Identification of two novel variants in NUS1 gene in two unrelated Chinese families with intellectual disorder and epilepsy

Identification of two novel variants in NUS1 gene in two unrelated Chinese families with intellectual disorder and epilepsy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of two novel variants in NUS1 gene in two unrelated Chinese families with intellectual disorder and epilepsy Yuling Kan, Haiyan Zhao, Hongxing Li, Chunli Rong, Nana Su, Yangyang Zhu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4158407/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Mutations in the NUS1 gene, which encodes a Nogo-B receptor (NgBR), are related to congenital disorder of glycosylation, epilepsy, and Parkinson’s disease. However, due to the limited number of cases with genotype and detailed clinical features, more cases are needed to better understand the functional and phenotypic characteristics of NUS1 variants. In this study, we reported two unrelated Chinese individuals suffering from intellectual disorder and epilepsy. Materials and methods Whole-exome sequencing (WES) was performed on the two patients to identify pathogenic variants, and copy number variation sequencing (CNV-Seq) was conducted on the patients 2. The candidate variants were subsequently validated using Sanger sequencing. Additionally, bioinformatics analyses were used to investigate the deleteriousness of the identified variants. Results WES identified two novel variants in the NUS1 gene [NM_138459.5: c.640A > T/p.K214*, c.278delC/p.L94Wfs*11] in the two unrelated individuals with myoclonus, epilepsy, and intellectual disability. These variants resulted in truncated NgBR proteins, which lost the cis-PTase domain. According to the American College of Medical Genetics and Genomics (ACMG) classification, p.K214* was evaluated as likely pathogenic and p.L94Wfs*11 was evaluated as pathogenic. CNV-Seq analysis revealed a 0.4Mb duplication of Xq27.2q27.2 in patient 2, which was considered uncertain significance. Conclusion Our findings strongly suggest that the two novel variants in NUS1 gene may be the cause of the patient's clinical characteristics, possibly due to the loss of cis-PTase activity. Furthermore, our study expanded the genotype-phenotype spectrum of the NUS1 gene. NUS1 NgBR WES intellectual disorder epilepsy Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Epilepsy is commonly associated with major comorbidities, and intellectual disability (ID) affects approximately 25% of children with epilepsy (Berg et al., 2008 ; Robertson, Hatton, Emerson, & Baines, 2015 ). When epilepsy and ID co-occur, they tend to be refractory and have a poor prognosis. Therefore, understanding the genetic etiology and pathogenesis of epilepsy and ID is essential for diagnosis, prognosis, genetic counseling, and treatment. The intellectual developmental disorder-55 with seizures (MRD55, OMIM: 617831) is a rare genetic disorder that ccombines delayed development, ID, delayed speech, and various types of epileptic seizures (Hamdan et al., 2017 ). It has been reported that MRD55 is mainly caused by pathogenic variants in the NUS1 gene (Den et al., 2019 ; Yu et al., 2021 ). NUS1 , located on 6q22.1, encodes Nogo-B receptor (NgBR), which interacts with the dehydrodolichyl diphosphate synthase complex subunit (DHDDS) to promote the activity of cisprenyltransferase ( cis-PTase ) (Park et al., 2014 ). The formed dehydrodolichyl diphosphate synthase (DDS) complex, together with DHDDS, plays a crucial role in the Dol-P biosynthetic machinery in eukaryotic cells (Harrison et al., 2011 ; Shridas, Rush, & Waechter, 2003 ). In the Human Mutation Database (HGMD, Q2 2023), 40 disease-causing variants of NUS1 have been documented, associated with various neurological disorders, including developmental and epileptic encephalopathy (Hamdan et al., 2017 ), dystonia (Wirth et al., 2020 ), Parkinson’s disease (Chen et al., 2020 ), congenital disorder of glycosylation (Park et al., 2014 ), progressive myoclonic epilepsy (Courage et al., 2021 ), and movement disorder (Yu et al., 2021 ). However, due to the limited number of cases with genotype and detailed clinical features, more cases are needed to gain a better understanding of the pathogenesis of MRD55. In this study, we conducted whole-exome sequencing (WES) analysis on two unrelated Chinese individuals who presented with myoclonus, epilepsy, and ID. As a result, we identified two novel variants in the NUS1 gene, expanding the genotype spectrum of NUS1 . Materials and methods Patient and clinical assessments Two patients affected with myoclonus, epilepsy, and ID were recruited for the study (Fig. 1 A and 1 C). The study was approved by the Ethics Committee of Binzhou People's Hospital. The peripheral blood samples, clinical information, magnetic resonance imaging (MRI), and electroencephalography (EEG) were collected after the families provided informed consent. Whole-exome sequencing Genomic DNA was extracted from whole blood samples using the Magnetic universal Genomic DNA Kit (TIANGEN, China). IDT xGen Exome Research Panel v 1.0 (Integrated DNA Technologies, Coralville, Iowa, United States) was used to enrich genomic DNA. Then, the samples were sequenced on an Illumina NovaSeq 6,000 (Illumina, CA, United States) at an average depth of 100× at YinFeng Gene Technology Co., Ltd. (Jinan, China). After removing low-quality reads, SNVs and indels were called using GATK (DePristo et al., 2011 ) and annotated by ANNOVAR (Wang, Li, & Hakonarson, 2010 ). The variants with > 0.1% minor allele frequency (MAF) in the 1000 Genomes Project, ExAC, and gnomAD databases were removed. The deleteriousness and conservation of low-frequency variants were predicted by various bioinformatics algorithms (MutationTaster, SIFT, PolyPhen-2, GERP++, CADD, Revel score, and M-CAP). Finally, the pathogenicity of the candidate variants was evaluated according to the American College of Medical Genetics (ACMG) guidelines (Richards et al., 2015 ). Sanger sequencing The extracted genomic DNA was used as the template for PCR amplification. The primers were as followers: NUS1 (c.640A > T): F: 5'-CAGTGTTATCTGAGAATCCCATC-3', R: 5'- AAGAATCAGGCTTTATGGTGTG-3'; NUS1 (c.278delC): F: 5'- GGTCCCATTCAGACAAGTCCC-3', R: 5'- TCACCTTGGTGGTCGTAGACG-3'. The amplification product was sequenced by YinFeng Gene Technology Co., Ltd. Copy number variation sequencing The low-depth whole genome copy number variation sequencing (CNV-seq) was performed in the patient 2. After DNA extraction and and library construction, the sample was sequenced on an Illumina NovaSeq 6,000 (Illumina) at YinFeng Gene Technology Co., Ltd.. The sequencing results were mapped to the UCSC hg19 human reference genome, and the pathogenicity of the detected CNVs was evaluated according to the ACMG standard (2019). All suspected deleted or duplicate regions are compared to the database, including DECIPHER ( https://decipher.sanger.ac.uk/browser ), Online Mendelian Inheritance in Man (OMIM) ( https://omim.org/ ), GeneReviews ( https://www.ncbi.nlm.nih.gov/books/NBK1116/ ), ClinVar ( http://www.ncbi.nlm.nih.gov/clinvar/ ), ClinGen ( http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/ ), Genome Aggregation Database ( https://gnomad.broadinstitute.org/ ), CNV Interpretation Scoring Rubric ( http://cnvcalc.clinicalgenome.org/cnvcalc/ ), and Database of Genomic Variants ( http://dgv.tcag.ca/dgv/app/home ). In silico analysis Multiple amino acid sequences of the NUS1 gene from different species were obtained from the Uniprot database ( https://www.uniprot.org/ ) and analyzed using MEGA software. The protein structural models for both the wild-type and the two mutated forms of NUS1 proteins were predicted using SWISS-MODEL. Results Clinical outcomes The patient 1 (Fig. 1 A), an 8-year-old girl, was the first child born to nonconsanguineous parents. She was born at full-term natural birth without suffocation, with a birth weight of 3.5kg. She stood alone at the age of 1, and experienced seizure at the age of 1 year and 6 months, manifested as intermittent blinking, slow reaction, and unstable standing, which lasted for 5 to 10 seconds, with daily episodes. The patient can walk without support at the age of 2-year-old, spoke "dad” and “mom" at 6 years of age, and can only say a few simple words until now. Her electroencephalograms (EEG) showed slowing down of background activity, with generalized spike slow waves, multi spike-slow waves, sharp slow waves, and slow waves (Supplementary Fig. 1A). Brain CT showed no abnormalities (Supplementary Fig. 1B). She was monitored to have multiple atonic seizures, several myoclonic seizures and atypical absence seizures during the awake period. Her father is healthy, but her mother had unsteady gait and ID. However, the patient's family refused to provide other information about the mother, including blood samples. The patient 2 in family 2 (Fig. 1 C) was a 28-year-old male who experienced his first seizure at 2 months old. This seizure presented as involuntary shaking of both upper limbs, lasting for 1–2 seconds and occurring intermittently. He achieved walking independently at 1 year and 4 months old and began speaking at 1.5 years old. At the age of 8 years, he began showing signs of poor balance and coordination, and his cognitive abilities were behind those of his peers. When he was 1 year old, he had seizures characterized by involuntary tremors or falling, which occurred 1 to several times a day. He received a diagnosis of "spasms" at