Incomplete penetrance of a KANSL1 loss of function variant resolved by RNA analysis,  a case report

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The extensive clinical and genetic variability in epilepsy makes accurate diagnosis a significant challenge. Case presentation In this study, we describe a girl with a clinical presentation of developmental and epileptic encephalopathy (DEE). Using whole genome sequencing (WGS), we identified several candidate variants in the HNRNPU , NIPBL , and KANSL1 genes with partial overlap with the patient clinical presentation. Subsequent analysis revealed that only the variant in the HNRNPU gene arose de novo while the others were inherited from an unaffected parents. However, the previously reported pathogenic loss of function variant in the KANSL1 gene, inherited from a healthy mother, complicated comprehensive family counseling. A thorough investigation using RNA analysis showed that the variant in the KANSL1 gene is located in a duplicated locus, which is not functional, explaining the assumed incomplete penetrance. Conclusions This case illustrates the importance of integrating WGS with additional analyses to accurately diagnose and understand the molecular basis of incomplete penetrance in the KANSL1 gene. KANSL1 HNRNPU Incomplete penetrance RNA analysis Figures Figure 1 Figure 2 Introduction Epilepsy is among the most prevalent neurological disorders, with an incidence rate of approximately 50 new cases per 100,000 people annually [1]. Genetic factors account for about 70–80% of epilepsy cases [2], and around 1% of the global population is affected by this condition. Approximately 75% of epilepsy cases manifest during childhood, highlighting the increased vulnerability of the developing brain to seizures [1]. Epilepsy encompasses various types and syndromes, classified based on clinical and EEG features, etiologies, and comorbidities. Developmental and epileptic encephalopathies (DEEs) represent the most severe spectrum of epilepsy disorders with a significant monogenic component [3], [4]. Due to the phenotypic and genetic heterogeneity of DEEs, a multigene screening approach is essential. Currently, 172 genes have been identified as responsible for DEEs [3]. Despite the routine use of panels and exome sequencing, the diagnostic efficiency for DEE remains limited and ranges from 18–48% [5]. WGS has the potential to enhance diagnostic outcomes by providing uniform coverage across the genome, detecting variants in noncoding regions and the mitochondrial genome, identifying repeat expansion variants, and offering more reliable detection of structural variants, especially those with breakpoints in repetitive regions and copy-neutral variants such as inversions and translocations [6]. In this study, we describe a female patient with early-onset epileptic encephalopathy. WGS identified a pathogenic variant in the HNRNPU gene. Additionally, a previously reported pathogenic variant in the KANSL1 gene, associated with Koolen-de Vries syndrome [7], was found to be inherited from the healthy mother. Through RNA analysis, we were able to establish the mechanism of incomplete penetrance of the disease and to explain the absence of a pathological phenotype in the proband and the proband's mother. Materials and methods Subject The proband, a two-year-old girl, and her parents underwent a detailed clinical examination at various medical institutions in Moscow (National Medical Research Center for Neurosurgery named after Academician N.N. Burdenko). DNA analysis and genetic counseling were performed at the Research Center for Medical Genetics, Russia. All research participants gave informed consent (or responsible consent form for the infant proband) for the clinical examination and publication of their anonymized data. The study was performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Research Center for Medical Genetics, Russia. Whole genome sequencing The whole genome sequencing of the proband's DNA sample, obtained from peripheral blood, was conducted in-house. DNA extraction from whole blood was carried out using the Quick-DNA Miniprep Kit (Zymo Research, California, USA), following the manufacturer's protocol. To assess DNA purity, absorbance measurements were taken at both 260/280 nm and 230/260 nm using a DS-11 FX + spectrophotometer/fluorometer (DeNovix). Library preparation (PCR-Free) was performed using MGI platforms following their respective protocols.. Subsequently, paired-end sequencing (2x150) was executed on the DNBSEQ-T7 sequencer from MGI with an average coverage of 30x. The data processing was carried out using the “NGSData-Genome” program (Beskorovainy N.S. Program “NGSData”//Certificate of State Registration of Computer Programs No. 2021662119.2021.). The reads were aligned to the reference genome hg19 using bwa v.0.7.17-r1188. Variant calling was performed with strelka2 v.2.9.10 and gatk v.4 algorithms. Copy number variations were assessed using cnvkit 0.9.9, while structural variants were detected with Manta v.1.6.0. Additionally, tandem repeats were analyzed using ExpansionHunterDenovo. Variant annotation was performed using SnpEff v5.0, annovar v.2017, and vep v.104.3. Splice predictors used included dbNSFP v.4, SPiP v.2.1, mmsplice v.2.3, spliceai v.1.3.1, and spidex v.1. For annotation of identified variants, automatic pathogenicity prediction algorithms (SIFT, PolyPhen2-HDIV, PolyPhen2-HVAR, MutationTaster, LRT) were used, as well as evolutionary conservation position calculation algorithms (PhyloP, PhastCons). To assess the population frequencies of the identified variants, data from the “1000 Genomes” project, ESP6500, Exome Aggregation Consortium, and gnomAD were used. Sanger sequencing Validation of the variants identified through WGS was performed using Sanger sequencing. DNA samples from the proband's and his parents' peripheral blood were used as templates. The PCR was conducted using the ProFlex PCR System (Applied Biosystems). Visualization of the obtained PCR products was carried out using agarose gel electrophoresis. The PCR products were sequenced by Sanger sequencing in-house (Evrogen). For the validation and segregation of the NM_015443.4( KANSL1 ):c.727C > T variant, the following primers were used 5’-GGAGACAGCCAGTTTTGGAG-3’ and 5’-GGTCTCACTGGCAGCTTTTC-3’. For the validation and segregation of the NM_031844.3( HNRNPU ):c.1665_1666del variant, the primers 5’-ATGGGAGGAAAGGCAAGATT-3’ and 5’-ATTTTGAGGACCGCATGTTC-3’ were used. For the validation and segregation of the NM_133433.4(NIPBL):c.8101A > G variant, the primers 5’-AAGAGTAAAGACACGGGTACAAA-3’ and 5’-TGAGAATCTGTTTCTGCTGCAC-3’ were used. RNA analysis of a variant in KANSL1 Analysis of mRNA structure of the KANSL1 locus, containing the c.727C > T variant, was performed using RNA extracted from peripheral blood mononuclear cells (PBMC). PBMCs were extracted from the proband's and mother's peripheral blood by centrifugation in a Ficoll gradient. RNA was extracted using the Extract RNA reagent (Evrogen, Russia). Reverse transcription was carried out using the Reverse Transcription System (Dialat, Russia) according to the manufacturer's recommendations. The quality of the obtained cDNA was assessed by performing qPCR of housekeeping genes. To determine the presence of the c.727C > T variant in the KANSL1 mRNA, the target locus was amplified using the following priners: 5’-AGCACCTGTAAATGGGTTGG-3’ and 5’-AAGGGACTGTGTGGAGGATG-3’ with further Sanger sequencing Results Clinical findings The proband was first consulted by a geneticist at the Research Centre For Medical Genetics at the age of 1 year and 4 months due to seizures. The patient's medical history revealed that she was born from the first pregnancy, which included the use of Duphaston during the first trimester and maternal toxicosis. At 20 weeks gestation, fetal ultrasound indicated ventriculomegaly. Spontaneous natural delivery occurred at 40–41 weeks of gestation. At birth, her weight was 2960 grams, length 50 cm, and her Apgar scores were 8–9. Early development was delayed. Seizures debuted at 12 mounts and were characterized by brief