{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T10:54:04Z","timestamp":1790679244991,"version":"4.1.0"},"reference-count":28,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,12,29]],"date-time":"2022-12-29T00:00:00Z","timestamp":1672272000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health, Institute of Child Health and Human Development","doi-asserted-by":"publisher","award":["HD110576"],"award-info":[{"award-number":["HD110576"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health, Institute of Child Health and Human Development","doi-asserted-by":"publisher","award":["RIN2018"],"award-info":[{"award-number":["RIN2018"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"name":"European Union","award":["HD110576"],"award-info":[{"award-number":["HD110576"]}]},{"name":"European Union","award":["RIN2018"],"award-info":[{"award-number":["RIN2018"]}]},{"name":"European Regional Development Fund (ERDF)","award":["HD110576"],"award-info":[{"award-number":["HD110576"]}]},{"name":"European Regional Development Fund (ERDF)","award":["RIN2018"],"award-info":[{"award-number":["RIN2018"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Asparagine Synthetase Deficiency (ASNSD) is a disease caused by mutations in asparagine synthetase (ASNS). Newborns exhibit microcephaly, intractable epileptic-like seizures, progressive brain atrophy, and axial hypotonia. ASNSD results in global developmental delays and premature death. The present report describes a 9-year-old child who is a compound heterozygote with ASNS mutations c.1439C &gt; T and c.239A &gt; G leading to variants p.S480F and p.N80S, respectively. When grown in a complete culture medium, primary fibroblasts from the child contained ASNS mRNA and protein levels similar to an unrelated wild-type fibroblast cell line. When the child\u2019s fibroblasts were cultured for up to 72 h in a medium lacking asparagine, proliferation was reduced by about 50%. Purification of ASNS proteins harboring either the S480F or the N80S substitution had reduced enzymatic activity by 80% and 50%, respectively. Ectopic expression of either variant in ASNS-null Jensen rat sarcoma (JRS) cells did not support proliferation in the absence of medium-supplied asparagine, whereas expression of wild-type enzyme completely restored growth. These studies add to the list of pathogenic ASNS variants and use enzyme activity and protein expression in ASNS-null cells to expand our knowledge of the biological impact of mutations in the ASNS gene.<\/jats:p>","DOI":"10.3390\/ijms24010559","type":"journal-article","created":{"date-parts":[[2022,12,29]],"date-time":"2022-12-29T02:56:50Z","timestamp":1672282610000},"page":"559","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Analysis of Enzyme Activity and Cellular Function for the N80S and S480F Asparagine Synthetase Variants Expressed in a Child with Asparagine Synthetase Deficiency"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1746-6320","authenticated-orcid":false,"given":"Stephen J.","family":"Staklinski","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610, USA"},{"name":"School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8382-056X","authenticated-orcid":false,"given":"Sarah","family":"Snanoudj","sequence":"additional","affiliation":[{"name":"Department of Metabolic Biochemistry, Rouen University Hospital, F-76000 Rouen, France"},{"name":"University Normandie UNIROUEN, CHU Rouen, INSERM U1245, F-76000 Rouen, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anne-Marie","family":"Guerrot","sequence":"additional","affiliation":[{"name":"University Normandie UNIROUEN, CHU Rouen, INSERM U1245, F-76000 Rouen, France"},{"name":"Department of Genetics and Reference Center for Developmental Disorders, Rouen University Hospital, F-76000 Rouen, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catherine","family":"Vanhulle","sequence":"additional","affiliation":[{"name":"Department of Neonatalogy, Pediatric Intensive Care and Neuropediatrics, Rouen University Hospital, F-76000 Rouen, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fran\u00e7ois","family":"Lecoquierre","sequence":"additional","affiliation":[{"name":"University Normandie UNIROUEN, CHU Rouen, INSERM U1245, F-76000 Rouen, France"},{"name":"Department of Genetics and Reference Center for Developmental Disorders, Rouen University Hospital, F-76000 Rouen, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6184-7270","authenticated-orcid":false,"given":"Soumeya","family":"Bekri","sequence":"additional","affiliation":[{"name":"Department of Metabolic Biochemistry, Rouen University Hospital, F-76000 Rouen, France"},{"name":"University Normandie UNIROUEN, CHU Rouen, INSERM U1245, F-76000 Rouen, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael S.","family":"Kilberg","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32610, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1146\/annurev.biochem.75.103004.142520","article-title":"Asparagine synthetase chemotherapy","volume":"75","author":"Richards","year":"2006","journal-title":"Annu. Rev. Biochem."},{"key":"ref_2","first-page":"2350","article-title":"Organization and expression of the cell cycle gene, ts 11, that encodes asparagine synthetase","volume":"9","author":"Greco","year":"1989","journal-title":"Mol. Cell. Biol."},{"key":"ref_3","first-page":"259","article-title":"Molecular structure of the human asparagine synthetase gene","volume":"4","author":"Zhang","year":"1989","journal-title":"Gene"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1159\/000133685","article-title":"Refined localization of the asparagine synthetase gene (ASNA) to chromosome 7, region q 21.3,and characterization of teh somatic cell hybrid line 4AF\/106\/KO15","volume":"66","author":"Heng","year":"1994","journal-title":"Cytogenet. Cell Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"E789","DOI":"10.1152\/ajpendo.00015.2013","article-title":"Asparagine Synthetase: Regulation by Cell Stress and Involvement in Tumor Biology","volume":"304","author":"Balasubramanian","year":"2013","journal-title":"Am. J. Physiology. Endocrinol. Metab."