{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T03:57:26Z","timestamp":1792468646358,"version":"4.5.0"},"reference-count":43,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T00:00:00Z","timestamp":1767744000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background: The STAT4 gene plays a key role in immune regulation and is associated with susceptibility to autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Objectives: The objective of this study is to analyze variants of uncertain significance (VUSs) in STAT4 using bioinformatics tools to predict their functional and structural impact. Methods: A total of 48,295 variants of the STAT4 gene (ENSG00000138378) were retrieved from the Ensembl database. A tiered filtering approach was used to assess VUS pathogenicity, integrating in silico prediction tools such as SIFT, PolyPhen, MutPred2, and Align-GVGD, as well as structural modeling platforms including Chimera, ModRefiner, Missense3D, HOPE, and DynaMut2. Results: Eighty missense VUSs were identified; of these, 13 were prioritized based on concordant signals across multiple computational predictors. These variants showed significant alterations in the physicochemical properties of the protein, including changes in hydrophobicity and disruption of hydrogen bonding. Notably, the rs140675301 (Glu128Val) variant lies within a conserved loop, and in silico analyses suggest that this mutation may alter kinase specificity regarding the phosphorylation of serine 130. Conclusions: The integrative use of the bioinformatic tools employed represents a valuable preliminary step prior to undertaking more complex and resource-intensive functional studies. This complementary strategy strengthens the interpretative framework for VUS, guiding subsequent experimental validation and supporting a structured assessment of variant relevance, particularly in the context of immune-related genes such as STAT4.<\/jats:p>","DOI":"10.3390\/genes17010072","type":"journal-article","created":{"date-parts":[[2026,1,7]],"date-time":"2026-01-07T08:26:56Z","timestamp":1767774416000},"page":"72","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["In Silico Functional and Structural Analysis of STAT4 Variants of Uncertain Significance"],"prefix":"10.3390","volume":"17","author":[{"given":"Karla Mayela","family":"Bravo-Villagra","sequence":"first","affiliation":[{"name":"Centro Universitario de Ciencias de la Salud, Instituto de Nutrigen\u00e9tica y Nutrigen\u00f3mica Traslacional, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"},{"name":"Programa de Doctorado en Gen\u00e9tica Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric Jonathan","family":"Maciel-Cruz","sequence":"additional","affiliation":[{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara 44340, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosa Michel","family":"Mart\u00ednez-Contreras","sequence":"additional","affiliation":[{"name":"Programa de Doctorado en Gen\u00e9tica Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"},{"name":"Centro de Investigaci\u00f3n Biom\u00e9dica de Occidente, Instituto Mexicano del Seguro Social, Guadalajara 44340, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6174-0609","authenticated-orcid":false,"given":"Itzae Adonai","family":"Guti\u00e9rrez-Hurtado","sequence":"additional","affiliation":[{"name":"Programa de Doctorado en Gen\u00e9tica Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"},{"name":"Departamento de Biolog\u00eda Molecular y Gen\u00f3mica, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-2443-0210","authenticated-orcid":false,"given":"Alexis Missael","family":"Vizca\u00edno-Quirarte","sequence":"additional","affiliation":[{"name":"Centro Universitario de Ciencias de la Salud, Instituto de Investigaci\u00f3n en Ciencias Biom\u00e9dicas, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"},{"name":"Programa de Doctorado en Psicolog\u00eda de la Salud, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2272-9260","authenticated-orcid":false,"given":"Jos\u00e9 Francisco","family":"Mu\u00f1oz-Valle","sequence":"additional","affiliation":[{"name":"Centro Universitario de Ciencias de la Salud, Instituto de Investigaci\u00f3n en Ciencias Biom\u00e9dicas, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5151-041X","authenticated-orcid":false,"given":"Andres","family":"L\u00f3pez-Quintero","sequence":"additional","affiliation":[{"name":"Centro Universitario de Ciencias de la Salud, Instituto de Nutrigen\u00e9tica y Nutrigen\u00f3mica Traslacional, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"},{"name":"Programa de Doctorado en Gen\u00e9tica Humana, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara 44340, Jalisco, Mexico"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"R62","DOI":"10.1186\/ar2975","article-title":"Influence of HLA DRB1 Alleles in the Susceptibility of Rheumatoid Arthritis and the Regulation of Antibodies against Citrullinated Proteins and Rheumatoid Factor","volume":"12","author":"Balsa","year":"2010","journal-title":"Arthritis Res. Ther."