Nrf2 on cell metastasis in cervical cancer.

BACKGROUND: Cervical cancer (CC) has poor prognosis and high mortality rate for its metastasis during the disease progression. Epithelial-mesenchymal transition (EMT) and anoikis are initial and pivotal steps during the metastatic process. Although higher levels of Nrf2 are associated with aggressive tumor behaviors in cervical cancer, the detailed mechanism of Nrf2 in cervical cancer metastasis, especially EMT and anoikis, remains unclear. METHODS: Immunohistochemistry (IHC) was used to examine Nrf2 expression in CC. Wound healing assay and transwell analysis were used to evaluate the migration ability of CC cells. Western blot, qTR-PCR and immunofluorescent staining were used to verify the expression level of Nrf2, the EMT associated markers and anoikis associated proteins. Flow cytometry assays and cell counting were used to detect the apoptosis of cervical cancer cells. The lung and lymph node metastatic mouse model were established for studies in vivo. The interaction between Nrf2 and Snail1 was confirmed by rescue-of-function assay. RESULTS: When compared with cervical cancer patients without lymph node metastasis, Nrf2 was highly expressed in patients with lymph node metastasis. And Nrf2 was proved to enhance the migration ability of HeLa and SiHa cells. In addition, Nrf2 was positively correlated with EMT processes and negatively associated with anoikis in cervical cancer. In vivo, a xenograft assay also showed that Nrf2 facilitated both pulmonary and lymphatic distant metastasis of cervical cancer. Rescue-of-function assay further revealed the mechanism that Nrf2 impacted the metastasis of CC through Snail1. CONCLUSION: Our fundings established Nrf2 plays a crucial role in the metastasis of cervical cancer by enhancing EMT and resistance to anoikis by promoting the expression of Snail1, with potential value as a therapeutic candidate.

Phosphorylation of EZH2 differs HER2-positive breast cancer invasiveness in a site-specific manner.

HER2-positive breast cancer (BC) invasiveness and drug-resistance issue is the critical treatment obstacle recently. We investigated the total and phosphorylated status EZH2 expression in database and BC tissue microarray. We demonstrated for the first time that EZH2 is distributed both in cytoplasm and nucleus of breast cancer cells in a phosphorylation site-specific manner. High expressed-EZH2 cases more frequently had an advanced clinical stage (lymph node metastasis) and aggressive features than EZH2-low cases, potentially indicating the high risk of HER2-positive BC (p < 0.05). Notably, highly expressed phosphorylated EZH2 is differently located in cytoplasm or nucleus in a site-specific manner in breast cancer cells. Nucleus-located pEZH2-S21 is expressed in invasive and lymph node metastatic HER2-positive BC cases (p = 0.144, p = 0.001). Cytoplasmic pEZH2-T487 is correlated with HER2 positive status (p = 0.014).In conclusion, high expression of nucleus-located EZH2 might be a predictor of invasive BC. Activation of phosphorylated EZH2-S21 site in nucleus would be a potential predictor of HER2-positve BC and poor efficacy of HER2-target therapy. These results point to a PRC2-independent non-epigenetic mechanism and therapeutic strategy of EZH2 in HER2-positive BC.

Bifunctional small molecules that mediate the degradation of extracellular proteins.

Targeted protein degradation (TPD) has emerged as a promising therapeutic strategy. Most TPD technologies use the ubiquitin-proteasome system, and are therefore limited to targeting intracellular proteins. To address this limitation, we developed a class of modular, bifunctional synthetic molecules called MoDE-As (molecular degraders of extracellular proteins through the asialoglycoprotein receptor (ASGPR)), which mediate the degradation of extracellular proteins. MoDE-A molecules mediate the formation of a ternary complex between a target protein and ASGPR on hepatocytes. The target protein is then endocytosed and degraded by lysosomal proteases. We demonstrated the modularity of the MoDE-A technology by synthesizing molecules that induce depletion of both antibody and proinflammatory cytokine proteins. These data show experimental evidence that nonproteinogenic, synthetic molecules can enable TPD of extracellular proteins in vitro and in vivo. We believe that TPD mediated by the MoDE-A technology will have widespread applications for disease treatment.

Characterization of the skin keloid microenvironment.

