Compound heterozygous deletions of the WWOX gene caused a WOREE syndrome associated with severe epileptic encephalopathy

Compound heterozygous deletions of the WWOX gene caused a WOREE syndrome associated with severe epileptic encephalopathy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Compound heterozygous deletions of the WWOX gene caused a WOREE syndrome associated with severe epileptic encephalopathy Xing-sheng Dong, Xiao-jun Wen, De-gang Wang, Yi Xiong, Zhi-ming Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1682290/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 Recent studies showed that germline, bi-allelic and pathogenic variants of the WWOX gene have been associated with spinocerebellar ataxia type 12 (SCAR12) and a severe WWOX-related epileptic encephalopathy (WOREE syndrome). The underlying mechanisms of the diseases are poorly understood. Here, we reported the case of a WOREE syndrome patient with early-onset refractory seizures and global neurodevelopmental delay who died at the age of two and a half years. The whole exon sequencing showed homozygous exon 6 deletions in the WWOX gene. Quantitative real-time polymerase chain reaction (PCR) confirmed that deletions were inherited from each parent. An exons 6–8 deletion was inherited from the mother, while an exon 6 deletion plus a microdeletion involving intron 5 was inherited from the father. Due to the structure of the WWOX locus encompassing the FRA16D fragile site, we confirmed the exact breakpoints using whole genomic sequencing combined with long-range PCR. Our findings extend the mutation spectrum of the WOREE syndrome and support an important role for the WWOX gene in neural development. breakpoints FRA16D fragile site germline WGS WOREE syndrome WWOX Figures Figure 1 Figure 2 Figure 3 Introduction The WW domain-containing oxidoreductase ( WWOX ) gene maps to chromosome 16q23.1-23.2 and spans the second most common chromosomal fragile site (FRA16D) frequently altered in cancer (Bednarek et al. 2000 ; Hussain et al. 2019 ). The WWOX gene encodes a 414-amino-acid protein consisting of two tandem WW domains in its N-terminus and an extended short-chain dehydrogenase/reductase (SDR) domain in its C-terminus (Bednarek et al. 2000 ; Del Mare et al. 2009 ). WWOX was initially thought to be a tumor suppressor (Aqeilan et al. 2014 ; Gardenswartz and Aqeilan 2014 ). However, in recent years, animal models demonstrated that pathogenic variants of WWOX are associated with two major autosomal recessive neurological disorders, a relatively mild spinocerebellar ataxia 12 (SCAR12) and a more severe early infantile WWOX-related epileptic encephalopathy (WOREE syndrome). The WOREE syndrome is also known as developmental and epileptic encephalopathy 28 (DEE28), depending on the type of mutation and its effect on WWOX expression (Mallaret et al. 2014 ; Piard et al. 2019 ). Two missense variants in the WWOX gene are associated with SCAR12, while two null alleles are associated with the WOREE syndrome. Genotypes with a missense variation and a null allele resulted in an intermediate phenotype (Piard et al. 2019 ). So far, a total of 56 patients have been reported with the WOREE syndrome. The clinical spectrum includes early onset of refractory seizures, encephalopathy, spasticity with hyperreflexia and hypokinesia, developmental delay, structural brain abnormalities and early death within the first years. Most of the patients with WOREE syndrome make no eye contact and are not able to sit, speak or walk (Piard et al. 2019 ; Banne et al. 2021 ). Regarding SCAR12, only 6 patients have been reported in two consanguineous families due to homozygous missense mutations. Patients with SCAR12 display a milder phenotype, including early-onset seizures, delayed psychomotor development with intellectual disability and cerebellar ataxia. They can be treated with antiepileptic drugs and no premature death has been reported (Piard et al. 2019 ; Banne et al. 2021 ). Brain abnormalities were found especially in the WORRE syndrome, such as hypoplasia of the corpus callosum, progressive cerebral atrophy, delayed myelination and optic nerve atrophy (Mignot et al. 2015 ; Ehaideb et al. 2018 ; Piard et al. 2019 ). The correlation between genotype and phenotype in these diseases is limited. In this study, we reported the case of a single patient with severe early-onset refractory seizures and two deletions involving exon 6 and exon 6 to 8 in the WWOX gene. We confirmed the exact breakpoints of these deletions using whole genomic sequencing combined with long-range polymerase chain reaction (PCR). Materials And Methods Blood samples were collected after informed consent had been obtained from the patient or their relatives. Whole exome sequencing The whole exome sequencing (WES) was performed as previously described (Wang et al. 2022). Briefly, genomic DNA of the proband was extracted from peripheral blood. KAPA HyperExome Probes (Roche NimbleGen, USA) were used for capturing sequences. The enriched library was sequenced with the MGIseq-2000 platform using a paired-end 100 bp sequencing strategy. Raw reads were filtered according to previously published criteria (Wei et al. 2011). Then, clean reads were mapped to the reference genome GRCh38 by using Burrows‐Wheeler Aligner (BWA). Single-nucleotide variants (SNVs), insertions and deletions (indels) were called using the Genome Analyses Tool Kit (GATK) and annotated with SnpEff software. All candidate variants were filtered for data interpretation with minor allele frequency < 0.05 in dbSNP (https://www.ncbi.nlm.nih.gov/SNP), HapMap (https://www.genome.gov/international-hapmap-project), 1000 Genomes Project (www.internationalgenome.org) and a database of 100 healthy Chinese adults. Copy number variations (CNVs) were detected using the ExomeDepth R package. According to the American College of Medical Genetics (ACMG) guideline (Richards et al. 2015), all mutations are classified as benign, likely benign, variants of unknown clinical significance (VUS), likely pathogenic and pathogenic. Quantitative real-time PCR To confirm the deletion