A Novel NODAL variant in a young embolic stroke patient with visceral heterotaxy

A Novel NODAL variant in a young embolic stroke patient with visceral heterotaxy | 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 Case Report A Novel NODAL variant in a young embolic stroke patient with visceral heterotaxy Kei Kaburagi, Yuta Hagiwara, Keiji Tachikawa, Noriko Miyake, Hisanao Akiyama, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2847400/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Apr, 2024 Read the published version in BMC Neurology → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Ischemic stroke in young adults can be caused by a variety of etiologies including the monogenic disorders. Visceral heterotaxy is a condition caused by abnormal left-right determinations during embryonic development. We aimed to determine the cause of a young ischemic stroke patient with visceral heterotaxy. Case presentation: We performed neurological, radiological, and genetic evaluations in a 17-year-old male patient presenting ischemic stroke and visceral heterotaxy to determine the underlying cause of this rare disease combination. Brain MRI showed evidence of embolic stroke, abdominal CT showed visceral heterotaxy, and echocardiogram showed cardiac anomaly with right-to-left-shunt (RLS). Whole genome sequencing (WGS) revealed a heterozygous missense variant (NM_018055.5: c.1016T > C, p.(Met339Val)) in the NODAL gene, which is essential to the determination of the left-right body axis. Conclusions: Our study highlights the importance of evaluating genetic causes in young ischemic stroke of unknown cause and the need for stroke risk management in visceral heterotaxy patients with RLS. To the best of our knowledge, we report the first genetically-confirmed case of visceral heterotaxy with young embolic stroke reported to date. Visceral heterotaxy Cryptogenic stroke Whole-genome sequencing Figures Figure 1 Figure 2 Background Ischemic stroke in young adults is occasionally reported in multiple conditions including monogenic disorders. Genetic testing can find the underlying genetic causes such as defects of coagulation factors, connective tissue abnormalities, or cardiovascular disorders 1 , 2 . Despite the impact of monogenic stroke on patients and their families and the importance of assessing genetic causes, patients with young ischemic stroke do not always undergo genetic investigation probably due to their rarity and lack of cost-effective evidence of high-throughput gene sequencing. Efforts have been made to utilize genetic analysis using stroke gene panels 2 – 4 or whole-exome sequencing 5 to manage monogenic stroke, but the detection rate of the causal genes varies and the clinical significance is unclear probably due to the heterogeneous nature of the stroke and lack of our knowledge when monogenic disorder should be suspected in ischemic stroke. Young adults with congenital cardiac disease have increased risk of cerebral ischemic stroke. While this is primarily due to electro-abnormalities, such as atrial fibrillation, embolic stroke from the venous system due to the right-to-left shunt may also be a factor even in the absence of arrythmia. Therefore, it may be important to consider the preventive medications in managing the patient. Here we report a patient with visceral heterotaxy and embolic stroke whose genetic testing was useful to identify a novel pathogenic variant in the NODAL gene, emphasizing the importance of performing genetic analysis in a patient with young ischemic stroke. Case presentation The patient is a 17-year-old male with asplenia syndrome (complete endocardial deficiency, double outlet right ventricle, and pulmonary artery stenosis) as a congenital heart malformation, although his symptoms are only mild cyanosis, and he is independent in activities of daily living (ADL). He was referred to our hospital with a sudden episode of transient left hemiparesis. He suddenly became aware of weakness on the left upper and lower limbs while defecating. The symptoms gradually improved within 3 hours, but the sensory disturbance remained. There was no marked family history and his parents were healthy. We suspected cerebrovascular disease, because he had a cardiac malformation that could cause a right-to-left shunt. The head magnetic resonance imaging (MRI) showed cerebral infarction on the right temporo-parietal robe, and magnetic resonance angiography (MRA) showed no vessel abnormalities (Fig. 1 A, B). Based on the findings of MRI, we concluded that the