Methylation of the 4q35 D4Z4 repeat defines disease status in facioscapulohumeral muscular dystrophy

Abstract

Genetic diagnosis of facioscapulohumeral muscular dystrophy (FSHD) remains a challenge in clinical practice as it cannot be detected by standard sequencing methods despite being the third most common muscular dystrophy. The conventional diagnostic strategy addresses the known genetic parameters of FSHD: the required presence of a permissive haplotype, a size reduction of the D4Z4 repeat of chromosome 4q35 (defining FSHD1), or a pathogenic variant in an epigenetic suppressor gene (consistent with FSHD2). Incomplete penetrance and epistatic effects of the underlying genetic parameters as well as epigenetic parameters (D4Z4 methylation) pose challenges to diagnostic accuracy and hinder prediction of clinical severity. In order to circumvent the known limitations of conventional diagnostics and to complement genetic parameters with epigenetic ones, we developed and validated a multistage diagnostic workflow that consists of a haplotype analysis and a high throughput methylation profile analysis (FSHD-MPA). FSHD-MPA determines the average global methylation level of the D4Z4 repeat array as well as the regional methylation of the most distal repeat unit by combining bisulfite conversion with next-generation sequencing (NGS) and a bioinformatics pipeline and uses these as diagnostic parameters. We applied the diagnostic workflow to a cohort of 148 patients and compared the epigenetic parameters based on FSHD-MPA to genetic parameters of conventional genetic testing. In addition, we studied the correlation of repeat length and methylation level within the most distal repeat unit with age-corrected clinical severity and age at disease onset in FSHD patients. The results of our study show that FSHD-MPA is a powerful tool to accurately determine the epigenetic parameters of FSHD, allowing discrimination between FSHD patients and healthy individuals, while simultaneously distinguishing FSHD1 and FSHD2. The strong correlation between methylation level and clinical severity indicates that the methylation level determined by FSHD-MPA accounts for differences in disease severity among individuals with similar genetic parameters. Thus, our findings further confirm that epigenetic parameters rather than genetic parameters represent FSHD disease status and may serve as a valuable biomarker for disease status.

D4Z4 Methylation Levels Combined with a Machine Learning Pipeline Highlight Single CpG Sites as Discriminating Biomarkers for FSHD Patients

“…By this way, they reported a reduced average methylation of the detected CpG sites in FSHD subjects, which is in accordance with our data. Moreover, they found the association of these methylation profiles with the disease severity, as also reported by previous studies, and propose the application of DNA methylation into the diagnostic workflow [ 19 ].…”

“…Indeed, both samples referred to patients harboring a 4qA/4qA genotype, which could overestimate the methylation levels due to the fact that the assay would detect the methylation levels of both alleles, in contrast to subjects with a single copy of 4qA that would provide a more precise measure. Of note, this similar issue has also been highlighted in the recent study by Erdmann et al, 2022 [ 19 ].…”

“…Indeed, various studies investigated the association of reduced D4Z4 methylation levels with the disease, though reporting variable results depending on different sample sizes, employed methodologies (BSS, long read sequencing, antibody-based methods and utilization of methylation-sensitive restriction enzymes) and analyzed region/CpG sites (whole D4Z4 unit, 5′ DUX4 -ORF, distal region of 4q35) [ 13 , 14 , 15 , 16 , 17 , 18 , 21 , 29 ]. On this subject, the study by Erdmann et al, 2022 performed an evaluation of D4Z4 methylation in a diagnostic workflow aimed at enhancing the interpretation of disease manifestations [ 19 ]. Importantly, the study is based on a BSS-NGS approach, focusing their attention on the 4q distal region and the entire repeated unit.…”

“…Considering that the DNA methylation status representative of D4Z4 locus has been recognized as a hallmark of the disease, several research studies tested it as a possible diagnostic biomarker. In particular, a number of protocols have been proposed to assess the methylation status, although different CpG sites/regions, biological sources and variable sample sizes have been used [ 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. Given these premises, mathylation analysis and Machine Learning (ML) pipelines were tested as possible methods to prioritize FSHD subjects for standard molecular testing and supporting the clinical diagnosis.…”

Association of 4qA-Specific Distal D4Z4 Hypomethylation With Disease Severity and Progression in Facioscapulohumeral Muscular Dystrophy

“…Similar issues with inconclusive diagnostic analysis of FSHD based on detection of 4qA methylation levels have also been noted in recent studies. 21,22…”