{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,28]],"date-time":"2026-10-28T18:12:08Z","timestamp":1793211128090,"version":"4.5.0"},"reference-count":20,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2025,11,11]],"date-time":"2025-11-11T00:00:00Z","timestamp":1762819200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Background. Hypophosphatasia (HPP) is a rare genetic disorder caused by impaired tissue non-specific alkaline phosphatase (ALPL\/TNSALP) activity that impacts the musculoskeletal and neurological systems. It is extremely variable, with up to six forms of increasing severity. The large phenotypic variability and the still remaining high number of variants of uncertain significance (VUS) in the ALPL gene represent a conundrum for clinicians dealing with people suspected to be suffering from HPP. Methods. We applied a multi-faceted bench-based and high-throughput bioinformatics analysis to investigate the effect of 21 ALPL variants (18 deleterious\u2014pathogenic or likely pathogenic\u2014and 3 VUS) on the structure and function of the mutated encoded protein. The results were compared with available clinical and biochemical data. Results. Most variants were downregulated or not expressed by Western blot analysis. Impairment of the enzymatic activity was confirmed in vitro for all variants by a specific colorimetric enzymatic assay. In silico prediction was in line with functional data and allowed for preliminary categorization of variants based on their impact on both the overall stability of the protein complex and local structural alterations. Coherence among bioinformatics, experimental and clinical data was documented for more than 70% of the variants. Conclusions. Functional and in silico characterizations of ALPL variants in people with a suspicion of HPP offer integrative strategies to genotyping in assisting clinicians for diagnosis confirmation in doubtful cases.<\/jats:p>","DOI":"10.3390\/cells14221768","type":"journal-article","created":{"date-parts":[[2025,11,12]],"date-time":"2025-11-12T00:33:17Z","timestamp":1762907597000},"page":"1768","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Functional and In Silico Characterization of ALPL Gene Variants Reveals Genotype\u2013Phenotype Correlations in Italian Hypophosphatasia Patients"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4823-0310","authenticated-orcid":false,"given":"Giulia","family":"Casamassima","sequence":"first","affiliation":[{"name":"UOC Genetica Medica, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-8722-020X","authenticated-orcid":false,"given":"Anna Maria","family":"Grieco","sequence":"additional","affiliation":[{"name":"UOC Genetica Medica, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4539-028X","authenticated-orcid":false,"given":"Tommaso","family":"Biagini","sequence":"additional","affiliation":[{"name":"Computational Biology and Bioinformatics Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00136 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giorgia","family":"Buono","sequence":"additional","affiliation":[{"name":"UOC Genetica Medica, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luigia","family":"Cinque","sequence":"additional","affiliation":[{"name":"UOC Genetica Medica, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Flavia","family":"Pugliese","sequence":"additional","affiliation":[{"name":"Unit of Endocrinology, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francesco Pio","family":"Guerra","sequence":"additional","affiliation":[{"name":"Laboratory of Oncology, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9478-7929","authenticated-orcid":false,"given":"Francesco","family":"Petrizzelli","sequence":"additional","affiliation":[{"name":"Laboratory of Bioinformatics, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3218-1646","authenticated-orcid":false,"given":"Mario","family":"Mastroianno","sequence":"additional","affiliation":[{"name":"Scientific Direction, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0434-8533","authenticated-orcid":false,"given":"Tommaso","family":"Mazza","sequence":"additional","affiliation":[{"name":"Computational Biology and Bioinformatics Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00136 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marco","family":"Castori","sequence":"additional","affiliation":[{"name":"UOC Genetica Medica, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfredo","family":"Scillitani","sequence":"additional","affiliation":[{"name":"Computational Biology and Bioinformatics Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00136 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vito","family":"Guarnieri","sequence":"additional","affiliation":[{"name":"UOC Genetica Medica, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, 71013 Foggia, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Villa-Su\u00e1rez, J.M., Garc\u00eda-Fontana, C., And\u00fajar-Vera, F., Gonz\u00e1lez-Salvatierra, S., de Haro-Mu\u00f1oz, T., Contreras-Bol\u00edvar, V., Garc\u00eda-Fontana, B., and Mu\u00f1oz-Torres, M. (2021). Hypophosphatasia: A Unique Disorder of Bone Mineralization. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22094303"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1250","DOI":"10.1002\/humu.24010","article-title":"Large-scale in vitro functional testing and novel variant scoring via protein modeling provide insights into alkaline phosphatase activity in hypophosphatasia","volume":"41","author":"Reynders","year":"2020","journal-title":"Hum. Mutat."