{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T17:53:02Z","timestamp":1782237182572,"version":"3.54.5"},"update-to":[{"DOI":"10.1371\/journal.pgen.1012167","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000}}],"reference-count":48,"publisher":"Public Library of Science (PLoS)","issue":"6","license":[{"start":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T00:00:00Z","timestamp":1781654400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000071","name":"National Institute of Child Health and Human Development","doi-asserted-by":"publisher","award":["R01HD114863"],"award-info":[{"award-number":["R01HD114863"]}],"id":[{"id":"10.13039\/100000071","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Urea Cycle Disorders Foundation"}],"content-domain":{"domain":["www.plosgenetics.org"],"crossmark-restriction":false},"short-container-title":["PLoS Genet"],"abstract":"<jats:p>\n                    Sequence variants in the urea cycle gene argininosuccinate synthase (\n                    <jats:italic>ASS1<\/jats:italic>\n                    ) cause Citrullinemia type 1 (CTLN1), a rare autosomal recessive disease. Mechanistically, reduction in argininosuccinate synthetase (ASS) enzyme activity impairs the urea cycle, leading to an accumulation of citrulline and neurotoxic ammonia. Disease severity varies according to the degree of enzyme impairment, ranging from severe neonatal forms (classic citrullinemia) to milder, late-onset forms that may manifest in childhood or adulthood. We established a high-throughput yeast functional assay of human ASS and individually measured the impact of 2,193 amino acid substitutions, representing 90% of all single nucleotide variant (SNV)-accessible substitutions. When benchmarked against existing clinical variant annotation, our assay distinguishes known benign variants from strong loss of function pathogenic variants, enabling identification of a functional score threshold below which variants show clinically relevant impairment of ASS activity. Using the ACMG OddsPath framework, our assay meets PS3_supporting criteria for pathogenicity classification and achieves full PS3-level strength when variants observed as homozygotes in other primates are used as benign proxies for calibration. These results provide direct functional evidence to inform reclassification of\n                    <jats:italic>ASS1<\/jats:italic>\n                    missense variants. Under the current ACMG guidelines, inclusion of our data yielded definitive classifications (pathogenic or likely pathogenic) for all 25 ClinVar VUS falling in the functionally impaired range of our assay. Mapping functional scores onto the protein structure, we confirmed that residues involved in catalysis are highly sensitive to substitution. In addition, we identified residues from adjacent subunits of the ASS homotetramer that form compound active sites. Assaying these positions revealed a capacity for intragenic complementation consistent with a variant sequestration model: a form of positive epistasis in which deleterious variants from different subunits are sequestered into only a subset of active sites, restoring function in the remaining variant-free sites. The discovery of intragenic complementation in ASS reveals a novel mode of functional interaction with clinical implications for interpreting variant combinations in heterozygous individuals.\n                  <\/jats:p>","DOI":"10.1371\/journal.pgen.1012167","type":"journal-article","created":{"date-parts":[[2026,6,17]],"date-time":"2026-06-17T17:36:51Z","timestamp":1781717811000},"page":"e1012167","update-policy":"https:\/\/doi.org\/10.1371\/journal.pgen.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional profiling of 2,193 ASS1 missense variants: Insights into variant pathogenicity and epistatic interactions in citrullinemia type I"],"prefix":"10.1371","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0369-7259","authenticated-orcid":true,"given":"Russell S.","family":"Lo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5265-1970","authenticated-orcid":true,"given":"Gareth A.","family":"Cromie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8779-8697","authenticated-orcid":true,"given":"Michelle","family":"Tang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy","family":"Sirr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9140-5585","authenticated-orcid":true,"given":"Ljubica","family":"Caldovic","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroki","family":"Morizono","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas","family":"Ah