Decoding common and complex genotypes of facioscapulohumeral muscular dystrophy using methylation profiling and long-read sequencing.
18 affiliations
- 1MGZ - Medizinisch Genetisches Zentrum, Munich 80335, Germany.
- 2Friedrich Baur Institute at the Department of Neurology, LMU University Hospital, Ludwig-Maximilians-Universität München, Munich 80336, Germany.
- 3Department of Medicine IV, LMU University Hospital, Ludwig-Maximilians-Universität München, Munich 80336, Germany.
- 4Department of Neurology, BG University Hospital Bergmannsheil, Ruhr-University Bochum, Bochum 44789, Germany.
- 5BG University Hospital Bergmannsheil, Heimer Institute for Muscle Research, Bochum 44789, Germany.
- 6Department of Neurology with Experimental Neurology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt Universität zu Berlin, and Berlin Institute of Health (BIH), Berlin 10117, Germany.
- 7Institute of Neurogenomics, Helmholtz Center Munich, Neuherberg 85764, Germany.
- 8Department of Neurology and Mannheim Center for Translational Neuroscience, Medical Faculty Mannheim, Heidelberg University, Mannheim 68167, Germany.
- 9Department of Neurology, University Hospital Carl Gustav Carus at TUD Dresden University of Technology, Dresden 01307, Germany.
- 10Department of Neurology, University Hospital Linz, Linz 4021, Austria.
- 11Institute for Clinical Genetics, University Hospital Carl Gustav Carus at TUD Dresden University of Technology, Dresden 01307, Germany.
- 12Department of Neuropediatrics, University Hospital Carl Gustav Carus at TUDDresden University of Technology, Dresden 01307, Germany.
- 13Department of Neurology and Center for Translational Neuro- and Behavioral Sciences (C-TNBS), University Hospital Essen, Essen 45147, Germany.
- 14Institute of Human Genetics, University Hospital Heidelberg, Heidelberg 69120, Germany.
- 15Neurologie Mingers, Übach-Palenberg 52531, Germany.
- 16Institute for Human Genetics, Hannover Medical School, Hannover 30625, Germany.
- 17Mitteldeutscher Praxisverbund Humangenetik, Leipzig 04103, Germany.
- 18Clinical Genetics and Genomic Medicine, University Hospital Würzburg, Würzburg 97080, Germany.
Abstract
Due to the complexity of the genomic region, the relationship between genotype, phenotype and epigenetics in facioscapulohumeral muscular dystrophy remains incompletely understood, and diagnostics are limited in precision. In this study, we applied Oxford Nanopore Technology long-read sequencing to characterize FSHD1 and FSHD2 patients with common and previously confirmed genotypes (n = 14). This approach enabled the simultaneous determination of allele-specific methylation profiles, haplotypes and repeat lengths. Our results confirm the presence of an asymmetric methylation gradient increasing from the proximal to the distal end of 4q35 repeat arrays with a steeper slope in FSHD1 (2.5-8.1%/repeat unit) compared to the two FSHD2 patients (0.3 and 1.2%/repeat unit) studied. The slope is, therefore, a potential marker to differentiate both types of facioscapulohumeral muscular dystrophy, being less biased by repeat length compared to parameters determined in current bisulfite sequencing-based methylation assays. By determining distal methylation allele-specifically, long-read sequencing overcomes a major limitation of such assays. Distal methylation of pathogenic (range 22-45%) and non-pathogenic repeat arrays (range 77-87%) (P < 0.0001) is clearly separated and shows a stronger correlation with age-corrected clinical severity (r = -0.81, 95% CI: -0.48 to -0.94) compared to studies based on bisulfite sequencing. Overall, this strengthens distal methylation as a superior diagnostic marker of facioscapulohumeral muscular dystrophy disease status. Detailed clinical and genetic characterization of a second cohort (n = 20) comprising patients with complex genotypes (n = 11) and FSHD2 (n = 9) was performed using our bisulfite sequencing-based methylation profile analysis, complementary methods and in three cases, long-read sequencing. Based on these results, we highlight underrecognized patient groups at risk of misdiagnosis when methylation analysis is not part of the diagnostic workup: (i) patients carrying a 4qA166 repeat array; (ii) patients with underrepresented population backgrounds; (iii) facioscapulohumeral muscular dystrophy patients carrying 11 repeat units in the absence of global hypomethylation; and (iv) FSHD2 patients without a detectable causative variant in known epigenetic suppressor genes (SMCHD1, DNMT3B and LRIF1). We hypothesize that unknown epigenetic modifiers explain the penetrance of repeat arrays with 8-10 repeat units in the European and North American populations, the pathogenicity of some repeat arrays with 11 repeat units and the hypomethylation of the entire 4q35 repeat array in some FSHD2 patients without a detected variant in known epigenetic suppressor genes. From our results, recommendations for standard diagnostics are derived. Finally, we hypothesize a preliminary model linking DNA methylation, known genetic parameters and disease severity.
Related
- Image-based assessment of muscle contractile performance in facioscapulohumeral muscular dystrophy: protocol of the MUSCLE+ observational study in the Netherlands
- Ultrasound Contrast between the Trapezius and Supraspinatus Muscles as a Potentially Specific Finding of Facioscapulohumeral Muscular Dystrophy
- Muscle strength, quantity and quality and muscle fat quantity and their association with oxidative stress in patients with facioscapulohumeral muscular dystrophy: Effect of antioxidant supplementation
- Protocol for genome-wide differential methylation using long-read DNA sequencing in the cortex of a mouse model of Alzheimer's disease.