the local hospital and was treated with "sodium valproate". This treatment stabilized his condition, and he stopped taking the medication around 9 years old. However, similar symptoms reappeared at 12 years old, occurring several times a day to once every few days. At our hospital, he underwent treatment with "Oxcarbazepine, Phenobarbital, and traditional Chinese medicine". His condition gradually stabilized, and he experienced no seizures for 6–7 years. Unfortunately, at 22 years old, he had recurrent seizures due to self-withdrawal of the medication. These seizures all occurred during wakefulness, and their frequency has increased over the past 2 months, happening 3–4 times a day to once every few days. Current examination showed that he had slow reactions, unclear speech, poor intelligence, mild trembling in both hands and feet, lack of coordination in voluntary movements, unstable finger-to-nose test, and lack of coordination in alternating hand movements. His bilateral Hoffmann's sign and Babinski sign were negative. Psychological assessment tests revealed that he has moderate depression. The Wisconsin Card Sorting Test (WCST) report confirmed cognitive impairments, weak cognitive shifting ability, and learning difficulties. Likewise, the Wechsler Adult Intelligence Scale (WAIS) and Wechsler Memory Scale (WMS) tests indicated delays in his general intellectual level and general memory level. EEG results showed intermittent epileptic discharge, primarily in the bilateral frontal and left frontal regions, as well as in the left occipital and midline regions (Supplementary Fig. 1C). The brain MRI revealed abnormal signals in the left frontal lobe and the cisterna magna (Supplementary Fig. 1D). Genetic investigations Proband-only WES was performed in patient 1 with an average depth of 143.07X. The single nucleotide variants (SNVs) and InDels were identified, whose minor allele frequency (MAF) > 1% in publicly available databases (dbSNP, 1000 Genomes Project, ExAC, and GnomAD databases) were filtered. Through WES analysis, a heterozygous variant c.640A > T (p.K214*, NM_138459.5) in NUS1 gene was discovered in the patient 1. This nonsense variant was located in the third exon out of 5 exons in the NUS1 gene and was predicted to lead to premature termination of translation. Importantly, this variant was not found in the gnomAD, ExAC, or 1000 Genomes databases and had not been previously reported. According to the ACMG guidelines, the variant c.640A > T was classified as likely pathogenic (PVS1 + PM2_Supporting). Sanger sequencing revealed that her father did not carry this variant, but it remained uncertain if it was inherited from her affected mother due to the unavailability of a maternal sample (Fig. 1 B). In addition, no CNVs was detected within the range covered by WES analysis in patient 1. Proband-only WES analysis identified a heterozygous variant in the NUS1 gene (c.278delC, p.L94Wfs*11, NM_138459.5) in patient 2, using the same analysis pipeline. This variant, located in the first exon out of five exons in the NUS1 gene, was predicted to alter the reading frame and result in a premature translation termination, leading to a truncated protein with 11 miscoded amino acids. Importantly, this variant was not present in the gnomAD, ExAC, or 1000 Genomes databases, and it had not been reported in the published literature. Subsequently, Sanger sequencing was performed to validate this variant, and as illustrated in Fig. 1 D, neither of the patient's parents carried the mutation, indicating that the identified variant was de novo . Collectively, this variant was classified as pathogenic under the ACMG guidelines (PVS1 + PM6 + PM2_Supporting). It was worth noting that no other pathogenic or likely pathogenic variants were found in any other genes accounting for the patient’s phenotypes. CNV-seq was conducted in patient 2, revealing a 0.4Mb duplication of Xq27.2q27.2 which was classified as uncertain significance (Supplemental Fig. 2). This duplication involved only one protein-coding gene called SPANXA and did not contain any known triplosensitivity genes or regions. It is worth noting that a single case (variant: esv3577446) with complete coverage of this duplicated region was documented in the Database of Genomic Variants (DGV) database. Additionally, neither the DECIPHER nor ClinVar databases included any cases associated with segmental duplication in this particular region. Pathogenic assessment The variant c.640A > T/p.K214* results in a truncated protein that includes complete transmembrane domains 1, 2, and 3 (TM, TM2, and TM3), as well as part of the cis-PTase domain. However, the latter half of the cis-PTase domain and the RXG motif are deleted (Fig. 2 A). To determine the effect of the c.640A > T/p.K214* variant, functional prediction programs including BayesDel, EIGEN, DANN, FATHMM-MKL, LRT, and MutationTaster were utilized. According to MutationTaster, the change was predicted to be “Disease causing” with a score of “1”, while BayesDel predicted the mutation was “Pathogenic Strong”. (Supplementary Table 1). Regarding the protein consequences of the NUS1 (p.K214*) mutation, a multiple sequence alignment (MSA) of the amino acids encoded by NUS1 revealed that the p.214 lysine residue was highly conserved (Fig. 3 A). The SWISS-MODEL was employed to predict the protein structure of the NUS1 protein. It indicated that the base shift from A to T resulted in a truncated protein and subsequent conformational changes (Fig. 4 A and B). The variant c.278delC/p.L94Wfs*11 causes a truncated protein which contains complete TM1 and TM2 domains, while the TM3 and cis-PTase domains are expected to be deleted, along with the RXG motif (Fig. 2 A). As a result, c.278delC leads to malformations of the NUS1 protein, resulting in insufficient functional haploids. Moreover, the multiple sequence alignment (MSA) of the amino acids encoded by NUS1 indicates that the p.94 leucine is highly conserved (Fig. 3 B). The SWISS-MODEL predicted that c.278delC leads to a frameshift mutation, causing a truncated protein with a deletion of the base (C) (Fig. 4 C and D). This mutant protein, consisting of only 105 amino acids, loses most of the functional domains of the NUS1 protein. Therefore, c.640A > T and c.278delC may cause malformations of the NUS1 protein, resulting in insufficient functional haploids. A total of 40 likely pathogenic/pathogenic SNVs and indels in the NUS1 gene were included in the HGMD database (as of Quarter 2, 2023), of which 5 were associated with myoclonic epilepsy, 1 with intellectual disability and epileptic seizures, 4 with developmental and epileptic encephalopathy, 2 with epilepsy (Supplementary Table 2), indicating the correlation between NUS1 and epilepsy. Furthermore, the ClinVar database (as of July 2023, Supplementary Table 3) reported 33 likely pathogenic/pathogenic SNVs and indels in the NUS1 gene, with 10 of these variants were associated with “Intellectual disability, autosomal dominant 55, with seizures”. Additionally, 13 truncated variants were reported, highlighting the potential role of truncated NUS1 proteins in disease etiology. Follow-up Patient 1 is currently taking Sodium valproate 0.2g tid, Topiramate 25mg bid, Clonazepam 1.5mg in the morning and 2mg in the evening. The patient 2 is currently taking Nanshing Quanxie Capsules 1.4g morning and 1.75g evening; Oxcarbazepine tablets 0.6g bid, Yinaoling Pills 2g bid. No obvious adverse reactions have been found for either patient. Discussion NgBR encoded by NUS1 , is one of the subunits of the mammalian cis-PTs, which constitute a large family of enzymes conserved during evolution and present in all domains of life (Grabińska, Edani, Park, Kraehling, & Sessa, 2017 ). It is primarily located in the endoplasmic reticulum. NgBR plays a crucial in lipid and cholesterol homeostasis through direct interaction with Nogo-B, and influences N-linked protein glycosylation by regulating the cis-PTase activity (Harrison et al., 2011 ; Li et al., 2018 ). Functional experiments showed that the loss of Drosophila NUS1 orthologous gene tango14 leads to motor deficits, reduces the number of apoptotic dopaminergic neurons and dopamine contents in Drosophila, and results in cholesterol accumulation in the Malpighian tubules and brain, as well as the formation of neurodegenerative brain vacuoles in an age dependent manner (Guo et al., 2018 ; Xue et al., 2022 ). Many studies have confirmed that NUS1 is associated with diseases such as Parkinson's disease, epilepsy and congenital glycosylation disorders (Guo et al., 2018 ; Ji, Zhao, Zhang, & Wang, 2023 ; Monfrini, Miller, Frucht, Di Fonzo, & Riboldi, 2022 ; Park et al., 2014 ). However, the current number of reported cases with NUS1 pathogenic variants is insufficient to establish a clear genotype/phenotypic correlation. Moreover, the molecular mechanism underlying the various diseases caused by NUS1 remains unclear. Therefore, further studies investigating more cases and conducting functional analyses are necessary. To date, a total of 13 variants of the NUS1 gene associated with epilepsy have been reported, with 10 pathogenic/likely pathogenic variant included in the ClinVar database as of July 2023 (Supplementary Table 2). ID, myoclonus, and movement disorders are commonly observed in addition to seizures. Zhang et al. reported a de novo variant c.51_54delTCTG (p.L18Tfs*31) in a Chinese patient with MRD55 (Zhang et al., 2021 ). Herein, the two patients we reported carried two novel truncated variants (c.640A > T/p.K214*, c.278delC/p.L94Wfs*11) in the NUS1 gene. So far, three NUS1 variants associated with MRD55 have been reported, all of which were found in Chinese patients. The p.K214* variant causes the loss of the latter half of the cis-PTase domain and the RXG motif, which are crucial for prenyltransferase activity and have the potential to induce nonsense-mediated decay (NMD) of the transcript. The p.L94Wfs*11 variant directly leads to the deletion of TM3, cis-PTase domain and the RXG motif, and it also causes NMD. Thus, haploinsufficiency of the NUS1 gene is the cause of MRD55. However, it is worth noting that not all abnormal transcripts undergo NMD. Den K et al. (Den et al., 2019 ) confirmed that the transcribed mRNA of the NUS1 gene variant [c.601_691del: p. (Arg202Glnfs*9)] was only partially subjected to NMD. Therefore, further verification is needed to determine the level of truncated NgBR protein in the patients. In this study, both patients presented with generalized myoclonic seizures and varying degrees of ID, consistent with previously reported patients (Zhang et al., 2021 ). However, patient 2 also exhibited tremor and ataxia. Moreover, the MRI of patient 2 showed abnormal signal in the left frontal lobe and cisterna occipitalis, suggesting the presence of cavernous hemangioma and arachnoid cyst. Fraiman et al. reported on a patient with the variant c.692-1G > A, who presented with progressive myoclonic epilepsy combined with psychotic symptoms, mainly visual and auditory hallucinations and persecution delusion, as well as aggression (Fraiman, Maia-de-Oliveira, Moreira-Neto, & Godeiro-Junior, 2021 ). In our study, patient 2 did not exhibit a similar mental disorder but instead had moderate depression. It is important to note that none of the previously reported patients with detailed clinical phenotypes had similar mental disorders, apart from these two patients. In the genomAD database, the pLI score for NUS1 is 0.98, indicating the intolerance of NUS1 to loss-of-function variants and the role of NUS1 haploinsufficiency in disease occurrence. A homozygous variant p.R290H located in the cis-PTase domain of the NUS1 gene was identified in siblings with congenital glycosylation disorders, congenital scoliosis, developmental delays, hearing deficit and visual impairment, and refractory epilepsy (Park et al., 2014 ). This variant affects the glycosylation of NPC2 by reducing cis-PTase activity, triggers defects in cellular cholesterol trafficking and dolichol biosynthesis (Park et al., 2014 ). Another frameshift variant c.22_23insA (p.Val8Aspfs*126) was found in patients with familial epilepsy, tremors, and cerebellar ataxia. Although this variant did not affect protein glycosylation, it reduced the protein level of NUS1 and altered protein localization (Araki et al., 2020 ). Moreover, a 50% reduction in the steady-state level of NgBR protein was observed in cell lines of patients with the c.752T > G (p.L251*) variant who presented with moderate ID, seizures, tremors, and mild gait ataxia (Yu et al., 2021 ). This variant affected the biosynthesis of total polyprenol and dolichol lipids, but not protein glycosylation (Yu et al., 2021 ). In addition, an increase in lysosomal cholesterol accumulation and multiple lysosomal defects were also observed in the patient's cell line, which may be related to motor deficits (Yu et al., 2021 ). Overall, these studies suggest that alterations in NUS1 cis-PTase activity, protein level and protein localization contribute to disease occurrence. Both variants (p.K214* and p.L94Wfs*11) reported in this study were predicted to cause NMD, leading to a reduction in protein dosage in patients. Conclusion In conclusion, we identified two novel truncated variants in the NUS1 gene, c.640A > T/p.K214* and c.278delC/p.L94Wfs*11, in two Chinese patients. Both patients exhibited similar phenotypes, including epilepsy, ID, and poor language abilities. Patient 2, however, also displayed additional symptoms such as tremor, dyskinesia, and moderate depression, suggesting clinical phenotypic heterogeneity in NUS1 -associated diseases. It remains to be determined whether these phenotypes are associated with different NUS1 variants. Nonetheless, our study makes a significant contribution to expanding the genotype-phenotype spectrum of the NUS1 gene. Declarations Ethics approval and consent to participate This study was approved by the Research Ethics Committee of the Binzhou People’s Hospital (Binzhou, China; approval no. LL-2022-010). All participants provided written informed consent. Consent for publication All authors agree to publish this article. Availability of data and materials The data are available from the corresponding author on reasonable request. Competing interests The authors declare no competing financial interests. Funding This study was supported by the Medical workers Science and Technology Innovation Plan of Shandong Province (grant no.SDYWZGKCJH2022044 and SDYWZGKCJHLH202212). Authors' contributions Yuling Kan, Writing-original draft; Haiyan Zhao and Hongxing Li , Writing-review & editing; Chunli Rong and Nana Su and Yangyang Zhu, Statistics data and prepared all figures; Xueping Gao and Jinghan Jiang prepared all tables; Junji Hu and Jian Zhang, Revise the article & supervise the subject. All authors reviewed the manuscript. Acknowledgements Not applicable. References Araki, K., Nakamura, R., Ito, D., Kato, K., Iguchi, Y., Sahashi, K., . . . Katsuno, M. (2020). NUS1 mutation in a family with epilepsy, cerebellar ataxia, and tremor. Epilepsy Res, 164 , 106371. doi:10.1016/j.eplepsyres.2020.106371 Berg, A. T., Langfitt, J. T., Testa, F. M., Levy, S. R., DiMario, F., Westerveld, M., & Kulas, J. (2008). Global cognitive function in children with epilepsy: a community-based study. Epilepsia, 49 (4), 608-614. doi:10.1111/j.1528-1167.2007.01461.x Chen, X., Xiao, Y., Zhou, M., Lin, Y., Guo, W., Huang, S., . . . Xu, P. (2020). Genetic analysis of NUS1 in Chinese patients with Parkinson's disease. Neurobiol Aging, 86 , 202 e205-202 e206. doi:10.1016/j.neurobiolaging.2019.09.002 Courage, C., Oliver, K. L., Park, E. J., Cameron, J. M., Grabińska, K. A., Muona, M., . . . Lehesjoki, A. E. (2021). Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes. Am J Hum Genet, 108 (4), 722-738. doi:10.1016/j.ajhg.2021.03.013 Den, K., Kudo, Y., Kato, M., Watanabe, K., Doi, H., Tanaka, F., . . . Matsumoto, N. (2019). Recurrent NUS1 canonical splice donor site mutation in two unrelated individuals with epilepsy, myoclonus, ataxia and scoliosis - a case report. BMC Neurol, 19 (1), 253. doi:10.1186/s12883-019-1489-x DePristo, M. A., Banks, E., Poplin, R., Garimella, K. V., Maguire, J. R., Hartl, C., . . . Daly, M. J. (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet, 43 (5), 491-498. doi:10.1038/ng.806 Fraiman, P., Maia-de-Oliveira, J. P., Moreira-Neto, M., & Godeiro-Junior, C. (2021). Psychosis in NUS1 de novo mutation: New phenotypical presentation. Clin Genet, 99 (3), 475-476. doi:10.1111/cge.13867 Grabińska, K. A., Edani, B. H., Park, E. J., Kraehling, J. R., & Sessa, W. C. (2017). A conserved C-terminal RXG motif in the NgBR subunit of cis-prenyltransferase is critical for prenyltransferase activity. J Biol Chem, 292 (42), 17351-17361. doi:10.1074/jbc.M117.806034 Guo, J. F., Zhang, L., Li, K., Mei, J. P., Xue, J., Chen, J., . . . Tang, B. S. (2018). Coding mutations in NUS1 contribute to Parkinson's disease. Proc Natl Acad Sci U S A, 115 (45), 11567-11572. doi:10.1073/pnas.1809969115 Hamdan, F. F., Myers, C. T., Cossette, P., Lemay, P., Spiegelman, D., Laporte, A. D., . . . Michaud, J. L. (2017). High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies. Am J Hum Genet, 101 (5), 664-685. doi:10.1016/j.ajhg.2017.09.008 Harrison, K. D., Park, E. J., Gao, N., Kuo, A., Rush, J. S., Waechter, C. J., . . . Sessa, W. C. (2011). Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation. EMBO J, 30 (12), 2490-2500. doi:10.1038/emboj.2011.147 Ji, C., Zhao, J., Zhang, J., & Wang, K. (2023). Novel NUS1 variant in a Chinese patient with progressive myoclonus epilepsy: a case report and systematic review. Neurol Sci . doi:10.1007/s10072-023-06851-4 Li, Y. K., Xie, Y. J., Wu, D. C., Long, S. L., Tang, S., & Mo, Z. C. (2018). Nogo‑B receptor in relevant carcinoma: Current achievements, challenges and aims (Review). Int J Oncol, 53 (5), 1827-1835. doi:10.3892/ijo.2018.4520 Monfrini, E., Miller, C., Frucht, S. J., Di Fonzo, A., & Riboldi, G. M. (2022). Progressive myoclonus without epilepsy due to a NUS1 frameshift insertion: Dyssynergia cerebellaris myoclonica revisited. Parkinsonism Relat Disord, 98 , 53-55. doi:10.1016/j.parkreldis.2022.03.016 Park, E. J., Grabińska, K. A., Guan, Z., Stránecký, V., Hartmannová, H., Hodaňová, K., . . . Sessa, W. C. (2014). Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, causes a congenital disorder of glycosylation. Cell Metab, 20 (3), 448-457. doi:10.1016/j.cmet.2014.06.016 Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., . . . Rehm, H. L. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med, 17 (5), 405-424. doi:10.1038/gim.2015.30 Robertson, J., Hatton, C., Emerson, E., & Baines, S. (2015). Prevalence of epilepsy among people with intellectual disabilities: A systematic review. Seizure, 29 , 46-62. doi:10.1016/j.seizure.2015.03.016 Shridas, P., Rush, J. S., & Waechter, C. J. (2003). Identification and characterization of a cDNA encoding a long-chain cis-isoprenyltranferase involved in dolichyl monophosphate biosynthesis in the ER of brain cells. Biochem Biophys Res Commun, 312 (4), 1349-1356. doi:10.1016/j.bbrc.2003.11.065 Wang, K., Li, M., & Hakonarson, H. (2010). ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res, 38 (16), e164. doi:10.1093/nar/gkq603 Wirth, T., Tranchant, C., Drouot, N., Keren, B., Mignot, C., Cif, L., . . . Chelly, J. (2020). Increased diagnostic yield in complex dystonia through exome sequencing. Parkinsonism Relat Disord, 74 , 50-56. doi:10.1016/j.parkreldis.2020.04.003 Xue, J., Zhu, Y., Wei, L., Huang, H., Li, G., Huang, W., . . . Duan, R. (2022). Loss of Drosophila NUS1 results in cholesterol accumulation and Parkinson's disease-related neurodegeneration. FASEB J, 36 (7), e22411. doi:10.1096/fj.202200212R Yu, S. H., Wang, T., Wiggins, K., Louie, R. J., Merino, E. F., Skinner, C., . . . Steet, R. (2021). Lysosomal cholesterol accumulation contributes to the movement phenotypes associated with NUS1 haploinsufficiency. Genet Med, 23 (7), 1305-1314. doi:10.1038/s41436-021-01137-6 Zhang, P., Cui, D., Liao, P., Yuan, X., Yang, N., Zhen, Y., . . . Huang, Q. (2021). Case Report: Clinical Features of a Chinese Boy With Epileptic Seizures and Intellectual Disabilities Who Carries a Truncated NUS1 Variant. Front Pediatr, 9 , 725231. doi:10.3389/fped.2021.725231 Additional Declarations No competing interests reported. Supplementary Files SupplementaryTable1.docx SupplementaryTable2.xlsx SupplementaryTable3.xlsx Supplementaryfigure1.tif Supplemental figure 1 The EEG results of patients. (A) EEG of the patient 1. (B) Brain CT image of patient 1. (C) EEG of the patient 2. Supplementaryfigure2.tif Supplemental figure 2 Schematic presentation of CNV-seq results for patient 2. The green arrow indicates the location of the duplication region. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4158407","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285437142,"identity":"eda2dd45-3982-4165-a681-8f9a8f8113c4","order_by":0,"name":"Yuling Kan","email":"","orcid":"","institution":"Binzhou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuling","middleName":"","lastName":"Kan","suffix":""},{"id":285437145,"identity":"35942872-d09c-45dc-aea8-54da533b8505","order_by":1,"name":"Haiyan Zhao","email":"","orcid":"","institution":"Zibo Changguo Hospital","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Zhao","suffix":""},{"id":285437147,"identity":"67efde09-e99e-43ca-8c55-36f07f06429c","order_by":2,"name":"Hongxing Li","email":"","orcid":"","institution":"Zibo Changguo Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hongxing","middleName":"","lastName":"Li","suffix":""},{"id":285437149,"identity":"5365f247-1288-4118-a7e3-70802ff5b040","order_by":3,"name":"Chunli Rong","email":"","orcid":"","institution":"Binzhou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunli","middleName":"","lastName":"Rong","suffix":""},{"id":285437152,"identity":"21454d0b-4d57-4412-8e71-fca8a9d6af29","order_by":4,"name":"Nana Su","email":"","orcid":"","institution":"Binzhou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Su","suffix":""},{"id":285437153,"identity":"479bdef4-1e72-4de8-a4f5-fc74a859df94","order_by":5,"name":"Yangyang Zhu","email":"","orcid":"","institution":"The first clinical College of Binzhou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Yangyang","middleName":"","lastName":"Zhu","suffix":""},{"id":285437155,"identity":"885a2f00-2c89-47a5-86e4-cdcc9f89932e","order_by":6,"name":"Xueping Gao","email":"","orcid":"","institution":"Yinfeng Gene Technology Co, Ltd","correspondingAuthor":false,"prefix":"","firstName":"Xueping","middleName":"","lastName":"Gao","suffix":""},{"id":285437157,"identity":"906aca15-0023-41e6-9564-636c64865be7","order_by":7,"name":"Jinghan Jiang","email":"","orcid":"","institution":"Yinfeng Gene Technology Co, Ltd","correspondingAuthor":false,"prefix":"","firstName":"Jinghan","middleName":"","lastName":"Jiang","suffix":""},{"id":285437158,"identity":"d835dd56-1d92-4601-9219-3828a692b9c4","order_by":8,"name":"Junji Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACAwaGBAYGHgk5fvbmAyAm8VqMJXuOJRCtBQwSN9zIMcCrEg7MJRKeSRfIWABtOfP5w8Mddgz87d34LbOckZAmPQPsl95tEolnkhkkzpzdgN9hN4BaeMB+ObuNIbGNmcFAIpc4LSC/PP6Q2FZPmhYGicS2w0RoOfMg2RrisGNmQC3HeQj75XhO4m3enjpQVD7++LOtWo6/vRe/FmA0JjAw9iBxCSgHAfYDDAw/iFA3CkbBKBgFIxcAAFmQRpkJENClAAAAAElFTkSuQmCC","orcid":"","institution":"Zibo Changguo Hospital","correspondingAuthor":true,"prefix":"","firstName":"Junji","middleName":"","lastName":"Hu","suffix":""},{"id":285437161,"identity":"ac7e017f-bdd6-436d-a80c-081b49f7c674","order_by":9,"name":"Jian Zhang","email":"","orcid":"","institution":"Binzhou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-03-24 14:29:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4158407/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4158407/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53962561,"identity":"85d129f2-41f1-4743-9f12-79fe204e7160","added_by":"auto","created_at":"2024-04-02 18:20:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":946680,"visible":true,"origin":"","legend":"\u003cp\u003eThe pedigree and Sanger sequencing validation of c.640A\u0026gt;T/p.K214* and c.278delC/p.L94Wfs*11 in the \u003cem\u003eNUS1 \u003c/em\u003egene. (A) Schematic presentation of the familial pedigrees of patients 1. Arrows indicate the proband. (B) DNA sequencing data of the variant c.640A\u0026gt;T/p.K214* identified in the patient 1. The arrow indicates the location of the mutation. (C) Schematic presentation of the familial pedigrees of patients 2. Arrows indicate the proband. (D) DNA sequencing data of the variant c.278delC/p.L94Wfs*11. (E) Brain MRI findings in patient 2.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/726d17f8aef85a30dc647bcb.png"},{"id":53962556,"identity":"abb476f6-afa0-46fc-bbb8-bf6270b7a921","added_by":"auto","created_at":"2024-04-02 18:20:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":167563,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of the NUS1 protein with the positions of the mutations found in the two families.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/0323e8f28f3373cd4f5bcdaf.png"},{"id":53962554,"identity":"7611c419-3eac-4bb0-a222-2ab4e90d0403","added_by":"auto","created_at":"2024-04-02 18:20:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":361887,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment of the NUS1 proteins of various species. Residues K214 (A) and L94 (B) (indicated by arrow) are highly conserved.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/406afa823f59b71c16fe441d.png"},{"id":53962555,"identity":"123f7bfb-4efa-498c-ab2f-146b0bb6b7c5","added_by":"auto","created_at":"2024-04-02 18:20:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":928225,"visible":true,"origin":"","legend":"\u003cp\u003eThe 3D modeling of the wild-type and variant protein indicated different structures (visualized by SWISS-MODEL). (A) The wild-type NUS1 protein. (B) the mutant p.K214* protein. Left panel: Overall 3D modeling of proteins. Right panel: Partial magnification of 3D models of proteins. Dark arrow indicated the position of the p.K214. (C) The wild-type NUS1 protein. Left panel: Overall 3D modeling of proteins. Right panel: Partial magnification of 3D model of wild-type protein. Dark arrow indicated the position of the p.L94. (D) the mutant p.L94Wfs*11 protein.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/37c606b3370038e02309ae49.png"},{"id":59798682,"identity":"937d72c1-355a-4f26-833a-7fca32feacec","added_by":"auto","created_at":"2024-07-07 11:32:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3083413,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/017dffb3-d0e5-41d3-bce8-0e190440ffa8.pdf"},{"id":53962563,"identity":"69838578-6375-4cc2-872f-bfc8e65ade61","added_by":"auto","created_at":"2024-04-02 18:20:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15732,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/f9b083e36dd976a3113bfdd6.docx"},{"id":53962559,"identity":"4117d7f5-c16f-41b9-9570-84c49bf9bb69","added_by":"auto","created_at":"2024-04-02 18:20:51","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13502,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/c94290647bf86bad45786363.xlsx"},{"id":53962560,"identity":"c361ca37-d2ca-43bc-8193-dbd4c9db7540","added_by":"auto","created_at":"2024-04-02 18:20:51","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18501,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/bdc39cf7e6a242e22a2d6abf.xlsx"},{"id":53962557,"identity":"853248c6-5c32-4d58-94a0-d5e0edcb5d6c","added_by":"auto","created_at":"2024-04-02 18:20:51","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15677950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental figure 1\u003c/strong\u003e The EEG results of patients. (A) EEG of the patient 1. (B) Brain CT image of patient 1. (C) EEG of the patient 2.\u003c/p\u003e","description":"","filename":"Supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/991ff0df0a5c03df7035d859.tif"},{"id":53962558,"identity":"1662afff-bbcd-4202-8314-8aa4eed2ce39","added_by":"auto","created_at":"2024-04-02 18:20:51","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":8848374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental figure 2 \u003c/strong\u003eSchematic presentation of CNV-seq results for patient 2. The green arrow indicates the location of the duplication region.\u003c/p\u003e","description":"","filename":"Supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4158407/v1/0d661917a62804efc02331e8.