A novel AXIN2 mutation and perinatal risk factors contribute to sagittal craniosynostosis: evidence from a monochorionic diamniotic twin family

A novel AXIN2 mutation and perinatal risk factors contribute to sagittal craniosynostosis: evidence from a monochorionic diamniotic twin family | 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 A novel AXIN2 mutation and perinatal risk factors contribute to sagittal craniosynostosis: evidence from a monochorionic diamniotic twin family Jin Xu, Qing Yan, Chengcheng Song, Jingjia Liang, Liang Zhao, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-57248/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 : Craniosynostosis, the premature fusion of one or more cranial sutures, affects approximately 1 in every 2000-2500 live births. The etiology of sagittal craniosynostosis, the most prevalent form of isolated craniosynostosis, involves interplay between genetic and environmental insults, such as perinatal risk factors. However, the detailed information still remains largely unknown. Methods : The proband, a female monochorionic twin diagnosed with sagittal craniosynostosis, as well as her healthy twin sister and parents, were enrolled in our study. The obstetric medical records were retrospectively reviewed. Genetic cause was investigated by whole exome sequencing (WES) and further confirmed by Sanger sequencing. Results : We identified a novel heterozygous mutation of AXIN2 (c.1181G>A) in monochorionic twins and their father. However, only the proband presented sagittal craniosynostosis. This mutation results in the replacement of Arg at residue 394 by His (p.R394H). Arg 394 is located at the GSK3β binding domain of the AXIN2 protein, which is highly conserved across species. The obstetric medical records revealed that the proband had additional persistent breech presentation and intrauterine growth restriction, except for the perinatal risk factors shared by the twins. Conclusions : Based on the role of AXIN2 in craniosynostosis development and deleterious prediction in silico of AXIN2 (c.1181G>A: p.R394H), we speculate that this particular mutation confers susceptibility to sagittal craniosynostosis, while extra environmental insults are also involved in the pathogenesis of this case. Our findings provide a new evidence for the gene-environment interplay in understanding pathogenesis of craniosynostosis. Medical Genetics Sagittal craniosynostosis AXIN2 perinatal risk factors mutation monochorionic diamniotic twin Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Craniosynostosis (CS), defined as premature fusion of one or more cranial sutures, affects approximately 1 in every 2000-2500 live births[1]. It usually involves as an isolated condition (non-syndromic craniosynostosis, NCS), but may also be associated with other malformations as part of complex syndromes (syndromic craniosynostosis)[2]. Sagittal craniosynostosis is the most prevalent form of NCS, which accounts for 40-58% of all NCS cases[3]. Sagittal suture premature closure restricts the widen of the skull, thus causing the scaphocephaly deformity and other adverse neurologic outcomes[4]. The pathoetiology of NCS, involving interplay between genetic and environmental factors, is still not well defined[5-8]. FGF (fibroblast growth factor), BMP (bone morphogenic protein), Wnt (wingless-type integration sites) pathways are the major regulators in suture biology[9, 10]. Mutations of pivotal components in these signaling pathways, including FGFR2 (fibroblast growth factor receptor 2), TWIST1 (twist, drosophila, homolog of 1), AXIN2 (axis inhibitor 2), have been identified the origins of craniosynostosis[1, 9]. Non-genetic risk factors, such as intrauterine constraint, twin gestation, low birth weight, malnutrition, premature delivery, maternal thyroid disorders, gestational diabetes, virus infectious, can either cause or exacerbate craniosynostosis[7, 11-14]. Although several findings demonstrate the interactions between genetic and environmental influences that contribute to prematurely fused sutures[5, 15], more evidence are still needed to delineate the institutional patterns. Monochorionic (MC) twins, sharing almost the same genome, offer a unique opportunity to study the gene/environment interactions, for the healthy twin as an ideal control. Discordant phenotypes and lack of 100% concordance between MC twins emphasize the interplay between genetic and environmental influences in the etiology of the disease[16]. In our study, we reported a female monochorionic twin, who was diagnosed with sagittal craniosynostosis, carried a novel heterozygous mutation of AXIN2 (c.1181G>A: p.R394H). This mutation also exists in the patient’s father and her monochorionic sister; however, neither of whom present craniosynostosis. Intriguingly, the obstetric history documented that the proband had a persistent breech presentation and intrauterine growth restriction. We speculate that this particular AXIN2 mutation confers susceptibility, and additional environmental insults eventually trigger the sagittal craniosynostosis to occur. Materials And Methods Clinic examination All participants signed the informed consent and received physical examination in the craniofacial clinic. Whole exome sequencing and data analysis Genomic DNA, extracted from peripheral blood samples (I-1, I-2, II-1 and II-2) and skull periosteum tissue (II-1), was subjected to whole-exome sequencing (WES) on the platform of Genergy Biotechnology, Shanghai, China. Raw reads were aligned to the human genome reference assembly (GRCh37/hg19) using the Burrows-Wheeler Aligner[17]. The Picard software was employed to remove PCR duplicates and evaluate the quality of variants. DNA variants was called and analyzed using the Genome Analysis Toolkit[18]. The variants with read depths less than 4× were filtered out. All variants were further annotated using the ANNOVAR tool with the information of the Exome Aggregation Consortium (ExAC) Browser, Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), MutationTaster, Online Mendelian Inheritance in Man (OMIM), Gene Ontology and KEGG Pathway databases[19-25]. The