A novel OPHN1 variant results in a mild form of OPHN1 syndrome characterized by cyclic strabismus

A novel OPHN1 variant results in a mild form of OPHN1 syndrome characterized by cyclic strabismus | 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 Article A novel OPHN1 variant results in a mild form of OPHN1 syndrome characterized by cyclic strabismus Sachiko Nishina, Satoshi Kofuji, Keiko Matsubara, Hazuki Anzai, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6684034/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2026 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract Individuals with cyclic strabismus experience the disease in a pattern in which days with or without strabismus alternate in a circadian rhythm-dependent manner. However, the molecular mechanism that underlies this rhythm is unknown. In this study, we link cyclic strabismus to a novel hemizygous variant of the OPHN1 gene, which encodes a Rho GAP protein, and mutations of which are known cause strabismus. Interestingly, this novel K306N variant affected the PH domain of OPHN1 and enhanced its ability to bind to the phosphatidylinositol phosphates (PIPs) PI4P and PI5P. Our work indicates that at least some cases of cyclic strabismus are due to a gain-of-function variant of OPHN1 . We propose that this alteration may change the subcellular localization of the OPHN1 Rho GAP protein such that its normal activity in an appropriate site is reduced, leading to cyclic strabismus. Health sciences/Diseases/Eye diseases Health sciences/Medical research/Paediatric research Cyclic strabismus Circadian rhythm OPHN1 Rho GAP PH domain Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Strabismus is a common eye disease that results in abnormal binocular function and so impairs quality of vision. Strabismus occurs in both children and adults, and although there are racial differences, the frequency of strabismus is as high as 2.1–3.6% both nationally and internationally 1 , 2 , 3 , 4 . Cyclic strabismus is a very rare form of strabismus with an incidence of about 3 per 3,500 strabismic patients 5 . In affected individuals, days or half-days with or without strabismus alternate in a 48 hr or 24 hr pattern. The disease presents mainly in children with esotropia (crossed eyes). As the patient grows, the cycling gradually breaks down and the strabismus becomes constant, after which it is generally resolved with surgical treatment. Strabismus of any type is thought to be due to both environmental and genetic factors 6 , 7 , but the latter are currently only partially understood. Cyclic strabismus is even more of a mystery. Circadian rhythms are presumably involved, but the causative genes and molecular mechanisms are completely unknown. Several types of congenital incomitant strabismus, where the eye misalignment changes with a different gaze direction, show Mendelian inheritance. For example, congenital fibrosis of the extraocular muscles (CFEOM) type 1 is caused by mutations in the KIF21A (12q12) gene encoding a kinesin motor protein, whereas CFEOM type 2 is linked to alterations to the PHOX2A (11q13) gene encoding a homeodomain transcription factor 8 . Duane syndrome type 2 has been mapped to its causative gene CHN1 (2q31) by linkage analysis of a large family. CHN1 encodes Chimerin-1, a GTPase activating protein (GAP) specific for the small GTP-binding protein RAC 9 . CHN1 is expressed primarily in the brain, and Chimerin-1 is thought to play an important role in signal transduction in the oculomotor axon pathway. However, none of these genes has been associated with cyclic strabismus. The oligophrenin-1 (OPHN1) gene is located on the human X chromosome and encodes a Rho GAP protein containing BAR and PH domains that bind to actin or phospholipids, respectively 10 . Such Rho family proteins are small GTP-binding proteins that activate intracellular signaling essential for cell migration and cell morphology. Mutations of OPHN1 result in “OPHN1 syndrome”, a rare disorder that mainly affects males and is characterized by intellectual disability, low muscle tone, developmental and cognitive delay, early-onset seizures, abnormal behavior, characteristic facial features, and inability to coordinate movements. Pertinently, strabismus is also a feature of OPHN1 syndrome. In the present study, we investigated the involvement of a novel OPHN1 variant in a case of cyclic strabismus and delved into potential causative molecular mechanisms. Results Patient history The patient was an 8-year-old boy who was referred to the Division of Ophthalmology of the National Center for Child Health and Development (NCCHD) for the treatment of cyclic esotropia (ET). He was born to healthy nonconsanguineous parents at 40 weeks gestational age and weighed 3,362 grams. When he was three years old, he developed nephrotic syndrome and was successfully treated. His psychomotor development since then has been normal with no signs of intellectual disability. His parents and younger brother show no obvious signs of disease. At the age of five, without any triggers, the patient suddenly developed ET in his left eye and complained of diplopia (double vision). He underwent an ophthalmological examination by a family physician, and an MRI scan of the patient’s head revealed no abnormalities. Subsequently, the ET resolved spontaneously but continued to recur very occasionally. However, at the age of 8 years, the patient showed periods of alternating ET and orthotropia. He visited the Hirakata Eye Clinic, after which his mother was instructed to record ET and non-ET days on a calendar. This tabulation revealed that the patient experienced ET and non-ET periods every half-day, which led to a diagnosis of cyclic ET in a 24-hour cycle. He was referred to NCCHD because the cycle gradually collapsed, and he increasingly complained of diplopia with ET. The initial examination of this patient at NCCHD revealed that, in the morning, his left eye showed a constant ET of 40 prism diopters (PD) at both near and far distances (Fig. 1 A). No stereoacuity was detected. However, by the afternoon of the same day, he exhibited orthotropia with fine stereoacuity of 40 seconds (Fig. 1 B). He had normal pupillary responses, and the decimal visual acuity was 1.5 in both the right and left eyes. The anterior segment and fundus were normal bilaterally. No other neurologic or systemic abnormalities were detected. This pattern was observed over repeated examinations. The patient then underwent strabismus surgery consisting of left recession of the medial rectus muscle of 5 mm, and left resection of the lateral rectus muscle of 6 mm. As documented in the pre-operative and post-operative nine-gaze photographs shown in Figs. 1 C and 1 D, after surgery, the patient was able to maintain orthophoria with stereoacuity of 40 seconds. The cycling has since resolved and there has been no recurrence in the following three years. A novel variant in the OPHN1 gene on the X chromosome To investigate the genetic cause of the patient’s disorder, we performed whole exome sequencing (WES) of DNA obtained from blood of the patient and his parents. This analysis identified a hemizygous missense variant (c.918G > T, p.K306N) in the PH domain of OPHN1 in the patient (Fig. 2 A–C). The patient's mother was found to be heterozygous for this variant. Notably, this substitution results in the loss of a positive charge at the 306th codon. The lysine residue at this position is highly conserved across multiple species, including human, Callithrix jacchus (marmoset), mouse, and zebrafish (Fig. 2 D). We noted that this OPHN1 variant was absent from population databases such as gnomAD ( https://gnomad.broadinstitute.org/ ) and that maintained by the Tohoku Medical Megabank Organization ( https://www.megabank.tohoku.ac.jp ). In silico prediction tools suggested its pathogenicity: SIFT score = 0.0418 (damaging), PolyPhen-2 (HumVar) score = 0.991 (probably damaging), and CADD score = 26.2 (a score ≥ 20 is