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of two novel variants in NUS1 gene in two unrelated Chinese families with intellectual disorder and epilepsy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is commonly associated with major comorbidities, and intellectual disability (ID) affects approximately 25% of children with epilepsy (Berg et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Robertson, Hatton, Emerson, \u0026amp; Baines, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). When epilepsy and ID co-occur, they tend to be refractory and have a poor prognosis. Therefore, understanding the genetic etiology and pathogenesis of epilepsy and ID is essential for diagnosis, prognosis, genetic counseling, and treatment. The intellectual developmental disorder-55 with seizures (MRD55, OMIM: 617831) is a rare genetic disorder that ccombines delayed development, ID, delayed speech, and various types of epileptic seizures (Hamdan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It has been reported that MRD55 is mainly caused by pathogenic variants in the \u003cem\u003eNUS1\u003c/em\u003e gene (Den et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eNUS1\u003c/em\u003e, located on 6q22.1, encodes Nogo-B receptor (NgBR), which interacts with the dehydrodolichyl diphosphate synthase complex subunit (DHDDS) to promote the activity of cisprenyltransferase (\u003cem\u003ecis-PTase\u003c/em\u003e) (Park et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The formed dehydrodolichyl diphosphate synthase (DDS) complex, together with DHDDS, plays a crucial role in the Dol-P biosynthetic machinery in eukaryotic cells (Harrison et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Shridas, Rush, \u0026amp; Waechter, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). In the Human Mutation Database (HGMD, Q2 2023), 40 disease-causing variants of \u003cem\u003eNUS1\u003c/em\u003e have been documented, associated with various neurological disorders, including developmental and epileptic encephalopathy (Hamdan et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), dystonia (Wirth et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Parkinson\u0026rsquo;s disease (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), congenital disorder of glycosylation (Park et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), progressive myoclonic epilepsy (Courage et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and movement disorder (Yu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, due to the limited number of cases with genotype and detailed clinical features, more cases are needed to gain a better understanding of the pathogenesis of MRD55.\u003c/p\u003e \u003cp\u003eIn this study, we conducted whole-exome sequencing (WES) analysis on two unrelated Chinese individuals who presented with myoclonus, epilepsy, and ID. As a result, we identified two novel variants in the NUS1 gene, expanding the genotype spectrum of \u003cem\u003eNUS1\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient and clinical assessments\u003c/h2\u003e \u003cp\u003eTwo patients affected with myoclonus, epilepsy, and ID were recruited for the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The study was approved by the Ethics Committee of Binzhou People's Hospital. The peripheral blood samples, clinical information, magnetic resonance imaging (MRI), and electroencephalography (EEG) were collected after the families provided informed consent.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWhole-exome sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA was extracted from whole blood samples using the Magnetic universal Genomic DNA Kit (TIANGEN, China). IDT xGen Exome Research Panel v 1.0 (Integrated DNA Technologies, Coralville, Iowa, United States) was used to enrich genomic DNA. Then, the samples were sequenced on an Illumina NovaSeq 6,000 (Illumina, CA, United States) at an average depth of 100\u0026times; at YinFeng Gene Technology Co., Ltd. (Jinan, China).\u003c/p\u003e \u003cp\u003eAfter removing low-quality reads, SNVs and indels were called using GATK (DePristo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and annotated by ANNOVAR (Wang, Li, \u0026amp; Hakonarson, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The variants with \u0026gt;\u0026thinsp;0.1% minor allele frequency (MAF) in the 1000 Genomes Project, ExAC, and gnomAD databases were removed. The deleteriousness and conservation of low-frequency variants were predicted by various bioinformatics algorithms (MutationTaster, SIFT, PolyPhen-2, GERP++, CADD, Revel score, and M-CAP). Finally, the pathogenicity of the candidate variants was evaluated according to the American College of Medical Genetics (ACMG) guidelines (Richards et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSanger sequencing\u003c/h2\u003e \u003cp\u003eThe extracted genomic DNA was used as the template for PCR amplification. The primers were as followers: \u003cem\u003eNUS1\u003c/em\u003e (c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T): F: 5'-CAGTGTTATCTGAGAATCCCATC-3', R: 5'- AAGAATCAGGCTTTATGGTGTG-3'; \u003cem\u003eNUS1\u003c/em\u003e (c.278delC): F: 5'- GGTCCCATTCAGACAAGTCCC-3', R: 5'- TCACCTTGGTGGTCGTAGACG-3'. The amplification product was sequenced by YinFeng Gene Technology Co., Ltd.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCopy number variation sequencing\u003c/h2\u003e \u003cp\u003eThe low-depth whole genome copy number variation sequencing (CNV-seq) was performed in the patient 2. After DNA extraction and and library construction, the sample was sequenced on an Illumina NovaSeq 6,000 (Illumina) at YinFeng Gene Technology Co., Ltd.. The sequencing results were mapped to the UCSC hg19 human reference genome, and the pathogenicity of the detected CNVs was evaluated according to the ACMG standard (2019). All suspected deleted or duplicate regions are compared to the database, including DECIPHER (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://decipher.sanger.ac.uk/browser\u003c/span\u003e\u003cspan address=\"https://decipher.sanger.ac.uk/browser\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Online Mendelian Inheritance in Man (OMIM) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/\u003c/span\u003e\u003cspan address=\"https://omim.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), GeneReviews (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/books/NBK1116/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/books/NBK1116/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ClinVar (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/clinvar/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/clinvar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), ClinGen (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/projects/dbvar/clingen/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Genome Aggregation Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://gnomad.broadinstitute.org/\u003c/span\u003e\u003cspan address=\"https://gnomad.broadinstitute.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), CNV Interpretation Scoring Rubric (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cnvcalc.clinicalgenome.org/cnvcalc/\u003c/span\u003e\u003cspan address=\"http://cnvcalc.clinicalgenome.org/cnvcalc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and Database of Genomic Variants (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://dgv.tcag.ca/dgv/app/home\u003c/span\u003e\u003cspan address=\"http://dgv.tcag.ca/dgv/app/home\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIn silico analysis\u003c/h2\u003e \u003cp\u003eMultiple amino acid sequences of the \u003cem\u003eNUS1\u003c/em\u003e gene from different species were obtained from the Uniprot database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and analyzed using MEGA software. The protein structural models for both the wild-type and the two mutated forms of NUS1 proteins were predicted using SWISS-MODEL.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eClinical outcomes\u003c/h2\u003e\n\u003cp\u003eThe patient 1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), an 8-year-old girl, was the first child born to nonconsanguineous parents. She was born at full-term natural birth without suffocation, with a birth weight of 3.5kg. She stood alone at the age of 1, and experienced seizure at the age of 1 year and 6 months, manifested as intermittent blinking, slow reaction, and unstable standing, which lasted for 5 to 10 seconds, with daily episodes. The patient can walk without support at the age of 2-year-old, spoke \"dad\u0026rdquo; and \u0026ldquo;mom\" at 6 years of age, and can only say a few simple words until now. Her electroencephalograms (EEG) showed slowing down of background activity, with generalized spike slow waves, multi spike-slow waves, sharp slow waves, and slow waves (Supplementary Fig.\u0026nbsp;1A). Brain CT showed no abnormalities (Supplementary Fig.\u0026nbsp;1B). She was monitored to have multiple atonic seizures, several myoclonic seizures and atypical absence seizures during the awake period. Her father is healthy, but her mother had unsteady gait and ID. However, the patient's family refused to provide other information about the mother, including blood samples.