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• W3097947115 abstract "NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development. NCKAP1/NAP1 regulates neuronal cytoskeletal dynamics and is essential for neuronal differentiation in the developing brain. Deleterious variants in NCKAP1 have been identified in individuals with autism spectrum disorder (ASD) and intellectual disability; however, its clinical significance remains unclear. To determine its significance, we assemble genotype and phenotype data for 21 affected individuals from 20 unrelated families with predicted deleterious variants in NCKAP1. This includes 16 individuals with de novo (n = 8), transmitted (n = 6), or inheritance unknown (n = 2) truncating variants, two individuals with structural variants, and three with potentially disruptive de novo missense variants. We report a de novo and ultra-rare deleterious variant burden of NCKAP1 in individuals with neurodevelopmental disorders which needs further replication. ASD or autistic features, language and motor delay, and variable expression of intellectual or learning disability are common clinical features. Among inherited cases, there is evidence of deleterious variants segregating with neuropsychiatric disorders. Based on available human brain transcriptomic data, we show that NCKAP1 is broadly and highly expressed in both prenatal and postnatal periods and demostrate enriched expression in excitatory neurons and radial glias but depleted expression in inhibitory neurons. Mouse in utero electroporation experiments reveal that Nckap1 loss of function promotes neuronal migration during early cortical development. Combined, these data support a role for disruptive NCKAP1 variants in neurodevelopmental delay/autism, possibly by interfering with neuronal migration early in cortical development. Recent large-scale genome-wide sequencing studies have implicated many high-impact autism spectrum disorder (ASD) (MIM: 209850) candidate genes.1De Rubeis S. He X. Goldberg A.P. Poultney C.S. Samocha K. Cicek A.E. Kou Y. Liu L. Fromer M. Walker S. et al.DDD StudyHomozygosity Mapping Collaborative for AutismUK10K ConsortiumSynaptic, transcriptional and chromatin genes disrupted in autism.Nature. 2014; 515: 209-215Crossref PubMed Scopus (1612) Google Scholar, 2Iossifov I. O’Roak B.J. Sanders S.J. Ronemus M. Krumm N. Levy D. Stessman H.A. Witherspoon K.T. Vives L. Patterson K.E. et al.The contribution of de novo coding mutations to autism spectrum disorder.Nature. 2014; 515: 216-221Crossref PubMed Scopus (1498) Google Scholar, 3Satterstrom F.K. Kosmicki J.A. Wang J. Breen M.S. De Rubeis S. An J.-Y. Peng M. Collins R. Grove J. Klei L. et al.Autism Sequencing ConsortiumiPSYCH-Broad ConsortiumLarge-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism.Cell. 2020; 180: 568-584.e23Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar However, due to the rarity of variants and the limited phenotypic data, the clinical significance and genotype-phenotype relationships for most candidate genes still remain to be determined. ASD shows a wide range of overlapping clinical features with other neurodevelopmental disorders (NDDs), such as intellectual disability (ID), language and motor developmental delay, and different psychiatric symptoms.4Lai M.C. Lombardo M.V. Baron-Cohen S. Autism.Lancet. 2014; 383: 896-910Abstract Full Text Full Text PDF PubMed Scopus (1374) Google Scholar As a result, it is both critical and challenging to define the particular genetic subtypes, especially among the most penetrant variants. International collaboration is key and has been pivotal in defining and establishing some recently described ASD/NDD genes, such as CHD8 (MIM: 610528),5Barnard R.A. Pomaville M.B. O’Roak B.J. Mutations and Modeling of the Chromatin Remodeler CHD8 Define an Emerging Autism Etiology.Front. Neurosci. 2015; 9: 477Crossref PubMed Scopus (57) Google Scholar ADNP (MIM: 611386),6Helsmoortel C. Vulto-van Silfhout A.T. Coe B.P. Vandeweyer G. Rooms L. van den Ende J. Schuurs-Hoeijmakers J.H. Marcelis C.L. Willemsen M.H. Vissers L.E. et al.A SWI/SNF-related autism syndrome caused by de novo mutations in ADNP.Nat. Genet. 