Erdmann Hannes, Scharf Florentine, Gehling Stefanie, Benet-Pagès Anna, Jakubiczka Sibylle, Becker Kerstin, Seipelt Maria, Kleefeld Felix, Knop Karl Christian, Prott Eva-Christina, Hiebeler Miriam, Montagnese Federica, Gläser Dieter, Vorgerd Matthias, Hagenacker Tim, Walter Maggie C, Reilich Peter, Neuhann Teresa, Zenker Martin, Holinski-Feder Elke, Schoser Benedikt, Abicht Angela
Medical Genetics Center (MGZ), 80335 Munich, Germany.
Friedrich-Baur-Institute, Department of Neurology, Klinikum der Universität, Ludwig-Maximilians-Universität, 80336 Munich, Germany.
Brain. 2023 Apr 19;146(4):1388-1402. doi: 10.1093/brain/awac336.
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 bisulphite conversion with next-generation sequencing 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.
面肩肱型肌营养不良症（FSHD）的基因诊断在临床实践中仍然是一项挑战，尽管它是第三大常见的肌营养不良症，但无法通过标准测序方法检测到。传统的诊断策略涉及FSHD已知的基因参数：允许单倍型的必需存在、4号染色体q35区域D4Z4重复序列的大小减少（定义为FSHD1）或表观遗传抑制基因中的致病变异（与FSHD2一致）。潜在基因参数以及表观遗传参数（D4Z4甲基化）的不完全外显率和上位效应给诊断准确性带来了挑战，并阻碍了临床严重程度的预测。为了规避传统诊断方法的已知局限性，并用表观遗传参数补充基因参数，我们开发并验证了一种多阶段诊断工作流程，该流程包括单倍型分析和高通量甲基化谱分析（FSHD-MPA）。FSHD-MPA通过将亚硫酸氢盐转化与下一代测序和生物信息学管道相结合，确定D4Z4重复序列阵列的平均全局甲基化水平以及最远端重复单元的区域甲基化，并将这些用作诊断参数。我们将该诊断工作流程应用于148名患者的队列，并将基于FSHD-MPA的表观遗传参数与传统基因检测的基因参数进行比较。此外，我们研究了FSHD患者中最远端重复单元内的重复长度和甲基化水平与年龄校正后的临床严重程度以及疾病发病年龄之间的相关性。我们的研究结果表明，FSHD-MPA是一种强大的工具，可准确确定FSHD的表观遗传参数，能够区分FSHD患者和健康个体，同时区分FSHD1和FSHD2。甲基化水平与临床严重程度之间的强相关性表明，FSHD-MPA确定的甲基化水平解释了具有相似基因参数的个体之间疾病严重程度的差异。因此，我们的研究结果进一步证实，表观遗传参数而非基因参数代表了FSHD疾病状态，并且可能作为疾病状态的有价值生物标志物。