\u003c/p\u003e\n\u003cp\u003eThe patient 2 in family 2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC) was a 28-year-old male who experienced his first seizure at 2 months old. This seizure presented as involuntary shaking of both upper limbs, lasting for 1\u0026ndash;2 seconds and occurring intermittently. He achieved walking independently at 1 year and 4 months old and began speaking at 1.5 years old. At the age of 8 years, he began showing signs of poor balance and coordination, and his cognitive abilities were behind those of his peers. When he was 1 year old, he had seizures characterized by involuntary tremors or falling, which occurred 1 to several times a day. He received a diagnosis of \"spasms\" at the local hospital and was treated with \"sodium valproate\". This treatment stabilized his condition, and he stopped taking the medication around 9 years old. However, similar symptoms reappeared at 12 years old, occurring several times a day to once every few days. At our hospital, he underwent treatment with \"Oxcarbazepine, Phenobarbital, and traditional Chinese medicine\". His condition gradually stabilized, and he experienced no seizures for 6\u0026ndash;7 years. Unfortunately, at 22 years old, he had recurrent seizures due to self-withdrawal of the medication. These seizures all occurred during wakefulness, and their frequency has increased over the past 2 months, happening 3\u0026ndash;4 times a day to once every few days. Current examination showed that he had slow reactions, unclear speech, poor intelligence, mild trembling in both hands and feet, lack of coordination in voluntary movements, unstable finger-to-nose test, and lack of coordination in alternating hand movements. His bilateral Hoffmann's sign and Babinski sign were negative. Psychological assessment tests revealed that he has moderate depression. The Wisconsin Card Sorting Test (WCST) report confirmed cognitive impairments, weak cognitive shifting ability, and learning difficulties. Likewise, the Wechsler Adult Intelligence Scale (WAIS) and Wechsler Memory Scale (WMS) tests indicated delays in his general intellectual level and general memory level. EEG results showed intermittent epileptic discharge, primarily in the bilateral frontal and left frontal regions, as well as in the left occipital and midline regions (Supplementary Fig.\u0026nbsp;1C). The brain MRI revealed abnormal signals in the left frontal lobe and the cisterna magna (Supplementary Fig.\u0026nbsp;1D).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003eGenetic investigations\u003c/h2\u003e\n\u003cp\u003eProband-only WES was performed in patient 1 with an average depth of 143.07X. The single nucleotide variants (SNVs) and InDels were identified, whose minor allele frequency (MAF)\u0026thinsp;\u0026gt;\u0026thinsp;1% in publicly available databases (dbSNP, 1000 Genomes Project, ExAC, and GnomAD databases) were filtered. Through WES analysis, a heterozygous variant c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T (p.K214*, NM_138459.5) in NUS1 gene was discovered in the patient 1. This nonsense variant was located in the third exon out of 5 exons in the \u003cem\u003eNUS1\u003c/em\u003e gene and was predicted to lead to premature termination of translation. Importantly, this variant was not found in the gnomAD, ExAC, or 1000 Genomes databases and had not been previously reported. According to the ACMG guidelines, the variant c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T was classified as likely pathogenic (PVS1\u0026thinsp;+\u0026thinsp;PM2_Supporting). Sanger sequencing revealed that her father did not carry this variant, but it remained uncertain if it was inherited from her affected mother due to the unavailability of a maternal sample (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, no CNVs was detected within the range covered by WES analysis in patient 1.\u003c/p\u003e\n\u003cp\u003eProband-only WES analysis identified a heterozygous variant in the \u003cem\u003eNUS1\u003c/em\u003e gene (c.278delC, p.L94Wfs*11, NM_138459.5) in patient 2, using the same analysis pipeline. This variant, located in the first exon out of five exons in the \u003cem\u003eNUS1\u003c/em\u003e gene, was predicted to alter the reading frame and result in a premature translation termination, leading to a truncated protein with 11 miscoded amino acids. Importantly, this variant was not present in the gnomAD, ExAC, or 1000 Genomes databases, and it had not been reported in the published literature. Subsequently, Sanger sequencing was performed to validate this variant, and as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD, neither of the patient's parents carried the mutation, indicating that the identified variant was \u003cem\u003ede novo\u003c/em\u003e. Collectively, this variant was classified as pathogenic under the ACMG guidelines (PVS1\u0026thinsp;+\u0026thinsp;PM6\u0026thinsp;+\u0026thinsp;PM2_Supporting). It was worth noting that no other pathogenic or likely pathogenic variants were found in any other genes accounting for the patient\u0026rsquo;s phenotypes.\u003c/p\u003e\n\u003cp\u003eCNV-seq was conducted in patient 2, revealing a 0.4Mb duplication of Xq27.2q27.2 which was classified as uncertain significance (Supplemental Fig.\u0026nbsp;2). This duplication involved only one protein-coding gene called \u003cem\u003eSPANXA\u003c/em\u003e and did not contain any known triplosensitivity genes or regions. It is worth noting that a single case (variant: esv3577446) with complete coverage of this duplicated region was documented in the Database of Genomic Variants (DGV) database. Additionally, neither the DECIPHER nor ClinVar databases included any cases associated with segmental duplication in this particular region.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003ePathogenic assessment\u003c/h2\u003e\n\u003cp\u003eThe variant c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T/p.K214* results in a truncated protein that includes complete transmembrane domains 1, 2, and 3 (TM, TM2, and TM3), as well as part of the \u003cem\u003ecis-PTase\u003c/em\u003e domain. However, the latter half of the \u003cem\u003ecis-PTase\u003c/em\u003e domain and the RXG motif are deleted (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). To determine the effect of the c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T/p.K214* variant, functional prediction programs including BayesDel, EIGEN, DANN, FATHMM-MKL, LRT, and MutationTaster were utilized. According to MutationTaster, the change was predicted to be \u0026ldquo;Disease causing\u0026rdquo; with a score of \u0026ldquo;1\u0026rdquo;, while BayesDel predicted the mutation was \u0026ldquo;Pathogenic Strong\u0026rdquo;. (Supplementary Table\u0026nbsp;1). Regarding the protein consequences of the \u003cem\u003eNUS1\u003c/em\u003e (p.K214*) mutation, a multiple sequence alignment (MSA) of the amino acids encoded by \u003cem\u003eNUS1\u003c/em\u003e revealed that the p.214 lysine residue was highly conserved (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). The SWISS-MODEL was employed to predict the protein structure of the NUS1 protein. It indicated that the base shift from A to T resulted in a truncated protein and subsequent conformational changes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA and B).\u003c/p\u003e\n\u003cp\u003eThe variant c.278delC/p.L94Wfs*11 causes a truncated protein which contains complete TM1 and TM2 domains, while the TM3 and \u003cem\u003ecis-PTase\u003c/em\u003e domains are expected to be deleted, along with the RXG motif (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). As a result, c.278delC leads to malformations of the NUS1 protein, resulting in insufficient functional haploids. Moreover, the multiple sequence alignment (MSA) of the amino acids encoded by \u003cem\u003eNUS1\u003c/em\u003e indicates that the p.94 leucine is highly conserved (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The SWISS-MODEL predicted that c.278delC leads to a frameshift mutation, causing a truncated protein with a deletion of the base (C) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). This mutant protein, consisting of only 105 amino acids, loses most of the functional domains of the NUS1 protein. Therefore, c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T and c.278delC may cause malformations of the NUS1 protein, resulting in insufficient functional haploids.\u003c/p\u003e\n\u003cp\u003eA total of 40 likely pathogenic/pathogenic SNVs and indels in the \u003cem\u003eNUS1\u003c/em\u003e gene were included in the HGMD database (as of Quarter 2, 2023), of which 5 were associated with myoclonic epilepsy, 1 with intellectual disability and epileptic seizures, 4 with developmental and epileptic encephalopathy, 2 with epilepsy (Supplementary Table\u0026nbsp;2), indicating the correlation between \u003cem\u003eNUS1\u003c/em\u003e and epilepsy. Furthermore, the ClinVar database (as of July 2023, Supplementary Table\u0026nbsp;3) reported 33 likely pathogenic/pathogenic SNVs and indels in the NUS1 gene, with 10 of these variants were associated with \u0026ldquo;Intellectual disability, autosomal dominant 55, with seizures\u0026rdquo;. Additionally, 13 truncated variants were reported, highlighting the potential role of truncated NUS1 proteins in disease etiology.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003eFollow-up\u003c/h2\u003e\n\u003cp\u003ePatient 1 is currently taking Sodium valproate 0.2g tid, Topiramate 25mg bid, Clonazepam 1.5mg in the morning and 2mg in the evening. The patient 2 is currently taking Nanshing Quanxie Capsules 1.4g morning and 1.75g evening; Oxcarbazepine tablets 0.6g bid, Yinaoling Pills 2g bid. No obvious adverse reactions have been found for either patient.