Missense Mutations in NKAP Cause a Disorder of Transcriptional Regulation Characterized by Marfanoid Habitus and Cognitive Impairment
2019; Elsevier BV; Volume: 105; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2019.09.009
ISSN1537-6605
AutoresSarah K. Fiordaliso, Aiko Iwata‐Otsubo, Alyssa Ritter, Mathieu Quesnel-Vallières, Katsunori Fujiki, Eriko Nishi, Miroslava Hančárová, Noriko Miyake, Jenny E.V. Morton, Sangmoon Lee, Karl Hackmann, Masashige Bando, Koji Masuda, Ryuichiro Nakato, Michiko Arakawa, Elizabeth Bhoj, Dong Li, Hákon Hákonarson, Ryojun Takeda, Margaret Harr, Beth Keena, Elaine H. Zackai, Nobuhiko Okamoto, Seiji Mizuno, Jung Min Ko, Alica Valachová, Darina Prchalová, Markéta Vlčková, Tommaso Pippucci, Christoph Seiler, Murim Choi, Naomichi Matsumoto, Nataliya Di Donato, Yoseph Barash, Zdeněk Sedláček, Katsuhiko Shirahige, Kosuke Izumi,
Tópico(s)Signaling Pathways in Disease
ResumoNKAP is a ubiquitously expressed nucleoplasmic protein that is currently known as a transcriptional regulatory molecule via its interaction with HDAC3 and spliceosomal proteins. Here, we report a disorder of transcriptional regulation due to missense mutations in the X chromosome gene, NKAP. These mutations are clustered in the C-terminal region of NKAP where NKAP interacts with HDAC3 and post-catalytic spliceosomal complex proteins. Consistent with a role for the C-terminal region of NKAP in embryogenesis, nkap mutant zebrafish with a C-terminally truncated NKAP demonstrate severe developmental defects. The clinical features of affected individuals are highly conserved and include developmental delay, hypotonia, joint contractures, behavioral abnormalities, Marfanoid habitus, and scoliosis. In affected cases, transcriptome analysis revealed the presence of a unique transcriptome signature, which is characterized by the downregulation of long genes with higher exon numbers. These observations indicate the critical role of NKAP in transcriptional regulation and demonstrate that perturbations of the C-terminal region lead to developmental defects in both humans and zebrafish. NKAP is a ubiquitously expressed nucleoplasmic protein that is currently known as a transcriptional regulatory molecule via its interaction with HDAC3 and spliceosomal proteins. Here, we report a disorder of transcriptional regulation due to missense mutations in the X chromosome gene, NKAP. These mutations are clustered in the C-terminal region of NKAP where NKAP interacts with HDAC3 and post-catalytic spliceosomal complex proteins. Consistent with a role for the C-terminal region of NKAP in embryogenesis, nkap mutant zebrafish with a C-terminally truncated NKAP demonstrate severe developmental defects. The clinical features of affected individuals are highly conserved and include developmental delay, hypotonia, joint contractures, behavioral abnormalities, Marfanoid habitus, and scoliosis. In affected cases, transcriptome analysis revealed the presence of a unique transcriptome signature, which is characterized by the downregulation of long genes with higher exon numbers. These observations indicate the critical role of NKAP in transcriptional regulation and demonstrate that perturbations of the C-terminal region lead to developmental defects in both humans and zebrafish. We have identified a cohort of ten affected males from eight families who carry missense mutations in the X chromosome gene NKAP (MIM: 300766). Two of these individuals, subjects 7 and 9, were previously reported with minimal clinical detail, one in a cohort of individuals with a potential Lujan-Fryns syndrome (MIM: 309520),1Hackmann K. Rump A. Haas S.A. Lemke J.R. Fryns J.P. Tzschach A. Wieczorek D. Albrecht B. Kuechler A. Ripperger T. et al.Tentative clinical diagnosis of Lujan-Fryns syndrome--A conglomeration of different genetic entities?.Am. J. Med. Genet. A. 2016; 170A: 94-102Crossref PubMed Scopus (8) Google Scholar and one in a cohort of individuals with intellectual disability.2Deciphering Developmental Disorders StudyPrevalence and architecture of de novo mutations in developmental disorders.Nature. 