{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T19:17:29Z","timestamp":1790709449163,"version":"4.1.0"},"reference-count":40,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2022,11,21]],"date-time":"2022-11-21T00:00:00Z","timestamp":1668988800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"AIRETT (Associazione Italiana Rett)","award":["08A621"],"award-info":[{"award-number":["08A621"]}]},{"name":"AIRETT (Associazione Italiana Rett)","award":["08A801"],"award-info":[{"award-number":["08A801"]}]},{"name":"Italian Ministry of Health","award":["08A621"],"award-info":[{"award-number":["08A621"]}]},{"name":"Italian Ministry of Health","award":["08A801"],"award-info":[{"award-number":["08A801"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Rett syndrome caused by MECP2 variants is characterized by a heterogenous clinical spectrum accounted for in 60% of cases by hot-spot variants. Focusing on the most frequent variants, we generated in vitro iPSC-neurons from the blood of RTT girls with p.Arg133Cys and p.Arg255*, associated to mild and severe phenotype, respectively, and of an RTT male harboring the close to p.Arg255*, p.Gly252Argfs*7 variant. Truncated MeCP2 proteins were revealed by Western blot and immunofluorescence analysis. We compared the mutant versus control neurons at 42 days for morphological parameters and at 120 days for electrophysiology recordings, including girls\u2019 isogenic clones. A precocious reduced morphological complexity was evident in neurons with truncating variants, while in p.Arg133Cys neurons any significant differences were observed in comparison with the isogenic wild-type clones. Reduced nuclear size and branch number show up as the most robust biomarkers. Patch clamp recordings on mature neurons allowed the assessment of cell biophysical properties, V-gated currents, and spiking pattern in the mutant and control cells. Immature spiking, altered cell capacitance, and membrane resistance of RTT neurons, were particularly pronounced in the Arg255* and Gly252Argfs*7 mutants. The overall results indicate that the specific markers of in vitro cellular phenotype mirror the clinical severity and may be amenable to drug testing for translational purposes.<\/jats:p>","DOI":"10.3390\/ijms232214491","type":"journal-article","created":{"date-parts":[[2022,11,22]],"date-time":"2022-11-22T02:59:22Z","timestamp":1669085962000},"page":"14491","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Modeling RTT Syndrome by iPSC-Derived Neurons from Male and Female Patients with Heterogeneously Severe Hot-Spot MECP2 Variants"],"prefix":"10.3390","volume":"23","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3096-2342","authenticated-orcid":false,"given":"Sara","family":"Perego","sequence":"first","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5428-7615","authenticated-orcid":false,"given":"Valentina","family":"Alari","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gianluca","family":"Pietra","sequence":"additional","affiliation":[{"name":"Laboratory of Biology BIO@SNS, Scuola Normale Superiore, Piazza dei Cavalieri, 7, 56126 Pisa, Italy"},{"name":"Institute of Neuroscience, National Research Council (CNR), Via Giuseppe Moruzzi, 1, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Lamperti","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alessandro","family":"Vimercati","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole","family":"Camporeale","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Garzo","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6073-5445","authenticated-orcid":false,"given":"Francesca","family":"Cogliati","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3087-8514","authenticated-orcid":false,"given":"Donatella","family":"Milani","sequence":"additional","affiliation":[{"name":"Fondazione IRCCS Ca Granda Ospedale Maggiore, Policlinico, Via Francesco Sforza, 28, 20122 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4638-4663","authenticated-orcid":false,"given":"Aglaia","family":"Vignoli","sequence":"additional","affiliation":[{"name":"Department of Health Sciences, Universit\u00e0 degli Studi di Milano, Struttura Complessa di Neuropsichiatria dell\u2019Infazia e Adolescenza ASST GOM