Detection and Discrimination between Deletional and Non-Deletional Prader-Willi and Angelman Syndromes by Methylation-Specific PCR and Quantitative Melting Curve Analysis

Abstract

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are clinically distinct neurological disorders caused by a lack of expression of oppositely imprinted genes in chromosomal region 15q11-13. The loss of expression can be due to parent-specific segmental deletions or can arise from non-deletional mechanisms, such as uniparental disomy of chromosome 15 or defects in imprinting. Most current diagnostic methods to distinguish PWS from AS require separate amplification and detection steps, and some methods cannot differentiate between deletional and non-deletional forms of these syndromes. We have developed a single-step, methylation-specific PCR, and quantitative melting curve analysis assay to identify methylation differences and copy number changes in PWS and AS. In this strategy, duplex amplification followed by melting curve analysis was performed to detect the maternally and paternally imprinted SNRPN alleles and LIS1 reference gene. To discriminate between deletional and non-deletional PWS and AS, relative peak height ratios of maternal or paternal SNRPN:LIS1 were determined, respectively. To validate the diagnostic accuracy of the analysis, methylation-specific multiplex ligation-dependent probe amplification was performed on all PWS and AS samples. Complete concordance between the melting curve analysis and methylation-specific multiplex ligation-dependent probe amplification results was observed for all PWS and AS samples. Methylation-specific PCR and quantitative melting curve analysis represents a simple, rapid, and robust alternative to methylation-specific multiplex ligation-dependent probe amplification for the detection of and discrimination between deletional and non-deletional forms of PWS and AS.