{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T03:39:34Z","timestamp":1792640374773,"version":"4.5.0"},"reference-count":40,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2022,12,18]],"date-time":"2022-12-18T00:00:00Z","timestamp":1671321600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"FSHD Society","award":["Winter2021-0992658837 (COD:FSHD)"],"award-info":[{"award-number":["Winter2021-0992658837 (COD:FSHD)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>The study describes a protocol for methylation analysis integrated with Machine Learning (ML) algorithms developed to classify Facio-Scapulo-Humeral Dystrophy (FSHD) subjects. The DNA methylation levels of two D4Z4 regions (DR1 and DUX4-PAS) were assessed by an in-house protocol based on bisulfite sequencing and capillary electrophoresis, followed by statistical and ML analyses. The study involved two independent cohorts, namely a training group of 133 patients with clinical signs of FSHD and 150 healthy controls (CTRL) and a testing set of 27 FSHD patients and 25 CTRL. As expected, FSHD patients showed significantly reduced methylation levels compared to CTRL. We utilized single CpG sites to develop a ML pipeline able to discriminate FSHD subjects. The model identified four CpGs sites as the most relevant for the discrimination of FSHD subjects and showed high metrics values (accuracy: 0.94, sensitivity: 0.93, specificity: 0.96). Two additional models were developed to differentiate patients with lower D4Z4 size and patients who might carry pathogenic variants in FSHD genes, respectively. Overall, the present model enables an accurate classification of FSHD patients, providing additional evidence for DNA methylation as a powerful disease biomarker that could be employed for prioritizing subjects to be tested for FSHD.<\/jats:p>","DOI":"10.3390\/cells11244114","type":"journal-article","created":{"date-parts":[[2022,12,19]],"date-time":"2022-12-19T07:27:30Z","timestamp":1671434850000},"page":"4114","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["D4Z4 Methylation Levels Combined with a Machine Learning Pipeline Highlight Single CpG Sites as Discriminating Biomarkers for FSHD Patients"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3503-3318","authenticated-orcid":false,"given":"Valerio","family":"Caputo","sequence":"first","affiliation":[{"name":"Genomic Medicine Laboratory-UILDM, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"},{"name":"Department of Biomedicine and Prevention, Tor Vergata University, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9517-0165","authenticated-orcid":false,"given":"Domenica","family":"Megalizzi","sequence":"additional","affiliation":[{"name":"Genomic Medicine Laboratory-UILDM, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"},{"name":"Department of Biomedicine and Prevention, Tor Vergata University, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7824-8423","authenticated-orcid":false,"given":"Carlo","family":"Fabrizio","sequence":"additional","affiliation":[{"name":"Data Science Unit, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4374-7430","authenticated-orcid":false,"given":"Andrea","family":"Termine","sequence":"additional","affiliation":[{"name":"Data Science Unit, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luca","family":"Colantoni","sequence":"additional","affiliation":[{"name":"Genomic Medicine Laboratory-UILDM, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cristina","family":"Bax","sequence":"additional","affiliation":[{"name":"Genomic Medicine Laboratory-UILDM, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7682-7088","authenticated-orcid":false,"given":"Juliette","family":"Gimenez","sequence":"additional","affiliation":[{"name":"Epigenetics and Genome Reprogramming Laboratory, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mauro","family":"Monforte","sequence":"additional","affiliation":[{"name":"Unit\u00e0 Operativa Complessa di Neurologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0849-9144","authenticated-orcid":false,"given":"Giorgio","family":"Tasca","sequence":"additional","affiliation":[{"name":"Unit\u00e0 Operativa Complessa di Neurologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy"},{"name":"John Walton Muscular Dystrophy Research Centre, Newcastle University and Newcastle Hospitals NHS Foundation Trusts, Newcastle Upon Tyne NE1 3BZ, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enzo","family":"Ricci","sequence":"additional","affiliation":[{"name":"Unit\u00e0 