periods of eye rolling lasting several seconds, with up to 14 episodes a day. Valproic acid therapy was unsuccessful in reducing seizure frequency. Video electroencephalography (EEG) monitoring showed epileptiform activity, and brain MRI confirmed ventriculomegaly. The girl was also seen by a neurologist, who diagnosed her with delayed psychomotor and speech development, non-progressive internal hydrocephalus and generalized idiopathic epilepsy, as well as hypotonic-atactic syndrome. Physical examination by a geneticist noted plagiocephaly along with several mild dysmorphic features including, hypertelorism, long eyelashes, epicanthus, hypoplasia of the eyebrow ridges, a "wine" stain on the forehead, short neck, and an incomplete transverse palmar crease. Molecular findings At the age of 1.5 years, due to the onset of seizures, developmental delay, and dysmorphisms, a genetic cause of the disease was suspected, and whole-genome sequencing was prescribed for the proband. WGS revealed three candidate variants: HNRNPU, NIPBL , and KANSL1 (Fig. 1 A, 1 B) (Table 1 ). Figure 1 . A. Pedigree of the reported family with genotypes. B. Validation of variants identified in the proband through WGS using Sanger sequencing. C. Presence of the c.717C > T variant in the KANSL1 gene in DNA samples from blood and buccal epithelial tissues of available family members. Table 1 Variants table. Gene Chromosome HGVS DNA reference HGVS protein reference Variant type Predicted effect Genotype HNRNPU 1 NM_031844.2:c.1665_1666del L556Afs*12 del Loss-of-function wt/del KANSL1 17 NM_015443.3: c.727C > T Q243* noncense Loss-of-function G/A NIPBL 5 NM_133433.3: c.8101A > G M2701V missense ? A/G N/A – not available A novel heterozygous missense variant in exon 47 of the NIPBL gene, NM_133433.4:c.8101A > G, resulting in an amino acid substitution at position 2701 of the protein (p.(Met2701Val)), was identified (Fig. 1 B). Heterozygous pathogenic variants, including missense, in the NIPBL gene are known to cause Cornelia de Lange syndrome type 1 (OMIM: 122470). This syndrome is characterized by intellectual disability, distinctive facial features, prenatal and postnatal growth retardation, and hirsutism. Congenital anomalies include malformations of the upper limbs, gastrointestinal malformations/rotations, pyloric stenosis, diaphragmatic hernia, heart defects, and genitourinary malformations (Boyle et al. , 2015). The neurological symptoms and facial dysmorphism of Cornelia de Lange syndrome partially matched the proband's phenotype (Table 2 ). Segregation analysis indicated that the variant was inherited from the proband’s heatlhy father. The second heterozygous variant in exon 9 of the HNRNPU gene, NM_031844.3:c.1665_1666del, was also identified, resulting in a frameshift and formation of a premature termination codon (p.(Leu556Alafs*12)) (Fig. 1 B). This substitution wasn’t described before. Heterozygous loss-of-function (LoF) variants in the HNRNPU gene have been reported in patients with developmental and epileptic encephalopathy type 54 (OMIM: 617391), often accompanied by variable craniofacial and skeletal anomalies [8] (Table 2 ). Segregation analysis confirmed the de novo status of the variant. Finally, WGS revealed a nonsense variant previously described as pathogenic in exon 2 of the KANSL1 gene, NM_015443.4:c.727C > T (p.(Gln243Ter)) (clinvar accession number SCV004703389) (Fig. 1 B). Loss-of-function variants in the KANSL1 gene are associated with Koolen-de Vries syndrome (OMIM: 610443), characterized by congenital malformations, developmental delay/intellectual disability, neonatal/childhood hypotonia, epilepsy, dysmorphisms, and behavioral features. Psychomotor developmental delay is noted in all individuals from an early age [7, 8]. It is important to note that the variant was detected in 18 out of 53 reads (mutant allele fraction of 34%). Segregation analysis showed that this variant was inherited from the healthy mother. The clinical presentation of Koolen-de Vries syndrome only partially matched the proband's clinical picture (Table 2 ), which may be a consequence of the incomplete penetrance of this syndrome. Table 2 Frequency of select features in patients afflicted by HNRNPU-related neurodevelopmental syndrome, Cornelia de Lange syndrome, Koolen-de Vries syndrome. The presence of these features in the proband. Symptom HNRNPU-related neurodevelopmental syndrome* Cornelia de Lange syndrome** Koolen-de Vries syndrome*** Proband’s symptoms Developmental delay 100% > 95% > 75% + Dysmorphic craniofacial features 97% 98% 75% + Seizure disorder 95% 25% 25%-49% + Intellectual disability 84% > 95% 75% + Speech delay 80% > 95% 75% + Hypotonia 79% 25%-49% 50%-75% + Feeding difficulties 57% > 95% 50%-75% + Behavioral issues 50% 25%-49% 50%-75% + Cardiac abnormalities 30% 30% 50%-75% + * [8] ** [9] *** [10] Study of the pathogenicity of the KANSL1 variant Since a pathogenic causative variant in the HNRNPU gene was identified in the patient, the presence of another variant described as pathogenic in the KANSL1 gene in both the patient and his healthy mother complicated the diagnosis and counseling of the patient. Since incomplete penetrance, as the most obvious cause, is not described for Koolen-de Vries syndrome, we began by investigating the likelihood of mosaicism for the variant. De novo mosaic variant has been previously described in the patient with atypical phenotype presentation [7, 8, 11]. The ratio of peaks of the heterozygous variant c.727C > T in the proband (wt/mut = 2/1) suggested the possibility of mosaicism. To test this hypothesis, we searched for the variant in genomic DNA samples extracted from the peripheral blood and buccal epithelial cells of the proband and the proband's mother. The presence of the variant with the same ratio of peaks was confirmed in all DNA samples tested, which ruled out mosaicism. Additionally, the variant was sought in all available family members (Fig. 1 C). Analysis revealed that the proband's mother and maternal grandmother is also a heterozygous carrier of the variant. In DNA samples from all family members carrying the variant, an unusual ratio of the heterozygous variant peaks c.727C > T (wt/mut = 2/1) was observed. Thus, the mosaic state of the c.727C > T variant was not confirmed The allelic imbalance observed in variants detected during WGS and Sanger sequencing in the proband prompted us to consider the presence of a duplication spanning the KANSL1 locus. Subsequent copy number variation (CNV) reanalysis of the WGS data confirmed the duplication of this region, delineated by approximate boundaries of chr17:44,165,260_44,651,550dup (156 kb). According to GnomAD, such a duplication is common and has a population frequency of approximately 20%. This common duplication encompasses exons 1 and 2 of the KANSL1 gene, including the region where the c.727C > T variant was identified. We hypothesized that the c.727C > T variant listed in the patient's WGS report is located not in the KANSL1 gene, but in its duplicated locus. This duplicated region of the gene is highly likely to be non-functional. To determine the expression of the duplicated KANSL1 locus, we designed a set of primers to amplify the mRNA locus of the KANSL1 gene spanning exons 1 to 3. Figure 2 . Diagram showing the layout of the KANSL1 gene, with the duplication region marked in blue. Primer locations for RNA analysis are indicated as F2-R3 and F2-R7. Sequencing chromatograms of the RT-PCR products are also provided. The boundaries of the locus were selected to ensure amplification occurs only from the main gene and not from the gene region included in the tandem duplication (Fig. 2 ). Sequencing of the RT-PCR product revealed that this mRNA locus is transcribed and contains the heterozygous c.727C > T variant. A subsequent review of the duplication boundaries in the patient’s genome data indicated that the patient's duplication differs from the common duplication registered in gnomAD, as it includes exon 3 and part of intron 3. We developed a new PCR system to amplify the KANSL1 mRNA from exon 2 to exon 7. The analysis did not show