2014; 46: 380-384Crossref PubMed Scopus (216) Google Scholar DYRK1A (MIM: 600855),7van Bon B.W.M. Coe B.P. Bernier R. Green C. Gerdts J. Witherspoon K. Kleefstra T. Willemsen M.H. Kumar R. Bosco P. et al.Disruptive de novo mutations of DYRK1A lead to a syndromic form of autism and ID.Mol. Psychiatry. 2016; 21: 126-132Crossref PubMed Scopus (104) Google Scholar POGZ (MIM: 614787),8Stessman H.A.F. Willemsen M.H. Fenckova M. Penn O. Hoischen A. Xiong B. Wang T. Hoekzema K. Vives L. Vogel I. et al.Disruption of POGZ Is Associated with Intellectual Disability and Autism Spectrum Disorders.Am. J. Hum. Genet. 2016; 98: 541-552Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar and TANC2 (MIM: 615047).9Guo H. Bettella E. Marcogliese P.C. Zhao R. Andrews J.C. Nowakowski T.J. Gillentine M.A. Hoekzema K. Wang T. Wu H. et al.University of Washington Center for Mendelian GenomicsDisruptive mutations in TANC2 define a neurodevelopmental syndrome associated with psychiatric disorders.Nat. Commun. 2019; 10: 4679Crossref Scopus (28) Google Scholar Although disruptive variants in any given gene explain only an extremely small proportion of individuals, defining the specific genetic subtypes not only increases the diagnostic yield of genetically defined cases but has the potential to optimize future clinical management. We previously prioritized 58 ASD candidate genes based on gene constraint and FMRP/RBFOX binding targets9Guo H. Bettella E. Marcogliese P.C. Zhao R. Andrews J.C. Nowakowski T.J. Gillentine M.A. Hoekzema K. Wang T. Wu H. et al.University of Washington Center for Mendelian GenomicsDisruptive mutations in TANC2 define a neurodevelopmental syndrome associated with psychiatric disorders.Nat. Commun. 2019; 10: 4679Crossref Scopus (28) Google Scholar for further consideration, including NCKAP1 (MIM: 604891). Knockout of Nckap1 in mice leads to embryonic lethal neural tube defects. Premature expression of Nckap1 retards the migration of neurons in the neocortex.10Yokota Y. Ring C. Cheung R. Pevny L. Anton E.S. Nap1-regulated neuronal cytoskeletal dynamics is essential for the final differentiation of neurons in cerebral cortex.Neuron. 2007; 54: 429-445Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Three de novo likely gene-disruptive (LGD) variants were originally reported in ASD probands from the Simons Simplex Collection (SSC) cohort (n = 2)2Iossifov I. O’Roak B.J. Sanders S.J. Ronemus M. Krumm N. Levy D. Stessman H.A. Witherspoon K.T. Vives L. Patterson K.E. et al.The contribution of de novo coding mutations to autism spectrum disorder.Nature. 2014; 515: 216-221Crossref PubMed Scopus (1498) Google Scholar and the Autism Clinical and Genetics Resources in China (ACGC) cohort (n = 1),11Guo H. Wang T. Wu H. Long M. Coe B.P. Li H. Xun G. Ou J. Chen B. Duan G. et al.Inherited and multiple de novo mutations in autism/developmental delay risk genes suggest a multifactorial model.Mol. Autism. 2018; 9: 64Crossref PubMed Scopus (72) Google Scholar with limited clinical information. In addition, a transmitted stop-gain variant was recently identified in a family with a multigenerational ID.12Anazi S. Maddirevula S. Salpietro V. Asi Y.T. Alsahli S. Alhashem A. Shamseldin H.E. AlZahrani F. Patel N. Ibrahim N. et al.Expanding the genetic heterogeneity of intellectual disability.Hum. Genet. 2017; 136: 1419-1429Crossref PubMed Scopus (87) Google Scholar Although previous findings implicate NCKAP1 as an ASD/ID risk gene,13Ruzzo E.K. Pérez-Cano L. Jung J.-Y. Wang L.