Fasiyoskapulohumeral musküler distrofi (FSHD)'de bisülfit dizilemesi ile DNA metilasyon profillemesi

Facioscapulohumeral Muscular Dystrophy (FSHD) is a progressive neuromuscular disorder caused by the aberrant expression of the DUX4 gene, which is normally epigenetically silenced in somatic cells. This aberrant expression is directly linked to hypomethylation of the D4Z4 macrosatellite repeat region at chromosome 4q35, a key epigenetic alteration that plays a critical role in disease pathogenesis. FSHD is classified into two subtypes: FSHD1, which results from contraction of the D4Z4 array from 11-150 repeat units down to 1-10 repeat units, and FSHD2, which is associated with pathogenic variants in epigenetic regulators such as SMCHD1, DNMT3B, and LRIF1. Despite their distinct genetic origins, both forms exhibit a significant loss of DNA methylation at the D4Z4 locus, leading to DUX4 activation and muscle degeneration. This study aimed to implement and optimize PCR-based bisulfite sequencing, a high-resolution, single-base method for assessing cytosine methylation patterns, in order to characterize D4Z4 methylation profiles in FSHD patients, enhance diagnostic accuracy, and evaluate CpG profiling as a biomarker for disease severity. A subset of 33 patients from a larger cohort of 112 individuals was selected for detailed methylation analysis, stratified by D4Z4 repeat size and clinical subtype. Six healthy individuals were included as controls. Methylation status was evaluated in the DR1 and DR2 regions, with special focus on CpG density and correlation to clinical and genetic backgrounds. Our findings showed that 17 FSHD1 and 6 FSHD2 patients exhibited significantly reduced methylation compared to controls, while 10 patients had borderline or normal methylation levels. These results support the utility of methylation profiling in distinguishing between FSHD subtypes and clarifying cases with ambiguous genetic findings. Additionally, integration of next-generation sequencing (NGS) data from a selected group of patients enabled further investigation into potential genetic modifiers. Methylation analysis in FSHD is crucial, as it reveals the epigenetic alterations that drive aberrant DUX4 expression, enabling precise differentiation between FSHD subtypes and enhancing diagnostic accuracy. Our study highlights the value of bisulfite sequencing as a complementary clinical and research tool that not only improves diagnostic precision but also contributes to a deeper understanding of the epigenetic landscape in FSHD, thereby informing targeted patient management and guiding potential therapeutic interventions.