the presence of the c.727C > T variant in the mRNA. Due to the complexity of the region, we were unable to determine the exact boundaries of the duplication on the DNA level. Based on coverage and allelic imbalance, we estimate its approximate boundaries to be chr17:44165365–44375856 with a size of 210 kb. Thus, we demonstrated that the variant c.727C > T in the KANSL1 gene resides within a duplication of this gene and is non-functional, enabling us to classify it as benign. The variant was submitted in ClinVar (accession number SCV005049516). Discussion Among hereditary diseases, a significant portion is represented by conditions with broad clinical presentations and variable expressivity. Clinical heterogeneity makes it impossible to diagnose these patients based solely on clinical symptoms. In such cases, an accurate diagnosis can only be made based on the identification of the causative genetic variant. Finding the molecular cause is often challenging, as many pathologies have similar clinical manifestations but result from disruptions in the expression of different genes. Whole genome sequencing is the method of choice for these patients. For a number of genetically heterogeneous conditions, WGS has dramatically improved the diagnostic yield [12]. However, WGS, like any diagnostic method, has its nuances and limitations [13]. In particular, the interpretation of WGS data often reveals multiple candidate variants, raising the question of how to prioritize them. Segregation analysis is commonly used for this purpose. This quick and straightforward approach determines whether a variant is inherited from one of the parents or arose de novo. However, segregation analysis is not an absolutely universal method, as many inheritance features, such as incomplete penetrance or mosaic inheritance, can complicate the interpretation. In our study, segregation analysis did not allow us to exclude the LoF variant in the KANSL1 gene from the list of candidate variants. This variant, in addition to the proband, was found in healthy family members across two generations. This circumstance and the unusual peak ratio of the heterozygous variant led to the hypothesis of mosaicism of the variant. One individual with a de novo mosaic nonsense KANSL1 variant (p.(Gln243ter)) was described recently [11, 13]. In our case the hypothesis of mosaicism was not confirmed, as the variant was found in all DNA samples taken from different tissues of the proband. Furthermore, segregation analysis cannot be relied upon if candidate genes exhibit incomplete penetrance. Although incomplete penetrance has not been described for KANSL1 , in the family we studied, two healthy members are carriers of a heterozygous nonsense variant. Loss-of-function variants in KANSL1 are a known mechanism for Koolen-de Vries syndrome [14]. Additionally, according to the population database GnomAD, KANSL1 has a pLI score of 1. These factors combined prevent the exclusion of the c.727C > T variant from the list of candidate genes and necessitate further analysis of the variant. Structural rearrangements present a particular challenge in the interpretation and further validation of WGS data [15],[16]. In addition to the obvious difficulties associated with detecting and annotating rearrangements, the presence of frequent population rearrangements significantly contributes to the search for the causative variant. Frequent duplications may contain transcriptionally inactive copies of genes or gene segments. Variants can accumulate in such loci and be annotated by bioinformatics algorithms as rare and pathogenic. One method for investigating the pathogenicity of such variants is RNA analysis. In our study, RNA analysis revealed that the variant in the KANSL1 gene is located within a duplicated region, which is non-functional and therefore cannot contribute to the development of a pathological phenotype. Using a comprehensive approach that included segregation and RNA analysis, we demonstrated that variants in the NIPBL (ClinVar accession number VCV002920673) and KANSL1 genes are not pathogenic and excluded them from the list of candidate genes. Given the clinical presentation of the proband and the de novo status of the variant in HNRNPU (ClinVar accession number VCV001343373), it was determined to be pathogenic and causative. Conclusions Whole genome sequencing is a powerful modern diagnostic tool that has greatly advanced the diagnosis of numerous genetic disorders. However, it does not always provide an exact diagnosis for patients, emphasizing the need for a comprehensive approach to accurately identify the molecular causes of complex genetic conditions. In our work, we used a combination of whole-genome sequencing methods, segregation analysis, and RNA analysis to diagnose the patient and describe the molecular mechanism of incomplete penetrance for the nonsense variant in the KANSL1 gene. Abbreviations DEE developmental and epileptic encephalopathy WGS whole genome sequencing PBMC peripheral blood mononuclear cells EEG electroencephalography CNV copy number variation LoF loss-of-function Declarations Ethics approval and consent to participate - This study was approved by the Institutional Review Board of the Research Centre for Medical Genetics, Russia and was performed in accordance with the Declaration of Helsinki. For this work written informed consent was obtained from all participants. For participants under 16 years old written informed consent was obtained from the participant’s parent. Consent for publication - All research participants gave written informed consent (or responsible consent form for infant proband) to the clinical examination and the publication of their anonymized data. Competing interests - The authors declare that they have no competing interests. Funding - The research was carried out within the state assignment of the Ministry of Science and Higher Education of the Russian Federation for RCMG. Author Contribution D.A. performed the segregation studies, functional analysis and wrote the manuscript. D.G. -wrote and revised the manuscript and performed the clinical examination of the patient. M.N. – analysed the NGS data. M.S. – wrote and revised the manuscript. All authors read and approved the final manuscript. Acknowledgement We thank Peter Sparber for his invaluable contribution for the quality of the manuscript Availability of data and materials - Data that support the findings of this study are available from the corresponding authors upon reasonable request References Stafstrom CE, Carmant L. Seizures and epilepsy: An overview for neuroscientists. Cold Spring Harb Perspect Biol. 2015;7. Myers CT, Mefford HC. Advancing epilepsy genetics in the genomic era. Genome Medicine. 2015;7. Guerrini R, Conti V, Mantegazza M, Balestrini S, Galanopoulou AS, Benfenati F. Developmental and epileptic encephalopathies: from genetic heterogeneity to phenotypic continuum. Physiological Reviews. 2023;103. Scheffer IE, Berkovic S, Capovilla G, Connolly MB, French J, Guilhoto L, et al. ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology. Epilepsia. 2017;58. Møller RS, Hammer TB, Rubboli G, Lemke JR, Johannesen KM. From next-generation sequencing to targeted treatment of non-acquired epilepsies. Expert Review of Molecular Diagnostics. 2019;19. Palmer EE, Sachdev R, Macintosh R, Melo US, Mundlos S, Righetti S, et al. Diagnostic Yield of Whole Genome Sequencing After Nondiagnostic Exome Sequencing or Gene Panel in Developmental and Epileptic Encephalopathies. Neurology. 2021;96. Koolen DA, Sharp AJ, Hurst JA, Firth H V., Knight SJL, Goldenberg A, et al. Clinical and molecular delineation of the 17q21.31 microdeletion syndrome. J Med Genet. 2008;45. Durkin A, Albaba S, Fry AE, Morton JE, Douglas A, Beleza A, et al. Clinical findings of 21 previously unreported probands with HNRNPU-related syndrome and comprehensive literature review. Am J Med Genet A. 2020;182. Cornelia de Lange Syndrome - GeneReviews® - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK1104/. Accessed 28 Jun 2024. Koolen-de Vries Syndrome - GeneReviews® - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK24676/. Accessed 28 Jun 2024. Awamleh Z, Choufani S, Wu W, Rots D, Dingemans AJM, Nadif Kasri N, et al. Correction: A new blood DNA methylation signature for Koolen-de Vries syndrome: Classification of missense KANSL1 variants and comparison to fibroblast cells. European Journal of Human Genetics. 