-K. Kashef-Haghighi D. Hartl C. Singh C. Xu J. Hoekstra J.N. Leventhal O. et al.Inherited and De Novo Genetic Risk for Autism Impacts Shared Networks.Cell. 2019; 178: 850-866.e26Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar its clinical significance is undetermined and the genotype-phenotype relationships have never been demonstrated, leaving the pathogenicity of NCKAP1 variants unclear. To address these issues, we focused on genotype-phenotype assessment of disruptive variants in NCKAP1. Based on our analysis of 21 individuals from 20 families with inherited and de novo deleterious NCKAP1 variants from a large-scale international consortium, we report a new NCKAP1-related NDD as associated with ASD. The affected individuals haboring NCKAP1 variants and their family members where available were recruited to different collaborating institutes from seven countries. For each affected individual, detailed clinical information was obtained through recontact or detailed review of medical records by neurologists, psychiatrists, pediatricians, geneticists, or genetic counselors. Genomic DNA was extracted from the whole blood of the affected individuals. Family members of the probands where available were also recruited for segregation analysis and phenotyping. We collected and reviewed detailed clinical data from 21 affected individuals from 20 families. Written informed consent was obtained from study participants or their parents or legal guardians, in line with local institutional review board (IRB) requirements at the time of collection. The IRB of the Central South University approved this study. All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national). In this cohort, NCKAP1 variants were detected by different methods. In short, 16 affected individuals/families underwent exome sequencing in either a clinical or research setting, two families underwent massively parallel targeted sequencing, one family (with microdeletion) underwent array comparative genomic hybridization, and one family (with chromosome inversion) underwent karyotyping coupled with mate-pair sequencing. Detailed methods for all families are documented in the Supplemental Subjects and Methods. We designed two genomic fragments containing exons and up to 300 bp of flanking 5′ and 3′ intronic sequences from genomic DNA. The genomic fragments connect with each other using oligonucleotides containing restriction enzyme sites at their 5′ and 3′ ends. PCRs were performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific) according to the manufacturer’s protocols, with primers carrying restriction sites. Inserts were cloned into the splicing vector pcDNA3.1myc-His(-)B, with the addition of T4 DNA Ligase (Thermo Scientific). The mutant plasmids were constructed by overlap-extension PCR with site-specific mutant primers. Wild-type and variant constructs were verified by Sanger sequencing. The HEK293 cells were cultured in DMEM containing 10% fetal bovine serum, 1% nonessential amino acids, 2 mM glutamine, and 1% penicillin/streptomycin stock solution at 37°C, 5% CO2, 95% humidity. Approximately 2 × 106 of HEK293 cells were grown to 90% confluency in 6-well plates. 4 μg plasmids (wild-type or variants) were transfected into HEK293 cells using Lipofectamine 2000 Reagent (Thermo Scientific) according to standard protocols. After 36 h of culturing, cellular RNA was extracted using the Trizol reagent (Invitrogen) according to the manufacturer’s protocols. Complementary DNA synthesis was carried out with 1 μg of RNA and the Revert Aid First Strand cDNA Synthesis Kit (Life Technologies) following the standard protocol. To evaluate splicing, PCR was performed using Phusion High-Fidelity DNA Polymerase (Thermo Scientific), with specialized primers. The PCR products were separated in 2% agarose gel. Transcripts were verified by Sanger sequencing. We introduced TADA to perform enrichment analysis for NCKAP1 de novo LGD variants. The fraction of ASD risk genes which was estimated by published researches14He X. Sanders S.J. Liu L. De Rubeis S. Lim E.T. Sutcliffe J.S. Schellenberg G.D. Gibbs R.A. Daly M.J. Buxbaum J.D. et al.Integrated model of de novo and inherited genetic variants yields greater power to identify risk genes.PLoS Genet. 