2017; 542: 433-438Crossref PubMed Scopus (772) Google Scholar The remaining eight individuals with NKAP mutations were identified independently by exome sequencing and recruited through GeneMatcher3Sobreira N. Schiettecatte F. Boehm C. Valle D. Hamosh A. New tools for Mendelian disease gene identification: PhenoDB variant analysis module; and GeneMatcher, a web-based tool for linking investigators with an interest in the same gene.Hum. Mutat. 2015; 36: 425-431Crossref PubMed Scopus (113) Google Scholar and collaborating clinicians. All individuals were enrolled in the research study under an institutional review board-protocol. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional review boards), and informed consent was obtained from the parents/guardians of the affected individuals. The clinical features of these ten affected males were highly conserved. All subjects had developmental delay/intellectual disability (ID), tall stature with Marfanoid habitus, and hypotonia. Shared facial dysmorphisms included: a long face (8/10), open-mouth appearance (7/10), midface hypoplasia (7/10), prominent or large ears (8/10), and a short philtrum (7/10). Musculoskeletal findings were common, with 5 of 10 reported to have pectus carinatum or excavatum, 6 of 10 with scoliosis, and 8 of 10 with arachnodactyly. Talipes equinovarus was identified in one subject. Other less common features included: genitourinary anomalies such as cryptorchidism (4/10), central obesity (5/10), and behavioral abnormalities that included attention deficit hyperactivity disorder (ADHD) or aggressive behaviors (5/10; Figure 1A, Tables 1 and S1, and Supplemental Note). Cardiac manifestations included mitral valve regurgitation (3/10), atrial septal defect (1/10), ventricular septal defect (1/10), small patent ductus arteriosus (1/10), and aortic dilatation (1/10). Although developmental delay or learning disability and tall stature/Marfanoid habitus were observed in all subjects with NKAP mutations, the other features, as noted above, were more variable. Mothers carrying NKAP mutations were unaffected or much less severely affected. While NKAP has been shown to play a role in chromosome alignment during mitosis,4Li T. Chen L. Cheng J. Dai J. Huang Y. Zhang J. Liu Z. Li A. Li N. Wang H. et al.SUMOylated NKAP is essential for chromosome alignment by anchoring CENP-E to kinetochores.Nat. Commun. 2016; 7: 12969Crossref PubMed Scopus (27) Google Scholar no chromosomal abnormalities were seen in the subjects with NKAP mutations. Similarly, although NKAP has been demonstrated to be a key developmental regulator of the hematopoietic and immune system,5Pajerowski A.G. Nguyen C. Aghajanian H. Shapiro M.J. Shapiro V.S. NKAP is a transcriptional repressor of notch signaling and is required for T cell development.Immunity. 2009; 30: 696-707Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar none of the subjects with NKAP mutations were known to have hematological or immunological manifestations.Table 1Clinical Features of Individuals with NKAP MutationsSubject 1Subject 2Subject 3Subject 4Subject 5Subject 6Subject 7Subject 8Subject 9Subject 10NKAP Mutationp.Arg330Cysp.Arg330Hisp.Arg333Glnp.Arg333Glnp.Arg333Glnp.Arg333Glnp.Ile337Thrp.Arg361Glnc.988C>Tc.989G>Ac.998G>Ac.998G>Ac.998G>Ac.998G>Ac.998G>Ac.998G>Ac.1010T>Cc.1082G>Ade novode novoapparently de novomaternally inheritedmaternally inheritedunkde novopresumably de novoNeurologicalDevelopmental delay/intellectual disabilityyesyesyesyesyesyesyesyesyesyesADHD/aggressive