Keloids are a fibroproliferative skin disorder that develops in people of all ages. Keloids exhibit some cancer-like behaviors, with similar genetic and epigenetic modifications in the keloid microenvironment. The keloid microenvironment is composed of keratinocytes, fibroblasts, myofibroblasts, vascular endothelial cells, immune cells, stem cells and collagen fibers. Recent advances in the study of keloids have led to novel insights into cellular communication among components of the keloid microenvironment as well as potential therapeutic targets for treating keloids. In this review, we summarized the nature of genetic and epigenetic regulation in keloid-derived fibroblasts, epithelial-to-mesenchymal transition of keratinocytes, immune cell infiltration into keloids, the differentiation of keloid-derived stem cells, endothelial-to-mesenchymal transition of vascular endothelial cells, extracellular matrix synthesis and remodeling, and uncontrolled angiogenesis in keloids with the aim of identifying new targets for therapeutic benefit. Video Abstract.

Drug-resistant idiopathic generalized epilepsy: A meta-analysis of prevalence and risk factors.

BACKGROUND: Idiopathic generalized epilepsy (IGE) is a common epilepsy syndrome with early age onset and generally good seizure outcomes. This study aims to determine the incidence and predictive risk factors for drug-resistant IGE. METHODS: We systematically searched three databases (PubMed, Embase, and Cochrane Library) in November 2022 and included 12 eligible studies which reported long-term outcomes (mean = 14.05) after antiseizure medications (ASMs) from 2001 to 2020. We defined drug resistance as the persistence of any seizure despite ASMs treatment (whether as monotherapies or in combination) given the criteria of drug resistance varied in original studies. A random-effects model was used to evaluate the prevalence of refractory IGE. Studies reporting potential poor prognostic factors were included for subsequent subgroup meta-analysis. RESULTS: The pooled prevalence of drug resistance in IGE cohorts was 27% (95% CI: 0.19-0.36). Subgroup analysis of the risk factors revealed that the psychiatric comorbidities (odds ratio (OR): 4.87, 95% confidence interval (CI): 2.97-7.98), combined three seizure types (absences, myoclonic jerks, and generalized tonic-clonic seizures) (OR: 5.37, 95% CI: 3.16-9.13), the presence of absence seizure (OR: 4.38, 95% CI: 2.64-7.28), generalized polyspike trains (GPT) (OR: 4.83, 95% CI: 2.42-9.64), sex/catamenial epilepsy (OR: 3.25, 95% CI: 1.97-5.37), and status epilepticus (OR: 5.94, 95% CI: 2.23-15.85) increased the risk of poor prognosis. Other factors, including age onset, family history, and side effects of ASMs, were insignificantly associated with a higher incidence of refractory IGE. CONCLUSION: Drug resistance is a severe complication of IGE. Further standardized research about clinical and electroencephalography factors is warranted.

Long Noncoding RNAs in Taxane Resistance of Breast Cancer.

Breast cancer is a common cancer in women and a leading cause of mortality. With the early diagnosis and development of therapeutic drugs, the prognosis of breast cancer has markedly improved. Chemotherapy is one of the predominant strategies for the treatment of breast cancer. Taxanes, including paclitaxel and docetaxel, are widely used in the treatment of breast cancer and remarkably decrease the risk of death and recurrence. However, taxane resistance caused by multiple factors significantly impacts the effect of the drug and leads to poor prognosis. Long noncoding RNAs (lncRNAs) have been shown to play a significant role in critical cellular processes, and a number of studies have illustrated that lncRNAs play vital roles in taxane resistance. In this review, we systematically summarize the mechanisms of taxane resistance in breast cancer and the functions of lncRNAs in taxane resistance in breast cancer. The findings provide insight into the role of lncRNAs in taxane resistance and suggest that lncRNAs may be used to develop therapeutic targets to prevent or reverse taxane resistance in patients with breast cancer.

Expressional activation and functional roles of human endogenous retroviruses in cancers.