Mew","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Gropman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3644-0625","authenticated-orcid":true,"given":"Aim\u00e9e M.","family":"Dudley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,6,17]]},"reference":[{"issue":"10","key":"pgen.1012167.ref001","doi-asserted-by":"crossref","first-page":"710","DOI":"10.1136\/jmedgenet-2016-103937","article-title":"Kinetic mutations in argininosuccinate synthetase deficiency: characterisation and in vitro correction by substrate supplementation","volume":"53","author":"C Diez-Fernandez","year":"2016","journal-title":"J Med Genet"},{"key":"pgen.1012167.ref002","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.ymgme.2013.07.008","article-title":"The incidence of urea cycle disorders","volume":"110","author":"ML Summar","year":"2013","journal-title":"Mol Genet Metab"},{"issue":"4","key":"pgen.1012167.ref003","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.ymgme.2008.05.004","article-title":"Cross-sectional multicenter study of patients with urea cycle disorders in the United States","volume":"94","author":"M Tuchman","year":"2008","journal-title":"Mol Genet Metab"},{"issue":"1","key":"pgen.1012167.ref004","doi-asserted-by":"crossref","DOI":"10.1111\/ped.70121","article-title":"Hyperammonemia in urea cycle disorders: a toxic metabolite for the brain","volume":"67","author":"J Kido","year":"2025","journal-title":"Pediatr Int"},{"issue":"9","key":"pgen.1012167.ref005","doi-asserted-by":"crossref","first-page":"1858","DOI":"10.1002\/acn3.50886","article-title":"Early prediction of phenotypic severity in Citrullinemia Type 1","volume":"6","author":"M Zielonka","year":"2019","journal-title":"Ann Clin Transl Neurol"},{"key":"pgen.1012167.ref006","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1007\/978-3-662-49771-5_19","article-title":"Disorders of the urea cycle and related enzymes.","volume-title":"Inborn Metabolic Diseases","author":"J H\u00e4berle","year":"2016"},{"issue":"3","key":"pgen.1012167.ref007","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1016\/j.ymgme.2003.08.002","article-title":"Mild citrullinemia in Caucasians is an allelic variant of argininosuccinate synthetase deficiency (citrullinemia type 1)","volume":"80","author":"J H\u00e4berle","year":"2003","journal-title":"Mol Genet Metab"},{"issue":"10","key":"pgen.1012167.ref008","doi-asserted-by":"crossref","first-page":"1222","DOI":"10.1002\/humu.20784","article-title":"Investigation of citrullinemia type I variants by in vitro expression studies","volume":"29","author":"C Berning","year":"2008","journal-title":"Hum Mutat"},{"key":"pgen.1012167.ref009","doi-asserted-by":"crossref","DOI":"10.1136\/bcr-2017-220193","article-title":"Hypomorphic citrullinaemia due to mutated ASS1 with episodic ataxia","volume":"2018","author":"AG Saini","year":"2018","journal-title":"BMJ Case Rep"},{"issue":"1","key":"pgen.1012167.ref010","first-page":"20","article-title":"Rare adult-onset citrullinemia type 1 in the postpartum period: a case report","volume":"7","author":"M Borsuk","year":"2023","journal-title":"Clin Pract Cases Emerg Med"},{"key":"pgen.1012167.ref011","article-title":"Urea Cycle Disorders Overview.","volume-title":"GeneReviews","author":"N Ah Mew","year":"1993"},{"issue":"5","key":"pgen.1012167.ref012","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":"S Richards","year":"2015","journal-title":"Genet Med"},{"key":"pgen.1012167.ref013","volume-title":"Methods in Yeast Genetics: A Laboratory Course Manual","author":"M Rose","year":"1990"},{"issue":"1","key":"pgen.1012167.ref014","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1038\/nprot.2007.13","article-title":"High-efficiency yeast transformation using the LiAc\/SS carrier DNA\/PEG method","volume":"2","author":"RD Gietz","year":"2007","journal-title":"Nat Protoc"},{"issue":"5","key":"pgen.1012167.ref015","doi-asserted-by":"crossref","first-page":"863","DOI":"10.1016\/j.ajhg.2023.03.019","article-title":"The functional impact of 1,570 individual amino acid substitutions in human OTC","volume":"110","author":"RS Lo","year":"2023","journal-title":"Am J Hum Genet"},{"key":"pgen.1012167.ref016"},{"key":"pgen.1012167.ref017"},{"issue":"5","key":"pgen.1012167.ref018","doi-asserted-by":"crossref","first-page":"471","DOI":"10.1002\/humu.23184","article-title":"Mutations in the human argininosuccinate synthetase (ASS1) gene, impact on patients, common changes, and structural considerations","volume":"38","author":"C Diez-Fernandez","year":"2017","journal-title":"Hum