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"19952","DOI":"10.1074\/jbc.R117.819060","article-title":"Asparagine synthetase: Function, structure, and role in disease","volume":"292","author":"Lomelino","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1038\/s42003-019-0587-z","article-title":"High-resolution crystal structure of human asparagine synthetase enables analysis of inhibitor binding and selectivity","volume":"2","author":"Zhu","year":"2019","journal-title":"Commun. Biol"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"16146","DOI":"10.1021\/bi9915768","article-title":"Three-dimensional structure of Escherichia coli asparagine synthetase B: A short journey from substrate to product","volume":"38","author":"Larsen","year":"1999","journal-title":"Biochemistry"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/S0003-9861(03)00118-8","article-title":"Revisiting the steady state kinetic mechanism of glutamine-dependent asparagine synthetase from Escherichia coli","volume":"413","author":"Tesson","year":"2003","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"429","DOI":"10.1016\/j.neuron.2013.08.013","article-title":"Deficiency of asparagine synthetase causes congenital microcephaly and a progressive form of encephalopathy","volume":"80","author":"Ruzzo","year":"2013","journal-title":"Neuron"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.cca.2022.04.989","article-title":"An intractable epilepsy phenotype of ASNS novel mutation in two patients with asparagine synthetase deficiency","volume":"531","author":"Liu","year":"2022","journal-title":"Clin. Chim. Acta"},{"key":"ref_12","unstructured":"Adam, M.P., Ardinger, H.H., Pagon, R.A., Wallace, S.E., Bean, L.J.H., Stephens, K., and Amemiya, A. (2018). Asparagine Synthetase Deficiency. GeneReviews\u00ae, University of Washington."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2071","DOI":"10.1038\/s41436-020-0919-x","article-title":"Clinical, molecular, and biochemical delineation of asparagine synthetase deficiency in Saudi cohort","volume":"22","author":"Alharby","year":"2020","journal-title":"Genet Med."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1016\/j.ymgme.2017.12.433","article-title":"Characterization of a novel variant in siblings with Asparagine Synthetase Deficiency","volume":"123","author":"Sacharow","year":"2018","journal-title":"Mol. Genet. Metab."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"178","DOI":"10.1016\/j.ymgme.2015.08.007","article-title":"Asparagine Synthetase Deficiency causes reduced proliferation of cells under conditions of limited asparagine","volume":"116","author":"Palmer","year":"2015","journal-title":"Mol. Genet. Metab."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1226","DOI":"10.1002\/jimd.12408","article-title":"In vitro functional analysis of four variants of human asparagine synthetase","volume":"44","author":"Matsumoto","year":"2021","journal-title":"J. Inherit. Metab. Dis."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"102385","DOI":"10.1016\/j.jbc.2022.102385","article-title":"Cellular and molecular characterization of two novel asparagine synthetase gene mutations linked to Asparagine Synthetase Deficiency","volume":"298","author":"Staklinski","year":"2022","journal-title":"J. Biol. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"218S","DOI":"10.1093\/jn\/136.1.218S","article-title":"Structure of the blood-brain barrier and its role in the transport of amino acids","volume":"136","author":"Hawkins","year":"2006","journal-title":"J. Nutr."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1055\/s-0036-1586222","article-title":"Epileptic Phenotype of Two Siblings with Asparagine Synthesis Deficiency Mimics Neonatal Pyridoxine-Dependent Epilepsy","volume":"47","author":"Gataullina","year":"2016","journal-title":"Neuropediatrics"},{"key":"ref_20","first-page":"1937","article-title":"Properties of asparagine synthetase in asparagine-independent variants of Jensen rat sarcoma cells induced by 5-azacytidine","volume":"3","author":"Sugiyama","year":"1983","journal-title":"Mol. Cell. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"589","DOI":"10.1093\/bioinformatics\/btp698","article-title":"Fast and accurate long-read alignment with Burrows-Wheeler transform","volume":"26","author":"Li","year":"2010","journal-title":"Bioinformatics"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.1101\/gr.107524.110","article-title":"The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data","volume":"20","author":"McKenna","year":"2010","journal-title":"Genome Res."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"80","DOI":"10.4161\/fly.19695","article-title":"A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3","volume":"6","author":"Cingolani","year":"2012","journal-title":"Fly"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"e97","DOI":"10.1093\/nar\/gku345","article-title":"CANOES: Detecting rare copy number variants from whole exome sequencing data","volume":"42","author":"Backenroth","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1038\/gim.2015.30","article-title":"Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology","volume":"17","author":"Richards","year":"2015","journal-title":"Genet. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"844","DOI":"10.1016\/j.str.2011.03.019","article-title":"A smoothed backbone-dependent rotamer library for proteins derived from adaptive kernel density estimates and regressions","volume":"19","author":"Shapovalov","year":"2011","journal-title":"Structure"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2\u2212\u0394\u0394CT Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1016\/1046-5928(92)90010-T","article-title":"High-level expression of human asparagine synthetase and production of monoclonal antibodies for enzyme purification","volume":"3","author":"Sheng","year":"1992","journal-title":"Prot. Exp. 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