Human endogenous retroviruses (HERVs) are widely believed to be remnants of ancestral germ line infections by exogenous retroviruses. Although HERVs are deemed as "nonfunctional DNAs" due to loss of most of their viral protein coding capacity during evolution as part of the human genome, cumulative evidences are showing the expressional activation and potential roles of HERVs in diseases especially cancers. Work by other researchers and us has observed the dysregulation of HERVs in cancers, identified new HERV-related genes, and revealed their potential importance in cancer development. Here, we summarized the current knowledge on the mechanisms of the expressional activation and functional roles of HERVs, with a focus on the H family HERV (HERV-H), in carcinogenesis. HERV expression is regulated by external chemical or physical substances and exogenous virus infection, as well as host factors such as epigenetic DNA methylation, transcription factors, cytokines, and small RNAs. Diverse roles of HERVs have been proposed by acting in the forms of noncoding RNAs, proteins, and transcriptional regulators during carcinogenesis. However, much remains to be learnt about the contributions of HERVs to human cancers. More investigation is warranted to elucidate the functions of these "fossil remnants" yet important viral DNAs in the human genome.

Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche in liver metastasis.

Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21-TLR7-IL-6 axis. Thus, sEVs-miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis.

Controlling Complex Nanoemulsion Morphology Using Asymmetric Cosurfactants for the Preparation of Polymer Nanocapsules.

Complex nanoemulsions, comprising multiphase nanoscale droplets, hold considerable potential advantages as vehicles for encapsulation and delivery as well as templates for nanoparticle synthesis. Although methods exist to controllably produce complex emulsions on the microscale, very few methods exist to produce them on the nanoscale. Here, we examine a recently developed method involving a combination of high-energy emulsification with conventional cosurfactants to produce oil-water-oil (O/W/O) complex nanoemulsions. Specifically, we study in detail how the composition of conventional ethoxylated cosurfactants Span80 and Tween20 influences the morphology and structure of the resulting complex nanoemulsions in the water-cyclohexane system. Using a combination of small-angle neutron scattering and cryo-electron microscopy, we find that the cosurfactant composition controls the generation of complex droplet morphologies including core-shell and multicore-shell O/W/O nanodroplets, resulting in an effective state diagram for the selection of nanoemulsion morphology. Additionally, the cosurfactant composition can be used to control the thickness of the water shell contained within the complex nanodroplets. We hypothesize that this degree of control, despite the highly nonequilibrium nature of the nanoemulsions, is ultimately determined by a competition between the opposing spontaneous curvature of the two cosurfactants, which strongly influences the interfacial curvature of the nanodroplets as a result of their ultralow interfacial tension. This is supported by a correlation between cosurfactant compositions that produces complex nanoemulsions and those that produce homogeneous mixed micelles in equilibrium surfactant-cyclohexane solutions. Ultimately, we show that the formation of complex O/W/O nanoemulsions is weakly perturbed upon the addition of hydrophilic polymer precursors, facilitating their use as templates for the formation of polymer nanocapsules.

Synthesis of Oil-Laden Poly(ethylene glycol) Diacrylate Hydrogel Nanocapsules from Double Nanoemulsions.

Multiple emulsions have received great interest due to their ability to be used as templates for the production of multicompartment particles for a variety of applications. However, scaling these complex droplets to nanoscale dimensions has been a challenge due to limitations on their fabrication methods. Here, we report the development of oil-in-water-in-oil (O1/W/O2) double nanoemulsions via a two-step high-energy method and their use as templates for complex nanogels comprised of inner oil droplets encapsulated within a hydrogel matrix. Using a combination of characterization methods, we determine how the properties of the nanogels are controlled by the size, stability, internal morphology, and chemical composition of the nanoemulsion templates from which they are formed. This allows for identification of compositional and emulsification parameters that can be used to optimize the size and oil encapsulation efficiency of the nanogels. Our templating method produces oil-laden nanogels with high oil encapsulation efficiencies and average diameters of 200-300 nm. In addition, we demonstrate the versatility of the system by varying the types of inner oil, the hydrogel chemistry, the amount of inner oil, and the hydrogel network cross-link density. These nontoxic oil-laden nanogels have potential applications in food, pharmaceutical, and cosmetic formulations.

Oil-in-Water-in-Oil Multinanoemulsions for Templating Complex Nanoparticles.