2013; 9: e1003671Crossref PubMed Scopus (162) Google Scholar is 1,000 (18,271 protein-coding genes in the genome) for the sake of performing TADA analysis of de novo variants. The prior parameters (gamma.mean.dn and beta.dn) for LGD variants were confirmed as Gamma (20, 1). In addition, the background mutation rate of total genes was obtained from previous studies.15Samocha K.E. Robinson E.B. Sanders S.J. Stevens C. Sabo A. McGrath L.M. Kosmicki J.A. Rehnström K. Mallick S. Kirby A. et al.A framework for the interpretation of de novo mutation in human disease.Nat. Genet. 2014; 46: 944-950Crossref PubMed Scopus (611) Google Scholar Through simulation, we learned the distribution of Bayes factor (BF) of genes and compared this to the observed BF of genes. p values were calculated based on 1 million simulations for robustness. p values were then corrected by Bonferroni method for 18,271 protein-coding genes in the genome. The full-length human NCKAP1 ORF (GenBank: NM_205842, No. HO205842) (YingRui Gene) was inserted into p.CAGGS-IRES-green fluorescent protein (GFP) vector, which was added a HA exogenous tag (5′-YPYDVPDYA-3′) at C-terminal of NCKAP1. Meanwhile, NCKAP1 variants were constructed into the same vector p.CAGGS-IRES-GFP via designing special primers. HEK293 cell lines were cultured with DMEM basic (1×) (GIBCO, C11995500BT), which had been added 10% fetal bovine serum (FBS, GIBCO, 12483020), and placed into 37°C and 5% CO2 incubator. We chose Lipofectamine 2000 Reagent (Invitrogen, 11668019) to transfect exogenous plasmids into cell lines. When the cells reached 70%–80% confluent, plasmid DNA-lipid complexes were prepared and added them to cells. For detailed operating steps and components, see lipofectamine 2000 Reagent protocol at Thermo Fisher Scientific Web. HEK293 cells were cultured on Microscope Cover Glass (Thermo Fisher Scientific, 12-545-82) in 24-well plates and transfected for 48 h. Cells were fixed by 4% PFA for 15 min, permeabilized by 0.1% Triton X-100 (1× PBS preparation) for 10 min, and blocked by 5% bovine serum albumin (1× PBS preparation) for 1 h. Then, glasses with cells were incubated with anti-HA (Cell Signaling Technology, 3724S) primary antibodies overnight at 4°C. Then, 1× PBS was used to wash glasses three times and stained Cy3 (Jackson ImmunoResearch, 115-165-003) secondary antibodies. 4,6-diamidino-2-phenylindole (DAPI) was used to label cell nuclei. The images were collected by TCS-SP5-II confocal microscope (Leica) and analyzed with ImageJ. RNA-seq data from BrainSpan was used to illustrate the dynamic expression of NCKAP1 across brain development. The age of brain samples ranged from 8 postconceptional weeks (PCW) to 40 years old. Normalized reads per kilobase million (RPKM) expression and sample meta-data were downloaded from BrainSpan. Univariate linear regression was used to analyze the relationship between NCKAP1 expressions and development periods. The linear regression analysis was conducted in brain regions separately. To investigate the cell-type-specific expression pattern of NCKAP1 in the developing human cerebral cortex, we introduced a single-cell RNA-seq dataset from in a previous publication.16Nowakowski T.J. Bhaduri A. Pollen A.A. Alvarado B. Mostajo-Radji M.A. Di Lullo E. Haeussler M. Sandoval-Espinosa C. Liu S.J. Velmeshev D. et al.Spatiotemporal gene expression trajectories reveal developmental hierarchies of the human cortex.Science. 2017; 358: 1318-1323Crossref PubMed Scopus (422) Google Scholar Briefly, the RNA-seq data were generated from 48 individuals. Processed normalized expression values were downloaded from the UCSC cell browser. Assignments of the cluster for each cell were derived from the source study. The biological interpretation for each broad cell type was derived from the previous publication. Statistical significance of enrichment or depletion for each cell type was calculated using the Wilcoxon Rank Sum Test. Bonferroni correction method was used to perform multiple testing correction. All analysis were performed in R stat (v.3.2.3). The mouse Nckap1 (GenBank: NM_016965) original shRNA reference sequences were obtained from MERCK (TRCN0000112255). We changed the restriction enzyme cutting sites (XbaI and