behaviornononoyesyesyesnonoyesyesHypotoniayesyesyesyesyesyesyesyesyesyesDysmorphismsLong faceyesyesyesyesyesnoyesunkyesyesShort philtrumyesyesyesyesnoyesyesunkyesnoOpen mouth appearanceyesyesyesyesyesyesyesunknonoMidface hypoplasianoyesyesyesyesyesyesunkyesnoProminent/large earsyesyesnoyesyesyesyesunkyesyesCardiacPathology (age at the last evaluation)atrial septal defect (10 yo)small patent ductal arteriosus (6 yo)mild mitral valve regurgitation (18 yo)mitral valve prolapse, mitral regurgitation and aortic root dilatation (10 yo)no (11 yo)no (5 yo)mitral valves prolapse with the minimal mitral regurgitation (21 yo)history of ventricular septal defect (7 yo)no (10 yo)no (16 yo)SkeletalTall statureyesyesyesyesyesyesyesyesyesyesScoliosisyesyesyesyesyesnoyesnononoPectusyesnononocarinatumcarinatumcarinatumnoexcavatumnoSlender limbsyesyesyesyesyesnoyesyesyesyesJoint laxityyesyesyesyesyesnoyesyesyesyesCamptodactylyyesnoyesnononoyesyesnonoArachnodactylyyesyesyesyesnonoyesyesyesyesUnk, unknown. Full table can be found as Table S1. Open table in a new tab Unk, unknown. Full table can be found as Table S1. Four affected males had apparently de novo NKAP variants, three had maternally inherited variants (subjects 5 and 6 and the unrelated subject 7), and inheritance for one subject (subject 8) was unknown. Two brothers (subject 3 and 4) apparently had de novo inheritance of the variant, consistent with maternal germline mosaicism. We identified five different missense mutations in the ten subjects (c.988C>T [p.Arg330Cys], 1 male; c.989G>A [p.Arg330His], 1 male; c.G998A [p.Arg333Gln], 6 males from 4 families; c.1010T>C [p.Ile337Thr], 1 male; and c.1082G>A [p.Arg361Gln], 1 male). All variants were located in exons 8 and 9 of NKAP (GenBank: NM_024528) encoding the C-terminal part of the protein, and all altered highly conserved amino acids (Table 1, Figure 1B). NKAP encodes a ubiquitously expressed, nuclear speckle protein, initially reported as a mediator of NF-κB signaling.6Chen D. Li Z. Yang Q. Zhang J. Zhai Z. Shu H.B. Identification of a nuclear protein that promotes NF-kappaB activation.Biochem. Biophys. Res. Commun. 2003; 310: 720-724Crossref PubMed Scopus (50) Google Scholar Subsequently, diverse biological roles of NKAP have been reported, including transcriptional regulation and chromosome alignment.4Li T. Chen L. Cheng J. Dai J. Huang Y. Zhang J. Liu Z. Li A. Li N. Wang H. et al.SUMOylated NKAP is essential for chromosome alignment by anchoring CENP-E to kinetochores.Nat. Commun. 2016; 7: 12969Crossref PubMed Scopus (27) Google Scholar, 5Pajerowski A.G. Nguyen C. Aghajanian H. Shapiro M.J. Shapiro V.S. NKAP is a transcriptional repressor of notch signaling and is required for T cell development.Immunity. 2009; 30: 696-707Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar NKAP was suggested to be involved in mRNA splicing,7Burgute B.D. Peche V.S. Steckelberg A.L. Glöckner G. Gaßen B. Gehring N.H. Noegel A.A. NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins.Nucleic Acids Res. 2014; 42: 3177-3193Crossref PubMed Scopus (29) Google Scholar and very recently, NKAP was shown to be a part of post-catalytic P-complex, a large splicing complex responsible for neighboring exonic ligation and processed mRNA release.8Fica S.M. Oubridge C. Wilkinson M.E. Newman A.J. Nagai K. A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation.Science. 