Complex nanoemulsions involving nanodroplets with a defined inner structure have great potential for encapsulation and templating applications. We report a method to form novel complex oil-in-water-in-oil nanoemulsions using a combination of high-energy processing with mixed nonionic surfactants that simultaneously achieve ultralow interfacial tension and frustrated curvature of the water-oil interface. The method produces multinanoemulsions possessing morphologies resembling water-swollen reverse vesicles with core-shell and multicore-shell morphologies of water in cyclohexane. A combination of macroscopic and microscopic characterization conclusively verifies and quantifies the complex morphologies, which vary systematically and reproducibly with water content for water volume fractions between 0.01 and 0.10. The complex morphologies are stable tens of hours, providing a route for their use as liquid templates for internally structured nanoparticles. As a demonstration, we test the complex nanoemulsions' ability to template complex polymer nanogels.

Binary diamondoid building blocks for molecular gels.

Adamantane is a type of diamondoid molecules that has a cage or globular shape with a diameter of 6.34 ± 0.04 Å.8 Anisotropic interactions between these truly nanoscopic particles can be induced through the derivatization of the diamondoid cage. Here we explore the gelation of paired systems of adamantane where attractions are introduced through van der Waals forces and hydrogen bonding. Gels are produced through the mixing of 1-adamantanecarboxylic acid (A1C) and 1-adamantylamine (A1N). Upon mixing dimethyl sulfoxide solutions of these molecules at vanishing concentrations, these diamondoid molecules rapidly precipitate. A space-filling gel of the resulting aggregates is observed at approximately 3% by weight. These resulting gels have elastic moduli of 10(2)-10(4) Pa in the 3-7 wt % concentration range. At a 1:1 mol ratio of 1-adamantanecarboxylic acid (A1C) and 1-adamantylamine (A1N), the gel's elastic modulus and yield stress increase as volume fractions ϕ(x) and ϕ(y) with x ≈ 4.2 and y ≈ 3.5. The dependencies of moduli and yield stress on the volume fraction display characteristics of colloidal gels. Transmission electron microscope (TEM) images indicate that the gels are formed from a network of interwoven and branched fibers which are composed of ∼30 nm crystallites that have undergone oriented aggregation to form fibers.

Risk assessment of arrhythmias related to three antiseizure medications: a systematic review and single-arm meta-analysis.

Objective: Antiseizure medications (ASMs) are first line therapy for seizure disorders. Their effects on arrhythmias, especially the risk of arrhythmias associated with lacosamide (LCM), levetiracetam (LEV), and perampanel (PER), have been intensely investigated. Methods: We searched four databases (PubMed, EMBASE, Cochrane Library, and Web of Science) until August 6, 2023. We used a common effects model and reported data as pooled incidence with 95% CIs. Meta-analyses were conducted to elucidate the risk of arrhythmias with different drugs, and Egger's regression was performed to detect publication bias analysis. Results: We included 11 clinical trials with 1,031 participants. The pooled incidence of arrhythmias in the LEV group was 0.005 (95% CI: 0.001-0.013), while it was 0.014 in the LCM group (95% CI: 0.003-0.030). Publication bias analyses indicated no significant bias in the LEV group (t = 0.02, df = 4, p-value = 0.9852) but a significant bias in the LCM group (t = 5.94, df = 3, p-value = 0.0095). We corrected for this bias in the LCM group using the trim-and-fill method, which yielded a similar pooled incidence of 0.0137 (95% CI: 0.0036-0.0280), indicating good reliability. Due to insufficient studies, we could not conduct a meta-analysis for PER, and we analyzed them in our systematic review. Conclusion: The use of LCM significantly elevated the risk of arrhythmias, while LEV had non-significant arrhythmogenic effects. As for the arrhythmogenic effects of PER, more clinical trials are needed in the future.

Epilepsy-associated genes: an update.