Operativa Complessa di Neurologia, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy"},{"name":"Istituto di Neurologia, Universit\u00e0 Cattolica del Sacro Cuore, 00168 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlo","family":"Caltagirone","sequence":"additional","affiliation":[{"name":"Department of Clinical and Behavorial Neurology, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emiliano","family":"Giardina","sequence":"additional","affiliation":[{"name":"Genomic Medicine Laboratory-UILDM, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"},{"name":"Department of Biomedicine and Prevention, Tor Vergata University, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raffaella","family":"Cascella","sequence":"additional","affiliation":[{"name":"Genomic Medicine Laboratory-UILDM, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"},{"name":"Department of Biomedicine and Prevention, Tor Vergata University, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1334-0920","authenticated-orcid":false,"given":"Claudia","family":"Strafella","sequence":"additional","affiliation":[{"name":"Genomic Medicine Laboratory-UILDM, Santa Lucia Foundation IRCCS, 00179 Rome, Italy"},{"name":"Department of Biomedicine and Prevention, Tor Vergata University, 00133 Rome, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1146\/annurev-genom-083118-014933","article-title":"The Genetics and Epigenetics of Facioscapulohumeral Muscular Dystrophy","volume":"20","author":"Himeda","year":"2019","journal-title":"Annu. Rev. Genom. Hum. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.3389\/fneur.2018.01027","article-title":"Digenic Inheritance of Shortened Repeat Units of the D4Z4 Region and a Loss-of-Function Variant in SMCHD1 in a Family With FSHD","volume":"9","author":"Cascella","year":"2018","journal-title":"Front. Neurol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3912","DOI":"10.1093\/hmg\/ddz239","article-title":"The Variability of SMCHD1 Gene in FSHD Patients: Evidence of New Mutations","volume":"28","author":"Strafella","year":"2019","journal-title":"Hum. Mol. Genet."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1101","DOI":"10.1016\/j.nmd.2021.09.010","article-title":"Facioscapulohumeral Muscular Dystrophy Type 2: An Update on the Clinical, Genetic, and Molecular Findings","volume":"31","author":"Jia","year":"2021","journal-title":"Neuromuscul. Disord."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"e2441","DOI":"10.1212\/WNL.0000000000009617","article-title":"Homozygous Nonsense Variant in LRIF1 Associated with Facioscapulohumeral Muscular Dystrophy","volume":"94","author":"Hamanaka","year":"2020","journal-title":"Neurology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1016\/j.ajhg.2013.08.004","article-title":"The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1","volume":"93","author":"Sacconi","year":"2013","journal-title":"Am. J. Hum. Genet."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1038\/ejhg.2014.191","article-title":"Diagnostic Approach for FSHD Revisited: SMCHD1 Mutations Cause FSHD2 and Act as Modifiers of Disease Severity in FSHD1","volume":"23","author":"Larsen","year":"2015","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1020","DOI":"10.1016\/j.ajhg.2016.03.013","article-title":"Mutations in DNMT3B Modify Epigenetic Repression of the D4Z4 Repeat and the Penetrance of Facioscapulohumeral Dystrophy","volume":"98","author":"Lemmers","year":"2016","journal-title":"Am. J. Hum. Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1136\/jmedgenet-2011-100101","article-title":"Patients with a Phenotype Consistent with Facioscapulohumeral Muscular Dystrophy Display Genetic and Epigenetic Heterogeneity","volume":"49","author":"Sacconi","year":"2012","journal-title":"J. Med. Genet."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"659","DOI":"10.1093\/hmg\/ddu486","article-title":"Inter-Individual Differences in CpG Methylation at D4Z4 Correlate with Clinical Variability in FSHD1 and FSHD2","volume":"24","author":"Lemmers","year":"2015","journal-title":"Hum. Mol. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1007\/s10048-019-00575-4","article-title":"Facioscapulohumeral Muscular Dystrophy (FSHD) Molecular Diagnosis: From Traditional Technology to the NGS Era","volume":"20","author":"Zampatti","year":"2019","journal-title":"Neurogenetics"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1353","DOI":"10.1056\/NEJMra1711801","article-title":"Next-Generation Sequencing to Diagnose Suspected Genetic Disorders","volume":"379","author":"Adams","year":"2018","journal-title":"N. Engl. J. Med."