2019; 363: 710-714Crossref PubMed Scopus (62) Google Scholar To analyze the effect of identified NKAP mutations, we measured NKAP mRNA and protein levels in lymphoblastoid cell line (LCL) samples from subjects with NKAP mutations compared to control samples and did not note any differences (Figure S1). Immunofluorescence showed no differences in intracellular distribution of NKAP between control and NKAP mutant samples (Figure S1). We could confirm previous findings that NKAP localizes in a nuclear speckled pattern, which contain nucleoplasmic granules enriched in mRNA processing proteins such as splicing factors.7Burgute B.D. Peche V.S. Steckelberg A.L. Glöckner G. Gaßen B. Gehring N.H. Noegel A.A. NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins.Nucleic Acids Res. 2014; 42: 3177-3193Crossref PubMed Scopus (29) Google Scholar Given the previously reported functions of NKAP, we hypothesized that these NKAP mutations alter transcription. Transcriptome analysis was performed using RNA sequencing (RNA-seq) of LCLs from case and control subjects. RNA-seq was initially performed using three LCLs with the p.Arg333Gln substitution and three control LCLs. Clustering analysis revealed that subjects with NKAP mutations exhibited a consistent unique, disrupted transcriptome profile, with 455 upregulated genes and 721 downregulated genes compared to control lines (FDR < 0.05) (Table S2). This suggests that the majority of differentially expressed genes (DEGs) are downregulated in NKAP mutant samples (Figures 2A and 2B ). DEGs include HES1 and JAG1, Notch signaling pathway genes. The RNA-seq results were validated by qRT-PCR of two DEGs (HES1 and APP) (Figure S2). RNA-seq was also performed using LCL with the p.Arg361Gln substitution, and it demonstrated a similar DEG profile (Figure S3). Pathway analysis for the DEGs identified in subjects with NKAP mutations was performed with DAVID Functional Annotation Bioinformatics Microarray Analysis (Table S3). Terms such as "extracellular matrix" were enriched in the NKAP mutant DEGs. Because individuals with NKAP mutations demonstrated features associated with connective tissue disorder, misexpression of these genes may be related to their clinical features. Previously, the Notch signaling pathway was shown to be a transcriptional target of NKAP and mediates the various signaling pathways, such as TGF-β, involved in regulating the extracellular matrix gene expression.5Pajerowski A.G. Nguyen C. Aghajanian H. Shapiro M.J. Shapiro V.S. NKAP is a transcriptional repressor of notch signaling and is required for T cell development.Immunity. 2009; 30: 696-707Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9LaFoya B. Munroe J.A. Mia M.M. Detweiler M.A. Crow J.J. Wood T. Roth S. Sharma B. Albig A.R. Notch: A multi-functional integrating system of microenvironmental signals.Dev. Biol. 2016; 418: 227-241Crossref PubMed Scopus (58) Google Scholar, 10Verrecchia F. Mauviel A. Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation.J. Invest. Dermatol. 2002; 118: 211-215Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar Therefore, alterations in the Notch signaling pathway may be involved in the mechanism of this transcriptome profile. NKAP localizes to the post-catalytic P-complex, whose function is exonic ligation and mRNA release after 5′ and 3′ splice site selection.8Fica S.M. Oubridge C. Wilkinson M.E. Newman A.J. Nagai K. A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation.Science. 2019; 363: 710-714Crossref PubMed Scopus (62) Google Scholar Therefore, although NKAP is a part of the spliceosomal complex, the dysfunction of NKAP may not cause increased aberrant or alternative splicing. Consistent with this notion, splicing analysis with MAJIQ11Vaquero-Garcia J. Barrera A. Gazzara M.R. González-Vallinas J. Lahens N.F. Hogenesch J.B. Lynch K.W. Barash Y. A new view of transcriptome complexity and regulation through the lens of local splicing variations.eLife. 