patient had an embolic mechanism through cardiac malformation. Abdominal Computer tomography (CT) scan showed visceral heterotaxy (Fig. 1 C). Transthoracic echocardiography revealed endocardial cushion defect (ECD) (Fig. 1 D). After admission at the age of 17, blood tests showed slightly low activity of antithrombin III (61%) and protein C (63%), and the presence of congenital coagulopathy was suspected, although genetic testing for SERPINC1 and PROC were negative. In addition, there was no evidence of antiphospholipid antibody syndrome or hyper-homocystinemia, which could cause juvenile cerebral infarction. There were no signs of infectious endocarditis and of deep vein thrombosis (DVT). However, the level of D-dimer FDP was slightly elevated 0.6µg/ml (normal range: 0-0.5µg/ml) and the Risk of Paradoxical Embolism (RoPE) score 6 was high (score = 10). Therefore, we diagnosed him as a paradoxical cerebral embolism caused by ECD and potential DVT with right-to-left shunt. To further investigate the cause of his stroke, whole-genome sequencing (WGS) was performed on the patient (See online method). WGS of the patient revealed a novel heterozygous variant NM_018055.5: c.1016T > C: p.(Met339Thr) (chr10:70432964–70432964 in the genomic coordinate of GRCh38) in the NODAL gene (NM_018055.5, MIM#601265), which is responsible for autosomal dominant visceral heterotaxy (MIM#270100) 7 . The obtained coverage of WGS was 33.5 x. The unaffected father also had this variant, suggesting incomplete penetrance as reported 7 (Fig. 1 A). The father underwent an abdominal CT scan that was completely normal. Sanger-sequencing validated the variant (Fig. 1 B). This variant has not been reported in the gnomAD database or the WGS database of 9850 Japanese control individuals 8 . The missense variant was predicted to be damaging with CADD-PHRED score of 26.2. This variant was located in the evolutionally-conserved Nodal active ligand (110 aa) (Fig. 2 C) and showed high conservation scores (GERP of 5.84, SIFT of 0, PhyloP of 4.603, PolyPhen-2 of 0.998, and PhastCons of 1). MutationTaster prediction was “Disease causing”. Alteration of the same amino acid, c.1015A > G, p.(Met339Val), was reported to cause a mild reduction in nodal signaling by using activin-responsive ARE-luciferase reporter assay in a zebrafish embryo, although detailed clinical description of the patient with the Met339Val variant was unavailable 9 . Protein structure of homodimerized Nodal and its co-receptor CFC1 (CFC1-EGF domain) was modeled by AlphaFold2 10 , which showed that Met339 and other previously reported pathogenic variants were located at the Nodal-CFC1 binding site (Fig. 2 D). We further searched for pathogenic variants of the PROC gene in the WGS data that may explain the decrease in Protein C in this patient, but were unable to find any candidate variants. Furthermore, we did not identify any pathogenic variants that were responsible for monogenic stroke panel genes 2 . Anticoagulation therapy was started with continuous intravenous unfractionated heparin and switched to warfarin. The patient showed no recurrence of ischemic stroke thereafter. Discussion and conclusions We described a novel NODAL missense variant in a patient presenting with young embolic stroke and visceral heterotaxy. This variant was located in a highly conserved Nodal active domain, and analysis of the protein structure suggested that it may have a role in CFC1 binding. It is noteworthy that the unaffected father has the same variant. Disruption of the left-right axis determination in embryonic development can result in random left-right selection 11 . In support of this, genetic analyses of heterotaxy family showed approximately 50% penetrance, even in monozygotic twins 12 . A heterotaxy family with a NODAL variant was reported, suggesting a ~ 50% penetrance rate 13 . Collectively, it is likely that the unaffected father with Met339Ther did not show heterotaxy due to random determination of the left-right axis. Family-based genetic analysis using high-throughput sequencers usually uses variant filtering based on a hypothesis of 100% penetrance. It may be important to consider this randomness of disease manifestation when performing genetic analysis in a family with visceral heterotaxy. The patient had an embolic stroke. We considered whether genetic abnormality and cerebral infarction are related. As echocardiography showed an apparent right-to-left shunt, it may be possible that a right-to-left shunt in the heart allowed clots in the venous system to