involving exon 6 of the WWOX gene, we developed a gene dosage assay based on quantitative real-time PCR (qRT-PCR). DNA samples from all family members and a healthy control were diluted in 50 ng/µl. The qRT-PCR was conducted in a total of 20 µl containing 10 µl 2× GoTaq® qPCR Master Mix (Promega, USA), 0.25 µl of each primer pairs (10 µM), 1 µl of DNA sample (50 ng/µl) and ddH 2 O. The PCR reaction was performed using the SLAN-96S real-time PCR System (Hongshi Med. Tec., China). The cycling conditions were as follows: 95℃ for 2 min, then 40 cycles of 95℃ for 15 s and 60℃ for 20 s. The WWOX-exon6 primer pairs were generated as forward primer (5′-TACCATGAACTACACTTGCTGT-3′) and reverse primer (5′-GATCTATAACCCTCCACTGGAAC-3′). The GAPDH gene was used as an internal reference and primer pairs were generated as forward primer (5′- GTCAGTGGTGGACCTGACCT-3′) and reverse primer (5′- TCGCTGTTGAAGTCAGAGGA-3′). The fold-change was calculated using the 2 - DD Ct method. Whole genome sequencing In order to confirm the deletions and accurately identify their breakpoints, a low-coverage whole genomic sequencing (30× WGS) was performed on the proband. The qualified genomic DNA was randomly fragmented by Covaris sonicator and the proper fragment (350 bp) was obtained after fragment selection. The WGS libraries were prepared by MGIEasy FS DNA Prep kit (BGI, China) according to the manufacturer’s instructions. Then, they were sequenced using DNBSEQ-T7 platforms generating 2 × 100 bp paired-end reads. The WGS data were processed using an in-house analysis pipeline. Briefly, the raw data quality check was conducted using FastQC (version 0.11.9). Reads were mapped to the reference genome (GRCh38) using BWA-MEM (version 0.7.17-r1188) and GATK (version 4.2.1.0). Duplicates were removed using Picard MarkDuplicates (version 2.21.2). The CNVs were called using the CNVnator (version 0.2.7) read-depth algorithm. The structural variations (SV) were detected using the BreakDancer-1.3.6. Long-range PCR analysis Deletion breakpoints in the WWOX gene were confirmed by long-range PCR and Sanger sequencing. PCR primers flanking the breakpoints were generated as follows: F1 (5′-TTTCTGGAGCAGTCTATTT-3′) and R1 (5′-TGAACAGCCAGCCAATAC-3′); F2 (5′-AGGGTATTAACATCTTGCA-3′) and R2 (5′-ATCTGCTCCGCTTAGTCA-3′); F3 (5′-TAAGGGCTCAGTAGCGTAG-3′) and R3 (5′- CAAATCTACCGACCTTAT-3′). The PCR products were analyzed by electrophoresis in agarose gel (1.2%) and specific fragments were sequenced. Sequencing results were queried online using the UCSC Genome Browser tool (Human GRCh38/hg38, https://genome.ucsc.edu) to identify breakpoints. After identifying breakpoints, sequences flanking the breakpoints were screened for repetitive elements using the RepeatMasker program (http://www.repeatmasker.org/) to identify repetitive sequences at breakpoint junctions. The R-loop forming sequences (RLFSs) were screened by R-loopDB (http://rloop.bii.a-star.edu.sg). Results The patient was born at term by normal spontaneous vaginal delivery, the weight was 3.39 kg. No history of hypoxia during the perinatal period was reported. Shortly after birth, he was transferred to the department of neonatology because of neonatal hyperbilirubinemia. Then, he was discharged one week later. At 15 days after birth, he presented infantile seizures characterized by closed teeth, cyanosis around the lips and tetanic twitch of the limbs. The symptoms lasted around 30 seconds, with a frequency of 10-15 times per day. The administration of antiepileptic drugs was not effective. He did several hospital admissions because of recurrent seizure attacks. The patient developed growth and development retardation and was unable to follow objects, roll over, sit alone or speak. Magnetic resonance imaging (MRI) showed white matter hypersignal and delayed myelination in the brain. He died at the age of two and a half years because of persistent seizures. At the moment of death, the patients was 69 cm in height and 8 kg in weight. The familiar medical history was unremarkable except for the maternal grandfather (I-3) who suffered from lung cancer and the paternal grandmother (I-2) who died of a cerebrovascular accident more than 10 years ago (Fig. 1a). Genetic findings The WES analysis revealed a homozygous deletion involving exon 6 of the WWOX gene in the proband (data not shown). The qRT-PCR indicated that the maternal grandfather (I3), father (II1) and mother (II2) were characterized by an heterozygous deletion involving exon 6 of the WWOX gene (Fig. 1b). Thus, the homozygous deletion was inherited from the parents. The WGS analysis revealed also the presence of three larger deletions involving the WWOX gene in the proband (Fig. 2). A large deletion about 177,200 bp in length involved exons 6-8 at chr16: 78331193 – 78508394 (hg38). Another deletion about 13,261 bp in length involved intron 5 at chr16: 78337740 – 78351002 (hg38). The last deletion about 53,904 bp in length involved exon 6 at chr16: 78368802 – 78422707 (hg38). A 800-bp PCR product was amplified by gap-PCR using primers F1 and R1 in I3, II2 and III1. The sequencing showed that the deletion was 177,204 bp in length with the deletion junction characterized by a 4-bp microhomology. The 5′ breakpoint was located within coordinates 78331189 and 78331193, the 3′ breakpoint was located within coordinates 78508394 and 78508398 (Fig. 3ad). Another 900-bp PCR product was amplified by gap-PCR using primers F2 and R2 in II1 and III1. The sequencing showed that the deletion was 13,260 bp in length. The 5′ breakpoint was located at 78337741, the 3′ breakpoint was located at 78351002 (Fig. 3bd). The last 1300-bp PCR product was amplified by gap-PCR using primers F3 and R3 in II1 and III1. The sequencing showed that the deletion was 53,904 bp in length. The 5′ breakpoint was located at 78368802, the 3′ breakpoint was located at 78422707, and the deletion junction insert ATACACACACAC. The 3′ flanking regions were (AT)n(AC)n repetitive sequences (Fig. 3cd). Discussion The clinical phenotypes associated with germline, bi-allelic and pathogenic variants of the WWOX gene are highly