BamHI) and inserted the sequences into FUGW-H1-GFP vector. Plasmids were deliquated to 2 μg/μL and mixed with 0.01% Fast Green (Sigma-Aldrich). Each pregnant mouse received 20 μL plasmids. We injected plasmids into lateral ventricles of embryonic 13.5/14.5-day mouse brains. Electroporation was operated using an Electro Square Porator (ECM 830) and the relevant parameter is 50 ms square pulses with 1,000 ms suspensions at 30v. The injected mouse brains were collected on embryonic 17.5/16.5 day, fixed into 4% PFA for 24 h, and gradually dehydrated into 15% sucrose for 24 h and 30% sucrose, which were compounded by 1∗PSB for 24 h until precipitated at the bottom of centrifuge tubes. Whole brains were embedded with embedding medium (the ratio of O.C.T. compound and 20% sucrose is 2:1) and frozen at −80°C. Study protocols comply with all relevant ethical regulations and were approved by the IRB of Central South University. For immunofluorescence experiments, frozen mouse brain tissue was cut to 20–30 μm thickness along coronal planes. The neurons, which were transfected with exogenous plasmids and expressed relevant proteins, were stained by Rabbit GFP first antibody (1:500) (Invitrogen, A11122) and corrective Alex 488 secondary antibody (1:250) and emitted green fluorescence under fluorescence microscopy. Two de novo LGD variants in NCKAP1 (GenBank: NM_205842.3) c.523_524insCA (p.Gly175Alafs∗14) and c.3262G>T (p.Glu1088∗) have been reported in the SSC cohort (Figure 1A). Through reanalysis of the SSC cohort genome-sequencing data, we identified a de novo intronic variant adjacent to the canonical splicing site (c.530+3A>G). By minigene assay, we demonstrated that this putative splicing variant affected normal splicing, leading to skipping of the 6th exon of NCKAP1 (Figure 1B). We then investigated de novo variants data from two other published large-scale ASD cohorts: the ASC cohort3Satterstrom F.K. Kosmicki J.A. Wang J. Breen M.S. De Rubeis S. An J.-Y. Peng M. Collins R. Grove J. Klei L. et al.Autism Sequencing ConsortiumiPSYCH-Broad ConsortiumLarge-Scale Exome Sequencing Study Implicates Both Developmental and Functional Changes in the Neurobiology of Autism.Cell. 2020; 180: 568-584.e23Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar (n = 4,046) and the MSSNG cohort17C Yuen R.K. Merico D. Bookman M. L Howe J. Thiruvahindrapuram B. Patel R.V. Whitney J. Deflaux N. Bingham J. Wang Z. et al.Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder.Nat. Neurosci. 2017; 20: 602-611Crossref PubMed Scopus (454) Google Scholar (n = 1,625); no additional de novo cases were identified in 5,671 trios, indicating the extreme rarity of NCKAP1 de novo variants in ASD. Loss of Nckap1 in mice leads to neural tube and neuronal differentiation and migration defects in mouse developing brain.10Yokota Y. Ring C. Cheung R. Pevny L. Anton E.S. Nap1-regulated neuronal cytoskeletal dynamics is essential for the final differentiation of neurons in cerebral cortex.Neuron. 2007; 54: 429-445Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar In the WAVE complex, NCKAP1 interacts directly with CYFIP2, which was recently reported to be associated with ID and autism.17C Yuen R.K. Merico D. Bookman M. L Howe J. Thiruvahindrapuram B. Patel R.V. Whitney J. Deflaux N. Bingham J. Wang Z. et al.Whole genome sequencing resource identifies 18 new candidate genes for autism spectrum disorder.Nat. Neurosci. 2017; 20: 602-611Crossref PubMed Scopus (454) Google Scholar, 18Zweier M. Begemann A. McWalter K. Cho M.T. Abela L. Banka S. Behring B. Berger A. Brown C.W. Carneiro M. et al.Deciphering Developmental Disorders (DDD) StudySpatially clustering de novo variants in CYFIP2, encoding the cytoplasmic FMRP interacting protein 2, cause intellectual disability and seizures.Eur. J. Hum. Genet. 2019; 27: 747-759Crossref Scopus (25) Google Scholar, 19Nakashima M. Kato M. Aoto K. Shiina M. Belal H. Mukaida S. Kumada S. Sato A. Zerem A. Lerman-Sagie T. et al.De novo hotspot variants in CYFIP2 cause early-onset epileptic encephalopathy.Ann. Neurol. 