enter the brain circulation and caused an embolic stroke. Although we could not detect DVT, we suspected that the abnormal finding of protein C level inspires the insidious occurrence of DVT. Although the patient showed a mildly reduced Protein C level (63%), no pathogenic variants were found in the PROC gene. It is reported that protein C deficiency due to pathogenic genetic variants can decrease Protain C level below 55%. Mild reductions (55–65%) are also seen in healthy individuals 14 . It may be caused by less effective genetic conditions, such as common eQTL variants in regulatory regions 15 , although we could not draw any conclusions about this possibility. Thus, we diagnosed the type of cerebral infarction as paradoxical cerebral embolism. We should be aware that patients with a NODAL variant may develop a stroke. In conclusion, we report a novel NODAL variant in a young embolic stroke patient with visceral heterotaxy. It would be important to investigate a monogenic disorder that may secondarily cause a young embolic stroke. It is also important to carefully evaluate unaffected family members when performing family-based WGS, as heterotaxy can have a penetrance of 50% due to random left-right selection. Declarations Ethics approval and consent to participate Written informed and disclosure consents were obtained from the patient. Institutional review board of St. Marianna University School of Medicine approved the study protocol (#4983). Consent for publication Consent for publication was obtained from the patient. Availability of data and materials WGS data is private and unavailable. Competing interests The authors have no competing interests to report. Acknowledgment We are grateful to the patient and his parents for participating in our study. We would like to thank to Ritsuko Oikawa and Yoshiko Miyake for their technical assistance and Yoshikazu Haramoto for his comments about Nodal. Additionally, we are thankful for the partial support provided by the supercomputer at ROIS National Institute of Genetics for computation. Funding This work was supported by grants from the Japan Agency for Medical Research and Development (AMED No.22ek0109493). We thank the patient and his parents for participating in our study. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics. References Ekker MS, Boot EM, Singhal AB, et al. Epidemiology, aetiology, and management of ischaemic stroke in young adults. Lancet Neurol 2018;17:790-801. Ilinca A, Puschmann A, Putaala J, et al. Updated Stroke Gene Panels: Rapid evolution of knowledge on monogenic causes of stroke. Eur J Hum Genet 2022. Tan RYY, Traylor M, Megy K, et al. How common are single gene mutations as a cause for lacunar stroke? A targeted gene panel study. Neurology 2019;93:e2007-e2020. Fang F, Xu Z, Suo Y, et al. Gene panel for Mendelian strokes. Stroke Vasc Neurol 2020;5:416-421. Ilinca A, Martinez-Majander N, Samuelsson S, et al. Whole-Exome Sequencing in 22 Young Ischemic Stroke Patients With Familial Clustering of Stroke. Stroke 2020;51:1056-1063. Kent DM, Ruthazer R, Weimar C, et al. An index to identify stroke-related vs incidental patent foramen ovale in cryptogenic stroke. Neurology 2013;81:619-625. Gebbia M, Ferrero GB, Pilia G, et al. X-linked situs abnormalities result from mutations in ZIC3. Nat Genet 1997;17:305-308. Kawai Y, Watanabe Y, Omae Y, et al. Exploring the genetic diversity of the Japanese Population: Insights from a Large-Scale Whole Genome Sequencing Analysis. bioRxiv 2023:2023.2001.2023.525133. Roessler E, Pei W, Ouspenskaia MV, et al. Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly. Mol Genet Metab 2009;98:225-234. Jumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021;596:583-589. Layton WM, Jr. Random determination of a developmental process: reversal of normal visceral asymmetry in the mouse. J Hered 1976;67:336-338. Noone PG, Bali D, Carson JL, et al. Discordant organ laterality in monozygotic twins with primary ciliary dyskinesia. Am J Med Genet 1999;82:155-160. Li AH, Hanchard NA, Azamian M, et al. Genetic architecture of laterality defects revealed by whole exome sequencing. Eur J Hum Genet 2019;27:563-573. Miletich J, Sherman L, Broze G, Jr. Absence of thrombosis in subjects with heterozygous protein C deficiency. N Engl J Med 1987;317:991-996. Gudjonsson A, Gudmundsdottir V, Axelsson GT, et al. A genome-wide association study of serum proteins reveals shared loci with common diseases. Nat Commun 2022;13:480. Yan YT, Liu JJ, Luo Y, et al. Dual roles of Cripto as a ligand and