2024;32. Bagger FO, Borgwardt L, Jespersen AS, Hansen AR, Bertelsen B, Kodama M, et al. Whole genome sequencing in clinical practice. BMC Medical Genomics. 2024;17. Rolando JC, Melkonian A V., Walt DR. The Present and Future Landscapes of Molecular Diagnostics. Annual Review of Analytical Chemistry. 2024;17. Koolen DA, Pfundt R, Linda K, Beunders G, Veenstra-Knol HE, Conta EH, et al. The Koolen-de Vries syndrome: A phenotypic comparison of patients with a 17q21.31 microdeletion versus a KANSL1 sequence variant. European Journal of Human Genetics. 2016;24. Hehir-Kwa JY, Tops BBJ, Kemmeren P. The clinical implementation of copy number detection in the age of next-generation sequencing. Expert Review of Molecular Diagnostics. 2018;18. Scarano C, Veneruso I, De Simone RR, Di Bonito G, Secondino A, D’Argenio V. The Third-Generation Sequencing Challenge: Novel Insights for the Omic Sciences. Biomolecules 2024, Vol 14, Page 568. 2024;14:568. Additional Declarations No competing interests reported. Supplementary Files CAREchecklistKANSL1.docx RNAanalysisKANSL1.jpg SegregationKANSL1.jpg Segregation.jpg 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-4710959","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":326882886,"identity":"67c7cdfd-e81b-47a2-9af8-a4ba630d903a","order_by":0,"name":"Daria Akimova","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYDACHgYDhg8HGOQYGBgbDxCthXHGAQZjoJYG4rUwcxxgSGwAsonTottzeONnhjN26WvbDzccYNxzmLAWs7NtxdIFN5Jzt51JBDrsGTFazvMYSM/4wJy77QBIywHitBj/5vlQn252/iGxWs72mEnz3DicYHaDaFvOHCuznHHmuOG2G0BbEg6kE6MlefOND8eq5c3Opz988OGANWEtqCCBVA2jYBSMglEwCrADAJIsSNOLVl8PAAAAAElFTkSuQmCC","orcid":"","institution":"Research Centre for Medical Genetics","correspondingAuthor":true,"prefix":"","firstName":"Daria","middleName":"","lastName":"Akimova","suffix":""},{"id":326882889,"identity":"df458e67-c922-4cb8-bc23-bc2fda661e46","order_by":1,"name":"Daria Guseva","email":"","orcid":"","institution":"Research Centre for Medical Genetics","correspondingAuthor":false,"prefix":"","firstName":"Daria","middleName":"","lastName":"Guseva","suffix":""},{"id":326882891,"identity":"ade9895e-18da-489b-8383-c071d4d1e76d","order_by":2,"name":"Maria Nefedova","email":"","orcid":"","institution":"Independent Clinical Bioinformatics Laboratory, Moscow, Russia","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Nefedova","suffix":""},{"id":326882893,"identity":"765ab88c-2c33-4469-ab76-1335d65cc39d","order_by":3,"name":"Mikhail Skoblov","email":"","orcid":"","institution":"Research Centre for Medical Genetics","correspondingAuthor":false,"prefix":"","firstName":"Mikhail","middleName":"","lastName":"Skoblov","suffix":""}],"badges":[],"createdAt":"2024-07-09 09:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4710959/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4710959/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":62218502,"identity":"c4893948-d1eb-4793-80fd-4cff6da38f0a","added_by":"auto","created_at":"2024-08-11 12:03:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":536732,"visible":true,"origin":"","legend":"\u003cp\u003eA. Pedigree of the reported family with genotypes. B. Validation of variants identified in the proband through WGS using Sanger sequencing. C. Presence of the c.717C\u0026gt;T variant in the KANSL1 gene in DNA samples from blood and buccal epithelial tissues of available family members.\u003c/p\u003e","description":"","filename":"Onlinefigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4710959/v1/4ffcd28b29e6f9f1cf79bb05.png"},{"id":62216770,"identity":"bd9d37b7-aad3-4101-858a-31468b1438a6","added_by":"auto","created_at":"2024-08-11 11:47:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":378345,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram showing the layout of the KANSL1 gene, with the duplication region marked in blue.\u003c/p\u003e","description":"","filename":"Onlinefigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4710959/v1/ff63013e708cc8a4c80e6268.png"},{"id":65085056,"identity":"27bd00eb-5be0-4f19-a003-6fe18594adf8","added_by":"auto","created_at":"2024-09-23 12:41:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1759863,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4710959/v1/4e00f73a-ec24-4d34-a16f-2eed8d01ab3b.pdf"},{"id":62216767,"identity":"70f1c806-6994-43e9-9f0a-51360ac8daa4","added_by":"auto","created_at":"2024-08-11 11:47:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":28351,"visible":true,"origin":"","legend":"","description":"","filename":"CAREchecklistKANSL1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4710959/v1/2eebdbc832e1759bdee16fd2.docx"},{"id":62217596,"identity":"ee1b8207-4c13-4a82-bfbf-617feeeb22d4","added_by":"auto","created_at":"2024-08-11 11:55:06","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":53964,"visible":true,"origin":"","legend":"","description":"","filename":"RNAanalysisKANSL1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4710959/v1/e28df6b575baeb4c44f54028.jpg"},{"id":62216771,"identity":"a990ec14-f15a-490a-b180-b69e2bdefee4","added_by":"auto","created_at":"2024-08-11 11:47:06","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":57642,"visible":true,"origin":"","legend":"","description":"","filename":"SegregationKANSL1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4710959/v1/6eaff1af1698956b27dcd629.jpg"},{"id":62216772,"identity":"b5c1a842-fec4-4b40-b43e-3a63e74fa71e","added_by":"auto","created_at":"2024-08-11 11:47:07","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":48413,"visible":true,"origin":"","legend":"","description":"","filename":"Segregation.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4710959/v1/ccff593195537dfb8dcb1eb2.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Incomplete penetrance of a KANSL1 loss of function variant resolved by RNA analysis, a case report","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEpilepsy is among the most prevalent neurological disorders, with an incidence rate of approximately 50 new cases per 100,000 people annually [1]. Genetic factors account for about 70\u0026ndash;80% of epilepsy cases [2], and around 1% of the global population is affected by this condition. Approximately 75% of epilepsy cases manifest during childhood, highlighting the increased vulnerability of the developing brain to seizures [1]. Epilepsy encompasses various types and syndromes, classified based on clinical and EEG features, etiologies, and comorbidities. Developmental and epileptic encephalopathies (DEEs) represent the most severe spectrum of epilepsy disorders with a significant monogenic component [3], [4].\u003c/p\u003e \u003cp\u003eDue to the phenotypic and genetic heterogeneity of DEEs, a multigene screening approach is essential. Currently, 172 genes have been identified as responsible for DEEs [3]. Despite the routine use of panels and exome sequencing, the diagnostic efficiency for DEE remains limited and ranges from 18\u0026ndash;48% [5]. WGS has the potential to enhance diagnostic outcomes by providing uniform coverage across the genome, detecting variants in noncoding regions and the mitochondrial genome, identifying repeat expansion variants, and offering more reliable detection of structural variants, especially those with breakpoints in repetitive regions and copy-neutral variants such as inversions and translocations [6].\u003c/p\u003e \u003cp\u003eIn this study, we describe a female patient with early-onset epileptic encephalopathy. WGS identified a pathogenic variant in the \u003cem\u003eHNRNPU\u003c/em\u003e gene. Additionally, a previously reported pathogenic variant in the \u003cem\u003eKANSL1\u003c/em\u003e gene, associated with Koolen-de Vries syndrome [7], was found to be inherited from the healthy mother. Through RNA analysis, we were able to establish the mechanism of incomplete penetrance of the disease and to explain the absence of a pathological phenotype in the proband and the proband's mother.