heterogeneous. Symptoms range from a mild phenotypic SCAR12 to a severe early infantile WWOX-related epileptic encephalopathy (WOREE syndrome), depending on the type of mutation and its effect on WWOX expression. Therefore, accurate diagnosis and treatment are hampered by the heterogeneous clinical presentation of individuals. In this study, we identified a patient with the WOREE syndrome and compound heterozygous WWOX deletions of exon 6 and exons 6–8. The exon 6 deletion allele was inherited from the father (II1), while the exons 6–8 deletion allele was inherited from the mother (II-2) and maternal grandfather (I-3). The exon 6 deletion (c.517_605ddel, H173Afs*67) is a common deletion predicted to cause a frameshift mutation resulting in a truncated protein with loss of function. The exon 6–8 deletion (c.517_1056del, His173_Met352del) theoretically produces a protein with normal WW domains but missing the SDR domain which may retain poor, if any, residual function (Mignot et al. 2015 ). The patient mainly presented with early-onset epilepsy, daily seizures and antiepileptic drug resistance, with no eye contact and developmental delay. The phenotype was consistent with previous reports (Piard et al. 2019 ). The patient died at the age of two and a half years. Different in vitro and in vivo functional studies have indicated WWOX´s role as a tumor suppressor. The reduction or absence of WWOX expression has been associated with several cancers, including breast cancer, thyroid cancer, oral cancer and lung cancer (Iliopoulos et al. 2005 ). WWOX expression is altered by deletion and/or aberrant synthesis in a significant number of non-small cell lung cancer (NSCLC) tumors (51.8%) (Yendamuri et al. 2003 ). Similar results have been described in breast cancer (Driouch et al. 2002 ). Moreover, a high incidence of exons 6–8 deletions has been reported in Chinese patients with NSCLC (63.6%), suggesting that exons 6–8 deletions might play a role in the tumorigenesis of NSCLC (Zhou et al. 2005 ). In this study, we hypothesized that the heterozygous WWOX exons 6–8 deletion observed in maternal grandfather (I-3, lung cancer patient) caused an abnormal WWOX expression and the consequent clinical phenotype. In similar clinical phenotypes, deletions have a higher frequency than other mutation types. In our case, compound heterozygous deletions caused the WOREE syndrome. A single allele was linked to an exon 6 deletion and an intron 5 deletion, indicating that the chromosome was broken and rearranged twice. This also reflects the WWOX gene encompassing FRA16D from introns 5 to 8, indicating that the WWOX gene is prone to breakages and rearrangements. Multiple models have tried to explain the underlying mechanisms behind the FRA16D instability, including longer AT repeats forming a cruciform and stall replication. Replication origins located within CFS sequences are less efficient and probably responsible for replication perturbation along fragile sites (Durkin and Glover 2007 ; Zhang and Freudenreich 2007 ; Hussain et al. 2019 ; Kaushal et al. 2019 ). Although CNVs can be identified by multiplex ligation-dependent probe amplification (MLPA), qRT-PCR, array-CGH and WES, the detection results do not affect the correlation analysis between genotype and phenotype. In order to explain how WWOX deletions involving FRA16D were generated, we used 30× WGS combined with long-range PCR to identify the breakpoints. We accurately identified the breakpoint junctions of three deletions. No R-loop forming sequences were found using R-loopDB to screen the sequences flanking the breakpoint junctions of the three deletions. The deletion involving exons 6–8 and another deletion involving intron 5 were presumably generated by the non-homologous end joining (NHEJ) repair pathway, because this model requires one to four base pairs or no homology between proximal and distal breakpoints. The 3´ flanking regions were (AT)n(AC)n repetitive sequences involving exon 6. The deletion might be caused by the abundance of AT-rich DNA forming complex secondary structures that slow down or block the progress of the replication machinery and ultimately leading to chromosome breakage (Zhang and Freudenreich 2007 ). Conclusions In conclusion, we reported compound heterozygous WWOX deletions of exon 6 and exons 6–8 causing a more severe early infantile WOREE syndrome. Furthermore, we confirmed the exact breakpoints of these deletions using WGS combined with long-range PCR. Our findings extend the mutation spectrum of the WOREE syndrome and support an important role for the WWOX gene in neural development. Declarations Acknowledgments We are grateful to the family members for their participation in the study. We acknowledge TopEdit LLC for linguistic editing and proofreading during the preparation of this article. Author Contribution DXS and ZML drafted the manuscript and contributed to conception and design of the study. XJW performed experiments. DGW and YX contributed to the data acquisition. ZML critically revised and gave final approval for publication of the paper. Fundings This work was funded by general program of social benefit and basic research project of Zhongshan city (2021B1084) and major Program of social benefit and basic research project of Zhongshan city (2021B3003) Availability of data and materials All data generated or analyzed during this study are included in this published article. Ethics Approval and Consent to Participate All procedures performed in studies involving human participants were in accordance with the Ethics Committee of Zhongshan Boai Hospital Affiliated to Southern Medical University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The written informed consent was taken from the participants or their guardians. Conflicts of Interest/Competing Interests The authors declare that they have no competing interests. References Aqeilan RI, Abu-Remaileh M, et al. (2014) The common fragile site FRA16D gene product WWOX: roles in tumor suppression and genomic stability. Cellular and molecular life sciences: CMLS 71(23): 4589–4599. doi 10.1007/s00018-014-1724-y Banne E, Abudiab B, et al. (2021) Neurological Disorders Associated with WWOX Germline Mutations-A Comprehensive Overview. Cells 10(4). doi 10.3390/cells10040824 Bednarek AK, Laflin KJ, et al. 