workflow of our genetic analysis is shown in Fig. 1. Sanger sequencing The AXIN2 mutation was validated by Sanger sequencing using the following primers: Forward: 5′-CGCACACCCTAACGCACCCCAT-3′ and Reverse: 5′-ACCGCCCACCTAGCCTGCTGAA-3′, which was performed by Tsingke, China. The data were then visualized and analyzed using FinchTV (Geospiza) software. Conservation analysis Multiple-species amino acid sequences were obtained from National Center for Biotechnology Information (NCBI), and analyzed by WebLogo (Version 2.8.2, http://weblogo.berkeley.edu)[26]. Structural analysis Three-dimensional models of the wild-type and mutant site (p.R394H) of AXIN2 protein were designed using the I-TASSER[27]. The models were manipulated and visualized using the PyMOL software (PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA). Results Clinical information The pedigree came from Wuhan, Hubei Province, China. The female proband (II-1), the elder monochorionic diamniotic (MCDA) twin (Fig. 2a-c and Addition file: Fig. S1-3), was diagnosed with sagittal craniosynostosis at the age of 9 months in the Department of Neurosurgery, Children’s Hospital of Nanjing Medical University. She was born to non-consanguineous parents without family history of craniosynostosis. Her mother, a 30-year-old Chinese female, conceived MCDA twins by frozen embryo transfer (FET) (Addition file: Fig. S1-3). Her father was 36-year-old at that time. The 26 days after FET, four-dimensional ultrasound scan confirmed two embryos were inside a gestational sac (approximately 22 mm × 13 mm); one embryo was 2.8 mm in length and the fetal heart rate was 107 per minute, the other embryo was 3.3 mm in length and the fetal heart rate was 118 per minute (Addition file: Fig. S1). At 13 weeks of gestation, IgG of cytomegalovirus and herpes simplex virus were detected in the mother’s serum by the toxoplasmosis, rubella, cytomegalovirus and herpes simplex (TORCH) test (Addition file: Table S1). At 14 weeks of gestations, maternal dietary assessment indicated that the mother had inadequate intakes of energy, protein, fat, vitamins and several other minerals (Addition file: Table S2); and non-invasive prenatal testing demonstrated low risks for Trisomy 13, 18 and 21 (Addition file: Table S3). At 17 weeks of gestations, elevated levels of thyroid-stimulating hormone (TSH) and urinary iodine were detected (Addition file: Table S4). At 27 weeks of gestations, the mother was diagnosed with gestational diabetes mellitus (fasting blood glucose level, 7.38 mmol/L; postprandial one-hour blood glucose level, 15.30 mmol/L; postprandial two-hour blood glucose level, 11.03 mmol/L). At 28 weeks of gestations, the proband (II-1) and her twin younger sister (II-2) were born via spontaneous vaginal delivery (Fig. 2a, b). The proband (II-1), who had been in persistent breech position on the left side of mother’s uterus (Fig. 2b), was first recorded by ultrasound scan at 23.5 weeks of gestation; her birth weight was 880 g (50-90 centile[28]). The younger sister (II-2), who had been in cephalic position on the right side of mother’s uterus (Fig. 2b), weighted 990 g (90-97 centile[28]) at birth. Only the proband presented the typical appearance of sagittal craniosynostosis (Fig. 2c). While no signs of craniofacial deformity were found in her parents (I-1, I-2) and sister (II-2) (Fig. 2c). Cranial index, which represents the ratio of maximum cranial width to maximum cranial length[29], is decreased in the head shape of sagittal craniosynostosis. In our case, the cranial index of the proband and her sister are 70.9%, 83.2%, respectively. Taken together, the clinical findings indicate that the proband, who developed sagittal craniosynostosis, had additional persistent breech presentation and intrauterine growth restriction, except for the perinatal risk factors shared by the twins. Mutation analysis The whole-exome sequencing (WES) was applied to uncover the potential genetic etiology leading to sagittal craniosynostosis in our case. Due to the low incidence rate of sagittal craniosynostosis[3], we focused on private and/or rare (minor allele frequency, MAF < 0.01) variants on exons or splicing sites (Fig. 1). However, none of candidate germline or somatic mutations were proband-specific. As gene-environment interactions have been revealed in the pathogenesis of craniosynostosis [5, 15], we wonder whether the adverse intrauterine exposures (environment) triggered the susceptible individual (II-1) to develop sagittal craniosynostosis. Based on this hypothesis, we re-analyzed our sequencing data and identified a heterozygous missense mutation of AXIN2 (c.1181G>A, p.R394H) in the leukocytes of subjects I-1, II-1 and II-2, and skull periosteum tissue of subject II-1. The findings were further validated by Sanger sequencing (Fig. 3a-b). The nucleotide sequence showed a heterozygous G to A transition at nucleotide 1181 (c.1181 G>A) of the coding sequence, which resulted in the replacement of Arg at residue 394 by His of AXIN2 protein. Conservation analysis Arg394, located at the GSK3β binding domain (amino acid 327 to 413 according to the UniProt Consortium) of the AXIN2 protein (Fig. 3c)[30], is conserved across species (Fig. 3d, e) and was predicted to be potentially deleterious by in silico analysis (Table 1). Structural analysis I-TASSER structural analysis predicted that the p. R394H substitution may affect the spatial structure of the GSK3β binding domain in AXIN2 protein (Fig. 4 a, b). Taken together, our results indicate that AXIN2 (c.1181G>A, p.R394H) mutation may confer deleterious of sagittal craniosynostosis. Discussion Craniosynostosis, a highly heterogeneous disease, is caused by genetic mutations, adverse exposures as well as their interactions. The study of gene/environment interactions provides the foundation for clarifying the pathogenesis of craniosynostosis, identifying susceptibility individuals, discerning modifiable environmental risk factors and establishing effective strategies for prevention and early diagnosis. In our study, we demonstrated that a novel heterozygous AXIN2 (c.1181G>A: p.R394H) mutation was presented in three