Development of a Locus-specific qPCR-HRM Method for DNA Methylation Analysis of the SHANK3 Gene

Purpose:DNA methylation within CpG islands is a major epigenetic mechanism regulating gene expression. SHANK3 encodes a synaptic scaffolding protein essential for neurodevelopment and synaptic function, and its aberrant methylation has been implicated in neuropsychiatric disorders. This study aimed to develop a reliable locus-specific high-resolution melting (HRM) assay for the analysis of SHANK3 methylation.Methods:In silico CpG island analysis identified a CpG-rich region upstream of exon 3 as a suitable locus for primer design. Bisulfite-converted fully methylated and unmethylated control DNAs were used to generate and test three primer sets. HRM assays were optimized and validated on two real-time PCR platforms (LightCycler® 480 and CFX96). Conversion efficiency and amplification specificity were confirmed using commercial methylated and unmethylated DNA controls.Results:Two primer sets successfully amplified the target region and produced distinct melting profiles, enabling clear discrimination between methylated and unmethylated templates. The assay demonstrated high reproducibility and consistent melting pattern separation across platforms.Conclusion:This locus-specific HRM assay provides a robust and cost-effective approach for qualitative analysis of SHANK3 DNA methylation. It offers a methodological basis for future validation studies and potential clinical investigations of epigenetic dysregulation in neurodevelopmental and neuropsychiatric disorders.