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(2014) The tumour suppressor gene WWOX is mutated in autosomal recessive cerebellar ataxia with epilepsy and mental retardation. Brain: a journal of neurology 137(Pt 2): 411–419. doi 10.1093/brain/awt338 Mignot C, Lambert L, et al. (2015) WWOX-related encephalopathies: delineation of the phenotypical spectrum and emerging genotype-phenotype correlation. Journal of medical genetics 52(1): 61–70. doi 10.1136/jmedgenet-2014-102748 Piard J, Hawkes L, et al. (2019) The phenotypic spectrum of WWOX-related disorders: 20 additional cases of WOREE syndrome and review of the literature. Genetics in medicine: official journal of the American College of Medical Genetics 21(6): 1308–1318. doi 10.1038/s41436-018-0339-3 Richards S, Aziz N, et al. (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in medicine: official journal of the American College of Medical Genetics 17(5): 405–424. doi 10.1038/gim.2015.30 Wang DG, Dong XS, et al. (2022) Identification of a novel TBX5 c.755 + 1 G > A variant and related pathogenesis in a family with Holt-Oram syndrome. American journal of medical genetics Part A 188(1): 58–70. doi 10.1002/ajmg.a.62488 Wei X, Ju X, et al. (2011) Identification of sequence variants in genetic disease-causing genes using targeted next-generation sequencing. PloS one 6(12): e29500. doi 10.1371/journal.pone.0029500 Yendamuri S, Kuroki T, et al. (2003) WW domain containing oxidoreductase gene expression is altered in non-small cell lung cancer. Cancer research 63(4): 878–881 Zhang H and Freudenreich CH (2007) An AT-rich sequence in human common fragile site FRA16D causes fork stalling and chromosome breakage in S. cerevisiae. Molecular cell 27(3): 367–379. doi 10.1016/j.molcel.2007.06.012 Zhou Y, Xu Y, et al. (2005) Deletion and mutation of WWOX exons 6–8 in human non-small cell lung cancer. Journal of Huazhong University of Science and Technology Medical sciences = Hua zhong ke ji da xue xue bao Yi xue Ying De wen ban = Huazhong keji daxue xuebao Yixue Yingdewen ban 25(2): 162–165. doi 10.1007/bf02873566 Additional Declarations No competing interests reported. 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-1682290","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":110224857,"identity":"45566063-49d3-421a-984d-b1a278114bda","order_by":0,"name":"Xing-sheng Dong","email":"","orcid":"","institution":"Prenatal Diagnosis Center, Affiliated Boai Hospital of Zhongshan, Southern Medical University, Zhongshan, Guangdong, China","correspondingAuthor":false,"prefix":"","firstName":"Xing-sheng","middleName":"","lastName":"Dong","suffix":""},{"id":110224858,"identity":"e600a10d-1003-4b8b-b3ba-739cdaf5b69f","order_by":1,"name":"Xiao-jun Wen","email":"","orcid":"","institution":"Reproductive Medicine Center, Affiliated Boai Hospital of Zhongshan, Southern Medical University, Zhongshan, Guangdong, China","correspondingAuthor":false,"prefix":"","firstName":"Xiao-jun","middleName":"","lastName":"Wen","suffix":""},{"id":110224859,"identity":"d6bd2778-7e41-4cbb-9238-d8e6ce405f41","order_by":2,"name":"De-gang Wang","email":"","orcid":"","institution":"Prenatal Diagnosis Center, Affiliated Boai Hospital of Zhongshan, Southern Medical University, Zhongshan, Guangdong, China","correspondingAuthor":false,"prefix":"","firstName":"De-gang","middleName":"","lastName":"Wang","suffix":""},{"id":110224860,"identity":"6b0ed09d-6a3e-4859-afd2-4b231ff0d7a6","order_by":3,"name":"Yi Xiong","email":"","orcid":"","institution":"Prenatal Diagnosis Center, Affiliated Boai Hospital of Zhongshan, Southern Medical University, Zhongshan, Guangdong, China","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Xiong","suffix":""},{"id":110224861,"identity":"981aaf1d-5987-4e02-a741-b8a4b8df037d","order_by":4,"name":"Zhi-ming Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYBACNhDxoECCwYCBsYGBoUJCTp6wFmYGhgQDmJYzFsaGDQTtAWthAGoBAsa2ikSGAwQ08En3H3yQYGAhb87e3Pjh4zyJBMYG5oePbuBzmMxhZgOgwwx39hxslpy5TSKPnYHN2DgHnxaJZDYJoJYEgxuJbcy82ySKGRt42KQJaGH/Adfyd45EYsMBwlrYGOBaGBuI02IMcpjhhjNAv/QckzA2bCbgF/kZiQ8/fKiokzc43v7ww4+aOjl59uaHj/FpwQKYSVM+CkbBKBgFowALAADJVESD44pIEQAAAABJRU5ErkJggg==","orcid":"","institution":"Prenatal Diagnosis Center, Affiliated Boai Hospital of Zhongshan, Southern Medical University, Zhongshan, Guangdong, China","correspondingAuthor":true,"prefix":"","firstName":"Zhi-ming","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2022-05-22 16:59:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1682290/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1682290/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22299730,"identity":"f77ba9a5-a66c-46a4-9261-190ba1a7cda5","added_by":"auto","created_at":"2022-06-06 14:10:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65131,"visible":true,"origin":"","legend":"\u003cp\u003ePedigree of the family. The proband is labeled with arrows. \u003cstrong\u003eb\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eQuantitative PCR validation of the exon 6 deletion of the \u003cem\u003eWWOX\u003c/em\u003e gene. The Y-axis represents the log R ratio, the X-axis indicates the exon 6.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1682290/v1/0250cdae6464ad75eb47bb40.png"},{"id":22300419,"identity":"ae73a3a1-5a31-4d55-a2a8-b7bef8728267","added_by":"auto","created_at":"2022-06-06 14:15:05","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":670658,"visible":true,"origin":"","legend":"\u003cp\u003eWhole genome sequencing confirmed the deletions of the \u003cem\u003eWWOX\u003c/em\u003e gene. The horizontal red lines in the red alignments represent deletions in alignments that otherwise match the reference sequence. Breakpoints can be inferred from the short reads by examination of the outer read extents and the depth in coverage. The black dotted arrow represents the breaking point, the red double arrow line represents the range of deletions.