Mutat"},{"issue":"1","key":"pgen.1012167.ref019","doi-asserted-by":"crossref","first-page":"4026","DOI":"10.1038\/s41467-024-48481-0","article-title":"Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants","volume":"15","author":"M Gr\u00f8nb\u00e6k-Thygesen","year":"2024","journal-title":"Nat Commun"},{"issue":"3","key":"pgen.1012167.ref020","doi-asserted-by":"crossref","DOI":"10.15252\/msb.20199265","article-title":"CRISPR\/Cas9 recombineering-mediated deep mutational scanning of essential genes in Escherichia coli","volume":"16","author":"A Choudhury","year":"2020","journal-title":"Mol Syst Biol"},{"key":"pgen.1012167.ref021"},{"key":"pgen.1012167.ref022","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1107\/S0907444907067455","article-title":"Structure of human argininosuccinate synthetase","volume":"64","author":"T Karlberg","year":"2008","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"issue":"15","key":"pgen.1012167.ref023","doi-asserted-by":"crossref","first-page":"13074","DOI":"10.1074\/jbc.M112436200","article-title":"Substrate induced conformational changes in argininosuccinate synthetase","volume":"277","author":"CT Lemke","year":"2002","journal-title":"J Biol Chem"},{"issue":"18","key":"pgen.1012167.ref024","doi-asserted-by":"crossref","first-page":"15890","DOI":"10.1074\/jbc.M112430200","article-title":"Crystal structure of argininosuccinate synthetase from Thermus thermophilus HB8. Structural basis for the catalytic action","volume":"277","author":"M Goto","year":"2002","journal-title":"J Biol Chem"},{"issue":"25","key":"pgen.1012167.ref025","doi-asserted-by":"crossref","first-page":"22964","DOI":"10.1074\/jbc.M213198200","article-title":"Structures of argininosuccinate synthetase in enzyme-ATP substrates and enzyme-AMP product forms: stereochemistry of the catalytic reaction","volume":"278","author":"M Goto","year":"2003","journal-title":"J Biol Chem"},{"issue":"4","key":"pgen.1012167.ref026","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1002\/prot.340200407","article-title":"A P-loop-like motif in a widespread ATP pyrophosphatase domain: implications for the evolution of sequence motifs and enzyme activity","volume":"20","author":"P Bork","year":"1994","journal-title":"Proteins"},{"key":"pgen.1012167.ref027","doi-asserted-by":"crossref","DOI":"10.1093\/nar\/gkae1090","article-title":"ClinVar: updates to support classifications of both germline and somatic variants","volume":"53","author":"MJ Landrum","year":"2025","journal-title":"Nucleic Acids Res"},{"issue":"1","key":"pgen.1012167.ref028","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/s13073-019-0690-2","article-title":"Recommendations for application of the functional evidence PS3\/BS3 criterion using the ACMG\/AMP sequence variant interpretation framework","volume":"12","author":"SE Brnich","year":"2019","journal-title":"Genome Med"},{"issue":"10","key":"pgen.1012167.ref029","doi-asserted-by":"crossref","first-page":"1734","DOI":"10.1002\/humu.24088","article-title":"Fitting a naturally scaled point system to the ACMG\/AMP variant classification guidelines","volume":"41","author":"SV Tavtigian","year":"2020","journal-title":"Hum Mutat"},{"key":"pgen.1012167.ref030"},{"key":"pgen.1012167.ref031","article-title":"The landscape of tolerated genetic variation in humans and primates","author":"H Gao","year":"2023","journal-title":"bioRxiv"},{"issue":"3","key":"pgen.1012167.ref032","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1016\/j.clinbiochem.2012.10.008","article-title":"Mutation spectrum of the ASS1 gene in Korean patients with citrullinemia type I","volume":"46","author":"HI Woo","year":"2013","journal-title":"Clin Biochem"},{"key":"pgen.1012167.ref033","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1186\/s13023-014-0187-4","article-title":"Urea cycle disorders in Spain: an observational, cross-sectional and multicentric study of 104 cases","volume":"9","author":"E Mart\u00edn-Hern\u00e1ndez","year":"2014","journal-title":"Orphanet J Rare Dis"},{"issue":"1","key":"pgen.1012167.ref034","doi-asserted-by":"crossref","first-page":"24","DOI":"10.1002\/humu.10230","article-title":"Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients","volume":"22","author":"H-Z Gao","year":"2003","journal-title":"Hum Mutat"},{"key":"pgen.1012167.ref035","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1258\/000456303321016259","article-title":"Moderate citrullinaemia without hyperammonaemia in a child with mutated and deficient argininosuccinate synthetase","volume":"40","author":"W