{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T14:13:56Z","timestamp":1772547236972,"version":"3.50.1"},"reference-count":40,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T00:00:00Z","timestamp":1772409600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>NKAP (NF-kappa-B-activating protein) is a ubiquitously expressed nuclear protein involved in multiple biological processes. Males with missense NKAP mutations have been reported to present with marfanoid features and behavioral and musculoskeletal abnormalities. We have previously reported that a disruptive NKAP mutation resulted in extremely skewed X chromosome inactivation (XCI), leading to phenotypic manifestation of hemophilia A (HA) in a HA carrier. In this study, with the aim of exploring the phenotypic manifestations of deleterious NKAP mutations in males, as well as their involvement in the mechanism of XCI regulation in females, we generated NKAP mutant mice using CRISPR\/Cas9 technology. Gait analysis studies conducted in male mice hemizygous for mutant NKAP by the CatWalk XT system revealed significant alterations in gait parameters, consistent with hypotonia reported in human mutant NKAP patients. By breeding mutant NKAP mice with HA mice, we generated a double heterozygous mutant NKAP\/HA mouse model, i.e., female mice carrying mutant NKAP with a WT F8 copy on one X chromosome, and WT NKAP with a mutant F8 copy on the other X chromosome. XCI pattern analysis using methylation-sensitive restriction enzymes demonstrated that mutant NKAP\/HA females exhibited significant XCI skewing of the X chromosome bearing the mutant NKAP copy. Furthermore, these females exhibited significantly reduced F8 mRNA levels and FVIII (factor VIII) antigen levels, as demonstrated by quantitative RT-PCR and ELISA, respectively. Murine embryonic fibroblasts (MEFs) derived from a hemizygous mutant NKAP embryo exhibited markedly reduced proliferation rate and increased senescence compared to WT NKAP MEFs, suggesting that XCI skewing induced by mutant NKAP results from secondary selection against cells with an active X chromosome bearing the mutant NKAP copy.<\/jats:p>","DOI":"10.3390\/ijms27052330","type":"journal-article","created":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T12:39:56Z","timestamp":1772455196000},"page":"2330","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Deleterious NKAP Mutations Are Associated with Musculoskeletal Abnormalities in Hemizygous Males and Skewed X Chromosome Inactivation in Heterozygous Females"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-6920-790X","authenticated-orcid":false,"given":"Einat","family":"Avishai","sequence":"first","affiliation":[{"name":"National Hemophilia Center, Sheba Medical Center, Ramat Gan 52621, Israel"},{"name":"Amalia Biron Research Institute of Thrombosis and Hemostasis, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rima","family":"Dardik","sequence":"additional","affiliation":[{"name":"National Hemophilia Center, Sheba Medical Center, Ramat Gan 52621, Israel"},{"name":"Amalia Biron Research Institute of Thrombosis and Hemostasis, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Linda","family":"Rubinstein","sequence":"additional","affiliation":[{"name":"The Joseph Sagol Neuroscience Center, Sheba Medical Center, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"},{"name":"The Gonda Multidisciplinary Brain Research Center, Bar Ilan University, Ramat Gan 5290002, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6652-2667","authenticated-orcid":false,"given":"Ivan","family":"Budnik","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Division of Hematology, Oncology, and Blood & Marrow Transplantation, The University of Iowa, Iowa City, IA 52242, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yair","family":"Ben Gera","sequence":"additional","affiliation":[{"name":"The Joseph Sagol Neuroscience Center, Sheba Medical Center, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rachel","family":"Twitto-Greenberg","sequence":"additional","affiliation":[{"name":"The Joseph Sagol Neuroscience Center, Sheba Medical Center, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gili","family":"Kenet","sequence":"additional","affiliation":[{"name":"National Hemophilia Center, Sheba Medical Center, Ramat Gan 52621, Israel"},{"name":"Amalia Biron Research Institute of Thrombosis and Hemostasis, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1279-2557","authenticated-orcid":false,"given":"Tami","family":"Livnat","sequence":"additional","affiliation":[{"name":"National Hemophilia Center, Sheba Medical Center, Ramat Gan 52621, Israel"},{"name":"Amalia Biron Research Institute of Thrombosis and Hemostasis, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3971-0336","authenticated-orcid":false,"given":"Sarina","family":"Levy-Mendelovich","sequence":"additional","affiliation":[{"name":"National Hemophilia Center, Sheba Medical Center, Ramat Gan 52621, Israel"},{"name":"Amalia Biron Research Institute of Thrombosis and Hemostasis, Gray School of Medicine, Tel Aviv University, Tel Aviv 52621, Israel"},{"name":"The Sheba Talpiot Medical Leadership Program, Sheba Medical Center, Tel Hashomer, Ramat Gan 52621, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"720","DOI":"10.1016\/j.bbrc.2003.09.074","article-title":"Identification of a nuclear protein that promotes NF-kappa B activation","volume":"310","author":"Chen","year":"2003","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"3177","DOI":"10.1093\/nar\/gkt1311","article-title":"NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins","volume":"42","author":"Burgute","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"4987","DOI":"10.4049\/jimmunol.1501653","article-title":"NKAP regulates invariant NKT cell proliferation and differentiation into ROR-\u03b3t-expressing NKT17 cells","volume":"196","author":"Thapa","year":"2016","journal-title":"J. Immunol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Liu, J., Zhang, M., Kan, Y., Wang, W., Liu, J., Gong, J., and Yang, J. (2021). Nuclear Factor-\u03baB Activating Protein plays an oncogenic role in neuroblastoma tumorigenesis and recurrence through the phosphatidylinositol 3-kinase\/protein kinase B signaling pathway. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.622793"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2684","DOI":"10.1182\/blood-2010-02-268391","article-title":"Adult hematopoietic stem cells require NKAP for maintenance and survival","volume":"116","author":"Pajerowski","year":"2010","journal-title":"Blood"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"987","DOI":"10.1016\/j.ajhg.2019.09.009","article-title":"Missense mutations in NKAP cause a disorder of transcriptional regulation characterized by Marfanoid habitus and cognitive impairment","volume":"105","author":"Fiordaliso","year":"2019","journal-title":"Am. J. Hum. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1002\/ajmg.a.37378","article-title":"Tentative clinical diagnosis of Lujan-Fryns syndrome\u2014A conglomeration of different genetic entities?","volume":"170A","author":"Hackmann","year":"2016","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1554","DOI":"10.1182\/blood.V67.6.1554.1554","article-title":"Carrier detection in hemophilia A: A cooperative international study. I. The carrier phenotype","volume":"67","author":"Graham","year":"1986","journal-title":"Blood"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e164","DOI":"10.1111\/hae.14186","article-title":"Genetic causes of haemophilia in women and girls","volume":"27","author":"Miller","year":"2021","journal-title":"Haemophilia"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Dardik, R., Avishai, E., Lalezari, S., Barg, A.A., Levy-Mendelovich, S., Budnik, I., Barel, O., Khavkin, Y., Kenet, G., and Livnat, T. (2021). Molecular mechanisms of skewed X-chromosome inactivation in female hemophilia patients - lessons from wide genome analyses. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22169074"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1016\/j.ydbio.2018.10.008","article-title":"Mosaicism in CRISPR\/Cas9-mediated genome editing","volume":"445","author":"Mehravar","year":"2019","journal-title":"Dev. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"16428","DOI":"10.1038\/s41598-022-20665-y","article-title":"Simultaneous monitoring of mouse grip strength, force profile, and cumulative force profile distinguishes muscle physiology following surgical, pharmacologic and diet interventions","volume":"12","author":"Munier","year":"2022","journal-title":"Sci. Rep."},{"key":"ref_13","first-page":"1229","article-title":"Methylation of HpaII and HhaI sites near the polymorphic CAG repeat in the human androgen receptor gene correlates with X chromosome inactivation","volume":"51","author":"Allen","year":"1992","journal-title":"Am. J. Hum. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5091","DOI":"10.2147\/CMAR.S178919","article-title":"NKAP functions as an oncogene and its expression is induced by CoCl2 treatment in breast cancer via AKT\/mTOR signaling pathway","volume":"10","author":"Liu","year":"2018","journal-title":"Cancer Manag. Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3451","DOI":"10.1158\/0008-5472.CAN-19-3663","article-title":"p27 as a transcriptional regulator: New roles in development and cancer","volume":"80","author":"Razavipour","year":"2020","journal-title":"Cancer Res."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Creff, J., and Besson, A. (2020). Functional versatility of the CDK Inhibitor p57Kip2. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.584590"},{"key":"ref_17","first-page":"2853","article-title":"Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor","volume":"6","author":"Patil","year":"2008","journal-title":"PLoS Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1798","DOI":"10.1038\/nprot.2009.191","article-title":"Protocols to detect senescence-associated beta-galactosidase (SA-\u03b2gal) activity, a biomarker of senescent cells in culture and in vivo","volume":"4","author":"Erusalimsky","year":"2009","journal-title":"Nat. Protoc."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"352","DOI":"10.4049\/immunohorizons.1900052","article-title":"The interaction between NKAP and HDAC3 is critical for T cell maturation","volume":"3","author":"Dash","year":"2019","journal-title":"Immunohorizons"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"696","DOI":"10.1016\/j.immuni.2009.02.011","article-title":"NKAP is a transcriptional repressor of notch signaling and is required for T cell development","volume":"30","author":"Pajerowski","year":"2009","journal-title":"Immunity"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.1084\/jem.20101874","article-title":"NKAP is required for T cell maturation and acquisition of functional competency","volume":"208","author":"Hsu","year":"2011","journal-title":"J. Exp. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1038\/s41419-022-04524-2","article-title":"RNA binding protein NKAP protects glioblastoma cells from ferroptosis by promoting SLC7A11 mRNA splicing in an m6A-dependent manner","volume":"13","author":"Sun","year":"2022","journal-title":"Cell Death Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e18305","DOI":"10.1111\/jcmm.18305","article-title":"From phenotype to mechanism: Prenatal spectrum of NKAP mutation-related disorder and its pathogenesis inducing congenital heart disease","volume":"28","author":"Xu","year":"2024","journal-title":"J. Cell. Mol. Med."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Timotius, I.K., Roelofs, R.F., Richmond-Hacham, B., Noldus, L.P.J.J., von H\u00f6rsten, S., and Bikovski, L. (2023). CatWalk XT gait parameters: A review of reported parameters in pre-clinical studies of multiple central nervous system and peripheral nervous system disease models. Front. Behav. Neurosci., 17.","DOI":"10.3389\/fnbeh.2023.1147784"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1089\/neu.2006.23.537","article-title":"CatWalk-assisted gait analysis in the assessment of spinal cord injury","volume":"23","author":"Hamers","year":"2006","journal-title":"J. Neurotrauma"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Maricelli, J.W., Lu, Q.L., Lin, D.C., and Rodgers, B.D. (2016). Trendelenburg-like gait, instability and altered step patterns in a mouse model for limb girdle muscular dystrophy 2i. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0161984"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Parvathy, S.S., and Masocha, W. (2013). Gait analysis of C57BL\/6 mice with complete Freund\u2019s adjuvant-induced arthritis using the CatWalk system. BMC Musculoskelet. Disord., 14.","DOI":"10.1186\/1471-2474-14-14"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"365","DOI":"10.1007\/s12031-019-01422-9","article-title":"Motor effects of minimal traumatic brain injury in mice","volume":"70","author":"Namdar","year":"2020","journal-title":"J. Mol. Neurosci."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Bernardes, D., and Oliveira, A.L.R. (2017). Comprehensive catwalk gait analysis in a chronic model of multiple sclerosis subjected to treadmill exercise training. BMC Neurol., 17.","DOI":"10.1186\/s12883-017-0941-z"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1016\/j.molcel.2020.09.022","article-title":"Characterization of metabolic patterns in mouse oocytes during meiotic maturation","volume":"80","author":"Li","year":"2020","journal-title":"Mol. Cell"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Shapiro, M.J., Anderson, J., Lehrke, M.J., Chen, M., Nelson Holte, M., and Shapiro, V.S. (2019). NKAP regulates senescence and cell death pathways in hematopoietic progenitors. Front. Cell. Dev. Biol., 7.","DOI":"10.3389\/fcell.2019.00214"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"804","DOI":"10.1002\/humu.23012","article-title":"Identification of intellectual disability genes in female patients with a skewed X-inactivation pattern","volume":"37","author":"Fieremans","year":"2016","journal-title":"Hum. Mutat."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1016\/j.jneumeth.2012.02.001","article-title":"Gait analysis as a method for assessing neurological outcome in a mouse model of stroke","volume":"206","author":"Hetze","year":"2012","journal-title":"J. Neurosci. Methods"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"21372","DOI":"10.1038\/s41598-021-00625-8","article-title":"Gait performance of adolescent mice assessed by the CatWalk XT depends on age, strain and sex and correlates with speed and body weight","volume":"11","author":"Pitzer","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1111\/j.1399-0004.2004.00310.x","article-title":"The dynamics of X-inactivation skewing as women age","volume":"66","author":"Hatakeyama","year":"2004","journal-title":"Clin. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"29762","DOI":"10.1038\/s41598-025-11822-0","article-title":"RQdeltaCT: An open-source R package for relative quantification of gene expression using delta Ct methods","volume":"15","author":"Zalewski","year":"2025","journal-title":"Sci. Rep."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Riedel, G., R\u00fcdrich, U., Fekete-Drimusz, N., Manns, M.P., Vondran, F.W., and Bock, M. (2014). An extended \u0394CT-method facilitating normalisation with multiple reference genes suited for quantitative RT-PCR analyses of human hepatocyte-like cells. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0093031"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Huang, E., Bi, Y., Jiang, W., Luo, X., Yang, K., Gao, J.L., Gao, Y., Luo, Q., Shi, Q., and Kim, S.H. (2012). Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0032428"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1002\/fft2.44","article-title":"Guidelines for cell viability assays","volume":"1","author":"Kamiloglu","year":"2020","journal-title":"Food Front."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2330\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T09:48:55Z","timestamp":1772531335000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/5\/2330"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,2]]},"references-count":40,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["ijms27052330"],"URL":"https:\/\/doi.org\/10.3390\/ijms27052330","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,2]]}}}