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1682290/v1/c3b534c51990b4b0f6b0725d.png"},{"id":22299729,"identity":"c8b37cb3-d993-4618-9fd2-17ddfa149ba1","added_by":"auto","created_at":"2022-06-06 14:10:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":431487,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the breakpoints of the three deletions. \u003cstrong\u003ea\u003c/strong\u003e. A 800-bp PCR product was amplified by gap-PCR using primers F1 and R1 in I3, II2 and III1. No PCR product was observed from I1, I4, II1 or the control. The sequencing results showed that the deletion junction was characterized by a 4-bp microhomology. The 5´ breakpoint was located within coordinates 78331189 and 78331193, the 3´ breakpoint was located within coordinates 78508394 and 78508398. M: marker, N: normal individual. \u003cstrong\u003eb\u003c/strong\u003e. A 900-bp PCR product was amplified by gap-PCR using primers F2 and R2 in II1 and III1. No PCR product was observed from I1, I3, I4, II2 or the control. The sequencing results showed that the 5´ breakpoint was located at 78337741, the 3´ breakpoint was located at 78351002. \u003cstrong\u003ec\u003c/strong\u003e. A 1300-bp PCR product was amplified by gap-PCR using primers F3 and R3 in II1 and III1. No PCR product was observed from I1, I3, I4, II2 or the control. The sequencing results showed that the 5´ breakpoint was located at 78368802, the 3´ breakpoint was located at 78422707. The deletion junction inserted ATACACACACAC. The 3´ flanking regions were characterized by (AT)n(AC)n\u003cstrong\u003e \u003c/strong\u003erepetitive sequences.\u003cstrong\u003e d\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eSchematic representation of the three deletions. The deletion inherited from proband’s mother is represented as a red bar. The other two deletion are represented as blue bars.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1682290/v1/7b46a9ea45597d3a0b92acab.png"},{"id":22300421,"identity":"c548d68b-da9d-4583-9b15-fa147d364c4c","added_by":"auto","created_at":"2022-06-06 14:15:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":619340,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1682290/v1/61a34f3b-0170-4cbc-8d1a-fe8e23ca9c93.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Compound heterozygous deletions of the WWOX gene caused a WOREE syndrome associated with severe epileptic encephalopathy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe WW domain-containing oxidoreductase (\u003cem\u003eWWOX\u003c/em\u003e) gene maps to chromosome 16q23.1-23.2 and spans the second most common chromosomal fragile site (FRA16D) frequently altered in cancer (Bednarek et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Hussain et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003eWWOX\u003c/em\u003e gene encodes a 414-amino-acid protein consisting of two tandem WW domains in its N-terminus and an extended short-chain dehydrogenase/reductase (SDR) domain in its C-terminus (Bednarek et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Del Mare et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). WWOX was initially thought to be a tumor suppressor (Aqeilan et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Gardenswartz and Aqeilan \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). However, in recent years, animal models demonstrated that pathogenic variants of WWOX are associated with two major autosomal recessive neurological disorders, a relatively mild spinocerebellar ataxia 12 (SCAR12) and a more severe early infantile WWOX-related epileptic encephalopathy (WOREE syndrome). The WOREE syndrome is also known as developmental and epileptic encephalopathy 28 (DEE28), depending on the type of mutation and its effect on WWOX expression (Mallaret et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Piard et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Two missense variants in the \u003cem\u003eWWOX\u003c/em\u003e gene are associated with SCAR12, while two null alleles are associated with the WOREE syndrome. Genotypes with a missense variation and a null allele resulted in an intermediate phenotype (Piard et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSo far, a total of 56 patients have been reported with the WOREE syndrome. The clinical spectrum includes early onset of refractory seizures, encephalopathy, spasticity with hyperreflexia and hypokinesia, developmental delay, structural brain abnormalities and early death within the first years. Most of the patients with WOREE syndrome make no eye contact and are not able to sit, speak or walk (Piard et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Banne et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Regarding SCAR12, only 6 patients have been reported in two consanguineous families due to homozygous missense mutations. Patients with SCAR12 display a milder phenotype, including early-onset seizures, delayed psychomotor development with intellectual disability and cerebellar ataxia. They can be treated with antiepileptic drugs and no premature death has been reported (Piard et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Banne et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Brain abnormalities were found especially in the WORRE syndrome, such as hypoplasia of the corpus callosum, progressive cerebral atrophy, delayed myelination and optic nerve atrophy (Mignot et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ehaideb et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Piard et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe correlation between genotype and phenotype in these diseases is limited. In this study, we reported the case of a single patient with severe early-onset refractory seizures and two deletions involving exon 6 and exon 6 to 8 in the \u003cem\u003eWWOX\u003c/em\u003e gene. We confirmed the exact breakpoints of these deletions using whole genomic sequencing combined with long-range polymerase chain reaction (PCR).\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003eBlood samples were collected after informed consent had been obtained from the patient or their relatives.