Ruitenbeek","year":"2003","journal-title":"Ann Clin Biochem"},{"issue":"3","key":"pgen.1012167.ref036","first-page":"231","article-title":"Clinical, laboratory data and outcomes of 17 Iranian citrullinemia type 1 patients: Identification of five novel ASS1 gene mutations","volume":"63","author":"S Moarefian","year":"2022","journal-title":"JIMD Rep"},{"issue":"22","key":"pgen.1012167.ref037","doi-asserted-by":"crossref","first-page":"2885","DOI":"10.1002\/ajmg.a.32527","article-title":"The role of molecular testing and enzyme analysis in the management of hypomorphic citrullinemia","author":"DP Dimmock","year":"2008","journal-title":"Am J Med Genet A"},{"issue":"3","key":"pgen.1012167.ref038","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1002\/humu.20847","article-title":"Mutations and polymorphisms in the human argininosuccinate synthetase (ASS1) gene","volume":"30","author":"K Engel","year":"2009","journal-title":"Hum Mutat"},{"issue":"2","key":"pgen.1012167.ref039","doi-asserted-by":"crossref","DOI":"10.1002\/mgg3.2058","article-title":"Analysis of ASS1 gene in ten unrelated middle eastern families with citrullinemia type 1 identifies rare and novel variants","volume":"11","author":"M Daou","year":"2023","journal-title":"Mol Genet Genomic Med"},{"key":"pgen.1012167.ref040","doi-asserted-by":"crossref","first-page":"46","DOI":"10.1016\/j.ymgme.2014.07.003","article-title":"Biochemical and molecular characteristics of patients with organic acidaemias and urea cycle disorders identified through newborn screening","volume":"113","author":"Newborn Screening Laboratory Staff","year":"2014","journal-title":"Mol Genet Metab"},{"issue":"6","key":"pgen.1012167.ref041","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1007\/s00431-003-1177-z","article-title":"Neonatal screening for citrullinaemia","volume":"162","author":"J Sander","year":"2003","journal-title":"Eur J Pediatr"},{"issue":"10","key":"pgen.1012167.ref042","article-title":"Constructing and interpreting a large-scale variant effect map for an ultrarare disease gene: comprehensive prediction of the functional impact of PSAT1 genotypes","volume":"19","author":"MJ Xie","year":"2023","journal-title":"PLoS Genet"},{"key":"pgen.1012167.ref043","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/S0022-2836(64)80156-X","article-title":"The theory of inter-allelic complementation","volume":"8","author":"FH Crick","year":"1964","journal-title":"J Mol Biol"},{"key":"pgen.1012167.ref044"},{"issue":"8","key":"pgen.1012167.ref045","doi-asserted-by":"crossref","DOI":"10.1002\/ajmg.a.63609","article-title":"When rare meets common: treatable genetic diseases are enriched in the general psychiatric population","volume":"194","author":"V Sriretnakumar","year":"2024","journal-title":"Am J Med Genet A"},{"issue":"2","key":"pgen.1012167.ref046","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.ejogrb.2009.11.014","article-title":"First manifestation of citrullinemia type I as differential diagnosis to postpartum psychosis in the puerperal period","volume":"149","author":"J H\u00e4berle","year":"2010","journal-title":"Eur J Obstet Gynecol Reprod Biol"},{"issue":"3","key":"pgen.1012167.ref047","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1001\/jama.2024.19662","article-title":"Expanded newborn screening using genome sequencing for early actionable conditions","volume":"333","author":"A Ziegler","year":"2025","journal-title":"JAMA"},{"issue":"1","key":"pgen.1012167.ref048","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1159\/000541935","article-title":"Co-creating the experience of consent for newborn genome sequencing: the generation study","volume":"27","author":"M Leblond","year":"2024","journal-title":"Public Health Genomics"}],"updated-by":[{"DOI":"10.1371\/journal.pgen.1012167","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T00:00:00Z","timestamp":1782172800000}}],"container-title":["PLOS Genetics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pgen.1012167","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,23]],"date-time":"2026-06-23T17:43:25Z","timestamp":1782236605000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pgen.1012167"}},"subtitle":[],"editor":[{"given":"Gregory M","family":"Findlay","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,6,17]]},"references-count":48,"journal-issue":{"issue":"6","published-online":{"date-parts":[[2026,6,17]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pgen.1012167","relation":{},"ISSN":["1553-7404"],"issn-type":[{"value":"1553-7404","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,17]]}}}