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/iji.12347","article-title":"Associative Role of HLA-DRB1 SNP Genotypes as Risk Factors for Susceptibility and Severity of Rheumatoid Arthritis: A North-East Indian Population-Based Study","volume":"45","author":"Das","year":"2018","journal-title":"Int. J. Immunogenet."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Lamana, A., L\u00f3pez-Santalla, M., Castillo-Gonz\u00e1lez, R., Ortiz, A.M., Mart\u00edn, J., Garc\u00eda-Vicu\u00f1a, R., and Gonz\u00e1lez-\u00c1lvaro, I. (2015). The Minor Allele of Rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0142683"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1070","DOI":"10.1136\/annrheumdis-2017-212794","article-title":"The STAT4 SLE Risk Allele Rs7574865[T] Is Associated with Increased IL-12-Induced IFN-\u03b3 Production in t Cells from Patients with SLE","volume":"77","author":"Hagberg","year":"2018","journal-title":"Ann. Rheum. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2909","DOI":"10.1007\/s10067-016-3320-z","article-title":"STAT4 Rs7574865 G\/T Polymorphism Is Associated with Rheumatoid Arthritis and Disease Activity, but Not with Anti-CCP Antibody Levels in a Mexican Population","volume":"35","year":"2016","journal-title":"Clin. Rheumatol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Bravo-Villagra, K.M., Mu\u00f1oz-Valle, J.F., Ba\u00f1os-Hern\u00e1ndez, C.J., Cerpa-Cruz, S., Navarro-Zarza, J.E., Parra-Rojas, I., Aguilar-Vel\u00e1zquez, J.A., Garc\u00eda-Arellano, S., and L\u00f3pez-Quintero, A. (2024). STAT4 Gene Variant Rs7574865 Is Associated with Rheumatoid Arthritis Activity and Anti-CCP Levels in the Western but Not in the Southern Population of Mexico. Genes, 15.","DOI":"10.3390\/genes15020241"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Esparza Guerrero, Y., Vazquez Villegas, M.L., Nava Valdivia, C.A., Ponce Guarneros, J.M., Perez Guerrero, E.E., Gomez Ramirez, E.E., Ramirez Villafa\u00f1a, M., Contreras Haro, B., Martinez Hernandez, A., and Cardona Mu\u00f1oz, E.G. (2023). Association of the STAT4 Gene Rs7574865 Polymorphism with IFN-\u03b3 Levels in Patients with Systemic Lupus Erythematosus. Genes, 14.","DOI":"10.3390\/genes14030537"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1575","DOI":"10.7150\/ijbs.41852","article-title":"STAT4: An Immunoregulator Contributing to Diverse Human Diseases","volume":"16","author":"Yang","year":"2020","journal-title":"Int. J. Biol. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Berg, A., Gr\u00e4ber, M., Schmutzler, S., Hoffmann, R., and Berg, T. (2022). A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of STAT4. Methods Protoc., 5.","DOI":"10.3390\/mps5060093"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1038\/s41392-021-00791-1","article-title":"The JAK\/STAT Signaling Pathway: From Bench to Clinic","volume":"6","author":"Hu","year":"2021","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1177\/1759720X18776224","article-title":"The Role of the JAK\/STAT Signal Pathway in Rheumatoid Arthritis","volume":"10","author":"Malemud","year":"2018","journal-title":"Ther. Adv. Musculoskelet. Dis."},{"key":"ref_12","first-page":"15","article-title":"Pain and the JAK-STAT Pathwsy","volume":"2018","author":"Salaffi","year":"2018","journal-title":"Pain Res. Manag."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.imlet.2016.05.003","article-title":"STAT4 Confers Risk for Rheumatoid Arthritis and Systemic Lupus Erythematosus in Mexican Patients","volume":"175","year":"2016","journal-title":"Immunol. Lett."