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole exome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe whole exome sequencing (WES) was performed as previously described (Wang et al. 2022). Briefly, genomic DNA of the proband was extracted from peripheral blood. KAPA HyperExome Probes (Roche NimbleGen, USA) were used for capturing sequences. The enriched library was sequenced with the MGIseq-2000 platform using a paired-end 100 bp sequencing strategy. Raw reads were filtered according to previously published criteria (Wei et al. 2011). Then, clean reads were mapped to the reference genome GRCh38 by using Burrows‐Wheeler Aligner (BWA). Single-nucleotide variants (SNVs), insertions and deletions (indels) were called using the Genome Analyses Tool Kit (GATK) and annotated with SnpEff software. All candidate variants were filtered for data interpretation with minor allele frequency \u0026lt; 0.05 in dbSNP (https://www.ncbi.nlm.nih.gov/SNP), HapMap (https://www.genome.gov/international-hapmap-project), 1000 Genomes Project (www.internationalgenome.org) and a database of 100 healthy Chinese adults. Copy number variations (CNVs) were detected using the ExomeDepth R package. According to the American College of Medical Genetics (ACMG) guideline (Richards et al. 2015), all mutations are classified as benign, likely benign, variants of unknown clinical significance (VUS), likely pathogenic and pathogenic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the deletion involving exon 6 of the \u003cem\u003eWWOX\u003c/em\u003e gene, we developed a gene dosage assay based on quantitative real-time PCR (qRT-PCR). DNA samples from all family members and a healthy control were diluted in 50 ng/\u0026micro;l. The qRT-PCR was conducted in a total of 20 \u0026micro;l containing 10 \u0026micro;l 2\u0026times; GoTaq\u0026reg; qPCR Master Mix (Promega, USA), 0.25 \u0026micro;l of each primer pairs (10 \u0026micro;M), 1 \u0026micro;l of DNA sample (50 ng/\u0026micro;l) and ddH\u003csub\u003e2\u003c/sub\u003eO. The PCR reaction was performed using the SLAN-96S real-time PCR System (Hongshi Med. Tec., China). The cycling conditions were as follows: 95℃ for 2 min, then 40 cycles of 95℃ for 15 s and 60℃ for 20 s. The WWOX-exon6 primer pairs were generated as forward primer (5\u0026prime;-TACCATGAACTACACTTGCTGT-3\u0026prime;) and reverse primer (5\u0026prime;-GATCTATAACCCTCCACTGGAAC-3\u0026prime;). The \u003cem\u003eGAPDH\u003c/em\u003e gene was used as an internal reference and primer pairs were generated as forward primer (5\u0026prime;- GTCAGTGGTGGACCTGACCT-3\u0026prime;) and reverse primer (5\u0026prime;- TCGCTGTTGAAGTCAGAGGA-3\u0026prime;). The fold-change was calculated using the 2\u003csup\u003e-\u003c/sup\u003e\u003csup\u003eDD\u003c/sup\u003e\u003csup\u003eCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to confirm the deletions and accurately identify their breakpoints, a low-coverage whole genomic sequencing (30\u0026times; WGS) was performed on the proband. The qualified genomic DNA was randomly fragmented by Covaris sonicator and the proper fragment (350 bp) was obtained after fragment selection. The WGS libraries were prepared by MGIEasy FS DNA Prep kit (BGI, China) according to the manufacturer\u0026rsquo;s instructions. Then, they were sequenced using DNBSEQ-T7 platforms generating 2 \u0026times; 100 bp paired-end reads. The WGS data were processed using an in-house analysis pipeline. Briefly, the raw data quality check was conducted using FastQC (version 0.11.9). Reads were\u003c/p\u003e\n\u003cp\u003emapped to the reference genome (GRCh38) using BWA-MEM (version 0.7.17-r1188) and GATK (version 4.2.1.0). Duplicates were removed using Picard MarkDuplicates (version 2.21.2). The CNVs were called using the CNVnator (version 0.2.7) read-depth algorithm. The structural variations (SV) were detected using the BreakDancer-1.3.6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLong-range PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDeletion breakpoints in the \u003cem\u003eWWOX\u003c/em\u003e gene were confirmed by long-range PCR and Sanger sequencing. PCR primers flanking the breakpoints were generated as follows: F1 (5\u0026prime;-TTTCTGGAGCAGTCTATTT-3\u0026prime;) and R1 (5\u0026prime;-TGAACAGCCAGCCAATAC-3\u0026prime;); F2 (5\u0026prime;-AGGGTATTAACATCTTGCA-3\u0026prime;) and R2 (5\u0026prime;-ATCTGCTCCGCTTAGTCA-3\u0026prime;); F3 (5\u0026prime;-TAAGGGCTCAGTAGCGTAG-3\u0026prime;) and R3 (5\u0026prime;- CAAATCTACCGACCTTAT-3\u0026prime;). The PCR products were analyzed by electrophoresis in agarose gel (1.2%) and specific fragments were sequenced. Sequencing results were queried online using the UCSC Genome Browser tool (Human GRCh38/hg38, https://genome.ucsc.edu) to identify breakpoints. After identifying breakpoints, sequences flanking the breakpoints were screened for repetitive elements using the RepeatMasker program (http://www.repeatmasker.org/) to identify repetitive sequences at breakpoint junctions. The R-loop forming sequences (RLFSs) were screened by R-loopDB (http://rloop.bii.a-star.edu.sg).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe patient was born at term by normal spontaneous vaginal delivery, the weight was 3.39 kg. No history of hypoxia during the perinatal period was reported. Shortly after birth, he was transferred to the department of neonatology because of neonatal hyperbilirubinemia. Then, he was discharged one week later. At 15 days after birth, he presented infantile seizures characterized by closed teeth, cyanosis around the lips and tetanic twitch of the limbs. The symptoms lasted around 30 seconds, with a frequency of 10-15 times per day. The administration of antiepileptic drugs was not effective. He did several hospital admissions because of recurrent seizure attacks. The patient developed growth and development retardation and was unable to follow objects, roll over, sit alone or speak. Magnetic resonance imaging (MRI) showed white matter hypersignal and delayed myelination in the brain. He died at the age of two and a half years because of persistent seizures. At the moment of death, the patients was 69 cm in height and 8 kg in weight. The familiar medical history was unremarkable except for the maternal grandfather (I-3) who suffered from lung cancer and the paternal grandmother (I-2) who died of a cerebrovascular accident more than 10 years ago (Fig. 1a).