2018; 83: 794-806Crossref PubMed Scopus (39) Google Scholar This interaction and the mouse knockout model warranted a more detailed search for additional individuals with potentially deleterious variants. Using GeneMatcher20Philippakis A.A. Azzariti D.R. Beltran S. Brookes A.J. Brownstein C.A. Brudno M. Brunner H.G. Buske O.J. Carey K. Doll C. et al.The Matchmaker Exchange: a platform for rare disease gene discovery.Hum. Mutat. 2015; 36: 915-921Crossref PubMed Scopus (293) Google Scholar and an international network of collaborators, we recruited a total of 18 individuals with NCKAP1 putative disruptive variants (including the three individuals from the SSC cohort) (Figure 1C, Tables 1 and S1). The group consists of one individual with a de novo microdeletion involving NCKAP1 and another two adjacent genes, DUSP19 (MIM: 611437) and NUP35 (MIM: 608140) (Figure 1D); one individual with a de novo chromosome inversion for which one of the breakpoints occurred in the first intron of NCKAP1 (Figure 1C); 14 individuals with LGD variants (8 de novo, 4 inherited, 2 undetermined) (Figure 1C); and two individuals with intronic variants (c.760−3A>C and c.2522−3C>G) adjacent to splice sites (Table 1, Figure 1C). As we did for the initial splicing variant (c.530+3A>G), we applied a minigene assay to the additional two putative splicing variants. Variant c.760−3A>C is de novo and was identified in an individual with ID. However, we did not detect abnormal splicing (Figure S1), although we cannot exclude the possibility of a tissue-specific splicing effect of this variant in vivo or quantitative changes in gene expression. The pathogenic effect for this variant is still to be determined. The second putative splicing variant, c.2522−3C>G, was identified in a family where the variant was transmitted from the affected mother. Our minigene assay revealed that this variant leads to a skipping of the 24th exon of NCKAP1 (Figure 1E).Table 1Summary of NCKAP1 Variants Identified in NDD-Affected IndividualsFamily IndexCohortCohort SizeMethodsgDNA Change (chr2, hg19)FunctionNT Changeaa ChangeInheritancegnomADClinical SignificanceLGD Variants1SSC2,508WESg.183866861_183866862insTGframeshiftc.523_524insCAp.Gly175Alafs∗14de novo0P3GeneDx31,111WESg.183860531_183860532insAframeshiftc.656_657insTp.Tyr220Ilefs∗9de novo0P5Lyon200WESg.183859579T>Astopgainc.796A>Tp.Lys266∗de novo0P6GeneDx31,111WESg.183826886C>Tsplicingc.1899+1G>A–unknown0LP7Parkville160WESg.183821230G>Astopgainc.2131C>Tp.Arg711∗paternal0P8ASID10,927targetg.183817939delframeshiftc.2292delp.Ile765Leufs∗18maternal0LP9Poitiers224–350WESg.183817632G>Astopgainc.2410C>Tp.Arg804∗not maternal0LP10Lausanne–WESg.183817632G>Astopgainc.2410C>Tp.Arg804∗not maternal0LP12ACGC2,926targetg.183792844C>Tsplicingc.3198+1G>A–de novo0P13Lyon200WESg.183791591_183791592dupframeshiftc.3240_3241dupp.Lys1081Ilefs∗15de novo0P14SSC2,508WESg.183791570G>Tstopgainc.3262G>Tp.Glu1088∗de novo0P15Riyadh105WESg.183790537C>Astopgainc.3298G>Tp.Glu1100∗paternal0PDe Novo Intronic Variants2SSC2,508WESg.183866852T>Csplicingc.530+3A>G–de novo0P4Riyadh2,219WESg.183859618T>Gsplicingc.760−3A>C–de novo0VUS11GeneDx31,111WESg.183817233G>Csplicingc.2522−3C>G–maternal0LPMicrodeletion16Odense–aCGHg.183762482-184182761delmicrodeletion240 kb deletion–de novo0PChromosome Inversion17Colorado–MPSinv(2)(2pter®p23.1(30340928)::2q32.1(183896661)®p23.1(30340928)::2q32.1(183896662) ®2qter).inversion––de novo–PDe Novo Missense Variants18GeneDx31,111WESg.183902823G>Cmissensec.5C>Gp.Ser2Trpde novo0VUS19GeneDx31,111WESg.183832053C>Tmissensec.1537G>Ap.Ala513Thrde novo1VUS20GeneDx31,111WESg.183790473G>Amissensec.3362C>Tp.Ala1121Valde novo0VUSNCKAP1 isoform is GenBank: NM_205842.3. MPS, mate pair sequencing of inversion; P, pathogenic; LP, likely pathogenic; VUS, variants of uncertain significance. Open table in a new tab NCKAP1 isoform is GenBank: NM_205842.3. MPS, mate pair sequencing of inversion; P, pathogenic; LP, likely pathogenic; VUS, variants of uncertain significance. NCKAP1 is a highly constrained gene, intolerant of variation; it is located in the top 3.9 percentile by residual variance intolerance score21Ruderfer D.M. Hamamsy T. Lek M. Karczewski K.J. Kavanagh D. Samocha K.E. Daly M.J. MacArthur D.G. Fromer M. Purcell S.M. Exome Aggregation ConsortiumPatterns of genic intolerance of rare copy number variation in 59,898 human exomes.Nat. Genet. 