members of the family, including the monochorionic twins and their father. However, only the female proband with an additional environmental insult, that is, persistent breech presentation and intrauterine growth restriction in our case, developed overt craniosynostosis. We assume that this AXIN2 mutation predisposes to sagittal craniosynostosis but needs extra environmental insults to initiate the disease. It has been well accepted that AXIN2 is essential for normal calvarial morphogenesis by directly targeting β-catenin, orchestrating the crosstalk of Wnt, BMP, FGF signaling pathways and maintaining suture cell stemness[31-33]. Deletion or mutation of AXIN2 attribute to premature suture closure and craniosynostosis in humans and mice[6, 32]. Moreover, phenotype data available in International Mouse Phenotyping Consortium (IMPC, https://www.mousephenotype.org/) show that all the female and male homozygous AXIN2 knockout mice present calvarial malformation. For heterozygous AXIN2 deletion mice, only 2/7 females develop an abnormal head shape; however, the males are not observed any noticeable distinctions, which indicates the incomplete penetrance of AXIN2 mutations. In this study, an AXIN2 heterozygous missense mutation (c.1181G>A) was identified in peripheral blood samples of subjects I-1, II-1 and II-2 (Fig. 1a), which indicates that the proband inherits the mutation from her father. The function of AXIN2 (c.1181G>A) has not been reported according to the lasted updates in ClinVar (Record last updated Dec 17, 2019, https://www.ncbi.nlm.nih.gov/clinvar/variation/234485/ ). Our findings indicate that this mutation is conserved across species and was likely to be deleterious by in silico prediction tools; however, only the proband suffered sagittal craniosynostosis. We assumed that the reason of phenotypic segregation in our case is probably because AXIN2 (c.1181G>A) mutation possesses incomplete penetrance, thus making it insufficiency to trigger the disease alone. However, further clinical observations and animal studies are still needed to validate our hypothesis. Prenatal risk factors, such as maternal thyroid disorders, gestational diabetes, drug use, malnutrition, virus infectious, intrauterine constraint, twin gestation, premature delivery, are associated with craniosynostosis and likely increase the susceptibility in an already genetically predisposed infant [7, 11-14]. Monochorionic (MC) twins provide exceptional chances to decipher the interplay among genetic and environment influences in the pathogenesis of premature suture fusion[34]. In our study, the mother of monochorionic twins suffered the majority of risk factors prenatally, however, only the infant with breech presentation and intrauterine growth restriction experienced sagittal craniosynostosis. Our findings corroborate another well-established gene-environment interaction model of NCS, which substantiates almost the same environmental insults ultimately determining phenotype[15]. In all, we highlight that the intrauterine growth restriction and breech position, deserves particularly attention in predicting sagittal craniosynostosis occurrence. However, our gene-environment interaction fashion was observed in the context of AXIN2 (c.1181G>A) mutation with small sample size and unforeseen bias, which is unlikely to provide accurate assessments for all types NCS. And we also cannot evaluate the epigenetic role in pathogenesis of this case. Further epidemiological and experimental studies are encouraged to give a more unambiguous pattern. Conclusion In summary, we described a monochorionic twin with AXIN2 (c.1181G>A) mutation suffered sagittal craniosynostosis in the insult of prenatal risk factors (intrauterine growth restriction and breech position). Our findings provide a new evidence for the gene-environment interplay in etiology of NCS, which will be informative in the molecular diagnosis and genetic counselling in clinic. Abbreviations WES: Whole exome sequencing; CS: Craniosynostosis; NCS: Non-syndromic craniosynostosis; FGF: Fibroblast growth factor; FGFR2: Fibroblast growth factor receptor 2; BMP: Bone morphogenic protein; TWIST1: Twist, drosophila, homolog of 1; AXIN2: Axis inhibitor 2; MC twins: Monochorionic (mc) twins; ExAC: Exome aggregation consortium; SIFT: Sorting intolerant from tolerant; PolyPhen-2: Polymorphism phenotyping v2; OMIM: Online mendelian inheritance in man; NCBI: National center for biotechnology information; PyMOL: Molecular graphics system, delano scientific; MCDA: Monochorionic diamniotic; FET: Frozen embryo transfer; MAF: Minor allele frequency Declarations Acknowledgments We thank Xi Chen for her kind help in drawing the schematic diagram of fetal position. Authors' contributions AHG, GW, JX designed the study. GW, QY, LZ provided patients’ data and performed clinical assessments. JX, CCS, JJL, ZKW, CX, QL, SQX, LYZ, YS conducted the experiments. JX, QY, CCS, JJL, XZ, ZKW analysed the data. JX, QY, CCS, JJL wrote the manuscript. All authors critically revised the manuscript for important intellectual content. AHG, GW supervised the study. Fundings This work was financially supported by National Key Research and Development Program of China (2019YFA0802701, 2018YFC1004203), the National Science Fund for Outstanding Young Scholars (81722040), the National Natural Science Foundation of China (91839102, 81703261), the Natural Science Foundation of Jiangsu Province (BK20171060), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJB330002), the Jiangsu Students’ Platform for Innovation and Entrepreneurship Training Program (201810312036Y). Availability of data and materials The data of whole-exome sequencing are available upon request. Ethical Approval and Consent to participate This research was carried out according to the principles of the Declaration of Helsinki and was approved by the Nanjing Medical University Ethics Committee. All participants have signed informed consent forms. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Timberlake AT, Persing JA: Genetics of Nonsyndromic Craniosynostosis. Plast Reconstr Surg 2018, 141(6):1508-1516. Paumard-Hernandez B, Berges-Soria J, Barroso E, Rivera-Pedroza CI, Perez-Carrizosa V, Benito-Sanz S et al: Expanding the mutation spectrum in 182 Spanish probands with craniosynostosis: identification and characterization of novel TCF12 variants. European journal of human genetics : EJHG 2015, 23(7):907-914. 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Plastic and reconstructive surgery 2012, 129(4):945-954. Table Table 1: In silico predictions of c.1181G>A (p.R394H) mutation on AXIN2 function AXIN2 c.1181G>A Allele Frequency in Human Populations Global in 1000 Genomes Project Not recorded Global in ExAC 0.0006369 Global in gnomAD 0.000127397 Conservation PhastCons 1 Phylop 5.36 Functional Prediction SIFT Deleterious PolyPhen-2 Possibly damaging MutationTaster Disease causing Supplementary Files Additionfile.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-57248","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":1426873,"identity":"feb0e490-3edc-47a4-8838-ced8d65f02f6","order_by":0,"name":"Jin Xu","email":"","orcid":"","institution":"Nanjing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Xu","suffix":""},{"id":1426874,"identity":"d0e799a6-234a-40e6-9a42-ceb36e48adcc","order_by":1,"name":"Qing Yan","email":"","orcid":"","institution":"Children's Hospital of Nanjing Medical 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University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Wang","suffix":""},{"id":1426887,"identity":"739dea82-052e-4e34-af4f-0f89ca7e7bfc","order_by":14,"name":"Aihua Gu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYDACZh4wJcfAcADMJV6LMQlaGCBaEhugJhDWwN/Oe0zix6/D6dsZT6dJMFRYJzawnz2AV4vEYb40yd6+w7k7G85uk2A4k57YwJOXgN+awzxmErw9h3M3HABqYWw7nNggwWOAV4c8UIvk357D6QZgLf+I0GIA1CLN8+NwAkRLAxFaDA/zGFvLNqQbAh222SLhWLpxG08Ofi1y588Y3nzzx1re4MbZjTc+1FjL9rOfwa8FDBjbgITEAQaGBCDNRlg9CPwBYv4G4tSOglEwCkbByAMA7sVIQBkdzzgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7576-9998","institution":"Nanjing Medical University","correspondingAuthor":true,"prefix":"","firstName":"Aihua","middleName":"","lastName":"Gu","suffix":""}],"badges":[],"createdAt":"2020-08-11 10:17:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-57248/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-57248/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":1962122,"identity":"804495e5-3f5d-4d3a-a6d1-f5a343d9915f","added_by":"auto","created_at":"2020-08-17 21:26:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":95434,"visible":true,"origin":"","legend":"Workflow for the genetic analyses using WES and Sanger sequencing in pedigree diagnosed with sagittal craniosynostosis.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-57248/v1/Figure1.png"},{"id":1962123,"identity":"62bcc41c-77bf-4d16-8060-23220d1ceabb","added_by":"auto","created_at":"2020-08-17 21:26:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":124832,"visible":true,"origin":"","legend":"Clinical characteristics of the proband with sagittal craniosynostosis. (a) The pedigree of the family. The proband is marked with an upward arrow. Open squares/circles denote unaffected individuals; squares denote males and circles denote females. (b) Schematic diagram of the fetal position before delivery. The proband (II-1) was in breech position, while her healthy sister (II-2) was in cephalic position. (c) the computerized tomographic (CT) scan revealed fused sagittal suture (solid circle) and digital impressions (arrowheads) in the proband (left panel). Cranial index of the proband is 70.9%. The proband’s sister was also received CT examination under the request of the parents for ruling out craniosynostosis. Her sagittal suture remains patent (dotted circle) and cranial index is 83.2% (right panel).","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-57248/v1/Figure2.png"},{"id":1962124,"identity":"b94e7fea-cecd-4334-8d6b-e5bf92f8f051","added_by":"auto","created_at":"2020-08-17 21:26:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310082,"visible":true,"origin":"","legend":"Sequence, conservation analyses of c.1181G\u003eA (p.R394H). (a, b) A heterozygous mutation, c.1181G\u003eA, was found in the proband as well as her father and sister (b), but not in her mother (a). (c) Schematic diagram depicts the protein structure of AXIN2. The 394th amino acid position, which resides in the GSK3β_binding domain, is mutated R394H. (d) Evolutionary conservation analysis revealed that the Arg394 site is conserved from zebrafish to humans. (e) WebLogo analysis showed that the Arg394 site was relatively conserved. TNKS_binding: Tankyrase binding N-terminal segment of axin; RGS: Regulator of G protein signaling; DIX: Disheveled and AXIN interacting.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-57248/v1/Figure3.png"},{"id":1962125,"identity":"9d648f6b-2fbf-4dd3-b64c-e87ef1e2acf3","added_by":"auto","created_at":"2020-08-17 21:26:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":666137,"visible":true,"origin":"","legend":"Spatial structure of the GSK3β binding domain of the AXIN2 protein. (a) Location of the Arg394 residues within the GSK3β binding domain of AXIN2. Arrowheads indicate the hydrogen bond in the domain. (b) Location of the His 394 residues within the GSK3β binding domain of AXIN2. ","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-57248/v1/Figure4.png"},{"id":13579495,"identity":"a643af37-955c-4d57-831a-a39233b2dd84","added_by":"auto","created_at":"2021-09-17 04:20:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1367910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-57248/v1/6e4a2967-e2fd-46cd-9afa-577704e42359.pdf"},{"id":1962127,"identity":"a4e5a58f-8a6a-42a8-86af-5a46a3e492fd","added_by":"auto","created_at":"2020-08-17 21:26:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5069629,"visible":true,"origin":"","legend":"","description":"","filename":"Additionfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-57248/v1/Additionfile.docx"}],"financialInterests":"","formattedTitle":"A novel AXIN2 mutation and perinatal risk factors contribute to sagittal craniosynostosis: evidence from a monochorionic diamniotic twin family","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCraniosynostosis (CS), defined as premature fusion of one or more cranial sutures, affects approximately 1 in every 2000-2500 live births[1]. It usually involves as an isolated condition (non-syndromic craniosynostosis, NCS), but may also be associated with other malformations as part of complex syndromes (syndromic craniosynostosis)[2]. Sagittal craniosynostosis is the most prevalent form of NCS, which accounts for 40-58% of all NCS cases[3]. Sagittal suture premature closure restricts the widen of the skull, thus causing the scaphocephaly deformity and other adverse neurologic outcomes[4].