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic findings\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe WES analysis revealed a homozygous deletion involving exon 6 of the \u003cem\u003eWWOX\u003c/em\u003e gene in the proband (data not shown). The qRT-PCR indicated that the maternal grandfather (I3), father (II1) and mother (II2) were characterized by an heterozygous deletion involving exon 6 of the \u003cem\u003eWWOX\u003c/em\u003e gene (Fig. 1b). Thus, the homozygous deletion was inherited from the parents. The WGS analysis revealed also the presence of three larger deletions involving the \u003cem\u003eWWOX\u003c/em\u003e gene in the proband (Fig. 2). A large deletion about 177,200 bp in length involved exons 6-8 at chr16: 78331193 \u0026ndash; 78508394 (hg38). Another deletion about 13,261 bp in length involved intron 5 at chr16: 78337740 \u0026ndash; 78351002 (hg38). The last deletion about 53,904 bp in length involved exon 6 at chr16: 78368802 \u0026ndash; 78422707 (hg38). A 800-bp PCR product was amplified by gap-PCR using primers F1 and R1 in I3, II2 and III1. The sequencing showed that the deletion was 177,204 bp in length with the deletion junction characterized by a 4-bp microhomology. The 5\u0026prime; breakpoint was located within coordinates 78331189 and 78331193, the 3\u0026prime; breakpoint was located within coordinates 78508394 and 78508398 (Fig. 3ad). Another 900-bp PCR product was amplified by gap-PCR using primers F2 and R2 in II1 and III1. The sequencing showed that the deletion was 13,260 bp in length. The 5\u0026prime; breakpoint was located at 78337741, the 3\u0026prime; breakpoint was located at 78351002 (Fig. 3bd). The last 1300-bp PCR product was amplified by gap-PCR using primers F3 and R3 in II1 and III1. The sequencing showed that the deletion was 53,904 bp in length. The 5\u0026prime; breakpoint was located at 78368802, the 3\u0026prime; breakpoint was located at 78422707, and the deletion junction insert ATACACACACAC. The 3\u0026prime; flanking regions were (AT)n(AC)n repetitive sequences (Fig. 3cd).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe clinical phenotypes associated with germline, bi-allelic and pathogenic variants of the \u003cem\u003eWWOX\u003c/em\u003e gene are highly heterogeneous. Symptoms range from a mild phenotypic SCAR12 to a severe early infantile WWOX-related epileptic encephalopathy (WOREE syndrome), depending on the type of mutation and its effect on WWOX expression. Therefore, accurate diagnosis and treatment are hampered by the heterogeneous clinical presentation of individuals. In this study, we identified a patient with the WOREE syndrome and compound heterozygous \u003cem\u003eWWOX\u003c/em\u003e deletions of exon 6 and exons 6\u0026ndash;8. The exon 6 deletion allele was inherited from the father (II1), while the exons 6\u0026ndash;8 deletion allele was inherited from the mother (II-2) and maternal grandfather (I-3). The exon 6 deletion (c.517_605ddel, H173Afs*67) is a common deletion predicted to cause a frameshift mutation resulting in a truncated protein with loss of function. The exon 6\u0026ndash;8 deletion (c.517_1056del, His173_Met352del) theoretically produces a protein with normal WW domains but missing the SDR domain which may retain poor, if any, residual function (Mignot et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The patient mainly presented with early-onset epilepsy, daily seizures and antiepileptic drug resistance, with no eye contact and developmental delay. The phenotype was consistent with previous reports (Piard et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The patient died at the age of two and a half years.\u003c/p\u003e \u003cp\u003eDifferent in vitro and in vivo functional studies have indicated WWOX\u0026acute;s role as a tumor suppressor. The reduction or absence of WWOX expression has been associated with several cancers, including breast cancer, thyroid cancer, oral cancer and lung cancer (Iliopoulos et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). WWOX expression is altered by deletion and/or aberrant synthesis in a significant number of non-small cell lung cancer (NSCLC) tumors (51.8%) (Yendamuri et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Similar results have been described in breast cancer (Driouch et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Moreover, a high incidence of exons 6\u0026ndash;8 deletions has been reported in Chinese patients with NSCLC (63.6%), suggesting that exons 6\u0026ndash;8 deletions might play a role in the tumorigenesis of NSCLC (Zhou et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In this study, we hypothesized that the heterozygous \u003cem\u003eWWOX\u003c/em\u003e exons 6\u0026ndash;8 deletion observed in maternal grandfather (I-3, lung cancer patient) caused an abnormal WWOX expression and the consequent clinical phenotype.