PURPOSE: To provide an updated list of epilepsy-associated genes based on clinical-genetic evidence. METHODS: Epilepsy-associated genes were systematically searched and cross-checked from the OMIM, HGMD, and PubMed databases up to July 2023. To facilitate the reference for the epilepsy-associated genes that are potentially common in clinical practice, the epilepsy-associated genes were ranked by the mutation number in the HGMD database and by case number in the China Epilepsy Gene 1.0 project, which targeted common epilepsy. RESULTS: Based on the OMIM database, 1506 genes were identified to be associated with epilepsy and were classified into three categories according to their potential association with epilepsy or other abnormal phenotypes, including 168 epilepsy genes that were associated with epilepsies as pure or core symptoms, 364 genes that were associated with neurodevelopmental disorders as the main symptom and epilepsy, and 974 epilepsy-related genes that were associated with gross physical/systemic abnormalities accompanied by epilepsy/seizures. Among the epilepsy genes, 115 genes (68.5%) were associated with epileptic encephalopathy. After cross-checking with the HGMD and PubMed databases, an additional 1440 genes were listed as potential epilepsy-associated genes, of which 278 genes have been repeatedly identified variants in patients with epilepsy. The top 100 frequently reported/identified epilepsy-associated genes from the HGMD database and the China Epilepsy Gene 1.0 project were listed, among which 40 genes were identical in both sources. SIGNIFICANCE: Recognition of epilepsy-associated genes will facilitate genetic screening strategies and be helpful for precise molecular diagnosis and treatment of epilepsy in clinical practice.

Determination of dimethylated nucleosides in serum from colorectal cancer patients by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

RNA modifications play a crucial regulatory role in a variety of biological processes and are closely related to numerous diseases, including cancer. The diversity of metabolites in serum makes it a favored biofluid for biomarkers discovery. In this work, a robust and accurate hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) approach was established for simultaneous determination of dimethylated nucleosides in human serum. Using the established method, we were able to accurately quantify the concentrations of N6-2'-O-dimethyladenosine (m6Am), N2,N2-dimethylguanosine (m2,2G), and 5,2'-O-dimethyluridine (m5Um) in serum samples from 53 healthy controls, 57 advanced colorectal adenoma patients, and 39 colorectal cancer (CRC) patients. The results showed that, compared with healthy controls and advanced colorectal adenoma patients, the concentrations of m6Am and m2,2G were increased in CRC patients, while the concentration of m5Um was decreased in CRC patients. These results indicate that these three dimethylated nucleosides could be potential biomarkers for early detection of colorectal cancer. Interestingly, the level of m5Um was gradually decreased from healthy controls to advanced colorectal adenoma patients to CRC patients, indicating m5Um could also be used to evaluate the level of malignancy of colorectal tumor. In addition, this study will contribute to the investigation on the regulatory mechanisms of RNA dimethylation in the onset and development of colorectal cancer.

Association of FAT1 with focal epilepsy and correlation between seizure relapse and gene expression stage.

PURPOSE: The FAT1 gene encodes FAT atypical cadherin 1, which is essential for foetal development, including brain development. This study aimed to investigate the relationship between FAT1 variants and epilepsy. METHODS: Trio-based whole-exome sequencing was performed on a cohort of 313 patients with epilepsy. Additional cases with FAT1 variants were collected from the China Epilepsy Gene V.1.0 Matching Platform. RESULTS: Four pairs of compound heterozygous missense FAT1 variants were identified in four unrelated patients with partial (focal) epilepsy and/or febrile seizures, but without intellectual disability/developmental abnormalities. These variants presented no/very low frequencies in the gnomAD database, and the aggregate frequencies in this cohort were significantly higher than those in controls. Two additional compound heterozygous missense variants were identified in two unrelated cases using the gene-matching platform. All patients experienced infrequent (yearly/monthly) complex partial seizures or secondary generalised tonic-clonic seizures. They responded well toantiseizure medication, but seizures relapsed in three cases when antiseizure medication were decreased or withdrawn after being seizure-free for three to six years, which correlated with the expression stage of FAT1. Genotype-phenotype analysis showed that epilepsy-associated FAT1 variants were missense, whereas non-epilepsy-associated variants were mainly truncated. The relationship between FAT1 and epilepsy was evaluated to be "Strong" by the Clinical Validity Framework of ClinGen. CONCLUSIONS: FAT1 is a potential causative gene of partial epilepsy and febrile seizures. Gene expression stage was suggested to be one of the considerations in determining the duration ofantiseizure medication. Genotype-phenotype correlation helps to explain the mechanisms underlying phenotypic variation.