coreceptor in the nodal signaling pathway. Mol Cell Biol 2002;22:4439-4449. Additional Declarations No competing interests reported. Supplementary Files AdditionalMaterials.docx Cite Share Download PDF Status: Published Journal Publication published 11 Apr, 2024 Read the published version in BMC Neurology → Version 1 posted Editorial decision: Revision requested 23 Jan, 2024 Reviews received at journal 11 Dec, 2023 Reviews received at journal 06 Dec, 2023 Reviewers agreed at journal 29 Nov, 2023 Reviewers agreed at journal 29 Nov, 2023 Reviewers invited by journal 27 Jun, 2023 Editor assigned by journal 27 Jun, 2023 Editor invited by journal 13 Jun, 2023 Submission checks completed at journal 13 Jun, 2023 First submitted to journal 22 Apr, 2023 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. 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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-2847400","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":199085074,"identity":"3240abb3-5246-4b8c-ba22-faa80d2fbf76","order_by":0,"name":"Kei Kaburagi","email":"","orcid":"","institution":"St. Marianna University School of Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kei","middleName":"","lastName":"Kaburagi","suffix":""},{"id":199085075,"identity":"2703fef0-50f8-4562-a617-a6e839b6a9ed","order_by":1,"name":"Yuta Hagiwara","email":"","orcid":"","institution":"St. Marianna University School 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05:14:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2847400/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2847400/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12883-024-03619-x","type":"published","date":"2024-04-11T15:00:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":37405903,"identity":"c4d56581-6114-4703-a119-34bfa3a0c39f","added_by":"auto","created_at":"2023-05-23 21:51:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":814466,"visible":true,"origin":"","legend":"\u003cp\u003eClinical and radiological features of the patient\u003c/p\u003e\n\u003cp\u003e(A) The head MRI (Diffusion weighted image) shows acute cerebral infarction on temporo-parietal robe. (B) MR angiography image shows no vessel abnormality. (C) Abdominal CT shows visceral heterotaxy. S: Stomach. L: Liver. (D) Echocardiogram. Echocardiogram shows ECD. RV: Right ventricle. LV: Left ventricle.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2847400/v1/29021c4acc91d20a3117044a.png"},{"id":37405904,"identity":"bf8fd221-cb45-4220-8696-70fbc3080c55","added_by":"auto","created_at":"2023-05-23 21:51:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265175,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic analysis of \u003cem\u003eNODAL\u003c/em\u003e variant.\u003c/p\u003e\n\u003cp\u003e(A) Family tree of the patient. WT: wild-type (B) Sanger-sequencing validated c.1016T\u0026gt;C variant in the patient and the father. (C) The scheme of NODAL protein. Nodal is post-translationally cleaved and forms mature active ligand (red). Met339 is located in the active ligand (arrow). (D) Met339 and nearby residues are conserved across species. Genome alignment was obtained from UCSC genome browser (http://genome.ucsc.edu). (E) Evaluation of protein structure of M394 (red) and other residues (blue) on the TGF-β domain, whose changes are reported to cause heterotaxy. Protein structures of homo-dimerized Nodal mature protein (gray) and CFC1-EGF domain of CFC1 (yellow) were modeled by Alphafold2. L, S and F (orange) in CFC1 represent the three residues reported to be essential for binding to Nodal in CFC1 of mouse Cripto\u003csup\u003e16\u003c/sup\u003e. The molecular structure was drawn by PyMOL (www.pymol.org). Amino acids reported to cause visceral heterotaxy including M339 cluster at the CFC1 and Nodal binding site.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2847400/v1/70ceb7ea35ecebcbf8f5e1c6.png"},{"id":54712674,"identity":"92bc0a34-2aa8-4996-9f66-f73473cc32f3","added_by":"auto","created_at":"2024-04-15 15:12:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1070693,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2847400/v1/9b2f7f1c-48aa-46ea-b4a3-57a6d5a0ac41.pdf"},{"id":37405902,"identity":"fe61757b-f952-4bf4-b1b5-5499485f7e46","added_by":"auto","created_at":"2023-05-23 21:51:58","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15076,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-2847400/v1/53bec88d5016a8d6e59a509d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A Novel NODAL variant in a young