Validation of a Multiplex Methylation-sensitive PCR Assay for the Diagnosis of Prader-Willi and Angelman’s Syndromes

Background: Prader-Willi (PWS) and Angelman’s (AS) syndromes are two distinct clinical entities caused by alterations in an identical but differentially methylated region of DNA on chromosome 15q. Highly complex laboratory tests are required for diagnosis because the disorders are caused by several genetic mechanisms. Methylation-specific PCR (MSPCR) is a relatively simple alternative method to detect the methylation status of the PWS/AS region. Methods and Results: DNA was treated with sodium bisulfite, with alterations to the published method in which a neutralization step after the alkali treatment of the modified DNA enabled the use of the modified product directly in the PCR, eliminating the need for ethanol precipitation. Multiplex MSPCR using primers to methylated and unmethylated DNA was optimized to yield equal amplification efficiency for both products. Complete concordance was observed during the clinical validation of 40 previously characterized samples, except for one patient with mosaic AS detected by fluorescence in situ hybridization. Conclusion: We have developed and validated a multiplex MSPCR assay with alterations of the original published protocol that is technically robust and reproducible and can be used as a screening assay to detect PWS and AS.