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubject\u003c/h2\u003e \u003cp\u003eThe proband, a two-year-old girl, and her parents underwent a detailed clinical examination at various medical institutions in Moscow (National Medical Research Center for Neurosurgery named after Academician N.N. Burdenko). DNA analysis and genetic counseling were performed at the Research Center for Medical Genetics, Russia. All research participants gave informed consent (or responsible consent form for the infant proband) for the clinical examination and publication of their anonymized data. The study was performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Research Center for Medical Genetics, Russia.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequencing\u003c/h2\u003e \u003cp\u003eThe whole genome sequencing of the proband's DNA sample, obtained from peripheral blood, was conducted in-house. DNA extraction from whole blood was carried out using the Quick-DNA Miniprep Kit (Zymo Research, California, USA), following the manufacturer's protocol. To assess DNA purity, absorbance measurements were taken at both 260/280 nm and 230/260 nm using a DS-11 FX\u0026thinsp;+\u0026thinsp;spectrophotometer/fluorometer (DeNovix).\u003c/p\u003e \u003cp\u003eLibrary preparation (PCR-Free) was performed using MGI platforms following their respective protocols.. Subsequently, paired-end sequencing (2x150) was executed on the DNBSEQ-T7 sequencer from MGI with an average coverage of 30x. The data processing was carried out using the \u0026ldquo;NGSData-Genome\u0026rdquo; program (Beskorovainy N.S. Program \u0026ldquo;NGSData\u0026rdquo;//Certificate of State Registration of Computer Programs No. 2021662119.2021.). The reads were aligned to the reference genome hg19 using bwa v.0.7.17-r1188. Variant calling was performed with strelka2 v.2.9.10 and gatk v.4 algorithms. Copy number variations were assessed using cnvkit 0.9.9, while structural variants were detected with Manta v.1.6.0. Additionally, tandem repeats were analyzed using ExpansionHunterDenovo. Variant annotation was performed using SnpEff v5.0, annovar v.2017, and vep v.104.3. Splice predictors used included dbNSFP v.4, SPiP v.2.1, mmsplice v.2.3, spliceai v.1.3.1, and spidex v.1.\u003c/p\u003e \u003cp\u003eFor annotation of identified variants, automatic pathogenicity prediction algorithms (SIFT, PolyPhen2-HDIV, PolyPhen2-HVAR, MutationTaster, LRT) were used, as well as evolutionary conservation position calculation algorithms (PhyloP, PhastCons). To assess the population frequencies of the identified variants, data from the \u0026ldquo;1000 Genomes\u0026rdquo; project, ESP6500, Exome Aggregation Consortium, and gnomAD were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSanger sequencing\u003c/h2\u003e \u003cp\u003eValidation of the variants identified through WGS was performed using Sanger sequencing. DNA samples from the proband's and his parents' peripheral blood were used as templates. The PCR was conducted using the ProFlex PCR System (Applied Biosystems). Visualization of the obtained PCR products was carried out using agarose gel electrophoresis. The PCR products were sequenced by Sanger sequencing in-house (Evrogen). For the validation and segregation of the NM_015443.4(\u003cem\u003eKANSL1\u003c/em\u003e):c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant, the following primers were used 5\u0026rsquo;-GGAGACAGCCAGTTTTGGAG-3\u0026rsquo; and 5\u0026rsquo;-GGTCTCACTGGCAGCTTTTC-3\u0026rsquo;. For the validation and segregation of the NM_031844.3(\u003cem\u003eHNRNPU\u003c/em\u003e):c.1665_1666del variant, the primers 5\u0026rsquo;-ATGGGAGGAAAGGCAAGATT-3\u0026rsquo; and 5\u0026rsquo;-ATTTTGAGGACCGCATGTTC-3\u0026rsquo; were used. For the validation and segregation of the NM_133433.4(NIPBL):c.8101A\u0026thinsp;\u0026gt;\u0026thinsp;G variant, the primers 5\u0026rsquo;-AAGAGTAAAGACACGGGTACAAA-3\u0026rsquo; and 5\u0026rsquo;-TGAGAATCTGTTTCTGCTGCAC-3\u0026rsquo; were used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA analysis of a variant in\u003c/b\u003e \u003cb\u003eKANSL1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of mRNA structure of the \u003cem\u003eKANSL1\u003c/em\u003e locus, containing the c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant, was performed using RNA extracted from peripheral blood mononuclear cells (PBMC). PBMCs were extracted from the proband's and mother's peripheral blood by centrifugation in a Ficoll gradient. RNA was extracted using the Extract RNA reagent (Evrogen, Russia). Reverse transcription was carried out using the Reverse Transcription System (Dialat, Russia) according to the manufacturer's recommendations. The quality of the obtained cDNA was assessed by performing qPCR of housekeeping genes. To determine the presence of the c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the \u003cem\u003eKANSL1\u003c/em\u003e mRNA, the target locus was amplified using the following priners: 5\u0026rsquo;-AGCACCTGTAAATGGGTTGG-3\u0026rsquo; and 5\u0026rsquo;-AAGGGACTGTGTGGAGGATG-3\u0026rsquo; with further Sanger sequencing\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eClinical findings\u003c/h2\u003e \u003cp\u003eThe proband was first consulted by a geneticist at the Research Centre For Medical Genetics at the age of 1 year and 4 months due to seizures. The patient's medical history revealed that she was born from the first pregnancy, which included the use of Duphaston during the first trimester and maternal toxicosis. At 20 weeks gestation, fetal ultrasound indicated ventriculomegaly. Spontaneous natural delivery occurred at 40\u0026ndash;41 weeks of gestation. At birth, her weight was 2960 grams, length 50 cm, and her Apgar scores were 8\u0026ndash;9. Early development was delayed. Seizures debuted at 12 mounts and were characterized by brief periods of eye rolling lasting several seconds, with up to 14 episodes a day. Valproic acid therapy was unsuccessful in reducing seizure frequency. Video electroencephalography (EEG) monitoring showed epileptiform activity, and brain MRI confirmed ventriculomegaly.\u003c/p\u003e \u003cp\u003eThe girl was also seen by a neurologist, who diagnosed her with delayed psychomotor and speech development, non-progressive internal hydrocephalus and generalized idiopathic epilepsy, as well as hypotonic-atactic syndrome. Physical examination by a geneticist noted plagiocephaly along with several mild dysmorphic features including, hypertelorism, long eyelashes, epicanthus, hypoplasia of the eyebrow ridges, a \"wine\" stain on the forehead, short neck, and an incomplete transverse palmar crease.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMolecular findings\u003c/h2\u003e \u003cp\u003eAt the age of 1.5 years, due to the onset of seizures, developmental delay, and dysmorphisms, a genetic cause of the disease was suspected, and whole-genome sequencing was prescribed for the proband.\u003c/p\u003e \u003cp\u003eWGS revealed three candidate variants: \u003cem\u003eHNRNPU, NIPBL\u003c/em\u003e, and \u003cem\u003eKANSL1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. \u003cem\u003eA. Pedigree of the reported family with genotypes. B. Validation of variants identified in the proband through WGS using Sanger sequencing. C. Presence of the c.717C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the KANSL1 gene in DNA samples from blood and buccal epithelial tissues of available family members.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVariants table.