embolic stroke patient with visceral heterotaxy","fulltext":[{"header":"Background","content":"\u003cp\u003eIschemic stroke in young adults is occasionally reported in multiple conditions including monogenic disorders. Genetic testing can find the underlying genetic causes such as defects of coagulation factors, connective tissue abnormalities, or cardiovascular disorders\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Despite the impact of monogenic stroke on patients and their families and the importance of assessing genetic causes, patients with young ischemic stroke do not always undergo genetic investigation probably due to their rarity and lack of cost-effective evidence of high-throughput gene sequencing. Efforts have been made to utilize genetic analysis using stroke gene panels\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e or whole-exome sequencing\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e to manage monogenic stroke, but the detection rate of the causal genes varies and the clinical significance is unclear probably due to the heterogeneous nature of the stroke and lack of our knowledge when monogenic disorder should be suspected in ischemic stroke.\u003c/p\u003e \u003cp\u003eYoung adults with congenital cardiac disease have increased risk of cerebral ischemic stroke. While this is primarily due to electro-abnormalities, such as atrial fibrillation, embolic stroke from the venous system due to the right-to-left shunt may also be a factor even in the absence of arrythmia. Therefore, it may be important to consider the preventive medications in managing the patient.\u003c/p\u003e \u003cp\u003eHere we report a patient with visceral heterotaxy and embolic stroke whose genetic testing was useful to identify a novel pathogenic variant in the \u003cem\u003eNODAL\u003c/em\u003e gene, emphasizing the importance of performing genetic analysis in a patient with young ischemic stroke.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003eThe patient is a 17-year-old male with asplenia syndrome (complete endocardial deficiency, double outlet right ventricle, and pulmonary artery stenosis) as a congenital heart malformation, although his symptoms are only mild cyanosis, and he is independent in activities of daily living (ADL). He was referred to our hospital with a sudden episode of transient left hemiparesis. He suddenly became aware of weakness on the left upper and lower limbs while defecating. The symptoms gradually improved within 3 hours, but the sensory disturbance remained. There was no marked family history and his parents were healthy.\u003c/p\u003e \u003cp\u003eWe suspected cerebrovascular disease, because he had a cardiac malformation that could cause a right-to-left shunt. The head magnetic resonance imaging (MRI) showed cerebral infarction on the right temporo-parietal robe, and magnetic resonance angiography (MRA) showed no vessel abnormalities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B). Based on the findings of MRI, we concluded that the patient had an embolic mechanism through cardiac malformation. Abdominal Computer tomography (CT) scan showed visceral heterotaxy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Transthoracic echocardiography revealed endocardial cushion defect (ECD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter admission at the age of 17, blood tests showed slightly low activity of antithrombin III (61%) and protein C (63%), and the presence of congenital coagulopathy was suspected, although genetic testing for \u003cem\u003eSERPINC1\u003c/em\u003e and \u003cem\u003ePROC\u003c/em\u003e were negative. In addition, there was no evidence of antiphospholipid antibody syndrome or hyper-homocystinemia, which could cause juvenile cerebral infarction. There were no signs of infectious endocarditis and of deep vein thrombosis (DVT). However, the level of D-dimer FDP was slightly elevated 0.6\u0026micro;g/ml (normal range: 0-0.5\u0026micro;g/ml) and the Risk of Paradoxical Embolism (RoPE) score\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e was high (score\u0026thinsp;=\u0026thinsp;10). Therefore, we diagnosed him as a paradoxical cerebral embolism caused by ECD and potential DVT with right-to-left shunt.\u003c/p\u003e \u003cp\u003eTo further investigate the cause of his stroke, whole-genome sequencing (WGS) was performed on the patient (See online method). WGS of the patient revealed a novel heterozygous variant NM_018055.5: c.1016T\u0026thinsp;\u0026gt;\u0026thinsp;C: p.