generally considered deleterious). These findings indicate that the biochemical properties of the OPHN1 PH domain are likely to be altered in a negative way by this variant. Due to its novelty, this variant was classified by the American College of Medical Genetics (ACMG) guidelines as a variant of uncertain significance (PM2) 11 . However, our examination of our patient suggests that this variant is indeed significant in the context of cyclic strabismus. The OPHN1 (K306N) variant increases PH domain binding activity to PI4P and PI5P To begin to define the changes to the biochemical properties of the OPHN1 PH domain that might be caused by the K306N variant, we used AlphaFold 3 12 to construct a model predicting how the wild-type (WT) PH domain binds to PI(4,5)P 2 . This analysis suggested that the K306 residue was located near the phospholipid-binding pocket of the PH domain, and that K306 might directly interact with the phosphate group of PI(4,5)P 2 (Fig. 3 A). The predicted structure is consistent with known interaction regions between other PH domains and PIPs 13 . We therefore speculated that the K to N substitution in the variant might alter the PH domain’s ability to bind to lipids. To examine the binding properties of a PH domain bearing the K306N variant, we expressed HA-tagged versions of the OPHN1 (WT) and variant OPHN1 (K306N) proteins in HEK293T cells and purified them using magnetic agarose beads. We then compared the binding abilities of these proteins to various PIPs-containing liposomes 14 . The results showed that, while OPHN1 (WT) and OPHN1 (K306N) did not differ in their capacity to bind to phosphatidylinositol-3-phosphate (PI3P), the ability of the variant protein to bind to PI4P and PI5P was enhanced compared to that of OPHN1 (WT) (Fig. 3 B, C). Thus, OPHN1 (K306N) is a gain-of-function variant that results in a PH domain with a higher affinity for PI4P and PI5P. Precisely why increased PI4P and PI5P binding leads to cyclic strabismus remains under investigation. Discussion The subject of this study was a young male patient with cyclic strabismus but without intellectual disability. WES of the patient’s family identified a missense variant of OPHN1 in the patient and his mother. Interestingly, the variant was not in the BAR or GAP domains, but rather in the PH domain. Once expressed as protein, this point-changed PH domain showed an increased ability to bind to PI4P and PI5P, making the alteration biochemically a gain-of-function change. To our knowledge, this is the first reported case of inherited cyclic strabismus presenting with an OPHN1 PH domain variant. In 2018, Moortgat et al. published an excellent paper describing a wide range of patients exhibiting OPHN1 syndrome 10 . The authors summarized findings on 20 OPHN1 mutations observed in 49 male and 14 female patients since 1998, and 4 novel OPHN1 mutations identified by the authors themselves in patients. Of these patients, 90% exhibited strabismus. Interestingly, all 24 OPHN1 mutations were missense mutations or deletions in the OPH1 BAR or GAP domains. Two types of missense mutations in the OPHN1 BAR domain as well as two types in the GAP domain were observed in patients exhibiting mild to moderate intellectual disability associated with seizures, cerebellar signs, ventriculomegaly and strabismus. These results suggested that these presumably loss-of-function mutations in the OPHN1 BAR and GAP domains were strongly related to intellectual disability and strabismus. In contrast, our novel OPHN1 (K306N) variant is a gain-of-function variant that affects the OPHN1 PH domain and results in only cyclic strabismus with no intellectual disability. Thus, our patient displays a very mild form of OPHN1 syndrome. The OPHN1 protein controls the activity-dependent maturation and plasticity of excitatory synapses, and mutations of the X-linked OPHN1 gene can lead to mental retardation. Nadif et al. showed that OPHN1 controls excitatory synapse features through its Rho-GAP activity, which locally modulates RhoA/Rho kinase activities and thus actin dynamics, thereby promoting the stabilization of synaptic AMPA receptors 15 . This finding implies that the subcellular localization of OPHN1 is important for regulating neural function. Interestingly, Valnegri et al. reported that synaptic activity induced the localization of the protein encoded by the clock gene Bmal1 to dendrites and spines, a process that is mediated by activated AMPA receptors that interact with OPHN1 16 . As predicted by our AlphaFold 3 analysis, the K306N variant in the OPHN1 PH domain altered its binding properties to PIPs. These alterations likely result from the change in the amino acid charge and sidechain length caused by the switch from K to N in the vicinity of the binding pocket. Our observations are another example of the utility of using AlphaFold 3 as an excellent tool for predicting protein complex 3D structure and biochemical properties. We found that the OPHN1 K306N protein showed enhanced binding to PI4P and PI5P. PI4P (10% of total PIs) is an important lipid component of the Golgi complex, plasma membrane, and endosomal compartments, and regulates vesicular transport among these structures 17 . PI4P thus ensures the integrity of several processes, including: i) proper location of enzymes resident in the Golgi apparatus; ii) induction of membrane deformation to form vesicles; iii) sorting of proteins to endosomes; and iv) transport of proteins from the trans-Golgi to the plasma membrane. On the other hand, the role of PI5P is largely unknown due to its very low concentration (0.1 to 0.5% of total PIs) in normal cells 18 . It has been recently reported that levels of lipid metabolites in the brain (including phospholipids) are regulated by circadian rhythms 19 . The expression of several lipid metabolism-associated genes, such as those encoding ABC transporters, CD36 (implicated in free acid uptake), and NPC1L1 (cholesterol transporter), has been detected in the brain’s suprachiasmatic nucleus (SCN); these gene expression data have been codified in a mammalian circadian gene expression profile database 20 . All of these genes show similar rhythmic activities over a 24 hr cycle. Notably, clock genes are conserved among Drosophila, mice and humans. In WT flies maintained on sugar-only medium under a light-dark cycle, the transport of lipid species, including diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs), showed a synchronized bimodal oscillation pattern, with maxima at the beginning and end of the light phase 21 . So, we speculate that PI4P and PI5P levels may also be under circadian rhythm control in human cells. If the levels of these lipids increase intracellularly (say, in the plasma membrane etc.), the variant OPHN1 (K306N) Rho GAP protein might be drawn to them in a circadian fashion due to its enhanced binding capacity, and so might alter its subcellular localization. This fluctuation or even loss of RhoGAP activity in the appropriate normal site might lead to cyclic neurophysiological dysfunction and strabismus (Fig. 4 ), just as we observed in our patient. Conclusion We have reported on a young male patient with cyclic strabismus but no intellectual disability. We identified the OPHN1 (K306N) variant as responsible for the patient’s cyclic strabismus and very mild form of OPHN1 syndrome. Furthermore, we conducted lipid binding studies supportive of a molecular mechanism that can explain the periodicity of the disease. To our knowledge, this is the first report of an OPHN1 gene variant that causes cyclic strabismus. Experiments are under way to confirm our proposed model shown in Fig. 4 , and we eagerly await the reporting of a second clinically similar case and/or the identification of genes linked to this important signaling pathway. Materials and methods Molecular analysis Trio-based WES was performed on DNA isolated from blood obtained from the patient and his parents using the