\u003c/p\u003e\n\u003cp\u003eThe pathoetiology of NCS, involving interplay between genetic and environmental factors, is still not well defined[5-8]. FGF (fibroblast growth factor), BMP (bone morphogenic protein), Wnt (wingless-type integration sites) pathways are the major regulators in suture biology[9, 10]. Mutations of pivotal components in these signaling pathways, including \u003cem\u003eFGFR2 \u003c/em\u003e(fibroblast growth factor receptor 2), \u003cem\u003eTWIST1\u003c/em\u003e (twist, drosophila, homolog of 1), \u003cem\u003eAXIN2\u003c/em\u003e (axis inhibitor 2), have been identified the origins of craniosynostosis[1, 9]. Non-genetic risk factors, such as intrauterine constraint, twin gestation, low birth weight, malnutrition, premature delivery, maternal thyroid disorders, gestational diabetes, virus infectious, can either cause or exacerbate craniosynostosis[7, 11-14]. Although several findings demonstrate the interactions between genetic and environmental influences that contribute to prematurely fused sutures[5, 15], more evidence are still needed to delineate the institutional patterns.\u003c/p\u003e\n\u003cp\u003eMonochorionic (MC) twins, sharing almost the same genome, offer a unique opportunity to study the gene/environment interactions, for the healthy twin as an ideal control. Discordant phenotypes and lack of 100% concordance between MC twins emphasize the interplay between genetic and environmental influences in the etiology of the disease[16].\u003c/p\u003e\n\u003cp\u003eIn our study, we reported a female monochorionic twin, who was diagnosed with sagittal craniosynostosis, carried a novel heterozygous mutation of \u003cem\u003eAXIN2\u003c/em\u003e (c.1181G\u0026gt;A: p.R394H). This mutation also exists in the patient\u0026rsquo;s father and her monochorionic sister; however, neither of whom present craniosynostosis. Intriguingly, the obstetric history documented that the proband had a persistent breech presentation and intrauterine growth restriction. We speculate that this particular \u003cem\u003eAXIN2 \u003c/em\u003emutation confers susceptibility, and additional environmental insults eventually trigger the sagittal craniosynostosis to occur.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eClinic examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll participants signed the informed consent and received physical examination in the craniofacial clinic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole exome sequencing and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA, extracted from peripheral blood samples (I-1, I-2, II-1 and II-2) and skull periosteum tissue (II-1), was subjected to whole-exome sequencing (WES) on the platform of Genergy Biotechnology, Shanghai, China. Raw reads were aligned to the human genome reference assembly (GRCh37/hg19) using the Burrows-Wheeler Aligner[17]. The Picard software was employed to remove PCR duplicates and evaluate the quality of variants. DNA variants was called and analyzed using the Genome Analysis Toolkit[18]. The variants with read depths less than 4\u0026times; were filtered out. All variants were further annotated using the ANNOVAR tool with the information of the Exome Aggregation Consortium (ExAC) Browser, Sorting Intolerant From Tolerant (SIFT), Polymorphism Phenotyping v2 (PolyPhen-2), MutationTaster, Online Mendelian Inheritance in Man (OMIM), Gene Ontology and KEGG Pathway databases[19-25]. The workflow of our genetic analysis is shown in Fig. 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSanger sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eAXIN2\u003c/em\u003e mutation was validated by Sanger sequencing using the following primers: Forward: 5\u0026prime;-CGCACACCCTAACGCACCCCAT-3\u0026prime; and Reverse: 5\u0026prime;-ACCGCCCACCTAGCCTGCTGAA-3\u0026prime;, which was performed by Tsingke, China. The data were then visualized and analyzed using FinchTV (Geospiza) software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConservation analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMultiple-species amino acid sequences were obtained from National Center for Biotechnology Information (NCBI), and analyzed by WebLogo (Version 2.8.2, http://weblogo.berkeley.edu)[26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree-dimensional models of the wild-type and mutant site (p.R394H) of AXIN2 protein were designed using the I-TASSER[27]. The models were manipulated and visualized using the PyMOL software (PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eClinical information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pedigree came from Wuhan, Hubei Province, China. The female proband (II-1), the elder monochorionic diamniotic (MCDA) twin (Fig. 2a-c and Addition file: Fig. S1-3), was diagnosed with sagittal craniosynostosis at the age of 9 months in the Department of Neurosurgery, Children\u0026rsquo;s Hospital of Nanjing Medical University. She was born to non-consanguineous parents without family history of craniosynostosis. Her mother, a 30-year-old Chinese female, conceived MCDA twins by frozen embryo transfer (FET) (Addition file: Fig. S1-3). Her father was 36-year-old at that time. The 26 days after FET, four-dimensional ultrasound scan confirmed two embryos were inside a gestational sac (approximately 22 mm \u0026times; 13 mm); one embryo was 2.8 mm in length and the fetal heart rate was 107 per minute, the other embryo was 3.3 mm in length and the fetal heart rate was 118 per minute (Addition file: Fig. S1).