Development of a locus-specific HRM assay for DNA methylation analysis of the SHANK3 gene

DNA methylation within CpG islands is a key epigenetic mechanism regulating gene expression. SHANK3 encodes a synaptic scaffolding protein essential for neurodevelopment and synaptic function, and aberrant SHANK3 methylation has been implicated in neuropsychiatric disorders. To enable reliable locus-specific investigation of SHANK3 epigenetic regulation, we developed a high-resolution melting (HRM) assay. In silico screening identified a CpG-rich region upstream of exon 3 as the most suitable locus for assay design. Bisulfite-converted sequences representing fully methylated and unmethylated states were used to generate three primer sets, of which two successfully amplified the target region and produced distinct melting profiles discriminating methylated from unmethylated templates. The assay was optimized on two HRM platforms (LightCycler® 480 and CFX96), and conversion efficiency was confirmed with commercial control DNAs. This locus-specific HRM assay provides a methodological framework for qualitative SHANK3 methylation analysis and represents a promising tool for future validation studies and potential clinical investigations in neurodevelopmental and neuropsychiatric disorders.