SureSelectXT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA). Captured libraries were sequenced on a NovaSeq 6000 platform (Illumina San Diego, CA, USA) with 150-bp paired-end reads. Data processing, variant calling, and annotation were conducted according to previously described methods 22 . The GRCh37/hg19 human reference genome ( https://genome.ucsc.edu ) and NM_002547 transcript ( https://www.ncbi.nlm.nih.gov/genbank ) were used as references. We screened for de novo , hemizygous, and biallelic variants of OPHN1 in the patient. Rare variants were defined as those with minor allele frequencies ≤ 0.01 in the following public databases and our in-house database: (1) the East Asian population of the Genome Aggregation Database (gnomAD genome and exome, EAS subsets) ( https://gnomad.broadinstitute.org/ ); and (2) the Japanese population reference panel (8.3KJPN) ( https://ijgvd.megabank.tohoku.ac.jp/ ). In silico predictions of pathogenicity of the OPHN1 variants of interest were performed using the following tools: (1) SIFT ( http://sift.jcvi.org/ ); (2) PolyPhen-2 (HumVar model) ( http://genetics.bwh.harvard.edu/pph2/ ); and (3) Combined Annotation-Dependent Depletion (CADD) ( http://cadd.gs.washington.edu/ ). Variants of interest were validated by Sanger sequencing. Primer sequences are available upon request. Sequence alignment of human , Callithix jacchus , mouse, and zebrafish OPHN1 PH domains Amino acid sequences of human, Callithrix jacchus , and mouse OPHN1 were obtained from the National Center for Biotechnology Information (NCBI) database. The zebrafish OPHN1 amino acid sequence was obtained from the Ensembl genome database. The accession numbers are as follows: human OPHN1 AAI40764.1, Callithrix jacchus OPHN1 XP_002762999.1, mouse OPHN1 NP_443208.1, and zebrafish OPHN1 ENSDART00000123562.5. A multiple sequence alignment of human, Callithrix jacchus , mouse, and zebrafish OPHN1 proteins was constructed using Clustal X alignment software 23 . The amino acid sequence of the human OPHN1 PH domain was obtained from the UniProt Knowledgebase (UniProtKB) (accession number: O60890). The locations identities of the Callithrix jacchus , mouse, and zebrafish OPHN1 PH domains were determined based on their homology to the PH domain of human OPHN1. Structure prediction using AlphaFold 3 The structure of the complex formed by the binding of the human OPHN1 PH domain to PI(4,5)P₂ diC8 was predicted using AlphaFold 3 (version 3.0.1) 12 . AlphaFold 3 was installed on a local machine, and structure prediction was performed using the official model parameters provided by Google DeepMind. Multiple sequence alignment for the human OPHN1 PH domain was generated using Jackhmmer 24 and HHblits 25 . Ten models were generated, and the structure with the highest score was selected for further analysis. Purification of recombinant OPHN1 proteins The human OPHN1 gene sequence plus an HA tag were subcloned into the pAAV-CMV vector (TaKaRa Bio). This plasmid was transfected into HEK293T cells using polyethyleneimine (Sigma-Aldrich). After 48 hr, the transfected cells were collected and lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% Triton X-100, 1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml pepstatin A, 5 µg/ml aprotinin, 50 mM sodium fluoride]. After centrifugation at 20,400 x g at 4°C for 10 min, the supernatant was incubated with anti-HA-tag mAb Magnetic Agarose Beads (MBL, M180-10) for 1 hr. The magnetic agarose beads were collected on a magnetic rack and washed with lysis buffer without Triton X-100. To elute proteins, the beads were incubated at 37°C for 10 min in phosphate-buffered saline containing 2 mg/mL HA peptide. The concentration of the eluted protein was measured using the BCA Protein Assay Kit (Pierce, 23227). The purity of the eluted protein was confirmed by Coomassie brilliant blue staining. PolyPIPosome binding assay Purified HA-tagged OPHN1 protein (0.5 µg) was diluted in 250 µl of PolyPIPosome binding buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA (pH 7.5), 1 mM MgCl 2 , 0.2 mM CaCl 2 , 5 mM KCl, 1 mg/ml fatty acid-free BSA]. PolyPIPosomes (Echelon ) were added to a final concentration of 3 µM, and the mixture was incubated at room temperature (RT) for 15 min. PolyPIPosomes used were: PI (Y-P000), PI(3)P (Y-P003), PI(4)P (Y-P004), and PI(5)P (Y-P005). Dynabeads MyOne Streptavidin C1 (Invitrogen, 65001) (10 µg) were added and the PolyPIPosome mixture incubated at RT for 30 min. The magnetic beads were collected on a magnetic rack, washed with PolyPIPosome binding buffer, and resuspended in 1.2 x Laemmli SDS sample buffer. Samples were incubated at 100°C for 1 min to elute proteins, which were then fractionated by SDS-PAGE. Gels were subjected to Western blotting using anti-HA antibody (Cell Signaling Technology, 3724). Band intensities were measured using Image J (NIH). Statistical analyses Analyses of statistical significance were performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA). The unpaired two-tailed Student's t -test was used for comparisons between two groups. Data were considered statistically significant at P < 0.05. Abbreviations OPHN1, oligophrenin-1; Rho GAP, Rho GTPase-activating protein; PH domain, pleckstrin homology domain; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate. Declarations Acknowledgments The authors thank the patient and his parents for participating in this study. Funding information This work was supported by a grant from the Japanese National Center for Child Health and Development (to S.N. and M.F.); a Health and Labour Sciences Research Grant for Research on Intractable Diseases (#20FC1055 to S.N.) from the Ministry of Health, Labour and Welfare, Tokyo, Japan; JSPS KAKENHI Grants #16K11309, #19K10004 and #22K09848 (to S.N.); JSPS KAKENHI Grants #20H03381 and #24K02177 (to H.N.); a Nanken-Kyoten grant from the Institute of Science Tokyo (to H.N.); a grant for the study of Multilayered Stress Diseases (JPMXP1323015483 to H.N.); and a grant from the SECOM Science and Technology Foundation (to H.N.). Author Contributions S.N., M.F. and H.N. conceived and designed the study. S.N., K.H. and H.A. performed clinical analyses. S.K. performed biochemical experiments and acquired the data. K.M. and M. F. performed genomic analyses. Y.H. and N.I. performed structural analyses. Y.O., J.H. and H.N. analyzed data. S.N., M.F. and H.N. wrote the manuscript. Data Availability The data of the variant have been deposited into ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and will be available after the publication under the number SUB15380213. The primary patient datasets, along with other data and materials used in this study are available from the corresponding authors upon reasonable request. Disclosure of Potential Conflicts of Interest The authors declare that no competing interests exist. Ethics statement The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Japanese National Centre for Child Health and Development (permit no. 686). Written informed consent was obtained from all subjects and/or their legal guardian(s) for publication of their images in an online open-access journal. References Multi-ethnic Pediatric Eye Disease Study Group. 