\u003c/p\u003e\n\u003cp\u003eAt 13 weeks of gestation, IgG of cytomegalovirus and herpes simplex virus were detected in the mother\u0026rsquo;s serum by the toxoplasmosis, rubella, cytomegalovirus and herpes simplex (TORCH) test (Addition file: Table S1). At 14 weeks of gestations, maternal dietary assessment indicated that the mother had inadequate intakes of energy, protein, fat, vitamins and several other minerals (Addition file: Table S2); and non-invasive prenatal testing demonstrated low risks for Trisomy 13, 18 and 21 (Addition file: Table S3). At 17 weeks of gestations, elevated levels of thyroid-stimulating hormone (TSH) and urinary iodine were detected (Addition file: Table S4). At 27 weeks of gestations, the mother was diagnosed with gestational diabetes mellitus (fasting blood glucose level, 7.38 mmol/L; postprandial one-hour blood glucose level, 15.30 mmol/L; postprandial two-hour blood glucose level, 11.03 mmol/L).\u003c/p\u003e\n\u003cp\u003eAt 28 weeks of gestations, the proband (II-1) and her twin younger sister (II-2) were born via spontaneous vaginal delivery (Fig. 2a, b). The proband (II-1), who had been in persistent breech position on the left side of mother\u0026rsquo;s uterus (Fig. 2b), was first recorded by ultrasound scan at 23.5 weeks of gestation; her birth weight was 880 g (50-90 centile[28]). The younger sister (II-2), who had been in cephalic position on the right side of mother\u0026rsquo;s uterus (Fig. 2b), weighted 990 g (90-97 centile[28]) at birth. Only the proband presented the typical appearance of sagittal craniosynostosis (Fig. 2c). While no signs of craniofacial deformity were found in her parents (I-1, I-2) and sister (II-2) (Fig. 2c). Cranial index, which represents the ratio of maximum cranial width to maximum cranial length[29], is decreased in the head shape of sagittal craniosynostosis. In our case, the cranial index of the proband and her sister are 70.9%, 83.2%, respectively. Taken together, the clinical findings indicate that the proband, who developed sagittal craniosynostosis, had additional persistent breech presentation and intrauterine growth restriction, except for the perinatal risk factors shared by the twins.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMutation analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole-exome sequencing (WES) was applied to uncover the potential genetic etiology leading to sagittal craniosynostosis in our case. Due to the low incidence rate of sagittal craniosynostosis[3], we focused on private and/or rare (minor allele frequency, MAF \u0026lt; 0.01) variants on exons or splicing sites (Fig. 1). However, none of candidate germline or somatic mutations were proband-specific. As gene-environment interactions have been revealed in the pathogenesis of craniosynostosis [5, 15], we wonder whether the adverse intrauterine exposures (environment) triggered the susceptible individual (II-1) to develop sagittal craniosynostosis. Based on this hypothesis, we re-analyzed our sequencing data and identified a heterozygous missense mutation of \u003cem\u003eAXIN2\u003c/em\u003e (c.1181G\u0026gt;A, p.R394H) in the leukocytes of subjects I-1, II-1 and II-2, and skull periosteum tissue of subject II-1. The findings were further validated by Sanger sequencing (Fig. 3a-b). The nucleotide sequence showed a heterozygous G to A transition at nucleotide 1181 (c.1181 G\u0026gt;A) of the coding sequence, which resulted in the replacement of Arg at residue 394 by His of AXIN2 protein.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConservation analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArg394, located at the GSK3\u0026beta; binding domain (amino acid 327 to 413 according to the UniProt Consortium) of the AXIN2 protein (Fig. 3c)[30], is conserved across species (Fig. 3d, e) and was predicted to be potentially deleterious by \u003cem\u003ein silico \u003c/em\u003eanalysis (Table 1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI-TASSER structural analysis predicted that the p. R394H substitution may affect the spatial structure of the GSK3\u0026beta; binding domain in AXIN2 protein (Fig. 4 a, b). Taken together, our results indicate that\u003cem\u003e AXIN2 \u003c/em\u003e(c.1181G\u0026gt;A, p.R394H) mutation may confer deleterious of sagittal craniosynostosis.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCraniosynostosis, a highly heterogeneous disease, is caused by genetic mutations, adverse exposures as well as their interactions. The study of gene/environment interactions provides the foundation for clarifying the pathogenesis of craniosynostosis, identifying susceptibility individuals, discerning modifiable environmental risk factors and establishing effective strategies for prevention and early diagnosis. In our study, we demonstrated that a novel heterozygous \u003cem\u003eAXIN2\u003c/em\u003e (c.1181G\u0026gt;A: p.R394H) mutation was presented in three members of the family, including the monochorionic twins and their father. However, only the female proband with an additional environmental insult, that is, persistent breech presentation and intrauterine growth restriction in our case, developed overt craniosynostosis. We assume that this \u003cem\u003eAXIN2\u003c/em\u003e mutation predisposes to sagittal craniosynostosis but needs extra environmental insults to initiate the disease.