2016; 5: e11752Crossref PubMed Scopus (191) Google Scholar did not identify major splicing pattern alterations with little to no alternative splicing, intron retention, or de novo (unannotated) splicing junction usage changes (Figure 3, Table S4). We found only 39 splicing variations with changes in percent spliced in (PSI) of more than 15 and with a probability that dPSI is above 15 (P(|dPSI|> = 0.15)) of more than 0.95 in control versus NKAP mutant LCLs (Table S4). Although 14 out of 15 splicing variations in which a retained intron has a delta percent spliced in (dPSI) of more than 15 display increased intron retention in NKAP mutants (Figure 3A), intron retention levels are not globally affected when looking at all splicing variations detected in the transcriptome (Figure 3B). NKAP mutant cells also show few splicing variations involving de novo junctions (unannotated, putatively involving cryptic splice sites) and no preference in the inclusion of these junctions (Figure 3C) and a global usage of de novo junctions similar to control cells (Figure 3D). Alternatively, we did note that long genes with a higher number of exons were downregulated. As such, we evaluated the differential expression levels of long genes. Expression levels of the genes whose coding region length is in the top 10 percentile were lower than other genes whose coding region length is below 90th percentile (p value = 2.2e−16) (Figure 4A). The coding region length of downregulated genes was significantly longer than that of upregulated genes (Figure 4B). Finally, we evaluated the differential expression levels of genes with higher exon numbers in the subjects with NKAP mutations. As the majority of human genes contains less than 40 exons,12Sakharkar M.K. Perumal B.S. Sakharkar K.R. Kangueane P. An analysis on gene architecture in human and mouse genomes.In Silico Biol. (Gedrukt). 2005; 5: 347-365PubMed Google Scholar we separately evaluated the level of differential gene expression among genes with ≤40 exons and genes with ≥41 exons (top 2 percentile). Expression levels of the genes with ≥41 exons were lower than those of the genes with ≤40 exons (p value = 3.59e−05) (Figure 4A). The exon numbers of downregulated genes were higher than those of upregulated genes, although the difference was not significant (p value = 0.196) (Figure 4B). These data indicate that NKAP dysfunction due to NKAP mutations results in misexpression of longer genes with larger numbers of exons. To evaluate the effects of NKAP mutation during embryogenesis, we established an nkap mutant zebrafish model using CRISPR/Cas9. Reciprocal BLAST of the human NKAP cDNA sequence (GenBank: NM_024528) against the zebrafish genome identified only one ortholog, nkap (GenBank: NM_001003414, with 68% identity to human). In fish, nkap is located on chromosome 14, with two copies of nkap present. We identified a gRNA targeting a region where human NKAP missense mutations reside (Figures 5A and S5) and have established F0 fish had a germline heterozygous 1 bp deletion in nkap (c.859delA; GenBank: NM_001003414), resulting in a frameshift mutation (p.Arg298GlufsTer6; GenBank: NP_001003414) (Figures 5A and S5). These heterozygous F0 1 bp deletion fish were then outcrossed to a wild-type fish, and 40 F1 heterozygous fish with the 1 bp deletion in nkap were identified (Figure S5). These heterozygous nkap mutant F1 zebrafish did not show a striking early developmental phenotype. Heterozygous nkap mutant F1 fish were incrossed to obtain an F2 generation that contained offspring with a homozygous 1 bp deletion in nkap (Figure 5A). Transmission of nkap 1 bp deletion followed a Mendelian inheritance pattern, with approximately 25% of F2 fish showing an obvious phenotype due to homozygous nkap 1 bp deletion. These homozygous nkap mutant fish consistently demonstrated striking early developmental defects, characterized by edema in the eye, intestinal tract, and heart with noticeable curvature of the notochord (Figure 5B). CNS necrosis was noticeable from 2 dpf. All homozygous nkap mutant fish lacked a heartbeat at 4 dpf and began to decompose; none survived beyond 4 