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Among them, facioscapulohumeral dystrophy (FSHD) is a muscular disorder characterized by the loss of repressive epigenetic features affecting the <jats:italic>D4Z4<\/jats:italic> locus (4q35). As a consequence, these alterations are responsible for DNA hypomethylation and a transcriptional\u2010active chromatin conformation change that, in turn, lead to the aberrant expression of <jats:italic>DUX4<\/jats:italic> in muscle cells. In the present study, methylation levels of 29 CpG sites of the DR1 region (within each repeat unit of the <jats:italic>D4Z4<\/jats:italic> macrosatellite) were assessed on 335 subjects by employing primers designed for enhancing the performance of the assay. First, the DR1 original primers were optimized by adding M13 oligonucleotide tails. Moreover, the DR1 reverse primer was replaced with a degenerate one. As a result, the protocol optimization allowed a better sequencing resolution and a more accurate evaluation of DR1 methylation levels. Moreover, the assessment of the repeatability of measurements proved the reliability and robustness of the assay. The optimized protocol emerges as an excellent method to detect methylation levels compatible with FSHD.<\/jats:p>","DOI":"10.1002\/elps.202300058","type":"journal-article","created":{"date-parts":[[2023,8,11]],"date-time":"2023-08-11T05:12:21Z","timestamp":1691730741000},"page":"1588-1594","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Epigenetic profiling of the <i>D4Z4<\/i> locus: Optimization of the protocol for studying DNA methylation at single CpG site level"],"prefix":"10.1002","volume":"44","author":[{"given":"Domenica","family":"Megalizzi","sequence":"first","affiliation":[{"name":"Genomic Medicine Laboratory\u2010UILDM Santa Lucia Foundation IRCCS  Rome Italy"},{"name":"Department of Biomedicine and Prevention Tor Vergata University  Rome Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giulia","family":"Trastulli","sequence":"additional","affiliation":[{"name":"Genomic Medicine 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