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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-6684034","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":502136069,"identity":"78bdd5a4-5e49-4266-98bb-d6f75cc957fa","order_by":0,"name":"Sachiko Nishina","email":"","orcid":"","institution":"National Center for Child Health and Development","correspondingAuthor":false,"prefix":"","firstName":"Sachiko","middleName":"","lastName":"Nishina","suffix":""},{"id":502136070,"identity":"e3c69157-6b53-4630-ac35-7c010c3051bf","order_by":1,"name":"Satoshi Kofuji","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Satoshi","middleName":"","lastName":"Kofuji","suffix":""},{"id":502136071,"identity":"1adc4e4d-34f2-43f4-840b-c103ac93177e","order_by":2,"name":"Keiko Matsubara","email":"","orcid":"","institution":"National Research Institute for Child Health and Development","correspondingAuthor":false,"prefix":"","firstName":"Keiko","middleName":"","lastName":"Matsubara","suffix":""},{"id":502136072,"identity":"12c5c9d0-3a6f-4ac2-ab36-1e3939294f2c","order_by":3,"name":"Hazuki Anzai","email":"","orcid":"","institution":"National Center for Child Health and Development","correspondingAuthor":false,"prefix":"","firstName":"Hazuki","middleName":"","lastName":"Anzai","suffix":""},{"id":502136073,"identity":"409dcc56-fc5b-44a6-8329-f257007d72ae","order_by":4,"name":"Kyoko Hirakata","email":"","orcid":"","institution":"Hirakata Eye Clinic","correspondingAuthor":false,"prefix":"","firstName":"Kyoko","middleName":"","lastName":"Hirakata","suffix":""},{"id":502136074,"identity":"b3990cfb-d649-4a55-a906-61cf5da3d2da","order_by":5,"name":"Yuya Hanazono","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Yuya","middleName":"","lastName":"Hanazono","suffix":""},{"id":502136075,"identity":"daf18b47-c513-4e19-99b5-c67f43771d87","order_by":6,"name":"Yoshimi Okamoto-Uchida","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Yoshimi","middleName":"","lastName":"Okamoto-Uchida","suffix":""},{"id":502136076,"identity":"1edcb12b-60ed-4698-825e-d85c48741abe","order_by":7,"name":"Jun Hirayama","email":"","orcid":"","institution":"Bunkyo University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Hirayama","suffix":""},{"id":502136077,"identity":"c0f2505a-1e93-410b-832a-023c61404ef0","order_by":8,"name":"Nobutoshi Ito","email":"","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Nobutoshi","middleName":"","lastName":"Ito","suffix":""},{"id":502136078,"identity":"d7a573e1-ddd6-49ba-971b-1bae94d588b2","order_by":9,"name":"Maki Fukami","email":"","orcid":"","institution":"National Research Institute for Child Health and Development","correspondingAuthor":false,"prefix":"","firstName":"Maki","middleName":"","lastName":"Fukami","suffix":""},{"id":502136079,"identity":"7b321409-445e-46fc-be98-666be056c35d","order_by":10,"name":"Hiroshi Nishina","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYDACZgY2hgQQ4wZjA8MHBgYeEFsCRAD5hLUwzgBpYSOkhQGkBKwFqJ0HypfA5y5zdvZnDx7usGPgu93c+Ng2554Mg3x34g2GGjsG5tnYrbFs5jE3SDyTzCB552Czce62YqDDeDdbMBxLZmCccwCrFoPDPGwSiW3MDAY3Etukc7clgLRsk2BgO8DAOCMBhxb2Z0At9RAtlnAt//BpYTADajkM0cII08LYhk8LD0jLcR6QXwx7gVrY2HI3WyT2JfPg9Mv5488kf7ZVy/Hdbn/44Oe2BHt+5rMbb3z4ZidniCPEYIAHzgJHE9BJPIYz8OrABuTxRugoGAWjYBSMIAAAaS1WrcPZFSoAAAAASUVORK5CYII=","orcid":"","institution":"Institute of Science Tokyo","correspondingAuthor":true,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Nishina","suffix":""}],"badges":[],"createdAt":"2025-05-17 02:23:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6684034/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6684034/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-48129-7","type":"published","date":"2026-04-24T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89385050,"identity":"c33882bd-7bae-4b48-85d9-33a92f1077f9","added_by":"auto","created_at":"2025-08-19 12:28:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1258143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotographs of the eyes of a young male patient with cyclic strabismus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eIn the morning, the patient showed left esotropia of 40 PD at near and far distances.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e In the afternoon of the same day, the patient exhibited orthotropia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Preoperative photograph of the patient’s nine-gaze indicating a 24-hr cycle of esotropia without abduction deficit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003ePostoperative photograph of the patient’s nine-gaze showing orthotropia and cycle resolution.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6684034/v1/48c0b5e805b05ecc499a52c2.png"},{"id":89386627,"identity":"74eafdbd-b5cd-445f-ba86-e5560d8538f9","added_by":"auto","created_at":"2025-08-19 12:36:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1350235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOPHN1 PH domain: novel variant of the human X-linked \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOPHN1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene and cross-species conservation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA)\u003c/strong\u003e Diagram of the domain structure and amino acid length of the normal human OPHN1 protein. The site of the K306N variant is indicated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Diagram of the DNA nucleotide sequences of the partial \u003cem\u003eOPHN1\u003c/em\u003e gene in the patient and his mother, showing the missense variant leading to the K306N alteration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The pedigree of the patient’s family showing unaffected, carrier and affected status.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Alignment of the amino acid sequences (single letter code) of the human, \u003cem\u003eCallithrix jacchus\u003c/em\u003e (callithrix), mouse, and zebrafish OPHN1 PH domains. *, amino acids identical in all four proteins. -, missing amino acids. Numbers, amino acid positions from the amino terminus of the human OPHN1 protein. Arrow, lysine substituted with asparagine (K306N) in the patient.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6684034/v1/a76a8bbe6216e3ac1ab03e99.png"},{"id":89385052,"identity":"1ebd4d08-f46f-4f65-b1dd-3bc701d47fe2","added_by":"auto","created_at":"2025-08-19 12:28:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":753224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical properties of WT OPHN1 vs. its K306N variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Model of the structure of the WT human OPHN1 PH domain bound to a ligand, in this case phosphatidylinositol 4,5-bisphosphate (PI(4,5)P\u003csub\u003e2\u003c/sub\u003e) diC8, as predicted by AlphaFold 3. In this model, the head group of the ligand binds to a shallow pocket in the protein, placing lysine (K) 306 in a position to directly interact with one of the phosphate groups on the inositol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Western blot to detect the binding of HA-OPHN1 (WT or K306N) to the indicated PolyPIPosomes. Results are representative of three trials. The original blot is presented in Supplementary Figure 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Quantification of the band intensities in (B) expressed as the ratio of each band’s intensity to that of PI. Data are the mean ± s.d. (\u003cem\u003en\u003c/em\u003e=7). P values were determined by unpaired two-tailed Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6684034/v1/bde63185e683c9221ffca3be.png"},{"id":89388853,"identity":"aa4a9c28-a5b8-4542-b5d5-5e1e238f9f25","added_by":"auto","created_at":"2025-08-19 12:44:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1009417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA hypothesis to explain why cyclic strabismus is associated with the OPHN1 (K306N) variant.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe WT OPHN1 (K306) RhoGAP protein (no mutation) shows no circadian rhythm in its activity. Under steady-state conditions (left panel), the variant OPHN1 (K306N) RhoGAP protein continues to regulate the conversion of Rho proteins from the GTP to GDP form in a normal fashion and so contributes to the maintenance of the orthophoria state. However, when a circadian rhythm-dependent increase in local intracellular production of PI4P and/or PI5P occurs (right panel), the OPHN1 (K306N) RhoGAP protein is drawn to these lipids, changes its localization due to its enhanced binding capacity, and neglects some of its normal functions in the normal site. The resulting transient local increase in RhoGTP may lead to cyclic neurophysiological dysfunction and strabismus.