Clinical Epigenetics (Oct 2024)

Integrating D4Z4 methylation analysis into clinical practice: improvement of FSHD molecular diagnosis through distinct thresholds for 4qA/4qA and 4qA/4qB patients

  • Claudia Strafella,
  • Domenica Megalizzi,
  • Giulia Trastulli,
  • Emma Proietti Piorgo,
  • Luca Colantoni,
  • Giorgio Tasca,
  • Mauro Monforte,
  • Stefania Zampatti,
  • Guido Primiano,
  • Cristina Sancricca,
  • Sara Bortolani,
  • Eleonora Torchia,
  • Beatrice Ravera,
  • Francesca Torri,
  • Giulio Gadaleta,
  • Barbara Risi,
  • Filomena Caria,
  • Francesca Gerardi,
  • Elena Carraro,
  • Valeria Gioiosa,
  • Matteo Garibaldi,
  • Laura Tufano,
  • Erica Frezza,
  • Roberto Massa,
  • Carlo Caltagirone,
  • Elena Maria Pennisi,
  • Antonio Petrucci,
  • Marika Pane,
  • Annalia Frongia,
  • Francesca Gragnani,
  • Marianna Scutifero,
  • Paola Mandich,
  • Marina Grandis,
  • Maria Antonietta Maioli,
  • Carlo Casali,
  • Elisabetta Manfroi,
  • Luisa Politano,
  • Luigia Passamano,
  • Roberta Petillo,
  • Carmelo Rodolico,
  • Alessia Pugliese,
  • Stefano Carlo Previtali,
  • Valeria Sansone,
  • Liliana Vercelli,
  • Tiziana Enrica Mongini,
  • Giulia Ricci,
  • Gabriele Siciliano,
  • Massimiliano Filosto,
  • Enzo Ricci,
  • Raffaella Cascella,
  • Emiliano Giardina,
  • FSHD Italian Clinical Group

DOI
https://doi.org/10.1186/s13148-024-01747-2
Journal volume & issue
Vol. 16, no. 1
pp. 1 – 13

Abstract

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Abstract Background Facioscapulohumeral dystrophy (FSHD) is a myopathy characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). The assessment of DNA methylation at two regions (DUX4-PAS and DR1) of D4Z4 locus proved to be an effective method to detect epigenetic signatures compatible with FSHD. The present study aims at validating the employment of this method into clinical practice and improving the protocol by refining the classification thresholds of 4qA/4qA patients. To this purpose, 218 subjects with clinical suspicion of FSHD collected in 2022–2023 were analyzed. Each participant underwent in parallel the traditional FSHD molecular testing (D4Z4 sizing) and the proposed methylation assay. The results provided by both analyses were compared to evaluate the concordance and calculate the performance metrics of the methylation test. Results Among the 218 subjects, the 4q variant type distribution was 54% 4qA/4qA, 43% 4qA/4qB and 3% 4qB/4qB. The methylation analysis was performed only on carriers of at least one 4qA allele. After refining the classification threshold, the test reached the following performance metrics: sensitivity = 0.90, specificity = 1.00 and accuracy = 0.93. These results confirmed the effectiveness of the methylation assay in identifying patients with genetic signature compatible with FSHD1 and FSHD2 based on their DUX4-PAS and DR1 profile, respectively. The methylation data were also evaluated with respect to the clinical information. Conclusions The study confirmed the ability of the method to accurately identify methylation profiles compatible with FSHD genetic signatures considering the 4q genotype. Moreover, the test allows the detection of hypomethylated profiles in asymptomatic patients, suggesting its potential application in identifying preclinical conditions in patients with positive family history and FSHD genetic signatures. Furthermore, the present work emphasizes the importance of interpreting methylation profiles considering the patients’ clinical data.

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