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1186\/s13148-015-0072-6","article-title":"Individual Epigenetic Status of the Pathogenic D4Z4 Macrosatellite Correlates with Disease in Facioscapulohumeral Muscular Dystrophy","volume":"7","author":"Jones","year":"2015","journal-title":"Clin. Epigenet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"733","DOI":"10.1212\/WNL.0000000000000708","article-title":"Differential DNA Methylation of the D4Z4 Repeat in Patients with FSHD and Asymptomatic Carriers","volume":"83","author":"Gaillard","year":"2014","journal-title":"Neurology"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1136\/jmedgenet-2015-103436","article-title":"Allele-Specific DNA Hypomethylation Characterises FSHD1 and FSHD2","volume":"53","author":"Calandra","year":"2016","journal-title":"J. Med. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"e372","DOI":"10.1212\/NXG.0000000000000372","article-title":"Methylation Hotspots Evidenced by Deep Sequencing in Patients with Facioscapulohumeral Dystrophy and Mosaicism","volume":"5","author":"Roche","year":"2019","journal-title":"Neurol. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Gould, T., Jones, T.I., and Jones, P.L. (2021). Precise Epigenetic Analysis Using Targeted Bisulfite Genomic Sequencing Distinguishes FSHD1, FSHD2, and Healthy Subjects. Diagnostics, 11.","DOI":"10.3390\/diagnostics11081469"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Nikolic, A., Jones, T.I., Govi, M., Mele, F., Maranda, L., Sera, F., Ricci, G., Ruggiero, L., Vercelli, L., and Portaro, S. (2020). Interpretation of the Epigenetic Signature of Facioscapulohumeral Muscular Dystrophy in Light of Genotype-Phenotype Studies. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21072635"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Erdmann, H., Scharf, F., Gehling, S., Benet-Pag\u00e8s, A., Jakubiczka, S., Becker, K., Seipelt, M., Kleefeld, F., Knop, K.C., and Prott, E.C. (2022). Methylation of the 4q35 D4Z4 Repeat Defines Disease Status in Facioscapulohumeral Muscular Dystrophy. Brain, awac336.","DOI":"10.1093\/brain\/awac336"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1186\/s12967-022-03743-7","article-title":"Simultaneous Measurement of the Size and Methylation of Chromosome 4qA-D4Z4 Repeats in Facioscapulohumeral Muscular Dystrophy by Long-Read Sequencing","volume":"20","author":"Hiramuki","year":"2022","journal-title":"J. Transl. Med."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"392","DOI":"10.1212\/WNL.0b013e31827f075c","article-title":"A Focal Domain of Extreme Demethylation within D4Z4 in FSHD2","volume":"80","author":"Hartweck","year":"2013","journal-title":"Neurology"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"854","DOI":"10.1002\/ana.24640","article-title":"Magnetic Resonance Imaging in a Large Cohort of Facioscapulohumeral Muscular Dystrophy Patients: Pattern Refinement and Implications for Clinical Trials","volume":"79","author":"Tasca","year":"2016","journal-title":"Ann. Neurol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1002\/1531-8249(199906)45:6<751::AID-ANA9>3.0.CO;2-M","article-title":"Progress in the Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy and Correlation between the Number of KpnI Repeats at the 4q35 Locus and Clinical Phenotype","volume":"45","author":"Ricci","year":"1999","journal-title":"Ann. Neurol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2604","DOI":"10.1111\/ene.14446","article-title":"Deep Phenotyping of Facioscapulohumeral Muscular Dystrophy Type 2 by Magnetic Resonance Imaging","volume":"27","author":"Giacomucci","year":"2020","journal-title":"Eur. J. Neurol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"2055","DOI":"10.1007\/s00415-021-10786-1","article-title":"Diagnostic Magnetic Resonance Imaging Biomarkers for Facioscapulohumeral Muscular Dystrophy Identified by Machine Learning","volume":"269","author":"Monforte","year":"2021","journal-title":"J. Neurol."},{"key":"ref_26","unstructured":"Kuhn, M. (2022, September 20). Caret: Classification and Regression Training; Astrophysics Source Code Library, 2015. 1505.003. Available online: https:\/\/www.semanticscholar.org\/paper\/caret%3A-Classification-and-Regression-Training-Kuhn\/258c7e3242b91e02e092e77e058f6275ba52b12d."