Niguarda, Piazza Ospedale Maggiore, 3, 20162 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1769-6548","authenticated-orcid":false,"given":"Angela","family":"Peron","sequence":"additional","affiliation":[{"name":"Child Neuropsychiatry Unit\u2014Epilepsy Center, Department of Health Sciences, ASST Santi Paolo e Carlo, San Paolo Hospital, Universit\u00e0 Degli Studi di Milano, 20142 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1367-7227","authenticated-orcid":false,"given":"Lidia","family":"Larizza","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tommaso","family":"Pizzorusso","sequence":"additional","affiliation":[{"name":"Laboratory of Biology BIO@SNS, Scuola Normale Superiore, Piazza dei Cavalieri, 7, 56126 Pisa, Italy"},{"name":"Institute of Neuroscience, National Research Council (CNR), Via Giuseppe Moruzzi, 1, 56124 Pisa, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5893-0193","authenticated-orcid":false,"given":"Silvia","family":"Russo","sequence":"additional","affiliation":[{"name":"Experimental Research Laboratory of Medical Cytogenetics and Molecular Genetics, IRCCS Istituto Auxologico Italiano, Via Ariosto 13, 20145 Milan, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,11,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1093\/hmg\/11.2.115","article-title":"Insight into Rett Syndrome: MeCP2 Levels Display Tissue- and Cell-Specific Differences and Correlate with Neuronal Maturation","volume":"11","author":"Shahbazian","year":"2002","journal-title":"Hum. Mol. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1016\/j.mcn.2004.07.006","article-title":"MECP2 Is Progressively Expressed in Post-Migratory Neurons and Is Involved in Neuronal Maturation Rather than Cell Fate Decisions","volume":"27","author":"Kishi","year":"2004","journal-title":"Mol. Cell. Neurosci."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"ddw269","DOI":"10.1093\/hmg\/ddw269","article-title":"Exclusive Expression of MeCP2 in the Nervous System Distinguishes between Brain and Peripheral Rett Syndrome-like Phenotypes","volume":"25","author":"Ross","year":"2016","journal-title":"Hum. Mol. Genet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"624290","DOI":"10.3389\/fgene.2021.624290","article-title":"The Molecular Functions of MeCP2 in Rett Syndrome Pathology","volume":"12","author":"Sharifi","year":"2021","journal-title":"Front. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"422","DOI":"10.1016\/j.neuron.2007.10.001","article-title":"The Story of Rett Syndrome: From Clinic to Neurobiology","volume":"56","author":"Chahrour","year":"2007","journal-title":"Neuron"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"240","DOI":"10.1002\/cne.22009","article-title":"Widespread Changes in Dendritic and Axonal Morphology in Mecp2 -Mutant Mouse Models of Rett Syndrome: Evidence for Disruption of Neuronal Networks","volume":"514","author":"Belichenko","year":"2009","journal-title":"J. Comp. Neurol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"436","DOI":"10.3174\/ajnr.A0857","article-title":"Selective Cerebral Volume Reduction in Rett Syndrome: A Multiple-Approach MR Imaging Study","volume":"29","author":"Carter","year":"2008","journal-title":"AJNR Am. J. Neuroradiol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Pascual-Alonso, A., Mart\u00ednez-Monseny, A.F., Xiol, C., and Armstrong, J. (2021). MECP2-Related Disorders in Males. Ijms, 22.","DOI":"10.3390\/ijms22179610"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1212\/01.wnl.0000291011.54508.aa","article-title":"Specific Mutations in Methyl-CpG-Binding Protein 2 Confer Different Severity in Rett Syndrome","volume":"70","author":"Neul","year":"2008","journal-title":"Neurology"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1136\/jmedgenet-2013-102113","article-title":"Methyl-CpG-Binding Protein 2 ( MECP2 ) Mutation Type Is Associated with Disease Severity in Rett Syndrome","volume":"51","author":"Cuddapah","year":"2014","journal-title":"J. Med. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Ananiev, G., Williams, E.C., Li, H., and Chang, Q. (2011). Isogenic Pairs of Wild Type and Mutant Induced Pluripotent Stem Cell (IPSC) Lines from Rett Syndrome Patients as In Vitro Disease Model. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0025255"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"847","DOI":"10.1006\/nbdi.2001.0428","article-title":"Gene Expression Profiling in Postmortem Rett Syndrome Brain: Differential Gene Expression and Patient Classification","volume":"8","author":"Colantuoni","year":"2001","journal-title":"Neurobiol. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1038\/85906","article-title":"Deficiency of Methyl-CpG Binding Protein-2 in CNS Neurons Results in a Rett-like Phenotype in Mice","volume":"27","author":"Chen","year":"2001","journal-title":"Nat. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"322","DOI":"10.1038\/85899","article-title":"A Mouse Mecp2-Null Mutation Causes Neurological Symptoms That Mimic Rett Syndrome","volume":"27","author":"Guy","year":"2001","journal-title":"Nat. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1007\/s00335-019-09793-5","article-title":"Treating Rett Syndrome: From Mouse Models to Human Therapies","volume":"30","author":"Vashi","year":"2019","journal-title":"Mamm. Genome"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"584","DOI":"10.1038\/s41593-020-00794-1","article-title":"Single-Cell Atlas of Early Human Brain Development Highlights Heterogeneity of Human Neuroepithelial Cells and Early Radial Glia","volume":"24","author":"Eze","year":"2021","journal-title":"Nat. Neurosci"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1038\/s41586-019-1289-x","article-title":"Individual Brain Organoids Reproducibly Form Cell Diversity of the Human Cerebral Cortex","volume":"570","author":"Velasco","year":"2019","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1016\/j.cell.2006.07.024","article-title":"Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors","volume":"126","author":"Takahashi","year":"2006","journal-title":"Cell"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1016\/j.cell.2010.10.016","article-title":"A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells","volume":"143","author":"Marchetto","year":"2010","journal-title":"Cell"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Gomes, A.R., Fernandes, T.G., Cabral, J.M.S., and Diogo, M.M. (2021). Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives. Ijms, 22.","DOI":"10.3390\/ijms22073751"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"698812","DOI":"10.3389\/fnins.2021.698812","article-title":"Pre-Clinical Investigation of Rett Syndrome Using Human Stem Cell-Based Disease Models","volume":"15","author":"Haase","year":"2021","journal-title":"Front. Neurosci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1038\/nature12517","article-title":"Cerebral Organoids Model Human Brain Development and Microcephaly","volume":"501","author":"Lancaster","year":"2013","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1016\/j.tcb.2019.11.004","article-title":"Organoid and Assembloid Technologies for Investigating Cellular Crosstalk in Human Brain Development and Disease","volume":"30","author":"Marton","year":"2020","journal-title":"Trends Cell Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1073\/pnas.1524013113","article-title":"KCC2 Rescues Functional Deficits in Human Neurons Derived from Patients with Rett Syndrome","volume":"113","author":"Tang","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Kim, J.J., Savas, J.N., Miller, M.T., Hu, X., Carromeu, C., Lavall\u00e9e-Adam, M., Freitas, B.C.G., Muotri, A.R., Yates, J.R., and Ghosh, A. (2019). Proteomic Analyses Reveal Misregulation of LIN28 Expression and Delayed Timing of Glial Differentiation in Human IPS Cells with MECP2 Loss-of-Function. PLoS ONE, 14.","DOI":"10.1371\/journal.pone.0212553"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1578","DOI":"10.1002\/biot.201400751","article-title":"Neural Commitment of Human Pluripotent Stem Cells under Defined Conditions Recapitulates Neural Development and Generates Patient-Specific Neural Cells","volume":"10","author":"Fernandes","year":"2015","journal-title":"Biotechnol. J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1453","DOI":"10.1016\/j.stemcr.2018.04.001","article-title":"Loss of MECP2 Leads to Activation of P53 and Neuronal Senescence","volume":"10","author":"Ohashi","year":"2018","journal-title":"Stem Cell Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.molcel.2020.05.016","article-title":"Dysregulation of BRD4 Function Underlies the Functional Abnormalities of MeCP2 Mutant Neurons","volume":"79","author":"Xiang","year":"2020","journal-title":"Mol. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"14169","DOI":"10.1073\/pnas.1018979108","article-title":"Neuronal Maturation Defect in Induced Pluripotent Stem Cells from Patients with Rett Syndrome","volume":"108","author":"Kim","year":"2011","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"504","DOI":"10.1016\/j.bbrc.2017.10.073","article-title":"The L1 Adhesion Molecule Normalizes Neuritogenesis in Rett Syndrome-Derived Neural Precursor Cells","volume":"494","author":"Yoo","year":"2017","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2982","DOI":"10.1002\/ajmg.a.61874","article-title":"Sleep Disordered Breathing and Daytime Hypoventilation in a Male with MECP2 Mutation","volume":"182","author":"Cacciatori","year":"2020","journal-title":"Am. J. Med. Genet."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3671","DOI":"10.1523\/JNEUROSCI.3735-16.2017","article-title":"CREB Signaling Is Involved in Rett Syndrome Pathogenesis","volume":"37","author":"Bu","year":"2017","journal-title":"J. Neurosci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"97","DOI":"10.3389\/fnana.2014.00097","article-title":"Dendritic Spine Dysgenesis in Rett Syndrome","volume":"8","author":"Xu","year":"2014","journal-title":"Front. Neuroanat."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"639","DOI":"10.1007\/s13238-020-00773-z","article-title":"Graded and Pan-Neural Disease Phenotypes of Rett Syndrome Linked with Dosage of Functional MeCP2","volume":"12","author":"Chen","year":"2021","journal-title":"Protein Cell"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1038\/s41586-019-1506-7","article-title":"Conserved Cell Types with Divergent Features in Human versus Mouse Cortex","volume":"573","author":"Hodge","year":"2019","journal-title":"Nature"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2128","DOI":"10.1002\/stem.1180","article-title":"Disease Modeling Using Embryonic Stem Cells: MeCP2 Regulates Nuclear Size and RNA Synthesis in Neurons","volume":"30","author":"Yazdani","year":"2012","journal-title":"Stem Cells"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"139","DOI":"10.1007\/s00401-020-02244-5","article-title":"Intellectual Disability: Dendritic Anomalies and Emerging Genetic Perspectives","volume":"141","author":"Quach","year":"2021","journal-title":"Acta Neuropathol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"101553","DOI":"10.1016\/j.scr.2019.101553","article-title":"Generation of Three IPSC Lines (IAIi002, IAIi004, IAIi003) from Rubinstein-Taybi Syndrome 1 Patients Carrying CREBBP Non Sense c.4435G>T, p.(Gly1479*) and c.3474G>A, p.(Trp1158*) and Missense c.4627G>T, p.(Asp1543Tyr) Mutations","volume":"40","author":"Alari","year":"2019","journal-title":"Stem Cell Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.scr.2018.05.019","article-title":"iPSC-Derived Neurons of CREBBP- and EP300 -Mutated Rubinstein-Taybi Syndrome Patients Show Morphological Alterations and Hypoexcitability","volume":"30","author":"Alari","year":"2018","journal-title":"Stem Cell Res."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Alari, V., Scalmani, P., Ajmone, P.F., Perego, S., Avignone, S., Catusi, I., Lonati, P.A., Borghi, M.O., Finelli, P., and Terragni, B. (2021). Histone Deacetylase Inhibitors Ameliorate Morphological Defects and Hypoexcitability of IPSC-Neurons from Rubinstein-Taybi Patients. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22115777"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/22\/14491\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:23:06Z","timestamp":1760145786000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/22\/14491"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,21]]},"references-count":40,"journal-issue":{"issue":"22","published-online":{"date-parts":[[2022,11]]}},"alternative-id":["ijms232214491"],"URL":"https:\/\/doi.org\/10.3390\/ijms232214491","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,21]]}}}