\u003c/p\u003e\n\u003cp\u003eIt has been well accepted that \u003cem\u003eAXIN2\u003c/em\u003e is essential for normal calvarial morphogenesis by directly targeting \u0026beta;-catenin, orchestrating the crosstalk of Wnt, BMP, FGF signaling pathways and maintaining suture cell stemness[31-33]. Deletion or mutation of \u003cem\u003eAXIN2 \u003c/em\u003eattribute to premature suture closure and craniosynostosis in humans and mice[6, 32]. Moreover, phenotype data available in International Mouse Phenotyping Consortium (IMPC, https://www.mousephenotype.org/) show that all the female and male homozygous\u003cem\u003e AXIN2\u003c/em\u003e knockout mice present calvarial malformation. For heterozygous \u003cem\u003eAXIN2\u003c/em\u003e deletion mice, only 2/7 females develop an abnormal head shape; however, the males are not observed any noticeable distinctions, which indicates the incomplete penetrance of \u003cem\u003eAXIN2 \u003c/em\u003emutations. In this study, an \u003cem\u003eAXIN2\u003c/em\u003e heterozygous missense mutation (c.1181G\u0026gt;A) was identified in peripheral blood samples of subjects I-1, II-1 and II-2 (Fig. 1a), which indicates that the proband inherits the mutation from her father. The function of\u003cem\u003e AXIN2\u003c/em\u003e (c.1181G\u0026gt;A) has not been reported according to the lasted updates in ClinVar (Record last updated Dec 17, 2019, \u003ca href=\"https://www.ncbi.nlm.nih.gov/clinvar/variation/234485/\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/variation/234485/\u003c/a\u003e). Our findings indicate that this mutation is conserved across species and was likely to be deleterious by\u003cem\u003e in silico \u003c/em\u003eprediction tools; however, only the proband suffered sagittal craniosynostosis. We assumed that the reason of phenotypic segregation in our case is probably because \u003cem\u003eAXIN2\u003c/em\u003e (c.1181G\u0026gt;A) mutation possesses incomplete penetrance, thus making it insufficiency to trigger the disease alone. However, further clinical observations and animal studies are still needed to validate our hypothesis.\u003c/p\u003e\n\u003cp\u003ePrenatal risk factors, such as maternal thyroid disorders, gestational diabetes, drug use, malnutrition, virus infectious, intrauterine constraint, twin gestation, premature delivery, are associated with craniosynostosis and likely increase the susceptibility in an already genetically predisposed infant [7, 11-14]. Monochorionic (MC) twins provide exceptional chances to decipher the interplay among genetic and environment influences in the pathogenesis of premature suture fusion[34]. In our study, the mother of monochorionic twins suffered the majority of risk factors prenatally, however, only the infant with breech presentation and intrauterine growth restriction experienced sagittal craniosynostosis. Our findings corroborate another well-established gene-environment interaction model of NCS, which substantiates almost the same environmental insults ultimately determining phenotype[15]. In all, we highlight that the intrauterine growth restriction and breech position, deserves particularly attention in predicting sagittal craniosynostosis occurrence.\u003c/p\u003e\n\u003cp\u003eHowever, our gene-environment interaction fashion was observed in the context of \u003cem\u003eAXIN2\u003c/em\u003e (c.1181G\u0026gt;A) mutation with small sample size and unforeseen bias, which is unlikely to provide accurate assessments for all types NCS. And we also cannot evaluate the epigenetic role in pathogenesis of this case. Further epidemiological and experimental studies are encouraged to give a more unambiguous pattern.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we described a monochorionic twin with \u003cem\u003eAXIN2\u003c/em\u003e (c.1181G\u0026gt;A) mutation suffered sagittal craniosynostosis in the insult of prenatal risk factors (intrauterine growth restriction and breech position). Our findings provide a new evidence for the gene-environment interplay in etiology of NCS, which will be informative in the molecular diagnosis and genetic counselling in clinic.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eWES: Whole exome sequencing; CS: Craniosynostosis; NCS: Non-syndromic craniosynostosis; FGF: Fibroblast growth factor; FGFR2: Fibroblast growth factor receptor 2; BMP: Bone morphogenic protein; TWIST1: Twist, drosophila, homolog of 1; AXIN2: Axis inhibitor 2; MC twins: Monochorionic (mc) twins; ExAC: Exome aggregation consortium; SIFT: Sorting intolerant from tolerant; PolyPhen-2: Polymorphism phenotyping v2; OMIM: Online mendelian inheritance in man; NCBI: National center for biotechnology information; PyMOL: Molecular graphics system, delano scientific; MCDA: Monochorionic diamniotic; FET: Frozen embryo transfer; MAF: Minor allele frequency\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Xi Chen for her kind help in drawing the schematic diagram of fetal position.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthors' contributions\u003c/p\u003e\n\u003cp\u003eAHG, GW, JX designed the study. GW, QY, LZ provided patients\u0026rsquo; data and performed clinical assessments. JX, CCS, JJL, ZKW, CX, QL, SQX, LYZ, YS conducted the experiments. JX, QY, CCS, JJL, XZ, ZKW analysed the data. JX, QY, CCS, JJL wrote the manuscript. All authors critically revised the manuscript for important intellectual content. AHG, GW supervised the study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFundings\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by National Key Research and Development Program of China (2019YFA0802701, 2018YFC1004203), the National Science Fund for Outstanding Young Scholars (81722040), the National Natural Science Foundation of China (91839102, 81703261), the Natural Science Foundation of Jiangsu Province (BK20171060), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (17KJB330002), the Jiangsu Students\u0026rsquo; Platform for Innovation and Entrepreneurship Training Program (201810312036Y).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data of whole-exome sequencing are available upon request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEthical Approval and Consent to participate\u003c/p\u003e\n\u003cp\u003eThis research was carried out according to the principles of the Declaration of Helsinki and was approved by the Nanjing Medical University Ethics Committee. All participants have signed informed consent forms.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n\u003cli\u003eTimberlake AT, Persing JA: Genetics of Nonsyndromic Craniosynostosis. Plast Reconstr Surg 2018, 141(6):1508-1516.\u003c/li\u003e\n\u003cli\u003ePaumard-Hernandez B, Berges-Soria J, Barroso E, Rivera-Pedroza CI, Perez-Carrizosa V, Benito-Sanz S et al: Expanding the mutation spectrum in 182 Spanish probands with craniosynostosis: identification and characterization of novel TCF12 variants. 