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHGVS DNA reference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHGVS protein reference\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eVariant type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePredicted effect\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGenotype\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHNRNPU\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_031844.2:c.1665_1666del\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL556Afs*12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003edel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLoss-of-function\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003ewt/del\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eKANSL1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_015443.3: c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQ243*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enoncense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eLoss-of-function\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eG/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNIPBL\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNM_133433.3: c.8101A\u0026thinsp;\u0026gt;\u0026thinsp;G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eM2701V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003emissense\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e?\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eA/G\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eN/A \u0026ndash; not available\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eA novel heterozygous missense variant in exon 47 of the NIPBL gene, NM_133433.4:c.8101A\u0026thinsp;\u0026gt;\u0026thinsp;G, resulting in an amino acid substitution at position 2701 of the protein (p.(Met2701Val)), was identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Heterozygous pathogenic variants, including missense, in the NIPBL gene are known to cause Cornelia de Lange syndrome type 1 (OMIM: 122470). This syndrome is characterized by intellectual disability, distinctive facial features, prenatal and postnatal growth retardation, and hirsutism. Congenital anomalies include malformations of the upper limbs, gastrointestinal malformations/rotations, pyloric stenosis, diaphragmatic hernia, heart defects, and genitourinary malformations (Boyle \u003cem\u003eet al.\u003c/em\u003e, 2015). The neurological symptoms and facial dysmorphism of Cornelia de Lange syndrome partially matched the proband's phenotype (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Segregation analysis indicated that the variant was inherited from the proband\u0026rsquo;s heatlhy father.\u003c/p\u003e \u003cp\u003eThe second heterozygous variant in exon 9 of the \u003cem\u003eHNRNPU\u003c/em\u003e gene, NM_031844.3:c.1665_1666del, was also identified, resulting in a frameshift and formation of a premature termination codon (p.(Leu556Alafs*12)) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This substitution wasn\u0026rsquo;t described before. Heterozygous loss-of-function (LoF) variants in the \u003cem\u003eHNRNPU\u003c/em\u003e gene have been reported in patients with developmental and epileptic encephalopathy type 54 (OMIM: 617391), often accompanied by variable craniofacial and skeletal anomalies [8] (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Segregation analysis confirmed the de novo status of the variant.\u003c/p\u003e \u003cp\u003eFinally, WGS revealed a nonsense variant previously described as pathogenic in exon 2 of the \u003cem\u003eKANSL1\u003c/em\u003e gene, NM_015443.4:c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.(Gln243Ter)) (clinvar accession number SCV004703389) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Loss-of-function variants in the \u003cem\u003eKANSL1\u003c/em\u003e gene are associated with Koolen-de Vries syndrome (OMIM: 610443), characterized by congenital malformations, developmental delay/intellectual disability, neonatal/childhood hypotonia, epilepsy, dysmorphisms, and behavioral features. Psychomotor developmental delay is noted in all individuals from an early age [7, 8]. It is important to note that the variant was detected in 18 out of 53 reads (mutant allele fraction of 34%). Segregation analysis showed that this variant was inherited from the healthy mother. The clinical presentation of Koolen-de Vries syndrome only partially matched the proband's clinical picture (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which may be a consequence of the incomplete penetrance of this syndrome.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequency of select features in patients afflicted by HNRNPU-related neurodevelopmental syndrome, Cornelia de Lange syndrome, Koolen-de Vries syndrome. The presence of these features in the proband.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSymptom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHNRNPU-related neurodevelopmental syndrome*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCornelia de Lange syndrome**\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKoolen-de Vries syndrome***\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProband\u0026rsquo;s symptoms\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDevelopmental delay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDysmorphic craniofacial features\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e97%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSeizure disorder\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25%-49%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntellectual disability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpeech delay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHypotonia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e79%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25%-49%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50%-75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeeding difficulties\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e57%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;95%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50%-75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBehavioral issues\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25%-49%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50%-75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCardiac abnormalities\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50%-75%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003cp\u003e* [8]\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003cp\u003e** [9]\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003cp\u003e*** [10]\u003c/p\u003e \u003cp\u003e \u003cb\u003eStudy of the pathogenicity of the\u003c/b\u003e \u003cb\u003eKANSL1\u003c/b\u003e \u003cb\u003evariant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSince a pathogenic causative variant in the \u003cem\u003eHNRNPU\u003c/em\u003e gene was identified in the patient, the presence of another variant described as pathogenic in the \u003cem\u003eKANSL1\u003c/em\u003e gene in both the patient and his healthy mother complicated the diagnosis and counseling of the patient. Since incomplete penetrance, as the most obvious cause, is not described for Koolen-de Vries syndrome, we began by investigating the likelihood of mosaicism for the variant. De novo mosaic variant has been previously described in the patient with atypical phenotype presentation [7, 8, 11]. The ratio of peaks of the heterozygous variant c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T in the proband (wt/mut\u0026thinsp;=\u0026thinsp;2/1) suggested the possibility of mosaicism. To test this hypothesis, we searched for the variant in genomic DNA samples extracted from the peripheral blood and buccal epithelial cells of the proband and the proband's mother. The presence of the variant with the same ratio of peaks was confirmed in all DNA samples tested, which ruled out mosaicism.