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6684034/v1/72fda039509e8bc356a8f5d6.png"},{"id":107927666,"identity":"c6009b96-3945-435e-a512-c0d1be51ff75","added_by":"auto","created_at":"2026-04-27 16:00:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4613771,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6684034/v1/ad758bb6-36dc-4627-a744-b14d7443b5d8.pdf"},{"id":89385048,"identity":"ad20c67d-60de-45da-b753-8e128df4c5b7","added_by":"auto","created_at":"2025-08-19 12:28:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":88746,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigNishinaSetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6684034/v1/9c2220f612344d502ffaf460.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"A novel OPHN1 variant results in a mild form of OPHN1 syndrome characterized by cyclic strabismus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStrabismus is a common eye disease that results in abnormal binocular function and so impairs quality of vision. Strabismus occurs in both children and adults, and although there are racial differences, the frequency of strabismus is as high as 2.1\u0026ndash;3.6% both nationally and internationally\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Cyclic strabismus is a very rare form of strabismus with an incidence of about 3 per 3,500 strabismic patients\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In affected individuals, days or half-days with or without strabismus alternate in a 48 hr or 24 hr pattern. The disease presents mainly in children with esotropia (crossed eyes). As the patient grows, the cycling gradually breaks down and the strabismus becomes constant, after which it is generally resolved with surgical treatment. Strabismus of any type is thought to be due to both environmental and genetic factors\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, but the latter are currently only partially understood. Cyclic strabismus is even more of a mystery. Circadian rhythms are presumably involved, but the causative genes and molecular mechanisms are completely unknown.\u003c/p\u003e\u003cp\u003eSeveral types of congenital incomitant strabismus, where the eye misalignment changes with a different gaze direction, show Mendelian inheritance. For example, congenital fibrosis of the extraocular muscles (CFEOM) type 1 is caused by mutations in the \u003cem\u003eKIF21A\u003c/em\u003e (12q12) gene encoding a kinesin motor protein, whereas CFEOM type 2 is linked to alterations to the \u003cem\u003ePHOX2A\u003c/em\u003e (11q13) gene encoding a homeodomain transcription factor\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Duane syndrome type 2 has been mapped to its causative gene \u003cem\u003eCHN1\u003c/em\u003e (2q31) by linkage analysis of a large family. \u003cem\u003eCHN1\u003c/em\u003e encodes Chimerin-1, a GTPase activating protein (GAP) specific for the small GTP-binding protein RAC\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eCHN1\u003c/em\u003e is expressed primarily in the brain, and Chimerin-1 is thought to play an important role in signal transduction in the oculomotor axon pathway. However, none of these genes has been associated with cyclic strabismus.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eoligophrenin-1 (OPHN1)\u003c/em\u003e gene is located on the human X chromosome and encodes a Rho GAP protein containing BAR and PH domains that bind to actin or phospholipids, respectively\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Such Rho family proteins are small GTP-binding proteins that activate intracellular signaling essential for cell migration and cell morphology. Mutations of \u003cem\u003eOPHN1\u003c/em\u003e result in \u0026ldquo;OPHN1 syndrome\u0026rdquo;, a rare disorder that mainly affects males and is characterized by intellectual disability, low muscle tone, developmental and cognitive delay, early-onset seizures, abnormal behavior, characteristic facial features, and inability to coordinate movements. Pertinently, strabismus is also a feature of OPHN1 syndrome. In the present study, we investigated the involvement of a novel \u003cem\u003eOPHN1\u003c/em\u003e variant in a case of cyclic strabismus and delved into potential causative molecular mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatient history\u003c/h2\u003e\u003cp\u003eThe patient was an 8-year-old boy who was referred to the Division of Ophthalmology of the National Center for Child Health and Development (NCCHD) for the treatment of cyclic esotropia (ET). He was born to healthy nonconsanguineous parents at 40 weeks gestational age and weighed 3,362 grams. When he was three years old, he developed nephrotic syndrome and was successfully treated. His psychomotor development since then has been normal with no signs of intellectual disability. His parents and younger brother show no obvious signs of disease.\u003c/p\u003e\u003cp\u003eAt the age of five, without any triggers, the patient suddenly developed ET in his left eye and complained of diplopia (double vision). He underwent an ophthalmological examination by a family physician, and an MRI scan of the patient\u0026rsquo;s head revealed no abnormalities. Subsequently, the ET resolved spontaneously but continued to recur very occasionally. However, at the age of 8 years, the patient showed periods of alternating ET and orthotropia. He visited the Hirakata Eye Clinic, after which his mother was instructed to record ET and non-ET days on a calendar. This tabulation revealed that the patient experienced ET and non-ET periods every half-day, which led to a diagnosis of cyclic ET in a 24-hour cycle. He was referred to NCCHD because the cycle gradually collapsed, and he increasingly complained of diplopia with ET.\u003c/p\u003e\u003cp\u003eThe initial examination of this patient at NCCHD revealed that, in the morning, his left eye showed a constant ET of 40 prism diopters (PD) at both near and far distances (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). No stereoacuity was detected. However, by the afternoon of the same day, he exhibited orthotropia with fine stereoacuity of 40 seconds (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). He had normal pupillary responses, and the decimal visual acuity was 1.5 in both the right and left eyes. The anterior segment and fundus were normal bilaterally. No other neurologic or systemic abnormalities were detected. This pattern was observed over repeated examinations. The patient then underwent strabismus surgery consisting of left recession of the medial rectus muscle of 5 mm, and left resection of the lateral rectus muscle of 6 mm. As documented in the pre-operative and post-operative nine-gaze photographs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, after surgery, the patient was able to maintain orthophoria with stereoacuity of 40 seconds. The cycling has since resolved and there has been no recurrence in the following three years.\u003c/p\u003e\u003cp\u003e\u003cb\u003eA novel variant in the\u003c/b\u003e \u003cb\u003eOPHN1\u003c/b\u003e \u003cb\u003egene on the X chromosome\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the genetic cause of the patient\u0026rsquo;s disorder, we performed whole exome sequencing (WES) of DNA obtained from blood of the patient and his parents. This analysis identified a hemizygous missense variant (c.918G\u0026thinsp;\u0026gt;\u0026thinsp;T, p.K306N) in the PH domain of OPHN1 in the patient (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;C). The patient's mother was found to be heterozygous for this variant. Notably, this substitution results in the loss of a positive charge at the 306th codon. The lysine residue at this position is highly conserved across multiple species, including human, \u003cem\u003eCallithrix jacchus\u003c/em\u003e (marmoset), mouse, and zebrafish (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eWe noted that this OPHN1 variant was absent from population databases such as gnomAD (\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) and that maintained by the Tohoku Medical Megabank Organization (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megabank.tohoku.ac.jp\u003c/span\u003e\u003cspan address=\"https://www.megabank.tohoku.ac.