2016; 48: 1107-1111Crossref PubMed Scopus (126) Google Scholar and has no reported truncating variants (the probability of being loss-of-function intolerant [pLI] = 1) in the gnomAD database.22Lek M. Karczewski K.J. Minikel E.V. Samocha K.E. Banks E. Fennell T. O’Donnell-Luria A.H. Ware J.S. Hill A.J. Cummings B.B. et al.Exome Aggregation ConsortiumAnalysis of protein-coding genetic variation in 60,706 humans.Nature. 2016; 536: 285-291Crossref PubMed Scopus (6555) Google Scholar No LGD variant within NCKAP1, for example, has been observed in 114,704 gnomAD samples who were not ascertained for having a neurological condition in a neurological case/control study (non-neuro subset).23Karczewski K.J. Francioli L.C. Tiao G. Cummings B.B. Alföldi J. Wang Q. Collins R.L. Laricchia K.M. Ganna A. Birnbaum D.P. et al.Genome Aggregation Database ConsortiumThe mutational constraint spectrum quantified from variation in 141,456 humans.Nature. 2020; 581: 434-443Crossref PubMed Scopus (3193) Google Scholar Based on our larger international cohort, we assessed statistical significance of our genetic findings. We first assessed an enrichment of de novo LGD variants among probands compared with random occurrence by applying TADA.14He X. Sanders S.J. Liu L. De Rubeis S. Lim E.T. Sutcliffe J.S. Schellenberg G.D. Gibbs R.A. Daly M.J. Buxbaum J.D. et al.Integrated model of de novo and inherited genetic variants yields greater power to identify risk genes.PLoS Genet. 2013; 9: e1003671Crossref PubMed Scopus (162) Google Scholar Six individuals with de novo NCKAP1 LGD variants were detected from four cohorts (SSC: c.523_524insCA [p.Gly175Alafs∗14], c.3262G>T [p.Glu1088∗]; GeneDx: c.656_657insT [p.Tyr220Ilefs∗9]; Lyon: c.796A>T [p.Lys266∗], c.3240_3241dup [p.Lys1081Ilefs∗15]; ACGC: c.3198+1G>A; Table 1) where a total of 36,745 individuals with NDD were tested. After combining three published ASD/NDD cohorts (ASC cohort, n = 4,046; MSSNG cohort, n = 1,625; and ASID cohort,24Stessman H.A.F. Xiong B. Coe B.P. Wang T. Hoekzema K. Fenckova M. Kvarnung M. Gerdts J. Trinh S. Cosemans N. et al.Targeted sequencing identifies 91 neurodevelopmental-disorder risk genes with autism and developmental-disability biases.Nat. Genet. 2017; 49: 515-526Crossref PubMed Scopus (310) Google Scholar n = 10,927) with no NCKAP1 de novo LGD variants identified, the TADA-denovo model reveals an exome-wide significant enrichment (p = 5.0 × 10−7, padj = 0.0091, Bonferroni correction). Second, since almost half of the LGD variants were transmitted, we investigated whether there is a significant burden of NCKAP1 LGD variants regardless of the inheritance status by performing a Fisher’s exact test using non-neuro gnomAD subset samples as controls. Eleven individuals with NCKAP1 LGD variants (SSC: c.523_524insCA [p.Gly175Alafs∗14], c.3262G>T [p.Glu1088∗]; GeneDx: c.656_657insT [p.Tyr220Ilefs∗9], c.1899+1G>A; Parkville: c.2131C>T [p.Arg711∗]; ASID: c.2292del [p.Ile765Leufs∗18]; Poitiers: c.2410C>T [p.Arg804∗]; Lyon: c.796A>T [p.Lys266∗], c.3240_3241dup [p.Lys1081Ilefs∗15]; ACGC: c.3198+1G>A; Riyadh: c.3298G>T [p.Glu1100∗]; Table 1) were detected from eight cohorts where both de novo and inherited data are available. We observe a genome-wide significant burden of NCKAP1 LGD variants in our international NDD cohorts (11 probands in 48,206 NDD individuals versus 0 individuals in 114,704" @default.
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• W3097947115 date "2020-11-01" @default.
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• W3097947115 title "NCKAP1 Disruptive Variants Lead to a Neurodevelopmental Disorder with Core Features of Autism" @default.
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