\u003c/p\u003e \u003cp\u003eAdditionally, the variant was sought in all available family members (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Analysis revealed that the proband's mother and maternal grandmother is also a heterozygous carrier of the variant. In DNA samples from all family members carrying the variant, an unusual ratio of the heterozygous variant peaks c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T (wt/mut\u0026thinsp;=\u0026thinsp;2/1) was observed. Thus, the mosaic state of the c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant was not confirmed\u003c/p\u003e \u003cp\u003eThe allelic imbalance observed in variants detected during WGS and Sanger sequencing in the proband prompted us to consider the presence of a duplication spanning the KANSL1 locus. Subsequent copy number variation (CNV) reanalysis of the WGS data confirmed the duplication of this region, delineated by approximate boundaries of chr17:44,165,260_44,651,550dup (156 kb). According to GnomAD, such a duplication is common and has a population frequency of approximately 20%. This common duplication encompasses exons 1 and 2 of the KANSL1 gene, including the region where the c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant was identified. We hypothesized that the c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant listed in the patient's WGS report is located not in the KANSL1 gene, but in its duplicated locus. This duplicated region of the gene is highly likely to be non-functional. To determine the expression of the duplicated KANSL1 locus, we designed a set of primers to amplify the mRNA locus of the \u003cem\u003eKANSL1\u003c/em\u003e gene spanning exons 1 to 3.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cem\u003eDiagram showing the layout of the KANSL1 gene, with the duplication region marked in blue. Primer locations for RNA analysis are indicated as F2-R3 and F2-R7. Sequencing chromatograms of the RT-PCR products are also provided.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe boundaries of the locus were selected to ensure amplification occurs only from the main gene and not from the gene region included in the tandem duplication (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Sequencing of the RT-PCR product revealed that this mRNA locus is transcribed and contains the heterozygous c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant.\u003c/p\u003e \u003cp\u003eA subsequent review of the duplication boundaries in the patient\u0026rsquo;s genome data indicated that the patient's duplication differs from the common duplication registered in gnomAD, as it includes exon 3 and part of intron 3. We developed a new PCR system to amplify the \u003cem\u003eKANSL1\u003c/em\u003e mRNA from exon 2 to exon 7. The analysis did not show the presence of the c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant in the mRNA.\u003c/p\u003e \u003cp\u003eDue to the complexity of the region, we were unable to determine the exact boundaries of the duplication on the DNA level. Based on coverage and allelic imbalance, we estimate its approximate boundaries to be chr17:44165365\u0026ndash;44375856 with a size of 210 kb.\u003c/p\u003e \u003cp\u003eThus, we demonstrated that the variant c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T in the \u003cem\u003eKANSL1\u003c/em\u003e gene resides within a duplication of this gene and is non-functional, enabling us to classify it as benign. The variant was submitted in ClinVar (accession number SCV005049516).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAmong hereditary diseases, a significant portion is represented by conditions with broad clinical presentations and variable expressivity. Clinical heterogeneity makes it impossible to diagnose these patients based solely on clinical symptoms. In such cases, an accurate diagnosis can only be made based on the identification of the causative genetic variant.\u003c/p\u003e \u003cp\u003eFinding the molecular cause is often challenging, as many pathologies have similar clinical manifestations but result from disruptions in the expression of different genes. Whole genome sequencing is the method of choice for these patients. For a number of genetically heterogeneous conditions, WGS has dramatically improved the diagnostic yield [12].\u003c/p\u003e \u003cp\u003eHowever, WGS, like any diagnostic method, has its nuances and limitations [13]. In particular, the interpretation of WGS data often reveals multiple candidate variants, raising the question of how to prioritize them. Segregation analysis is commonly used for this purpose. This quick and straightforward approach determines whether a variant is inherited from one of the parents or arose de novo. However, segregation analysis is not an absolutely universal method, as many inheritance features, such as incomplete penetrance or mosaic inheritance, can complicate the interpretation.\u003c/p\u003e \u003cp\u003eIn our study, segregation analysis did not allow us to exclude the LoF variant in the \u003cem\u003eKANSL1\u003c/em\u003e gene from the list of candidate variants. This variant, in addition to the proband, was found in healthy family members across two generations. This circumstance and the unusual peak ratio of the heterozygous variant led to the hypothesis of mosaicism of the variant. One individual with a de novo mosaic nonsense \u003cem\u003eKANSL1\u003c/em\u003e variant (p.(Gln243ter)) was described recently [11, 13]. In our case the hypothesis of mosaicism was not confirmed, as the variant was found in all DNA samples taken from different tissues of the proband.\u003c/p\u003e \u003cp\u003eFurthermore, segregation analysis cannot be relied upon if candidate genes exhibit incomplete penetrance. Although incomplete penetrance has not been described for \u003cem\u003eKANSL1\u003c/em\u003e, in the family we studied, two healthy members are carriers of a heterozygous nonsense variant. Loss-of-function variants in \u003cem\u003eKANSL1\u003c/em\u003e are a known mechanism for Koolen-de Vries syndrome [14]. Additionally, according to the population database GnomAD, \u003cem\u003eKANSL1\u003c/em\u003e has a pLI score of 1. These factors combined prevent the exclusion of the c.727C\u0026thinsp;\u0026gt;\u0026thinsp;T variant from the list of candidate genes and necessitate further analysis of the variant.\u003c/p\u003e \u003cp\u003eStructural rearrangements present a particular challenge in the interpretation and further validation of WGS data [15],[16]. In addition to the obvious difficulties associated with detecting and annotating rearrangements, the presence of frequent population rearrangements significantly contributes to the search for the causative variant. Frequent duplications may contain transcriptionally inactive copies of genes or gene segments. Variants can accumulate in such loci and be annotated by bioinformatics algorithms as rare and pathogenic. One method for investigating the pathogenicity of such variants is RNA analysis. In our study, RNA analysis revealed that the variant in the \u003cem\u003eKANSL1\u003c/em\u003e gene is located within a duplicated region, which is non-functional and therefore cannot contribute to the development of a pathological phenotype.\u003c/p\u003e \u003cp\u003eUsing a comprehensive approach that included segregation and RNA analysis, we demonstrated that variants in the \u003cem\u003eNIPBL\u003c/em\u003e (ClinVar accession number VCV002920673) and \u003cem\u003eKANSL1\u003c/em\u003e genes are not pathogenic and excluded them from the list of candidate genes. Given the clinical presentation of the proband and the de novo status of the variant in \u003cem\u003eHNRNPU\u003c/em\u003e (ClinVar accession number VCV001343373), it was determined to be pathogenic and causative.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWhole genome sequencing is a powerful modern diagnostic tool that has greatly advanced the diagnosis of numerous genetic disorders. However, it does not always provide an exact diagnosis for patients, emphasizing the need for a comprehensive approach to accurately identify the molecular causes of complex genetic conditions.\u003c/p\u003e \u003cp\u003eIn our work, we used a combination of whole-genome sequencing methods, segregation analysis, and RNA analysis to diagnose the patient and describe the molecular mechanism of incomplete penetrance for the nonsense variant in the \u003cem\u003eKANSL1\u003c/em\u003e gene.