(Met339Thr) (chr10:70432964\u0026ndash;70432964 in the genomic coordinate of GRCh38) in the \u003cem\u003eNODAL\u003c/em\u003e gene (NM_018055.5, MIM#601265), which is responsible for autosomal dominant visceral heterotaxy (MIM#270100)\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The obtained coverage of WGS was 33.5 x. The unaffected father also had this variant, suggesting incomplete penetrance as reported\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The father underwent an abdominal CT scan that was completely normal. Sanger-sequencing validated the variant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This variant has not been reported in the gnomAD database or the WGS database of 9850 Japanese control individuals\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The missense variant was predicted to be damaging with CADD-PHRED score of 26.2. This variant was located in the evolutionally-conserved Nodal active ligand (110 aa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) and showed high conservation scores (GERP of 5.84, SIFT of 0, PhyloP of 4.603, PolyPhen-2 of 0.998, and PhastCons of 1). MutationTaster prediction was \u0026ldquo;Disease causing\u0026rdquo;. Alteration of the same amino acid, c.1015A\u0026thinsp;\u0026gt;\u0026thinsp;G, p.(Met339Val), was reported to cause a mild reduction in nodal signaling by using activin-responsive ARE-luciferase reporter assay in a zebrafish embryo, although detailed clinical description of the patient with the Met339Val variant was unavailable\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Protein structure of homodimerized Nodal and its co-receptor CFC1 (CFC1-EGF domain) was modeled by AlphaFold2\u003csup\u003e10\u003c/sup\u003e, which showed that Met339 and other previously reported pathogenic variants were located at the Nodal-CFC1 binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). We further searched for pathogenic variants of the \u003cem\u003ePROC\u003c/em\u003e gene in the WGS data that may explain the decrease in Protein C in this patient, but were unable to find any candidate variants. Furthermore, we did not identify any pathogenic variants that were responsible for monogenic stroke panel genes\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnticoagulation therapy was started with continuous intravenous unfractionated heparin and switched to warfarin. The patient showed no recurrence of ischemic stroke thereafter.\u003c/p\u003e"},{"header":"Discussion and conclusions","content":"\u003cp\u003eWe described a novel \u003cem\u003eNODAL\u003c/em\u003e missense variant in a patient presenting with young embolic stroke and visceral heterotaxy. This variant was located in a highly conserved Nodal active domain, and analysis of the protein structure suggested that it may have a role in CFC1 binding. It is noteworthy that the unaffected father has the same variant. Disruption of the left-right axis determination in embryonic development can result in random left-right selection\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In support of this, genetic analyses of heterotaxy family showed approximately 50% penetrance, even in monozygotic twins\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. A heterotaxy family with a \u003cem\u003eNODAL\u003c/em\u003e variant was reported, suggesting a\u0026thinsp;~\u0026thinsp;50% penetrance rate\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Collectively, it is likely that the unaffected father with Met339Ther did not show heterotaxy due to random determination of the left-right axis. Family-based genetic analysis using high-throughput sequencers usually uses variant filtering based on a hypothesis of 100% penetrance. It may be important to consider this randomness of disease manifestation when performing genetic analysis in a family with visceral heterotaxy.\u003c/p\u003e \u003cp\u003eThe patient had an embolic stroke. We considered whether genetic abnormality and cerebral infarction are related. As echocardiography showed an apparent right-to-left shunt, it may be possible that a right-to-left shunt in the heart allowed clots in the venous system to enter the brain circulation and caused an embolic stroke. Although we could not detect DVT, we suspected that the abnormal finding of protein C level inspires the insidious occurrence of DVT. Although the patient showed a mildly reduced Protein C level (63%), no pathogenic variants were found in the \u003cem\u003ePROC\u003c/em\u003e gene. It is reported that protein C deficiency due to pathogenic genetic variants can decrease Protain C level below 55%. Mild reductions (55\u0026ndash;65%) are also seen in healthy individuals\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. It may be caused by less effective genetic conditions, such as common eQTL variants in regulatory regions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, although we could not draw any conclusions about this possibility. Thus, we diagnosed the type of cerebral infarction as paradoxical cerebral embolism. We should be aware that patients with a \u003cem\u003eNODAL\u003c/em\u003e variant may develop a stroke.