dpf. The 1 bp deletion in this nkap mutant line is expected to cause a frameshift mutation (Figure 5A). It is in exon 8, the penultimate exon, leaving the possibility that the mutant nkap mRNA is escaping nonsense-mediated decay. To examine this possibility, we evaluated nkap expression levels in the F2 homozygous mutant zebrafish at 2 dpf using qRT-PCR. The RNA expression levels of nkap were not significantly altered at either the 5′ or 3′ regions of nkap in the homozygous mutant zebrafish compared to wild-type, indicating that the mutant nkap transcripts likely escape nonsense-mediated decay (Figure 5C). Therefore, as a consequence of the deletion, this nkap mutant zebrafish likely produced a C-terminal-truncated NKAP. The striking developmental phenotype of these zebrafish supports a critical role of the C-terminal region of NKAP during embryogenesis. Interestingly, the ZFIN database reports a homozygous transgenic zebrafish that has an insertion at the nkap start codon (nkaphi1477Tg).13Amsterdam A. Nissen R.M. Sun Z. Swindell E.C. Farrington S. Hopkins N. Identification of 315 genes essential for early zebrafish development.Proc. Natl. Acad. Sci. USA. 2004; 101: 12792-12797Crossref PubMed Scopus (658) Google Scholar, 14Golling G. Amsterdam A. Sun Z. Antonelli M. Maldonado E. Chen W. Burgess S. Haldi M. Artzt K. Farrington S. et al.Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development.Nat. Genet. 2002; 31: 135-140Crossref PubMed Scopus (457) Google Scholar Its developmental defects closely resemble those of homozygous 1 bp nkap deletion zebrafish embryos, including CNS necrosis, curvature of the notochord, and pericardial edema with most dying between 2 dpf and 5 dpf. Because the C-terminal truncation causes effects comparable to those of start codon elimination, the C-terminal NKAP region is likely essential for developmental regulation governed by NKAP. Both the highly conserved clinical phenotype characterized by developmental delay and Marfanoid habitus identified in this study and the unique transcriptome profile identified in subject-derived LCLs establish a distinct clinical entity. Germline truncating mutations in NKAP have never been observed in the public database, gnomAD, among healthy individuals, indicating that loss-of-function NKAP mutations are likely detrimental to human health and development.15Lek 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 (6602) Google Scholar Consistent with an X-linked recessive condition, NKAP mutations resulted in symptoms only in males, and females with heterozygous NKAP mutations appeared to be unaffected or significantly less affected. In fact, NKAP undergoes X-inactivation.16Tukiainen T. Villani A.C. Yen A. Rivas M.A. Marshall J.L. Satija R. Aguirre M. Gauthier L. Fleharty M. Kirby A. et al.GTEx ConsortiumLaboratory, Data Analysis &Coordinating Center (LDACC)—Analysis Working GroupStatistical Methods groups—Analysis Working GroupEnhancing GTEx (eGTEx) groupsNIH Common FundNIH/NCINIH/NHGRINIH/NIMHNIH/NIDABiospecimen Collection Source Site—NDRIBiospecimen Collection Source Site—RPCIBiospecimen Core Resource—VARIBrain Bank Repository—University of Miami Brain Endowment BankLeidos Biomedical—Project ManagementELSI StudyGenome Browser Data Integration &Visualization—EBIGenome Browser Data Integration &Visualization—UCSC Genomics Institute, University of California Santa CruzLandscape of X chromosome inactivation across human tissues.Nature. 2017; 550: 244-248Crossref PubMed Scopus (474) Google Scholar Similar to human NKAP, the mouse Nkap also resides on the X chromosome, and hematopoietic lineage-specific hemizygous Nkap-null mutant male mice show embryonic lethality.17Pajerowski A.G. Shapiro M.J. Gwin K. Sundsbak R. Nelson-Holte M. Medina K. Shapiro V.S. Adult hematopoietic stem cells require NKAP for maintenance and survival.Blood. 