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003eIn silico\u003c/em\u003e prediction tools suggested its pathogenicity: SIFT score\u0026thinsp;=\u0026thinsp;0.0418 (damaging), PolyPhen-2 (HumVar) score\u0026thinsp;=\u0026thinsp;0.991 (probably damaging), and CADD score\u0026thinsp;=\u0026thinsp;26.2 (a score\u0026thinsp;\u0026ge;\u0026thinsp;20 is generally considered deleterious). These findings indicate that the biochemical properties of the OPHN1 PH domain are likely to be altered in a negative way by this variant. Due to its novelty, this variant was classified by the American College of Medical Genetics (ACMG) guidelines as a variant of uncertain significance (PM2)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. However, our examination of our patient suggests that this variant is indeed significant in the context of cyclic strabismus.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eThe OPHN1 (K306N) variant increases PH domain binding activity to PI4P and PI5P\u003c/h3\u003e\n\u003cp\u003eTo begin to define the changes to the biochemical properties of the OPHN1 PH domain that might be caused by the K306N variant, we used AlphaFold 3\u003csup\u003e12\u003c/sup\u003e to construct a model predicting how the wild-type (WT) PH domain binds to PI(4,5)P\u003csub\u003e2\u003c/sub\u003e. This analysis suggested that the K306 residue was located near the phospholipid-binding pocket of the PH domain, and that K306 might directly interact with the phosphate group of PI(4,5)P\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The predicted structure is consistent with known interaction regions between other PH domains and PIPs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We therefore speculated that the K to N substitution in the variant might alter the PH domain\u0026rsquo;s ability to bind to lipids. To examine the binding properties of a PH domain bearing the K306N variant, we expressed HA-tagged versions of the OPHN1 (WT) and variant OPHN1 (K306N) proteins in HEK293T cells and purified them using magnetic agarose beads. We then compared the binding abilities of these proteins to various PIPs-containing liposomes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The results showed that, while OPHN1 (WT) and OPHN1 (K306N) did not differ in their capacity to bind to phosphatidylinositol-3-phosphate (PI3P), the ability of the variant protein to bind to PI4P and PI5P was enhanced compared to that of OPHN1 (WT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). Thus, OPHN1 (K306N) is a gain-of-function variant that results in a PH domain with a higher affinity for PI4P and PI5P. Precisely why increased PI4P and PI5P binding leads to cyclic strabismus remains under investigation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe subject of this study was a young male patient with cyclic strabismus but without intellectual disability. WES of the patient\u0026rsquo;s family identified a missense variant of \u003cem\u003eOPHN1\u003c/em\u003e in the patient and his mother. Interestingly, the variant was not in the BAR or GAP domains, but rather in the PH domain. Once expressed as protein, this point-changed PH domain showed an increased ability to bind to PI4P and PI5P, making the alteration biochemically a gain-of-function change. To our knowledge, this is the first reported case of inherited cyclic strabismus presenting with an OPHN1 PH domain variant.\u003c/p\u003e\u003cp\u003eIn 2018, Moortgat \u003cem\u003eet al.\u003c/em\u003e published an excellent paper describing a wide range of patients exhibiting OPHN1 syndrome\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The authors summarized findings on 20 \u003cem\u003eOPHN1\u003c/em\u003e mutations observed in 49 male and 14 female patients since 1998, and 4 novel \u003cem\u003eOPHN1\u003c/em\u003e mutations identified by the authors themselves in patients. Of these patients, 90% exhibited strabismus. Interestingly, all 24 \u003cem\u003eOPHN1\u003c/em\u003e mutations were missense mutations or deletions in the OPH1 BAR or GAP domains. Two types of missense mutations in the OPHN1 BAR domain as well as two types in the GAP domain were observed in patients exhibiting mild to moderate intellectual disability associated with seizures, cerebellar signs, ventriculomegaly and strabismus. These results suggested that these presumably loss-of-function mutations in the OPHN1 BAR and GAP domains were strongly related to intellectual disability and strabismus. In contrast, our novel OPHN1 (K306N) variant is a gain-of-function variant that affects the OPHN1 PH domain and results in only cyclic strabismus with no intellectual disability. Thus, our patient displays a very mild form of \u003cem\u003eOPHN1\u003c/em\u003e syndrome.\u003c/p\u003e\u003cp\u003eThe OPHN1 protein controls the activity-dependent maturation and plasticity of excitatory synapses, and mutations of the X-linked \u003cem\u003eOPHN1\u003c/em\u003e gene can lead to mental retardation. Nadif \u003cem\u003eet al.\u003c/em\u003e showed that OPHN1 controls excitatory synapse features through its Rho-GAP activity, which locally modulates RhoA/Rho kinase activities and thus actin dynamics, thereby promoting the stabilization of synaptic AMPA receptors\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This finding implies that the subcellular localization of OPHN1 is important for regulating neural function. Interestingly, Valnegri \u003cem\u003eet al.\u003c/em\u003e reported that synaptic activity induced the localization of the protein encoded by the clock gene \u003cem\u003eBmal1\u003c/em\u003e to dendrites and spines, a process that is mediated by activated AMPA receptors that interact with OPHN1\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAs predicted by our AlphaFold 3 analysis, the K306N variant in the OPHN1 PH domain altered its binding properties to PIPs. These alterations likely result from the change in the amino acid charge and sidechain length caused by the switch from K to N in the vicinity of the binding pocket. Our observations are another example of the utility of using AlphaFold 3 as an excellent tool for predicting protein complex 3D structure and biochemical properties.\u003c/p\u003e\u003cp\u003eWe found that the OPHN1 K306N protein showed enhanced binding to PI4P and PI5P. PI4P (10% of total PIs) is an important lipid component of the Golgi complex, plasma membrane, and endosomal compartments, and regulates vesicular transport among these structures\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. PI4P thus ensures the integrity of several processes, including: i) proper location of enzymes resident in the Golgi apparatus; ii) induction of membrane deformation to form vesicles; iii) sorting of proteins to endosomes; and iv) transport of proteins from the trans-Golgi to the plasma membrane. On the other hand, the role of PI5P is largely unknown due to its very low concentration (0.1 to 0.5% of total PIs) in normal cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIt has been recently reported that levels of lipid metabolites in the brain (including phospholipids) are regulated by circadian rhythms\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. The expression of several lipid metabolism-associated genes, such as those encoding ABC transporters, CD36 (implicated in free acid uptake), and NPC1L1 (cholesterol transporter), has been detected in the brain\u0026rsquo;s suprachiasmatic nucleus (SCN); these gene expression data have been codified in a mammalian circadian gene expression profile database\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. All of these genes show similar rhythmic activities over a 24 hr cycle. Notably, clock genes are conserved among Drosophila, mice and humans. In WT flies maintained on sugar-only medium under a light-dark cycle, the transport of lipid species, including diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs), showed a synchronized bimodal oscillation pattern, with maxima at the beginning and end of the light phase\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. So, we speculate that PI4P and PI5P levels may also be under circadian rhythm control in human cells. If the levels of these lipids increase intracellularly (say, in the plasma membrane etc.), the variant OPHN1 (K306N) Rho GAP protein might be drawn to them in a circadian fashion due to its enhanced binding capacity, and so might alter its subcellular localization. This fluctuation or even loss of RhoGAP activity in the appropriate normal site might lead to cyclic neurophysiological dysfunction and strabismus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), just as we observed in our patient.