\u003c/p\u003e \u003cp\u003eIn conclusion, we report a novel \u003cem\u003eNODAL\u003c/em\u003e variant in a young embolic stroke patient with visceral heterotaxy. It would be important to investigate a monogenic disorder that may secondarily cause a young embolic stroke. It is also important to carefully evaluate unaffected family members when performing family-based WGS, as heterotaxy can have a penetrance of 50% due to random left-right selection.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed and disclosure consents were obtained from the patient. Institutional review board of St. Marianna University School of Medicine approved the study protocol (#4983).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsent for publication was obtained from the patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWGS data is private and unavailable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to report.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the patient and his parents for participating in our study. We would like to thank to Ritsuko Oikawa and Yoshiko Miyake for their technical assistance and Yoshikazu Haramoto for his comments about Nodal. Additionally, we are thankful for the partial support provided by the supercomputer at ROIS National Institute of Genetics for computation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the Japan Agency for Medical Research and Development (AMED No.22ek0109493). We thank the patient and his parents for participating in our study. Computations were partially performed on the NIG supercomputer at ROIS National Institute of Genetics.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEkker MS, Boot EM, Singhal AB, et al. Epidemiology, aetiology, and management of ischaemic stroke in young adults. Lancet Neurol 2018;17:790-801.\u003c/li\u003e\n\u003cli\u003eIlinca A, Puschmann A, Putaala J, et al. Updated Stroke Gene Panels: Rapid evolution of knowledge on monogenic causes of stroke. Eur J Hum Genet 2022.\u003c/li\u003e\n\u003cli\u003eTan RYY, Traylor M, Megy K, et al. How common are single gene mutations as a cause for lacunar stroke? A targeted gene panel study. Neurology 2019;93:e2007-e2020.\u003c/li\u003e\n\u003cli\u003eFang F, Xu Z, Suo Y, et al. Gene panel for Mendelian strokes. Stroke Vasc Neurol 2020;5:416-421.\u003c/li\u003e\n\u003cli\u003eIlinca A, Martinez-Majander N, Samuelsson S, et al. Whole-Exome Sequencing in 22 Young Ischemic Stroke Patients With Familial Clustering of Stroke. Stroke 2020;51:1056-1063.\u003c/li\u003e\n\u003cli\u003eKent DM, Ruthazer R, Weimar C, et al. An index to identify stroke-related vs incidental patent foramen ovale in cryptogenic stroke. Neurology 2013;81:619-625.\u003c/li\u003e\n\u003cli\u003eGebbia M, Ferrero GB, Pilia G, et al. X-linked situs abnormalities result from mutations in ZIC3. Nat Genet 1997;17:305-308.\u003c/li\u003e\n\u003cli\u003eKawai Y, Watanabe Y, Omae Y, et al. Exploring the genetic diversity of the Japanese Population: Insights from a Large-Scale Whole Genome Sequencing Analysis. bioRxiv 2023:2023.2001.2023.525133.\u003c/li\u003e\n\u003cli\u003eRoessler E, Pei W, Ouspenskaia MV, et al. Cumulative ligand activity of NODAL mutations and modifiers are linked to human heart defects and holoprosencephaly. Mol Genet Metab 2009;98:225-234.\u003c/li\u003e\n\u003cli\u003eJumper J, Evans R, Pritzel A, et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021;596:583-589.\u003c/li\u003e\n\u003cli\u003eLayton WM, Jr. Random determination of a developmental process: reversal of normal visceral asymmetry in the mouse. J Hered 1976;67:336-338.\u003c/li\u003e\n\u003cli\u003eNoone PG, Bali D, Carson JL, et al. Discordant organ laterality in monozygotic twins with primary ciliary dyskinesia. Am J Med Genet 1999;82:155-160.\u003c/li\u003e\n\u003cli\u003eLi AH, Hanchard NA, Azamian M, et al. Genetic architecture of laterality defects revealed by whole exome sequencing. Eur J Hum Genet 2019;27:563-573.