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"150584","DOI":"10.1016\/j.bbrc.2024.150584","article-title":"In-Silico Assessment of Novel Peptidomimetics Inhibitor Targeting STAT3 and STAT4 N-Terminal Domain Dimerization: A Comprehensive Study Using Molecular Docking, Molecular Dynamics Simulation, and Binding Free Energy Analysis","volume":"733","author":"Shree","year":"2024","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1002\/humu.24298","article-title":"Annotating and Prioritizing Genomic Variants Using the Ensembl Variant Effect Predictor-A Tutorial","volume":"43","author":"Hunt","year":"2022","journal-title":"Hum. Mutat."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"bay119","DOI":"10.1093\/database\/bay119","article-title":"Ensembl Variation Resources","volume":"2018","author":"Hunt","year":"2018","journal-title":"Database"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"The UniProt Consortium (2019). UniProt: A Worldwide Hub of Protein Knowledge. Nucleic Acids Res., 47, D506\u2013D515.","DOI":"10.1093\/nar\/gky1049"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"evab151","DOI":"10.1093\/gbe\/evab151","article-title":"Hunting for Beneficial Mutations: Conditioning on SIFT Scores When Estimating the Distribution of Fitness Effect of New Mutations","volume":"14","author":"Chen","year":"2022","journal-title":"Genome Biol. Evol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1002\/humu.21490","article-title":"Prediction of Missense Mutation Functionality Depends on Both the Algorithm and Sequence Alignment Employed","volume":"32","author":"Hicks","year":"2011","journal-title":"Hum. Mutat."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1317","DOI":"10.1093\/nar\/gkj518","article-title":"Computational Approaches for Predicting the Biological Effect of P53 Missense Mutations: A Comparison of Three Sequence Analysis Based Methods","volume":"34","author":"Mathe","year":"2006","journal-title":"Nucleic Acids Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1136\/jmg.2005.033878","article-title":"Comprehensive Statistical Study of 452 BRCA1 Missense Substitutions with Classification of Eight Recurrent Substitutions as Neutral","volume":"43","author":"Tavtigian","year":"2006","journal-title":"J. Med. Genet."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5918","DOI":"10.1038\/s41467-020-19669-x","article-title":"Inferring the Molecular and Phenotypic Impact of Amino Acid Variants with MutPred2","volume":"11","author":"Pejaver","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2014A Visualization System for Exploratory Research and Analysis","volume":"25","author":"Pettersen","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2525","DOI":"10.1016\/j.bpj.2011.10.024","article-title":"Improving the Physical Realism and Structural Accuracy of Protein Models by a Two-Step Atomic-Level Energy Minimization","volume":"101","author":"Xu","year":"2011","journal-title":"Biophys. J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"31","DOI":"10.18036\/estubtdc.1378676","article-title":"Protein structure prediction: An in-depth comparison of approaches and tools","volume":"13","author":"Altunkulah","year":"2024","journal-title":"Eski\u015fehir Tek. \u00dcniversitesi Bilim Ve Teknol. Derg.-C Ya\u015fam Bilim. Ve Biyoteknoloji"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"2197","DOI":"10.1016\/j.jmb.2019.04.009","article-title":"Can Predicted Protein 3D Structures Provide Reliable Insights into Whether Missense Variants Are Disease Associated?","volume":"431","author":"Ittisoponpisan","year":"2019","journal-title":"J. Mol. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Venselaar, H., te Beek, T.A., Kuipers, R.K., Hekkelman, M.L., and Vriend, G. (2010). Protein Structure Analysis of Mutations Causing Inheritable Diseases. An e-Science Approach with Life Scientist Friendly Interfaces. BMC Bioinform., 11.","DOI":"10.1186\/1471-2105-11-548"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"60","DOI":"10.1002\/pro.3942","article-title":"DynaMut2: Assessing Changes in Stability and Flexibility upon Single and Multiple Point Missense Mutations","volume":"30","author":"Rodrigues","year":"2021","journal-title":"Protein Sci."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Caballero-Avenda\u00f1o, A., Guti\u00e9rrez-Angulo, M., Ayala-Madrigal, M.d.l.L., Moreno-Ortiz, J.M., Gonz\u00e1lez-Mercado, A., and Peregrina-Sandoval, J. (2024). In Silico Analysis of the Missense Variants of Uncertain Significance of CTNNB1 Gene Reported in GnomAD Database. Genes, 15.","DOI":"10.3390\/genes15080972"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1549","DOI":"10.1007\/s00439-022-02457-6","article-title":"Genome Interpretation Using in Silico Predictors of Variant Impact","volume":"141","author":"Katsonis","year":"2022","journal-title":"Hum. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1016\/j.csbj.2023.12.031","article-title":"Structural and Functional Prediction, Evaluation, and Validation in the Post-Sequencing Era","volume":"23","author":"Li","year":"2024","journal-title":"Comput. Struct. Biotechnol. J."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Cannon, S., Williams, M., Gunning, A.C., and Wright, C.F. (2023). Evaluation of in Silico Pathogenicity Prediction Tools for the Classification of Small In-Frame Indels. BMC Med. Genom., 16.","DOI":"10.1186\/s12920-023-01454-6"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Kamal, E., Kaddam, L.A., Ahmed, M., and Alabdulkarim, A. (2025). Integrating Artificial Intelligence and Bioinformatics Methods to Identify Disruptive STAT1 Variants Impacting Protein Stability and Function. Genes, 16.","DOI":"10.3390\/genes16030303"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Ernst, C., Hahnen, E., Engel, C., Nothnagel, M., Weber, J., Schmutzler, R.K., and Hauke, J. (2018). Performance of in Silico Prediction Tools for the Classification of Rare BRCA1\/2 Missense Variants in Clinical Diagnostics. BMC Med. Genom., 11.","DOI":"10.1186\/s12920-018-0353-y"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"11114","DOI":"10.1038\/s41598-021-88586-w","article-title":"In Silico Analysis of BRCA1 and BRCA2 Missense Variants and the Relevance in Molecular Genetic Testing","volume":"11","author":"Poon","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Young, C.C., Feng, B.-J., Mackenzie, C.B., Girard, E., Hu, D., Iwasaki, Y., Momozawa, Y., Lesueur, F., Ziv, E., and Neuhausen, S.L. (2019). Evaluation of ACMG Rules for In Silico Evidence Strength Using An Independent Computational Tool Absent of Circularities on ATM and CHEK2 Breast Cancer Cases and Controls. bioRxiv.","DOI":"10.1101\/835264"},{"key":"ref_37","first-page":"e3787","article-title":"In-Silico Method for Predicting Pathogenic Missense Variants Using Online Tools: AURKA Gene A as a Model","volume":"22","author":"MacielCruz","year":"2024","journal-title":"Iran. J. Biotechnol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1016\/j.jbiotec.2021.05.002","article-title":"Hydrophobicity, Amphilicity, and Flexibility: Relation between Molecular Protein Properties and the Macroscopic Effects of Surface Activity","volume":"334","author":"Koop","year":"2021","journal-title":"J. Biotechnol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"140557","DOI":"10.1016\/j.bbapap.2020.140557","article-title":"Low Barrier Hydrogen Bonds in Protein Structure and Function","volume":"1869","author":"Kemp","year":"2021","journal-title":"Biochim. Biophys. Acta BBA Proteins Proteom."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1186\/s13059-017-1353-5","article-title":"Evaluation of in Silico Algorithms for Use with ACMG\/AMP Clinical Variant Interpretation Guidelines","volume":"18","author":"Ghosh","year":"2017","journal-title":"Genome Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"148279","DOI":"10.1016\/j.gene.2024.148279","article-title":"Large-Scale DNA Sequencing Identifies Rare Variants Associated with Systemic Lupus Erythematosus Susceptibility in Known Risk Genes","volume":"907","author":"Latini","year":"2024","journal-title":"Gene"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1085","DOI":"10.1136\/annrheumdis-2021-221754","article-title":"Multiomics Analysis of Rheumatoid Arthritis Yields Sequence Variants That Have Large Effects on Risk of the Seropositive Subset","volume":"81","author":"Saevarsdottir","year":"2022","journal-title":"Ann. Rheum. Dis."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Peng, Y., Alexov, E., and Basu, S. (2019). Structural Perspective on Revealing and Altering Molecular Functions of Genetic Variants Linked with Diseases. Int. J. Mol. Sci., 20.","DOI":"10.20944\/preprints201812.0273.v2"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/1\/72\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,8]],"date-time":"2026-01-08T09:16:03Z","timestamp":1767863763000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/17\/1\/72"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,7]]},"references-count":43,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["genes17010072"],"URL":"https:\/\/doi.org\/10.3390\/genes17010072","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,7]]}}}