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eWe have reported on a young male patient with cyclic strabismus but no intellectual disability. We identified the OPHN1 (K306N) variant as responsible for the patient\u0026rsquo;s cyclic strabismus and very mild form of \u003cem\u003eOPHN1\u003c/em\u003e syndrome. Furthermore, we conducted lipid binding studies supportive of a molecular mechanism that can explain the periodicity of the disease. To our knowledge, this is the first report of an \u003cem\u003eOPHN1\u003c/em\u003e gene variant that causes cyclic strabismus. Experiments are under way to confirm our proposed model shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, and we eagerly await the reporting of a second clinically similar case and/or the identification of genes linked to this important signaling pathway.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eMolecular analysis\u003c/h2\u003e\u003cp\u003eTrio-based WES was performed on DNA isolated from blood obtained from the patient and his parents using the SureSelectXT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, USA). Captured libraries were sequenced on a NovaSeq 6000 platform (Illumina San Diego, CA, USA) with 150-bp paired-end reads. Data processing, variant calling, and annotation were conducted according to previously described methods\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The GRCh37/hg19 human reference genome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://genome.ucsc.edu\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and NM_002547 transcript (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/genbank\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/genbank\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used as references.\u003c/p\u003e\u003cp\u003eWe screened for \u003cem\u003ede novo\u003c/em\u003e, hemizygous, and biallelic variants of OPHN1 in the patient. Rare variants were defined as those with minor allele frequencies\u0026thinsp;\u0026le;\u0026thinsp;0.01 in the following public databases and our in-house database: (1) the East Asian population of the Genome Aggregation Database (gnomAD genome and exome, EAS subsets) (\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); and (2) the Japanese population reference panel (8.3KJPN) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ijgvd.megabank.tohoku.ac.jp/\u003c/span\u003e\u003cspan address=\"https://ijgvd.megabank.tohoku.ac.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). \u003cem\u003eIn silico\u003c/em\u003e predictions of pathogenicity of the OPHN1 variants of interest were performed using the following tools: (1) SIFT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://sift.jcvi.org/\u003c/span\u003e\u003cspan address=\"http://sift.jcvi.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); (2) PolyPhen-2 (HumVar model) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genetics.bwh.harvard.edu/pph2/\u003c/span\u003e\u003cspan address=\"http://genetics.bwh.harvard.edu/pph2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e); and (3) Combined Annotation-Dependent Depletion (CADD) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cadd.gs.washington.edu/\u003c/span\u003e\u003cspan address=\"http://cadd.gs.washington.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Variants of interest were validated by Sanger sequencing. Primer sequences are available upon request.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSequence alignment of human\u003c/b\u003e, \u003cb\u003eCallithix jacchus\u003c/b\u003e, \u003cb\u003emouse, and zebrafish OPHN1 PH domains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAmino acid sequences of human, \u003cem\u003eCallithrix jacchus\u003c/em\u003e, and mouse OPHN1 were obtained from the National Center for Biotechnology Information (NCBI) database. The zebrafish OPHN1 amino acid sequence was obtained from the Ensembl genome database. The accession numbers are as follows: human OPHN1 AAI40764.1, \u003cem\u003eCallithrix jacchus\u003c/em\u003e OPHN1 XP_002762999.1, mouse OPHN1 NP_443208.1, and zebrafish OPHN1 ENSDART00000123562.5. A multiple sequence alignment of human, \u003cem\u003eCallithrix jacchus\u003c/em\u003e, mouse, and zebrafish OPHN1 proteins was constructed using Clustal X alignment software\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The amino acid sequence of the human OPHN1 PH domain was obtained from the UniProt Knowledgebase (UniProtKB) (accession number: O60890). The locations identities of the \u003cem\u003eCallithrix jacchus\u003c/em\u003e, mouse, and zebrafish OPHN1 PH domains were determined based on their homology to the PH domain of human OPHN1.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStructure prediction using AlphaFold 3\u003c/h3\u003e\n\u003cp\u003eThe structure of the complex formed by the binding of the human OPHN1 PH domain to PI(4,5)P₂ diC8 was predicted using AlphaFold 3 (version 3.0.1)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. AlphaFold 3 was installed on a local machine, and structure prediction was performed using the official model parameters provided by Google DeepMind. Multiple sequence alignment for the human OPHN1 PH domain was generated using Jackhmmer\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e and HHblits\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Ten models were generated, and the structure with the highest score was selected for further analysis.\u003c/p\u003e\n\u003ch3\u003ePurification of recombinant OPHN1 proteins\u003c/h3\u003e\n\u003cp\u003eThe human OPHN1 gene sequence plus an HA tag were subcloned into the pAAV-CMV vector (TaKaRa Bio). This plasmid was transfected into HEK293T cells using polyethyleneimine (Sigma-Aldrich). After 48 hr, the transfected cells were collected and lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5% Triton X-100, 1 mM PMSF, 20 \u0026micro;g/ml leupeptin, 20 \u0026micro;g/ml pepstatin A, 5 \u0026micro;g/ml aprotinin, 50 mM sodium fluoride]. After centrifugation at 20,400 x g at 4\u0026deg;C for 10 min, the supernatant was incubated with anti-HA-tag mAb Magnetic Agarose Beads (MBL, M180-10) for 1 hr. The magnetic agarose beads were collected on a magnetic rack and washed with lysis buffer without Triton X-100. To elute proteins, the beads were incubated at 37\u0026deg;C for 10 min in phosphate-buffered saline containing 2 mg/mL HA peptide. The concentration of the eluted protein was measured using the BCA Protein Assay Kit (Pierce, 23227). The purity of the eluted protein was confirmed by Coomassie brilliant blue staining.