\u003c/li\u003e\n\u003cli\u003eMiletich J, Sherman L, Broze G, Jr. Absence of thrombosis in subjects with heterozygous protein C deficiency. N Engl J Med 1987;317:991-996.\u003c/li\u003e\n\u003cli\u003eGudjonsson A, Gudmundsdottir V, Axelsson GT, et al. A genome-wide association study of serum proteins reveals shared loci with common diseases. Nat Commun 2022;13:480.\u003c/li\u003e\n\u003cli\u003eYan YT, Liu JJ, Luo Y, et al. Dual roles of Cripto as a ligand and coreceptor in the nodal signaling pathway. Mol Cell Biol 2002;22:4439-4449.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-neurology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nurl","sideBox":"Learn more about [BMC Neurology](http://bmcneurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nurl","title":"BMC Neurology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Visceral heterotaxy, Cryptogenic stroke, Whole-genome sequencing","lastPublishedDoi":"10.21203/rs.3.rs-2847400/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2847400/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eIschemic stroke in young adults can be caused by a variety of etiologies including the monogenic disorders. Visceral heterotaxy is a condition caused by abnormal left-right determinations during embryonic development. We aimed to determine the cause of a young ischemic stroke patient with visceral heterotaxy.\u003c/p\u003e\u003ch2\u003eCase presentation:\u003c/h2\u003e \u003cp\u003eWe performed neurological, radiological, and genetic evaluations in a 17-year-old male patient presenting ischemic stroke and visceral heterotaxy to determine the underlying cause of this rare disease combination. Brain MRI showed evidence of embolic stroke, abdominal CT showed visceral heterotaxy, and echocardiogram showed cardiac anomaly with right-to-left-shunt (RLS). Whole genome sequencing (WGS) revealed a heterozygous missense variant (NM_018055.5: c.1016T\u0026thinsp;\u0026gt;\u0026thinsp;C, p.(Met339Val)) in the \u003cem\u003eNODAL\u003c/em\u003e gene, which is essential to the determination of the left-right body axis.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eOur study highlights the importance of evaluating genetic causes in young ischemic stroke of unknown cause and the need for stroke risk management in visceral heterotaxy patients with RLS. To the best of our knowledge, we report the first genetically-confirmed case of visceral heterotaxy with young embolic stroke reported to date.\u003c/p\u003e","manuscriptTitle":"A Novel NODAL variant in a young embolic stroke patient with visceral heterotaxy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-05-23 21:51:53","doi":"10.21203/rs.3.rs-2847400/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-01-23T15:25:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-11T14:57:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-12-06T05:19:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8c78383e-9ba7-4726-b8f3-b48cc0399411","date":"2023-11-30T02:46:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1651d7cd-05b0-441f-9efd-0e01d16d3a83","date":"2023-11-29T22:52:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-06-28T00:18:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-06-28T00:15:16+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2023-06-13T06:07:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-06-13T06:05:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Neurology","date":"2023-04-22T05:04:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-neurology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nurl","sideBox":"Learn more about [BMC Neurology](http://bmcneurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nurl","title":"BMC Neurology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"72955e81-727c-42d0-91a7-c767f044073a","owner":[],"postedDate":"May 23rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-04-15T15:05:19+00:00","versionOfRecord":{"articleIdentity":"rs-2847400","link":"https://doi.org/10.1186/s12883-024-03619-x","journal":{"identity":"bmc-neurology","isVorOnly":false,"title":"BMC Neurology"},"publishedOn":"2024-04-11 15:00:36","publishedOnDateReadable":"April 11th, 2024"},"versionCreatedAt":"2023-05-23 21:51:53","video":"","vorDoi":"10.1186/s12883-024-03619-x","vorDoiUrl":"https://doi.org/10.1186/s12883-024-03619-x","workflowStages":[]},"version":"v1","identity":"rs-2847400","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2847400","identity":"rs-2847400","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}