- Arabidopsis nanopore direct RNA sequencing data for mutants in splicing, polyadenylation, and m6A methylation.
BibTeX
@article{scharf2026decoding,
title = {{Decoding common and complex genotypes of facioscapulohumeral muscular dystrophy using methylation profiling and long-read sequencing.}},
author = {Florentine Scharf and Morghan C Lucas and Stefanie Gehling and Felix Kleefeld and Veronika Schönrock and Anna Benet-Pagès and Hans-Werner Rausch and Lucas Schirmer and Jochen Schäfer and Annika Saak and Joseph Porrmann and Sylvia Hütter and Christina Emde and Anne Boblest and Svenja Neuhoff and Tim Hagenacker and Sebastian Burkart and Sebastian Mingers and Ann-Cathrine Berking and Lisa Olfe and Corinna Hendrich and Kristin Schröck and Elizabeta Sauer and Agata A Mossakowski and Kai Sendelbach and Udo Koehler and Kerstin Becker and Tobias Bartolomaeus and Ariane Hallermayr and Barbara Klink and Teresa Neuhann and Maggie C Walter and Benedikt Schoser and Angela Abicht and Hannes Erdmann},
journal = {Brain communications},
year = {2026},
month = oct,
doi = {10.1093/braincomms/fcag370},
url = {https://europepmc.org/article/MED/42909895},
note = {CC BY},
}
RIS
TY - JOUR
TI - Decoding common and complex genotypes of facioscapulohumeral muscular dystrophy using methylation profiling and long-read sequencing.
AU - Florentine Scharf
AU - Morghan C Lucas
AU - Stefanie Gehling
AU - Felix Kleefeld
AU - Veronika Schönrock
AU - Anna Benet-Pagès
AU - Hans-Werner Rausch
AU - Lucas Schirmer
AU - Jochen Schäfer
AU - Annika Saak
AU - Joseph Porrmann
AU - Sylvia Hütter
AU - Christina Emde
AU - Anne Boblest
AU - Svenja Neuhoff
AU - Tim Hagenacker
AU - Sebastian Burkart
AU - Sebastian Mingers
AU - Ann-Cathrine Berking
AU - Lisa Olfe
AU - Corinna Hendrich
AU - Kristin Schröck
AU - Elizabeta Sauer
AU - Agata A Mossakowski
AU - Kai Sendelbach
AU - Udo Koehler
AU - Kerstin Becker
AU - Tobias Bartolomaeus
AU - Ariane Hallermayr
AU - Barbara Klink
AU - Teresa Neuhann
AU - Maggie C Walter
AU - Benedikt Schoser
AU - Angela Abicht
AU - Hannes Erdmann
PY - 2026
DA - 2026/10/10
JO - Brain communications
DO - 10.1093/braincomms/fcag370
UR - https://europepmc.org/article/MED/42909895
AB - Due to the complexity of the genomic region, the relationship between genotype, phenotype and epigenetics in facioscapulohumeral muscular dystrophy remains incompletely understood, and diagnostics are limited in precision. In this study, we applied Oxford Nanopore Technology long-read sequencing to characterize FSHD1 and FSHD2 patients with common and previously confirmed genotypes (n = 14). This approach enabled the simultaneous determination of allele-specific methylation profiles, haplotypes and repeat lengths. Our results confirm the presence of an asymmetric methylation gradient increasing from the proximal to the distal end of 4q35 repeat arrays with a steeper slope in FSHD1 (2.5-8.1%/repeat unit) compared to the two FSHD2 patients (0.3 and 1.2%/repeat unit) studied. The slope is, therefore, a potential marker to differentiate both types of facioscapulohumeral muscular dystrophy, being less biased by repeat length compared to parameters determined in current bisulfite sequencing-based methylation assays. By determining distal methylation allele-specifically, long-read sequencing overcomes a major limitation of such assays. Distal methylation of pathogenic (range 22-45%) and non-pathogenic repeat arrays (range 77-87%) (P < 0.0001) is clearly separated and shows a stronger correlation with age-corrected clinical severity (r = -0.81, 95% CI: -0.48 to -0.94) compared to studies based on bisulfite sequencing. Overall, this strengthens distal methylation as a superior diagnostic marker of facioscapulohumeral muscular dystrophy disease status. Detailed clinical and genetic characterization of a second cohort (n = 20) comprising patients with complex genotypes (n = 11) and FSHD2 (n = 9) was performed using our bisulfite sequencing-based methylation profile analysis, complementary methods and in three cases, long-read sequencing. Based on these results, we highlight underrecognized patient groups at risk of misdiagnosis when methylation analysis is not part of the diagnostic workup: (i) patients carrying a 4qA166 repeat array; (ii) patients with underrepresented population backgrounds; (iii) facioscapulohumeral muscular dystrophy patients carrying 11 repeat units in the absence of global hypomethylation; and (iv) FSHD2 patients without a detectable causative variant in known epigenetic suppressor genes (SMCHD1, DNMT3B and LRIF1). We hypothesize that unknown epigenetic modifiers explain the penetrance of repeat arrays with 8-10 repeat units in the European and North American populations, the pathogenicity of some repeat arrays with 11 repeat units and the hypomethylation of the entire 4q35 repeat array in some FSHD2 patients without a detected variant in known epigenetic suppressor genes. From our results, recommendations for standard diagnostics are derived. Finally, we hypothesize a preliminary model linking DNA methylation, known genetic parameters and disease severity.
SN - 2632-1297
ER -