Development of a High-Resolution Melting (HRM) Assay for DNA Methylation Analysis of the SHANK3 Gene
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Background
DNA methylation within CpG islands is a key epigenetic mechanism regulating gene expression. SHANK3 encodes a synaptic scaffolding protein with critical roles in neurodevelopment and synaptic function, and aberrant SHANK3 methylation has been implicated in neuropsychiatric disorders. Reliable locus-specific assays are required to investigate its epigenetic regulation.
Methods
The SHANK3 sequence (NCBI Gene ID: 85358) was screened in silico to identify CpG islands. A CpG-rich region upstream of exon 3 was selected for assay development. Bisulfite-converted sequences representing fully methylated and unmethylated states were aligned, and three primer sets were designed accordingly. Bisulfite conversion was performed using the EZ DNA Methylation™ Kit, and conversion efficiency was confirmed with the DAPK1 control assay and commercially available methylated and unmethylated genomic DNAs. High-resolution melting (HRM) analyses were carried out on LightCycler® 480 and CFX96 platforms.
Results
Primer Sets 1 and 3 consistently amplified the target region and produced distinct melting profiles that discriminated methylated from unmethylated templates, whereas Primer Set 2 was less efficient. Gradient PCR optimization identified 54 °C as the optimal annealing temperature. Non-template controls showed late-cycle amplification and nonspecific products, but these were distinguished by melting temperatures lower than those of the expected amplicon and did not interfere with interpretation.
Conclusion
A locus-specific HRM assay for SHANK3 methylation analysis was successfully developed and optimized. The method enables qualitative discrimination of methylated and unmethylated templates, and provides a robust framework for future validation studies and potential application to clinical investigations of SHANK3 epigenetic regulation.