\u003c/p\u003e \u003cp\u003eIn similar clinical phenotypes, deletions have a higher frequency than other mutation types. In our case, compound heterozygous deletions caused the WOREE syndrome. A single allele was linked to an exon 6 deletion and an intron 5 deletion, indicating that the chromosome was broken and rearranged twice. This also reflects the \u003cem\u003eWWOX\u003c/em\u003e gene encompassing FRA16D from introns 5 to 8, indicating that the \u003cem\u003eWWOX\u003c/em\u003e gene is prone to breakages and rearrangements. Multiple models have tried to explain the underlying mechanisms behind the FRA16D instability, including longer AT repeats forming a cruciform and stall replication. Replication origins located within CFS sequences are less efficient and probably responsible for replication perturbation along fragile sites (Durkin and Glover \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zhang and Freudenreich \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Hussain et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kaushal et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Although CNVs can be identified by multiplex ligation-dependent probe amplification (MLPA), qRT-PCR, array-CGH and WES, the detection results do not affect the correlation analysis between genotype and phenotype. In order to explain how WWOX deletions involving FRA16D were generated, we used 30\u0026times; WGS combined with long-range PCR to identify the breakpoints. We accurately identified the breakpoint junctions of three deletions. No R-loop forming sequences were found using R-loopDB to screen the sequences flanking the breakpoint junctions of the three deletions. The deletion involving exons 6\u0026ndash;8 and another deletion involving intron 5 were presumably generated by the non-homologous end joining (NHEJ) repair pathway, because this model requires one to four base pairs or no homology between proximal and distal breakpoints. The 3\u0026acute; flanking regions were (AT)n(AC)n repetitive sequences involving exon 6. The deletion might be caused by the abundance of AT-rich DNA forming complex secondary structures that slow down or block the progress of the replication machinery and ultimately leading to chromosome breakage (Zhang and Freudenreich \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we reported compound heterozygous \u003cem\u003eWWOX\u003c/em\u003e deletions of exon 6 and exons 6\u0026ndash;8 causing a more severe early infantile WOREE syndrome. Furthermore, we confirmed the exact breakpoints of these deletions using WGS combined with long-range PCR. Our findings extend the mutation spectrum of the WOREE syndrome and support an important role for the \u003cem\u003eWWOX\u003c/em\u003e gene in neural development.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to the family members for their participation in the study. We acknowledge TopEdit LLC for linguistic editing and proofreading during the preparation of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDXS and ZML drafted the manuscript and contributed to conception and design of the study. XJW performed experiments. DGW and YX contributed to the data acquisition. ZML critically revised and gave final approval for publication of the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by general program of social benefit and basic research project of Zhongshan city (2021B1084) and major Program of social benefit and basic research project of Zhongshan city (2021B3003)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval and Consent to Participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the Ethics Committee of Zhongshan Boai Hospital Affiliated to Southern Medical University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The written informed consent was taken from the participants or their guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest/Competing Interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAqeilan RI, Abu-Remaileh M, et al. (2014) The common fragile site FRA16D gene product WWOX: roles in tumor suppression and genomic stability. Cellular and molecular life sciences: CMLS 71(23): 4589\u0026ndash;4599. doi \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00018-014-1724-y\u003c/span\u003e\u003cspan address=\"10.1007/s00018-014-1724-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanne E, Abudiab B, et al. 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Journal of Huazhong University of Science and Technology Medical sciences = Hua zhong ke ji da xue xue bao Yi xue Ying De wen ban = Huazhong keji daxue xuebao Yixue Yingdewen ban 25(2): 162\u0026ndash;165. doi \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/bf02873566\u003c/span\u003e\u003cspan address=\"10.1007/bf02873566\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"breakpoints, FRA16D fragile site, germline, WGS, WOREE syndrome, WWOX","lastPublishedDoi":"10.21203/rs.3.rs-1682290/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1682290/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRecent studies showed that germline, bi-allelic and pathogenic variants of the \u003cem\u003eWWOX\u003c/em\u003e gene have been associated with spinocerebellar ataxia type 12 (SCAR12) and a severe WWOX-related epileptic encephalopathy (WOREE syndrome). The underlying mechanisms of the diseases are poorly understood. Here, we reported the case of a WOREE syndrome patient with early-onset refractory seizures and global neurodevelopmental delay who died at the age of two and a half years. The whole exon sequencing showed homozygous exon 6 deletions in the \u003cem\u003eWWOX\u003c/em\u003e gene. Quantitative real-time polymerase chain reaction (PCR) confirmed that deletions were inherited from each parent. An exons 6\u0026ndash;8 deletion was inherited from the mother, while an exon 6 deletion plus a microdeletion involving intron 5 was inherited from the father. Due to the structure of the \u003cem\u003eWWOX\u003c/em\u003e locus encompassing the FRA16D fragile site, we confirmed the exact breakpoints using whole genomic sequencing combined with long-range PCR. Our findings extend the mutation spectrum of the WOREE syndrome and support an important role for the \u003cem\u003eWWOX\u003c/em\u003e gene in neural development.\u003c/p\u003e","manuscriptTitle":"Compound heterozygous deletions of the WWOX gene caused a WOREE syndrome associated with severe epileptic encephalopathy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-06 14:10:03","doi":"10.21203/rs.3.rs-1682290/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":"dfece01f-f6ad-44dc-ac02-86edb79e4e0e","owner":[],"postedDate":"June 6th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-06-06T14:10:04+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-06 14:10:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1682290","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1682290","identity":"rs-1682290","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}