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Lancet Glob Health 2015, 3(11):e681-691.\u003c/li\u003e\n\u003cli\u003eBendon CL, Sheerin FB, Wall SA, Johnson D: The relationship between scaphocephaly at the skull vault and skull base in sagittal synostosis. J Craniomaxillofac Surg 2014, 42(3):245-249.\u003c/li\u003e\n\u003cli\u003eRen J, Wen L, Gao X, Jin C, Xue Y, Yao X: DOG 1.0: illustrator of protein domain structures. Cell Res 2009, 19(2):271-273.\u003c/li\u003e\n\u003cli\u003eLiu B, Yu HM, Hsu W: Craniosynostosis caused by Axin2 deficiency is mediated through distinct functions of beta-catenin in proliferation and differentiation. Dev Biol 2007, 301(1):298-308.\u003c/li\u003e\n\u003cli\u003eYu H-MI, Jerchow B, Sheu T-J, Liu B, Costantini F, Puzas JE et al: The role of Axin2 in calvarial morphogenesis and craniosynostosis. Development 2005, 132(8):1995-2005.\u003c/li\u003e\n\u003cli\u003eMaruyama T, Jeong J, Sheu T-J, Hsu W: Stem cells of the suture mesenchyme in craniofacial bone development, repair and regeneration. Nat Commun 2016, 7:10526-10526.\u003c/li\u003e\n\u003cli\u003eLakin GE, Sinkin JC, Chen R, Koltz PF, Girotto JA: Genetic and epigenetic influences of twins on the pathogenesis of craniosynostosis: a meta-analysis. Plastic and reconstructive surgery 2012, 129(4):945-954.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e\u003cem\u003e In silico\u003c/em\u003e predictions of c.1181G\u0026gt;A (p.R394H) mutation on AXIN2 function\u003c/p\u003e\n\u003ctable\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"520\"\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAXIN2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e c.1181G\u0026gt;A\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"520\"\u003e\n\u003cp\u003e\u003cstrong\u003eAllele Frequency in Human Populations\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003eGlobal in 1000 Genomes Project\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003eNot recorded\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003eGlobal in ExAC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003e0.0006369\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003eGlobal in gnomAD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003e0.000127397\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"520\"\u003e\n\u003cp\u003e\u003cstrong\u003eConservation\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003ePhastCons\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003ePhylop\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003e5.36\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"2\" width=\"520\"\u003e\n\u003cp\u003e\u003cstrong\u003eFunctional Prediction\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003eSIFT\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003eDeleterious\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003ePolyPhen-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003ePossibly damaging\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"302\"\u003e\n\u003cp\u003eMutationTaster\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"217\"\u003e\n\u003cp\u003eDisease causing\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\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":"Sagittal craniosynostosis, AXIN2, perinatal risk factors, mutation, monochorionic diamniotic twin ","lastPublishedDoi":"10.21203/rs.3.rs-57248/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-57248/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Craniosynostosis, the premature fusion of one or more cranial sutures, affects approximately 1 in every 2000-2500 live births. The etiology of sagittal craniosynostosis, the most prevalent form of isolated craniosynostosis, involves interplay between genetic and environmental insults, such as perinatal risk factors. However, the detailed information still remains largely unknown. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: The proband, a female monochorionic twin diagnosed with sagittal craniosynostosis, as well as her healthy twin sister and parents, were enrolled in our study. The obstetric medical records were retrospectively reviewed. Genetic cause was investigated by whole exome sequencing (WES) and further confirmed by Sanger sequencing.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: We identified a novel heterozygous mutation of AXIN2 (c.1181G\u0026gt;A) in monochorionic twins and their father. However, only the proband presented sagittal craniosynostosis. This mutation results in the replacement of Arg at residue 394 by His (p.R394H). Arg 394 is located at the GSK3β binding domain of the AXIN2 protein, which is highly conserved across species. The obstetric medical records revealed that the proband had additional persistent breech presentation and intrauterine growth restriction, except for the perinatal risk factors shared by the twins.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e: Based on the role of AXIN2 in craniosynostosis development and deleterious prediction in silico of AXIN2 (c.1181G\u0026gt;A: p.R394H), we speculate that this particular mutation confers susceptibility to sagittal craniosynostosis, while extra environmental insults are also involved in the pathogenesis of this case. Our findings provide a new evidence for the gene-environment interplay in understanding pathogenesis of craniosynostosis.\u003c/p\u003e","manuscriptTitle":"A novel AXIN2 mutation and perinatal risk factors contribute to sagittal craniosynostosis: evidence from a monochorionic diamniotic twin family","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-08-17 21:26:09","doi":"10.21203/rs.3.rs-57248/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":"a0a455d9-1113-45cb-ac1d-ae4e82830b71","owner":[],"postedDate":"August 17th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":300995,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2020-10-15T11:35:46+00:00","versionOfRecord":[],"versionCreatedAt":"2020-08-17 21:26:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-57248","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-57248","identity":"rs-57248","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}