2010; 116: 2684-2693Crossref PubMed Scopus (27) Google Scholar In zebrafish, nkap is located on chromosome 14. We observed developmental defects only in homozygous nkap mutant zebrafish, while defects were not obvious in heterozygous animals. These observations suggest that elimination of non-mutated NKAP is required to impair development. The clinical findings of individuals with NKAP mutations demonstrate essential roles of NKAP in neural development as well as in bone and connective tissue. This notion is supported by the striking developmental defects seen in nkap mutant zebrafish, which include spine deformities, a common finding also in human subjects with NKAP mutations. In addition, our gene ontology analysis of NKAP mutant cells revealed enrichment of extracellular matrix genes. Although the transcriptional target genes of NKAP may differ between LCLs and connective tissues, misexpression of extracellular matrix genes may result in defective connective tissue development in subjects with NKAP mutations. This connective tissue role is supported by the clinical phenotype of this disorder which overlaps with connective tissue disorders, such as Marfan syndrome (MIM: 154700)18Dietz H.C. Cutting G.R. Pyeritz R.E. Maslen C.L. Sakai L.Y. Corson G.M. Puffenberger E.G. Hamosh A. Nanthakumar E.J. Curristin S.M. et al.Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene.Nature. 1991; 352: 337-339Crossref PubMed Scopus (1605) Google Scholar, 19Neptune E.R. Frischmeyer P.A. Arking D.E. Myers L. Bunton T.E. Gayraud B. Ramirez F. Sakai L.Y. Dietz H.C. Dysregulation of TGF-beta activation contributes to pathogenesis in Marfan syndrome.Nat. Genet. 2003; 33: 407-411Crossref PubMed Scopus (1163) Google Scholar and Beals syndrome (MIM: 121050),20Putnam E.A. Zhang H. Ramirez F. Milewicz D.M. Fibrillin-2 (FBN2) mutations result in the Marfan-like disorder, congenital contractural arachnodactyly.Nat. Genet. 1995; 11: 456-458Crossref PubMed Scopus (224) Google Scholar with the presence of tall stature and joint contractures. In addition, Lujan-Fryns syndrome,1Hackmann K. Rump A. Haas S.A. Lemke J.R. Fryns J.P. Tzschach A. Wieczorek D. Albrecht B. Kuechler A. Ripperger T. et al.Tentative clinical diagnosis of Lujan-Fryns syndrome--A conglomeration of different genetic entities?.Am. J. Med. Genet. A. 2016; 170A: 94-102Crossref PubMed Scopus (8) Google Scholar characterized by Marfanoid habitus, tall stature, and intellectual disability was considered in one individual. Thus, NKAP alterations should be considered for individuals with suspected Marfan, Beals, or Lujan-Fryns syndromes. The missense mutations identified in subjects were strictly clustered toward the C-terminal end of NKAP. This C-terminal end of NKAP is highly conserved, and missense mutations in the region between Pro296 and Glu394 (exon 7 to exon 9) are also entirely absent in gnomAD,15Lek 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 (6602) Google Scholar underscoring the critical importance of this region (Figure S4). In addition, the lethal mutations caused by C-terminal truncations in zebrafish also support that this region of NKAP is essential in embryogenesis. The C-terminal end of NKAP (aa 273–415), where the subjects have missense mutations, harbors the HDAC3-interacting domain.5Pajerowski A.G. Nguyen C. Aghajanian H. Shapiro M.J. Shapiro V.S. NKAP is a transcriptional repressor of notch signaling and is required for T cell development.Immunity. 