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNgBR encoded by \u003cem\u003eNUS1\u003c/em\u003e, is one of the subunits of the mammalian cis-PTs, which constitute a large family of enzymes conserved during evolution and present in all domains of life (Grabińska, Edani, Park, Kraehling, \u0026amp; Sessa, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is primarily located in the endoplasmic reticulum. NgBR plays a crucial in lipid and cholesterol homeostasis through direct interaction with Nogo-B, and influences N-linked protein glycosylation by regulating the \u003cem\u003ecis-PTase\u003c/em\u003e activity (Harrison et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Functional experiments showed that the loss of Drosophila \u003cem\u003eNUS1\u003c/em\u003e orthologous gene tango14 leads to motor deficits, reduces the number of apoptotic dopaminergic neurons and dopamine contents in Drosophila, and results in cholesterol accumulation in the Malpighian tubules and brain, as well as the formation of neurodegenerative brain vacuoles in an age dependent manner (Guo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Xue et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Many studies have confirmed that \u003cem\u003eNUS1\u003c/em\u003e is associated with diseases such as Parkinson's disease, epilepsy and congenital glycosylation disorders (Guo et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ji, Zhao, Zhang, \u0026amp; Wang, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Monfrini, Miller, Frucht, Di Fonzo, \u0026amp; Riboldi, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Park et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, the current number of reported cases with \u003cem\u003eNUS1\u003c/em\u003e pathogenic variants is insufficient to establish a clear genotype/phenotypic correlation. Moreover, the molecular mechanism underlying the various diseases caused by \u003cem\u003eNUS1\u003c/em\u003e remains unclear. Therefore, further studies investigating more cases and conducting functional analyses are necessary.\u003c/p\u003e \u003cp\u003eTo date, a total of 13 variants of the \u003cem\u003eNUS1\u003c/em\u003e gene associated with epilepsy have been reported, with 10 pathogenic/likely pathogenic variant included in the ClinVar database as of July 2023 (Supplementary Table\u0026nbsp;2). ID, myoclonus, and movement disorders are commonly observed in addition to seizures. Zhang et al. reported a de novo variant c.51_54delTCTG (p.L18Tfs*31) in a Chinese patient with MRD55 (Zhang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Herein, the two patients we reported carried two novel truncated variants (c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T/p.K214*, c.278delC/p.L94Wfs*11) in the \u003cem\u003eNUS1\u003c/em\u003e gene. So far, three \u003cem\u003eNUS1\u003c/em\u003e variants associated with MRD55 have been reported, all of which were found in Chinese patients. The p.K214* variant causes the loss of the latter half of the \u003cem\u003ecis-PTase\u003c/em\u003e domain and the RXG motif, which are crucial for prenyltransferase activity and have the potential to induce nonsense-mediated decay (NMD) of the transcript. The p.L94Wfs*11 variant directly leads to the deletion of TM3, \u003cem\u003ecis-PTase\u003c/em\u003e domain and the RXG motif, and it also causes NMD. Thus, haploinsufficiency of the \u003cem\u003eNUS1\u003c/em\u003e gene is the cause of MRD55. However, it is worth noting that not all abnormal transcripts undergo NMD. Den K et al. (Den et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) confirmed that the transcribed mRNA of the \u003cem\u003eNUS1\u003c/em\u003e gene variant [c.601_691del: p. (Arg202Glnfs*9)] was only partially subjected to NMD. Therefore, further verification is needed to determine the level of truncated NgBR protein in the patients. In this study, both patients presented with generalized myoclonic seizures and varying degrees of ID, consistent with previously reported patients (Zhang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, patient 2 also exhibited tremor and ataxia. Moreover, the MRI of patient 2 showed abnormal signal in the left frontal lobe and cisterna occipitalis, suggesting the presence of cavernous hemangioma and arachnoid cyst. Fraiman et al. reported on a patient with the variant c.692-1G\u0026thinsp;\u0026gt;\u0026thinsp;A, who presented with progressive myoclonic epilepsy combined with psychotic symptoms, mainly visual and auditory hallucinations and persecution delusion, as well as aggression (Fraiman, Maia-de-Oliveira, Moreira-Neto, \u0026amp; Godeiro-Junior, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In our study, patient 2 did not exhibit a similar mental disorder but instead had moderate depression. It is important to note that none of the previously reported patients with detailed clinical phenotypes had similar mental disorders, apart from these two patients.\u003c/p\u003e \u003cp\u003eIn the genomAD database, the pLI score for \u003cem\u003eNUS1\u003c/em\u003e is 0.98, indicating the intolerance of \u003cem\u003eNUS1\u003c/em\u003e to loss-of-function variants and the role of \u003cem\u003eNUS1\u003c/em\u003e haploinsufficiency in disease occurrence. A homozygous variant p.R290H located in the cis-PTase domain of the \u003cem\u003eNUS1\u003c/em\u003e gene was identified in siblings with congenital glycosylation disorders, congenital scoliosis, developmental delays, hearing deficit and visual impairment, and refractory epilepsy (Park et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This variant affects the glycosylation of NPC2 by reducing \u003cem\u003ecis-PTase\u003c/em\u003e activity, triggers defects in cellular cholesterol trafficking and dolichol biosynthesis (Park et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Another frameshift variant c.22_23insA (p.Val8Aspfs*126) was found in patients with familial epilepsy, tremors, and cerebellar ataxia. Although this variant did not affect protein glycosylation, it reduced the protein level of \u003cem\u003eNUS1\u003c/em\u003e and altered protein localization (Araki et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Moreover, a 50% reduction in the steady-state level of NgBR protein was observed in cell lines of patients with the c.752T\u0026thinsp;\u0026gt;\u0026thinsp;G (p.L251*) variant who presented with moderate ID, seizures, tremors, and mild gait ataxia (Yu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This variant affected the biosynthesis of total polyprenol and dolichol lipids, but not protein glycosylation (Yu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, an increase in lysosomal cholesterol accumulation and multiple lysosomal defects were also observed in the patient's cell line, which may be related to motor deficits (Yu et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overall, these studies suggest that alterations in \u003cem\u003eNUS1 cis-PTase\u003c/em\u003e activity, protein level and protein localization contribute to disease occurrence. Both variants (p.K214* and p.L94Wfs*11) reported in this study were predicted to cause NMD, leading to a reduction in protein dosage in patients.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we identified two novel truncated variants in the \u003cem\u003eNUS1\u003c/em\u003e gene, c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T/p.K214* and c.278delC/p.L94Wfs*11, in two Chinese patients. Both patients exhibited similar phenotypes, including epilepsy, ID, and poor language abilities. Patient 2, however, also displayed additional symptoms such as tremor, dyskinesia, and moderate depression, suggesting clinical phenotypic heterogeneity in \u003cem\u003eNUS1\u003c/em\u003e-associated diseases. It remains to be determined whether these phenotypes are associated with different \u003cem\u003eNUS1\u003c/em\u003e variants. Nonetheless, our study makes a significant contribution to expanding the genotype-phenotype spectrum of the \u003cem\u003eNUS1\u003c/em\u003e gene.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Research Ethics Committee of the Binzhou People\u0026rsquo;s Hospital (Binzhou, China; approval no. LL-2022-010). All participants provided written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch4\u003eCompeting interests\u003c/h4\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Medical workers Science and Technology Innovation Plan of Shandong Province (grant no.SDYWZGKCJH2022044 and SDYWZGKCJHLH202212).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYuling Kan, Writing-original draft; Haiyan Zhao and Hongxing Li , Writing-review \u0026amp; editing; Chunli Rong and Nana Su and Yangyang Zhu, Statistics data and prepared all figures; Xueping Gao and Jinghan Jiang prepared all tables; Junji Hu and Jian Zhang, Revise the article \u0026amp; supervise the subject. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAraki, K., Nakamura, R., Ito, D., Kato, K., Iguchi, Y., Sahashi, K., . . . Katsuno, M. (2020). NUS1 mutation in a family with epilepsy, cerebellar ataxia, and tremor. \u003cem\u003eEpilepsy Res, 164\u003c/em\u003e, 106371. doi:10.1016/j.eplepsyres.2020.106371\u003c/li\u003e\n \u003cli\u003eBerg, A. T., Langfitt, J. T., Testa, F. M., Levy, S. R., DiMario, F., Westerveld, M., \u0026amp; Kulas, J. (2008). Global cognitive function in children with epilepsy: a community-based study. \u003cem\u003eEpilepsia, 49\u003c/em\u003e(4), 608-614. doi:10.1111/j.1528-1167.2007.01461.x\u003c/li\u003e\n \u003cli\u003eChen, X., Xiao, Y., Zhou, M., Lin, Y., Guo, W., Huang, S., . . . Xu, P. (2020). Genetic analysis of NUS1 in Chinese patients with Parkinson\u0026apos;s disease. \u003cem\u003eNeurobiol Aging, 86\u003c/em\u003e, 202 e205-202 e206. doi:10.1016/j.neurobiolaging.2019.09.002\u003c/li\u003e\n \u003cli\u003eCourage, C., Oliver, K. L., Park, E. J., Cameron, J. M., Grabińska, K. A., Muona, M., . . . Lehesjoki, A. E. (2021). Progressive myoclonus epilepsies-Residual unsolved cases have marked genetic heterogeneity including dolichol-dependent protein glycosylation pathway genes. \u003cem\u003eAm J Hum Genet, 108\u003c/em\u003e(4), 722-738. doi:10.1016/j.ajhg.2021.03.013\u003c/li\u003e\n \u003cli\u003eDen, K., Kudo, Y., Kato, M., Watanabe, K., Doi, H., Tanaka, F., . . . Matsumoto, N. (2019). Recurrent NUS1 canonical splice donor site mutation in two unrelated individuals with epilepsy, myoclonus, ataxia and scoliosis - a case report. \u003cem\u003eBMC Neurol, 19\u003c/em\u003e(1), 253. doi:10.1186/s12883-019-1489-x\u003c/li\u003e\n \u003cli\u003eDePristo, M. A., Banks, E., Poplin, R., Garimella, K. V., Maguire, J. R., Hartl, C., . . . Daly, M. J. (2011). A framework for variation discovery and genotyping using next-generation DNA sequencing data. \u003cem\u003eNat Genet, 43\u003c/em\u003e(5), 491-498. doi:10.1038/ng.806\u003c/li\u003e\n \u003cli\u003eFraiman, P., Maia-de-Oliveira, J. P., Moreira-Neto, M., \u0026amp; Godeiro-Junior, C. (2021). Psychosis in NUS1 de novo mutation: New phenotypical presentation. \u003cem\u003eClin Genet, 99\u003c/em\u003e(3), 475-476. doi:10.1111/cge.13867\u003c/li\u003e\n \u003cli\u003eGrabińska, K. A., Edani, B. H., Park, E. J., Kraehling, J. R., \u0026amp; Sessa, W. C. (2017). A conserved C-terminal RXG motif in the NgBR subunit of cis-prenyltransferase is critical for prenyltransferase activity. \u003cem\u003eJ Biol Chem, 292\u003c/em\u003e(42), 17351-17361. doi:10.1074/jbc.M117.806034\u003c/li\u003e\n \u003cli\u003eGuo, J. F., Zhang, L., Li, K., Mei, J. P., Xue, J., Chen, J., . . . Tang, B. S. (2018). Coding mutations in NUS1 contribute to Parkinson\u0026apos;s disease. \u003cem\u003eProc Natl Acad Sci U S A, 115\u003c/em\u003e(45), 11567-11572. doi:10.1073/pnas.1809969115\u003c/li\u003e\n \u003cli\u003eHamdan, F. F., Myers, C. T., Cossette, P., Lemay, P., Spiegelman, D., Laporte, A. D., . . . Michaud, J. L. (2017). High Rate of Recurrent De Novo Mutations in Developmental and Epileptic Encephalopathies. \u003cem\u003eAm J Hum Genet, 101\u003c/em\u003e(5), 664-685. doi:10.1016/j.ajhg.2017.09.008\u003c/li\u003e\n \u003cli\u003eHarrison, K. D., Park, E. J., Gao, N., Kuo, A., Rush, J. S., Waechter, C. J., . . . Sessa, W. C. (2011). Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation. \u003cem\u003eEMBO J, 30\u003c/em\u003e(12), 2490-2500. doi:10.1038/emboj.2011.147\u003c/li\u003e\n \u003cli\u003eJi, C., Zhao, J., Zhang, J., \u0026amp; Wang, K. (2023). Novel NUS1 variant in a Chinese patient with progressive myoclonus epilepsy: a case report and systematic review. \u003cem\u003eNeurol Sci\u003c/em\u003e. doi:10.1007/s10072-023-06851-4\u003c/li\u003e\n \u003cli\u003eLi, Y. K., Xie, Y. J., Wu, D. C., Long, S. L., Tang, S., \u0026amp; Mo, Z. C. (2018). Nogo‑B receptor in relevant carcinoma: Current achievements, challenges and aims (Review). \u003cem\u003eInt J Oncol, 53\u003c/em\u003e(5), 1827-1835. doi:10.3892/ijo.2018.4520\u003c/li\u003e\n \u003cli\u003eMonfrini, E., Miller, C., Frucht, S. J., Di Fonzo, A., \u0026amp; Riboldi, G. M. (2022). Progressive myoclonus without epilepsy due to a NUS1 frameshift insertion: Dyssynergia cerebellaris myoclonica revisited. \u003cem\u003eParkinsonism Relat Disord, 98\u003c/em\u003e, 53-55. doi:10.1016/j.parkreldis.2022.03.016\u003c/li\u003e\n \u003cli\u003ePark, E. J., Grabińska, K. A., Guan, Z., Str\u0026aacute;neck\u0026yacute;, V., Hartmannov\u0026aacute;, H., Hodaňov\u0026aacute;, K., . . . Sessa, W. C. (2014). Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, causes a congenital disorder of glycosylation. \u003cem\u003eCell Metab, 20\u003c/em\u003e(3), 448-457. doi:10.1016/j.cmet.2014.06.016\u003c/li\u003e\n \u003cli\u003eRichards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., . . . Rehm, H. L. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. \u003cem\u003eGenet Med, 17\u003c/em\u003e(5), 405-424. doi:10.1038/gim.2015.30\u003c/li\u003e\n \u003cli\u003eRobertson, J., Hatton, C., Emerson, E., \u0026amp; Baines, S. (2015). Prevalence of epilepsy among people with intellectual disabilities: A systematic review. \u003cem\u003eSeizure, 29\u003c/em\u003e, 46-62. doi:10.1016/j.seizure.2015.03.016\u003c/li\u003e\n \u003cli\u003eShridas, P., Rush, J. S., \u0026amp; Waechter, C. J. (2003). Identification and characterization of a cDNA encoding a long-chain cis-isoprenyltranferase involved in dolichyl monophosphate biosynthesis in the ER of brain cells. \u003cem\u003eBiochem Biophys Res Commun, 312\u003c/em\u003e(4), 1349-1356. doi:10.1016/j.bbrc.2003.11.065\u003c/li\u003e\n \u003cli\u003eWang, K., Li, M., \u0026amp; Hakonarson, H. (2010). ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. \u003cem\u003eNucleic Acids Res, 38\u003c/em\u003e(16), e164. doi:10.1093/nar/gkq603\u003c/li\u003e\n \u003cli\u003eWirth, T., Tranchant, C., Drouot, N., Keren, B., Mignot, C., Cif, L., . . . Chelly, J. (2020). Increased diagnostic yield in complex dystonia through exome sequencing. \u003cem\u003eParkinsonism Relat Disord, 74\u003c/em\u003e, 50-56. doi:10.1016/j.parkreldis.2020.04.003\u003c/li\u003e\n \u003cli\u003eXue, J., Zhu, Y., Wei, L., Huang, H., Li, G., Huang, W., . . . Duan, R. (2022). Loss of Drosophila NUS1 results in cholesterol accumulation and Parkinson\u0026apos;s disease-related neurodegeneration. \u003cem\u003eFASEB J, 36\u003c/em\u003e(7), e22411. doi:10.1096/fj.202200212R\u003c/li\u003e\n \u003cli\u003eYu, S. H., Wang, T., Wiggins, K., Louie, R. J., Merino, E. F., Skinner, C., . . . Steet, R. (2021). Lysosomal cholesterol accumulation contributes to the movement phenotypes associated with NUS1 haploinsufficiency. \u003cem\u003eGenet Med, 23\u003c/em\u003e(7), 1305-1314. doi:10.1038/s41436-021-01137-6\u003c/li\u003e\n \u003cli\u003eZhang, P., Cui, D., Liao, P., Yuan, X., Yang, N., Zhen, Y., . . . Huang, Q. (2021). Case Report: Clinical Features of a Chinese Boy With Epileptic Seizures and Intellectual Disabilities Who Carries a Truncated NUS1 Variant. \u003cem\u003eFront Pediatr, 9\u003c/em\u003e, 725231. doi:10.3389/fped.2021.725231\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NUS1, NgBR, WES, intellectual disorder, epilepsy","lastPublishedDoi":"10.21203/rs.3.rs-4158407/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4158407/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMutations in the \u003cem\u003eNUS1\u003c/em\u003e gene, which encodes a Nogo-B receptor (NgBR), are related to congenital disorder of glycosylation, epilepsy, and Parkinson\u0026rsquo;s disease. However, due to the limited number of cases with genotype and detailed clinical features, more cases are needed to better understand the functional and phenotypic characteristics of \u003cem\u003eNUS1\u003c/em\u003e variants. In this study, we reported two unrelated Chinese individuals suffering from intellectual disorder and epilepsy.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e \u003cp\u003eWhole-exome sequencing (WES) was performed on the two patients to identify pathogenic variants, and copy number variation sequencing (CNV-Seq) was conducted on the patients 2. The candidate variants were subsequently validated using Sanger sequencing. Additionally, bioinformatics analyses were used to investigate the deleteriousness of the identified variants.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWES identified two novel variants in the \u003cem\u003eNUS1\u003c/em\u003e gene [NM_138459.5: c.640A\u0026thinsp;\u0026gt;\u0026thinsp;T/p.K214*, c.278delC/p.L94Wfs*11] in the two unrelated individuals with myoclonus, epilepsy, and intellectual disability. These variants resulted in truncated NgBR proteins, which lost the \u003cem\u003ecis-PTase\u003c/em\u003e domain. According to the American College of Medical Genetics and Genomics (ACMG) classification, p.K214* was evaluated as likely pathogenic and p.L94Wfs*11 was evaluated as pathogenic. CNV-Seq analysis revealed a 0.4Mb duplication of Xq27.2q27.2 in patient 2, which was considered uncertain significance.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur findings strongly suggest that the two novel variants in \u003cem\u003eNUS1\u003c/em\u003e gene may be the cause of the patient's clinical characteristics, possibly due to the loss of \u003cem\u003ecis-PTase\u003c/em\u003e activity. Furthermore, our study expanded the genotype-phenotype spectrum of the \u003cem\u003eNUS1\u003c/em\u003e gene.\u003c/p\u003e","manuscriptTitle":"Identification of two novel variants in NUS1 gene in two unrelated Chinese families with intellectual disorder and epilepsy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-02 18:20:33","doi":"10.21203/rs.3.rs-4158407/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e684ed0-e2bb-482e-bf78-55e2a4e519db","owner":[],"postedDate":"April 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-07T11:24:51+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-02 18:20:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4158407","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4158407","identity":"rs-4158407","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}