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePolyPIPosome binding assay\u003c/h2\u003e\u003cp\u003ePurified HA-tagged OPHN1 protein (0.5 \u0026micro;g) was diluted in 250 \u0026micro;l of PolyPIPosome binding buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA (pH 7.5), 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 5 mM KCl, 1 mg/ml fatty acid-free BSA]. PolyPIPosomes (Echelon\u003cb\u003e)\u003c/b\u003e were added to a final concentration of 3 \u0026micro;M, and the mixture was incubated at room temperature (RT) for 15 min. PolyPIPosomes used were: PI (Y-P000), PI(3)P (Y-P003), PI(4)P (Y-P004), and PI(5)P (Y-P005). Dynabeads MyOne Streptavidin C1 (Invitrogen, 65001) (10 \u0026micro;g) were added and the PolyPIPosome mixture incubated at RT for 30 min. The magnetic beads were collected on a magnetic rack, washed with PolyPIPosome binding buffer, and resuspended in 1.2 x Laemmli SDS sample buffer. Samples were incubated at 100\u0026deg;C for 1 min to elute proteins, which were then fractionated by SDS-PAGE. Gels were subjected to Western blotting using anti-HA antibody (Cell Signaling Technology, 3724). Band intensities were measured using Image J (NIH).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eAnalyses of statistical significance were performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA). The unpaired two-tailed Student's \u003cem\u003et\u003c/em\u003e-test was used for comparisons between two groups. Data were considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOPHN1, oligophrenin-1; Rho GAP, Rho GTPase-activating protein; PH domain, pleckstrin homology domain; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the patient and his parents for participating in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the Japanese National Center for Child Health and Development (to S.N. and M.F.); a Health and Labour Sciences Research Grant for Research on Intractable Diseases (#20FC1055 to S.N.) from the Ministry of Health, Labour and Welfare, Tokyo, Japan; JSPS KAKENHI Grants #16K11309, #19K10004 and #22K09848 (to S.N.); JSPS KAKENHI Grants #20H03381 and #24K02177 (to H.N.); a Nanken-Kyoten grant from the Institute of Science Tokyo (to H.N.); a grant for the study of Multilayered Stress Diseases (JPMXP1323015483 to H.N.); and a grant from the SECOM Science and Technology Foundation (to H.N.). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.N., M.F. and H.N. conceived and designed the study. S.N., K.H. and H.A. performed clinical analyses. S.K. performed biochemical experiments and acquired the data. K.M. and M. F. performed genomic analyses. Y.H. and N.I. performed structural analyses. Y.O., J.H. and H.N. analyzed data. S.N., M.F. and H.N. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data of the variant have been deposited into ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and will be available after the publication under the number SUB15380213. The primary patient datasets, along with other data and materials used in this study are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of Potential Conflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no competing interests exist.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Japanese National Centre for Child Health and Development (permit no. 686). Written informed consent was obtained from all subjects and/or their legal guardian(s) for publication of their images in an online open-access journal.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMulti-ethnic Pediatric Eye Disease Study Group. 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Circadian influences on brain lipid metabolism and neurodegenerative diseases. Metabolites 14, 723 (2024). \u003c/li\u003e\n\u003cli\u003ePizarro, A., Hayer, K., Lahens, N. F. \u0026amp; Hogenesch, J. B. CircaDB: a database of mammalian circadian gene expression profiles. Nucleic Acids Res. 41, D1009\u0026ndash;D1013 (2012). \u003c/li\u003e\n\u003cli\u003eAmatobi, K. M. et al. The circadian clock is required for rhythmic lipid transport in Drosophila in interaction with diet and photic condition. J. Lipid Res. 64, 100417 (2023). \u003c/li\u003e\n\u003cli\u003eMiyado, M. et al. Germline-derived gain-of-function variants of Gs\u0026alpha;-coding GNAS gene identified in nephrogenic syndrome of inappropriate antidiuresis. J. Am. Soc. Nephrol. 30, 877\u0026ndash;889 (2019).\u003c/li\u003e\n\u003cli\u003eLarkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947\u0026ndash;2948 (2007). \u003c/li\u003e\n\u003cli\u003eJohnson, L.S., Eddy, S.R. \u0026amp; Portugaly, E. Hidden Markov model speed heuristic and iterative HMM search procedure. BMC Bioinformatics 11, 431 (2010).\u003c/li\u003e\n\u003cli\u003eRemmert, M., Biegert, A., Hauser, A. et al. HHblits: lightning-fast iterative protein sequence searching by HMM-HMM alignment. Nat Methods 9, 173\u0026ndash;175 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cyclic strabismus, Circadian rhythm, OPHN1, Rho GAP, PH domain","lastPublishedDoi":"10.21203/rs.3.rs-6684034/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6684034/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIndividuals with cyclic strabismus experience the disease in a pattern in which days with or without strabismus alternate in a circadian rhythm-dependent manner. However, the molecular mechanism that underlies this rhythm is unknown. In this study, we link cyclic strabismus to a novel hemizygous variant of the \u003cem\u003eOPHN1\u003c/em\u003e gene, which encodes a Rho GAP protein, and mutations of which are known cause strabismus. Interestingly, this novel K306N variant affected the PH domain of \u003cem\u003eOPHN1\u003c/em\u003e and enhanced its ability to bind to the phosphatidylinositol phosphates (PIPs) PI4P and PI5P. Our work indicates that at least some cases of cyclic strabismus are due to a gain-of-function variant of \u003cem\u003eOPHN1\u003c/em\u003e. We propose that this alteration may change the subcellular localization of the OPHN1 Rho GAP protein such that its normal activity in an appropriate site is reduced, leading to cyclic strabismus.\u003c/p\u003e","manuscriptTitle":"A novel OPHN1 variant results in a mild form of OPHN1 syndrome characterized by cyclic strabismus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 12:28:11","doi":"10.21203/rs.3.rs-6684034/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-10T12:57:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-09T17:26:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T22:21:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T16:22:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136279537668702139683791304993290556392","date":"2025-12-01T08:00:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21262308682703490715963624616258366278","date":"2025-11-29T09:31:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"180478643358657698080309905322690405153","date":"2025-11-29T03:32:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-24T21:55:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197385863483441454629523121504367625441","date":"2025-11-11T14:38:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275169498362044993857825947029922471781","date":"2025-06-20T04:59:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-16T19:00:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-13T15:54:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-12T14:33:02+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-12T04:13:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-05-17T02:21:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"91c7deb3-6bee-4308-aef5-8bed78bb60e7","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53335096,"name":"Health sciences/Diseases/Eye diseases"},{"id":53335097,"name":"Health sciences/Medical research/Paediatric research"}],"tags":[],"updatedAt":"2026-04-27T16:00:36+00:00","versionOfRecord":{"articleIdentity":"rs-6684034","link":"https://doi.org/10.1038/s41598-026-48129-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-04-24 15:57:14","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2025-08-19 12:28:11","video":"","vorDoi":"10.1038/s41598-026-48129-7","vorDoiUrl":"https://doi.org/10.1038/s41598-026-48129-7","workflowStages":[]},"version":"v1","identity":"rs-6684034","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6684034","identity":"rs-6684034","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}