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edevelopmental and epileptic encephalopathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ewhole genome sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBMC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eperipheral blood mononuclear cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEEG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eelectroencephalography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCNV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecopy number variation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLoF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eloss-of-function\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate -\u003c/strong\u003e \u003cp\u003eThis study was approved by the Institutional Review Board of the Research Centre for Medical Genetics, Russia and was performed in accordance with the Declaration of Helsinki. For this work written informed consent was obtained from all participants. For participants under 16 years old written informed consent was obtained from the participant\u0026rsquo;s parent.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003e \u003cb\u003eConsent for publication\u003c/b\u003e -\u003c/strong\u003e \u003cp\u003eAll research participants gave written informed consent (or responsible consent form for infant proband) to the clinical examination and the publication of their anonymized data.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003e \u003cb\u003eCompeting interests\u003c/b\u003e -\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding -\u003c/h2\u003e \u003cp\u003eThe research was carried out within the state assignment of the Ministry of Science and Higher Education of the Russian Federation for RCMG.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eD.A. performed the segregation studies, functional analysis and wrote the manuscript. D.G. -wrote and revised the manuscript and performed the clinical examination of the patient. M.N. \u0026ndash; analysed the NGS data. M.S. \u0026ndash; wrote and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Peter Sparber for his invaluable contribution for the quality of the manuscript\u003c/p\u003e\u003ch2\u003eAvailability of data and materials -\u003c/h2\u003e \u003cp\u003eData that support the findings of this study are available from the corresponding authors upon reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStafstrom CE, Carmant L. Seizures and epilepsy: An overview for neuroscientists. Cold Spring Harb Perspect Biol. 2015;7.\u003c/li\u003e\n\u003cli\u003eMyers CT, Mefford HC. Advancing epilepsy genetics in the genomic era. Genome Medicine. 2015;7.\u003c/li\u003e\n\u003cli\u003eGuerrini R, Conti V, Mantegazza M, Balestrini S, Galanopoulou AS, Benfenati F. Developmental and epileptic encephalopathies: from genetic heterogeneity to phenotypic continuum. Physiological Reviews. 2023;103.\u003c/li\u003e\n\u003cli\u003eScheffer IE, Berkovic S, Capovilla G, Connolly MB, French J, Guilhoto L, et al. ILAE classification of the epilepsies: Position paper of the ILAE Commission for Classification and Terminology. Epilepsia. 2017;58.\u003c/li\u003e\n\u003cli\u003eM\u0026oslash;ller RS, Hammer TB, Rubboli G, Lemke JR, Johannesen KM. From next-generation sequencing to targeted treatment of non-acquired epilepsies. Expert Review of Molecular Diagnostics. 2019;19.\u003c/li\u003e\n\u003cli\u003ePalmer EE, Sachdev R, Macintosh R, Melo US, Mundlos S, Righetti S, et al. Diagnostic Yield of Whole Genome Sequencing After Nondiagnostic Exome Sequencing or Gene Panel in Developmental and Epileptic Encephalopathies. Neurology. 2021;96.\u003c/li\u003e\n\u003cli\u003eKoolen DA, Sharp AJ, Hurst JA, Firth H V., Knight SJL, Goldenberg A, et al. Clinical and molecular delineation of the 17q21.31 microdeletion syndrome. J Med Genet. 2008;45.\u003c/li\u003e\n\u003cli\u003eDurkin A, Albaba S, Fry AE, Morton JE, Douglas A, Beleza A, et al. Clinical findings of 21 previously unreported probands with HNRNPU-related syndrome and comprehensive literature review. Am J Med Genet A. 2020;182.\u003c/li\u003e\n\u003cli\u003eCornelia de Lange Syndrome - GeneReviews\u0026reg; - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK1104/. Accessed 28 Jun 2024.\u003c/li\u003e\n\u003cli\u003eKoolen-de Vries Syndrome - GeneReviews\u0026reg; - NCBI Bookshelf. https://www.ncbi.nlm.nih.gov/books/NBK24676/. Accessed 28 Jun 2024.\u003c/li\u003e\n\u003cli\u003eAwamleh Z, Choufani S, Wu W, Rots D, Dingemans AJM, Nadif Kasri N, et al. Correction: A new blood DNA methylation signature for Koolen-de Vries syndrome: Classification of missense KANSL1 variants and comparison to fibroblast cells. European Journal of Human Genetics. 2024;32.\u003c/li\u003e\n\u003cli\u003eBagger FO, Borgwardt L, Jespersen AS, Hansen AR, Bertelsen B, Kodama M, et al. Whole genome sequencing in clinical practice. BMC Medical Genomics. 2024;17.\u003c/li\u003e\n\u003cli\u003eRolando JC, Melkonian A V., Walt DR. The Present and Future Landscapes of Molecular Diagnostics. Annual Review of Analytical Chemistry. 2024;17.\u003c/li\u003e\n\u003cli\u003eKoolen DA, Pfundt R, Linda K, Beunders G, Veenstra-Knol HE, Conta EH, et al. The Koolen-de Vries syndrome: A phenotypic comparison of patients with a 17q21.31 microdeletion versus a KANSL1 sequence variant. European Journal of Human Genetics. 2016;24.\u003c/li\u003e\n\u003cli\u003eHehir-Kwa JY, Tops BBJ, Kemmeren P. The clinical implementation of copy number detection in the age of next-generation sequencing. Expert Review of Molecular Diagnostics. 2018;18.\u003c/li\u003e\n\u003cli\u003eScarano C, Veneruso I, De Simone RR, Di Bonito G, Secondino A, D\u0026rsquo;Argenio V. The Third-Generation Sequencing Challenge: Novel Insights for the Omic Sciences. Biomolecules 2024, Vol 14, Page 568. 2024;14:568.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"KANSL1, HNRNPU, Incomplete penetrance, RNA analysis","lastPublishedDoi":"10.21203/rs.3.rs-4710959/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4710959/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eEpilepsy is a prevalent neurological disorder, affecting approximately 1% of the global population. The extensive clinical and genetic variability in epilepsy makes accurate diagnosis a significant challenge.\u003c/p\u003e\u003ch2\u003eCase presentation\u003c/h2\u003e \u003cp\u003eIn this study, we describe a girl with a clinical presentation of developmental and epileptic encephalopathy (DEE). Using whole genome sequencing (WGS), we identified several candidate variants in the \u003cem\u003eHNRNPU\u003c/em\u003e, \u003cem\u003eNIPBL\u003c/em\u003e, and \u003cem\u003eKANSL1\u003c/em\u003e genes with partial overlap with the patient clinical presentation. Subsequent analysis revealed that only the variant in the \u003cem\u003eHNRNPU\u003c/em\u003e gene arose de novo while the others were inherited from an unaffected parents. However, the previously reported pathogenic loss of function variant in the \u003cem\u003eKANSL1\u003c/em\u003e gene, inherited from a healthy mother, complicated comprehensive family counseling. A thorough investigation using RNA analysis showed that the variant in the \u003cem\u003eKANSL1\u003c/em\u003e gene is located in a duplicated locus, which is not functional, explaining the assumed incomplete penetrance.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis case illustrates the importance of integrating WGS with additional analyses to accurately diagnose and understand the molecular basis of incomplete penetrance in the \u003cem\u003eKANSL1\u003c/em\u003e gene.\u003c/p\u003e","manuscriptTitle":"Incomplete penetrance of a KANSL1 loss of function variant resolved by RNA analysis, a case report","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-11 11:47:02","doi":"10.21203/rs.3.rs-4710959/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":"4a8cbb63-06fc-48ae-8089-7484bcccbc32","owner":[],"postedDate":"August 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T12:32:56+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-11 11:47:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4710959","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4710959","identity":"rs-4710959","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}