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"9569","DOI":"10.1038\/s41598-019-46004-2","article-title":"Epidemiological, Clinical and Genetic Study of Hypophosphatasia in A Spanish Population: Identification of Two Novel Mutations in the Alpl Gene","volume":"9","author":"Real","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"ziaf044","DOI":"10.1093\/jbmrpl\/ziaf044","article-title":"The ALPL gene variant project: Results of the first 100 reclassified variants","volume":"6","author":"Farman","year":"2025","journal-title":"JBMR Plus"},{"key":"ref_5","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":"5","author":"Richards","year":"2015","journal-title":"Genet. Med."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Cinque, L., Pugliese, F., Salcuni, A.S., Trombetta, D., Battista, C., Biagini, T., Augello, B., Nardella, G., Conti, F., and Corbetta, S. (2023). Clinical and molecular description of the first Italian cohort of 33 subjects with hypophosphatasia. Front. Endocrinol., 14.","DOI":"10.3389\/fendo.2023.1205977"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1016\/j.bone.2013.02.017","article-title":"Compound heterozygosity of two functional null mutations in the ALPL gene associated with deleterious neurological outcome in an infant with hypophosphatasia","volume":"1","author":"Hofmann","year":"2013","journal-title":"Bone"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1159\/000354467","article-title":"Characterization of six missense mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in Chinese children with hypophosphatasia","volume":"3","author":"Yang","year":"2013","journal-title":"Cell. Physiol. Biochem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"968","DOI":"10.1093\/oxfordjournals.jbchem.a022032","article-title":"Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162-->Thr mutation associated with lethal hypophosphatasia","volume":"5","author":"Shibata","year":"1998","journal-title":"J. Biochem."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1002\/humu.1154","article-title":"Twelve novel mutations in the tissue-nonspecific alkaline phosphatase gene (ALPL) in patients with various forms of hypophosphatasia","volume":"18","author":"Taillandier","year":"2001","journal-title":"Hum. Mutat."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1016\/j.bbadis.2012.01.007","article-title":"Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C201-Y or C489-S substitution associated with severe hypophosphatasia","volume":"1822","author":"Satou","year":"2012","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1016\/j.biochi.2007.09.013","article-title":"Targeting ubiquitin specific proteases for drug discovery","volume":"2","author":"Daviet","year":"2008","journal-title":"Biochimie"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1038\/s41592-022-01488-1","article-title":"ColabFold: Making protein folding accessible to all","volume":"19","author":"Mirdita","year":"2022","journal-title":"Nat. Methods"},{"key":"ref_14","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_15","doi-asserted-by":"crossref","first-page":"W382","DOI":"10.1093\/nar\/gki387","article-title":"The FoldX web server: An online force field","volume":"33","author":"Schymkowitz","year":"2005","journal-title":"Nucleic Acids Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1007\/s004180050103","article-title":"Immunolocalization of tissue non-specific alkaline phosphatase in mice","volume":"107","author":"Hoshi","year":"1997","journal-title":"Histochem. Cell Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1186\/s13052-025-01883-2","article-title":"Safety and efficacy of long term asfotase alfa treatment in childhood hypophosphatasia","volume":"51","author":"Lauriola","year":"2025","journal-title":"Ital. J. Pediatr."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Fauvert, D., Brun-Heath, I., Lia-Baldini, A.S., Bellazi, L., Taillandier, A., Serre, J.L., de Mazancourt, P., and Mornet, E. (2009). Mild forms of hypophosphatasia mostly result from dominant negative effect of severe alleles or from compound heterozygosity for severe and moderate alleles. BMC Med. Genet., 10.","DOI":"10.1186\/1471-2350-10-51"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.job.2019.07.003","article-title":"Molecular and cellular basis of hypophosphatasia","volume":"61","author":"Komaru","year":"2019","journal-title":"J. Oral Biosci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"31171","DOI":"10.1074\/jbc.M102788200","article-title":"Structural evidence for a functional role of human tissue nonspecific alkaline phosphatase in bone mineralization","volume":"33","author":"Mornet","year":"2001","journal-title":"J. Biol. Chem."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/22\/1768\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,13]],"date-time":"2025-11-13T05:30:27Z","timestamp":1763011827000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/14\/22\/1768"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,11]]},"references-count":20,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["cells14221768"],"URL":"https:\/\/doi.org\/10.3390\/cells14221768","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,11]]}}}