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Kuhn, M., and Johnson, K. (2013). Applied Predictive Modeling, Springer.","DOI":"10.1007\/978-1-4614-6849-3"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"799","DOI":"10.1111\/cge.13726","article-title":"Consequences of Epigenetic Derepression in Facioscapulohumeral Muscular Dystrophy","volume":"97","author":"Greco","year":"2020","journal-title":"Clin. Genet."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1186\/1868-7083-6-23","article-title":"Identifying Diagnostic DNA Methylation Profiles for Facioscapulohumeral Muscular Dystrophy in Blood and Saliva Using Bisulfite Sequencing","volume":"6","author":"Jones","year":"2014","journal-title":"Clin. Epigenet."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1038\/ng1262","article-title":"Hypomethylation of D4Z4 in 4q-Linked and Non-4q-Linked Facioscapulohumeral Muscular Dystrophy","volume":"35","author":"Lemmers","year":"2003","journal-title":"Nat. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"21648","DOI":"10.1038\/s41598-020-78578-7","article-title":"Large Genotype-Phenotype Study in Carriers of D4Z4 Borderline Alleles Provides Guidance for Facioscapulohumeral Muscular Dystrophy Diagnosis","volume":"10","author":"Ricci","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"628","DOI":"10.1016\/j.ajhg.2012.02.019","article-title":"Large-Scale Population Analysis Challenges the Current Criteria for the Molecular Diagnosis of Fascioscapulohumeral Muscular Dystrophy","volume":"90","author":"Scionti","year":"2012","journal-title":"Am. J. Hum. Genet."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Huichalaf, C., Micheloni, S., Ferri, G., Caccia, R., and Gabellini, D. (2014). DNA Methylation Analysis of the Macrosatellite Repeat Associated with FSHD Muscular Dystrophy at Single Nucleotide Level. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0115278"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"e2273","DOI":"10.1212\/WNL.0000000000007456","article-title":"FSHD1 and FSHD2 Form a Disease Continuum","volume":"92","author":"Sacconi","year":"2019","journal-title":"Neurology"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"27","DOI":"10.3389\/fmed.2020.00027","article-title":"Artificial Intelligence in Medicine: Today and Tomorrow","volume":"7","author":"Briganti","year":"2020","journal-title":"Front. Med."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Caputo, V., Megalizzi, D., Fabrizio, C., Termine, A., Colantoni, L., Caltagirone, C., Giardina, E., Cascella, R., and Strafella, C. (2022). Update on the Molecular Aspects and Methods Underlying the Complex Architecture of FSHD. Cells, 11.","DOI":"10.3390\/cells11172687"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Fabrizio, C., Termine, A., Caputo, V., Megalizzi, D., Zampatti, S., Falsini, B., Cusumano, A., Eandi, C.M., Ricci, F., and Giardina, E. (2022). WARE: Wet AMD Risk-Evaluation Tool as a Clinical Decision-Support System Integrating Genetic and Non-Genetic Factors. J. Pers. Med., 12.","DOI":"10.3390\/jpm12071034"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Zampatti, S., Fabrizio, C., Ragazzo, M., Campoli, G., Caputo, V., Strafella, C., Pellicano, C., Cascella, R., Spalletta, G., and Petrosini, L. (2021). Precision Medicine into Clinical Practice: A Web-Based Tool Enables Real-Time Pharmacogenetic Assessment of Tailored Treatments in Psychiatric Disorders. J. Pers. Med., 11.","DOI":"10.3390\/jpm11090851"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"992.e11","DOI":"10.1016\/j.neuron.2021.12.019","article-title":"Genome-Wide Identification of the Genetic Basis of Amyotrophic Lateral Sclerosis","volume":"110","author":"Zhang","year":"2022","journal-title":"Neuron"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"104623","DOI":"10.1016\/j.compbiomed.2021.104623","article-title":"Deep Learning Segmentation of Transverse Musculoskeletal Ultrasound Images for Neuromuscular Disease Assessment","volume":"135","author":"Marzola","year":"2021","journal-title":"Comput. Biol. Med."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/11\/24\/4114\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:43:40Z","timestamp":1760147020000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/11\/24\/4114"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,18]]},"references-count":40,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2022,12]]}},"alternative-id":["cells11244114"],"URL":"https:\/\/doi.org\/10.3390\/cells11244114","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,18]]}}}