2009; 30: 696-707Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Since HDAC3 is an important transcriptional regulatory molecule, NKAP mutations may affect the HDAC3-mediated transcriptional regulatory role of NKAP, although we did not detect any changes in HDAC3 dosage or intracellular distribution in the subjects (Figure S1). Very recently, NKAP was shown to be a part of the post-catalytic P-complex, which is responsible for the last steps of splicing, and a crystal structural analysis revealed that the C-terminal region of NKAP (aa 329–358) interacts with SLU7 and PRP8, subunits of P-complex.8Fica S.M. Oubridge C. Wilkinson M.E. Newman A.J. Nagai K. A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation.Science. 2019; 363: 710-714Crossref PubMed Scopus (62) Google Scholar Therefore, the disruption of exon ligation or processed mRNA release is another candidate mechanism explaining transcriptional alteration seen in this diagnosis. Consistent with the specific role of NKAP in the P-complex, we did not identify an increase of aberrant or alternative splicing events in the LCL samples with NKAP mutations. RNA-seq analysis of LCL samples with NKAP mutations revealed that longer genes with more exons are more often downregulated. This suggests that genes which require more exonic ligation/mRNA release steps before the complete processing of the entire transcript may be more sensitive to NKAP dysfunction. Downregulation of long gene expression may be of key importance for normal neurodevelopment, as a large number of neuronal lineage-specific genes are characterized by long coding regions, and several neurological disorders, such as Rett syndrome (MIM: 312750) and autism, have been shown to be associated with the dysregulation of long genes.21Gabel H.W. Kinde B. Stroud H. Gilbert C.S. Harmin D.A. Kastan N.R. Hemberg M. Ebert D.H. Greenberg M.E. Disruption of DNA-methylation-dependent long gene repression in Rett syndrome.Nature. 2015; 522: 89-93Crossref PubMed Scopus (362) Google Scholar, 22King I.F. Yandava C.N. Mabb A.M. Hsiao J.S. Huang H.S. Pearson B.L. Calabrese J.M. Starmer J. Parker J.S. Magnuson T. et al.Topoisomerases facilitate transcription of long genes linked to autism.Nature. 2013; 501: 58-62Crossref PubMed Scopus (267) Google Scholar Although intriguing, further work is needed to determine the role of NKAP in transcriptional regulation of long genes, as long genes are more sensitive to many factors including splicing, transcription efficiency, rate of transit of the transcriptional machinery, and mRNA stabilization. In conclusion, we report a neurodevelopmental disorder with musculoskeletal features caused by NKAP missense mutations that result in transcriptional disruption. Our study supports pleiotropic roles for NKAP in the development of several organ systems and underscores the essential role for NKAP in vertebrate embryogenesis. The authors declare no competing interests. Download .pdf (34.99 MB) Help with pdf files Document S1. Supplemental Note, Figures S1–S5, Tables S1, S5, S6, S8, and S9, Supplemental Material and Methods, and Supplemental Acknowledgments Download .xlsx (.12 MB) Help with xlsx files Table S2. Differentially Expressed Genes Identified in Individuals with NKAP Mutations Download .xlsx (.05 MB) Help with xlsx files Table S3. DAVID Gene Ontology Analysis of the Differentially Expressed Genes of the Samples with NKAP Mutations Download .xlsx (.02 MB) Help with xlsx files Table S4. List of Splicing Pattern Alteration Events Download .xlsx (.04 MB) Help with xlsx files Table S7. Rare Variants Identified in the Subjects with NKAP Mutations Download .xlsx (.01 MB) Help with xlsx files Table S10. RNA-Seq Statistics DAVID Functional Annotation Bioinformatics Microarray Analysis, https://david.ncifcrf.gov/DECIPHER, https://decipher.sanger.ac.ukGenBank, https://www.ncbi.nlm.nih.gov/genbank/GeneBase 1.1, http://apollo11.isto.unibo.it/software/GeneMatcher, https://www.genematcher.org/gnomAD, https://gnomad.broadinstitute.org/MAJIQ, https://majiq.biociphers.org/OMIM, https://www.omim.org/ZFIN, https://zfin.org/
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